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).