Cytotoxicity Of γδT Cells Research Articles

HLA-G Expression/Secretion and T-Cell Cytotoxicity in Missed Abortion in Comparison to Normal Pregnancy.

The main role of HLA-G is to protect the semi-allogeneic embryo from immune rejection by proper interaction with its cognate receptors on the maternal immune cells. Spontaneous abortion is the most common adverse pregnancy outcome, with an incidence rate between 10% and 15%, with immunologic dysregulation being thought to play a role in some of the cases. In this study, we aimed to detect the membrane and soluble HLA-G molecule at the maternal-fetal interface (MFI) and in the serum of women experiencing missed abortion (asymptomatic early pregnancy loss) in comparison to the women experiencing normal early pregnancy. In addition, the proportion of T cells and their cytotoxic profile was evaluated. We observed no difference in the spatial expression of HLA-G at the MFI and in its serum levels between the women with missed abortions and those with normal early pregnancy. In addition, comparable numbers of peripheral blood and decidual total T and γδT cells were found. In addition, as novel data we showed that missed abortion is not associated with altered extravilous invasion into uterine blood vessels and increased cytotoxicity of γδT cells. A strong signal for HLA-G on non-migrating extravilous trophoblast in the full-term normal placental bed was detected. In conclusion, HLA-G production at the MFI or in the blood of the women could not be used as a marker for normal pregnancy or missed abortions.

The cytotoxicity of γδT cells in non-small cell lung cancer mediated via coordination of the BCL-2 and AKT pathways.

The effectiveness and mechanisms of γδT-cell immunotherapy in lung cancer remain unclear. In this study, we assessed the effects of continuous, low-dose γδT-cell intervention on lung cancer cells. We cultured γδT cells with a lung cancer cell line (A549) and replaced the γδT-cell population every 48 hours. The killing effect of γδTcells on A549 cells and the Half-maximal inhibitory concentration (IC50) value were detected by the cholecystokinin octapeptide (CCK-8) method. The levels of perforin, granzyme B and the inflammatory factors interleukin-6 (IL-6), interferon (IFN)-γ, and tumor necrosis factor-alpha (TNF-a), in the supernatants of cocultured cells were measured by ELISA. The protein expression of Bcl-2, Bax, PI3K and Akt was detected by western blotting. Our results indicated that γδT-cell treatment decreased the protein expression of Bcl-2, PI3K, and AKT but upregulated that of Bax. Moreover, γδT-cell treatment increased perforin and granzyme B release related to the Bax/Bcl-2 signaling pathway. In addition, γδT-cell-mediated cytolysis for A549 cells involved the PI3K/AKT pathway. In vivo results were consistent with the in vitro results. γδT-cell immunotherapy integrated regulation of a signaling pathway network involving the mutual regulation of apoptosis and proliferation. γδT-cell immunotherapy could be used to enhance the cytotoxic killing of lung cancer cells.

Enhancing the effectiveness of γδ T cells by mRNA transfection of chimeric antigen receptors or bispecific T cell engagers

Adoptive cell therapy (ACT) utilizing γδ Tcells is becoming a promising option for the treatment of cancer, because it offers an off-the-shelf allogeneic product that is safe, potent, and clinically effective. Approaches to engineer or enhance immune-competent cells for ACT, like expression of chimeric antigen receptors (CARs) or combination treatments with bispecific Tcell engagers, have improved the specificity and cytotoxic potential of ACTs and have shown great promise in preclinical and clinical settings. Here, we test whether electroporation of γδ Tcells with CAR or secreted bispecific Tcell engager (sBite) mRNA is an effective approach to improve the cytotoxicity of γδ Tcells. Using a CD19-specific CAR, approximately 60% of γδ Tcells are modified after mRNA electroporation and these cells show potent anticancer activity invitro and invivo against two CD19-positive cancer cell lines. In addition, expression and secretion of a CD19 sBite enhances γδ Tcell cytotoxicity, both invitro and invivo, and promotes killing of target cells by modified and unmodified γδ Tcells. Taken together, we show that transient transfection of γδ Tcells with CAR or sBite mRNA by electroporation can be an effective treatment platform as a cancer therapeutic.

