Clonal CD8+ T cell expansion in CAVD

Background: Calcific aortic valve disease (CAVD) is a complex cardiovascular pathology, culminating in aortic stenosis, heart failure and premature mortality, with no comprehensive treatment strategy, except invasive valve replacement. While inflammation may contribute to valvular fibro-calcific pathology and T cells have been identified within the leaflets, their contribution to CAVD pathogenesis remains unclear. Methods: To elucidate the heterogenous phenotype of the immune populations present in CAVD patients, deep phenotypic screens of paired valve and peripheral blood cells were conducted via flow cytometry (n=20) and immunohistochemistry (n=10). Following identification of a significant population of memory T cells; specifically, CD8+ T cells within the valve, single cell RNA sequencing and paired single T cell receptor sequencing was conducted on an additional 4 patients on CD45+ CD3+, CD4+ or CD8+ T cells. Results: Through unsupervised clustering, six T cell clusters were identified within the patient. Tissue resident memory (TRM) T cells were detected for the first time within the valve, exhibiting a highly cytotoxic, activated, and terminally differentiated phenotype. This pan-pro-inflammatory signal was differentially identified in T cells originating from the valve, and not observed in the blood, indicative of an adaptive, local not-systemic inflammatory signature in CAVD patients. T cell receptor analysis identified hyperexpanded clones within the CD8+ T cell central memory (TCM) population, with TRM cells comprising most of the large and medium clonal expansion within the T cell population. Clonal expansion occurred within the valve more than the blood, with clonally expanded receptors remaining within their tissue of origin. Clonal interaction network analysis demonstrated the greatest proportion of clones originating from CD8+ TCM and TRM populations, exhibiting a tissue resident, cytotoxic environment within the valve. Conclusion: Taken together, the data identified T cell sub-populations within the context of CAVD and further predicted possible epitopes responsible for the clonal expansion of the valvular T cells, which may be important for propagating autoimmune driven inflammation in CAVD.