Pathogenesis of Immune checkpoint inhibitors-induced myocarditis

Project Summary Immune checkpoint inhibitors (ICIs) blocking PD-1, PD-L1, CTLA-4 and LAG-3 are groundbreaking therapies for cancer. Approximately 50% of cancer patients in the US are eligible for ICI therapy. However, ICI treatment can cause various immune-related adverse events (irAEs), which present as inflammation targeting numerous organs including the heart. ICI-associated myocarditis (ICI-myocarditis) occurs in 1-2% of patients treated with ICI therapy and is fatal in up to 50% of cases. More ICI inhibitors are in the testing phase and combining two or more ICI inhibitors has been shown to increase ICI-myocarditis incidence. Thus, there is an exceptionally high need to understand ICI myocarditis pathogenesis in order to aid the development of new targeted therapies, given the high mortality of ICI-myocarditis and its increasing incidence. We generated a clinically relevant mouse model for human ICI-myocarditis by injecting αPD-1 blocking antibody into the immunocompetent A/J mouse strain. Our mouse model showed multi-organ irAEs, with significant inflammation in the heart. Resembling ICI- myocarditis in humans, we observed cardiac troponin elevation, arrhythmia, and massive CD8+T cell infiltration in the heart. We showed that ICI-myocarditis disease was mediated by autoreactive myosin-specific (Myhc+) effector T(eff) cells. We found that Myhc+PD-1+ CD69+ T cells are present in naive mice hearts as well. We further investigated the residency of Myhc+ T cells and found through flow cytometry, intravascular labeling, qPCR, and scRNA sequencing that they upregulated tissue residency markers and resided in the heart as tissue resident memory T (TRM) cells. Upon further characterization, we found that myosin-specific TRM cells were increased in mice with previous cardiac injury, aged mice, and male mice. We observed with 2D imaging that they localize along the perimyocardium and atria. We hypothesize that ICI-myocarditis is driven by Myhc+ PD-1+ autoreactive TRM cells, which increase in number in the heart after cardiac injury, and are also dependent on biological factors such as age and sex. In AIM 1, to investigate how biological variables modulate ICI-myocarditis risk, we will induce ICI-myocarditis in mice that recovered from ischemic reperfusion (I/R) injury or modified experimental autoimmune myocarditis (mEAM). We anticipate that previous cardiac injury, older age or male sex will increase the number of PD-1+ Myhc+ TRM cells in the heart leading to increased vulnerability to ICI-myocarditis. In AIM 2, we will comprehensively characterize the 3D localization and transcriptional profile of cardiac myosin-specific autoreactive TRM cells in naïve mice, mice with previous mEAM and during ICI-myocarditis progression. We will use CODA, a high-dimensional deep learning algorithm that enables the 3D reconstruction of a heart’s cardiac conductive system, immune foci (including the TRM cells), and blood vessels at micron and single-cell resolutions. In AIM 3, we will investigate if cardiac myosin-specific autoreactive TRM cells are necessary for ICI-myocarditis development through a parabiosis surgery mouse model. Lastly, we propose autoreactive TRM cell depletion by neutralizing IL-15, which is essential for TRM maintenance, as a novel method for the prevention of ICI-myocarditis.