Absence of LTα does not alter donor T-cell function or inflammatory cytokine generation. (A) B6D2F₁ recipients of B6.Lta^−/− or B6.WT grafts were bled at day 7, and serum cytokines were analyzed by cytokine bead array, as described in “Cytokine analysis.” (B) Donor T cells were enumerated from the spleens of B6D2F₁ recipients 14 days after transplantation. Data shown are absolute number ± SEM per spleen (n ≥ 9). (C) CD4⁺ and CD8⁺ T cells were assessed by flow cytometry 14 days after transplantation for expression of the activation markers CD25 and CD69 (n = 5-8 per group). (D) Donor splenocytes were cultured for 2 hours with soluble CD3 in the presence of brefeldin A and assessed for production of the inflammatory cytokines IFN-γ and TNF by intracellular cytokine staining, as described in “Cytokine analysis” (n = 5-8 per group). (E) Donor CD4⁺ T cells were sort purified 14 days after transplantation and plated in mixed lymphocyte assays, as described in “Mixed lymphocyte culture.” Proliferation was determined by ³H thymidine uptake. Data are mean ± SEM from triplicate wells and are representative of 3 replicate experiments. (F) CD8⁺ T cells were sort purified on day 14 after transplantation and plated in chromium release assays with both allogeneic (P815) and syngeneic (EL4) tumor cell targets. Data are mean ± SEM from triplicate wells and are representative of 2 replicate experiments.