Cytogenetic and DNA microarray analysis of human glioma cell models resistant to alloreactive cytotoxic T lymphocytes

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Genetic instability and heterogeneity within gliomas may allow intrinsically immunoresistant cells to escape local adoptive immunotherapy with alloreactive cytotoxic T lymphocytes (aCTL), sensitized to patient HLA antigens. To study the mechanism(s) by which glioma cells resist aCTL, we isolated two immunoresistant (IR) glioma cell clones (13-06-IR29 and 13-06-IR30) from a glioblastoma cell explant that received continuous immunoselective pressure from multiple aCTL preparations. We previously reported that relative to parental cells (13-06-MG), the IR clones resisted aCTL lysis. In addition, flow cytometric analysis revealed that downregulation of HLA class I expression was not likely a mechanism of resistance to aCTL-mediated cell damage (Proceedings of the AACR, Volume 45, March 2004). We have expanded characterization of the IR clones by cytogenetic and molecular cytogenetic techniques. By G-banding and spectral karyotype analysis, a gain of chromosome 7 and loss of chromosomes 10 and 17p were observed in parental and immunoselected cells and the IR clones. The number of chromosomes per cell for parental and immunoselected cells (range 72 to 87 chromosomes/cell) was higher than in the IR cells (range 55 to 76 chromosomes/cell). In addition, preferential loss of chromosomes 2q, 4p, 6, and gain of 20p were observed in the majority of IR cells analyzed. Based upon the cytogenetic findings, a hypothetical model is proposed that traces the origins of the IR clones back to clonal variants within the immunoselected and parental cell populations. Cell injury data, obtained by in-vitro quantitative morphologic and 7-aminoactinomycin D flow cytometric assays, revealed reduced apoptotic cell death when IR cells were coincubated with aCTL cells. Since changes in apoptosis were observed, we examined the expression patterns of apoptosis-related genes in parental and IR cells by cDNA microarray analysis. Interestingly, we found a complete lack of Apaf-1 expression in clone 13-06-IR30 and a 20-fold downregulation in clone 13-06-IR29. The data suggested that Apaf-1 might play a role in determining the sensitivity of glioma cells to aCTL-mediated damage. In agreement with this role, we observed a 4-fold or greater upregulation of Apaf-1 in an immunosensitive clone, 13-06-IS52, relative to the parental and IR cells. We are exploring if modulation of Apaf-1 expression regulates the sensitivity of glioma cells to aCTL-mediated damage. Support: NIH F31 CA94834 & RO1 NS56798