Adoptive transfer of allogeneic tumor-specific T cells mediates effective regression of large tumors across major histocompatibility barriers by Andrea Boni, Pawel Muranski, Lydie Cassard, Claudia Wrzesinski, Chrystal M. Paulos, Douglas C. Palmer, Luca Gattinoni, Christian S. Hinrichs, Chi-Chao Chan, Steven A. Rosenberg, and Nicholas P. Restifo Blood Volume 112(12):4746-4754 December 1, 2008 ©2008 by American Society of Hematology

Allogeneic antitumor T lymphocyte persistence in vivo. Allogeneic antitumor T lymphocyte persistence in vivo. C57BL/6 mice bearing B16 tumors were irradiated with 5 or 9 Gy TBI and injected on day 0 with 106 allogeneic pmel-1b/d cells, vaccinia virus encoding hgp10025-33, and exogenous rhIL-2 (alloPVI) or with 106 syngeneic pmel-1b/b cells, vaccinia virus encoding hgp10025-33, and exogenous rhIL-2 (synPVI). All groups received syngeneic BMT with 106 unsorted bone marrow cells the day after the transfer of the effector cells. At the indicated time points, mice were killed, and the spleens were analyzed by flow cytometry for the presence of the transferred cells. (A) The dot plots show the percentage of CD8+ H-2 Dd+ cells. Allogeneic cells were detectable up to day 24 after transfer. (B) Absolute numbers of CD8+Vβ13+ cells present in the spleens of the animals. Each bar represents 3 mice plus or minus SE. Data are representative of 3 independent experiments. Andrea Boni et al. Blood 2008;112:4746-4754 ©2008 by American Society of Hematology

Allogeneic tumor-specific lymphocytes can mediate tumor regression after intensive lymphodepleting preparatory regimen. Allogeneic tumor-specific lymphocytes can mediate tumor regression after intensive lymphodepleting preparatory regimen. C57BL/6 mice bearing B16 tumors were left untreated (A) or irradiated with 9 Gy TBI (B). Mice were left untreated as a control (NT) or injected on day 0 with vaccinia virus encoding hgp100 and exogenous IL-2 (VI) or injected with 106 or 107 (as indicated) allogeneic pmel-1b/d cells, vaccine, and IL-2 (PVI). *P < .001. **P < .05. All groups received syngeneic BMT with 106 unsorted bone marrow cells the day after the transfer of the effector cells (day 1). Results of tumor area are the mean of measurements of at least 5 mice per group (± SEM). Data are representative of 4 independent experiments. Andrea Boni et al. Blood 2008;112:4746-4754 ©2008 by American Society of Hematology

Therapeutic efficacy of allogeneic cells correlates with the intensity of the lymphodepleting preparatory regimen. Therapeutic efficacy of allogeneic cells correlates with the intensity of the lymphodepleting preparatory regimen. C57BL/6 mice bearing B16 tumors were irradiated with 5, 7, 9, or 11 Gy TBI. For each irradiation dose, mice were left untreated as a control (NT) or injected on day 0 with 106 allogeneic pmel-1b/d cells, vaccinia virus encoding hgp100, and exogenous rhIL-2 (PVI). All groups received syngeneic BMT with 106 unsorted bone marrow cells the day after the transfer of the effector cells. Statistical results are as follows: 5 Gy NT versus 5 Gy PVI, not significant; 11 Gy NT versus 11 Gy PVI, P < .02; 5 Gy PVI versus 7 Gy PVI, P < .04; 7 Gy PVI versus 9 Gy PVI, P < .03. There was no statistical difference between mice receiving 9 Gy versus 11 Gy plus PVI. Results of tumor area are the mean of measurements of at least 5 mice per group (± SEM). Data are representative of 2 independent experiments. Andrea Boni et al. Blood 2008;112:4746-4754 ©2008 by American Society of Hematology

