Opposing T cell responses in experimental autoimmune encephalomyelitis

Abstract

Experimental autoimmune encephalomyelitis is a model for multiple sclerosis. Here we show that induction generates successive waves of clonally expanded CD4+, CD8+ and γδ+ T cells in the blood and central nervous system, similar to gluten-challenge studies of patients with coeliac disease. We also find major expansions of CD8+ T cells in patients with multiple sclerosis. In autoimmune encephalomyelitis, we find that most expanded CD4+ T cells are specific for the inducing myelin peptide MOG35–55. By contrast, surrogate peptides derived from a yeast peptide major histocompatibility complex library of some of the clonally expanded CD8+ T cells inhibit disease by suppressing the proliferation of MOG-specific CD4+ T cells. These results suggest that the induction of autoreactive CD4+ T cells triggers an opposing mobilization of regulatory CD8+ T cells.

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Fig. 1: Concomitant activation of all T cells after EAE immunization.
Fig. 2: CD4+, CD8+ and γδ+ T cells are clonally expanded after EAE.
Fig. 3: Clonally expanded CD8 TCRs are not responsive to myelin.
Fig. 4: Clonally expanded EAE CD8 TCRs bind to novel peptides.
Fig. 5: CD8+ T cell-specific SP immunization ameliorates EAE.
Fig. 6: CD8+ T-cell-specific SP immunization suppresses MOG35–55-specific CD4+ T cells.

Data availability

RNA-seq data and yeast pMHC selection data are deposited in the Gene Expression Omnibus (GEO) data repository with accession number GSE130975. Source Data for each figure are provided. Other data that support the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank members of the Davis, Chien, Garcia and Steinman laboratories for discussions. We thank A. Han and M. M. Mamedov for help designing mouse TCR primers, and V. Mallajyosulla for help with the H2-Db library constructs. We thank the Stanford Functional Genomics Facility and the Human Immune Monitoring Center for DNA and RNA sequencing (NIH shared facility equipment grant, S10OD018220). We thank C. Carswell, B. Gomez and the Stanford Shared FACS facility for assistance in sorting cells and providing access to the equipment. We also thank A. Nau, L. Steinman and C. Tato for critical reading of the manuscript and discussions. We thank C. Bohlen for myelin proteins. N.S. was supported by a Postdoctoral Fellowship from National Multiple Sclerosis Society (NMSS) and also a Career Transition Grant from NMSS. F.Z. was supported by the Ovarian Cancer Research Fund Alliances. Human samples collection was supported by a grant from NMSS RG-1611-26299 (J.R.O and S.L.H). J.I. is funded by the US National Institutes of Health (NIH) grant 1DP2AR069953. R.A.F. is supported by The Wellcome Trust for the Sir Henry Wellcome Fellowship (WT101609MA). M.M.D. and K.C.G. are funded by the US NIH (U19 AI057229) and the Howard Hughes Medical Institute. K.C.G. is also funded by the US NIH (R01AI103867) and Mathers Foundation. Additional early support also came from the Simons Foundation (to M.M.D.).

Author information

N.S. and M.M.D. conceived the study. N.S., W.S.S., R.A.F., W.Y. and F.Z. performed experiments. N.S. and D.M.L. analysed TCR sequencing data. J.R.O. and S.L.H. provided PBMCs from newly diagnosed patients with MS. J.I. generated bone-marrow-derived dendritic cells. V.B.M. performed histopathology and analysed uveitis data. X.J. assisted in sequencing TCRs and RNA-seq analysis. L.M.S. guided and M.J.S. analysed RNA-seq data. K.C.G. and R.A.F. guided experiments involving mouse yeast–pMHC display and R.A.F. analysed yeast–peptide MHC sequencing data. Y.-H.C. guided experiments that involve γδ TCR sequencing and analysis. N.S. and M.M.D. wrote the manuscript with input from all the authors.

Correspondence to Mark M. Davis.

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Competing interests

The authors declare no competing interests.

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Peer review information Nature thanks Hartmut Wekerle and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Fig. 1 Massive clonal expansion of all T cells after EAE immunization.

ae, C57BL/6J mice were immunized for EAE induction, and cells from blood, draining LN, spleen and the CNS were isolated and stained with a cocktail of cell-surface antibodies on different days after immunization (D0 (unimmunized), D3, D5, D7, D10, D12, D15, D17, D19, D21, D23 and D3). a, Infiltrating CD4+, CD8+ and γδ+ T cells were single-cell-sorted on D0 (unimmunized), D7, D10, D15 and D19 after immunization. The cells underwent single-cell paired TCR sequencing. (n = 3 mice per group/time point). In total, we sequenced 1,302 (CD4+), 1,660 (CD8+), and 1,451 (γδ+) paired TCR sequences. bd, Average percentage clonal expansion of CD4+ (b) CD8+ (c) and γδ+ (d) T cells among unimmunized and immunized mice in all days and tissues combined together. Data are mean ± s.e.m. e, Percentage of identical CD4+, CD8+ and γδ+ TCR sequences shared between blood and the CNS within each day after immunization. f, Frequency of major groups (groups 1–4) of tTγδ17 cells in blood and CNS on different days after immunization. g, Corresponding paired TCRγ and TCRδ sequences that define each major group (groups 1–4) of tTγδ17 cells. Source data

Extended Data Fig. 2 Concomitant activation of all T cells after EAE immunization, and clonally expanded CD8 TCRs are not specific to myelin peptides or proteins.

af, C57BL/6J mice were immunized for EAE induction and cells from blood, draining LN and spleen were isolated and analysed for total frequency of activated (CD44high) (a, c, e) and naive (CD62Lhigh) (b, d, f) CD4+, CD8+ cells. Data are mean ± s.e.m. and representative of two independent experiments. *P = 0.046; **P = 0.0023; ***P = 0.0002; ****P < 0.0001, one-way ANOVA followed by Dunnett’s post hoc multiple comparison test. g, Nine clonally expanded CD8 TCRs (EAE1-CD8 to EAE9-CD8) were retrovirally transduced to express on 58 αβ−/− cells. Untransduced and transduced cell lines were stained with fluorochrome-labelled anti-TCRβ and anti-CD3 to determine the surface expression of TCRs. h, Untransduced and transduced EAE-CD8 TCR cell lines were stimulated with plate-bound anti-CD3 and soluble anti-CD28 for 12–16 h and surface-stained with activation marker CD69. i, Untransduced 58 αβ−/− or OT-1 TCR transduced cell lines were stimulated with bone-marrow-derived dendritic cells pulsed with SIINFEKL peptide or whole OVA protein for 12–16 h, washed, and stained with CD69. j, Unstimulated 58 αβ−/− or EAE-CD8 TCR transduced cell lines were stimulated with pool of peptides (PP1–PP7) from MOG, MBP, PLP, MAG and SIINFEKL peptides, and examined for expression of CD69 (CD69 expression shown in figure for EAE1-CD8 TCR). Peptides are of variable lengths (8–12 amino acids). Each peptide pool contained 50 peptides. Data are representative of three independent experiments.

Extended Data Fig. 3 Generation and functional validation of a H2-Db yeast peptide–MHC library.

a, Schematic of the mouse class I MHC H2-Db displayed on yeast as β2m, α1, α2 and α3 with peptide covalently linked to the MHC N terminus. b, Design of the peptide library displayed by H2-Db. Design is based on the structure of the 6218 TCR bound to H2-Db-restricted acid polymerase peptide 224–233 (SSLENFRAYV, DbPA224) (RCSB Protein Data Bank accession 3PQY)19. c, Mutation required for proper folding of the H2-Db displayed on yeast (α2-W131 to α2-G131). Mutations were derived from error-prone mutagenesis. d, Design for two different lengths of H2-Db libraries. For the nine-amino-acid (9 MER) library, residues from P1 to P9 were randomized, with limited diversity at MHC anchor positions P5 (Asn, N) and P9 (Met, Ile and Leu, M/I/L). For the ten-amino-acid (10 MER) library, residues from P1 to P10 were randomized, with limited diversity at MHC anchor positions P5 (Asn, N) and P10 (Met, Ile and Leu, M/I/L). TCR contact residues are coloured pink and MHC anchor residues are coloured red or blue. eg, Selection of PA224-H2-Db error-prone library with 6218 soluble TCR. Increased MYC expression among induced yeast peptide-H2-Db error-prone library at different rounds (RD1–RD4) of selection (e, g) and 6218 soluble TCR tetramer staining on the post-RD4 error-prone H2-Db library (g). 6218 TCR was screened on the error-prone yeast library once.

Extended Data Fig. 4 In vitro and in vivo characterization of CD8+ T cells specific for surrogate peptides after EAE.

a, Jurkat αβ−/− T cells expressing 6218, EAE6 and EAE7-CD8 TCRs were stained with corresponding yeast library-enriched pMHC tetramers (SSLENFRAYV, ASR, SMRP, YQP and HDR). b, From unimmunized mice (n = 4), or mice immunized with MOG (n = 5), MOG + SP (n = 5), or SP (n = 5), spleen and LN cells were isolated, and cells were enriched for SP-specific CD8+ T cells with pMHC tetramers. Representative flow cytometry gating strategy is shown for different cell surface markers and tetramer-specific cells. c, Representative flow cytometry data are shown for activation status (defined as CD44+CD62L) on CD8+ T cells specific for SP (ASR, HDR, SMRP and YQP-tet+) from wild-type and different immunization groups (MOG, MOG + SP, and SP). d, Activated/effector phenotype of CD8+ T cells specific for SP (ASR, HDR, SMRP and YQP-tet+) from wild-type (n = 5) and different immunization groups (MOG (n = 3), MOG + SP (n = 4), and SP (n = 3)) is quantified (n = 5 mice per group). *P = 0.0169; **P = 0.0020; ****P < 0.0001, one-way ANOVA followed by Tukey’s post hoc multiple comparison test. Data are mean ± s.e.m. e, C57BL/6J mice were immunized for EAE with an emulsion containing MOG35–55, CFA plus PTX with (n = 10) or without (n = 10) influenza peptide (SSLENFRAYV). The clinical scores after immunization were recorded. Data are mean ± s.e.m. and representative of two independent experiments. Source data

Extended Data Fig. 5 CD8+ T cell-specific SP immunization suppresses MOG35–55-specific CD4+ T cells and induces CD8+ T cells with a regulatory phenotype.

ac, C57BL/6J mice were immunized with an emulsion containing MOG35–55, CFA and PTX (n = 5), or MOG35–55, CFA, PTX and SP (n = 5). From unimmunized mice (a) and mice ten days after immunization (b, c) spleen and LN cells were isolated, stained and enriched for MOG35–55 I-Ab pMHC-specific CD4+ T cells and an irrelevant tetramer. Representative FACS plots for different groups are shown. dg, Spleen and LN cells were isolated from unimmunized mice (n = 5) (d) and mice 10 days after immunization with MOG (n = 5) (g), MOG plus SP (n = 5) (f) or SP alone (n = 4) (e), and then stained and enriched for SP-specific CD8+ T cells using the pMHC tetramer. Representative FACS dot plots for CD8+ T cell with a regulatory phenotype (CD44+CD122+Ly49+) from each group are shown. h, Tetramer-positive (that is, ASR, HDR, SMRP and YQP-tet+) CD8+ T cells were sub-gated for CD122, CD44 and Ly49, and the frequency of CD122+CD44+Ly49+ cells among SP-specific cells are shown among different immunization groups. ***P = 0.0002, one-way ANOVA followed by Tukey’s post hoc multiple comparison test. Data are mean ± s.e.m. and representative of two independent experiments.

