Interleukin 35-producing B cells prolong the survival of GVHD mice by secreting exosomes with membrane-bound IL-35 and upregulating PD-1/LAG-3 checkpoint proteins.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective treatment for aggressive hematologic malignancies. However, the risk of developing graft-versus-host disease (GVHD) is a significant barrier to allo-HSCT. GVHD is a debilitating condition with high mortality rates and current therapeutic options for GVHD are limited, with corticosteroids being the standard treatment. However, the adverse effects of steroids make prolonged use difficult, necessitating the development of safer therapies. IL-35-producing B-cells (i35-Bregs) have emerged as critical regulators of immunity during autoimmune diseases. In this study, we investigated whether i35-Bregs immunotherapy can suppress and mitigate GVHD. We administered a single dose of i35-Bregs (1.5×10) to mice undergoing allo-HSCT and monitored disease severity and survival of GVHD mice over 90 days post-transplantation. We discovered that i35-Bregs secrete exosomes containing membrane-bound IL-35 (i35-Exosomes) and investigated whether generated i35-exosomes can be used as stand-alone immunotherapy for GVHD. i35-Breg-induced expression of cytokines or checkpoint proteins (PD-1, LAG-3, CTLA-4) was analyzed by Flow cytometry, ELISA, and RNA-seq analysis. Characterization of membrane-bound IL-35 was by Proximity ligation assay (PLA), immunohistochemistry/Confocal microscopy and Alpha Fold-Multimer modeling. A single dose of 1.5×10 i35-Breg reduced severity of GVHD and prolonged GVHD survival, with more than 70% i35-Breg-treated mice surviving beyond day-90 post-transplantation while observing 100% mortality among untreated mice by day-45. Contrary to the view that IL-35 is secreted cytokine, we show here that i35-Bregs mitigate GVHD via membrane-bound IL-35 and by secreting i35-exosomes. Furthermore, i35-Bregs or generated i35-exosomes induce alloreactive T-cells to upregulate checkpoint proteins associated with T-cell exhaustion and anergy, inhibiting alloreactive responses and propagating infectious-tolerance mechanisms that suppress GVHD. Importantly, i35-Bregs or i35-exosomes suppresses GVHD by increasing bystander lymphocytes coated with immunosuppressive i35-exosomes. This study demonstrates that i35-Bregs and i35-exosomes play a critical role in mitigating GVHD. The combination of i35-Breg and i35-exosome immunotherapy may be an effective strategy for treating GVHD and other inflammatory diseases.