Stimulation Of Γδ Research Articles

An optimized cultivation method for future in vivo application of γδ T cells.

γδ T cells, with their properties of both the innate and acquired immune systems, are suitable candidates for cellular immunotherapy in cancer. Because of their non-major histocompatibility complex (MHC) binding T cell receptor, allogenic transfer is feasible without relevant graft versus host reactions. In recent years, much experience has been gained with ex vivo expansion and stimulation of γδ T cells using bisphosphonates and Interleukin 2. Unfortunately, many current stimulation protocols are based on the use of xenogenic materials and other potentially hazardous supplements, which conflicts with basic principles of Good Manufacturing Practice (GMP). Adherence to the concept and current guidelines of GMP is state of the art for production of Advanced Therapy Medicinal Products (ATMP) like cell therapeutics and a necessity for clinical use under a regulatory perspective. In this study, we developed a new stimulation protocol that induces a marked increase of γδ T cell counts and allows for an easier transition from research to clinical applications with minimized regulatory workload. It reliably leads to a cell product with a purity of more than 90% γδ T cells and improved in vitro anti-tumor activity compared to our previous standard procedure. Furthermore, by investigating correlations between properties of unstimulated γδ T cells and proliferation rate as well as degranulation ability of stimulated γδ T cells, we can draw conclusions about suitable donors. Finally, we examined if expansion can be improved by pulsing zoledronate and/or using Interleukin 15 with or without Interleukin 2. Significant improvements can be achieved with respect to intrinsic and antibody-dependent cell-mediated cytotoxicity. Our results demonstrate that the stimulation protocol presented here leads to an improved γδ T cell product for future clinical applications.

Immune-Stimulatory Effects of Rapamycin Are Mediated by Stimulation of Antitumor γδ T Cells.

The FDA-approved mTOR inhibitor rapamycin mediates important immune effects, but its contributions to the anticancer effects of the drug are unclear. Here we report evidence that rapamycin-mediated cancer protection relies upon stimulation of γδ T cells. In a well-established mouse model of carcinogen and inflammation-driven skin carcinogenesis, IFNγ recruited γδ TCRmid T cells to the epidermis where rapamycin boosted their perforin-dependent antitumor properties. These antitumor cells were mostly Vγ5-Vγ4-Vγ1- in phenotype. IFNγ signals were required in both hematopoietic and nonhematopoietic cells for rapamycin to optimally promote epidermal infiltration of γδ TCRmid T cells, as mediated by CXCR3-CXCL10 interactions, along with the antitumor effects of these cells. In mouse xenograft models of human squamous cell carcinoma, rapamycin improved human γδ T-cell-mediated cancer cell killing. Our results identify immune mechanisms for the cancer prevention and treatment properties of rapamycin, challenging the paradigm that mTOR inhibition acts primarily by direct action on tumor cells. Cancer Res; 76(20); 5970-82. ©2016 AACR.

Current approaches to bone-targeted therapy in genitourinary malignancies

Bone metastases in patients with genitourinary cancers are associated with increased risk for skeletal-related events including pathologic fractures, spinal cord compression, and the requirement for surgery or palliative radiotherapy to bone. The nitrogen-containing bisphosphonate zoledronic acid and the monoclonal antibody against RANK, denosumab, are approved for the prevention of skeletal-related events in genitourinary cancers. These agents have different mechanisms of action and pharmacokinetic profiles, and while both are effective in reducing the risk of skeletal-related events, other clinical effects differ. There is evidence for direct and indirect anticancer activity with zoledronic acid from preclinical studies and emerging data from clinical studies suggesting an effect on patient survival. Potential anticancer mechanisms include inhibition of angiogenesis, enhanced immune surveillance via stimulation of γδ T cells, and reduction of circulating tumor cells. A synergistic effect of chemotherapy plus zoledronic acid has also been suggested. Further research is ongoing regarding the roles of these antiresorptive therapies in patients with bone metastases or at high risk for malignant spread to skeletal sites.

Heat Shock Protein 90 Expression in Epstein-Barr Virus-Infected B Cells Promotes γδ T-Cell Proliferation In Vitro

The aim of this study was to elucidate the in vitro response of gammadelta T cells to Epstein-Barr virus (EBV)-infected B cells and to determine whether EBV-induced heat shock proteins (HSPs) might serve as gammadelta T-cell stimulants. Cytofluorometric analysis revealed HSP90 cell surface expression in 12% of the EBV-immortalized B-cell population in all four of the B-cell lines tested. HSP27, HSP60, and HSP70 were not detected on the cell surface by cytofluorometry in these same B-cell lines. HSP90 and HSP60, but not HSP70 or HSP27, were detected on the cell surface after 125I cell surface labeling and immunoprecipitation with anti-human HSP monoclonal antibodies. In vitro kinetic studies indicated that gammadelta T cells increased at least twofold by day 11 postinfection in cultures of EBV-seronegative peripheral blood lymphocytes infected with EBV, whereas percentages of alphabeta T cells in these same cultures either decreased slightly or remained relatively unchanged in response to EBV infection. Addition of anti-human HSP90 monoclonal antibody to the EBV-infected lymphocyte cultures inhibited gammadelta T-cell expansion by 92%. The inhibition of gammadelta T-cell expansion by anti-HSP90 antibody was reversed upon treatment with exogenous HSP90. Taken together, these results indicate that HSP90 played an important role in the stimulation of gammadelta T cells during EBV infection of B cells in vitro and may serve as an important immunomodulator of gammadelta T cells during acute EBV infection.

Leptospira interrogansActivation of Human Peripheral Blood Mononuclear Cells: Preferential Expansion of TCRγδ+ T Cells vs TCRαβ+ T Cells

Innate and adaptive immune responses induced by leptospirosis have not been well characterized. In this study we show that in vitro exposure of naive human PBMC to Leptospira interrogans results in cell proliferation and the production of IFN-gamma, IL-12, and TNF-alpha. Cell proliferation was highest when using high numbers of Leptospira. Optimal cell proliferation occurred at 6-8 days, and the majority of cells contained in these cultures were gamma/delta T cells. These cultures showed a 10- to 50-fold expansion of gamma/delta T cells compared with the initial cellular input. Additionally, these cultures contained elevated numbers of NK cells. In contrast, exposure of PBMC to low numbers of Leptospira failed to induce gammadelta T cell or NK cell expansion, but induced significant alphabeta T cell expansion. Vgamma9/Vdelta2 were expressed on all gamma/delta T cells expanded by exposure of PBMC to Leptorspira: Leptospira stimulation of purified TCRgammadelta(+) T cells, obtained from 8-day cultures of Leptospira-stimulated PBMC, induced high levels of IFN-gamma production, but no cell proliferation, suggesting that such stimulation of gammadelta T cells did not depend on specialized accessory cells or Ag processing. Finally, in patients with acute leptospirosis, there was a significant (4- to 5-fold) increase in the number of peripheral blood TCRgammadelta(+) T cells. These results indicate that Leptospira can activate gammadelta T cells and alphabeta T cells and will guide further investigations into the roles of these T cell populations in host defense and/or the pathology of leptospirosis.

Stimulation of γδ T cells by aminobisphosphonates and induction of antiplasma cell activity in multiple myeloma

AbstractBisphosphonates are well-known inhibitors of osteoclastic bone resorption, but recent clinical reports support the possibility of direct or indirect antitumor effects by these compounds. Because bisphosphonates share structural homologies with recently identified γδ T-cell ligands, we examined the stimulatory capacity of bisphosphonates to γδ T cells and determined whether γδ T-cell stimulation by bisphosphonates could be exploited to generate antiplasma cell activity in multiple myeloma (MM). All tested aminobisphosphonates (alendronate, ibandronate, and pamidronate) induced significant expansion of γδ T cells (Vγ9Vδ2 subset) in peripheral blood mononuclear cell cultures of healthy donors at clinically relevant concentrations (half-maximal activity, 0.9-4 μmol/L). The proliferative response of γδ T cells to aminobisphosphonates was IL-2 dependent, whereas activation of γδ T cells (up-regulation of CD25 and CD69) occurred in the absence of exogenous cytokines. Pamidronate-activated γδ T cells produced cytokines (ie, interferon [IFN]-γ) and exhibited specific cytotoxicity against lymphoma (Daudi) and myeloma cell lines (RPMI 8226, U266). Pamidronate-treated bone marrow (BM) cultures of 24 patients with MM showed significantly reduced plasma cell survival compared with untreated cultures, especially in cultures in which activation of BM-γδ T cells was evident (14 of 24 patients with MM). γδ T-cell depletion from BM cultures completely abrogated the cytoreductive effect on myeloma cells in 2 of 3 tested patients with MM. These results show that aminobisphosphonates stimulating γδ T cells have pronounced effects on the immune system, which might contribute to the antitumor effects of these drugs.

