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

Abstract

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.

An Atlas of Immune Cell Exhaustion in HIV-Infected Individuals Revealed by Single-Cell Transcriptomics

“…Importantly, the absolute CD4 + T cell counts in the clinical blood samples and the numbers estimated from the scRNA-seq analysis showed a strong correlation (R 2 =0.8963, Figure 1F), indicating that the scRNA-seq datasets accurately reflect the cell clusters present in the original blood samples. Figure S2A), which is consistent with a recently described CD4-Tpm cytotoxic cell cluster 35…”

CReSCENT: CanceR Single Cell ExpressioN Toolkit

“…This is reflected by both the intensity of the cluster's red colour in Figure 2B and the dominant distribution of the red violin in Figure 2C when CMC1 is selected from the DEGs menu. Other DEGs within cluster 3 include CST7, NKG7, KLDR1, GNLY, and GZMH, several of which have been previously associated with cytotoxic T cell function (27). This coincides with the analysis from the original authors of this dataset (26), who identified cells belonging to cluster 3 enriched for CD8+ effector memory CD45RA (CD8 TEMRA ) cells ( Figure 2D) and expression of CCL5 ( Figure 2E), which is a marker of CD8+ TEM cells (26,28).…”

Comprehensive analysis of antiviral adaptive immunity formation and reactivation down to single-cell level

“…Very similar clusters of genes were found in single-cell RNAseq analysis of CD4-cytotoxic lymphocytes EMRA cells [33]. The expression pattern of granzymes and killer-like receptors in our clusters suggests that cells in cluster 2 may be the precursors of cells in cluster 1.…”