Volume 15, Issue 8, Pages 1558-1563 (August 2007) Chemotherapy Enhances CD8+ T Cell-mediated Antitumor Immunity Induced by Vaccination With Vaccinia Virus  Chung Kil Song, Hee Dong Han, Kyung Hee Noh, Tae Heung Kang, Yong Sung Park, Jin Hee Kim, Eun Suk Park, Byung Cheol Shin, Tae Woo Kim  Molecular Therapy  Volume 15, Issue 8, Pages 1558-1563 (August 2007) DOI: 10.1038/sj.mt.6300221 Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 1 Combined therapy of intraperitoneally (IP)-injected Vac-Sig/E7/LAMP-1 and oral epigallocatechin-3-gallate (EGCG) treatment on the growing tumor. C57BL/6 mice were inoculated with 5 × l05 TC-1 tumor cells. Mice received a vaccination with 3 × l06 plaque-forming units/mouse of Vac-WT or Vac-Sig/E7/LAMP-1 via the IP route at 7 days after tumor inoculation. Three days after vaccination, EGCG was administered in the drinking water at a concentration of 0.5 mg/ml for 5 days. (a) Tumor volumes (mm3) from each group were recorded for 14 days after vaccination. (b) The experimental schedule of the vaccination and EGCG treatment. The data presented in this figure are from one representative experiment of the three performed. Vac-Sig/E7/LAMP-1, vaccinia virus-encoding E7 linked to the sorting signal of the lysosome-associated membrane protein 1. Molecular Therapy 2007 15, 1558-1563DOI: (10.1038/sj.mt.6300221) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 2 E7-specific CD8+ T cell responses after chemoimmunotherapy using epigallocatechin-3-gallate (EGCG) as an anticancer drug. Splenocytes from the treated mice in Figure 1 were harvested 14 days, cultured in vitro with major histocompatibility complex class I-restricted E7 (amino acids 49–57) peptide overnight and stained for both CD8 and intracellular interferon-γ (IFN-γ). (a) Representative flow cytometry data. (b) The bar graph depicting the number of antigen-specific IFN-γ secreting CD8+ T cells precursors/3 × 105 splenocytes (mean ± SD). The data presented in this figure are from one representative experiment of the two performed. Sig/E7/LAMP-1, DNA vaccine-encoding E7 linked to the sorting signal of the lysosome-associated membrane protein 1. Molecular Therapy 2007 15, 1558-1563DOI: (10.1038/sj.mt.6300221) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 3 In vivo antibody depletions of mice treated with the combination of epigallocatechin-3-gallate and Vac-Sig/E7/LAMP-1. TC-1 bearing mice (five per group) were treated as described in Figure 1b, CD4, natural killer (NK) and CD8 depletions were initiated 5 days after vaccination and lasted till the end of the experiment. The bar graph represents the volume of tumor 24 days after TC-1 tumor cells challenge. The data presented in this figure are from one representative experiment of the two performed. Molecular Therapy 2007 15, 1558-1563DOI: (10.1038/sj.mt.6300221) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 4 Synergistic antitumor therapeutic effects after combined Vac-Sig/E7/LAMP-1 vaccination and conventional anticancer drug treatment. C57BL/6 mice (five per group) were vaccinated with Vac-WT or Vac-Sig/E7/LAMP-17 days after tumor inoculation and then further treated with or without conventional anticancer drugs 3 days after vaccination. (a) Cisplatin (CDDP); (b) cyclophosphamide (CTX); (c) doxorubicin (DOX). Tumor volumes (mm3) from each group were recorded for 25 days after tumor inoculation. The data presented in this figure are from one representative experiment of the two performed. Vac-Sig/E7/LAMP, vaccinia virus-encoding E7 linked to the sorting signal of the lysosome-associated membrane protein 1. Molecular Therapy 2007 15, 1558-1563DOI: (10.1038/sj.mt.6300221) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 5 Combined Vac-Sig/E7/LAMP-1 vaccination and conventional anticancer drug treatment generated an enhanced E7-specific CD8+ T cell immune response compared to monotherapy alone. Splenocytes from the treated mice in Figure 4 were harvested 14 days after vaccination, and cultured in vitro with major histocompatibility complex class I-restricted E7 (amino acids 49–57) peptide overnight and stained for both CD8 and intracellular interferon (IFN)-γ. (a) Cisplatin (CDDP); (b) cyclophosphamide (CTX); (c) doxorubicin (DOX). The bar graph depicts the number of E7-specific IFN-γ-secreting CD8+ T cells/3 × 105 splenocytes (mean ± SD). The data presented in this figure are from one representative experiment of the there performed. Sig/E7/LAMP-1, DNA vaccine-encoding E7 linked to the sorting signal of the lysosome-associated membrane protein 1. Molecular Therapy 2007 15, 1558-1563DOI: (10.1038/sj.mt.6300221) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 6 Synergistic antitumor therapeutic effects after combined Vac-Sig/E7/LAMP-1 vaccination and cyclophosphamide (CTX) treatment. TC-1 P3 (A15)-bearing C57BL/6 mice (five per group) were vaccinated with Vac-WT or Vac-Sig/E7/LAMP-17 days after tumor inoculation and then further treated with or without CTX 3 days after vaccination. (a) Tumor volumes (mm3) from each group were recorded for 25 days after tumor inoculation. (b) The bar graph depicting the number of E7-specific interferon (IFN)-γ-secreting CD8+ T cells/3 × 105 splenocytes 14 days after vaccination. The data presented in this figure are from one representative experiment of the two performed. Sig/E7/LAMP-1, DNA vaccine E7 linked to the sorting signal of the lysosome-associated membrane protein 1. Molecular Therapy 2007 15, 1558-1563DOI: (10.1038/sj.mt.6300221) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions