Expanding group 1 ILCs sustain early CNS defense against Toxoplasma gondii during impaired adaptive response

Expanding group 1 ILCs sustain early CNS defense against Toxoplasma gondii during impaired adaptive response | 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 Research Article Expanding group 1 ILCs sustain early CNS defense against Toxoplasma gondii during impaired adaptive response Johannes Steffen, Nina Sophie Bellersheim, Caio Andreeta Figueiredo, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8278466/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 12 You are reading this latest preprint version Abstract Specialized immune interfaces of the central nervous system (CNS), as the choroid plexus and meninges, serve as dynamic gatekeepers coordinating the transition from innate to adaptive immunity during neuroinflammatory responses. While this transition is essential for resolving CNS inflammation, the precise temporal and functional bridging of these phases remains incompletely characterized. To elucidate the role of local innate effectors in maintaining CNS protection we employed the model of Toxoplasma gondii -induced neuroinflammation and applied Fingolimod (FTY720) to pharmacologically modulate the S1P receptor and prevent lymphocyte recirculation and CNS infiltration. Despite resulting in impaired T cell infiltration, CNS parasite control was preserved during the acute phase, coinciding with expansion of group 1 innate lymphoid cells (ILCs), including tissue-resident NK cells and type 1 ILCs (ILC1s). High-dimensional profiling indicated that brain-derived ILC1s acquire distinct, tissue-adapted signatures and sustain robust type 1 cytokine responses ( Ifng , Tnf , Irf8 ) crucial for microglial activation and early antiparasitic defense. Collectively, rapidly adapting innate effectors supported early defense in the infected CNS when adaptive immunity was compromised, providing detailed insight into the context-specific orchestration of CNS protection. These results refine the understanding of the innate–adaptive transition during neuroinflammation and position group 1 ILCs as promising targets for neuroprotective immunomodulation. innate lymphoid cells CNS immunity ILC1s IFN-γ type 1 cytokines neuroinflammation FTY720 (Fingolimod) innate-adaptive transition T cell-independent immunity brain-resident ILCs choroid plexus meninges Toxoplasma gondii adaptive immunity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Once considered strictly immune privileged, the central nervous system (CNS) is now recognized to rely on compartmentalized neuroimmune communication networks that actively shape its development, function, and maintenance ( 1 ). CNS barrier interfaces, such as the choroid plexus and meninges, not only function as physical barriers but as neuroimmune signaling hubs that relay contextual somatic and environmental cues to the CNS ( 1 , 2 ). Innate effectors positioned at these interfaces rapidly detect infection, injury, or neurodegeneration, initiating a local response that propagates and amplifies inflammatory signals, modulates barrier permeability, and facilitates infiltration of peripheral immune cells ( 3 ). Among them, an early cytokine and chemokine-mediated response shapes the recruitment, activation, and effector functions of adaptive immune cells that govern the trajectory, severity, and persistence of chronic neuroinflammation ( 4 , 5 ). While this tightly regulated spatiotemporal cascade ensures a response precisely tuned to the CNS microenvironment, functional bridging between innate and adaptive responses remains poorly defined. The rapid innate response offers a temporal advantage to ensure protection until the adaptive response is fully established. In situ proliferation of resident ILCs ( 6 ), together with local signals that promote tissue residency and effector differentiation, such as IL-2 ( 7 ), IL-15, and TGF-β ( 8 ), may support cytotoxic responses in the CNS ( 9 ) to temporally bridge impairments in adaptive immunity. Deciphering the spatiotemporal dynamics and functional capacities of innate immune cells is therefore crucial for resolving gaps in the understanding of neuroinflammation. The protozoan parasite Toxoplasma gondii ( T. gondii ) offers a unique model to dissect the spatiotemporal dynamics of CNS immunity. After crossing the CNS barriers, T. gondii infects parenchymal cells, triggering a robust and defined sequence of innate and adaptive immune responses ( 10 ). Effective CNS immunity requires a balanced response, controlling the infection without causing irreversible damage to a tissue with limited regenerative capacity. While long-term control of T. gondii relies on adaptive immunity, particularly T cell-mediated responses ( 11 – 13 ), the early containment of the parasite primarily depends on a swift response from brain-resident innate effectors ( 14 – 16 ). Among these, group 1 innate lymphoid cells (ILCs), comprising conventional natural killer (cNK) cells, tissue-resident NK (trNK) cells, and ILC1s, are critical contributors to early immune response. While cNK are patrolling cells, it was demonstrated that they can leave the circulation and establish tissue-residency following infection. This was shown by the acquisition of ILC1 markers, such as CD49a and CD69 (trNK cells) ( 17 , 18 ). ILC1s are permanently integrated in the fabric of the tissue and rapidly produce IFN-γ and TNF, limiting parasite expansion and shaping downstream immunity ( 19 , 20 ). Whether these cells can transiently compensate for the reduced presence of adaptive lymphocytes in the CNS and sustain sufficient response during the acute infection phase remains unclear. To dissect the capacity of innate immune cells to bridge delayed or reduced adaptive responses, we pharmacologically restricted lymphocyte recirculation and CNS infiltration using FTY720, a sphingosine-1-phosphate receptor modulator ( 21 ), during acute T. gondii infection. This approach allowed us to focus on the local immune compartment when assessing ILC expansion, spatial distribution, and functional contributions to early host defense. In the absence of effective T cell infiltration, ILCs robustly expanded within the CNS, maintaining a proinflammatory, antiparasitic microenvironment sufficient to suppress parasite replication. High-dimensional profiling revealed that CNS-associated ILC1s acquire a distinct, tissue-adapted phenotype, distinguishing them from their peripheral counterparts. These findings highlight the immune system’s functional plasticity, demonstrating that the innate immune compartment rapidly responds and temporarily compensates for numerical impairments in the adaptive immune compartment to sustain effective early CNS defense. Materials and methods 3.1 Mice Experiments were performed using female, wild-type C57BL/6J mice (8–14 weeks old) bred and group-housed under specific pathogen free (SPF) conditions with a 12-h light/dark cycle at 22°C and ad libitum access to food and water. All animal procedures were approved by the local authorities in accordance to German and European legislation. 3.2 Infection T. gondii tissue cysts (type II ME49 strain) were obtained from the brains of female NMRI mice previously infected with T. gondii 6–12 months earlier. Brain tissue was mechanically homogenized in 1 mL sterile phosphate-buffered saline (PBS), and cysts number was quantified by light microscopy in ≥ 110 µL homogenate. The homogenate was subsequently diluted in sterile PBS, and animals were infected by intraperitoneal administration of two cysts ( 20 , 22 ). Uninfected control mice received an equivalent volume of sterile PBS as a mock control. To determine the disease score, the animals were scored daily based on different categories (see Table S1 ) 3.3 Tissue collection Mice received a single intravenous bolus of 50 µg FITC-labeled anti-CD45 antibody 10 min before anesthesia. Following euthanasia, peripheral blood samples were collected, and the animals were transcardially perfused with 60 mL ice-cold sterile PBS. The brain (including pia mater), choroid plexus, meninges (dura meninges and partially arachnoid meninges), spleen, liver, and small intestine were removed. For gene expression analysis, samples were incubated in RNAlater at 4 ° C overnight, and then stored at -20°C until further processing. For cell isolation, brain, spleen, and liver were stored in sterile ice-cold PBS; small intestine was stored in ice-cold PBS + PS (PBS, 100 U/mL Penicillin, 100 µg/mL Streptomycin); dural meninges and choroid plexus were stored in 150 µL RPMI + PS (RPMI 1640, 100 U/mL Penicillin, 100 µg/mL Streptomycin). 3.4 Cell isolation Cells were isolated as previously described for brain, liver, and spleen ( 20 ), small intestine ( 23 ), and meninges ( 24 ) with slight modifications. Brains were minced, homogenized in dissection buffer (HBSS, 50 mM glucose, 13 mM HEPES, pH 7.3), filtered through a 70 µm cell strainer, and centrifuged (400 g , 20 min, 4°C). The mononuclear cells were separated via discontinuous, isotonic Percoll gradient (70% / 30%) centrifugation (800 g , 35 min, 4°C, minimal acceleration / deceleration). Choroid plexus and meninges were digested with 1 mg/mL DNAse I and 1.5 mg/mL Collagenase D (600 rpm, 25 min, 37°C) in 200 µL of RPMI + PS. Following digestion, 800 µL RPMI + PS were added, mechanical dissociation was performed by 20 repeated passages through a pipette tip. Cell suspension was filtered through 40 µm strainer, and centrifuged (400 g , 10 min, 4°C). For small intestine preparation, tissue was cleared of fat and feces, Peyer’s patches were removed, the tissue was opened longitudinally, and washed three times with 20 mL ice-cold PBS + PS by vortexing for 15 sec and filtering through a metal strainer to remove mucus. Tissue was transferred into 20 mL dissociation solution (HBSS without Ca2 + /Mg2 + , 5 mM EDTA, 10 mM HEPES, 100 U/mL Penicillin, 100 µg/mL Streptomycin, 1 mM DTT), incubated (100 rpm, 20 min, RT), vortexed for 15 sec, and filtered through metal strainer. The filtrate containing epithelial cells and intraepithelial leukocytes was discarded. Remaining tissue was incubated with dissociation solution, vortexed for 15 sec, and filtered through metal strainer. Tissue was washed twice with 30 mL PBS + PS (first wash: 100 rpm, 20 min, RT and vortexing for 15 sec; second wash: vortexing for 15 sec), and filtered through metal strainer. Tissue was minced with scissors, transferred into 10 mL digestion solution (RPMI 1640, 4% FCS, 100 U/mL Penicillin, 100 µg/mL Streptomycin, 0.25 mg/mL Collagenase D (Roche), 0.2 mg/mL Dispase II (Roche), 0.25 mg/mL DNAse I (Sigma-Aldrich)), incubated (100 rpm, 37°C for 15 min), vortexed for 15 sec and filtered through 100 µm strainer. Lamina propria leukocyte (LPL)-containing filtrate was stored on ice, while remaining tissue was digested and filtered once more through 100 µm strainer to pool cells. LPL suspensions were filtered through 40 µm strainer and centrifuged (800 g , 15 min, RT), resuspended in 33% isotonic Percoll and separated via discontinuous, isotonic Percoll gradient (80%/44%/33%) centrifugation (600 g , 20 min, 20°C, minimal acceleration / deceleration). The enriched leukocytes on the 44%/80% interphase were harvested and washed with PBS + 2mM EDTA (600 g , 10 min, 4°C). Livers were mechanically dissociated by passage through a 70 µm cell strainer, washed with PBS, centrifuged (400 g , 20 min, 4°C), and mononuclear cells further enriched by discontinuous, isotonic Percoll gradient (70% / 35%) centrifugation (800 g , 35 min, 4°C, minimal acceleration / deceleration). Spleens were mechanically homogenized by passage through a 40 µm cell strainer. Erythrocytes in the blood and spleen samples were lysed using RBC lysis buffer (10 min), subsequently washed with PBS, centrifuged (400 g , 10 min, 4°C). 3.5 Flow cytometry Single-cell suspensions were resuspended in FACS buffer (PBS, 2% (v/v) FCS, 2 mM EDTA) and incubated with 1 µg anti-mouse CD16/32 antibody and a viability dye (20 min, 4°C). Surface staining was performed using fluorochrome-conjugated antibodies against CD3ε, CD8a, CD5, CD19, Ly6G, CD45, NK1.1, NKp46, CD49a, CD49b, CD69, and CXCR3 (30 min, 4°C). Cells were washed (FACS buffer) and centrifuged (400 g , 10 min 4°C) twice. For intracellular staining, cells were fixed and permeabilized with the Foxp3 / Transcription Factor Staining Buffer Set (60 min, 4°C), washed / centrifuged twice, and stained using fluorochrome-conjugated antibodies against Eomes and T-bet (40 min, 4°C). Cells were washed / centrifuged twice, resuspended in FACS buffer, acquired using an Attune™ NxT flow cytometer (Thermo Fisher Scientific) with FMO controls assessing background fluorescence. Computational analyses, including FlowSOM clustering ( 25 ), Uniform Manifold Approximation and Projection (UMAP) for dimensionality reduction ( 26 ), and data visualization via violin plots ( 27 ), were performed using FlowJo software (v10.8.2; BD Biosciences) and R 4.4.3 ( 28 ). 3.6 RNA and DNA isolation, qPCR and RT-qPCR Tissue samples were removed from RNAlater and homogenized (4,350 rpm, 3x30 s) with RNA-Solv®Reagent (Omega Bio-Tek, R6830) and Zirconium oxide beads (Precellys, P000926-LYSK0-A) in a BeadBug 6 homogenizer (Biozym). Chloroform (20% v/v) was added to the lysate to facilitate phase separation. DNA was extracted from the interphase using DNA-binding columns (VWR, 13-HCR-02) according to the manufacturer’s instructions, while RNA was recovered from the aqueous phase via isopropanol precipitation. Nucleic acid concentrations and purity were determined using a NanoDrop 2000 spectrophotometer (ThermoFisher), and samples stored at -80°C until analysis. Quantitative gene expression analysis was performed using 30 ng of isolated RNA, TaqMan™ RNA-to-CT™ 1-Step Kit, TaqMan™ probes for transforming growth factor beta 1 ( Tgfb1 ), interleukin 15 ( Il15 ), interferon gamma (Ifng ) , tumor necrosis factor ( Tnf ), interferon regulatory factor 8 ( Irf8 ), interleukin 7 ( Il7 ), interleukin 12a ( Il12a ), and interleukin-18 ( Il18 ), and LightCycler® 96 System (Roche Diagnostics) (Table S2) ( 20 ). Mean Cq values were obtained (LightCycler® 96, v1.1.0.1320), and relative expression to hypoxanthine guanine phosphoribosyl transferase ( Hprt ) was calculated using 2 −ΔCq where ΔCq = (Cq (Target gene) − Cq ( Hprt )) ( 29 ) in Excel® 2019 (v16.0.10413.20020, Microsoft®). Parasite burden was assessed in using 100 ng of isolated DNA, FastStart Essential DNA Green Master (Roche), and LightCycler® 96 System (Roche Diagnostics), as previously described ( 30 ). Amplification of the conserved T. gondii B1 gene was used to assess parasite load, with murine argininosuccinate lyase ( Asl) serving as the reference gene (Table S3). Parasite burden was calculated using 2 −ΔCq where ΔCq = (Cq ( B1 ) − Cq ( Asl )) as described above. 3.7 Statistical analysis The statistical tests and sample sizes are provided in the figure legends. Data normality (D’Agostino-Pearson for n ≥ 8, Shapiro-Wilk normality test for n < 8) and equality of variances (F test) were assessed in advance for selection of the statistical test. Sample sizes were based on literature and prior experience to ensure statistical power. Investigators were blinded during experiments and analysis when possible. Statistical calculations were performed in Prism (v10.4.1, GraphPad) and considered significant if p ≤ 0.05. Unless otherwise stated, data are presented as arithmetic mean ± SD. Results 4.1 FTY720 leads to expanded group 1 ILCs cells in the CNS during infection To assess the contribution of adaptive lymphocyte trafficking during infection, we treated a subset of infected animals with FTY720 (Fig. 1 A), a sphingosine-1-phosphate receptor (S1PR) modulator that impedes lymphocyte egress from lymphoid tissues ( 21 ). FTY720 treatment started at day 7 post-infection (Fig. 1 A), to avoid interfering with immune cell redistribution necessary for anti-parasitic response in peripheral tissues, while still effectively restricting lymphocyte recruitment to the CNS. We analyzed the distribution and dynamics of group 1 innate lymphoid cells and divided them into conventional NK (cNK) cells, tissue-resident NK (trNK) cells, and ILC1s, as previously described ( 8 , 17 ), across the brain, the choroid plexus, and the meninges (Fig. 1 B-I). FTY720 treatment significantly increased both frequency and total number of brain-resident group 1 innate lymphocytes, suggesting that S1P-dependent changes are associated with enhanced expansion of innate lymphoid populations. While Eomes⁺CD49a⁻ cNK cell populations remained stable, both Eomes⁺CD49a⁺ trNK cells and Eomes⁻CD49a⁺ ILC1s were significantly elevated in infected animals, with further expansion in FTY720-treated cohorts (Fig. 1 C, D, E, F). These findings align with a recent data implicating TGF-β1 and IL-15 in the induction of NK cell residency and cytotoxic programming ( 8 ), pathways both upregulated in the inflamed brain during T. gondii infection (Fig. 1 G). Expression of key cytokines and host-defense factors Ifng , Tnf , Irf8 , and Il7 remained unaffected upon FTY720 treatment (Fig. 1 G). CNS barrier compartments, such as the meninges and the choroid plexus, are crucial immunological hubs that undergo significant changes during inflammation ( 31 , 32 ), functioning as proliferative and immunoregulatory niches and crucial entry points for peripheral immune cells, as reflected by increased frequencies of CD45⁺ cell (Fig. 1 H, I). In the choroid plexus, trNK cells and ILC1s markedly expanded upon infection, independent of FTY720 treatment (Fig. 1 H). Conversely, the meninges rather exhibited an overall decline group 1 ILC subsets during acute infection, with FTY720 selectively reducing cNK and trNK cells at baseline and early timepoints, whereas percentage and number of ILC1s were preserved (Fig. 1 I). These compartment-specific dynamics indicate that tissue-resident group 1 ILCs, particularly ILC1s, are poised to rapidly expand within the CNS and border regions independent of S1P-mediated trafficking, reinforcing their contribution to early neuroinflammatory response. ( A ) C57Bl/6J mice were T. gondii (ME49) or mock-infected (Ctrl) intraperitoneally, followed daily oral treatment with FTY720 (FTY) or diluent starting 7 days post-infection (dpi). Representative flow cytometry plots and quantification of cNK, trNK, and ILC1s in the ( B , D ) brain parenchyma, the ( H ) choroid plexus, and the ( I ) meninges. ( E , F ) Temporal changes in composition of group 1 ILCs during infection. ( G ) Relative mRNA levels of indicated genes in the brain parenchyma. Data are pooled from four independent experiments (n = 7–10 mice per group). Individual values and mean are plotted. The differences between groups (FTY720 vs. diluent) were analyzed by ordinary two-way ANOVA followed by Šídák’s multiple comparisons test (corresponding p-values are provided for p < 0.1 and considered significant for p < 0.05). Following the onset of neuroinflammation, activated microglia not only exert direct effector functions but also play a key role in sustaining inflammation and promoting the recruitment of peripheral immune cells ( 33 ). To assess whether FTY720-dependent redistribution impairs these myeloid contributions, we analyzed brain-resident populations as well as recruited immune cells. T. gondii infection induced a robust expansion of CD45⁺CD11b⁺ myeloid cells in the brain by day 14, including CX 3 CR1 + microglia, Ly6C⁺ monocytes, and Ly6G⁺ neutrophils (Fig. S1 E, H). FTY720 treatment had no apparent effect on microglial expansion or activation. The upregulated MHC class I and II by day 10 (Fig. S1 F) indicates intact IFN-γ-STAT1 signaling in microglia ( 33 ). Although infection-induced downregulation of CX 3 CR1 was evident in both infected groups, the decrease was slightly delayed in FTY720-treated mice (Fig. S1 F). Beyond their direct effector functions, microglia shape neuroinflammatory milieu by production of CXCL9 and CXCL10, orchestrating the recruitment of innate lymphoid cells and peripheral immune cells via CXCR3-dependent mechanisms ( 33 , 34 ).While T. gondii infection significantly increased Cxcl9 and Cxcl10 transcription relative to mock-infected controls, FTY720 treatment did not affect expression levels of these chemokines (Fig. S1 H). We next explored infiltrating myeloid subsets, important contributors in CNS parasite control ( 35 – 37 ). While Ly6C hi monocytes were slightly reduced in FTY720-treated animals, no significant differences were observed in Ly6C int or Ly6C low monocytes or in neutrophils until day 14 post-infection (Fig. S1 G). Finally, we analyzed brain-infiltrating leukocytes, revealing a marked reduction in both the frequency and absolute number of CD4⁺ and CD8 + T cells in FTY720-treated animals at day 14 post-infection compared to infected diluent-treated animals (Fig. S1 B-D), confirming the suppression of S1P-dependent migration during infection. Despite this pharmacological blockade, the course of the infection and the parasite burden in the brain parenchyma and the periphery remained mostly unaltered (Fig. 2 A-E). Collectively, these data demonstrate that innate myeloid cell activation and infiltration are largely preserved despite impaired T cell recruitment, highlighting the critical role of non-adaptive immunity in mounting a protective neuroinflammatory response and controlling infection during the acute phases of cerebral toxoplasmosis. Wild-type C57Bl/6 mice with T. gondii or mock-infected (Control) and orally treated with FTY720 or diluent (see also Fig. 1 A). Absolute intake of ( A ) food and ( B ) water upon infection. ( C ) Body weight of animals (relative to weight at day of infection). ( D ) Disease score of animals. ( E ) Parasite burden in indicated tissues at 10 and 14 days post-infection (dpi). Absolute intake of food (A) and water (B) were recorded per cage and divided by number of animals for comparison. The differences between groups (FTY720 vs. diluent) were analyzed by (A-D) mixed model followed by Tukey’s multiple comparisons test (p-values are provided and considered significant for p < 0.05) or (E) ordinary two-way ANOVA followed by Šídák’s multiple comparisons test (p-values are provided for p < 0.10 and considered significant for p < 0.05). 4.2 Peripheral ILC1 Dynamics in Liver and Small Intestine Show Transient Expansion During Acute Infection To determine whether CNS accumulation of group 1 innate lymphoid cells reflects systemic redistribution ( 38 – 41 ) or a coordinated local response ( 6 ), we analyzed cNK, trNK, and ILC1 populations in the liver and small intestine. In the liver, their expansion by day 10 post-infection was followed by their contraction by day 14 (Fig. 3 B), indicating a transient peripheral response. In the small intestine, the expansion of group 1 ILCs persisted through day 14, indicating a more sustained immune activation in gut-associated tissues (Fig. 3 F). These tissue-specific temporal dynamics show that the resolution of peripheral response coincides with CNS accumulation of ILC1s and trNK cells, raising the possibility of migratory seeding from peripheral reservoirs to the CNS and its border regions. ( A ) Representative flow cytometry plots from the liver and quantification of ( B , E ) cNK, trNK, and ILC1s, and CD45 + cells ( C , F ) in the liver and the small intestine. ( D , G ) Temporal changes in composition of group 1 ILCs during infection. Data are pooled from four independent experiments (n = 7–10 mice per group). (B, C, E, F) Individual values and mean, or (D, G) mean values are plotted. The differences between groups (FTY720 vs. diluent) were analyzed by ordinary two-way ANOVA followed by Šídák’s multiple comparisons test (corresponding p-values are provided for p < 0.1 and considered significant for p < 0.05). 4.3 Type 1 Innate Lymphoid Cells Localize Predominantly to the Parenchyma in the Infected CNS To resolve whether group 1 ILCs are infiltrating the parenchyma or remain confined in the vasculature, intravascular labeling was performed prior to transcardial perfusion tissue harvest using fluorescent anti-CD45 antibodies (Fig. 4 A). Efficiency of labeling and perfusion are reflected by quantification FITC + cells in blood and CNS compartments (Fig. 4 ). Group 1 ILCs showed distinct compartmental localization patterns. In the brain, vascular labelling of cNK cells sharply declined during infection, indicating parenchymal infiltration. In contrast, trNK cells and ILC1s consistently lacked vascular labeling, confirming their extravascular localization (Fig. 4 B). In the meninges, vascular labeling of CD45⁺ cells and group 1 ILCs increased progressively with infection, likely reflecting elevated vascular permeability during neuroinflammation (Fig. 4 C). In the choroid plexus, vascular labeling of CD45⁺ cells remained low across conditions, and trNK cells and ILC1s displayed low vascular labeling, consistent with their stromal localization (Fig. 4 D). These data confirm the infection-induced parenchymal infiltration of circulating cNK cells and the predominant tissue residency of trNK cells and ILC1s. Their accumulation within parenchymal and stromal compartments (Fig. 1 C, D, H), largely devoid of direct vascular exposure (Fig. 4 B, D), supports the hypothesis of active tissue infiltration and/or local retention/expansion during infection. The positioning group 1 ILCs in close proximity to infected cells facilitates rapid effector cytokine responses and engagement in early immune defenses. FITC-labelled anti-CD45 antibody was intravenously injected prior to transcardial perfusion to label leukocytes accessible from the vasculature. ( A ) Representative flow cytometry plot and quantification of FITC + fraction shows efficient labelling in blood. Vascular labeling of total CD45⁺ cells, cNK, trNK, and ILC1 populations in ( B ) the brain, ( C ) the meninges, and ( D ) the choroid plexus at day 0, 10, and 14 post-infection. Samples from animals with vascular labelling (FITC + ) < 90% in blood were excluded from analysis. Mean values are plotted. 4.4 Phenotypic profiling reveals CNS-specific adaptation of ILC1s To characterize the tissue-specific adaptation of type 1 innate lymphocytes during T. gondii infection, we performed high-dimensional phenotypic profiling using UMAP clustering of concatenated group 1 ILCs (CD45 + Lin − T-bet + NK1.1 + NKp46 + ) from brain, choroid plexus, meninges, liver, blood, small intestine, and spleen of mock-infected and infected animals (Fig. 5 A). Clustering analysis using FlowSOM identified three phenotypically distinct populations (Fig. 5 B, C), similar to salivary gland ( 8 ). Population 1 (Eomes⁺CD49b⁺CXCR3 low CD49a⁻CD69⁻) corresponds to conventional NK cells and converges into Population 2 (Eomes⁺CD49b⁺CXCR3 low CD49a⁺CD69⁺) with a trNK phenotype. A third, more distinct, population (Population 3) exhibited an Eomes⁻CD49b⁻CXCR3 low CD49a⁺CD69⁺ profile consistent with ILC1s (Fig. 5 B, C). All three populations expressed similar levels of canonical type 1 innate markers NK1.1, NKp46, and T-bet (Fig. 5 D). Tissue projection showed that CNS compartments were predominantly enriched in Populations 2 and 3 (Fig. 5 E), with brain-derived cells predominantly overlapping with those from the choroid plexus and meninges, but limited overlap with liver, blood, small intestine, or spleen, suggesting CNS-specific adaptation of type 1 ILCs. To further explore this compartmentalization, we performed hierarchical clustering of Population 3 (∼ILC1s) centroids across tissues in UMAP space (Fig. 5 F). The resulting dendrogram resolved three main organ clusters (Fig. 5 G), highlighting shared phenotypic signatures among CNS-associated ILC1s (brain parenchyma, choroid plexus, and meninges) and distinguishing them from peripheral counterparts (small intestine, spleen, blood, and liver). While surface marker profiles were generally conserved across tissues, CNS ILC1s displayed elevated expression of residency and activation markers CD49a and CD69 (Fig. 5 H), suggesting compartment-specific phenotypic imprinting driven by microenvironmental cues during infection. (A) UMAP projection and surface marker expression of concatenated group 1 ILCs (NK1.1⁺NKp46⁺) derived from brain, choroid plexus, meninges, liver, blood, small intestine, and spleen of mock-infected and T. gondii -infected animals. (B) UMAP projection, (C) expression heatmap (relative MFI), and (D) expression violin plots of metaclusters generated FlowSOM. (E) Tissue distribution of group 1 ILCs in UMAP projection. (F) UMAP projection and (G) hierarchical clustering of Population 3 (∼ILC1) based on mean UMAP coordinates. (H) Expression violin plots heatmap of indicated surface markers in brain parenchyma (Br), choroid plexus (CP), meninges (M), liver (Li), blood (Bl), small intestine (SI), and spleen (Sp). Discussion Host defense against T. gondii infection follows a conserved spatiotemporal sequence of innate and adaptive immune responses. In the CNS, long-term control of T. gondii relies on adaptive immunity, particularly CD4 + and CD8 + T cell-mediated responses ( 11 , 13 , 42 ), while resident innate immune cells orchestrate early pathogen control and subsequent T cell-mediated immunity ( 14 , 16 , 43 , 44 ). Among these, NK cells and ILC1 act as crucial early responders. ILC1s serve as a non-redundant early source IFN-γ and TNF, limiting parasite expansion and shaping successive immune response ( 20 ). However, whether innate lymphoid cells can transiently compensate for impaired adaptive responses and sustain sufficient parasite control in the CNS remains elusive. Acute T. gondii infection elicits a compartmentalized immune response within the CNS, with distinct cellular and molecular signatures emerging in the parenchyma, meninges, and choroid plexus ( 10 , 15 , 20 ). This reflects the anatomical and functional specialization of these microenvironments, facilitating a coordinated parasite control that minimizes immunopathology. The choroid plexus serves as an early sentinel, rapidly detecting peripheral and local inflammatory cues and shaping the initial CNS response ( 15 ). In contrast, the meninges function as a critical priming site for adaptive immunity ( 45 ), orchestrating T cell activation and entry to ensure parasite control in the parenchyma, a crucial site of parasite persistence through cyst latency. However, these site-specific differences imply an unequal reliance on resident and recruited immune cells, raising the question of their respective contributions to host protection and pathology. The marked expansion of ILC1s and trNK cells in the model of infection-induced neuroinflammation exemplifies a programmed flexibility in the innate compartment, dynamically adapting to disrupted adaptive reinforcements. This likely reflects a conserved strategy to ensure continuity of host defense during periods of immunological vulnerability. Convergent evidence across different infections demonstrates that group 1 ILCs provide early protection prior to full adaptive immunity ( 19 , 46 – 49 ). The Innate and adaptive systems provide an integrated response that is dynamically cross-regulated through the competition of shared cytokines, metabolites, and stromal cues ( 7 , 50 , 51 ) allowing ILC1s and trNK cells to bridge any gaps to adaptive programs to maintain tissue function and integrity protect against immune-mediated pathology ( 52 ). Tissue-specific cues further imprint ILC1s with CNS-adapted profiles ( 53 ), expressing high levels of functional tissue-retention markers (CD49a and CD69), presumably driven by cytokines such as TGF-β and IL-15, as previously described for the periphery ( 54 – 56 ). Despite the pharmacological blockade of T cell recruitment via FTY720, Ifng and Tnf transcript levels remained elevated and parasite burden was stable, suggesting group 1 ILCs as compensatory effectors in the early phase of infection. Sustained microglial activation (evidenced by MHC-I/II upregulation), further suggests that IFN-γ produced by group 1 ILCs contributes to functional activation of microglial and preserved inflammatory tone in the context of impaired adaptive response. Temporal mapping revealed that expansion of group 1 ILCs coincided with preserved proinflammatory tone and parasite control, consistent with our previous study showing that depletion or ablation of ILC1s impairs the CNS resistance to T. gondii ( 20 ). However, since FTY720 mechanistically differs from fatal systemic T cell depletion ( 11 ), it only moderately effects peripheral T cell responses while restricting T cell entry to the CNS. Consequently, durability of an isolated ILC response in the CNS remains unresolved. In summary, our results highlight the temporal and compartmental specialization of immune responses during infection-induced acute neuroinflammation, with innate immune cells orchestrating the early defense and adaptive immune cells ensuring long-term parasite control. Group 1 ILCs contribute to restraining acute parasite replication and facilitating transition toward long-term parasite control by tissue-resident memory CD8⁺ T cells ( 57 ), collectively illustrating how the CNS immune architecture leverages the dynamic and synergistic interplay between innate and adaptive immunity to ensure timely containment and long-term neuroprotection. 5.1 Limitations Analysis of fixed timepoints (10 and 14 days post-infection) inherently limits temporal resolution, potentially masking transient immune dynamics. Initiation of FTY720 treatment at 7 dpi minimizes peripheral effects but might not entirely cover the relevant time frame of T cell priming or CNS infiltration. FTY720 targets four of five S1P receptors (S1PRs) ( 21 ), mainly inhibiting lymphatic egress via S1PR1 but exhibiting off-target effects via S1PRs. Beyond trafficking, FTY720 modulates T cell functions via TCF-1 upregulation, suppressing IFN-γ and granzyme B expression and promoting an exhausted-like phenotype ( 58 ). Although considered minimal, we cannot exclude contribution of FTY720 off-target effects beyond canonical S1P modulation. Quantifications whole-brain homogenates does not allow identification of cell type-specific contributions. While our prior work implicates ILC1s in cytokine production ( 20 ), single-cell or spatial transcriptomics could improve resolution. Sustained microglial activation suggests a contribution of ILC1-derived IFN-γ, but remains to be verified. Although peripheral expansion suggests that migration might supplement local proliferation, the origin of CNS group 1 ILCs remains unresolved and requires further fate-mapping and cell tracking approaches. Despite limitations in modeling human immune diversity and kinetics, our results highlight capacity of innate lymphocytes to preserve early CNS defense during impaired adaptive response and outline critical directions for mechanistic investigation. Declarations Ethical Approval All experiments were approved by the local authorities in accordance to German and European legislation. Consent for publication Not applicable. Data availability All data generated are included in this article and its supplementary information files, and are available from the corresponding author on reasonable request. Conflicts of interest and disclosures JS owns equity in Innate Pharma and GSK, companies which may be involved in areas relevant to this work. Funding This work was supported by grants from the German Research Foundation to JS (start-up funding by DFG SPP1937), AD (DFG SPP1937), CSNK (KL2963/2-1, KL2963/3-1), TS (SCHU2326/2-2), and IRD (DFG SPP1937, DU1112/5–1). Authors contributions Johannes Steffen: Methodology, Validation, Formal analysis, Investigation, Data Curation, Writing - Original Draft, Writing - Review & Editing, Visualization, Funding acquisition, Nina Sophie Bellersheim: Validation, Formal analysis, Investigation, Data Curation, Caio Andreeta Figueiredo: Methodology, Investigation, Writing - Review & Editing , Laura Knop: Investigation, Vladyslava Dovhan : Investigation, Andreas Diefenbach : Resources, Christoph S. N. Klose: Writing - Review & Editing, Thomas Schüler: Conceptualization, Resources, Writing - Review & Editing, Ildiko R. 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17:11:18","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":139625,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8278466/v1/7287bfd91a5d55c0bf5dcbe0.png"},{"id":98606655,"identity":"e9d74be2-d81e-4328-9c41-0dd536b9b2ed","added_by":"auto","created_at":"2025-12-19 13:45:13","extension":"xml","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":165451,"visible":true,"origin":"","legend":"","description":"","filename":"8daa91c7539c4d3fb28834c8d55c3ad81structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8278466/v1/3c154bd2f0802ee608d7751e.xml"},{"id":98628908,"identity":"3e2ec897-69f2-4a44-a18f-d9f04e2577b8","added_by":"auto","created_at":"2025-12-19 17:12:47","extension":"html","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":178615,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8278466/v1/080a4d805c2106316d179a93.html"},{"id":98606634,"identity":"dcede846-edff-4506-ac21-deafda04275e","added_by":"auto","created_at":"2025-12-19 13:45:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":705228,"visible":true,"origin":"","legend":"\u003cp\u003eFTY720 expands group 1 innate lymphoid cells in the CNS during \u003cem\u003eT. gondii\u003c/em\u003e infection.\u003c/p\u003e\n\u003cp\u003e(A) C57Bl/6J mice were \u003cem\u003eT. gondii\u003c/em\u003e (ME49) or mock-infected (Ctrl) intraperitoneally, followed daily oral treatment with FTY720 (FTY) or diluent starting 7 days post-infection (dpi). Representative flow cytometry plots and quantification of cNK, trNK, and ILC1s in the (B, D) brain parenchyma, the (H) choroid plexus, and the (I) meninges. (E, F) Temporal changes in composition of group 1 ILCs during infection. (G) Relative mRNA levels of indicated genes in the brain parenchyma. Data are pooled from four independent experiments (n = 7–10 mice per group). Individual values and mean are plotted. The differences between groups (FTY720 vs. diluent) were analyzed by ordinary two-way ANOVA followed by Šídák’s multiple comparisons test (corresponding p-values are provided for p \u0026lt; 0.1 and considered significant for p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8278466/v1/1594084d5786b9c572e2c79f.png"},{"id":98628519,"identity":"2b5ed75a-136c-43d9-83fb-dd8386f59485","added_by":"auto","created_at":"2025-12-19 17:11:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":272006,"visible":true,"origin":"","legend":"\u003cp\u003eFTY720 does not alter the course of \u003cem\u003eT. gondii\u003c/em\u003e infection\u003c/p\u003e\n\u003cp\u003eWild-type C57Bl/6 mice with \u003cem\u003eT. gondii\u003c/em\u003e or mock-infected (Control) and orally treated with FTY720 or diluent (see also Fig. 1A). Absolute intake of (A) food and (B) water upon infection. (C) Body weight of animals (relative to weight at day of infection). (D) Disease score of animals. (E) Parasite burden in indicated tissues at 10 and 14 days post-infection (dpi). Absolute intake of food (A) and water (B) were recorded per cage and divided by number of animals for comparison. The differences between groups (FTY720 vs. diluent) were analyzed by (A-D) mixed model followed by Tukey’s multiple comparisons test (p-values are provided and considered significant for p \u0026lt; 0.05) or (E) ordinary two-way ANOVA followed by Šídák’s multiple comparisons test (p-values are provided for p \u0026lt; 0.10 and considered significant for p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8278466/v1/6b76e165f42932f050fd54b3.png"},{"id":98629196,"identity":"c0249c1b-ff25-4f9c-9ff2-2097d9b063d0","added_by":"auto","created_at":"2025-12-19 17:13:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":274028,"visible":true,"origin":"","legend":"\u003cp\u003eTransient hepatic and sustained intestinal expansion of group 1 ILCs\u003c/p\u003e\n\u003cp\u003e(A) Representative flow cytometry plots from the liver and quantification of (B, E) cNK, trNK, and ILC1s, and CD45\u003csup\u003e+\u003c/sup\u003e cells (C, F) in the liver and the small intestine. (D, G)\u0026nbsp;Temporal changes in composition of group\u0026nbsp;1 ILCs during infection. Data are pooled from four independent experiments (n\u0026nbsp;=\u0026nbsp;7–10 mice per group). (B, C, E, F) Individual values and mean, or (D, G) mean values are plotted. The differences between groups (FTY720 vs. diluent) were analyzed by ordinary two-way ANOVA followed by Šídák’s multiple comparisons test (corresponding p-values are provided for p\u0026nbsp;\u0026lt;\u0026nbsp;0.1 and considered significant for p\u0026nbsp;\u0026lt;\u0026nbsp;0.05).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8278466/v1/5843824b12f6cfee05f30486.png"},{"id":98606640,"identity":"b15a0980-491f-4f7d-8624-2e0abda97709","added_by":"auto","created_at":"2025-12-19 13:45:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":277149,"visible":true,"origin":"","legend":"\u003cp\u003eCompartment-specific vascular accessibility.\u003c/p\u003e\n\u003cp\u003eFITC-labelled anti-CD45 antibody was intravenously injected prior to transcardial perfusion to label leukocytes accessible from the vasculature. (A)\u0026nbsp;Representative flow cytometry plot and quantification of FITC\u003csup\u003e+\u003c/sup\u003e fraction shows efficient labelling in blood. Vascular labeling of total CD45⁺ cells, cNK, trNK, and ILC1 populations in (B) the brain, (C) the meninges, and (D) the choroid plexus at day 0, 10, and 14 post-infection. Samples from animals with vascular labelling (FITC\u003csup\u003e+\u003c/sup\u003e) \u0026lt;90% in blood were excluded from analysis. Mean values are plotted.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8278466/v1/166537da1a498bab30d69c1a.png"},{"id":98628349,"identity":"10423ad5-de0b-463d-9c60-974dcd288486","added_by":"auto","created_at":"2025-12-19 17:11:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3584050,"visible":true,"origin":"","legend":"\u003cp\u003eHigh-dimensional phenotypic profiling reveals compartment-specific adaptation of group 1 innate lymphoid cells during \u003cem\u003eT. gondii\u003c/em\u003einfection.\u003c/p\u003e\n\u003cp\u003e(A) UMAP projection and surface marker expression of concatenated group 1 ILCs (NK1.1⁺NKp46⁺) derived from brain, choroid plexus, meninges, liver, blood, small intestine, and spleen of mock-infected and \u003cem\u003eT. gondii\u003c/em\u003e-infected animals. (B) UMAP projection, (C) expression heatmap (relative MFI), and (D) expression violin plots of metaclusters generated FlowSOM. (E) Tissue distribution of group 1 ILCs in UMAP projection. (F) UMAP projection and (G) hierarchical clustering of Population 3 (∼ILC1) based on mean UMAP coordinates. (H) Expression violin plots heatmap of indicated surface markers in brain parenchyma (Br), choroid plexus (CP), meninges (M), liver (Li), blood (Bl), small intestine (SI), and spleen (Sp).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8278466/v1/ce8ab11439c213586a5fde2e.png"},{"id":98632156,"identity":"d953ca9e-e559-484d-b53e-e99e4f9eaed3","added_by":"auto","created_at":"2025-12-19 17:21:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5771440,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8278466/v1/28353ae0-8a6f-4df1-a73d-cb0578d6538e.pdf"},{"id":98629397,"identity":"b7016f90-a809-41ae-aa20-593e032a936a","added_by":"auto","created_at":"2025-12-19 17:13:50","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":801568,"visible":true,"origin":"","legend":"","description":"","filename":"SupplFig10.15.png","url":"https://assets-eu.researchsquare.com/files/rs-8278466/v1/006d7cf64c7f09ae0d0eba9d.png"},{"id":98628335,"identity":"666b02dc-7598-457e-bb25-5d59c3d7b8ba","added_by":"auto","created_at":"2025-12-19 17:11:17","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":458317,"visible":true,"origin":"","legend":"","description":"","filename":"Supplement.docx","url":"https://assets-eu.researchsquare.com/files/rs-8278466/v1/c75925fa5f09361c42786258.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Expanding group 1 ILCs sustain early CNS defense against Toxoplasma gondii during impaired adaptive response","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOnce considered strictly immune privileged, the central nervous system (CNS) is now recognized to rely on compartmentalized neuroimmune communication networks that actively shape its development, function, and maintenance (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). CNS barrier interfaces, such as the choroid plexus and meninges, not only function as physical barriers but as neuroimmune signaling hubs that relay contextual somatic and environmental cues to the CNS (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Innate effectors positioned at these interfaces rapidly detect infection, injury, or neurodegeneration, initiating a local response that propagates and amplifies inflammatory signals, modulates barrier permeability, and facilitates infiltration of peripheral immune cells (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Among them, an early cytokine and chemokine-mediated response shapes the recruitment, activation, and effector functions of adaptive immune cells that govern the trajectory, severity, and persistence of chronic neuroinflammation (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). While this tightly regulated spatiotemporal cascade ensures a response precisely tuned to the CNS microenvironment, functional bridging between innate and adaptive responses remains poorly defined. The rapid innate response offers a temporal advantage to ensure protection until the adaptive response is fully established. In situ proliferation of resident ILCs (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), together with local signals that promote tissue residency and effector differentiation, such as IL-2 (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), IL-15, and TGF-β (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), may support cytotoxic responses in the CNS (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e) to temporally bridge impairments in adaptive immunity. Deciphering the spatiotemporal dynamics and functional capacities of innate immune cells is therefore crucial for resolving gaps in the understanding of neuroinflammation.