Volume 13, Issue 1, Pages 183-193 (January 2006) Recombinant Vaccinia Virus Expressing Interleukin-2 Invokes Anti-tumor Cellular Immunity in an Orthotopic Murine Model of Head and Neck Squamous Cell Carcinoma  Santanu Dasgupta, Malaya Bhattacharya-Chatterjee, Bert W. O'Malley, Sunil K. Chatterjee  Molecular Therapy  Volume 13, Issue 1, Pages 183-193 (January 2006) DOI: 10.1016/j.ymthe.2005.06.481 Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Increased number of T cells in splenocytes and TDLN cells in mice vaccinated with rvv-IL-2. Immune cells obtained from spleen and TDLN of individual mice on day 12 were assayed. Each group contained four mice. Cells were stained with PE-conjugated anti-mouse CD3 and FITC-conjugated CD4 or CD8 antibodies (bicolor). Isotype-matched PE- and FITC-conjugated irrelevant antibodies were used in each case as negative controls. Gates were set on CD3+ T cell population coexpressing CD4+ or CD8+. The percentage of CD3+ CD4+ or CD3+ CD8+ T cells is presented in the upper right quadrant for each group and mean ± SE values of four mice analyzed independently are given on the top of the quadrant. The percentage of CD3+ CD4+ or CD3+ CD8+ T cells was significantly higher in both spleen and TDLN in the rvv-IL-2-vaccinated group compared to the control rvv-lacZ group. This experiment was repeated two more times with similar results. Molecular Therapy 2006 13, 183-193DOI: (10.1016/j.ymthe.2005.06.481) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Induction of cytolytic activities of immune cells from spleen and TDLN by rvv vaccination. Mice were vaccinated on days 7 and 10 after tumor implantation. Cytolytic activities of (A, B) splenocytes and (C, D) TDLN cells were measured on days 9 and 12, respectively. The immune cells harvested from each mouse were cultured in the presence of irradiated SCCVII cells and rhIL-2 for 7 days as described under Materials and Methods. In the cytolytic assay, immune cells from PBS group were incubated with 51Cr-labeled target SCCVII/SF cells only, whereas rvv-lacZ- (empty symbols) or rvv-IL-2- (filled symbols) vaccinated mice were incubated with 51Cr-labeled SCCVII/SF as well as control targets AG104A fibrosarcoma (H-2kk) and colorectal cells, C15 (K-2b). Percentage lysis is presented as the mean ± SE of the four mice analyzed in each group. Tumor-specific cytolytic activities were significantly higher in rvv-IL-2-vaccinated mice compared to those vaccinated with rvv-lacZ. This experiment was repeated two more times with similar results. Molecular Therapy 2006 13, 183-193DOI: (10.1016/j.ymthe.2005.06.481) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 3 CD8+ T cells mediated SCCVII/SF tumor cell lysis following rvv-IL-2 vaccination. Cytolytic activities were determined with purified CD4+ and CD8+ T cells harvested from (A) spleen and (B) TDLN of rvv-IL-2-vaccinated mice. Mice were vaccinated on day 7 after tumor implantation. Splenocytes and TDLN cells from individual mice were harvested on day 9, and CD4+/CD8+ T cells were isolated by anti-CD4/CD8 magnetic beads as described under Materials and Methods. Before cytolytic assay, purified cells from each mouse were cultured separately in the presence of irradiated SCCVII/SF cells and rhIL-2 (1 ng/well) for 7 days in a tissue culture incubator. Values are means ± SE of four mice analyzed in each group. Only purified CD8+ T cells from both spleen and TDLN induced significant lysis of SCCVII/SF target cells. This experiment was repeated two more times with similar results. Molecular Therapy 2006 13, 183-193DOI: (10.1016/j.ymthe.2005.06.481) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Tumor-specific cytolytic activities are mediated by MHC Class I restricted CD8+ T cells. Cytolytic activities were determined with (A, B) splenocytes and (C, D) immune cells obtained from regional lymph nodes of rvv-IL-2-vaccinated mice. Mice were vaccinated on day 7. Splenocytes and TDLN cells obtained from individual mice were harvested on day 9 and cultured in the presence of irradiated SCCVII/SF cells and rhIL-2 (1 ng/well) for 7 days as