Treg cells play an important role in the CNS during multiple infections as well as autoimmune inflammation, but the behavior of this cell type in the CNS has not been explored. In mice, infection with Toxoplasma gondii leads to a Th1-polarized parasite-specific effector T cell response in the brain. Similarly, the Treg cells in the CNS during T. gondii infection are Th1-polarized, exemplified by T-bet, CXCR3, and IFN-γ expression. Unlike effector CD4+ T cells, an MHC Class II tetramer reagent specific for T. gondii did not recognize Treg cells isolated from the CNS. Likewise, TCR sequencing revealed minimal overlap in TCR sequence between effector and regulatory T cells in the CNS. Whereas effector T cells are found in the brain parenchyma where parasites are present, Treg cells were restricted to the meninges and perivascular spaces. The use of intravital imaging revealed that activated CD4+ T cells within the meninges were highly migratory, while Treg cells moved more slowly and were found in close association with CD11c+ cells. To test whether the behavior of Tregs in the meninges is influenced by interactions with CD11c+ cells, mice were treated with anti-LFA-1 antibodies to reduce the number of CD11c+ cells in this space. The anti-LFA-1 treatment led to fewer contacts between Tregs and the remaining CD11c+ cells and increased the speed of Treg cell migration. These data suggest that Treg cells are anatomically restricted within the CNS and the interaction with CD11c+ populations regulates their local behavior during T. gondii infection.
Regulatory T cells (Treg cells) have potent suppressive capacity capable of limiting effector T cell responses and immune-mediated pathology in the context of immune homeostasis as well as in infectious and non-infectious inflammatory processes. While multiple suppressive mechanisms have been attributed to Treg cells (1, 2), only a limited number of reports have examined Treg cell behavior in vivo, where Tregs have been imaged in the bone marrow, spleen, lymph nodes in diabetes and graft-versus-host models, and in tumors (3–7). In many CNS inflammatory conditions Treg cells are recruited to the brain, where it has been proposed that their presence represents one mechanism to limit the catastrophic consequences of inflammation in this site (8, 9). For example, in mice infected intracranially with murine hepatitis virus, the depletion of Treg cells leads to an increase in self-reactive T cell responses and more severe pathology in the brain (10). While the importance of Treg cells in many experimental models that involve the CNS has been demonstrated (10–15), the behavior of these cells within the brain remains unexplored.
Toxoplasma gondii is a protozoan parasite that establishes a chronic infection within the CNS. In mice, cytotoxic T cells and T cell-production of IFN-γ are required to control parasite replication within the brain (16–18). Several studies have established that Treg cells contribute to the regulation of effector T cells during acute toxoplasmosis (19–21) and that during many intracellular infections there is the emergence of a population of Th1-like Treg cells that express T-bet, IFN-γ, IL-10, and CXCR3 (20–22), but there are open questions about the specificity of these populations(23, 24). During acute toxoplasmosis, expansion of the Treg cell population is associated with an increase in parasite burden within the brain (21, 25). These latter observations suggest that Treg cells can suppress the protective T cell response required to control T. gondii but it is unclear if this is a general regulatory effect or mediated locally within the brain. The studies presented here reveal that, unlike parasite-specific effector T cells, during TE, Treg cells were localized predominantly to the meninges and perivascular cuffs where they maintained interactions with CD11c-expressing cells, which influence the migratory behavior of Treg cells. These observations suggest that Treg-dendritic cell interactions are an important component of Treg cell function during toxoplasmic encephalitis (TE) and this may be broadly relevant to Treg cell functions in other inflammatory settings that affect the CNS.
MATERIALS AND METHODS
Infections and treatments
C57BL/6, CD11c-YFP, actin-CFP, and IL-10eGFP “Tiger” mice were purchased from The Jackson Laboratory (Bar Harbor, ME). FoxP3-GFP were originally obtained from Vijay Kuchroo of Harvard University and crossed to the CD11c-YFP strain. IL-10eGFP reporter VertX mice were obtained from Christopher Karp of Cincinnati Children’s Hospital. All procedures were performed in accordance to the guidelines of the University of Pennsylvania and University of Virginia Institutional Animal Care and Use Committee. Ovalbumin-expressing Prugnauid strain parasites expressing Tomato fluorescent protein (PruOVATOM) were generated as previously described (26) and maintained by serial passage in human fibroblast (HFF) cell monolayers in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum. Prior to infections, parasites were purified from HFF culture by filtration through a 5.0μm filter (Nucleopore, Clifton, NJ). Mice were infected with 103 tachyzoites in 200 μl PBS i.p. Anti-LFA-1 antibodies (BioXcell) and normal rat IgG (Sigma) were administered i.p. in PBS four hours prior to imaging experiments.
Single cell suspensions were generated from spleen and lymph node by macerating the tissues through 40 μm nylon mesh filters (BD Falcon, Bedford, MA). Spleen samples were subjected to hypotonic red blood cell lysis. Brain mononuclear cells (BMNCs) were isolated as previously described (27). Briefly, perfused brains were cut into small pieces, passed through an 18 gauge needle, and digested with collagenase/dispase and DNase (Roche) for 90 minutes. Following the digestion, the cells were washed and strained through a 70 μm filter. Subsequently, cells were resuspended in 60% percoll, overlayed with 30% percoll, and centrifuged at room temperature for 25 minutes at 2000 rpm. BMNCs were collected from the interface, washed, and enumerated. For flow cytometry, 1–2 × 106 cells were washed with FACs buffer (1X PBS, 0.2% BSA, and 2mM EDTA) and incubated in Fc block (0.1 μg/ml 24G2 antibody) for 15 minutes prior to surface staining with CD4-FITC, ICAM-PE, CD11a-PE, CD25-PE, CD8-PerCpCy5.5, CD8-eFlour780, CD45-APC, CD11c-PECy7, CD11b-AF780, CD3-FITC, CD19-FITC, NK1.1-FITC, and MHC Class II-eFluor450 (eBioscience). T. gondii-specific cells were identified with a PE-conjugated I-Ab -AVEIHRPVPGTAPPS tetramer reagent (NIH Tetramer Facility, Atlanta, GA). For intracellular cytokine staining, cells were cultured for four hours in the presence of PMA, ionomycin, and brefeldin A. Following surface staining, cells were fixed in eBiosceince fix/perm buffer. Cytokines were detected with IFN-γ-PE-Cy7 and IL-10 PE. For GFP staining, cells were stained with rabbit anti-GFP (eBioscience) followed by a goat anti-rabbit Alexa 488 (Life Technologies) antibody. For transcription factor staining, cells were fixed with Fix/Perm buffer and stained with FoxP3-Pacific Blue, FoxP3-AF488, or T-bet-PE (eBioscience). Flow cytometry was performed on a BD LSRII Fortessa or FACsCanto using FACsDIVA 6.0 software (BD Biosciences, San Jose, CA). Analysis was performed using FloJo software (Treestar Inc., Ashland, OR).
TCR sequencing and analysis
Immune cells were isolated from the meninges of T. gondii infected mice as previously described (28). Cells were stained with antibodies against CD3, CD4, and FoxP3 and sorted on a Becton Dickinson Infux Cell Sorter. DNA from FoxP3− and FoxP3+ CD4+ T cells was purified using a Qiagen DNA Micro Kit. The TCR-β CDR3 regions were sequenced with Immunoseq Assay from Adaptive Biotechnologies (Seattle, WA).
The TCR-β CDR3 sequences obtained were analyzed in the following ways: First, the presence/absence of amino acid sequences in the TCR-seq was determined, giving us a binary matrix of zeros and ones, with a one indicating that the sequence was measured at least once in that sample. The Jaccard index was used to quantify the similarity of the detected sequences in one sample versus another and was visualized with the R package corrplot (29). The binary matrix was then used to create the binary heatmap showing the presence/absence of amino acid sequences. Before creating the heatmap, amino acid sequences with low counts across all samples were removed; specifically, a sequence was removed if it did not make up 0.3% of the total measured sequences in at least one of the six samples. The heatmap plot itself was produced with the R package pheatmap (30). The UpSet plot (31) used to visualize the overlap of amino acid sequences between the samples (set comparisons) was created with the R package UpSetR (32). As opposed to the binary heatmap, all of the measured sequences were used to create the UpSet plot.
For immunohistochemistry, organs were embedded in OCT and flash frozen. Six μm sections were cut using a Leica 3050M cryostat (Leica Microsystems. Sections were fixed with a solution of 75% acetone and 25% ethanol. Anti-laminin (Cedarlane), anti-FoxP3 (eBioscience), and anti-CD4 (eBioscience). Anti-rabbit Alexa 488 (Invitrogen), anti-rat Cy3, or biotinylated anti-rat (Jackson Immunoresearch) were used as secondary antibodies for fluorescence staining. A Cy3 tyramide signal amplification kit (Perkin Elmer) was used to amplify FoxP3 staining. DAPI (Invitrogen) was used to visualize nuclei. Images were captured using standard fluorescence microscopy using a Nikon Eclipse E600 microscope (Melville, NY) equipped with a Photometrics Cool Snap EZ CCD camera (Tucson, AZ) or an inverted Leica DMI4000 B microscope equipped with a Hammamatsu camera. Metamorph and Nikon NIS Elements software was used to captures images and Imaris (Bitplane), or Image J image analysis software was used to overlay images.
Effector T cells
CD4+ CD25− cells from mice expressing cyan fluorescent protein (CFP) under the actin promoter were isolated using enrichment columns (Miltenyi, San Diego, CA). The cells were activated with plate-bound anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) and cultured with anti-IL-4 (1 μg/ml) and 20 U/ml recombinant human IL-2 (proleukin) for 4 days. One million activated CFP+ cells were transferred to mice chronically infected with T. gondii and imaged seven days later.
Mice were sacrificed by CO2 asphyxiation and the brains were removed immediately, with minimal mechanical disruption and placed in heated chamber where specimens were constantly perfused with warmed (37°C), oxygenated media (phenol-red free RPMI 1640 supplemented with 10% FBS, Gibco). The temperature in the imaging chamber was maintained at 37°C using heating elements and monitored using a temperature control probe. Intravital imaging experiments were performed using the thinned skull technique, as previously described (33). Briefly, mice were anesthetized and a region of skull bone of 0.5–1 mm in diameter was thinned with a dental drill and surgical blade until approximately 30μm of skull remained. All imaging was performed using a Leica SP5 2-photon microscope system (Leica Microsystems, Mannheim, Germany) equipped with a picosecond laser (Coherent Inc., Santa Clara, CA) and external non-descan detectors that allow simultaneous detection of emissions of different wavelengths and second harmonic signals (SHG, ~460nm). Enhanced GFP, YFP, and QDots were excited using laser light of 920 nm. Images were obtained using a 20X water-dipping lens. Four-dimensional imaging data was collected by obtaining images from the x-, y-, and z-planes, with a z- thickness of 68 μm and step size of 4 μm to allow for the capture of a complete z-series every 20 seconds for period of 15 minutes. The resulting images were analyzed with Volocity (PerkinElmer, Waltham, MA) or Imaris software. Movies of T cell migration, mean migratory velocity, and cell contact duration were calculated using the software.
Statistical analyses were performed using Prism software. P values of less than 0.05 were considered significant. The tests used in each experiment are denoted in the figure legends.