P-005: Novel strategy of elotuzumab and zoledronic acid with Th1-like γδT cells against myeloma

<h3>Background</h3> We are now in the new era of immunotherapies against multiple myeloma (MM) such as anti-CD38 and anti-SLAMF7 therapeutic monoclonal antibodies (mAbs); however, MM progression falls into attenuation and exhaustion of effector cells via immune checkpoint axes such as PD-1/PD-L1 and TIGIT/CD155, leading to hamper the clinical efficacy of these therapeutic mAbs. <h3>Methods</h3> To overcome this issue, we have been studying the utilization of Vδ2 γδT cells in peripheral blood mononuclear cells, which can be expanded and induced as Th1-like phenotype ex vivo using aminobisphosphonates in combination with IL-2, as effectors for MM treatment. The present study was aimed to develop the novel therapeutic strategies of γδT cells in combination with anti-MM mAbs. <h3>Results</h3> The next generation IMiDs, cereblon E3 ligase modulators (CELMoDs) of iberdomide and CC-92480 have been developed. CELMoDs was able to expand and activate γδT cells at much lower dose compared to IMiDs of lenalidomide and pomalidomide, in combination with zoledronic acid (ZA) ex vivo. Since the expanded Th1-like γδT cells highly expressed CD16, FcγRIIIa without expression of checkpoint molecules such as PD-1, TIGIT, CTLA-4, and LAG-3, we next examined whether anti-MM mAbs, elotuzumab (ELO), daratumumab (DARA), and isatuximab (ISA), induced ADCC in the presence of the γδT cells. DARA and ISA did not induce cytotoxicity against CD38-expressing MM cells in the presence of the γδT cells; however, addition of ELO further increased cytotoxicity of the γδT cells against SLAMF7-expressing MM.1S and OPM-2 cells but not RPMI 8226 cells with marginal SLAMF7 expression, suggesting induction of ELO's ADCC with the γδT cells. We next examined the cytotoxic effect of the combinatory treatment on osteoclasts (OCs) because MM cells resides in the bone marrow and the interaction between MM cells and OCs forms vicious cycle of MM cell growth and bone destruction. Of note, OCs, which can be induced from monocytes using M-CSF and RANK ligand, highly expressed SLAMF7 along with elevation of NFATc1 transcription factor. Although the expanded γδT cells can target OCs, addition of ELO augmented the cytotoxic effect of γδT cells on OCs, suggesting the emergence of ELO's ADCC with Th1-like γδT cells towards OCs. As γδT cells recognize not only neoantigens but also phosphoantigens (pAgs), we treated MM cells and OCs with ZA to enhance the expression of pAgs, and γδT cells then incubated with the ZA-treated cells. ZA-stimulation augmented the cytotoxicity of γδT cells towards MM cells and OCs even in the presence of ELO. <h3>Conclusions</h3> Taken together, our findings demonstrated the effective usage of ELO and Th1-like γδT cells with ZA against MM cells and ambient cells in MM bone marrow. Further study is warranted on the effective revitalization and expansion of Th1-like γδT cells using CELMoDs.

Gamma/Delta T-Cells Enhance Carboplatin-induced Cytotoxicity Towards Advanced Bladder Cancer Cells.

Bladder cancer (BLCA, urothelial bladder cancer) is one of the most common malignancies with increasing incidence and mortality worldwide. Poor diagnosis and the limitation of treatment is still an unmet need in clinical practice. γδ T-Cells have been paid increasing attention because of their potent cytotoxicity against tumors. Herein, we investigated the cytolytic effect of γδ T-cells in combination with the chemotherapeutic drug, carboplatin, against BLCA cells. The standard protocol for the induction and expansion of peripheral blood mononuclear cell-derived γδ T-cells was a zoledronic acid/interleukin-2-based medium system for 2 weeks. The cytotoxicity of γδ T-cells with and without carboplatin against BLCA cells was examined. After incubation, T-cell receptor-positive γδ T-cells showed a natural killer cell-like phenotypic characteristic and dose-dependently increased cytotoxicity against BLCA cells. Interestingly, we found that in advanced BLCA cells, which were more resistant to carboplatin, the cell viability was significantly (p<0.05) reduced in the presence of γδ T-cells. Our findings showed that γδ T-cell therapy has potent benefit in cancer treatment.

Beneficial Effect of Antibiotics and Microbial Metabolites on Expanded Vδ2Vγ9 T Cells in Hepatocellular Carcinoma Immunotherapy.