Extended Data Fig. 6 CD8+ T cells elicited after MOG + SP immunization are specific, their suppression is mediated by perforin, adoptive transfer of CD122+CD44+Ly49+ abrogates EAE, and SP triggers a more severe, inflammatory retinal uveitis than IRBP peptide alone.

ag, C57BL/6J mice were immunized with an emulsion containing MOG35–55, CFA and PTX (a), MOG35–55, CFA, PTX and SP (b), or MOG35–55, CFA, PTX and influenza peptide (e). Spleen and LN cells were isolated from mice ten days after immunization, and cells were enriched for CD4+ or CD8+ T cells or APCs by FACS. CD4+ T cells from MOG-immunized mice were labelled with CellTrace Violet (CTV) and co-cultured with APCs from MOG-immunized mice in the absence (a) or presence of CD8+ T cells from wild-type mice (c) or mice immunized with MOG plus SP (b), CFA plus PTX (d), MOG plus influenza peptide (e), or CD8+ T cells from perforin-knockout (PFNKO) mice immunized with MOG plus SP (f). g, CTV-labelled CD4+ T cells from mice immunized with MOG35–55, CFA plus PTX were co-cultured with CD8+ T cells from mice immunized with MOG35–55, CFA, PTX plus SP in the presence of anti-Qa-1b antibody (10 μg ml−1). h, CTV-labelled CD4+ T cells from mice immunized with OVA329–337, CFA plus PTX were co-cultured with CD8+ T cells from mice immunized with MOG35–55, CFA, PTX plus SP. Seven days after co-culture, cells were washed and stained with surface markers and analysed for CD4+ T cell proliferation (CTV dilution). Representative data are from two independent experiments. i, C57BL/6J mice were immunized with MOG35–55, CFA, PTX plus SP (n = 10) and ten days after immunization spleen and LN cells were isolated, stained and enriched for CD8+ T cells followed by FACS for Ly49+ and Ly49 cells. Sorted Ly49+ and Ly49 cells were adoptively transferred (8 million cells per mouse) to C57BL/6J mice (n = 5 mice per group) at the time of immunization. The clinical scores after adoptive transfer and immunization are shown. ****P < 0.0001, regression analysis with two-way ANOVA followed by Bonferroni post hoc multiple comparison test. Data are mean ± s.e.m. and representative of two independent experiments. j, In the wild-type, untreated mouse eye, the retina shows a normal laminar pattern and there are no leukocytes in the vitreous. k, After subcutaneous injection of IRBP peptide antigen, there was only a mild inflammatory response in 40% of eyes with activated leukocyte invasion of the vitreous (red arrow) and mild disruption of the retina outer nuclear layer photoreceptors (black arrow). l, After subcutaneous injection of both IRBP and SP there was a severe inflammatory response in 80% of eyes with activated leukocyte invasion of the vitreous (red arrows) and severe disruption of the retina photoreceptors (black arrows). INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; RGC, retinal ganglion cell layer; RPE, retinal plexiform layer. Five C57BL/6J mice were examined for each condition. EAU was induced, and mice were euthanized on day 21 after immunization. Mouse eyes were enucleated, fixed and pupil-optic nerve sections were examined by histology. mo, C57BL/6J mice were immunized with IRBP, CFA and PTX with or without SP. Spleen and LN cells were isolated from mice 10 days after immunization, and cells were enriched for CD4+ or CD8+ T cells, or APCs by FACS. CTV-labelled CD4+ T cells from IRBP-immunized mice were co-cultured with APCs from IRBP-immunized mice and purified Ly49 T cells (n), Ly49+ T cells (o) or without CD8+ T cells (m) from mice immunized with IRBP and SP. Seven days after co-culture, cells were washed and stained with surface markers and analysed for CD4+ T cell proliferation. Source data

Extended Data Fig. 7 Transcriptional profiling of Ly49+ versus Ly49 cells.

C57BL/6J mice were immunized with SP, CFA and PTX (n = 3). Spleen and LN cells were isolated from D10 mice, and enriched for CD8+ T cells by FACS and sorting for Ly49 and Ly49+ cells, followed by bulk RNA-seq analysis. a, A heat map of differentially expressed genes (log2-transformed fold change > 2, and adjusted P < 0.005) in Ly49+/Ly49 RNA-seq samples. Columns show samples, and rows and columns are ordered based on hierarchical clustering. Normalized gene expression values are centred for each gene by subtracting the average value of all samples from each sample value (2–3 mice per group). b, Gene Ontology enrichment analysis of differentially expressed genes (log2-transformed fold change > 2, and two-tailed Benjamini–Hochberg adjusted P < 0.005 from DESeq2) between Ly49+ and Ly49 RNA-seq samples. The y axis represents the top-30 enriched Gene Ontologies (genes from Gene Ontologies highlighted in green are in Supplementary Table 6). The x-axis value is the fraction of genes in that ontology that are differentially expressed. The colour of the dot represents that significance of the Gene Ontology enrichment (one-tailed Fisher’s exact test), and the size of the dot represents the number of genes differentially expressed. The plot was made with the R package clusterProfiler. c, Volcano plot representing gene-expression differences between the Ly49+ and Ly49 samples (three mice per group). Each point is a gene. List of genes specifically expressed in CD4+ T regulatory cells25 are coloured red if they are expressed in both MOG and SP RNA-seq samples, and green if not. The horizontal dotted line is made at −log10(0.05), and the two vertical dotted lines represent a fold change of log2(2). Genes with a negative fold change are highly expressed in Ly49+ cells.

Extended Data Fig. 8 Major clonal expansion of CD8+ T cells in newly diagnosed patients with MS.

ac, PBMCs from healthy controls (HC) (n = 4) and patients with MS (n = 18) were stained and analysed by flow cytometry to determine the frequency of T cells. The frequency of CD4+ (a), CD8+ (b) and γδ+ (c) T cells is shown. Data are mean ± s.e.m. d, e, Brain homing and activated (CD49d+CD29+HLA-DR+CD38+) CD8+ T cells were single-cell-sorted from PBMCs of healthy controls (d) and newly diagnosed patients with MS (e). The cells underwent single-cell paired TCR sequencing. Pie charts depicting clonal expansion of CD8+ T cells among healthy controls (n = 10) and patients with MS (n = 18). The number of cells with β chain successfully identified is shown above its pie chart. For each TCR clone expressed by two or more cells (clonally expanded), the absolute number of cells expressing that clone (≥2, ≥5, ≥10, ≥20 and ≥50) is shown by a distinct coloured section.

Extended Data Fig. 9 TCR repertoire of brain homing activated CD4+ T cells in newly diagnosed patients with MS.

a, b, Brain homing and activated (CD49d+CD29+HLA-DR+CD38+) CD4+ T cells were single-cell-sorted from PBMCs of healthy controls (a) and newly diagnosed patients with MS (b). The cells underwent single-cell paired TCR sequencing. Pie charts depicting clonal expansion of CD4+ T cells among healthy controls (n = 10) and patients with MS (n = 18). The number of cells with β chain successfully identified is shown above its pie chart. For each TCR clone expressed by two or more cells (clonally expanded), the absolute number of cells expressing that clone (≥2, ≥5, ≥10, ≥20 and ≥50) is shown by a distinct coloured section. Source data

Extended Data Fig. 10 TCR repertoire of brain-homing-activated γδ+ T cells in newly diagnosed patients with MS.

a, b, Brain homing and activated (CD49d+CD29+HLA-DR+CD38+) γδ+ T cells were single-cell-sorted from PBMCs of healthy controls (a) and newly diagnosed patients with MS (b). The cells underwent single-cell paired TCR sequencing. Pie charts depicting clonal expansion of γδ+ T cells among healthy controls (n = 10) and patients with MS (n = 18). The number of cells with δ chain successfully identified is shown above its pie chart. For each TCR clone expressed by two or more cells (clonally expanded), the absolute number of cells expressing that clone (≥2, ≥5, ≥10, ≥20 and ≥50) is shown by a distinct coloured section. From the single-cell sorted γδ+ T cells, RAR-related orphan receptor (ROR) transcripts were amplified using gene-specific primers and sequenced simultaneously with γ and δ chains. c, The number of γδ+ T cells positive for the RORC transcript is shown among healthy controls (n = 10) and patients with MS (n = 18). *P = 0.0301, paired t-test. Data are mean ± s.e.m.

Supplementary information

Supplementary Information

This file contains Legends for Supplementary Tables 1-9.

