Trastuzumab Enhanced The Cytotoxicity Of Vγ9Vδ2 T Cells Against Zoledronate Sensitized Osteosarcoma Cells

Osteosarcoma (OS) is the most common bone malignancy affecting children and adolescents. Effect of treatment has been improved greatly using surgery with neochemotherapy, but 30-50 percent of local OS will recurrence eventually. However, treatment for patients with recurrence or metastatic disease is particularly challenging, and the prognosis is very poor. In spite of the advances in multimodality therapy, the survival outcome has plateaued over the last 3 decades. Novel treatment is urgently needed to improve the survival rate and therapeutic ratio for OS patients.Immunotherapy has been proposed as the alternative strategy for cancer patients following surgery, chemotherapy and radiotheraoy. We previously demonstrated that Vγ9Vδ2 T cell efficiently killed ZOL pretreated osteosarcoma cells. Nevertheless little is known whether they could exert cytolytic activities to human CS cells. We hypothesize that immunotherapy based on Vγ9Vδ2 T cells, which recognize and kill tumor targets via a number of different mechanisms, would bypass conventional therapy resistant mechanisms and lead to novel clinical approach.In this paper, we sough to determine whether trastuzumab enhanced the cytotoxicity of Vγ9Vδ2 T cells against zoledronate sensitized osteosarcoma cells. We first examined phenotype of Vγ9Vδ2 T cells expanded from peripheral blood mononuclear cells (PBMCs) of healthy donors (HDs) stimulated by Zol and IL-2. We found that the phenotype of Vy9V82 T cells swithed from naive T cells to effector-memory T cells and terminally differentiated T cells with increasing cytotoxity. Secondly, we try to find whether trastuzumab enhanced the cytotoxicity of Vγ9Vδ2 T cells against osteosarcoma cells. We found that HER2 expression was a prerequisite for TTZ binding to OS cells. The surface expression of HER2 was determined by flow cytometry. OS cell line U2OS displayed intermediate levels of HER2 surface expression, whereas only a low expression was determined for HOS. TTZ alone was not able to inhibit the proliferation of OS cells even in a 72-h treatment period. On the other side, co-incubation with TTZ induced ADCC in cell line U2OS, increasing levels of cytotoxicity. However no obvious ADCC effect was observed in HOS with very little HER-2 expression. Finnally, we investigated whether trastuzumab enhanced the cytotoxicity of Vγ9Vδ2 T cells against zoledronate sensitized osteosarcoma cells. Results of the study show that in vitro expanded Vy9V82 T cells have ADCC potential, and their anti-tumor activity can be dramatically enhanced when tumor cells are pre-incubated with therapeutic mAbs.TTZ is a humanized mAb that binds to the extracellular domain of human epidermal growth factor receptor 2 (HER2). Vγ9Vδ2 T cells produce greater cytotoxicity against HER2-positive cells incombination with TTZ.Part 1 Vγ9Vδ2 T cell expansion, purification and phenotype analysisObjective:To expand and purify Vγ9Vδ2 T cells from peripheral blood mononuclear cells and to analyze the phenotype of these cells at the different time points.Methods:PBMCs from healthy donors were stimulated with IL-2 or ZOL+IL-2, the phenotype of ex vivo expanded Vγ9Vδ2 T cells were assessed by flow cytometry at the different time points. Vy9V82 T cells were purified by immunomagnetic beads and cytotoxicity against target cells was analyzed using a standard lactate dehydrogenase release assay.Results:Vγ9Vδ2 T cells were selectively expanded after PBMCs were stimulated with Zol+IL-2, with increasing percentage and cytotoxcity at the first 12-18 days and then reached the plateau weeks. The phenotype of Vγ9Vδ2 T cells switched from naive T cells to effector-memory T cells and terminally differentiated T cells.Conclusions:The cytotoxcity of Vγ9Vδ2 T cells expanded and stimulated