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_EMRA (effector memory T cells expressing CD45RA) subset, which is highly enriched for CD4-CTLs, compared with CD4⁺ T cells in the central memory (T_CM) and effector memory (T_EM) subsets. Simultaneous T cell antigen receptor (TCR) analysis in single cells and bulk subsets revealed that CD4-T_EMRA cells show marked clonal expansion compared with T_CM and T_EM cells and that most of CD4-T_EMRA were dengue virus (DENV)-specific in donors with previous DENV infection. The profile of CD4-T_EMRA 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_EMRA 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.