Effect of irradiation on cytokine production, MHC antigen expression, and vaccine potential of interleukin-2 and interferon-gamma gene-modified melanoma cells.

Recent studies have shown that tumor cells transduced with interleukin-2 (IL-2) or interferon-gamma (IFN-gamma) genes stimulated a potent and specific antitumor immunity in experimental animals. For use as a human vaccine, tumor cells must be inactivated by irradiation to ensure the arrest of their growth. This study was undertaken to examine the effects of irradiation (10,000 rad) on the growth characteristics and vaccine potential of IL-2 and IFN-gamma-modified human melanomas and B16 murine melanoma. Irradiation caused cessation of cell growth and gradual reduction of cell number. Irradiated melanoma cells displayed 1.5 to 10-fold increases in the surface expression of MHC class I and/or class II antigens. The increases in MHC antigens persisted for 7-14 days postirradiation and then declined thereafter. Furthermore, IL-2- and IFN-gamma-transduced melanoma cells showed enhanced expression of the cytokine mRNA and increased cytokine secretion after irradiation. The effect of irradiation on the vaccine potential of the transduced cells was examined in C57BL/ 6 mice by prophylactic immunization and immunotherapy, and in nude mice by mixed transplantation assays. The irradiated, cytokine-transduced B16 cell vaccine was as or more effective than the unirradiated vaccine. These irradiated vaccines protected the animals against a challenging tumorigenic dose of B16 parental cells and suppressed the growth of 4-day-established B16 lung metastases. The ability of the irradiated IL-2-transduced human melanomas to inhibit the growth of admixed parental melanoma cells was retained but was less efficacious than unirradiated cells. The results suggest that irradiation does not abrogate the vaccine potential of IL-2- and IFN-gamma-transduced melanomas. These findings have implications for designing specific active immunotherapy protocols utilizing cytokine gene-modified tumor cells.

Transduction of human renal carcinoma cells with human gamma-interferon gene via retroviral vector.

We used a retroviral vector containing a human gamma-interferon (gamma-IFN) gene to transduce 13 renal carcinoma cell lines. The transduction efficiencies ranged from 0% to 60%, as determined by using an analogous vector containing the LacZ marker gene. In addition, gene-transferred resistance to the antibiotic neomycin was used to select for transduced cells. Nine of 13 lines were successfully transduced. Transduction was associated with the morphologic change of elongation, and there was a marked decrease in cell growth rate. Transduced cells secreted varying amounts (20-1076 pg/10(6) cells/d) of gamma-IFN as measured by enzyme-linked immunosorbent assay for at least 2 to 3 weeks after transduction (including 1 day of transduction, 6-7 days of selection, and an additional 8-12 days before the first passage of the transduced cells). Human leukocyte antigen (HLA) class II expression was markedly increased in six of seven cell lines; HLA class I expression was significantly increased in two of eight lines. Transduced cells that were subjected to cryopreservation after irradiation still produced gamma-IFN and expressed HLA class I and II antigens, although generally at lower levels than before these manipulations. This study confirms that retroviral vector transduction of the human gamma-IFN gene into renal carcinoma cells is feasible and associated with persistent production of gamma-IFN and increased expression of HLA class I and II molecules, and these effects are retained after irradiation and cryopreservation. This suggests that an autologous tumor cell vaccine trial with irradiated gamma-IFN gene-transduced renal carcinoma cell is rationale and feasible.

Interferon gamma (IFNgamma) gene transfer of an EMT6 tumor that is poorly responsive to IFNgamma stimulation: increase in tumor immunogenicity is accompanied by induction of a mouse class II transactivator and class II MHC.

