Volume 17, Issue 5, Pages 906-913 (May 2009) Adoptive Transfer of Human Papillomavirus E7-specific CTL Enhances Tumor Chemoresponse Through the Perforin/Granzyme-mediated Pathway  Jeong-Im Sin, Jung-Min Kim, Sung Hwa Bae, In Hee Lee, Jong Sup Park, Hun Mo Ryoo  Molecular Therapy  Volume 17, Issue 5, Pages 906-913 (May 2009) DOI: 10.1038/mt.2009.32 Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 1 Evaluation of tumor cell lytic activity of CTL effector cells in vitro, determination of CD8+ T cells as an effector cell for tumor cell killing, the percentage of CD8+ T cells in CTL effector cells, and therapeutic effects of adoptive therapy using CTL effector cells and chemotherapy against established tumor. For generation of E7-specific CTL effector cells, animals were immunized s.c. with E7 subunit vaccines at 0 and 2 weeks, At 5 weeks, mice were killed and spleen was obtained. Splenocytes were subsequently stimulated for 5 days with E7 CTL peptides and rIL-2. These CTL effector cells were evaluated for in vitro lytic activity against TC-1 and nonspecific target B16 cells (a), and tested for cell populations responsible for lytic activity by T-cell subset depletion (b). (b) Whole splenocytes from animals immunized as shown in a, as well as the CD4+ T cell- and CD8+ T cell-depleted populations were stimulated in vitro for 5 days with E7 CTL peptides and rIL-2 and then used for testing lytic activity at the E:T ratio of 30:1. (c) The percentage of CD8+ T cells in the CTL effector cell populations was measured by flow cytometry. (d) For determination of antitumor therapeutic activity, tumor (7 mm)-bearing mice (n = 5/group) were injected i.t. with cisplatin (2.5 mg/kg) at days 0 and 7, and/or administered i.v. with 1 × 107 CTL effector cells at days 0 and 1. Tumor size was measured biweekly using a caliper and mean tumor sizes were recorded. The numbers in (/) denote the number of mice showing a complete tumor regression/the number of mice tested and represent a combination of two experiments. Values and bars represent mean CTL lytic activity and tumor sizes, and the SD, respectively. This was repeated with similar results. *P < 0.05 compared to negative control. **P < 0.05 compared to cisplatin. ***P < 0.05 compared to cisplatin or adoptive therapy alone. CTL, cytotoxic T lymphocyte; E, effector; Neg. control, negative control; T, target. Molecular Therapy 2009 17, 906-913DOI: (10.1038/mt.2009.32) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 2 Evaluation of antitumor protective responses to parental tumor cell rechallenges. (a) All five tumor-regressed animals of Figure 1d were rechallenged s.c. with 4 ×105 TC-1 cells per mouse at 60 days after the first treatment with cisplatin plus adoptive CTL therapy. Naive control shows age-matched naive animals. (b,c) Another group of tumor-regressed animals (n = 5) of Figure 1d (b) and naive controls (c) were rechallenged s.c. with 4 × 105 TC-1 cells per mouse in the right abdomen and with 4 × 105 B16 cells per mouse in the left abdomen at 60 days after the first treatment. Tumor size was measured biweekly using a caliper and mean tumor sizes were recorded. Values and bars represent mean tumor sizes and the SD, respectively. CTL, cytotoxic T lymphocyte. Molecular Therapy 2009 17, 906-913DOI: (10.1038/mt.2009.32) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 3 Expression levels of cellular surface markers and HPV-16 E7 antigens in TC-1 cells by treatment with cisplatin. TC-1 tumor cells were treated for 48 hours with the indicated doses of cisplatin. Cells were then analyzed for the mRNA expression levels of cell surface markers (Fas, ICAM-1, MHC class I, β2-microglobulin) and HPV-16 E7 antigens by RT-PCR (a). (b–d) Tumor cells were treated for 48 hours with cisplatin at the indicated doses and then reacted with anti-Fas-PE, anti-ICAM-1-PE, anti-H-2Kb-FITC, and isotype control-PE/FITC to analyze the expression levels of cell surface markers (Fas, ICAM-1, MHC class I) by flow cytometry. This was repeated with similar results. FITC, fluorescein isothiocyanate; HPV, human papillomavirus; ICAM-1, intercellular adhesion molecule-1; MHC, major histocompatibility complex; PE, phycoerythrin; RT-PCR, reverse transcription PCR. Molecular Therapy 2009 17, 906-913DOI: (10.1038/mt.2009.32) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 4 No blockage of increased sensitivity of cisplatin-treated tumor cells to CTL-mediated killing by treatment with anti-FasL and anti-ICAM-1 Abs. TC-1 tumor cells were