Abstract
Multiple sclerosis (MS) is an inflammatory disease of the CNS that causes the demyelination of nerve cells and destroys oligodendrocytes, neurons, and axons. Historically, MS has been thought to be a CD4 T cell–mediated autoimmune disease of CNS white matter. However, recent studies identified CD8 T cell infiltrates and gray matter lesions in MS patients. These findings suggest that CD8 T cells and CNS Ags other than myelin proteins may be involved during the MS disease process. In this article, we show that CD8 T cells reactive to glial fibrillary acidic protein (GFAP), a protein expressed in astrocytes, can avoid tolerance mechanisms and, depending upon the T cell–triggering event, drive unique aspects of inflammatory CNS autoimmunity. In GFAP-specific CD8 TCR-transgenic (BG1) mice, tissue resident memory-like CD8 T cells spontaneously infiltrate the gray matter and white matter of the CNS, resulting in a relapsing–remitting CNS autoimmunity. The frequency, severity, and remissions from spontaneous disease are controlled by the presence of polyclonal B cells. In contrast, a viral trigger induces GFAP-specific CD8 T effector cells to exclusively target the meninges and vascular/perivascular space of the gray and white matter of the brain, causing a rapid, acute CNS disease. These findings demonstrate that the type of CD8 T cell–triggering event can determine the presentation of distinct CNS autoimmune disease pathologies.
This article is featured in In This Issue, p.2935
Introduction
Multiple sclerosis (MS) is an inflammatory T cell–mediated autoimmune disease of the CNS that causes the demyelination of nerve cells and destroys oligodendrocytes, neurons, and axons (1, 2). MS is thought to be primarily a CD4 T cell–mediated disease. Disease susceptibility linkage to MHC class II genes, the study of myelin-reactive CD4 T cells from MS patients, and models of experimental autoimmune encephalomyelitis (EAE) clearly indicate that myelin-reactive CD4 T cells have a central role in MS disease pathogenesis (3–8). However, CD4 T cells are unlikely to be the sole mediators of disease pathogenicity because treatments specifically targeting these cells have failed to limit the rate of disease relapses or new lesion formation, whereas therapies that deplete or inhibit CNS infiltration of all lymphocyte subsets have been more successful (9–11).
Over the past several years, strong evidence has been accumulating to suggest that CD8 T cells also contribute to MS disease. Studies showed that CD8 T cells are found in both white matter and gray matter MS plaques. In addition, these CD8 T cells are often oligoclonal and can outnumber CD4 T cells, regardless of the stage or activity of disease (2, 12–16). However, the Ag specificity of these CNS-infiltrating CD8 T cells remains unclear. In addition, the function of these T cells has been proposed to be either pathogenic or protective.
In support of CD8 T cells having a pathogenic role in the MS disease process, myelin-specific CD8 T cells have been isolated from MS patients that are capable of killing neuronal cells in vitro (17–21). In addition, MS disease susceptibility shows some genetic linkage to particular MHC class I alleles (22, 23). In animal models of CNS disease, CD8 T cells specific for myelin basic protein (MBP), myelin oligodendrocyte protein (MOG), and proteolipid protein (PLP) were shown to be pathogenic (24–28). The clinical symptoms induced by CNS-reactive CD8 T cells can be diverse. Mice carrying activated MBP-specific CD8 T cells succumb to a nonparalytic, acute demyelinating CNS autoimmunity that is clinically and histologically different from classic CD4-EAE. These atypical-EAE disease pathologies have similarities to MS patients with upper motor neuron disease (24). Experiments with MOG and PLP-specific CD8 T cells, in contrast, induced CNS disease symptoms similar to classical EAE (25–28). These data suggest that myelin-specific CD8 T cells may contribute to some of the disease heterogeneity observed in MS patients.
In contrast to a pathogenic role, many studies suggested that CD8 T cells are suppressive to CNS disease. In animal models, early studies found that polyclonal CD8 T cells can limit disease severity and relapses of CD4 T cell–mediated EAE (29, 30). The ability of CD8 T cells to regulate CNS autoimmune disease may occur from CD8 T cells targeting activated CD4 T cells through the recognition of peptide displayed on MHC class I and Ib molecules, as well as by secreting IL-10 and other anti-inflammatory soluble mediators (5, 31–33). Consistent with these findings, CD8 T cell clones that can lyse myelin-specific CD4 T cells were detected in MS patients (34–36), and longitudinal magnetic resonance imaging analysis showed a negative correlation between the percentage of Tc2 cytokine-producing CD8 T cells in the periphery of MS patients and the development of lesions (37). Thus, CD8 T cells, like their CD4 counterparts, can be pathogenic or immunoregulatory.
To contribute to the CNS autoimmune disease process, autoreactive CD8 T cells have to avoid negative selection within the thymus and be exported to the peripheral T cell repertoire. Several CNS proteins, and in particular myelin proteins that are often the target of encephalogenic CD4 T cells, are predominately expressed behind the blood–brain barrier. The lack of or minimal expression of these CNS proteins within the thymus is thought to allow encephalogenic T cells to develop. However, some myelin epitopes are expressed and presented in the thymus, and developing T cells that are reactive to these ligands can be subject to thymic deletion or be skewed toward low-avidity or suppressive responses (6, 38–41). Following export to the mature T cell repertoire, activation events result in CD8 T cells differentiating into a number of effector and suppressor lineages, depending upon the TCR–peptide/MHC interaction and the local inflammatory environment (42). Thus, some of the diverse contributions of CD8 T cells to CNS autoimmune disease may be a product of CD8 T cells that have differentiated into different effector or suppressor lineages or that target different CNS Ags.
Experiments presented in this article demonstrate that the clinical and histological features of CD8 T cell–mediated CNS disease are dependent upon how the CD8 T cells are activated and correlate with the CD8 T cells lineage that are present within the CNS. We observe that, in C57BL/6 mice, CD8 T cells reactive to multiple CNS proteins are present in the mature T cell repertoire. For CD8 T cells that target glial fibrillary acidic protein (GFAP), a protein expressed in astrocytes, we show that high-avidity CD8 T cells that target a stable GFAP peptide/MHC complex can avoid tolerance mechanisms and, when activated, induce white matter and gray matter CNS autoimmune pathology. Furthermore, spontaneous relapsing–remitting and chronic disease mediated by GFAP-specific CD8 T cells are associated with CD8 T cells with tissue-resident memory-like phenotypes infiltrating the CNS parenchyma and are regulated by polyclonal B cells. In contrast, rapid acute disease following a viral trigger stems from GFAP-specific CD8 T cells with a T effector (TEFF) and T effector-memory (TEM) lineage that are located primarily within the meninges and vascular/perivascular space, with minimal parenchymal infiltration. Differences in the composition and location of the lesions, as well as clinical symptoms created during spontaneous disease versus a viral induction, indicate that the triggering event that activates autoreactive CD8 T cells can contribute to heterogeneity in CNS autoimmunity and clinical disease course.
Materials and Methods
Mice
C57BL/6, C57BL/6.SJL, Rag1−/−, μMT, Nur77-GFP, and Gfap−/− mice were purchased from The Jackson Laboratory (Bar Harbor, ME). BG1-transgenic (Tg) mouse lines were created by injecting TCR-encoding plasmids directly into C57BL/6 oocytes. All mice were maintained in a pathogen-free environment in accordance with institutional guidelines in the Animal Care Facility at the University of Massachusetts Medical School.
Peptides, tetramers, and construction of recombinant viruses
The ability of 8–10mer peptide sequences from CNS proteins to bind MHC was evaluated using the consensus binding prediction implemented in the Immune Epitope Database (http://www.iedb.org). All peptides were purchased from A&A Laboratory (San Diego, CA). Recombinant replication-deficient human adenovirus (Ad)5 expressing the full-length GFAP, MAG, MBP, MOG, or PLP cDNA was constructed using the AdEasy XL Adenoviral Vector System (Stratagene). Recombinant vaccinia virus (Vac) expressing the full-length GFAP, MAG, MBP, MOG, or PLP cDNA and protein truncations fused to GFP were inserted into the thymidine kinase gene (43). H2-Db and H2-Kb tetramers presenting GFAP264–272 were constructed using published methods (44).
Isolation and characterization of the CD8 T cell clone and cloning of the BG1 TCR
Four-week-old C57BL/6 mice were infected i.p. with 2 × 107 PFU recombinant human Ad5 or Vac expressing full-length GFAP, MAG, MBP, MOG, or PLP. Following 3 wk of infection, 2 × 107 spleen and lymph node cells were isolated and stimulated in vitro with irradiated spleen cells loaded with 1 μg/ml a pool of five peptides. Following 1 wk in culture, responding T cells were cloned by limiting dilution with irradiated spleen cells pulsed with 1 μg/ml peptide pools and 50 U IL-2. Cultures that demonstrated T cell expansion were expanded by weekly restimulation and tested for specificity as read out by the ability to produce IFN-γ in response to target cells loaded with GFAP peptides.
TCR Vβ-chains were identified by staining with a set of Vβ-specific Abs (BD Biosciences), and the TCRα-chains were identified by PCR analysis using a panel of TCR Vα primers that collectively amplify all TCR Vα gene families. GFAP-specific T cells expressed TCRs carrying Vα4:Vβ9 and Vα5:Vβ9 sequences; MOG-specific T cells expressed TCRs carrying Vα4:Vβ4, Vα8:Vβ4, and Vα10:Vβ11 sequences; and MAG-specific T cells expressed different Vα17:Vβ11 clonotypes.
The rearranged cDNAs for BG1 TCR Vα4.5 were cloned XhoI to BspEI using the primers Vα4.5 sense 5′-GGGCTCGAGAAGATGGACTCTTCTCCA-3′ and TCR Cα antisense 5′-CTGGTACACAGCAGGTTCCGGATTCTGGATGT-3′. The rearranged cDNAs for Vβ9.1 gene were cloned EcoRI to Bgl2 using the primers Vβ9.1 sense 5′-TCGACGAATTCGAGAGGAAGCATGGATCCTAGACTTCTTTGCTG-3′ and TCR Cβ antisense 5′-CTTGGGTGGAGTCACATTTCTCAGATCTTC-3′.
All of the TCR genes were sequenced, and error-free full-length cDNAs were subcloned into the human CD2 promoter transgene cassette for T cell–specific expression (45). TCR-Tg mouse lines were established by injecting C57BL/6 oocytes with the TCR-Tg plasmids.
Characterization of CD8 T cell specificity
The specificity of the T cell clones was assessed by the ability to produce IFN-γ following Ag recognition. The CD8 T cell clones were restimulated for 5 d, and then 3 × 105 CD8 T cells were incubated for 4 h at 37°C with 1 × 105 bone marrow–derived dendritic cells (BM-DCs) infected at a multiplicity of infection of 3 with Vac:GFAP, MAG, MOG, or vaccinia expressing GFP viruses or pulsed with GFAP, MAG, or MOG peptides in the presence of GolgiStop and GolgiPlug (1 μl/ml; BD Biosciences). BM-DCs were differentiated in vitro by growing bone marrow with GM-CSF for 7 d (43). For analysis of IFN-γ production, T cells were surfaced stained with anti-CD8 and Thy1.2, washed, fixed in 2% formaldehyde (Fischer Scientific), and stained for intracellular IFN-γ using a Cytofix/Cytoperm kit (BD Biosciences), according to the manufacturer’s protocol. To clarify MHC restriction, CD8 T cell clones were tested for the ability to recognize GFAP, MAG, or MOG peptides presented by BM-DCs expanded from NOD mice (H2-Kd and H2-Db) or a fibroblast cell line isolated from H2-Kb−/−H2-Db−/− mice that was retrovirally transduced with either H2-Kb or H2-Db. Flow cytometry (LSR II; BD Biosciences) of CD8 T cell responses was analyzed using FlowJo software, version 8.3 (TreeStar).
RMA-S stabilization assay
To detect ability of peptides to bind to MHC class I molecules, 5 × 105 RMA-S cells, which are Ag-processing defective T cell lymphoma cells derived from C57BL/6 mice, were mixed with peptides at various concentrations and incubated at 25°C overnight. Cells were cultured at 37°C for 2 h, washed, and stained for MHC class I molecule surface expression using anti–H2-Kb–PE (AF6-885) and anti–H2-Db–FITC (KH95) (both from BD Biosciences).
Clinical scoring scale for classical and nonclassical CNS autoimmunity
Classical EAE was scored on a scale of 0–5 following traditional scoring methods (46). Nonclassical EAE symptoms were scored on a scale of 0–12 using a method that tests for cerebellar dysfunction. Scores of 0–3 are given for balancing on a ledge, hind limb clasping, gait, and kyphosis (47).
Isolation of T cells from CNS and analysis of lymphocyte populations
To isolate lymphocytes from the CNS, mice were deeply anesthetized with 125 mg/kg ketamine and 10 mg/kg xylazine and perfused through the caudal vena cava with 40 ml heparinized PBS. Brain and spinal cord were isolated, manually disassociated, and passed through a 40-μm cell strainer. Lymphocyte populations were isolated from single-cell suspensions using a 30 and 70% Percoll density gradient. Lymphocyte populations were cell surface stained and analyzed by flow cytometry. In some experiments, cells were fixed in 2% formaldehyde and analyzed by intracellular staining. To identify the cytokine profile, lymphocytes were stimulated in vitro with 50 ng/ml PMA and 750 ng/ml ionomycin in the presence of GolgiStop/GolgiPlug (1:1000) for 4 h at 37°C and analyzed for intracellular cytokine expression.
