Astrocytic but not Microglial Antigen Presentation Shapes Protective Immunity to Toxoplasma gondii in the Brain

Astrocytic but not Microglial Antigen Presentation Shapes Protective Immunity to Toxoplasma gondii in the Brain | 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 Astrocytic but not Microglial Antigen Presentation Shapes Protective Immunity to Toxoplasma gondii in the Brain Sydney A. Labuzan, Anne E. Schuster, Michael A. Kovacs, Maureen N. Cowan, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9544619/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract T cells play a pivotal role in orchestrating immune defense within the central nervous system (CNS) during many infections. Toxoplasma gondii , a brain-trophic protozoan parasite, establishes lifelong CNS infection that remains largely subclinical in immunocompetent hosts but can cause severe encephalitis in immunocompromised individuals. While CD8⁺ T cells are essential for controlling T. gondii during chronic infection through both cytokine production and cytolytic killing, the CNS-resident cells that functionally present antigen in the brain to promote T cell function or serve as cytolytic targets remain incompletely defined. Here, we investigated the contributions of CNS-resident macrophages and astrocytes, two key CNS-resident cell types, antigen presentation during chronic T. gondii infection. Using mice lacking MHCI or MHCII in CNS-resident macrophages, we found no impairment of immune responses or ability of the brain to control parasite, indicating dispensable function of resident macrophages as APCs during infection. However, deletion of MHCI on astrocytes led to deficits in parasite control, in turn promoting elevated CD4⁺ T cell cytokine production and recruitment of iNOS⁺ inflammatory monocytes. We observed increased presence of lytic parasite within the brain, which suggests that astrocyte MHCI may be necessary to control parasite replication throughout the CNS. Our findings underscore a previously underappreciated role for astrocytic MHCI within the CNS during infection and highlights the dispensability of CNS-resident macrophages to this process. Antigen Presentation Microglia Astrocytes Neuroimmunology Toxoplasma gondii Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 BACKGROUND T cells emerge as critical orchestrators of the immune response during many central nervous system (CNS) infections, shaping host-protective mechanisms essential for host survival 1 , 2 . One prominent brain-tropic pathogen is Toxoplasma gondii , an intracellular protozoan parasite that establishes a lifelong chronic infection in the CNS across a range of mammalian hosts, including humans 3 , 4 . In immunocompetent individuals, T. gondii infection is typically asymptomatic due to effective immune control. However, in immunocompromised populations, such as individuals with AIDS or organ transplant recipients, reactivation of the parasite can lead to toxoplasmic encephalitis (TE), a potentially fatal uncontrolled infection of the brain 5 . In murine models, deficiencies in T cell responses lead to unchecked parasite proliferation and lethal TE, underscoring the indispensable role of T cells in controlling chronic T. gondii infection 1 , 3 , 6 – 14 . Although the CNS is no longer considered strictly immune-privileged, it retains unique immunological features 15 – 17 . Under homeostatic conditions, the brain exhibits minimal expression of antigen-presenting machinery and lacks conventional antigen-presenting cells (APCs), such as dendritic cells 15 , 16 . In the setting of a neurotropic pathogen, like T. gondii , this prompts a central question of how CD8⁺ T cell responses are sustained and how infected cells are recognized and eliminated within the CNS. Studies have shown that microglia, the resident innate immune cells of the brain, upregulate antigen presentation machinery following an insult to the brain, including in the context infection or neurodegeneration 18 – 26 . This suggests one mechanism by which the brain becomes a more immunologically reactive tissue. The blood-brain barrier (BBB) at steady-state serves as a stringent gatekeeper to limit immune cell infiltration into the brain parenchyma 27 . During infection with T. gondii , the brain vasculature becomes activated to allow the robust recruitment of parasite-specific T cells into the brain parenchyma 28 – 30 . Of note, once recruited, T cells can only persist within the tissue if their cognate antigen is expressed at this site 31 , 32 . There is growing interest in understanding the dual role of CD8 + T cells in the brain, where they can act as both pathogenic and protective agents during infection, neurodegeneration, and injury 20 , 27 , 33 – 39 . It is known that coordinated interferon-gamma (IFNγ) production by both CD4 + and CD8 + T cells is a critical component of the immune response to T. gondii 1 , 7 , 9 , 14 , 40 – 42 . In addition to cytokine production, CD8⁺ T cells mediate host-protection through cytolytic killing, recognizing infected cells via Major histocompatibility complex I (MHCI) antigen presentation, and eliminating them through perforin- and granzyme-dependent mechanisms 1 , 41 , 43 – 45 . Perforin production by CD8 + T cells was found to be dispensable in the peripheral phase of T. gondii infection, but necessary for control of cyst burden in the chronic brain phase of infection 13 . This demonstrates that cytolytic killing by CD8 + T cells is an integral component of immunity to T. gondii in the brain. Conventional type 1 dendritic cells (cDC1s) have been shown to be a key antigen-presenting cell type that promotes expansion of parasite-specific T cells at peripheral sites during early T. gondii infection 1 , 7 , 46 , 47 . However, these cells largely remain at the brain borders during infection and are rarely found within deeper brain parenchyma, leaving it an open question as to which cells interact with CD8 + T cells once they transit into the brain 48 . CNS antigen presentation to date has been most thoroughly studied in experimental autoimmune encephalitis (EAE) where antigen-specific T cells infiltrate the CNS parenchyma and drive disease pathogenesis 49 , 50 . In this context, dendritic cells recruited into the CNS parenchyma have been identified as critical APCs via Major histocompatibility complex class II (MHCII) while microglial presentation proved dispensable 51 , 52 . Recent literature has expanded our understanding of antigen presentation within the CNS, revealing unexpected complexity among resident and infiltrating cell types. Microglia have been shown to upregulate MHCI and II molecules during infection and to putatively cross-present antigen to CD8⁺ T cells in the context of vesicular stomatitis virus 18 , 20 , 36 . Despite rarely found actively infected by T. gondii , this cross-presentation would allow microglia to display phagocytosed antigen via MHCI or II to stimulate T cell function or be targeted for cytolytic killing 3 , 25 , 53 . Several studies have implicated microglia, perivascular macrophages, and brain endothelial cells (BECs) in priming CD8⁺ T cells for CNS entry during viral infections 20 , 25 , 34 , 54 . Although microglia are widely recognized as professional APCs in the CNS, the potential for other resident cells to present antigen for targeted cytolytic killing remains unknown. Astrocytes are glial cells well known for their homeostatic functions in maintaining the BBB, metabolizing glutamate, stabilizing extracellular concentration of potassium, and producing trophic survival factors for neurons and other glia 55 . Foundational studies, largely in vitro , recognized astrocytes as non-traditional APCs that upregulate antigen presentation machinery in autoimmune disorders and infection 55 – 61 . In the context of T. gondii , astrocytes play a critical role in parasite containment through IFN-driven responses and form heterogeneous reactive subpopulations during chronic infection 62 – 64 . Neurons are the predominant cell type with active infection of T. gondii , with a smaller subset of astrocytes observed harboring parasite throughout the course of infection 53 . As such, neurons and astrocytes represent potential sources of T. gondii antigen for presentation via MHCI to CD8⁺ T cells, with the capacity to either promote T cell function, act as targets of cytolytic killing, or contribute to both processes. Using a mouse model in which neurons express the H2-L d allele, an MHCI variant known to drive an immunodominant, protective CD8 + T cell response, studies have implicated neuronal antigen presentation in controlling parasite burden during T. gondii infection 65 . Neurons have also been shown, along with microglia, to contribute to the shaping of resident memory T cell populations in a model of latent T. gondii infection 24 , 65 . These studies point to potential roles of antigen presentation by infected cells during chronic neuroinflammatory states. Despite these recent advances, significant gaps remain in our understanding of how CNS-resident cells use antigen presentation to shape CD8⁺ T cell function and control of parasite during chronic neuroinflammation. In this study, we aimed to define how CNS-resident macrophage and astrocyte antigen presentation modulates T cell responses and parasite control during chronic T. gondii infection. To address this question, we employed cell type-specific knockouts of the B2m gene, a critical component of the MHCI complex, on astrocytes and CNS-resident macrophages. We hypothesized that ablation of MHCI expression in one or both cell types would impair CD8⁺ T cell–mediated immunity or targeted killing, leading to increased parasite burden in the brain. We additionally generated a CNS-specific macrophage knockout of MHCII to examine how this would impact CD4 + T cell function and parasite burden in the brain. MHCI or MHCII-deficient CNS-resident macrophages displayed no immune impairment, suggesting dispensable function or compensation by other cells presenting antigen within the CNS. We found that loss of MHCI in astrocytes resulted in impaired parasite control during chronic infection. We also observed heightened immune activity, including elevated cytokine production by infiltrating CD4⁺ T cells and increased production of iNOS⁺ by inflammatory monocytes. These findings reveal a unique role for astrocyte antigen presentation in parasite control during chronic CNS infection and provide new insight into the specialized immunological landscape of the brain. RESULTS Microglia upregulate antigen presentation machinery during chronic Toxoplasma gondii infection. To examine the timing of this upregulation during infection, RT-qPCR analysis of whole brain homogenate from mice harvested at various timepoints from day 0 (naive) to day 42 (D42) post-infection was performed. Interferon-γ (IFNγ) promotes the upregulation of antigen presentation machinery, including MHC molecules, in brain-resident cells during inflammation 18 , 21 , 36 , 66 . When we examined dynamics of gene expression of Ifng across infection, we observed an increased pattern of expression from 8 dpi up until 42 dpi ( Fig. 1 C ) . The gene β2m , a key component of the MHCI complex, followed a similar pattern of expression across infection in the brain ( Fig. 1 D ) . Further, gene expression of H2-Aa , a key MHC Class II gene, by RT-qPCR showed a similar upregulation, with a peak during chronic infection ( Fig. 1 E ) . Together, these data indicate that CNS infection is accompanied by a coordinated, sustained induction of IFNγ and both MHCI and MHCII gene expression, peaking during chronic infection. Observing these changes in the whole brain across infection, we aimed to hone in on in which CNS-resident cells’ antigen presentation is functionally relevant for control of infection. Due to their role as the innate immune cell of the brain, we hypothesized microglia would serve as robust antigen presenters during chronic infection. We used ROSA26 Ai6/Ai6 x Cx3cr1 CreERT2+/− mice, which express ZsGreen fluorescence specifically in Cx3Cr1 + cells following cre activity induced by the application of tamoxifen 67 . We administered five intraperitoneal (i.p.) injections of tamoxifen. We then waited four weeks for peripheral monocyte turnover, leaving labeling confined to longer-lived CNS-resident macrophages. We then mock-infected with PBS or infected mice i.p. with 10 cysts of the Type II strain of T. gondii Me49 ( Fig. 1 F ) . Brains were harvested at 4 weeks post-infection (4 wpi) and flow cytometry was performed to examine antigen presentation markers (Fig. S1 A). Upon infection, we measured robust populations of MHCI and MHCII expressing cells not present in the naïve brain. Preparing brain tissue for flow cytometric analyses results in a single cell suspension predominantly composed of immune cells and lacking CNS-resident cell types. To explore the immune cell populations expressing MHCI and MHCII during infection we categorized individual immune populations. At the whole brain level, concurrent with our RT-qPCR results, we observe very low expression of MHCI in the naïve state, but robust upregulation during infection (Fig. S1 B). Within the infected brain, as we expected, infiltrating immune cells abundantly express MHCI (~ 44% Ly6C hi and ~ 19% Ly6C low monocytes), and microglia upregulate MHCI expression ( Fig. 1 G-H, S1C ) . Correspondingly, we observe low expression of MHCII in the naïve state, but robust upregulation during infection (Fig. S1 D). Within the MHCII + population in the infected brain, we see this population comprised predominantly of infiltrating monocytes (~ 42% Ly6C hi and ~ 25% Ly6C low monocytes) and smaller populations of B cells and microglia (~ 7% B cells and ~ 6% microglia) (Fig. S1 E) . Small numbers of dendritic cells (cDC1s and cDC2s) expressing both MHCI and MHCII are found within the meninges at this time point but are rarely found in the deeper brain parenchyma 32 . Narrowing in on the CD45 int CD11b + ZsGreen + microglial population, as expected, we observed minimal expression of MHCI or MHCII molecules in the naïve state (~ 2–3%) but significant upregulation of these molecules during chronic infection in the brain (~ 98–99%), reflecting a similar pattern previously observed as early as 12dpi in the brain (Fig. 1 G-I, Fig. S1 A ) 18 . Although microglia comprised a relatively minor fraction of MHCI and MHCII-expressing cells compared to infiltrating immune cells, their drastic shift in expression of antigen presentation machinery highlighted them as candidates for promoting T cell function during CNS infection. CNS-resident macrophage MHCI- antigen presentation is dispensable for control of T. gondii during chronic infection. Since we observe this robust upregulation of MHCI during infection by microglia, we wanted to elucidate the functional relevance of this to promoting T cell function and control of parasite. To address this question, we generated ROSA26 Ai6/Ai6 x Cx3cr1 CreERT2+/− x B2m fl/fl mice, hereafter referred to as MG B2m , to ablate MHCI from Cx3Cr1 + cells and allow fluorescent ZsGreen-labeling of these cells. We induced deletion of the β2m gene via five doses of tamoxifen administration. We waited four weeks to make the deletion specific to CNS-resident macrophages (i.e. microglia and border associated macrophages) and infected mice i.p. with 10 cysts of the Me49 T. gondii strain. Mice progressed to the chronic phase of infection and brains were analyzed at 6 wpi (Fig. 2 A ) . We examined immune responses in the brain at this timepoint using flow cytometry (Fig. S2A). We first examined efficacy of the genetic deletion and observed that compared to controls (MG WT ) (~ 98%), on average only ~ 13% of microglia in the brains of knockout mice (MG B2m ) expressed MHCI during chronic infection (Fig. 2 B-C). Microglia lacking MHCI had no change in sufficiency for expression of MHCII ( Fig. S3A ). By flow cytometry, we observed no differences in the total number of TCRβ + T cells, or TCRβ + CD4 + and TCRβ + CD8 + T cells in the brains of knockout mice during chronic infection ( Fig. S3B-D ). Established mechanisms of parasite control in the brain include T cell–derived IFNγ and tumor necrosis factor–α (TNFα), along with downstream inducible nitric oxide synthase (iNOS) production by infiltrating inflammatory monocytes 3 . When assessing functionality of these CD8 + T cells to produce cytokine, we plated and incubated cells ex vivo with Brefeldin A (BFA). We observed decreased production of IFNγ when mice lack MHCI on CNS-resident macrophages by percentage of the overall CD8 + population (Fig. 2 D-E) but not by number (Fig. 2 F). At the transcriptional level, we observed no difference in Ifng levels in the brains of knockout mice compared to WT mice (Fig. 2 G). Additionally, we observed no differences in TNFα production by CD8 + T cells ( Fig. S3E-F) or at the whole brain RNA level ( Fig. S3G ). We observed within the CD4 + T cell population a decrease in production of both IFNγ and TNFα in our knockout mice by frequency but not by overall number present in the brain ( Fig. S3H-K) . Collectively, these results demonstrate that loss of MHCI in CNS-resident macrophages does not alter T cell effector function, as measured by cytokine production. As a further measure of immune control, we measured production of iNOS by Ly6C hi infiltrating myeloid cells and found decreased percentage in the knockout mice (Fig. 2 H-I ). However, this did not correspond to a decrease in overall number of Ly6C hi iNOS + infiltrating myeloid cells (Fig. 2 J) or any difference in overall Nos2 RNA level in the brains of these mice (Fig. 2 K). These results suggested an overall unimpaired immune response when CNS-resident macrophages lack MHCI, likely due to dispensable function or compensatory antigen presentation by other cells in the brain. Finally, to assess how microglial antigen presentation affects control of parasite, we used three measures of parasite burden: cyst counts performed by brightfield microscopy, qPCR assay of parasite genomic DNA, and parasite Act1 gene expression from whole brain samples. While observing no significant differences in the quantity of cysts or abundance of parasitic genomic DNA (Fig. 2 L-M), we did observe a decrease in gene expression of parasite Act1 in the knockout mice (Fig. 2 N ) . Given the lack of increased parasite burden and overall preserved immune responses, this suggests that MHCI by CNS-resident macrophages is dispensable for parasite control during chronic infection. Loss of MHC Class II expression in CNS-resident macrophages does not