Cytokine gene therapy of gliomas: Induction of reactive CD4(+) T cells by interleukin-4-transfected 9L gliosarcoma is essential for protective immunity

Tumor cells genetically modified to secrete cytokines stimulate potent immu ne responses against peripheral and central nervous system tumors; however, variable results on the efficacy of this strategy for therapeutic interven tion against established intracranial neoplasia have been reported. We have Found that vaccination with rat 9L gliosarcoma cells expressing interleuki n 4 (9LmIL4) induced a specific, protective, immune response against rechal lenge with parental 9L tumors, In naive rats, sham-transfected 9L (9Lneo) t umors and 9LmIL4 tumors grew at comparable rates for 12-14 days, and then 9 LmIL4 tumors regressed. After regression of 9LmIL4 tumors, rats were resist ant to rechallenge with parental 9L cells. To investigate the mechanism(s) responsible for 9LmIL4-induced immunity, the phenotype and function of tumo r-infiltrating lymphocytes (TILs) in 9Lneo and 9LmIL4 tumors were compared. In flow cytometric analyses, it was determined that CD4(+) T cells were th e predominant cell type in both 9Lneo and 9LmIL4 tumors at day 10, However, at the onset of regression (day 14), 9LmIL4 tumors were infiltrated predom inantly by CD8(+) T cells. To investigate functional aspects of the anti-9L tumor responses, we assessed the capacity of 9LmIL4 TILs to mediate specif ic lytic function or production of cytokines. In response to parental 9L, T ILs isolated from day 14 9LmIL4 tumors were demonstrated to produce substan tially greater amounts of IFN-gamma than did TILs from 9Lneo tumors. Althou gh freshly isolated TILs from 9LmIL4 or control tumors did not lyse 9L cell s in Cr-51-release cytotoxicity assays, specific cytotoxicity was demonstra ble using TILs from day 14 9LmIL4 or splenocytes from 9LmIL4-bearing rats a fter their restimulation for 5 days with parental 9L tumor cells in vitro. Antibody blocking studies demonstrated that cytokine production and lytic a ctivity by TILs, or splenocytes from 9LmIL4-immunized rats, were mediated i n a T-cell receptor-dependent fashion. Because interleukin-4 also promotes humoral responses, quantity and isotype of immunoglobulins in sera from 9Ln eo or 9LmIL4-immunized rats were compared. The amount of IgG1 antibodies wa s significantly increased in sera from 9LmIL4-immunized rats compared to se ra from 9Lneo-bearing rats. Experiments using sublethally irradiated, naive rats adoptively transferred with splenocytes and/or sera from 9LmIL4-immun ized or naive rats demonstrated that immune cells, with or without immune s era, protected recipients from challenge with parental 9L. Immune sera prov ided no protection when given with lymphocytes from naive rats, and it did not enhance protection against parental 9L when given in conjunction with l ymphocytes for 9LmIL4-immunized rats. In additional adoptive transfer exper iments, an essential role for CD4(+) T cells in immunity was observed becau se their depletion from among splenocytes of 9LmIL4-immunized rats eliminat ed the protective effective against 9L, whereas depletion of CD8(+) cells r esulted in a more limited effect on protection against 9L, These data sugge st that strategies for inducing systemic, long-term tumor-specific reactivi ty among CD4(+) T cells will be critical for the development of immunothera py of gliomas.