\u003c/p\u003e \u003cp\u003eThe protozoan parasite \u003cem\u003eToxoplasma gondii\u003c/em\u003e (\u003cem\u003eT. gondii\u003c/em\u003e) offers a unique model to dissect the spatiotemporal dynamics of CNS immunity. After crossing the CNS barriers, \u003cem\u003eT. gondii\u003c/em\u003e infects parenchymal cells, triggering a robust and defined sequence of innate and adaptive immune responses (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Effective CNS immunity requires a balanced response, controlling the infection without causing irreversible damage to a tissue with limited regenerative capacity. While long-term control of \u003cem\u003eT. gondii\u003c/em\u003e relies on adaptive immunity, particularly T cell-mediated responses (\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e), the early containment of the parasite primarily depends on a swift response from brain-resident innate effectors (\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Among these, group 1 innate lymphoid cells (ILCs), comprising conventional natural killer (cNK) cells, tissue-resident NK (trNK) cells, and ILC1s, are critical contributors to early immune response. While cNK are patrolling cells, it was demonstrated that they can leave the circulation and establish tissue-residency following infection. This was shown by the acquisition of ILC1 markers, such as CD49a and CD69 (trNK cells) (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). ILC1s are permanently integrated in the fabric of the tissue and rapidly produce IFN-γ and TNF, limiting parasite expansion and shaping downstream immunity (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Whether these cells can transiently compensate for the reduced presence of adaptive lymphocytes in the CNS and sustain sufficient response during the acute infection phase remains unclear.\u003c/p\u003e \u003cp\u003eTo dissect the capacity of innate immune cells to bridge delayed or reduced adaptive responses, we pharmacologically restricted lymphocyte recirculation and CNS infiltration using FTY720, a sphingosine-1-phosphate receptor modulator (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), during acute \u003cem\u003eT. gondii\u003c/em\u003e infection. This approach allowed us to focus on the local immune compartment when assessing ILC expansion, spatial distribution, and functional contributions to early host defense. In the absence of effective T cell infiltration, ILCs robustly expanded within the CNS, maintaining a proinflammatory, antiparasitic microenvironment sufficient to suppress parasite replication. High-dimensional profiling revealed that CNS-associated ILC1s acquire a distinct, tissue-adapted phenotype, distinguishing them from their peripheral counterparts. These findings highlight the immune system\u0026rsquo;s functional plasticity, demonstrating that the innate immune compartment rapidly responds and temporarily compensates for numerical impairments in the adaptive immune compartment to sustain effective early CNS defense.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Mice\u003c/h2\u003e \u003cp\u003eExperiments were performed using female, wild-type C57BL/6J mice (8\u0026ndash;14 weeks old) bred and group-housed under specific pathogen free (SPF) conditions with a 12-h light/dark cycle at 22\u0026deg;C and ad libitum access to food and water. All animal procedures were approved by the local authorities in accordance to German and European legislation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Infection\u003c/h2\u003e \u003cp\u003e \u003cem\u003eT. gondii\u003c/em\u003e tissue cysts (type II ME49 strain) were obtained from the brains of female NMRI mice previously infected with \u003cem\u003eT. gondii\u003c/em\u003e 6\u0026ndash;12 months earlier. Brain tissue was mechanically homogenized in 1 mL sterile phosphate-buffered saline (PBS), and cysts number was quantified by light microscopy in \u0026ge;\u0026thinsp;110 \u0026micro;L homogenate. The homogenate was subsequently diluted in sterile PBS, and animals were infected by intraperitoneal administration of two cysts (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Uninfected control mice received an equivalent volume of sterile PBS as a mock control. To determine the disease score, the animals were scored daily based on different categories (see Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Tissue collection\u003c/h2\u003e \u003cp\u003eMice received a single intravenous bolus of 50 \u0026micro;g FITC-labeled anti-CD45 antibody 10 min before anesthesia. Following euthanasia, peripheral blood samples were collected, and the animals were transcardially perfused with 60 mL ice-cold sterile PBS. The brain (including pia mater), choroid plexus, meninges (dura meninges and partially arachnoid meninges), spleen, liver, and small intestine were removed. For gene expression analysis, samples were incubated in RNAlater at 4\u003cb\u003e\u0026deg;\u003c/b\u003eC overnight, and then stored at -20\u0026deg;C until further processing. For cell isolation, brain, spleen, and liver were stored in sterile ice-cold PBS; small intestine was stored in ice-cold PBS\u0026thinsp;+\u0026thinsp;PS (PBS, 100 U/mL Penicillin, 100 \u0026micro;g/mL Streptomycin); dural meninges and choroid plexus were stored in 150 \u0026micro;L RPMI\u0026thinsp;+\u0026thinsp;PS (RPMI 1640, 100 U/mL Penicillin, 100 \u0026micro;g/mL Streptomycin).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Cell isolation\u003c/h2\u003e \u003cp\u003eCells were isolated as previously described for brain, liver, and spleen (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), small intestine (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), and meninges (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e) with slight modifications. Brains were minced, homogenized in dissection buffer (HBSS, 50 mM glucose, 13 mM HEPES, pH 7.3), filtered through a 70 \u0026micro;m cell strainer, and centrifuged (400 \u003cem\u003eg\u003c/em\u003e, 20 min, 4\u0026deg;C). The mononuclear cells were separated via discontinuous, isotonic Percoll gradient (70% / 30%) centrifugation (800 \u003cem\u003eg\u003c/em\u003e, 35 min, 4\u0026deg;C, minimal acceleration / deceleration).\u003c/p\u003e \u003cp\u003eChoroid plexus and meninges were digested with 1 mg/mL DNAse I and 1.5 mg/mL Collagenase D (600 rpm, 25 min, 37\u0026deg;C) in 200 \u0026micro;L of RPMI\u0026thinsp;+\u0026thinsp;PS. Following digestion, 800 \u0026micro;L RPMI\u0026thinsp;+\u0026thinsp;PS were added, mechanical dissociation was performed by 20 repeated passages through a pipette tip. Cell suspension was filtered through 40 \u0026micro;m strainer, and centrifuged (400 \u003cem\u003eg\u003c/em\u003e, 10 min, 4\u0026deg;C).\u003c/p\u003e \u003cp\u003eFor small intestine preparation, tissue was cleared of fat and feces, Peyer\u0026rsquo;s patches were removed, the tissue was opened longitudinally, and washed three times with 20 mL ice-cold PBS\u0026thinsp;+\u0026thinsp;PS by vortexing for 15 sec and filtering through a metal strainer to remove mucus. Tissue was transferred into 20 mL dissociation solution (HBSS without Ca2\u003csup\u003e+\u003c/sup\u003e/Mg2\u003csup\u003e+\u003c/sup\u003e, 5 mM EDTA, 10 mM HEPES, 100 U/mL Penicillin, 100 \u0026micro;g/mL Streptomycin, 1 mM DTT), incubated (100 rpm, 20 min, RT), vortexed for 15 sec, and filtered through metal strainer. The filtrate containing epithelial cells and intraepithelial leukocytes was discarded. Remaining tissue was incubated with dissociation solution, vortexed for 15 sec, and filtered through metal strainer. Tissue was washed twice with 30 mL PBS\u0026thinsp;+\u0026thinsp;PS (first wash: 100 rpm, 20 min, RT and vortexing for 15 sec; second wash: vortexing for 15 sec), and filtered through metal strainer. Tissue was minced with scissors, transferred into 10 mL digestion solution (RPMI 1640, 4% FCS, 100 U/mL Penicillin, 100 \u0026micro;g/mL Streptomycin, 0.25 mg/mL Collagenase D (Roche), 0.2 mg/mL Dispase II (Roche), 0.25 mg/mL DNAse I (Sigma-Aldrich)), incubated (100 rpm, 37\u0026deg;C for 15 min), vortexed for 15 sec and filtered through 100 \u0026micro;m strainer. Lamina propria leukocyte (LPL)-containing filtrate was stored on ice, while remaining tissue was digested and filtered once more through 100 \u0026micro;m strainer to pool cells. LPL suspensions were filtered through 40 \u0026micro;m strainer and centrifuged (800 \u003cem\u003eg\u003c/em\u003e, 15 min, RT), resuspended in 33% isotonic Percoll and separated via discontinuous, isotonic Percoll gradient (80%/44%/33%) centrifugation (600 \u003cem\u003eg\u003c/em\u003e, 20 min, 20\u0026deg;C, minimal acceleration / deceleration). The enriched leukocytes on the 44%/80% interphase were harvested and washed with PBS\u0026thinsp;+\u0026thinsp;2mM EDTA (600 \u003cem\u003eg\u003c/em\u003e, 10 min, 4\u0026deg;C).\u003c/p\u003e \u003cp\u003eLivers were mechanically dissociated by passage through a 70 \u0026micro;m cell strainer, washed with PBS, centrifuged (400 \u003cem\u003eg\u003c/em\u003e, 20 min, 4\u0026deg;C), and mononuclear cells further enriched by discontinuous, isotonic Percoll gradient (70% / 35%) centrifugation (800 \u003cem\u003eg\u003c/em\u003e, 35 min, 4\u0026deg;C, minimal acceleration / deceleration).\u003c/p\u003e \u003cp\u003eSpleens were mechanically homogenized by passage through a 40 \u0026micro;m cell strainer. Erythrocytes in the blood and spleen samples were lysed using RBC lysis buffer (10 min), subsequently washed with PBS, centrifuged (400 \u003cem\u003eg\u003c/em\u003e, 10 min, 4\u0026deg;C).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Flow cytometry\u003c/h2\u003e \u003cp\u003eSingle-cell suspensions were resuspended in FACS buffer (PBS, 2% (v/v) FCS, 2 mM EDTA) and incubated with 1 \u0026micro;g anti-mouse CD16/32 antibody and a viability dye (20 min, 4\u0026deg;C). Surface staining was performed using fluorochrome-conjugated antibodies against CD3ε, CD8a, CD5, CD19, Ly6G, CD45, NK1.1, NKp46, CD49a, CD49b, CD69, and CXCR3 (30 min, 4\u0026deg;C). Cells were washed (FACS buffer) and centrifuged (400 \u003cem\u003eg\u003c/em\u003e, 10 min 4\u0026deg;C) twice. For intracellular staining, cells were fixed and permeabilized with the Foxp3 / Transcription Factor Staining Buffer Set (60 min, 4\u0026deg;C), washed / centrifuged twice, and stained using fluorochrome-conjugated antibodies against Eomes and T-bet (40 min, 4\u0026deg;C). Cells were washed / centrifuged twice, resuspended in FACS buffer, acquired using an Attune\u0026trade; NxT flow cytometer (Thermo Fisher Scientific) with FMO controls assessing background fluorescence. Computational analyses, including FlowSOM clustering (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), Uniform Manifold Approximation and Projection (UMAP) for dimensionality reduction (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), and data visualization via violin plots (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), were performed using FlowJo software (v10.8.2; BD Biosciences) and R 4.4.3 (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.6 RNA and DNA isolation, qPCR and RT-qPCR\u003c/h2\u003e \u003cp\u003eTissue samples were removed from RNAlater and homogenized (4,350 rpm, 3x30 s) with RNA-Solv\u0026reg;Reagent (Omega Bio-Tek, R6830) and Zirconium oxide beads (Precellys, P000926-LYSK0-A) in a BeadBug 6 homogenizer (Biozym). Chloroform (20% v/v) was added to the lysate to facilitate phase separation. DNA was extracted from the interphase using DNA-binding columns (VWR, 13-HCR-02) according to the manufacturer\u0026rsquo;s instructions, while RNA was recovered from the aqueous phase via isopropanol precipitation. Nucleic acid concentrations and purity were determined using a NanoDrop 2000 spectrophotometer (ThermoFisher), and samples stored at -80\u0026deg;C until analysis.