Animal experiments and clinical trials have shown that the gut microbiota modulates host immunity and immune checkpoint-mediated responses to tumor cells. However, it remains unclear whether microbiota can also play a role in the tumor immune response of γδT cells, a kind of cell that targets cancer directly. Here, we report that microbiota dysbiosis induced by antibiotics enhanced γδT cell efficacy during tumor therapy in a mouse model. Further microbiota and metabolite analysis revealed that the alteration of γδT cell cytotoxicity might be closely associated with specific metabolites, which are produced by intestinal bacteria and stimulate γδT cells to release more cytotoxic cytokines, such as granzyme B and perforin. Among the metabolites that we analyzed, 3-indopropionic acid (IPA) showed the highest concentration in antibiotic-treated mice and can improve the cytotoxic ability of γδT cells both in vitro and in vivo. Our research determined how the gut microbiota can influence the antitumor ability of γδT cells and identified potential intermediate molecules that connect the gut microbiota and γδT cells.

Hypoxia regulates the differentiation and anti-tumor effector functions of γδT cells in oral cancer.

Hypoxia within the tumor microenvironment (TME) is a key factor contributing to immunosuppression in tumors, co-relating with poor treatment outcome and decreased overall survival in advanced oral cancer (OC) patients. Vδ2 is a dominant subset of gamma delta T cells (γδT cells) present in the peripheral blood which exhibits potent anti-tumor cytotoxicity and is evolving as a key player of anti-cancer cellular therapy. However, the fate of γδT cells in hypoxic oral tumors remains elusive. In the present study, we compared the effect of hypoxia (1% O2 ) and normoxia (21% O2 ) on the expansion, proliferation, activation status, cytokine secretion and cytotoxicity of γδT cells isolated from OC patients and healthy individuals. Hypoxia-exposed γδT cells exhibited reduced cytotoxicity against oral tumor cells. Our data demonstrated that hypoxia reduces the calcium efflux and the expression of degranulation marker CD107a in γδT cells, which explains the decreased anti-tumor cytotoxicity of γδT cells observed under hypoxia. Hypoxia-exposed γδT cells differentiated to γδT17 [γδ T cells that produce interleukin (IL)-17] cells, which corroborated our observations of increased γδT17 cells observed in the oral tumors. Co-culture of γδT cells with CD8 T cells in the presence of hypoxia showed that programmed cell death ligand 1 (PD-L1)high γδT cells brought about apoptosis of programmed cell death 1 (PD-1)high CD8 T cells which could be significantly reversed upon blocking PD-1. Thus, future immunotherapeutic treatment modality for oral cancer may use a combined approach of blocking the PD-1/PD-L1 signaling and targeting hypoxia-inducible factor 1α, which may help in reversing hypoxia-induced immunosuppression.

Cytokine Pre-activated γδT cells in Cancer Adoptive Immunotherapy

Abstract Adoptive cell therapy harnesses the power of immune system by stimulating and expanding immune cells to combat cancer. The application of tumour-specific αβT cell-based therapy may be hindered by limited tumour antigens. γδT cells are promising candidates for cellular immunotherapy because of their abundant IFN-γ or IL-17 production and MHC-independent cytotoxicity against a broad spectrum of tumors. Numerous attempts to use γδT cells in cancer immunotherapy have been made and resulted in variable efficacy and an overall good safety profile. Better expansion strategies are needed to improve the efficacy of the clinical trials using γδT cell-based immunotherapies. In the current project, we aim to explore the efficacy of cytokine pre-activated γδT cells in cancer immunotherapy. Pre-activated γδT cells by a combination of cytokines were characterised for their surface activation marker expression, cytokine production, and cytotoxicity against tumour cells. We found that IL-12/18, IL-12/15/18, IL-12/18/21 and IL-12/15/18/21 are the most effective cytokine combinations for in vitro expansion of γδT cells in terms of activation and cytokine production. IL-21 upregulated their cytotoxicity against tumour cells in murine systems. All four combinations showed upregulated cytotoxicity of human γδT cells. These findings were extended to in vivo studies using murine hepatocellular carcinoma model.

Regulatory Interactions Between Neutrophils, Tumor Cells and T Cells.

Apart from their activity in combating infections, neutrophils play an important role in regulating the tumor microenvironment. Neutrophils can directly kill (antibody-coated) cancer cells, and support other immune anti-tumoral strategies. On the other hand, neutrophils can also exert pro-tumorigenic activities via the production of factors which promote cancer growth, angiogenesis and metastasis formation. The balance of anti- and pro-cancer activity is influenced by the particularly delicate interplay that exists between neutrophils and T lymphocytes. In murine models, it has been reported that γδ T cells are a major source of IL-17 that drives the recruitment and pro-tumorigenic differentiation of neutrophils. This, however, contrasts with the well-studied anti-tumor activity of γδ T cells in experimental models and the anti-tumor activity of human γδ T cells. In this article, we first review the reciprocal interactions between neutrophils, tumor cells and T lymphocytes with a special focus on their interplay with γδ T cells, followed by the presentation of our own recent results. We have previously shown that zoledronic acid (ZOL)-activated neutrophils inhibit γδ T-cell proliferation due to the production of reactive oxygen species, arginase-1 and serine proteases. We now demonstrate that killing of ductal pancreatic adenocarcinoma (PDAC) cells by freshly isolated resting human γδ T cells was reduced in the presence of neutrophils and even more pronounced so after activation of neutrophils with ZOL. In contrast, direct T-cell receptor-dependent activation by γδ T cell-specific pyrophosphate antigens or by bispecific antibodies enhanced the cytotoxic activity and cytokine/granzyme B production of resting human γδ T cells, thereby overriding the suppression by ZOL-activated neutrophils. Additionally, the coculture of purified neutrophils with autologous short-term expanded γδ T cells enhanced rather than inhibited γδ T-cell cytotoxicity against PDAC cells. Purified neutrophils alone also exerted a small but reproducible lysis of PDAC cells which was further enhanced in the presence of γδ T cells. The latter set-up was associated with improved granzyme B and IFN-γ release which was further increased in the presence of ZOL. Our present results demonstrate that the presence of neutrophils can enhance the killing capacity of activated γδ T cells. We discuss these results in the broader context of regulatory interactions between neutrophils and T lymphocytes.

Abstract 1421: Low dose gemcitabine increases the sensitivity of human Vgamma9Vdelta2T cell mediated cytotoxicity through NKG2D-NKG2D ligands axis in bladder cancer cells

Abstract INTRODUCTION AND OBJECTIVES: In clinical trials, Zoledronic acid (ZOL) pretreatment in cancer cells is widely used to improve the efficacy of human γδT cell immunotherapy. We herein provide new strategies for the enhancement of γδT cell function by anticancer pretreatment in urinary bladder cancer (UBC) cells. The aim of this study was whether pretreatment of cancer cells with standard anticancer agents would increase the cytotoxicity of γδT cells. METHODS: The surface expression of NKG2D, TCRVγ9, TCRVδ2 and the intracellular levels of perforin and Granzyme B in γδT cells were examined. In in vitro assays, UBC cell lines T24, TCCSUP, and UMUC3 were used. Flow cytometric analysis of Carboxyfluorescein diacetate succinimidyl ester (CFSE) /propidium iodide (PI) staining was used in in vitro cytotoxicity assays. Imaging of cancer cells lysed by human γδT cell was captured by laser microscopy. UBC cells were treated with various standard anticancer agents at sub-lethal concentrations, including cisplatin (CDDP), gemcitabine (GEM), methotrexate (MTX), vinblastine (VBL), adriamycin (ADR), and mitomycin C (MMC). The expressions of MICA/B, ULBP1, and ULBP2/5/6 in UBC cells were investigated. In in vivo experiments, the efficacy of intravesical administration of ex vivo-expanded γδT cells was examined in an orthotopic xenograft model using In Vivo Imaging System (IVIS). RESULTS: Ex vivo-expanded γδT cell expressed NKG2D receptor on the cell surface and both TCRVγ9, TCRVδ2 positive lineage were well expanded. Activation of ex vivo-expanded γδT cell was confirmed by intracellular staining of granules, containing perforin and Granzyme B. We examined the synergistic effects of anticancer agents and γδT cells in vitro. We found that these anticancer agents upregulated the expressions of MICA/B and ULBP family in UBC cells, which γδT cells use to recognize cancer cells and resulted in the increased cytotoxicity of γδT cells. The cytotoxicity was abrogated by induction of siRNA against MICA/B. These findings were also confirmed in MICA/B or NKG2D blocking experiments. We also found that intravesical administration of γδT cells showed potent cytotoxicity using an orthotopic xenograft. Finally, we revealed that low dose gemcitabine pretreatment synergistically increased the cytotoxicity of γδT cells in vivo. CONCLUSIONS: These results indicated that γδT cell adoptive immunotherapy in combination with standard anticancer agents is an effective strategy and may be a promising approach to the treatment of UBC. Citation Format: Teruki Shimizu, Mako Tomogane, Yusuke Sano, Daiki Shimizu, Masatsugu Miyashita, Atsuko Fujihara, Fumiya Hongo, Osamu Ukimura, Eishi Ashihara. Low dose gemcitabine increases the sensitivity of human Vgamma9Vdelta2T cell mediated cytotoxicity through NKG2D-NKG2D ligands axis in bladder cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1421.

Donor Vγ4+T cells promote graft-versus-leukemia effect and ameliorate acute graftversus- host disease in allogeneic hematopoietic stem cell transplantation

Abstract Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is one of the most effective ways of treating leukemia and other malignant diseases. However, disease recurrence and GVHD are still the major causes of death after allo-HSCT. Previous studies showed that immune cells involved in graft versus leukemia (GVL) effect traditionally include CD8+ T, CD4+ T and NK cells. Unfortunately, some of these T-cell subsets also cause graft-versus- host disease (GVHD). The role of donor γδT cells after transplantation is less well defined. We detected direct anti-leukemia cytotoxicity of γδT cells in vitro and the cytotoxicity of Vγ1 subset was stronger than the Vγ4 subset. To evaluate the role of donor γδT cells in aGVHD and GVL, we established the murine HLA fully mismatched allo-HSCT GVL/GVHD model. Our results showed that TCRδ−/− donor bone marrow cells caused enhanced disease progression and aggravated aGVHD compared with wildtype donors. Furthermore, flow cytometry analysis demonstrated γδT cells mediated anti-leukemia effects through promoting IFN-γ production by CD8+ T cells while suppressed aGVHD by inhibiting the activation of CD4+ T cells in liver, lung and IEL. The experiments of adoptive transfer of in vitro expanded γδT/Vγ1/Vγ4 cells and the in vivo depletion of Vγ1/Vγ4 cells indicated that the subset of donor γδT cells to alleviate murine aGVHD and enhance GVL effect might be Vγ4 γδT cells. The GVL effect of Vγ4 cells in vivo could be mediated by the production of IL-17A and regulation of CD8+ T cell function. These findings suggest donor γδT cells infusion could be a novel strategy to improve patients’ overall survival after allo-HSCT.

Emerging role of γδ T cells in vaccine-mediated protection from infectious diseases.

γδ T cells are fascinating cells that bridge the innate and adaptive immune systems. They have long been known to proliferate rapidly following infection; however, the identity of the specific γδ T cell subsets proliferating and the role of this expansion in protection from disease have only been explored more recently. Several recent studies have investigated γδ T‐cell responses to vaccines targeting infections such as Mycobacterium, Plasmodium and influenza, and studies in animal models have provided further insight into the association of these responses with improved clinical outcomes. In this review, we examine the evidence for a role for γδ T cells in vaccine‐induced protection against various bacterial, protozoan and viral infections. We further discuss results suggesting potential mechanisms for protection, including cytokine‐mediated direct and indirect killing of infected cells, and highlight remaining open questions in the field. Finally, building on current efforts to integrate strategies targeting γδ T cells into immunotherapies for cancer, we discuss potential approaches to improve vaccines for infectious diseases by inducing γδ T‐cell activation and cytotoxicity.

Effects of fetal umbilical cord blood mesenchymal stem cells on γδT lymphocyte function and its related mechanism

Objective To explore the effects of fetal umbilical cord blood mesenchymal stem cells on γδT lymphocyte function and its related mechanism. Methods The Human peripheral blood mononuclear cells (PBMCs) or γδT cells were activated and expanded with 2 μg/ml pamidronate and 100 IU/ml interleukin-2 (IL-2) with or without the presence Fetal umbilical cord blood mesenchymal stem cells (UCMSCs). Flow cytometry was performed to evaluate the effects of UC-MSCs on proliferation, cytokine expression and cytotoxicity of γδT cells; besides, flow cytometry was used to detect the effects of UC-MSCs on Fas-L and TRAIL expression in γδT cells and γδT cell apoptosis as well. Results UC-MSCs inhibited γδT cell proliferation in a dose-dependent but cell-contact independent manner; when PBMCs and UC-MSCs were cultured at a ratio of 3∶1 and 40∶1, IFN-γ+ cells decreased by 46.7% and 31.4%, respectively (P=0.001, P=0.021), granzyme B positive cells increased by 79.7%, 165%, respectively, while the ratio of TNF-α and IL-10 positive cells did not change significantly compared with the control group (P=0.062); Cytotoxicity test results showed that, UCMSCs inhibited the cytotoxicity ofγδT cells against influenza virus H1N1 infected A549 cells, and when PBMCs and UC-MSCs were cultured at a ratio of 3∶1 and 40∶1, Fas-L+ γδT cells decreased by 87.6% and 37.5% (P=0.000, 0.009), the proportion of TRIAL+ γδT cells decreased by 43.2% and 18.7% (P=0.037, 0.042), however, UC-MSCs did not affect γδ T cell apoptosis (P=0.070, 0.064). Conclusion UC-MSCs can suppress γδT cells proliferation and cytotoxicity and modulated their cytokine production, and Fas-L and TRAIL were involved in the regulation. Key words: Umbilical cord blood mesenchymal stem cells; γ δ T cells; Proliferation; Cytokines; Apoptosis

MP54-02 DEVELOPMENT OF INTRAVESICAL HUMAN γΔT CELL BASED CHEMO-IMMUNOTHERAPY AGAINST URINARY BLADDER CANCER CELLS IN AN ORTHOTOPIC XENOGRAFT MODEL

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I1 Apr 2018MP54-02 DEVELOPMENT OF INTRAVESICAL HUMAN γΔT CELL BASED CHEMO-IMMUNOTHERAPY AGAINST URINARY BLADDER CANCER CELLS IN AN ORTHOTOPIC XENOGRAFT MODEL Teruki Shimizu, Mako Tomogane, Masatsugu Miyashita, Osamu Ukimura, and Eishi Ashihara Teruki ShimizuTeruki Shimizu More articles by this author , Mako TomoganeMako Tomogane More articles by this author , Masatsugu MiyashitaMasatsugu Miyashita More articles by this author , Osamu UkimuraOsamu Ukimura More articles by this author , and Eishi AshiharaEishi Ashihara More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.1693AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Intravesical BCG treatment continues to play a central role in the treatment of non-muscle invasive urinary bladder cancer (UBC). There is currently no gold standard for salvage intravesical therapy after BCG treatment failure. To develop the substitute for BCG treatment failure, we focused on γδT cell immunotherapy. γδT cells exert potent cytotoxicity towards various cancer cells and can recognize isopentenyl pyrophosphate (IPP) through the TCRγδ receptor, and zoredronic acid (ZOL) pretreatment is widely used to induce IPP accumulation in cancer cells. In this study, we hilighted two issues; combined use of anticancer agents along with ZOL and a bladder instillation approach. The aims are to improve recognition of cancer cells by γδT cells and to increase E/T (Effector/Target) ratio. METHODS We examined the efficacy of ex vivo-expanded γδT cell immunotherapy. Flow cytometric analysis of CFSE/propidium iodide (PI) staining was used in in vitro cytotoxicity assays.After co-culture for 4 h, CFSE/PI double positive population was measured as apoptotic cancer cells. In in vivo experiment, the efficacy of ex vivo-expanded γδT cell immunotherapy was examined in an orthotopic xenograft model using In Vivo Imaging System (IVIS). RESULTS Ex vivo-expanded γδT cells showed potent cytotoxicity against UBC cells in in vitro assays. Combination treatment with standard anticancer agents showed that low dose gemcitabine pretreatment significantly enhanced the cytotoxicity of γδT cells by upregulating the expression of MICA and MICB (MICA/B), which are tumor-associated antigens recognized by γδT cells. These effects were abrogated by small interfering RNA-mediated knockdown of MICA/B in UBC cells. IVIS analysis revealed the potent cytotoxicity of weekly intravesical administration of γδT cells (Figure. 1), and weekly gemcitabine pretreatment enhanced the cytotoxicity of γδT cells in vivo. CONCLUSIONS Low dose gemcitabine pretreatment significantly enhanced the cytotoxicity of γδT cells by upregulating the expression MICA/B in in vitro. Intravesical γδT cell immunotherapy exerted potent cytotoxicity against UBC and intravesical γδT cell immunotherapy in combination with low dose gemcitabine may be a promising strategy in UBC. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e712 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Teruki Shimizu More articles by this author Mako Tomogane More articles by this author Masatsugu Miyashita More articles by this author Osamu Ukimura More articles by this author Eishi Ashihara More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Low dose gemcitabine increases the cytotoxicity of human Vγ9Vδ2 T cells in bladder cancer cells in vitro and in an orthotopic xenograft model

ABSTRACTHuman γδT cell immunotherapy is well tolerated and has shown promising results in clinical trials; however, its antitumor efficacy is limited, including results in solid tumors. Ex-vivo expanded γδT cell stimulated by zoledronic acid (ZOL) activates the γδT cell subpopulation of so called Vγ9Vδ2 T cells. To improve the clinical outcomes of Vγ9Vδ2 T cell (abbreviated as γδT cell here) immunotherapy, we aimed to increase the cytotoxicity of γδT cells by focusing on two issues: recognition of tumor cells by γδT cells and the effector (γδT cell)-to-target (tumor cell) (E/T) ratio. Ex vivo-expanded γδT cells showed potent cytotoxicity against urinary bladder cancer (UBC) cells in in vitro assays. Combination treatment with standard anticancer agents showed that low dose gemcitabine pretreatment significantly enhanced the cytotoxicity of γδT cells by upregulating the expression of MICA and MICB (MICA/B), which are tumor-associated antigens recognized by γδT cells. These effects were abrogated by small interfering RNA-mediated knockdown of MICA/B in UBC cells, suggesting that pre-exposing cancer cells to anticancer agents could be a promising strategy. A bladder instillation approach was used to increase the E/T ratio. The efficacy of ex vivo-expanded γδT cell immunotherapy was examined in an orthotopic xenograft model. In Vivo Imaging System analysis revealed the potent cytotoxicity of weekly intravesical administration of γδT cells, and weekly gemcitabine pretreatment enhanced the cytotoxicity of γδT cells in vivo. In conclusion, intravesical γδT cell immunotherapy and combination therapy with low dose gemcitabine may be a promising strategy in UBC.

The expression levels of PD-L1 in cancer cells affect γδT cell cytotoxicity

The expression levels of PD-L1 in cancer cells affect γδT cell cytotoxicity

Zoledronic acid boosts γδ T-cell activity in children receiving αβ+ T and CD19+ cell-depleted grafts from an HLA-haplo-identical donor

ABSTRACTWe demonstrated that γδ T cells of patients given HLA-haploidentical HSCT after removal of αβ+ T cells and CD19+ B cells are endowed with the capacity of killing leukemia cells after ex vivo treatment with zoledronic acid (ZOL). Thus, we tested the hypothesis that infusion of ZOL in patients receiving this type of graft may enhance γδ T-cell cytotoxic activity against leukemia cells. ZOL was infused every 28 d in 43 patients; most were treated at least twice. γδ T cells before and after ZOL treatments were studied in 33 of these 43 patients, till at least 7 mo after HSCT by high-resolution mass spectrometry, flow-cytometry, and degranulation assay. An induction of Vδ2-cell differentiation, paralleled by increased cytotoxicity of both Vδ1 and Vδ2 cells against primary leukemia blasts was associated with ZOL treatment. Cytotoxic activity was further increased in Vδ2 cells, but not in Vδ1 lymphocytes in those patients given more than one treatment. Proteomic analysis of γδ T cells purified from patients showed upregulation of proteins involved in activation processes and immune response, paralleled by downregulation of proteins involved in proliferation. Moreover, a proteomic signature was identified for each ZOL treatment. Patients given three or more ZOL infusions had a better probability of survival in comparison to those given one or two treatments (86% vs. 54%, respectively, p = 0.008). Our data indicate that ZOL infusion in pediatric recipients of αβ T- and B-cell-depleted HLA-haploidentical HSCT promotes γδ T-cell differentiation and cytotoxicity and may influence the outcome of patients.

The effect of activated Mϕ1 on γδT cell-mediated killing of gastric cancer cells in vitro.

