RAPA and IL-2 Ab complex expanded CD8⁺FoxP3⁺ T_reg are functional and protect from gut GVHD. (A) Recipient B6D2F1 mice were lethally irradiated and 24 hours later transplanted with BM and T cells from B6.FoxP3-GFP mice. Recipients received RAPA daily (1.5 mg/kg intraperitoneally) and IL-2 Ab complexes at day 0 and day 4 after transplantation. At day 7 after transplantation, LN and spleens were removed, CD8⁺Foxp3⁺ cells sorted and cultured with CFSE-labeled CD45.1⁺CD3⁺ T cells, CD45.2⁺ DCs, and 1 μg/mL CD3 with CFSE dilution quantified in CD45.1⁺ cells 3 days later. (B) B6D2F1 recipients were lethally irradiated and received BM and 0.5 × 10⁶ sorted CD4⁺FoxP3⁻ T cells from B6.FoxP3-GFP donors in conjunction with 3 × 10⁶ sorted CD8⁺FoxP3⁻ cells from FoxP3.LuciDTR-4 donors (luciferase, GFP, and the DT receptor-driven of the FoxP3 promoter). All recipients received RAPA (daily) and IL-2 Ab complexes (day 0 and day 4). DT (160 ng intraperitoneally) or saline was administered daily from day 3 until day 6. Bioluminescent imaging was performed and quantified at day 7; n = 6 per group. **P < .01. (C) Representative images of small intestine taken at day 7 after transplantation (original magnification ×200) and GVHD histopathology scores; n = 6 per group. **P < .01. (D) B6D2F1 recipients were lethally irradiated and 24 hours later transplanted with BM and T cells from B6.FoxP3-GFP mice. Recipients received RAPA or cyclosporine daily. Spleen and mLN were removed at day 7 after transplantation and FoxP3⁺ populations analyzed. Representative plots are shown of the mLN for CD8⁺ enumeration or the spleen for CD4⁺ enumeration. Absolute numbers were quantitated, and graphs represent mean ± SEM; n = 7 or 8 per group. *P < .05. ***P < .005.