Fig. 4: Murine IL-2pa-self-sufficient Tregs are suppressive in vitro and in vivo.

A In vitro suppression assay. The percentage of suppression represents Tconv inhibition of proliferation by Tregs. Transduced Tregs were sorted three days post-transduction for the experiment (n = 2 per group). B Schematic design of the autoimmunity induction experiment in twelve- to twenty-week-old male B6 Foxp3^DTR Thy1.2 mice expressing the diphtheria toxin receptor (DTR) in endogenous Tregs. Negative control mice did not receive Tregs and were not treated with diphtheria toxin (DT) (n = 4), positive control mice did not receive Tregs and were treated with DT (n = 6), Tregs-IL2pa mice received a 9 × 10⁵ injection of Tregs-IL2pa and were treated with DT 20 days post-injection (n = 6). Mice were weighed at least three times per week and were bled on days 3, 10, and 17 for flow cytometry analysis. Data are pooled from two independent experiments. Schematic created in BioRender⁴⁹. C Follow-up of endogenous CD4 Tregs and D injected cells in the blood. Statistically significant differences were observed in the percentage of endogenous Tregs between DT-untreated and treated control mice on days 3 and 10 (p = 0.0357). E Mice weight follow-up during the autoimmunity experiment. Statistically significant differences were found between DT-untreated control mice and treated mice (p = 0.0095), as well as between DT-treated control mice and DT-treated mice receiving Tregs-IL2pa (p = 0.0260). F Percentage of mice with diarrhea and G spleen weights at the end of the autoimmunity induction experiment on day 22. Spleen weights were significantly higher in DT-treated control mice compared to DT-untreated mice (p = 0.0357). H Histological score corresponding to the sum of grades assigned for lymphocyte infiltration in each organ. Statistically significant differences were observed in the colon between DT-treated control mice and untreated mice (p = 0.0179) and between DT-treated control mice and mice receiving Tregs-IL2pa (p = 0.0079). In the liver, a statistically significant difference was observed between DT-treated control mice and untreated mice (p = 0.0357), as well as between DT-treated mice and mice that received Tregs-IL2pa (p = 0.0397). I Representative images of hematoxylin and eosin-stained sections of the colon, lungs, and liver at 200× magnification. One slide per group and per organ was analyzed, with n = 3 in the Ctrl without DT group, n = 5 in the Ctrl with DT group, and n = 4 in the Tregs-IL2pa with DT group. In the colon, the arrows indicate inflammatory cell infiltration, and the star indicates crypt regeneration. In the lungs, the arrows indicate interstitial lymphocytic infiltrate. In the liver, the arrow indicates lymphocytic infiltrate in a portal tract and the star indicates intrasinusoidal lymphocytic infiltrate. Data are expressed as mean ± SEM in the panels (A, C, D, E, G, H) and compared using a two-tailed Mann-Whitney test in the panels (C, E, G) and (H) (*p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001).