Single-cell transcriptional analyses reveal tissue-driven segregation of T cell function in secondary antiviral response

Virus-specific memory T cells generated during primary infection and maintained in tissues and circulation are capable of initiating a quick and robust response upon secondary exposure to viral antigens. To understand the nature of this recall response and the factors that determine antiviral T cell function, we performed single-cell transcriptome profiling of virus-specific CD8+ T cells across diverse tissue sites following stimulation with viral antigens using pooled library amplification for transcriptome expression sequencing (PLATE-seq)(Bush et al., 2017). From this analysis, we found that antigen-responsive cells primarily cluster based on tissue and not virus specificity, with the exception of CMV-specific T cells from the BM. These findings suggest tissue-regulated antiviral T cell function, with specific CMV-driven influences in the BM. Overall design: Single-cell suspensions from blood, BM, spleen, lung, and LLN from a deceased human donor were stimulated in vitro with either Flu or CMV CD8+ T cell epitopes synthesized in the form of megapools (MPs) that allow simultaneous presentation of a large number of virus-specific epitopes predicted utilizing the Immune Epitope Database and Analysis Resource (IEDB) and bioinformatic approaches. After 24 hr in vitro stimulation with MPs, antigen-responding CD8+ T cells (CD69+ IFN-?+) were individually sorted into wells of 96-well plates, followed by single-cell RNA sequencing (scRNA-seq) using pooled library amplification for transcriptome expression sequencing (PLATE-seq)(Bush et al., 2017).