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• W4313477592 abstract "Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract The discovery of meningeal lymphatic vessels that drain the CNS has prompted new insights into how immune responses develop in the brain. In this study, we examined how T cell responses against CNS-derived antigen develop in the context of infection. We found that meningeal lymphatic drainage promotes CD4+ and CD8+ T cell responses against the neurotropic parasite Toxoplasma gondii in mice, and we observed changes in the dendritic cell compartment of the dural meninges that may support this process. Indeed, we found that mice chronically, but not acutely, infected with T. gondii exhibited a significant expansion and activation of type 1 and type 2 conventional dendritic cells (cDC) in the dural meninges. cDC1s and cDC2s were both capable of sampling cerebrospinal fluid (CSF)-derived protein and were found to harbor processed CSF-derived protein in the draining deep cervical lymph nodes. Disrupting meningeal lymphatic drainage via ligation surgery led to a reduction in CD103+ cDC1 and cDC2 number in the deep cervical lymph nodes and caused an impairment in cDC1 and cDC2 maturation. Concomitantly, lymphatic vessel ligation impaired CD4+ and CD8+ T cell activation, proliferation, and IFN-γ production at this site. Surprisingly, however, parasite-specific T cell responses in the brain remained intact following ligation, which may be due to concurrent activation of T cells at non-CNS-draining sites during chronic infection. Collectively, our work reveals that CNS lymphatic drainage supports the development of peripheral T cell responses against T. gondii but remains dispensable for immune protection of the brain. Editor's evaluation Kovacs et al. provide important and valuable data on the role of meningeal lymphatic drainage in T cell responses during chronic Toxoplasma gondii (T.g.) infection in mice. They present compelling data showing dendritic cell (DC) accumulation in the dura and CSF at 6 weeks post-infection, which matches with the replication peak of T.g. in the brain, and with T cell expansion/activation in the draining lymph node (dCLN). They also convincingly show that during chronic infection, antigen-specific T cells are generated not only in the dCLN but also in the periphery (ILN), which could account for the presence of T cells in the brain after surgical blockade of the lymphatics. This study highlights also how the CNS is protected against environmental challenges. https://doi.org/10.7554/eLife.80775.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Although the presence of T cells in the CNS can be pathological, T cells play an essential role in orchestrating host-protective immune responses in the CNS during infection (Ellwardt et al., 2016; Korn and Kallies, 2017). At present, the biological mechanisms that drive the formation of T cell responses in the CNS remain poorly defined. One well-studied brain-tropic pathogen is Toxoplasma gondii, an intracellular protozoan parasite that causes chronic, lifelong infection in a wide range of mammalian hosts, including humans (Hill and Dubey, 2002; Pappas et al., 2009). Although the course of infection is typically asymptomatic in humans, severe neurologic disease can manifest in immunocompromised individuals, including HIV/AIDS patients (Luft and Remington, 1992). Consistent with this susceptibility, experimental studies have demonstrated that mice deficient in T cell responses are unable to protect the host from fatal disease (Gazzinelli et al., 1992). For this reason, murine infection with T. gondii is a useful model for investigating how T cell responses develop in the CNS. At homeostasis, the immunologically quiescent brain harbors very few T cells, but in response to T. gondii infection, parasite-specific T cells are actively and continuously recruited to the brain (Harris et al., 2012). The process is tightly regulated, as circulating T cells that have been activated in the periphery can only persist within the brain when their cognate antigen is expressed in the brain (Schaeffer et al., 2009; Wilson et al., 2009). Disrupting entry of newly activated T cells into the brain leads to impaired parasite