reached the plateau at 2-4 weeks and thereby suitable for adoptive immunotherapy.Part 2 In vitro Trastuzumab enhanced the cytotoxcity of Vγ9Vδ2 T cells against chondrosarcoma cellsObjective:To investigate the cytotoxcity of Vγ9Vδ2 T cells in combination with TZZ against osteosarcoma cells in vitro.Methods:PBMCs(peripheral blood mononuclear cells) from healthy donors were stimulated with IL-2 or ZOL+IL-2, the phenotype of ex vivo expanded Vγ9Vδ2 T cells were assessed by flow cytometry at the different time points. To test HER2 surface expression lever of OS cells. In vitro cytotoxicity of expanded Vγ9Vδ2 T cells combined with TZZ against OS cells was analyzed using a standard MTS. The level of CD 107a and IFN-γ of Vγ9Vδ2 T cells was evaluated by flow cytometry.Results:OS cell line U2OS displayed intermediate levels of HER2 surface expression, whereas only a low expression was determined for HOS. TTZ alone was not able to inhibit the proliferation of OS cells even in a 72-h treatment period. On the other side, co-incubation with TTZ induced ADCC in cell line U2OS, increasing levels of cytotoxicity. However no obvious ADCC effect was observed in HOS with very little HER-2 expression.Conclusions:Trastuzumab can enhance the cytotoxicity of Vγ9Vδ2 T cells against osteosarcoma cells in vitro. Co-incubation with TTZ induced ADCC in cell line U2OS, increasing levels of cytotoxicity. However no obvious ADCC effect was observed in HOS with very little HER-2 expression.Part 3 In vitro Trastuzumab enhanced the cytotoxcity of Vγ9Vδ2 T cells against zoledronate sensitized chondrosarcoma cellsObjective:To investigate the cytotoxcity of Vγ9Vδ2 T cells in combination with TZZ against zoledronate sensitized osteosarcoma cells in vitro.Methods:PBMCs(peripheral blood mononuclear cells) from healthy donors were stimulated with IL-2 or ZOL+IL-2, the phenotype of ex vivo expanded Vy9V82 T cells were assessed by flow cytometry at the different time points. To test HER2 surface expression lever of OS cells. In vitro cytotoxicity of expanded Vy9V82 T cells combined with TZZ against OS cells was analyzed using a standard MTS. The level of CD107a and IFN-γ of Vγ9Vδ2 T cells was evaluated by flow cytometry. To investigate mechanisms of recognition and killing of CS cells, Vγ9Vδ2 T cells were incubated with blocking Abs in order to block TCR, NKG2D, perforin, TRAIL, and Fas pathways.Results:We mimic the clinical scenario tumor cells were pretreated by 10 μM ZOL (which would be reached following 16 mg ZOL infusions in cancer patients) for 2 h. Vγ9Vδ2 T cells were able to mediate cytotoxicity against ZOL sensitized U2OS targets with cell death averaging 32.0% at an E:T ratio of 5:1 following ZOL pre-treatment. The addition of TTZ increased Vγ9Vδ2 T cell cytotoxicity of ZOL-sensitized U2OS. In addition, TTZ markedly enhanced CD 107a expression on Vγ9Vδ2 T cells. These data collectively showed that Vγ9Vδ2 T cell-mediated killing can be further increased by therapeutic TTZ and ZOL. The antibody was added to Vγ9Vδ2T cells 30 minutes prior to co-culture.Treatment of effector cells with CMA and anti-pan γδTCR revealed that Vγ9Vδ2 T cell cytotoxicity was TCR-dependent and was mediated by perforin (means of 78.4% and 83.4% inhibition respectively). Cytotoxic inhibition using anti-CD16 blocking mAb indicated that the increased cytotoxicity of Vγ9Vδ2 T cells by TTZ against ZOL-sensitized U2OS was mediated by CD 16. There was only 6.9% reduction in cytotoxicity using anti-NKG2D antibody, indicating that NKG2D was not involved in the enhanced cytotoxicity of Vγ9Vδ2 T cells by TTZ and ZOL.Conclusions:Trastuzumab can enhance the cytotoxicity of Vγ9Vδ2 T cells against zoledronate sensitized osteosarcoma cells. Vγ9Vδ2 T cell cytotoxicity was TCR-dependent and was mediated by perforin, and NKG2D was not involved in the enhanced cytotoxicity of Vγ9Vδ2 T cells by TTZ and ZOL.