Protection against GVHD after TLI/ATS is dependent on both donor CD4⁺CD25⁺ Tregs and host invariant NKT cells. (A) Representative FACS patterns of the donor transplant inoculum before (PRE) and after (POST) CD25⁺ T-cell depletion. Percentage of CD4⁺CD25⁺ T cells among gated CD4⁺TCRαβ⁺ T cells is shown enclosed in boxes. (B) Survival of wild-type BALB/c hosts after TLI/ATS conditioning, followed by transplantation of either undepleted (UNDEP DONOR, WT HOST; n = 8) or CD25⁺ T cell–depleted (CD25-DEP DONOR, WT HOST; n = 9) donor grafts consisting of 50 × 10⁶ bone marrow and 60 × 10⁶ splenocytes from wild-type C57BL/6 donors. Also shown is survival of NKT cell–deficient Jα18^−/− (n = 10) hosts conditioned with TLI/ATS and injected with 50 × 10⁶ untreated whole bone marrow and 60 × 10⁶ untreated splenocytes from wild-type C57BL/6 donors (UNT DONOR, Jα18^−/− HOST). Each group represents survival combined from 2 to 3 separate experiments. (C) Mean body weight (± SE) of TLI/ATS-conditioned wild-type BALB/c hosts receiving either undepleted or CD25⁺ T cell–depleted donor grafts or TLI/ATS-conditioned NKT cell–deficient Jα18^−/− hosts receiving untreated donor grafts shown in panels A and B, at serial time points after transplantation. + indicates analysis was stopped when 2 hosts remained in the group. (D) Mean (± SE) histopathologic GVHD scores of colon, skin, and liver at day 6 from wild-type (WT) hosts given untreated or CD25-depleted transplants or Jα18^−/− hosts given untreated transplants. n = 5 for all groups. (E) Microscopic examination of hematoxylin/eosin-stained sections of colon at day 6 in a representative host from each group shown in panel C. Comparison of WT BALB/c host given TLI/ATS and an undepleted transplant and WT BALB/c host given TLI/ATS and a CD25⁺ T cell–depleted transplant demonstrates severe crypt apoptosis (→), inflammatory infiltrates (*), and crypt atrophy with loss of goblet cells in the latter compared with the former. NKT cell–deficient Jα18^−/− host given TLI/ATS and untreated transplant reveals severe crypt apoptosis (→), inflammatory infiltrates (*), and crypt atrophy with loss of goblet cells. All micrographs are at 300× final magnification.