Ex vivo activation and expansion of γδ T cells for immunotherapy of glioblastoma.

2553 Background: Activated γδ T cells will efficiently kill established glioblastoma (GBM) cell lines in vitro. However, their potential for cell therapy has been limited by their relatively small numbers in the peripheral blood and their sensitivity to activation induced cell death (AICD) during ex vivo expansion. Our laboratory developed a method whereby γδ T cells, when stimulated by IFN-γ, IL- 12, and anti-CD2, acquire resistance to AICD and can be expanded in culture with anti-CD3 and IL-2. The γδ T cells expanded by this method are highly cytotoxic to GBM. We have now sought to adapt the laboratory method for human use. METHODS: The laboratory method and selection procedure were modified to employ pharmaceutical-grade reagents. Peripheral blood mononuclear cells (1.0 × 106/ml) were stimulated overnight with media containing cGMP grade human serum, IFN-γ, IL-12, and anti-CD2. Media containing human serum, IL-2 and anti-CD3 was then added (1 v/v) and refreshed 1 v/v 3× weekly. The cultures were harvested after two weeks followed by depletion of CD4+ and CD8+ T cells, thereby enriching γδ T cells. Cytotoxicity of expanded of γδ T cells was evaluated against GBM primary tumor cultures, established GBM cell lines, and cultured astrocytes using a commercial flow cytometric method. RESULTS: After two weeks in culture, γδ T cells expanded up to 1000 fold (usually 200-400 fold), consistent with results from our previous research-scale method. Positive selection yielded a γδ T cell product with >80% purity and >70% recovery. The cell products exhibited incremental cytotoxicity against several primary GBM cultures and established GBM cell lines. Astrocytoma cells were not killed. CONCLUSIONS: Apoptosis-resistant γδ T cells can be expanded and selected using clinically approvable reagents and in numbers sufficient for immunotherapy of malignant brain tumors. Initial data show that expanded γδ T cells retain cytotoxicity against the GBM primary cultures and spare normal astrocytes. No significant financial relationships to disclose.

Duke Scholars

Author Richard D Lopez Medicine, Hematologic Malignancies and Cellular Therapy