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).