Figure 6
Figure 6. Host DCs, IL-12, and IFN-γ are critical for the hyperacute GVHD induced by KRN7000. (A) Irradiated B6.WT or B6.IL-12−/− mice received whole spleen from G-CSF–mobilized syngeneic B6 or allogeneic BALB/c donors, followed by control diluent or KRN7000 IP on days +1 and +4. Overall survival by Kaplan-Meier analysis, combined data from 2 experiments (n = 4, syn KRN7000; n = 16, allo B6.WT groups; n = 13, allo KRN7000 B6.IL-12−/− group). *P < .005 allo KRN7000 B6.WT vs allo KRN7000 B6.IL-12−/− and allo control B6.WT. (B) Irradiated B6 mice received whole spleen from G-CSF–mobilized BALB/c donors, followed by control diluent, C20:2, or KRN7000 IP on day +1. DCs were sort-purified from spleens pooled from 4 mice per group 6 hours after glycolipid injection, and their IL12a expression relative to β2m was determined by real-time PCR. (C) Irradiated B6.WT or B6.IFN-γR−/− recipients were transplanted with G-CSF–mobilized BALB/c grafts and KRN7000 or control diluent administered at day +1 (n = 6 per group). Numbers of host (H-2Dd−) DCs remaining in the spleen were elucidated 3 days after transplant. **P < .01 vs other groups. Combined data from 2 replicate experiments. (D) B6.IFN-γR−/−→B6.WT chimeras were transplanted with whole spleen from G-CSF–mobilized syngeneic B6 or allogeneic BALB/c donors, followed by control diluent or KRN7000 on days +1 and +4. Overall survival by Kaplan-Meier analysis (n = 4, syn group; n = 8, allo groups). P = .33 between allo groups. (E) Irradiated B6.WT or B6.CD11c.DTR mice were injected with diphtheria toxin IP, transplanted with whole spleen from G-CSF–mobilized BALB/c donors 1 hour later, and given KRN7000 IP on day +1 (24 hours later). Effective depletion of splenic DCs was confirmed 24 hours after KRN7000 administration. Representative FACS plots are shown, with the mean ± SE of residual DC percentages given alongside (n = 9-10). (F) Serum IL-12 was determined at 6 hours, and serum IFN-γ at 24 hours, after KRN7000 injection, in transplant recipients from panel E. Data show mean ± SE of combined data from 2 identical experiments (n = 8-10). **P < .001. (G) Hepatic lymphocytes from transplants described in panel E were pooled from 5 mice per group at 24 hours after glycolipid injection and gradient-purified, and IFN-γ secretion by cell subsets was determined by cytokine secretion assay. One of 2 representative experiments is shown.

Host DCs, IL-12, and IFN-γ are critical for the hyperacute GVHD induced by KRN7000. (A) Irradiated B6.WT or B6.IL-12−/− mice received whole spleen from G-CSF–mobilized syngeneic B6 or allogeneic BALB/c donors, followed by control diluent or KRN7000 IP on days +1 and +4. Overall survival by Kaplan-Meier analysis, combined data from 2 experiments (n = 4, syn KRN7000; n = 16, allo B6.WT groups; n = 13, allo KRN7000 B6.IL-12−/− group). *P < .005 allo KRN7000 B6.WT vs allo KRN7000 B6.IL-12−/− and allo control B6.WT. (B) Irradiated B6 mice received whole spleen from G-CSF–mobilized BALB/c donors, followed by control diluent, C20:2, or KRN7000 IP on day +1. DCs were sort-purified from spleens pooled from 4 mice per group 6 hours after glycolipid injection, and their IL12a expression relative to β2m was determined by real-time PCR. (C) Irradiated B6.WT or B6.IFN-γR−/− recipients were transplanted with G-CSF–mobilized BALB/c grafts and KRN7000 or control diluent administered at day +1 (n = 6 per group). Numbers of host (H-2Dd−) DCs remaining in the spleen were elucidated 3 days after transplant. **P < .01 vs other groups. Combined data from 2 replicate experiments. (D) B6.IFN-γR−/−→B6.WT chimeras were transplanted with whole spleen from G-CSF–mobilized syngeneic B6 or allogeneic BALB/c donors, followed by control diluent or KRN7000 on days +1 and +4. Overall survival by Kaplan-Meier analysis (n = 4, syn group; n = 8, allo groups). P = .33 between allo groups. (E) Irradiated B6.WT or B6.CD11c.DTR mice were injected with diphtheria toxin IP, transplanted with whole spleen from G-CSF–mobilized BALB/c donors 1 hour later, and given KRN7000 IP on day +1 (24 hours later). Effective depletion of splenic DCs was confirmed 24 hours after KRN7000 administration. Representative FACS plots are shown, with the mean ± SE of residual DC percentages given alongside (n = 9-10). (F) Serum IL-12 was determined at 6 hours, and serum IFN-γ at 24 hours, after KRN7000 injection, in transplant recipients from panel E. Data show mean ± SE of combined data from 2 identical experiments (n = 8-10). **P < .001. (G) Hepatic lymphocytes from transplants described in panel E were pooled from 5 mice per group at 24 hours after glycolipid injection and gradient-purified, and IFN-γ secretion by cell subsets was determined by cytokine secretion assay. One of 2 representative experiments is shown.

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