restriction, indicating that ongoing T cell stimulation in the periphery is essential (Harris et al., 2012; Wilson et al., 2009). However, because the brain parenchyma does not harbor lymphatic vessels, it remains poorly understood how peripheral T cells become alerted to the presence of microbial antigen in the brain. In other organs, lymphatic vessels serve as conduits for the transport of tissue-derived antigen and dendritic cells to lymph nodes, where naive and memory T cells are optimally positioned for detection of their cognate antigen (Thomas et al., 2016; Gasteiger et al., 2016). The recent discovery of functional lymphatic vessels in the dura mater layer of meninges has prompted a significant reconsideration of how the CNS engages the peripheral immune system (Louveau et al., 2015b; Aspelund et al., 2015). Meningeal lymphatic vessels are observed in rodents, primates, and humans (Absinta et al., 2017; Albayram et al., 2022), and in experimental models of brain cancer and autoimmunity these vessels have been shown to play an integral role in regulating T cell responses in the CNS (Song et al., 2020; Louveau et al., 2018b). Mouse studies have demonstrated that meningeal lymphatic vessels convey macromolecules and immune cells from the meninges and cerebrospinal fluid (CSF) to the deep cervical lymph nodes (Louveau et al., 2018b). Indeed, when model antigens like ovalbumin (OVA) are injected into the brain, these molecules travel from the brain interstitium into the CSF via glymphatic flow (Iliff et al., 2012) and have the potential to be presented to T cells in the deep cervical lymph nodes (Ling et al., 2003; Harris et al., 2014). In a murine model of acute brain infection, it was shown that meningeal lymphatic drainage contributes to the clearance of Japanese encephalitis virus from the brain and promotes host survival (Li et al., 2022). However, questions remain regarding how the meningeal lymphatic system affects T cell responses against brain-derived microbial antigen and the specific role that the meninges and meningeal lymphatic drainage play during chronic brain infection. In this study, we report an expansion of type 1 and type 2 conventional dendritic cell (cDC) populations in the dural meninges following infection with T. gondii. cDC2s in particular displayed broad upregulation of co-stimulatory molecules and MHC class II, while both cDC1s and cDC2s were capable of sampling CSF-derived protein. Lymphatic vessel ligation experiments revealed that meningeal lymphatic drainage is required for dendritic cell responses in the deep cervical lymph nodes and promotes robust T cell activation, proliferation, and cytokine production at this site. However, in contrast to the finding that meningeal lymphatic drainage is host-protective during acute viral infection of the brain (Li et al., 2022), we observed that meningeal lymphatic drainage is dispensable for controlling T. gondii infection of the brain, with the T cell response in the brain remaining intact following surgical disruption of meningeal lymphatic outflow. Concurrent activation of T cells at alternative sites, including lymph nodes that do not drain the CNS, potentially explains the durability of the T cell response in the brain. Overall, our findings highlight the role of the dural meninges in the immune response to chronic brain infection, providing new evidence that meningeal lymphatic drainage promotes T cell responses against antigen expressed in the brain. Results Expansion of type 1 and type 2 conventional dendritic cell populations in the dural meninges during chronic brain infection The dura mater layer of meninges has emerged as an important anatomic site for immune surveillance of the CNS (Alves de Lima et al., 2020; Rua and McGavern, 2018). Soluble brain- and CSF-derived molecules accumulate within the meninges around the dural sinuses and can be captured by local antigen-presenting cells (APCs) (Louveau et al., 2018b; Rustenhoven et al., 2021). Moreover, in contrast to the brain, which is devoid of lymphatic vessels, the dural meninges develop an extensive network of lymphatic vessels which directly transport soluble brain- and CSF-derived molecules to peripheral lymph nodes (Aspelund et al., 2015; Louveau et al., 2018b). At homeostasis, a small number of APCs in the dural meninges surround these lymphatic vessels, and upon stimulation these cells can traffic to the deep cervical lymph nodes (Louveau et al., 2018b). Although CD11chiMHC IIhi APCs accumulate in the brain during T. gondii infection (John et al., 2011; Fischer et al., 2000), there is only limited evidence to suggest that, in the absence of local lymphatic vasculature, these cells are able to migrate out of the brain to transport antigen directly to peripheral lymph nodes (Carare et al., 2008; Clarkson et al., 2017). By contrast, professional APCs in the dural meninges are uniquely positioned to sample CNS material and traffic through lymphatic vessels to lymph nodes for T cell activation. Supporting a role for these cells in the immune response to brain infection, we report a striking accumulation of CD11c+ cells in the dural meninges of CD11cYFP reporter mice chronically infected with the ME49 strain of T. gondii (Figure 1a). CD11c+ cells localized preferentially around the dural sinuses near meningeal lymphatic vessels (Figure 1b), increasing local area coverage from 9% in naïve mice to 44% in chronically infected mice (Figure 1c). This accumulation of CD11c+ cells around the sinuses was associated with a threefold increase in the number of CD11c+ cells co-localized to LYVE1+ meningeal lymphatic vessels in infected mice compared to naïve mice (Figure 1d–e). This observation suggests that dural APCs access lymphatic vessels in greater numbers in response to brain infection and is consistent with trafficking studies performed previously (Louveau et al., 2018b). Figure 1 with 3 supplements see all Download asset Open asset Conventional dendritic cells accumulate in the dural meninges during chronic brain infection and sample CSF-derived protein. Mice were infected with 10 cysts of the ME49 strain of T. gondii intraperitoneally (i.p.) and analyzed at 6 weeks post-infection. (a) Confocal microscopy was used to image whole-mount dural meninges dissected from the skullcaps of naïve or chronically infected CD11cYFP reporter mice. Images are representative of three independent experiments. Scale bar, 2000 μm. (b) Representative images of CD11c+ cells (yellow) in the region surrounding the dural sinuses. Scale bar, 150 μm. (c) Quantification of area coverage by CD11c+ cells in the region surrounding the dural sinuses. Data compiled from three experiments (n = 10-11 mice per group). (d-e) Representative images (d) and quantification (e) of CD11c+ cells (yellow) present within LYVE1+ meningeal lymphatic vessels (white). Data compiled from three experiments (n = 10-11 mice per group). Scale bar, 50 μm. (f) Quantification by spectral flow cytometry of total cDC1 number (CD45+Lin-CD11chiMHC IIhiCD64-CD26+XCR1+SIRPα-) in the dural meninges of naïve, acutely infected, or chronically infected C57BL/6 mice. Data compiled from two experiments (n = 7 mice per group). (g) Quantification by spectral flow cytometry of total cDC2 number (CD45+Lin-CD11chiMHC IIhiCD64-CD26+XCR1-SIRPα+) in the dural meninges of naive, acutely infected, or chronically infected C57BL/6 mice. Data compiled from two experiments (n = 7 mice per group). (h) Quantification of T. gondii gDNA in brain and dural meninges by real-time PCR. Data compiled from two experiments (n = 3-8 mice per group). (i–k) Quantification of the geometric mean fluorescence intensity (MFI) of CD80 (i), CD86 (j), and MHC class II (k) expressed by cDC1s and cDC2s in the dural meninges. Data compiled from two experiments (n = 7 mice per group). (l) Schematic diagram illustrating intra-cisterna magna (i.c.m.) injection of DQ-OVA into the CSF. (m) Representative images of MHC class II-expressing antigen-presenting cells (magenta) co-labeling with fluorescent DQ-OVA cleavage products (green) in the vicinity of (closed arrows) or present within (open arrows) meningeal lymphatic vessels (LYVE1+, white). Three independent experiments were performed. Scale bar, 50 μm. Data are represented as mean values ± s.e.m. For c and e, statistical significance was measured using randomized block ANOVA (two-way), with p<0.001 (***). For (f and g) statistical significance was measured using a one-way ANOVA with post-hoc Tukey multiple comparison testing, with p<0.05 (*), p<0.01 (**), and p<0.001 (***). For (h–k) statistical significance was measured using a two-way ANOVA, with Tukey’s multiple comparison test to assess differences across timepoints [p<0.01 (##) and p<0.001 (###)] and Sidak’s multiple comparison test to assess differences between tissues (h) or cell type (i–k) [ns = not significant, p<0.05 (*), p<0.01 (**), and p<0.001 (***)]. To determine whether dendritic cells represent a portion of this emergent population of CD11c+ cells, and to more precisely characterize changes in distinct conventional dendritic cell (cDC) subsets in response to brain infection, we performed spectral flow cytometry on cells purified from the dural meninges of naïve, acutely infected (2 weeks post-infection), or chronically infected (6 weeks post-infection) C57BL/6 mice (Figure 1—figure supplement 1). Acute infection represents the period of time, lasting 2–3 weeks, when the parasite spreads systemically throughout the host’s peripheral tissues as fast-replicating tachyzoites. Once the parasite is cleared from peripheral tissues by a Th1-driven immune response, chronic infection takes hold and the parasite becomes largely confined to the CNS and skeletal muscle as slow-replicating, cyst-forming bradyzoites (Saeij et al., 2005). Our analysis centered on cDC1s, which play a specialized role in the cross-presentation (Durai and Murphy, 2016) and are essential for generating early immunity against T. gondii (Poncet et al., 2019), and cDC2s, which are important activators of CD4 +T cells (Durai and Murphy, 2016) but whose role in T. gondii infection remains unresolved. While there was no difference in the number of cDC1s (CD45+Lin-CD11chiMHC IIhiCD64-CD26+XCR1+SIRPα-) or cDC2s (CD45+Lin-CD11chiMHC IIhiCD64-CD26+XCR1-SIRPα+) in naive and acutely infected mice, by 6 weeks post-infection (wpi) there was a threefold increase in the number of cDC1s and sixfold increase in the number of cDC2s in the dural meninges (Figure 1f–g). Moreover, there was a shift in the proportion of cDC1s and cDC2s (Figure 1—figure supplement 2a-e). At baseline, cDC2s made up only 20% of the CD26+ cDC population in the dural meninges, but by 6 wpi cDC2s made up 40% of the CD26+ population (Figure 1—figure supplement 2c). Intriguingly, the expansion of the dendritic cell compartment in the dural meninges during chronic infection tracked with increasing parasite burden in the brain, which was found to peak at 6 wpi by real-time PCR (Figure 1h). By contrast, parasite could not be detected in the dural meninges at 6 wpi by either real-time PCR or immunostaining of PFA-fixed tissue (Figure 1h, Figure 1—figure supplement 2d). These data suggest that the accumulation of cDC1s and cDC2s in the dural meninges occurred independently of ongoing infection of the dural meninges. This finding reinforces an emerging view that immune cells at border tissues of the CNS, including the dural meninges, play a key role in responding to pathology within the brain parenchyma (Alves de Lima et al., 2020; Rua and McGavern, 2018; Rustenhoven et al., 2021; Cugurra et al., 2021; Pulous et al., 2022). We next examined the maturation status of dendritic cells (Hammer and Ma, 2013) in the dural meninges (Figure 1i–k). Consistent with the increase in number of cDC1s and cDC2s at 6 wpi, upregulation of co-stimulatory molecules and MHC class II occurred largely after progression to chronic brain infection (Figure 1i–k, Figure 1—figure supplement 2e-g). Additionally, cDC2s displayed a much broader degree of activation compared to cDC1s, upregulating expression of CD80, CD86, and MHC class II (Figure 1i–k). By contrast, cDC1s expressed minimal levels of CD80 (Figure 1i) and showed an increase in CD86 expression but not MHC class II expression (Figure 1j–k). These data suggest that chronic brain infection promotes maturation of dendritic cells in the dural meninges but has a greater effect on the activation status of cDC2s than cDC1s. Because activated dendritic cells introduced into the CSF exit the CNS by accessing meningeal lymphatic vessels (Louveau et al., 2018b), it is possible that some of the dendritic cells that we observed in the meninges of infected mice came from the CSF. Therefore, we performed flow cytometry on CSF pooled from 4 to 5 naive or