Fig. 5: In vitro characterization of human Tregs-IL2pa.
A Schematic design of the in vitro survival assay. Schematic created in BioRender49. Sorted Tregs were cultured and transduced on day 7 with lentiviruses to generate Ctrl Tregs (production of GFP alone) and Tregs-IL2pa (production of the IL-2 partial agonist and GFP). On day 9, unsorted transduced cells (approximately 35% of transduced cells for both vectors), were cultured in fresh medium under three different conditions: B with a CD3/CD28 restimulation on day 9 and addition of exogenous IL-2 on days 9 and 11, C with a restimulation and without the addition of IL-2, and D without restimulation and without the addition of exogenous IL-2. Data were obtained from two different experiments (n = 2 per group). E Multidimensional scaling (MDS) representation illustrating the similarities and differences in gene expression profiles between fresh and cultured Tregs from three different blood donors. The proximity of the dots reflects the degree of similarity in their transcriptomic profiles. Convex hulls delineate each condition. Tregs were obtained from three different donors (n = 3). F Boxplot showing the gene expression levels of some Treg activation and functional markers (n = 3). G Volcano plot illustrating differential gene expression between transduced sorted Ctrl Tregs and Tregs-IL2pa. Genes upregulated in Ctrl Tregs are shown in green, while those upregulated in Tregs-IL2pa are shown in red. H Bar plot illustrating the results of functional enrichment analysis. Each horizontal bar represents a biological pathway from the BioPlanet database, with the length proportional to its statistical significance, indicated by the -log10 of the p-value. I In vitro suppression assay. Data were extracted from two separate experiments (n = 6 per group). Data are presented as mean ± SEM in panels (B–D), and (I), with comparisons performed using a two-tailed unpaired t-test in panel (F) and a two-tailed Mann–Whitney test in panel (I) (*p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001). The boxplots in panel (F) show the median (Q2) as the central line, the first and third quartiles (Q1 and Q3) as the bounds of the box, and whiskers extending to 1.5 times the interquartile range; points beyond are outliers. For panel (G), differential expression analysis was performed using DESeq2 with two-sided tests and unadjusted p-values. For panel (H), statistical analysis was performed using a two-sided Fisher’s exact test, and p-values were adjusted for multiple comparisons.
