Single-cell T cell receptor sequencing of paired tissue and blood samples reveals clonal expansion of CD8+ effector T cells in patients with calcific aortic valve disease

Abstract

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 valve replacement. While T cells have been identified within the valve, their contribution to pathogenesis remains unclear. To elucidate the heterogeneous phenotype of the immune populations present within patients with CAVD, 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 a further 4 patients on CD45+ CD3+, CD4+ or CD8+ T cells. Through unsupervised clustering, 7 T cell populations were identified within the blood and 10 identified within the valve. 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 the majority of large and medium clonal expansion within the entire T cell population. Clonal interaction network analysis demonstrated the greatest proportion of clones originating from CD8+ T cell effector memory (TEM) and CD4+ naive / TCM populations and ending in the CD8+ TRM and CD8+ TCM clusters, suggesting a clonal expansion and predicted trajectory of T cells towards a tissue resident, cytotoxic environment within the valve. CDR3 epitope predictive analysis identified 7 potential epitope targets, of which GALNT4 and CR1L have previously been implicated in a cardiovascular context as mediators of inflammation. 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 inflammation in CAVD.

Altered inflammatory state and mitochondrial function identified by transcriptomics in paediatric congenital heart patients prior to surgical repair

“…Bulk transcriptomic data was deconvoluted via granulator using the single cell matrix from healthy RV dataset GSE145154 8 . Raw data was downloaded through the SRA toolkit, and Seurat object produced as previously described 13 . Seurat object data was normalized with scTRANSFORM and celltype identified as dictated by the published data.…”