Figure 2: Adaptive plasticity of IL-10⁺ and IL-35⁺ T_reg cells.

a, scRNAseq tSNE plots of bulk T_reg cells from naive LNs or 14 days post inoculation B16 tumors (TIL T_reg cells) from Foxp3^Cre-YFP mice.

b, scRNAseq tSNE plot depicting the expression of Il10 and Ebi3 in individual T_reg cells overlaid on the same tSNE plot as in (a).

c, Heat maps contrasting the top 30 genes selected based on the differential expression analysis of T_reg cells utilizing the two-sided Negative Binomial Exact test, demonstrating the lack of distinct transcriptional signatures. p-values were adjusted to control the false discovery rate (FDR) set at 0.05. Treg cells were first stratified into IL-10⁻Ebi3⁻, IL-10⁺Ebi3⁻, IL-10⁻Ebi3⁺, and IL-10⁺Ebi3⁺ as described in (b). (Naive LN): IL-10⁻Ebi3⁻ (n=717 cells), IL-10⁺Ebi3⁻ (n=3 cells), IL-10⁻Ebi3⁺ (n=83 cells), and IL-10⁺Ebi3⁺ (n=3 cells). (TIL): IL-10⁻Ebi3⁻ (n=491 cells), IL-10⁺Ebi3⁻ (n=88 cells), IL-10⁻Ebi3⁺ (n=491 cells), and IL-10⁺Ebi3⁺ (n=111 cells).

d, Bar graphs with overlaid scatter-dots depicting the TCR V_β gene usage, comparing the T_reg cells subpopulations and effector T cells. Three independent B16 tumor-bearing Il10^GFP.Ebi3^Tom.Foxp3^Cre-YFP mice were used to harvest T_reg cell subpopulations for sequencing without pooling. Bars represent mean values.

e, In vitro tracing of adaptive plasticity in cytokine expression by TCR-stimulation. (top): Naive T_reg cells from LN and spleens were double-sort purified and stimulated with anti-CD3/CD28-coated beads in the presence of hIL2 and CD11c⁺ cells for 72 hours, followed by FACS analysis. (bottom): Stacked bar graph summarizing four independent experiments. Statistical significance was determined by Two-way ANOVA with Holm-Sidak multiple comparisons (^#p=0.0073, *p=0.0016, **=P=0.0017, ***p=0.0002, ****p<0.0001).

f, Diffusion pseudo-time analysis depicting the stochastic oscillation of IL-10 and IL-35 (Ebi3) expression based on the transcriptomic features of sequenced T_reg cells from the scRNAseq experiment.

g, Stacked-bar graph demonstrating the distribution of indicated T_reg subpopulations along the Pseudotime projection as analyzed in (f). The Pseudotime projection was evenly divided into 10 fractions and the percent distribution of each T_reg cell subpopulation was calculated.