A clear understanding of the interactions between classically activated macrophages (Mϕ1) and γδT cells may improve current therapeutic approaches, including that of immunotherapy for treating certain types of cancer. The present study aimed to expand the current knowledge by showing the effect of culture supernatants of Mϕ1 on the proliferation, cell surface marker expression and tumor suppression effects of γδT cells, and by exploring the potential mechanisms involved. In vitro, Mϕ1 were cultured by GM-CSF and IFN-γ. The isopentenyl pyrophosphate method was used to amplify human peripheral blood γδT cells. The surface markers of macrophages and γδT cells were detected by flow cytometry. The proliferation of γδT cells induced by the culture supernatants of Mϕ1 was investigated using the MTT assay. The lactate dehydrogenase method was used to detect the cytotoxicity of γδT cells on the SGC-7901 gastric cancer cell line. Ten days after cultivation, the percentage of γδT cells from the repertoire of naive cells, expanded from 4.21 to 91.27%. The percentage of cells expressing CD44 was 94%. The percentage of CD68 on cultured Mϕ1 was increased from 17.7 to 73.2%. The culture supernatants of Mϕ1 increased the proliferation of γδT cells compared with the control group (33.8% vs. 0, P<0.01). The culture supernatants of Mϕ1 increased the cytotoxicity of γδT cells compared with the control group (70.18 vs. 47.25%, P<0.01). In conclusion, the supernatant of cultured Mϕ1 promotes the proliferation of γδT cells and their cytotoxic effect on the SGC-7901 gastric cancer cell line.

Five Layers of Receptor Signaling in γδ T-Cell Differentiation and Activation.

The contributions of γδ T-cells to immunity to infection or tumors critically depend on their activation and differentiation into effectors capable of secreting cytokines and killing infected or transformed cells. These processes are molecularly controlled by surface receptors that capture key extracellular cues and convey downstream intracellular signals that regulate γδ T-cell physiology. The understanding of how environmental signals are integrated by γδ T-cells is critical for their manipulation in clinical settings. Here, we discuss how different classes of surface receptors impact on human and murine γδ T-cell differentiation, activation, and expansion. In particular, we review the role of five receptor types: the T-cell receptor (TCR), costimulatory receptors, cytokine receptors, NK receptors, and inhibitory receptors. Some of the key players are the costimulatory receptors CD27 and CD28, which differentially impact on pro-inflammatory subsets of γδ T-cells; the cytokine receptors IL-2R, IL-7R, and IL-15R, which drive functional differentiation and expansion of γδ T-cells; the NK receptor NKG2D and its contribution to γδ T-cell cytotoxicity; and the inhibitory receptors PD-1 and BTLA that control γδ T-cell homeostasis. We discuss these and other receptors in the context of a five-step model of receptor signaling in γδ T-cell differentiation and activation, and discuss its implications for the manipulation of γδ T-cells in immunotherapy.

Resistance of cyclooxygenase-2 expressing pancreatic ductal adenocarcinoma cells against γδ T cell cytotoxicity

The prostaglandin (PG) synthetase cyclooxygenase 2 (Cox-2) promotes tumorigenesis, tumor progression, and metastasis in a variety of human cancer entities including pancreatic ductal adenocarcinoma (PDAC). In this study, we demonstrate that in PDAC cells such as Colo357 cells, enhanced Cox-2 expression and increased release of the Cox-2 metabolite prostaglandin E2 (PGE2) promotes resistance against γδ T cell-mediated lysis. Co-culture with activated γδ T cells induced an upregulation of Cox-2 expression in Colo357 cells, and thereby an enhanced PGE2 release, in response to tumor necrosis factor α (TNFα) secretion from γδ T cells. The PGE2-mediated inhibition of γδ T cell cytotoxicity against Cox-2-expressing PDAC cells can be partially overcome by Cox-2 inhibitors. Our results show that differences between PDAC cells in regards to sensitivity to γδ T-cell cytotoxicity can be due to distinct levels of Cox-2 expression associated with varying amounts of PGE2 release. While γδ T cell cytotoxicity against PDAC cells expressing low levels of Cox-2 can be effectively enhanced by tribody [(Her2)2×Vγ9] with specificity for Vγ9 T cell receptor and HER-2/neu on PDAC cells, a combination of tribody [(Her2)2×Vγ9] and Cox-2 inhibitor is necessary to induce complete lysis of Cox-2 high expressing Colo357. In conclusion, our results suggest that the application of tribody [(Her2)2×Vγ9] that enhances γδ T-cell cytotoxicity and Cox-2 inhibitors that overcome PGE2-mediated resistance of PDAC cells to the cytotoxic activity of γδ T cells might offer a promising combined immunotherapy for pancreatic cancer.