Analysis of Vγ9Vδ2 T lymphocytes response to human glioma cell lines: possible implications for therapy

Abstract

T cells link innate and acquired immunity. In humans 90% of circulating T cells express the V9V2 TCR rearrangement and recognize non peptidic antigens in a MHC-unrestricted manner. After antigen recognition, activated V9V2 T cells rapidly proliferate, produce high levels of cytokines and chemokines and can differentiate in cytotoxic effector cells. Specifically, V9V2 T cells recognize unprocessed non-peptidic compounds such as isopentenyl pyrophosphate (IPP), which are produced through the isoprenoid biosynthesis pathway. Moreover, V9V2 T cells can also be activated by aminobisphosphonates drugs through an indirect mechanism: they inhibit farnesyl pyrophosphate synthase, an enzyme of cholesterol biosynthesis, acting downstream of IPP synthesis; this inhibition, in turn, leads to the accumulation of endogenous IPP, directly recognized by V9V2 T cells. A specific feature of V2 T cell biology is their ability to recognize tumor cells presenting a dis-regulation in mevalonate pathway, resulting in an increase of phosphorilated metabolites such as isopentenyl-pyrophosphate (IPP). Thus, the increased isoprenoid metabolism in cancer cells induces V2 T cell activation through cellular IPP accumulation. Mevalonate cycle is present in all eucaryotic cells and produce cholesterol and prenyl-compounds. The main two enzymes (HMG-CoA reductase and FPP synthase) in the mevalonate pathway are carefully regulated and can be farmacologically modulated by different drugs (mevastatin and aminobisphosphonates respectively). Several studies show that V2 T cells recognize and kill several cancer cells, such as lymphoma, colon-, lung-, renal, breast carcinoma, and glioma. In renal cancer patients, a V2- based immunotherapy with a synthetic phosphorilated compound is in course with promising results. Similarly, aminobisphosphonates (Zoledronic Acid) is currently used for bone metastases in prostate cancer patients. In this context, the possibility to massively activate and expand in vitro a relatively large number of cells opens new interesting prospects in the immunotherapy of cancer disease. Gliomas are tumors arising from glia or their precursors within the central nervous system. Unfortunately, the majority of patients with glioma tumors die in less then of one year; in these patients, new treatment strategies are therefore hardly needed. Aim of this study was to analyse the activity of human V2 T cells against glioma cancer cells and to verify the possibility to target these innate cells in new immunotherapeutical strategies. In a first set of experiments, we set up an in vitro protocol able to expand human V2 T cells by using IPP and IL-2. After 12 days the expanded V2 T cell lines (80-95% of purity) were analysed for their differentiation phenotype, (as expression of CD27 and CD45RA markers of T, B, NK cells), cytokines production (IFN and TNF) and natural cytotoxicity capability (Perforin). Results showed that in vitro expanded V2 T cell lines present an effector memory phenotype and have high functionality both in terms of cytokines production and Perforin release. We then studied three different glioma cell lines: T70, U373 and U251 by analyzing GFAP expression on cell surface by direct immunofluorescence. Resulted showed that all glioma cells was positive for GFAP. In a second set of experiments, V2 T cell lines were co-cultured with glioma cells in order to analyse the activation of V2 T cells and the effects on the viability of glioma cells. In our system, V2 T cell lines were able to recognized glioma cells (T70, U373, U251) by specifically differentiate in effector memory cells, and release Perforin. In contrast, they did not produce cytokines. In order to verify the cytotoxic effect of V2 T cells on glioma cells, we performed a viability test on glioma cells in the absence and in the presence of V2 T cell lines. Briefly, glioma cells were labelled with Annexin/Propidium Iodide and were analysed by flow cytometry. Interestingly, V2 T cells were able to kill glioma cells through an apoptotic mechanism, demonstrating their antitumoral activity. We then decided to study if Zoledronic Acid (ZOL) treatment of glioma cells could improve V2 T lines response. Glioma cells were treated with ZOL in vitro for two hours, and co-cultured with V2 T cell lines, analyzing V2 T cells response by flow cytometry. Results showed that V2 T cell lines were able to recognize glioma cells by releasing high amount of IFN and TNF. V2 T cells activation was mediated by ZOL-induced IPP accumulation, since the incubation with mevastatin was able to completely block this biological effect. Finally, we studied the direct effect of different concentrations of ZOL on glioma cells viability before and after the co-culture with V2 T lines. We observed that treatment with ZOL induced necrosis on glioma cells, but only the co-culture with V2 T lines together to the treatment with ZOL increased both the apoptosis and necrosis of glioma cells. Altogether, our results suggest that the induction of a strong antitumoral response of V2 T cells by using aminobisphosphonates could represent a new interesting immunotherapy approach for glioma care.