Phenotype and localization of Treg cells during Toxoplasmic encephalitis
To characterize Treg cell responses during chronic toxoplasmosis, mononuclear cell preparations were isolated from the spleen, cervical lymph nodes (CLN), and brains of mice infected with T. gondii. The numbers and phenotype of these populations were assessed using flow cytometry for intracellular Foxp3 (Figure 1). Based on the use of MHC class I and II tetramers that contain defined parasite epitopes, activated antigen-specific CD4+ and CD8+ effector cells were readily detected in the spleen and cervical lymph nodes and the Treg cells represented 12–18% of the total CD4+ population (data not shown). In the brains of uninfected mice, T cells are confined to the meningeal spaces (28, 34) and BMNC preparations contained very few conventional effector/memory T cells, Treg cells, or DC (Figure 1a and data not shown). During early stages of infection, a sizable population of effector CD4+ T cells was recruited to the brain by day 14 (Figure 1a). Tregs comprised approximately 1.5% of the CD4+ T cell compartment at day 14 and 21 post-infection. During chronic infection (day 28 post-infection), Treg cells comprise approximately 8–10% of the total CD4+ T cell population and these cells express high levels of CD25 (Figure 1b). To further characterize this population, IL-10-eGFP reporter mice (VertX or Tiger mice) were infected and IL-10 and IFN-γ production was assessed and compared to basal IL-10 production in the naïve spleen. We observed negligible IL-10 production from conventional CD4+ T cells in the spleens of naïve and infected mice. On the other hand, Tregs in the spleen did produce some IL-10 at baseline (approximately 5%) that increased to nearly 20% during infection (Figure 1c). In the infected CNS, approximately 4% of FoxP3− CD4+ cells were eGFP+, whereas 45% of Tregs in the CNS expressed IL-10-eGFP (Figure 1c). While the number of FoxP3+ and FoxP3− IL-10-producing cells are similar, the MFI of IL-10-eGFP was higher in the Treg cells isolated from the infected brain (Figure 1d). Analysis of IFN-γ production revealed that while 57% of effector CD4+ T cells in the infected brain produced IFN-γ, only 12% of the Tregs produced IFN-γ at this site (Figure 1c). This analysis also highlighted the presence of a small sub-population of Treg cells that produce both cytokines in the brain, which was not observed in peripheral tissues at this time point (Figure 1c). Consistent with the highly polarized Th1 response that is generated against T. gondii, the Treg cells in the brain during TE express the canonical Th1 transcription factor T-bet (Figure 1e) and the T-bet-dependent chemokine receptor CXCR3 (Figure 1f–g). Moreover, Tregs isolated from the infected CNS express higher levels of CXCR3 in comparison to effector CD4+ and CD8+ T cells (Figure 1f) and the CXCR3 expression on CNS Tregs was higher in the brain in comparison to Tregs isolated from the spleen and cervical lymph nodes of infected mice (Figure 1g). Together these data demonstrate that a Th1 polarized Treg is recruited to the CNS during chronic T. gondii infection.
Open in a separate window
Treg cells are present in the CNS during chronic T. gondii infection and produce cytokines
Mononuclear cells were isolated from the brains of C57BL/6 mice infected with T. gondii for 28 days. Numbers of Foxp3+ and Foxp3- CD4 T cells infiltrating the brain were measured on days 0, 7, 21, and 28 post-infection (a). FoxP3 and CD25 expression was measured on CD4+ cells isolated from the CNS by flow cytometry on day 28 post-infection (b). IL-10 expression was measured by flow cytometry in IL-10-eGFP reporter mice on day 28 post-infection. The co-expression of IL-10 and IFN-γ in CD4+ T cells was measured in the naïve and infected spleen and the infected brain (c). The mean florescence intensity (MFI) of IL-10-eGFP expression in FoxP3+ and FoxP3− CD4+ T cells isolated from the brain (d). Expression of T-bet by CD4+Foxp3− and CD4+Foxp3+ cells (e). CXCR3 expression was measured on CD4+Foxp3− (FoxP3−), CD4+Foxp3+ (FoxP3+), and CD8+ T cells (f) and Treg CXCR3 expression was compared between the CNS, spleen, and cervical lymph nodes (g). The presented data is representative of five independent experiments with 4 animals per group (b) or two independent experiments with 3 mice per group (a, c–g). *** denotes p<0.001 as determined by the students t-test.
Specificity and sequencing of Treg cells isolated from the meninges during TE
During infection with T. gondii, parasite-specific CD4+ and CD8+ T cells, as well as Tregs accumulate in the CNS, but little is known about the specificity of the local Treg populations. To address whether the Tregs may be specific for parasite antigen, immune cells isolated from the CNS were stained with an MHC Class II tetramer reagent specific for the AVEIHRPVPGTAPPS peptide. Parasite-specific CD4+ effector cells were detected but very few CD4+ T cells were tetramer-positive and FoxP3+ (0.018±0.0059%) (Figure 2a). Although this analysis was performed using a single tetramer, these results suggest that Treg cells in the CNS may not recognize the same antigens as the Toxoplasma-specific effector CD4+ T cells. To further explore the clonality and potential specificity of the Tregs in the CNS, effector and regulatory CD4+ T cells were sorted from meninges of infected mice and the CDR3 regions of the TCRβ gene were sequenced. The TCR sequences of effector T cells and Tregs from three individual experiments were compared at the amino acid level. First, the similarity of the TCR sequences was compared between samples, where the presence of a sequence was considered, but not the frequency of the sequence within the population. While many unique sequences were detected in each population, the most similarity was found in the effector CD4+ T cell populations (Figure 2b–c and Supplementary Table 1). When comparing TCR sequences from effector T cells and Tregs, we found very little overlap (approximately 1%) between the populations (Figure 2d), which is in agreement with results from MHC II tetramer staining. Moreover, if a sequence was detected in both effector and Treg populations, the number of reads for that sequence was overrepresented primarily in the Treg population (Figure 2e). These results suggest that Treg cells found in the CNS during chronic infection are rarely from the same clonal lineage as the effector population and some Tregs may lose FoxP3 expression and resemble effector T cells. Unexpectedly, we found that the CDR3 regions of Treg TCRs had very little sequence overlap (0.2–0.65%) between three separate T. gondii infections (Figure 2b–c and and2f),2f), suggesting that the Treg repertoire recruited to the CNS during infection varies greatly between experimental infections. While the TCR sequences do not identify the antigen specificity of the Treg cells, these experiments reveal that during each individual infection in C57BL/6 mice the presence of a unique repertoire of Treg cells that is largely distinct from the effector cells arises in the CNS.
