Both Functions of Antigen Uptake/Presentation and Anti-Tumor Cytotoxicity Are Possessed by Human Blood γδ T Cells.

Abstract

Immune competent cells which possess both abilities to present tumor antigens to effector αβ T cells and to act by themselves as effector cells of anti-tumor immune reaction, if they would exist, are thought to be useful cells for establishing an efficient anti-tumor cellular immunotherapy. We identified human Vγ2Vδ2 T cells to be the multi-functional cells with the abilities of antigen presentation for antigen-specific immune reaction and anti-tumor cytotoxicity using non-peptides as antigens. Human γδ T cells were expanded from human peripheral blood mononuclear cells by the culture with zoledronate or isopentenyl pyrophosphate (IPP) and a low concentration of IL-2 for 1–2 weeks. Immature dendritic cells were generated from blood non-adherent cells or anti-CD14 microbead-separated monocytes by the culture with GM-CSF and IL-4 for 6–8 days and mature dendritic cells were induced from immature dendritic cells by the additional 2 days-culture with TNF-α, IL-1α, IL-6 and PGE2. Cultured γδ T cell-containing cells were analyzed for the expression of surface phenotypes and cytoplasmic molecules by flow cytometry. The supernatant of the culture of anti-γδ TCR microbead-separated γδ T cells were assayed for the concentration of IFN-γ, TNF-α, IL-4, IL-5, and IL-2 by cytometric bead array. The uptake ability of γδ T cells were investigated by flow cytometry using FITC-dextran and Lucifer yellow. Antigen presenting ability of γδ T cells was evaluated by mixed lymphocyte culture by using purified γδ T cells as stimulator cells and blood non-adherent cells or naïve αβ T cells as responder cells in both autologous and allogeneic settings. In autologous settings, KLH or PPD were added as exogenous antigens to γδ T cells or dendritic cells for the whole duration of the culture. Anti-tumor cytotoxicity of γδ T cells was evaluated by 51Cr-release assay by using purified γδ T cells as effector cells and leukemia/myeloma cell lines or fresh acute leukemia leukemia cells as target cells. Seventy to eighty percent of the cultured cells generated from blood mononuclear cells were positive for γδ TCR and almost all of the γδ TCR+ cells possessed Vδ2 TCR. Ten to twenty percient of γδ T cells were positive for CD40/CD80 and most of γδ T cells were positive for CD86/CD54/CD58/HLA-DR. The expanded γδT cells were shown to produce a remarkable amount of IFN-γ and a considerable amount of TNF-α and the cytokine production was much increased by the addition of a low dose of IL-2. Cultured γδ T cells were shown to be able to uptake FITC-Dextran and lucifer yellow at a comparable level of immature dendritic cells. Purified and cultured γδ T cells were demonstrated to possess an allogeneic and antigen-specific autologous antigen presenting ability at a similar level of mature dendritic cells. Zoledronate/IL-2-expanded γδ T cells were demonstrated to be cytotoxic against leukemia or myeloma cell lines and fresh acute leukemia leukemia cells at effector to target ratio dependent. The present study revealed that human γδ T cells, which could be markedly expanded by the culture with zoledronate or IPP and IL-2, possess both functions of antigen presentation and anti-tumor cytotoxicity and might be efficiently applied to anti-tumor cellular immunotherapy.