Dissecting The Mechanisms of Transitional B Cell Tolerance

Abstract

The study of B cell tolerance has remained elusive for many years. However, patients with primary immunodeficiencies resulting from monogenic causes who present with autoimmune manifestations can be studied to elucidate the mechanisms of peripheral B cell tolerance. Based on our group’s previous findings that there is a developmental block from transitional to follicular B cells in patients with activated PI3Kδ syndrome (APDS), we hypothesized that peripheral tolerance is mediated in the transitional B cell stage. To identify the transitional B lymphocyte subset where this checkpoint occurs, we used multi-parameter flow cytometry to study B cell populations obtained from the peripheral blood of patients with common variable immunodeficiencies and healthy individuals. We then performed a multimodal single-cell analysis consisting of single-cell RNA sequencing, single-cell B cell receptor sequencing (scBCR-seq), and cellular indexing of transcriptomes and epitopes by sequencing on peripheral blood-derived B cells from APDS patients. We observed an expansion in early transitional B cells (transitional 1 (T1), T2, and T3a) coupled with a pathological block in the T3b/follicular subset. Upon analyzing the proportion of B cells expressing VH4-34 (which is known to have intrinsic autoreactive properties) from the scBCR-seq data, we found that the T2 cluster in healthy individuals contains a high percentage of these cells compared to all other clusters, whereas in APDS patients, all B cell populations contain a high percentage of these cells compared to the same clusters in healthy individuals. We also found that early transitional B cell subsets, activated naïve, and CD45RB+ double negative B cells were highly clonal in APDS patients, suggesting antigen-specific proliferation. Isotype analysis of patient B cell clusters revealed reduced class switching in activated naïve and CD45RB+ double negative B cells. Collectively, our data suggest a peripheral tolerance checkpoint in the T2 stage, which appears to be dysfunctional in APDS patients, leading to the production of IgM antibodies that are presumably directed against self-antigens.