impact immune control of T. gondii in the brain In addition to upregulating MHCI, we observed that CNS-resident macrophages robustly upregulate expression of MHCII during chronic T. gondii infection (Fig. 1 G-I) 18 . Previous work showed that when microglia lack the transcription factor Stat1 and cannot respond to IFN-signaling, they do not upregulate MHCII and succumb to T. gondii infection 18 . Having observed no significant deficit in immune control when CNS-resident macrophages lack MHCI expression, we hypothesized that these cells may preferentially present phagocytosed parasitic antigen via the MHCII pathway. To test this hypothesis, we generated Rosa26 Ai6/Ai6 x Cx3cr1 CreERT2+/− x Iab fl/fl (MG MHCII ) mice that ablate MHCII from CNS-resident macrophages. We administered tamoxifen at 4–6 weeks of age, waited for 4 weeks, and mice were then subsequently infected. Mice were analyzed at a chronic infection timepoint of 4wpi by flow cytometry ( Fig. 3 A, Fig. S2A) . We examined efficacy of the genetic deletion and observed that compared to controls (MG WT ) (~ 98%), on average only ~ 10% of microglia in the brains of knockout mice (MG MHCII ) expressed MHCII during chronic infection (Fig. 3 B-C ). We found MG MHCII mice remained sufficient for MHCI expression ( Fig. S4A ). We hypothesized that if CNS-resident macrophage MHCII presentation plays a role in sustaining T cell responses during chronic T. gondii infection, a disruption in CNS-resident macrophage MHCII expression would result in reduced effector functions of CD4 T cells. Thus, we analyzed this population first by flow cytometry. By flow cytometry, we observed no differences in the total number of TCRβ + T cells, or TCRβ + CD4 + and TCRβ + CD8 + T cells in the brains of knockout mice during chronic infection ( Fig. S4B-D ). We observed no deficits in production of IFNγ by CD4 + T cells either by measures of frequency of total CD4 + T cells (Fig. 3 D-E ) or in number of CD4 + IFNγ + T cells (Fig. 3 F). Additionally, we observed no differences in overall level of Ifng in the brains of these mice at the RNA level (Fig. 3 G ) . When assessing production of the cytokine TNFα, we observe an increase in CD4 + TNFα + T cells by frequency of all CD4 + T cells in MG MHCII brains, but no difference in overall number ( Fig. S4E-F ). At the whole brain level, there is also no difference in Tnf gene expression between MG MHCII and MG WT mice ( Fig. S4G) . We also observed no change in CD8 + T cell IFNγ and TNFα production by flow cytometry when microglia lack MHCII ( Fig. S4H-K) . Taken together, these results demonstrate that loss of MHCII in microglia does not affect T cell effector function, as measured by cytokine production. We further examined downstream production of iNOS by Ly6C hi infiltrating inflammatory monocytes and observed no changes by percentage of iNOS + monocytes (Fig. 3 H-I ) or by total number present in the brain (Fig. 3 J ). Further confirming these results, we found no difference in Nos2 expression at the RNA level in our microglial knockouts (Fig. 3 K ) . Additionally, we observed no difference in parasite burden by any measures when CNS-resident macrophages lacked MHCII expression ( Fig. 3 L-N ). These results from our CNS-resident macrophage MHCI and MHCII knockouts suggest a dispensable role for microglial antigen presentation in the context of T. gondii infection and prompted us to question a role for other CNS-resident cells within the CNS. Astrocytes upregulate MHC Class I expression during chronic T. gondii infection. While traditionally appreciated for their roles in maintenance of neuronal health and homeostasis, astrocytes have recently emerged as critical players in neuroimmunity 55 , 56 , 62 , 68 , 69 . Despite being shown to upregulate antigen presentation machinery and stimulate T cell proliferation in vitro , the capacity of astrocytes to act as antigen presenters in vivo remains ambiguous 56 , 60 , 61 . To investigate whether astrocytes are a relevant cell type in presenting antigen to CD8 + T cells during T. gondii infection, C57BL/6 mice were either mock-infected with PBS or infected with 10 cysts of the Type II strain Me49 and brains harvested 6 weeks later to assess expression of astrocytic MHCI. Through flow cytometry analysis, we found GLAST+ cells had very little expression of MHCI (~ 2%) in the PBS-injected naïve group, compared to a significant increase in MHCI during infection (~ 60%) (p < 0.001) ( Fig. 4 A-B, Fig. S5A). Further, by Mean Fluorescence Intensity (MFI) we observe a significant increase in the abundance of MHCI present on the GLAST + population during infection (Fig. 4 C). Additionally, in brains stained by immunofluorescence from chronically infected mice, we observe colocalization of GFAP + cells with MHCI staining, further indicative of upregulation of MHCI by astrocytes ( Fig. 4 D ) . To determine how astrocytic MHCI expression dictates CD8 + T cell function in the brain during infection, we crossed Gfap-77.6 - cre to B2m fl/fl mice to constitutively excise the gene B2m from astrocytes. Gfap77.6 -cre −/− - B2m fl/fl (Cre-) control and Gfap77.6 -cre +/− - B2m fl/fl (Cre+) mice were infected with 10 cysts of the type II Me49 strain of T. gondii and brains harvested 6 weeks later (Fig. 4 E). To confirm excision efficiency of the B2m gene in the knockout mice, ACSA2 + astrocytes were purified, and RT-qPCR was performed for the gene B2m . We observed an approximate ~ 50% reduction in B2m gene expression in Gfap77.6 -cre +/− - B2m fl/fl mice (Cre+) compared to control Gfap77.6 -cre −/− - B2m fl/fl (Cre-) mice (Fig. 4 F). To examine intact capacity to present by more traditional APC populations, CD45 int CD11b + CNS-resident macrophages and infiltrating CD45 hi CD11b + macrophages were assessed by flow cytometry ( Fig. S5B). We found canonical APCs were unaffected in Gfap -cre +/− B2m fl/fl (Cre+) mice and were sufficient for MHCI expression comparable to controls (Cre-) mice ( Fig. S5C-F ). Mice deficient in astrocytic MHC class I display impaired parasitic control during chronic infection. To first assess how knockdown in astrocytic MHCI antigen presentation would affect control of parasite, we used three measures of parasite burden. We observed no difference in cyst counts between knockout (Cre+) and wildtype (Cre-) mice (Fig. 5 A). However, by total parasite genomic DNA and Act1 gene expression, we observed increased parasite burden in the brains of mice with deletion of MHCI from astrocytes ( Fig. 5 B-C ). Further, we performed qPCR of Sag1 and Bag1 , genes specific to tachyzoite and bradyzoite parasite stages, respectively. We observe an increased ratio of Sag1/Bag1 present when mice lack astrocytic MHCI, indicating an increase in the tachyzoite form of the parasite (Fig. 5 D). This suggests an impairment of control of parasite replication. To visualize parasite in the brains of these mice, immunofluorescence with an astrocytic marker (GFAP) and an antibody against the Me49 parasite was performed. In agreement with our quantitative parasite burden data, we observed notable areas of tachyzoites (Fig. 5 E-F) but no difference in the cyst form of the parasite (Fig. 5 G-H). Astrocyte MHC Class I-deficiency leads to increased CD4 + T cell cytokine production. Observing the increase in parasite burden in knockout mice, we next assessed CD8 + and CD4 + T cell function when mice lack astrocytic MHCI. We hypothesized if astrocyte MHCI expression played a key role in coordinating CD8 + T cell function, we would observe decreased effector T cell functions, such as cytokine (IFNγ and TNFα) and Granzyme B production. To assess their function, flow cytometric analysis was performed for immune cell populations at the 6-week timepoint. We observed no overall differences in the number of overall TCRß + T cells (Fig. 6 A), TCRß + CD4 + T cells (Fig. 6 B), and TCRß + CD8 + T cells in the brains of knockout mice (Fig. 6 C). When we assessed production of the key cytokine IFNγ, we observed no deficits in production by TCRß + CD8 + T cells either by frequency (Fig. 6 D-E) or by overall number (Fig. 6 F). We also observed no differences in the production of TNFα by TCRß + CD8 + T cells when mice lack astrocytic MHCI ( Fig. S6A-B ). Intriguingly, we did observe increases in TCRß + CD4 + T cells producing IFNγ by frequency and number (Fig. 6 G-I), as well as those producing the cytokine TNFα ( Fig. S6C-D) . To confirm astrocyte antigen presentation does not impact the capacity of T cells to produce cytokine, brain cells were stimulated ex vivo with PMA/ionomycin. We observed no differences in TCRß + CD8 + capacity to produce IFNγ and TNFα ( Fig. S6E-F). PMA/ionomycin stimulated TCRß + CD4 + cells from brains lacking astrocytic MHCI did possess higher capacity to produce IFNγ but not TNFα when compared to controls ( Fig. S6G-H). To examine deficits in cytotoxic function by CD8 + T cells when astrocytes lack MHCI, staining was additionally performed for Granzyme B (GrzmB). We observed no difference in the production of Granzyme B by frequency, number, or MFI of TCRß + CD8 + T cells ( Fig. S6I-K ). Further, at the whole brain RNA level, we observed no change in the amount of GrzmB transcript in the Cre+ animals ( Fig. S6L ). Overall, these results suggest that there is not a deficit in cytotoxic potential or cytokine production by CD8 + T cells. These results suggest that CD8⁺ T cell function is not directly affected, whereas altered CD4⁺ T cell responses point to increased inflammation as an indirect consequence of astrocyte MHCI deletion, due to increased parasite burden. Since we observed increases in cytokine production by CD4 + T cells, we hypothesized we would observe downstream effects on IFNγ-dependent processes, including the production of anti-parasitic iNOS by infiltrating monocytes. We observed no change in the total number of infiltrating myeloid cells when mice lack astrocytic MHCI (Fig. S7A) . To assess production of iNOS by these cells, we performed flow cytometric analysis and observed increases in both the frequency of iNOS + Ly6c hi inflammatory monocytes ( Fig. 6 J ) , as well as total number in the brains of mice lacking astrocytic MHCI ( Fig. 6 K ) . Further, we observed increased level of Nos2 gene expression in the brain when mice lack astrocyte MHCI ( Fig. 6 L ) . Further, we found a decreased frequency in CD4 + Foxp3 + regulatory T cells within the total CD4 + T cell population, but ultimately similar number of regulatory T cells between groups ( Fig. S7C-D ). This increased proportion of CD4 + effector T cells supports findings of an overall enhanced inflammatory response to the increased parasite in the knockout mice. We further found increased expression in IFN-driven genes, including chemoattractants Cxcl9 and Cxcl10 and adhesion molecules Icam and Vcam , which correspond with overall enhanced inflammation in the brains of the knockout mice ( Fig. 6 M ) . When assessing the spleen as an indicator of systemic immune activation in Cre+ knockout mice, we found no differences in the overall numbers of TCRß + T cells, or TCRß + CD4 + and TCRß + CD8 + subsets ( Fig. S7E-F ). We observed no difference in the numbers of proliferative CD4 + and CD8 + T cells within the spleens of these mice compared to controls at this timepoint ( Fig. S7F ). Taken together, this suggests that the enhanced immune response is specific to the brains of these knockout animals. These observations indicate that when astrocytes lack the ability to present antigen to cytotoxic T cells, parasite replication increases, and leads to an enhanced immune response driven by cytokine producing CD4 + T cells. DISCUSSION Our study reveals that MHCI antigen presentation by astrocytes, but not CNS-resident macrophages, is important in control of Toxoplasma gondii replication within the brain. We found that when CNS-resident macrophages lacked MHCI or MHCII expression, we observed no deficits in parasite control or immune responses. However, we demonstrate that the absence of MHCI expression on astrocytes results in an increase in parasite. The increase in parasite led to enhanced rather than deficient immune responses driven by CD4 + T cell cytokine production and downstream monocyte iNOS production. These findings highlight a previously undescribed role for how astrocytes present antigen to promote control of T. gondii in the CNS. These results provide not only greater insight into host–parasite interactions in the brain, but also how T cell recognition of infected astrocytes plays a role in control of pathogen. Much of the work in the neuroimmune space has emphasized microglia as the central APCs and modulators of neuroinflammatory responses within the CNS 19 – 21 , 25 , 26 , 36 , 51 , 56 , 70 – 74 . More recently, this perspective has expanded, as interest grows in how T cells contribute to brain immunity across diverse contexts, from infectious diseases to neurodegenerative disorders. This shift reflects a broader recognition that antigen presentation in the CNS is more complex and multifaceted than previously thought. Our group previously demonstrated that interferon signaling in microglia is essential for control of T. gondii , as mice lacking Stat1 in microglia succumb early in chronic infection 18 . Interferon signaling drives robust upregulation of antiparasitic effector pathways, including Irg and Gbp family genes, along with antigen presentation machinery, highlighting the importance of interferon in enabling microglia to mount an effective response 18 . Despite rarely being found infected in vivo in mice, when microglia lack Stat1 signaling, microglia were found to be harboring parasite, suggesting an innate ability to clear parasite by these cells 18 . While these findings establish microglia as indispensable for parasite control, the results of the present study indicate that antigen presentation itself is a dispensable component of this protective role. Findings across disease models illustrate the context-dependent nature of microglial antigen presentation in the CNS. In EAE, dendritic cells rather than microglia appear to be the dominant APCs coordinating CD4⁺ T cell responses, consistent with our observation that microglial MHCII is dispensable during chronic T. gondii infection 51 , 52 . Moreover, our data align with previous findings demonstrating that loss of Tap in microglia does not affect total T cell populations or cytokine production in the brains of mice during latent T. gondii infection 24 . In peripheral tissues, infected cDC1s are shown to play a key role in presentation of T. gondii to promote an immune response, however they largely remain at the brain borders during chronic infection 47 , 48 , 75 . Our data demonstrate that CD11b⁺CD45 hi Ly6C low and Ly6C hi infiltrating peripheral populations constitute the largest contributors to the overall pool of MHCI⁺ and MHCII⁺ cells in the brain during infection. Thus, these cells could be poised to act as the predominant APCs throughout T. gondii infection, compensating for any microglial dysfunction. By contrast, in a viral model of Theiler’s murine encephalomyelitis virus (TMEV), microglia and perivascular macrophages act as APCs to promote CD8 + T cell infiltration into the brain 20 . Similarly, studies in tauopathy have shown that microglia regulate T cell entry and function in ways that exacerbate disease pathology 76 . A key distinction between these models is the degree of peripheral myeloid cell infiltration into the brain. Some viral infections and neurodegenerative models exhibit limited recruitment of peripheral APCs to the brain parenchyma, whereas chronic T. gondii infection and EAE involve substantial infiltration of monocytes and dendritic cells, respectively. Together, these findings suggest that microglia may play a more prominent APC role in settings where infiltrating professional APCs are scarce, while in highly inflammatory contexts with abundant peripheral APCs their contribution may be comparatively dispensable. Neurons, as the principal cell observed with active infection, would be expected to encounter the highest levels of parasite-derived antigen. It is now appreciated that MHCI expression by neurons during development is crucial for synaptic pruning and refinement, but whether neurons upregulate and use MHCI functionally during disease remains unclear 77 , 78 . Recent studies have aimed to understand how MHCI expression by neurons may play a role in control of T. gondii . In vitro primary murine neurons respond to IFNγ to upregulate Irg s, Gbp2 , Stat1 , and Mhc1 , as well as pretreatment with IFNγ leads to decreased levels of T. gondii infected neurons 79 . In a latent infection model of T. gondii where C57BL/6 mice were generated to possess a floxed MHCI immunoprotective H2-L d allele, knocking out H2-L d from neurons results in greater cerebral parasite burden 65 . These findings raise additional questions about how this process occurs in vivo in C57BL/6 mice, which express the H2-Dᵇ and H2-Kᵇ MHCI alleles, and whether neurons that present antigen can directly interact with cytotoxic CD8⁺ T cells. Although neurons represent the predominant cell type harboring T. gondii in the brain, a small subset of infected astrocytes has been identified through the use of a Cre-secreting parasite 53 . In this system, T. gondii is engineered to secrete Cre-recombinase into host cells, enabling fluorescence in cells injected with parasite effector proteins during invasion 53 . The relative rarity of infected astrocytes despite widespread parasite exposure suggests that these cells possess intrinsic or extrinsic immune-mediated mechanisms for parasite clearance, a topic that has received increasing attention in the field. Previous work has shown the necessity of Stat1 -mediated interferon-signaling in astrocytes in restricting T. gondii burden in the brain 80 . This finding was inferred to be a deficit in the ability to upregulate anti-parasitic machinery, thus allowing astrocytes to serve as a residential niche for parasite 80 . Recently, it was shown that astrocytes do not use caspase 8-mediated apoptosis as a parasite restriction mechanism during T. gondii infection 81 . While in vitro studies have observed upregulation of MHCI by astrocytes and interactions in co-cultures with CD8 + T cells, to date it has not been thoroughly explored how antigen presentation by astrocytes may contribute to host defense from pathogens in vivo within the CNS 60 , 61 , 66 ,8283 . Our findings directly address this gap by demonstrating that astrocytic MHCI upregulation is one mechanism for pathogen restriction during CNS infection. We propose