Study On The Immunotherapy For Chondrosarcoma With Vγ9Vδ2T Cells And Zoledronate

Chondrosarcoma (CS) is characterized by the production of cartilaginous matrix arising from cartilaginous tissues. It represents the second most common neoplasm of bone only after osteosarcoma. Chondrosarcoma is relatively chemo-and radiotherapy resistant. The majority of these tumors grow slowly and rarely metastasize, and wide surgical excision remains the best available treatment option. However, treatment for patients with unresectable or metastatic disease is particularly challenging. Novel therapeutic strategies are needed to improve the survival rate and therapeutic ratio.Immunotherapy has been proposed as the alternative strategy for cancer patients following surgery, chemotherapy and radiotheraoy. We previously demonstrated that Vγ9Vδ2T 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 Vγ9Vδ2T 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 Vy9V82T cells could exert cytolytic activities to human CS cells. We first examined phenotype of Vy9V82T cells expanded from peripheral blood mononuclear cells (PBMCs) of healthy donors (HDs) stimulated by Zol and IL-2. We found that the phenotype of Vγ9Vδ2T cells swithed from naive T cells to effector-memory T cells and terminally differentiated T cells with increasing cytotoxity. Secondly, we found that although CS cell line SW1353and primary cells were only little or moderate sensitive to y8T cell cytotoxicity, Zol sensitized CS cells to Vγ9Vδ2T cells cytotoxicity dramatically. Finnally, we investigated the role of Vy9V52T cells in combination with zoledronic acid in the treatment of CS xenograft tumor in vivo. We found that Vy9V82T cells were able to infiltrate tumors and significantly suppressed the development of SW1353xenografts.Part1Vγ9Vδ2T cell expansion, purification and phenotype analysisObjective:To expand and purify Vy9V82T 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-2or ZOL+IL-2, the phenotype of ex vivo expanded Vy9V82T 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:Vγ9Vδ2T cells were selectively expanded after PBMCs were stimulated with Zol+IL-2, with increasing percentage and cytotoxcity at the first12-18days and then reached the plateau weeks. The phenotype of Vy9V82T cells switched from naive T cells to effector-memory T cells and terminally differentiated T cells.Conclusions:The cytotoxcity of Vγ9Vδ2T cells expanded and stimulated reached the plateau at2-4weeks and thereby suitable for adoptive immunotherapy.Part2In vitro Cytotoxcity of Vγ9Vδ2T cells against chondrosarcoma cellsObjective:To investigate the cytotoxcity of Vγ9Vδ2T cells in combination with ZOL against chondrosarcoma cells in vitro.Methods:In vitro cytotoxicity of expanded Vy9V82T cells against untreated and ZOL-pretreated CS cells was analyzed using a standard lactate dehydrogenase release assay. The level of CD107a and IFN-a of Vγ9Vγ2T cells was evaluated by flow cytometry. To investigate mechanisms of recognition and killing of CS cells, Vy9V82T cells were incubated with blocking Abs in order to block TCR, NKG2D, perform, TRAIL, and Fas pathways. Results:1. CS cell line SW1353and primary cells were only little or moderate sensitive to Va9Va2T cell cytotoxicity, ZOL sensitized CS cells to Va9Va2T cells cytotoxicity dramatically..2. IFN-a was secreted from Va9Va2T cells after co-cultured with CS cells and were paralleled with CD107a expression.3. The mechanism of ZOL-enhanced cytotoxicity was dependent on the granule exocytose and was mainly TCR-mediated, NKG2D and TRAIL pathways only played a minor role.Conclusions:Va9Va2T cells exerted cytotoxicity against CS cells in vitro.Part3In vivo anti-tumor effect of Vγ9Vδ2T cells in CS xenografts animal modelsObjective:To determined the In vivo anti-tumor effect of Vy9V82T cells in CS xenografts animal modelsMethods:CS xenografts animal models were established by inoculated subcutannously with CS cells line SW1353to4-week old nude mice. Adoptive immunotherapy with zoledronate and Vy9V82T cells were initiated3days or2weeks after CS cells inculation. Control groups consisted of mice receiving zoledronate or Vγ9Vδ2T cells only. Mice were evaluated by tumor size and survival.Results:Combination therapy with zoledronate and Vy9V82T cells showed limited effect to established tumors. However, when adoptive immunotherapy was initiated3days after CS cells inoculation, zoledronate and Vy9V82T cells significantly prolonged the survival and suppress the growth of subcutaneous xenografts. Similar results were obtained in orthotopic mouse model.Conclusions:Vγ9Vδ2T cells and zoledronate inhibited the growth of transplanted tumors and prolonged the survival of mice.