Regulatory T cells that accumulate in the CNS during T. gondii infection have little TCR sequence similarity with effector T cells and vary greatly between experiments
Brains from chronically infected C57Bl/6 mice were harvested and processed for flow cytometry 28 days post infection. Cells were gated on CD3+CD4+ live lymphocytes and T. gondii tetramer-specific effector (CD4+Foxp3−) and regulatory (CD4+Foxp3+) T cells are shown (a). T cells isolated from the meninges were FACS sorted to isolate effector (CD4+Foxp3−) and Treg (CD4+Foxp3+) populations. CDR3 regions of the TCRβ chains were sequenced from each population. The resulting sequences were compared between populations and experiments to identify the degree of overlap by Jaccard index (b). Sequences that represented at least 0.3% of the population were displayed by binary heatmap (blue=present and white=absent), indicating overlap between samples (c). The amount of overlap between effector T cells (FoxP3−) and Tregs (FoxP3+) from a single experiment is shown by upset plot, where joining lines indicate sequences detected both samples, with the number of sequences indicated above the bar (d). Within the overlapping samples the ratio (fold change) of reads between FoxP3+ and FoxP3− samples is depicted (e). The overlap among Tregs between experiments is shown by upset plot (f).
Treg localization in the CNS during chronic T. gondii infection
In order to understand the spatial organization of the Treg cells during TE, IHC was performed for CD4, parasite antigen, Foxp3, and laminin to identify basement membranes and thus demarcate the meninges and blood vessels and identify CD4+ T cell location within the brain. Staining for CD4 revealed the presence of T cells in the meninges and perivascular cuffs, but the largest numbers of effector T cells were present in the parenchyma (around 75.7%), where parasite cysts and replicating tachyzoites are found (Figure 3a). In contrast, Foxp3+ cells were largely (89%) localized to the inflamed meninges and perivascular cuffs, and thus were more rarely detected in the brain parenchyma in comparison to effector CD4+ T cells (Figure 3b–e). Together with the data presented in Figure 1, these data indicate that the Treg cells present in the CNS during TE are Th1 polarized like the effector population, but their distribution within the brain is distinct from effector CD4+ Th1 cells.
Tregs are absent from the brain parenchyma
Sections of brain from T. gondii-infected C57BL/6 mice were stained with antibodies against CD4 (red) and T. gondii (green) (a), FoxP3 (red) and laminin (green) to detect basement membranes (b–c), or FoxP3 (red) and CD4 (green) (d). DAPI was used to detect nuclei (blue). Quantification of parenchymal and perivascular Foxp3+ (n=163) and Foxp3- (n=189) CD4+ cells is shown in (e).
Imaging Treg cell populations during toxoplasmic encephalitis
Because dendritic cell (DC) populations are critical regulators of Treg homeostasis and are known targets of Treg suppression (2, 5, 35), the Foxp3-GFP mice were crossed with CD11c-YFP reporter mice and mice expressing both reporters were used for intravital imaging studies (Figure 4a, Supplementary movie 1). Firstly, these studies showed that there was close association of Foxp3+ cells with blood vessels in the CNS of mice with TE and that these cells were frequently co-localized with CD11c+ cells (Figure 4a), confirming results obtained with IHC (data not shown). Next, imaging of Foxp3+ cells in the CLN of infected mice revealed that they were highly motile and had many short lived interactions with CD11c+ cells (Figure 4b–c, Supplementary movie 2), similar to the behavior of Treg cells in the lymph nodes of uninfected mice (3). However, the Treg cells localized within the meninges of infected mice had a slower velocity and formed long-lived associations with CD11c+ populations in this microenvironment in comparison to the spleen (Figure 4d, Supplementary 3). In contrast to naïve T cell interactions with dendritic cells during priming (36), Foxp3+ cells were not stationary and did not round up while interacting with CD11c+ cells but were frequently observed to move from one CD11c+ cell to the next, while maintaining contact with these cells (Supplementary Movies 3). It is relevant to note that when CD4+ T cells from CFP-expressing mice were activated and expanded in vitro and transferred to infected FoxP3-GFP reporter mice, this CD4+CD44hiCD62LloFoxP3− population was present in the same area as Treg cells but remained highly migratory (Figure 4e, Supplementary Movie 4) and did not form sustained interactions with the CD11c-YFP cells (data not shown). These data highlight that the Treg cells in the CNS during TE display a pattern of behavior that is distinct from Treg cell populations in the CLN or effector CD4+ T cells present in the meninges (Figure 4f).
Tregs form long-lived contacts with CD11c+ cells in the brain
FoxP3-GFP CD11c-YFP mice were infected with T. gondii for 28 days. Intravital imaging was performed through thinned skull (blue) to detect FoxP3-GFP (green), CD11c-YFP (yellow), and the vasculature highlighted by a fluorescent vascular tracer (red) (a). On day 28 post-infection, explant lymph nodes from CD11c-YFP x FoxP3-GFP mice were imaged by MP microscopy, with secondary harmonic generation (SHG, blue) is shown in blue (b). The duration of contact between FoxP3-GFP-expressing cells (green) and YFP-expressing cells (yellow) was measured using Volocity software (c–d). CD4+CD25− T cells from a CFP-expressing mouse were activated in vitro and transferred to a chronically infected FoxP3-GFP mouse. Explant brains were imaged using multiphoton microscopy and individual cell paths of Tregs (green) and effector cells (blue) were tracked (e-f). The lymph nodes from naïve and chronically infected FoxP3-GFP mice were also imaged using MP microscopy. More than 100 GFP-expressing cells in each tissue from 4 or more movies and 4 independent experiments were tracked. The track velocity of Tregs in the lymph node and CNS and effector cells in the CNS was calculated (f). Results are denoted as non-significant (n.s.), ** for p<0.01, and *** for p<0.001 as measured by one way ANOVA with a Tukey’s multiple comparison post-test.
Anti-LFA-1 treatment disrupts Treg interactions with CD11c-expressing cells in the meninges
The reduced migratory phenotype of Treg cells observed in the CNS during TE may be explained by prolonged interactions with CD11c+ cells. Previous studies have demonstrated that treatment with anti-LFA-1 antibodies leads to a loss of dendritic cells in the CNS (37). Indeed, treatment with anti-LFA-1 antibodies for four hours leads to a significant decrease in DCs (CD3−CD19−NK1.1−CD11c+MHCIIhi) in the meninges (Figure 5a). To determine if Treg cell interactions with DCs in vivo affects their migratory behavior, mice were treated with LFA-1 blocking antibodies four hours prior to imaging (Supplementary Movie 5). This loss of dendritic cells resulted in a reduced duration of contacts between Treg cells and the remaining CD11c+ cells, with fewer Tregs maintaining contact for the duration of the imaging period and more cells making contacts of short duration (Figure 5b). The average contact time was significantly reduced from 11.5 to 8.5 minutes. Moreover, antibody blockade resulted in a significant increase in the speed of FoxP3+ cell migration from to 2.2 to 3.3 μm/min (Figure 5c). Together these results suggest that interactions between Treg cells and CD11c+ cells limit the migratory speed of Treg cells in the CNS.
Dendritic cells shape the migratory behavior of FoxP3-GFP cells in the meninges
FoxP3-GFPxCD11c-YFP mice were chronically infected with T. gondii. On day 28 post-infection, mice received 200 μg of control or anti-LFA-1 blocking antibodies by intraperitoneal injection. The number of dendritic cells (live CD3−NK1.1−CD19− CD45hi CD11chiMHC class IIhi) remaining after four hours of antibody treatment was measured by flow cytometry (a). Explant brains were imaged 4 hours post-antibody injection. The contact duration between cell types in each condition was measured (b). The track velocity of FoxP3-GFP cells was tracked in each condition (c). 148 cells in control-treated mice and 181 cells in anti-LFA-1 treated mice were tracked from two independent experiments. * denotes p<0.05 and *** p<0.001 by student’s t-test.
Multiple studies have associated the presence of Treg cells in the CNS with the ability to limit inflammation in the context of infection (West Nile Virus, Murine Hepatitis Virus, and Coronaviruses) and autoimmunity (EAE and MS) (10–14, 38, 39), but little is known about the localization and behavior of Treg cells in these different disease settings. In addition, in many cases it has been difficult to discern local effects within the CNS versus a role for this regulatory population in peripheral events (40). The studies presented here reveal that during TE, unlike the effector CD4+ T cells, Foxp3+ T cells were restricted to the perivascular spaces and the meninges. Precedent exists for this observation, as Campbell and colleagues reported a similar localization of Treg cells in the CNS in a model of EAE (41), and suggested that signaling through CXCR3 is critical to prevent Treg cell entry into the inflamed brain parenchyma. Consistent with this idea, Treg cells present in the CNS during TE express high levels of CXCR3 and we have observed that in CXCR3 knockout mice infected with T. gondii, the localization of Treg cells is altered and that these populations are now present in the brain parenchyma (Harris unpublished observation). Thus, while recent studies have proposed that CXCR3 expression on Treg cells during Th1 inflammation allows these populations to access sites of Th1 inflammation (42), these observations suggest that CXCR3 is not only involved in entry to the tissue, but can also regulate Treg cell location within the CNS.
Whether the exclusion of Treg cells from the brain parenchyma is biologically relevant remains unclear but this may be one mechanism that allows effector T cells present in the brain parenchyma to operate independently of the suppressive effects of Treg cells and thereby better control parasite replication. In previous reports, the use of IL-2 complexes to expand Treg cell populations in infected mice led to an increased parasite burden in the CNS (21, 25), further suggesting an important role for Treg cells in controlling effector T cell responses and resulting parasite burden. There are several possible ways that Treg cells in perivascular sites might influence parasite specific effector responses in the parenchyma: In models of EAE and viral encephalitis the ability of effector T cells to interact with antigen presenting cells within these perivascular compartments allows effector T cells to proliferate and be retained in the CNS (43, 44). The ability of Treg cells to limit DC activity at this site of T cell entry to the parenchyma of the brain, perhaps through the production of IL-10, may allow Treg cells to serve as “gatekeepers” to the CNS.
Recent studies have shown that TCR signaling is required for Treg cell suppressor capacity in vivo (45) but it remains unclear whether Treg cell populations in the brain during TE are specific for parasite-derived antigens or whether these are self-reactive populations. While reagents to detect parasite-specific CD4+ T cells are currently limited to a single tetramer reagent, our results do not indicate that Tregs are specific for this parasite antigen. Moreover, the results from TCR sequencing performed in this study suggest that if Tregs are indeed parasite-specific, they rarely share a clonal lineage with effector T cells. We also found that when a TCR sequence is shared between effector T cells and Tregs, more reads were typically detected in the Treg population, which may suggest the loss of FoxP3 expression and acquisition of an effector T cell phenotype. In addition, unique Treg clones were identified in the CNS in each independent experimental infection, suggesting that variable(s) other than infection shape the repertoire of Tregs in the CNS. Our results are in agreement with several studies that also did not detect overlap in TCR sequences between regulatory and effector CD4+ T cell populations in diverse settings of tissue inflammation (46–49).