that this effect is mediated through interactions with cytotoxic CD8⁺ T cells, enabling detection of and elimination of infected astrocytes. CONCLUSION Together, our findings establish astrocytes as a functionally important source of antigen presentation required for effective control of T. gondii in the CNS. Although microglia robustly upregulate antigen presentation machinery during infection, their antigen presentation is dispensable for parasite control. By contrast, astrocyte MHCI expression plays a role in limiting pathogen burden, likely through display of parasite antigen by infected cells. This work provides novel insight into host–pathogen dynamics within the infected brain, where professional immune cells such as microglia, while highly reactive, do not appear to function as critical APCs in promoting T cell function. Instead, effective immune control appears to depend in part on antigen presentation by infected parenchymal cells themselves. Given their abundance and association with neurons and sites of parasite reactivation, astrocytes are positioned to serve as sites of CD8 + T cell immune surveillance and targeted pathogen clearance. However, this has not been thoroughly explored in vivo , a gap addressed in the present study. Our finding gives new insight into host-pathogen dynamics and likely extends to other disease contexts in which astrocytes are directly infected, underscoring their broader role as sources of antigen that drive pathogen control through MHCI upregulation. METHODS Animals and Treatments: Gfap Cre77.6 (#024098), Cx3cr1 CreERT2 (#020940), Iab1 fl/fl (#037709), ROSA26 Ai6/Ai6 (#007906), and CBA/J (#000656) strains were obtained from the Jackson Laboratory and maintained within UVA’s animal facility. Swiss Webster (#024) mice were purchased from Charles River Laboratories. B2m fl/fl mice were generously provided by Dr. Wayne Yokoyama from Washington University. Cre lines were bred with B2m fl/fl mice to produce Gfap cre/+ x B2m fl/fl , ROSA26 Ai6/Ai6 x Cx3cr1 CreERT+/−2 x B2m fl/fl , and ROSA26 Ai6/Ai6 x Cx3cr1 CreERT2+/− x H2-Ab1 fl/fl mouse lines. The Me49 type II strain of T. gondii was maintained in vivo and passaged through chronically infected (3–12 months) Swiss Webster and CBA/J mice. For experimental infections with the Me49 strain, tissue cysts were prepared from homogenized brains of chronically infected (3–8 weeks) CBA/J mice. Mice were then inoculated i.p. with 10 tissue cysts of Me49 in 200 µl of 1X PBS (Gibco Cat#14190144). Mice infected and used for studies were monitored and euthanized if they showed weight loss greater than 20% of their pre-infection bodyweight. Tamoxifen treatment: To induce cre-expression and excision of B2m and Iab1 for the Cre ERT2 driven mouse lines ( Cx3cr1 CreERT2 ) , tamoxifen (Sigma-Aldrich Cat#T5645) was dissolved in corn oil (Sigma-Aldrich Cat#C8267) and filtered through a 0.45 µm filter (Millipore Cat#SLGSM33SS). At four to six weeks old, age and sex-matched mice were i.p. injected with tamoxifen (200 mg/kg) every other day for a total of five injections. Four weeks was allowed for turnover of peripheral macrophages prior to parasite infection. RNA sequencing analysis: RNA reads from FASTQ files were trimmed and filtered using Trimmomatic (v0.39) paired end set to phred 33 quality scoring. Adapters were trimmed, and reads with a minimum quality score of 15, leading and trailing quality scores of 3, and minimum fragment length of 36 were used for analysis. FastQC (v0.11.9) was used to verify quality of sample reads. Trimmed and filtered reads were aligned to thee GENCODE M13 reference genome using Salmon (v0.8.2) and output as sam files. Transcript abundance files were imported into R (v4.1.1) and converted to gene abundances using Tximport (v1.24.0). The R Bioconductor package, DESeq2 (v1.36.0), was used to perform differential expression analysis. DESeq2-normalized data was visualized using the following R packages: ComplexHeatmap (v2.25.2) and ggplot2 (v4.0.2). Gene names were converted from mouse ENSEMBL gene identifiers to gene symbols using the Bioconductor BiomaRT (v2.52.0) database. Labeled genes were manually selected from significantly differentially expressed genes from the DESeq2 results data frame. All genes with a Benjamini-Hochberg (BH) adjusted p-value below 0.05 were considered significantly upregulated if they had a log2FC > 0.5, and downregulated if they had a log2FC < -0.5. Enrichment score is reported as the -log10 of enrichment p value, based on Kolmogorov-Smirnov (KS) analysis. For targeted analysis of antigen presentation and processing genes, the GO term was used, and the top 30 expressed genes were plotted. Parasite burden quantification: DNA was isolated from whole brain homogenate using the Isolate II Genomic DNA Kit (Bioline, BIO-52067). Prior to isolation, brains were first homogenized in 1X PBS using the Omni TH tissue homogenizer (Omni International). Amplification of T. gondii 529 bp repeat region using the SensiFAST Probe No-Rox Kit (Bioline, BIO-86005) and CFX384 Real-Time System (Bio-Rad) was performed as previously described 84 . Tissue DNA (500 ng) was loaded into each reaction. T. gondii isolated from human foreskin fibroblasts (HFFs) was used to generate a serial standard curve from 3-300,000 genome copies and determine the number of T. gondii genomes per µg of tissue DNA. To measure brain parasite by cyst counts, whole brains were first placed in 4 mL of complete RMPI and passed through an 18-gauge and then 23-gauge (BD, Cat# 305155) needle to homogenize tissue. 30 µL of brain homogenate was then mounted on a slide and T. gondii cysts were manually counted using a DM2000 LED bright-field microscope. RT-qPCR: Brain homogenate was inoculated in Trizol (Fisher Scientific Cat#15-596-026). RNA was extracted according to manufacturer’s (Invitrogen) protocol. cDNA was then generated using a High-Capacity Reverse Transcription Kit (Applied Biosystems Cat# 4374967). Quantitative PCR was performed using 2X Taq based Master Mix (Bioline Cat#21105) and Taq Man gene expression assays (ThermoScientific Cat#4331182). Samples were run on a CFX384 Real-Time System thermocycler (Bio-Rad Laboratories). Genes were normalized to murine Hprt and the 2 (−ΔΔCT) method was used to analyze relative expression 85 . The following Thermofisher mouse gene probes were used: Hprt (Mm00446968_m1), Ifng (Mm01168134_m1), B2m (Mm00437762_m1), H2-Aa (Mm00439211_m1), Grzmb (Mm00442837_m1), Tnf (Mm00443258_m1), Nos2 (Mm00440502_m1), Cxcl9 (Mm00434946_m1), Cxcl10 (Mm00445235_m1), Icam (Mm00516023_m1), Vcam (Mm01320970_m1). Custom primers for used for analyzing T. gondii genomic DNA and gene expression were used as previously described 18 . Tissue processing for flow cytometry: After transcardiac perfusion of mice using 20 mL of cold 1X PBS, brains were collected into cold complete RPMI media (cRPMI; 10% FBS [Gibco], 1% penicillin/streptomycin [Gibco], 1% sodium pyruvate [Gibco], 1% non-essential amino acids [Gibco], and 0.1% 2-Mercaptoethanol [Life Technologies]). Brains were then passed through an 18-gauge and 23-gauge needle for mechanical homogenization. For immune cell isolation, tissue was digested in a solution containing collagenase/dispase (0.227 mg/mL, Sigma-Aldrich) and DNase (50 U/ml, Roche) at 37°C for 45 minutes. For isolating astrocytes, tissue was instead triturated using a 10 mL pipette, then digested in a solution containing Papain (4 U/mL) (Worthington Biochemical, Cat#LS003126) at 37°C for 45 minutes, with repeated trituration every 15 minutes. Digested brains were then passed through a 70-µm strainer (Corning) and washed with cRPMI. Myelin was separated out from mononuclear cells by resuspending samples in 20 mL of 40% Percoll (Cytiva, Cat#17-0891-02) and centrifuging at 650g for 25 minutes. Myelin was aspirated, and the remaining cell pellets were washed in cRPMI, resuspended, and kept on ice until plating. For ex vivo cytokine stimulation, cells were resuspended in cRPMI with Brefeldin A (20 µg/ml) (Selleckchem, Cat#S7046) or Brefeldin A, Phorbol 12-Myristate 13-Acetate (PMA) (200 ng/ml) (Sigma Aldrich, Cat#P1585), and Ionomycin (1 µg/ml) (Sigma Aldrich, Cat#I0634) for 5 hours at 37° C. Spleens were harvested into cold cRPMI, mechanically homogenized, and passed through a 40-µm strainer (Fisher Scientific, Cat#08-771-1). Cells were resuspended in red-blood cell (RBC) lysis buffer (0.16M NH 4 Cl) for 2 minutes. Samples were washed and resuspended with cRPMI and kept on ice until plating. Cells counts were acquired by diluting 1:10 in 0.4% trypan blue solution (Sigma-Aldrich Cat#T8154) and counted on a hemocytometer (Hausser Scientific Cat#3110) using a DM 2000 LED brightfield microscope (Leica). Flow Cytometry: Single cell suspensions were plated in a 96 well plate and subsequently resuspended in Fc Block, comprised of FACS buffer (1X PBS, 0.2% BSA, and 2mM EDTA) with 0.1 µg/mL 2.4G2 Ab (BioXCell, Cat#CUS-HB-197) and 0.1% rat gamma globulin (Jackson Immunoresearch, Cat#012-000-002) for 10 minutes. Cells were stained for surface markers and, for extracellular T cell and myeloid panels, eBioscience fixable live/dead viability dye 780 (1:800, Thermo Fisher Scientific, Cat#50-112-9035) or, for intracellular cytokine staining panel, eBioscience fixable live/dead viability dye efluor506 (1:800, Thermo Fisher Scientific, Cat#65-0866-14) for 30 minutes at 4°C. Cells were then washed twice with 50 µL FACS buffer. For intracellular staining, cells were fixed with fixation/permeabilization solution (eBioscience, 00-5123-43 and 00-5223-56) overnight at 4°C. Cells were then washed twice with 50 µL permeabilization buffer (eBioscience, 00-8333-56) and stained for intracellular markers in 1X perm buffer for 30 minutes at room temperature. Subsequently, they were washed twice with 1X perm buffer and finally resuspended in 200 µL FACS buffer. They were then acquired on a 3 or 5 laser Cytek Aurora Flow Cytometry System or the Gallios Flow Cytometer. Data was analyzed using FlowJo software v10.9.0. The following antibodies at 1:200 were used: CD45-AF700 (BioLegend, Cat#103128), CD45-eFlour 450 (Thermo Scientific, Cat#48-0451-82) CD11b-PerCP Cy5.5 (Thermo Fisher Scientific, Cat#45-0112-80), iNOS-APC (Thermo Fisher Scientific, Cat#17-5920-82), MHCII-Super Bright 780 (Thermo Fisher Scientific, Cat#78-5321-82), CD4-BV650 (Thermo Fisher Scientific, Cat#563232), CD8-BV421 (Thermo Fisher Scientific, Cat#563898), CD8-PerCP-Cy5.5 (Thermo Fisher Scientific, Cat#45-0081-82 ), Foxp3-eFlour 450 (Thermo Fisher Scientific, Cat#48-5773-82), IFNγ-PerCPCy5.5 (Thermo Fisher Scientific, Cat#45-7311-82), TNFα-PE (Thermo Fisher Scientific, Cat#12-7321-81), TCRβ-APC (Thermo Fisher Scientific, Cat#17-5961-81), Ly6C-PE (Thermo Fisher Scientific, Cat#12-5932-82), Ly6C-PE/Cy7 (Thermo Fisher Scientific, Cat#25-5932-82), Ki67-PE/Cy7 (Thermo Fisher Scientific, Cat#25-5698-82), B220-PE-Cy5 (Thermo Fisher Scientific, Cat#15-0452-82), NK1.1-SB780 (Thermo Scientific, Cat#78-5941-82), Ly6g-BV711 (Biolegend, Cat#127643), H2K b /H2D b -PE/Cy7 (BioLegend, Cat#114616), H2K b /H2D b -PE (BioLegend, Cat# 114608). The following antibodies were used at 1:50 dilution: GLAST-PE (Miltenyi Biotech, Cat#130-118-344). The follow antibody was used at a 1:20 dilution: Granzyme-B-APC (Thermo Fisher Scientific, Cat#GRB05). Astrocyte Purification: For purification of astrocytes, brains were harvested and processed as described above. ACSA2 + astrocytes were then isolated by magnetic bead enrichment according to manufacturer protocol (Miltenyi Biotech, Cat# 130-097-678). Immunofluorescence: For immunofluorescence, brains were bisected along the sagittal midline and either immediately fresh frozen on dry ice or fixed in cold 4% PFA (EMS Cat#15710-S) for 24 hr at 4° C. Fixed brains were then cryoprotected in 30% sucrose for 24 hr at 4° C, embedded in OCT (Tissue Tek Cat#25608-930), and frozen on dry ice. Tissue blocks were stored at -20°C until needed for further analysis. 30–50 µm fixed sections were then prepared using a CM1950 cryostat (Leica) and stored in 1X PBS as free-floating sections. For fresh frozen tissue, 10–15 µm sections were immediately mounted onto charged glass slides (Fisher Scientific Cat#1255015) and allowed to dry at room temperature overnight prior to staining. To immunostain brain sections, the slices were first incubated in a blocking solution [2% normal donkey serum] (Jackson ImmunoResearch Cat#017-000-121), 1% BSA, 0.05% Tween 20 (Fisher Scientific Cat#BP337), and 0.5% Triton X-100 (Sigma-Aldrich Cat#028SK001) in 1 X PBS) at room temperature for 1 hour. Then, tissue was stained for 1 hour at room temperature or overnight at 4° C with primary antibodies in blocking solution. Samples were washed three times in 0.05% Tween 20 solution and stained with secondary antibodies for 1 hr at room temperature in blocking solution. Finally, tissues were washed three times and mounted onto glass slides using AquaMount (Polysciences Cat#18606), and coverslipped (Globe Scientific Cat#1419). In some experiments, the tissue was counter-stained with DAPI (ThermoScientific Cat#62248) and washed just before mounting onto slides. Slides were dried, AquaMount (Polysciences Cat#18606) was applied, and coverslipped (Globe Scientific Cat#1419). Primary antibodies included: anti-Me49 (1:10,000 dilution) (gift from Fausto Araujo), GFAP (1:200 dilution) (DAKO Cat#Z0334) or (Invitrogen Cat#130300), MHCI (1:100 dilution) (Abcam Cat#ab15681). Secondary antibodies were used at 1:400 dilution. To stain Me49 and GFAP (rabbit): donkey anti-rabbit- AF594 (Jackson Cat#711585152); to stain MHCI and GFAP (rat): donkey anti-rat-AF647 (Jackson Cat#712605150). Images were acquired using a Leica Stellaris 5 confocal microscope and processed using Fiji software 86 . Statistical Analysis: All data was graphed in GraphPad Prism 9. Statistical analyses were performed using Prism software (v8.4) or RStudio (v 4.4.2) statistical packages. A two-tailed Student’s t-test was used to compare two independent groups. To account for biological variation between experiments, data compiled from experimental replicates was analyzed in R using a randomized block ANOVA, where experimental groups were modeled as a fixed effect and experimental day as a random effect 87 . For time course experiment data, gene expression data were log₂-transformed to improve normality, and differences across timepoints were evaluated using one-way ANOVA with Tukey’s post hoc multiple comparisons test. Outliers were identified and removed using ROUTs method with a Q value of 1 88 . Data from flow cytometric analyses and qPCR results were graphed using Graph-Pad Prism and data related to transcriptomic analyses were graphed using R. Error bars indicate standard error of the mean (s.e.m). The test used for each experiment is denoted in the figure legend, and p -values are denoted with ns = not significant, p < 0.05(*), p < 0.01(**), and p < 0.001 (***). Abbreviations CNS Central Nervous System TE Toxoplasmic encephalitis MHCI Major histocompatibility complex I MHCII Major histocompatibility complex II DEGs Differentially expressed genes GO Gene ontology qPCR Quantitative polymerase chain reaction APC Antigen presenting cell iNOS Inducible nitric oxide synthase AIDS Acquired Immunodeficiency Syndrome BBB Blood-brain barrier IFNg Interferon-gamma cDC1s Conventional type 1 dendritic cells cDC2s Conventional type 2 dendritic cells EAE Experimental autoimmune encephalitis BEC Brain endothelial cell b2m Beta-2 microglobulin PBS Phosphate buffered saline RT-qPCR Reverse transcription-quantitative polymerase chain reaction Wpi Weeks post infection RNA Ribonucleic acid TNFa Tumor necrosis factor alpha DNA Deoxyribonucleic acid MFI Mean fluorescence intensity GFAP Glial fibrillary acidic protein GrzmB Granzyme-B TMEV Theiler’s murine encephalomyelitis virus HFFs Human foreskin fibroblasts BFA Brefeldin A PMA Phorbol 12-Myristate 13-Acetate FACS Fluorescence-activated cell sorting ANOVA Analysis of Variance Declarations Ethics approval and consent to participate: All procedures involving animal care and use were approved by and conducted in accordance with the University of Virginia’s Institutional Animal Care and Use Committee (IACUC) under protocol number 3968. Consent for publication: Not applicable Availability of Data and Materials: All data needed to support the conclusions of this paper are present in the paper and/or the Supplementary Materials, with all data points shown. The dataset supporting the conclusions of this article will be available in FigShare upon publication. Bulk RNA-sequencing data will be available on GEO upon publication. Competing Interests: The authors have no financial or personal conflicts of interest to declare. Funding: National Institutes of Health grants R01NS112516 and R01NS134747 (THH); 5T32NS115657 (SAL); F30AI154740, 5T32AI007496 and 5T32GM007267 (MAK); 5T32GM008715 (LAS); T32AI007496 (MNC, IWB, and AEM); T32AI007046 (AGK). This work was also funded by the University of Virginia Harrison Undergraduate Award (AES), Wagner Fellowship (SAL), Virginia Brain Institute Fellowship (SAL), Pinn Scholars Award (THH), Shannon Fellowship (THH), and Strategic Investment Fund (THH). Author Contributions: Conceptualization: SAL, MAK, MNC, THH; Methodology: SAL, MAK, MNC, THH; Investigation: SAL, AES, MAK, MNC, LAS, AGK, AEM; Formal Analysis: SAL, MAK, MNC, MJL, LAS; Data Curation: SAL, MJL; Resources: AGK, THH; Writing-original draft: SAL; Writing-review and editing: SAL, AES, MAK, MNC, MJL, IWB, LAS, AGK, AEM, THH; Visualization: SAL, MJL; Project Administration: THH; Supervision: THH; Funding Acquisition: THH. All authors read and approved the final manuscript. Acknowledgements: We would like to thank members of the Center for Brain Immunology and Glia (BIG) and Department of Neuroscience at the University of Virginia for their scientific input throughout this project and access to instrumentation. We thank Stephanie Moy for help with initial pilot experiments. We thank Marieke K. Jones for her guidance with statistical analyses and R programming. 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Cowan","email":"","orcid":"","institution":"University of Virginia","correspondingAuthor":false,"prefix":"","firstName":"Maureen","middleName":"N.","lastName":"Cowan","suffix":""},{"id":632560784,"identity":"c4fe3d20-2f0f-4c28-851c-908f8e1cc48f","order_by":4,"name":"Mark J. Lawson","email":"","orcid":"","institution":"University of Virginia","correspondingAuthor":false,"prefix":"","firstName":"Mark","middleName":"J.","lastName":"Lawson","suffix":""},{"id":632560785,"identity":"9825406d-ff56-49f8-823f-d1fe864b7bc5","order_by":5,"name":"Isaac W. Babcock","email":"","orcid":"","institution":"University of Virginia","correspondingAuthor":false,"prefix":"","firstName":"Isaac","middleName":"W.","lastName":"Babcock","suffix":""},{"id":632560786,"identity":"c66710fc-78fc-466f-bc65-9ff96bc6d28a","order_by":6,"name":"Lydia A. 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Harris","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCklEQVRIiWNgGAWjYHCChAOMDUCKmYHxAYMBAwMbA5SLC/AgaWE2IFYLA0QNULEEkgRuLfbsBx4e+LjDLt+8nf1ZNU/BPbs+6ebGDwwV1okNuGzhSUg4OPNMsuWcwzxmt3kMipPbZA42SzCcScethSEh4TBvG7OBBDMP280ZBgnJbBKJDRKMbYdxa+F/kHD4b1s9UAv7s0KoluYfjP/waJEA2gI0E6iFwYzhg0GCHVBLmwRjAx4tNx4kHOxtOw5ymLEEUEsCSItFwrF0Y1xa2Ptzkj/8bKs2kOA//vBDwp8Ee/kZ6Y9vfKixlsWlBWhPAgoX4p4ETHXI9hxA4drjVTwKRsEoGAUjEgAAQ8BXA3W699sAAAAASUVORK5CYII=","orcid":"","institution":"University of Virginia","correspondingAuthor":true,"prefix":"","firstName":"Tajie","middleName":"H.","lastName":"Harris","suffix":""}],"badges":[],"createdAt":"2026-04-27 16:54:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9544619/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9544619/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109468002,"identity":"6e8c8d16-ddf6-4551-a960-161e70f5357d","added_by":"auto","created_at":"2026-05-18 12:31:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":960603,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntigen presentation machinery is upregulated in the CNS in response to \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. gondii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection. \u003c/strong\u003eC57BL/6 mice were mock-infected with PBS or infected intraperitoneally (i.p.) with 10 cysts of the Me49 strain of \u003cem\u003eT. gondii\u003c/em\u003e, and whole brains were harvested at 4 weeks post-infection (4wpi) for bulk RNA-sequencing. \u003cstrong\u003eA)\u003c/strong\u003e Volcano plot indicating differential gene expression between whole brain samples for naïve and chronically infected mice, with top 30 DEGs from the GO Term “Antigen processing and presentation” (GO: 0019882) indicated on plot. \u003cstrong\u003eB)\u003c/strong\u003e Heatmap displaying these top 30 DEGs in naïve and infected brains. \u003cstrong\u003e(C-E)\u003c/strong\u003e RT-qPCR analysis of Ifng\u003cem\u003e \u003c/em\u003e\u003cstrong\u003e(C)\u003c/strong\u003e\u003cem\u003e,\u003c/em\u003e B2m\u003cem\u003e \u003c/em\u003e\u003cstrong\u003e(D)\u003c/strong\u003e, and H2-Aa \u003cstrong\u003e(E) \u003c/strong\u003eexpression in whole mouse brain across harvested at seven timepoints from naïve to 42dpi. Data normalized to naïve timepoint. \u003cstrong\u003eF)\u003c/strong\u003e Experimental paradigm for labeling of ZsGreen+ CNS-resident macrophages and infection. \u003cstrong\u003eG)\u003c/strong\u003e Representative flow cytometry gating for CD11b+CD45intZsGreen+ microglia MHCI and MHCII expression in naïve and infected samples. \u003cstrong\u003e(H-I)\u003c/strong\u003e Flow cytometric quantification of microglial MHCI and MHCII expression at naïve and 4wpi. n=4 mice/group (\u003cstrong\u003eA-B\u003c/strong\u003e), n=2-3 mice/timepoint (\u003cstrong\u003eC-E\u003c/strong\u003e), in n=3-4 mice/group (\u003cstrong\u003eG-I\u003c/strong\u003e). For \u003cstrong\u003eA-B\u003c/strong\u003e statistical significance was defined in the differential gene expression analysis as an adjusted p value \u0026lt; 0.05. For \u003cstrong\u003eC-E \u003c/strong\u003edata were log₂-transformed and evaluated using one-way ANOVA with Tukey’s post hoc multiple comparisons test. For \u003cstrong\u003eH-I\u003c/strong\u003e, statistical significance determined using unpaired t-test, Data are presented as mean ± s.e.m., *** = p \u0026lt; 0.001. \u003cstrong\u003e(F)\u003c/strong\u003e made using Biorender.com.\u003c/p\u003e","description":"","filename":"Figure1Antigenpresentationgeneral1.png","url":"https://assets-eu.researchsquare.com/files/rs-9544619/v1/da649e997eb055b36042899c.png"},{"id":109468003,"identity":"cf4c067b-429d-4f66-b180-6c539242b56c","added_by":"auto","created_at":"2026-05-18 12:31:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":722993,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCNS-resident macrophage MHCI is dispensable for control of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. gondii \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003einfection\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ewithin the brain. A)\u003c/strong\u003e Experimental schematic for tamoxifen administration and infection of MGWT and MGB2m groups. \u003cstrong\u003eB)\u003c/strong\u003e Representative histogram of MHCI expression on ZsGreen+ microglia. \u003cstrong\u003eC)\u003c/strong\u003e Frequency of ZsGreen+ microglia expressing MHCI to examine efficacy of excision in MGB2m compared to MGWT mice. \u003cstrong\u003eD)\u003c/strong\u003e Representative flow gating for CD8+IFNg+ populations. Flow cytometric quantification of \u003cstrong\u003eE)\u003c/strong\u003e frequency of IFNg+ within the TCRb+CD8+ population and \u003cstrong\u003eF)\u003c/strong\u003e total number of TCRb+CD8+IFNg+ cells in the brains in MGWT and MGB2m mice. \u003cstrong\u003eG)\u003c/strong\u003e RT-qPCR analysis of Ifng\u003cem\u003e \u003c/em\u003ein whole mouse brain of MGWT and MGB2m mice. \u003cstrong\u003eH)\u003c/strong\u003e Representative flow gating for iNOS+ (CD45hiCD11b+Ly6G-Ly6ChiNOS+) populations. Flow cytometric quantification of \u003cstrong\u003eI)\u003c/strong\u003e frequency and \u003cstrong\u003eJ)\u003c/strong\u003e total number of iNOS+ within the CD45hiCD11b+Ly6G-Ly6Chi population in the brains of MGWT and MGB2m mice. \u003cstrong\u003eK)\u003c/strong\u003e RT-qPCR analysis of Nos2\u003cem\u003e \u003c/em\u003ein whole brains of MGWT and MGB2m mice. To quantify parasite burden: \u003cstrong\u003eL)\u003c/strong\u003e manual cyst counts \u003cstrong\u003eM)\u003c/strong\u003e RT-qPCR for genomic parasite DNA and \u003cstrong\u003eN)\u003c/strong\u003e RT-qPCR for \u003cem\u003eT. gondii\u003c/em\u003e-specific Act1. Statistical significance was determined via randomized block ANOVA compiled from 3-4 experiments with n=10-12 mice/group \u003cstrong\u003e(C-F),\u003c/strong\u003e and n=15 mice/group \u003cstrong\u003e(G, I-N).\u003c/strong\u003e Data are presented as mean ± s.e.m; * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001, ns; not significant. \u003cstrong\u003e(A)\u003c/strong\u003e made using Biorender.com.\u003c/p\u003e","description":"","filename":"Figure2MicrogliaMHCIPresentation1.png","url":"https://assets-eu.researchsquare.com/files/rs-9544619/v1/95bb28b1e3ef6046357585c6.png"},{"id":109468005,"identity":"1b65414e-fa54-48a2-af2c-83b6bde4942d","added_by":"auto","created_at":"2026-05-18 12:31:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":891703,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCNS-resident macrophage MHCII deletion does not impact immune responses or control of CNS parasite burden. A)\u003c/strong\u003e Experimental schematic for tamoxifen administration and infection. \u003cstrong\u003eB)\u003c/strong\u003e Representative histogram of MHCII expression by ZsGreen+ microglia in MGWT and MGMHCII and \u003cstrong\u003eC)\u003c/strong\u003e frequency of ZsGreen+ microglia expressing MHCII to examine efficacy of knockdown of MHCII in MGMHCII mice compared to MGWT. \u003cstrong\u003eD)\u003c/strong\u003e Representative flow gating for TCRb+CD4+IFNg+ populations. Flow cytometric quantification of \u003cstrong\u003eE)\u003c/strong\u003e frequency and \u003cstrong\u003eF) \u003c/strong\u003etotal number of IFNg+ within TCRb+CD4+ cells in the brain. \u003cstrong\u003eG)\u003c/strong\u003e RT-qPCR analysis of Ifng\u003cem\u003e \u003c/em\u003ein whole mouse brain. \u003cstrong\u003eH)\u003c/strong\u003e Representative flow gating for iNOS+ (CD45hiCD11b+Ly6G-Ly6ChiNOS+) populations. Flow cytometric quantification of \u003cstrong\u003eI)\u003c/strong\u003e frequency and \u003cstrong\u003eJ)\u003c/strong\u003e total number of iNOS+ within the CD45hiCD11b+Ly6G-Ly6Chi population in the brain. \u003cstrong\u003eK)\u003c/strong\u003e RT-qPCR analysis of Nos2\u003cem\u003e \u003c/em\u003ein whole mouse brain. To quantify parasite burden: \u003cstrong\u003eL)\u003c/strong\u003e manual cyst counts \u003cstrong\u003eM)\u003c/strong\u003e RT-qPCR for genomic parasite DNA and \u003cstrong\u003eN)\u003c/strong\u003e RT-qPCR for \u003cem\u003eT. gondii\u003c/em\u003e-specific Act1.\u0026nbsp; Statistical significance was determined via randomized block ANOVA compiled from 3-4 experiments with n=9-10 mice/group \u003cstrong\u003e(C),\u003c/strong\u003e and n=13-16 mice/group \u003cstrong\u003e(E-G, I-N).\u003c/strong\u003e Data are presented as mean ± s.e.m;\u0026nbsp; ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001, and ns; not significant.\u003cstrong\u003e (A)\u003c/strong\u003e made using Biorender.com.\u003c/p\u003e","description":"","filename":"Figure3MicrogliaMHCIIPresentation1.png","url":"https://assets-eu.researchsquare.com/files/rs-9544619/v1/c17116b5cecf02711b1fe54e.png"},{"id":109468019,"identity":"36f5e21f-bd66-4056-a343-01bdc95901cc","added_by":"auto","created_at":"2026-05-18 12:34:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1546376,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAstrocytes upregulate MHCI in response to \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. gondii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection. A)\u003c/strong\u003e Representative flow cytometry plots showing MHCI expression on GLAST⁺ astrocytes from naïve and 6wpi infected brains, pre-gated on singlets, live cells, and CD45⁻CD31⁻ populations. \u003cstrong\u003eB)\u003c/strong\u003e Frequency and \u003cstrong\u003eC)\u003c/strong\u003e Mean Fluorescence Intensity (MFI) of GLAST⁺ astrocytes expressing MHCI in naïve and infected mice. \u003cstrong\u003eD)\u003c/strong\u003e Representative IHC images showing MHCI (yellow) expression and GFAP⁺ astrocytes (white) in infected brain tissue. Scale bar= 100 μm. Inset highlight colocalization of MHCI⁺ GFAP+ astrocytes (arrowheads). \u003cstrong\u003eE)\u003c/strong\u003e Experimental schematic for astrocyte-specific deletion of \u003cem\u003eB2m\u003c/em\u003e using \u003cem\u003eGFAP-Cre × B2mfl/fl\u003c/em\u003e mice during chronic infection. \u003cstrong\u003eF)\u003c/strong\u003e RT-qPCR analysis examining B2m expression in purified ACSA2-astrocytes from Cre⁺ mice compared to Cre⁻ controls. n= 4-5 mice/group \u003cstrong\u003e(B-C)\u003c/strong\u003e and n= 5-6 mice/group \u003cstrong\u003e(F).\u003c/strong\u003e Statistical significance determined using unpaired t-test, data presented as mean ± s.e.m. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure4AstrocytesupregulateMHCI1.png","url":"https://assets-eu.researchsquare.com/files/rs-9544619/v1/89099d7b044eba60144ffdb5.png"},{"id":109468029,"identity":"57719a83-f74a-4a89-a112-30925a5304d0","added_by":"auto","created_at":"2026-05-18 12:35:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1940343,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAstrocyte-specific MHCI deletion increases \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. gondii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e parasite burden in the brain. (A)\u003c/strong\u003e Total counts of parasite cysts per brain in \u003cem\u003eGFAP-Cre-/- B2mfl/fl\u003c/em\u003e (Cre-) and \u003cem\u003eGFAP-Cre+/- B2mfl/fl\u003c/em\u003e (Cre+) mice. \u003cstrong\u003e(B)\u003c/strong\u003e qPCR of \u003cem\u003eT. gondii\u003c/em\u003e genomic DNA isolated from the brain. \u003cstrong\u003e(C)\u003c/strong\u003e RT-qPCR of parasite-specific \u003cstrong\u003eAct1\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003efrom whole brain RNA\u003c/strong\u003e. \u003cstrong\u003e(D)\u003c/strong\u003e Ratio of parasite \u003cstrong\u003eSag1\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(tachyzoite-specific gene) to Bag1\u003c/strong\u003e (bradyzoite-specific gene) normalized to Act1 expression. \u003cstrong\u003e(E–H)\u003c/strong\u003e Representative immunofluorescence images of infected brain tissue from \u003cem\u003eGFAP-Cre⁻/-\u003c/em\u003e (\u003cstrong\u003eE,G\u003c/strong\u003e) and \u003cem\u003eGFAP-Cre+/-\u003c/em\u003e (\u003cstrong\u003eF,H)\u003c/strong\u003e \u003cem\u003eB2m\u003c/em\u003efl/fl mice showing parasites (Me49, red), astrocytes (GFAP, green), and nuclei (DAPI, blue). Scale bar = 50 μm. \u003cstrong\u003e(A-D) \u003c/strong\u003eStatistical significance was determined via randomized block ANOVA compiled from 4 experiments with n=16-19 mice/group. Data are presented as mean ± s.e.m; *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure5Astrocyteparasiteburden1.png","url":"https://assets-eu.researchsquare.com/files/rs-9544619/v1/5585b9aed6a56d61e9bca356.png"},{"id":109468008,"identity":"a02a45e7-b92c-409b-a2c8-8fe70a6b5cb1","added_by":"auto","created_at":"2026-05-18 12:31:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":788743,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAstrocyte MHCI deletion enhances CD4⁺ T cell–driven immune responses. A)\u003c/strong\u003e Total numbers of TCRβ⁺ T cells, \u003cstrong\u003eB)\u003c/strong\u003e CD4⁺ TCRβ⁺ T cells, and \u003cstrong\u003eC)\u003c/strong\u003e CD8⁺ TCRβ⁺ T cells in the brains of \u003cem\u003eGFAP-Cre-/-\u003c/em\u003e and \u003cem\u003eGFAP-Cre\u003c/em\u003e+/-\u003cem\u003eB2mfl/fl\u003c/em\u003e mice at 6wpi. \u003cstrong\u003eD)\u003c/strong\u003e Representative flow plots and quantification of the \u003cstrong\u003eE)\u003c/strong\u003e frequency and \u003cstrong\u003eF)\u003c/strong\u003e total number of CD8⁺IFNγ⁺ T cells. \u003cstrong\u003eG) \u003c/strong\u003eRepresentative flow plots and quantification of the \u003cstrong\u003eH)\u003c/strong\u003e frequency and \u003cstrong\u003eI)\u003c/strong\u003e total number of IFNγ⁺ CD4⁺ T cells. \u003cstrong\u003eJ) \u003c/strong\u003eFrequency and \u003cstrong\u003eK)\u003c/strong\u003e total number of iNOS⁺ Ly6Chi inflammatory monocytes in the brain. \u003cstrong\u003eL)\u003c/strong\u003e RT-qPCR analysis of Nos2 expression in whole brain homogenate. \u003cstrong\u003eM)\u003c/strong\u003e RT-qPCR expression of chemokine and adhesion molecule genes (\u003cstrong\u003eCxcl9, Cxcl10, Icam1, Vcam1\u003c/strong\u003e) in whole brain tissue. Statistical significance was determined via randomized block ANOVA compiled from 2-4 experiments with n=16-19 mice/group (\u003cstrong\u003eA-C, E-F, H-I, J-L\u003c/strong\u003e), n=9-14 mice/group (\u003cstrong\u003eM\u003c/strong\u003e) data are represented as mean ± s.e.m. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003e\u003cstrong\u003ep \u003c/strong\u003e\u003c/em\u003e\u0026lt; 0.01; ns, not significant.\u003c/p\u003e","description":"","filename":"Figure6Astrocyteimmuneresponses1.png","url":"https://assets-eu.researchsquare.com/files/rs-9544619/v1/cefb7ce7e1d979ec5bc753b6.png"},{"id":109469352,"identity":"cdb22245-0091-4b8d-9d8d-413af09b86d7","added_by":"auto","created_at":"2026-05-18 12:41:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6241321,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9544619/v1/482b3a08-63ad-47fd-8a2d-f25263882ec9.pdf"},{"id":108949997,"identity":"ec0cd90d-530e-42a2-9b4c-3b120068b0f9","added_by":"auto","created_at":"2026-05-11 07:01:09","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":8194417,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaldatacombinedfinal.docx","url":"https://assets-eu.researchsquare.com/files/rs-9544619/v1/27e33f9735c0449cf1bca7c0.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Astrocytic but not Microglial Antigen Presentation Shapes Protective Immunity to Toxoplasma gondii in the Brain","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eT cells emerge as critical orchestrators of the immune response during many central nervous system (CNS) infections, shaping host-protective mechanisms essential for host survival\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. One prominent brain-tropic pathogen is \u003cem\u003eToxoplasma gondii\u003c/em\u003e, an intracellular protozoan parasite that establishes a lifelong chronic infection in the CNS across a range of mammalian hosts, including humans\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. In immunocompetent individuals, \u003cem\u003eT. gondii\u003c/em\u003e infection is typically asymptomatic due to effective immune control. However, in immunocompromised populations, such as individuals with AIDS or organ transplant recipients, reactivation of the parasite can lead to toxoplasmic encephalitis (TE), a potentially fatal uncontrolled infection of the brain\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. In murine models, deficiencies in T cell responses lead to unchecked parasite proliferation and lethal TE, underscoring the indispensable role of T cells in controlling chronic \u003cem\u003eT. gondii\u003c/em\u003e infection\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10 CR11 CR12 CR13\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAlthough the CNS is no longer considered strictly immune-privileged, it retains unique immunological features\u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Under homeostatic conditions, the brain exhibits minimal expression of antigen-presenting machinery and lacks conventional antigen-presenting cells (APCs), such as dendritic cells\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In the setting of a neurotropic pathogen, like \u003cem\u003eT. gondii\u003c/em\u003e, this prompts a central question of how CD8⁺ T cell responses are sustained and how infected cells are recognized and eliminated within the CNS. Studies have shown that microglia, the resident innate immune cells of the brain, upregulate antigen presentation machinery following an insult to the brain, including in the context infection or neurodegeneration\u003csup\u003e\u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22 CR23 CR24 CR25\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. This suggests one mechanism by which the brain becomes a more immunologically reactive tissue. The blood-brain barrier (BBB) at steady-state serves as a stringent gatekeeper to limit immune cell infiltration into the brain parenchyma\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. During infection with \u003cem\u003eT. gondii\u003c/em\u003e, the brain vasculature becomes activated to allow the robust recruitment of parasite-specific T cells into the brain parenchyma\u003csup\u003e\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Of