TNF-α priming induces Treg activation, promote their proliferation, and enhances their ability to suppress GVHD. (A-G) Tcons were primed for 48 hours with standard culture conditions (white bars) or with the addition of interferon-γ (light gray bars), IL-6 (medium gray bars), IL-12 (dark gray bars), or TNF-α (black bars) at different concentrations (L = low concentration = 1 ng/mL, M = medium concentration = 10 ng/mL, and H = high concentration = 100 ng/mL). TNF-α priming resulted in increased Treg expression of FoxP3 (A), CD25 (B), CD69 (C), surface CTLA4 (D), LAG3 (E), CD62L (F), and LAP (G). Data have been collected after gating live Tcon cells for CD4⁺FoxP3⁺ Tregs and CD4⁺FoxP3⁻ T cells. Data are representative of 1 of 5 experiments. (H) IL-10 concentration was measured in supernatants of cultured Tcons and resulted higher when cells were incubated with TNF-α (black) than with standard conditions (white). Data are representative of 1 of 2 experiments. (I) Sorted unprimed Tregs, TNF-α–primed Tregs, TNF-α–primed Tregs in the presence of TNFRI blocking antibody, and TNF-α–primed Tregs in the presence of TNFRII blocking antibody were stained with CellTrace Violet proliferation dye (Thermo Fisher Scientific) and incubated with irradiated allogeneic splenocytes in the presence of IL-2. Sample gating analysis showing CellTrace Violet dye dilution due to cell proliferation is reported for all the different conditions. TNF-α priming increased the Treg proliferative response to the allogeneic stimulus, TNFRI-blocking antibody did not interfere with such response, whereas TNFRII abrogated it. Data are representative of 1 of 3 consecutive experiments. (J) C57BL/6 luc⁺ TNF-α–primed Tregs were washed and injected into lethally irradiated BALB/c recipients that also received allogeneic C57BL/6 TCD-BM and Tcons at the Treg:Tcon 1:10 ratio. BLI sample images are shown (i). Red crosses represent mice that did not survive the experiment. BLI revealed increased in vivo proliferation of luc⁺ TNF-α–primed Tregs (black bars) at day +6 and at day +9 after transplantation in comparison with unprimed Tregs (white bars) (ii). Data are representative of 1 of 3 experiments. Statistical analysis was performed with the 2-tailed Student t test. *P < .05; **P < .01; ***P < .001. (K-M) C57BL/6 allogeneic TNF-α–primed Tregs were injected together with TCD-BM and Tcons (1 × 10⁶) from C57BL/6 allogeneic donor mice at day 0 into lethally irradiated BALB/c recipients. Survival (K), GVHD score (L), and percentage of weight recovery (M) after transplantation of mice that received only lethal irradiation (dotted line, ⁠), TCD-BM (dashed line, ⁠), TCD-BM + Tcons (⁠⁠), TCD-BM + Tcons + unprimed Tregs at the Treg:Tcon ratio of 1:2 (⁠⁠), TCD-BM + Tcons + unprimed Tregs at the Treg:Tcon ratio of 1:10 (⁠⁠), and TCD-BM + Tcons + TNF-α–primed Tregs at the Treg:Tcon ratio of 1:10 (⁠⁠) are shown. Data are the result of 2 pooled experiments. In survival experiments, statistical analysis has been performed with the Log-rank test, whereas in GVHD score and weight variation experiments, the 2-way ANOVA test was used. *P < .01; ***P < .001. ANOVA, analysis of variance; ns, not significant; SS, side scatter; TNFRI, TNF receptor I; TNFRII, TNR receptor II.