Several observations suggest that the unique behavior of Treg cells in the CNS (compared with activated effector CD4+ T cells in the same areas or Treg cells in secondary lymphoid organs) may be a function of the sustained interactions with CD11c+ populations. Indeed, several in vitro studies have also examined the influence of Treg cells on DCs and found that Treg cells decrease levels of MHC class II and expression of co-stimulatory molecules (18, 35, 50). This observation is consistent with early studies that demonstrated that the ability of Treg cells to interact with CD11c+ cells is central to Treg cell suppression of effector T cell responses (3, 5, 51). In our studies, the loss of dendritic cells from the meninges led to increased Treg cell velocity, suggesting that interactions between these two cell types influence Treg behavior at this site. The Treg interaction with DCs in this space may be important for suppressing DC function by down-regulating the co-stimulatory capacity of these antigen presenting cells or their production of cytokine, thereby regulating local effector T cell responses. Alternatively, recent studies have also demonstrated that DCs in peripheral tissues provide survival signals for Treg cells that differ from those in the lymph nodes (52) and that these cells may promote the survival of Treg cells in the brain. If these interactions are disrupted long-term, it is possible Treg survival could be affected. Regardless, the ability to visualize how Treg cells behave in inflamed tissues and determine the cell types they interact with provides insight into how these cells operate to limit inflammatory processes and a better understanding of how local effector responses are regulated at sites of inflammation.
Click here to view.(34K, pdf)
Click here to view.(1.0M, pdf)
The authors would like to thank Gordon Ruthel and the Penn Vet Imaging Core for assistance with microscopy experiments, Vijay Kuchroo and Christopher Karp for kindly providing mouse strains, and the members of our laboratories for their feedback during the development of this manuscript.
1This work was funded by National Institutes of Health grants NS-091067 to T.H.H., F32-AI-007496 to C.A.O, AI-110201 and AI-41158 to C.A.H., and P30-CA044579–23 in support of the University of Virginia Flow Cytometry Core; German Research Foundation (DFG) grant KO4609/1-1 to C.K.; and the Commonwealth of Pennsylvania.
- Bettini M, Vignali DA. Regulatory T cells and inhibitory cytokines in autoimmunity. Curr Opin Immunol. 2009;21:612–618. [PMC free article][PubMed] [Google Scholar]
- Vignali DA, Collison LW, Workman CJ. How regulatory T cells work. Nature reviews Immunology. 2008;8:523–532. [PMC free article][PubMed] [Google Scholar]
- Tang Q, Adams JY, Tooley AJ, Bi M, Fife BT, Serra P, Santamaria P, Locksley RM, Krummel MF, Bluestone JA. Visualizing regulatory T cell control of autoimmune responses in nonobese diabetic mice. Nature immunology. 2006;7:83–92. [PMC free article][PubMed] [Google Scholar]
- Fujisaki J, Wu J, Carlson AL, Silberstein L, Putheti P, Larocca R, Gao W, Saito TI, Celso C Lo, Tsuyuzaki H, Sato T, Cote D, Sykes M, Strom TB, Scadden DT, Lin CP. In vivo imaging of Treg cells providing immune privilege to the haematopoietic stem-cell niche. Nature. 2011;474:216–219. [PMC free article][PubMed] [Google Scholar]
- Bauer CA, Kim EY, Marangoni F, Carrizosa E, Claudio NM, Mempel TR. Dynamic Treg interactions with intratumoral APCs promote local CTL dysfunction. J Clin Invest. 2014;124:2425–2440. [PMC free article][PubMed] [Google Scholar]
- Lin KL, Fulton LM, Berginski M, West ML, Taylor NA, Moran TP, Coghill JM, Blazar BR, Bear JE, Serody JS. Intravital imaging of donor allogeneic effector and regulatory T cells with host dendritic cells during GVHD. Blood. 2014;123:1604–1614. [PMC free article][PubMed] [Google Scholar]
- Camirand G, Wang Y, Lu Y, Wan YY, Lin Y, Deng S, Guz G, Perkins DL, Finn PW, Farber DL, Flavell RA, Shlomchik WD, Lakkis FG, Rudd CE, Rothstein DM. CD45 ligation expands Tregs by promoting interactions with DCs. J Clin Invest. 2014;124:4603–4613. [PMC free article][PubMed] [Google Scholar]
- Lowther DE, Hafler DA. Regulatory T cells in the central nervous system. Immunol Rev. 2012;248:156–169. [PubMed] [Google Scholar]
- Anderton SM. Treg and T-effector cells in autoimmune CNS inflammation: a delicate balance, easily disturbed. Eur J Immunol. 2010;40:3321–3324. [PubMed] [Google Scholar]
- Cervantes-Barragan L, Firner S, Bechmann I, Waisman A, Lahl K, Sparwasser T, Thiel V, Ludewig B. Regulatory T cells selectively preserve immune privilege of self-antigens during viral central nervous system infection. Journal of immunology. 2012;188:3678–3685. [PubMed] [Google Scholar]
- de Aquino MT, Puntambekar SS, Savarin C, Bergmann CC, Phares TW, Hinton DR, Stohlman SA. Role of CD25(+) CD4(+) T cells in acute and persistent coronavirus infection of the central nervous system. Virology. 2013;447:112–120. [PMC free article][PubMed] [Google Scholar]