note, once recruited, T cells can only persist within the tissue if their cognate antigen is expressed at this site\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThere is growing interest in understanding the dual role of CD8\u003csup\u003e+\u003c/sup\u003e T cells in the brain, where they can act as both pathogenic and protective agents during infection, neurodegeneration, and injury\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan additionalcitationids=\"CR34 CR35 CR36 CR37 CR38\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. It is known that coordinated interferon-gamma (IFNγ) production by both CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells is a critical component of the immune response to \u003cem\u003eT. gondii\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. In addition to cytokine production, CD8⁺ T cells mediate host-protection through cytolytic killing, recognizing infected cells via Major histocompatibility complex I (MHCI) antigen presentation, and eliminating them through perforin- and granzyme-dependent mechanisms\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Perforin production by CD8\u003csup\u003e+\u003c/sup\u003e T cells was found to be dispensable in the peripheral phase of \u003cem\u003eT. gondii\u003c/em\u003e infection, but necessary for control of cyst burden in the chronic brain phase of infection\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. This demonstrates that cytolytic killing by CD8\u003csup\u003e+\u003c/sup\u003e T cells is an integral component of immunity to \u003cem\u003eT. gondii\u003c/em\u003e in the brain. Conventional type 1 dendritic cells (cDC1s) have been shown to be a key antigen-presenting cell type that promotes expansion of parasite-specific T cells at peripheral sites during early \u003cem\u003eT. gondii\u003c/em\u003e infection\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. However, these cells largely remain at the brain borders during infection and are rarely found within deeper brain parenchyma, leaving it an open question as to which cells interact with CD8\u003csup\u003e+\u003c/sup\u003e T cells once they transit into the brain\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCNS antigen presentation to date has been most thoroughly studied in experimental autoimmune encephalitis (EAE) where antigen-specific T cells infiltrate the CNS parenchyma and drive disease pathogenesis\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. In this context, dendritic cells recruited into the CNS parenchyma have been identified as critical APCs via Major histocompatibility complex class II (MHCII) while microglial presentation proved dispensable\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Recent literature has expanded our understanding of antigen presentation within the CNS, revealing unexpected complexity among resident and infiltrating cell types. Microglia have been shown to upregulate MHCI and II molecules during infection and to putatively cross-present antigen to CD8⁺ T cells in the context of vesicular stomatitis virus\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Despite rarely found actively infected by \u003cem\u003eT. gondii\u003c/em\u003e, this cross-presentation would allow microglia to display phagocytosed antigen via MHCI or II to stimulate T cell function or be targeted for cytolytic killing\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Several studies have implicated microglia, perivascular macrophages, and brain endothelial cells (BECs) in priming CD8⁺ T cells for CNS entry during viral infections\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Although microglia are widely recognized as professional APCs in the CNS, the potential for other resident cells to present antigen for targeted cytolytic killing remains unknown.\u003c/p\u003e \u003cp\u003eAstrocytes are glial cells well known for their homeostatic functions in maintaining the BBB, metabolizing glutamate, stabilizing extracellular concentration of potassium, and producing trophic survival factors for neurons and other glia\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Foundational studies, largely \u003cem\u003ein vitro\u003c/em\u003e, recognized astrocytes as non-traditional APCs that upregulate antigen presentation machinery in autoimmune disorders and infection\u003csup\u003e\u003cspan additionalcitationids=\"CR56 CR57 CR58 CR59 CR60\" citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. In the context of \u003cem\u003eT. gondii\u003c/em\u003e, astrocytes play a critical role in parasite containment through IFN-driven responses and form heterogeneous reactive subpopulations during chronic infection\u003csup\u003e\u003cspan additionalcitationids=\"CR63\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Neurons are the predominant cell type with active infection of \u003cem\u003eT. gondii\u003c/em\u003e, with a smaller subset of astrocytes observed harboring parasite throughout the course of infection\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. As such, neurons and astrocytes represent potential sources of \u003cem\u003eT. gondii\u003c/em\u003e antigen for presentation via MHCI to CD8⁺ T cells, with the capacity to either promote T cell function, act as targets of cytolytic killing, or contribute to both processes. Using a mouse model in which neurons express the H2-L\u003csup\u003ed\u003c/sup\u003e allele, an MHCI variant known to drive an immunodominant, protective CD8\u003csup\u003e+\u003c/sup\u003e T cell response, studies have implicated neuronal antigen presentation in controlling parasite burden during \u003cem\u003eT. gondii\u003c/em\u003e infection\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. Neurons have also been shown, along with microglia, to contribute to the shaping of resident memory T cell populations in a model of latent \u003cem\u003eT. gondii\u003c/em\u003e infection\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. These studies point to potential roles of antigen presentation by infected cells during chronic neuroinflammatory states. Despite these recent advances, significant gaps remain in our understanding of how CNS-resident cells use antigen presentation to shape CD8⁺ T cell function and control of parasite during chronic neuroinflammation.\u003c/p\u003e \u003cp\u003eIn this study, we aimed to define how CNS-resident macrophage and astrocyte antigen presentation modulates T cell responses and parasite control during chronic \u003cem\u003eT. gondii\u003c/em\u003e infection. To address this question, we employed cell type-specific knockouts of the \u003cem\u003eB2m\u003c/em\u003e gene, a critical component of the MHCI complex, on astrocytes and CNS-resident macrophages. We hypothesized that ablation of MHCI expression in one or both cell types would impair CD8⁺ T cell\u0026ndash;mediated immunity or targeted killing, leading to increased parasite burden in the brain. We additionally generated a CNS-specific macrophage knockout of MHCII to examine how this would impact CD4\u003csup\u003e+\u003c/sup\u003e T cell function and parasite burden in the brain. MHCI or MHCII-deficient CNS-resident macrophages displayed no immune impairment, suggesting dispensable function or compensation by other cells presenting antigen within the CNS. We found that loss of MHCI in astrocytes resulted in impaired parasite control during chronic infection. We also observed heightened immune activity, including elevated cytokine production by infiltrating CD4⁺ T cells and increased production of iNOS⁺ by inflammatory monocytes. These findings reveal a unique role for astrocyte antigen presentation in parasite control during chronic CNS infection and provide new insight into the specialized immunological landscape of the brain.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eMicroglia upregulate antigen presentation machinery during chronic\u003c/b\u003e \u003cb\u003eToxoplasma gondii\u003c/b\u003e \u003cb\u003einfection.\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo examine the timing of this upregulation during infection, RT-qPCR analysis of whole brain homogenate from mice harvested at various timepoints from day 0 (naive) to day 42 (D42) post-infection was performed. Interferon-γ (IFNγ) promotes the upregulation of antigen presentation machinery, including MHC molecules, in brain-resident cells during inflammation\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. When we examined dynamics of gene expression of \u003cem\u003eIfng\u003c/em\u003e across infection, we observed an increased pattern of expression from 8 dpi up until 42 dpi \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. The gene \u003cem\u003eβ2m\u003c/em\u003e, a key component of the MHCI complex, followed a similar pattern of expression across infection in the brain \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. Further, gene expression of \u003cem\u003eH2-Aa\u003c/em\u003e, a key MHC Class II gene, by RT-qPCR showed a similar upregulation, with a peak during chronic infection \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e. Together, these data indicate that CNS infection is accompanied by a coordinated, sustained induction of IFNγ and both MHCI and MHCII gene expression, peaking during chronic infection. Observing these changes in the whole brain across infection, we aimed to hone in on in which CNS-resident cells\u0026rsquo; antigen presentation is functionally relevant for control of infection.\u003c/p\u003e \u003cp\u003eDue to their role as the innate immune cell of the brain, we hypothesized microglia would serve as robust antigen presenters during chronic infection. We used \u003cem\u003eROSA26\u003c/em\u003e\u003csup\u003eAi6/Ai6\u003c/sup\u003e x \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003eCreERT2+/\u0026minus;\u003c/sup\u003e mice, which express ZsGreen fluorescence specifically in \u003cem\u003eCx3Cr1\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e cells following cre activity induced by the application of tamoxifen\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. We administered five intraperitoneal (i.p.) injections of tamoxifen. We then waited four weeks for peripheral monocyte turnover, leaving labeling confined to longer-lived CNS-resident macrophages. We then mock-infected with PBS or infected mice i.p. with 10 cysts of the Type II strain of \u003cem\u003eT. gondii\u003c/em\u003e Me49 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e. Brains were harvested at 4 weeks post-infection (4 wpi) and flow cytometry was performed to examine antigen presentation markers \u003cb\u003e(Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA).\u003c/b\u003e\u003c/p\u003e \u003cp\u003eUpon infection, we measured robust populations of MHCI and MHCII expressing cells not present in the na\u0026iuml;ve brain. Preparing brain tissue for flow cytometric analyses results in a single cell suspension predominantly composed of immune cells and lacking CNS-resident cell types. To explore the immune cell populations expressing MHCI and MHCII during infection we categorized individual immune populations. At the whole brain level, concurrent with our RT-qPCR results, we observe very low expression of MHCI in the na\u0026iuml;ve state, but robust upregulation during infection \u003cb\u003e(Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB).\u003c/b\u003e Within the infected brain, as we expected, infiltrating immune cells abundantly express MHCI (~\u0026thinsp;44% Ly6C\u003csup\u003ehi\u003c/sup\u003e and ~\u0026thinsp;19% Ly6C\u003csup\u003elow\u003c/sup\u003e monocytes), and microglia upregulate MHCI expression \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-H, S1C\u003cb\u003e)\u003c/b\u003e. Correspondingly, we observe low expression of MHCII in the na\u0026iuml;ve state, but robust upregulation during infection \u003cb\u003e(Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD).\u003c/b\u003e Within the MHCII\u003csup\u003e+\u003c/sup\u003e population in the infected brain, we see this population comprised predominantly of infiltrating monocytes (~\u0026thinsp;42% Ly6C\u003csup\u003ehi\u003c/sup\u003e and ~\u0026thinsp;25% Ly6C\u003csup\u003elow\u003c/sup\u003e monocytes) and smaller populations of B cells and microglia (~\u0026thinsp;7% B cells and ~\u0026thinsp;6% microglia) \u003cb\u003e(Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eE)\u003c/b\u003e. Small numbers of dendritic cells (cDC1s and cDC2s) expressing both MHCI and MHCII are found within the meninges at this time point but are rarely found in the deeper brain parenchyma\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNarrowing in on the CD45\u003csup\u003eint\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eZsGreen\u003csup\u003e+\u003c/sup\u003e microglial population, as expected, we observed minimal expression of MHCI or MHCII molecules in the na\u0026iuml;ve state (~\u0026thinsp;2\u0026ndash;3%) but significant upregulation of these molecules during chronic infection in the brain (~\u0026thinsp;98\u0026ndash;99%), reflecting a similar pattern previously observed as early as 12dpi in the brain (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-I, \u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA\u003c/b\u003e)\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Although microglia comprised a relatively minor fraction of MHCI and MHCII-expressing cells compared to infiltrating immune cells, their drastic shift in expression of antigen presentation machinery highlighted them as candidates for promoting T cell function during CNS infection.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCNS-resident macrophage MHCI- antigen presentation is dispensable for control of\u003c/b\u003e \u003cb\u003eT. gondii\u003c/b\u003e \u003cb\u003eduring chronic infection.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSince we observe this robust upregulation of MHCI during infection by microglia, we wanted to elucidate the functional relevance of this to promoting T cell function and control of parasite. To address this question, we generated \u003cem\u003eROSA26\u003c/em\u003e\u003csup\u003eAi6/Ai6\u003c/sup\u003e x \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreERT2+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003ex B2m\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice, hereafter referred to as MG\u003csup\u003eB2m\u003c/sup\u003e, to ablate MHCI from \u003cem\u003eCx3Cr1\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e cells and allow fluorescent ZsGreen-labeling of these cells. We induced deletion of the \u003cem\u003eβ2m\u003c/em\u003e gene via five doses of tamoxifen administration. We waited four weeks to make the deletion specific to CNS-resident macrophages (i.e. microglia and border associated macrophages) and infected mice i.p. with 10 cysts of the Me49 \u003cem\u003eT. gondii\u003c/em\u003e strain. Mice progressed to the chronic phase of infection and brains were analyzed at 6 wpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. We examined immune responses in the brain at this timepoint using flow cytometry \u003cb\u003e(Fig. S2A).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe first examined efficacy of the genetic deletion and observed that compared to controls (MG\u003csup\u003eWT\u003c/sup\u003e) (~\u0026thinsp;98%), on average only\u0026thinsp;~\u0026thinsp;13% of microglia in the brains of knockout mice (MG\u003csup\u003eB2m\u003c/sup\u003e) expressed MHCI during chronic infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C). Microglia lacking MHCI had no change in sufficiency for expression of MHCII (\u003cb\u003eFig. S3A\u003c/b\u003e). By flow cytometry, we observed no differences in the total number of TCRβ\u003csup\u003e+\u003c/sup\u003e T cells, or TCRβ\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e and TCRβ\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells in the brains of knockout mice during chronic infection (\u003cb\u003eFig. S3B-D\u003c/b\u003e). Established mechanisms of parasite control in the brain include T cell\u0026ndash;derived IFNγ and tumor necrosis factor\u0026ndash;α (TNFα), along with downstream inducible nitric oxide synthase (iNOS) production by infiltrating inflammatory monocytes\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. When assessing functionality of these CD8\u003csup\u003e+\u003c/sup\u003e T cells to produce cytokine, we plated and incubated cells \u003cem\u003eex vivo\u003c/em\u003e with Brefeldin A (BFA). We observed decreased production of IFNγ when mice lack MHCI on CNS-resident macrophages by percentage of the overall CD8\u003csup\u003e+\u003c/sup\u003e population (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-E) but not by number (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). At the transcriptional level, we observed no difference in \u003cem\u003eIfng\u003c/em\u003e levels in the brains of knockout mice compared to WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Additionally, we observed no differences in TNFα production by CD8\u003csup\u003e+\u003c/sup\u003e T cells (\u003cb\u003eFig. S3E-F)\u003c/b\u003e or at the whole brain RNA level (\u003cb\u003eFig. S3G\u003c/b\u003e). We observed within the CD4\u003csup\u003e+\u003c/sup\u003e T cell population a decrease in production of both IFNγ and TNFα in our knockout mice by frequency but not by overall number present in the brain (\u003cb\u003eFig. S3H-K)\u003c/b\u003e. Collectively, these results demonstrate that loss of MHCI in CNS-resident macrophages does not alter T cell effector function, as measured by cytokine production.\u003c/p\u003e \u003cp\u003eAs a further measure of immune control, we measured production of iNOS by Ly6C\u003csup\u003ehi\u003c/sup\u003e infiltrating myeloid cells and found decreased percentage in the knockout mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH-I\u003cb\u003e).\u003c/b\u003e However, this did not correspond to a decrease in overall number of Ly6C\u003csup\u003ehi\u003c/sup\u003eiNOS\u003csup\u003e+\u003c/sup\u003e infiltrating myeloid cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ) or any difference in overall \u003cem\u003eNos2\u003c/em\u003e RNA level in the brains of these mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK). These results suggested an overall unimpaired immune response when CNS-resident macrophages lack MHCI, likely due to dispensable function or compensatory antigen presentation by other cells in the brain.\u003c/p\u003e \u003cp\u003eFinally, to assess how microglial antigen presentation affects control of parasite, we used three measures of parasite burden: cyst counts performed by brightfield microscopy, qPCR assay of parasite genomic DNA, and parasite \u003cem\u003eAct1\u003c/em\u003e gene expression from whole brain samples. While observing no significant differences in the quantity of cysts or abundance of parasitic genomic DNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL-M), we did observe a decrease in gene expression of parasite \u003cem\u003eAct1\u003c/em\u003e in the knockout mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eN\u003cb\u003e)\u003c/b\u003e. Given the lack of increased parasite burden and overall preserved immune responses, this suggests that MHCI by CNS-resident macrophages is dispensable for parasite control during chronic infection.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLoss of MHC Class II expression in CNS-resident macrophages does not impact immune control of\u003c/b\u003e \u003cb\u003eT. gondii\u003c/b\u003e \u003cb\u003ein the brain\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn addition to upregulating MHCI, we observed that CNS-resident macrophages robustly upregulate expression of MHCII during chronic \u003cem\u003eT. gondii\u003c/em\u003e infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-I)\u003csup\u003e18\u003c/sup\u003e. Previous work showed that when microglia lack the transcription factor \u003cem\u003eStat1\u003c/em\u003e and cannot respond to IFN-signaling, they do not upregulate MHCII and succumb to \u003cem\u003eT. gondii\u003c/em\u003e infection\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Having observed no significant deficit in immune control when CNS-resident macrophages lack MHCI expression, we hypothesized that these cells may preferentially present phagocytosed parasitic antigen via the MHCII pathway. To test this hypothesis, we generated \u003cem\u003eRosa26\u003c/em\u003e\u003csup\u003eAi6/Ai6\u003c/sup\u003e x \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreERT2+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003ex Iab\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e (MG\u003csup\u003eMHCII\u003c/sup\u003e) mice that ablate MHCII from CNS-resident macrophages. We administered tamoxifen at 4\u0026ndash;6 weeks of age, waited for 4 weeks, and mice were then subsequently infected. Mice were analyzed at a chronic infection timepoint of 4wpi by flow cytometry \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cb\u003eFig. S2A)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eWe examined efficacy of the genetic deletion and observed that compared to controls (MG\u003csup\u003eWT\u003c/sup\u003e) (~\u0026thinsp;98%), on average only\u0026thinsp;~\u0026thinsp;10% of microglia in the brains of knockout mice (MG\u003csup\u003eMHCII\u003c/sup\u003e) expressed MHCII during chronic infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-C\u003cb\u003e).\u003c/b\u003e We found MG\u003csup\u003eMHCII\u003c/sup\u003e mice remained sufficient for MHCI expression (\u003cb\u003eFig. S4A\u003c/b\u003e). We hypothesized that if CNS-resident macrophage MHCII presentation plays a role in sustaining T cell responses during chronic \u003cem\u003eT. gondii\u003c/em\u003e infection, a disruption in CNS-resident macrophage MHCII expression would result in reduced effector functions of CD4 T cells. Thus, we analyzed this population first by flow cytometry. By flow cytometry, we observed no differences in the total number of TCRβ\u003csup\u003e+\u003c/sup\u003e T cells, or TCRβ\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e and TCRβ\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells in the brains of knockout mice during chronic infection (\u003cb\u003eFig. S4B-D\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eWe observed no deficits in production of IFNγ by CD4\u003csup\u003e+\u003c/sup\u003e T cells either by measures of frequency of total CD4\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-E\u003cb\u003e)\u003c/b\u003e or in number of CD4\u003csup\u003e+\u003c/sup\u003eIFNγ\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Additionally, we observed no differences in overall level of \u003cem\u003eIfng\u003c/em\u003e in the brains of these mice at the RNA level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG\u003cb\u003e)\u003c/b\u003e. When assessing production of the cytokine TNFα, we observe an increase in CD4\u003csup\u003e+\u003c/sup\u003eTNFα\u003csup\u003e+\u003c/sup\u003e T cells by frequency of all CD4\u003csup\u003e+\u003c/sup\u003e T cells in MG\u003csup\u003eMHCII\u003c/sup\u003e brains, but no difference in overall number (\u003cb\u003eFig. S4E-F\u003c/b\u003e). At the whole brain level, there is also no difference in \u003cem\u003eTnf\u003c/em\u003e gene expression between MG\u003csup\u003eMHCII\u003c/sup\u003e and MG\u003csup\u003eWT\u003c/sup\u003e mice (\u003cb\u003eFig. S4G)\u003c/b\u003e. We also observed no change in CD8\u003csup\u003e+\u003c/sup\u003e T cell IFNγ and TNFα production by flow cytometry when microglia lack MHCII (\u003cb\u003eFig. S4H-K)\u003c/b\u003e. Taken together, these results demonstrate that loss of MHCII in microglia does not affect T cell effector function, as measured by cytokine production.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further examined downstream production of iNOS by Ly6C\u003csup\u003ehi\u003c/sup\u003e infiltrating inflammatory monocytes and observed no changes by percentage of iNOS\u003csup\u003e+\u003c/sup\u003e monocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH-I\u003cb\u003e)\u003c/b\u003e or by total number present in the brain (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ\u003cb\u003e).\u003c/b\u003e Further confirming these results, we found no difference in \u003cem\u003eNos2\u003c/em\u003e expression at the RNA level in our microglial knockouts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK\u003cb\u003e)\u003c/b\u003e. Additionally, we observed no difference in parasite burden by any measures when CNS-resident macrophages lacked MHCII expression \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL-N\u003cb\u003e).\u003c/b\u003e These results from our CNS-resident macrophage MHCI and MHCII knockouts suggest a dispensable role for microglial antigen presentation in the context of \u003cem\u003eT. gondii\u003c/em\u003e infection and prompted us to question a role for other CNS-resident cells within the CNS.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAstrocytes upregulate MHC Class I expression during chronic\u003c/b\u003e \u003cb\u003eT. gondii\u003c/b\u003e \u003cb\u003einfection.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWhile traditionally appreciated for their roles in maintenance of neuronal health and homeostasis, astrocytes have recently emerged as critical players in neuroimmunity\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e,\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e,\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. Despite being shown to upregulate antigen presentation machinery and stimulate T cell proliferation \u003cem\u003ein vitro\u003c/em\u003e, the capacity of astrocytes to act as antigen presenters \u003cem\u003ein vivo\u003c/em\u003e remains ambiguous\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. To investigate whether astrocytes are a relevant cell type in presenting antigen to CD8\u003csup\u003e+\u003c/sup\u003e T cells during \u003cem\u003eT. gondii\u003c/em\u003e infection, C57BL/6 mice were either mock-infected with PBS or infected with 10 cysts of the Type II strain Me49 and brains harvested 6 weeks later to assess expression of astrocytic MHCI. Through flow cytometry analysis, we found GLAST+ cells had very little expression of MHCI (~\u0026thinsp;2%) in the PBS-injected na\u0026iuml;ve group, compared to a significant increase in MHCI during infection (~\u0026thinsp;60%) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B, \u003cb\u003eFig. S5A).\u003c/b\u003e Further, by Mean Fluorescence Intensity (MFI) we observe a significant increase in the abundance of MHCI present on the GLAST\u003csup\u003e+\u003c/sup\u003e population during infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Additionally, in brains stained by immunofluorescence from chronically infected mice, we observe colocalization of GFAP\u003csup\u003e+\u003c/sup\u003e cells with MHCI staining, further indicative of upregulation of MHCI by astrocytes \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eTo determine how astrocytic MHCI expression dictates CD8\u003csup\u003e+\u003c/sup\u003e T cell function in the brain during infection, we crossed \u003cem\u003eGfap-77.6\u003c/em\u003e- cre to \u003cem\u003eB2m\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e mice to constitutively excise the gene \u003cem\u003eB2m\u003c/em\u003e from astrocytes. \u003cem\u003eGfap77.6\u003c/em\u003e-cre\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e-\u003cem\u003eB2m\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e (Cre-) control and \u003cem\u003eGfap77.6\u003c/em\u003e-cre\u003csup\u003e+/\u0026minus;\u003c/sup\u003e-\u003cem\u003eB2m\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e (Cre+) mice were infected with 10 cysts of the type II Me49 strain of \u003cem\u003eT. gondii\u003c/em\u003e and brains harvested 6 weeks later (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). To confirm excision efficiency of the \u003cem\u003eB2m\u003c/em\u003e gene in the knockout mice, ACSA2\u003csup\u003e+\u003c/sup\u003e astrocytes were purified, and RT-qPCR was performed for the gene \u003cem\u003eB2m\u003c/em\u003e. We observed an approximate\u0026thinsp;~\u0026thinsp;50% reduction in \u003cem\u003eB2m\u003c/em\u003e gene expression in \u003cem\u003eGfap77.6\u003c/em\u003e-cre\u003csup\u003e+/\u0026minus;\u003c/sup\u003e-\u003cem\u003eB2m\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e mice (Cre+) compared to control \u003cem\u003eGfap77.6\u003c/em\u003e-cre\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e-\u003cem\u003eB2m\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e (Cre-) mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). To examine intact capacity to present by more traditional APC populations, CD45\u003csup\u003eint\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e CNS-resident macrophages and infiltrating CD45\u003csup\u003ehi\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e macrophages were assessed by flow cytometry (\u003cb\u003eFig. S5B).\u003c/b\u003e We found canonical APCs were unaffected in \u003cem\u003eGfap\u003c/em\u003e-cre\u003csup\u003e+/\u0026minus;\u003c/sup\u003e\u003cem\u003eB2m\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e (Cre+) mice and were sufficient for MHCI expression comparable to controls (Cre-) mice (\u003cb\u003eFig. S5C-F\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMice deficient in astrocytic MHC class I display impaired parasitic control during chronic infection.\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo first assess how knockdown in astrocytic MHCI antigen presentation would affect control of parasite, we used three measures of parasite burden. We observed no difference in cyst counts between knockout (Cre+) and wildtype (Cre-) mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). However, by total parasite genomic DNA and \u003cem\u003eAct1\u003c/em\u003e gene expression, we observed increased parasite burden in the brains of mice with deletion of MHCI from astrocytes \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-C\u003cb\u003e).\u003c/b\u003e Further, we performed qPCR of \u003cem\u003eSag1\u003c/em\u003e and \u003cem\u003eBag1\u003c/em\u003e, genes specific to tachyzoite and bradyzoite parasite stages, respectively. We observe an increased ratio of \u003cem\u003eSag1/Bag1\u003c/em\u003e present when mice lack astrocytic MHCI, indicating an increase in the tachyzoite form of the parasite (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). This suggests an impairment of control of parasite replication. To visualize parasite in the brains of these mice, immunofluorescence with an astrocytic marker (GFAP) and an antibody against the Me49 parasite was performed. In agreement with our quantitative parasite burden data, we observed notable areas of tachyzoites (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-F) but no difference in the cyst form of the parasite (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-H).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAstrocyte MHC Class I-deficiency leads to increased CD4\u0026thinsp;+\u0026thinsp;T cell cytokine production.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eObserving the increase in parasite burden in knockout mice, we next assessed CD8\u003csup\u003e+\u003c/sup\u003e and CD4\u003csup\u003e+\u003c/sup\u003e T cell function when mice lack astrocytic MHCI. We hypothesized if astrocyte MHCI expression played a key role in coordinating CD8\u003csup\u003e+\u003c/sup\u003e T cell function, we would observe decreased effector T cell functions, such as cytokine (IFNγ and TNFα) and Granzyme B production. To assess their function, flow cytometric analysis was performed for immune cell populations at the 6-week timepoint. We observed no overall differences in the number of overall TCR\u0026szlig;\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), TCR\u0026szlig;\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), and TCR\u0026szlig;\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells in the brains of knockout mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). When we assessed production of the key cytokine IFNγ, we observed no deficits in production by TCR\u0026szlig;\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells either by frequency (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-E) or by overall number (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). We also observed no differences in the production of TNFα by TCR\u0026szlig;\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells when mice lack astrocytic MHCI (\u003cb\u003eFig. S6A-B\u003c/b\u003e). Intriguingly, we did observe increases in TCR\u0026szlig;\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cells producing IFNγ by frequency and number (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG-I), as well as those producing the cytokine TNFα (\u003cb\u003eFig. S6C-D)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eTo confirm astrocyte antigen presentation does not impact the capacity of T cells to produce cytokine, brain cells were stimulated \u003cem\u003eex vivo\u003c/em\u003e with PMA/ionomycin. We observed no differences in TCR\u0026szlig;\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e capacity to produce IFNγ and TNFα (\u003cb\u003eFig. S6E-F).\u003c/b\u003e PMA/ionomycin stimulated TCR\u0026szlig;\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e cells from brains lacking astrocytic MHCI did possess higher capacity to produce IFNγ but not TNFα when compared to controls (\u003cb\u003eFig. S6G-H).\u003c/b\u003e To examine deficits in cytotoxic function by CD8\u003csup\u003e+\u003c/sup\u003e T cells when astrocytes lack MHCI, staining was additionally performed for Granzyme B (GrzmB). We observed no difference in the production of Granzyme B by frequency, number, or MFI of TCR\u0026szlig;\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells (\u003cb\u003eFig. S6I-K\u003c/b\u003e). Further, at the whole brain RNA level, we observed no change in the amount of \u003cem\u003eGrzmB\u003c/em\u003e transcript in the Cre+ animals (\u003cb\u003eFig. S6L\u003c/b\u003e). Overall, these results suggest that there is not a deficit in cytotoxic potential or cytokine production by CD8\u003csup\u003e+\u003c/sup\u003e T cells. These results suggest that CD8⁺ T cell function is not directly affected, whereas altered CD4⁺ T cell responses point to increased inflammation as an indirect consequence of astrocyte MHCI deletion, due to increased parasite burden.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSince we observed increases in cytokine production by CD4\u003csup\u003e+\u003c/sup\u003e T cells, we hypothesized we would observe downstream effects on IFNγ-dependent processes, including the production of anti-parasitic iNOS by infiltrating monocytes. We observed no change in the total number of infiltrating myeloid cells when mice lack astrocytic MHCI \u003cb\u003e(Fig. S7A)\u003c/b\u003e. To assess production of iNOS by these cells, we performed flow cytometric analysis and observed increases in both the frequency of iNOS\u003csup\u003e+\u003c/sup\u003eLy6c\u003csup\u003ehi\u003c/sup\u003e inflammatory monocytes \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ\u003cb\u003e)\u003c/b\u003e, as well as total number in the brains of mice lacking astrocytic MHCI \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK\u003cb\u003e)\u003c/b\u003e. Further, we observed increased level of \u003cem\u003eNos2\u003c/em\u003e gene expression in the brain when mice lack astrocyte MHCI \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL\u003cb\u003e)\u003c/b\u003e. Further, we found a decreased frequency in CD4\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003e regulatory T cells within the total CD4\u003csup\u003e+\u003c/sup\u003e T cell population, but ultimately similar number of regulatory T cells between groups (\u003cb\u003eFig. S7C-D\u003c/b\u003e). This increased proportion of CD4\u003csup\u003e+\u003c/sup\u003e effector T cells supports findings of an overall enhanced inflammatory response to the increased parasite in the knockout mice. We further found increased expression in IFN-driven genes, including chemoattractants \u003cem\u003eCxcl9\u003c/em\u003e and \u003cem\u003eCxcl10\u003c/em\u003e and adhesion molecules \u003cem\u003eIcam\u003c/em\u003e and \u003cem\u003eVcam\u003c/em\u003e, which correspond with overall enhanced inflammation in the brains of the knockout mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eM\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eWhen assessing the spleen as an indicator of systemic immune activation in Cre+ knockout mice, we found no differences in the overall numbers of TCR\u0026szlig;\u003csup\u003e+\u003c/sup\u003e T cells, or TCR\u0026szlig;\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e and TCR\u0026szlig;\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e subsets (\u003cb\u003eFig. S7E-F\u003c/b\u003e). We observed no difference in the numbers of proliferative CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells within the spleens of these mice compared to controls at this timepoint (\u003cb\u003eFig. S7F\u003c/b\u003e). Taken together, this suggests that the enhanced immune response is specific to the brains of these knockout animals. These observations indicate that when astrocytes lack the ability to present antigen to cytotoxic T cells, parasite replication increases, and leads to an enhanced immune response driven by cytokine producing CD4\u003csup\u003e+\u003c/sup\u003e T cells.