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ImmunologyNature Immunology
Peer-reviewed journal article

Antigen-specificity of clonally-enriched CD8+ T cells in multiple sclerosis

Fumie HayashiiD, Kristen Mittl, Ravi DandekariD, Josiah Gerdts, Ebtesam Hassan, Ryan D. SchubertiD, Lindsay OshiroiD, Rita P. LoudermilkiD, Ariele L. GreenfieldiD, Danillo G. AugustoiD, Gregory C Havton, Shriya Anumarlu, Arhan Surapaneni, Akshaya RameshiD, Edwina B. TraniD, Kanishka KoshaliD, Kerry Kizer, Joanna Dreux, Alaina K. Cagalingan, Florian Schustek, Lena Flood, Tamson V. MooreiD, Lisa L. KirkemoiD, Isabelle FisheriD, Tiffany P. CooperiD, , Joseph J. SabatinoiD

Abstract

CD8+ T cells are the dominant lymphocyte population in multiple sclerosis (MS) lesions where they are highly clonally expanded. The clonal identity, function, and antigen specificity of CD8+ T cells in MS are not well understood. Here we report a comprehensive single-cell RNA-seq and T cell receptor (TCR)-seq analysis of the cerebrospinal fluid (CSF) and blood from a cohort of treatment-naive MS patients and control participants. A small subset of highly expanded and activated CD8+ T cells were enriched in the CSF in MS that displayed high activation, cytotoxicity and tissue-homing transcriptional profiles. Using a combination of unbiased and targeted antigen discovery approaches, MS-derived CD8+ T cell clonotypes recognizing Epstein-Barr virus (EBV) antigens and multiple novel mimotopes were identified. These findings shed vital insight into the role of CD8+ T cells in MS and pave the way towards disease biomarkers and therapeutic targets. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/611010v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1a0b738org.highwire.dtl.DTLVardef@5cf5c2org.highwire.dtl.DTLVardef@10abdccorg.highwire.dtl.DTLVardef@32c7b2_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical SummaryC_FLOATNO Created in BioRender. Sabatino, J. (2024) BioRender.com/e66l598 C_FIG

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In-depth summary

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Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 2 (17 Oct 2024) · CC BY 4.0

Paired CSF and blood scRNA/TCR-seq in treatment-naive MS shows a small set of expanded, cytotoxic CSF-enriched CD8+ clonotypes, two of which recognize EBV epitopes.

The antigen specificity of clonally expanded CD8+ T cells in MS lesions and CSF is poorly understood, and earlier CSF single-cell studies did not analyze the antigens of expanded cells in detail. This work pairs CSF clonal profiling with three parallel antigen discovery strategies.

Key results

  1. ShownA small group of highly expanded, CSF-enriched T cell clonotypes was identified, mostly CD8+. Frequencies above 2% occurred only in MS/CIS participants, and one patient (MS6) contributed a large share.

    33 clonotypes with CSF frequency ≥0.75% and at least two-fold above blood; more than 70% CD8+; 0.76-4.9% of an individual's CSF repertoire; MS6 had 11 clonotypes, nearly 20%; mean frequencies MS/CIS vs HC/OND not significantly different. · Selective enrichment of highly expanded T cell…

  2. ShownTwo CSF-enriched CD8+ clonotypes were validated as EBV-specific by tetramer binding and cytokine production. A screen of other viral epitopes found no further hits, and the two TCRs did not react to the self-peptide homologs tested.

    TCR 86333_1456 (MS8) matched EBNA3A193-201 FLRGRAYGL on HLA-B*08:01; TCR 69317_24418 (MS6) matched BZLF154-64 EPLPQGQLTAY on HLA-B*35:01. Nineteen TCRs vs 98 peptides across 8 MHC I alleles gave no other hits. TCR 86333_17042 failed validation. · Probing viral specificity of clonally expanded…; Lack of self-antigen cross-reactivity of…

  3. ShownUnbiased pMHC yeast display produced mimotopes that several TCRs bound as tetramers, but only one gave a functional response. No natural foreign or human peptide homolog triggered cytokine release, so no genuine target antigen emerged.

