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. 2020 Mar 24;117(12):6686-6696.
doi: 10.1073/pnas.1913940117. Epub 2020 Mar 11.

CD29 identifies IFN-γ-producing human CD8+ T cells with an increased cytotoxic potential

Benoît P Nicolet  1   2 , Aurélie Guislain  1   2 , Floris P J van Alphen  3 , Raquel Gomez-Eerland  4 , Ton N M Schumacher  4 , Maartje van den Biggelaar  3   5 , Monika C Wolkers  6   2
Affiliations

CD29 identifies IFN-γ-producing human CD8+ T cells with an increased cytotoxic potential

Benoît P Nicolet et al. Proc Natl Acad Sci U S A. .

Abstract

Cytotoxic CD8+ T cells can effectively kill target cells by producing cytokines, chemokines, and granzymes. Expression of these effector molecules is however highly divergent, and tools that identify and preselect CD8+ T cells with a cytotoxic expression profile are lacking. Human CD8+ T cells can be divided into IFN-γ- and IL-2-producing cells. Unbiased transcriptomics and proteomics analysis on cytokine-producing fixed CD8+ T cells revealed that IL-2+ cells produce helper cytokines, and that IFN-γ+ cells produce cytotoxic molecules. IFN-γ+ T cells expressed the surface marker CD29 already prior to stimulation. CD29 also marked T cells with cytotoxic gene expression from different tissues in single-cell RNA-sequencing data. Notably, CD29+ T cells maintained the cytotoxic phenotype during cell culture, suggesting a stable phenotype. Preselecting CD29-expressing MART1 TCR-engineered T cells potentiated the killing of target cells. We therefore propose that CD29 expression can help evaluate and select for potent therapeutic T cell products.

Keywords: CD29; IFN-γ; IL-2; T cells; cytotoxicity.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
RNA-seq analysis on CD8+ T cells after intracellular cytokine staining. (A) Scheme to isolate RNA and protein from cytokine-producing, fixed T cells. Primary human CD8+ T cells were activated for 48 h with α-CD3/α-CD28, and rested for 4 d in the presence of 10 ng/mL rhIL-15. T cells were reactivated for 4 h with 10 ng/mL PMA and 1 µM ionomycin. Intracellular cytokine staining was performed in RNA-protecting buffers. Cells were FACS-sorted under RNA-protecting conditions. Total RNA and protein was recovered for RNA-seq and mass spectrometry analysis (Fig. 2) by reversing formaldehyde cross-linking. IL2: IL-2 single producers; IFNG: IFN-γ single producers; DP: double positive: IL-2 and IFN-γ coproducers; DN: double negative for IL-2 and IFN-γ production. (B–G) RNA-seq analysis of CD8+ T cells with a differential IL-2 and/or IFN-γ production profile. (B) Gene biotypes and (C) samplewise Pearson correlation coefficient of genes (n = 2,349) that are differentially expressed between all four T cell populations. (D) Heatmap of differentially expressed protein-coding genes (n = 1,984), numbers indicate k-means clusters (k = 5). (E) Heatmap of differentially expressed TFs with >100 DESeq2 normalized counts, (F) lncRNAs, and (G) RBPs with >10 DESeq2 normalized counts. (E–G) Expression levels of biological replicates were averaged (n = 3 per population). Color scale of heatmaps represents Z-score.
Fig. 2.
Fig. 2.
Mass spectrometry analysis on cytokine producing, fixed CD8+ T cells. (A) Number of proteins identified by MS analysis of FACS-sorted T cell populations per sample (bars) and of all 12 samples combined after filtering (red line; n = 3,833). (B) Samplewise Pearson correlation coefficient of differentially expressed proteins (n = 81). (C) Heatmap of differentially expressed proteins, numbers indicate k-means clusters (k = 9). Proteins showing differential expression in both mRNA and protein are indicated in blue. Color scale represents log2 centered intensity. (D) Venn diagram of differentially expressed proteins and genes (Fig. 1).
Fig. 3.
Fig. 3.
IL2 and IFNG producers express unique sets of secreted proteins. (A and B) Heatmap of differentially expressed genes (A, n = 67) and proteins (B, n = 7) that are annotated as secreted proteins (see Materials and Methods). Expression levels of biological replicates were averaged (n = 3 per population). Gray: not detected. Color scale of heatmaps represents Z-score. (C) Validation of secreted effector molecules identified in A and B in four new individual donors. α-CD3/α-CD28 activated T cells were rested for 4 d prior to 4-h reactivation with PMA–ionomycin in the presence of monensin. Cytokine production was determined by intracellular cytokine staining. Plots depict the production of effector molecules in DN, IL2, IFNG, and DP producers. Repeated measurement ANOVA with Tukey posttest (*P < 0.05, **P < 0.01, ***P < 0.001).
Fig. 4.
Fig. 4.
CD29 and CD38 identify IFNG and IL2 producers. (A) Heatmap of differentially expressed CD molecules identified by MS analysis with LFC >1.5 (gray: not detected). Expression levels of biological replicates were averaged (n = 3 per population). Color scale represents Z-score. (B) CD29 (ITGB1; Left) and CD38 (Right) expression of DN, IL2, IFNG, and DP producers in α-CD3/α-CD28 activated T cells that were rested for 4 d and then reactivated for 4 h with PMA–ionomycin (n = 6 donors). (C) Representative CD29 and CD38 expression profile of nonactivated CD8+ T cells. (D) Representative IL-2 and IFN-γ production after 4-h reactivation with PMA–ionomycin of CD8+ T cells (total), and of CD29+ and CD38+ T cells, based on gating shown in C. Resting CD8+ T cells (Left) served as control. (Right) Compiled data of 12 donors. (E and F) CD8+ T cells were FACS-sorted at day 7 of culture based on CD29 and CD38 expression. Cells were reactivated for 2 d with α-CD3/α-CD28 and rested for another 14 d with rhIL-15 (day 23). (E) Representative CD29 and CD38 expression. (F) Representative IFN-γ and IL-2 production and compiled data of four donors by CD29+ and CD38+ after 4-h reactivation with PMA–ionomycin. Paired ratio t test (*P < 0.05; ****P < 0.0001, ns: not significant).
Fig. 5.
Fig. 5.
CD29 marks ex vivo nonnaïve CD8+ T cells with a cytotoxic signature. (A) Representative expression profile of CD29 and CD38 of peripheral blood-derived CD8+ T cells ex vivo. (B) Percentage of CD29+CD8+ T cells in naïve (TN: CD27+CD45RA+), memory (TMem: CD27+CD45RA−), effector memory (TEM: CD27−CD45RA−), and effector (TEff: CD27−CD45RA+, n = 8 donors) peripheral blood-derived CD8+ T cells. ***P < 0.001, ****P < 0.0001. (C, Left) Representative dot plot of CD45RA and CD27 expression in CD8+ T cells that were activated for 4 h with PMA–ionomycin. (C, Right) IFN-γ production and CD29 expression of TN. Representative of five donors. (D) Representative expression of CD49d by CD29+ (black) and CD29− (light gray) peripheral blood-derived CD8+ TN (n = 4 donors). (E and F) Peripheral blood-derived CD8+ T cells were activated for 6 h with MHC-I restricted peptide pools for CMV (E) or EBV (F). (Left) Representative dot plots of IFN-γ and CD29 expression and (Right) compilation of eight donors. Ratio t test. Numbers depict P value. (G) Single-cell gene expression analysis from ref. of peripheral blood-derived CD8+ T cells excluding naïve T cells (for details see SI Appendix, Fig. S4 E–H). (Left) tSNE projection of ITGB1 (CD29) expression in nonnaïve CD8+ T cells [n = 1,023, log2(TPM)]. (Right) Volcano plot of differentially expressed genes in ITGB1high and ITGB1low CD8+ T cells. Cutoff for ITGB1 gene expression was determined as depicted in SI Appendix, Fig. S4I. (H) Venn diagram of the core signature of CD29+CD8+ T cells from blood and lung (the top 50 up-regulated genes in each tissue). Differential expression with absolute LFC >0.5 and p-adjusted <0.05.
Fig. 6.
Fig. 6.
CD29 gene signature is associated with better survival, and CD29+T cells are superior in killing tumor cells. (A and B) Kaplan–Meier plots of overall survival of (A) all patients suffering from SKCM or (B) of patients with high CD8+ T cell infiltrate estimates. Patients were stratified for a high or low CD29+ gene signature as defined in Fig. 5H (see Materials and Methods). Numbers indicate the numbers of patients at risk at each time point. The indicated P value was calculated using a log-rank test. (C) CD29+ or CD38+ MART1 TCR-engineered FACS-sorted CD8+ T cells were cultured for 6 h with MART1+ or MART1− tumor cells at an effector:target (E:T) ratio of 3:1. Representative dot plot of IFN-γ and IL-2 production as measured by ICCS. (D and E) MART1-TCR-expressing CD8+ T cells (total CD8+ T cells, or FACS-sorted CD29+ and CD38+) were cocultured for 20 h with CFSE-labeled MART1+ (Upper) or MART1− (Lower) tumor cells at an E:T ratio of 3:1. Dead tumor cells were determined by near-IR live-dead marker. (D) Representative dot plot, and (E) compiled data of six donors. Repeated measurement ANOVA with Tukey posttest. Numbers indicate P value. (F) Correlation of tumor killing of total CD8+ MART1-TCR–expressing T cells with the percentage of CD29+CD8+ T cells present in the T cell product (n = 34, linear regression).
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