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 markers and the triggering of pro-inflammatory cytokine expression. Here, by uncovering the critical signaling pathways in Breg induction by mesenchymal stromal cells (MSCs), we first established an efficient Breg induction system based on MSCs and GSK-3β blockage (CHIR-99021), which had a robust capacity to induce IL-10(+) Bregs while suppressing tumor necrosis factor α (TNF-α) expression. Furthermore, these Breg populations 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 chemically 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 peripheral blood mononuclear cells and the inflammatory cytokine secretion of T cells. When adoptively transferred into a humanized mouse model of GVHD, induced CD1c(+) Bregs effectively alleviated GVHD. Overall, we established an efficient ex vivo induction system for human Bregs, which has implications for developing novel Bregs-based therapies for GVHD.