Bromohydrin pyrophosphate-stimulated Vgamma9delta2 T cells expanded ex vivo from patients with poor-prognosis neuroblastoma lyse autologous primary tumor cells.

Gamma/delta T cells (Vgamma9delta2) contribute to innate immunity and exert natural cytotoxicity against a variety of tumors. Using a synthetic phosphoantigen (Bromohydrin Pyrophosphate, BrHPP), we amplified Vgamma9delta2 T cells in vitro from neuroblastoma patients. In the presence of BrHPP and low doses of IL-2, robust proliferation of Vgamma9delta2 T cells was obtained from peripheral blood mononuclear cells (PBMC) harvested at diagnosis. Moderate proliferation was observed from PBMC harvested after stem cell transplantation, whereas modest levels of Vgamma9delta2 T cells were obtained from PBMC harvested after induction therapy. Proliferation was observed after a single in vitro stimulation with BrHPP. After 21 days in culture, Vgamma9delta2 T cells represented more than 80% of cultured cells (a 50-fold expansion from baseline). Moreover, BrHPP-amplified Vgamma9delta2 T cells from patients-expressed activation markers and were able to lyse allogeneic and autologous neuroblasts. This cytotoxic activity was gammadelta T-cell receptor-dependent. Clinical trials using BrHPP are warranted in patients with poor-prognosis neuroblastoma, either to expand patient-derived Vgamma9delta2 T cells ex vivo or by direct administration to in vivo to boost the pool of resident Vgamma9delta2 T cells in vivo.

Large scale expansion of Vgamma9Vdelta2 T lymphocytes from human peripheral blood mononuclear cells after a positive selection using MACS "TCR gamma/delta+ T cell isolation kit".

Interest in gamma9delta2 T cells has increased greatly in the past decade. While several protocols allowed the amplification of a large proportion of these cells in vitro, the purity of the final preparation is usually heterogeneous between different donors. Functional studies of this population are often controversial due to the presence of other populations such as NK cells which share a wide range of characteristics. Here, the gamma9delta2 T cells labelled-fraction is purified and mixed with the irradiated unlabelled fraction followed by a single stimulation with phosphoantigen, in turn followed by a classical step of amplification in the presence of interleukin 2. In this study, we describe a straightforward protocol to amplify pure populations of gamma9delta2 T cells which could be useful in fundamental research or in the development of a new generation of gammadelta cell therapy protocol.

Bromohydrin pyrophosphate enhances antibody-dependent cell-mediated cytotoxicity induced by therapeutic antibodies.

In human blood, 1% to 5% of lymphocytes are gammadelta T cells; they mostly express the gammadelta T-cell receptor (TCR)Vgamma9, recognize nonpeptide phosphoantigens (PAgs) produced by microbes and tumor cells, and mediate different modes of lytic activities directed against tumor target cells. Antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by cytolytic lymphoid cells is essential for the clinical activity of anticancer monoclonal antibodies (mAbs), but whether PAgs affect ADCC by gammadelta T cells is unknown. Here we report that, in association with the CD20(+)-specific mAb rituximab (RTX), the synthetic PAg bromohydrin pyrophosphate (BrHPP) increased TCRVgamma9(+) cell binding to CD20(+) lymphoma cells in vitro. This combination activated phospho-ZAP70 and phospho-ERK1/2 signaling in TCRVgamma9(+) cells and strongly enhanced their ADCC activity. We obtained similar results with BrHPP in the context of the mAbs alemtuzumab and trastuzumab. Furthermore, BrHPP enhanced RTX-mediated depletion of CD20(+) cells in vitro from peripheral blood mononuclear cells of healthy subjects and enhanced ADCC by gammadelta T cells from patients with chronic lymphocytic leukemia. In cynomolgus macaques, a regimen combining RTX, BrHPP, and IL2 activated TCRVgamma9(+) lymphocytes and enhanced B-cell depletion from blood and lymph nodes. Thus, the combination with BrHPP PAg is able to improve the efficacy of cancer immunotherapy by therapeutic mAbs.