Ex vivo Expanded PD-L1⁺ Bregs Protect Against Transplant Rejection

Achieving immune tolerance is a key goal in organ transplantation, as it eliminates the need for long-term immunosuppression. Regulatory B cells (Bregs) present a promising strategy for inducing tolerance. Our previous findings demonstrate that the adoptive transfer of ex vivo-expanded murine splenic B regulatory cells, referred to as TLR-Bregs (TLR9/TLR4 stimulation), induces tolerance to allografts. Here, we identify that circulating T cells increase PD-1 expression following TLR-Bregs cellular therapy. To investigate whether PD-L1-PD-1 signaling is involved in tolerance induction by TLR-Bregs, we generate TLR-Bregs from wild-type (WT) or PD-L1-deficient (^-/-, KO) B6 splenocytes and assess their regulatory functions. Our findings reveal that TLR-Bregs express high levels of PD-L1 and extend graft survival via a PD-L1-dependent mechanism. Mechanistically, PD-L1 enhances the activation and survival of TLR-Bregs; PD-L1 is required for TLR-Bregs to induce PD-1 expression and an optimal inhibition of effector CD4⁺T cells while promoting the formation of functional Foxp3^hiCCR7^hiCTLA4^hiTregs. This study highlights the foundational role of PD-L1 in inducing tolerance by ex-vivo-expanded TLR-Bregs.