Introduction: This study checks if PD-L1 transferred from Toll-Like-Receptor-Stimulated Bregs (TLR-Bregs) to activated CD4⁺T cells is functional.
Method: Bregs were generated from purified splenic B cells of wild-type C57BL/6 (B6) mice and were ex-vivo stimulated via TLR9 and TLR4 for TLR-Bregs and TLR9 alone for control CpG-Bregs. 1.5X10⁵ CD3-28 beads stimulated CD4⁺T cells from PD-L1 deficient (KO) mice were co-cultured with these Bregs, and proliferation was assessed through mixed lymphocyte reaction and CFSE trace. We distinguished PD-L1 expression and proliferation in CD4⁺T cells sourced separately from splenocytes and peripheral lymph nodes. Wells with Bregs alone were used as the control to estimate Breg reduction in the presence of CD4⁺T cells.
Results: Without Bregs, the CD4⁺T cell proliferation was similar between lymphatic-derived and splenic-derived. PD-L1 transfer was observed from TLR-Bregs to activated CD4⁺T cells derived from both splenocytes and lymphatic cells. CpG-Bregs did not transfer PD-L1 to CD4⁺T cells. Both CpG-Bregs and TLR-Bregs suppressed lymphatic CD4⁺T cells with dose-dependency, but TLR-Bregs showed more robust suppressive function than the same dose of CpG-Bregs. These results suggested that PD-L1 transfer from TLR-Bregs to lymphatic CD4⁺T cells is not functional. In contrast, A high dose (1.5X10⁵) of CpG-Bregs slightly suppresses splenic-derived CD4⁺T cells, while a low dose (1X10⁵) of CpG-Bregs cannot inhibit splenic-derived CD4⁺T cell proliferation, indicating splenic-derived CD4⁺T cells were more resistant to CpG-Bregs induced inhibition than lymphatic-derived CD4⁺T cells. Interestingly, low doses of TLR-Bregs could not inhibit the proliferation of splenic-derived CD4⁺T cells, while high doses of TLR-Bregs still inhibit the proliferation of splenic-derived CD4⁺T cells. For proliferating splenic-derived CD4⁺T cells, the CD4⁺PD-L1⁺ cell proportion and PD-L1 MFI were comparable after co-culturing with low-dose and high-dose TLR-Bregs. Notably, the live cell number of TLR-Bregs decreased more significant in a low-dose group than in the high-dose group after co-culturing with CD4⁺T cells. After co-culturing with CD4⁺T cells, a higher proportion of TLR-Bregs in the low-dose group became PD1⁺, while PD-L1 upregulation on TLR-Bregs in the low-dose group is less than counterparts in the high-dose group. These results indicated that the transferred PD-L1 on splenic CD4⁺T cells mediates suppression to TLR-Bregs in the low-dose group via PD-L1-PD1 signaling, resulting in more significant decreased live TLR-Bregs number and regulation function than TLR-Bregs in the high-dose group.
Conclusion: We present the first evidence of functional PD-L1 transfer from TLR-Bregs to splenic CD4⁺T cells. Our previous date observed PD-L1 transfer from CD4⁺T cells to CpG-Bregs and TLR-Bregs. If the PD-L1 transfer to Bregs is also functional, it may enhance the regulatory function of TLR-Bregs as the suppressive function of TLR-Bregs is PD-L1 dependent.