Precursors of human CD4 cytotoxic T lymphocytes identified by single-cell transcriptome analysis.

CD4 cytotoxic T lymphocytes (CD4-CTLs) have been reported to play a protective role in several viral infections. However, little is known in humans about the biology of CD4-CTL generation, their functional properties, and heterogeneity, especially in relation to other well-described CD4 memory T cell subsets. We performed single-cell RNA sequencing in more than 9000 cells to unravel CD4-CTL heterogeneity, transcriptional profile, and clonality in humans. Single-cell differential gene expression analysis revealed a spectrum of known transcripts, including several linked to cytotoxic and costimulatory function that are expressed at higher levels in the T (effector memory T cells expressing CD45RA) subset, which is highly enriched for CD4-CTLs, compared with CD4 T cells in the central memory (T) and effector memory (T) subsets. Simultaneous T cell antigen receptor (TCR) analysis in single cells and bulk subsets revealed that CD4-T cells show marked clonal expansion compared with T and T cells and that most of CD4-T were dengue virus (DENV)-specific in donors with previous DENV infection. The profile of CD4-T was highly heterogeneous across donors, with four distinct clusters identified by the single-cell analysis. We identified distinct clusters of CD4-CTL effector and precursor cells in the T subset; the precursor cells shared TCR clonotypes with CD4-CTL effectors and were distinguished by high expression of the interleukin-7 receptor. Our identification of a CD4-CTL precursor population may allow further investigation of how CD4-CTLs arise in humans and, thus, could provide insights into the mechanisms that may be used to generate durable and effective CD4-CTL immunity.