Host IL-4 is required for protection against GVHD, NKT-cell regulatory activity, and proliferation of donor Tregs after transplantation. (A) Wild-type (WT) versus IL-4^−/− BALB/c host survival after TLI/ATS, followed by transplantation of 50 × 10⁶ bone marrow and 60 × 10⁶ splenocytes from wild-type C57BL/6 donors (IL-4^−/−, n = 10; WT, n = 7). Each group represents survival combined from 2 to 3 separate experiments. (B) Mean body weight (± SE) at serial time points after transplantation in hosts shown in panel A. + indicates analysis was stopped when 2 hosts remained in the group. (C) Microscopic examination of hematoxylin/eosin-stained sections of colon at day 6 in one representative host from each group shown in panels A,B. IL-4^−/− BALB/c host given TLI/ATS with WT donor cells shows evidence of inflammatory infiltrate (*) in colonic crypts and multiple areas of apoptotic crypt nuclei (→). WT BALB/c host given TLI/ATS conditioning with WT donor cells shows normal appearance. Specimens shown are at ×300 final magnification. (D) Representative FACS analyses of CD4 versus intracellular Foxp3 staining of gated H-2K^b+CD4⁺ splenocytes (top panels) and CD4 versus CD8 staining of gated H-2K^b+TCRαβ⁺ splenocytes (bottom panels) 6 days after transplantation of untreated donor cells into TLI/ATS-treated IL-4^−/− BALB/c hosts with and without injections of 0.5 × 10⁶ sorted wild-type BALB/c NKT cells. NKT cells were injected 4 hours before donor cell transplantation. (E) Mean (± SE; ×10⁵) absolute number of H-2K^b+CD4⁺CD25⁺Foxp3⁺ cells isolated from spleen at day 6 from groups of transplanted BALB/c hosts from Figure 3D. n = 4-6 per group. Values for additional control wild-type BALB/c hosts given untreated donor cell transplants are shown for comparison (n = 5). (F) Representative histograms from 1 of 2 to 3 similar experiments showing CFSE-staining intensity versus cell number for gated CD45.1⁺CD4⁺TCRαβ⁺Foxp3⁺ cells and CD45.1⁺CD8⁺TCRαβ⁺ cells from the pretransplant donor cell inoculum (DONOR C57BL/6 (PRE)) and cells isolated from spleen of transplanted TLI/ATS-conditioned hosts at day 6. SYN, Wild-type CD45.2 C57BL/6 TLI/ATS-conditioned hosts given C57BL/6 CD45.1⁺ splenocytes and CD45.2⁺ marrow cells; IL-4^−/− CD45.2 BALB/c TLI/ATS-conditioned hosts were given no NKT cells (IL-4^−/− BALB/c HOST), 0.5 × 10⁶ sorted wild-type BALB/c NKT cells (IL-4^−/− BALB/c HOST + WT NKT), or 0.5 × 10⁶ sorted IL-4^−/− BALB/c NKT cells (IL-4^−/− BALB/c HOST + IL-4^−/− NKT) 4 hours before untreated donor cell transplantation. n = 2-3 pooled animals per group. (G) Mean ± SD (%) gated CD45.1⁺CD4⁺TCRαβ⁺Foxp3⁺ cells and CD45.1⁺CD8⁺TCRαβ⁺ CFSE^low cells in the groups shown in panel F. Mean of 2 to 3 experiments. n = 2-3 pooled animals per group.