treated for 48 hours with cisplatin (1.0 µg/ml) and then used as target cells, which were coincubated with CTL effector cells at the indicated E:T ratios in the presence of isotype Ab controls, anti-FasL, and anti-ICAM-1 Abs. Abs were used at 10 µg/ml. Values and bars indicate mean %CTL and the SD, respectively. This was repeated twice with similar results. Ab, antibody; CTL, cytotoxic T lymphocyte; E, effector; ICAM-1, intercellular adhesion molecule-1; T, target. Molecular Therapy 2009 17, 906-913DOI: (10.1038/mt.2009.32) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 5 Induction of FasL on CTL effector cells and its effect on antitumor activity in vitro and in vivo, and the functional role of Fas expressed on cisplatin-treated tumor cells. (a) CTL effector cells were prepared as shown in Figure 1. These cells were treated for additional 3 hours with or without 10 ng/ml PMA and 3 µg/ml ionomycin at the final stage of 5-day stimulation periods. Cells were washed and stained with FITC-labeled anti-CD8 and either PE-labeled anti-FasL or PE-labeled isotype control Abs. FITC-stained CD8+ T cells (1 × 104) were gated and read on the relative intensity of PE displaying the expression levels of FasL on the CD8+ T-cell surface. nontreatment (isotype-PE); nontreatment (anti-FasL-PE); PMA/ionomycin treatment (isotype-PE); PMA/ionomycin treatment (anti-FasL-PE). (b) TC-1 tumor cells were treated for 48 hours with 1.0 µg/ml cisplatin and then used as target cells, which were cocultured with CTL effector cells obtained after PMA/ionomycin-treatment (FasL-induction) or nontreatment (noninduction) for measuring in vitro tumor lytic activity at the indicated E:T ratios. (c) Tumor (7 mm)-bearing mice (n = 5/group) were injected i.t. with cisplatin (2.5 mg/kg) at days 0 and 7, and administered i.v. with 3 × 106 CTL effector cells previously treated for 3 hours with PMA/ionomycin (FasL induction) or without PMA/ionomycin (noninduction) at day 0. Tumor size was measured biweekly, and mean tumor sizes were recorded. (d) TC-1 tumor cells (1.3 × 104 cells/well), previously treated for 48 hours with 1 µg/ml cisplatin, were added with agonist anti-Fas Abs at the indicated doses. Cells were incubated at 37 °C for 15 hours and then cell supernatants were taken to evaluate the levels of lytic activity using cytotoxicity assay. Values and bars indicate mean %lytic activity, tumor sizes, %cytotoxicity, and the SD, respectively. This was repeated twice with similar results. *P < 0.05 compared to negative control. **P < 0.05 compared to cisplatin alone. Ab, antibody; CTL, cytotoxic T lymphocyte; FITC, fluorescein isothiocyanate; Neg. control, negative control; PE, phycoerythrin; PMA, phorbol 12-myristate 13-acetate. Molecular Therapy 2009 17, 906-913DOI: (10.1038/mt.2009.32) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 6 Blockage of increased sensitivity of cisplatin-treated tumor cells to CTL-mediated killing by treatment with EGTA, and antitumor effects of adoptive therapy using CTL effector cells from wild type versus perforin KO mice in combination with cisplatin. (a) For in vitro CTL sensitivity assay, TC-1 tumor cells were treated for 48 hours with cisplatin (1.0 µg/ml) and then used as target cells, which were coincubated with CTL effector cells at the indicated E:T ratios in the presence or absence of 4 mmol/l EGTA/2 mmol/l MgCl2. Values and bars indicate mean %CTL and the SD, respectively. This was repeated twice with similar results. (b,c) For animal studies, each group (n = 12) of wild type and perforin KO mice was immunized s.c. with E7 subunit vaccines at 0 and 2 weeks, At 5 weeks, mice were killed and spleen was obtained. Splenocytes were subsequently stimulated for 5 days with E7 CTL peptides and rIL-2. To examine antitumor activity, tumor (5 mm)-bearing mice (n = 5/group) were injected i.t. with cisplatin (2.5 mg/kg) at days 0 and 7, and/or administered i.v. with 1 × 107 CTL effector cells from wild type (b) and perforin KO mice (c) at days 0 and 1. Tumor size was measured biweekly using a caliper and mean tumor sizes were recorded. Values and bars represent mean tumor sizes and the SD, respectively. This was repeated twice with similar results. *P < 0.05 compared to negative control. **P < 0.05 compared to cisplatin, adoptive therapy or E7 vaccines alone. CTL, cytotoxic T lymphocyte; EGTA, ethylene glycol tetraacetic acid; KO, knockout. Molecular Therapy 2009 17, 906-913DOI: (10.1038/mt.2009.32) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions