T:B cell cooperation in ectopic lymphoid follicles propagates CNS autoimmunity

Meningeal ectopic lymphoid follicle (eLF)-like structures have been described in multiple sclerosis, but their role in central nervous system (CNS) autoimmunity is unclear. Here, we used a T helper 17 (TH17) adoptive transfer experimental autoimmune encephalomyelitis model featuring formation of eLFs. Single-cell RNA sequencing revealed that clusters of activated B cells and B1/marginal zone-like B cells were overrepresented in the CNS and identified B cells poised for undergoing germinal center reactions and clonal expansion in the CNS. Using intravital imaging to directly visualize TH17-B cell interactions, we demonstrated that T and B cells form long-lasting antigen-specific contacts in meningeal eLFs that result in reactivation of autoreactive T cells. CNS T cells depended on CNS B cells to maintain a proinflammatory cytokine profile. Our study reveals that extensive T-B cell cooperation occurs in meningeal eLFs, promoting both B cell differentiation and T cell reactivation, and may thereby propagate smoldering inflammation in the CNS. For each mouse, B cells from CNS, spleen and cLNs were pooled (90.000 B cells in total), centrifuged and, after a washing step, resuspended in PBS containing 0.04 % BSA at a concentration of 1000 cells/μl. The viability of cells was confirmed by staining with trypan blue solution. Further processing of samples was performed using the 10x Chromium Single Cell 5` Solution (Chromium Next GEM Single Cell V(D)J v1.1 with Feature Barcoding Technology for Cell Surface Protein, 10x Genomics) according to the manufacturer`s instructions. Prior to loading on the chip, 5000 cells of each compartment were labeled with anti-mouse hashtag oligonucleotide (HTO) antibodies (TotalSeq™-C0301/C0302/C0303, Biolegend) and pooled together. cDNA libraries were pooled and send for sequencing. scRNA-seq and scBCR-seq libraries were sequenced on NovaSeq S4 Flowcells using 150-bp paired-end reads and 8 bp for the i7 index, aiming for 50,000 reads per cell for gene expression and 5,000 reads per cell for BCR enrichment. Cell hashing libraries were sequenced on NovaSeq S1 Flowcells using 50-bp paired-end reads and 8 bp for the i7 index, aiming for 10,000 reads per cell.