Figure 3.

Effect of metabolic inhibitors and CTLA4Ig on pre-activated CD8+ T cells and a model of acute skin allograft rejection. (A) Previously activated and resting in vitro T cells were re-stimulated for 48 h with plate-bound anti-CD3 (1 μg/ml) and soluble anti-CD28 (2 μg/ml) in the presence of CTLA4Ig and MI as in Figure 1C. At 36 h, brefeldin A was added to each culture and IFN-gamma (FN-g) and Granzyme B (GzB) production was compared to eFluor 450 dilution in CD8+ T cells. (B) Schematic of transplant and treatment model. FVB-Tg(CAG-luc, -GFP)L2G85Chco/J (H2q) to B6(Cg)-Tyrc-2J/J (H2b) complete MHC-mismatched full thickness skin transplantation. MI therapy was started from the day of transplantation (day 0) until POD 10 and stopped for 3 days to induce acute rejection. On POD 14, treatment was resumed with either CTLA4Ig or MI. MI: metabolic inhibitors: 2DG+ metformin + DON. 2DG, 500 mg/kg once daily; metformin 150 mg/kg once daily; DON 1.6 mg/kg once every other day; CTLA4Ig 0.5 mg every other day starting on day 14. (C) Representative images from a single mouse for each of the indicated time points. (D) Skin graft viability measured by luminescent light intensity (photons/sec). (E) Skin graft appearance on POD 18. (F) Hematoxylin and eosin stain (X200) of skin grafts on POD 18. **p < 0.01 (2-way ANOVA). Data are representative of three experiments (A) and two experiments (B–D).