Vac-GFAP induction of CNS autoimmunity
CD8 T cells (CD45.2+) were positively isolated from Rag−/− Gfap−/− BG1 mice using MACS. Titrating numbers of BG1 CD8 T cells were transferred i.v. into recipient C57BL/6.SJL (CD45.1+) mice, followed by infection using 1 × 107 PFU Vac:GFAP on day 0. Mice were monitored for 6 wk (only 2 wk is shown in figures). Disease scores are based on atypical EAE symptoms. Transferred BG1 CD8 T cells in spleen and CNS were analyzed by flow cytometry on days 4, 6, and 14.
Histologic analysis of brain and spinal cords
For formalin-fixed paraffin sections, brain and spinal cord were dissected, fixed in 10% Millonig’s modified phosphate-buffered formalin for 48 h, sectioned, and embedded in paraffin wax. To ensure reproducibility, a rodent brain matrix (Electron Microscopy Sciences) was used for all brain sectioning. Six serial sections of five brain cross-sections and three longitudinal sections (C1–6, T3–13, L3–cauda equina) per mouse were stained with H&E, Luxol Fast Blue (LFB), or mAb specific for GFAP (Vector Laboratories). Sections from other major organs from these mice were stained with H&E.
For immunohistochemistry analysis, mice were deeply anesthetized and perfused with 40 ml ice-cold heparinized PBS with 4% paraformaldehyde. Brain and spinal cords were dissected and cryopreserved in 10% sucrose for 2 h, 20% sucrose for 2 h, and 30% sucrose overnight at 4°C. Sectioned tissues were frozen in Optimal Cutting Temperature compound (Tissue-Tek). The brain sections were cut, fixed in acetone, and blocked with anti-CD16/32 Ab (2.4G2), followed by 5% goat serum plus blocking buffer (PBS containing 2% BSA, 0.1% Triton X-100, and 0.02% sodium azide). After washing, the sections were stained with the following Abs in blocking buffer for 1 h at room temperature and then overnight at 4°C: anti-CD4–FITC (RM4.5), anti-B220–allophycocyanin (RA3-6B2), anti-CD11c–Biotin (N418), and anti-CD19–Biotin (eBio1D3) (all from eBioscience); anti-CD3e–FITC (145-2C11), anti-CD3e–allophycocyanin (145-2C11), and anti-CD4–allophycocyanin (RM4.5) (all from BD Biosciences); anti-CD8–allophycocyanin (2.43; Bio X Cell); rat anti-GFAP (2.2B10; Vector Laboratories); and goat anti-Rat IgG–Texas Red, Streptavidin-FITC (all from Molecular Probes). For preservation, the labeled sections were mounted in ProLong Gold (Molecular Probes), with or without DAPI, which stains nuclei. Images were acquired on a Leica TCS SP5 II confocal microscope using HCX PL APO 63×/1.40–0.60 and HCX PL APO 20×/0.70 objective lenses. Signals were acquired with LAS AF software (Leica), and Photoshop CS4 (Adobe) was used for image processing.
Results
Isolation of CD8 T cells reactive to GFAP, MAG, and MOG
To identify CNS-reactive CD8 T cells that avoid negative selection within the thymus and populate the T cell repertoire, C57BL/6 mice were infected with recombinant Vac or Ad expressing the full-length cDNA of GFAP, MAG, MBP, MOG, or PLP. After 3 wk, splenocytes from infected mice were restimulated in vitro with GFAP, MAG, MBP, MOG, or PLP peptides that carry H-2b MHC class I binding motifs and cloned by limiting dilution. Using this protocol, we isolated CD8 T cell clones specific for GFAP264–272 (AASRNAELL) bound to H2-Db, MAG444–451 (VICTSRNL) bound to H2-Kb, and MOG81–88 (VTLRIQNV) bound to H2-Kb (Fig. 1A–I). However, we were unable to identify MBP- or PLP-specific CD8 T cells from C57BL/6 mice. GFAP-specific CD8 T cells reacted strongly with APCs presenting soluble GFAP264–272, with an EC50 value of 40 nM, whereas MAG- and MOG-specific CD8 T cells showed a weaker response, with EC50 values of 3 and 2 μM, respectively (Fig. 1A, 1D, 1G). Correlating with the strong CD8 T cell response, the GFAP264–272 peptide stably bound H2-Db, as read out using an RMA-S stabilization assay (Fig. 1J). In contrast, both MAG444–451 and MOG81–88 epitopes stabilized H2-Kb poorly (Fig. 1K). These data indicate that C57BL/6 mice carry CD8 T cells reactive to multiple CNS Ags, including strong-avidity CD8 T cells that target a stable GFAP peptide/MHC complex.
Isolation and characterization of CD8 T cell clone specific for GFAP, MAG, and MOG. CD8+ T cells were isolated from spleen and lymph nodes of Ad-GFAP, Ad-MAG, and Ad-MOG or Vac-GFAP, Vac-MAG, and Vac-MOG infected C57BL/6 mice, restimulated with different peptides predicted to bind to either H2-Kb or H2-Db, and cloned by limiting dilution. Four GFAP264–272-specific (A, B, and C), MAG444–451-specific (D, E, and F), or MOG81–88-specific (G, H, and I) CD8 T cell clones were tested for the ability to produce IFN-γ in response to APCs presenting titrating concentrations of peptide Ag and the ability to recognize virally infected target cells or target cells expressing only H2-Kb or H2-Db. RMA-S cells were incubated overnight at 25°C with different concentrations of GFAP264-272, LCMV NP396–404, or DMK138–146 (J), or MAG444–451, MOG81–88, or OVA257–264 (K), and RMA-S cells were shifted to 37°C for 2 h and stained with Abs specific for H2-Db or H2-Kb. Data shown are representative of at least three individual experiments. The horizontal lines in (B), (E), and (H) represent the average value. Error bars in graphs represent SEM. *p < 0.05, ***p < 0.001.
Generation of BG1 TCR-Tg mice specific for GFAP264–272
To identify how strong-avidity CD8 T cells reactive to GFAP avoid tolerance mechanisms, we constructed TCR-Tg mice expressing the Vα4.5 and Vβ9.1 TCR chains from the GFAP-specific CD8 T cell clone, BG1. Several founder lines were created, each of which displayed a similar phenotype, and one line was chosen for comprehensive analysis.
GFAP is expressed within the brain and spinal cord and, at a lower level, in some peripheral tissues, including the thymus, intestine, and pancreas (48). Thus, it is possible that developing BG1 CD8 T cells could engage cells presenting the GFAP peptide and undergo deletion or inactivation. To determine whether tolerance was occurring, CD8 T cells isolated from BG1 mice were compared with CD8 T cells isolated from GFAP-deficient (Gfap−/−) BG1 mice, which do not express the self-Ag that BG1 T cells recognize (Fig. 2). Central or peripheral tolerance was not observed in 4-wk-old BG1 mice; wild-type (WT) and Gfap−/− BG1 mice have a similar thymic cellularity, produce similar numbers of CD8 single-positive thymocytes and mature CD8 T cells that stain equivalently with H2-Db/GFAP264–272 tetramers (Fig. 2A–D), and have a similar ability to produce TNF-α and IFN-γ in response to titrating amounts of GFAP264–272 peptide (Fig. 2E, 2F).
Development of GFAP-specific CD8+ T cells in BG1 mice. Thymocytes (A and B) or splenocytes (C, D, E, and F) from 4-wk-old WT BG1, Rag−/− BG1, Gfap−/− BG1, and Rag−/−Gfap−/− BG1 mice were stained with indicated Abs, including H2-Db:GFAP264–272 tetramer. Reactivity to H2-Db:GFAP262–272 tetramer is shown for CD8 single-positive thymocytes (B) or mature CD8+ T cells (D) (open graph). Shaded graphs show H2-Db:GFAP262–272 tetramer staining of CD8 T cells isolated from C57BL/6 mice. Horizontal lines in (D) represent tetramer-positive T cells. The numbers in the graph are the percent positive T cells. (E and F) CD8 T cell splenocytes from BG1 mice produce IFN-γ and TNF-α following activation with APCs presenting titrating concentrations of GFAP264–272, shown in (E) in response to 1 μM GFAP264–272. Each result is representative of at least five mice/group, and each experiment was performed at least twice. (G) Sequence of the BG1 TRAV6-6*01 (Vα4.5) TCRα-chain and TRB17*01 (Vβ9.1) TCRβ-chain.
Consistent with the hypothesis that BG1 CD8 T cells are primarily ignorant of GFAP within secondary lymphoid organs, BG1 CD8 T cells isolated from Gfap−/− mice did not undergo proliferation when transferred into GFAP-expressing C57BL/6 mice (Fig. 3A, 3B). BG1 CD8 T cells strongly proliferated if recipient mice were infected with Vac:GFAP, indicating that the transferred CD8 T cells did not become unresponsive to T cell activation (Fig. 3C, 3D). These data indicate that GFAP-reactive CD8 T cells that are not subject to overt thymic deletion or the development of unresponsiveness can develop in C57BL/6 mice. However, CD8 T cells spontaneously enter into the brain and spinal cord of BG1 mice. Within the CNS tissue, the majority of BG1 CD8 T cells receive signals through their TCR, because they strongly express the TCR-signaling–dependent protein, Nur77 (49), as well as the activation markers CD69 and CD25 (Fig. 3E, 3F). In contrast, BG1 CD8 T cells were not observed to generate strong TCR-mediated signals in peripheral lymphoid organs or non-CNS organs (Fig. 3E–H).
BG1 CD8 T cells are primarily ignorant of endogenous GFAP Ag within the secondary lymphoid organs. Naive Gfap−/− CD45.2+ BG1 CD8 T cells were labeled with CFSE and adoptively transferred into Gfap+/+ CD45.1+ (A) or Gfap−/− CD45.1+ (B) recipients. (C and D) CD45.2+ CD8 T cells were analyzed for CFSE dilution 3 d posttransfer or infected with Vac:GFAP (open graph) or Vac:PLP (shaded graph) and analyzed 3 d postinfection. CD8 T cells from 8-wk-old BG1 TCR-Tg mice carrying the TCR-signaling reporter transgene Nur77-GFP were analyzed for GFP expression and the activation markers CD69 (E) or CD25 (F) within the secondary lymphoid organs and CNS. Quantitation of Nur77-GFP expression in BG1 CD8 T cells (G) and CD4 T cells (H) in lymphoid and nonlymphoid tissues. Lines in (G) and (H) represent the average value. Results are representative of at least two independent experiments, with three mice/group.
BG1 mice succumb to spontaneous relapsing–remitting CNS autoimmunity
To determine whether BG1 mice maintain quiescence to GFAP over their lifetime, a cohort of BG1 mice on a C57BL/6 background (WT BG1), Rag1−/− BG1 and Gfap−/− BG1 mice, were analyzed for clinical signs of CNS disease as they aged. We observed that BG1 mice do not maintain ignorance of GFAP: ∼47% (44/93) of WT BG1 mice and 100% (22/22) of Rag−/− BG1 mice succumb to spontaneous clinical signs of CNS autoimmunity by 6 mo of age (Fig. 4A).
WT BG1 and Rag−/− BG1 mice succumb to spontaneous relapsing–remitting CNS autoimmunity. (A) Incidence of first clinical signs of spontaneous EAE-like disease. Disease incidence is statistically significantly different between WT BG1 (n = 93) and Rag−/− BG1 (n = 22) mice (p = 0.003, log-rank test). No Gfap−/− BG1 mice (n = 15) developed signs of disease. (B) Two examples of a clinical disease course for WT BG1 mice (left panels) and Rag−/− BG1 mice (right panels). Note that some of the Rag−/− BG1 mice did not survive to 180 d-old because of progressive disease.
WT BG1 and Rag−/− BG1 mice developed two distinguishable forms of clinical symptoms (Table I). The majority of diseased WT BG1 mice (34/44) and Rag1−/− BG1 mice (22/22) developed balancing defects, lethargy, uneven gait, and ataxia (Supplemental Video 1). These symptoms are referred to as atypical disease and were scored using a scale of 0–12 that tests for cerebellar dysfunction. Scores of 0–3 are given for balancing on a ledge, hind limb clasping, gait, and kyphosis (47). Most diseased WT BG1 mice (27/44) and some Rag−/− BG1 mice (9/22) also succumbed to mild ascending flaccid paralysis, symptoms referred to as classical EAE. These mice were scored on a scale of 0–5, following classical EAE protocol. Some of the diseased WT BG1 mice (10/44) only displayed classical EAE symptoms (Table I). In both WT BG1 and Rag1−/− BG1 mice, clinical symptoms began as episodic bouts of functional impairment, with many mice displaying severe CNS dysfunction (e.g., classical disease scores of 3, atypical scores of 6–8) and then remitting to unobservable functional defects (Fig. 3B, 3C). Rag1−/− BG1 mice developed more severe bouts of disease and had more relapses than did WT BG1 mice (Table I). As Rag1−/− BG1 mice aged, (21/22) mice developed chronic disease (defined as ≥30 d of continual functional impairment), with a small subset (4/22) progressing to morbidity. In contrast, only 1 of 44 WT BG1 mice converted to chronic disease.
