Introduction: We aim to elucidate the crucial role of PDL1-PD1 signaling in the modulation of anti-graft responses by TLR-Bregs.
Method:  We expanded Bregs ex-vivo from the splenic B cells of wild-type C57BL/6 (WT) or PDL1 deficient (KO) mice. TLR-Bregs were generated via TLR9 and TLR4 signaling, while CpG-Bregs (TLR9 stimulation) served as controls. We compared these putative Bregs to evaluate their impact on graft survival, modulation of T cell exhaustion, Treg induction, and regulation of activated CD4⁺T cells.
Results: WT TLR-Bregs, but not PDL1^KOTLR-Bregs, significantly extended islet and skin graft survival in WT B6 recipients. Phenotypic analysis revealed all Bregs were in the transitional stage and manifested rare PDL1 but higher PD1 expression in ^KOTLR-Bregs than WT. ^KOTLR-Bregs were less effective in modulating higher PD1 and lower PDL1 expression (exhaustion) in recipient circulating T cells. WT TLR-Bregs induced a greater proportion of Foxp3^hiCCR7^hiCTLA4^hiTregs in the draining lymph node, while ^KOBregs generated fewer Tregs with less functional markers. Notably, PDL1 transfer was observed in-vivo and in-vitro between activating TLR-Bregs and CD4⁺T, affecting the expression profiles in KO cells. Uniquely, WT TLR-Bregs suppressed the PD1-expressing activating WT CD4⁺T in-vivo and in-vitro in a dose-dependent manner. Conversely, WT CpG-Bregs and TLR-Bregs dose-dependently suppress activating ^KOCD4⁺T. Specifically, WT TLR-Bregs showed further improved graft survival, enhanced inhibitory function, and increased Breg survival with ^KOCD4⁺T than WT CD4⁺T. We then discovered that activating WT CD4⁺T (especially engaged TLR-Bregs) highly expressed PDL1, while activating Bregs were PD1 expressing. Post in-vivo and in-vitro contact with PDL1^hiCD4⁺T, ^KOBregs manifest reduced cell survival, less PDL1 expression, and lower PD1⁺cell proportion but higher PD1 protein than WT TLR-Bregs. Moreover, PDL1 expression on WT TLR-Bregs further increased after suppressing PDL1^hiCD4⁺T, and WT TLR-Bregs up-regulated PD1 protein on activating WT PDL1^hiCD4⁺T. Dynamic expression changes in PD1 and PDL1 on Bregs and CD4⁺T cells indicated activated PDL1^hiCD4⁺T counteract PD1⁺Bregs; while WT Breg-TLR, but not ^KOBregs, secure their regulatory capacity by up-regulating self PDL1 and PD1 on CD4⁺T cells.
Conclusion: High PDL1 expression on TLR-Bregs is foundational to their regulatory function. PDL1 deficiency in TLR-Bregs impairs their ability to suppress CD4⁺T cell activation, promotes T cell exhaustion, induces functional iTregs, and prevents the antagonistic effects of activated PDL1^hiCD4⁺T cells. Conversely, PDL1 deficiency on CD4⁺T enhances Bregs' regulatory capacity. This study underscores the therapeutic potential of targeting the PDL1-PD1 pathway on Bregs in enhancing allograft tolerance.