✍🏼 Margaret L. Axelrod, Wouter C. Meijers, Elles M. Screever, Juan Qin, Mary Grace Carroll, Xiaopeng Sun, Elie Tannous, Yueli Zhang, Ayaka Sugiura, Brandie C. Taylor, Ann Hanna, Shaoyi Zhang, Kaushik Amancherla, Warren Tai, Jordan J. Wright, Spencer C. Wei, Susan R. Opalenik, Abigail L. Toren, Jeffrey C. Rathmell, P. Brent Ferrell, Elizabeth J. Phillips, Simon Mallal, Douglas B. Johnson, James P. Allison, Justin M. Balko

🏠 Department of Medicine, Vanderbilt University Medical Center, Nashville, TN

📑 nature (2022)

Abstract

Immune-related adverse events, particularly severe toxicities such as myocarditis, are major challenges to the utility of immune checkpoint inhibitors (ICIs) in anticancer therapy¹. The pathogenesis of ICI-associated myocarditis (ICI-MC) is poorly understood. Pdcd1^–/–Ctla4^+/– mice recapitulate clinicopathological features of ICI-MC, including myocardial T cell infiltration². Here, using single-cell RNA and T cell receptor (TCR) sequencing of cardiac immune infiltrates from Pdcd1^–/–Ctla4^+/– mice, we identify clonal effector CD8⁺ T cells as the dominant cell population. Treatment with anti-CD8-depleting, but not anti-CD4-depleting, antibodies improved the survival of Pdcd1^–/–Ctla4^+/– mice. Adoptive transfer of immune cells from mice with myocarditis induced fatal myocarditis in recipients, which required CD8⁺ T cells. The cardiac-specific protein α-myosin, which is absent from the thymus^3,4, was identified as the cognate antigen source for three major histocompatibility complex class I-restricted TCRs derived from mice with fulminant myocarditis. Peripheral blood T cells from three patients with ICI-MC were expanded by α-myosin peptides. Moreover, these α-myosin-expanded T cells shared TCR clonotypes with diseased heart and skeletal muscle, which indicates that α-myosin may be a clinically important autoantigen in ICI-MC. These studies underscore the crucial role for cytotoxic CD8⁺ T cells, identify a candidate autoantigen in ICI-MC and yield new insights into the pathogenesis of ICI toxicity.

How the WOLF was used in this study
Researchers used a WOLF Cell Sorter to enrich genetically modified T cells for functional and single-cell analyses. After retroviral transduction of reporter cells with T-cell receptors (TCRs) of interest, surface expression of the transduction marker Thy1.1 was confirmed by flow cytometry. The WOLF sorter was then used to select and collect Thy1.1-positive cells, achieving over 90% purity post-sort. Additionally, live CD3⁺ T cells were sorted using the WOLF with viability staining to exclude dead cells before downstream sequencing and analysis. This cell sorting step was crucial to obtain homogeneous populations of viable, TCR-expressing T cells, ensuring accurate functional assays and transcriptomic profiling in studies of immune mechanisms underlying checkpoint-inhibitor-associated myocarditis.