Abstract Interferon gamma (IFNgamma) is an important cytokine with immunomodulatory properties that include activation of immune cells and induction of class I and class II major histocompatibility complex antigens. In this study a retroviral vector was used to introduce the IFNgamma gene into EMT6 tumor cells to assess the effect of IFNgamma gene expression on tumor immunogenicity. Transfectants were selected in G418-containing tissue-culture medium and were determined to express the inserted IFNgamma gene by reverse transcriptase/polymerase chain reaction. Flow-cytometric analysis revealed that parental unmodified EMT6 cells constitutively expressed only class I MHC and were poorly responsive to exogenous IFNgamma stimulation, whereas class II MHC was induced in IFNgamma-transfected cells. The induction of class II MHC in IFNgamma-transfected cells correlated with the expression of a mouse class II transactivator gene that was dormant in unmodified or mock-transfected cells. In addition, IFNgamma-gene-transfected tumor cells were found to secrete up to 17 ng IFN (equivalent to 75 units/10(6) cells) by enzyme-linked immunosorbent assay (ELISA). Whereas parental EMT6 cells grew unchecked, the growth of genetically modified tumor cells was significantly inhibited in immunocompetent mice. Rechallenge of animals that rejected an IFNgamma-transfected EMT6 clone (EMT6-B17) with parental EMT6 cells resulted in tumor rejection, suggesting that IFNgamma-transfected EMT6 cells were able to induce long-term immunity. Mixing experiments using gene-transfected and unmodified tumor cells demonstrated that 10% of IFNgamma-transfected cells in the population was sufficient to protect mice against subsequent challenge with tumorigenic EMT6 cells. These studies demonstrate that the immunogenicity of tumor cells that are poorly responsive to exogenous IFNgamma can be enhanced by inserting and expressing the IFNgamma transgene. These findings also suggest a role for class II MHC in reducing tumorigenicity of the EMT6 tumor and inducing long-term tumor immunity.

Transduction of human melanoma cells with the gamma interferon gene enhances cellular immunity.

Human tumor cells transduced with the gamma interferon (gamma IFN) gene are currently used in specific active immunotherapy protocols to enhance the antitumor immune responses of cancer patients. This in vitro study was undertaken to examine the initial events in the cellular immune response that may occur following the administration of the gamma IFN-transduced cell vaccine. Human melanoma tumor cell lines were transduced with a MoMLV-based retroviral vector carrying the human gamma IFN gene. The transduced cells expressed the cytokine gene, secreted biologically active gamma IFN, and exhibited enhanced expression of MHC class I and class II (HLA-DR), and ICAM-1 surface antigens. The gamma IFN-transduced and corresponding parental melanoma cells were used for the induction of short-term lymphocyte cultures. Peripheral blood lymphocytes or lymph node cells from 20 melanoma patients were stimulated for 5 to 15 days with autologous or MHC class I-matched allogeneic parental or gamma IFN-transduced melanoma cells. Seven of the 20 lymphocyte cultures showed substantial increases in lytic activity following stimulation with the transduced melanoma cells in comparison to control lymphocyte cultures stimulated with unmodified parental melanoma. The cytolytic activity stimulated with gamma IFN-modified melanomas was mediated partly by MHC-restricted cytotoxic T lymphocytes and partly by NK cells. Lymphocyte cultures that displayed increases in cytotoxicity after stimulation with the gamma IFN-transduced melanoma cells also exhibited enhanced expression or induction of one or more of the following lymphokines: IL-4, IL-1 alpha, IL-1 beta, gamma IFN, and TNF-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Retrovirus-mediated gene transfer of the human gamma-IFN gene: a therapy for cancer.

A retroviral vector-mediated gene transfer system was used to introduce m gamma-IFN and h gamma-IFN genes into mouse and human tumor cells, respectively. Murine tumor cell lines and primary human melanoma tumor cells were successfully transduced with gamma-IFN vector, and these transduced cells secreted measurable levels of biologically active m gamma-IFN and h gamma-IFN, respectively. Both murine and human tumor cell lines that expressed gamma-IFN exhibited increased surface expression of HLA class I antigens when tested by Western blot and FACS analysis. gamma-IFN--transduced human melanoma cells were more active in stimulating tumor-specific cytolytic activity of CTLs from melanoma patients in vitro. m gamma-IFN--transduced tumor cells were substantially less tumorigenic than the corresponding parent tumor cell lines in immune-competent mice. In addition, injection of m gamma-IFN--transduced tumor cells resulted in activation of tumor-specific CTL in vivo. We plan to use gamma-IFN--transduced autologous tumor cells to boost host immune responses as a potential therapy for human melanoma.