Reporting Summary

Supplementary Table 1

List of Phase 1, 2, and 3 mouse TCR primers used for paired sequencing.

Supplementary Table 2

EAE-CD4 TCRs used for generating T cell transductants. Mouse (M1, M2, and M3), Blood (BL), Central nervous system (CNS), and day (D).

Supplementary Table 3

EAE-CD8 TCRs used for generating T cell transductants and soluble TCRs. Mouse (M1, M2, and M3), Blood (BL), Central nervous system (CNS), and day (D).

Supplementary Table 4

Myelin peptides used for TCR cell line stimulation. 350 peptides derived from myelin oligodendrocyte glycoprotein (MOG), proteolipid protein (PLP), myelin basic protein (MBP), myelin associated glycoprotein (MAG). These peptides mixed into 7 peptide pools (PP1-7) and used for TCR cell line stimulation.

Supplementary Table 5

Enrichment of unique peptides per round for 6218, EAE6, and EAE7-CD8 TCR selections on yeast H2-Db Library. Summary of total number of Illumina reads by round (RD) for 6218, EAE6, and EAE7-CD8 TCR selections. Unique peptide sequences corresponded to reads that were in-frame with no stop codons. Fraction of unique peptides refers to total sequencing reads per RD divided by unique peptide sequences for that RD. Corrected fold enrichment refers to fold enrichment of peptides per RD selection normalized to the total number of reads from naïve RD.

Supplementary Table 6

EAE clinical traits associated with MOG, MOG + SP, and SP Immunization. CDS, cumulative disease score over 30 days of experiment; DA, days affected; SI, severity index (cumulative disease score/days affected); SP, surrogate peptides; PS, peak score; DO, day of onset in affected animals; NS, not significant. Values are shown as means ± SEM. Significance of differences for the trait values among the experimental conditions was assessed by c2 analysis (overall incidence) or One-ANOVA analysis, followed by Tukey’s post hoc multiple comparisons; P values are as indicated. aPercentage affected. Animals were considered affected if clinical scores ³ 1 were apparent for ³ 2 consecutive days.

Supplementary Table 7

EAE clinical traits associated with MOG, SP challenge, and MOG challenge. CDS, cumulative disease score over 30 days of experiment; DA, days affected; SI, severity index (cumulative disease score/days affected); SP, surrogate peptides; PS, peak score; DO, day of onset in affected animals; NS, not significant. Values are shown as means ± SEM. Significance of differences for the trait values among the experimental conditions was assessed by c2 analysis (overall incidence) or One-ANOVA analysis, followed by Tukey’s post hoc multiple comparisons; P values are as indicated. aPercentage affected. Animals were considered affected if clinical scores ³ 1 were apparent for ³ 2 consecutive days.

Supplementary Table 8

Differentially expressed genes between Ly49+ and Ly49- CD8+ cells sorted based on gene ontology.

Supplementary Table 9

Demographic and clinical features of the multiple sclerosis and healthy control dataset. Study participants form an on-going prospective observational study at the University of California, San Francisco, CA Multiple Sclerosis Center. Patients were recruited when suspected of having MS or presented with an initial event indicative of MS within 24 hours and no later than 90 days. Patients with Clinically Isolated Syndromes (CIS) were also included if they fulfilled the Magnetic Resonance Imaging in Multiple Sclerosis (MAGNIMS) criteria (Polman et al., 2011). Eligibility criteria also included no prior treatment with MS disease modifying therapies or board spectrum immune suppressants and no treatment with corticosteroids within the last 30 days. Peripheral blood lymphocytes were prepared by ficol gradient and frozen in liquid nitrogen within 2 hours of phlebotomy. Age matched and sex matched healthy control PBMCs were obtained from Stanford Blood Center, Stanford University, Stanford, CA. Ancestry; 1-European American, 2-African American, 3-Hispanic, 4-Asian. HLA-DRB1*15:01; 1-carrier and 2-non-carrier. PP primary progressive MS, RR relapsing remitting MS, CIS clinical isolated syndrome. HC, healthy control.

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