Materials and Methods
Reagents and cell lines Recombinant human IL-1b, -2, -4, and -6 were kindly provided by Ohtsuka Pharmaceutical Co., Ltd. (Tokushima), Shionogi & Co., Ltd (Osaka), Ono Pharmaceutical Co., Ltd. (Osaka), and Ajinomoto, Inc. (Kawasaki), respectively. RHAMa medium was used for culture of human lymphocytes.25) The enzyme solution used for dissociation of tumor tissues consisted of collagenase type V (500 mg/liter), hyaluronidase type V (50 mg/liter), DNAse type I (12.5 mg/liter), penicillin (2x105 IU/liter)), streptomycin (100 mg/liter), and 10%(v/v) FBS in RPMI1640 medium. Monoclonal antibodies for HLA-typing were purchased from One Lamda (California) except those against A2 (MA2.2 clone, Nonesuch RD, ME) and Bw4 / A24 / A32 (HLA-01 clone, Sanbio Inc., The Netherlands). For detection of MHC-class I, the monoclonal antibody, W32/64, was used within 1 week of the primary culture of the tumor cells. The NK-sensitive cell line K562, the LAK-sensitive cell line Daudi, and the renal cancer cell lines OS-RC-2 and Hpt.10 were provided by RIKEN Cell Bank. A gastric carcinoma cell line, GT3TKB, submitted for the preliminary experiment was also obtained from routine cultures in RIKEN Cell Bank.
Target renal carcinoma cells Surgically resected specimens of renal carcinomas and associated normal renal tissues removed from a location as far as possible from the carcinoma were minced and dissociated with the enzyme solution at room temperature for 1 h. The mixture was placed on a layered gradient of Ficoll-Paque solutions (100% and 75% Ficoll layers) and centrifuged. Cells at the interface of the 100% and 75% Ficoll layers were collected and cultured in DMEM with 10% FBS. Normal cells and carcinoma cells were subcultured for one passage, and preserved frozen in liquid nitrogen until use in induction of CTL and subsequent experiments. Total cell numbers obtained depended on the tumor tissue size and cell viability that was affected by the latent time of the operation and transportation. Usually, 106 to 107 cells can be obtained either for normal cells and tumor cells from resected specimens. However, most of the cells died within next several days in the primary culture. Some of the carcinoma cells were maintained in DMEM with 10% FBS for several months, with the aim of establishing carcinoma cell lines.
Lymphocyte culture For LAK and NK culture, PBMC were collected by the conventional method of Ficoll-Paque gradient centrifugation. LAK were prepared by culturing PBMC (1x106 cells/ml) in RHAMa medium supplemented with 10% heat-inactivated FBS and 500 U/ml IL-2 for 2 weeks. NK were prepared by culturing PBMC (1x106 cells/ml) in the presence of X-ray-irradiated Daudi cells (1x105 cells/ml) and IL-2 (50 U/ml) for 2 weeks, as described elsewhere.26) Before addition of the PBMC for CTL induction, confluent renal carcinoma cells maintained in a 6-well plate were irradiated with 30 Gy of X-rays. The PBMC (106 cells/ml) were then cultured on these autologous carcinoma cells in the induction medium, i. e., RHAMa medium supplemented just before use with 5% autologous plasma and the IL-cocktail (IL-1b (167 U/ml), IL-2 (67 U/ml), IL-4 (67 U/ml), and IL-6 (134 U/ml)). The CTL induction culture was continued with appropriate changes of the medium (at least half of the medium was changed every 2 days) until the target cells disappeared completely. The CTL were then routinely subcultured on unirradiated autologous target cells at an E/T ratio of 10 in RHAMa medium supplemented with 5% autologous plasma, or 5% heat-inactivated FBS, and the IL-cocktail. The target cells were renewed once a week. Cytotoxic activity of CTL, LAK and NK was determined as described22) by co-culturing lymphocytes and the target tumor cells for 24 h, except in the cases indicated. The target cells adhering to the bottom of the wells were measured as surviving cells by crystal violet staining. If the lymphocytes were strongly cytotoxic, this assay is as sensitive for assessment of the killing activity as the standard 51Cr-release cytotoxicity assay when it is applied to the adherent target cells (see Fig. 1 of ref. 22). To exclude the possibility of simple growth inhibition of the tumor cells by the CTL, we separately measured the OD570 of each well of control target cells at the start of the killing assay and this value was taken as 100% in calculation of the percentage of surviving target cells. Note that the tumor cells at the E/T of 0 grew during the 24-h incubation period and therefore showed values higher than 100%. Each point shown is the mean of triplicate observations ± SD.
Flow cytometry and HLA-typing CTL and tumor cells were stained with FITC- or PE-labelled antibodies (against human CD3, CD4, CD8, CD56 and MHC class-I) according to the manufacturer's protocol. The stained cells were analysed by FACScan with LYSIS II software (Becton Dickinson, CA). Serological HLA-typing was performed by flow cytometry after staining with mouse monoclonal antibodies against HLA-A2, Bw4 / A24 / A32, A1 / A11 / A26, A11 / A24 / B41 / Cw1, A25 / A26, Bw4 / A9 / A32 A9 / A32 / A25, A23 / A24, and A33 / B8, and FITC-labelled goat secondary antibody against mouse IgG or IgM. HLA-subtypes, if required, were determined by PCR with HLA-A2402 allele-specific primer sets.27)
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|Liu, SQ; (1998). High rate of induction of human autologous cytotoxic T lymphocytes against renal caricinoma cells cultured with an interleukin cocktail. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Available at URL http://www.mcmaster.ca/inabis98/|
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