*Purpose: Regulatory B cells (Breg) participate in control of alloimmune responses in murine models of graft tolerance and are associated with improved outcomes in clinical transplant recipients. However, our understanding of human Breg characteristics, and the potential to harness them for therapeutic purposes, has been hampered by the reported variability of their phenotype and function. Here we developed a custom mass cytometry panel and pipeline for deep profiling and utilized it to investigate IL-10-producing human B cells.

*Methods: PBMC from healthy donors were stimulated with 11 different cocktails of TLR ligands, anti-CD40 and cytokines to identify optimal conditions for eliciting IL-10-producing human B cells. Immunophenotype and functional features were determined using antibodies against 24 surface and 14 intracellular proteins and analysis by mass cytometry (CyTOF). Live cell tracing was used to map the origin of IL-10-producing B cells. PBMC from healthy donors, transplant recipients and operationally tolerant (OT) transplant recipients were analyzed for IL-10-producing B cells.

*Results: The combination of CpG, anti-CD40, IL-2, IL-21 and IL-35 was most effective in eliciting IL-10-producing B cells (mean=34.1% of CD19⁺ B cells)). High dimensionality reduction with UMAP revealed that seven previously identified Breg phenotypes including CD19⁺CD24^hiCD38^hi and CD19⁺CD25^hiCD71^hiCD73^– B cells captured some, but not all, IL-10-producing B cells. Live cell tracking indicates that multiple B cell subsets can give rise to IL-10-producing cells but the CD45RB⁺CD27⁺ B cell memory subset and the CD24⁺CD38⁺ transitional B cell subset yielded the highest proportion of IL-10⁺ B cells. While B cells that exclusively produced IL-10 could be detected, the majority of IL-10⁺ B cells also produced TNFα and IL-6 with lesser proportions of IL-10⁺ B cells also producing IL-4, Granzyme B and the p35 subunit of IL-35. OT transplant recipients and healthy controls had increased IL-10⁺TNFα^– B cells (8% and 10% respectively) compared to transplant recipients (2%). IL-10⁺ B cells were particularly enriched in CD9⁺ B cells in OT patients but not in transplant controls.

*Conclusions: Human IL-10-producing B cell are heterogeneous with respect to phenotype and function and can emerge from multiple canonical B cell subsets. OT transplant recipients have increased proportions of IL-10⁺ B cells compared to transplant recipients on immunosuppression. High dimensional, single cell analysis of human IL-10⁺ B cells provides important insight for understanding immune regulation in transplantation and for harnessing Breg subsets for therapeutic use.

« Back to 2022 American Transplant Congress