GFAP-specific CD8 T cells infiltrate the brain of BG1 mice
The neurologic impairments observed in diseased BG1 mice indicated that pathogenic CD8 T cells were infiltrating the CNS. To identify the time frame in which CD8 T cells enter into the CNS, we analyzed cellular infiltrates in BG1 mice at different ages. In WT BG1 mice and Rag−/− BG1 mice, CD8 T cells were found within the brain at 30 d of age and were present throughout life (Fig. 5A). Because not all WT BG1 mice succumb to spontaneous clinical symptoms, T cell trafficking to the CNS is not the sole arbiter of whether overt clinical disease occurs. In addition to CD8 T cells, CD4 T cells and B cells were found within the brain of WT BG1 mice at all ages (Fig. 5B, 5C). Similar lymphocyte populations were observed within the spinal cord, albeit at a lower frequency than in the brain (Fig. 5D–F). These frequencies correspond to a 10-fold increase in T cells and 100-fold increase in B cells that infiltrate the brain compared with the spinal cord on a per weight basis. These lymphocyte accumulations within the CNS require CD8 T cell recognition of GFAP, because Gfap−/− BG1 mice carry few T cells or B cells within the brain.
Brain-infiltrating BG1 CD8+ T cells enter into the CNS of young mice and have memory-like phenotypes with limited inflammatory and cytotoxic function. Absolute numbers of lymphocytes in the brain (A, B, and C) and spinal cord (D, E, and F) were determined for three age groups. CD8 T cells (Thy1.2+ CD8+ CD4− B220−), CD4 T cells (Thy1.2+ CD8− CD4+ B220−), and B cells (Thy1.2− CD8− CD4− B220+) were identified by flow cytometry. The horizontal lines in (A)–(F) represent the average value. (G) BG1 CD8 T cell subsets within the brain were determined by the expression of CD62L, CD44, CD127, KLRG1, CD103, and CD69, as shown in Supplemental Fig. 2. CD8 T cells were defined as T central memory (TCM) (CD62L+ CD44+ KLRG1−), T effector-memory (TEM) (CD62L− CD44+ KLRG1−), T effector (TEFF) (CD62L− CD44+ KLRG1+), or TRM (CD44+ CD69+ CD103+). BG1 CD8 T cells within the brain of 45–60-d-old BG1 mice are poor secretors of IFN-γ and IL-17A (H) and lack GZB production (I). The lines in (I) show the GZB-positive cells. The numbers are the percent GZB-positive. Data shown are from at least six individual mice/group. Error bars in graphs represent SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. ns, not significant.
To identify the CD8 T cell effector populations that target the CNS during spontaneous CNS disease, the lineage and effector function of brain-resident CD8 T cells were determined. The CD8 T cells present within the CNS of WT BG1 mice and Rag−/− BG1 mice show a similar, mixed expression of CD44 and CD62L, as well as a coincident lack of KLRG1 or CD127 expression (Fig. 5G, Supplemental Fig. 2). In addition, a large frequency of the CD8 T cells residing within the brain express the activation marker, CD69, and the αE integrin, CD103. Based on these phenotypic markers, many of the brain-resident CD8 T cells resemble antiviral tissue-resident memory (TRM) cells that populate peripheral tissues following viral challenges (Fig. 5G) (50, 51). Functionally, however, only low frequencies of CD8 T cells within the CNS are capable of producing IFN-γ, IL-17, or granzyme B (GZB), indicating that many of the BG1 CD8 T cells present within the brain are not classic effector CD8 T cells (Fig. 5H, 5I). Collectively, these data indicate that BG1 CD8 T cells that spontaneously enter into the brain recognize cells presenting GFAP264–272, with many adapting to the brain environment without gaining inflammatory cytokine expression or cytotoxic effector functions.
BG1 CD8 T cells target the cerebellum, midbrain, and spinal cord early in spontaneous disease
At the onset of clinical disease, lesions are present in the cerebellum that shows apoptosis and necrosis, loss of neutropil, some edema, and extensive myelin loss. Prominent glial cell responses are present, suggesting that the lesions have been present for some time, even in young mice (Fig. 6A–E). Moderate lesions occur near the hippocampus/thalamus with ventricular involvement, as well as some scattered gray matter lesions in the mid brain (Fig. 6F, 6G). Demyelination occurs in areas surrounding sites of inflammation (Fig. 6D, 6E). In older WT BG1 mice, as well as Rag−/− BG1 mice that have converted to chronic disease, CNS inflammation is associated with effacement of nervous tissue, leaving stromal elements and dense accumulations, principally composed of lymphocytes (Fig. 6H–K). Essentially no white matter lesions are seen within the cerebral cortex. Mice that developed paralytic disease have diffuse meningeal, submeningeal, and central canal lymphocyte infiltration along most of the length of the spinal cord that is more severe in the caudal region. In this area, infiltration occurs in the neuronal tissue, and rarefied tissue is observed (Fig. 6L). Consistent with minimal BG1 T cell signaling in peripheral organs (Fig. 3G, 3H), no histological damage is observed outside of the CNS.
BG1 mice develop lesions within the gray and white matter of the brain and spinal cord. (A) Cerebellum of a healthy C57BL/6 mouse. (B) Earlier phases of disease in BG1 mice are characterized by involvement of broad areas of cerebellar meninges and gray and white matter, with necrosis and apoptosis accompanied by a dense glial and inflammatory response (arrows). LFB stain of healthy (C) and diseased (D) area of the cerebellum of a BG1 mouse shows that the inflammatory response is intermixed with necrotic and apoptotic cells and results in loss of myelin (loss of dark blue stain) along with other elements of the neuropil. (E) CNS lesions show extensive focal regions of necrosis and apoptosis accompanied by mixed inflammatory cell populations. The inflammatory cellular component can vary within the site but is typically composed of mixtures of neutrophils (thick arrow), lymphocytes (thin arrows), and microglial elements. At this stage it is common to find extension of the inflammatory response from colliculus meninges (F) and choroid plexus (G) into adjacent gray matter nervous tissue. In late stages of disease, the cerebellum and other affected sites become distinguished by the presence of dense accumulations of lymphocytes [arrows (H)] and the replacement of nervous tissue elements by a vascular-rich stroma (thick arrows (J)] containing scattered inflammatory cells [thin arrows (I and J)]. (K) The extensive loss of myelin within the cerebellum is visualized by a lack of LFB stain. (L) Lesions of the spinal cord are less dramatic but consistent with a pattern of meningeal and perivascular accumulations of predominately lymphocytes extending into the adjacent neuropil. Images are representative examples of six WT BG1 and Rag−/− BG1 mice with early-onset or late spontaneous CNS disease. Diseased WT BG1 mice (M–Q) and Rag−/− BG1 mice (R–V) develop T cell accumulations surrounded and intermixed with GFAP+ astrocytes. Lymphocyte aggregates in WT BG1 Tg mice contain CD8, CD4 T cells (M) and B cells (P) and are ringed with CD11c+ cells (Q). The yellow-stained cells in (O) are not T cells, because no CD4+ CD8+ double-positive T cells are observed in BG1 mice; they likely represent either nonspecific binding or staining of cellular debris. (R, S, T, and U) Lesions in diseased Rag−/− BG1 mice contain CD8 T cells, without CD4 T cells or B cells. (V) Lesions are ringed with CD11c+ and GFAP+ astrocytes. Images are representative of three WT BG1 mice and four Rag−/− BG1 mice analyzed. Original magnification ×4 for (A), (B), and (H); ×10 for (C), (D), (F), (G), (I), and (K); ×20 for (J) and (L); and ×40 for (E). H&E stains are shown in (A), (B), (E)–(J), and (L). LFB stains are shown in (C), (D), and (K). Scale bar, 250 μm in (M) and (R); scale bar, 60 μm in (N)–(Q) and (S)–(V).
