Ex vivo-generated human CD1c+ regulatory B cells by a chemically defined system suppress immune responses and alleviate graft-versus-host disease

IL-10+ regulatory B cells (Bregs) show great promise in treating graft versus host disease (GVHD), a life-threatening complication of post-hematopoietic stem cell transplantation. However, obtaining high-quality human IL-10+ Bregs in vitro remains a challenge due to the lack of unique specific marker and the triggering of pro-inflammatory cytokines expression. Here, by uncovering the critical signaling pathways in Bregs induction by mesenchymal stromal cells (MSCs), we firstly established an efficient Bregs induction system based on MSCs and GSK-3β blockage (CHIR-99021), which had a robust capacity to induce IL-10+ Bregs while suppress TNF-α expression. Furthermore, these Bregs population could be identified and enriched by CD1c+. Mechanistically, MSCs induced the expansion of Bregs through the PKA-mediated phosphorylation of cAMP response element binding protein (CREB). Thus, we developed a chemical-defined inducing protocol by PKA-CREB agonist, instead of MSCs, which can also effectively induce CD1c+ Bregs with lower TNF-α expression. Importantly, induced CD1c+ Bregs suppressed the proliferation of PBMC and the inflammatory cytokines secretion of T cells. When adoptive transferred to a humanized mouse model of GVHD, induced CD1c+ Bregs effectively alleviated GVHD. Overall, we establish an efficient ex vivo induction system for human Bregs, which has implications in developing novel Bregs-based therapies for GVHD.