Identification of human exTreg cells as CD16+CD56+ cytotoxic CD4+ T cells

Abstract

In atherosclerosis, some regulatory T (T_reg) cells become exT_reg cells. We crossed inducible T_reg and exT_reg cell lineage-tracker mice (FoxP3^{eGFP-Cre-ERT2}ROSA26^{CAG-fl-stop-fl-tdTomato}) to atherosclerosis-prone Apoe^-/- mice, sorted T_reg cells and exT_reg cells and determined their transcriptomes by bulk RNA sequencing (RNA-seq). Genes that were differentially expressed between mouse T_reg cells and exT_reg cells and filtered for their presence in a human single-cell RNA-sequencing (scRNA-seq) panel identified exT_reg cell signature genes as CST7, NKG7, GZMA, PRF1, TBX21 and CCL4. Projecting these genes onto the human scRNA-seq with CITE-seq data identified human exT_reg cells as CD3⁺CD4⁺CD16⁺CD56⁺, which was validated by flow cytometry. Bulk RNA-seq of sorted human exT_reg cells identified them as inflammatory and cytotoxic CD4⁺T cells that were significantly distinct from both natural killer and T_reg cells. DNA sequencing for T cell receptor-β showed clonal expansion of T_reg cell CDR3 sequences in exT_reg cells. Cytotoxicity was functionally demonstrated in cell killing and CD107a degranulation assays, which identifies human exT_reg cells as cytotoxic CD4⁺T cells.

Extended Data Fig. 1 |. Experimental controls for lineage-tracker atherosclerotic mouse model and differentially expressed genes in mouse Treg cells vs exTregs.

(a,b) Eight week-old female Foxp3^{eGFP-Cre-ERT2} ROSA26^{fl-STOP-fl-tdTomato} Apoe^−/− mice were injected with Tamoxifen twice for 5 days each, at week 1 and 6, then fed Western diet (WD) for 12 weeks. a) gating strategy, b) representative plots and quantification of exTreg and Treg cells among CD4⁺T cells (black circles) in lymph nodes (LNs) and spleen, harvested after 12 weeks of WD from 3 independent mice. Non-T cells (orange open squares) and CD4⁻T cells (green open circles) are negative controls. Frequencies of exTregs and Treg cells among the parent subsets were plotted as mean ± SEM. Statistical comparisons were done using 2-way ANOVA with Dunnett’s multiple comparison test. **** p < 0.0001. c) Volcano plot representing significantly differentially expressed genes between mouse Treg cells and exTregs from 20-week old FoxP3^{eGFP-Cre-ERT2} ROSA26^{CAG-fl-stop-fl-tdTomato} Apoe^−/− mice (lymph nodes and spleen pooled). Left, up in Treg cells (blue). Right, up in exTregs (red). Horizontal dotted line is at -log₁₀ (p adjusted) = 1.3 (p_adj = 0.05). The top 60 exTreg and 60 Treg classifying genes from the SVM model are annotated. Canonical Treg genes Il2ra and Foxp3 are shown in black boxes. Statistical analyses of DE genes using two-tailed Wald test with Benjamini-Hochberg correction for multiple comparisons.

Extended Data Fig. 2 |. Expression of exTreg candidate genes in scRNAseq data and validation by qRT-PCR from sorted human cells.

(a) Feature maps showing the gene expression of the single gene markers CST7, NKG7, GZMA, PRF1, TBX21 and CCL4 in the human single-cell dataset for all CD4 T cell clusters. (b) Combinations 1–4 and 6 of exTreg candidate genes are highlighted in red on UMAP embeddings of CD4 T clusters from the scRNA-Seq. (c) UMAP embeddings of CD4 T clusters. Black outline marks cluster CD4T_7; cells that express either CD56 (left) or CD16 (right) are shown as red dots. (d) Gating strategy to identify exTreg and Treg cells in human PBMCs. Dump channel: CD14, CD19. (e) Gene expression analysis of CST7, NKG7, GZMA, PRF1, TBX21 and CCL4 in sorted human Treg cells (blue circles) and exTregs (red circles) by qRT-PCR. Gene-specific Ct values were normalized (ΔCt) based on actin (ACTB). Relative expression was calculated by the 1/ΔCt method. n = 7. 33.33% male, 66.67% female donors; age: 21–54 yrs. Data shown as mean ± SEM. Each dot represents a biological replicate from an independent donor. Statistical comparisons by two-tailed Mann Whitney U test. **p = 0.0012,***p = 0.0006.

Extended Data Fig. 3 |. Human bulk RNAseq.

(a) Gating strategy used to sort human exTregs and Treg cells to perform bulk RNA-seq. (b) gene set enrichment analysis (GSEA) of bulk RNA-seq transcriptomes of sorted human exTreg cells against CD4T_7 (left) and all other clusters (right). Normalized enrichment score (NES) and FDR q values are indicated. (c) Significantly (adjusted p < 0.05) enriched pathways in human exTreg cells, based on genes expressed at significantly higher levels in human exTreg than in Treg cells. Analysis by Bioplanet2019 from the EnrichR suite. Dotted line indicates adjusted p = 0.05 (-log₁₀ padj=1.3). Statistical comparisons with two-tailed Fisher’s exact test and Benjamini- Hochberg adjustment of p-values. (d) Gating strategy to identify exTregs and NK cells in human PBMCs. Dump channel: CD14, CD19.

Extended Data Fig. 4 |. Mouse bulk RNAseq.

(a) Comparative gene signature analysis between mouse exTregs and Treg cells. Genes were filtered for significant differential expression in mouse and human dataset. Gene expression shown here is from FoxP3^{eGFP-Cre-ERT2} ROSA26^{CAG-fl-stop-fl-tdTomato} Apoe^−/− mice. Low-expressed genes (<7 raw reads in all samples) in our dataset were filtered out. Technical replicates were averaged, biological replicates shown as columns. Analysis of differentially expressed (DE) genes was done using DESeq2. Curated list of significant DE (log₂FC ± 1, adjusted p < 0.05) genes are shown on normalized heatmaps, scaled by row (z scores). (b) Gene set enrichment analysis (GSEA) of mouse exTreg genes from bulk RNA-seq transcriptomes against human exTreg (left) and Treg cells (right) from the human bulk RNA-seq data set. Mouse orthologs of human genes, filtered for those present in the human scRNA-Seq targeted gene panel, were used to calculate enrichment for mouse bulk RNA-seq dataset. (c) Comparative gene signature analysis between mouse exTreg and NK cells. An external dataset was used for mouse NK cells (3 samples): GSE122597, GSE116177, and GSE52043. EdgeR was used to normalize the counts by applying the trimmed mean of M-values (TMM) method and counts per million (CPM) conversion. All other data processing and filtering steps were same as in a. Curated list of significant DE (log₂FC ± 1, adjusted p < 0.05) genes are shown on normalized heatmaps, scaled by row (z scores). Statistical analyses of DE genes (a,c) using two-tailed Wald test with Benjamini-Hochberg correction for p-value adjustment. All data from independent biological replicates.

Extended Data Fig. 5 |. Assessment of proliferation in mouse Treg cells and exTregs.

(a) Eight week-old female Foxp3^{eGFP-Cre-ERT2} ROSA26^{fl-STOP-fl-tdTomato} Apoe^−/− mice were injected with Tamoxifen twice for 5 days each, at week 1 and 6, then fed Western diet (WD) for 12 weeks. BrDU (0.8 mg/mL) was incorporated in the drinking water for the last 9 days of WD (n = 6). (b) Gating scheme for CD4⁺T cells. (c) Ki67 FMO control. (d) Representative plots and quantification of proliferating Treg cells (blue circles, %Ki67⁺BrDU⁺CD4⁺Foxp3⁺RFP⁺) and exTregs (red circles, %Ki67⁺BrDU⁺CD4⁺Foxp3⁻RFP⁺) in the spleen (n = 6), as identified by anti-BrDU and anti-Ki-67 Abs. Data shown as mean ± SEM. Each animal is an independent biological replicate. Gates were set by FMO for Ki67 and by no BrdU controls for BrdU. Background from “No BrDU” control was subtracted for normalization. The percentage of proliferating cells was compared by two-tailed Mann-Whitney U test, **p = 0.0087.

Extended Data Fig. 6 |. Treg marker expression on human exTregs.

Representative contour FACS plots showing the expressions of PD-1, GITR, LAG3 and TIGIT in exTreg cells (left). Corresponding FMO controls were used to set the gates. Right, contour plots showing the expression of these markers in all CD4⁺T cells and in Treg cells from the same donor.

Extended Data Fig. 7 |. Cytotoxic and T cell activation marker expression on stimulated human exTreg vs NK cells.

Contour plots show intracellular expression of CD40L (X-axis) and Perforin (Y-axis) in exTregs and NK cells from unstimulated and PMA+ionomycin stimulated PBMCs. Data from three independent donors. 33.33% male, 66.67% female donors, age: 25–43 yrs.

Extended Data Fig. 8 |. Gating strategy and representative plots.

(a) Gating strategy used to analyze granzyme B, perforin and TNF in Treg cells and exTregs. (b) Contour plots show surface expression of chemokine receptors CCR5, CXCR2, CXCR3, CXCR4 and CX3CR1 on exTreg cells (left) and their corresponding expression in all CD4⁺T cells (right). Individual FMO controls were used to set the gate for expression of each receptor.

Fig. 1 |. Deep transcriptomes from mouse exT_reg cells and T_reg cells identify differentially expressed candidate genes.

a, Frequency of mouse exT_reg cells (red) and T_reg cells (blue) among all CD4⁺T cells in spleen and LNs of FoxP3^{eGFP-Cre-ERT2}ROSA26^{CAG-fl-stop-fl-tdTomato} Apoe^−/− mice at 4 (T_reg n = 7; exT_reg n = 5), 8 (T_reg n = 4; exT_reg n = 4), 12 (T_reg n = 5; exT_reg n = 5) and 20 (T_reg n = 6; exT_reg n = 5) weeks after tamoxifen injection. All mice were on regular CD. b, Median fluorescence intensity (MFI) of CD25 expression on exT_reg cells (red circles), T_reg cells (blue circles) and conventional CD4⁺T cells (T_con, black circles) in spleen (n = 4) and LNs (n = 6) from 16-week-old FoxP3^{eGFP-Cre-ERT2}ROSA26^{CAG-fl-stop-fl-tdTomato} Apoe^−/− mice on CD. Results (a and b) are represented as the mean ± s.e.m. Spleen (b) *P = 0.0286; LN (b) **P = 0.0022; two-tailed Mann–Whitney U test. c, PCA of bulk RNA-seq data from sorted mouse exT_reg cells and T_reg cells from spleen and LNs of 20-week-old FoxP3^{eGFP-Cre-ERT2}ROSA26^{CAG-fl-stop-fl-tdTomato} Apoe^−/− mice on CD. Results (a–c) are from independent biological replicates. d, Volcano plot of differentially expressed mouse exT_reg and T_reg cell-classifying genes, identified by the SVM trained on mouse transcripts with human orthologs and filtered for those present in the human scRNA-seq targeted gene panel. y and x axes capped at 20 (P = 10⁻²⁰) and ±4 (log₂FC), respectively. Horizontal line at −log₁₀ (P adjusted) = 1.3 (same as P_adj = 0.05). Statistical analyses were performed using a two-tailed Wald test with Benjamini–Hochberg P-value adjustment. e, Mouse aortas with carotid artery branches from FoxP3^{eGFP-Cre-ERT2}ROSA26^{CAG-fl-stop-fl-tdTomato} Apoe^−/− mice were fixed and imaged using a Leica SP8 multiphoton microscope. GFP⁺ T_reg cells (pseudocolored green) and tdTomato⁺ exT_reg cells (pseudocolored pink) in the adventitia (top). Bottom image is a zoomed-in view of the white box. Blue-green indicates a second harmonic generation microscopy analysis of extracellular matrix. Scale bar, 100 μm. Data are representative of four independent experiments.

Fig. 2 |. Mouse exT_reg cell classifier genes identify human exT_reg cell candidate genes and surface markers in a human scRNA-seq and CITE-seq dataset of CD4⁺T cells.

a, Differentially expressed mouse exT_reg classifier genes were examined among all CD4⁺ T cell clusters from a published single-cell human dataset from 61 men and women (aged 40–80 years). Statistical significance for the enrichment of each gene in one cluster versus all others was assessed. Average log₂ fold change (log₂FC, dot color) and positive cell proportions (pct.1, dot size) for significantly (P < 0.05) enriched genes are shown. Six highly expressed exT_reg genes (CST7, NKG7, GZMA, PRF1, TBX21 and CCL4) enriched in human cluster CD4T_7 (red boxes). b, Feature plot showing expression of FOXP3 gene (blue dots) in the human CD4⁺ T single-cell dataset. T_reg cell cluster CD4T_17, previously identified as T_reg cells, highlighted. c, Frequencies of cells that expressed each of the exT_reg cell signature genes in CD4T_7 (red circles) and CD4T_17 (T_reg cells, blue circles). n = 61. d, Cells expressing optimal exT_reg cell candidate gene combination (CST7 + NKG7 + GZMA + PRF1 + TBX21) are highlighted in red on UMAP embeddings of CD4⁺ T clusters from the scRNA-seq data. All other cells are in gray. e, Differentially expressed surface markers (CITE-seq antibodies) on cells expressing candidate gene combinations. Average log₂FC (dot color) and log₂(pct.1/pct.₂) × avg_log₂FC (dot size) for significant (P < 0.05) differentially expressed protein markers on candidate cells versus CD4T_17 (T_reg cells) are shown. Enriched exT_reg markers CD127, CD16 and CD56 are marked with red boxes. The second-to-last combination is candidate 5 (CST7 + NKG7 + GZMA + PRF1 + TBX21). f, UMAP embeddings of CD4⁺ T clusters. Black outline marks cluster CD4T_7; cells that coexpressed CD16 and CD56 are shown as red dots. g, Representative plots from flow cytometry (FACS) showing CD16⁺CD56⁺CD4⁺T cells (exT_reg cells, red) and CD25⁺CD127^loCD4⁺T cells (T_reg cells, blue) with FMO controls. h, MFI of CD16 (n = 6), CD56 (n = 6), CD127 (n = 3) and FOXP3 (n = 6) expression in exT_reg cells (red circles) and T_reg cells (blue circles). 20% male, 80% female donors; ages 23–64 years. Results (c and h) are represented as the mean ± s.e.m. Each dot (c and h) represents a biological replicate from an independent human donor. Statistical comparisons using a two-tailed Mann–Whitney U test (c and h) and a two-tailed Wilcoxon’s rank-sum test with Benjamini–Hochberg correction for multiple comparisons (a and e). In c, ****P < 0.000000000000001 for CST7, NKG7, GZMA, PRF1 and ****P = 3.86 × 10⁻¹³ for TBX21. In h, *P = 0.0121, **P = 0.0022.

Fig. 3 |. Deep transcriptomes from sorted human CD3⁺CD4⁺CD16⁺CD56⁺ exT_reg cells contrasted with T_reg cells and NK cells.

a, PCA plot of bulk RNA-seq data from sorted human exT_reg (CD3⁺CD4⁺CD16⁺CD56⁺) and T_reg (CD3⁺CD4⁺CD25⁺CD127^lo) cells. n = 7. Donor details for human bulk RNA-seq in Supplementary Table 13. b,c, Comparative gene signature analysis between human exT_reg cells and T_reg cells (b) or NK (c) cells. Genes were filtered for significant differential expression, consistent in both mouse and human datasets (only human shown here). An external dataset was used for human NK cells: GSE133383 (samples GSM3907331, GSM3907341 and GSM3907351). Lowly expressed genes (<7 raw reads in all samples) in our dataset were filtered out. EdgeR was used to normalize the counts by applying the trimmed mean of M-values method and counts-per-million conversion. Analysis of DEGs was done using DESeq2. Curated lists of significant DEGs (log₂FC ± 1, adjusted P < 0.05) genes are shown on normalized heat maps, scaled by row (z scores). d, Representative FACS plots showing exT_reg cells as CD3⁺CD4⁺CD16⁺CD56⁺ T cells and NK cells as CD3⁻CD4⁻CD16⁺CD56⁺ non-T cells in hPBMCs. e, Histograms showing the fluorescence intensities of conjugated antibodies against TCRαβ, CCR7, CD127 and CD27 on NK cells (gray) and exT_reg cells (red). The scaled y axis was normalized to mode. f, MFI values of the specified markers (n = 12 for each) in NK cells (gray circles) and exT_reg cells (red circles) were plotted as the mean ± s.e.m. 40% male and 60% female donors, aged 20–69 years. Each dot represents a biological replicate from an independent human donor. Statistical comparisons were done using a two-tailed Wald test with Benjamini–Hochberg correction for multiple testing (b and c) and a two-tailed Mann–Whitney U test (f). ****P = 7.396 × 10⁻⁷ in f.

Fig. 4 |. Oligoclonal human exT_reg cells are clonally expanded from proliferating T_reg cells.

a, Productive Simpson’s clonality of TCRβ sequences from sorted naïve T cells (black circles), T_reg cells (blue circles) and exT_reg cells (red circles). n = 4. Donor details for human TCR-seq in Supplementary Table 13. b, Frequency of rearrangements shared between exT_reg cells and naïve or between exT_reg cells and T_reg cells was measured using Morisita’s index. n = 4. Results (a and b) are represented as the mean ± s.e.m. c, GLIPH2-analyzed conserved amino acid motifs in TCRβ sequences. Groups with exT_reg TCRs were filtered for statistically significant expansion score (P < 0.05). 178 of 345 expanded exT_reg GLIPH2 groups were shared by T_reg cells. Relative abundance of T_reg and exT_reg TCRs in these 178 T_reg/exT_reg groups was compared and DEG patterns (log₂FC ± 1, two-sided Poisson test P < 0.05) are shown as a volcano plot. Horizontal line at −log₁₀ (P value) = 1.3 (same as P = 0.05). Vertical lines at |log₂FC| = 1. d, Violin plots showing normalized expression levels (transcripts per million) of proliferation genes MKI67, TOP2A, CCNA2, CCNB1, CCNB2, MCM2, MCM3, MCM5 and MCM6 in human bulk transcriptomes from sorted human T_reg cells (blue dots) and exT_reg cells (red dots). n = 7. 33.33% male and 66.67% female donors, aged 21–54 years. e, Frequency of PD-1 (n = 16), GITR (n = 4), LAG3 (n = 16) and TIGIT (n = 16) expressing cells by FACS, percentage of parent (all CD4⁺T cells (black circles), exT_reg cells (red circles), T_reg cells (blue circles); mean ± s.e.m.). 50% male and 50% female donors, aged 20–69 years. Each dot represents a biological replicate from an independent donor. Statistical comparisons were done using one-way analysis of variance (ANOVA) with Dunnett’s multiple-comparison test (a), a two-tailed Mann–Whitney U test (b and d) and a Kruskal–Wallis test, adjusted with Dunn’s multiple-comparison testing (e). In a, exT_reg cells versus naive **P = 0.0012; versus T_reg cells **P = 0.0068. In b, *P = 0.0286. In d, *P = 0.0111 for MKI67, CCNB1; *P = 0.0175 for MCM6; **P = 0.007 for CCNA2 and MCM3; **P = 0.0012 for CCNB2; ***P = 0.0006 for TOP2A, MCM2, MCM5. In e, PD-1 **P = 0.0023, GITR **P = 0.0047, LAG3 ****P = 3.21 × 10⁻⁷, TIGIT ****P = 5.227 × 10⁻⁵ for exT_reg cells versus CD4⁺T cells; PD-1 *P = 0.0329, GITR P = 0.3396, LAG3 ****P = 1.659 × 10⁻⁵, TIGIT P = 0.423 for exT_reg cells versus T_reg cells. NS, not significant.

Fig. 5 |. Human exT_reg cells are not suppressive but are cytotoxic.

a, CFSE-labeled CD3⁺CD4⁺CD25⁻T cells (T_eff) were co-cultured at 1:1 ratio with either CTV-labeled T_reg cells or exT_reg cells, in the presence of anti-CD3/CD28/CD2-coated beads. Left, representative histograms from three independent experiments showing CFSE in T_eff cells alone (black line), in T_eff cells cultured with T_reg cells (blue line) and in T_eff cells cultured with exT_reg cells (red line). Right, proportion of dividing T_eff cells after 5 d of co-culture with T_reg cells (blue circles, n = 5) or ex T_reg cells (red circles, n = 3). 40% male and 60% female donors; aged 23–43 years. b, Heat map of DEGs (P < 0.01 and |log₂FC| > 2, based on a two-tailed Wald test with Benjamini–Hochberg P-value adjustment) between human exT_reg versus T_reg transcriptomes from Fig. 3. Cytotoxic and T_reg signature genes are highlighted. c, FACS analysis of cytotoxic proteins FASLG, perforin and granzyme B (genes are labeled red in b) in human exT_reg cells, compared to bulk CD4⁺T cells. Left, representative histograms of intensities of fluorochrome-conjugated antibodies against FASLG, perforin and granzyme B on exT_reg cells (red) and all CD4⁺T cells (black). Right, MFIs of marker protein expression in exT_reg cells. n = 6. 50% male and 50% female donors, aged 25–37 years. d, Representative contour plots (left) and quantification (right) of basal and anti-CD3-induced degranulation in exT_reg cells (red) and NK cells (gray), as measured by surface mobilization of the degranulation marker CD107a by FACS. hPBMCs were co-cultured with uncoated P815 cells (target alone, open circles) or 5 μg ml⁻¹ anti-CD3-coated P815 cells (target + anti-CD3, filled circles) for 6 h at a 10:1 PBMC:P815 ratio. n = 3. 33.33% male and 66.67% female donors; aged 25–38 years. e, Anti-CD3-loaded P815 cells were co-cultured with CD8 CTLs (black circles, n = 5), T_reg cells (blue circles, n = 5) or exT_reg cells (red circles, n = 4) at a 1:5 P815:effector cells ratio for 16 h. 60% male and 40% female donors, aged 21–45 years. Cytotoxicity was assessed by measuring lactate dehydrogenase amounts in the supernatant. Results (a and c–e) are represented as the mean ± s.e.m. Each dot represents a biological replicate from an independent donor. Statistical comparisons were done using a two-tailed Mann–Whitney U test (a and e) and a two-tailed unpaired t-test (d). *P = 0.0357 (a). ***P = 0.0008 (d, exT_reg cells); P = 0.7707 (d; NKs). **P = 0.0079 (e, T_reg cells versus CTLs); *P = 0.0159 (e; T_reg cells versus exT_reg cells).

Fig. 6 |. Human exT_reg cells express cytotoxic proteins, inflammatory cytokines, chemokines and chemokine receptors.

a, Frequencies of GzmB⁺perforin⁺ and TNF⁺ cells in T_reg cells (blue circles) and exT_reg cells (red circles) were assessed in an ICS assay by FACS. n = 6. 50% male and 50% female donors; aged 24–39 years. b, Representative contour plots (left) showing intracellular expression of IFN-γ under unstimulated and PMA–ionomycin-stimulated conditions in exT_reg cells and T_reg cells. Right, frequency of PMA-induced IFN-γ in exT_reg cells (red circles) and T_reg cells (blue circles). n = 5. 40% male and 60% female donors; aged 23–39 years. c, hPBMCs were stained for intracellular expression of CCL3 (MIP-1α), CCL4 (MIP-1β) and CCL5 (RANTES). Top, representative contour FACS plots showing expression among all CD4⁺T cells and in exT_reg cells. Bottom, frequencies of CCL3⁺, CCL4⁺ and CCL5⁺ cells among the parent subset (all CD4⁺T cells (black circles), exT_reg cells (red circles)). n = 6. 50% male and 50% female donors, aged 26–33 years. d, Frequencies of CCR5 (n = 12), CXCR2 (n = 5), CXCR3 (n = 12), CXCR4 (n = 12) and CX3CR1 (n = 13) chemokine receptor-expressing exT_reg cells (red circles) and all CD4⁺T cells (black circles). 40% male and 60% female donors, aged 20–69 years. Results (a–d) were plotted as the mean ± s.e.m. Each dot represents a biological replicate from an independent donor. Statistical comparisons were performed using a two-tailed Mann–Whitney U test (a–d). **P = 0.0022 (a), **P = 0.0079 (b), **P = 0.0022 (c). In d, **P = 0.0068 (CCR5), **P = 0.0079 (CXCR2), ****P = 7.396 × 10⁻⁷ (CXCR3), **P = 0.0023 (CXCR4), ****P = 1.92 × 10⁻⁷ (CX3CR1).

Fig. 7 |. Inflammatory and cytotoxic human exT_reg genes overexpressed in individuals with coronary artery disease.

a, Genes that were significantly upregulated in human exT_reg cells compared to T_reg cells (2,803 genes) in the human bulk RNA-seq data (Fig. 3) were intersected with the genes present in the published human scRNA-seq panel (Fig. 2). Donor details for human bulk RNA-seq are in Supplementary Table 13 and those for scRNA-seq are in ref. . b, Mean expression of genes in CD16⁺CD56⁺ exT_reg cells from the scRNA-seq dataset that were significantly increased in CAD⁺ non-diabetic (brown circles in top, n = 7) or diabetic (red squares in bottom, n = 11) individuals in comparison to control CAD⁻ non-diabetic (black circles, n = 12) individuals. Results are shown as the mean ± s.e.m. Each point represents data from exT_reg cells from an independent donor. Statistical comparisons were done using a two-tailed Mann–Whitney U test. Top, **P = 0.0064 (CNOT2), *P = 0.0414 (CCL4), **P = 0.009 (IL18RAP), *P = 0.0361 (KLRG1), *P = 0.0163 (KLRC1), *P = 0.0371 (LYN), **P = 0.0095 (SAMD3), *P = 0.0395 (SYNE1). Bottom, *P = 0.0126 (CCL5), *P = 0.0354 (FGFBP2), *P = 0.0373 (ITGA4), *P = 0.018 (ITGAM), *P = 0.031 (KLRB1), *P = 0.032 (KLRC1). c, log₂FC and adjusted P values of a DEG analysis (based on a two-tailed Wald test with Benjamini–Hochberg P-value adjustment) between exT_reg cells and T_reg cells for the 13 CAD-relevant exT_reg genes from b.