described under Materials and Methods. Murine anti-CD4, anti-CD8, anti-MHC-I (H-2Kk), and anti-MHC-II (1Ak) antibodies were used as blockades for the cytolytic activities. Isotype-matched irrelevant antibodies were used as controls. Values are means ± SE of four mice analyzed in each group. Cytolysis of SCCVII/SF cells by both the splenocytes and the TDLN cells from rvv-IL-2-vaccinated mice was significantly inhibited by anti-CD8 and -MHC I antibodies. This experiment was repeated two more times with similar results. Molecular Therapy 2006 13, 183-193DOI: (10.1016/j.ymthe.2005.06.481) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 5 Tumor-specific helper T cell activities induced by vaccination with rvv-IL-2. T cell proliferation assays were performed and stimulation indices of (A, B) bulk splenocytes and (C, D) purified immune cells harvested from spleen and TDLN were determined as described under Materials and Methods. Data are presented as means ± SE of four mice analyzed in each vaccinated group. Vaccination was administered on days 7 and 10 after tumor implantation. Bulk or purified immune cells from both spleen and TDLN were harvested on day 12 from individual mice and stimulated for 5 days in a tissue culture incubator using irradiated SCCVII/SF or AG104A or C15 tumor cells as stimulant. Concanavalin A was used as a positive control stimulant, whereas culture medium served as the background stimulant. Stimulation indices of (A) bulk splenocytes or (B) TDLN cells from the rvv-IL-2 group were significantly higher compared to that of the control rvv-lacZ group. Stimulation of purified CD4+ T cells was significantly higher in the presence of irradiated SCCVII/SF cells compared to the control irradiated C15 cells. Stimulation of the purified CD4+ T cells was also higher by irradiated SCCVII/SF cells compared to the purified CD8+ T cells. The experiments were repeated at least three times with similar results. Molecular Therapy 2006 13, 183-193DOI: (10.1016/j.ymthe.2005.06.481) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 6 Higher frequencies of IFN-γ-secreting T cells are induced in vivo by rvv-IL-2 vaccination. An IFN-γ ELISPOT assay was performed with (A) splenocytes and (B) immune cells obtained from TDLN of rvv-vaccinated mice on day 12. Cells were stimulated in the presence or absence of relevant SCCVII or irrelevant AG104A cells in a 96-well IFN-γ-coated plate. Data are reported as the average number of spots per 1 ± 105 responders ± SE of four mice analyzed individually in each group. Representative wells from ELISPOT plates are shown below the graph. Numbers of IFN-γ spots were significantly higher in the rvv-IL-2-vaccinated group compared to the control rvv-lacZ group when SCCVII cells were used as the stimulant. This experiment was repeated two more times with similar results. Molecular Therapy 2006 13, 183-193DOI: (10.1016/j.ymthe.2005.06.481) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 7 Diminished anti-tumor responses in rvv-IL-2-vaccinated mice following antibody-mediated depletion of CD8+ and CD4+ T cells. After tail vein injection of antibodies as described under Materials and Methods, depletion of the specific T cells was confirmed by flow cytometry. Tumor implantation was performed on day 0. Mice were vaccinated with rvv-IL-2 on days 7, 10, and 14 and depletion status was maintained with anti-CD4 and -CD8 antibodies before and during the treatment with rvv vaccines. On days 7, 9, 12, and 16, three mice from each treatment group were sacrificed and tumor weights were taken (mg ± SE). (A) Survival was significantly decreased in the vaccinated mice in which either CD8+ or CD4+ T cells or both were depleted compared to the control IgG2-injected or nondepleted groups (P < 0.0001). (B) Tumor sizes were also significantly larger in the depleted groups at different time points compared to the IgG2-treated or nondepleted groups. This experiment was repeated two more times with similar results. Molecular Therapy 2006 13, 183-193DOI: (10.1016/j.ymthe.2005.06.481) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions