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.