The dura mater maintains B cell lymphopoietic capacity during chronic Trypanosoma brucei infection

The dura mater maintains B cell lymphopoietic capacity during chronic Trypanosoma brucei infection | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The dura mater maintains B cell lymphopoietic capacity during chronic Trypanosoma brucei infection Matthew C. Sinton, Alice Costain, Chloe Barnes, Olivia Shorthouse, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8621800/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The dura mater hosts a rich population of B cell progenitors, but its capacity to sustain B cell development during systemic lymphopenia remains unclear. Using a murine model of chronic Trypanosoma brucei infection, which induces peripheral B cell depletion, we demonstrate that the dura mater maintains intact B cell lymphopoiesis independently of bone marrow and spleen in both male and female mice, in a process involving various chemotactic and pro-survival factors derived from the dura mater stroma. Furthermore, dura mater-derived B cells exhibit a distinct immunoglobulin repertoire that is distinct from the splenic repertoire and is dominated by Ighv1 family members. The immunoglobulins produce locally at the CNS borders are polyreactive and able to recognise both CNS and parasite antigens. Lastly, adoptive transfer of dura mater B cells, or their cognate antibodies, into B cell-deficient mice delays parasitemia onset, whereas splenic B cells from infected hosts fail to confer similar protection. These findings identify, for the first time, the dura mater as a resilient, autonomous B cell lymphopoietic niche that shapes specialized humoral responses during chronic infection, highlighting its potential role in coordinating immune defense when conventional lymphoid organs are compromised. Biological sciences/Immunology/Infection Health sciences/Diseases/Infectious diseases Biological sciences/Neuroscience/Neuroimmunology B cell lymphopoiesis CNS immune response Trypanosoma brucei infection CXCR4/CXCL12 signalling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Under homeostasis, the dura mater hosts a wide array of resident early haematopoietic precursors including those responsible for de novo B cell lymphopoiesis 1 – 3 . The vascular connections between the skull bone marrow in the calvaria and the underlying dura mater layers form an anatomical bridge 4 , 5 , suggesting that many of the early precursor populations originate from the calvaria before seeding the dura mater. However, the impact of infections that affect the central nervous system (CNS) borders on these resident precursor populations remains to be determined. Furthermore, whether the mechanisms providing protection against infection in the dura mater result in the same type of B cell responses generated in conventional lymphoid organs such as the spleen is unknown. Trypanosoma brucei parasites, the causative agents of sleeping sickness, successfully colonise a myriad of tissues during the chronic stages of infection, including the CNS borders and the brain parenchyma 6 – 11 . In the periphery, T. brucei infection results in severe peripheral B cell lymphopenia 12 – 14 , including NK cell- and neutrophil- mediated killing of conventional B-2 B cells in the spleen 15 , 16 , compromising subsequent humoral responses necessary to control the infection. This raises the question of whether alternative tissue sites can support B cell lymphopoiesis when canonical lymphoid tissues such as the bone marrow and the spleen are compromised. We previously showed that the dura mater acquires properties typically associated with ectopic lymphoid clusters allowing the generation of high affinity antibodies during the chronic stages of T. brucei infection 10 . Critically, the presence of these dura-associated ectopic lymphoid tissues under homeostasis has been independently reported in mice and humans 17 , highlighting conservation of these immunological hubs across species. In this study, we asked whether chronic T. brucei infection also disrupts dura mater B cell development, as observed in bona fide lymphopoietic organs such as the bone marrow and the spleen in response to T. brucei infection, resulting in systemic B cell lymphopenia. Here, we show that the dura mater supports B cell lymphopoiesis when peripheral lymphopoietic sites are impaired in a sex-independent manner. Mechanistically, we report that signals from the dura mater stroma, particularly those derived from fibroblasts, endothelial cells, and sensory neurons provide environmental cues for the survival, replication, and development of B cells in this tissue compartment. We revealed that this process is dependent on CXCR4/CXCL12 and IL-7/STAT5 signalling which results in sustained B cell chemotaxis and replication capacity. Critically, we show that the repertoire of B cells developing in the dura mater undergoes extensive clonal selection that is distinct from those found in the spleen. Within the IgG + B cell compartment, dura mater B cells predominantly express various members of the Ighv1 family, in particular Ighv1-15, Ighv1-26 , and Ighv1-76 compared to splenic IgG + B cells. These polyreactive dura mater-enriched IgG clones recognise a wide range of CNS and T. brucei intracellular antigens, mainly flagellar and metabolic proteins. Lastly, through adoptive transfer experiments, we demonstrate that dura mater B cells, as well as the antibodies they they produce, but not splenic B cells from infected mice, significantly delay the appearance of parasites in the circulation in B cell-deficient mice, indicating that, in µMT recipient mice, dura mater B cells delay the onset of parasitaemia more efficiently than splenic B cells from infected mice. Collectively, our data show that the dura mater can support B cell development and selection with a distinct repertoire when peripheral lymphopoietic organs are compromised. This work highlights the dura as a site of cellular and humoral responses against a pathogen that highjacks the peripheral immune system to promote chronicity. Results The dura mater supports B cell lymphopoiesis during chronic T. brucei infection During homeostatic conditions, the dura mater harbours a rich population of precursor cells, including haematopoietic stem cells (HSCs) and B cell progenitors 2 , 3 , 18 . However, the impact of infection on these tissue-resident populations remains to be elucidated. Thus, we first set out to characterise the impact of T. brucei infection, an extracellular pathogen that actively colonises the CNS and brain borders during the chronic stage of the infection (> 25 days post-infection) 9 , 10 on the B cell compartment within the dura mater using flow cytometry. Extravascular lymphocytes were identified following intravenous injection of anti-CD45 antibody ( Supplementary Fig. 1A ). Consistent with previous reports 12 , 15 , we observed a significant reduction in the B cell counts within the femur bone marrow, spleen, and skull bone marrow, indicative of a widespread B cell lymphopenia in peripheral comparments, as reported previously 12 . Unexpectedly, the number of B cells in the dura mater remained unchanged (Fig. 1A and B ) even when peripheral sites supporting B cell development are blunted. These data indicate that B cell depletion in response to T. brucei infection is tissue-dependent. To further understand the impact of infection on B cell development, we initially set out to test define B cell subsets using the Hardy fraction system 19 , but failed to reliably obtain enough resolution within the B cell compartment in the dura mater, owing perhaps to the relatively lower cell counts compared to other organs. To circumvent this issue, we employed a gating strategy previously reported 2 , 19 for the identification of dura mater B cell progenitors. This approach relies on the expression of the bona fide B cell markers B220 and CD43 to broadly classify these cells into three main stages: CD43 + B220 Low early progenitors (equivalent to Hardy fraction A encompassing pro-B cells and pro-pre B cells), CD43 Low B220 Low late progenitors (equivalent to Hardy fractions B-D encompassing precursor B cells, including small and large pre-B cells), and CD43 - B220 + mature B cells (equivalent to Hardy E, encompassing B cells ready to enter the transitional stages) ( Supplementary Fig. 1B ). The early and late progenitors were further characterised by the expression of CD93/AA4.1, IL-7 receptor, and CXCR4, whereas the late and mature stages express progressively lower levels of CD93/AA4.1 and higher levels of CD23 and IgM ( Supplementary Fig. 1B ). In naïve mice, we detected all B cell developmental stages in the femur and skull bone marrow, as well as in the spleen and dura mater, consistent with previous reports 2 , 3 , 18 (Fig. 1C). However, upon T. brucei infection, most of the B cell developmental stages in the bone marrow and the spleen decreased significantly, whereas these compartments remained intact in the dura mater (Fig. 1C, D ). These deficiencies were not restricted to bona fide B lymphopoietic organs, as B cells were also partially depleted in the gastrointestinal tract ( Supplementary Fig. 1C and D ). Additionally, there was a significant increase in V(D)J recombination events in B cells purified from the dura mater during T. brucei infection, as measured by Kappa-deleting Recombination Excision Circle (KREC) assay ( Supplementary Fig. 1E and 1F ), demonstrating that B cell maturation events within the dura mater increase during chronic T. brucei infection. These changes within the B cell compartment do not seem to be related to defects in early hematopoietic precursors, given that the frequency and abundance of cKIT + SCA1 Low (LK), cKIT + SCA1 + (LSK), and cKIT Low SCA1 Low (LS Low K Low ) hematopoietic stem cell precursors, including CD135 + CD127 + common lymphoid progenitors (CLPs), did not significantly change in response to infection in these compartments ( Supplementary Fig. 1G-I) . Furthermore, these changes were restricted to the B cell linage, as the extravascular CD3 + T cell compartment significantly increased or remained unchanged in response to infection in all these organs ( Supplementary Fig. 2F ), including in blood ( Supplementary Fig. 2G-H ), indicating that the effects of infection are limited to developing B cells in lymphopoietic organs and not due to a broad depletion of haematopoietic progenitors. Critically, we observed a significant reduction in both the frequency of intravascular and extravascular CD45 + cells in the femur bone marrow and the spleen during T. brucei infection, whereas only the intravascular CD45 + compartment changed in the dura mater ( Supplementary Fig. 2A-B ). Furthermore, both intra- and extravascular CD19 + B220 + B cells were significantly less frequent in the femur bone marrow and spleen, but the extravascular B cells did not change in the dura mater during infection ( Supplementary Fig. 2C-E ). Lastly, B cell frequencies and counts (Fig. 2A-C), as well as developmental competencies (Fig. 2D-F) were similarly detected in both males and females, indicating that these effects are sex-independent. Together, these data demonstrate that the murine dura mater maintains early, late, and mature B cell compartments during chronic T. brucei infection, independently of sex and contribution from other bona fide B cell lymphoietic organs. The dura mater is characterised by a unique set of cytokine-cytokine receptor interactions during chronic T. brucei infection. To better understand how these canonical and non-canonical lymphopoietic sites respond to infection, we initially conducted a comparative whole tissue bulk transcriptomics analysis of the dura mater, local (skull) and distal (femur) bone marrow, and spleen from naïve and T. brucei -infected mice. Pairwise analysis revealed a total of 2,216, 6,236, 805, and 8,640 differentially expressed genes during infection in the skull bone marrow, dura mater, femur bone marrow, and spleen, respectively, when compared to naïve controls ( Supplementary Fig. 3A and Supplementary table 1 and 2 ). In response to chronic T. brucei infection, the bone marrow transcriptional landscape was defined by an upregulation of gene pathways associated with antigenic presentation and allograft ( padj 1.79 − 12 and padj 5.73 − 11 ; e.g., H2-Ob, Tap1, H2-Q7, Cd8b1, H2-DMb2, H2-K1 ), and a downregulation of gene pathways related to hematopoietic cell linage ( padj 2.51 − 06 ;e.g., Cd5, Cd4, Cd3e, H2-Aa, Fcgr1, H2-DMa, Cd44, Il4ra, Il3ra, Csf1r ) and B cell homeostasis ( padj 6.06 − 05 ;e.g., Blk, Cd19, Cr2, Cd79a, Blnk, Cd22, Fos, Cd79b ), consistent with our flow cytometric analyses ( Supplementary Fig. 3B and supplementary Fig. 4 ). In contrast, we observed a significant enrichment in various pathways associated with inflammatory responses such as cytokine-cytokine receptor interactions and Th17 differentiation ( Il12rb1, Rorc, Il27 ) and chemotaxis in the dura mater, consistent with our previous reports 10 ( Supplmentary Fig. 3B and C ), and a concomitant dysregulation of gene pathways associated with extracellular matrix (ECM)-receptor interactions ( padj 1.04 − 06 ;e.g., Col4a5, Col2a1, Col4a6, Vwf, Itga7, Tnn, Tnxb, Vtn, Itgb7 ) and various synaptic innervations such as GABAergic synapse ( padj 3.05 − 04 ;e.g., Adcy1, Slc38a2, Slc38a3, Gabrd ), Glutamatergic synapse ( padj 3.84 − 05 ;e.g., Grin2d, Grin2c, Gnb3, Slc17a6, Sl17a8 ), and neuroactive ligand-receptor interactions ( padj 1.85 − 06 ;e.g., P2rx4, Adora2a, Htr7, Adra1a, Ghr, Calcr, Vip, Ntsr1 ), owing perhaps to disruptions in the the synaptic innervations of the dura mater from the trigeminal ganglia 20 , 21 . These datasets highlight broad differential transcriptional responses triggered by infection between the dura mater and canonical lymphopoietic sites. To gain additional insights into which stromal cell types may be involved in promoting B cell differentiation in the dura mater, we performed comparative single cell transcriptomic analyses of stromal and CD45 + immune cells from the dura mater and the adjacent bone marrow in the calvaria in response to chronic T. brucei infection. After removing low quality cells, we obtained a total of 74,696 high quality cells, split across the skull bone marrow (46,966 cells) and the dura mater (27,730 cells) from naïve and T. brucei infected mice (Fig. 3A and B ). These cells segregrated into 17 transcriptional clusters encompassing both immune and stromal cells. Within the immune compartment, we identified clusters expressing bona fide markers for myeloid cells (e.g., Ctsc, Lyz2, C1qa, Ccr5, Mertk, Chil3, Mpo, Ccr2, Cd177, F13a1 ), common erythroid progenitors (e.g., Sox6, Hba-a1, Hba-a2, Scl4a1 ), neutrophils (e.g., S100a8, S100a9, Lcn2, Mmp8 ), granulocytes (e.g., Cxcl12, S100a8, S100a9, Pdzm4 ), Early B cell stages (e.g., Bach2, Aff3, Ebf1, Prkch, Pax5 ), various innate and conventional T cell subsets (e.g., Cd4, Cd8a, Cd274, Tox, Ctla4 ), plasma cells (e.g., Igkc, Ighm, Jchain, Xbp1, Pou2af1 ), and hematopoietic stem cells/common myeloid progenitors (e.g., Cd86, H2-Eb1, Flt3, Etv6 ) (Fig. 3A-C). Within the stroma, we identified various vascular-associated cells subsets including lymphatic endothelial cells (e.g., Pdpn, Pecam1, Col1a1, Col1a2, Il33 ), blood endothelial cells (e.g., Pecam1, Esam, Tek, Adgrl4, Flt1 ), choroid plexus-like epithelial cells (e.g., Ttr, Enpp2, Htrc2, Slc4a10, Col9a3 ) similar to those previously reported at the dura-arachnoid interface 22 , 23 , radial glia-like cells 24 (e.g., Sox9, Pax9, Ncam1, Chl1 ), and various sensory neuron subsets (e.g., Rbfox1, Grid2, Trpv1, Dcx, Grip1, Syt1 ), likely representing innervations from the trigeminal ganglia 21 , 25 , 26 (Fig. 3A-C). We further subset the B cell compartment and identified a total of 4,450 high-quality B cells from the skull bone marrow (3,088 cells) and the dura mater (1,362 cells) (Fig. 3D and E ). These cells encompassed hematopoietic stem cells/common lymphoid progenitors (e.g., Runx2, Tcf4, Siglech, Irf8 ), early/late B progenitors (e.g., Bach2, Aff3, Ebf1, Bank1, Pax5, Ikzf3 ), as well as mature B cells (e.g., Cd19, Ms4a1, Ebf1 ), myeloid-like B cells (e.g., Saa3, Igkc, Ighm, Cd74, Itgam ) that we have detected in the dura mater previously 10 , and plasmablasts/plasma cells (e.g., Igkc, Ighm, Jchain, Inpp4b ) (Fig. 3D-F). As expected, we found HSCs/CLPs as well as three distinct transcriptional clusters of early/late B cell stages (Early/late B 1 to B 3), mature B cells, and plasmablasts/plasma cells in the skull bone marrow of naïve animals, whereas in the dura mater we identified HSCs/CLPs and cells within the early/late B cell clusters 2 and 3, characterised by the expression of genes associated with the final stages of B cell maturation such as Ebf1, Pax5, Bank1 , and Ikzf3 (encoding the transcription factor AIOLOS) 27 (Fig. 3E). In contrast, during chronic T. brucei infection, these early precursors, in particular cells within early/late B cell clusters 1, were not detected in the skull bone marrow, whereas early/late B cell clusters 2 expanded in the dura mater (Fig. 3E), in agreement with our flow cytometry results. These early/late stages B cell stages were dominated by gene pathways associated with cell cycle and apoptosis, whereas the ones in the dura mater were enriched in gene pathways associated with antigenic presentation, endocytosis, and hematopoietic cell linage commitment (Fig. 3G). These results might suggest that the signals within each tissue compartment either promote apoptosis and impaired differentiation (skull bone marrow) or activation and lineage commitment (dura mater). B cell development is intimately linked to stromal-derived cues to be successfully completed. We therefore conducted cell-cell interaction analysis using CellChat 28 to identify the potential pathways that might be involved in sustaining B cell development in the dura mater during chronic T. brucei infection. This analysis revealed that the lymphatic endothelial cells and, to a lesser extent, blood endothelial cells and myeloid cells, are predicted to be key coordinators of the interactions occurring in the skull bone marrow and dura mater during T. brucei infection ( Supplementary Fig. 4A and B ). Interestingly, previous studies have highlighted that fibroblasts mediate dura mater B cell recruitment and retention via CXCR4/CXCL12 signalling axis under homeostasis 2 , 29 , but so far this has not been tested in the context of an inflammatory meningeal disease. Our in silico prediction highlights lymphatic endothelial cells, as well as granulocytes, provide several chemoattactant cues to various immune cells in the skull bone marrow and dura mater during T. brucei infection, mediated primarily by Cxcr4-Cxcl12, Cxcr2/Cxcl12 , and Ppbp-Cxcr2 (Fig. 4A, and Supplementary Fig. 4C ). To better characterise this stroma-B cell interaction axis in the dura mater and bone marrow during T. brucei infection, we next asked whether these tissues increase their expression of CXCL12. Indeed, we observed that the production of CXCL12 is significantly increased in the dura mater in response to T. brucei infection (Fig. 4B), potentially suggesting a higher chemotactic drive towards cell retention in these tissue compartments. We then asked whether the expression of CXCR4, the main receptor for CXCL12 driving B cell recruitment during development 30 , also increased in various B cell developmental stages within these tissue compartments. CXCR4 expression did not change significantly in early, late, and mature B cells in the skull bone marrow in response to infection, whereas early B cell stages in the dura mater expressed significantly higher levels of CXCR4 during infection (Fig. 4D and E ), indicating a higher chemotactic and/or retention drive of dura mater early B cell precursors towards a CXCL12 gradient within this compartment. To functionally test whether bone marrow- and dura mater-derived B cells have a similar chemotaxis capacity mediated by the CXCL12/CXCR4 signalling axis, we established a transwell system to measure B cells to migrate towards a CXCL12 gradient. In naïve mice, we observed that both femur bone marrow- and dura mater-derived B cells efficiently migrated towards a gradient of CXCL12 to similar levels (chemotaxis index of ~ 2 in both cases) in a process dependent on CXCR4 signalling, as demonstrated by the significant reduction in migration in the presence of CXCR4 blocking antibody ( Supplementary Fig. 4D ). However, B cells obtained from the bone marrow of infected mice, but not from the dura mater, showed a diminished capacity to sense and migrate towards a CXCL12 compared to B cells from naïve bone marrow ( Supplementary Fig. 4D ), despite an increased in CXCR4 expression, indicating impaired chemotaxis in the B cells derived from the bone marrow. Unexpectedly, the CXCL-12-mediated chemotaxis of dura mater-dwelling B cells seems to be independent of CXCR4 during infection, as CXCR4 blocking antibody did not significantly reduce the migration of these cells ( Supplementary Fig. 4D ), owing perhaps to an overall higher abundance of CXCR4 in dura mater-derived B cells during infection compared to bone marrow-derived B cells. This observation, together with an increased expression of CXCL12, indicate a higher chemotaxis and retention potential in the dura mater during T. brucei infection. Once retained within the tissue, early B cell stages require molecules such as IL-7 provided by their niches to promote their proliferation, survival, and the genomic reorganisation during development 31 , 32 . We observed no significant changes in the expression of IL-7Ra (CD127) in the early B cell stages in the skull bone marrow or dura mater in response to the infection (Fig. 4F and G ). However, we observed a significant decrease in the levels of phospho-STAT5, which is a cognate IL-7Ra signal transducer, in the early, late, and mature stages of B cell development in the skull bone marrow (Fig. 4H and I ). In the dura mater, early stage B cells showed a significant increased in the levels of phospho-STAT5 in response to infection, and this remained unchanged throughout B cell development in this tissue compartment (Fig. 4H and I ), indicating that the dura mater provides critical pro-survival and replicative cues that are disrupted in the skull bone marrow. Consistent with this, chronic T. brucei infection also resulted in a comparatively higher frequency of Ki67 + B cells in the dura mater B cells compared to those in the femur bone marrow (Fig. 4I and J ). Together, these results indicate that the dura mater provide chemotactic and pro-survival cues supporting local B cell development during chronic T. brucei infection, while these niches are disrupted in the bone marrow. Dura mater B cells have a unique and expanded B cell receptor repertoire during T. brucei infection Having established that the dura mater provides a nurturing environment for the survival and development of dura mater-dwelling B cells during chronic T. brucei infection, we next asked whether these B cells are phenotypically different from those found peripherally. We first profiled the diversity of antibody isotypes detected in the spleen and dura mater in response to chronic T. brucei infection by flow cytometry. We observed that in response to T. brucei infection there was a significant increase in the number of IgM + , IgG + , and IgA + extravascular B cells in the dura mater of female mice (Fig. 5A-C). In the dura mater of male mice, only IgA + extravascular B cells significantly diminished in numbers, whereas IgM + and IgG + B cells did not significantly change in response to infection ( Supplementary Fig. 5A-C ). These responses at the brain border were in sharp contrast to those observed in the spleen, where IgM + B cells were significantly reduced in numbers in both females and male mice, without noticeable changes in the IgA + B cell counts (Fig. 5A-C and Supplementary Fig. 5A-C ). Male mice also displayed a significant reduction in the number of IgG + B cells in the spleen in response to infection that was not detected in female mice, further highlighting potential sex-dependent humoral responses to infection (Fig. 5A-C and Supplementary Fig. 5A-C ). We scarcely detected IgE + B cells in the extravascular compartment in both tissues across sexes and experimental conditions (Fig. 5A-C and Supplementary Fig. 5A-C ). These results highlight tissue-dependent effects on the humoral responses to T. brucei infection, with a broad expansion of B cell subsets occurring specifically in the dura mater of female mice. We previously reported that T. brucei infection results in the accumulation of IgG + autorreactive B cells in the dura mater of female mice 10 , consistent with our results so far. Given the potential role of IgG deposition and IgG autoantibodies in the neuropathology observed in this experimental system, we then asked whether the population of dura mater-dwelling IgG + B cells are clonally related to those found in the spleen. We also asked whether chronic T. brucei infection shapes the B cell diversity independently of other organs such as the spleen. To do this, we performed high-throughput, IgG-focussed B cell receptor (BCR) sequencing, including the heavy and light chains, comparing B cells from the spleen and dura mater of naïve mice or during the chronic stage of T. brucei infection. All dura mater and splenic BCR repertoire libraires were prepared and sequenced in parallel with identical primer pools and batches to account for potential confounders introduced by primer biases. Based on the Ighv profiling, we observed that the number of unique B cell IgG clones, as determined by the complementary-determining region 3 (CDR3) sequence, significantly expanded in the spleen during infection from ~ 17 distinct BCR clones in naïve animals to ~ 87 different Ighv clones during T. brucei infection ( Supplementary Fig. 6A ). However, in the dura mater, the overall number of B cell IgG clones significantly diminished, from ~ 112 unique BCR IgG clones based on Ighv gene detection in naïve animals to ~ 27 clones in T. brucei infected animals ( Supplementary Fig. 6A ). The same splenic clonal expansion was evident when exploring Igkv clonality, without significant changes in the dura mater repertoire ( Supplementary Fig. 6B ). Additionally, no significant differences were observed in the number of unique Iglv B cell clones in either the spleen or dura mater ( Supplementary Fig. 6C ). Overall, of the 71 and 73 Ighv genes detected in the dura mater and spleen, respectively, 69.4% (59 genes) were shared between tissues, whereas 14.4% (12 genes) were exclusively detected in the dura mater, including several members of the Ighv1 family ( Ighv1-78, Ighv1-66, Ighv1-58, Ighv1-36 ), Ighv5 ( Ighv5-6, Ighv5-12-1 ). Additionally, 16.5% (14 genes) were exclusively detected in the spleen ( Supplementary Fig. 6D ). Across matched cohorts processed with identical library preparations and IMGT-based mapping, most of the clones detected in the dura mater during infection had a significant bias towards the expression of the Ighv1 family, including Ighv1-76, Ighv1-26 , and Ighv1-15 , relative to splenic BCRs (Fig. 5D and supplementary Fig. 5E ). B cells expressing Ighv1-76, Ighv1-26 , and Ighv1-15 became more abundant in the dura mater in response to infection, from approximately 1.26%, 2.64%, and 3.08% under homeostasis to ~ 4.84%, 17.27%, and 15.08% during infection, respectively (Fig. 5D, E, and Supplementary table 5 and 6) . Further examination of the Ighv repertoire revelaed that these are public clones, as these were independently sequenced in > 2 mice within each experimental group (Fig. 5E). We observed no selection bias for particular Ighv clones in the spleen between naïve and infected animals ( Supplementary Fig. 6F ). During B cell activation, the complementary-determining region (CDR), in particular CDR3, undergo activation-induced deaminase (AID)-dependent somatic hypermutation and affinity maturation. In naïve animals, the CDR3 amino acid length was comparable between B cell clones in the dura mater and the spleen, both around ~ 12–13 amino acids in leght (Fig. 5F and G, and supplementary table 7 ). However, during chronic T. brucei infection the length of the IgH CDR3 sequences of B cell clones in the dura mater became significantly shorter (~ 11 amino acids) compared to those in the spleen (~ 13 amino acids), where no differences were detected (Fig. 5F and G, and supplementary table 7 ). This indicates that distinct insertions/deletions mechanisms for affinity maturation are at play in the dura mater compared to the spleen during chronic T. brucei infection. The usage of particular Igkv chain genes were also diverse in the dura mater during infection compared to the spleen (Fig. 5H, supplementary Fig. 5G, and Supplementary table 5 and 6 ). For instance, B cells expressing members of the Igkv6 family, including Igkv6-15, Igkv6-23, and Igkv6-32 , become more abundant in the dura mater in response to infection, from approximately 8.08%, 3.30%, and 1.54% under homeostasis to ~ 15.81%, 11.39%, and 5.33% during infection (Fig. 5I and supplementary Fig. 5G ). In contrast, there was no significant expansion of Igkv6-15 in the spleen during infection, and a more modest expansion from 1.95%, and 1.84% under homeostasis to 8.05%, and 6.7% for Igkv6-23, and Igkv6-32 , respectively (Fig. 5H and 5I , Supplementary Fig. 5H, and Supplementary table 5 and 6 ). The IgK CDR3 amino acid length of the B cells in the dura mater was significantly longer than those in the spleen in naïve animals (Fig. 5J and K, and Supplementary table 7 ), and became significantly shorter (~ 11.5 amino acids in naïve to ~ 10.5 amino acids in infected) in response to infection (Fig. 5J and K ), without noticeable changes in the splenic B cell clones. Taken together, our depth-normalised sequencing analysis demonstrate that the IgG BCR repertoire in the dura mater is uniquely enriched for members of the Ighv1 family during chronic T. brucei infection. Furthermore, these rearrangement studies support the notion that dura mater-dwelling B cells display distinctive features, including clonal selection and BCR sequence maturation, that are distinct from B cells in peripheral organs such as the spleen during chronic CNS infections. This possibly reflects the development of ectopic structures in the dura mater, which support germinal centre-like reactions during chronic T. brucei infection 10 . Dura mater B cell-associated antibodies are polyreactive and recognise a wide range of CNS antigens We previously demonstrated that chronic T. brucei infection triggers a broad autoimmune response resulting in IgG deposition in the brain 10 . Having established that three members of the Ighv1 family, namely Ighv1-15, Ighv1-26 , and Ighv1-76 , and Igkv6-32 are significantly overrepresented in the dura mater during infection when compared to the splenic BCR repertoire, we next examined whether these immunoglobulins were able to recognise host antigens. Both heavy and light chain candidates expanded in the dura mater during infection provided us with a rational for downstream functional testing. To address this, we generated recombinant monoclonal antibodies, pairing the various members of the Ighv1 identified in our screening with Igkv6-32. Once generated, we used these recombinant antibodies to screen for potential binding capacity against host antigens using a targeted array of 120 brain and neuronal-associated antigens. We observed that Ig1-15, Ig1-26, and Ig1-76 were able to significantly recognise 81% (94 antigens) of the antigens in the array, including several proteins involved with neurotransmission (GAD1/GAD67, GAD2, GAD65, GRID2, GLUD2, AchRm2, GRIA2,AchR3, mGluR1, mGluR2), CNS autoimmunity (MBP, MOG, MOBP, MAG, AQP4) and inflammation (Lactoferrin, GAPDH, S100A8/A9, LY6H, and Lipocalin-2), amongst others (Fig. 6A and 6B and Supplementary table 8 ). Interestingly, these recombinant monoclonal antibodies also recognise myelin basic protein (MBP), which we previously detected in serum and cerebrospinal fluid of chronically infected mice and humans 10 . Furthermore, Ig1-76, and to a lesser extentextent Ig1-26 recognised systemic antigens such as cytokines (e.g., IFN-α, MPO) complement (e.g., C1q), surface antigens (e.g., CD4, CD8a, b2-glycoprotein 1), viral particles (e.g., EBV, Influenza), and intracellular antigens (e.g., histones, nucleosome, dsDNA) ( Supplementary Fig. 7 ), further highlighting the broad range of antigenic recognition. To validate these findings, we developed an immunoassay with two selected antigens detected in these screenings: Lactoferrin, which is the top hit in the three monoclonal antibody screenings and is thought to be exploited by T. brucei to acquire iron from their microenvironment 33 , and myelin oligodendrocyte glycoprotein (MOG), which we previously reported being associated with T. brucei infection 10 . As negative controls, we included recombinant Activated Leukocyte Cell Adhesion Molecule (ALCAM) and Bovine Serum Albumin (BSA). Consistent with the antigen array, we observed a high binding affinity of Ig1-26 for MOG and Lactoferrin, whereas Ig1-76 seemed to preferentially detect Lactoferrin (Fig. 6C). We failed to detect a robust signal with Ig1-15 (Fig. 6C). Importantly, none of these antibodies recognised ALCAM or BSA, highlighting their potential specificity for the antigens identified in the screening. Taken together, these studies demonstrate that Ighv1/Igkv6-32 recombinant antibodies, which are overrepresented in the dura mater during T. brucei infection, recognise brain and neuronal-associated antigens. The antibodies produced by dura mater B cells recognise metabolic and flagellar T. brucei antigens Having established that the antibodies produced by dura mater B cells during chronic infection are able to bind host neuronal-associated antigens, we next asked whether these antibodies also recognise T. brucei -associated antigens. To address this, we employed a a two-step protein purification approach (gel filtration and anion exchange) from total T. brucei lysate (Fig. 7A). This combined approach enabled us to improve resolution to identify bona fide chromatographic fractions of interest using an ELISA-based screening. The initial gel exclusion chromatography step identified five fractions that showed robust positivity using either Ig1-15, Ig1-26, or Ig-76 (Fig. 7A and supplementary Fig. 8A ). hese were all confirmed to contain proteins with a wide range of molecular weights ( Supplementary Fig. 8B ). In particular, the T. brucei antigens identified by Ig1-76 and Ig1-26 spanned proteins ranging from 250 kDa to 20 kDa, whereas the ones detected by Ig1-15 were ~ 10–15 kDa ( Supplementary Fig. 8B ). Upon anion exchange chromatography, these were further resolved into nine fractions of interest that were preferentially detected by either Ig1-15 or Ig1-26 (Fig. 7A and supplementary Fig. 8C ), but none were reactive when using Ig1-76 so this antibody was excluded from downstream analyses. Downstream in silico analysis of the LC-MS/MS data from these nine fractions identified a total of 181 significant peptide hits (-Log10 p value > 20) encompassing 56 glycosomal (e.g., fructose-biphosphate aldolase, glycerol-3-phosphate dehydrogenase), flagellar (e.g., paraflagellar rod protein), nuclear and/or kinetoplastid (e.g., nucleolar protein), and cytoplasmic (e.g., heat shock proteins, alpha tubulin, polyubiquitin, calpain) T. brucei antigens (Fig. 7B and supplementary table 9 ). Unexpectedly, these antibodies had limited reactivity to variant surface glyproteins (VSGs), which are recognised to be the main immunogenic T. brucei molecules, indicating that intracellular antigens also elicit robust immune responses. Most of these antigens were detected in fractions two, three, and five, which were preferentially recognised by Ig1-26, and to lesser extent by Ig1-15 (Fig. 7B and supplementary table 9 ), and were associated with metabolic, flagellar, or stress response pathways (Fig. 7C). Indeed, using immunofluorescence and flow cytometry on cultured-adapted T. brucei , we identified that both Ig1-15 and Ig1-26 bound to intracellular antigens compared to surface antigens (Fig. 7D and E, and supplementary Fig. 8D ). Furthermore, the affinity of these antibodies to T. brucei Fructose-biphosphate-aldolase (TbFBA) and heat-shock protein 70 (TbHSP70), but not the murine counterparts, was independently confirmed by ELISA using recombinant proteins (Fig. 7F and 7G, respectively), confirming our proteomics analysis. Altoghether, these results demonstrate that the antibodies generated in the dura mater during chronic T. brucei infection can also recognise a wide range of intracellular T. brucei antigens. Dura mater B cells and the antibodies they produce delay the onset of T. brucei infection Having established that the antibodies produced by dura mater-dwelling B cells recognise T. brucei antigens, we then asked whether these B cells were able to delay the onset of infection in vivo . For this, we adoptively transferred B cells from the dura mater or spleen obtained from infected mice or naïve controls into µMT mice, which lack mature B cells and immunoglobulins, as previously reported 34 . We chose this model to explore specific B cell-dependent effects on the control of T. brucei infection systemically. Once transferred, we monitored the appearance of parasites in circulation and the levels of anti- T. brucei IgG titres in serum in these various experimental conditions. As expected, parasites were detected in circulation within ~ 48 hours in µMT mice that received PBS only (Fig. 7H) but were protected for a significantly longer period of time when the µMT mice received splenic B cells from naïve controls compared to the PBS-treated group ( supplementary Fig. 8E ), owing perhaps to the production of natural IgM by splenic naïve B cells 35 . However, chronic T. brucei infection resulted in an inability of splenic B cells to control the onset of the infection in µMT mice, as demonstrated by no significant changes in parasitaemia compared to the PBS-treated group ( Supplementary Fig. 8E ), indicating that the splenic B cells are functionally impaired, as previously reported 12 , 13 . In contrast, we found that when µMT mice received B cells from the dura mater of either naïve and T. brucei -infected animals, they displayed lower levels of parasitaemia and remained undetectable for longer, as evidenced by the sustained control of the parasite numbers for a period of ~ 72 hours (Fig. 7H and supplementary Fig. 8F ). Indeed, µMT mice that received dura mater B cells from chronically infected C57BL/6 animals showed significantly lower levels of parasitaemia compared to the matched splenic B cells counterparts (Fig. 7H), indicating that dura mater B cells are able to delay the onset of the infection more effectively than splenic B cells in the µMT model. We also noticed that the µMT mice reconstituted with dura mater B cells intraperitoneally from infected donor mice showed higher levels of anti- T. brucei IgG2a in serum compared with those that received splenic B cells (Fig. 7I). Lastly, passive transfer of recombinant Ig1-15, Ig1-26, or a combination of both, resulted in significantly lower levels of parasites in circulation compared to mice that received irrelevant mouse IgG2a control (Fig. 7J). demonstrating that antibodies contribute to early control. Taken together, these studies demonstrate that the B cells nurtured in the dura mater during chronic infection, and potentially the antibodies they produce, can delay the early onset of T. brucei parasitaemia. Discussion In this study, we demonstrate, for the first time, that the dura mater acts as an autonomous B cell niche supporting immune responses during chronic systemic inflammatory challenges, such as those triggered by Trypanosoma brucei infection. Using a flow cytometry approach to identify early, late, and mature B cell subsets 2 , we demonstrated that all B cells stages in the dura mater remain unchanged during chronic T. brucei whilst the femur and skull bone marrows, and the spleen B cell compartment, are all impaired in both male and female mice, albeit with potential sex-dependent effects on the type of humoral responses in each of these tissues. Furthermore, using whole tissue and single cell transcriptomics coupled with in vivo and ex vivo functional studies, we demonstrate that the dura mater B cells retention and survival during chronic T. brucei infection involves CXCR4/CXCL12 and IL-7/STAT5 signalling. Furthermore, we found that the B cells residing in the dura mater express a distinct BCR repertoire compared to splenic B cells during chronic T. brucei infection, resulting in the generation of polyreactive antibodies able to recognise a broad range of host and parasite proteins. Lastly, using passive and active adoptive transfer experiments, we showed that dura mater-derived B cells, but not splenic-derived B cells, from infected mice significantly delay the onset of the infection in B cell-deficient mice, highlighting the potential protective capacity of the B cells generated at the brain borders whereas splenic B cells seem functionally impaired. Given that the early B cell precursors are likely derived from the skull bone marrow and seed the dura mater through skull channels 2 – 4 , 36 , it is tempting to speculate that an increased capacity of skull bone-derived marrow B cells to migrate towards a CXCL12 gradient, in particular early B cell progenitors, results in continuous seeding of the meningeal microenvironment. Once in the dura mater, additional pro-survival factors derived from the stromal cells such as IL-7 sustain B cell development and maturation. Indeed, we detected an overrepresentation of genes associated with apoptosis and mitochondrial metabolism in the early B cell developmental stages in the skull bone marrow, whereas the same populations in the dura mater expressed genes associated with activation, antigenic presentation, and hematopoietic commitment, consistent with the notion that the bone marrow fails to provide enough survival factors, resulting in B cell depletion. These processes are likely to occur in a sex-independent manner, as the dura mater from both male and female mice showed no signs of infection-induced B cell lymphopenia. However, our results do not rule out the possibility that a fraction of mature B cells in the dura mater, in particular mature IgA + B cells, migrate to and seed the meninges compartment from other tissues such as the nasal turbinates 37 or the gastrointestinal tract 38 . We observed a strong bias towards IgA + B cell accumulation in the spleen and dura mater of male mice but not in females, indicating a sex-dependent bias in the humoral responses to infection. Future work addressing the role and impact of sex hormones on the adaptive and humoral responses to infection at the brain borders merits further investigation. We have previously reported an IL-17 signalling sex-dependent bias in the Th17 responses and weight loss elicited in male mice in response to infection 39 , and our data further uncover sex-dependent immunological responses to T. brucei infection. IL-17A signalling promotes the production of IgA in muscosal sites 40 , 41 , and indeed we observed an upregulation of genes associated with Th17 responses in the infected dura mater. Whether IL-17 signalling is critical for promoting IgA + accumulation in the spleen and dura mater of infected male mice, or whether these IgA + B cells are derived from the nasorostral and/or gastrointestinal tract and the role of IgA + B cells in the control of T. brucei infection and tissue immunopathology, merits future investigation. Our previous work highlighted the accumulation of IgG + autoreactive B cells at the brain border of female mice 10 . In the present study, we further characterised the antibody repertoire and found that it is dominated by a significant bias in the Ighv usage towards specific members of the Ighv1 family, including Ighv1-15, Ighv1-26 , and Ighv1-76 . Some of these autoantibodies recognise proteins that could play a key role in host-pathogen interactions. For instance, lactoferrin, an iron-binding glycoprotein belonging to the transferrin family, might be exploited by T. brucei for survival in microenvironments with low nutrient content such as the CSF 33 . Interestingly, some members of the Ighv1 immunoglobulin family, in particular Ighv1-69 , are thought to result in the production of anti-influenza antibodies directed against the hemagglutinin stem 42 , 43 . Using host antigen microarrays, we demonstrated that the antibodies overrepresented in the dura mater during infection were polyreactive and recognised various CNS and systemic antigens, including myelin-related proteins, which we have previously reported in the murine dura mater and in the CSF of sleeping sickness patients 10 , in addition to proteins involved in neurotransmission (e.g., AchRm2, GRIA2, AchR3, mGluR1, mGluR2), or bind to antigens that have been previously associated infection-induced narcolepsy, such as Influenza A H1N1 44–46 . Although the role of autoantibodies in the immunopathology of narcolepsy remains incompletely understood and controversial, it is tempting to speculate that some of the autoantibodies detected in dura mater in this model of infection, in particular those recognising neurotransmitter receptors, could potentially disrupt normal brain function. Future work addressing this aspect would certainly be of relevance to better understand the brain immunopathology triggered by T. brucei infection and the ensuing sleep disorders described in mice and humans 47 – 49 . The autoantibodies generated in the dura mater might play a significant role in the host-parasite interactions at the brain border. Indeed, using a wide range of orthogonal approaches we also identified that Ig1-26 and Ig1-15 also recognise a range of T. brucei antigens. Unexpectedly these were mostly associated with intracellular metabolic and flagellar proteins, and we demonstrated that these were specific against parasite but not host antigens in independent studies. These observations are intriguing as it was long assumed that most of the antibodies generated against T. brucei recognise surface antigens, in particular VSGs, with limited work describing the antigenicity of intracellular proteins 50 , 51 . Given the predominance of intracellular parasite antigens recognised by these antibodies and limited VSG reactivity, we interpret the early, partial reduction in parasitaemia as consistent with effector-mediated mechanisms rather than direct neutralisation. We did not formally test complement or Fc receptor dependence on this process, and therefore cannot yet describe the observed protection to specific humoral effector pathways. Future work will test whether complement activation and/or Fc receptor engagement are required for this effect. Our work broadens our understanding of parasite-derived factors that may participate in the immune response, and further highlights the importance of understanding tissue-specific responses to this infection. Lastly, using adoptive B cell transfer experiments and passive transfer of dura mater-enriched recombinant antibodies, we demonstrated delayed onset of T. brucei infection in µMT mice. Furthermore, the protective effects observed in transfer experiments relied on measuring parasites in circulation and we interpret these findings as evidence that dura mater-derived humoral effector molecules can potentially influence systemic parasitaemia. However, we did not quantify parasite burden or antibody deposition at the dura mater-CNS interface after adoptive transfer experiments in mMT mice, and this cannot directly ascribe effects at the CNS stage. Although the mechanisms involved in the observed protection in µMT mice are likely to be diverse, it is possible that the autoantibodies generated against host antigens such as lactoferrin might indeed represent a host strategy to limit parasite replication and survival in the CNS. Future work is required to test whether the generation of autoantibodies can indeed impose a bottleneck for parasite survival in various tissues. Since B cells are key players in neuroinflammatory and autoimmune disorders, our findings bring us closer to understanding the origin and functional relevance of the B cells generated at the brain borders in the context of pathological conditions that affect brain health and resilience during infection. Our study provides novel insights and functional relevance of dura mater B cells in the context of infection-induced systemic B cell lymphopenia. However, it is important to acknowledge key limitations. Firstly, the abundance of dura mater B cells compared to other tissues precluded us from gaining additional granularity. However, the studies presented here are consistent with previous observations under homeostasis and highlight the physiological relevance of the B cells developing at the brain borders compared to canonical lymphopoietic sites 3 . Secondly, our BCR repertoire profiling targeted class-switched IgG + B cells. We did not generate IgM or IgA BCR repertoires; thus, the V-gene usage cannot be generalised to non-IgG compartments without additional profiling and this will need to be addressed in future work. Whether additional heavy-light chain pairings, result in similar polyreactivity merits further investigation. A direct comparison of dura mater versus splenic antibody preparations were not performed due to suboptiomal recombinant yields from putative splenic clones. However, we used the IgG-targeted BCR information to start exploring the role of dura mater-enriched immunoglobulins in the recognition of host and parasite antigens, significantly expanding our current knowledge of the reactivity of meningeal B cells during chronic neuroinflammation. Lastly, we were not able to purify enough specific B cell subsets from dura mater (e.g., B-1 cells) for adoptive transfer experiments using µMT mice. All of the adoptive transfer experiments resulted in systemic effects that could be attributed to more than one B cell subset (e.g., IgM-producing B cells, innate B-1 cells, class-switched B cells), and therefore we could not disentangle their relative contribution to the observed effects in infected mice. Future studies combining fate mapping and conditional knockouts for these various subsets would enable us to understand these processess in more details. In summary, we provided a comprehensive overview of the B cell landscape in the dura mater in the context of infection-induced impaired B cell lymphopoiesis, providing evidence of the nature of the antibodies produced locally at the brain borders and their potential role in sustaining critical host-pathogen interactions mediating both anti-parasitic and autoimmune responses. Future work exploring the relationship between these locally-generated antibodies and the behavioural changes observed in this infection model will further unravel the mechanistic links between cellular and humoral immunity and behaviour. Materials and methods Ethical statement. All animal experiments were approved by the University of Glasgow Ethical Review Committee and performed in accordance with the Home Office guidelines, UK Animals (Scientific Procedures) Act, 1986 and EU directive 2010/63/EU. All experiments were conducted under SAPO regulations and UK Home Office project licence number PP5602024 to Juan F. Quintana. The in vivo work presented in this study was conducted between 25-30-days post-infection (dpi) and correlated with increased clinical scores and procedural severity. Murine infections with Trypanosoma brucei . Six- to eight-week-old female C57BL/6J mice (JAX, stock 000664), mMT mice (JAX, stock) , were inoculated by intra-peritoneal injection with ~2 x 10 3 parasites of strain T. brucei brucei Antat 1.1E 52 . Parasitaemia was monitored by regular sampling from tail venesection and examined using phase microscopy and the rapid “matching” method 53 . Uninfected mice of the same strain, sex and age served as uninfected controls. Mice were fed ad libitum and kept on a 12 h light–dark cycle. All the experiments were conducted between 8h and 12h. For sample collection, we focussed on 25-30 days post-infection unless stated otherwise, as this has previously been shown to correlate with parasite infiltration in the epidural space 54,55 . Tissue processing and flow cytometry analysis. To discriminate circulating versus brain-resident immune cells, we performed intravascular staining of peripheral CD45 + immune cells, as previously reported 56 . Briefly, a total of 2 mg of anti-mouse CD45 antibody in 100 ml of 1X PBS was injected intravenously ~3 minutes prior culling. Mice were euthanised as described above and transcardially perfused with ice-cold 0.025% (wt/vol) EDTA in 1X PBS. Whole dura mater were enzymatically digested with Collagenase VIII (1 mg/ml) and DNAse I (1 mg/ml; Sigma) in 1X PBS (HSBB) (Invitrogen) for ~30 minutes at 37 °C and with agitation at 200 r.p.m., according to previously published protocols 57 . Femur samples were cleared from muscle and skin prior to bone marrow isolation. Skull caps were cut into ~1mm pieces. These tissues were then placed on a 0.5 mL eppendorf tube with a narrow opening at the bottom. This tube was then placed inside a 1.5mL Eppendorf and centrifuge at 800g for 10 minutes to harvest either femur or skull bone marrow. Spleen tissue were first pressed onto 70 mm nylon mesh filters to obtain single cell suspensions that were then washed twice with complete RPMI 1640. Red blood cells from both splenic and marrow cell suspensions were lysed using ACK lysis buffer (Thermo) on ice for a total 10 minutes. ACK lysis buffer was then diluted ten times with 1X PBS and cells peletted by centrifugation at 800g for 10 minutes at 4 o C. Large intestines were opened longitudinally and washed in 1x HBSS before incubation for 30 mins at 37 o C at 200 rpm in strip media (2 mM HEPES, 5 mM EDTA and 1mM DTT). The tissue was removed and placed into digest media (2 mg/mL Dispase and 2 mg/mL Collagenase I) and incubated for 30 mins at 37 o C at 200 rpm. Cells were then passed through a 100 mM strainer and pelleted by centrifugation at 400g for 5 minutes at 4 o C. For flow cytometry analysis, single cell suspensions from blood, femur bone marrow, skull bone marrow, spleen, and dura mater were resuspended in ice-cold FACS buffer (2 mM EDTA, 5 U/ml DNAse I, 25 mM HEPES and 2.5% Foetal calf serum (FCS) in 1X PBS) containing Horizon Brilliant stain buffer (25 ml/sample; BD Biosciences) and stained for extracellular markers. We used the following commercially available antibodies from Biolegend: CD45-APC-Cy7 (clone 30-F11, 2 mg/100 ml 1X PBS i.v.), TER-119-APC-Cy7 (clone TER-119; 1/400), CD19-APC-Cy7 (clone 1D3/CD19; 1/400), F4/80-APC-Cy7 (clone BM8; 1/400), CD45-BV510 (clone 30-F11; 1/400), CD45-BV785 (clone 30-F11; 1/400), CD19-PE-Cy5 (clone 6D5; 1/400), CD19-FITC (clone 6D5; 1/400), B220-BV605 (clone RA-6B2; 1/400), B220-BV650 (clone RA3-6B2; 1/400), CD43-APC (clone S11; 1/400), CD43-AF700 (clone S11; 1/400), cKIT-AF488 (clone 2B8; 1/200), SCA1-APC (clone D7; 1/100), CD135-PE-Cy5 (clone A2F10; 1/200), CD127/IL-7Ra-PE (clone S1006K; 1/200), CD3-APC-eFluor700 (clone 17A2; 1/400), CXCR4-BV421 (clone L276F12; 1/400), IgD-BV421 (clone 11-26c.2a; 1/800), IgM-BV711 (clone RMM-1; 1/800). For intracellular staining, we used the True-Phos Perm buffer set (Biolegend) to fix cells for 10 minutes on ice, followed by permeabilization for 30 minutes on ice. Cells were then pelleted at 500g for 10 minutes, and resuspended in 100 mL of permeabilization buffer containing Horizon Brilliant stain buffer as above. The following antibodies were used for intracellular statining: Ki67-PE-Cy7 (Biolegend, clone 11F6, 1/500), pSTAT5-PE (BD Biosciences, clone 47/Stat5-pY694, 1/500), and IgA-PE (clone TRFK5; 1/800). Samples were incubated overnight at 4 o C protected from light, and then washed twice with FACS buffer and resuspended in 100 mL of FACS buffer. Samples were run on a flow cytometer LSRFortessa (BD Biosciences) and analysed using FlowJo software version 10 (Treestar). KREC assay. We followed a protocol previously published for KREC assay in the dura mater 3 . Briefly, after perfusion, the dura mater was peeled off from the skull and immediately placed in lysing buffer (10 mM Tris-HCl pH 8.0) containing 100 mg/ml of proteinase K and total DNA was further extracted using the DNeasy Blood & tissue Kit (Qiagen). Total gDNA was eluted in 30 ml of ultrapure water (Qiagen) and quantified using a Nanodrop. A total of 50 ng of total DNA was used as input for PCR using the primers specific for KREC (Forward: 5’-GGAGTGGATTCAGGACACTGCT, Reverse: 5’-CTCCAATAAGTCACCCTTTCCTTGT). Gapdh was used as loading control (Forward: 5’-ACCACAGTCCATGCCATCAC, Reverse: 5’-TCCACCACCCTGTTGCTGTA). All primers were adquired from Integrated DNA Technologies. PCR reactions were perfomed for 30 cycles (94 o C for 3 minutes, 55 o C for 30 seconds, and 72 o C for 1 minute) using Q5 high-fidelity DNA polymerase with high GC content buffer (New England Biolabs). PCR products were further resolved by electrophoresis in 1% agarose gel. For quantitative PCR, we used the sample input gDNA and primers but used the Luna Universal qPCR Master Mix (New England Biolabs) with the same PCR conditions using QuantStudio qPCR system (Thermo Fisher). Whole mount dura mater preparation and immunofluorescence . After euthanasia, the skull caps were carefully removed using fine tweezers and scissors and placed immediately in 10% neutral buffered Formalin (NFB) for 10 minutes at room temperature. Following fixation of the skull caps, for single molecule fluorescent in situ hybridisation (smFISH) experiments, mounted dura mater specimens were dehydrated in 50, 70 and 100% ethanol. RNAscope 2.5 Assay (Advanced Cell Diagnostics) was used for all smFISH experiments according to the manufacturer’s protocols. We used RNAscope probes against mouse Col1a2 on channel 1 (Cat No. 585461), Pdgfrb on channel 2 (Cat. No. 411381-C2), and Cxcl12 on channel 3 (Cat. No. 422711-C3). All RNAscope smFISH probes were designed and validated by Advanced Cell Diagnostics. Images were acquired using a snapshot widefield fluorescent microscope (Zeiss) and adjusted for brightness and contrast using Fiji. Single stain and unstained controls to set up signal-to-noise threshold during post-acquisition analyses. Ex vivo chemotaxis assay. Transmigration of precursor B cells was assessed in 6.5-mm diameter 24-Transwell chemotaxis chambers (Costar, Cambridge, MA) with a pore size of 5 mm. Briefly, freshly prepared skull bone marrow and dura mater preparations were resuspended in RPMI-1640 medium supplemented with 10% foetal calf serum and 1/100 Penicillin/Streptomycin, and 100 ml of cell suspension at a concentration of 2.5x10 5 cells were seeded loaded in the upper chamber of the Transwell culture insert. Filters were transferred into the wells containing RPMI-1640 medium supplemented with 10% foetal calf serum and 1/100 Penicillin/Streptomycin and 100 ng/ml of recombinant mouse CXCL12 (R&D systems, 460-SD-050). The chambers were incubated for 3 hours at 37 °C in 5% carbon dioxide. After incubation, cells remaining in the upper chamber, as well as those that had migrated into the lower chamber, were harvested, counted using an automated Countess II Cell Counter (Thermo Fisher), and analysed by flow cytometry in a Fortessa II (BD Bioscience) to specifically quantify the number early, late, and mature B cells in all the fractions harvested. ELISA. After preparing single cell suspensions of skull bone marrow and dura mater, CD45 + immune cells were depleted using mouse CD45 magnetic beads (Miltenyi, 130-052-301). The flowthrough, considered to be depleted of CD45 + cells, was pelleted at 10,000g for 10 minutes at 4 o C, resuspended in 200 ml of RIPA buffer (ThermoFisher, 89900) supplemented with cOmplete EDTA-free Protease Inhibitor cocktail (Roche, 11836170001). Total protein was quantified using a nanodrop and 50 ml aliquots were prepared and stored at -80oC until analysis. Mouse CXCL12 (Biolegend, 444207) ELISA kit was used to determine the levels of these two molecules in tissue, according to the manufacturer’s instructions. 450nm absorbance was measured using a Varioscan ELISA plate reader, and the quantification was performed against a battery of standards of known concentrations. Data structure and statistical test were employed to determine normality and skewness of the data prior to assessment of statistical significance. Whole tissue processing for bulk transcriptomics. Mice were euthanised by cardiac puncture and perfused with 30-40 mL of ice-cold 1X PBS containing 2 mM EDTA to avoid clotting. Following euthanasia, skull and femur bone marrow, as well as spleen and dura materwere rapidly dissected. Any remaining blood was lysed with ACK lysis buffer (Thermo Fisher, cat. No. A1049201) for 10 minutes on ice, and the remaining cells were pelleted at 500g for 10 minutes at 4 o C and resuspended in Trizol (Invitrogen, cat. No. 15596026). Total RNA was then purified using the RNeasy Kit (Qiagen) as per the manufacturer’s recommendations. The RNA was purified in 30 µL of DPEC-treated water (Thermo Fisher, R0601), and RNA concentration measured on a NanoDrop™ 2000 (Thermo Fisher Scientific). Samples were shipped to Novogene (Cambridge, UK) to undergo quality control, library preparation and sequencing. RNA integrity was assessed using an RNA Nano 6000 Assay Kit (Agilent Technologies) with a TapeStation (Agilent Technologies), as per the manufacturer’s instructions. Samples with an RNA integrity number (RIN) of >6.0 were qualified for RNA sequencing. Bulk transcriptomics Library preparation. Library preparation was performed by Novogene (Cambridge, UK). Messenger RNA (mRNA) was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in a First-Strand Synthesis Reaction Buffer (5X). First-strand cDNA was synthesised using random hexamer primers and M-MuLV Reverse Transcriptase (RNase H-). Second-strand cDNA synthesis was then performed using DNA Polymerase I and RNase H. Remaining overhangs were converted to blunt ends via exonuclease/polymerase activity. Following adenylation of 3’ ends of DNA fragments, adaptors with hairpin loop structures were ligated. To select cDNA fragments of 370–420 bp in length, library fragments were purified using AMPure XP beads (Beckman Coulter), as per the manufacturer’s instructions. PCR was then performed using Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) primers. Finally, PCR products were purified (AMPure XP system) using AMPure XP beads (Beckman Coulter), as per the manufacturer’s instructions, and library quality was assessed using a Bioanalyzer 2100 (Agilent Technologies). Bulk transcriptomics sequencing and data analysis. Clustering of the index-coded samples was performed on a cBot Cluster Generation System using a TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, libraries were sequenced on an Illumina Novaseq 6000 platform and 150 bp paired-end reads were generated. Raw reads in fastq format were processed through proprietary Perl scripts developed by Novogene (Cambridge, UK). Clean reads were obtained by removing reads containing adaptors, poly-N, or low-quality reads from raw data. Concurrently, the Q20, Q30 and GC content of the clean data was calculated ( supplementary table 1A ). Genome and genome annotation files (Genome Reference Consortium Mouse Build; GRCm39) were downloaded. An index of the reference genome was built using Hisat2 (v2.0.5) and paired-end clean reads were aligned to the reference genome using Hisat2 (v2.0.5). The featureCounts (v. 1.5.0-p3) package was used to count read numbers mapped to each gene, before calculating the Fragments Per Kilobase of transcript sequence per Millions base pairs (FPKM) of each gene using the length of the gene and reads count mapped to this gene. Differential expression analysis was performed using the DESeq2 R package (v. 1.20.0). The resulting P values were adjusted using the Benjamini and Hochberg approach to control false discovery rate. Genes with an adjusted P value of <0.05 were assigned as differentially expressed. Pathway enrichment analysis of differentially expressed genes was performed using the DAVID platform, mapping genes to the KEGG database. KEGG terms with an adjusted P value < 0.05 were considered significantly enriched ( supplementary table 1B ). Heatmaps were generated using the pheatmap (Version 1.0.12) and Tidyverse packages in R (Version 4.2.1). Samples were clustered by Euclidean distance. PCA analysis was performed using ggplot2, with naive and infected male and female mice plotted separately. Comparative single cell transcriptomics profiling of the murine skull bone marrow and dura mate. Single cell suspensions from the calvaria bone marrow and dura mater were obtained as described above. Once single cell suspensions were obtained from the whole dura mater, calvaria bone marrow, and femur bone marrow, cells were counted using a hemocytometer, fixed using the EvercodeTM Cell Fixation kit v2 (Parse Bioscience). Cells were pelleted for 10 minutes at 600g and 4 o C in 15 ml Falcon tubes pre-coated with 1% BSA to minimise cell adherence and losses. Cell pellets were resuspended in 750 ml of Cell Prefixation buffer, followed by passage through a 40 mm cell strainer to remove cell clumps and addition of 250 ml of Fixation solution. The cells were then incubated for 10 minutes on ice, resuspended in 150 ml of Cell buffer and diluted at a final density of ~500 cells/ml (dura mater) or 10,000 cells/ml (calvaria and femur bone marrow). Libraries were prepared using a split-pool combinatorial barcoding strategy using the Evercode whole transcriptome v2 (Parse Bioscience). The final libraries (~300-500 bp in length) were eluted from SPRI beads using molecular grade water and analysed on a TapeStation. The resulting libraries were sequenced on a Novaseq X plus Illumina platform using a 450 million read, 150bp paired-end sequencing strategy at a depth of around 30,000-50,000 reads per cell. The sequencing files were first converted to FASTQ format using BCL2Fastq. These FASTQ files were then processed with Parse Pipeline v1.6.2. The pipeline demultiplexes reads from the Evercode single-cell RNA-seq libraries using the four-round combinatorial barcodes and unique molecular identifiers (UMIs), followed by alignment to the Parse Biosciences custom GRCm39 reference genome. After processing, all eight libraries were merged using the pipeline’s comb option. The resulting uniquely mapped UMI counts were output as a gene-by-cell count matrix in sparse format. All downstream analyses were performed using Scanpy v1.11.2. Low-quality cells were removed to minimize technical noise and ensure reliable downstream analysis. Cell quality was assessed using three metrics: the number of UMIs per cell barcode (library size), the number of detected genes per cell, and the proportion of UMIs mapped to mitochondrial genes. We first excluded genes not expressed in at least five cells, then filtered out cells with fewer than 300 detected genes. Violin plots of the three quality metrics were generated for each sample to identify outliers that might represent doublets or multiplets. Cells with more than 50,000 total UMIs were removed as potential doublets, and those with more than 6,000 detected genes were also excluded to maintain comparable gene expression distributions across samples. After filtering, the following cell counts remained for downstream analysis: 9,619 and 9,915 cells from the naïve and infected skull bone marrow, respectively, and 4,156 and 13,607 cells from the naïve and infected dura mater, respectively. The first step in visualization and clustering was to identify highly variable genes (HVGs). To do this, we used Scanpy’s highly_variable_genes() function with the parameters min_mean = 0.0125 , max_mean = 3 , and min_disp = 0.25 . These HVGs were then used for dimensionality reduction via principal component analysis (PCA). The dataset was further reduced to two dimensions using either t-distributed stochastic neighbor embedding (t-SNE) or UMAP, using the first 30 principal components as input. Finally, we applied Leiden clustering to group the cells. To identify the marker genes for a cluster, we compared that cluster with all other clusters using t-test. We used the ‘ rank_gene_groups’ function from Scanpy package to do this differential expression. We then reported top 200 genes that were differentially expressed in that cluster as the marker for the cluster. These marker genes were then used to annotate the cell types of a cluster. To identify the B-cell subclusters, we first subsetted the aggregated population by selecting only Early B cells and B/plasma cells. We then recalculated PCA and performed Leiden clustering on this B-cell subset. Finally, we applied a t-test using the rank_genes_groups function to identify marker genes for the resulting B-cell subclusters. Cell-cell communication prediction analysis. We performed Cell–cell communication analysis using CellChat v.2.2.0 28 in R v.4.5.2. CellChatinfers intercellular signaling networks from single-cell RNA-sequencing data. After preprocessing and normalization of the expression matrix, cells were grouped according to their sample+annotated cell types. CellChat objects were constructed following the standard workflow. We applied the CellChat database of ligand–receptor interactions to identify statistically significant signaling events, using default parameters for overexpression analysis and permutation testing to assess interaction probability. Communication networks were quantified and visualized to compare signaling strength and patterns among cell populations. Pathway-level aggregation was then used to determine dominant outgoing and incoming signaling pathways for each cell group. All analyses were conducted using CellChat v.2.2.0 28 , and visualization functions within the package were used to generate network and pathway plots included in the study. B cell receptor profiling. Dura mater and splenic B cells from naïve and infected mice ( n = 6 mice/group/tissue) were purified using a magnetic pan-B cell Isolation kit (Miltenyi, Cat. No. 130-095-813). Magnetically sorted cells were washed with ice-cold 1X PBS supplemented with 0.5% BSA and 2 mM EDTA, and cells resuspended in Trizol and kept at -20 o C for subsequent RNA extraction. Total RNA was extracted using the RNeasy mini kit (Qiagen, 74104) and eluted in 30 ml of DPEC-treated water. The RNA integrity was assessed the RNA ScreenTape (Agilent, cat. No. 5067-5576) on a TapeStation. High-quality RNA (integrity value > 7.5) samples from matched spleen and dura materB cells from naïve and infected animals at 28 days post-infection ( n = 6 samples/tissue/time point) was submitted for IgG-specific heavy and light chain B cell receptor library preparation, sequencing, and analysis to Genewiz using the company’s immunogenomics pipeline. On average, we obtained 144,740,272 raw reads with a Q30 value of 90%. Upon filtering low-quality reads, we obtained a total of 125,241,964 clean reads with a Q30 value of 93% ( supplementary table 2A ). The assembled reads were blasted against IMGT reference database to identify the best match of germline V(D)J genes, and CDR1, CDR2, CDR3 variable region sequences. During the mapping and clustering, the clones’ abundance was calculated for each type of chain. For a detected CDR3 sequence, if CDR1 and CDR2 sequences were not detected from input assembled reads, ‘N’ and ‘X’ will be assigned according to nucleotide sequence and amino acid sequence. V(D)J diversity was estimated by mapping against the IMGT database 58 and represented as frequency of the total clones detected. To analyse CDR3 amino acid usage frequency, CDR3 sequences were clustered according to similarity (threshold: 0.8). Weblogo software 59 was used for visualising CDR3 amino acid patterns of each cluster. Each logo consists of stacks of symbols and the size of letter represents its frequency from unique and abundance levels. All tables and weblogo pictures can be accessed in supplementary table 2B . Adoptive and passive transfer experiments. We followed a previous report to set up the dura mater B cell adoptive transfer experiments 34 . B cells were magnetically sorted from spleen and dura mater from naïve and animals infected with T. brucei Antat1.1E at day 25 post-infection. To increase the cell recovery, we pooled 7-9 dura mater prior to B cell purification. Purified B cells were then resuspended in sterile 1X PBS to a final density of ~10 4 B cells/ml and were then adoptively transferred into mMT recipient mice ( n = 4-5 mice per group) via intraperitoneal injection. As a control group, mMT mice received sterile 1X PBS alone. Recipient mMT mice were infected with 10 3 T. brucei Antat1.1E intraperitoneally (~1 trypanosome per every ten B cells) ~18 hours after cell transfer. Mice then were monitored daily for the presence of parasites in blood via tail venepuncture, as well as for body weight and food intake. The experiment was terminated when all the mice displayed patent parasitaemia. For IgG transfer experiments, mMT recipient mice ( n = 4 mice per group) were inoculated via intraperitoneal injection with 5 mg/kg of bodyweight using Ig1-15, Ig1-26, both recombinant antibodies, or irrelevant mouse IgG2a one day prior to and on the day of infection. Mice were then infected with 10 3 T. brucei Antat1.1E intraperitoneally, and monitored daily for the presence of parasites in blood via tail venepuncture, bodyweight, and food intake. The experiment was terminated when all the mice reached ~10 8 parasites/ml. Detection of host antigens detected by autoantigens Recombinant monoclonal antibodies were generated by Genescript by selecting the most abundant heavy chains detected during infection in the dura mater tissue ( Ighv1-15, Ighv1-26 , and Ighv1-76 ) and the most abundant light chain ( Ighv6-85 ). Selected plasmids were transfected into CHO cells lines and expressed in a small scale (~4 mL) as soluble proteins. We attempted to generate further monoclonal antibodies with additional pairings (e.g., combining Ighv1-5 and Ighv8-5 with Igkv6-15) but these pairings resulted in poor expression and secretion, and we therefore decided not to pursue these. Once generated, these recombinant monoclonal antibodies were assessed using a commercial microarray-based platform (GeneCopoeia, PA002). Briefly, antibody concentrations ranging from 5 mg to 0.05 mg were hybridised to distinct microarray spots containing 120 native brain and neuronal-associated antigens spotted onto nitrocellulose fibres (adhered to glass slides). Next, the slides were incubated with a fluorescently-coupled anti-IgG secondary antibody, and microarrays were scanned using a GenePix 4400A microarray scanner. Raw fluorescence data was normalized to PBS controls on each slide. The data presented in the heatmaps are normalised signal-to-noise (SNR) ratios. Selected candidates were anlyse independently using ELISA. Briefly, 5 mg/ml of recombinant mouse MOG (RDsystems), ALCAM (RDsystems), Lactoferrin (Abcam), or BSA (Thermo) were use to coat ELISA plates in carbonate buffer (Biolegend) overnight at 4 o C. Next day, plates were washed at least five times with ELISA wash buffer (Biolegend), blocked with blocking buffer (1X PBS containing 2% BSA) for 2 hours at room temperature, followed by 3 washed with ELISA wash buffer, and incubation with recombinant monoclonal Ig1-15, Ig1-26, or Ig1-76 coupled to horseradish peroxidase serially diluted in blockling buffer for 1 hour at room temperature. Plates were then washed at least 5 times with ELISA wash buffer and incubated with 3,3’,5,5’-tetramethylbenzidine (TMB) for the development of a colorimetric reaction at room temperature. Reactions were stopped using 1X Stop buffer (Biolegend) and the plates were read at 405 nm with a 567 nm correction readout using a Varioscan ELISA plate scanner. Trypanosoma brucei immunofluorescence and flow cytometry In addition to the host antigen screening, we also assessed the capacity of the recombinant antibodies to potentially recognise T. brucei antigens. T. brucei Lister 427 were cultured in HMI-9 supplemented with 10% FBS and 1% Penicillin/Streptomycin at 37 o C and 5% CO 2 . Prior to harvesting, parasites were incubated with 5 mM MitoSox Red for 10 minutes at 37 o C and 5% CO 2 that we used here as an intracellular marker. Parasites were harvested during the log phase of growth (~0.75-1x10 6 parasites/ml) by centrifugation at 500g for 10 minutes and washed twice with ice-cold 1X PBS. Once washed, parasites were fixed with 4% PFA on ice for 10 minutes and seeded on poly-lysine-coated slides for ~30 minutes at room temperature protected from light. Where indicated, the parasites were then permeabilised with 1X PBS containing 1% Triton X-100 for 10 minutes at room temperature. Once permeabilised, the cells were blocked with 1X PBS containing 1% BSA for 30 minutes at room temperature, following by incubation with recombinant monoclonal antibodies, previously labelled with ReadyLabel antibody labelling kit with Alexa Fluor 647 (Thermo), diluted 1/50 in blocking buffer (1X PBS containing 1% BSA) overnight at 4 o C. Irrelevant mouse IgG2a antibody (Nobus Biologicals) were coupled to Alexa Fluor 647 an antibody labelling kit (Thermo Fisher) and were included as controls. Slides were then washed 5 times with ice-cold 1X PBS and mounted using Antifade Gold containing DAPI (Vectashield). Images were acquired using a snapshot widefield fluorescent microscope (Zeiss) and adjusted for brightness and contrast using Fiji. Single stain and unstained controls to set up signal-to-noise threshold during post-acquisition analyses. For flow cytometry analysis, the parasites were resuspended in ice-cold 1X PBS after harvesting, fixed with 4% PFA and washed twice with ice-cold 1X PBS. Samples were split into two fractions, one containing Triton X-100 in 1X PBS for intracellular staining or without Triton X-100 for surface staining. Samples were then blocked with 1X PBS containing 1% BSA for 30 minutes at room temperature, followed by incubation with the recombinant monoclonal antibodies or irrelevant mouse IgG2a antibodies coupled to Alexa Fluor 647 for 1 hour on ice. Samples were then washed three times with FACS buffer (1X PBS containing 2.5 mM EDTA and 2.5% FBS) prior to analysis on a BD Bioscience LSR Fortessa. FCS files were then processed and analysed using FloJo version 9.0. Trypanosoma brucei protein purification, fractionation, and antigen discovery T. brucei Lister 427 were harvested during the log phase of growth (~0.75-1x10 6 parasites/ml) by centrifugation at 500g for 10 minutes and resuspended in modified TEN buffer (150 mM NaCl, 10 mM MgCl2, 1 mM DTT, 50 mM Tris-HCl pH 7.4, 5 mM EDTA, 1% Triton X-100) containing 1X Complete protease Inhibitor cocktail (Roche). The cells were vortexed at full speed (~3,200 rpm) three times for 5 minutes each, followed by sonication three times for 20 secons each at 40% intensity. Cell lysates were cleared by centrifugation at 10,000rpm at 4 o C for 10 minutes to remove any cell debris. The protein content of the cleared supernatant was quantified using the BCA protein assay (Thermo). T. brucei lysates were diluted in carbonate buffer pH 9.5 to a final concentration of 50 mg/ml and used to coat Maxisorb ELISA plates (Biolegend) overnight at 4 o C. Plates were then washed with 1X ELISA wash buffer (Biolegend) prior to blocked with blocking buffer (1X PBS containing 2% BSA) for 2 hours at room temperature. Serial dilutions of recombinant monoclonal antibodies, previously labelled with horseradish peroxidase using a commercially available kit (Thermo), were incubated on the coated plates for 1 hour at room temperature. The plates were then extensively washed five to seven times with 1X ELISA wash buffer and incubated with 3,3’,5,5’-tetramethylbenzidine (TMB) for the development of a colorimetric reaction at room temperature. Reactions were stopped using 1X Stop buffer (Biolegend) and the plates were read at 405 nm with a 567 nm correction readout using a Varioscan ELISA plate scanner. For protein fractionation and antigen discovery, we initially performed a gel filtration step of 1 ml (1.5 mg protein/ml) using a Superdex 200 10/300 NGC column at a flow rate of 0.75 ml/min using TEN buffer (150 mM NaCl, 10 mM MgCl2, 1 mM DTT, 50 mM Tris-HCl pH 7.4, 5 mM EDTA). The resulting fractions (0.5 mL) were screened for reactivity against recombinant monoclonal antibodies Ig1-15, Ig1-26, and Ig1-76 coupled to HRP using ELISA. Fractions with a positive signal above background were pooled together and taken forward for a second round of protein fractionation using a Resource Q Anion exchange column. Samples were pre-diluted six times to reduce the concentration of NaCl from 150 mM to 25 mM prior to anion exchange. The column was initially washed with run buffer (50 mM Tris-HCl, 1 mM EDTA, pH 8.0) and samples run at a flow rate of 1.6 ml/minute in run buffer, and then eluted into 0.5 ml fractions in elution buffer (50 mM Tris-HCl, 1 mM EDTA, 2 mM NaCl, pH 8.0). Selected fractions of interest based on ELISA results were then subjected to untargeted LC/MS. For sample separation, separation was performed on a Thermo Vanquish Neo UHPLC system configured with buffer A as 0.1% formic acid in water and buffer B as 0.1% formic acid in 80% acetonitrile. A specified injection volume was loaded on to the analytical column (IonOpticks Aurora series Rapid TS, 8cm x 75 mm ID, 1.7 mm C18) kept at 35 o C at An initial rate of 800 nl/min which was then dropped to 200 nl/min in 0.25 min to rapidly re-pressurise the column. The separation was also started during this time, with a gradient of 1% B to 5% B over this period. The next step was 5% B to 35% B over 20.25 minutes, 35% B to 100% B over 0.5 minutes before washing for 2 minutes at 100% B and dropping down to 1% B in 0.5 minute. The complete method time was 30 minutes. Mass spectrometry was conducted using an Orbitrap Astral coupled to a Vanquish Neo UHPLC system to identify T. brucei candidate antigens likely to be recognised by these recombinant antibodies. Data was acquired in a data dependent manner using a fixed cycle time of 2 sec, an expected peak width of 15 sec and a default charge state of 2. Full MS data was acquired in positive mode over a scan range of 300 to 1750 Th, with a resolution of 120,000, a normalised AGC target of 300% and an automatic max fill time for a single microscan. Fragmentation data was obtained from signals with a charge state of +2 to +4 and an intensity over 5,000 and they were dynamically excluded from further analysis for a period of 15 sec after a single acquisition within a 10ppm window. Fragmentation spectra were acquired with a resolution of 15,000 with a normalised collision energy of 30%, a normalised AGC target of 300%, first mass of 110 Th and a max fill time of 25 mS for a single microscan. All data was collected in profile mode. Data was acquired in a data independent manner with an expected peak width of 6 sec and a default charge state of 2. Full MS data was acquired in positive mode over a scan range of 350 to 1750 Th, with a resolution of 240,000, a standard (automatic) normalised AGC target and a maximum injection time of 10 ms for a single microscan. Fragmentation data was obtained according to a table of 150 m/z windows, with 3 Th m/z widths. The DIA window type was set to automatic, and window optimisation was selected. These were acquired in the astral, with a normalised collision energy of 25%. The m/z range was 145-1450. A normalised AGC target of 500% was used with a maximum injection time of 7 ms for a single microscan. All data was collected in centroid mode. Data availability The whole tissue transcriptome data generated in this study have been deposited in the Gene Expression Omnibus uder the accession number GSE297581. The B cell receptor profiling dataset have been deposited in the Gene Expression Omnibus uder the accession number GSE300949. Additional data and files can also be sourced via Supplementary Tables. The raw files for the single cell experiments has been deposited in ArrayExpress under the accession number 847907. The processed .rds files and accompanying script can be access via Zenodo (DOI: 10.5281/zenodo.18188237). Statistical analysis All statistical analyses were performed using Graph Prism Version 8.0 for Windows or macOS, GraphPad Software (La Jolla California USA). The data distribution was determined by normality testing using the Shapiro-Wilks test. Depending on data distribution and normality we applied various statistical methods that are reported in each figure caption. For the in vivo experiments, we matched sex and age of the mice in experimental batches using a block design including randomisation of experimental units. Data collection and analysis were not performed blindly to the conditions of the experiment due to the specific requirements of the UK Home Office project licence. Declarations Acknowledgement We thank all the staff members of the University of Manchester Biological Services facility for their thoughtful contribution to the manintenace of our animal colonies. We also thanks the staff members of the University of Manchester professional and technical services (imaging, flow cytometry, and genomics) for their technical support. We are grateful to Gloria Lopez Castejon (University of Manchester, UK) for providing feedback on this manuscript. This work was funded through a Sir Henry Wellcome postdoctoral fellowship (221640/Z/20/Z to JFQ), a Wellcome Trust Career Development Award (309148/Z/24/Z to JFQ), and an Academy of Medical Sciences Springboard Award (SBF009/1079). MCS is supported by a Wellcome Early Career Award Fellowship (303388/Z/23/Z). CB is funded by an UKRI MRC research grant (MR/W018497/1). NAM is supported by an Institute Strategic Programme grant funding from the UKRI Biotechnology and Biological Sciences Research Council (BBS/E/RL/230002B). Author contributions statement Conceptualisation: JFQ , MCS, CB, NAM, AAS. Methodology: MCS, AC, CB, OS, KW, AM, JC, SW. Bioinformatics analysis : SMB. Formal analysis : JFQ, MCS, AC, CB, JCPF, AAS. Writing – original draft: JFQ . Writing – reviewing and editing: JFQ, MCS, AC, CB, CB, NAM. Writing – final edits: MCS, JFQ. Funding acquisition: JFQ. The authors declare that they have no competing interests, commercial or otherwise. Correspondence and requests for materials should be addressed to Juan F. Quintana ( [email protected] ). Competing interests statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. References Schafflick D et al (2021) Single-cell profiling of CNS border compartment leukocytes reveals that B cells and their progenitors reside in non-diseased meninges. Nat Neurosci 24:1225–1234 Brioschi S et al (2021) Heterogeneity of meningeal B cells reveals a lymphopoietic niche at the CNS borders. Science 373:eabf9277 Wang Y et al (2021) Early developing B cells undergo negative selection by central nervous system-specific antigens in the meninges. Immunity 54:2784–2794e6 Herisson F et al (2018) Direct vascular channels connect skull bone marrow and the brain surface enabling myeloid cell migration. Nat Neurosci 21:1209–1217 Shi K et al (2022) Bone marrow hematopoiesis drives multiple sclerosis progression. Cell 185:2234–2247e17 De Niz M et al (2021) Organotypic endothelial adhesion molecules are key for Trypanosoma brucei tropism and virulence. Cell Rep 36:109741 Beaver AK et al (2024) Tissue spaces are reservoirs of antigenic diversity for Trypanosoma brucei. Nature 636:430–437 Trindade S et al (2022) Slow growing behavior in African trypanosomes during adipose tissue colonization. Nat Commun 13:7548 Quintana JF et al (2022) Single cell and spatial transcriptomic analyses reveal microglia-plasma cell crosstalk in the brain during Trypanosoma brucei infection. Nat Commun 13:5752 Quintana JF et al (2023) The murine meninges acquire lymphoid tissue properties and harbour autoreactive B cells during chronic Trypanosoma brucei infection. PLOS Biol 21:e3002389 Alfituri OA et al (2020) To the Skin and Beyond: The Immune Response to African Trypanosomes as They Enter and Exit the Vertebrate Host. Front Immunol 11 Bockstal V et al (2011) T. brucei Infection Reduces B Lymphopoiesis in Bone Marrow and Truncates Compensatory Splenic Lymphopoiesis through Transitional B-Cell Apoptosis. PLoS Pathog 7:e1002089 De Trez C, Khan S, Magez S (2020) T. brucei infections abrogate diverse plasma cell-mediated effector B cell responses, independently of their specificity, affinity and host genetic background. PLoS Negl Trop Dis 14:e0008358 Cnops J et al (2016) Maintenance of B cells during chronic murine Trypanosoma brucei gambiense infection. Parasite Immunol 38:642–647 Frenkel D et al (2016) Trypanosoma brucei Co-opts NK Cells to Kill Splenic B2 B Cells. PLOS Pathog 12:e1005733 Pham HTT et al (2023) Neutrophil metalloproteinase driven spleen damage hampers infection control of trypanosomiasis. Nat Commun 14:5418 Fitzpatrick Z et al (2024) Venous-plexus-associated lymphoid hubs support meningeal humoral immunity. Nature 1–8. 10.1038/s41586-024-07202-9 Niu C et al (2022) Identification of hematopoietic stem cells residing in the meninges of adult mice at steady state. Cell Rep 41:111592 Hardy RR et al (2000) B-cell commitment, development and selection. Immunol Rev 175:23–32 Mecheri B, Paris F, Lübbert H (2018) Histological investigations on the dura mater vascular system of mice. Acta Histochem 120:846–857 Kaag Rasmussen M et al (2024) Trigeminal ganglion neurons are directly activated by influx of CSF solutes in a migraine model. Science 385:80–86 Shah T et al (2023) Arachnoid granulations are lymphatic conduits that communicate with bone marrow and dura-arachnoid stroma. J Exp Med 220:e20220618 The origin of subdural neomembranes I. Fine structure of the dura-arachnoid interface in man. - PMC. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2018597/ Bifari F et al (2017) Neurogenic Radial Glia-like Cells in Meninges Migrate and Differentiate into Functionally Integrated Neurons in the Neonatal Cortex. Cell Stem Cell 20:360–373e7 Terrier L et al (2021) The trigeminal system: The meningovascular complex— A review. J Anat 239:1–11 Noseda R, Melo-Carrillo A, Nir R-R, Strassman AM, Burstein R (2019) Non-Trigeminal Nociceptive Innervation of the Posterior Dura: Implications to Occipital Headache. J Neurosci 39:1867–1880 Matthias P, Rolink AG (2005) Transcriptional networks in developing and mature B cells. Nat Rev Immunol 5:497–508 Jin S et al (2021) Inference and analysis of cell-cell communication using CellChat. Nat Commun 12:1088 Schmidt TH, Bannard O, Gray EE, Cyster JG (2013) CXCR4 promotes B cell egress from Peyer’s patches. J Exp Med 210:1099–1107 Giorgiutti S, Rottura J, Korganow A-S, Gies V (2024) CXCR4: from B-cell development to B cell–mediated diseases. Life Sci Alliance 7:e202302465 Clark MR, Mandal M, Ochiai K, Singh H (2014) Orchestrating B cell lymphopoiesis through interplay of IL-7 receptor and pre-B cell receptor signalling. Nat Rev Immunol 14:69–80 Nemazee D (2017) Mechanisms of central tolerance for B cells. Nat Rev Immunol 17:281–294 Gilabert Carbajo C et al (2021) Novel aspects of iron homeostasis in pathogenic bloodstream form Trypanosoma brucei. PLOS Pathog 17:e1009696 Roth R, Mamula MJ (1997) Trafficking of Adoptively Transferred B Lymphocytes in B-Lymphocyte-Deficient Mice. J Exp Biol 200:2057–2062 Yang Y, Tung JW, Ghosn EEB, Herzenberg LA, Herzenberg LA (2007) Division and differentiation of natural antibody-producing cells in mouse spleen. Proc. Natl. Acad. Sci. 104, 4542–4546 Mazzitelli JA et al (2023) Skull bone marrow channels as immune gateways to the central nervous system. Nat Neurosci 26:2052–2062 Liu J et al (2024) Turbinate-homing IgA-secreting cells originate in the nasal lymphoid tissues. Nature 632:637–646 Fitzpatrick Z et al (2020) Gut-educated IgA plasma cells defend the meningeal venous sinuses. Nature 587:472–476 Sinton MC et al (2022) IL-17 signalling is critical for controlling subcutaneous adipose tissue dynamics and parasite burden during chronic murine Trypanosoma brucei infection. Nat. Commun. 14, 09.23.509158 (2022) Cao AT, Yao S, Gong B, Elson CO, Cong Y (2012) Th17 Cells Upregulate Polymeric Ig Receptor and Intestinal IgA and Contribute to Intestinal Homeostasis. J Immunol 189:4666–4673 Dann SM et al (2015) IL-17A promotes protective IgA responses and expression of other potential effectors against the lumen-dwelling enteric parasite Giardia. Exp Parasitol 156:68–78 Avnir Y et al (2016) IGHV1-69 polymorphism modulates anti-influenza antibody repertoires, correlates with IGHV utilization shifts and varies by ethnicity. Sci Rep 6:20842 Teo QW et al (2023) Stringent and complex sequence constraints of an IGHV1-69 broadly neutralizing antibody to influenza HA stem. Cell Rep 42:113410 Sarkanen TO, Alakuijala APE, Dauvilliers YA, Partinen MM (2018) Incidence of narcolepsy after H1N1 influenza and vaccinations: Systematic review and meta-analysis. Sleep Med Rev 38:177–186 Tesoriero C et al (2016) H1N1 influenza virus induces narcolepsy-like sleep disruption and targets sleep–wake regulatory neurons in mice. Proc. Natl. Acad. Sci. 113 Vuorela A et al (2021) Enhanced influenza A H1N1 T cell epitope recognition and cross-reactivity to protein-O-mannosyltransferase 1 in Pandemrix-associated narcolepsy type 1. Nat Commun 12:2283 Rijo-Ferreira F et al (2018) Sleeping sickness is a circadian disorder. Nat Commun 9:62 Rijo-Ferreira F et al (2020) Sleeping Sickness Disrupts the Sleep-Regulating Adenosine System. J Neurosci Off J Soc Neurosci 40:9306–9316 Pg K (2013) Clinical features, diagnosis, and treatment of human African trypanosomiasis (sleeping sickness). Lancet Neurol. 12 Mugnier MR, Stebbins CE, Papavasiliou FN (2016) Masters of Disguise: Antigenic Variation and the VSG Coat in Trypanosoma brucei. PLOS Pathog 12:e1005784 Stijlemans B, Radwanska M, De Trez C, Magez S (2017) African Trypanosomes Undermine Humoral Responses and Vaccine Development: Link with Inflammatory Responses? Front Immunol 8:582 Le Ray D, Barry JD, Easton C, Vickerman K (1977) First tsetse fly transmission of the ‘AnTat’ serodeme of Trypanosoma brucei. Ann Soc Belg Med Trop 57:369–381 Herbert WJ, Lumsden WH (1976) Trypanosoma brucei: a rapid ‘matching’ method for estimating the host’s parasitemia. Exp Parasitol 40:427–431 Vlaminck KD et al (2022) Differential plasticity and fate of brain-resident and recruited macrophages during the onset and resolution of neuroinflammation. Immunity 55, 2085–2102.e9 Coles JA, Stewart-Hutchinson PJ, Myburgh E, Brewer JM (2017) The mouse cortical meninges are the site of immune responses to many different pathogens, and are accessible to intravital imaging. Methods San Diego Calif 127:53–61 Kristin G, Anderson K, Mayer-Barber H, Sung L, Beura BR, James JJ, Taylor L, Qunaj, Thomas S, Griffith Vaiva Vezys, Daniel L BarberDavid Masopust. Intravascular staining for discrimination of vascular and tissue leukocytes | Springer Nature Experiments. https://experiments.springernature.com/articles/ 10.1038/nprot.2014.005 Manglani M, Gossa S, McGavern DB (2018) Leukocyte Isolation from Brain, Spinal Cord, and Meninges for Flow Cytometric Analysis. Curr Protoc Immunol 121:e44 Manso T et al (2022) IMGT® databases, related tools and web resources through three main axes of research and development. Nucleic Acids Res 50:D1262–D1272 Crooks GE, Hon G, Chandonia J-M, Brenner SE (2004) WebLogo: A Sequence Logo Generator: Fig. 1. Genome Res 14:1188–1190 Additional Declarations There is NO Competing Interest. Supplementary Files Supplementaryfigure1v07012026.jpg Supplementary figure 1. Identification of B cell developmental stages across tissues during T. brucei infection. A) Gating strategy employed for the identification of extravascular CD19 + B cells for the downstream identification of B cell developmental stages. B) Representative flow cytometry for the detection of extravascular early (E), late (L), and mature (M) B cell stages based on the expression of CD43 and B220 within the extravascular CD19 + B220 + B cell compartment in the femur bone marrow (fBM), spleen, skull bone marrow (skBM), and dura mater. including the expression level of markers associated with early developmental stages (CD93, IL-7R, CXCR4) and mature stages (CD23, IgM), indicating the geometric mean fluorescence intensity for each of these markers. C) Representative flow cytometry to identify early, late, and mature B cell stages in the large intestine of female mice during chronic T. brucei infection based on the expression of CD43 and B220 within the extravascular CD19 + B cell compartment. D) Quantification of flow cytometry based on percentage of extravascular CD45 + immune cells (left), total counts (middle), or percentage of each of the various B cell stages (left). E) Representative end-point PCR of Kappa-deleting Recombination Excision Circle (KREC) from gDNA purified from perfused dura mater from naïve and infected mice. The gene Aicda was used as loading control. F) KREC quantification by qPCR from tissues collected from naïve (teal) and T. brucei -infected (purple) dura mater ( n = 6 mice/group). Parametric T -test; A p value < 0.05 was considered significant. G) Representative gating strategy to identify extravascular hematopoetic stem cells (HSCs), including common lymphoid progenitors (CLPs). H) Quantification of various HSCs, based on the expression of c-KIT and SCA1, as well as Lineage - SCA1 Low cKIT Low CD127 + CD135 + CLPs (I) in the dura mater, skBM, spleen, and fBM. Pulled data from across two independent replicates and n = 6-8 mice/group. Mixed-effect analysis; A p value < 0.05 was considered significant. Representative flow cytometry analysis (J) and quantification (K) of circulating B220 + B cells and CD3 + within the intravascular CD45+ cells in blood sampels from naïve and T. brucei -infected female mice. Mixed-effect analysis; A p value < 0.05 was considered significant. Supplementary2v07012026.jpg Supplementary figure 2. T. brucei -induced changes in the B cell compartment occur in an extravascular- and tissue-dependent manner. A-B) Gating strategy to identify live cells, from which intravascular CD45 + immune cells are further discriminated from extravascular CD45 + based on differential staining. Representative flow cytometry data to identify B cell or T cells in the femur bone marrow (fBM), dura mater, and spleen in the based on the expression of B220 and CD3, respectively, within the intravascular (C) or extravascular (D) CD45 + immune cell compartment. Quantification of B220 + B cells (E) and CD3 + T cells (F) within the intravascular and extracellular CD45 + immune cell compartment during chronic T. brucei infection. Supplementaryfigure3v13012026.jpg Supplementary figure 3. Bulk transcriptomics analysis underscore tissue-specific signatures associated with B cell chemotaxis and survival within the dura mater during T. brucei infection. A) Number of differentially expressed genes (DEGs) in CNS border-associated tissues (skBM, dura mater) and peripheral B cell lymphopotietic organs (femur bone marrow, spleen) from naïve and T. brucei -infected mice ( n =4 mice/tissue/condition). DEGs were defined as those with a Log 2 fold change (Log 2 FC) > 1 and an adjusted p value < 0.05. B) Gene ontology analysis showing significant pathways enriched in the femur bone marrow (fBM), spleen, and skull bone marrow (skBM) from naïve (teal) and T. brucei -infected (purple) mice. Gene pathways were considered to be significantly enriched if they had an adjusted p adjusted < 0.05. C) Heatmap depicting the normalise gene counts (fragment per kilobase per million reads) for genes within the cytokine-cytokine receptor interaction pathway in naïve and infected femur bone marrow (fBM), skull bone marrow (skBM), dura mater and spleen. Supplementaryfigure413012026.jpg Supplementary Figure 4. Cell-cell interaction analysis reveal a rich network supporting B cell development in the dura mater during chronic T. brucei infection. Predicted global cell-cell interaction analysis in the skull bone marrow (A) and the dura mater (B) in naïve animals (left) and T. brucei -infected mice (right). The thickness of the arrows represent the likelihood and predicted strength of the interaction. C) Circos plot representing significant heterotypic cell-cell interactions mediated by chemokine ligand-receptor communication between skull bone marrow (top) and dura mater B cells (bottom) during chronic T. brucei infection. The thickness of the arrows represent the likelihood and predicted strength of the interaction. D) Ex vivo CXCL12-mediated chemotaxis assay using fBM and dura mater preparations from naïve and infected animals ( n = 6 mice across two independent experiments). Isolated cells were added to the upper chamber of a transwell chamber and subjected to a 3-hour migration period. After this period, cells were harvested from the upper and lower chambers and analysed by flow cytometry to specifically determine the frequency and number of B cells. The chemotactic effect of CXCL12 was measured in the presence and absence of an anti-CXCR4 blocking antibody. The chemotaxis index was calculated as the proportion of cells found in the lower chamber of the transwell compared to those found in the upper chamber after the incubation period. Brown-Forsythe test and Welch ANOVA test; A p value < 0.05 was considered significant. skBM, skull bone marrow; LEC, lymphatic endothelial cells; BEC, blood endothelial cells; CEP, common erythroid progenitors; HSC, hematopoietic stem cells; CMP, common myeloid progenitor; RG, radial glia; PB, plasmablasts; PCs, plasma cells; EpiCs, Choroid plexus-like epithelial cells. Supplementaryfigure513012026.jpg Supplementary figure 5. Sex-dependent effects of T. brucei infection in antibody repertoire across tissues. A) Gating strategy for the indentification of extravascular CD19 + B220 + B cells. B) Representative flow cytometry analysis of IgM and IgG expression within the extravascular CD19 + B220 + B cell compartment in the spleen and dura mater from male and female mice. IgD + IgM + B cells were considered to be naïve B cells, whereas the IgD - IgM - double negative (DNs) B cells constitute class-switched B cells. C) Representative flow cytometry of IgG, IgA, and IgE expression within the DNs B cells shown in (A). D) Normalised cell counts of all the immunoglobulin isotypes measured by flow cytometry in (B) and (C) in spleen and dura mater from naïve (teal) and chronically T. brucei -infected (purple) female mice. Representative data from two independent experiments ( n = 4-5 mice/group). Mixed-effect analysis; A p value < 0.05 was considered significant. Supplementaryfigure613012026.jpg Supplementary figure 6. Dura mater B cells have a unique and expanded B cell receptor repertoire during T. brucei infection. A) Number of unique Ighv clones detected in the spleen and dura mater of naïve (teal) and infected (purple) animals (n = 6 animals/group). Data presented as average and standard error. Welch’s T test; A p value < 0.05 was considered significant. B)Number of unique Igkv clones detected in the spleen and dura mater of naïve (teal) and infected (purple) animals ( n = 6 animals/group). Data presented as average and standard error. Welch’s T test; A p value < 0.05 was considered significant. C) Number of unique Iglv clones detected in the spleen and dura mater of naïve (teal) and infected (purple) animals (n = 6 animals/group). Data presented as average and standard error. Brown-Forsythe test and Welch ANOVA test; ns = not significant. D)Venn diagram depicting the number of Ighv genes and percentages that overlap between the dura mater(green) and spleen (orange). Ighv gene usage in the dura mater (E) or spleen (F) of naïve (teal) and infected (purple) mice. Only sequences consistently detected in two or more mice were included in the analysis. Multiple pair T test; A p value < 0.05 was considered significant. Non-significant pairwise comparisons are not highlighted in the figure. Igkv gene utilisation in the dura mater (G) or spleen (H) of naïve (teal) and infected (purple) mice. Only sequences consistently detected in two or more mice were included in the analysis. Multiple pair T test; A p value < 0.05 was considered significant. For simplicity, non-significant pairwise comparisons are not highlighted in the figure. Supplementaryfigure7v14012026.jpg Supplementary figure 7. Dura mater-enriched IgGs expressed during chronic T. brucei infection recognise a wide range of systemic antigens. Heatmap depicting the normalised fluorescent signal-to-noise ratio for 120 brain and neuronal-enriched antigens significantly detected by recombinant monoclonal antibodies Ig1-15, Ig1-26, and Ig1-76. Antibody concentrations ranging from 5 to 0.05 mg of protein were used for the analysis. Supplementaryfigure816012026.jpg Supplementary figure 8. Dura mater B cells and their cognate antibodies recognise a wide range of intracellular T. brucei antigens and confer protection against infection. A) Immunoassay to identify antibody reactivity against T. brucei protein fractions obtain from size exclusion chromatography. B) SDS-PAGE electrophoresis of selected size exclusion chromatography fractions of interest based on the immunoassay in (A). C) Immunoassay to identify antibody reactivity against T. brucei protein fractions obtain from anion exchange chromatography. D) Immunofluorescence to determine the capacity of recombinant Ig1-15 and Ig1-26 to recognise T. brucei Lister 427 antigens in situ comparing parasites with and without permeabilization prior to antibody labelling. An irrelevant mouse IgG2a was included as negative control. Scale bar = 25 mm. E) Measurement of parasitaemia over time following adoptive transfer experiments in which B cell-deficient mMT mice acted as recipients of B cells purified from naïve or T. brucei -infected splenocytes. F)Parasitaemia as in (E) but using dura mater-derived B cells. Non-parametric ANOVA with multiple corrections. The experimental groups are statistically compared against animals that received PBS alone prior to infection with T. brucei Antat 1.1E; A p value < 0.05 was considered significant. QuintanaSupplementarytablesv14012026.xlsx Supplementary tables Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8621800","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":576637288,"identity":"a3f56988-f34d-40e7-8272-1cb0a4898b23","order_by":0,"name":"Matthew C. Sinton","email":"","orcid":"https://orcid.org/0000-0003-1292-799X","institution":"University of Manchester","correspondingAuthor":false,"prefix":"","firstName":"Matthew","middleName":"C.","lastName":"Sinton","suffix":""},{"id":576637295,"identity":"4d72ed05-f52b-43ad-86df-600f84de3968","order_by":1,"name":"Alice Costain","email":"","orcid":"","institution":"University of Manchester","correspondingAuthor":false,"prefix":"","firstName":"Alice","middleName":"","lastName":"Costain","suffix":""},{"id":576637302,"identity":"143ca9fa-663c-4446-8420-c5d0931a88a0","order_by":2,"name":"Chloe Barnes","email":"","orcid":"","institution":"University of Manchester","correspondingAuthor":false,"prefix":"","firstName":"Chloe","middleName":"","lastName":"Barnes","suffix":""},{"id":576637303,"identity":"a3a834ec-faa1-4cd9-9edb-68d3d908963c","order_by":3,"name":"Olivia Shorthouse","email":"","orcid":"","institution":"University of Manchester","correspondingAuthor":false,"prefix":"","firstName":"Olivia","middleName":"","lastName":"Shorthouse","suffix":""},{"id":576637307,"identity":"b3cbcaa1-682a-4c16-844f-1f9b694de551","order_by":4,"name":"Syed Murtuza Baker","email":"","orcid":"https://orcid.org/0000-0002-6633-333X","institution":"University of Manchester","correspondingAuthor":false,"prefix":"","firstName":"Syed","middleName":"Murtuza","lastName":"Baker","suffix":""},{"id":576637315,"identity":"c4627025-4adf-43d7-a4b9-3558272d7f1b","order_by":5,"name":"Jose Carlos Paredes","email":"","orcid":"","institution":"University of Notre Dame","correspondingAuthor":false,"prefix":"","firstName":"Jose","middleName":"Carlos","lastName":"Paredes","suffix":""},{"id":576637316,"identity":"a0fb905b-70d2-4849-a115-ea926aa93217","order_by":6,"name":"Katrine Wonsbek","email":"","orcid":"","institution":"University of Manchester","correspondingAuthor":false,"prefix":"","firstName":"Katrine","middleName":"","lastName":"Wonsbek","suffix":""},{"id":576637318,"identity":"316fea76-843b-4010-9223-e64900964011","order_by":7,"name":"Anmoyul Mohon","email":"","orcid":"","institution":"University of Manchester","correspondingAuthor":false,"prefix":"","firstName":"Anmoyul","middleName":"","lastName":"Mohon","suffix":""},{"id":576637319,"identity":"1e3d647b-5901-49f4-a8d6-846ab1f4adde","order_by":8,"name":"Jessica Costa","email":"","orcid":"","institution":"University of Manchester","correspondingAuthor":false,"prefix":"","firstName":"Jessica","middleName":"","lastName":"Costa","suffix":""},{"id":576637322,"identity":"a8e3f05f-5f3e-4a7b-85b6-9380cacc1c58","order_by":9,"name":"Alvaro Acosta-Serrano","email":"","orcid":"","institution":"University of Notre Dame","correspondingAuthor":false,"prefix":"","firstName":"Alvaro","middleName":"","lastName":"Acosta-Serrano","suffix":""},{"id":576637324,"identity":"70cfaf07-5300-4b8f-a275-ff474b04a9ee","order_by":10,"name":"Cécile Bénézech","email":"","orcid":"","institution":"EDINBURGH UNIVERSITY","correspondingAuthor":false,"prefix":"","firstName":"Cécile","middleName":"","lastName":"Bénézech","suffix":""},{"id":576637325,"identity":"b79b5c3e-cadc-4dda-a436-a278208aa754","order_by":11,"name":"Neil A. Mabbott","email":"","orcid":"https://orcid.org/0000-0001-7395-1796","institution":"The Roslin Institute \u0026 Royal (Dick) School of Veterinary Sciences","correspondingAuthor":false,"prefix":"","firstName":"Neil","middleName":"A.","lastName":"Mabbott","suffix":""},{"id":576637280,"identity":"c7b17303-b2a9-46b0-9734-2d893435527f","order_by":12,"name":"Juan F. Quintana","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-5092-5576","institution":"University of Manchester","correspondingAuthor":true,"prefix":"","firstName":"Juan","middleName":"F.","lastName":"Quintana","suffix":""}],"badges":[],"createdAt":"2026-01-16 19:25:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8621800/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8621800/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":100748574,"identity":"60e2e842-e0b2-476e-a6d8-a9c80d05869c","added_by":"auto","created_at":"2026-01-21 04:07:03","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":186379,"visible":true,"origin":"","legend":"","description":"","filename":"Sintonetalv16012026.docx","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/92217559f3cc3c0f41193f43.docx"},{"id":100796572,"identity":"a79dbb82-6c81-41c8-a1df-a1807bed156d","added_by":"auto","created_at":"2026-01-21 13:44:16","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2452946,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1v07012026.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/3950bc8dbc84844d1a652b3a.jpg"},{"id":100796579,"identity":"a199a759-0400-407b-9b31-27e4d7ce05ad","added_by":"auto","created_at":"2026-01-21 13:44:19","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3286694,"visible":true,"origin":"","legend":"","description":"","filename":"Figure2v16012026.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/f940ea6c553b241e18595d41.jpg"},{"id":100748566,"identity":"ec6cec4e-acfb-4ce1-a3e5-5bdcb812ad1c","added_by":"auto","created_at":"2026-01-21 04:07:03","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2925961,"visible":true,"origin":"","legend":"","description":"","filename":"Figure3v13012025.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/094f942aec058f5606d113f2.jpg"},{"id":100748591,"identity":"66b1e912-7a78-4924-a4cb-4d32e3201368","added_by":"auto","created_at":"2026-01-21 04:07:04","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4591910,"visible":true,"origin":"","legend":"","description":"","filename":"QuintanaSupplementarytablesv14012026.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/4eed488e278e28219b273011.xlsx"},{"id":100796717,"identity":"dcb55523-b2fb-4220-8151-3ab60e17e0e4","added_by":"auto","created_at":"2026-01-21 13:45:17","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2155913,"visible":true,"origin":"","legend":"","description":"","filename":"Figure416012026.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/218daf205f27783b1c477c15.jpg"},{"id":100796728,"identity":"ff72b531-96f1-4f63-a2be-f3dcb8b133fb","added_by":"auto","created_at":"2026-01-21 13:45:20","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2886392,"visible":true,"origin":"","legend":"","description":"","filename":"Figure513012026.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/2446c2611da5cdf2d133aec7.jpg"},{"id":100748589,"identity":"206b3a13-90a1-4953-a553-46809ed1fe2f","added_by":"auto","created_at":"2026-01-21 04:07:04","extension":"jpg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1860334,"visible":true,"origin":"","legend":"","description":"","filename":"Figure6v14012026.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/7bc38e112f32e5636d6180d8.jpg"},{"id":100748587,"identity":"24e6d0a1-b611-4c52-bd57-2d7126b8c4d0","added_by":"auto","created_at":"2026-01-21 04:07:04","extension":"jpg","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2331484,"visible":true,"origin":"","legend":"","description":"","filename":"Figure7v16012026.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/2a60d677158fd64fb65e3c7c.jpg"},{"id":100748593,"identity":"f17bf8cf-f2c9-4aa8-a5d1-a9f33d920763","added_by":"auto","created_at":"2026-01-21 04:07:04","extension":"json","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":13073,"visible":true,"origin":"","legend":"","description":"","filename":"NCOMMS26004787.json","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/629221e47ce40f948a370cf2.json"},{"id":100748578,"identity":"b2177e35-f823-4752-8b66-70f8cb825bf8","added_by":"auto","created_at":"2026-01-21 04:07:03","extension":"xml","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":223600,"visible":true,"origin":"","legend":"","description":"","filename":"NCOMMS260047870enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/a5422465b5efaa7fb05b5861.xml"},{"id":100796681,"identity":"10e2571b-06e4-4f50-8f8e-43d40087e713","added_by":"auto","created_at":"2026-01-21 13:45:02","extension":"jpg","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2452946,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1v07012026.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/beaaab7f9caeebf2e73877a5.jpg"},{"id":100796711,"identity":"7c74ed78-6dbe-4d68-ad6a-f61fe9876724","added_by":"auto","created_at":"2026-01-21 13:45:15","extension":"jpg","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3286694,"visible":true,"origin":"","legend":"","description":"","filename":"Figure2v16012026.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/54ce80eb49087c35349f18d1.jpg"},{"id":100748590,"identity":"bb665fea-2d12-4691-b23c-30366a9bdcbc","added_by":"auto","created_at":"2026-01-21 04:07:04","extension":"jpg","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2925961,"visible":true,"origin":"","legend":"","description":"","filename":"Figure3v13012025.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/71a7590eaab5e53924402339.jpg"},{"id":100748595,"identity":"bd77976d-e637-47da-8061-e3c719e93256","added_by":"auto","created_at":"2026-01-21 04:07:04","extension":"jpg","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2155913,"visible":true,"origin":"","legend":"","description":"","filename":"Figure416012026.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/9f5e093a635231d61945b7f5.jpg"},{"id":100748569,"identity":"615240d5-5e03-42fd-af8a-9f310a6bca1f","added_by":"auto","created_at":"2026-01-21 04:07:03","extension":"jpg","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2886392,"visible":true,"origin":"","legend":"","description":"","filename":"Figure513012026.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/ae9c351703ceaa6ddddc3866.jpg"},{"id":100748594,"identity":"c00bf2b0-66be-419b-aba2-77482198061d","added_by":"auto","created_at":"2026-01-21 04:07:04","extension":"jpg","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1860334,"visible":true,"origin":"","legend":"","description":"","filename":"Figure6v14012026.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/a8670f705d45816bb88283cc.jpg"},{"id":100748601,"identity":"047108d2-db3d-4698-a824-4b7e7188bcca","added_by":"auto","created_at":"2026-01-21 04:07:05","extension":"jpg","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2331484,"visible":true,"origin":"","legend":"","description":"","filename":"Figure7v16012026.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/9cf7b422ff13f0e5df1a46aa.jpg"},{"id":100748592,"identity":"235961cd-0d43-40f5-ab6d-788e361125fd","added_by":"auto","created_at":"2026-01-21 04:07:04","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":297867,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure1v07012026.png","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/9ee60a0fe7ccb17200e4d169.png"},{"id":100748596,"identity":"773102e3-a4c4-4599-8f6d-4cbc0da97640","added_by":"auto","created_at":"2026-01-21 04:07:05","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":387130,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure2v16012026.png","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/272a253d1250aeeea0fac431.png"},{"id":100796688,"identity":"01b183f2-afb7-411d-913c-1860ccbc9a95","added_by":"auto","created_at":"2026-01-21 13:45:10","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":457828,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure3v13012025.png","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/b4461b8554f6a473b61ff415.png"},{"id":100748579,"identity":"2e2a9587-2c04-48ca-99e2-0fd00be91de0","added_by":"auto","created_at":"2026-01-21 04:07:03","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":315118,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure416012026.png","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/0d4ac21dddc25bfa95fd7f2b.png"},{"id":100857801,"identity":"f687f677-df4b-4dcc-b4a7-5393d1e1600c","added_by":"auto","created_at":"2026-01-22 07:22:46","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":382800,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure513012026.png","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/7108060a9bea15ad5860cf2a.png"},{"id":100748603,"identity":"f5d1d2ef-5f7d-405b-8486-0a3cfc530405","added_by":"auto","created_at":"2026-01-21 04:07:05","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":287519,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure6v14012026.png","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/6755a4809da42f9aa66b1c1a.png"},{"id":100748598,"identity":"f08830f8-9569-405f-a8bb-dbdafc9f6642","added_by":"auto","created_at":"2026-01-21 04:07:05","extension":"png","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":337983,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure7v16012026.png","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/7d368d2aaa1c14c34af533c2.png"},{"id":100748582,"identity":"c2038092-605a-4b67-ab4d-849b5d14ada4","added_by":"auto","created_at":"2026-01-21 04:07:03","extension":"xml","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":219967,"visible":true,"origin":"","legend":"","description":"","filename":"NCOMMS260047870structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/bb1555bbc6b6c28d701b65be.xml"},{"id":100748600,"identity":"c285bcc2-7135-4f73-8b57-17f9f218e1b0","added_by":"auto","created_at":"2026-01-21 04:07:05","extension":"html","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":243188,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/6828337dfee82526418154e9.html"},{"id":100748562,"identity":"b6d98604-6a45-45c8-a2d2-e6ce85f83dc0","added_by":"auto","created_at":"2026-01-21 04:07:02","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2452946,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe murine dura mater sustains B cell lymphopoiesis during chronic \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. brucei\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection.\u003c/strong\u003e\u0026nbsp; \u003cstrong\u003eA)\u003c/strong\u003e Representative flow cytometry for the detection of extravascular CD19\u003csup\u003e+\u003c/sup\u003e B220\u003csup\u003e+\u003c/sup\u003e B cells in the femur bone marrow (fBM), spleen, skull bone marrow (skBM), and dura mater of naïve and \u003cem\u003eT. brucei\u003c/em\u003e-infected animals at 25 days post-infection. \u003cstrong\u003eB)\u003c/strong\u003e Quantification of extravascular CD19\u003csup\u003e+\u003c/sup\u003e B220\u003csup\u003e+\u003c/sup\u003e B cells in the tissues reported in (A), reported as percentage of extravascular CD45\u003csup\u003e+\u003c/sup\u003e cells (left) our total counts (right) shown in. Pulled data from across four independent replicates and \u003cem\u003en \u003c/em\u003e= 20-25 mice/group. Mixed-effect analysis; A \u003cem\u003ep\u003c/em\u003e value \u0026lt; 0.05 was considered significant. \u003cstrong\u003eC)\u003c/strong\u003e Representative flow cytometry for the detection of extravascular early, late, and mature B cell stages in the femur bone marrow (fBM), spleen, skull bone marrow (skBM), and dura mater from naïve (teal)\u0026nbsp; and \u003cem\u003eT. brucei\u003c/em\u003e-infected mice at 25 days post-infection (purple). The percentage of each of these populations (to the extravascular CD19\u003csup\u003e+\u003c/sup\u003e B cell compartment) is also included. \u003cstrong\u003eD)\u003c/strong\u003e Quantification of early, late, and mature B cell stages in the tissues reported in (C) from tissues obtained from naïve (teal) and \u003cem\u003eT. brucei\u003c/em\u003e-infected animals at 25 days post-infection (purple). Pulled data from across four independent replicates and \u003cem\u003en \u003c/em\u003e= 20-25 mice/group. Mixed-effect analysis; A \u003cem\u003ep\u003c/em\u003e value \u0026lt; 0.05 was considered significant. skBM, skull bone marrow; fBM, femur bone marrow.\u003c/p\u003e","description":"","filename":"Figure1v07012026.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/246d9b18a70bb769362b723a.jpg"},{"id":100748560,"identity":"8e969191-3619-4bf7-abe7-427a9d696303","added_by":"auto","created_at":"2026-01-21 04:07:02","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3286694,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. brucei\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection in the B cell compartment are independent of sex. A)\u003c/strong\u003e Representative flow cytometry for the detection of extravascular CD19\u003csup\u003e+\u003c/sup\u003e B220\u003csup\u003e+\u003c/sup\u003e B cells in the femur bone marrow (fBM), spleen, skull bone marrow (skBM), and dura mater of male and female naïve and \u003cem\u003eT. brucei\u003c/em\u003e-infected animals at 25 days post-infection. Frequency (\u003cstrong\u003eB\u003c/strong\u003e) and counts (\u003cstrong\u003eC\u003c/strong\u003e) of extravascular CD19\u003csup\u003e+\u003c/sup\u003e B220\u003csup\u003e+\u003c/sup\u003e B cells reported in (A). Representative data from two independent replicates; n\u003cem\u003e \u003c/em\u003e= 4-5 mice/group. Mixed-effect analysis; A \u003cem\u003ep\u003c/em\u003e value \u0026lt; 0.05 was considered significant. \u003cstrong\u003eD)\u003c/strong\u003e Representative flow cytometry for the detection of extravascular early, late, and mature B cell stages based on the expression of CD43 and B220 within the extravascular CD19\u003csup\u003e+\u003c/sup\u003e B cell compartment\u0026nbsp; in the femur bone marrow (fBM), spleen, skull bone marrow (skBM), and dura mater from naïve and \u003cem\u003eT. brucei\u003c/em\u003e infected male and female mice. Quantification of early, late, and mature B cell stages in males (\u003cstrong\u003eE\u003c/strong\u003e) and females (\u003cstrong\u003eF\u003c/strong\u003e) across the tissues reported in (D). Representative data from two independent replicates; n\u003cem\u003e \u003c/em\u003e= 4-5 mice/group. Mixed-effect analysis; A \u003cem\u003ep\u003c/em\u003e value \u0026lt; 0.05 was considered significant.\u003c/p\u003e","description":"","filename":"Figure2v16012026.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/cab8b8969c9025007d033873.jpg"},{"id":100748567,"identity":"48ac70a9-9437-42c8-84e4-6427f949982a","added_by":"auto","created_at":"2026-01-21 04:07:03","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2925961,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative single cell transcriptomics analysis of the skull bone marrow and dura mater during \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. brucei\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection. A) \u003c/strong\u003eCombined UMAP plot for 74,696 high-quality cells obtained from naïve and \u003cem\u003eT. brucei\u003c/em\u003e-infected female skull bone marrow (46,966 cells) and dura mater (27,730 cells). A total of seventeen clusters were identified encompassing immune (myeloid cells, granulocytes, mixed T cells, neuotrophils, haematopoietic progenitors, and B cells) and stromal cells (LECs, BECs, sensory neurons, RG-like cells, epithelial cells). \u003cstrong\u003eB)\u003c/strong\u003e UMAP plot highlighting the cell distribution across the tissues examined and between experimental conditions. \u003cstrong\u003eC)\u003c/strong\u003e Heatmap depicting the normalised expression for the top 5 marker genes for each of the clusters represented in (A) and (B). \u003cstrong\u003eD)\u003c/strong\u003e Combined UMAP plot for the B cells after subsetting cells from (A). A total of seven B cell clusters were identified after dimensional reduction, encompassing early and late B cell developmental stages, as well as mature B cells, antibody-secreting cells (PB/PCs), and atypical CD11b\u003csup\u003e+\u003c/sup\u003e B cells. \u003cstrong\u003eE)\u003c/strong\u003e UMAP plot highlighting B cell subset distribution across the tissues examined and between experimental conditions. \u003cstrong\u003eF)\u003c/strong\u003e Heatmap depicting the normalised expression for the top 5 marker genes for each of the B cell clusters represented in (D) and (E). \u003cstrong\u003eG)\u003c/strong\u003e KEGG pathway enrichment analysis of the early/late B cell stages obtained from the skull bone marrow and dura mater during \u003cem\u003eT. brucei\u003c/em\u003e infection. Example of genes within each of the pathways is provided for each category. skBM, skull bone marrow; LEC, lymphatic endothelial cells; BEC, blood endothelial cells; CEP, common erythroid progenitors; HSC, hematopoietic stem cells; CMP, common myeloid progenitor; RG, radial glia; PB, plasmablasts; PCs, plasma cells; EpiCs, Choroid plexus-like epithelial cells.\u003c/p\u003e","description":"","filename":"Figure3v13012025.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/ad78838f2b231d13c57dab29.jpg"},{"id":100748570,"identity":"8de57e36-9b76-4cfc-bfab-011c7c4c279e","added_by":"auto","created_at":"2026-01-21 04:07:03","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2155913,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell-cell interaction analysis reveal a rich network supporting B cell development in the dura mater during chronic \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. brucei\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection. A) \u003c/strong\u003eCircos plot representing significant heterotypic cell-cell interactions mediated by chemokine ligand-receptor communication between dura mater B cells and other cells within the niche in naïve and \u003cem\u003eT. brucei\u003c/em\u003e-infected female. \u003cstrong\u003eB)\u003c/strong\u003e Quantification of CXCL12 from tissue lysates (25 mg of total protein) obtained from the dura mater from naïve and \u003cem\u003eT. brucei\u003c/em\u003e-infected animals at 25 days post-infection by ELISA. \u003cem\u003en\u003c/em\u003e = 5-6 mice/group. Welch’s \u003cem\u003eT\u003c/em\u003e test; A \u003cem\u003ep\u003c/em\u003e value \u0026lt; 0.05 was considered significant. \u003cstrong\u003eC) \u003c/strong\u003eRepresentative flow cytometry analysis of CXCR4 expression in early, late, and mature B cells from the skBM (left panel) and dura mater (right panel) obtained from naïve and infected animals. A fluorescent minus one control (FMO) was included for gating purposes. \u003cstrong\u003eD)\u003c/strong\u003e Quantification of CXCR4 expression based on geometric mean fluorescence intensity (gMFI) in early, late, and mature B cells from the skBM (left panel) and dura mater (right panel) obtained from naïve and infected animals. Pooled data from two independent experiments (\u003cem\u003en\u003c/em\u003e = 9-10 mice/group). Mixed-effect analysis; A \u003cem\u003ep\u003c/em\u003e value \u0026lt; 0.05 was considered significant. \u003cstrong\u003eE)\u003c/strong\u003e Representative flow cytometry experiments to measure the expression of CD127/IL-7Ra in tissue-dwelling early, late, and mature B cells from skBM (left panel) and dura mater (right panel), including quantification of mean fluorescent intensity (\u003cstrong\u003eF\u003c/strong\u003e). Pooled data from two independent experiments (\u003cem\u003en\u003c/em\u003e = 9-10 mice/group). Mixed-effect analysis; A \u003cem\u003ep\u003c/em\u003e value \u0026lt; 0.05 was considered significant.\u003cstrong\u003e G) \u003c/strong\u003eRepresentative flow cytometry experiments to measure the expression of phosphorylated STAT5 (pSTAT5) in tissue-dwelling early, late, and mature B cells in skBM (left panel) and dura mater (right panel) from naïve and \u003cem\u003eT. brucei\u003c/em\u003e-infected animals, including quantification of mean fluorescent intensity (\u003cstrong\u003eH\u003c/strong\u003e). Pooled data from two independent experiments (\u003cem\u003en\u003c/em\u003e = 4-6 mice/group). Mixed-effect analysis; A \u003cem\u003ep\u003c/em\u003e value \u0026lt; 0.05 was considered significant.\u003cstrong\u003e I) \u003c/strong\u003eRepresentative flow cytometry experiments to measure the expression of the replication factor Ki67 in tissue-dwelling B cells from skBM (left panel) and dura mater (right panel), including quantification of the percentage of Ki67\u003csup\u003e+\u003c/sup\u003e within the extravascular B cell compartment (\u003cstrong\u003eJ\u003c/strong\u003e) and mean fluorescence intensity (\u003cstrong\u003eJ\u003c/strong\u003e). Non-parametric one-way ANOVA; A \u003cem\u003ep\u003c/em\u003e value \u0026lt; 0.05 was considered significant. skBM, skull bone marrow; LEC, lymphatic endothelial cells; BEC, blood endothelial cells; CEP, common erythroid progenitors.\u003c/p\u003e","description":"","filename":"Figure416012026.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/c9f090a0d3b1a34cf58a4a7e.jpg"},{"id":100748588,"identity":"fcdfdd7b-0bbb-425a-821a-5c58acd36640","added_by":"auto","created_at":"2026-01-21 04:07:04","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2886392,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDura mater B cells have a unique and expanded B cell receptor repertoire during \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. brucei\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection. A)\u003c/strong\u003e Representative flow cytometry analysis of IgM and IgD expression within the extravascular CD19\u003csup\u003e+\u003c/sup\u003e B220\u003csup\u003e+\u003c/sup\u003e B cells in the spleen and dura mater. IgD\u003csup\u003e+\u003c/sup\u003e IgM\u003csup\u003e+\u003c/sup\u003e B cells were considered to be naïve B cells, whereas the IgD\u003csup\u003e-\u003c/sup\u003e IgM\u003csup\u003e-\u003c/sup\u003e double negative (DNs) B cells constitute class-switched B cells. \u003cstrong\u003eB\u003c/strong\u003e) Representative flow cytometry of IgG, IgA, and IgE expression within the DNs B cells shown in (A). We did not detect IgE expression in these cells, so these class-switched IgD\u003csup\u003e-\u003c/sup\u003e IgM\u003csup\u003e+\u003c/sup\u003e IgA\u003csup\u003e-\u003c/sup\u003e cells were assigned as IgG\u003csup\u003e+\u003c/sup\u003e B cells. C) Quantification of all the immunoglobulin classes measured by flow cytometry in (A) and (B) in dura mater and spleen from naïve (teal) and chronically \u003cem\u003eT. brucei\u003c/em\u003e-infected (purple) female mice. Representative data from two independent experiments (\u003cem\u003en\u003c/em\u003e = 4-5 mice/group). Data presented as average and standard error. Mixed-effect analysis; A \u003cem\u003ep\u003c/em\u003e value \u0026lt; 0.05 was considered significant. \u003cstrong\u003eD)\u003c/strong\u003e Average frequency of \u003cem\u003eIghv \u003c/em\u003eusage in the dura mater (top row) and spleen (bottom row) in naïve and infected animals. The number in the centre represents the average clonal count across independent biological replicates (\u003cem\u003en\u003c/em\u003e = 6 animals/group).\u003cstrong\u003e E)\u003c/strong\u003e Clonal frequency of selected \u003cem\u003eIghv1\u003c/em\u003e family members in naïve and infected dura mater (top) and spleen (bottom). Kruskal-Wallis test; A \u003cem\u003ep\u003c/em\u003e value \u0026lt; 0.05 was considered significant. \u003cstrong\u003eF)\u003c/strong\u003e \u003cem\u003eIgh\u003c/em\u003e chain CDR3 amino acid length distribution in B cells from matched dura mater and spleen of naïve and \u003cem\u003eT. brucei\u003c/em\u003e-infected mice (\u003cem\u003en\u003c/em\u003e = 6 mice/group). Mixed-effect analysis; A \u003cem\u003ep\u003c/em\u003e value \u0026lt; 0.05 was considered significant. E). \u003cstrong\u003eG)\u003c/strong\u003e \u003cem\u003eIgh\u003c/em\u003e chain CDR3 amino acid length distribution in B cells from dura mater (left panel) and spleen (right panel) of naïve and \u003cem\u003eT. brucei\u003c/em\u003e-infected mice (\u003cem\u003en\u003c/em\u003e = 6 mice/group). Data presented as average and standard error. Parametric \u003cem\u003eT\u003c/em\u003e-test; A \u003cem\u003ep\u003c/em\u003e value \u0026lt; 0.05 was considered significant. \u003cstrong\u003e\u0026nbsp;H)\u003c/strong\u003e Average frequency of \u003cem\u003eIgkv \u003c/em\u003eusage in the dura mater (top row) and spleen (bottom row) in naïve and infected animals. The number in the centre represents the average clonal count across independent biological replicates (n = 6 animals/group). Mixed-effect analysis; A \u003cem\u003ep\u003c/em\u003e value \u0026lt; 0.05 was considered significant. \u003cstrong\u003eI)\u003c/strong\u003e Clonal frequency of selected \u003cem\u003eIgkv6\u003c/em\u003e family members in naïve and infected dura mater(top) and spleen (bottom). Kruskal-Wallis test; A \u003cem\u003ep\u003c/em\u003e value \u0026lt; 0.05 was considered significant.\u003cstrong\u003e J)\u003c/strong\u003e \u003cem\u003eIgk\u003c/em\u003e chain CDR3 amino acid length distribution in B cells from matched dura mater and spleen of naïve and \u003cem\u003eT. brucei\u003c/em\u003e-infected mice (\u003cem\u003en\u003c/em\u003e = 6 mice/group). Mixed-effect analysis; A \u003cem\u003ep\u003c/em\u003e value \u0026lt; 0.05 was considered significant. E). \u003cstrong\u003eK)\u003c/strong\u003e \u003cem\u003eIgk\u003c/em\u003e chain CDR3 amino acid length distribution in B cells from dura mater (left panel) and spleen (right panel) of naïve and \u003cem\u003eT. brucei\u003c/em\u003e-infected mice (\u003cem\u003en\u003c/em\u003e = 6 mice/group). Data presented as average and standard error. Parametric \u003cem\u003eT\u003c/em\u003e-test; A \u003cem\u003ep\u003c/em\u003e value \u0026lt; 0.05 was considered significant.\u003c/p\u003e","description":"","filename":"Figure513012026.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/15e54d9a8497d345c24a8146.jpg"},{"id":100748563,"identity":"fdd091f6-4c71-4cba-bfb5-1de308bd0436","added_by":"auto","created_at":"2026-01-21 04:07:02","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1860334,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDura mater-enriched immunoglobulins expressed during chronic \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. brucei\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection recognise a wide range of brain and neuronal-associated antigens. A) \u003c/strong\u003eHeatmap depicting the normalised fluorescent signal-to-noise ratio for 120 brain and neuronal-enriched antigens significantly detected by recombinant monoclonal antibodies Ig1-15, Ig1-26, and Ig1-76. Antibody concentrations ranging from 5 to 0.05 mg of protein were used for the analysis. The star symbol detones selected candidates used for downstream validation. \u003cstrong\u003eB)\u003c/strong\u003e Ven diagram depicting the host antigens identified in this screening that were commonly detected by the recombinant monoclonal antibodies used in (A). A table summarising the nature of the antigens in this array is also included. \u003cstrong\u003eC)\u003c/strong\u003e ELISA analysis to validate the results from the host antigen array. For this, serially diluted recombinant monoclonal antibodies were used on plates coated with myelin oligodendrocyte glycoprotein (MOG) or Lactoferrin, both at 50 mg/ml. Recombinant Activated Leukocyte Cell Adhesion Molecule (ALCAM) and Bovine Serum Albumin (BSA) at 50 mg/ml were included in the assay as negative controls. Plates were analysed on a plate reader at 450 nm and the data is reported as normalised to a 570 nm background read.\u003c/p\u003e","description":"","filename":"Figure6v14012026.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/274628c247958ed2fe371912.jpg"},{"id":100748586,"identity":"c0d77908-0b64-4a2d-a998-134ba41a59dd","added_by":"auto","created_at":"2026-01-21 04:07:04","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2331484,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDura mater B cells and their cognate antibodies recognise a wide range of intracellular \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. brucei\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e antigens and delay the onset of parasitaemia. A) \u003c/strong\u003eSchematic representation of the strategy to identify putative \u003cem\u003eT. brucei\u003c/em\u003e antigens recognised by dura mater-associated antibodies. A combined size exclusion and anion exchanged chromatography coupled with LS/MS was used to identified candidates for downstream validation. \u003cstrong\u003eB)\u003c/strong\u003e Heatmap (normalised peptide counts) depicting the 56 \u003cem\u003eT. brucei\u003c/em\u003e proteins identified in our screening that were recognised Ig1-15 and Ig1-26. Only those proteins with \u0026gt;1 peptide and an adjusted \u003cem\u003ep \u003c/em\u003evalue \u0026lt;0.05 were included in this analysis. \u003cstrong\u003eC)\u003c/strong\u003e Pathway enrichment analysis of the \u003cem\u003eT. brucei\u003c/em\u003e proteins identified in (B). \u003cstrong\u003eD)\u003c/strong\u003e Immunofluorescence to determine the capacity of recombinant Ig1-15 and Ig1-26 to recognise \u003cem\u003eT. brucei\u003c/em\u003e Lister 427 antigens \u003cem\u003ein situ\u003c/em\u003e. An irrelevant mouse IgG2a was included as negative control. Scale bar = 10 mm. \u003cstrong\u003eE)\u003c/strong\u003e \u003cstrong\u003eTop panel: \u003c/strong\u003eFlow cytometry staining of \u003cem\u003eT. brucei\u003c/em\u003e Lister 427 parasites with or without permeabilization to determine whether recombinant Ig1-15 and Ig1-26 recognise intracellular vs surface antigens, respectively. \u003cstrong\u003eBottom panel: \u003c/strong\u003eQuantification of flow cytometry data, reported as geometric mean fluorescent intensity for the signal detected with each of the recombinant antibodies with or without permeabilization. An irrelevant mouse IgG2a was included as negative control. \u003cstrong\u003eF-G)\u003c/strong\u003eELISA assay to determine the reactivity of Ig1-15 and Ig1-26 against selected protein \u003cem\u003eT. brucei\u003c/em\u003e protein candidates identified in (B), namely Fructose Biphosphate Aldolase (FBA) or Heat-Shock Protein 70 (HSP70). The murine counterparts of these candidate proteins were also included as controls and to determine cross-reactivity. \u003cstrong\u003eH)\u003c/strong\u003e Measurement of parasitaemia over time following adoptive transfer experiments in which B cell-deficient mMT mice acted as recipients of B cells purified from infected spleens or dura mater tissue. Non-parametric ANOVA with multiple corrections. The experimental groups are statistically compared against animals that received PBS alone prior to infection with \u003cem\u003eT. brucei\u003c/em\u003e Antat 1.1E; A \u003cem\u003ep\u003c/em\u003e value \u0026lt; 0.05 was considered significant. \u003cstrong\u003eI)\u003c/strong\u003eMeasurement of parasitaemia over time in passively-transferred mMT mice with recombinant Ig1-15, Ig1-26, or both antibodies prior to infection with \u003cem\u003eT. brucei\u003c/em\u003e. Non-parametric ANOVA with multiple corrections. The experimental groups are statistically compared against animals that received an irrelevant IgG2a antibody as control prior to infection with \u003cem\u003eT. brucei\u003c/em\u003e Antat 1.1E; A \u003cem\u003ep\u003c/em\u003e value \u0026lt; 0.05 was considered significant.\u003c/p\u003e","description":"","filename":"Figure7v16012026.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/995fd79e954b541afcfe5eb5.jpg"},{"id":105794621,"identity":"f467f058-456b-434b-a9d0-0f7dc8e75a95","added_by":"auto","created_at":"2026-03-31 08:28:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18535211,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/09342bce-2f33-4087-92c1-454240d75124.pdf"},{"id":100857741,"identity":"5b9415f1-55f5-4f78-ad3f-b143bb04d65b","added_by":"auto","created_at":"2026-01-22 07:21:26","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2982884,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary figure 1. Identification of B cell developmental stages across tissues during \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. brucei\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection.\u003c/strong\u003e\u0026nbsp; \u003cstrong\u003eA)\u003c/strong\u003e Gating strategy employed for the identification of extravascular CD19\u003csup\u003e+\u003c/sup\u003e B cells for the downstream identification of B cell developmental stages. \u003cstrong\u003eB)\u003c/strong\u003e Representative flow cytometry for the detection of extravascular early (E), late (L), and mature (M) B cell stages based on the expression of CD43 and B220 within the extravascular CD19\u003csup\u003e+\u003c/sup\u003e B220\u003csup\u003e+\u003c/sup\u003e B cell compartment\u0026nbsp; in the femur bone marrow (fBM), spleen, skull bone marrow (skBM), and dura mater. including the expression level of markers associated with early developmental stages (CD93, IL-7R, CXCR4) and mature stages (CD23, IgM), indicating the geometric mean fluorescence intensity for each of these markers. \u003cstrong\u003eC)\u003c/strong\u003e Representative flow cytometry to identify early, late, and mature B cell stages in the large intestine of female mice during chronic \u003cem\u003eT. brucei\u003c/em\u003e infection based on the expression of CD43 and B220 within the extravascular CD19\u003csup\u003e+\u003c/sup\u003e B cell compartment. \u003cstrong\u003eD)\u003c/strong\u003e Quantification of flow cytometry based on percentage of extravascular CD45\u003csup\u003e+\u003c/sup\u003e immune cells (left), total counts (middle), or percentage of each of the various B cell stages (left). \u003cstrong\u003eE)\u003c/strong\u003e Representative end-point PCR of Kappa-deleting Recombination Excision Circle (KREC) from gDNA purified from perfused dura mater from naïve and infected mice. The gene \u003cem\u003eAicda\u003c/em\u003e was used as loading control. \u003cstrong\u003eF) \u003c/strong\u003eKREC quantification by qPCR from tissues collected from naïve (teal) and \u003cem\u003eT. brucei\u003c/em\u003e-infected (purple) dura mater (\u003cem\u003en\u003c/em\u003e = 6 mice/group). Parametric \u003cem\u003eT\u003c/em\u003e-test; A \u003cem\u003ep\u003c/em\u003e value \u0026lt; 0.05 was considered significant. \u003cstrong\u003eG)\u003c/strong\u003e Representative gating strategy to identify extravascular hematopoetic stem cells (HSCs), including common lymphoid progenitors (CLPs). \u003cstrong\u003eH)\u003c/strong\u003e Quantification of various HSCs, based on the expression of c-KIT and SCA1, as well as Lineage\u003csup\u003e-\u003c/sup\u003e SCA1\u003csup\u003eLow\u003c/sup\u003e cKIT\u003csup\u003eLow\u003c/sup\u003e CD127\u003csup\u003e+\u003c/sup\u003e CD135\u003csup\u003e+\u003c/sup\u003e CLPs (\u003cstrong\u003eI\u003c/strong\u003e) in the dura mater, skBM, spleen, and fBM. Pulled data from across two independent replicates and \u003cem\u003en \u003c/em\u003e= 6-8 mice/group. Mixed-effect analysis; A \u003cem\u003ep\u003c/em\u003e value \u0026lt; 0.05 was considered significant. Representative flow cytometry analysis (\u003cstrong\u003eJ\u003c/strong\u003e) and quantification (\u003cstrong\u003eK\u003c/strong\u003e) of circulating B220\u003csup\u003e+\u003c/sup\u003e B cells and CD3\u003csup\u003e+\u003c/sup\u003e within the intravascular CD45+ cells in blood sampels from naïve and \u003cem\u003eT. brucei\u003c/em\u003e-infected female mice. Mixed-effect analysis; A \u003cem\u003ep\u003c/em\u003e value \u0026lt; 0.05 was considered significant.\u003c/p\u003e","description":"","filename":"Supplementaryfigure1v07012026.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/7881d2ce1a0a8fd6968be5c0.jpg"},{"id":100748564,"identity":"2bb8cf0d-6e40-4367-84b1-34b351c98d96","added_by":"auto","created_at":"2026-01-21 04:07:03","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2840815,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary figure 2. \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. brucei\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-induced changes in the B cell compartment occur in an extravascular- and tissue-dependent manner.\u003c/strong\u003e\u0026nbsp; \u003cstrong\u003eA-B)\u003c/strong\u003e Gating strategy to identify live cells, from which intravascular CD45\u003csup\u003e+\u003c/sup\u003e immune cells are further discriminated from extravascular CD45\u003csup\u003e+\u003c/sup\u003e based on differential staining. Representative flow cytometry data to identify B cell or T cells in the femur bone marrow (fBM), dura mater, and spleen in the based on the expression of B220 and CD3, respectively, within the intravascular \u003cstrong\u003e(C)\u003c/strong\u003e or extravascular \u003cstrong\u003e(D)\u003c/strong\u003e CD45\u003csup\u003e+\u003c/sup\u003e immune cell compartment. Quantification of B220\u003csup\u003e+\u003c/sup\u003e B cells (\u003cstrong\u003eE\u003c/strong\u003e) and CD3\u003csup\u003e+\u003c/sup\u003e T cells (\u003cstrong\u003eF\u003c/strong\u003e) within the intravascular and extracellular CD45\u003csup\u003e+\u003c/sup\u003e immune cell compartment during chronic \u003cem\u003eT. brucei\u003c/em\u003e infection.\u003c/p\u003e","description":"","filename":"Supplementary2v07012026.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/9ed207f909e7077240e96b5e.jpg"},{"id":100748575,"identity":"719c1773-9cf1-4c57-ad6a-8bfed2c575ca","added_by":"auto","created_at":"2026-01-21 04:07:03","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2241227,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary figure 3. Bulk transcriptomics analysis underscore tissue-specific signatures associated with B cell chemotaxis and survival within the dura mater during \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. brucei\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection. A) \u003c/strong\u003eNumber of differentially expressed genes (DEGs) in CNS border-associated tissues (skBM, dura mater) and peripheral B cell lymphopotietic organs (femur bone marrow, spleen) from naïve and \u003cem\u003eT. brucei\u003c/em\u003e-infected mice (\u003cem\u003en\u003c/em\u003e=4 mice/tissue/condition). DEGs were defined as those with a Log\u003csub\u003e2\u003c/sub\u003e fold change (Log\u003csub\u003e2\u003c/sub\u003eFC) \u0026gt; 1 and an adjusted \u003cem\u003ep \u003c/em\u003evalue \u0026lt; 0.05. \u003cstrong\u003eB)\u003c/strong\u003e Gene ontology analysis showing significant pathways enriched in the femur bone marrow (fBM), spleen, and skull bone marrow (skBM) from naïve (teal) and \u003cem\u003eT. brucei\u003c/em\u003e-infected (purple) mice. Gene pathways were considered to be significantly enriched if they had an adjusted \u003cem\u003ep\u003c/em\u003e adjusted \u0026lt; 0.05. \u003cstrong\u003eC)\u003c/strong\u003e Heatmap depicting the normalise gene counts (fragment per kilobase per million reads) for genes within the cytokine-cytokine receptor interaction pathway in naïve and infected femur bone marrow (fBM), skull bone marrow (skBM), dura mater and spleen.\u003c/p\u003e","description":"","filename":"Supplementaryfigure3v13012026.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/30190ccbf07d4930ee9c8459.jpg"},{"id":100796488,"identity":"828b7c29-46d7-4894-8529-a8a7124d1583","added_by":"auto","created_at":"2026-01-21 13:43:35","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":2623736,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 4. Cell-cell interaction analysis reveal a rich network supporting B cell development in the dura mater during chronic \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. brucei\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection. \u003c/strong\u003ePredicted global cell-cell interaction analysis in the skull bone marrow (\u003cstrong\u003eA\u003c/strong\u003e) and the dura mater (\u003cstrong\u003eB\u003c/strong\u003e) in naïve animals (left) and \u003cem\u003eT. brucei\u003c/em\u003e-infected mice (right). The thickness of the arrows represent the likelihood and predicted strength of the interaction.\u003cstrong\u003e C) \u003c/strong\u003eCircos plot representing significant heterotypic cell-cell interactions mediated by chemokine ligand-receptor communication between skull bone marrow (top) and dura mater B cells (bottom) during chronic \u003cem\u003eT. brucei\u003c/em\u003e infection. The thickness of the arrows represent the likelihood and predicted strength of the interaction. \u003cstrong\u003eD\u003c/strong\u003e) \u003cem\u003eEx vivo\u003c/em\u003e CXCL12-mediated chemotaxis assay using fBM and dura mater preparations from naïve and infected animals (\u003cem\u003en\u003c/em\u003e = 6 mice across two independent experiments). Isolated cells were added to the upper chamber of a transwell chamber and subjected to a 3-hour migration period. After this period, cells were harvested from the upper and lower chambers and analysed by flow cytometry to specifically determine the frequency and number of B cells. The chemotactic effect of CXCL12 was measured in the presence and absence of an anti-CXCR4 blocking antibody. The chemotaxis index was calculated as the proportion of cells found in the lower chamber of the transwell compared to those found in the upper chamber after the incubation period. Brown-Forsythe test and Welch ANOVA test; A \u003cem\u003ep\u003c/em\u003e value \u0026lt; 0.05 was considered significant. skBM, skull bone marrow; LEC, lymphatic endothelial cells; BEC, blood endothelial cells; CEP, common erythroid progenitors; HSC, hematopoietic stem cells; CMP, common myeloid progenitor; RG, radial glia; PB, plasmablasts; PCs, plasma cells; EpiCs, Choroid plexus-like epithelial cells.\u003c/p\u003e","description":"","filename":"Supplementaryfigure413012026.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/d5c39c0d50b32eb75ea34b8a.jpg"},{"id":100748584,"identity":"2a28c289-ebfb-4afb-b1a0-04c83dc43406","added_by":"auto","created_at":"2026-01-21 04:07:04","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":2098365,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary figure 5. Sex-dependent effects of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. brucei\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection in antibody repertoire across tissues. A) \u003c/strong\u003eGating strategy for the indentification of extravascular CD19\u003csup\u003e+\u003c/sup\u003e B220\u003csup\u003e+\u003c/sup\u003e B cells\u003cstrong\u003e.\u0026nbsp; B)\u003c/strong\u003e Representative flow cytometry analysis of IgM and IgG expression within the extravascular CD19\u003csup\u003e+\u003c/sup\u003e B220\u003csup\u003e+\u003c/sup\u003e B cell compartment in the spleen and dura mater from male and female mice. IgD\u003csup\u003e+\u003c/sup\u003e IgM\u003csup\u003e+\u003c/sup\u003e B cells were considered to be naïve B cells, whereas the IgD\u003csup\u003e-\u003c/sup\u003e IgM\u003csup\u003e-\u003c/sup\u003e double negative (DNs) B cells constitute class-switched B cells. \u003cstrong\u003eC\u003c/strong\u003e) Representative flow cytometry of IgG, IgA, and IgE expression within the DNs B cells shown in (A). \u003cstrong\u003eD)\u003c/strong\u003e Normalised cell counts of all the immunoglobulin isotypes measured by flow cytometry in (B) and (C) in spleen and dura mater from naïve (teal) and chronically \u003cem\u003eT. brucei\u003c/em\u003e-infected (purple) female mice. Representative data from two independent experiments (\u003cem\u003en\u003c/em\u003e = 4-5 mice/group). Mixed-effect analysis; A \u003cem\u003ep\u003c/em\u003e value \u0026lt; 0.05 was considered significant.\u003c/p\u003e","description":"","filename":"Supplementaryfigure513012026.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/d4851e383aa39f1535a592a3.jpg"},{"id":100748572,"identity":"cdb45e09-befd-413d-88ab-54ad5ab09862","added_by":"auto","created_at":"2026-01-21 04:07:03","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":3938113,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary figure 6. Dura mater B cells have a unique and expanded B cell receptor repertoire during \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. brucei\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection. A) \u003c/strong\u003eNumber of unique \u003cem\u003eIghv\u003c/em\u003e clones detected in the spleen and dura mater of naïve (teal) and infected (purple) animals (n = 6 animals/group). Data presented as average and standard error. Welch’s \u003cem\u003eT\u003c/em\u003e test; A \u003cem\u003ep\u003c/em\u003e value \u0026lt; 0.05 was considered significant.\u003cstrong\u003e B)\u003c/strong\u003eNumber of unique \u003cem\u003eIgkv\u003c/em\u003e clones detected in the spleen and dura mater of naïve (teal) and infected (purple) animals (\u003cem\u003en\u003c/em\u003e = 6 animals/group). Data presented as average and standard error. Welch’s \u003cem\u003eT\u003c/em\u003e test; A \u003cem\u003ep\u003c/em\u003evalue \u0026lt; 0.05 was considered significant. \u003cstrong\u003eC) \u003c/strong\u003eNumber of unique \u003cem\u003eIglv\u003c/em\u003eclones detected in the spleen and dura mater of naïve (teal) and infected (purple) animals (n = 6 animals/group). Data presented as average and standard error. Brown-Forsythe test and Welch ANOVA test; ns = not significant. \u003cstrong\u003eD)\u003c/strong\u003eVenn diagram depicting the number of \u003cem\u003eIghv\u003c/em\u003e genes and percentages that overlap between the dura mater(green) and spleen (orange). \u003cem\u003eIghv\u003c/em\u003e gene usage in the dura mater (\u003cstrong\u003eE\u003c/strong\u003e) or spleen (\u003cstrong\u003eF\u003c/strong\u003e) of naïve (teal) and infected (purple) mice. Only sequences consistently detected in two or more mice were included in the analysis. Multiple pair \u003cem\u003eT\u003c/em\u003e test; A \u003cem\u003ep\u003c/em\u003evalue \u0026lt; 0.05 was considered significant. Non-significant pairwise comparisons are not highlighted in the figure. \u003cem\u003eIgkv\u003c/em\u003e gene utilisation in the dura mater (\u003cstrong\u003eG\u003c/strong\u003e) or spleen (\u003cstrong\u003eH\u003c/strong\u003e) of naïve (teal) and infected (purple) mice. Only sequences consistently detected in two or more mice were included in the analysis. Multiple pair \u003cem\u003eT\u003c/em\u003e test; A \u003cem\u003ep\u003c/em\u003e value \u0026lt; 0.05 was considered significant. For simplicity, non-significant pairwise comparisons are not highlighted in the figure.\u003c/p\u003e","description":"","filename":"Supplementaryfigure613012026.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/03fb30d5a66cf78042a14b2a.jpg"},{"id":100857737,"identity":"c9775d1f-99fb-450e-973c-b81bac3fd403","added_by":"auto","created_at":"2026-01-22 07:21:22","extension":"jpg","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":1008588,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary figure 7. Dura mater-enriched IgGs expressed during chronic \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. brucei\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection recognise a wide range of systemic antigens. \u003c/strong\u003eHeatmap depicting the normalised fluorescent signal-to-noise ratio for 120 brain and neuronal-enriched antigens significantly detected by recombinant monoclonal antibodies Ig1-15, Ig1-26, and Ig1-76. Antibody concentrations ranging from 5 to 0.05 mg of protein were used for the analysis.\u003c/p\u003e","description":"","filename":"Supplementaryfigure7v14012026.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/6b63e9e02021e7d1ec025106.jpg"},{"id":100796529,"identity":"f5c683b1-0f73-4b5c-8bca-8cace0e8a9a8","added_by":"auto","created_at":"2026-01-21 13:43:56","extension":"jpg","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":2731605,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary\u003c/strong\u003e \u003cstrong\u003efigure 8. Dura mater B cells and their cognate antibodies recognise a wide range of intracellular \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. brucei\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e antigens and confer protection against infection. A) \u003c/strong\u003eImmunoassay to identify antibody reactivity against \u003cem\u003eT. brucei\u003c/em\u003e protein fractions obtain from size exclusion chromatography. \u003cstrong\u003eB)\u003c/strong\u003e SDS-PAGE electrophoresis of selected size exclusion chromatography fractions of interest based on the immunoassay in (A). \u003cstrong\u003eC)\u003c/strong\u003e Immunoassay to identify antibody reactivity against \u003cem\u003eT. brucei\u003c/em\u003eprotein fractions obtain from anion exchange chromatography. \u003cstrong\u003eD) \u003c/strong\u003eImmunofluorescence to determine the capacity of recombinant Ig1-15 and Ig1-26 to recognise \u003cem\u003eT. brucei\u003c/em\u003e Lister 427 antigens \u003cem\u003ein situ\u003c/em\u003e comparing parasites with and without permeabilization prior to antibody labelling. An irrelevant mouse IgG2a was included as negative control. Scale bar = 25 mm. \u003cstrong\u003eE)\u003c/strong\u003e Measurement of parasitaemia over time following adoptive transfer experiments in which B cell-deficient mMT mice acted as recipients of B cells purified from naïve or \u003cem\u003eT. brucei\u003c/em\u003e-infected splenocytes. \u003cstrong\u003eF)\u003c/strong\u003eParasitaemia as in (E) but using dura mater-derived B cells. Non-parametric ANOVA with multiple corrections. The experimental groups are statistically compared against animals that received PBS alone prior to infection with \u003cem\u003eT. brucei\u003c/em\u003e Antat 1.1E; A \u003cem\u003ep\u003c/em\u003e value \u0026lt; 0.05 was considered significant.\u003c/p\u003e","description":"","filename":"Supplementaryfigure816012026.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/5cb95a26c7e5325de0da8f56.jpg"},{"id":100748597,"identity":"a2db0c7f-2659-4218-8f60-ab8a961e8f1c","added_by":"auto","created_at":"2026-01-21 04:07:05","extension":"xlsx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":4591910,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary tables\u003c/p\u003e","description":"","filename":"QuintanaSupplementarytablesv14012026.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8621800/v1/cff7cfb9d3d01fc04aa831a9.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"The dura mater maintains B cell lymphopoietic capacity during chronic Trypanosoma brucei infection","fulltext":[{"header":"Introduction","content":"\u003cp\u003eUnder homeostasis, the dura mater hosts a wide array of resident early haematopoietic precursors including those responsible for \u003cem\u003ede novo\u003c/em\u003e B cell lymphopoiesis\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The vascular connections between the skull bone marrow in the calvaria and the underlying dura mater layers form an anatomical bridge\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, suggesting that many of the early precursor populations originate from the calvaria before seeding the dura mater. However, the impact of infections that affect the central nervous system (CNS) borders on these resident precursor populations remains to be determined. Furthermore, whether the mechanisms providing protection against infection in the dura mater result in the same type of B cell responses generated in conventional lymphoid organs such as the spleen is unknown.\u003c/p\u003e \u003cp\u003e \u003cem\u003eTrypanosoma brucei\u003c/em\u003e parasites, the causative agents of sleeping sickness, successfully colonise a myriad of tissues during the chronic stages of infection, including the CNS borders and the brain parenchyma\u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In the periphery, \u003cem\u003eT. brucei\u003c/em\u003e infection results in severe peripheral B cell lymphopenia\u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, including NK cell- and neutrophil- mediated killing of conventional B-2 B cells in the spleen\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, compromising subsequent humoral responses necessary to control the infection. This raises the question of whether alternative tissue sites can support B cell lymphopoiesis when canonical lymphoid tissues such as the bone marrow and the spleen are compromised. We previously showed that the dura mater acquires properties typically associated with ectopic lymphoid clusters allowing the generation of high affinity antibodies during the chronic stages of \u003cem\u003eT. brucei\u003c/em\u003e infection\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Critically, the presence of these dura-associated ectopic lymphoid tissues under homeostasis has been independently reported in mice and humans\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, highlighting conservation of these immunological hubs across species.\u003c/p\u003e \u003cp\u003eIn this study, we asked whether chronic \u003cem\u003eT. brucei\u003c/em\u003e infection also disrupts dura mater B cell development, as observed in \u003cem\u003ebona fide\u003c/em\u003e lymphopoietic organs such as the bone marrow and the spleen in response to \u003cem\u003eT. brucei\u003c/em\u003e infection, resulting in systemic B cell lymphopenia. Here, we show that the dura mater supports B cell lymphopoiesis when peripheral lymphopoietic sites are impaired in a sex-independent manner. Mechanistically, we report that signals from the dura mater stroma, particularly those derived from fibroblasts, endothelial cells, and sensory neurons provide environmental cues for the survival, replication, and development of B cells in this tissue compartment. We revealed that this process is dependent on CXCR4/CXCL12 and IL-7/STAT5 signalling which results in sustained B cell chemotaxis and replication capacity. Critically, we show that the repertoire of B cells developing in the dura mater undergoes extensive clonal selection that is distinct from those found in the spleen. Within the IgG\u003csup\u003e+\u003c/sup\u003e B cell compartment, dura mater B cells predominantly express various members of the \u003cem\u003eIghv1\u003c/em\u003e family, in particular \u003cem\u003eIghv1-15, Ighv1-26\u003c/em\u003e, and \u003cem\u003eIghv1-76\u003c/em\u003e compared to splenic IgG\u003csup\u003e+\u003c/sup\u003e B cells. These polyreactive dura mater-enriched IgG clones recognise a wide range of CNS and \u003cem\u003eT. brucei\u003c/em\u003e intracellular antigens, mainly flagellar and metabolic proteins. Lastly, through adoptive transfer experiments, we demonstrate that dura mater B cells, as well as the antibodies they they produce, but not splenic B cells from infected mice, significantly delay the appearance of parasites in the circulation in B cell-deficient mice, indicating that, in \u0026micro;MT recipient mice, dura mater B cells delay the onset of parasitaemia more efficiently than splenic B cells from infected mice. Collectively, our data show that the dura mater can support B cell development and selection with a distinct repertoire when peripheral lymphopoietic organs are compromised. This work highlights the dura as a site of cellular and humoral responses against a pathogen that highjacks the peripheral immune system to promote chronicity.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eThe dura mater supports B cell lymphopoiesis during chronic\u003c/b\u003e \u003cb\u003eT. brucei\u003c/b\u003e \u003cb\u003einfection\u003c/b\u003e\u003c/p\u003e \u003cp\u003eDuring homeostatic conditions, the dura mater harbours a rich population of precursor cells, including haematopoietic stem cells (HSCs) and B cell progenitors\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. However, the impact of infection on these tissue-resident populations remains to be elucidated. Thus, we first set out to characterise the impact of \u003cem\u003eT. brucei\u003c/em\u003e infection, an extracellular pathogen that actively colonises the CNS and brain borders during the chronic stage of the infection (\u0026gt;\u0026thinsp;25 days post-infection)\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e on the B cell compartment within the dura mater using flow cytometry. Extravascular lymphocytes were identified following intravenous injection of anti-CD45 antibody (\u003cb\u003eSupplementary Fig.\u0026nbsp;1A\u003c/b\u003e). Consistent with previous reports\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, we observed a significant reduction in the B cell counts within the femur bone marrow, spleen, and skull bone marrow, indicative of a widespread B cell lymphopenia in peripheral comparments, as reported previously\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Unexpectedly, the number of B cells in the dura mater remained unchanged (Fig.\u0026nbsp;1A \u003cb\u003eand B\u003c/b\u003e) even when peripheral sites supporting B cell development are blunted. These data indicate that B cell depletion in response to T. brucei infection is tissue-dependent.\u003c/p\u003e \u003cp\u003eTo further understand the impact of infection on B cell development, we initially set out to test define B cell subsets using the Hardy fraction system\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, but failed to reliably obtain enough resolution within the B cell compartment in the dura mater, owing perhaps to the relatively lower cell counts compared to other organs. To circumvent this issue, we employed a gating strategy previously reported\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e for the identification of dura mater B cell progenitors. This approach relies on the expression of the \u003cem\u003ebona fide\u003c/em\u003e B cell markers B220 and CD43 to broadly classify these cells into three main stages: CD43\u003csup\u003e+\u003c/sup\u003e B220\u003csup\u003eLow\u003c/sup\u003e early progenitors (equivalent to Hardy fraction A encompassing pro-B cells and pro-pre B cells), CD43\u003csup\u003eLow\u003c/sup\u003e B220\u003csup\u003eLow\u003c/sup\u003e late progenitors (equivalent to Hardy fractions B-D encompassing precursor B cells, including small and large pre-B cells), and CD43\u003csup\u003e-\u003c/sup\u003e B220\u003csup\u003e+\u003c/sup\u003e mature B cells (equivalent to Hardy E, encompassing B cells ready to enter the transitional stages) (\u003cb\u003eSupplementary Fig.\u0026nbsp;1B\u003c/b\u003e). The early and late progenitors were further characterised by the expression of CD93/AA4.1, IL-7 receptor, and CXCR4, whereas the late and mature stages express progressively lower levels of CD93/AA4.1 and higher levels of CD23 and IgM (\u003cb\u003eSupplementary Fig.\u0026nbsp;1B\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eIn na\u0026iuml;ve mice, we detected all B cell developmental stages in the femur and skull bone marrow, as well as in the spleen and dura mater, consistent with previous reports\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;1C). However, upon \u003cem\u003eT. brucei\u003c/em\u003e infection, most of the B cell developmental stages in the bone marrow and the spleen decreased significantly, whereas these compartments remained intact in the dura mater (Fig.\u0026nbsp;1C, \u003cb\u003eD\u003c/b\u003e). These deficiencies were not restricted to \u003cem\u003ebona fide\u003c/em\u003e B lymphopoietic organs, as B cells were also partially depleted in the gastrointestinal tract (\u003cb\u003eSupplementary Fig.\u0026nbsp;1C and D\u003c/b\u003e). Additionally, there was a significant increase in V(D)J recombination events in B cells purified from the dura mater during \u003cem\u003eT. brucei\u003c/em\u003e infection, as measured by Kappa-deleting Recombination Excision Circle (KREC) assay (\u003cb\u003eSupplementary Fig.\u0026nbsp;1E and 1F\u003c/b\u003e), demonstrating that B cell maturation events within the dura mater increase during chronic \u003cem\u003eT. brucei\u003c/em\u003e infection. These changes within the B cell compartment do not seem to be related to defects in early hematopoietic precursors, given that the frequency and abundance of cKIT\u003csup\u003e+\u003c/sup\u003e SCA1\u003csup\u003eLow\u003c/sup\u003e (LK), cKIT\u003csup\u003e+\u003c/sup\u003e SCA1\u003csup\u003e+\u003c/sup\u003e (LSK), and cKIT\u003csup\u003eLow\u003c/sup\u003e SCA1\u003csup\u003eLow\u003c/sup\u003e (LS\u003csup\u003eLow\u003c/sup\u003eK\u003csup\u003eLow\u003c/sup\u003e) hematopoietic stem cell precursors, including CD135\u003csup\u003e+\u003c/sup\u003e CD127\u003csup\u003e+\u003c/sup\u003e common lymphoid progenitors (CLPs), did not significantly change in response to infection in these compartments (\u003cb\u003eSupplementary Fig.\u0026nbsp;1G-I)\u003c/b\u003e. Furthermore, these changes were restricted to the B cell linage, as the extravascular CD3\u003csup\u003e+\u003c/sup\u003e T cell compartment significantly increased or remained unchanged in response to infection in all these organs (\u003cb\u003eSupplementary Fig.\u0026nbsp;2F\u003c/b\u003e), including in blood (\u003cb\u003eSupplementary Fig.\u0026nbsp;2G-H\u003c/b\u003e), indicating that the effects of infection are limited to developing B cells in lymphopoietic organs and not due to a broad depletion of haematopoietic progenitors.\u003c/p\u003e \u003cp\u003eCritically, we observed a significant reduction in both the frequency of intravascular and extravascular CD45\u003csup\u003e+\u003c/sup\u003e cells in the femur bone marrow and the spleen during \u003cem\u003eT. brucei\u003c/em\u003e infection, whereas only the intravascular CD45\u003csup\u003e+\u003c/sup\u003e compartment changed in the dura mater (\u003cb\u003eSupplementary Fig.\u0026nbsp;2A-B\u003c/b\u003e). Furthermore, both intra- and extravascular CD19\u003csup\u003e+\u003c/sup\u003e B220\u003csup\u003e+\u003c/sup\u003e B cells were significantly less frequent in the femur bone marrow and spleen, but the extravascular B cells did not change in the dura mater during infection (\u003cb\u003eSupplementary Fig.\u0026nbsp;2C-E\u003c/b\u003e). Lastly, B cell frequencies and counts (Fig.\u0026nbsp;2A-C), as well as developmental competencies (Fig.\u0026nbsp;2D-F) were similarly detected in both males and females, indicating that these effects are sex-independent. Together, these data demonstrate that the murine dura mater maintains early, late, and mature B cell compartments during chronic \u003cem\u003eT. brucei\u003c/em\u003e infection, independently of sex and contribution from other \u003cem\u003ebona fide\u003c/em\u003e B cell lymphoietic organs.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe dura mater is characterised by a unique set of cytokine-cytokine receptor interactions during chronic\u003c/b\u003e \u003cb\u003eT. brucei\u003c/b\u003e \u003cb\u003einfection.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo better understand how these canonical and non-canonical lymphopoietic sites respond to infection, we initially conducted a comparative whole tissue bulk transcriptomics analysis of the dura mater, local (skull) and distal (femur) bone marrow, and spleen from na\u0026iuml;ve and \u003cem\u003eT. brucei\u003c/em\u003e-infected mice. Pairwise analysis revealed a total of 2,216, 6,236, 805, and 8,640 differentially expressed genes during infection in the skull bone marrow, dura mater, femur bone marrow, and spleen, respectively, when compared to na\u0026iuml;ve controls (\u003cb\u003eSupplementary Fig.\u0026nbsp;3A and Supplementary table 1 and 2\u003c/b\u003e). In response to chronic \u003cem\u003eT. brucei\u003c/em\u003e infection, the bone marrow transcriptional landscape was defined by an upregulation of gene pathways associated with antigenic presentation and allograft (\u003cem\u003epadj\u003c/em\u003e 1.79\u003csup\u003e\u0026minus;\u0026thinsp;12\u003c/sup\u003e and \u003cem\u003epadj\u003c/em\u003e 5.73\u003csup\u003e\u0026minus;\u0026thinsp;11\u003c/sup\u003e; e.g., \u003cem\u003eH2-Ob, Tap1, H2-Q7, Cd8b1, H2-DMb2, H2-K1\u003c/em\u003e), and a downregulation of gene pathways related to hematopoietic cell linage (\u003cem\u003epadj\u003c/em\u003e 2.51\u003csup\u003e\u0026minus;\u0026thinsp;06\u003c/sup\u003e;e.g., \u003cem\u003eCd5, Cd4, Cd3e, H2-Aa, Fcgr1, H2-DMa, Cd44, Il4ra, Il3ra, Csf1r\u003c/em\u003e) and B cell homeostasis (\u003cem\u003epadj\u003c/em\u003e 6.06\u003csup\u003e\u0026minus;\u0026thinsp;05\u003c/sup\u003e;e.g., \u003cem\u003eBlk, Cd19, Cr2, Cd79a, Blnk, Cd22, Fos, Cd79b\u003c/em\u003e), consistent with our flow cytometric analyses (\u003cb\u003eSupplementary Fig.\u0026nbsp;3B and supplementary Fig.\u0026nbsp;4\u003c/b\u003e). In contrast, we observed a significant enrichment in various pathways associated with inflammatory responses such as cytokine-cytokine receptor interactions and Th17 differentiation (\u003cem\u003eIl12rb1, Rorc, Il27\u003c/em\u003e) and chemotaxis in the dura mater, consistent with our previous reports\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e (\u003cb\u003eSupplmentary Fig.\u0026nbsp;3B and C\u003c/b\u003e), and a concomitant dysregulation of gene pathways associated with extracellular matrix (ECM)-receptor interactions (\u003cem\u003epadj\u003c/em\u003e 1.04\u003csup\u003e\u0026minus;\u0026thinsp;06\u003c/sup\u003e;e.g., \u003cem\u003eCol4a5, Col2a1, Col4a6, Vwf, Itga7, Tnn, Tnxb, Vtn, Itgb7\u003c/em\u003e) and various synaptic innervations such as GABAergic synapse (\u003cem\u003epadj\u003c/em\u003e 3.05\u003csup\u003e\u0026minus;\u0026thinsp;04\u003c/sup\u003e;e.g., \u003cem\u003eAdcy1, Slc38a2, Slc38a3, Gabrd\u003c/em\u003e), Glutamatergic synapse (\u003cem\u003epadj\u003c/em\u003e 3.84\u003csup\u003e\u0026minus;\u0026thinsp;05\u003c/sup\u003e;e.g., \u003cem\u003eGrin2d, Grin2c, Gnb3, Slc17a6, Sl17a8\u003c/em\u003e), and neuroactive ligand-receptor interactions (\u003cem\u003epadj\u003c/em\u003e 1.85\u003csup\u003e\u0026minus;\u0026thinsp;06\u003c/sup\u003e;e.g., \u003cem\u003eP2rx4, Adora2a, Htr7, Adra1a, Ghr, Calcr, Vip, Ntsr1\u003c/em\u003e), owing perhaps to disruptions in the the synaptic innervations of the dura mater from the trigeminal ganglia\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. These datasets highlight broad differential transcriptional responses triggered by infection between the dura mater and canonical lymphopoietic sites.\u003c/p\u003e \u003cp\u003eTo gain additional insights into which stromal cell types may be involved in promoting B cell differentiation in the dura mater, we performed comparative single cell transcriptomic analyses of stromal and CD45\u003csup\u003e+\u003c/sup\u003e immune cells from the dura mater and the adjacent bone marrow in the calvaria in response to chronic \u003cem\u003eT. brucei\u003c/em\u003e infection. After removing low quality cells, we obtained a total of 74,696 high quality cells, split across the skull bone marrow (46,966 cells) and the dura mater (27,730 cells) from na\u0026iuml;ve and \u003cem\u003eT. brucei\u003c/em\u003e infected mice (Fig.\u0026nbsp;3A \u003cb\u003eand B\u003c/b\u003e). These cells segregrated into 17 transcriptional clusters encompassing both immune and stromal cells. Within the immune compartment, we identified clusters expressing \u003cem\u003ebona fide\u003c/em\u003e markers for myeloid cells (e.g., \u003cem\u003eCtsc, Lyz2, C1qa, Ccr5, Mertk, Chil3, Mpo, Ccr2, Cd177, F13a1\u003c/em\u003e), common erythroid progenitors (e.g., \u003cem\u003eSox6, Hba-a1, Hba-a2, Scl4a1\u003c/em\u003e), neutrophils (e.g., \u003cem\u003eS100a8, S100a9, Lcn2, Mmp8\u003c/em\u003e), granulocytes (e.g., \u003cem\u003eCxcl12, S100a8, S100a9, Pdzm4\u003c/em\u003e), Early B cell stages (e.g., \u003cem\u003eBach2, Aff3, Ebf1, Prkch, Pax5\u003c/em\u003e), various innate and conventional T cell subsets (e.g., \u003cem\u003eCd4, Cd8a, Cd274, Tox, Ctla4\u003c/em\u003e), plasma cells (e.g., \u003cem\u003eIgkc, Ighm, Jchain, Xbp1, Pou2af1\u003c/em\u003e), and hematopoietic stem cells/common myeloid progenitors (e.g., \u003cem\u003eCd86, H2-Eb1, Flt3, Etv6\u003c/em\u003e) (Fig.\u0026nbsp;3A-C). Within the stroma, we identified various vascular-associated cells subsets including lymphatic endothelial cells (e.g., \u003cem\u003ePdpn, Pecam1, Col1a1, Col1a2, Il33\u003c/em\u003e), blood endothelial cells (e.g., \u003cem\u003ePecam1, Esam, Tek, Adgrl4, Flt1\u003c/em\u003e), choroid plexus-like epithelial cells (e.g., \u003cem\u003eTtr, Enpp2, Htrc2, Slc4a10, Col9a3\u003c/em\u003e) similar to those previously reported at the dura-arachnoid interface\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, radial glia-like cells\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e (e.g., \u003cem\u003eSox9, Pax9, Ncam1, Chl1\u003c/em\u003e), and various sensory neuron subsets (e.g., \u003cem\u003eRbfox1, Grid2, Trpv1, Dcx, Grip1, Syt1\u003c/em\u003e), likely representing innervations from the trigeminal ganglia\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;3A-C). We further subset the B cell compartment and identified a total of 4,450 high-quality B cells from the skull bone marrow (3,088 cells) and the dura mater (1,362 cells) (Fig.\u0026nbsp;3D \u003cb\u003eand E\u003c/b\u003e). These cells encompassed hematopoietic stem cells/common lymphoid progenitors (e.g., \u003cem\u003eRunx2, Tcf4, Siglech, Irf8\u003c/em\u003e), early/late B progenitors (e.g., \u003cem\u003eBach2, Aff3, Ebf1, Bank1, Pax5, Ikzf3\u003c/em\u003e), as well as mature B cells (e.g., \u003cem\u003eCd19, Ms4a1, Ebf1\u003c/em\u003e), myeloid-like B cells (e.g., \u003cem\u003eSaa3, Igkc, Ighm, Cd74, Itgam\u003c/em\u003e) that we have detected in the dura mater previously\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, and plasmablasts/plasma cells (e.g., \u003cem\u003eIgkc, Ighm, Jchain, Inpp4b\u003c/em\u003e) (Fig.\u0026nbsp;3D-F). As expected, we found HSCs/CLPs as well as three distinct transcriptional clusters of early/late B cell stages (Early/late B 1 to B 3), mature B cells, and plasmablasts/plasma cells in the skull bone marrow of na\u0026iuml;ve animals, whereas in the dura mater we identified HSCs/CLPs and cells within the early/late B cell clusters 2 and 3, characterised by the expression of genes associated with the final stages of B cell maturation such as \u003cem\u003eEbf1, Pax5, Bank1\u003c/em\u003e, and \u003cem\u003eIkzf3\u003c/em\u003e (encoding the transcription factor AIOLOS)\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;3E). In contrast, during chronic \u003cem\u003eT. brucei\u003c/em\u003e infection, these early precursors, in particular cells within early/late B cell clusters 1, were not detected in the skull bone marrow, whereas early/late B cell clusters 2 expanded in the dura mater (Fig.\u0026nbsp;3E), in agreement with our flow cytometry results. These early/late stages B cell stages were dominated by gene pathways associated with cell cycle and apoptosis, whereas the ones in the dura mater were enriched in gene pathways associated with antigenic presentation, endocytosis, and hematopoietic cell linage commitment (Fig.\u0026nbsp;3G).\u003c/p\u003e \u003cp\u003eThese results might suggest that the signals within each tissue compartment either promote apoptosis and impaired differentiation (skull bone marrow) or activation and lineage commitment (dura mater). B cell development is intimately linked to stromal-derived cues to be successfully completed. We therefore conducted cell-cell interaction analysis using CellChat\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e to identify the potential pathways that might be involved in sustaining B cell development in the dura mater during chronic \u003cem\u003eT. brucei\u003c/em\u003e infection. This analysis revealed that the lymphatic endothelial cells and, to a lesser extent, blood endothelial cells and myeloid cells, are predicted to be key coordinators of the interactions occurring in the skull bone marrow and dura mater during \u003cem\u003eT. brucei\u003c/em\u003e infection (\u003cb\u003eSupplementary Fig.\u0026nbsp;4A and B\u003c/b\u003e). Interestingly, previous studies have highlighted that fibroblasts mediate dura mater B cell recruitment and retention \u003cem\u003evia\u003c/em\u003e CXCR4/CXCL12 signalling axis under homeostasis\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, but so far this has not been tested in the context of an inflammatory meningeal disease. Our \u003cem\u003ein silico\u003c/em\u003e prediction highlights lymphatic endothelial cells, as well as granulocytes, provide several chemoattactant cues to various immune cells in the skull bone marrow and dura mater during \u003cem\u003eT. brucei\u003c/em\u003e infection, mediated primarily by \u003cem\u003eCxcr4-Cxcl12, Cxcr2/Cxcl12\u003c/em\u003e, and \u003cem\u003ePpbp-Cxcr2\u003c/em\u003e (Fig.\u0026nbsp;4A, \u003cb\u003eand Supplementary Fig.\u0026nbsp;4C\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eTo better characterise this stroma-B cell interaction axis in the dura mater and bone marrow during \u003cem\u003eT. brucei\u003c/em\u003e infection, we next asked whether these tissues increase their expression of CXCL12. Indeed, we observed that the production of CXCL12 is significantly increased in the dura mater in response to \u003cem\u003eT. brucei\u003c/em\u003e infection (Fig.\u0026nbsp;4B), potentially suggesting a higher chemotactic drive towards cell retention in these tissue compartments. We then asked whether the expression of CXCR4, the main receptor for CXCL12 driving B cell recruitment during development\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, also increased in various B cell developmental stages within these tissue compartments. CXCR4 expression did not change significantly in early, late, and mature B cells in the skull bone marrow in response to infection, whereas early B cell stages in the dura mater expressed significantly higher levels of CXCR4 during infection (Fig.\u0026nbsp;4D \u003cb\u003eand E\u003c/b\u003e), indicating a higher chemotactic and/or retention drive of dura mater early B cell precursors towards a CXCL12 gradient within this compartment. To functionally test whether bone marrow- and dura mater-derived B cells have a similar chemotaxis capacity mediated by the CXCL12/CXCR4 signalling axis, we established a transwell system to measure B cells to migrate towards a CXCL12 gradient. In na\u0026iuml;ve mice, we observed that both femur bone marrow- and dura mater-derived B cells efficiently migrated towards a gradient of CXCL12 to similar levels (chemotaxis index of ~\u0026thinsp;2 in both cases) in a process dependent on CXCR4 signalling, as demonstrated by the significant reduction in migration in the presence of CXCR4 blocking antibody (\u003cb\u003eSupplementary Fig.\u0026nbsp;4D\u003c/b\u003e). However, B cells obtained from the bone marrow of infected mice, but not from the dura mater, showed a diminished capacity to sense and migrate towards a CXCL12 compared to B cells from na\u0026iuml;ve bone marrow (\u003cb\u003eSupplementary Fig.\u0026nbsp;4D\u003c/b\u003e), despite an increased in CXCR4 expression, indicating impaired chemotaxis in the B cells derived from the bone marrow. Unexpectedly, the CXCL-12-mediated chemotaxis of dura mater-dwelling B cells seems to be independent of CXCR4 during infection, as CXCR4 blocking antibody did not significantly reduce the migration of these cells (\u003cb\u003eSupplementary Fig.\u0026nbsp;4D\u003c/b\u003e), owing perhaps to an overall higher abundance of CXCR4 in dura mater-derived B cells during infection compared to bone marrow-derived B cells. This observation, together with an increased expression of CXCL12, indicate a higher chemotaxis and retention potential in the dura mater during \u003cem\u003eT. brucei\u003c/em\u003e infection.\u003c/p\u003e \u003cp\u003eOnce retained within the tissue, early B cell stages require molecules such as IL-7 provided by their niches to promote their proliferation, survival, and the genomic reorganisation during development\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. We observed no significant changes in the expression of IL-7Ra (CD127) in the early B cell stages in the skull bone marrow or dura mater in response to the infection (Fig.\u0026nbsp;4F \u003cb\u003eand G\u003c/b\u003e). However, we observed a significant decrease in the levels of phospho-STAT5, which is a cognate IL-7Ra signal transducer, in the early, late, and mature stages of B cell development in the skull bone marrow (Fig.\u0026nbsp;4H \u003cb\u003eand I\u003c/b\u003e). In the dura mater, early stage B cells showed a significant increased in the levels of phospho-STAT5 in response to infection, and this remained unchanged throughout B cell development in this tissue compartment (Fig.\u0026nbsp;4H \u003cb\u003eand I\u003c/b\u003e), indicating that the dura mater provides critical pro-survival and replicative cues that are disrupted in the skull bone marrow. Consistent with this, chronic \u003cem\u003eT. brucei\u003c/em\u003e infection also resulted in a comparatively higher frequency of Ki67\u003csup\u003e+\u003c/sup\u003e B cells in the dura mater B cells compared to those in the femur bone marrow (Fig.\u0026nbsp;4I \u003cb\u003eand J\u003c/b\u003e). Together, these results indicate that the dura mater provide chemotactic and pro-survival cues supporting local B cell development during chronic \u003cem\u003eT. brucei\u003c/em\u003e infection, while these niches are disrupted in the bone marrow.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDura mater B cells have a unique and expanded B cell receptor repertoire during\u003c/b\u003e \u003cb\u003eT. brucei\u003c/b\u003e \u003cb\u003einfection\u003c/b\u003e\u003c/p\u003e \u003cp\u003eHaving established that the dura mater provides a nurturing environment for the survival and development of dura mater-dwelling B cells during chronic \u003cem\u003eT. brucei\u003c/em\u003e infection, we next asked whether these B cells are phenotypically different from those found peripherally. We first profiled the diversity of antibody isotypes detected in the spleen and dura mater in response to chronic \u003cem\u003eT. brucei\u003c/em\u003e infection by flow cytometry. We observed that in response to \u003cem\u003eT. brucei\u003c/em\u003e infection there was a significant increase in the number of IgM\u003csup\u003e+\u003c/sup\u003e, IgG\u003csup\u003e+\u003c/sup\u003e, and IgA\u003csup\u003e+\u003c/sup\u003e extravascular B cells in the dura mater of female mice (Fig.\u0026nbsp;5A-C). In the dura mater of male mice, only IgA\u003csup\u003e+\u003c/sup\u003e extravascular B cells significantly diminished in numbers, whereas IgM\u003csup\u003e+\u003c/sup\u003e and IgG\u003csup\u003e+\u003c/sup\u003e B cells did not significantly change in response to infection (\u003cb\u003eSupplementary Fig.\u0026nbsp;5A-C\u003c/b\u003e). These responses at the brain border were in sharp contrast to those observed in the spleen, where IgM\u003csup\u003e+\u003c/sup\u003e B cells were significantly reduced in numbers in both females and male mice, without noticeable changes in the IgA\u003csup\u003e+\u003c/sup\u003e B cell counts (Fig.\u0026nbsp;5A-C \u003cb\u003eand Supplementary Fig.\u0026nbsp;5A-C\u003c/b\u003e). Male mice also displayed a significant reduction in the number of IgG\u003csup\u003e+\u003c/sup\u003e B cells in the spleen in response to infection that was not detected in female mice, further highlighting potential sex-dependent humoral responses to infection (Fig.\u0026nbsp;5A-C \u003cb\u003eand Supplementary Fig.\u0026nbsp;5A-C\u003c/b\u003e). We scarcely detected IgE\u003csup\u003e+\u003c/sup\u003e B cells in the extravascular compartment in both tissues across sexes and experimental conditions (Fig.\u0026nbsp;5A-C \u003cb\u003eand Supplementary Fig.\u0026nbsp;5A-C\u003c/b\u003e). These results highlight tissue-dependent effects on the humoral responses to \u003cem\u003eT. brucei\u003c/em\u003e infection, with a broad expansion of B cell subsets occurring specifically in the dura mater of female mice.\u003c/p\u003e \u003cp\u003eWe previously reported that \u003cem\u003eT. brucei\u003c/em\u003e infection results in the accumulation of IgG\u003csup\u003e+\u003c/sup\u003e autorreactive B cells in the dura mater of female mice\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, consistent with our results so far. Given the potential role of IgG deposition and IgG autoantibodies in the neuropathology observed in this experimental system, we then asked whether the population of dura mater-dwelling IgG\u003csup\u003e+\u003c/sup\u003e B cells are clonally related to those found in the spleen. We also asked whether chronic \u003cem\u003eT. brucei\u003c/em\u003e infection shapes the B cell diversity independently of other organs such as the spleen. To do this, we performed high-throughput, IgG-focussed B cell receptor (BCR) sequencing, including the heavy and light chains, comparing B cells from the spleen and dura mater of na\u0026iuml;ve mice or during the chronic stage of \u003cem\u003eT. brucei\u003c/em\u003e infection. All dura mater and splenic BCR repertoire libraires were prepared and sequenced in parallel with identical primer pools and batches to account for potential confounders introduced by primer biases. Based on the \u003cem\u003eIghv\u003c/em\u003e profiling, we observed that the number of unique B cell IgG clones, as determined by the complementary-determining region 3 (CDR3) sequence, significantly expanded in the spleen during infection from ~\u0026thinsp;17 distinct BCR clones in na\u0026iuml;ve animals to ~\u0026thinsp;87 different \u003cem\u003eIghv\u003c/em\u003e clones during \u003cem\u003eT. brucei\u003c/em\u003e infection (\u003cb\u003eSupplementary Fig.\u0026nbsp;6A\u003c/b\u003e). However, in the dura mater, the overall number of B cell IgG clones significantly diminished, from ~\u0026thinsp;112 unique BCR IgG clones based on \u003cem\u003eIghv\u003c/em\u003e gene detection in na\u0026iuml;ve animals to ~\u0026thinsp;27 clones in \u003cem\u003eT. brucei\u003c/em\u003e infected animals (\u003cb\u003eSupplementary Fig.\u0026nbsp;6A\u003c/b\u003e). The same splenic clonal expansion was evident when exploring \u003cem\u003eIgkv\u003c/em\u003e clonality, without significant changes in the dura mater repertoire (\u003cb\u003eSupplementary Fig.\u0026nbsp;6B\u003c/b\u003e). Additionally, no significant differences were observed in the number of unique \u003cem\u003eIglv\u003c/em\u003e B cell clones in either the spleen or dura mater (\u003cb\u003eSupplementary Fig.\u0026nbsp;6C\u003c/b\u003e). Overall, of the 71 and 73 \u003cem\u003eIghv\u003c/em\u003e genes detected in the dura mater and spleen, respectively, 69.4% (59 genes) were shared between tissues, whereas 14.4% (12 genes) were exclusively detected in the dura mater, including several members of the \u003cem\u003eIghv1\u003c/em\u003e family (\u003cem\u003eIghv1-78, Ighv1-66, Ighv1-58, Ighv1-36\u003c/em\u003e), \u003cem\u003eIghv5\u003c/em\u003e (\u003cem\u003eIghv5-6, Ighv5-12-1\u003c/em\u003e). Additionally, 16.5% (14 genes) were exclusively detected in the spleen (\u003cb\u003eSupplementary Fig.\u0026nbsp;6D\u003c/b\u003e). Across matched cohorts processed with identical library preparations and IMGT-based mapping, most of the clones detected in the dura mater during infection had a significant bias towards the expression of the \u003cem\u003eIghv1\u003c/em\u003e family, including \u003cem\u003eIghv1-76, Ighv1-26\u003c/em\u003e, and \u003cem\u003eIghv1-15\u003c/em\u003e, relative to splenic BCRs (Fig.\u0026nbsp;5D \u003cb\u003eand supplementary Fig.\u0026nbsp;5E\u003c/b\u003e). B cells expressing \u003cem\u003eIghv1-76, Ighv1-26\u003c/em\u003e, and \u003cem\u003eIghv1-15\u003c/em\u003e became more abundant in the dura mater in response to infection, from approximately 1.26%, 2.64%, and 3.08% under homeostasis to ~\u0026thinsp;4.84%, 17.27%, and 15.08% during infection, respectively (Fig.\u0026nbsp;5D, \u003cb\u003eE, and Supplementary table 5 and 6)\u003c/b\u003e. Further examination of the \u003cem\u003eIghv\u003c/em\u003e repertoire revelaed that these are public clones, as these were independently sequenced in \u0026gt;\u0026thinsp;2 mice within each experimental group (Fig.\u0026nbsp;5E). We observed no selection bias for particular \u003cem\u003eIghv\u003c/em\u003e clones in the spleen between na\u0026iuml;ve and infected animals (\u003cb\u003eSupplementary Fig.\u0026nbsp;6F\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eDuring B cell activation, the complementary-determining region (CDR), in particular CDR3, undergo activation-induced deaminase (AID)-dependent somatic hypermutation and affinity maturation. In na\u0026iuml;ve animals, the CDR3 amino acid length was comparable between B cell clones in the dura mater and the spleen, both around ~\u0026thinsp;12\u0026ndash;13 amino acids in leght (Fig.\u0026nbsp;5F \u003cb\u003eand G, and supplementary table 7\u003c/b\u003e). However, during chronic \u003cem\u003eT. brucei\u003c/em\u003e infection the length of the IgH CDR3 sequences of B cell clones in the dura mater became significantly shorter (~\u0026thinsp;11 amino acids) compared to those in the spleen (~\u0026thinsp;13 amino acids), where no differences were detected (Fig.\u0026nbsp;5F \u003cb\u003eand G, and supplementary table 7\u003c/b\u003e). This indicates that distinct insertions/deletions mechanisms for affinity maturation are at play in the dura mater compared to the spleen during chronic \u003cem\u003eT. brucei\u003c/em\u003e infection.\u003c/p\u003e \u003cp\u003eThe usage of particular \u003cem\u003eIgkv\u003c/em\u003e chain genes were also diverse in the dura mater during infection compared to the spleen (Fig.\u0026nbsp;5H, \u003cb\u003esupplementary Fig.\u0026nbsp;5G, and Supplementary table 5 and 6\u003c/b\u003e). For instance, B cells expressing members of the \u003cem\u003eIgkv6\u003c/em\u003e family, including \u003cem\u003eIgkv6-15, Igkv6-23, and Igkv6-32\u003c/em\u003e, become more abundant in the dura mater in response to infection, from approximately 8.08%, 3.30%, and 1.54% under homeostasis to ~\u0026thinsp;15.81%, 11.39%, and 5.33% during infection (Fig.\u0026nbsp;5I \u003cb\u003eand supplementary Fig.\u0026nbsp;5G\u003c/b\u003e). In contrast, there was no significant expansion of \u003cem\u003eIgkv6-15\u003c/em\u003e in the spleen during infection, and a more modest expansion from 1.95%, and 1.84% under homeostasis to 8.05%, and 6.7% for \u003cem\u003eIgkv6-23, and Igkv6-32\u003c/em\u003e, respectively \u003cem\u003e(Fig.\u0026nbsp;5H and 5I\u003c/em\u003e, \u003cb\u003eSupplementary Fig.\u0026nbsp;5H, and Supplementary table 5 and 6\u003c/b\u003e). The IgK CDR3 amino acid length of the B cells in the dura mater was significantly longer than those in the spleen in na\u0026iuml;ve animals (Fig.\u0026nbsp;5J \u003cb\u003eand K, and Supplementary table 7\u003c/b\u003e), and became significantly shorter (~\u0026thinsp;11.5 amino acids in na\u0026iuml;ve to ~\u0026thinsp;10.5 amino acids in infected) in response to infection (Fig.\u0026nbsp;5J \u003cb\u003eand K\u003c/b\u003e), without noticeable changes in the splenic B cell clones. Taken together, our depth-normalised sequencing analysis demonstrate that the IgG BCR repertoire in the dura mater is uniquely enriched for members of the \u003cem\u003eIghv1\u003c/em\u003e family during chronic \u003cem\u003eT. brucei\u003c/em\u003e infection. Furthermore, these rearrangement studies support the notion that dura mater-dwelling B cells display distinctive features, including clonal selection and BCR sequence maturation, that are distinct from B cells in peripheral organs such as the spleen during chronic CNS infections. This possibly reflects the development of ectopic structures in the dura mater, which support germinal centre-like reactions during chronic \u003cem\u003eT. brucei\u003c/em\u003e infection\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDura mater B cell-associated antibodies are polyreactive and recognise a wide range of CNS antigens\u003c/h2\u003e \u003cp\u003eWe previously demonstrated that chronic \u003cem\u003eT. brucei\u003c/em\u003e infection triggers a broad autoimmune response resulting in IgG deposition in the brain\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Having established that three members of the \u003cem\u003eIghv1\u003c/em\u003e family, namely \u003cem\u003eIghv1-15, Ighv1-26\u003c/em\u003e, and \u003cem\u003eIghv1-76\u003c/em\u003e, and \u003cem\u003eIgkv6-32\u003c/em\u003e are significantly overrepresented in the dura mater during infection when compared to the splenic BCR repertoire, we next examined whether these immunoglobulins were able to recognise host antigens. Both heavy and light chain candidates expanded in the dura mater during infection provided us with a rational for downstream functional testing. To address this, we generated recombinant monoclonal antibodies, pairing the various members of the \u003cem\u003eIghv1\u003c/em\u003e identified in our screening with \u003cem\u003eIgkv6-32.\u003c/em\u003e Once generated, we used these recombinant antibodies to screen for potential binding capacity against host antigens using a targeted array of 120 brain and neuronal-associated antigens. We observed that Ig1-15, Ig1-26, and Ig1-76 were able to significantly recognise 81% (94 antigens) of the antigens in the array, including several proteins involved with neurotransmission (GAD1/GAD67, GAD2, GAD65, GRID2, GLUD2, AchRm2, GRIA2,AchR3, mGluR1, mGluR2), CNS autoimmunity (MBP, MOG, MOBP, MAG, AQP4) and inflammation (Lactoferrin, GAPDH, S100A8/A9, LY6H, and Lipocalin-2), amongst others (Fig.\u0026nbsp;6A and 6B \u003cb\u003eand Supplementary table 8\u003c/b\u003e). Interestingly, these recombinant monoclonal antibodies also recognise myelin basic protein (MBP), which we previously detected in serum and cerebrospinal fluid of chronically infected mice and humans\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Furthermore, Ig1-76, and to a lesser extentextent Ig1-26 recognised systemic antigens such as cytokines (e.g., IFN-α, MPO) complement (e.g., C1q), surface antigens (e.g., CD4, CD8a, b2-glycoprotein 1), viral particles (e.g., EBV, Influenza), and intracellular antigens (e.g., histones, nucleosome, dsDNA) (\u003cb\u003eSupplementary Fig.\u0026nbsp;7\u003c/b\u003e), further highlighting the broad range of antigenic recognition. To validate these findings, we developed an immunoassay with two selected antigens detected in these screenings: Lactoferrin, which is the top hit in the three monoclonal antibody screenings and is thought to be exploited by \u003cem\u003eT. brucei\u003c/em\u003e to acquire iron from their microenvironment\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, and myelin oligodendrocyte glycoprotein (MOG), which we previously reported being associated with \u003cem\u003eT. brucei\u003c/em\u003e infection\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. As negative controls, we included recombinant Activated Leukocyte Cell Adhesion Molecule (ALCAM) and Bovine Serum Albumin (BSA). Consistent with the antigen array, we observed a high binding affinity of Ig1-26 for MOG and Lactoferrin, whereas Ig1-76 seemed to preferentially detect Lactoferrin (Fig.\u0026nbsp;6C). We failed to detect a robust signal with Ig1-15 (Fig.\u0026nbsp;6C). Importantly, none of these antibodies recognised ALCAM or BSA, highlighting their potential specificity for the antigens identified in the screening. Taken together, these studies demonstrate that \u003cem\u003eIghv1/Igkv6-32\u003c/em\u003e recombinant antibodies, which are overrepresented in the dura mater during \u003cem\u003eT. brucei\u003c/em\u003e infection, recognise brain and neuronal-associated antigens.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe antibodies produced by dura mater B cells recognise metabolic and flagellar\u003c/b\u003e \u003cb\u003eT. brucei\u003c/b\u003e \u003cb\u003eantigens\u003c/b\u003e\u003c/p\u003e \u003cp\u003eHaving established that the antibodies produced by dura mater B cells during chronic infection are able to bind host neuronal-associated antigens, we next asked whether these antibodies also recognise \u003cem\u003eT. brucei\u003c/em\u003e-associated antigens. To address this, we employed a a two-step protein purification approach (gel filtration and anion exchange) from total \u003cem\u003eT. brucei\u003c/em\u003e lysate (Fig.\u0026nbsp;7A). This combined approach enabled us to improve resolution to identify \u003cem\u003ebona fide\u003c/em\u003e chromatographic fractions of interest using an ELISA-based screening. The initial gel exclusion chromatography step identified five fractions that showed robust positivity using either Ig1-15, Ig1-26, or Ig-76 (Fig.\u0026nbsp;7A \u003cb\u003eand supplementary Fig.\u0026nbsp;8A\u003c/b\u003e). hese were all confirmed to contain proteins with a wide range of molecular weights (\u003cb\u003eSupplementary Fig.\u0026nbsp;8B\u003c/b\u003e). In particular, the \u003cem\u003eT. brucei\u003c/em\u003e antigens identified by Ig1-76 and Ig1-26 spanned proteins ranging from 250 kDa to 20 kDa, whereas the ones detected by Ig1-15 were ~\u0026thinsp;10\u0026ndash;15 kDa (\u003cb\u003eSupplementary Fig.\u0026nbsp;8B\u003c/b\u003e). Upon anion exchange chromatography, these were further resolved into nine fractions of interest that were preferentially detected by either Ig1-15 or Ig1-26 (Fig.\u0026nbsp;7A \u003cb\u003eand supplementary Fig.\u0026nbsp;8C\u003c/b\u003e), but none were reactive when using Ig1-76 so this antibody was excluded from downstream analyses. Downstream \u003cem\u003ein silico\u003c/em\u003e analysis of the LC-MS/MS data from these nine fractions identified a total of 181 significant peptide hits (-Log10 \u003cem\u003ep\u003c/em\u003e value\u0026thinsp;\u0026gt;\u0026thinsp;20) encompassing 56 glycosomal (e.g., fructose-biphosphate aldolase, glycerol-3-phosphate dehydrogenase), flagellar (e.g., paraflagellar rod protein), nuclear and/or kinetoplastid (e.g., nucleolar protein), and cytoplasmic (e.g., heat shock proteins, alpha tubulin, polyubiquitin, calpain) \u003cem\u003eT. brucei\u003c/em\u003e antigens (Fig.\u0026nbsp;7B \u003cb\u003eand supplementary table 9\u003c/b\u003e). Unexpectedly, these antibodies had limited reactivity to variant surface glyproteins (VSGs), which are recognised to be the main immunogenic \u003cem\u003eT. brucei\u003c/em\u003e molecules, indicating that intracellular antigens also elicit robust immune responses. Most of these antigens were detected in fractions two, three, and five, which were preferentially recognised by Ig1-26, and to lesser extent by Ig1-15 (Fig.\u0026nbsp;7B \u003cb\u003eand supplementary table 9\u003c/b\u003e), and were associated with metabolic, flagellar, or stress response pathways (Fig.\u0026nbsp;7C). Indeed, using immunofluorescence and flow cytometry on cultured-adapted \u003cem\u003eT. brucei\u003c/em\u003e, we identified that both Ig1-15 and Ig1-26 bound to intracellular antigens compared to surface antigens (Fig.\u0026nbsp;7D \u003cb\u003eand E, and supplementary Fig.\u0026nbsp;8D\u003c/b\u003e). Furthermore, the affinity of these antibodies to \u003cem\u003eT. brucei\u003c/em\u003e Fructose-biphosphate-aldolase (TbFBA) and heat-shock protein 70 (TbHSP70), but not the murine counterparts, was independently confirmed by ELISA using recombinant proteins (Fig.\u0026nbsp;7F and 7G, respectively), confirming our proteomics analysis. Altoghether, these results demonstrate that the antibodies generated in the dura mater during chronic \u003cem\u003eT. brucei\u003c/em\u003e infection can also recognise a wide range of intracellular \u003cem\u003eT. brucei\u003c/em\u003e antigens.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDura mater B cells and the antibodies they produce delay the onset of\u003c/b\u003e \u003cb\u003eT. brucei\u003c/b\u003e \u003cb\u003einfection\u003c/b\u003e\u003c/p\u003e \u003cp\u003eHaving established that the antibodies produced by dura mater-dwelling B cells recognise \u003cem\u003eT. brucei\u003c/em\u003e antigens, we then asked whether these B cells were able to delay the onset of infection \u003cem\u003ein vivo\u003c/em\u003e. For this, we adoptively transferred B cells from the dura mater or spleen obtained from infected mice or na\u0026iuml;ve controls into \u0026micro;MT mice, which lack mature B cells and immunoglobulins, as previously reported\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. We chose this model to explore specific B cell-dependent effects on the control of \u003cem\u003eT. brucei\u003c/em\u003e infection systemically. Once transferred, we monitored the appearance of parasites in circulation and the levels of anti-\u003cem\u003eT. brucei\u003c/em\u003e IgG titres in serum in these various experimental conditions. As expected, parasites were detected in circulation within ~\u0026thinsp;48 hours in \u0026micro;MT mice that received PBS only (Fig.\u0026nbsp;7H) but were protected for a significantly longer period of time when the \u0026micro;MT mice received splenic B cells from na\u0026iuml;ve controls compared to the PBS-treated group (\u003cb\u003esupplementary Fig.\u0026nbsp;8E\u003c/b\u003e), owing perhaps to the production of natural IgM by splenic na\u0026iuml;ve B cells\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. However, chronic \u003cem\u003eT. brucei\u003c/em\u003e infection resulted in an inability of splenic B cells to control the onset of the infection in \u0026micro;MT mice, as demonstrated by no significant changes in parasitaemia compared to the PBS-treated group (\u003cb\u003eSupplementary Fig.\u0026nbsp;8E\u003c/b\u003e), indicating that the splenic B cells are functionally impaired, as previously reported\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. In contrast, we found that when \u0026micro;MT mice received B cells from the dura mater of either na\u0026iuml;ve and \u003cem\u003eT. brucei\u003c/em\u003e-infected animals, they displayed lower levels of parasitaemia and remained undetectable for longer, as evidenced by the sustained control of the parasite numbers for a period of ~\u0026thinsp;72 hours (Fig.\u0026nbsp;7H \u003cb\u003eand supplementary Fig.\u0026nbsp;8F\u003c/b\u003e). Indeed, \u0026micro;MT mice that received dura mater B cells from chronically infected C57BL/6 animals showed significantly lower levels of parasitaemia compared to the matched splenic B cells counterparts (Fig.\u0026nbsp;7H), indicating that dura mater B cells are able to delay the onset of the infection more effectively than splenic B cells in the \u0026micro;MT model. We also noticed that the \u0026micro;MT mice reconstituted with dura mater B cells intraperitoneally from infected donor mice showed higher levels of anti-\u003cem\u003eT. brucei\u003c/em\u003e IgG2a in serum compared with those that received splenic B cells (Fig.\u0026nbsp;7I). Lastly, passive transfer of recombinant Ig1-15, Ig1-26, or a combination of both, resulted in significantly lower levels of parasites in circulation compared to mice that received irrelevant mouse IgG2a control (Fig.\u0026nbsp;7J). demonstrating that antibodies contribute to early control. Taken together, these studies demonstrate that the B cells nurtured in the dura mater during chronic infection, and potentially the antibodies they produce, can delay the early onset of \u003cem\u003eT. brucei\u003c/em\u003e parasitaemia.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we demonstrate, for the first time, that the dura mater acts as an autonomous B cell niche supporting immune responses during chronic systemic inflammatory challenges, such as those triggered by \u003cem\u003eTrypanosoma brucei\u003c/em\u003e infection. Using a flow cytometry approach to identify early, late, and mature B cell subsets \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, we demonstrated that all B cells stages in the dura mater remain unchanged during chronic \u003cem\u003eT. brucei\u003c/em\u003e whilst the femur and skull bone marrows, and the spleen B cell compartment, are all impaired in both male and female mice, albeit with potential sex-dependent effects on the type of humoral responses in each of these tissues. Furthermore, using whole tissue and single cell transcriptomics coupled with \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003eex vivo\u003c/em\u003e functional studies, we demonstrate that the dura mater B cells retention and survival during chronic \u003cem\u003eT. brucei\u003c/em\u003e infection involves CXCR4/CXCL12 and IL-7/STAT5 signalling. Furthermore, we found that the B cells residing in the dura mater express a distinct BCR repertoire compared to splenic B cells during chronic \u003cem\u003eT. brucei\u003c/em\u003e infection, resulting in the generation of polyreactive antibodies able to recognise a broad range of host and parasite proteins. Lastly, using passive and active adoptive transfer experiments, we showed that dura mater-derived B cells, but not splenic-derived B cells, from infected mice significantly delay the onset of the infection in B cell-deficient mice, highlighting the potential protective capacity of the B cells generated at the brain borders whereas splenic B cells seem functionally impaired.\u003c/p\u003e \u003cp\u003eGiven that the early B cell precursors are likely derived from the skull bone marrow and seed the dura mater through skull channels\u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, it is tempting to speculate that an increased capacity of skull bone-derived marrow B cells to migrate towards a CXCL12 gradient, in particular early B cell progenitors, results in continuous seeding of the meningeal microenvironment. Once in the dura mater, additional pro-survival factors derived from the stromal cells such as IL-7 sustain B cell development and maturation. Indeed, we detected an overrepresentation of genes associated with apoptosis and mitochondrial metabolism in the early B cell developmental stages in the skull bone marrow, whereas the same populations in the dura mater expressed genes associated with activation, antigenic presentation, and hematopoietic commitment, consistent with the notion that the bone marrow fails to provide enough survival factors, resulting in B cell depletion. These processes are likely to occur in a sex-independent manner, as the dura mater from both male and female mice showed no signs of infection-induced B cell lymphopenia. However, our results do not rule out the possibility that a fraction of mature B cells in the dura mater, in particular mature IgA\u003csup\u003e+\u003c/sup\u003e B cells, migrate to and seed the meninges compartment from other tissues such as the nasal turbinates\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e or the gastrointestinal tract\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe observed a strong bias towards IgA\u003csup\u003e+\u003c/sup\u003e B cell accumulation in the spleen and dura mater of male mice but not in females, indicating a sex-dependent bias in the humoral responses to infection. Future work addressing the role and impact of sex hormones on the adaptive and humoral responses to infection at the brain borders merits further investigation. We have previously reported an IL-17 signalling sex-dependent bias in the Th17 responses and weight loss elicited in male mice in response to infection\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, and our data further uncover sex-dependent immunological responses to \u003cem\u003eT. brucei\u003c/em\u003e infection. IL-17A signalling promotes the production of IgA in muscosal sites\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, and indeed we observed an upregulation of genes associated with Th17 responses in the infected dura mater. Whether IL-17 signalling is critical for promoting IgA\u003csup\u003e+\u003c/sup\u003e accumulation in the spleen and dura mater of infected male mice, or whether these IgA\u003csup\u003e+\u003c/sup\u003e B cells are derived from the nasorostral and/or gastrointestinal tract and the role of IgA\u003csup\u003e+\u003c/sup\u003e B cells in the control of \u003cem\u003eT. brucei\u003c/em\u003e infection and tissue immunopathology, merits future investigation.\u003c/p\u003e \u003cp\u003eOur previous work highlighted the accumulation of IgG\u003csup\u003e+\u003c/sup\u003e autoreactive B cells at the brain border of female mice\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In the present study, we further characterised the antibody repertoire and found that it is dominated by a significant bias in the \u003cem\u003eIghv\u003c/em\u003e usage towards specific members of the \u003cem\u003eIghv1\u003c/em\u003e family, including \u003cem\u003eIghv1-15, Ighv1-26\u003c/em\u003e, and \u003cem\u003eIghv1-76\u003c/em\u003e. Some of these autoantibodies recognise proteins that could play a key role in host-pathogen interactions. For instance, lactoferrin, an iron-binding glycoprotein belonging to the transferrin family, might be exploited by \u003cem\u003eT. brucei\u003c/em\u003e for survival in microenvironments with low nutrient content such as the CSF\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Interestingly, some members of the \u003cem\u003eIghv1\u003c/em\u003e immunoglobulin family, in particular \u003cem\u003eIghv1-69\u003c/em\u003e, are thought to result in the production of anti-influenza antibodies directed against the hemagglutinin stem\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Using host antigen microarrays, we demonstrated that the antibodies overrepresented in the dura mater during infection were polyreactive and recognised various CNS and systemic antigens, including myelin-related proteins, which we have previously reported in the murine dura mater and in the CSF of sleeping sickness patients\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, in addition to proteins involved in neurotransmission (e.g., AchRm2, GRIA2, AchR3, mGluR1, mGluR2), or bind to antigens that have been previously associated infection-induced narcolepsy, such as Influenza A H1N1\u003csup\u003e44\u0026ndash;46\u003c/sup\u003e. Although the role of autoantibodies in the immunopathology of narcolepsy remains incompletely understood and controversial, it is tempting to speculate that some of the autoantibodies detected in dura mater in this model of infection, in particular those recognising neurotransmitter receptors, could potentially disrupt normal brain function. Future work addressing this aspect would certainly be of relevance to better understand the brain immunopathology triggered by \u003cem\u003eT. brucei\u003c/em\u003e infection and the ensuing sleep disorders described in mice and humans\u003csup\u003e\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe autoantibodies generated in the dura mater might play a significant role in the host-parasite interactions at the brain border. Indeed, using a wide range of orthogonal approaches we also identified that Ig1-26 and Ig1-15 also recognise a range of \u003cem\u003eT. brucei\u003c/em\u003e antigens. Unexpectedly these were mostly associated with intracellular metabolic and flagellar proteins, and we demonstrated that these were specific against parasite but not host antigens in independent studies. These observations are intriguing as it was long assumed that most of the antibodies generated against \u003cem\u003eT. brucei\u003c/em\u003e recognise surface antigens, in particular VSGs, with limited work describing the antigenicity of intracellular proteins\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Given the predominance of intracellular parasite antigens recognised by these antibodies and limited VSG reactivity, we interpret the early, partial reduction in parasitaemia as consistent with effector-mediated mechanisms rather than direct neutralisation. We did not formally test complement or Fc receptor dependence on this process, and therefore cannot yet describe the observed protection to specific humoral effector pathways. Future work will test whether complement activation and/or Fc receptor engagement are required for this effect. Our work broadens our understanding of parasite-derived factors that may participate in the immune response, and further highlights the importance of understanding tissue-specific responses to this infection.\u003c/p\u003e \u003cp\u003eLastly, using adoptive B cell transfer experiments and passive transfer of dura mater-enriched recombinant antibodies, we demonstrated delayed onset of \u003cem\u003eT. brucei\u003c/em\u003e infection in \u0026micro;MT mice. Furthermore, the protective effects observed in transfer experiments relied on measuring parasites in circulation and we interpret these findings as evidence that dura mater-derived humoral effector molecules can potentially influence systemic parasitaemia. However, we did not quantify parasite burden or antibody deposition at the dura mater-CNS interface after adoptive transfer experiments in mMT mice, and this cannot directly ascribe effects at the CNS stage. Although the mechanisms involved in the observed protection in \u0026micro;MT mice are likely to be diverse, it is possible that the autoantibodies generated against host antigens such as lactoferrin might indeed represent a host strategy to limit parasite replication and survival in the CNS. Future work is required to test whether the generation of autoantibodies can indeed impose a bottleneck for parasite survival in various tissues. Since B cells are key players in neuroinflammatory and autoimmune disorders, our findings bring us closer to understanding the origin and functional relevance of the B cells generated at the brain borders in the context of pathological conditions that affect brain health and resilience during infection.\u003c/p\u003e \u003cp\u003eOur study provides novel insights and functional relevance of dura mater B cells in the context of infection-induced systemic B cell lymphopenia. However, it is important to acknowledge key limitations. Firstly, the abundance of dura mater B cells compared to other tissues precluded us from gaining additional granularity. However, the studies presented here are consistent with previous observations under homeostasis and highlight the physiological relevance of the B cells developing at the brain borders compared to canonical lymphopoietic sites\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Secondly, our BCR repertoire profiling targeted class-switched IgG\u003csup\u003e+\u003c/sup\u003e B cells. We did not generate IgM or IgA BCR repertoires; thus, the V-gene usage cannot be generalised to non-IgG compartments without additional profiling and this will need to be addressed in future work. Whether additional heavy-light chain pairings, result in similar polyreactivity merits further investigation. A direct comparison of dura mater versus splenic antibody preparations were not performed due to suboptiomal recombinant yields from putative splenic clones. However, we used the IgG-targeted BCR information to start exploring the role of dura mater-enriched immunoglobulins in the recognition of host and parasite antigens, significantly expanding our current knowledge of the reactivity of meningeal B cells during chronic neuroinflammation. Lastly, we were not able to purify enough specific B cell subsets from dura mater (e.g., B-1 cells) for adoptive transfer experiments using \u0026micro;MT mice. All of the adoptive transfer experiments resulted in systemic effects that could be attributed to more than one B cell subset (e.g., IgM-producing B cells, innate B-1 cells, class-switched B cells), and therefore we could not disentangle their relative contribution to the observed effects in infected mice. Future studies combining fate mapping and conditional knockouts for these various subsets would enable us to understand these processess in more details. In summary, we provided a comprehensive overview of the B cell landscape in the dura mater in the context of infection-induced impaired B cell lymphopoiesis, providing evidence of the nature of the antibodies produced locally at the brain borders and their potential role in sustaining critical host-pathogen interactions mediating both anti-parasitic and autoimmune responses. Future work exploring the relationship between these locally-generated antibodies and the behavioural changes observed in this infection model will further unravel the mechanistic links between cellular and humoral immunity and behaviour.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eEthical statement.\u0026nbsp;\u003c/strong\u003eAll animal experiments were approved by the University of Glasgow Ethical Review Committee and performed in accordance with the Home Office guidelines, UK Animals (Scientific Procedures) Act, 1986 and EU directive 2010/63/EU. \u0026nbsp;All experiments were conducted under SAPO regulations and UK Home Office project licence number PP5602024 to Juan F. Quintana. The \u003cem\u003ein vivo\u003c/em\u003e work presented in this study was conducted between 25-30-days post-infection (dpi) and correlated with increased clinical scores and procedural severity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMurine infections with\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eTrypanosoma brucei\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e Six- to eight-week-old female C57BL/6J mice (JAX, stock 000664),\u0026nbsp;mMT mice (JAX, stock) , were inoculated by intra-peritoneal injection with ~2 x 10\u003csup\u003e3\u003c/sup\u003e parasites of strain \u003cem\u003eT. brucei brucei\u003c/em\u003e Antat 1.1E\u003csup\u003e52\u003c/sup\u003e.\u0026nbsp;\u0026nbsp;Parasitaemia was monitored by regular sampling from tail venesection and examined using phase microscopy and the rapid \u0026ldquo;matching\u0026rdquo; method\u003csup\u003e53\u003c/sup\u003e. Uninfected mice of the same strain, sex and age served as uninfected controls.\u0026nbsp;\u0026nbsp;Mice were fed \u003cem\u003ead libitum\u003c/em\u003e and kept on a 12 h light\u0026ndash;dark cycle. All the experiments were conducted between 8h and 12h.\u0026nbsp;For sample collection, we focussed on 25-30 days post-infection unless stated otherwise, as this has previously been shown to correlate with parasite infiltration in the epidural space\u003csup\u003e54,55\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTissue processing and flow cytometry analysis.\u0026nbsp;\u003c/strong\u003eTo discriminate\u0026nbsp;circulating versus brain-resident immune cells, we performed intravascular staining of peripheral CD45\u003csup\u003e+\u003c/sup\u003e immune cells, as previously reported\u003csup\u003e56\u003c/sup\u003e. Briefly, a total of 2\u0026nbsp;mg of anti-mouse CD45 antibody in 100\u0026nbsp;ml of 1X PBS was injected intravenously ~3 minutes prior culling. Mice were euthanised as described above and transcardially perfused with ice-cold 0.025% (wt/vol) EDTA in 1X PBS. Whole dura mater were\u0026nbsp;enzymatically digested with Collagenase VIII (1 mg/ml) and DNAse I (1 mg/ml; Sigma) in 1X PBS (HSBB) (Invitrogen) for ~30 minutes at 37 \u0026deg;C and with agitation at 200 r.p.m., according to previously published protocols\u003csup\u003e57\u003c/sup\u003e. Femur samples were cleared from muscle and skin prior to bone marrow isolation. Skull caps were cut into ~1mm pieces. These tissues were then placed on a 0.5 mL eppendorf tube with a narrow opening at the bottom. This tube was then placed inside a 1.5mL Eppendorf and centrifuge at 800g for 10 minutes to harvest either femur or skull bone marrow. Spleen tissue were first pressed onto 70\u0026nbsp;mm nylon mesh filters to obtain single cell suspensions that were then washed twice with complete RPMI 1640. Red blood cells from both splenic and marrow cell suspensions were lysed using ACK lysis buffer (Thermo) on ice for a total 10 minutes. ACK lysis buffer was then diluted ten times with 1X PBS and cells peletted by centrifugation at 800g for 10 minutes at 4\u003csup\u003eo\u003c/sup\u003eC. Large intestines were opened longitudinally and washed in 1x HBSS before incubation for 30 mins at 37\u003csup\u003eo\u003c/sup\u003eC at 200 rpm in strip media (2 mM HEPES, 5 mM EDTA and 1mM DTT). The tissue was removed and placed into digest media (2 mg/mL Dispase and 2 mg/mL Collagenase I) and incubated for 30 mins at 37\u003csup\u003eo\u003c/sup\u003eC at 200 rpm. Cells were then passed through a 100\u0026nbsp;mM strainer and pelleted by centrifugation at 400g for 5 minutes at 4\u003csup\u003eo\u003c/sup\u003eC.\u0026nbsp;For flow cytometry analysis, single cell suspensions from blood, femur bone marrow, skull bone marrow, spleen, and\u0026nbsp;dura mater were resuspended in ice-cold FACS buffer (2\u0026thinsp;mM EDTA, 5\u0026thinsp;U/ml DNAse I, 25\u0026thinsp;mM HEPES and 2.5% Foetal calf serum (FCS) in 1X PBS) containing Horizon Brilliant stain buffer (25\u0026nbsp;ml/sample; BD Biosciences) and stained for extracellular markers. We used the following commercially available antibodies from Biolegend: CD45-APC-Cy7 (clone 30-F11, 2\u0026nbsp;mg/100\u0026nbsp;ml 1X PBS i.v.), TER-119-APC-Cy7 (clone TER-119; 1/400), CD19-APC-Cy7 (clone 1D3/CD19; 1/400), F4/80-APC-Cy7 (clone BM8; 1/400), CD45-BV510 (clone 30-F11; 1/400),\u0026nbsp;CD45-BV785 (clone 30-F11; 1/400), CD19-PE-Cy5 (clone 6D5; 1/400), CD19-FITC (clone 6D5; 1/400), B220-BV605 (clone RA-6B2; 1/400), B220-BV650 (clone RA3-6B2; 1/400), \u0026nbsp;CD43-APC (clone S11; 1/400), CD43-AF700 (clone S11; 1/400), \u0026nbsp; cKIT-AF488 (clone 2B8; 1/200), SCA1-APC (clone D7; 1/100), CD135-PE-Cy5 (clone A2F10; 1/200), CD127/IL-7Ra-PE (clone S1006K; 1/200), CD3-APC-eFluor700 (clone 17A2; 1/400), CXCR4-BV421 (clone L276F12; 1/400), IgD-BV421 (clone 11-26c.2a; 1/800), IgM-BV711 (clone RMM-1; 1/800). For intracellular staining, we used the True-Phos Perm buffer set (Biolegend) to fix cells for 10 minutes on ice, followed by permeabilization for 30 minutes on ice. Cells were then pelleted at 500g for 10 minutes, and resuspended in 100\u0026nbsp;mL of permeabilization buffer containing\u0026nbsp;Horizon Brilliant stain buffer as above. The following antibodies were used for intracellular statining: Ki67-PE-Cy7 (Biolegend, clone 11F6, 1/500), pSTAT5-PE (BD Biosciences, clone 47/Stat5-pY694, 1/500), and IgA-PE (clone TRFK5; 1/800). Samples were incubated overnight at 4\u003csup\u003eo\u003c/sup\u003eC protected from light, and then washed twice with FACS buffer and resuspended in 100\u0026nbsp;mL of FACS buffer.\u0026nbsp;Samples were run on a flow cytometer LSRFortessa (BD Biosciences) and analysed using FlowJo software version 10 (Treestar).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKREC assay.\u0026nbsp;\u003c/strong\u003eWe followed a protocol previously published for KREC assay in the dura mater\u003csup\u003e3\u003c/sup\u003e.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eBriefly,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eafter perfusion, the dura mater was peeled off from the skull and immediately placed in lysing buffer (10 mM Tris-HCl pH 8.0) containing 100 mg/ml of proteinase K and total DNA was further extracted using the DNeasy Blood \u0026amp; tissue Kit (Qiagen). Total gDNA was eluted in 30 ml of ultrapure water (Qiagen) and quantified using a Nanodrop. A total of 50 ng of total DNA was used as input for PCR using the primers specific for KREC (Forward: 5\u0026rsquo;-GGAGTGGATTCAGGACACTGCT, Reverse: 5\u0026rsquo;-CTCCAATAAGTCACCCTTTCCTTGT). \u003cem\u003eGapdh\u003c/em\u003e was used as loading control (Forward: 5\u0026rsquo;-ACCACAGTCCATGCCATCAC, Reverse: 5\u0026rsquo;-TCCACCACCCTGTTGCTGTA). All primers were adquired from Integrated DNA Technologies. PCR reactions were perfomed for 30 cycles (94\u003csup\u003eo\u003c/sup\u003eC for 3 minutes, 55\u003csup\u003eo\u003c/sup\u003eC for 30 seconds, and 72\u003csup\u003eo\u003c/sup\u003eC for 1 minute) using Q5 high-fidelity DNA polymerase with high GC content buffer (New England Biolabs). PCR products were further resolved by electrophoresis in 1% agarose gel. For quantitative PCR, we used the sample input gDNA and primers but used the Luna Universal qPCR Master Mix (New England Biolabs) with the same PCR conditions using QuantStudio qPCR system (Thermo Fisher).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWhole mount dura mater preparation and immunofluorescence\u003c/strong\u003e\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eAfter euthanasia, the skull caps were carefully removed using fine tweezers and scissors and placed immediately in 10% neutral buffered Formalin (NFB) for 10 minutes at room temperature. Following fixation of the skull caps, for single molecule fluorescent \u003cem\u003ein situ\u003c/em\u003e hybridisation (smFISH) experiments, mounted dura mater specimens were dehydrated in 50, 70 and 100% ethanol. RNAscope 2.5 Assay (Advanced Cell Diagnostics) was used for all smFISH experiments according to the manufacturer\u0026rsquo;s protocols.\u0026nbsp;We used RNAscope probes against mouse\u0026nbsp;\u003cem\u003eCol1a2\u003c/em\u003e on channel 1 (Cat No. 585461),\u0026nbsp;\u003cem\u003ePdgfrb\u003c/em\u003e on channel 2 (Cat. No. 411381-C2), and\u0026nbsp;\u003cem\u003eCxcl12\u0026nbsp;\u003c/em\u003eon channel 3 (Cat. No. 422711-C3).\u0026nbsp;All RNAscope smFISH probes were designed and validated by Advanced Cell Diagnostics.\u0026nbsp;Images were acquired using a snapshot widefield fluorescent microscope (Zeiss) and adjusted for brightness and contrast using Fiji. Single stain and unstained controls to set up signal-to-noise threshold during post-acquisition analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEx vivo\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;chemotaxis assay.\u003c/strong\u003e Transmigration of precursor B cells was assessed in 6.5-mm diameter 24-Transwell chemotaxis chambers (Costar, Cambridge, MA) with a pore size of 5\u0026nbsp;mm. Briefly, freshly prepared skull bone marrow and dura mater preparations were resuspended in RPMI-1640 medium supplemented with 10% foetal calf serum and 1/100 Penicillin/Streptomycin, and 100\u0026nbsp;ml of cell suspension at a concentration of 2.5x10\u003csup\u003e5\u003c/sup\u003e cells were seeded loaded in the upper chamber of the Transwell culture insert. Filters were transferred into the wells containing RPMI-1640 medium supplemented with 10% foetal calf serum and 1/100 Penicillin/Streptomycin and 100 ng/ml of recombinant mouse CXCL12 (R\u0026amp;D systems, 460-SD-050). The chambers were incubated for 3 hours at 37 \u0026deg;C in 5% carbon dioxide. After incubation, cells remaining in the upper chamber, as well as those that had migrated into the lower chamber, were harvested, counted using an automated Countess II Cell Counter (Thermo Fisher), and analysed by flow cytometry in a Fortessa II (BD Bioscience) to specifically quantify the number early, late, and mature B cells in all the fractions harvested.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eELISA.\u003c/strong\u003e After preparing single cell suspensions of skull bone marrow and dura mater, CD45\u003csup\u003e+\u003c/sup\u003e immune cells were depleted using mouse CD45 magnetic beads (Miltenyi, 130-052-301). The flowthrough, considered to be depleted of CD45\u003csup\u003e+\u003c/sup\u003e cells, was pelleted at 10,000g for 10 minutes at 4\u003csup\u003eo\u003c/sup\u003eC, resuspended in 200\u0026nbsp;ml of RIPA buffer (ThermoFisher, 89900) supplemented with cOmplete EDTA-free Protease Inhibitor cocktail (Roche, 11836170001). Total protein was quantified using a nanodrop and 50\u0026nbsp;ml aliquots were prepared and stored at -80oC until analysis. Mouse CXCL12 (Biolegend, 444207) ELISA kit was used to determine the levels of these two molecules in tissue, according to the manufacturer\u0026rsquo;s instructions. 450nm absorbance was measured using a Varioscan ELISA plate reader, and the quantification was performed against a battery of standards of known concentrations. Data structure and statistical test were employed to determine normality and skewness of the data prior to assessment of statistical significance. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWhole tissue processing for bulk transcriptomics.\u003c/strong\u003e Mice were euthanised by cardiac puncture and perfused with 30-40 mL of ice-cold 1X PBS containing 2 mM EDTA to avoid clotting. Following euthanasia, skull and femur bone marrow, as well as spleen and dura materwere rapidly dissected. Any remaining blood was lysed with ACK lysis buffer (Thermo Fisher, cat. No. A1049201) for 10 minutes on ice, and the remaining cells were pelleted at 500g for 10 minutes at 4\u003csup\u003eo\u003c/sup\u003eC and resuspended in Trizol (Invitrogen, cat. No. 15596026).\u0026nbsp;Total RNA was then purified using the RNeasy Kit (Qiagen) as per the manufacturer\u0026rsquo;s recommendations. The RNA was purified in 30\u0026thinsp;\u0026micro;L of DPEC-treated water (Thermo Fisher, R0601), and RNA concentration measured on a NanoDrop\u0026trade; 2000 (Thermo Fisher Scientific). Samples were shipped to Novogene (Cambridge, UK) to undergo quality control, library preparation and sequencing. RNA integrity was assessed using an RNA Nano 6000 Assay Kit (Agilent Technologies) with a TapeStation (Agilent Technologies), as per the manufacturer\u0026rsquo;s instructions. Samples with an RNA integrity number (RIN) of \u0026gt;6.0 were qualified for RNA sequencing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBulk transcriptomics Library preparation.\u003c/strong\u003e Library preparation was performed by Novogene (Cambridge, UK). Messenger RNA (mRNA) was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in a First-Strand Synthesis Reaction Buffer (5X). First-strand cDNA was synthesised using random hexamer primers and M-MuLV Reverse Transcriptase (RNase H-). Second-strand cDNA synthesis was then performed using DNA Polymerase I and RNase H. Remaining overhangs were converted to blunt ends via exonuclease/polymerase activity. Following adenylation of 3\u0026rsquo; ends of DNA fragments, adaptors with hairpin loop structures were ligated. To select cDNA fragments of 370\u0026ndash;420\u0026thinsp;bp in length, library fragments were purified using AMPure XP beads (Beckman Coulter), as per the manufacturer\u0026rsquo;s instructions. PCR was then performed using Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) primers. Finally, PCR products were purified (AMPure XP system) using AMPure XP beads (Beckman Coulter), as per the manufacturer\u0026rsquo;s instructions, and library quality was assessed using a Bioanalyzer 2100 (Agilent Technologies).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBulk transcriptomics sequencing and data analysis.\u003c/strong\u003e Clustering of the index-coded samples was performed on a cBot Cluster Generation System using a TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer\u0026rsquo;s instructions. After cluster generation, libraries were sequenced on an Illumina Novaseq 6000 platform and 150\u0026thinsp;bp paired-end reads were generated. Raw reads in fastq format were processed through proprietary Perl scripts developed by Novogene (Cambridge, UK). Clean reads were obtained by removing reads containing adaptors, poly-N, or low-quality reads from raw data. Concurrently, the Q20, Q30 and GC content of the clean data was calculated (\u003cstrong\u003esupplementary table 1A\u003c/strong\u003e). Genome and genome annotation files (Genome Reference Consortium Mouse Build; GRCm39) were downloaded. An index of the reference genome was built using Hisat2 (v2.0.5) and paired-end clean reads were aligned to the reference genome using Hisat2 (v2.0.5).\u003c/p\u003e\n\u003cp\u003eThe featureCounts (v. 1.5.0-p3) package was used to count read numbers mapped to each gene, before calculating the Fragments Per Kilobase of transcript sequence per Millions base pairs (FPKM) of each gene using the length of the gene and reads count mapped to this gene. Differential expression analysis was performed using the DESeq2 R package (v. 1.20.0). The resulting \u003cem\u003eP\u003c/em\u003e values were adjusted using the Benjamini and Hochberg approach to control false discovery rate. Genes with an adjusted \u003cem\u003eP\u003c/em\u003e value of \u0026lt;0.05 were assigned as differentially expressed. Pathway enrichment analysis of differentially expressed genes was performed using the DAVID platform, mapping genes to the KEGG database. KEGG terms with an adjusted \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significantly enriched (\u003cstrong\u003esupplementary table 1B\u003c/strong\u003e). Heatmaps were generated using the pheatmap (Version 1.0.12) and Tidyverse packages in R (Version 4.2.1). Samples were clustered by Euclidean distance. PCA analysis was performed using ggplot2, with naive and infected male and female mice plotted separately.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComparative single cell transcriptomics profiling of the murine skull bone marrow and dura mate.\u0026nbsp;\u003c/strong\u003eSingle cell suspensions from the calvaria bone marrow and dura mater were obtained as described above.\u0026nbsp;Once single cell suspensions were obtained from the whole dura mater, calvaria bone marrow, and femur bone marrow, cells were counted using a hemocytometer, fixed using the EvercodeTM Cell Fixation kit v2 (Parse Bioscience). Cells were pelleted for 10 minutes at 600g and 4\u003csup\u003eo\u003c/sup\u003eC in 15 ml Falcon tubes pre-coated with 1% BSA to minimise cell adherence and losses. Cell pellets were resuspended in 750\u0026nbsp;ml of Cell Prefixation buffer, followed by passage through a 40\u0026nbsp;mm cell strainer to remove cell clumps and addition of 250\u0026nbsp;ml of Fixation solution. The cells were then incubated for 10 minutes on ice, resuspended in 150\u0026nbsp;ml of Cell buffer and diluted at a final density of ~500 cells/ml (dura mater) or 10,000 cells/ml (calvaria and femur bone marrow). Libraries were prepared using a split-pool combinatorial barcoding strategy using the Evercode whole transcriptome v2 (Parse Bioscience). The final libraries (~300-500 bp in length) were eluted from SPRI beads using molecular grade water and analysed on a TapeStation. The resulting libraries were sequenced on a Novaseq X plus Illumina platform using a 450 million read, 150bp paired-end sequencing strategy at a depth of around 30,000-50,000 reads per cell. The sequencing files were first converted to FASTQ format using BCL2Fastq. These FASTQ files were then processed with Parse Pipeline v1.6.2. The pipeline demultiplexes reads from the Evercode single-cell RNA-seq libraries using the four-round combinatorial barcodes and unique molecular identifiers (UMIs), followed by alignment to the Parse Biosciences custom GRCm39 reference genome. After processing, all eight libraries were merged using the pipeline\u0026rsquo;s \u003cem\u003ecomb\u003c/em\u003e option. The resulting uniquely mapped UMI counts were output as a gene-by-cell count matrix in sparse format. All downstream analyses were performed using Scanpy v1.11.2. Low-quality cells were removed to minimize technical noise and ensure reliable downstream analysis. Cell quality was assessed using three metrics: the number of UMIs per cell barcode (library size), the number of detected genes per cell, and the proportion of UMIs mapped to mitochondrial genes. We first excluded genes not expressed in at least five cells, then filtered out cells with fewer than 300 detected genes. Violin plots of the three quality metrics were generated for each sample to identify outliers that might represent doublets or multiplets. Cells with more than 50,000 total UMIs were removed as potential doublets, and those with more than 6,000 detected genes were also excluded to maintain comparable gene expression distributions across samples. After filtering, the following cell counts remained for downstream analysis: 9,619 and 9,915 cells from the na\u0026iuml;ve and infected skull bone marrow, respectively, and 4,156 and 13,607 cells from the na\u0026iuml;ve and infected dura mater, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe first step in visualization and clustering was to identify highly variable genes (HVGs). To do this, we used Scanpy\u0026rsquo;s \u003cem\u003ehighly_variable_genes()\u003c/em\u003e function with the parameters \u003cem\u003emin_mean = 0.0125\u003c/em\u003e, \u003cem\u003emax_mean = 3\u003c/em\u003e, and \u003cem\u003emin_disp = 0.25\u003c/em\u003e. These HVGs were then used for dimensionality reduction via principal component analysis (PCA). The dataset was further reduced to two dimensions using either t-distributed stochastic neighbor embedding (t-SNE) or UMAP, using the first 30 principal components as input. Finally, we applied Leiden clustering to group the cells. To identify the marker genes for a cluster, we compared that cluster with all other clusters using t-test. We used the \u0026lsquo;\u003cem\u003erank_gene_groups\u0026rsquo;\u003c/em\u003e function from Scanpy package to do this differential expression. We then reported top 200 genes that were differentially expressed in that cluster as the marker for the cluster. These marker genes were then used to annotate the cell types of a cluster. To identify the B-cell subclusters, we first subsetted the aggregated population by selecting only Early B cells and B/plasma cells. We then recalculated PCA and performed Leiden clustering on this B-cell subset. Finally, we applied a t-test using the \u003cem\u003erank_genes_groups\u003c/em\u003e function to identify marker genes for the resulting B-cell subclusters.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell-cell communication prediction analysis.\u0026nbsp;\u003c/strong\u003eWe performed Cell\u0026ndash;cell communication analysis using \u003cstrong\u003eCellChat v.2.2.0\u003c/strong\u003e\u003csup\u003e28\u003c/sup\u003e in R v.4.5.2. CellChatinfers intercellular signaling networks from single-cell RNA-sequencing data. After preprocessing and normalization of the expression matrix, cells were grouped according to their sample+annotated cell types. CellChat objects were constructed following the standard workflow. We applied the CellChat database of ligand\u0026ndash;receptor interactions to identify statistically significant signaling events, using default parameters for overexpression analysis and permutation testing to assess interaction probability. Communication networks were quantified and visualized to compare signaling strength and patterns among cell populations. Pathway-level aggregation was then used to determine dominant outgoing and incoming signaling pathways for each cell group. All analyses were conducted using CellChat \u003cstrong\u003ev.2.2.0\u003c/strong\u003e\u003csup\u003e28\u003c/sup\u003e, and visualization functions within the package were used to generate network and pathway plots included in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB cell receptor profiling.\u0026nbsp;\u003c/strong\u003eDura mater and splenic B cells from na\u0026iuml;ve and infected mice (\u003cem\u003en\u003c/em\u003e = 6 mice/group/tissue) were purified using a magnetic pan-B cell Isolation kit (Miltenyi, Cat. No. 130-095-813). Magnetically sorted cells were washed with ice-cold 1X PBS supplemented with 0.5% BSA and 2 mM EDTA, and cells resuspended in Trizol and kept at -20\u003csup\u003eo\u003c/sup\u003eC for subsequent RNA extraction. Total RNA was extracted using the RNeasy mini kit (Qiagen, 74104) and eluted in 30 ml of DPEC-treated water. The RNA integrity was assessed the RNA ScreenTape (Agilent, cat. No. 5067-5576) on a TapeStation. High-quality RNA (integrity value \u0026gt; 7.5) samples from matched spleen and dura materB cells from na\u0026iuml;ve and infected animals at 28 days post-infection (\u003cem\u003en\u003c/em\u003e = 6 samples/tissue/time point) was submitted for IgG-specific heavy and light chain B cell receptor library preparation, sequencing, and analysis to Genewiz using the company\u0026rsquo;s immunogenomics pipeline. On average, we obtained 144,740,272 raw reads with a Q30 value of 90%. Upon filtering low-quality reads, we obtained a total of 125,241,964 clean reads with a Q30 value of 93% (\u003cstrong\u003esupplementary table 2A\u003c/strong\u003e). The assembled reads were blasted against IMGT reference database to identify the best match of germline V(D)J genes, and CDR1, CDR2, CDR3 variable region sequences. During the mapping and clustering, the clones\u0026rsquo; abundance was calculated for each type of chain. For a detected CDR3 sequence, if CDR1 and CDR2 sequences were not detected from input assembled reads, \u0026lsquo;N\u0026rsquo; and \u0026lsquo;X\u0026rsquo; will be assigned according to nucleotide sequence and amino acid sequence. V(D)J diversity was estimated by mapping against the IMGT database\u003csup\u003e58\u003c/sup\u003e and represented as frequency of the total clones detected. To analyse CDR3 amino acid usage frequency, CDR3 sequences were clustered according to similarity (threshold: 0.8). Weblogo software\u003csup\u003e59\u003c/sup\u003e was used for visualising CDR3 amino acid patterns of each cluster. Each logo consists of stacks of symbols and the size of letter represents its frequency from unique and abundance levels. All tables and weblogo pictures can be accessed in \u003cstrong\u003esupplementary table 2B\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdoptive and passive transfer experiments.\u0026nbsp;\u003c/strong\u003eWe followed a previous report to set up the dura mater B cell adoptive transfer experiments\u003csup\u003e34\u003c/sup\u003e\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eB cells were magnetically sorted from spleen and dura mater from na\u0026iuml;ve and animals infected with \u003cem\u003eT. brucei\u003c/em\u003e Antat1.1E at day 25 post-infection. To increase the cell recovery, we pooled 7-9 dura mater prior to B cell purification. Purified B cells were then resuspended in sterile 1X PBS to a final density of ~10\u003csup\u003e4\u003c/sup\u003e B cells/ml and were then adoptively transferred into\u0026nbsp;mMT recipient mice (\u003cem\u003en\u0026nbsp;\u003c/em\u003e= 4-5 mice per group) via intraperitoneal injection. As a control group,\u0026nbsp;mMT mice received sterile 1X PBS alone. Recipient\u0026nbsp;mMT mice were infected with 10\u003csup\u003e3\u003c/sup\u003e \u003cem\u003eT. brucei\u003c/em\u003e Antat1.1E intraperitoneally (~1 trypanosome per every ten B cells) ~18 hours after cell transfer. Mice then were monitored daily for the presence of parasites in blood via tail venepuncture, as well as for body weight and food intake. The experiment was terminated when all the mice displayed patent parasitaemia. For IgG transfer experiments,\u0026nbsp;mMT recipient mice (\u003cem\u003en\u0026nbsp;\u003c/em\u003e= 4 mice per group) were inoculated via intraperitoneal injection with 5 mg/kg of bodyweight using Ig1-15, Ig1-26, both recombinant antibodies, or irrelevant mouse IgG2a one day prior to and on the day of infection. Mice were then infected with 10\u003csup\u003e3\u003c/sup\u003e \u003cem\u003eT. brucei\u003c/em\u003e Antat1.1E intraperitoneally, and monitored daily for the presence of parasites in blood via tail venepuncture, bodyweight, and food intake. The experiment was terminated when all the mice reached ~10\u003csup\u003e8\u003c/sup\u003e parasites/ml.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of host antigens detected by autoantigens\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRecombinant monoclonal antibodies were generated by Genescript by selecting the most abundant heavy chains detected during infection in the dura mater tissue (\u003cem\u003eIghv1-15, Ighv1-26\u003c/em\u003e, and \u003cem\u003eIghv1-76\u003c/em\u003e) and the most abundant light chain (\u003cem\u003eIghv6-85\u003c/em\u003e). Selected plasmids were transfected into CHO cells lines and expressed in a small scale (~4 mL) as soluble proteins. We attempted to generate further monoclonal antibodies with additional pairings (e.g., combining Ighv1-5 and Ighv8-5 with Igkv6-15) but these pairings resulted in poor expression and secretion, and we therefore decided not to pursue these. Once generated, these recombinant monoclonal antibodies were assessed using a commercial microarray-based platform (GeneCopoeia, PA002). Briefly, antibody concentrations ranging from 5\u0026nbsp;mg to 0.05\u0026nbsp;mg were hybridised to distinct microarray spots containing 120 native\u0026nbsp;brain and neuronal-associated antigens\u0026nbsp;spotted onto nitrocellulose fibres (adhered to glass slides). Next, the slides were incubated with a fluorescently-coupled anti-IgG secondary antibody, and microarrays were scanned using a GenePix 4400A microarray scanner. Raw fluorescence data was normalized to PBS controls on each slide. The data presented in the heatmaps are normalised signal-to-noise (SNR) ratios. Selected candidates were anlyse independently using ELISA. Briefly, 5\u0026nbsp;mg/ml of recombinant mouse MOG (RDsystems), ALCAM (RDsystems), Lactoferrin (Abcam), or BSA (Thermo) were use to coat ELISA plates in carbonate buffer (Biolegend) overnight at 4\u003csup\u003eo\u003c/sup\u003eC. Next day, plates were washed at least five times with ELISA wash buffer (Biolegend), blocked with blocking buffer (1X PBS containing 2% BSA) for 2 hours at room temperature, followed by 3 washed with ELISA wash buffer, and incubation with recombinant monoclonal Ig1-15, Ig1-26, or Ig1-76 coupled to horseradish peroxidase serially diluted in blockling buffer for 1 hour at room temperature. Plates were then washed at least 5 times with ELISA wash buffer and incubated with 3,3\u0026rsquo;,5,5\u0026rsquo;-tetramethylbenzidine (TMB) for the development of a colorimetric reaction at room temperature. Reactions were stopped using 1X Stop buffer (Biolegend) and the plates were read at 405 nm with a 567 nm correction readout using a Varioscan ELISA plate scanner.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTrypanosoma brucei\u003c/em\u003e immunofluorescence and flow cytometry\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn addition to the host antigen screening, we also assessed the capacity of the recombinant antibodies to potentially recognise \u003cem\u003eT. brucei\u003c/em\u003e antigens. \u003cem\u003eT. brucei\u003c/em\u003e Lister 427 were cultured in HMI-9 supplemented with 10% FBS and 1% Penicillin/Streptomycin at 37\u003csup\u003eo\u003c/sup\u003eC and 5% CO\u003csub\u003e2\u003c/sub\u003e. Prior to harvesting, parasites were incubated with 5\u0026nbsp;mM MitoSox Red for 10 minutes at 37\u003csup\u003eo\u003c/sup\u003eC and 5% CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003ethat we used here as an intracellular marker. Parasites were harvested during the log phase of growth (~0.75-1x10\u003csup\u003e6\u003c/sup\u003e parasites/ml) by centrifugation at 500g for 10 minutes and washed twice with ice-cold 1X PBS. Once washed, parasites were fixed with 4% PFA on ice for 10 minutes and seeded on poly-lysine-coated slides for ~30 minutes at room temperature protected from light. Where indicated, the parasites were then permeabilised with 1X PBS containing 1% Triton X-100 for 10 minutes at room temperature. Once permeabilised, the cells were blocked with 1X PBS containing 1% BSA for 30 minutes at room temperature, following by incubation with recombinant monoclonal antibodies, previously labelled with ReadyLabel antibody labelling kit with Alexa Fluor 647 (Thermo), diluted 1/50 in blocking buffer (1X PBS containing 1% BSA) overnight at 4\u003csup\u003eo\u003c/sup\u003eC. Irrelevant mouse IgG2a antibody (Nobus Biologicals) were coupled to Alexa Fluor 647 an antibody labelling kit (Thermo Fisher) and were included as controls. Slides were then washed 5 times with ice-cold 1X PBS and mounted using Antifade Gold containing DAPI (Vectashield). Images were acquired using a snapshot widefield fluorescent microscope (Zeiss) and adjusted for brightness and contrast using Fiji. Single stain and unstained controls to set up signal-to-noise threshold during post-acquisition analyses. For flow cytometry analysis, the parasites were resuspended in ice-cold 1X PBS after harvesting, fixed with 4% PFA and washed twice with ice-cold 1X PBS. Samples were split into two fractions, one containing Triton X-100 in 1X PBS for intracellular staining or without Triton X-100 for surface staining. Samples were then blocked with 1X PBS containing 1% BSA for 30 minutes at room temperature, followed by incubation with the recombinant monoclonal antibodies or irrelevant mouse IgG2a antibodies coupled to Alexa Fluor 647 for 1 hour on ice. Samples were then washed three times with FACS buffer (1X PBS containing 2.5 mM EDTA and 2.5% FBS) prior to analysis on a BD Bioscience LSR Fortessa. FCS files were then processed and analysed using FloJo version 9.0.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTrypanosoma brucei\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eprotein purification, fractionation, and antigen discovery\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eT. brucei\u003c/em\u003e Lister 427 were harvested during the log phase of growth (~0.75-1x10\u003csup\u003e6\u003c/sup\u003e parasites/ml) by centrifugation at 500g for 10 minutes and resuspended in modified TEN buffer (150 mM NaCl, 10 mM MgCl2, 1 mM DTT, 50 mM Tris-HCl pH 7.4, 5 mM EDTA, 1% Triton X-100) containing 1X Complete protease Inhibitor cocktail (Roche). The cells were vortexed at full speed (~3,200 rpm) three times for 5 minutes each, followed by sonication\u0026nbsp;three times for 20 secons each at 40% intensity. Cell lysates were cleared by centrifugation at 10,000rpm at 4\u003csup\u003eo\u003c/sup\u003eC for 10 minutes to remove any cell debris. The protein content of the cleared supernatant was quantified using the\u0026nbsp;BCA protein assay\u0026nbsp;(Thermo).\u003cem\u003e\u0026nbsp;T. brucei\u003c/em\u003e lysates were diluted in carbonate buffer pH 9.5 to a final concentration of 50\u0026nbsp;mg/ml and used to coat Maxisorb ELISA plates (Biolegend) overnight at 4\u003csup\u003eo\u003c/sup\u003eC. Plates were then washed with 1X ELISA wash buffer (Biolegend) prior to blocked with blocking buffer (1X PBS containing 2% BSA) for 2 hours at room temperature. Serial dilutions of recombinant monoclonal antibodies, previously labelled with horseradish peroxidase using a commercially available kit (Thermo), were incubated on the coated plates for 1 hour at room temperature. The plates were then extensively washed five to seven times with 1X ELISA wash buffer and incubated with 3,3\u0026rsquo;,5,5\u0026rsquo;-tetramethylbenzidine (TMB) for the development of a colorimetric reaction at room temperature. Reactions were stopped using 1X Stop buffer (Biolegend) and the plates were read at 405 nm with a 567 nm correction readout using a Varioscan ELISA plate scanner.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor protein fractionation and antigen discovery, we initially performed a gel filtration step of 1 ml (1.5 mg protein/ml) using a Superdex 200 10/300 NGC column at a flow rate of 0.75 ml/min using\u0026nbsp;TEN buffer (150 mM NaCl, 10 mM MgCl2, 1 mM DTT, 50 mM Tris-HCl pH 7.4, 5 mM EDTA). The resulting fractions (0.5 mL)\u0026nbsp;were screened for reactivity against recombinant monoclonal antibodies Ig1-15, Ig1-26, and Ig1-76 coupled to HRP using ELISA. Fractions with a positive signal above background were pooled together and taken forward for a second round of protein fractionation using a Resource Q Anion exchange column. Samples were pre-diluted six times to reduce the concentration of NaCl from 150 mM to 25 mM prior to anion exchange. The column was initially washed with run buffer (50 mM Tris-HCl, 1 mM EDTA, pH 8.0) and samples run at a flow rate of 1.6 ml/minute in run buffer, and then eluted into 0.5 ml fractions in elution buffer (50 mM Tris-HCl, 1 mM EDTA, 2 mM NaCl, pH 8.0).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSelected fractions of interest based on ELISA results were then subjected to untargeted LC/MS. For sample separation, separation was performed on a Thermo Vanquish Neo UHPLC system configured with buffer A as 0.1% formic acid in water and buffer B as 0.1% formic acid in 80% acetonitrile. A specified injection volume \u0026nbsp;was loaded on to the analytical column (IonOpticks Aurora series Rapid TS, 8cm x 75 mm ID, 1.7\u0026nbsp;mm C18)\u0026nbsp;kept at 35\u0026nbsp;\u003csup\u003eo\u003c/sup\u003eC at An initial rate of 800 nl/min which was then dropped to 200 nl/min in 0.25 min to rapidly re-pressurise the column. The separation was also started during this time, with a gradient of 1% B to 5% B over this period. The next step was 5% B to 35% B over 20.25 minutes, 35% B to 100% B over 0.5 minutes before washing for 2 minutes at 100% B and dropping down to 1% B in 0.5 minute. The complete method time was 30 minutes. Mass spectrometry was conducted\u0026nbsp;using an Orbitrap Astral coupled to a Vanquish Neo UHPLC system to identify \u003cem\u003eT. brucei\u003c/em\u003e candidate antigens likely to be recognised by these recombinant antibodies.\u0026nbsp;Data was acquired in a data dependent manner using a fixed cycle time of 2 sec, an expected peak width of 15 sec and a default charge state of 2. Full MS data was acquired in positive mode over a scan range of 300 to 1750 Th, with a resolution of 120,000, a normalised AGC target of 300% and an automatic max fill time for a single microscan. Fragmentation data was obtained from signals with a charge state of +2 to +4 and an intensity over 5,000 and they were dynamically excluded from further analysis for a period of 15 sec after a single acquisition within a 10ppm window. Fragmentation spectra were acquired with a resolution of 15,000 with a normalised collision energy of 30%, a normalised AGC target of 300%, first mass of 110 Th and a max fill time of 25 mS for a single microscan. All data was collected in profile mode.\u003c/p\u003e\n\u003cp\u003eData was acquired in a data independent manner with an expected peak width of 6 sec and a default charge state of 2. Full MS data was acquired in positive mode over a scan range of 350 to 1750 Th, with a resolution of 240,000, a standard (automatic) normalised AGC target and a maximum injection time of 10 ms for a single microscan. Fragmentation data was obtained according to a table of 150 \u003cem\u003em/z\u003c/em\u003e windows, with 3 Th \u003cem\u003em/z\u003c/em\u003e widths. The DIA window type was set to automatic, and window optimisation was selected. These were acquired in the astral, with a normalised collision energy of 25%. \u0026nbsp;The \u003cem\u003em/z\u003c/em\u003e range was 145-1450. A normalised AGC target of 500% was used with a maximum injection time of 7 ms for a single microscan. All data was collected in centroid mode.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe whole tissue transcriptome data generated in this study have been deposited in the Gene Expression Omnibus uder the accession number GSE297581. The B cell receptor profiling dataset have been deposited in the Gene Expression Omnibus uder the accession number GSE300949. Additional data and files can also be sourced via Supplementary Tables. The raw files for the single cell experiments has been deposited in ArrayExpress under the accession number 847907. The processed .rds files and accompanying script can be access via Zenodo (DOI:\u0026nbsp;10.5281/zenodo.18188237).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical analyses were performed using Graph Prism Version 8.0 for Windows or macOS, GraphPad Software (La Jolla California USA). The data distribution was determined by normality testing using the Shapiro-Wilks test. Depending on data distribution and normality we applied various statistical methods that are reported in each figure caption. For the \u003cem\u003ein vivo\u003c/em\u003e experiments, we matched sex and age of the mice in experimental batches using a block design including randomisation of experimental units. Data collection and analysis were not performed blindly to the conditions of the experiment due to the specific requirements of the UK Home Office project licence.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank all the staff members of the University of Manchester Biological Services facility for their thoughtful contribution to the manintenace of our animal colonies. We also thanks the staff members of the University of Manchester professional and technical services (imaging, flow cytometry, and genomics) for their technical support. We are grateful to Gloria Lopez Castejon (University of Manchester, UK) for providing feedback on this manuscript. \u0026nbsp;This work was funded through a Sir Henry Wellcome postdoctoral fellowship (221640/Z/20/Z to JFQ), a\u0026nbsp;Wellcome Trust Career Development Award (309148/Z/24/Z to JFQ), and an Academy of Medical Sciences Springboard Award (SBF009/1079). MCS is supported by a Wellcome Early Career Award Fellowship (303388/Z/23/Z). CB is funded by an UKRI MRC research grant (MR/W018497/1). NAM is supported by an Institute Strategic Programme grant funding from the UKRI Biotechnology and Biological Sciences Research Council (BBS/E/RL/230002B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConceptualisation:\u0026nbsp;\u003c/strong\u003eJFQ\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003eMCS, CB, NAM, AAS. \u003cstrong\u003eMethodology:\u003c/strong\u003e MCS, AC, CB, OS, KW, AM, JC, SW. \u003cstrong\u003eBioinformatics analysis\u003c/strong\u003e: SMB. \u003cstrong\u003eFormal analysis\u003c/strong\u003e: JFQ, MCS, AC, CB, JCPF, AAS. \u003cstrong\u003eWriting \u0026ndash; original draft:\u003c/strong\u003e JFQ\u003cstrong\u003e. Writing \u0026ndash; reviewing and editing:\u003c/strong\u003e JFQ, MCS, AC, CB, CB, NAM. \u003cstrong\u003eWriting \u0026ndash; final edits:\u003c/strong\u003e MCS, JFQ. \u003cstrong\u003eFunding acquisition:\u003c/strong\u003e JFQ. The authors declare that they have no competing interests, commercial or otherwise. Correspondence and requests for materials should be addressed to Juan F. Quintana ([email protected]).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSchafflick D et al (2021) Single-cell profiling of CNS border compartment leukocytes reveals that B cells and their progenitors reside in non-diseased meninges. Nat Neurosci 24:1225\u0026ndash;1234\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrioschi S et al (2021) Heterogeneity of meningeal B cells reveals a lymphopoietic niche at the CNS borders. Science 373:eabf9277\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y et al (2021) Early developing B cells undergo negative selection by central nervous system-specific antigens in the meninges. Immunity 54:2784\u0026ndash;2794e6\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHerisson F et al (2018) Direct vascular channels connect skull bone marrow and the brain surface enabling myeloid cell migration. Nat Neurosci 21:1209\u0026ndash;1217\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi K et al (2022) Bone marrow hematopoiesis drives multiple sclerosis progression. Cell 185:2234\u0026ndash;2247e17\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Niz M et al (2021) Organotypic endothelial adhesion molecules are key for Trypanosoma brucei tropism and virulence. Cell Rep 36:109741\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeaver AK et al (2024) Tissue spaces are reservoirs of antigenic diversity for Trypanosoma brucei. Nature 636:430\u0026ndash;437\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTrindade S et al (2022) Slow growing behavior in African trypanosomes during adipose tissue colonization. Nat Commun 13:7548\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuintana JF et al (2022) Single cell and spatial transcriptomic analyses reveal microglia-plasma cell crosstalk in the brain during Trypanosoma brucei infection. Nat Commun 13:5752\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuintana JF et al (2023) The murine meninges acquire lymphoid tissue properties and harbour autoreactive B cells during chronic Trypanosoma brucei infection. PLOS Biol 21:e3002389\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlfituri OA et al (2020) To the Skin and Beyond: The Immune Response to African Trypanosomes as They Enter and Exit the Vertebrate Host. Front Immunol 11\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBockstal V et al (2011) T. brucei Infection Reduces B Lymphopoiesis in Bone Marrow and Truncates Compensatory Splenic Lymphopoiesis through Transitional B-Cell Apoptosis. PLoS Pathog 7:e1002089\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Trez C, Khan S, Magez S (2020) T. brucei infections abrogate diverse plasma cell-mediated effector B cell responses, independently of their specificity, affinity and host genetic background. PLoS Negl Trop Dis 14:e0008358\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCnops J et al (2016) Maintenance of B cells during chronic murine Trypanosoma brucei gambiense infection. Parasite Immunol 38:642\u0026ndash;647\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrenkel D et al (2016) Trypanosoma brucei Co-opts NK Cells to Kill Splenic B2 B Cells. PLOS Pathog 12:e1005733\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePham HTT et al (2023) Neutrophil metalloproteinase driven spleen damage hampers infection control of trypanosomiasis. Nat Commun 14:5418\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFitzpatrick Z et al (2024) Venous-plexus-associated lymphoid hubs support meningeal humoral immunity. Nature 1\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41586-024-07202-9\u003c/span\u003e\u003cspan address=\"10.1038/s41586-024-07202-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNiu C et al (2022) Identification of hematopoietic stem cells residing in the meninges of adult mice at steady state. Cell Rep 41:111592\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHardy RR et al (2000) B-cell commitment, development and selection. Immunol Rev 175:23\u0026ndash;32\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMecheri B, Paris F, L\u0026uuml;bbert H (2018) Histological investigations on the dura mater vascular system of mice. Acta Histochem 120:846\u0026ndash;857\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaag Rasmussen M et al (2024) Trigeminal ganglion neurons are directly activated by influx of CSF solutes in a migraine model. Science 385:80\u0026ndash;86\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShah T et al (2023) Arachnoid granulations are lymphatic conduits that communicate with bone marrow and dura-arachnoid stroma. J Exp Med 220:e20220618\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThe origin of subdural neomembranes I. Fine structure of the dura-arachnoid interface in man. - PMC. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2018597/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2018597/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBifari F et al (2017) Neurogenic Radial Glia-like Cells in Meninges Migrate and Differentiate into Functionally Integrated Neurons in the Neonatal Cortex. Cell Stem Cell 20:360\u0026ndash;373e7\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTerrier L et al (2021) The trigeminal system: The meningovascular complex\u0026mdash; A review. J Anat 239:1\u0026ndash;11\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNoseda R, Melo-Carrillo A, Nir R-R, Strassman AM, Burstein R (2019) Non-Trigeminal Nociceptive Innervation of the Posterior Dura: Implications to Occipital Headache. J Neurosci 39:1867\u0026ndash;1880\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatthias P, Rolink AG (2005) Transcriptional networks in developing and mature B cells. Nat Rev Immunol 5:497\u0026ndash;508\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJin S et al (2021) Inference and analysis of cell-cell communication using CellChat. Nat Commun 12:1088\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchmidt TH, Bannard O, Gray EE, Cyster JG (2013) CXCR4 promotes B cell egress from Peyer\u0026rsquo;s patches. J Exp Med 210:1099\u0026ndash;1107\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGiorgiutti S, Rottura J, Korganow A-S, Gies V (2024) CXCR4: from B-cell development to B cell\u0026ndash;mediated diseases. Life Sci Alliance 7:e202302465\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClark MR, Mandal M, Ochiai K, Singh H (2014) Orchestrating B cell lymphopoiesis through interplay of IL-7 receptor and pre-B cell receptor signalling. Nat Rev Immunol 14:69\u0026ndash;80\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNemazee D (2017) Mechanisms of central tolerance for B cells. Nat Rev Immunol 17:281\u0026ndash;294\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGilabert Carbajo C et al (2021) Novel aspects of iron homeostasis in pathogenic bloodstream form Trypanosoma brucei. PLOS Pathog 17:e1009696\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoth R, Mamula MJ (1997) Trafficking of Adoptively Transferred B Lymphocytes in B-Lymphocyte-Deficient Mice. J Exp Biol 200:2057\u0026ndash;2062\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Y, Tung JW, Ghosn EEB, Herzenberg LA, Herzenberg LA (2007) Division and differentiation of natural antibody-producing cells in mouse spleen. \u003cem\u003eProc. Natl. Acad. Sci.\u003c/em\u003e 104, 4542\u0026ndash;4546\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMazzitelli JA et al (2023) Skull bone marrow channels as immune gateways to the central nervous system. Nat Neurosci 26:2052\u0026ndash;2062\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu J et al (2024) Turbinate-homing IgA-secreting cells originate in the nasal lymphoid tissues. Nature 632:637\u0026ndash;646\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFitzpatrick Z et al (2020) Gut-educated IgA plasma cells defend the meningeal venous sinuses. Nature 587:472\u0026ndash;476\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSinton MC et al (2022) IL-17 signalling is critical for controlling subcutaneous adipose tissue dynamics and parasite burden during chronic murine Trypanosoma brucei infection. \u003cem\u003eNat. Commun.\u003c/em\u003e 14, 09.23.509158 (2022)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao AT, Yao S, Gong B, Elson CO, Cong Y (2012) Th17 Cells Upregulate Polymeric Ig Receptor and Intestinal IgA and Contribute to Intestinal Homeostasis. J Immunol 189:4666\u0026ndash;4673\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDann SM et al (2015) IL-17A promotes protective IgA responses and expression of other potential effectors against the lumen-dwelling enteric parasite Giardia. Exp Parasitol 156:68\u0026ndash;78\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAvnir Y et al (2016) IGHV1-69 polymorphism modulates anti-influenza antibody repertoires, correlates with IGHV utilization shifts and varies by ethnicity. Sci Rep 6:20842\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTeo QW et al (2023) Stringent and complex sequence constraints of an IGHV1-69 broadly neutralizing antibody to influenza HA stem. Cell Rep 42:113410\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSarkanen TO, Alakuijala APE, Dauvilliers YA, Partinen MM (2018) Incidence of narcolepsy after H1N1 influenza and vaccinations: Systematic review and meta-analysis. Sleep Med Rev 38:177\u0026ndash;186\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTesoriero C et al (2016) H1N1 influenza virus induces narcolepsy-like sleep disruption and targets sleep\u0026ndash;wake regulatory neurons in mice. \u003cem\u003eProc. Natl. Acad. Sci.\u003c/em\u003e 113\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVuorela A et al (2021) Enhanced influenza A H1N1 T cell epitope recognition and cross-reactivity to protein-O-mannosyltransferase 1 in Pandemrix-associated narcolepsy type 1. Nat Commun 12:2283\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRijo-Ferreira F et al (2018) Sleeping sickness is a circadian disorder. Nat Commun 9:62\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRijo-Ferreira F et al (2020) Sleeping Sickness Disrupts the Sleep-Regulating Adenosine System. J Neurosci Off J Soc Neurosci 40:9306\u0026ndash;9316\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePg K (2013) Clinical features, diagnosis, and treatment of human African trypanosomiasis (sleeping sickness). Lancet Neurol. 12\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMugnier MR, Stebbins CE, Papavasiliou FN (2016) Masters of Disguise: Antigenic Variation and the VSG Coat in Trypanosoma brucei. PLOS Pathog 12:e1005784\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStijlemans B, Radwanska M, De Trez C, Magez S (2017) African Trypanosomes Undermine Humoral Responses and Vaccine Development: Link with Inflammatory Responses? Front Immunol 8:582\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLe Ray D, Barry JD, Easton C, Vickerman K (1977) First tsetse fly transmission of the \u0026lsquo;AnTat\u0026rsquo; serodeme of Trypanosoma brucei. Ann Soc Belg Med Trop 57:369\u0026ndash;381\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHerbert WJ, Lumsden WH (1976) Trypanosoma brucei: a rapid \u0026lsquo;matching\u0026rsquo; method for estimating the host\u0026rsquo;s parasitemia. Exp Parasitol 40:427\u0026ndash;431\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVlaminck KD et al (2022) Differential plasticity and fate of brain-resident and recruited macrophages during the onset and resolution of neuroinflammation. Immunity 55, 2085\u0026ndash;2102.e9\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eColes JA, Stewart-Hutchinson PJ, Myburgh E, Brewer JM (2017) The mouse cortical meninges are the site of immune responses to many different pathogens, and are accessible to intravital imaging. Methods San Diego Calif 127:53\u0026ndash;61\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKristin G, Anderson K, Mayer-Barber H, Sung L, Beura BR, James JJ, Taylor L, Qunaj, Thomas S, Griffith Vaiva Vezys, Daniel L BarberDavid Masopust. Intravascular staining for discrimination of vascular and tissue leukocytes | Springer Nature Experiments. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://experiments.springernature.com/articles/\u003c/span\u003e\u003cspan address=\"https://experiments.springernature.com/articles/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nprot.2014.005\u003c/span\u003e\u003cspan address=\"10.1038/nprot.2014.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eManglani M, Gossa S, McGavern DB (2018) Leukocyte Isolation from Brain, Spinal Cord, and Meninges for Flow Cytometric Analysis. Curr Protoc Immunol 121:e44\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eManso T et al (2022) IMGT\u0026reg; databases, related tools and web resources through three main axes of research and development. Nucleic Acids Res 50:D1262\u0026ndash;D1272\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCrooks GE, Hon G, Chandonia J-M, Brenner SE (2004) WebLogo: A Sequence Logo Generator: Fig. 1. Genome Res 14:1188\u0026ndash;1190\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"B cell lymphopoiesis, CNS immune response, Trypanosoma brucei infection, CXCR4/CXCL12 signalling","lastPublishedDoi":"10.21203/rs.3.rs-8621800/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8621800/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe dura mater hosts a rich population of B cell progenitors, but its capacity to sustain B cell development during systemic lymphopenia remains unclear. Using a murine model of chronic \u003cem\u003eTrypanosoma brucei\u003c/em\u003e infection, which induces peripheral B cell depletion, we demonstrate that the dura mater maintains intact B cell lymphopoiesis independently of bone marrow and spleen in both male and female mice, in a process involving various chemotactic and pro-survival factors derived from the dura mater stroma. Furthermore, dura mater-derived B cells exhibit a distinct immunoglobulin repertoire that is distinct from the splenic repertoire and is dominated by \u003cem\u003eIghv1\u003c/em\u003e family members. The immunoglobulins produce locally at the CNS borders are polyreactive and able to recognise both CNS and parasite antigens. Lastly, adoptive transfer of dura mater B cells, or their cognate antibodies, into B cell-deficient mice delays parasitemia onset, whereas splenic B cells from infected hosts fail to confer similar protection. These findings identify, for the first time, the dura mater as a resilient, autonomous B cell lymphopoietic niche that shapes specialized humoral responses during chronic infection, highlighting its potential role in coordinating immune defense when conventional lymphoid organs are compromised.\u003c/p\u003e","manuscriptTitle":"The dura mater maintains B cell lymphopoietic capacity during chronic Trypanosoma brucei infection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-21 04:06:57","doi":"10.21203/rs.3.rs-8621800/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dc1ba2df-365a-4036-83dd-f4a110d7d2cb","owner":[],"postedDate":"January 21st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":61346817,"name":"Biological sciences/Immunology/Infection"},{"id":61346818,"name":"Health sciences/Diseases/Infectious diseases"},{"id":61346819,"name":"Biological sciences/Neuroscience/Neuroimmunology"}],"tags":[],"updatedAt":"2026-03-31T08:28:05+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-21 04:06:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8621800","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8621800","identity":"rs-8621800","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}