\u003c/p\u003e \u003cp\u003eQuantitative gene expression analysis was performed using 30 ng of isolated RNA, TaqMan\u0026trade; RNA-to-CT\u0026trade; 1-Step Kit, TaqMan\u0026trade; probes for transforming growth factor beta 1 (\u003cem\u003eTgfb1\u003c/em\u003e), interleukin 15 (\u003cem\u003eIl15\u003c/em\u003e), interferon gamma (Ifng\u003cem\u003e)\u003c/em\u003e, tumor necrosis factor (\u003cem\u003eTnf\u003c/em\u003e), interferon regulatory factor 8 (\u003cem\u003eIrf8\u003c/em\u003e), interleukin 7 (\u003cem\u003eIl7\u003c/em\u003e), interleukin 12a (\u003cem\u003eIl12a\u003c/em\u003e), and interleukin-18 (\u003cem\u003eIl18\u003c/em\u003e), and LightCycler\u0026reg; 96 System (Roche Diagnostics) (Table S2) (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Mean Cq values were obtained (LightCycler\u0026reg; 96, v1.1.0.1320), and relative expression to hypoxanthine guanine phosphoribosyl transferase (\u003cem\u003eHprt\u003c/em\u003e) was calculated using 2\u003csup\u003e\u0026minus;ΔCq\u003c/sup\u003e where ΔCq = (Cq (Target gene)\u0026thinsp;\u0026minus;\u0026thinsp;Cq (\u003cem\u003eHprt\u003c/em\u003e)) (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e) in Excel\u0026reg; 2019 (v16.0.10413.20020, Microsoft\u0026reg;).\u003c/p\u003e \u003cp\u003eParasite burden was assessed in using 100 ng of isolated DNA, FastStart Essential DNA Green Master (Roche), and LightCycler\u0026reg; 96 System (Roche Diagnostics), as previously described (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Amplification of the conserved \u003cem\u003eT. gondii B1\u003c/em\u003e gene was used to assess parasite load, with murine argininosuccinate lyase (\u003cem\u003eAsl)\u003c/em\u003e serving as the reference gene (Table S3). Parasite burden was calculated using 2\u003csup\u003e\u0026minus;ΔCq\u003c/sup\u003e where ΔCq = (Cq (\u003cem\u003eB1\u003c/em\u003e)\u0026thinsp;\u0026minus;\u0026thinsp;Cq (\u003cem\u003eAsl\u003c/em\u003e)) as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe statistical tests and sample sizes are provided in the figure legends. Data normality (D\u0026rsquo;Agostino-Pearson for n\u0026thinsp;\u0026ge;\u0026thinsp;8, Shapiro-Wilk normality test for n\u0026thinsp;\u0026lt;\u0026thinsp;8) and equality of variances (F test) were assessed in advance for selection of the statistical test. Sample sizes were based on literature and prior experience to ensure statistical power. Investigators were blinded during experiments and analysis when possible. Statistical calculations were performed in Prism (v10.4.1, GraphPad) and considered significant if p\u0026thinsp;\u0026le;\u0026thinsp;0.05. Unless otherwise stated, data are presented as arithmetic mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.1 FTY720 leads to expanded group 1 ILCs cells in the CNS during infection\u003c/h2\u003e \u003cp\u003eTo assess the contribution of adaptive lymphocyte trafficking during infection, we treated a subset of infected animals with FTY720 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), a sphingosine-1-phosphate receptor (S1PR) modulator that impedes lymphocyte egress from lymphoid tissues (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). FTY720 treatment started at day 7 post-infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), to avoid interfering with immune cell redistribution necessary for anti-parasitic response in peripheral tissues, while still effectively restricting lymphocyte recruitment to the CNS.\u003c/p\u003e \u003cp\u003eWe analyzed the distribution and dynamics of group 1 innate lymphoid cells and divided them into conventional NK (cNK) cells, tissue-resident NK (trNK) cells, and ILC1s, as previously described (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), across the brain, the choroid plexus, and the meninges (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-I). FTY720 treatment significantly increased both frequency and total number of brain-resident group 1 innate lymphocytes, suggesting that S1P-dependent changes are associated with enhanced expansion of innate lymphoid populations.\u003c/p\u003e \u003cp\u003eWhile Eomes⁺CD49a⁻ cNK cell populations remained stable, both Eomes⁺CD49a⁺ trNK cells and Eomes⁻CD49a⁺ ILC1s were significantly elevated in infected animals, with further expansion in FTY720-treated cohorts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D, E, F). These findings align with a recent data implicating TGF-β1 and IL-15 in the induction of NK cell residency and cytotoxic programming (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), pathways both upregulated in the inflamed brain during \u003cem\u003eT. gondii\u003c/em\u003e infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Expression of key cytokines and host-defense factors \u003cem\u003eIfng\u003c/em\u003e, \u003cem\u003eTnf\u003c/em\u003e, \u003cem\u003eIrf8\u003c/em\u003e, and \u003cem\u003eIl7\u003c/em\u003e remained unaffected upon FTY720 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eCNS barrier compartments, such as the meninges and the choroid plexus, are crucial immunological hubs that undergo significant changes during inflammation (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e), functioning as proliferative and immunoregulatory niches and crucial entry points for peripheral immune cells, as reflected by increased frequencies of CD45⁺ cell (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH, I). In the choroid plexus, trNK cells and ILC1s markedly expanded upon infection, independent of FTY720 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Conversely, the meninges rather exhibited an overall decline group 1 ILC subsets during acute infection, with FTY720 selectively reducing cNK and trNK cells at baseline and early timepoints, whereas percentage and number of ILC1s were preserved (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003eThese compartment-specific dynamics indicate that tissue-resident group 1 ILCs, particularly ILC1s, are poised to rapidly expand within the CNS and border regions independent of S1P-mediated trafficking, reinforcing their contribution to early neuroinflammatory response.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(\u003cb\u003eA\u003c/b\u003e) C57Bl/6J mice were \u003cem\u003eT. gondii\u003c/em\u003e (ME49) or mock-infected (Ctrl) intraperitoneally, followed daily oral treatment with FTY720 (FTY) or diluent starting 7 days post-infection (dpi). Representative flow cytometry plots and quantification of cNK, trNK, and ILC1s in the (\u003cb\u003eB\u003c/b\u003e, \u003cb\u003eD\u003c/b\u003e) brain parenchyma, the (\u003cb\u003eH\u003c/b\u003e) choroid plexus, and the (\u003cb\u003eI\u003c/b\u003e) meninges. (\u003cb\u003eE\u003c/b\u003e, \u003cb\u003eF\u003c/b\u003e) Temporal changes in composition of group 1 ILCs during infection. (\u003cb\u003eG\u003c/b\u003e) Relative mRNA levels of indicated genes in the brain parenchyma. Data are pooled from four independent experiments (n\u0026thinsp;=\u0026thinsp;7\u0026ndash;10 mice per group). Individual values and mean are plotted. The differences between groups (FTY720 vs. diluent) were analyzed by ordinary two-way ANOVA followed by Š\u0026iacute;d\u0026aacute;k\u0026rsquo;s multiple comparisons test (corresponding p-values are provided for p\u0026thinsp;\u0026lt;\u0026thinsp;0.1 and considered significant for p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eFollowing the onset of neuroinflammation, activated microglia not only exert direct effector functions but also play a key role in sustaining inflammation and promoting the recruitment of peripheral immune cells (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). To assess whether FTY720-dependent redistribution impairs these myeloid contributions, we analyzed brain-resident populations as well as recruited immune cells.\u003c/p\u003e \u003cp\u003e \u003cem\u003eT. gondii\u003c/em\u003e infection induced a robust expansion of CD45⁺CD11b⁺ myeloid cells in the brain by day 14, including CX\u003csub\u003e3\u003c/sub\u003eCR1\u003csup\u003e+\u003c/sup\u003e microglia, Ly6C⁺ monocytes, and Ly6G⁺ neutrophils (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eE, H). FTY720 treatment had no apparent effect on microglial expansion or activation. The upregulated MHC class I and II by day 10 (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eF) indicates intact IFN-γ-STAT1 signaling in microglia (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Although infection-induced downregulation of CX\u003csub\u003e3\u003c/sub\u003eCR1 was evident in both infected groups, the decrease was slightly delayed in FTY720-treated mice (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBeyond their direct effector functions, microglia shape neuroinflammatory milieu by production of CXCL9 and CXCL10, orchestrating the recruitment of innate lymphoid cells and peripheral immune cells via CXCR3-dependent mechanisms (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e).While \u003cem\u003eT. gondii\u003c/em\u003e infection significantly increased \u003cem\u003eCxcl9\u003c/em\u003e and \u003cem\u003eCxcl10\u003c/em\u003e transcription relative to mock-infected controls, FTY720 treatment did not affect expression levels of these chemokines (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003eWe next explored infiltrating myeloid subsets, important contributors in CNS parasite control (\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). While Ly6C\u003csup\u003ehi\u003c/sup\u003e monocytes were slightly reduced in FTY720-treated animals, no significant differences were observed in Ly6C\u003csup\u003eint\u003c/sup\u003e or Ly6C\u003csup\u003elow\u003c/sup\u003e monocytes or in neutrophils until day 14 post-infection (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eFinally, we analyzed brain-infiltrating leukocytes, revealing a marked reduction in both the frequency and absolute number of CD4⁺ and CD8\u003csup\u003e+\u003c/sup\u003e T cells in FTY720-treated animals at day 14 post-infection compared to infected diluent-treated animals (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB-D), confirming the suppression of S1P-dependent migration during infection. Despite this pharmacological blockade, the course of the infection and the parasite burden in the brain parenchyma and the periphery remained mostly unaltered (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-E).\u003c/p\u003e \u003cp\u003eCollectively, these data demonstrate that innate myeloid cell activation and infiltration are largely preserved despite impaired T cell recruitment, highlighting the critical role of non-adaptive immunity in mounting a protective neuroinflammatory response and controlling infection during the acute phases of cerebral toxoplasmosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWild-type C57Bl/6 mice with \u003cem\u003eT. gondii\u003c/em\u003e or mock-infected (Control) and orally treated with FTY720 or diluent (see also Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Absolute intake of (\u003cb\u003eA\u003c/b\u003e) food and (\u003cb\u003eB\u003c/b\u003e) water upon infection. (\u003cb\u003eC\u003c/b\u003e) Body weight of animals (relative to weight at day of infection). (\u003cb\u003eD\u003c/b\u003e) Disease score of animals. (\u003cb\u003eE\u003c/b\u003e) Parasite burden in indicated tissues at 10 and 14 days post-infection (dpi). Absolute intake of food (A) and water (B) were recorded per cage and divided by number of animals for comparison. The differences between groups (FTY720 vs. diluent) were analyzed by (A-D) mixed model followed by Tukey\u0026rsquo;s multiple comparisons test (p-values are provided and considered significant for p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) or (E) ordinary two-way ANOVA followed by Š\u0026iacute;d\u0026aacute;k\u0026rsquo;s multiple comparisons test (p-values are provided for p\u0026thinsp;\u0026lt;\u0026thinsp;0.10 and considered significant for p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Peripheral ILC1 Dynamics in Liver and Small Intestine Show Transient Expansion During Acute Infection\u003c/h2\u003e \u003cp\u003eTo determine whether CNS accumulation of group 1 innate lymphoid cells reflects systemic redistribution (\u003cspan additionalcitationids=\"CR39 CR40\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e) or a coordinated local response (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), we analyzed cNK, trNK, and ILC1 populations in the liver and small intestine. In the liver, their expansion by day 10 post-infection was followed by their contraction by day 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), indicating a transient peripheral response. In the small intestine, the expansion of group 1 ILCs persisted through day 14, indicating a more sustained immune activation in gut-associated tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eThese tissue-specific temporal dynamics show that the resolution of peripheral response coincides with CNS accumulation of ILC1s and trNK cells, raising the possibility of migratory seeding from peripheral reservoirs to the CNS and its border regions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(\u003cb\u003eA\u003c/b\u003e) Representative flow cytometry plots from the liver and quantification of (\u003cb\u003eB\u003c/b\u003e, \u003cb\u003eE\u003c/b\u003e) cNK, trNK, and ILC1s, and CD45\u003csup\u003e+\u003c/sup\u003e cells (\u003cb\u003eC\u003c/b\u003e, \u003cb\u003eF\u003c/b\u003e) in the liver and the small intestine. (\u003cb\u003eD\u003c/b\u003e, \u003cb\u003eG\u003c/b\u003e) Temporal changes in composition of group 1 ILCs during infection. Data are pooled from four independent experiments (n\u0026thinsp;=\u0026thinsp;7\u0026ndash;10 mice per group). (B, C, E, F) Individual values and mean, or (D, G) mean values are plotted. The differences between groups (FTY720 vs. diluent) were analyzed by ordinary two-way ANOVA followed by Š\u0026iacute;d\u0026aacute;k\u0026rsquo;s multiple comparisons test (corresponding p-values are provided for p\u0026thinsp;\u0026lt;\u0026thinsp;0.1 and considered significant for p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Type 1 Innate Lymphoid Cells Localize Predominantly to the Parenchyma in the Infected CNS\u003c/h2\u003e \u003cp\u003eTo resolve whether group 1 ILCs are infiltrating the parenchyma or remain confined in the vasculature, intravascular labeling was performed prior to transcardial perfusion tissue harvest using fluorescent anti-CD45 antibodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Efficiency of labeling and perfusion are reflected by quantification FITC\u003csup\u003e+\u003c/sup\u003e cells in blood and CNS compartments (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGroup 1 ILCs showed distinct compartmental localization patterns. In the brain, vascular labelling of cNK cells sharply declined during infection, indicating parenchymal infiltration. In contrast, trNK cells and ILC1s consistently lacked vascular labeling, confirming their extravascular localization (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In the meninges, vascular labeling of CD45⁺ cells and group 1 ILCs increased progressively with infection, likely reflecting elevated vascular permeability during neuroinflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). In the choroid plexus, vascular labeling of CD45⁺ cells remained low across conditions, and trNK cells and ILC1s displayed low vascular labeling, consistent with their stromal localization (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eThese data confirm the infection-induced parenchymal infiltration of circulating cNK cells and the predominant tissue residency of trNK cells and ILC1s. Their accumulation within parenchymal and stromal compartments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D, H), largely devoid of direct vascular exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, D), supports the hypothesis of active tissue infiltration and/or local retention/expansion during infection. The positioning group 1 ILCs in close proximity to infected cells facilitates rapid effector cytokine responses and engagement in early immune defenses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFITC-labelled anti-CD45 antibody was intravenously injected prior to transcardial perfusion to label leukocytes accessible from the vasculature. (\u003cb\u003eA\u003c/b\u003e) Representative flow cytometry plot and quantification of FITC\u003csup\u003e+\u003c/sup\u003e fraction shows efficient labelling in blood. Vascular labeling of total CD45⁺ cells, cNK, trNK, and ILC1 populations in (\u003cb\u003eB\u003c/b\u003e) the brain, (\u003cb\u003eC\u003c/b\u003e) the meninges, and (\u003cb\u003eD\u003c/b\u003e) the choroid plexus at day 0, 10, and 14 post-infection. Samples from animals with vascular labelling (FITC\u003csup\u003e+\u003c/sup\u003e)\u0026thinsp;\u0026lt;\u0026thinsp;90% in blood were excluded from analysis. Mean values are plotted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Phenotypic profiling reveals CNS-specific adaptation of ILC1s\u003c/h2\u003e \u003cp\u003eTo characterize the tissue-specific adaptation of type 1 innate lymphocytes during \u003cem\u003eT. gondii\u003c/em\u003e infection, we performed high-dimensional phenotypic profiling using UMAP clustering of concatenated group 1 ILCs (CD45\u003csup\u003e+\u003c/sup\u003eLin\u003csup\u003e\u0026minus;\u003c/sup\u003eT-bet\u003csup\u003e+\u003c/sup\u003eNK1.1\u003csup\u003e+\u003c/sup\u003eNKp46\u003csup\u003e+\u003c/sup\u003e) from brain, choroid plexus, meninges, liver, blood, small intestine, and spleen of mock-infected and infected animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Clustering analysis using FlowSOM identified three phenotypically distinct populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C), similar to salivary gland (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Population 1 (Eomes⁺CD49b⁺CXCR3\u003csup\u003elow\u003c/sup\u003eCD49a⁻CD69⁻) corresponds to conventional NK cells and converges into Population 2 (Eomes⁺CD49b⁺CXCR3\u003csup\u003elow\u003c/sup\u003eCD49a⁺CD69⁺) with a trNK phenotype. A third, more distinct, population (Population 3) exhibited an Eomes⁻CD49b⁻CXCR3\u003csup\u003elow\u003c/sup\u003eCD49a⁺CD69⁺ profile consistent with ILC1s (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C). All three populations expressed similar levels of canonical type 1 innate markers NK1.1, NKp46, and T-bet (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eTissue projection showed that CNS compartments were predominantly enriched in Populations 2 and 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), with brain-derived cells predominantly overlapping with those from the choroid plexus and meninges, but limited overlap with liver, blood, small intestine, or spleen, suggesting CNS-specific adaptation of type 1 ILCs.\u003c/p\u003e \u003cp\u003eTo further explore this compartmentalization, we performed hierarchical clustering of Population 3 (\u0026sim;ILC1s) centroids across tissues in UMAP space (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). The resulting dendrogram resolved three main organ clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eG), highlighting shared phenotypic signatures among CNS-associated ILC1s (brain parenchyma, choroid plexus, and meninges) and distinguishing them from peripheral counterparts (small intestine, spleen, blood, and liver). While surface marker profiles were generally conserved across tissues, CNS ILC1s displayed elevated expression of residency and activation markers CD49a and CD69 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eH), suggesting compartment-specific phenotypic imprinting driven by microenvironmental cues during infection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(A) UMAP projection and surface marker expression of concatenated group 1 ILCs (NK1.1⁺NKp46⁺) derived from brain, choroid plexus, meninges, liver, blood, small intestine, and spleen of mock-infected and \u003cem\u003eT. gondii\u003c/em\u003e-infected animals. (B) UMAP projection, (C) expression heatmap (relative MFI), and (D) expression violin plots of metaclusters generated FlowSOM. (E) Tissue distribution of group 1 ILCs in UMAP projection. (F) UMAP projection and (G) hierarchical clustering of Population 3 (\u0026sim;ILC1) based on mean UMAP coordinates. (H) Expression violin plots heatmap of indicated surface markers in brain parenchyma (Br), choroid plexus (CP), meninges (M), liver (Li), blood (Bl), small intestine (SI), and spleen (Sp).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHost defense against \u003cem\u003eT. gondii\u003c/em\u003e infection follows a conserved spatiotemporal sequence of innate and adaptive immune responses. In the CNS, long-term control of \u003cem\u003eT. gondii\u003c/em\u003e relies on adaptive immunity, particularly CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cell-mediated responses (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e), while resident innate immune cells orchestrate early pathogen control and subsequent T cell-mediated immunity (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Among these, NK cells and ILC1 act as crucial early responders. ILC1s serve as a non-redundant early source IFN-γ and TNF, limiting parasite expansion and shaping successive immune response (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). However, whether innate lymphoid cells can transiently compensate for impaired adaptive responses and sustain sufficient parasite control in the CNS remains elusive.\u003c/p\u003e \u003cp\u003eAcute \u003cem\u003eT. gondii\u003c/em\u003e infection elicits a compartmentalized immune response within the CNS, with distinct cellular and molecular signatures emerging in the parenchyma, meninges, and choroid plexus (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). This reflects the anatomical and functional specialization of these microenvironments, facilitating a coordinated parasite control that minimizes immunopathology. The choroid plexus serves as an early sentinel, rapidly detecting peripheral and local inflammatory cues and shaping the initial CNS response (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). In contrast, the meninges function as a critical priming site for adaptive immunity (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e), orchestrating T cell activation and entry to ensure parasite control in the parenchyma, a crucial site of parasite persistence through cyst latency. However, these site-specific differences imply an unequal reliance on resident and recruited immune cells, raising the question of their respective contributions to host protection and pathology.\u003c/p\u003e \u003cp\u003eThe marked expansion of ILC1s and trNK cells in the model of infection-induced neuroinflammation exemplifies a programmed flexibility in the innate compartment, dynamically adapting to disrupted adaptive reinforcements. This likely reflects a conserved strategy to ensure continuity of host defense during periods of immunological vulnerability. Convergent evidence across different infections demonstrates that group 1 ILCs provide early protection prior to full adaptive immunity (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan additionalcitationids=\"CR47 CR48\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). The Innate and adaptive systems provide an integrated response that is dynamically cross-regulated through the competition of shared cytokines, metabolites, and stromal cues (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e) allowing ILC1s and trNK cells to bridge any gaps to adaptive programs to maintain tissue function and integrity protect against immune-mediated pathology (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). Tissue-specific cues further imprint ILC1s with CNS-adapted profiles (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e), expressing high levels of functional tissue-retention markers (CD49a and CD69), presumably driven by cytokines such as TGF-β and IL-15, as previously described for the periphery (\u003cspan additionalcitationids=\"CR55\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite the pharmacological blockade of T cell recruitment via FTY720, \u003cem\u003eIfng\u003c/em\u003e and \u003cem\u003eTnf\u003c/em\u003e transcript levels remained elevated and parasite burden was stable, suggesting group 1 ILCs as compensatory effectors in the early phase of infection. Sustained microglial activation (evidenced by MHC-I/II upregulation), further suggests that IFN-γ produced by group 1 ILCs contributes to functional activation of microglial and preserved inflammatory tone in the context of impaired adaptive response.\u003c/p\u003e \u003cp\u003eTemporal mapping revealed that expansion of group 1 ILCs coincided with preserved proinflammatory tone and parasite control, consistent with our previous study showing that depletion or ablation of ILC1s impairs the CNS resistance to \u003cem\u003eT. gondii\u003c/em\u003e (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). However, since FTY720 mechanistically differs from fatal systemic T cell depletion (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), it only moderately effects peripheral T cell responses while restricting T cell entry to the CNS. Consequently, durability of an isolated ILC response in the CNS remains unresolved.\u003c/p\u003e \u003cp\u003eIn summary, our results highlight the temporal and compartmental specialization of immune responses during infection-induced acute neuroinflammation, with innate immune cells orchestrating the early defense and adaptive immune cells ensuring long-term parasite control. Group 1 ILCs contribute to restraining acute parasite replication and facilitating transition toward long-term parasite control by tissue-resident memory CD8⁺ T cells (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e), collectively illustrating how the CNS immune architecture leverages the dynamic and synergistic interplay between innate and adaptive immunity to ensure timely containment and long-term neuroprotection.\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e5.1 Limitations\u003c/h2\u003e \u003cp\u003eAnalysis of fixed timepoints (10 and 14 days post-infection) inherently limits temporal resolution, potentially masking transient immune dynamics. Initiation of FTY720 treatment at 7 dpi minimizes peripheral effects but might not entirely cover the relevant time frame of T cell priming or CNS infiltration. FTY720 targets four of five S1P receptors (S1PRs) (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), mainly inhibiting lymphatic egress via S1PR1 but exhibiting off-target effects via S1PRs. Beyond trafficking, FTY720 modulates T cell functions via TCF-1 upregulation, suppressing IFN-γ and granzyme B expression and promoting an exhausted-like phenotype (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). Although considered minimal, we cannot exclude contribution of FTY720 off-target effects beyond canonical S1P modulation.\u003c/p\u003e \u003cp\u003eQuantifications whole-brain homogenates does not allow identification of cell type-specific contributions. While our prior work implicates ILC1s in cytokine production (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), single-cell or spatial transcriptomics could improve resolution. Sustained microglial activation suggests a contribution of ILC1-derived IFN-γ, but remains to be verified. Although peripheral expansion suggests that migration might supplement local proliferation, the origin of CNS group 1 ILCs remains unresolved and requires further fate-mapping and cell tracking approaches.\u003c/p\u003e \u003cp\u003eDespite limitations in modeling human immune diversity and kinetics, our results highlight capacity of innate lymphocytes to preserve early CNS defense during impaired adaptive response and outline critical directions for mechanistic investigation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthical Approval\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were approved by the local authorities in accordance to German and European legislation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for publication \u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData availability \u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated are included in this article and its supplementary information files, and are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003ch2\u003eConflicts of interest and disclosures\u003c/h2\u003e\n\u003cp\u003eJS owns equity in Innate Pharma and GSK, companies which may be involved in areas relevant to this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the German Research Foundation to JS (start-up funding by DFG SPP1937), AD (DFG SPP1937), CSNK (KL2963/2-1, KL2963/3-1), TS (SCHU2326/2-2), and IRD (DFG SPP1937, DU1112/5\u0026ndash;1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthors contributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJohannes Steffen:\u003c/strong\u003e Methodology, Validation, Formal analysis, Investigation, Data Curation, Writing - Original Draft, Writing - Review \u0026amp; Editing, Visualization, Funding acquisition, \u003cstrong\u003eNina Sophie Bellersheim:\u003c/strong\u003e Validation, Formal analysis, Investigation, Data Curation, \u003cstrong\u003eCaio Andreeta Figueiredo:\u003c/strong\u003e Methodology, Investigation, Writing - Review \u0026amp; Editing\u003cstrong\u003e, Laura Knop:\u003c/strong\u003e Investigation, \u003cstrong\u003eVladyslava Dovhan\u003c/strong\u003e: Investigation, \u003cstrong\u003eAndreas Diefenbach\u003c/strong\u003e: Resources, \u003cstrong\u003eChristoph S. N. Klose:\u003c/strong\u003e Writing - Review \u0026amp; Editing, \u003cstrong\u003eThomas Sch\u0026uuml;ler:\u003c/strong\u003e Conceptualization, Resources, Writing - Review \u0026amp; Editing, \u003cstrong\u003eIldiko R. Dunay:\u003c/strong\u003e Conceptualization, Resources, Writing - Review \u0026amp; Editing, Supervision, Project administration, Funding acquisition\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eWe thank Petra Gr\u0026uuml;neberg for the exceptional technical assistance.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKovacs M, Dominguez-Belloso A, Ali-Moussa S, Deczkowska A. Immune control of brain physiology. Nat Rev Immunol. 2025.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmyth LCD, Kipnis J. Redefining CNS immune privilege. Nat Rev Immunol. 2025.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBecher B, Derfuss T, Liblau R. 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Front Pharmacol. 2022;13:807639.\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"journal-of-neuroinflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jneu","sideBox":"Learn more about [Journal of Neuroinflammation](http://jneuroinflammation.biomedcentral.com)","snPcode":"12974","submissionUrl":"https://submission.nature.com/new-submission/12974/3","title":"Journal of Neuroinflammation","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"innate lymphoid cells, CNS immunity, ILC1s, IFN-γ, type 1 cytokines, neuroinflammation, FTY720 (Fingolimod), innate-adaptive transition, T cell-independent immunity, brain-resident ILCs, choroid plexus, meninges, Toxoplasma gondii, adaptive immunity","lastPublishedDoi":"10.21203/rs.3.rs-8278466/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8278466/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSpecialized immune interfaces of the central nervous system (CNS), as the choroid plexus and meninges, serve as dynamic gatekeepers coordinating the transition from innate to adaptive immunity during neuroinflammatory responses. While this transition is essential for resolving CNS inflammation, the precise temporal and functional bridging of these phases remains incompletely characterized.\u003c/p\u003e \u003cp\u003eTo elucidate the role of local innate effectors in maintaining CNS protection we employed the model of \u003cem\u003eToxoplasma gondii\u003c/em\u003e-induced neuroinflammation and applied Fingolimod (FTY720) to pharmacologically modulate the S1P receptor and prevent lymphocyte recirculation and CNS infiltration. Despite resulting in impaired T cell infiltration, CNS parasite control was preserved during the acute phase, coinciding with expansion of group 1 innate lymphoid cells (ILCs), including tissue-resident NK cells and type 1 ILCs (ILC1s). High-dimensional profiling indicated that brain-derived ILC1s acquire distinct, tissue-adapted signatures and sustain robust type 1 cytokine responses (\u003cem\u003eIfng\u003c/em\u003e, \u003cem\u003eTnf\u003c/em\u003e, \u003cem\u003eIrf8\u003c/em\u003e) crucial for microglial activation and early antiparasitic defense.\u003c/p\u003e \u003cp\u003eCollectively, rapidly adapting innate effectors supported early defense in the infected CNS when adaptive immunity was compromised, providing detailed insight into the context-specific orchestration of CNS protection. These results refine the understanding of the innate\u0026ndash;adaptive transition during neuroinflammation and position group 1 ILCs as promising targets for neuroprotective immunomodulation.\u003c/p\u003e","manuscriptTitle":"Expanding group 1 ILCs sustain early CNS defense against Toxoplasma gondii during impaired adaptive response","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-19 13:45:08","doi":"10.21203/rs.3.rs-8278466/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-18T06:25:20+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-15T22:01:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-11T23:15:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-07T02:22:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"312910655094361659172499671774091369580","date":"2025-12-19T18:33:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"249453045808242301190806112009826222577","date":"2025-12-19T10:18:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"288193191830937324919052070521733312309","date":"2025-12-18T18:23:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"99546897366358006018477789951681745578","date":"2025-12-17T16:53:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-17T05:57:40+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-16T00:23:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-15T13:21:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Neuroinflammation","date":"2025-12-04T10:46:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-neuroinflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jneu","sideBox":"Learn more about [Journal of Neuroinflammation](http://jneuroinflammation.biomedcentral.com)","snPcode":"12974","submissionUrl":"https://submission.nature.com/new-submission/12974/3","title":"Journal of Neuroinflammation","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0cfa0ccc-6d43-4de9-b80f-f42933bfe447","owner":[],"postedDate":"December 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-01-18T06:38:31+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-19 13:45:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8278466","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8278466","identity":"rs-8278466","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}