Stimulation of γδ T cells by aminobisphosphonates and induction of antiplasma cell activity in multiple myeloma

Bisphosphonates are well-known inhibitors of osteoclastic bone resorption, but recent clinical reports support the possibility of direct or indirect antitumor effects by these compounds. Because bisphosphonates share structural homologies with recently identified γδ T-cell ligands, we examined the stimulatory capacity of bisphosphonates to γδ T cells and determined whether γδ T-cell stimulation by bisphosphonates could be exploited to generate antiplasma cell activity in multiple myeloma (MM). All tested aminobisphosphonates (alendronate, ibandronate, and pamidronate) induced significant expansion of γδ T cells (Vγ9Vδ2 subset) in peripheral blood mononuclear cell cultures of healthy donors at clinically relevant concentrations (half-maximal activity, 0.9-4 μmol/L). The proliferative response of γδ T cells to aminobisphosphonates was IL-2 dependent, whereas activation of γδ T cells (up-regulation of CD25 and CD69) occurred in the absence of exogenous cytokines. Pamidronate-activated γδ T cells produced cytokines (ie, interferon [IFN]-γ) and exhibited specific cytotoxicity against lymphoma (Daudi) and myeloma cell lines (RPMI 8226, U266). Pamidronate-treated bone marrow (BM) cultures of 24 patients with MM showed significantly reduced plasma cell survival compared with untreated cultures, especially in cultures in which activation of BM-γδ T cells was evident (14 of 24 patients with MM). γδ T-cell depletion from BM cultures completely abrogated the cytoreductive effect on myeloma cells in 2 of 3 tested patients with MM. These results show that aminobisphosphonates stimulating γδ T cells have pronounced effects on the immune system, which might contribute to the antitumor effects of these drugs.

Growth arrest of gammadelta T cells induced by monoclonal antibody against WC1 correlates with activation of multiple tyrosine phosphatases and dephosphorylation of MAP kinase erk2.

WC1 is a 215-kDa type 1 transmembrane glycoprotein, the expression of which is restricted to gammadelta T lymphocytes. The binding of an anti-WC1 monoclonal antibody (mAb) (SC-29) induces reversible growth arrest in proliferating interleukin (IL)-2-dependent gammadalta T lymphocytes and this study has examined the relevant biochemical mechanisms. WC1 binding activates multiple protein tyrosine phosphatases causing specific tyrosine dephosphorylation in the absence of calcium mobilization. One of the dephosphorylated proteins was identified as the MAP kinase erk2. Another phosphotyrosine protein of 70 kDa, found to coprecipitate with p85 phosphoinositol (PI)3-kinase was either dephosphorylated or uncoupled from the p85 PI 3-kinase immunoprecipitate after WC1 receptor binding by mAb SC-29. The anti-WC1-induced tyrosine dephosphorylation was reversed by stimulation of gammadelta T cells with concanavalin A or anti-CD3 mAb, demonstrating that at the biochemical level, mitogen stimulation is dominant to the growth-arresting effects of anti-WC1. It is therefore proposed that the activation of tyrosine phosphatases by WC1 binding and the resultant dephosphorylation of certain key signaling protein such as erk2 correlates with and may cause the induction of growth arrest in IL-2-dependent gammadelta T cells, without affecting the cells ability to respond to antigen. Possible mechanisms, which include the inhibition of IL-2 signal transduction pathways, are discussed.

Stimulation of γδ T cells by phosphoantigens

Stimulation of γδ T cells by phosphoantigens