Graft-Versus-Leukemia Effect and Graft-Versus-Host Disease Can Be Differentiated by Cytotoxic Mechanisms in a Murine Model of Allogeneic Bone Marrow Transplantation by Nobuhiro Tsukada, Tetsuji Kobata, Yoshifusa Aizawa, Hideo Yagita, and Ko Okumura Blood Volume 93(8):2738-2747 April 15, 1999 ©1999 by American Society of Hematology

Mortality and body weight during acute GVHD after allo-BMT. Mortality and body weight during acute GVHD after allo-BMT. (A) Lethal GVHD was induced by transfer of B6 BM cells plus 2.5 × 107 spleen cells (B6 BMS) into lethally irradiated BDF1 mice. Recipients of B6 BM cells (B6 BM), B6 T-cell–depleted BM cells (B6 TCD-BM), or syngeneic BDF1 BM cells plus spleen cells (BDF1 BMS) manifested no signs of GVHD and almost all recipients survived more than 80 days, except for 1 recipient of B6 BM. (B and C) Contribution of TNF--, FasL-, and perforin-mediated cytotoxic pathways to lethal acute GVHD. Lethally irradiated BDF1 mice were transplanted with BM cells plus 2.5 × 107 spleen cells from wild-type B6 (B6 BMS), B6-gld (gld BMS), or perforin-deficient (PKO BMS) mice. In another group, B6 BMS recipients were administered with anti–TNF- MoAb (B6 BMS + anti–TNF-). Mortality (B) and body weight (C) were monitored at the indicated days after BMT. Administration of anti–TNF- MoAb to the B6 BMS almost completely ameliorated the mortality and the body weight loss resulted from acute GVHD. Survival of the gld BMS and PKO BMS recipients was 100% and 80%, respectively, on day 80 after BMT. The data represent the results of three similar experiments. Nobuhiro Tsukada et al. Blood 1999;93:2738-2747 ©1999 by American Society of Hematology

Characterization of L1210 murine leukemia and P815 murine mastcytoma cells. Characterization of L1210 murine leukemia and P815 murine mastcytoma cells. (A) L1210 and P815 cells were cultured in the presence of recombinant TNF- for 24 hours and then cell viability was analyzed using the alamar Blue method. Both L1210 and P815 were resistant to TNF-, whereas L929 was susceptible to TNF-. (B)51Cr-labeled L1210 or P815 cells were cocultured with murine FasL transfectant (mFasL/L5178Y) cells at the indicated E/T ratios for 6 hours and then cytotoxicity was measured by51Cr-release assay. (C) Proliferative response of spleen cells obtained from wild-type, gld, or PKO B6 mice against allogeneic L1210 or P815 cells. Spleen cells (2 × 105) were cultured with irradiated L1210 or P815 cells (2 × 104) for 5 days and pulsed with 3H-TdR during the last 16 hours. Spleen cells were prepared from one mouse of each strain and were used immediately after preparation. Data are represented as the mean ± SD of triplicated samples. Spleen cells obtained from each mouse responded equally to L1210 or P815 cells. (D) Killing of L1210 cells by CTL derived from wild-type, gld, and PKO B6 mice. Spleen cells were collected after 5 days of coculture with irradiated L1210 cells as described in (B) and restimulated with irradiated L1210 cells in the presence of 10 U/mL IL-2 for 5 days. CTL activity was tested against 51Cr-labeled L1210 or P815 target cells at the indicated E/T ratios. Although killing activity of CTL derived from gld mice was only slightly diminished as compared with that from wild-type mice, PKO-derived CTL exhibited no significant cytotoxicity against L1210 cells. Data are represented as the mean ± SD of triplicated samples. A representative of three experiments is shown. Nobuhiro Tsukada et al. Blood 1999;93:2738-2747 ©1999 by American Society of Hematology

GVL effect after allo-BMT. GVL effect after allo-BMT. GVL effect was evaluated in BDF1 mice that were inoculated IV with 1 × 104 L1210 cells (A) or 1 × 105 P815 cells (B) 2 days before the BMT as described in Fig 1A. (A) Mice inoculated with L1210 and P815 cells died within 8 and 10 days, respectively, when they received irradiation alone without BMT (no BMT). Transfer of B6 TCD-BM or syngeneic BDF1 BMS resulted in early leukemia relapse. Recipients of B6 BM or BMS survived longer than the above-noted three groups, but all mice died within 60 days. Data represent the results of three similar experiments. Nobuhiro Tsukada et al. Blood 1999;93:2738-2747 ©1999 by American Society of Hematology

Histopathological examination of the liver obtained from normal BDF1 and the recipients of B6 TCD-BM, B6 BM, or B6 BMS in the GVL experiment shown in Fig 3A. Histopathological examination of the liver obtained from normal BDF1 and the recipients of B6 TCD-BM, B6 BM, or B6 BMS in the GVL experiment shown in Fig 3A. (A) Liver section from an age-matched normal BDF1 mouse. (B) Massive leukemia cell infiltration was observed in the liver section from the recipients of B6 TCD-BM at the time of death. (C) Liver section obtained from the recipients of B6 BM on day 28 after BMT showed residual leukemia cells among normal liver tissue. (D) Liver section from the recipients of B6 BMS on day 28 after BMT showed severe mononuclear cell infiltration, but there was no residual leukemia cell in any fields of the section. Each figure shows a representative of three similar experiments. Similar results were also observed by using another tumor P815 cells. Original magnification × 400. Nobuhiro Tsukada et al. Blood 1999;93:2738-2747 ©1999 by American Society of Hematology

Contribution of TNF-–, FasL-, or perforin-mediated cytotoxicity to the GVL effect. Contribution of TNF-–, FasL-, or perforin-mediated cytotoxicity to the GVL effect. GVL effect was evaluated in BDF1 mice that were inoculated IV with 1 × 104 L1210 or 1 × 105 P815 cells 2 days before the BMT as described in Fig1B. (A) Administration of anti–TNF- MoAb to the recipients of B6 BMS resulted in early death within 16 days. Six of 10 gld BMS recipients survived more than 80 days, whereas all B6 BMS or PKO BMS recipients died within 51 days. (B) Similar results were also observed by using another tumor P815 cells. Administration of anti–TNF- MoAb resulted in early death within 24 days. Six of 8 gld BMS recipients survived more than 80 days, whereas all B6 BMS or PKO BMS recipients died within 47 days. Data represent the results of three similar experiments. Nobuhiro Tsukada et al. Blood 1999;93:2738-2747 ©1999 by American Society of Hematology

Histopathological examination of the GVL experiments shown in Fig 5A. Histopathological examination of the GVL experiments shown in Fig 5A. (A) Administration of anti–TNF- MoAb resulted in early death with marked hepatosplenomegaly (left) compared with an age-matched normal mouse (right). The liver section obtained at the time of death showed massive leukemia infiltration (B). (C) Liver section obtained from gld BMS recipients on day 28 showed focal mononuclear infiltration around portal area, but there was no residual leukemia cells in any fields of the section. Residual leukemia cell was not detected in spleen, BM, or peripheral blood. (D) Residual leukemia cells were observed in small areas of the liver section from the PKO BMS recipients. Each figure shows a representative of three similar experiments. Similar results were also observed by using another tumor P815 cells. Original magnification × 400. Nobuhiro Tsukada et al. Blood 1999;93:2738-2747 ©1999 by American Society of Hematology