gms | German Medical Science

Objective: A pilot study evaluating the clinical outcome of stereotactic interstitial photodynamic therapy (iPDT) as primary treatment in selected GBM patients resulted in a surprisingly favorable long-term progression free survival. We hypothesized that treatment-induced modulation of the adaptive immune response might account for these effects. We thus set out to identify the impact of PDT-treated GBM cells on the effector functions of primary human T cells.

Method: After incubation with 5-ALA for 4 hours, the human glioblastoma cell line U87 was subjected to PDT-treatment (3 Joule/cm²). CD3/CD28-activated primary blood mononuclear cells obtained from healthy donors were co-cultivated with PDT-treated U87 or untreated controls for 48 hours. Thereafter, CD4+ and CD8+T cells were separated by magnetic cell separation. Expression of cytokines and activation markers were measured by real-time PCR (qPCR) and flow cytometry. Cytotoxicity was determined by ELISPOT and CalceinAM assays. Differences between groups were analyzed using Student's t test or Mann-Whitney U test, respectively. Values given represent means ± SEM.

Results: After co-culturing with PDT-treated U87 cells, in vitro activated primary human CD4+T cells did not exhibit significant changes in both cytokine profiles and activation markers. In contrast, in CD8+ T cells significantly increased mRNA and protein expression of Interferon-γ and Perforin were found (qPCR 2.26-fold ± 0,36 for Perforin and 4.9-fold ± 0.12 for Interferon-γ; n=8, p<0.001; Perforin-ELISPOT 3.5-fold ± 0.78, n=3, p=0.03). Cytotoxic activity was enhanced by 13 ± 4.4%, accordingly (CalceinAM fluorescence of supernatants, n=5, p=0.01). Moreover, flow cytometry revealed a significant decrease of the co-inhibitory T-Cell marker CTLA4 (23 ± 3.5%, n=5, p<0.001).

Conclusions: These results provide evidence that PDT treatment of GBM cells increases the cytotoxic potential of human CD8+ T cells. This implies that the favorable clinical response of GBM patients after iPDT may – at least partially – result from enhanced cytotoxic capabilities of CD8+ T cells thereby contributing to long-term tumor control.