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Cytokine Gene Therapy of Gliomas: Induction of Reactive CD4⁺ T Cells by Interleukin-4-transfected 9L Gliosarcoma Is Essential for Protective Immunity¹

Brain Tumor Center, University of Pittsburgh Cancer Institute [K. M. G-S., H. O., C. S. B-S., L. A. V., J. A., M. T. L., I. F. P., M. E. B., W. H. C.], and Departments of Neurological Surgery [K. M. G-S., H. O., J. A., I. F. P., M. E. B.], Pathology [C. S. B-S., L. A. V., W. H. C.], Surgery [M. T. L.], and Molecular Genetics and Biochemistry [M. T. L.], University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213

Tumor cells genetically modified to secrete cytokines stimulate potent immune responses against peripheral and central nervous system tumors; however, variable results on the efficacy of this strategy for therapeutic intervention against established intracranial neoplasia have been reported. We have found that vaccination with rat 9L gliosarcoma cells expressing interleukin 4 (9LmIL4) induced a specific, protective, immune response against rechallenge with parental 9L tumors. In naive rats, sham-transfected 9L (9Lneo) tumors and 9LmIL4 tumors grew at comparable rates for 12–14 days, and then 9LmIL4 tumors regressed. After regression of 9LmIL4 tumors, rats were resistant to rechallenge with parental 9L cells. To investigate the mechanism(s) responsible for 9LmIL4-induced immunity, the phenotype and function of tumor-infiltrating lymphocytes (TILs) in 9Lneo and 9LmIL4 tumors were compared. In flow cytometric analyses, it was determined that CD4⁺ T cells were the predominant cell type in both 9Lneo and 9LmIL4 tumors at day 10. However, at the onset of regression (day 14), 9LmIL4 tumors were infiltrated predominantly by CD8⁺ T cells. To investigate functional aspects of the anti-9L tumor responses, we assessed the capacity of 9LmIL4 TILs to mediate specific lytic function or production of cytokines. In response to parental 9L, TILs isolated from day 14 9LmIL4 tumors were demonstrated to produce substantially greater amounts of IFN- than did TILs from 9Lneo tumors. Although freshly isolated TILs from 9LmIL4 or control tumors did not lyse 9L cells in ⁵¹Cr-release cytotoxicity assays, specific cytotoxicity was demonstrable using TILs from day 14 9LmIL4 or splenocytes from 9LmIL4-bearing rats after their restimulation for 5 days with parental 9L tumor cells in vitro. Antibody blocking studies demonstrated that cytokine production and lytic activity by TILs, or splenocytes from 9LmIL4-immunized rats, were mediated in a T-cell receptor-dependent fashion. Because interleukin-4 also promotes humoral responses, quantity and isotype of immunoglobulins in sera from 9Lneo or 9LmIL4-immunized rats were compared. The amount of IgG1 antibodies was significantly increased in sera from 9LmIL4-immunized rats compared to sera from 9Lneo-bearing rats. Experiments using sublethally irradiated, naive rats adoptively transferred with splenocytes and/or sera from 9LmIL4-immunized or naive rats demonstrated that immune cells, with or without immune sera, protected recipients from challenge with parental 9L. Immune sera provided no protection when given with lymphocytes from naive rats, and it did not enhance protection against parental 9L when given in conjunction with lymphocytes for 9LmIL4-immunized rats. In additional adoptive transfer experiments, an essential role for CD4⁺ T cells in immunity was observed because their depletion from among splenocytes of 9LmIL4-immunized rats eliminated the protective effective against 9L, whereas depletion of CD8⁺ cells resulted in a more limited effect on protection against 9L. These data suggest that strategies for inducing systemic, long-term tumor-specific reactivity among CD4⁺ T cells will be critical for the development of immunotherapy of gliomas.

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