To elucidate the cellular organization of immune cells within the brain of WT BG1 and Rag−/− BG1 mice, the cerebella of diseased mice were stained with cell-specific Abs. In both WT BG1 and Rag−/− BG1 mice, T cells were found within the lesions in the CNS (Fig. 6M–V). Most of the T cells were detected adjacent to GFAP+ astrocytes or around the vessels (Fig. 6M, 6N, 6R, 6S). Although CD8 T cells are the dominant lymphocyte population in the cerebellum of Rag−/− BG1 mice, lesions in WT BG1 mice contain CD4 T cells and B cells, as well (Fig. 6O, 6P, 6T, 6U). In the cerebellum of both WT BG1 and Rag−/− BG1 mice, CD11c+ APCs are densely located at the lesion edge, suggesting that these lesions remain in a pathologically active state (Fig. 6Q, 6V).
B cells regulate spontaneous relapsing–remitting disease course in BG1 mice
Differences in the frequency and severity of spontaneous disease between WT BG1 and Rag−/− BG1 mice suggested that CD8 T cell–extrinsic mechanisms contribute to the regulation of CD8 T cell–mediated CNS disease. Rag−/− BG1 mice, which show a greater susceptibility to spontaneous CNS disease than do WT BG1 mice, lack B cells and nontransgenic T cells. To identify whether B cell or CD4 T cell populations contribute to the regulation of CD8 T cell–mediated CNS autoimmunity, we generated MHC II−/− (IAbβ−/−) BG1 and B cell−/− (μMT−/−) BG1 mice. We observed that μMT−/− BG1 mice are highly susceptible to spontaneous CNS disease, displaying clinical symptoms at a higher rate than littermate heterozygous controls (Fig. 7, Table I). In addition to the increase in frequency, 83% of μMT−/− BG1 mice developed chronic clinical disease compared with the relapsing–remitting disease most often observed in WT BG1 and Rag−/− BG1 mice (Fig. 7C). MHC II−/− BG1 mice showed a mild increase in the frequency of spontaneous CNS disease compared with littermate heterozygous controls (Fig. 7D, Table I). The increase in clinical symptom frequency, severity, and changes in disease course in μMT−/− BG1 mice occurs even though heterozygous littermate controls have similar numbers of CD8 T cells that target the CNS (Fig. 7E–J). These data indicate that B cells can contribute to the regulation of CD8 T cell-mediated CNS autoimmunity.
B cell–deficient BG1 mice are highly susceptible to chronic atypical EAE-like disease. Incidence of first clinical signs of EAE-like disease in μMT−/− BG1 (n = 25) or μMT+/− BG1 (n = 21) mice (A) and MHC II−/− BG1 (n = 27) or MHC II+/− BG1 (n = 17) mice (B). Representative examples of clinical disease in μMT−/− BG1 (C) and MHC II−/− BG1 (D) mice. Absolute numbers of lymphocytes in the brain (E, F, and G) or spinal cord (H, I, and J) were determined. The horizontal lines in (E)–(J) represent the average value. Each cell population was identified as shown in Fig. 4. Results are from at least five individual mice. Error bars represent SEM.
Virally activated BG1 CD8 T cells induce CNS autoimmunity with pathological features different from mice with spontaneous disease
The prolonged clinical disease course, along with T cell entry into the CNS parenchyma and associated inflammatory response observed in BG1 mice, is clinically and histologically distinct from the disease pathologies induced by activated MBP-specific CD8 T cells. Activated MBP-specific CD8 T cells induce a rapid clinical disease that targets the white matter throughout the brain, forming perivascular cuffs composed of lymphocytes, macrophages, and a few neutrophils. These perivascular cuffs are associated with necrosis and apoptosis of surrounding neuronal tissue and demyelination (24). The global differences in the CNS disease pathologies created by MBP- versus GFAP-specific CD8 T cells could be a result of the T cells targeting different Ags or a product of how disease was induced (spontaneous versus active induction). To test whether activated BG1 CD8 T cells target the CNS and induce CNS disease that resembles the spontaneous disease in BG1 mice or disease created by activated MBP-specific CD8 T cells, BG1 CD8 T cells were transferred into C57BL/6 recipient mice, and recipient mice were infected with Vac:GFAP.
We observed that Vac:GFAP infection of C57BL/6 mice carrying a small population of BG1 CD8 T cells resulted in severe ataxia and lethargy within 7 d, clinical symptoms that are very similar to those induced by activated MBP-specific CD8 T cells (Supplemental Video 2). No mice displayed evidence of ascending paralysis. The severity of the disease was commensurate with the number of BG1 CD8 T cells transferred into the C57BL/6 recipient mice. C57BL/6 mice that received ≥5 × 105 CD8 T cells succumbed to morbidity, whereas mice that received 1.25 × 105 or 2.5 × 105 CD8 T cells developed an acute disease that dissipated after a week (Fig. 8A). No onset of disease was observed in mice infected with Vac:GFAP that did not receive BG1 CD8 T cells, when recipient mice were Gfap−/−, or when recipient mice were infected with Vac:Neg virus. During the peak of Vac:GFAP-induced CNS disease, BG1 CD8 T cells within the CNS were strongly biased toward KLGR1+ short-term T effector (TEFF) cells and T effector-memory (TEM) cells and expressed high levels of IFN-γ, GZB, and IRF-4 (Fig. 8C–E, Supplemental Fig. 2).
Viral activation of the naive BG1 CD8 T cell induces CNS infiltration and autoimmunity. Vac:GFAP infection of C57BL/6 mice carrying small populations of BG1 CD8 T cells induces typical EAE disease symptoms (A) and weight loss (B) (n = 7–9 mice/group). For clarity, SD error bars are shown only for mice receiving 2.5 × 105 BG1 CD8+ cells. (C) Identification of brain-resident BG1 CD8 T cell subsets following Vac:GFAP infection (see Supplemental Fig. 1). BG1 CD8 T cells present within the brain following Vac:GFAP activation are strong producers of IFN-γ (D) and express GZB (E). Lines in (E) show the GZB-positive cells. Numbers are the percent GZB-positive. Phenotypic results are derived from or are a representative example of at least four individual mice. Error bars represent SEM. *p ≤ 0.05, **p ≤ 0.01. ns, not significant.
Within the brain of mice with virally triggered disease, affected areas are widely disseminated and included gray and white matter of the brainstem, midbrain, and cerebellum (Fig. 9). Vac:GFAP-activated BG1 CD8 T cells also targeted the cerebral cortex, a site not affected during spontaneous CNS disease in BG1 mice. Within the brain, infiltrating lymphocytes localized to the meninges and vascular/perivascular space, with minimal parenchymal infiltration. Surrounding the perivascular and meningeal infiltrates, moderate acute necrosis and apoptosis were observed, with some demyelination and neuronal cell loss, without a significant parenchymal inflammatory cell response. Commensurate with the lack of ascending paralytic disease, infiltration within the spinal cord was scant to very mild and almost exclusively meningeal. T cell infiltrates were not observed in organs other than the CNS (data not shown).
Vac:GFAP-activated BG1 CD8 T cells that target the CNS are typically confined to meningeal, perivascular, or other localized sites within the gray and white matter of brain. (A and B) Targeting of the cerebellum causes lymphocytic perivascular cuffing and extensive focal areas of cellular apoptosis and necrosis of the granular cell layer [arrow in (B)] but only mild inflammatory cell accumulation. (C and D) In targeted areas, some associated demyelination (loss of LFB stain) occurs [arrow in (D)]. (E and F) In animals that survive acute disease, loss of neurons are observed [pale area, arrow in (E)], along with the presence of widely cuffed vessels, vacuolated white matter, and glial cells but only scant parenchymal lymphocytes. Lymphocytic inflammation within the choroid plexus (G) and nearby vessels (surrounded by apoptotic bodies and lymphocytes) (H) does not extend into the adjacent nervous tissues. Diffuse meningitis and perivasculitis (I and J), including a region of the habenular nucleus (K), shows scattered apoptosis and individual cell necrosis with a mild, mixed inflammatory cell accumulation. (L) Vessels within the cerebral cortex are occasionally marginated and cuffed with lymphocytes (some of which are apoptotic within adjacent parenchyma), but extension into the surrounding nervous tissue is minimal. Original magnification ×4 in (E); ×10 in (A)–(D), (F), and (G); ×20 in (J) and (L); ×40 in (I) and (K); and ×60 in (H). H&E stains are shown in (A), (B), and (E)–(L); LFB stains are shown in (C) and (D). Images are representative examples of six mice with Vac:GFAP-induced CNS disease.
These data demonstrate that Vac:GFAP activation of BG1 CD8 T cells generated primarily classical TEFF and TEM CD8 T cells that targeted the brain and induced a rapid, acute, and clinically severe CNS autoimmunity. Many of the histological features and clinical symptoms of this CNS autoimmunity are similar to in vitro–activated MBP-specific CD8 T cell–induced disease, and they stand in contrast to the spontaneous relapsing–remitting disease that developed in BG1 mice.
Discussion
The experiments described in this article demonstrate that CD8 T cells that target a protein expressed in astrocytes can avoid tolerance mechanisms prior to entering into the CNS and causing autoimmune damage. GFAP-specific CD8 T cells were observed to target both the gray matter and white matter of the brain and spinal cord. Furthermore, the composition and location of the lesions, as well as the clinical symptoms of disease, were dependent upon the triggering event that activated the CNS-reactive CD8 T cells. These findings indicate that CD8 T cells that target astrocytes can induce CNS autoimmune disease and strongly suggest that different CD8 T cell effector lineages contribute to heterogeneity in CNS lesion formation and clinical disease course.
To contribute to the CNS autoimmune disease process, self-reactive CD8 T cells have to avoid thymic and peripheral tolerance mechanisms and, when activated, target the CNS. Similar to several myelin proteins, a splice variant of GFAP is expressed within the thymus (48, 52). However, this thymic expression appears to be below the detection of developing BG1 thymocytes, because negative selection was not observed in BG1 mice that express GFAP, even though BG1 CD8 T cells recognize target cells loaded with nanomolar concentrations of GFAP264–272 peptide. Demonstrating that GFAP-specific CD8 T cells target the CNS is in contrast to mice that express the neoantigen HA under the GFAP promoter, which, when challenged with HA-specific CD8 T cells, either develop immune tolerance or enterocolitis (53, 54) but not CNS autoimmunity. The HA-specific CL4 T cell was originally isolated from a non–HA-expressing mouse infected with influenza. Thus, it is possible that the HA-specific CL4-TCR is more sensitive to the viral neoantigen than are the BG1 CD8 T cells to endogenous GFAP. Consistent with the hypothesis, mice that doubly express the CL4 TCR and GFAP-HA die by 6 d of age, a rapid death that may occur prior to the ability of significant numbers of CD8 T cells to migrate to the CNS (53). Furthermore, when naive CL4 CD8 T cells are adoptively transferred into GFAP-HA mice, a strong proliferative response ensues that is directed at the HA neoantigen, a phenotype that we do not observe in BG1 adoptive transfers (54) (Fig. 3). Supporting the hypothesis that astrocyte-specific T cells can escape tolerance induction and drive CNS autoimmunity, S100β-specific CD4 T cells (which also target astrocytes) can develop, and the immunization of Lewis rats with S100β peptides induces CNS autoimmunity with gray and white matter pathology (55, 56). Thus, when activated, both CD4 and CD8 T cells that target astrocytes can contribute to CNS autoimmunity.
The observation that BG1 CD8 T cells are largely ignorant of endogenous GFAP protein is similar to studies analyzing a human TCR specific for an epitope of PLP and in contrast to studies of CD8 T cells in C3H mice that target MBP. PLP-specific CD8 T cells were found to develop in mice expressing HLA-A3 and remained primarily ignorant of PLP, with few mice succumbing to spontaneous CNS disease. When activated, these PLP-specific CD8 T cells induce CNS autoimmunity, indicating that the PLP epitope is presented within the CNS (27). In contrast, MBP-specific CD8 T cells are subject to tolerance induction, with some pathogenic MBP-specific T cell clonotypes using a variety of strategies to avoid thymic and peripheral tolerance mechanisms, including stealing the MBP epitope from APCs and expressing two TCRα-chains (57, 58). CD8 T cells specific for MOG were shown to induce CNS disease, indicating that these T cells develop; however, the phenotype of these T cells prior to activation has not been determined. In addition, CD8 T cells specific for multiple myelin epitopes have been identified in both MS patients and healthy controls, indicating that CD8 T cell tolerance to CNS proteins in incomplete. Thus, similar to CD4 T cell responses targeting CNS Ags (6, 38–41), the thymic expression of CNS proteins does not fully purge pathogenic CD8 T cells from the peripheral T cell repertoire.
