CD4-CTL precursor cells in the TEMRA subset.

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CD4-CTL precursor cells in the TEMRA subset. CD4-CTL precursor cells in the TEMRA subset. (A) tSNE 2D plots of single-cell RNA-seq data show the expression of the indicated differentially expressed transcripts between the four clusters (SCDE and MAST analysis, adjusted P < 0.05 and ≥2-fold change). (B) Violin plots show the single-cell expression pattern of the indicated differentially expressed transcripts across four TEMRA clusters along with TCM. Color scale shows the log2(TPM + 1) expression values (A) and mean expression values (B). The shapes represent the distribution of cells based on their log2(TPM + 1) expression values (y axis) (B). (C) Venn diagram comparing the transcripts enriched in precursor clusters (clusters 3 and 4) versus effector clusters (clusters 1 and 2) and transcripts enriched in TCM versus TEMRA (left); transcripts enriched in precursor clusters (clusters 3 and 4) versus TCM and transcripts enriched in TEMRA versus TCM (right) (gene list obtained from SCDE and MAST analysis, Benjamini-Hochberg adjusted P < 0.05 and ≥2-fold change). Examples of overlapping genes are shown to the right. (D) Percentage of the CD4-TEMRA subset in the CD4+ T cell population across 104 samples from 89 individuals. (E) Contour plots show the surface expression of CD45RA and CCR7 (left) or CD127 (IL-7R) (right) in live and singlet-gated CD3+CD4+ T cells obtained from PBMCs (left). Contour plots show coexpression of CD45RA and CD127 in TEMRA and TCM subsets (right). The gating strategy shows CD127− and CD127high proportion. (F) Proportion of total CD4-TEMRA (left), CD127− CD4-TEMRA (middle), and CD127high CD4-TEMRA cells (right) in CD4+ T cells from 15 donors with two longitudinal samples (V1 and V2). Donor #16, marked in red color, shows marked increase in the proportion of CD4-TEMRA and CD127− CD4-TEMRA (effectors) cells from V1 to V2. (G) Contour plots show the surface expression of CD45RA and CCR7 (top) or CD127 (bottom) in live and singlet-gated CD3+CD4+ cells obtained from PBMCs of donor #16 at two time points (168 days apart) [V1 (left) and V2 (right)]. Bottom panels show the expression of CD127 in the CD4-TEMRA subset for the same donor. ns, not significant; from Student’s two-tailed paired t test. Numbers inside the contour plots show the proportion of the indicated cell type. (H) Bulk RNA-seq analysis of the IL-7Rhigh TEMRA subset (CD4-CTL precursors), IL-7R− TEMRA (CD4-CTL effectors), and TCM cells shows the row-wise normalized TPM counts of top 200 (100 up-regulated and down-regulated, based on fold change) differentially expressed transcripts obtained by pairwise comparison of IL-7R− TEMRA (CD4-CTL effectors) versus TCM from the five donors (DESeq2 analysis, Benjamini-Hochberg adjusted P < 0.05 and ≥2-fold change). The panel above the heat map identifies the cell type, donor, and visit. (I) Contour plots show the coexpression of CD244 or GPR56 with CD28 in TCM (red), IL-7Rhigh TEMRA (CD4-CTL precursors) (magenta), and IL-7R− TEMRA (CD4-CTL effectors) (blue) in singlet-gated CD3+CD4+ T lymphocytes. Veena S. Patil et al. Sci. Immunol. 2018;3:eaan8664 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works