Dynamic regulation of hypoxia-associated regulatory CD39+CD73+ and IL35+ B cells in pancreatic cancer.

B cells can suppress anti-tumor T cell responses, representing a potential mechanism of resistance to immunotherapy. Multiple Breg subsets have been described in humans and mice. Bregs inhibit anti-tumor immunity through cytokine secretion, immune checkpoint expression, and potentially adenosine generation, which collectively suppress effector T cells, and strengthen immunosuppressive signaling. Hypoxia is a hallmark of pancreatic ductal adenocarcinoma (PDAC) and contributes to immune dysregulation within the tumor microenvironment (TME); however, the effects of hypoxia on B cell subsets remain poorly defined. In this study, we investigated how hypoxic stress shapes regulatory B cell phenotype using murine PDAC models. We show that nearly half of intratumoral B cells are hypoxic, which also increased across systemic B cell compartments with tumor burden. Hypoxic B cells were enriched for CD39 and CD73 ectonucleotidases and suppressed T cell activation via adenosine receptor-dependent mechanisms. Hypoxia preferentially induced CD39+CD73+ expression in peritoneal-derived B cells, indicating tissue-specific regulation, while splenic B cells showed limited responsiveness ex vivo. Conversely, hypoxia reduced IL-35 production by splenic Bregs through decreased Ebi3 expression and reduced activation marker levels. Within tumors, hypoxia correlated with CD11b+ and CD39+CD73+ B cells but not IL-35-producing subsets. Mechanistically, Ebi3 loss enhanced CD39 expression under hypoxia, whereas recombinant IL-35 suppressed CD39+CD73+ induction, suggesting reciprocal regulation between IL-35 and adenosine pathways. These findings reveal hypoxia-driven plasticity in immunosuppressive B cell programs in PDAC, characterized by suppression of IL-35-producing Bregs and promotion of adenosine-generating CD39+CD73+ B cells, highlighting potential therapeutic targets to modulate tumor-associated immunosuppression.