Abstract Background The Programmed cell death protein 1 (PD1) immune checkpoint is densely expressed on cardiac endothelial cells. The increasing clinical application of immune checkpoint inhibitor therapy targeting PD1 for the treatment of advanced malignancies has revealed profound cardiovascular side effects that have recently paralleled in a preclinical model. However, the effects of PD1 deficiency during baseline conditions and myocardial injury are so far unknown. Purpose This study aims to assess the impact of PD1 deficiency on myocardia immunity, and to evaluate the relevance of PD1 signalling in cardiac disease. Methods C57BL/6J wild-type mice and C57BL/6J Pdcd1−/− mice were purchased and bred at the animal facility. In-vivo ischaemia/reperfusion (I/R) injury was applied to assess the response in cardiac injury. In brief, mice were anesthetised followed by lateral thoracotomy and ligation of the left coronary artery for 45 min. For flow cytometry, hearts were removed and subjected to an enzymatic digestion. Single cell solutions were stained with different antibody panels to assess cardiac immune cells and the expression of programmed cell death protein 1 ligand 1 (PDL1). Western blot was conducted after homogenization of snap-frozen heart tissue using specific primary and corresponding secondary antibodies. Immunofluorescence and conventional haematoxylin and eosin stain from 4 μm sections were used to visualize the distribution of PDL1 in cardiac tissue. Results While no overt cardiac phenotype was observed in Pdcd1−/− mice, a profound upregulation of pro-inflammatory cytokines was determined during baseline conditions, including interleukin (IL) 1α IL4, and extracellular-signal regulated kinase (ERK) 1/2. NADPH oxidase 1 (NOX1) which is involved in production of reactive oxygen species and endothelial injury response was downregulated in Pdcd1−/− mice. Following I/R injury, a significant decline in endothelial PDL1 expression was observed, which was attributed to changes in the area at risk, as shown by immunofluorescence staining. The infarct size following in-vivo I/R injury was not altered upon PD1-deficiency as determined by TTC staining. However, flow cytometry determined increased cell numbers of distinct leukocyte subsets during reperfusion-related inflammation. Conclusion Deficiency of PD1/PDL1 signalling shows distinct inflammatory changes in cardiac tissue at baseline and I/R injury. The results indicate that relevant PD1-related detrimental effects are not limited to complications from cancer therapy but can be expected in various forms of cardiovascular disease, hence requiring further investigations. Funding Acknowledgement Type of funding sources: Public hospital(s). Main funding source(s): Department of Cardiology and Vascular Medicine, West German Heart and Vascular Center, University Hospital Essen, Germany

The programmed cell death protein 1 (PD1) immune checkpoint prevents inflammatory tissue damage by inhibiting immune reactions. Understanding the relevance of cardiac PD1 signaling may provide new insights into the inflammatory events under baseline conditions and disease. Here, we demonstrate distinct immunological changes upon PD1 deficiency in healthy hearts and during reperfused acute myocardial infarction (repAMI). In PD1-deficient mice, upregulated inflammatory cytokines were identified under baseline conditions including cardiac interleukins and extracellular signal-related kinase 1/2 (ERK1/2). A murine in vivo repAMI model to determine inflammatory changes in the early phase showed downregulation of the ligand PDL1, paralleled by an endothelial injury, indicated by loss of the CD31 signal. Immunofluorescence imaging showed decreased PDL1 expression specifically in the infarct zone, highlighting an involvement in PDL1 in myocardial injury response. Pharmacological depletion of PD1 prior to repAMI did not alter the area of infarction but led to increased numbers of CD8(+) T cells in treated mice. We conclude that PD1/PDL1 signaling plays a significant role in healthy hearts and repAMI, emphasizing the relevance of adaptive immunity during myocardial injury. The findings highlight the risk for adverse outcomes from acute myocardial infarction in the growing group of patients receiving immune checkpoint inhibitor therapy.

AIMS:Cardiac immune-related adverse events (irAEs) from immune checkpoint inhibition (ICI) targeting programmed death 1 (PD1) are of growing concern. Once cardiac irAEs become clinically manifest, fatality rates are high. Cardio-oncology aims to prevent detrimental effects before manifestation of severe complications by targeting early pathological changes. We therefore aimed to investigate early consequences of PD1 inhibition for cardiac integrity to prevent the development of overt cardiac disease.METHODS AND RESULTS:We investigated cardiac-specific consequences from anti-PD1 therapy in a combined biochemical and in vivo phenotyping approach. Mouse hearts showed broad expression of the ligand PDL1 on cardiac endothelial cells as a main mediator of immune-crosstalk. Using a novel melanoma mouse model, we assessed that anti-PD1 therapy promoted myocardial infiltration with CD4+ and CD8+ T cells, the latter being markedly activated. Left ventricular (LV) function was impaired during pharmacological stress, as shown by pressure-volume catheterization. This was associated with a dysregulated myocardial metabolism, including the proteome and the lipidome. Analogous to the experimental approach, in patients with metastatic melanoma (n = 7) receiving anti-PD1 therapy, LV function in response to stress was impaired under therapy. Finally, we identified that blockade of tumour necrosis factor alpha (TNFα) preserved LV function without attenuating the anti-cancer efficacy of anti-PD1 therapy.CONCLUSIONS:Anti-PD1 therapy induces a disruption of cardiac immune homeostasis leading to early impairment of myocardial functional integrity, with potential prognostic effects on the growing number of treated patients. Blockade of TNFα may serve as an approach to prevent the manifestation of ICI-related cardiotoxicity.