Abstract MP49: Single Cell Multiplex Immunophenotyping Using Mass Cytometry And CITE-Seq Reveals Decreases In Circulating PD-1 + CD8 + Memory T Cells With Features Of Exhaustion In Human Hypertension

Abstract

Emerging evidence from animal models has demonstrated the importance of multiple innate and adaptive immune cells in hypertension. We hypothesized that the abundance and phenotype of circulating immune cell subsets are altered in human hypertension. To test this, we performed high dimensional single cell profiling of human peripheral blood mononuclear cells using mass cytometry. Unsupervised computational analysis revealed a 40% decrease in CD8 + memory T cells in hypertensive individuals. Using Phenograph to identify subsets of these cells revealed a selective 60% decrease in PD-1 + CD8 + memory T cells in hypertension. This observation was confirmed in a validation cohort using flow cytometry in which PD-1 + CD8 + memory T cells were significantly decreased 44% in hypertensive compared to control individuals. To determine the phenotype of these PD-1 + CD8 + memory T cells, we performed Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) on four control and four hypertensive individuals. Using antibodies to identify PD-1 + and PD-1 - CD8 + memory T cells, gene set enrichment analysis of the coordinate single cell transcriptomic data revealed that PD-1 + cells exhibit over-representation of features of both immunologically active effector T cells and hypofunctional exhausted T cells. Thus, clustering analysis of PD-1 + CD8 + memory T cells was performed which demonstrated 4 distinct subclusters. One of these subclusters was decreased in hypertension and exhibited selective expression of multiple inhibitory receptors characteristic of exhausted T cells. At the protein level, this subcluster was marked by expression of the inhibitory receptor LAG3 and low levels of CD57. Combining these markers to identify PD-1 + LAG3 + CD57 - CD8 + memory T cells permitted identification of exhausted cells which demonstrated a significant 35% decrease in hypertensive compared to control individuals using flow cytometry. Taken together, these results demonstrate novel and reproducible decreases in circulating PD-1 + CD8 + memory T cells with features of exhaustion in human hypertension. These findings provide new insights into the pathogenesis of human hypertension including loss and/or re-invigoration of exhausted T cells.