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOur study reveals that MHCI antigen presentation by astrocytes, but not CNS-resident macrophages, is important in control of \u003cem\u003eToxoplasma gondii\u003c/em\u003e replication within the brain. We found that when CNS-resident macrophages lacked MHCI or MHCII expression, we observed no deficits in parasite control or immune responses. However, we demonstrate that the absence of MHCI expression on astrocytes results in an increase in parasite. The increase in parasite led to enhanced rather than deficient immune responses driven by CD4\u003csup\u003e+\u003c/sup\u003e T cell cytokine production and downstream monocyte iNOS production. These findings highlight a previously undescribed role for how astrocytes present antigen to promote control of \u003cem\u003eT. gondii\u003c/em\u003e in the CNS. These results provide not only greater insight into host\u0026ndash;parasite interactions in the brain, but also how T cell recognition of infected astrocytes plays a role in control of pathogen.\u003c/p\u003e \u003cp\u003eMuch of the work in the neuroimmune space has emphasized microglia as the central APCs and modulators of neuroinflammatory responses within the CNS\u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan additionalcitationids=\"CR71 CR72 CR73\" citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. More recently, this perspective has expanded, as interest grows in how T cells contribute to brain immunity across diverse contexts, from infectious diseases to neurodegenerative disorders. This shift reflects a broader recognition that antigen presentation in the CNS is more complex and multifaceted than previously thought.\u003c/p\u003e \u003cp\u003eOur group previously demonstrated that interferon signaling in microglia is essential for control of \u003cem\u003eT. gondii\u003c/em\u003e, as mice lacking \u003cem\u003eStat1\u003c/em\u003e in microglia succumb early in chronic infection\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Interferon signaling drives robust upregulation of antiparasitic effector pathways, including \u003cem\u003eIrg\u003c/em\u003e and \u003cem\u003eGbp\u003c/em\u003e family genes, along with antigen presentation machinery, highlighting the importance of interferon in enabling microglia to mount an effective response\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Despite rarely being found infected \u003cem\u003ein vivo\u003c/em\u003e in mice, when microglia lack \u003cem\u003eStat1\u003c/em\u003e signaling, microglia were found to be harboring parasite, suggesting an innate ability to clear parasite by these cells\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. While these findings establish microglia as indispensable for parasite control, the results of the present study indicate that antigen presentation itself is a dispensable component of this protective role.\u003c/p\u003e \u003cp\u003eFindings across disease models illustrate the context-dependent nature of microglial antigen presentation in the CNS. In EAE, dendritic cells rather than microglia appear to be the dominant APCs coordinating CD4⁺ T cell responses, consistent with our observation that microglial MHCII is dispensable during chronic \u003cem\u003eT. gondii\u003c/em\u003e infection\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Moreover, our data align with previous findings demonstrating that loss of \u003cem\u003eTap\u003c/em\u003e in microglia does not affect total T cell populations or cytokine production in the brains of mice during latent \u003cem\u003eT. gondii\u003c/em\u003e infection\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In peripheral tissues, infected cDC1s are shown to play a key role in presentation of \u003cem\u003eT. gondii\u003c/em\u003e to promote an immune response, however they largely remain at the brain borders during chronic infection\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e. Our data demonstrate that CD11b⁺CD45\u003csup\u003ehi\u003c/sup\u003eLy6C\u003csup\u003elow\u003c/sup\u003e and Ly6C\u003csup\u003ehi\u003c/sup\u003e infiltrating peripheral populations constitute the largest contributors to the overall pool of MHCI⁺ and MHCII⁺ cells in the brain during infection. Thus, these cells could be poised to act as the predominant APCs throughout \u003cem\u003eT. gondii\u003c/em\u003e infection, compensating for any microglial dysfunction.\u003c/p\u003e \u003cp\u003eBy contrast, in a viral model of Theiler\u0026rsquo;s murine encephalomyelitis virus (TMEV), microglia and perivascular macrophages act as APCs to promote CD8\u003csup\u003e+\u003c/sup\u003e T cell infiltration into the brain\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Similarly, studies in tauopathy have shown that microglia regulate T cell entry and function in ways that exacerbate disease pathology\u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e. A key distinction between these models is the degree of peripheral myeloid cell infiltration into the brain. Some viral infections and neurodegenerative models exhibit limited recruitment of peripheral APCs to the brain parenchyma, whereas chronic \u003cem\u003eT. gondii\u003c/em\u003e infection and EAE involve substantial infiltration of monocytes and dendritic cells, respectively. Together, these findings suggest that microglia may play a more prominent APC role in settings where infiltrating professional APCs are scarce, while in highly inflammatory contexts with abundant peripheral APCs their contribution may be comparatively dispensable.\u003c/p\u003e \u003cp\u003eNeurons, as the principal cell observed with active infection, would be expected to encounter the highest levels of parasite-derived antigen. It is now appreciated that MHCI expression by neurons during development is crucial for synaptic pruning and refinement, but whether neurons upregulate and use MHCI functionally during disease remains unclear\u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e,\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e. Recent studies have aimed to understand how MHCI expression by neurons may play a role in control of \u003cem\u003eT. gondii\u003c/em\u003e. \u003cem\u003eIn vitro\u003c/em\u003e primary murine neurons respond to IFNγ to upregulate \u003cem\u003eIrg\u003c/em\u003es, \u003cem\u003eGbp2\u003c/em\u003e, \u003cem\u003eStat1\u003c/em\u003e, and \u003cem\u003eMhc1\u003c/em\u003e, as well as pretreatment with IFNγ leads to decreased levels of \u003cem\u003eT. gondii\u003c/em\u003e infected neurons\u003csup\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e. In a latent infection model of \u003cem\u003eT. gondii\u003c/em\u003e where C57BL/6 mice were generated to possess a floxed MHCI immunoprotective \u003cem\u003eH2-L\u003c/em\u003e\u003csup\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sup\u003e allele, knocking out \u003cem\u003eH2-L\u003c/em\u003e\u003csup\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sup\u003e from neurons results in greater cerebral parasite burden\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. These findings raise additional questions about how this process occurs \u003cem\u003ein vivo\u003c/em\u003e in C57BL/6 mice, which express the \u003cem\u003eH2-Dᵇ\u003c/em\u003e and \u003cem\u003eH2-Kᵇ\u003c/em\u003e MHCI alleles, and whether neurons that present antigen can directly interact with cytotoxic CD8⁺ T cells.\u003c/p\u003e \u003cp\u003eAlthough neurons represent the predominant cell type harboring \u003cem\u003eT. gondii\u003c/em\u003e in the brain, a small subset of infected astrocytes has been identified through the use of a Cre-secreting parasite\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. In this system, \u003cem\u003eT. gondii\u003c/em\u003e is engineered to secrete Cre-recombinase into host cells, enabling fluorescence in cells injected with parasite effector proteins during invasion\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. The relative rarity of infected astrocytes despite widespread parasite exposure suggests that these cells possess intrinsic or extrinsic immune-mediated mechanisms for parasite clearance, a topic that has received increasing attention in the field. Previous work has shown the necessity of \u003cem\u003eStat1\u003c/em\u003e-mediated interferon-signaling in astrocytes in restricting \u003cem\u003eT. gondii\u003c/em\u003e burden in the brain\u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e. This finding was inferred to be a deficit in the ability to upregulate anti-parasitic machinery, thus allowing astrocytes to serve as a residential niche for parasite\u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e. Recently, it was shown that astrocytes do not use caspase 8-mediated apoptosis as a parasite restriction mechanism during \u003cem\u003eT. gondii\u003c/em\u003e infection\u003csup\u003e\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e. While \u003cem\u003ein vitro\u003c/em\u003e studies have observed upregulation of MHCI by astrocytes and interactions in co-cultures with CD8\u003csup\u003e+\u003c/sup\u003e T cells, to date it has not been thoroughly explored how antigen presentation by astrocytes may contribute to host defense from pathogens \u003cem\u003ein vivo\u003c/em\u003e within the CNS\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e,\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e,8283\u003c/sup\u003e. Our findings directly address this gap by demonstrating that astrocytic MHCI upregulation is one mechanism for pathogen restriction during CNS infection. We propose that this effect is mediated through interactions with cytotoxic CD8⁺ T cells, enabling detection of and elimination of infected astrocytes.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eTogether, our findings establish astrocytes as a functionally important source of antigen presentation required for effective control of \u003cem\u003eT. gondii\u003c/em\u003e in the CNS. Although microglia robustly upregulate antigen presentation machinery during infection, their antigen presentation is dispensable for parasite control. By contrast, astrocyte MHCI expression plays a role in limiting pathogen burden, likely through display of parasite antigen by infected cells. This work provides novel insight into host\u0026ndash;pathogen dynamics within the infected brain, where professional immune cells such as microglia, while highly reactive, do not appear to function as critical APCs in promoting T cell function. Instead, effective immune control appears to depend in part on antigen presentation by infected parenchymal cells themselves. Given their abundance and association with neurons and sites of parasite reactivation, astrocytes are positioned to serve as sites of CD8\u003csup\u003e+\u003c/sup\u003e T cell immune surveillance and targeted pathogen clearance. However, this has not been thoroughly explored \u003cem\u003ein vivo\u003c/em\u003e, a gap addressed in the present study. Our finding gives new insight into host-pathogen dynamics and likely extends to other disease contexts in which astrocytes are directly infected, underscoring their broader role as sources of antigen that drive pathogen control through MHCI upregulation.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and Treatments:\u003c/h2\u003e \u003cp\u003e \u003cem\u003eGfap\u003c/em\u003e \u003csup\u003e \u003cem\u003eCre77.6\u003c/em\u003e \u003c/sup\u003e (#024098), \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreERT2\u003c/em\u003e\u003c/sup\u003e (#020940), \u003cem\u003eIab1\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e (#037709), ROSA26\u003csup\u003eAi6/Ai6\u003c/sup\u003e (#007906), and CBA/J (#000656) strains were obtained from the Jackson Laboratory and maintained within UVA\u0026rsquo;s animal facility. Swiss Webster (#024) mice were purchased from Charles River Laboratories. \u003cem\u003eB2m\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice were generously provided by Dr. Wayne Yokoyama from Washington University. Cre lines were bred with \u003cem\u003eB2m\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice to produce \u003cem\u003eGfap\u003c/em\u003e\u003csup\u003ecre/+\u003c/sup\u003e x \u003cem\u003eB2m\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eROSA26\u003c/em\u003e \u003csup\u003eAi6/Ai6\u003c/sup\u003e x \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreERT+/\u0026minus;2\u003c/em\u003e\u003c/sup\u003e \u003cem\u003ex B2m\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e, and \u003cem\u003eROSA26\u003c/em\u003e \u003csup\u003eAi6/Ai6\u003c/sup\u003e x \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreERT2+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003ex H2-Ab1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mouse lines.\u003c/p\u003e \u003cp\u003eThe Me49 type II strain of \u003cem\u003eT. gondii\u003c/em\u003e was maintained \u003cem\u003ein vivo\u003c/em\u003e and passaged through chronically infected (3\u0026ndash;12 months) Swiss Webster and CBA/J mice. For experimental infections with the Me49 strain, tissue cysts were prepared from homogenized brains of chronically infected (3\u0026ndash;8 weeks) CBA/J mice. Mice were then inoculated i.p. with 10 tissue cysts of Me49 in 200 \u0026micro;l of 1X PBS (Gibco Cat#14190144). Mice infected and used for studies were monitored and euthanized if they showed weight loss greater than 20% of their pre-infection bodyweight.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTamoxifen treatment:\u003c/h3\u003e\n\u003cp\u003eTo induce cre-expression and excision of \u003cem\u003eB2m\u003c/em\u003e and \u003cem\u003eIab1\u003c/em\u003e for the Cre\u003csup\u003eERT2\u003c/sup\u003e driven mouse lines (\u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreERT2\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e)\u003c/em\u003e, tamoxifen (Sigma-Aldrich Cat#T5645) was dissolved in corn oil (Sigma-Aldrich Cat#C8267) and filtered through a 0.45 \u0026micro;m filter (Millipore Cat#SLGSM33SS). At four to six weeks old, age and sex-matched mice were i.p. injected with tamoxifen (200 mg/kg) every other day for a total of five injections. Four weeks was allowed for turnover of peripheral macrophages prior to parasite infection.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRNA sequencing analysis:\u003c/h2\u003e \u003cp\u003eRNA reads from FASTQ files were trimmed and filtered using Trimmomatic (v0.39) paired end set to phred 33 quality scoring. Adapters were trimmed, and reads with a minimum quality score of 15, leading and trailing quality scores of 3, and minimum fragment length of 36 were used for analysis. FastQC (v0.11.9) was used to verify quality of sample reads. Trimmed and filtered reads were aligned to thee GENCODE M13 reference genome using Salmon (v0.8.2) and output as sam files. Transcript abundance files were imported into R (v4.1.1) and converted to gene abundances using Tximport (v1.24.0). The R Bioconductor package, DESeq2 (v1.36.0), was used to perform differential expression analysis. DESeq2-normalized data was visualized using the following R packages: ComplexHeatmap (v2.25.2) and ggplot2 (v4.0.2). Gene names were converted from mouse ENSEMBL gene identifiers to gene symbols using the Bioconductor BiomaRT (v2.52.0) database. Labeled genes were manually selected from significantly differentially expressed genes from the DESeq2 results data frame. All genes with a Benjamini-Hochberg (BH) adjusted p-value below 0.05 were considered significantly upregulated if they had a log2FC\u0026thinsp;\u0026gt;\u0026thinsp;0.5, and downregulated if they had a log2FC \u0026lt; -0.5. Enrichment score is reported as the -log10 of enrichment p value, based on Kolmogorov-Smirnov (KS) analysis. For targeted analysis of antigen presentation and processing genes, the GO term was used, and the top 30 expressed genes were plotted.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eParasite burden quantification:\u003c/h3\u003e\n\u003cp\u003eDNA was isolated from whole brain homogenate using the Isolate II Genomic DNA Kit (Bioline, BIO-52067). Prior to isolation, brains were first homogenized in 1X PBS using the Omni TH tissue homogenizer (Omni International). Amplification of \u003cem\u003eT. gondii\u003c/em\u003e 529 bp repeat region using the SensiFAST Probe No-Rox Kit (Bioline, BIO-86005) and CFX384 Real-Time System (Bio-Rad) was performed as previously described\u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e. Tissue DNA (500 ng) was loaded into each reaction. \u003cem\u003eT. gondii\u003c/em\u003e isolated from human foreskin fibroblasts (HFFs) was used to generate a serial standard curve from 3-300,000 genome copies and determine the number of \u003cem\u003eT. gondii\u003c/em\u003e genomes per \u0026micro;g of tissue DNA. To measure brain parasite by cyst counts, whole brains were first placed in 4 mL of complete RMPI and passed through an 18-gauge and then 23-gauge (BD, Cat# 305155) needle to homogenize tissue. 30 \u0026micro;L of brain homogenate was then mounted on a slide and \u003cem\u003eT. gondii\u003c/em\u003e cysts were manually counted using a DM2000 LED bright-field microscope.