Traditionally, MS has been thought to be an autoimmune disease of the white matter of the CNS. However, more recent studies (2, 59–62) identified gray matter lesions in MS patients that appear at the earliest stages and accumulate over time. The observation that CD8 T cells are present within gray matter lesions of MS patients suggests that CD8 T cells reactive to Ags other than myelin proteins may contribute to MS disease progression (2, 62). Astrocytes reside within the white and gray matter of the CNS. GFAP, an intermediate filament protein, is a prototypical astrocyte-specific Ag that is expressed throughout the gray matter and white matter of the brain and spinal cord (63). Normally, astrocytes express low levels of MHC class I, which increases during inflammation (64). In MS lesions, for example, the expression level of GFAP increases, and peptides derived from GFAP are presented by MHC class I and class II molecules (65–67). Data presented in this article indicate that GFAP-specific CD8 T cells can target the CNS and induce gray matter and white matter pathology, inducing apoptosis and necrosis of neuropil and extensive myelin loss. Although GFAP-specific T cells isolated from MS patients have not been studied, GFAP-specific CD8 T cells have been isolated from patients with type 1 diabetes, indicating that autoimmune-prone individuals carry T cells with this reactivity pattern (68). In addition, CD8 T cells that target astrocytes and neurons were suggested in Rasmussen encephalitis cells (69).
The clinical signs of MS are highly variable and often include symptoms of upper motor neuron disease, such as hyperreflexia, ataxia, spasticity, and visual defects. In some cases there is evidence of lower motor neuron disease, such as sensory defects and partial or complete paralysis. In the majority of patients these symptoms manifest as a relapsing–remitting disease, usually converting over time to a chronic progressive stage (1, 4, 8). The idea that different T cell lineages associate with unique aspects of CNS disease is well documented for encephalogenic CD4 T cells. The detailed analysis of CD4 T cells responding to several neuroantigens showed that the effector lineage and activation status of CD4 T cells within the CNS regulate disease location, severity, and clinical outcome (70–72).
Our genetic studies suggest that CD4 T cells and B cells have complex roles in regulating CD8 T cell–initiated CNS autoimmunity. In contrast to the relapsing–remitting disease course observed in WT BG1 and Rag−/− BG1 mice, B cell−/− BG1 mice succumbed to a chronic disease course. This fundamental change in the manifestation of spontaneous CNS disease indicates that B cells can limit CD8 T cell–induced CNS autoimmunity and promote or allow for disease remissions. These data also suggest that CD4 T cells, absent in Rag−/− BG1 mice but present in B cell−/− BG1 mice, may promote CD8 T cell–induced CNS autoimmunity, possibly by providing CD8 T cell help. The elimination of B cells, resulting in enhanced CNS autoimmunity, may seem counterintuitive considering that rituximab, B cell–depletion therapy, shows promise in treating MS patients with relapsing–remitting disease (73). However, studies of CD4 T cell–mediated EAE models showed that B cells can impact CNS autoimmunity in multiple ways, detrimentally through the production of CNS-specific Abs and acting as APCs for pathogenic CD4 T cells, as well as in a suppressive manner by producing anti-inflammatory cytokines (73–79). Because B cells do not efficiently cross-present Ags on MHC I molecules to CD8 T cells (80), B cells may play a primarily suppressive role in CD8 T cell–initiated CNS disease.
How different CD8 T cell effector and memory lineages impact the MS disease process is poorly understood. Within the cerebrospinal fluid and CNS of MS patients, CD8 T cells can express IFN-γ and/or IL-17 and have T central memory (TCM) or TEM phenotypes (71–75, 81–85). The population of TRM phenotype CD8 T cells has not been studied in MS patients. In the experiments presented in this article, we observed that GFAP-specific CD8 T cells induce distinct CNS disease pathologies and a clinical disease course, depending upon how they are activated. BG1 CD8 T cells responding to a viral infection generate a population of highly cytotoxic effector CD8 T cells that rapidly enter into the CNS and localize to the meninges and vascular/perivascular space within both gray and white matter, causing localized necrosis and apoptosis. Clinically, these mice succumb to acute ataxia, spasticity, and lethargy. In contrast to this acute disease, BG1 T cells that are spontaneously recruited to the CNS and cause relapsing–remitting disease show a diversity of effector lineages. These primarily include lymphocytes that phenotypically resemble TRM and other poorly defined subsets of CD8 T cells, with a paucity of GZB- or IFN-γ–expressing TEFF-, TEM-, or TCM-lineage CD8 T cells. The memory-phenotype CD8 T cells that do develop enter into the CNS parenchyma as well as localize to the meninges and perivascular space and induce significant inflammatory responses. Why these different CD8 T cell subsets localize differently and how CD8 T cells that do not make conventional inflammatory cytokines induce CNS disease is unclear. In models of CD4-EAE, the expression of IFN-γ can limit the ability of CD4 T cells to enter into the parenchyma and affect T cell–trafficking patterns (86). In addition, the extensive inflammatory response during spontaneous CNS disease suggests that BG1 T cells can directly or indirectly induce NF-κB signaling in glial cells that may propagate the inflammatory response (87, 88).
In summary, the experiments described in this article indicate that CD8 T cells that target nonmyelin CNS Ags can avoid tolerance mechanisms and drive unique aspects of inflammatory CNS autoimmunity, including the targeting of gray matter and white matter of the brain. The composition and location of the lesions, as well as clinical symptoms created during spontaneous disease versus a viral induction, strongly suggest that the triggering event that activates autoreactive CD8 T cells contributes to heterogeneity in CNS disease and clinical disease course.
Disclosures
The authors have no financial conflicts of interest.
Acknowledgments
We thank Drs. Brian Stadinski for helpful discussions and Rebecca Smith for technical assistance with some of the experiments.
Footnotes
This work was supported by a Beckman Young Investigator Award and National Institutes of Health Grants RAI088495A and DK095077 (to E.S.H.). K.S. was supported by a fellowship from the Japanese Society for the Promotion of Science. E.S.H. is a member of the University of Massachusetts Medical School Diabetes and Endocrinology Research Center (National Institutes of Health Grant DK32520).
The online version of this article contains supplemental material.
Abbreviations used in this article:
- Ad
- adenovirus
- BM-DC
- bone marrow–derived dendritic cell
- EAE
- experimental autoimmune encephalomyelitis
- GFAP
- glial fibrillary acidic protein
- GZB
- granzyme B
- LFB
- Luxol Fast Blue
- MBP
- myelin basic protein
- MOG
- myelin oligodendrocyte protein
- MS
- multiple sclerosis
- PLP
- proteolipid protein
- TCM
- T central memory
- TEFF
- T effector
- TEM
- T effector-memory
- Tg
- transgenic
- TRM
- tissue-resident memory
- Vac
- vaccinia virus
- WT
- wild-type.
- Received October 30, 2013.
- Accepted January 28, 2014.
- Copyright © 2014 by The American Association of Immunologists, Inc.
References
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