    15 of 23 CD8+ TCRs tested; four TCRs showed peptide enrichment (3 MS/CIS, 1 HC); three of four bound tetramer; only clonotype 54189_65570 responded to CSERNPWTFY; no peptide homolog elicited cytokines above background. · Antigen discovery of highly expanded…

  4. ShownHighly expanded CSF T cells showed a cytotoxic effector-memory and tissue-homing program with reduced naive genes. The enriched clonotypes fell mostly in one CSF cluster defined by cytotoxic CD8+ markers.

    Higher CD8A, CD8B, NKG7, GZMA, GZMH, GZMK, EOMES, CCL5, CCL4; lower IL7R, LTB, LDHB vs unexpanded cells; the overwhelming majority of the 33 clonotypes were in cluster 1 of 11 CSF T cell clusters. · Single-cell transcriptomics of CSF-expanded T…

  5. ShownGLIPH2 grouped some enriched clonotypes into small networks of related TCRs. These were nearly all CD8+, mostly within a single individual, and mainly in MS/CIS participants.

    19 clonally related networks comprising 44 clonotypes; most networks had two clonotypes, two networks had five each; only 21 identical TCRs were shared between the blood of different individuals and 3 between blood and CSF. · Clonal relationships of expanded CSF-enriched T…

Methods, data and reproducibility

Paired 10X 5' scRNA-seq and scTCR-seq of CSF and blood defined clonotypes enriched in CSF (≥0.75% of CSF repertoire, at least two-fold over blood). Antigens were probed by pMHC yeast display, TCR database queries and viral tetramer panels, then validated by TCR knock-in tetramer and cytokine assays.

  • Paired CSF and blood from treatment-naive MS/CIS patients, OND patients and healthy controls (11 RR-MS, 2 CIS, 2 OND, 3 HC) · human · n = 18 participants
  • T cells with paired TCRαβ and scRNA-seq: 33,349 CD4+ and 15,119 CD8+ (31,756 and 10,825 unique clonotypes respectively) · human · n = 48,468 T cells
  • pMHC I yeast display libraries screened with CSF-enriched CD8+ TCRs · human (TCRs and HLA alleles) · n = 15 of 23 CD8+ TCRs tested
  • Viral epitope pMHC I tetramer panel screened against CSF-enriched CD8+ TCRs · human (TCRs and HLA alleles) · n = 19 TCRs; 98 peptides; 8 MHC I alleles
Methods & tools
Cell Ranger (v3.0.1, v3.1.0), Seurat (v3.1.2 – v4.3.0), SingleR (v1.1.7), DoubletFinder (v2.0.2), Immcantation (v3.1.0), Change-O (v0.4.6), alakazam, GLIPH2, VDJdb, TCRex, TCRmatch, pMHC yeast display, CRISPR/Cas9 TRAC TCR knock-in, pMHC I tetramers
Code
https://github.com/UCSF-Wilson-Lab/SingleCellVDJdecontamination
Reagents & environment
Cell Ranger v3.0.1 and v3.1.0; Seurat v3.1.2 – v4.3.0; STAR v2.5.1; SingleR v1.1.7; DoubletFinder v2.0.2; Immcantation v3.1.0; Change-O v0.4.6; GRCh38.v93 and v94 references.
Reproducibility gaps
No repository accession is given for raw sequencing data; the text says all data are in the main text or supplement, and the scRNA-seq was reported as previously published. Only a QC helper repository is linked.
Limitations (5)
  • The study had a smaller control group than the MS group, so follow-up with larger, well-matched MS and control cohorts is needed to identify disease-relevant T cell populations. · stated by the authors
  • Antigen testing was not exhaustive and covered a limited range of antigens; most enriched clonotypes still have unknown specificity. · stated by the authors
  • Screening of partially homologous self-peptides found no reactivity for the two EBV-specific TCRs, but this does not fully exclude self-antigen cross-reactivity. Pathogenicity of the cells also needs in vitro and in vivo testing. · stated by the authors
  • MS6 alone contributed 11 of the 33 enriched clonotypes, so cohort-level patterns may be driven by one individual. No sensitivity analysis of the 0.75% and two-fold thresholds is reported. · noted in this summary
  • The non-MS comparator group (n = 5) mixes OND patients and healthy controls, and one OND patient was on TNFα inhibitor therapy, which confounds the MS/CIS vs control comparisons. · noted in this summary

Who should read:Read this if you study T cell antigen discovery in autoimmunity and want a case study of tetramer versus functional validation of CSF-expanded CD8+ TCRs.

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