chronically infected mice and saw that, in fact, a population of CD11chiMHC IIhi APCs not present in naive mice emerged in the CSF of chronically infected mice (Figure 1—figure supplement 3a-b), expressing high levels of CD80 and CD86 (Figure 1—figure supplement 3c-e). Interestingly, the concentration of CSF-borne APCs was consistent with that reported by a study of human CSF samples isolated from patients with Lyme neuroborreliosis (Pashenkov et al., 2001). We next sought to determine whether APCs in the dural meninges are able to transport CSF-derived protein during infection. CSF has been shown to function as a sink for brain-derived macromolecules (Abbott, 2004; Plog and Nedergaard, 2018) and capturing CSF-borne protein represents a potential mechanism by which APCs in the dural meninges could sample brain-derived antigen and transport it to lymph nodes in the periphery. To address this question, soluble DQ-OVA was injected into the CSF of chronically infected mice via intra-cisterna magna (i.c.m.) injection (Figure 1l). DQ-OVA is a self-quenched conjugate of ovalbumin labeled with BODIPY dyes that only emits fluorescence after proteolytic cleavage. DQ-OVA has been commonly used to examine uptake and processing of soluble antigen by dendritic cells (Ling et al., 2003; Gerner et al., 2017; Sixt et al., 2005). Within 2 hr of i.c.m. injection, we found that 30% of cDC1s and cDC2s in the dural meninges harbored DQ-OVA cleavage products (Figure 1—figure supplement 2h-j). After 24 hr, cleavage products were detected in ~5% of cDC1s and cDC2s (Figure 1—figure supplement 2h-j), which may reflect egress of DQ-OVA +dendritic cells from the meninges or loss of the BODIPY fluorescent signal over time. Imaging of whole mount dural meninges revealed that DQ-OVA+ cells were distributed along and within lymphatic vessels after 12 hr, suggesting that dural APCs capture and transport CSF-borne antigen during chronic brain infection (Figure 1m). All together, these findings highlight significant changes in the APC compartment of the dural meninges in response to T. gondii brain infection. Type 1 and type 2 conventional dendritic cells increased in number in the dural meninges, displayed a more activated phenotype, and were able to sample CSF-borne protein in the vicinity of meningeal lymphatic vessels. T cell responses in the deep cervical lymph nodes peak following progression to chronic brain infection Initial studies in mice, and later in humans, have demonstrated that meningeal lymphatic vessels drain directly to the deep cervical lymph nodes (DCLN) (Albayram et al., 2022; Louveau et al., 2018b). Even before the immunologic function of the meningeal lymphatic system started coming into focus, these lymph nodes were strongly implicated in regulating CNS immunity. For example, injection of antigen into rodent brains elicited strong humoral responses in the DCLNs (Harling-Berg et al., 1989), and surgical excision of the DCLNs contributed to a reduction in severity of experimental autoimmune encephalomyelitis (van Zwam et al., 2009; Furtado, 2008). To date, few studies have examined the contribution of the deep cervical lymph nodes to immunity against CNS pathogens. To address the role of the DCLNs during T. gondii infection, we first confirmed that during chronic infection CSF-derived protein could be sampled by APCs within these lymph nodes. Upon i.c.m. injection of DQ-OVA into chronically infected mice, 3% of CD11chiMHC IIhi APCs in the DCLNs were found to harbor digested protein products after 5 hr (Figure 2a–b). By contrast, processed DQ-OVA was not detected in the inguinal lymph nodes (ILN), which drain peripheral tissue (Figure 2a–b). Further characterization of the DQ-OVA+ APC populations in the DCLNs revealed that equal proportions of cDC1s and cDC2s harbored digested protein products both at 2 hr and 24 hr after i.c.m. injection (Figure 2—figure supplement 1a-c), the latter time point representing when migratory dendritic cells trafficking from the meninges have been shown to reach the DCLNs (Louveau et al., 2018b). Figure 2 with 4 supplements see all Download asset Open asset Expansion of T cells in the deep cervical lymph nodes occurs primarily during the chronic stage of infection and tracks with parasite burden