Background: Myocardial inflammation is a hallmark of ischemia/reperfusion (I/R) injury and determines myocardial remodelling and healing, modulated by stimulating and inhibitory signalling cascades. The lymphocyte immune checkpoint Programmed death 1 (PD1) and its ligand PDL1 modulate the critical phase of T cell activation. Genetic or pharmacological ablation of PD1/PDL1 crosstalk has already been associated with cardiotoxic effects. Early evidence indicates a potential involvement of PD1/PDL1 in cardiac diseases, but the role of PD1/PDL1 in I/R injury has not yet been evaluated. Here, we aim to characterize how PD1/PDL1 is involved in myocardial I/R injury in a preclinical model.

Methods: Using a flow cytometry panel to identify cardiomyocytes, cardiac endothelial cells, fibroblasts, and leucocytes, expression of PD1 and PDL1 was examined in wild-type mice receiving anti-PD1 antibody therapy or immunoglobulin control. I/R injury was induced in vivo by temporary ligation of the left coronary artery for 45 min followed by reperfusion for up to 7 days. We used tetrazolium chloride (TTC) and Evan’s blue staining to determine infarct size. Infiltrating lymphocytes were analysed in anti-PD1 treated mice compared to immunoglobulin controls using flow cytometry. Cardiomyocyte apoptosis was assessed by terminal deoxynucleotidyl transferase (TdT) dUTP nick-End labelling (TUNEL) assay and we used mouse echocardiography to determine LV function.

Results: The analysis of PD1 and PDL1 expression revealed that PDL1 was mainly expressed on cardiac endothelial cells while PD1 expression was restricted to a low number of CD4⁺ and CD8⁺ T cells. PD1 blockade induced upregulation of PDL1 during baseline conditions. Following I/R injury, the expression of PDL1 on endothelial cells was downregulated at 24 h and 3 days (p = 0.001). I/R injury in anti-PD1 treated mice was associated with increased apoptosis within the reperfused area as determined by TUNEL staining followed by enhanced infiltration of CD8+ T cells (p = 0.036), while the infarct size did not significantly change. Interestingly, PDL1 expression was upregulated in anti-PD1 treated mice compared to immunoglobulin controls (p = 0.037). Mouse echocardiography revealed significantly reduced LV ejection fraction in anti-PD1 treated mice subjected to I/R injury.

Conclusions: The results indicate a distinct role of PD1/PDL1 signalling in preserving myocardial integrity upon I/R injury. Anti-PD1 treated mice showed increased apoptosis and an increased infiltration of CD8+ T cells with subsequent worsening of left ventricular function following I/R injury. Future studies are now needed to determine the impact of PD1/PDL1 signalling on long-term outcome after I/R injury and whether a PD1-agonizing therapy serves to promote a favourable reponse to I/R injury aiming to improve immune-modulated myocardial healing and repair.