Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) in which autoreactive and cross-reactive T cells are believed to play a central role in initiating and sustaining tissue damage. However, it remains poorly understood which antigen-specific T cell clones present in the blood actually gain access to the CNS, and whether this process differs between people with MS (pwMS) and healthy individuals. Addressing this question requires linking antigen specificity, transcriptional state, and T cell receptor (TCR) sequence at single-cell resolution, and matching these features between peripheral blood and cerebrospinal fluid (CSF). In this project, we will use single-cell RNA sequencing (scRNAseq) to profile antigen-specific T cells isolated from the blood of pwMS (n=13) and healthy controls (n=3), and compare their TCR repertoires directly with paired CSF T cell populations from the same donors. Antigen-specific T cells will first be enriched from blood samples by stimulation with a panel of candidate antigens, including Epstein-Barr virus (EBV) and the known CNS autoantigens ANO2 and CRYAB, as well as newly identified CNS targets, using a workflow we recently established and published in Cell (Thomas et al. 2026). Enriched cells will then undergo single-cell transcriptome and TCR sequencing to define their antigen specificity, activation state, and functional properties. In total we plan to sequence approximately 320,000 T cells across all donors and sample types, comprising 16 sequencing reactions. This resource will allow us, for the first time, to directly identify which autoreactive and cross-reactive T cell clones access the CNS in MS, and to determine how this compares to the healthy immune repertoire. The expected outcome is a library of antigen-resolved TCRs derived from the blood of pwMS and healthy donors, paired with matched CSF TCR repertoires from the same individuals. By integrating these single-cell immune profiles with detailed clinical and genetic data available through the Swedish MS Registry, we will further examine how CNS-infiltrating T cell clones relate to disease activity, disease progression, and individual patient characteristics. All blood and CSF samples required for this project have already been collected, and the associated clinical and registry data are readily available, so the proposed computational and sequencing work can begin immediately. This project builds directly on our recently published methodology for antigen-specific T cell profiling (Thomas et al. 2026) and extends it to a systematic, paired blood-CSF comparison in MS, addressing a fundamental open question in MS immunology: which T cells drive CNS entry, and how this process goes wrong in disease.