Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). It is caused by immune-mediated damage to myelin and neurons. While both CD4⁺ and CD8⁺ T cells infiltrate MS lesions, CD8⁺ T cells constitute the dominant lymphocyte population in the active lesions. These cells are frequently clonally expanded, suggesting antigen-driven immune responses. Moreover, genetic associations with major histocompatibility complex (MHC) class I alleles further implicate CD8⁺ T cells in MS pathogenesis. However, the antigen specificity and functional phenotype of these clonally expanded CD8⁺ T cells within the CNS remain poorly defined. Cerebrospinal fluid (CSF) provides an accessible window into immune processes occurring in the CNS because direct CNS tissue sampling is highly invasive. Earlier studies have shown clonal overlap between CSF T cells and those present in MS lesions, which supports the relevance of CSF-based immune profiling.
A study published in Nat Immunol aims to detect clonally expanded CD8⁺ T cells enriched in the CSF of patients with early, untreated MS and to determine their transcriptional characteristics and antigen specificity with a particular focus on EBV. It also established whether EBV-specific CD8⁺ T cells are selectively expanded in the CSF of MS patients compared with controls and whether these cells exhibit distinct functional phenotypes that may inform MS pathogenesis.
Paired CSF and blood samples were collected from individuals with relapsing-remitting MS or clinically isolated syndrome (CIS) and healthy controls, and patients with other neuro-inflammatory disorders. Single-cell RNA sequencing (scRNA-seq) combined with single-cell T cell receptor sequencing (scTCR-seq) was performed to enable high-resolution transcriptional profiling and clonal tracking of individual T cells. Clonally expanded and CSF-enriched CD8⁺ T cell populations were detected based on TCR frequency and compartmental enrichment. Antigen specificity was investigated by using complementary approaches, which include unbiased peptide-MHC yeast display, database-guided TCR alignment using VDJdb, GLIPH2 clustering to detect shared TCR motifs, and functional validation by using Jurkat reporter assays expressing defined TCRαβ pairs. Potential cross-reactivity with self-peptides was evaluated using panels of homologous peptides. The presence of Epstein-Barr virus (EBV) in CSF was assessed using PCR, droplet digital PCR, and transcript analysis of latent and lytic EBV genes.
The results showed that the most highly expanded and CSF-enriched T cell clonotypes in MS were predominantly CD8⁺ T cells, though a higher overall number of CD4⁺ T cells were analyzed. These CD8⁺ T cells exhibited transcriptional signatures consistent with antigen experience, cytotoxicity, and CNS trafficking closely resembling CD8⁺ T cells previously described in MS lesions. The study detected 3 distinct highly expanded CD8⁺ T cell clonotypes from different MS patients that were definitively specific for EBV antigens by using rigorous antigen discovery and validation strategies. EBV-specific CD8⁺ T cells were enriched in the CSF compared with blood and were observed exclusively in MS/CIS patients. In contrast, no enrichment of cytomegalovirus-specific CD8⁺ T cells was detected.
Transcriptional profiling revealed that EBV-specific CD8⁺ T cells displayed a phenotype distinct from other expanded CSF CD8⁺ T cells, characterized by increased expression of CXCR5, CD27, and other genes linked to follicular homing, memory differentiation, and B cell interaction, instead of tissue-resident memory markers. Functional assays showed no detectable cross-reactivity with homologous self-peptides, arguing against molecular mimicry as the dominant mechanism for these EBV-specific CD8⁺ T cells. Analysis of CSF revealed widespread detection of EBV DNA with significantly higher viral abundance and increased expression of BamHI-W transcripts in MS/CIS patients, which indicate improve EBV reactivation in MS and provides a plausible driver for EBV-specific CD8⁺ T cell expansion.
In conclusion, this study demonstrates that a significant subset of clonally expanded CD8⁺ T cells in the CSF of MS patient, which are specific for EBV. These cells show an effector phenotype associated with follicular homing and potential interactions with EBV-infected B cells, suggesting a role in immune activation in MS rather than direct cytotoxicity or autoreactivity. By integrating single-cell transcriptional profiling with clonal and antigen-specific analyses, this work advances understanding of CD8⁺ T cell dynamics in MS and reinforces the connection between EBV and MS pathogenesis, with important implications for future diagnostic and therapeutic strategies.
Reference: Hayashi F, Mittl K, Dandekar R, et al. Antigen specificity of clonally enriched CD8+ T cells in multiple sclerosis. Nat Immunol. 2026. doi:10.1038/s41590-025-02412-3