in the brain. (a-b) DQ-OVA was injected into the CSF of chronically infected mice by i.c.m. injection and fluorescent emission of proteolytically cleaved DQ-OVA was measured in CD11chiMHC IIhi antigen-presenting cells of the deep cervical lymph nodes (DCLNs) and inguinal lymph nodes (ILNs) by flow cytometry. (a) Representative contour plots of DQ-OVA+ antigen-presenting cells in the DCLNs or ILNs at 6 wpi. CD11chiMHC IIhi cells were pre-gated on singlets/live/TCRβ-/NK1.1-/CD19-. (b) Quantification of frequency of DQ-OVA+ antigen-presenting cells in the DCLNs or ILNs at 6 wpi. Data are compiled from three experiments (n = 11 mice per group) and are represented as mean values ± s.e.m. Statistical significance was measured using randomized block ANOVA (two-way), with p<0.001 (***). (c–d) C57BL/6 mice were infected i.p. with 10 cysts of the ME49 strain of T. gondii and total number of activated CD4+ T cells (c) or activated CD8+ T cells (d) in the DCLNs or ILNs was quantified at multiple time points over the course of acute and chronic infection (red dots). The steady-state number of activated CD4+ and CD8+ T cells in the different lymph node compartments was measured in naïve mice at corresponding time points (black dots). Activated T cells displayed a CD44hiCD62Llo phenotype. Data are compiled from three experiments and are represented as mean values ± s.e.m. (n = 6-14 mice per group per timepoint). (e–f) C57BL/6 mice were infected i.p. with 1,000 tachyzoites of the Pru-OVA strain of T. gondii. (e) Total number of SIINFEKL-specific CD8+ T cells in the DCLNs or ILNs was quantified at multiple time points over the course of acute and chronic infection using tetramer reagent. Data are compiled from two experiments and are represented as mean values ± s.e.m. (n = 7 mice per timepoint). (f) Quantification of T. gondii gDNA in brain (red), dural meninges (orange), deep cervical lymph nodes (black), inguinal lymph nodes (blue), subcutaneous adipose tissue isolated from the flank (green), and quadriceps femoris skeletal muscle tissue (gray) by real-time PCR. Data are compiled from two experiments and are represented as mean values ± s.e.m. (n = 5-6 mice per tissue per timepoint). For (c–e), statistical significance of differences across time points in infected mice was measured using one-way ANOVA with post-hoc Tukey multiple comparison testing. p<0.05 (*) and p<0.01 (**). For (f) statistical analysis was performed using a two-way ANOVA with Tukey’s multiple comparison test to assess differences across timepoints. Statistically significant differences are indicated, with p<0.05 (*) and p<0.001 (***). Figure 2—source data 1 CD4+ T cell activation in the DCLNs and ILNs over the course T. gondii infection. https://cdn.elifesciences.org/articles/80775/elife-80775-fig2-data1-v2.xlsx Download elife-80775-fig2-data1-v2.xlsx Figure 2—source data 2 CD8+ T cell activation in the DCLNs and ILNs over the course of T. gondii infection. https://cdn.elifesciences.org/articles/80775/elife-80775-fig2-data2-v2.xlsx Download elife-80775-fig2-data2-v2.xlsx Figure 2—source data 3 SIINFEKL-specific CD8+ T cell responses in the DCLNs and ILNs over the course of T. gondii infection. https://cdn.elifesciences.org/articles/80775/elife-80775-fig2-data3-v2.xlsx Download elife-80775-fig2-data3-v2.xlsx Figure 2—source data 4 Parasite burden in the brain and peripheral tissues over the course of T. gondii infection. https://cdn.elifesciences.org/articles/80775/elife-80775-fig2-data4-v2.xlsx Download elife-80775-fig2-data4-v2.xlsx Based on these results, we next sought to understand the kinetics of T cell activation in the deep cervical lymph nodes. For comparison, we also examined the kinetics of T cell activation in the inguinal lymph nodes. When C57BL/6 mice were infected with the ME49 strain of T. gondii, activated (CD44hiCD62Llo) CD4+ and CD8+ T cell number in the ILNs was greatest during acute infection (2 wpi) (Figure 2c–d, Figure 2—figure supplement 2a), consistent with the broad distribution of parasite in peripheral tissues at this time point. Conversely, only a small number of activated CD4+ and CD8+ T cells was detected in the DCLNs at 2 wpi (Figure 2c–d). Expansion of CD4+ and CD8+ T cells in the DCLNs became more pronounced after progression to the chronic stage of infection, with the peak in number of activated CD4+ and CD8+ T cells occurring at 6–8 wpi (Figure 2c–d). To better understand the stage-dependent activation of T cells in the DCLNs, we used MHC class I tetramer to track a parasite epitope (SIINFEKL)-specific population of CD8+ T cells generated in response to infection with Pru-OVA, a recombinant type II strain of T. gondii engineered to express a secreted, truncated form of the model antigen ovalbumin (Pepper et al., 2004; Figure 2—figure supplement 2b). Consistent with the polyclonal CD8+ T cell response observed in response to ME49 infection (Figure 2d), the peak SIINFKEKL-specific CD8+ T cell response in the ILNs occurred during acute infection (2–3 wpi), when very few parasite-specific T cells were detectable in the DCLNs (Figure 2e). By contrast, expansion of parasite-specific CD8+ T cells in the DCLNs was greatest during chronic infection, increasing significantly between 3 and 6 wpi (Figure 2e). These results likely reflect the distinct sources of antigen that each lymph node site drains during the different stages of infection. In support of this, we found that parasite-specific T cell responses in the DCLNs tracked with parasite burden in the brain (Figure 2f), whereas parasite-specific T cell responses in the ILNs tracked with parasite burden in the ILNs. During acute infection, ILNs appear to drain parasite from infected peripheral tissues or become directly infected themselves (Figure 2f). Notably, the DCLNs did not show high levels of parasite burden during acute infection, suggesting that hematogenous routes of dissemination did not provide significant access of the parasite to the DCLNs. To understand how the quality of T cell responses generated in the DCLNs and brain change as infection progresses from the acute to chronic stage, we assessed expression of co-inhibitory molecules, which can become upregulated in the setting of persistent infection (Attanasio and Wherry, 2016), and markers that define the effector and memory T cell subsets during T. gondii infection (Chu et al., 2016; Landrith et al., 2017; Figure 2—figure supplement 2c). We observed minimal expression of co-inhibitory molecules by endogenous and transferred SIINFEKL-specific CD8 + cells (Figure 2—figure supplement 3), and as expected we observed an increase in the proportion of memory-like T cell subsets during chronic infection (Figure 2—figure supplement 4). Meningeal lymphatic drainage promotes peripheral T cell responses against T. gondii To directly test the function of meningeal lymphatic drainage during T. gondii brain infection, we surgically ligated the collecting vessels afferent to the deep cervical lymph nodes. This approach has been used to test the function of meningeal lymphatic drainage in experimental models of multiple sclerosis, glioblastoma, and acute viral infection of the brain (Song et al., 2020; Louveau et al., 2018b; Li et al., 2022). We performed ligation at 3 wpi, when T cells begin to display a response against the parasite in the DCLNs (Figure 2), and analyzed mice 3 weeks later (Figure 3a). Unless otherwise stated, experiments were performed using the ME49 strain of T. gondii. Tracer studies using Evans blue or Alexa Fluor 594-conjugated ovalbumin (OVA-AF594) confirmed the efficacy of the procedure and demonstrated a greater than 95% reduction in outflow of CSF-derived components to the DCLNs in ligated animals (Figure 3—figure supplement 1a-b). Importantly, ligation did not affect outflow of CSF to the superficial cervical lymph nodes, another drainage site for CSF-borne protein (Figure 3—figure supplement 1c; Ma et al., 2017). Figure 3 with 1 supplement see all Download asset Open asset Restricting meningeal lymphatic drainage disrupts dendritic cell activation in the deep cervical lymph nodes. Chronically infected C57BL/6 mice were subjected to surgical ligation of collecting vessels afferent to the DCLNs or sham su" @default.
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• W4313477592 title "Author response: Meningeal lymphatic drainage promotes T cell responses against Toxoplasma gondii but is dispensable for parasite control in the brain" @default.
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