Fig. 3: Deep immunoprofiling of murine IL-2pa-self-sufficient Tregs.

A UMAP representation of single-cell transcriptome profiles of sorted Treg samples obtained from splenocytes of a 32-week-old B6 Thy1.2 mouse that received Tregs-IL2pa. Splenocyte samples include purified IL-2pa-stimulated endogenous Tregs (orange) and transduced Tregs-IL2pa (blue). Tregs from an unmanipulated B6 Foxp3-GFP control mouse (Ctrl Tregs, green) were used as controls. B Colored UMAP representation based on the expression of selected Treg activation/function markers, scaled from lowest gene expression in blue to highest in red. C UMAP representation of KLRG1, LAG3, and TIGIT senescence/exhaustion markers. D Representative expression profiles of Treg activation and functional markers from blood flow cytometry staining. Ctrl Tregs and Ctrl Tconv were obtained from unmanipulated mice (n = 3), and IL-2pa-stimulated endogenous Tregs and Tregs-IL2pa were obtained from mice receiving Tregs-IL2pa (n = 3). Stainings were performed 5 to 7 months post-injection. E TCR diversity Renyi profiles for each condition (n = 2 per group). Colored circles represent the mean across samples within the same group and shaded areas represent the standard deviation (SD). F Barplot representation of the proportion of each defined clone count interval within the total repertoire for each condition (n = 2 per group). Error bars indicate the standard error (SE). G Methylation analysis of the Treg Specific Demethylated Region (TSDR) with the average percentage of methylation for 14 CpG sites at the top and data for each CpG residue at the bottom, in Tconv (n = 2), IL-2pa-stimulated endogenous Tregs (n = 2) and Tregs-IL2pa (n = 2), and in Ctrl Tregs (n = 3). Statistically significant difference was found between Tconv and Ctrl Tregs (p < 0.0001). H Treg phenotype of injected cells after their transfer into RAG immunodeficient mice. Six- to seven-week-old male RAG mice received 9 × 10⁵ transduced unsorted Ctrl Tregs or Tregs-IL2pa with n = 3 per group. Cell phenotype was monitored in the blood. The percentage of CD25⁺Foxp3⁺ cells among the injected cells is shown on the left, while a pseudocolor plot representing CD25⁺Foxp3⁺ staining 36 days post-injection is shown on the right. Schematic created in BioRender⁴⁹. Statistically significant differences were observed between Tregs-IL2pa and Ctrl Tregs on days 16 (p = 0.0002), 28, and 36 (p < 0.0001). Data are presented as mean ± SEM in the panels (G, H) and compared using a two-tailed Mann-Whitney test in panel (G) and a two-tailed unpaired t-test in panel (H) (*p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001).