Tr cells generated with DC_VIPs prevent acute GVHD. DCs were generated from mouse BM cells in the absence (DC_controls) or presence (DC_VIPs) of VIP and activated with LPS to induce DC maturation. CD4 and CD8 T cells (H-2^b) were exposed to allogeneic (H-2^d)DC_controls or DC_VIPs to generate potential Tr cells (Tr_controls and Tr_VIPs). (A) Balb/c mice (H-2^d) received transplants of T-depleted BM cells supplemented with 1.5 × 10⁶ spleen mononuclear cells (BMS, 1.5 × 10⁷ cells/mouse) from B6 (H-2^b) mice (left panel) and B6 mice (H-2^b) received transplants of T cell-depleted BM cells supplemented with 1.5 × 10⁶ spleen mononuclear cells (BMS, 1.5 × 10⁷ cells/mouse) from Balb/c (H-2^d) mice (right panel). Medium (untreated, •), CD4Tr_controls (○) or CD4Tr_VIPs (▾) (H-2^b) were injected (1.5 × 10⁶ cells/mouse) in both recipients 2 days after allogeneic transplantation, and survival was monitored (10 animals/group). ^*P < .01 versus untreated recipients. (B) The effect of CD4Tr_VIPs is dose-dependent. Balb/c mice (H-2^d) received transplants of T-depleted BM cells supplemented with 1.5 × 10⁶ spleen mononuclear cells (BMS, 1.5 × 10⁷ cells/mouse) from B6 (H-2^b) mice and CD4Tr_VIPs (H-2^b) were injected in recipients together with transplantation at different numbers: 1.5 × 10⁶ cells, BMS/Tr_VIPs ratio of 10:1 (○); 3 × 10⁵ cells, BMS/Tr_VIPs ratio 50:1 (▾); or 1.5 × 10⁵ cells, BMS/Tr_VIPs ratio 100:1 (▿). Untreated control mice (•, n = 10). ^*P < .01 versus untreated recipients. (C) Balb/c mice that were (•) or that had received injections of CD4Tr_VIPs and been treated with control immunoglobulin (○), anti-IL-10 (▾), anti-TGF-β (▿), or anti-IL-10 plus anti-TGFβ (▪) antibodies after BMS B6 transplantation (n = 10). ^*P < .01 versus control immunoglobulin-treated CD4Tr_VIPs recipients. (D) Balb/c mice (H-2^d) were given transplants of T cell-depleted BM cells supplemented with 1.5 × 10⁶ spleen mononuclear cells (BMS, 1.5 × 10⁷ cells/mouse) from B6 (H-2^b) mice. Recipients were treated with medium (untreated, •), CD8Tr_controls (○) or CD8Tr_VIPs (▾) (5 × 10⁶ cells) at time of allogeneic transplantation and survival was monitored (5 animals/group). ^*P < .01 versus untreated recipients.