CITEseq analysis of human CSF cells in Multiple sclerosis

In CSF bulk RNA sequence, patients with MS (pwMS) exhibited higher T cell receprot (TCR) clonality and specificity to EBV lytic proteins compared to controls. TCRs from pwMS showed greater clustering coefficient (i.e. TCR similarity) in complementarity-determining regions 3 (CDR3), particularly those predicted to target EBV antigens. Importantly, TCR clustering coefficient was associated with the expression of cytotoxic CD8 module genes in WGCNA, such as CD8A, GZMH, GZMK, and NKG7 and with interferon signaling module. scRNA-seq revealed a subpopulation of GZMK+GZMH+ double positive (DP) CD8 T cells expressing genes related to identified cytotoxicity and interferon signaling modules. These DP CD8 T cells had significantly higher predicted specificity for EBV proteins than other CD8 cells. CSF was collected on ice and processed according to a written standard operating procedure by investigators blinded to diagnoses, clinical, and imaging outcomes. Aliquots were assigned alphanumeric identifiers and centrifuged for 10 minutes at 300g at 4°C within 30 minutes of collection. The pelleted CSF cells were processed for single cell RNA sequencing. Cells were labelled with 79 Totalseq B antibodies (Biolegend, USA) and washed with PBS with 1% Bovine Serum Albumin (Thermo Fisher Scientific, USA). Labeled cells were fixed by using fixed RNA feature barcode kit (10x Genomics, USA) and stored at -80 degree.