Human regulatory memory B cells defined by expression of TIM-1 and TIGIT are dysfunctional in Multiple Sclerosis.

Regulatory B cells (Bregs) play a pivotal role in suppressing immune responses, yet there is still a lack of cell surface markers that can rigorously identify them. In mouse models for multiple sclerosis (MS), TIM-1 or TIGIT expression on B cells is required for maintaining self-tolerance and regulating autoimmunity to the central nervous system. Here we investigated the activities of human memory B cells that differentially express TIM-1 and TIGIT to determine their potential regulatory function in healthy donors and patients with relapsing-remitting (RR) MS. FACS-sorted TIM-1+/-TIGIT+/- memory B (memB) cells co-cultured with allogenic CD4+ T cells were analyzed for proliferation and induction of inflammatory markers using flow cytometry and cytokine quantification, to determine Th1/Th17 cell differentiation. Transcriptional differences were assessed by SMARTSeq2 RNA sequencing analysis. TIM-1-TIGIT- double negative (DN) memB cells strongly induce T cell proliferation and pro-inflammatory cytokine expression. The TIM-1+ memB cells enabled low levels of CD4+ T cell activation and gave rise to T cells that co-express IL-10 with IFNg and IL-17A or FoxP3. T cells cultured with the TIM-1+TIGIT+ double positive (DP) memB cells exhibited reduced proliferation and IFNg, IL-17A, TNFa, and GM-CSF expression, and exhibited strong regulation in Breg suppression assays. The functional activity suggests the DP memB cells are a bonafide Breg population. However, MS DP memB cells were less inhibitory than HC DP memB cells. A retrospective longitudinal study of anti-CD20 treated patients found that post-treatment DP memB cell frequency and absolute number were associated with response to therapy. Transcriptomic analyses indicated that the dysfunctional MS-derived DP memB/Breg population exhibited increased expression of genes associated with T cell activation and survival (CD80, ZNF10, PIK3CA), and had distinct gene expression compared to the TIGIT+ or TIM-1+ memB cells. These findings demonstrate that TIM-1/TIGIT expressing memory B cell subsets have distinct functionalities. Co-expression of TIM-1 and TIGIT defines a regulatory memory B cell subset that is functionally impaired in MS. Peripheral blood was obtained from RRMS (Natalizumab treated) patients and age- and sex-matched healthy volunteers with signed informed consent as approved by the Institutional Review Board of Brigham and Women’s Hospital. Peripheral blood mononuclear cells (PBMCs) were isolated, incubated with fluorochrome-labelled monoclonal antibodies, and then sorted on FACS Aria III. 1000 cells of each TIM-1/TIGIT expressing memory B cells were sorted and collected. The plates were stored at -80 C and processed by the BROAD Institute Genomics platform for Smart-Seq2 RNA-seq. The Trombetta protocol was used sequentially to clean up the lysate, reverse transcribe the mRNA, amplify the transcriptome, PCR clean up, Nextera XT sequencing-library construction, and DNA SPRI bead cleanup. Illumina NextSeq500 was used to run 2 × 38bp paired sequencing at the BROAD Genomics platform. Sequencing reads were quality-controlled, mapped and quantified using the bcbio-nextgen bulk RNA-seq pipeline (version 1.2.8-1c563f1).