GVHD induction by KRN7000 is NK cell–dependent. (A) B6 donors received isotype control or anti-asialo GM1, as described in “Cellular depletion,” and specific NK-cell depletion of the graft confirmed before transplantation by FACS. (B) Irradiated B6D2F1 mice received G-CSF–mobilized spleen from B6 donors pretreated with isotype control or anti-asialo GM1 (corrected to transfer equivalent numbers of T cells to each group) or T-cell–depleted spleen, followed by control diluent or KRN7000 IP on days +1 and +4. (TCD, n = 5; all other groups, n = 21). Combined data from 3 experiments shown. Overall survival by Kaplan-Meier analysis. *P < .05 vs all other groups. Differences between all other T-cell–replete groups are not significant. (C) Irradiated B6 mice received whole spleen from G-CSF–mobilized BALB/c allogeneic donors, followed by control diluent, C20:2, or KRN7000 IP on day +1, and IL-12 levels were measured in sera 6 hours later (n = 8-12 per group combined from 2 experiments). Data expressed as mean ± SE; *P = .002 vs control; #P = .02 vs C20:2. (D) Irradiated B6.Jα18−/− mice received whole spleen from G-CSF–mobilized BALB/c.Jα18−/− donors, followed by control diluent, C20:2, or KRN7000 IP on day +1. IL-12 levels were measured in sera 6 hours later (n = 5 per group). Data are expressed as mean ± SE. (E) Irradiated B6.WT or B6.IL-12−/− mice received whole spleen from G-CSF–mobilized BALB/c allogeneic donors, followed by control diluent, C20:2, or KRN7000 IP on day +1, and IFN-γ levels were measured in sera 24 hours later (n = 8-14 per group, combined from 3 experiments). Data are expressed as mean ± SE; **P = .001, KRN7000 B6.WT vs B6.IL-12−/− recipients. (F) IFN-γ secretion assay on hepatic NK- and T-cell populations 24 hours after glycolipid administration in transplants, as described in panel E, one of 3 representative experiments shown.
