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

Human CD4+ cytotoxic T lymphocytes that play a vital role in antiviral immunity are highly heterogeneous. Committing to cytotoxicity CD4+ cytotoxic T lymphocytes (CD4-CTLs) were initially identified in patients with chronic viral infections, including dengue virus (DENV) infection, and these cells have been associated with protection in the context of severe DENV infection. Here, Patil et al. have carried out single RNA-seq and sequenced the T cell receptors (TCRs) of CD4+ T cells from human blood to identify precursors that give rise to CD4-CTLs. They report that CD4-CTLs undergo significant clonal expansion and that CD4-CTL precursor cells are characterized by high expression of interleukin-7 receptor. By defining the gene expression signature of CD4-CTLs and their precursors, their studies should facilitate improved vaccine design in the context of chronic viral infections. 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 TEMRA (effector memory T cells expressing CD45RA) subset, which is highly enriched for CD4-CTLs, compared with CD4+ T cells in the central memory (TCM) and effector memory (TEM) subsets. Simultaneous T cell antigen receptor (TCR) analysis in single cells and bulk subsets revealed that CD4-TEMRA cells show marked clonal expansion compared with TCM and TEM cells and that most of CD4-TEMRA were dengue virus (DENV)–specific in donors with previous DENV infection. The profile of CD4-TEMRA 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 TEMRA 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.