- Yu P, Gregg RK, Bell JJ, Ellis JS, Divekar R, Lee HH, Jain R, Waldner H, Hardaway JC, Collins M, Kuchroo VK, Zaghouani H. Specific T regulatory cells display broad suppressive functions against experimental allergic encephalomyelitis upon activation with cognate antigen. Journal of immunology. 2005;174:6772–6780. [PubMed] [Google Scholar]
- Kohm AP, Carpentier PA, Miller SD. Regulation of experimental autoimmune encephalomyelitis (EAE) by CD4+CD25+ regulatory T cells. Novartis Found Symp. 2003;252:45–52. discussion 52–44, 106–114. [PubMed] [Google Scholar]
- Reddy J, Illes Z, Zhang X, Encinas J, Pyrdol J, Nicholson L, Sobel RA, Wucherpfennig KW, Kuchroo VK. Myelin proteolipid protein-specific CD4+CD25+ regulatory cells mediate genetic resistance to experimental autoimmune encephalomyelitis. Proceedings of the National Academy of Sciences of the United States of America. 2004;101:15434–15439. [PMC free article][PubMed] [Google Scholar]
- Walsh JT, Zheng J, Smirnov I, Lorenz U, Tung K, Kipnis J. Regulatory T cells in central nervous system injury: a double-edged sword. J Immunol. 2014;193:5013–5022. [PMC free article][PubMed] [Google Scholar]
- Gazzinelli R, Xu Y, Hieny S, Cheever A, Sher A. Simultaneous depletion of CD4+ and CD8+ T lymphocytes is required to reactivate chronic infection with Toxoplasma gondii. J Immunol. 1992;149:175–180. [PubMed] [Google Scholar]
- Gazzinelli RT, Wysocka M, Hayashi S, Denkers EY, Hieny S, Caspar P, Trinchieri G, Sher A. Parasite-induced IL-12 stimulates early IFN-gamma synthesis and resistance during acute infection with Toxoplasma gondii. J Immunol. 1994;153:2533–2543. [PubMed] [Google Scholar]
- Onishi Y, Fehervari Z, Yamaguchi T, Sakaguchi S. Foxp3+ natural regulatory T cells preferentially form aggregates on dendritic cells in vitro and actively inhibit their maturation. Proceedings of the National Academy of Sciences of the United States of America. 2008;105:10113–10118. [PMC free article][PubMed] [Google Scholar]
- Couper KN, Blount DG, Wilson MS, Hafalla JC, Belkaid Y, Kamanaka M, Flavell RA, de Souza JB, Riley EM. IL-10 from CD4CD25Foxp3CD127 adaptive regulatory T cells modulates parasite clearance and pathology during malaria infection. PLoS Pathog. 2008;4:e1000004. [PMC free article][PubMed] [Google Scholar]
- Hall AO, Beiting DP, Tato C, John B, Oldenhove G, Lombana CG, Pritchard GH, Silver JS, Bouladoux N, Stumhofer JS, Harris TH, Grainger J, Wojno ED, Wagage S, Roos DS, Scott P, Turka LA, Cherry S, Reiner SL, Cua D, Belkaid Y, Elloso MM, Hunter CA. The cytokines interleukin 27 and interferon-gamma promote distinct Treg cell populations required to limit infection-induced pathology. Immunity. 2012;37:511–523. [PMC free article][PubMed] [Google Scholar]
- Oldenhove G, Bouladoux N, Wohlfert EA, Hall JA, Chou D, Dos Santos L, O’Brien S, Blank R, Lamb E, Natarajan S, Kastenmayer R, Hunter C, Grigg ME, Belkaid Y. Decrease of Foxp3+ Treg cell number and acquisition of effector cell phenotype during lethal infection. Immunity. 2009;31:772–786. [PMC free article][PubMed] [Google Scholar]
- Koch MA, Thomas KR, Perdue NR, Smigiel KS, Srivastava S, Campbell DJ. T-bet(+) Treg cells undergo abortive Th1 cell differentiation due to impaired expression of IL-12 receptor beta2. Immunity. 2012;37:501–510. [PMC free article][PubMed] [Google Scholar]
- Maizels RM, Smith KA. Regulatory T cells in infection. Adv Immunol. 2011;112:73–136. [PMC free article][PubMed] [Google Scholar]
- Belkaid Y. Role of Foxp3-positive regulatory T cells during infection. Eur J Immunol. 2008;38:918–921. [PMC free article][PubMed] [Google Scholar]
- Benson A, Murray S, Divakar P, Burnaevskiy N, Pifer R, Forman J, Yarovinsky F. Microbial infection-induced expansion of effector T cells overcomes the suppressive effects of regulatory T cells via an IL-2 deprivation mechanism. J Immunol. 2012;188:800–810. [PMC free article][PubMed] [Google Scholar]
- John B, Harris TH, Tait ED, Wilson EH, Gregg B, Ng LG, Mrass P, Roos DS, Dzierszinski F, Weninger W, Hunter CA. Dynamic Imaging of CD8(+) T cells and dendritic cells during infection with Toxoplasma gondii. PLoS pathogens. 2009;5:e1000505. [PMC free article][PubMed] [Google Scholar]
- Wilson EH, Wille-Reece U, Dzierszinski F, Hunter CA. A critical role for IL-10 in limiting inflammation during toxoplasmic encephalitis. J Neuroimmunol. 2005;165:63–74. [PubMed] [Google Scholar]
- Derecki NC, Cardani AN, Yang CH, Quinnies KM, Crihfield A, Lynch KR, Kipnis J. Regulation of learning and memory by meningeal immunity: a key role for IL-4. The Journal of experimental medicine. 2010;207:1067–1080. [PMC free article][PubMed] [Google Scholar]
- Wei T, Simko V. corrplot: Visualization of a Correlation Matrix. 2016 R package version 0.77. https://CRAN.R-project.org/package=corrplot.
- Kolde R. pheatmap: Pretty Heatmaps. 2015 R package version 1.0.8. https://CRAN.R-project.org/package=pheatmap.
- Lex A, Gehlenborg N, Strobelt H, Vuillemot R, Pfister H. UpSet: Visualization of Intersecting Sets. IEEE transactions on visualization and computer graphics. 2014;20:1983–1992. [PMC free article][PubMed] [Google Scholar]
- Gehlenborg N. UpsetR: A More Scalable Alternative to Venn and Euler Diagrams for Visualizing Intersecting Sets. 2016 R package 1.2.2. https://CRAN.R-project.org/package=UpSetR.
