Correlation of Professional Antigen-Presenting Tbet+CD11c+ B Cells With Bone Destruction in Untreated Rheumatoid Arthritis.

OBJECTIVE: Subsets of CD21-/low memory B cells (MBCs), including double-negative (DN, CD27-IgD-) and Tbet+CD11c+ cells, are expanded in chronic inflammatory diseases. In rheumatoid arthritis (RA), CD21-/low MBCs correlate with joint destruction. However, whether this is due to the Tbet+CD11c+ subset, its function and pathogenic contribution to RA are unknown. This study aims to investigate the association between CD21-/lowTbet+CD11c+ MBCs and joint destruction as well as other clinical parameters and to elucidate their functional properties in patients with untreated RA (uRA). METHODS: Clinical observations were combined with flow cytometry (n = 36) and single-cell RNA sequencing (scRNA-seq) and V(D)J sequencing (n = 4) of peripheral blood (PB) MBCs from patients with uRA. The transcriptome of circulating Tbet+CD11c+ MBCs was compared with scRNA-seq data of synovial B cells. In vitro coculture of Tbet+CD11c+ B cells with T cells was used to assess costimulatory capacity. RESULTS: CD21-/lowTbet+CD11c+ MBCs in PB correlated with bone destruction but no other clinical parameters analyzed. The Tbet+CD11c+ MBCs have undergone clonal expansion and express somatically mutated V genes. Gene expression analysis of these cells identified a unique signature of more than 150 up-regulated genes associated with antigen presentation functions, including B cell receptor activation and clathrin-mediated antigen internalization; regulation of actin filaments, endosomes, and lysosomes; antigen processing, loading, presentation, and costimulation; a transcriptome mirrored in their synovial tissue counterparts. In vitro, Tbet+CD11c+ B cells induced retinoic acid receptor-related orphan nuclear receptor γT expression in CD4+ T cells, thereby polarizing to Th17 cells, a T cell subset critical for osteoclastogenesis and associated with bone destruction. CONCLUSION: This study suggests that Tbet+CD11c+ MBCs contribute to the pathogenesis of RA by promoting bone destruction through antigen presentation, T cell activation, and Th17 polarization.

Single-cell RNA sequencing analyses: interference by the genes that encode the B-cell and T-cell receptors.

B and T cells are integral parts of the immune system and are implicated in many diseases, e.g. autoimmunity. Towards understanding the biology of B and T cells and subsets thereof, their transcriptomes can be analyzed using single-cell RNA sequencing. In some studies, the V(D)J transcripts encoding the variable regions of the B- and T-cell antigen receptors have been removed before the analyses. However, a systematic analysis of the effects of including versus excluding these genes is currently lacking. We have investigated the effects of these transcripts on unsupervised clustering and down-stream analyses of single-cell RNA sequencing data from B and T cells. We found that exclusion of the B-/T-cell receptor genes prior to unsupervised clustering resulted in clusters that represented biologically meaningful subsets, such as subsets of memory B and memory T cells. Furthermore, pseudo-time and trajectory inference analyses of early B-lineage cells resulted in a developmental pathway from progenitor to immature B cells. In contrast, when the B-/T-cell receptor genes were not removed, with the PCs used for clustering consisting of up to 70% V-genes, this resulted in some clusters being defined exclusively by V-gene segments. These did not represent biologically meaningful subsets; for instance in the early B-lineage cells, these clusters contained cells representing all developmental stages. Thus, in studies of B and T cells, to derive biologically meaningful results, it is imperative to remove the gene sequences that encode B- and T-cell receptors.

Human CD38 regulates B cell antigen receptor dynamic organization in normal and malignant B cells.

CD38 is a multifunctional protein expressed on the surface of B cells in healthy individuals but also in B cell malignancies. Previous studies have suggested a connection between CD38 and components of the IgM class B cell antigen receptor (IgM-BCR) and its coreceptor complex. Here, we provide evidence that CD38 is closely associated with CD19 in resting B cells and with the IgM-BCR upon engagement. We show that targeting CD38 with an antibody, or removing this molecule with CRISPR/Cas9, inhibits the association of CD19 with the IgM-BCR, impairing BCR signaling in normal and malignant B cells. Together, our data suggest that CD38 is a new member of the BCR coreceptor complex, where it exerts a modulatory effect on B cell activation upon antigen recognition by regulating CD19. Our study also reveals a new mechanism where α-CD38 antibodies could be a valuable option in therapeutic approaches to B cell malignancies driven by aberrant BCR signaling.