Allogeneic tumor-specific CD4+ cells cause tumor regression and ocular autoimmunity in lethally irradiated hosts. Allogeneic tumor-specific CD4+ cells cause tumor regression and ocular autoimmunity in lethally irradiated hosts. C57BL/6 mice bearing B16 tumors established for 12 days were irradiated with 9 Gy TBI. Mice were left untreated as a control (NT) or injected on day 0 with 106 allogeneic TRP-1b/d cells and exogenous IL-2 (AlloTI) or 106 syngeneic TRP-1b/b cells and exogenous IL-2 (SynTI). All groups received syngeneic BMT with 106 unsorted bone marrow cells the day after the transfer of the effector cells. (A) Spleens analyzed for the presence of transferred T cells at the indicated time points. The experiment was independently repeated with similar results. (B) B16 tumor growth in the different treatment groups. Results of tumor area are the mean of measurements of at least 5 mice per group (± SEM) (n = 5, 9, and 10 mice for NT, AlloTI, and SynTI, respectively, in the experiment shown). Data are representative of 3 independent experiments. (C) Hematoxylin and eosin staining of ocular tissue of mice killed at day 14. Images were obtained using a Nikon Eclipse E400 microscope equipped with Nuance Multispectral Imaging System VIS (original magnification ×100). Images were processed using Adobe Photoshop, version 7, as described in “Histology.” Andrea Boni et al. Blood 2008;112:4746-4754 ©2008 by American Society of Hematology

Bone marrow–derived lymphocytes are not necessary for allogeneic effector cell rejection. Bone marrow–derived lymphocytes are not necessary for allogeneic effector cell rejection. (A) C57BL/6 mice bearing B16 tumors were irradiated with 9 Gy TBI and injected on day 0 with 106 allogeneic pmel-1b/d cells, vaccinia virus encoding hgp100, and exogenous rhIL-2 (alloPVI) or with 106 syngeneic pmel-1b/b cells, vaccinia virus encoding hgp100, and exogenous rhIL-2 (synPVI). On the day after cell transfer, the mice received syngeneic BMT with 106 unsorted bone marrow cells derived either from wild-type animals (wt BM) or Rag1−/− (Rag1−/− BM) animals. Results of tumor area are the mean of measurements of at least 5 mice per group (± SEM). Data are representative of 2 independent experiments. (B) After 30 days, mice that received allogeneic effector cells were killed, and the spleens were analyzed by flow cytometry for the presence of the transferred cells. Allogeneic CD8+ H-2d+ cells were undetectable at day 30 in both allogeneic PVI groups independently of the type of bone marrow cells they received. Andrea Boni et al. Blood 2008;112:4746-4754 ©2008 by American Society of Hematology

GVHD-like reactions are doubtful when the TCR repertoire is limited. GVHD-like reactions are doubtful when the TCR repertoire is limited. (A,B). C57BL/6–pmel-1 (H-2b/b) cells were stimulated with the relevant peptide and cultured for 1 week in IL-2 and subsequently challenged in overnight cocultures against irradiated splenocytes derived from different inbred mouse strains (SJL, H-2s/s and DBA, H-2d/d) and F1 mice (B6-A F1, H-2b/a; B6-C3H F1, H-2b/k; and B6-BALB/c F1, H-2b/d) displaying different allogeneic MHC haplotypes in the presence or the absence of the relevant peptide gp10025-33 (+ pep). Syngeneic C57BL/6 H-2b/b irradiated splenocytes in the presence or the absence of the relevant gp10025-33 peptide were used as positive and negative controls, respectively. Panels A and B are representative of 2 independent experiments. (C) pmel-1 cells were generated on a B6-C3H F1 background (H-2b/k) and used in combination with vaccinia virus encoding hgp100 and exogenous rhIL-2 to treat B16 tumors established for 10 days in either B6-C3H F1 (SynPVI) or in B6-DBA F1 mice (H-2d/b) (AlloPVI). All groups were irradiated with 9 Gy TBI and given autologous BMT. Some groups also received different doses (104 or 105) of open repertoire B6-C3H F1 CD8+ naive T cells in conjunction with pmel-1 cells, vaccine, and rhIL-2 (AlloPVI + 105 CD8 and AlloPVI + 104 CD8). Results of tumor area are the mean of measurements of at least 5 mice per group (± SEM). Data are representative of 2 independent experiments. (D) Percentage of initial weight of mouse groups is shown. Andrea Boni et al. Blood 2008;112:4746-4754 ©2008 by American Society of Hematology