- Davalos D, Grutzendler J, Yang G, Kim JV, Zuo Y, Jung S, Littman DR, Dustin ML, Gan WB. ATP mediates rapid microglial response to local brain injury in vivo. Nat Neurosci. 2005;8:752–758. [PubMed] [Google Scholar]
- Radjavi A, Smirnov I, Derecki N, Kipnis J. Dynamics of the meningeal CD4(+) T-cell repertoire are defined by the cervical lymph nodes and facilitate cognitive task performance in mice. Molecular psychiatry. 2014;19:531–533. [PMC free article][PubMed] [Google Scholar]
- Liang B, Workman C, Lee J, Chew C, Dale B, Colonna L, Flores M, Li N, Schweighoffer E, Greenberg S, Tybulewicz V, Vignali D, Clynes R. Regulatory T cells inhibit dendritic cells by lymphocyte activation gene-3 engagement of MHC class II. Journal of immunology. 2008;180:5916–5926. [PubMed] [Google Scholar]
- Mempel TR, Henrickson SE, Von Andrian UH. T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases. Nature. 2004;427:154–159. [PubMed] [Google Scholar]
- John B, Ricart B, Tait Wojno ED, Harris TH, Randall LM, Christian DA, Gregg B, De Almeida DM, Weninger W, Hammer DA, Hunter CA. Analysis of behavior and trafficking of dendritic cells within the brain during toxoplasmic encephalitis. PLoS pathogens. 2011;7:e1002246. [PMC free article][PubMed] [Google Scholar]
- Koutrolos M, Berer K, Kawakami N, Wekerle H, Krishnamoorthy G. Treg cells mediate recovery from EAE by controlling effector T cell proliferation and motility in the CNS. Acta neuropathologica communications. 2014;2:163. [PMC free article][PubMed] [Google Scholar]
- Lanteri MC, O’Brien KM, Purtha WE, Cameron MJ, Lund JM, Owen RE, Heitman JW, Custer B, Hirschkorn DF, Tobler LH, Kiely N, Prince HE, Ndhlovu LC, Nixon DF, Kamel HT, Kelvin DJ, Busch MP, Rudensky AY, Diamond MS, Norris PJ. Tregs control the development of symptomatic West Nile virus infection in humans and mice. J Clin Invest. 2009;119:3266–3277. [PMC free article][PubMed] [Google Scholar]
- Graham JB, Da Costa A, Lund JM. Regulatory T cells shape the resident memory T cell response to virus infection in the tissues. J Immunol. 2014;192:683–690. [PMC free article][PubMed] [Google Scholar]
- Muller M, Carter SL, Hofer MJ, Manders P, Getts DR, Getts MT, Dreykluft A, Lu B, Gerard C, King NJ, Campbell IL. CXCR3 signaling reduces the severity of experimental autoimmune encephalomyelitis by controlling the parenchymal distribution of effector and regulatory T cells in the central nervous system. J Immunol. 2007;179:2774–2786. [PubMed] [Google Scholar]
- Koch MA, Tucker-Heard G, Perdue NR, Killebrew JR, Urdahl KB, Campbell DJ. The transcription factor T-bet controls regulatory T cell homeostasis and function during type 1 inflammation. Nature immunology. 2009;10:595–602. [PMC free article][PubMed] [Google Scholar]
- Bartholomaus I, Kawakami N, Odoardi F, Schlager C, Miljkovic D, Ellwart JW, Klinkert WE, Flugel-Koch C, Issekutz TB, Wekerle H, Flugel A. Effector T cell interactions with meningeal vascular structures in nascent autoimmune CNS lesions. Nature. 2009;462:94–98. [PubMed] [Google Scholar]
- Kang SS, Herz J, Kim JV, Nayak D, Stewart-Hutchinson P, Dustin ML, McGavern DB. Migration of cytotoxic lymphocytes in cell cycle permits local MHC I-dependent control of division at sites of viral infection. J Exp Med. 2011;208:747–759. [PMC free article][PubMed] [Google Scholar]
- Levine AG, Arvey A, Jin W, Rudensky AY. Continuous requirement for the TCR in regulatory T cell function. Nat Immunol. 2014;15:1070–1078. [PMC free article][PubMed] [Google Scholar]
- Lord J, Chen J, Thirlby RC, Sherwood AM, Carlson CS. T-cell receptor sequencing reveals the clonal diversity and overlap of colonic effector and FOXP3+ T cells in ulcerative colitis. Inflammatory bowel diseases. 2015;21:19–30. [PMC free article][PubMed] [Google Scholar]
- Nguyen P, Liu W, Ma J, Manirarora JN, Liu X, Cheng C, Geiger TL. Discrete TCR repertoires and CDR3 features distinguish effector and Foxp3+ regulatory T lymphocytes in myelin oligodendrocyte glycoprotein-induced experimental allergic encephalomyelitis. J Immunol. 2010;185:3895–3904. [PMC free article][PubMed] [Google Scholar]
- Liu X, Nguyen P, Liu W, Cheng C, Steeves M, Obenauer JC, Ma J, Geiger TL. T cell receptor CDR3 sequence but not recognition characteristics distinguish autoreactive effector and Foxp3(+) regulatory T cells. Immunity. 2009;31:909–920. [PMC free article][PubMed] [Google Scholar]
- Burzyn D, Kuswanto W, Kolodin D, Shadrach JL, Cerletti M, Jang Y, Sefik E, Tan TG, Wagers AJ, Benoist C, Mathis D. A special population of regulatory T cells potentiates muscle repair. Cell. 2013;155:1282–1295. [PMC free article][PubMed] [Google Scholar]
- Wing K, Onishi Y, Prieto-Martin P, Yamaguchi T, Miyara M, Fehervari Z, Nomura T, Sakaguchi S. CTLA-4 control over Foxp3+ regulatory T cell function. Science. 2008;322:271–275. [PubMed] [Google Scholar]
- Tang Q, Krummel MF. Imaging the function of regulatory T cells in vivo. Curr Opin Immunol. 2006;18:496–502. [PubMed] [Google Scholar]
- Smigiel KS, Richards E, Srivastava S, Thomas KR, Dudda JC, Klonowski KD, Campbell DJ. CCR7 provides localized access to IL-2 and defines homeostatically distinct regulatory T cell subsets. J Exp Med. 2014;211:121–136. [PMC free article][PubMed] [Google Scholar]