Administration of a tumor necrosis factor (TNF) inhibitor-encoding adenoviral vector decreases the severity of colonic inflammation in a DBA/2-->B6D2F1 murine model of colonic graft-versus-host disease (GVHD). The present studies evaluated the effect of TNF blockade on the splenic and colonic T-cell responses. cDNA encoding an artificial fusion protein consisting of the extracellular domain of the human 55-kDa receptor for TNF fused to a mouse IgG heavy chain was subcloned into an E1a-deficient adenoviral vector. Following transfer of DBA/2 T cells and bone marrow cells into irradiated B6D2F1 mice, the mice then received either the control adenovirus or the TNF inhibitor-encoding adenovirus. Splenic and colonic lymphocytes were isolated, stained with anti-H-2b, anti-H-2d, anti-CD3, anti-CD4, anti-CD8, and anti-CD45RB antibodies, and analyzed by flow cytometry. Splenic and colonic lymphocyte cytokine profiles also were assessed. More colonic T cells of donor origin were isolated from the control adenovirus recipients than from recipients of the TNF inhibitor encoding adenovirus (P = .027). Fewer CD4+ and CD8+ T cells were observed in colon but not in the spleen in the TNF inhibitor recipients. Fewer CD45RBlow (memory) T cells were observed in the CD4+ colonic lymphocytes isolated from the TNF inhibitor recipients than from controls. Importantly, lower levels of interleukin-2(IL-2) and interferon-gamma (INF-gamma) but not of IL-4 were observed in the lamina propria lymphocyte RNA isolated from the TNF inhibitor recipients. Infiltration and expansion of donor T cells and T-cell activation in the colon appear to be regulated by TNF during murine DBA/2 --> B6D2F1 gut GVHD.