Introduction: Although immune checkpoint inhibitors (ICIs) have been approved as anti-cancer drugs, they have negative effects on transplant patients who require immune tolerance. Therefore, this study investigated the potential of PD-L1-Fc fusion Ab in inducing immune tolerance in allogeneic heart transplantation in mice.

Methods: Allogeneic heterotopic heart transplantation was performed using male BALB/c mice as donors and male C57BL/6 mice as recipients. Graft survival was monitored by detecting heartbeats, and at 7 days after transplantation, peripheral blood was analyzed for phenotype and graft pathology was examined through immunohistochemistry (IHC). ELISA was performed to measure IFN-gamma levels in the blood on the same day. The immunosuppressant drugs included no treatment (control), PD-L1-Fc, anti-CD154 Ab, and anti-CD154 Ab+PD-L1-Fc (N=15 for each treatment).

Results: In the group received with a combination of anti-CD154 Ab and PD-L1-Fc, all individuals survived for over 180 days, while the graft survival rate in the PD-L1-Fc group was similar to that of the control group and only lasted for about 10 days. Flow cytometry analysis showed that the proportion of effector memory CD8 T cells and the expression of both CD154 and LAG3 molecules increased in the blood cells of the PD-L1-Fc group. IHC results showed that PD-L1-Fc group had high levels of inflammatory cell infiltration, while anti-CD154 Ab+PD-L1-Fc group had low levels of inflammatory cell infiltration. Compared to the group that received only anti-CD154 Ab, the anti-CD154 Ab+PD-L1-Fc group showed a 50% reduction in the loss of myocardial cells. The PD-L1-Fc group had an interstitial hemorrhage score of 2, while no interstitial hemorrhage was observed in the anti-CD154 Ab+PD-L1-Fc group. These results suggest that the combined treatment of anti-CD154 Ab and PD-L1-Fc may prolong the survival time of transplanted hearts while minimizing side effects. Additionally, ELISA results indicated a decrease in IFN-gamma levels in all groups treated with anti-CD154 Ab, while it remained high in the PD-L1-Fc group similar to the control group.

Conclusion: The use of PD-L1-Fc alone to induce T cell inhibition via the PD-1 signaling pathway in heart transplantation was less effective compared to the combined therapy with anti-CD154 Ab and PD-L1-Fc. This may be due to the upregulation of CD154 costimulatory molecules in T cells, which promotes T cell activation and diminished the inhibitory effect of the PD-1/PD-L1 interaction. These findings suggest that although the PD-1/PD-L1 interaction plays a crucial role in heart transplantation, its inhibitory negative feedback may be limited by costimulatory signals. Further investigation is warranted to determine the feasibility of applying this immunosuppressive mechanism in the field of xenotransplantation.