Administration of TNFR:Fc construct after transplantation attenuates GVHD in the absence of donor-derived TNF. (A) The b.END3 cell line (which is dependent on TNF/LTα3 signaling for the up-regulation of surface VCAM-1 and ICAM-1) was cultured in the presence of recombinant mouse TNF or LTα3 that had been preincubated with either TNFR:Fc or control IgG. Preincubation of recombinant TNF and LTα3 with human TNFR:Fc construct returned expression of these 2 molecules to the basal level seen in untreated cells, whereas incubation with control IgG allowed up-regulation of VCAM-1 and ICAM-1 to occur as expected. VCAM-1 and ICAM-1 surface expression was analyzed by flow cytometry. Plots shown are representative from 2 separate replicate experiments. (B) A total of 5 × 10⁶ BM and 10⁶ CD3⁺ T cells from B6.WT or B6Tnf^−/− donor mice was transplanted into lethally irradiated BALB/c recipients (n = 13-14), which were then treated with TNFR:Fc or control IgG on alternate days commencing at day +3 after transplantation and continuing until day 30. TCD bone marrow from B6.WT donors was transplanted into BALB/c recipients (n = 10) as non-GVHD controls (P = .004, WT.B6 donors, TNFR:Fc vs IgG; P = .023, Tnf^−/− donors, TNFR:Fc vs IgG). Data combined from 2 replicate experiments. (C) A total of 5 × 10⁶ BM and 2 × 10⁶ CD3⁺ T cells from B6.Tnf^−/− donor mice was transplanted into lethally irradiated B6D2F₁ recipients. Recipient mice were treated as described in panel B. Data shown are combined from 2 separate experiments, n = 16, BM plus T groups; n = 7, TCD control group. P = .001, TNFR:Fc treated versus IgG control (T cell–replete groups).