Neutralization of TGF-β attenuates antihost cytotoxicity and GVL. (A) Splenocytes from transplant recipients containing equal numbers of CD8⁺ T cells (based on the CD8 staining of the input cells) were used as effector cells against chromium-labeled P815 (host) and EL4 (donor) target cells. Percentage of specific lysis was determined in standard chromium-release assays, as described in “Materials and methods” against host-type P815 targets (open symbols) or donor-type EL4 targets (filled symbols) in allogeneic recipients of anti-TGF-β antibody (squares) or control antibody (circles). Data presented as mean ± SE of triplicate wells from 1 of 2 identical experiments. (B) At day +12 after transplantation, B6D2F1 recipients of G-CSF-mobilized allogeneic B6 (CD45.2) splenocytes that received anti-TGF-β or control antibody were injected intravenously with congenic B6 PTPRC^A (CD45.1) and CFSE-labeled B6D2F1 (CD45.2) splenocytes. Sixteen hours later, CD45.1⁺ and CFSE⁺ peripheral-blood mononuclear cells from transplant recipients were quantified by FACS. (Left) In vivo cytotoxicity index was determined in allogeneic recipients as the ratio between remaining syngeneic (CD45.1⁺) and allogeneic (CFSE⁺) cells, as described in “Materials and methods” (allogeneic plus anti-TGF-β, □, n = 10; allogeneic plus control antibody, ▪, n = 12). Cytotoxicity was negligible (ratio of approximately 1.0) in B6D2F1 recipients of syngeneic B6D2F1 splenocytes, irrespective of the antibody treatment (syngeneic plus anti-TGF-β antibody, ▤, n = 3; syngeneic plus control antibody, ▨, n = 3). Data presented as mean ± SE. ^**P < .005, allogeneic plus anti-TGF-β antibody compared with allogeneic plus control antibody. (Right) FACS plots from representative syngeneic recipient (top) and allogeneic recipients of control antibody (middle) and anti-TGF-β antibody (bottom), illustrating the in vivo cytotoxicity index. (C) Purified B6 T cells were stimulated with irradiated B6D2F1 splenocytes in the absence (control) or presence of TGF-β1 (2.5 ng/mL). Seven days later equivalent numbers of T cells were restimulated with irradiated B6D2F1 splenocytes for an additional 3 days in the absence of exogenous TGF-β. Absolute numbers of CD8⁺ cells in MLC after secondary stimulation (control, □; TGF-β, ▪). Data represent mean ± SE from 3 identical experiments. ^*P = .05; TGF-β compared with control. (D) T cells were stimulated in MLC, as in panel C, and, after secondary stimulation, equal numbers of CD8⁺ cells were used as effectors in standard chromium release assays. Percentage of specific lysis against allogenic P815 (control, □; TGF-β, ▪) and syngeneic EL4 targets (control, ○; TGF-β, •). Data represent mean ± SE from quadruplicate wells combined from 3 identical experiments. ^**P < .01; TGF-β compared with control.