Single-cell RNA sequencing identifies distinct transcriptomic signatures between PMA/Ionomycin and aCD3/aCD28 agonistic antibodies stimulation on primary human T cells.

Abstract

Abstract Immunologists have activated T cells in-vitro using various stimulation methods including PMA/Ionomycin and anti-CD3/CD28 agonistic antibodies. PMA stimulates protein kinase C, which is followed by activation of NF-κB, and Ionomycin translocates to the plasma membrane and increases intracellular calcium levels, resulting in activation of NFAT. On the other hand, anti-CD3/CD28 agonistic antibodies can activate T cells through ZAP-70, which phosphorylate LAT and SLP-76 to activate T cells. Despite the two different in vitro T cell activation methods that have been utilized for decades, the differential effects on primary human T cells following chemical-based or antibody-based activation have not been comprehensively described. As single-cell RNA sequencing (scRNA-seq) technologies enable the dissection of gene expression at single-cell resolution unbiasedly, we decided to compare the transcriptome profiles of the non-physiological and physiologic activation methods on four independent human PBMC-derived T cells. Remarkable differences in expression of cytokine and their receptor’s transcriptomes were identified. Also we identify activated CD4 T cell subsets (CD55+) enriched specifically by PMA/Ionomycin activation. We found that expression of CD40L gene was elevated on PMA/Ionomycin-treated CD4 T cells while expression of OX40 gene was elevated on aCD3/aCD28 agonistic antibody-treated CD4 T cells. We believe this human T cell transcriptome atlas derived from two different activation methods will enhance our understanding of the in vitro T cell activation assays.