\u003c/p\u003e\n\u003ch3\u003eRT-qPCR:\u003c/h3\u003e\n\u003cp\u003eBrain homogenate was inoculated in Trizol (Fisher Scientific Cat#15-596-026). RNA was extracted according to manufacturer\u0026rsquo;s (Invitrogen) protocol. cDNA was then generated using a High-Capacity Reverse Transcription Kit (Applied Biosystems Cat# 4374967). Quantitative PCR was performed using 2X Taq based Master Mix (Bioline Cat#21105) and Taq Man gene expression assays (ThermoScientific Cat#4331182). Samples were run on a CFX384 Real-Time System thermocycler (Bio-Rad Laboratories). Genes were normalized to murine \u003cem\u003eHprt\u003c/em\u003e and the 2\u003csup\u003e(\u0026minus;ΔΔCT)\u003c/sup\u003e method was used to analyze relative expression\u003csup\u003e\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u003c/sup\u003e. The following Thermofisher mouse gene probes were used: \u003cem\u003eHprt\u003c/em\u003e (Mm00446968_m1), \u003cem\u003eIfng\u003c/em\u003e (Mm01168134_m1), \u003cem\u003eB2m\u003c/em\u003e (Mm00437762_m1), \u003cem\u003eH2-Aa\u003c/em\u003e (Mm00439211_m1), \u003cem\u003eGrzmb\u003c/em\u003e (Mm00442837_m1), \u003cem\u003eTnf\u003c/em\u003e (Mm00443258_m1), \u003cem\u003eNos2\u003c/em\u003e (Mm00440502_m1), \u003cem\u003eCxcl9\u003c/em\u003e (Mm00434946_m1), \u003cem\u003eCxcl10\u003c/em\u003e (Mm00445235_m1), \u003cem\u003eIcam\u003c/em\u003e (Mm00516023_m1), \u003cem\u003eVcam\u003c/em\u003e (Mm01320970_m1). Custom primers for used for analyzing \u003cem\u003eT. gondii\u003c/em\u003e genomic DNA and gene expression were used as previously described\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTissue processing for flow cytometry:\u003c/h2\u003e \u003cp\u003eAfter transcardiac perfusion of mice using 20 mL of cold 1X PBS, brains were collected into cold complete RPMI media (cRPMI; 10% FBS [Gibco], 1% penicillin/streptomycin [Gibco], 1% sodium pyruvate [Gibco], 1% non-essential amino acids [Gibco], and 0.1% 2-Mercaptoethanol [Life Technologies]). Brains were then passed through an 18-gauge and 23-gauge needle for mechanical homogenization. For immune cell isolation, tissue was digested in a solution containing collagenase/dispase (0.227 mg/mL, Sigma-Aldrich) and DNase (50 U/ml, Roche) at 37\u0026deg;C for 45 minutes. For isolating astrocytes, tissue was instead triturated using a 10 mL pipette, then digested in a solution containing Papain (4 U/mL) (Worthington Biochemical, Cat#LS003126) at 37\u0026deg;C for 45 minutes, with repeated trituration every 15 minutes. Digested brains were then passed through a 70-\u0026micro;m strainer (Corning) and washed with cRPMI. Myelin was separated out from mononuclear cells by resuspending samples in 20 mL of 40% Percoll (Cytiva, Cat#17-0891-02) and centrifuging at 650g for 25 minutes. Myelin was aspirated, and the remaining cell pellets were washed in cRPMI, resuspended, and kept on ice until plating. For \u003cem\u003eex vivo\u003c/em\u003e cytokine stimulation, cells were resuspended in cRPMI with Brefeldin A (20 \u0026micro;g/ml) (Selleckchem, Cat#S7046) or Brefeldin A, Phorbol 12-Myristate 13-Acetate (PMA) (200 ng/ml) (Sigma Aldrich, Cat#P1585), and Ionomycin (1 \u0026micro;g/ml) (Sigma Aldrich, Cat#I0634) for 5 hours at 37\u0026deg; C.\u003c/p\u003e \u003cp\u003eSpleens were harvested into cold cRPMI, mechanically homogenized, and passed through a 40-\u0026micro;m strainer (Fisher Scientific, Cat#08-771-1). Cells were resuspended in red-blood cell (RBC) lysis buffer (0.16M NH\u003csub\u003e4\u003c/sub\u003eCl) for 2 minutes. Samples were washed and resuspended with cRPMI and kept on ice until plating.\u003c/p\u003e \u003cp\u003eCells counts were acquired by diluting 1:10 in 0.4% trypan blue solution (Sigma-Aldrich Cat#T8154) and counted on a hemocytometer (Hausser Scientific Cat#3110) using a DM 2000 LED brightfield microscope (Leica).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFlow Cytometry:\u003c/h2\u003e \u003cp\u003eSingle cell suspensions were plated in a 96 well plate and subsequently resuspended in Fc Block, comprised of FACS buffer (1X PBS, 0.2% BSA, and 2mM EDTA) with 0.1 \u0026micro;g/mL 2.4G2 Ab (BioXCell, Cat#CUS-HB-197) and 0.1% rat gamma globulin (Jackson Immunoresearch, Cat#012-000-002) for 10 minutes. Cells were stained for surface markers and, for extracellular T cell and myeloid panels, eBioscience fixable live/dead viability dye 780 (1:800, Thermo Fisher Scientific, Cat#50-112-9035) or, for intracellular cytokine staining panel, eBioscience fixable live/dead viability dye efluor506 (1:800, Thermo Fisher Scientific, Cat#65-0866-14) for 30 minutes at 4\u0026deg;C. Cells were then washed twice with 50 \u0026micro;L FACS buffer. For intracellular staining, cells were fixed with fixation/permeabilization solution (eBioscience, 00-5123-43 and 00-5223-56) overnight at 4\u0026deg;C. Cells were then washed twice with 50 \u0026micro;L permeabilization buffer (eBioscience, 00-8333-56) and stained for intracellular markers in 1X perm buffer for 30 minutes at room temperature. Subsequently, they were washed twice with 1X perm buffer and finally resuspended in 200 \u0026micro;L FACS buffer. They were then acquired on a 3 or 5 laser Cytek Aurora Flow Cytometry System or the Gallios Flow Cytometer. Data was analyzed using FlowJo software v10.9.0.\u003c/p\u003e \u003cp\u003eThe following antibodies at 1:200 were used: CD45-AF700 (BioLegend, Cat#103128), CD45-eFlour 450 (Thermo Scientific, Cat#48-0451-82) CD11b-PerCP Cy5.5 (Thermo Fisher Scientific, Cat#45-0112-80), iNOS-APC (Thermo Fisher Scientific, Cat#17-5920-82), MHCII-Super Bright 780 (Thermo Fisher Scientific, Cat#78-5321-82), CD4-BV650 (Thermo Fisher Scientific, Cat#563232), CD8-BV421 (Thermo Fisher Scientific, Cat#563898), CD8-PerCP-Cy5.5 (Thermo Fisher Scientific, Cat#45-0081-82 ), Foxp3-eFlour 450 (Thermo Fisher Scientific, Cat#48-5773-82), IFNγ-PerCPCy5.5 (Thermo Fisher Scientific, Cat#45-7311-82), TNFα-PE (Thermo Fisher Scientific, Cat#12-7321-81), TCRβ-APC (Thermo Fisher Scientific, Cat#17-5961-81), Ly6C-PE (Thermo Fisher Scientific, Cat#12-5932-82), Ly6C-PE/Cy7 (Thermo Fisher Scientific, Cat#25-5932-82), Ki67-PE/Cy7 (Thermo Fisher Scientific, Cat#25-5698-82), B220-PE-Cy5 (Thermo Fisher Scientific, Cat#15-0452-82), NK1.1-SB780 (Thermo Scientific, Cat#78-5941-82), Ly6g-BV711 (Biolegend, Cat#127643), H2K\u003csup\u003eb\u003c/sup\u003e/H2D\u003csup\u003eb\u003c/sup\u003e-PE/Cy7 (BioLegend, Cat#114616), H2K\u003csup\u003eb\u003c/sup\u003e/H2D\u003csup\u003eb\u003c/sup\u003e-PE (BioLegend, Cat# 114608). The following antibodies were used at 1:50 dilution: GLAST-PE (Miltenyi Biotech, Cat#130-118-344). The follow antibody was used at a 1:20 dilution: Granzyme-B-APC (Thermo Fisher Scientific, Cat#GRB05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAstrocyte Purification:\u003c/h2\u003e \u003cp\u003eFor purification of astrocytes, brains were harvested and processed as described above. ACSA2\u0026thinsp;+\u0026thinsp;astrocytes were then isolated by magnetic bead enrichment according to manufacturer protocol (Miltenyi Biotech, Cat# 130-097-678).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence:\u003c/h2\u003e \u003cp\u003eFor immunofluorescence, brains were bisected along the sagittal midline and either immediately fresh frozen on dry ice or fixed in cold 4% PFA (EMS Cat#15710-S) for 24 hr at 4\u0026deg; C. Fixed brains were then cryoprotected in 30% sucrose for 24 hr at 4\u0026deg; C, embedded in OCT (Tissue Tek Cat#25608-930), and frozen on dry ice. Tissue blocks were stored at -20\u0026deg;C until needed for further analysis. 30\u0026ndash;50 \u0026micro;m fixed sections were then prepared using a CM1950 cryostat (Leica) and stored in 1X PBS as free-floating sections. For fresh frozen tissue, 10\u0026ndash;15 \u0026micro;m sections were immediately mounted onto charged glass slides (Fisher Scientific Cat#1255015) and allowed to dry at room temperature overnight prior to staining. To immunostain brain sections, the slices were first incubated in a blocking solution [2% normal donkey serum] (Jackson ImmunoResearch Cat#017-000-121), 1% BSA, 0.05% Tween 20 (Fisher Scientific Cat#BP337), and 0.5% Triton X-100 (Sigma-Aldrich Cat#028SK001) in 1 X PBS) at room temperature for 1 hour. Then, tissue was stained for 1 hour at room temperature or overnight at 4\u0026deg; C with primary antibodies in blocking solution. Samples were washed three times in 0.05% Tween 20 solution and stained with secondary antibodies for 1 hr at room temperature in blocking solution. Finally, tissues were washed three times and mounted onto glass slides using AquaMount (Polysciences Cat#18606), and coverslipped (Globe Scientific Cat#1419). In some experiments, the tissue was counter-stained with DAPI (ThermoScientific Cat#62248) and washed just before mounting onto slides. Slides were dried, AquaMount (Polysciences Cat#18606) was applied, and coverslipped (Globe Scientific Cat#1419).\u003c/p\u003e \u003cp\u003ePrimary antibodies included: anti-Me49 (1:10,000 dilution) (gift from Fausto Araujo), GFAP (1:200 dilution) (DAKO Cat#Z0334) or (Invitrogen Cat#130300), MHCI (1:100 dilution) (Abcam Cat#ab15681). Secondary antibodies were used at 1:400 dilution. To stain Me49 and GFAP (rabbit): donkey anti-rabbit- AF594 (Jackson Cat#711585152); to stain MHCI and GFAP (rat): donkey anti-rat-AF647 (Jackson Cat#712605150). Images were acquired using a Leica Stellaris 5 confocal microscope and processed using Fiji software\u003csup\u003e\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis:\u003c/h2\u003e \u003cp\u003eAll data was graphed in GraphPad Prism 9. Statistical analyses were performed using Prism software (v8.4) or RStudio (v 4.4.2) statistical packages. A two-tailed Student\u0026rsquo;s t-test was used to compare two independent groups. To account for biological variation between experiments, data compiled from experimental replicates was analyzed in R using a randomized block ANOVA, where experimental groups were modeled as a fixed effect and experimental day as a random effect\u003csup\u003e\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e\u003c/sup\u003e. For time course experiment data, gene expression data were log₂-transformed to improve normality, and differences across timepoints were evaluated using one-way ANOVA with Tukey\u0026rsquo;s post hoc multiple comparisons test. Outliers were identified and removed using ROUTs method with a Q value of 1\u003csup\u003e88\u003c/sup\u003e. Data from flow cytometric analyses and qPCR results were graphed using Graph-Pad Prism and data related to transcriptomic analyses were graphed using R. Error bars indicate standard error of the mean (s.e.m). The test used for each experiment is denoted in the figure legend, and \u003cem\u003ep\u003c/em\u003e-values are denoted with ns\u0026thinsp;=\u0026thinsp;not significant, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05(*), p\u0026thinsp;\u0026lt;\u0026thinsp;0.01(**), and p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 (***).\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCNS Central Nervous System\u003c/p\u003e\n\u003cp\u003eTE Toxoplasmic encephalitis\u003c/p\u003e\n\u003cp\u003eMHCI Major histocompatibility complex I\u003c/p\u003e\n\u003cp\u003eMHCII Major histocompatibility complex II\u003c/p\u003e\n\u003cp\u003eDEGs Differentially expressed genes\u003c/p\u003e\n\u003cp\u003eGO Gene ontology\u003c/p\u003e\n\u003cp\u003eqPCR Quantitative polymerase chain reaction\u003c/p\u003e\n\u003cp\u003eAPC Antigen presenting cell\u003c/p\u003e\n\u003cp\u003eiNOS Inducible nitric oxide synthase\u003c/p\u003e\n\u003cp\u003eAIDS Acquired Immunodeficiency Syndrome\u003c/p\u003e\n\u003cp\u003eBBB Blood-brain barrier\u003c/p\u003e\n\u003cp\u003eIFNg\u0026nbsp;Interferon-gamma\u003c/p\u003e\n\u003cp\u003ecDC1s Conventional type 1 dendritic cells\u003c/p\u003e\n\u003cp\u003ecDC2s Conventional type 2 dendritic cells\u003c/p\u003e\n\u003cp\u003eEAE Experimental autoimmune encephalitis\u003c/p\u003e\n\u003cp\u003eBEC Brain endothelial cell\u003c/p\u003e\n\u003cp\u003eb2m Beta-2 microglobulin\u003c/p\u003e\n\u003cp\u003ePBS Phosphate buffered saline\u003c/p\u003e\n\u003cp\u003eRT-qPCR Reverse transcription-quantitative polymerase chain reaction\u003c/p\u003e\n\u003cp\u003eWpi Weeks post infection\u003c/p\u003e\n\u003cp\u003eRNA Ribonucleic acid\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTNFa\u0026nbsp;Tumor necrosis factor alpha\u003c/p\u003e\n\u003cp\u003eDNA Deoxyribonucleic acid\u003c/p\u003e\n\u003cp\u003eMFI Mean fluorescence intensity\u003c/p\u003e\n\u003cp\u003eGFAP Glial fibrillary acidic protein\u003c/p\u003e\n\u003cp\u003eGrzmB Granzyme-B\u003c/p\u003e\n\u003cp\u003eTMEV Theiler\u0026rsquo;s murine encephalomyelitis virus\u003c/p\u003e\n\u003cp\u003eHFFs Human foreskin fibroblasts\u003c/p\u003e\n\u003cp\u003eBFA Brefeldin A\u003c/p\u003e\n\u003cp\u003ePMA Phorbol 12-Myristate 13-Acetate\u003c/p\u003e\n\u003cp\u003eFACS Fluorescence-activated cell sorting\u003c/p\u003e\n\u003cp\u003eANOVA Analysis of Variance\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures involving animal care and use were approved by and conducted in accordance with the University of Virginia\u0026rsquo;s Institutional Animal Care and Use Committee (IACUC) under protocol number 3968.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials: \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data needed to support the conclusions of this paper are present in the paper and/or the Supplementary Materials, with all data points shown. The dataset supporting the conclusions of this article will be available in FigShare upon publication. Bulk RNA-sequencing data will be available on GEO upon publication. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no financial or personal conflicts of interest to declare.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFunding: \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNational Institutes of Health grants R01NS112516 and R01NS134747 (THH); 5T32NS115657 (SAL); F30AI154740, 5T32AI007496 and 5T32GM007267 (MAK); 5T32GM008715 (LAS); T32AI007496 (MNC, IWB, and AEM); T32AI007046 (AGK). This work was also funded by the University of Virginia Harrison Undergraduate Award (AES), Wagner Fellowship (SAL), Virginia Brain Institute Fellowship (SAL), Pinn Scholars Award (THH), Shannon Fellowship (THH), and Strategic Investment Fund (THH). \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: SAL, MAK, MNC, THH; Methodology: SAL, MAK, MNC, THH; Investigation: SAL, AES, MAK, MNC, LAS, AGK, AEM; Formal Analysis: SAL, MAK, MNC, MJL, LAS; Data Curation: SAL, MJL; Resources: AGK, THH; Writing-original draft: SAL; Writing-review and editing: SAL, AES, MAK, MNC, MJL, IWB, LAS, AGK, AEM, THH; Visualization: SAL, MJL; Project Administration: THH; Supervision: THH; Funding Acquisition: THH. All authors read and approved the final manuscript.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank members of the Center for Brain Immunology and Glia (BIG) and Department of Neuroscience at the University of Virginia for their scientific input throughout this project and access to instrumentation. We thank Stephanie Moy for help with initial pilot experiments. We thank Marieke K. Jones for her guidance with statistical analyses and R programming. We thank Fausto Araujo at Palo Alto Medical Foundation for gifting us the rabbit anti-Me49 antibody used in this study. Parts of Fig. 1, 2, and 3 were generated using Biorender.com. \u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGigley JP, Bhadra R, Khan IA. CD8 T Cells and \u003cem\u003eToxoplasma gondii\u003c/em\u003e: A New Paradigm. \u003cem\u003eJ. Parasitol. 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BMC Bioinformatics. 2006;7:123.\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":"Antigen Presentation, Microglia, Astrocytes, Neuroimmunology, Toxoplasma gondii","lastPublishedDoi":"10.21203/rs.3.rs-9544619/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9544619/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eT cells play a pivotal role in orchestrating immune defense within the central nervous system (CNS) during many infections. \u003cem\u003eToxoplasma gondii\u003c/em\u003e, a brain-trophic protozoan parasite, establishes lifelong CNS infection that remains largely subclinical in immunocompetent hosts but can cause severe encephalitis in immunocompromised individuals. While CD8⁺ T cells are essential for controlling \u003cem\u003eT. gondii\u003c/em\u003e during chronic infection through both cytokine production and cytolytic killing, the CNS-resident cells that functionally present antigen in the brain to promote T cell function or serve as cytolytic targets remain incompletely defined. Here, we investigated the contributions of CNS-resident macrophages and astrocytes, two key CNS-resident cell types, antigen presentation during chronic \u003cem\u003eT. gondii\u003c/em\u003e infection. Using mice lacking MHCI or MHCII in CNS-resident macrophages, we found no impairment of immune responses or ability of the brain to control parasite, indicating dispensable function of resident macrophages as APCs during infection. However, deletion of MHCI on astrocytes led to deficits in parasite control, in turn promoting elevated CD4⁺ T cell cytokine production and recruitment of iNOS⁺ inflammatory monocytes. We observed increased presence of lytic parasite within the brain, which suggests that astrocyte MHCI may be necessary to control parasite replication throughout the CNS. Our findings underscore a previously underappreciated role for astrocytic MHCI within the CNS during infection and highlights the dispensability of CNS-resident macrophages to this process.\u003c/p\u003e","manuscriptTitle":"Astrocytic but not Microglial Antigen Presentation Shapes Protective Immunity to Toxoplasma gondii in the Brain","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-11 07:01:00","doi":"10.21203/rs.3.rs-9544619/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-11T16:00:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"305197734389997563567078039531155219487","date":"2026-04-30T15:02:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"288193191830937324919052070521733312309","date":"2026-04-30T13:24:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"215015332922177360969493715715633903808","date":"2026-04-29T17:07:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"323875715316629512644778341002556043537","date":"2026-04-28T13:06:14+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-28T12:23:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-28T11:56:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-28T10:32:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Neuroinflammation","date":"2026-04-27T16:39:25+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":"5b2618f9-40ce-4d8c-ac77-e3728faf8baf","owner":[],"postedDate":"May 11th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-11T16:00:12+00:00","index":18,"fulltext":""},{"type":"reviewerAgreed","content":"305197734389997563567078039531155219487","date":"2026-04-30T15:02:02+00:00","index":17,"fulltext":""},{"type":"reviewerAgreed","content":"288193191830937324919052070521733312309","date":"2026-04-30T13:24:56+00:00","index":16,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T12:41:06+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-11 07:01:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9544619","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9544619","identity":"rs-9544619","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}