*Purpose: The PD-1: PD-L1 signaling has inhibitory properties and is a potential therapeutic target in transplantation. We have previously demonstrated that overexpression of PD-1 on T cells (PD-1 Tg) when combined with costimulation blockade (CTLA-4-Ig), promotes allograft tolerance in a fully MHC-mismatched cardiac transplant model. Thus, the modulation of PD-1 expression is a promising therapeutic target for long-term allograft acceptance. In this study, we aim to identify the mechanisms underlying the long-term graft survival in PD-1 Tg mice.

*Methods: BALB/c (H-2^d) hearts were transplanted into WT or PD-1 Tg B6 (H-2^b) mice treated with a single dose of CTLA-4-Ig. In some experiments, Tregs were depleted by treating animals with an anti-CD25 antibody on days -7 and -1. We assessed ICOS potential role in vivo by treating animals with an anti-ICOS blocking antibody or isotype control on days 0, 2, 4 and 6. Animals were euthanized at POD21 for mechanistic analyses. Graft, spleen and draining lymph nodes were analyzed by flow cytometry and ELISPOT. Flow-sorted PD-1 Tg Tregs (CD4⁺Foxp3-GFP⁺) were further analyzed using an RT-PCR array and an in vitro suppression assay. We analyzed over 90 functional markers on the Tregs.

*Results: We identified that Tregs from PD-1 Tg mice were required for tolerance induction (MST=89 days vs >100 days, p=0.0174). PD-1 Tg Tregs had higher expression of ICOS at both mRNA and protein levels when compared to WT Tregs. Prolongation of allograft survival in PD-1 Tg recipients was abrogated upon ICOS blockade, indicating a critical role for ICOS in the tolerance induction (MST: 23 vs >100 days, p=0.0133). Compared to WT Tregs, intragraft and splenic Tregs from PD-1 Tg recipients had a higher expression of IL-10 (p=0.0207) and LAP (p=0.0374), which was decreased by anti-ICOS treatment (p=0.0008 and p=0.0421) expression at day 21 post-transplant. We verified by ELISPOT that the increased IL-10 production in PD-1 Tg recipients (p<0.0001) was allo-specific, and it was inhibited by ICOS blockade (p=0.0009). Moreover, we flow-sorted Tregs from PD-1 Tg heart recipients had a superior ability to inhibit T cell proliferation (p=0.0445). This effect was abrogated by the anti-ICOS treatment (p=0.0145). Importantly, ICOS blockade had minimal effects on effector T cell responses, suggesting that the anti-ICOS treatment has a dominant inhibitory effect on Treg suppressive function.

*Conclusions: In sum, our data suggest that the PD-L1:PD-1 pathway is a promising target in transplantation, especially in synergy with costimulation blockade. PD-1 overexpression on T cells leads to an increase of ICOS expression on Tregs and its superior suppressive functions are dependent on this molecule.

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