A CD40-Targeted IL-21 fusokine enables rapid generation of human IL-10⁺granzyme B⁺ regulatory B cells

Regulatory B cells (Bregs) suppress pathogenic immune responses and maintain immune homeostasis, but their rarity and poor ex vivo expandability limit clinical translation as cellular therapeutics. Here, we describe a feeder-free platform for rapid generation of human IL-10⁺Granzyme B⁺ regulatory B cells using a CD40-targeted IL-21 fusion protein (CD40scFv–IL-21) combined with TLR9 activation. Human peripheral blood B cells stimulated with CD40scFv–IL-21 and CpG acquired a distinct CD19⁺CD25⁺CD71⁺CD73 low phenotype and robustly expressed IL-10 and granzyme B in more than 95% of input cells. Mechanistically, CD40scFv–IL-21 activated canonical CD40-associated NF-κB and p38 signaling while preserving IL-21–dependent STAT activation. Transcriptomic profiling identified a coordinated regulatory gene program distinct from CpG stimulation alone, including increased expression of IL10 , GZMB , CD274 , PDCD1LG2 , EBI3 , PRDM1 , and IRF4 . Conditioned-media chemokine profiling further supported acquisition of a distinct immunomodulatory secretory phenotype. Functionally, induced Bregs suppressed activated human T-cell proliferation in vitro and significantly prolonged survival in a xenogeneic graft-versus-host disease model. Together, these findings establish a scalable and translationally adaptable strategy for generation of human regulatory B cells for potential application in immune-mediated diseases and cellular immunotherapy.