Exposure to PB of GVHD-affected animals induces Treg activation and enhances Treg in vivo function. (A) Experimental scheme. PB from BALB/c mice affected by aGVHD was harvested at day +6 after hematopoietic cell transplantation and incubated with allogeneic Tregs obtained from healthy C57BL/6 animals. After 20 to 24 hours of culture, Tregs were washed and used for in vitro or in vivo experiments. (B-E) Graphs that report expression in gated CD4⁺FoxP3⁺ Tregs of intracellular FoxP3 (B), surface CD25 (C), surface CTLA4 (D), and TGF-β (measured through the surface expression of TGF-β LAP) (E) are shown. Unprimed Tregs (cultured with standard culture conditions only) are shown in white, Tregs primed with irradiated aGVHD PB in black, and Tregs primed with only the cellular fraction of irradiated aGVHD PB in gray. Sample staining histograms are also reported where unprimed Tregs stained with isotype control antibody (dashed line), unprimed Tregs (white), PB-primed Tregs (black), and PB cellular fraction-primed Tregs (gray) are shown. Data are representative of 1 of 3 independent experiments. Statistical analysis has been performed with 2-tailed Student t test. (F-G) C57BL/6 allogeneic aGVHD PB-primed Tregs were injected in a mouse model of GVHD, together with TCD-BM and Tcons from C57BL/6 allogeneic donor mice at day 0 into lethally irradiated BALB/c recipients. Survival (F) and percentage of weight recovery (G) after transplantation of mice that received only lethal irradiation (dotted line, ⁠), mice that received TCD-BM (dashed line, ⁠), mice that received TCD-BM + Tcons (⁠⁠), mice that received TCD-BM + Tcons + unprimed Tregs at the Treg:Tcon ratio of 1:2 (⁠⁠), mice that received TCD-BM + Tcons + unprimed Tregs at the Treg:Tcon ratio of 1:10 (⁠⁠), and mice that received TCD-BM + Tcons + aGVHD PB-primed Tregs at the Treg:Tcon ratio of 1:10 (⁠⁠) are reported. Shown data are the results of 2 pooled independent experiments. (H-I) C57BL/6 allogeneic aGVHD PB-primed Tregs were injected in a mouse model of GVHD 6 to 7 days after injection of C57BL/6 allogeneic TCD-BM and luc⁺ Tcons into lethally irradiated BALB/c recipients that showed clear signs and symptoms of aGVHD with a minimum GVHD score of 4, and had Tcon proliferation in vivo analyzed by BLI. The Treg:Tcon ratio used was 1:2. Survival (H) and percentage of weight recovery (I) after transplantation are reported of mice that received only lethal irradiation (dotted line, ⁠), mice that received TCD-BM (dashed line, ⁠), mice that received TCD-BM + Tcons (⁠⁠), mice that received TCD-BM + Tcons + unprimed Tregs (⁠⁠), and mice that received TCD-BM + Tcons + aGVHD PB-primed Tregs (⁠⁠). Reported data are the result of 4 pooled independent experiments. In survival experiments statistical analysis has been performed with the Log-rank test, whereas weight variations were assessed by 2-way ANOVA test. *P < .05; **P < .01; ***P < .001. ANOVA, analysis of variance; BLI, bioluminescent imaging; LAP, latency associated peptide; MFI, mean fluorescence intensity; ns, not significant.