Research

Overview

Our group’s interest in cardio-oncology and cardio-immunology came about after we defined new cardiovascular clinical syndromes associated with immune checkpoint inhibitors (ICI) including ICI-associated myocarditis and other complications. We have utilized our expertise as myocyte and mouse biologist to generate several pre-clinical models of ICI-associated myocarditis. These models suggest a fundamental role for immune checkpoints (e.g., CTLA-4, PD-1, and LAG-3) in cardiovascular homeostasis. Using novel single-cell platforms and spatial transcriptomics, we are currently elucidating specific immune populations that cause ICI-myocarditis.

Immunofluorescence staining of immune-infiltrated mouse heart tissue following treatment with ICI.

Cardiac-Immune Tolerance

A fundamental question that our laboratory is interested in is how the heart maintains immune tolerance. Both central tolerance (e.g., deletion of self-antigen-specific T cells in the thymus) and peripheral tolerance (e.g., regulatory T cells) play critical roles. Our group, for example, has shown that T cell specific for a-myosin (Myh6), which is not expressed in the thymus are critical drivers of mouse models of myocarditis and a subset of patients with myocarditis (Nature, PMID 36385524). We are interested in understanding why certain cardiac genes (e.g., Myh6) are not expressed in the thymus and what evolutionary advantage (if any) this brings. More globally, the lack of negative selection of T cells targeting a-myosin (Myh6) may have relevance to various cardiovascular diseases, a concept we are pursuing in the laboratory. Importantly, we hope to use the latest genome-engineering technologies to modulate the immune system for therapeutic benefit.

NFAT–GFP reporter activity in mouse models, measured by flow cytometry and shown as geometric mean fluorescence intensity (gMFI), of all TCR cell lines stimulated independently with 172, 10–20 amino-acid-long SBK2, ANP, BNP or α-myosin peptides. TCRs to the left of the dotted line are derived from single-cell sequencing data

Axelrod, M.L., Meijers, W.C., Screever, E.M. et al. 2022, *Nature* (PMID 36385524)

Immune Checkpoints and the Heart

In 2016, our group identified a new clinical syndrome of myotoxicity, including myocarditis, associated with immune checkpoint inhibitors (ICI) (NEJM, PMID 27806233; Lancet, PMID 29536852; Lancet Oncol, PMID 30442497). We have utilized our expertise as myocyte and mouse biologists to generate pre-clinical models of ICI-myocarditis which recapitulate the clinical syndrome (Cancer Discovery, PMID 33257470). Using novel single-cell platforms and spatial transcriptomics, we are currently elucidating specific immunologic and antigenic drivers of ICI-myocarditis (Nature, PMID 36385524; Circulation, PMID 37746718). We have also identified novel cardiac protective mechanisms against inflammatory damage (Science Translational Medicine, PMID 36322628) that may explain how sex hormones affect susceptibility to myocarditis. We believe we have uncovered a fundamental and previously unappreciated role for immune checkpoints (e.g., CTLA-4, PD-1, LAG-3) in the heart which may have relevance to other cardiac diseases. Current projects in the lab address and extend this concept.

Single-cell TNA sequencing data of sorted CD45+ immune cells from hearts of control wild-type mouse and *Pdcd1-/-Ctla4+/-* mice with myocarditis.

Axelrod et al. 2022, *Nature* (PMID 36385524)

Immune-checkpoint molecules dampen T cell activation as a mechanism of peripheral tolerance.

**Munir, A.Z., Gutierrez, A., Qin, J**. et al. 2024, *Nat Rev Cancer* (PMID 38982146)

Novel Diagnostic and Therapeutics for Inflammatory Heart Disease

In 2023, we established the UCSF Myocarditis Center, which has emerged as a leading center for diagnosis and treatment of myocarditis. We have leveraged this clinical program to better understand inflammatory cardiomyopathies. Besides ICI-myocarditis, our center treats patients with other inflammatory cardiomyopathies – from arrhythmogenic genetic cardiomyopathies to vaccine-associate myocarditis. We are prospectively collecting samples including blood and heart specimen and are applying novel molecular biology and immunologic techniques to better develop diagnostic and treatment strategies for patients. We are utilizing cutting-edge techniques to identify cell-specific transcriptional programs in various types of inflammatory heart disease. There are no approved medical treatments for acute myocarditis. Using mouse models, we have shown that CTLA-4 signaling plays a causal role in the development of myocarditis. In the mouse models, abatacept (recombinant CTLA4–Ig) blocks T-cell co-stimulation by binding to CD80/CD86 ligands and significantly attenuates myocarditis in mice (Cancer Discovery, 2021, PMID 33257470). We successfully treated the first patient with ICI-myocarditis (NEJM, 2019, PMID 31189043). However, we have found that a combination of Janus kinase (JAK) inhibition and CTLA4-Ig potentiates response. In collaboration with colleagues in France, we have successfully treated a case series of patients with ICI-myocarditis with excellent results (Cancer Discovery, 2023, PMID 33257470). Current projects in the Moslehi laboratory will be utilizing genomics and genome engineering technologies to better understand cardiovascular-immune interactions and harness reprogrammed immune cells to treat cardiovascular diseases.

Kaplan–Meier survival curve of female *Ctla4*^+/− *Pdcd1*^−/− mice treated with either vehicle (n = 51) or abatacept (n = 19).

Wei SC...Moslehi et al. 2021, *Cancer Discovery* (PMID 3257470)

Lymphocytic infiltration of the myocardium in patient treated with ipilimumab and nivolumab.

Johnson et al. 2016, *NJEM* (PMID 27806233)

Gene expression of JAK and STAT family in patients with ICI myocarditis (n = 9), in comparison with ICI-treated patients without myocarditis (ICI control, n = 4).

**Salem** et al. 2023, *Cancer Discovery* (PMID 36815259)

Kinase Signaling

Kinase inhibitors used in oncology have been transformative in cancer treatment but can lead to cardiovascular sequelae. Our group has especially been interested in covalent kinase inhibitors and their cardiovascular effects. For example, ibrutinib is a Bruton tyrosine kinase (BTK) inhibitor, which has treatment for a subset of leukemias and lymphoma. Our group has shown that ibrutinib is associated with atrial fibrillation and other arrhythmia (Haematologica, PMID 28751558; Blood, PMID 28223277; JACC, PMID 31558250). Ibrutinib is a first in class covalent kinase inhibitor, binding a free cysteine near the ATP-binding site. While probably more potent and selective with respect to kinase activity, there is the possibility that non-kinase proteins are being targeted. Using cell-based and animal models, our early data suggest a novel mechanism of signaling in atrial fibrillation. By understanding this better, we hope to modulate this signaling pathway for better arrhythmia drugs.

Moslehi Laboratory