IMPS-15PDT-TREATED GBM CELLS INCREASE EFFECTOR FUNCTIONS OF HUMAN CD8+ T-CELLS

Abstract

OBJECTIVE: Photodynamic therapy (PDT)-induced modulation of the adaptive immune response might account for long-term survival observed in glioblastoma (GBM) patients after PDT. We here analyze the impact of PDT-treated GBM cells on the effector functions of primary human T cells. METHODS: After incubation with 5-aminolevulinic acid (5-ALA) for 4 hours, the human glioblastoma cell line U87 was subjected either to PDT (3 Joule/cm2) or temozolomide (350 mM). PDT and temozolomide doses were equivalent regarding the evoked cell death rates. CD3/CD28-activated primary blood mononuclear cells (PBMCs) obtained from healthy donors were co-cultivated with PDT- or temozolomide-treated U87 or untreated controls for 48 hours. Thereafter, CD4+ and CD8 + T cells were separated by magnetic cell separation. Expression of cytokines, cytotoxicity and activation markers was measured by quantitative RT-PCR (qPCR) and flow cytometry. Cytotoxicity was determined by ELISPOT and CalceinAM fluorescence assays. Differences between groups were analyzed using Student's t test (parametric distribution) or Mann-Whitney U test (non-parametric distribution). Values are means+/-SEM. RESULTS: In vitro activated primary human CD4 + T cells did not exhibit changes in their cytokine profiles upon coculturing with PDT-treated U87 cells. In CD8+ T cells, however, significantly increased expression levels of Interferon-gamma (qPCR, 1.8-fold+/-0.34; n = 14, p < 0.001) and Perforin (qPCR, 1.6-fold+/-0.14, n = 14, p < 0.001; Perforin-ELISPOT 1.3-fold+/-0.11, n = 8, p = 0.003) were seen. Cytotoxic activity was enhanced by 13+/-4.4% (CalceinAM fluorescence of supernatants, n = 5, p = 0.017). Flow cytometry revealed a significant decrease of the co-inhibitory T cell marker CTLA4 (23+/-3.5%, n = 5, p < 0.001). Following temozolomide treatment, CD8+ cytotoxic activity was not enhanced (n = 4, p = 0.7). CONCLUSION: 5-ALA PDT but not temozolomide treatment of glioblastoma cells increases the cytotoxic activity of human CD8+ T cells. This might contribute to long-term tumor control in a considerable number of glioblastoma patients after PDT.