Skewed Differentiation of Circulating Vγ9Vδ2 T Lymphocytes in Melanoma and Impact on Clinical Outcome

Objective

The aim of this study was to evaluate over time circulating γδ T lymphocytes in melanoma patients in terms of frequency, effector functions, and relationship with clinical stage and evolution, by comparing preoperative values to those obtained at a mean follow-up of 36 months or in the event of recurrence or disease progression, and to those of healthy controls. Also, we correlated the presence of tumor-infiltrating γδ T lymphocytes with clinical evolution of melanoma.

Results

Mean frequencies of circulating γδ T cells before and after melanoma removal were very similar and comparable to healthy subjects, but patients who progressed to stage III or IV showed a significantly decreased frequency of circulating Vγ9Vδ2 T cells. The distribution of Vγ9Vδ2 memory and effector subsets was similar in healthy subjects and melanoma patients at diagnosis, but circulating γδ T cells of patients after melanoma removal had a skewed terminally-differentiated effector memory phenotype. Highly suggestive of progressive differentiation toward a cytotoxic phenotype, Vγ9Vδ2T cells from patients at follow up had increased cytotoxic potential and limited cytokine production capability, while the opposite pattern was detected in Vγ9Vδ2T cells from patients before melanoma removal.

Conclusions

Follow-up data also showed that tumor infiltrating γδ T cells were significantly associated with lower mortality and relapse rates, suggesting that they may serve as a prognostic biomarker, for human melanoma.     

HIV Infection of Monocytes-Derived Dendritic Cells Inhibits Vγ9Vδ2 T Cells Functions

DCs act as sentinel cells against incoming pathogens and represent the most potent antigen presenting cells, having the unique capability to prime naïve T cells. In addition to their role in induction of adaptive immune responses, DC are also able to activate innate cells as γδ T cells; in particular, a reciprocal crosstalk between DC and γδ T cells was demonstrated. However, whether HIV infection may alter DC-Vγ9Vδ2 T cells cross-talk was not yet described. To clarify this issue, we cultured activated Vγ9Vδ2 T cells with HIV infected monocyte derived DC (MoDC). After 5 days we evaluated MoDC phenotype, and Vγ9Vδ2 T cells activation and proliferation. In our model, Vγ9Vδ2 T cells were not able to proliferate in response to HIV-infected MoDC, although an up-regulation of CD69 was observed. Upon phosphoantigens stimulation, Vγ9Vδ2 T cells proliferation and cytokine production were inhibited when cultured with HIV-infected MoDC in a cell-contact dependent way. Moreover, HIV-infected MoDC are not able to up-regulate CD86 molecules when cultured with activated Vγ9Vδ2 T cells, compared with uninfected MoDC. Further, activated Vγ9Vδ2 T cells are not able to induce HLA DR up-regulation and CCR5 down-regulation on HIV-infected MoDC. These data indicate that HIV-infected DC alter the capacity of Vγ9Vδ2 T cells to respond to their antigens, pointing out a new mechanisms of induction of Vγ9Vδ2 T cells anergy carried out by HIV, that could contribute to immune evasion.     

Chemotherapy and zoledronate sensitize solid tumour cells to Vγ9Vδ2 T cell cytotoxicity

Combinations of cellular immune-based therapies with chemotherapy and other antitumour agents may be of significant clinical benefit in the treatment of many forms of cancer. Gamma delta (γδ) T cells are of particular interest for use in such combined therapies due to their potent antitumour cytotoxicity and relative ease of generation in vitro. Here, we demonstrate high levels of cytotoxicity against solid tumour-derived cell lines with combination treatment utilizing Vγ9Vδ2 T cells, chemotherapeutic agents and the bisphosphonate, zoledronate. Pre-treatment with low concentrations of chemotherapeutic agents or zoledronate sensitized tumour cells to rapid killing by Vγ9Vδ2 T cells with levels of cytotoxicity approaching 90%. In addition, zoledronate enhanced the chemotherapy-induced sensitization of tumour cells to Vγ9Vδ2 T cell cytotoxicity resulting in almost 100% lysis of tumour targets in some cases. Vγ9Vδ2 T cell cytotoxicity was mediated by perforin following TCR-dependent and isoprenoid-mediated recognition of tumour cells. Production of IFN-γ by Vγ9Vδ2 T cells was also induced after exposure to sensitized targets. We conclude that administration of Vγ9Vδ2 T cells at suitable intervals after chemotherapy and zoledronate may substantially increase antitumour activities in a range of malignancies. Financial support and conflicts of interest: This study was supported by grants from Medinet (Japan), and Suncorp Metway and Gallipoli Research Foundation (Australia). No financial or commercial interests arise from this study. Informed consent: This study was approved by Human Research Ethics Committees of the University of Queensland and Greenslopes Private Hospital and informed consent was obtained from all subjects.     

Human Vγ9Vδ2 T cells specifically recognize and kill acute myeloid leukemic blasts

Vγ9Vδ2 T cells are attractive candidates for antileukemic activity. The analysis of Vγ9Vδ2 T cells in newly diagnosed acute myeloid leukemia (AML) patients revealed that their absolute cell numbers were normal in the blood as well as in the bone marrow but showed a striking imbalance in the differentiation subsets, with preponderance of the effector memory population. This unusual phenotype was restored after removal of leukemic cells in patients, which reached complete remission after chemotherapy, suggesting that leukemic cells might be involved in the alteration of γδ T cell development in AML. Accordingly, coculture between AML cells and Vγ9Vδ2 T cells induced selection of effector cells. In accordance with their effector memory status, in vitro proliferation of Vγ9Vδ2 T cells was reduced compared with normal controls. Nevertheless, Vγ9Vδ2 T cells efficiently killed autologous AML blasts via the perforin/granzyme pathway. The ligands for DNAM-1 were expressed by AML cells. We showed that killing of AML blasts was TCR and DNAM-1 dependent. Using a xenotransplantation murine model, we showed that Vγ9Vδ2 T cells homed to the bone marrow in close proximity of engrafted leukemic cells and enhanced survival. These data demonstrate that Vγ9Vδ2 T cells are endowed with the ability to interact with and eradicate AML blasts both in vitro and in a mouse model. Collectively, our data revealed that Vγ9Vδ2 T cells have a potent antileukemic activity provided that optimal activation is achieved, such as with synthetic TCR agonists. This study enhances the interest of these cells for therapeutic purposes such as AML treatment.     

Type 1 responses of human Vγ9Vδ2 T cells to influenza A viruses

γδ T cells are essential constituents of antimicrobial and antitumor defenses. We have recently reported that phosphoantigen isopentenyl pyrophosphate (IPP)-expanded human Vγ9Vδ2 T cells participated in anti-influenza virus immunity by efficiently killing both human and avian influenza virus-infected monocyte-derived macrophages (MDMs) in vitro. However, little is known about the noncytolytic responses and trafficking program of γδ T cells to influenza virus. In this study, we found that Vγ9Vδ2 T cells expressed both type 1 cytokines and chemokine receptors during influenza virus infection, and IPP-expanded cells had a higher capacity to produce gamma interferon (IFN-γ). Besides their potent cytolytic activity against pandemic H1N1 virus-infected cells, IPP-activated γδ T cells also had noncytolytic inhibitory effects on seasonal and pandemic H1N1 viruses via IFN-γ but had no such effects on avian H5N1 or H9N2 virus. Avian H5N1 and H9N2 viruses induced significantly higher CCL3, CCL4, and CCL5 production in Vγ9Vδ2 T cells than human seasonal H1N1 virus. CCR5 mediated the migration of Vγ9Vδ2 T cells toward influenza virus-infected cells. Our findings suggest a novel therapeutic strategy of using phosphoantigens to boost the antiviral activities of human Vγ9Vδ2 T cells against influenza virus infection.     

Vγ9Vδ2 T cell-mediated recognition of human solid tumors. Potential for immunotherapy of hepatocellular and colorectal carcinomas

Introduction Vγ9Vδ2 T lymphocytes are reported to participate in the anti-tumor immune surveillance in human. They are known to recognize phosphoantigens and molecules expressed on cells undergoing neoplasic transformation. In this study, we investigated phenotype and anti-tumor cytotoxicity of ex vivo expanded Vγ9Vδ2 T cells in view of adoptive immunotherapy. Materials and Methods Experiments were performed with peripheral blood samples from eleven patients [six colorectal carcinoma (CRC), four hepatocellular carcinoma (HCC), one sarcoma] and sixteen healthy donors. Results/Discussion Ex vivo expansion of Vγ9Vδ2 T cells could be achieved by a single dose of phosphoantigen, either bromohydrin pyrophosphate or zoledronate, and supported by exogenous IL-2. After 2 weeks, expanded Vγ9Vδ2 T lymphocytes acquired the effector memory phenotype CD45RA⁻CD45RO^highCD27⁻. They expressed NKG2D and CD161 and the proinflammatory CXCR3 and CCR5 chemokine receptors. Vγ9Vδ2 T cells displayed a strong lytic activity toward a broad panel of tumor cell lines or primary cultures. Interestingly, HCC and CRC primary cells could be lysed by autologous Vγ9Vδ2 T cells whereas autologous normal cells were not sensitive to the lysis. mAbs blocking assays demonstrated that TCR was the most important receptor involved in the lysis of tumor cells. However, NKG2D receptor could deliver a costimulatory signal enhancing the lysis of HCC and CRC tumors expressing MICA/B. Treatment of tumor cells by the mevalonate pathway inhibitor, zoledronate, enhanced the killing of both HCC and CRC. Expansion index of Vγ9Vδ2 T cells was in similar levels in healthy donors or in cancer patients and total expansion was suitable for adoptive immunotherapy. Conclusion These results provide a rationale for the clinical evaluation of Vγ9Vδ2 T lymphocytes in HCC and CRC.     

Recognition by human Vγ9/Vδ2 T cells of melanoma cells upon fusion with Daudi cells

 Daudi Burkitt’s lymphoma cells, unlike other tumor cell lines, stimulate human T cells coexpressing the variable (V) region genes TCRG-V9 and V TCRD-V2 to proliferate and secrete lymphokines. Hybrids, derived by the fusion of Daudi cells with the human melanoma cell line MZ2-MEL 2.2, retain the morphology of melanoma cells. Unlike the parental melanoma cell line, these Daudi × MZ2-MEL 2.2 hybrids stimulate secretion of tumor necrosis factor (TNF) and granulocyte/macrophage colony stimulating factor (GM-CSF) by CD4-positive Vγ9/Vδ2 T-cell clones. Whereas the stimulator phenotype of Daudi cells behaves as a dominant trait in Daudi × melanoma hybrids, the expression of B-cell differentiation markers is suppressed. Thus, the γ/δ T-cell ligand expressed by Daudi cells behaves as a dominant tumor antigen in Daudi × melanoma hybrids and is unrelated to the differentiated B-cell phenotype. Dominant expression of the Daudi ligand for human Vγ9/Vδ2 T cells in these hybrids may provide a basis for defining the stimulatory principle at the molecular level. Received: 2 May 1996 / Revised: 15 July 1996     

Dual regulation effect and mechanism of human myeloid-derived suppressor cells on anticolorectal cancer cells activity of Vγ9Vδ2 T cells

Myeloid-derived suppressor cells (MDSC) are a group of immature inhibitory cells of bone marrow origin. Human γδ T cells (mainly Vγ9Vδ2 T cells) have emerged as dominant candidates for cancer immunotherapy because of their unique recognition pattern and broad killing activity against tumor cells. Intestinal mucosal intraepithelial lymphocytes are almost exclusively γδ T cells, so it plays an important role in inhibiting the development of colorectal cancer. In this study, we investigated the effects and molecular mechanism of human MDSC on anticolorectal cancer cells activity of Vγ9Vδ2 T cells. Our results suggested that MDSC can reduce the NKG2D expression of Vγ9Vδ2 T cells through direct cell–cell contact, which is associated with membrane-type transforming growth factor-β. In contrast, MDSC can increase Vγ9Vδ2 T cells activation and production of IFN-γ, perforin, Granzyme B through direct cell–cell contact. This may be related to the upregulation of T-bet in Vγ9Vδ2 T cells by MDSC. However, MDSC had a dominant negative regulatory effect on the anticolorectal cancer cells activity of Vγ9Vδ2 T cells. Our study provides a theoretical basis for the immune regulatory function of human MDSC on γδ T cells. This will be conducive to the clinical development of a new antitumor therapy strategy.     

Full Restoration of Brucella-Infected Dendritic Cell Functionality through Vγ9Vδ2 T Helper Type 1 Crosstalk

Vγ9Vδ2 T cells play an important role in the immune response to infectious agents but the mechanisms contributing to this immune process remain to be better characterized. Following their activation, Vγ9Vδ2 T cells develop cytotoxic activity against infected cells, secrete large amounts of cytokines and influence the function of other effectors of immunity, notably cells playing a key role in the initiation of the adaptive immune response such as dendritic cells. Brucella infection dramatically impairs dendritic cell maturation and their capacity to present antigens to T cells. Herein, we investigated whether V T cells have the ability to restore the full functional capacities of Brucella-infected dendritic cells. Using an in vitro multicellular infection model, we showed that: 1/Brucella-infected dendritic cells activate Vγ9Vδ2 T cells through contact-dependent mechanisms, 2/activated Vγ9Vδ2 T cells induce full differentiation into IL-12 producing cells of Brucella-infected dendritic cells with functional antigen presentation activity. Furthermore, phosphoantigen-activated Vγ9Vδ2 T cells also play a role in triggering the maturation process of dendritic cells already infected for 24 h. This suggests that activated Vγ9Vδ2 T cells could be used to modulate the outcome of infectious diseases by promoting an adjuvant effect in dendritic cell-based cellular therapies.     

Indirect stimulation of human Vγ2Vδ2 T cells through alterations in isoprenoid metabolism

Human Vγ2Vδ2 T cells monitor isoprenoid metabolism by recognizing (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), an intermediate in the 2-C-methyl-d-erythritol-4-phosphate pathway used by microbes, and isopentenyl pyrophosphate (IPP), an intermediate in the mevalonate pathway used by humans. Aminobisphosphonates and alkylamines indirectly stimulate Vγ2Vδ2 cells by inhibiting farnesyl diphosphate synthase (FDPS) in the mevalonate pathway, thereby increasing IPP/triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester that directly stimulate. In this study, we further characterize stimulation by these compounds and define pathways used by new classes of compounds. Consistent with FDPS inhibition, stimulation of Vγ2Vδ2 cells by aminobisphosphonates and alkylamines was much more sensitive to statin inhibition than stimulation by prenyl pyrophosphates; however, the continuous presence of aminobisphosphonates was toxic for T cells and blocked their proliferation. Aminobisphosphonate stimulation was rapid and prolonged, independent of known Ag-presenting molecules, and resistant to fixation. New classes of stimulatory compounds-mevalonate, the alcohol of HMBPP, and alkenyl phosphonates-likely stimulate differently. Mevalonate, a rate-limiting metabolite, appears to enter cells to increase IPP levels, whereas the alcohol of HMBPP and alkenyl phosphonates are directly recognized. The critical chemical feature of bisphosphonates is the amino moiety, because its loss switched aminobisphosphonates to direct Ags. Transfection of APCs with small interfering RNA downregulating FDPS rendered them stimulatory for Vγ2Vδ2 cells and increased cellular IPP. Small interfering RNAs for isopentenyl diphosphate isomerase functioned similarly. Our results show that a variety of manipulations affecting isoprenoid metabolism lead to stimulation of Vγ2Vδ2 T cells and that pulsing aminobisphosphonates would be more effective for the ex vivo expansion of Vγ2Vδ2 T cells for adoptive cancer immunotherapy.     

Expansion,Reexpansion, and Recall-Like Expansion of Vγ2Vδ2 T Cells in Smallpox Vaccination and Monkeypox Virus Infection

Little is known about the in vivo kinetics of T-cell responses in smallpox/monkeypox. We showed that macaque Vγ2Vδ2 T cells underwent 3-week-long expansion after smallpox vaccine immunization and displayed simple reexpansion in association with sterile anti-monkeypox virus (anti-MPV) immunity after MPV challenge. Virus-activated Vγ2Vδ2 T cells exhibited gamma interferon-producing effector function after phosphoantigen stimulation. Surprisingly, like αβ T cells, suboptimally primed Vγ2Vδ2 T cells in vaccinia virus/cidofovir-covaccinated macaques mounted major recall-like expansion after MPV challenge. Finally, Vγ2Vδ2 T cells localized in inflamed lung tissues for potential regulation. Our studies provide the first in vivo evidence that viruses, despite their inability to produce exogenous phosphoantigen, can induce expansion, reexpansion, and recall-like expansion of Vγ2Vδ2 T cells and stimulate their antimicrobial cytokine response.Human γδ T cells appear to contribute to both innate and adaptive immune responses (4, 6, 10, 19). Vγ2Vδ2 T cells exist only in primates, and in humans, they constitute 60 to 95% of total blood γδ T cells. The capacity of Vγ2Vδ2 T cells to undergo major clonal expansion in primary infection and to mount rapid recall expansion upon reinfection has been proposed as an adaptive immune response (6), which is consistent with memory phenotypes of Vγ2Vδ2 T cells (7), long-term expansion of memory-like Vδ2 T cells, and in vitro recall expansion of blood γδ T cells in vaccinated or infected humans (1, 15a, 16, 17, 25). It is important to note that the microbial antigen recognized by Vγ2Vδ2 T cells is temporally limited to (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), commonly referred to as phosphoantigen, produced in the newly discovered 2-C-methyl-d-erythritol-4-phosphate pathway of isoprenoid biosynthesis in bacteria, but not viruses (8). Our recent study has demonstrated that HMBPP is presented by a putative molecule on antigen-presenting cell membranes and recognized by Vγ2Vδ2 T-cell receptor (TCR) (24). Since viruses do not produce exogenous HMBPP recognized by Vγ2Vδ2 T cells, in vivo Vγ2Vδ2 T-cell expansion usually occurs only in HMBPP-producing bacterial or protozoal infections.Monkeypox virus (MPV) (an orthopoxvirus) has biological features similar to those of smallpox virus, and MPV infection is clinically similar to smallpox in humans (9, 12, 22). Immune responses of Vγ2Vδ2 T cells during lethal MPV infection have not been studied, although some laboratories have undertaken in vitro studies of γδ T-cell immune responses to vaccinia virus (1, 2, 15). We presume that initial vaccinia virus immunization and subsequent MPV challenge of macaques would provide an ideal in vivo setting in which to determine whether Vγ2Vδ2 T cells can mount innate-like or recall-like responses to orthopoxvirus infections. We made a novel observation indicating expansion, reexpansion, and recall-like expansion of Vγ2Vδ2 T cells with effector function in response to smallpox vaccination and MPV infection in macaques.     

The capacity of CD4+ Vγ9Vδ2 T cells to kill cancer cells correlates with co-expression of CD56

Human Vγ9Vδ2 T cells are a unique T-cell type, and data from recent studies of Vγ9Vδ2 T cells emphasize their potential relevance to cancer immunotherapy. Vγ9Vδ2 T cells exhibit dual properties since they are both antigen-presenting cells and cytotoxic toward cancer cells. The majority of Vγ9Vδ2 T cells are double-negative for the co-receptors CD4 and CD8, and only 20–30% express CD8. Even though they are mostly neglected, a small fraction of Vγ9Vδ2 T cells also express the co-receptor CD4. Here the authors show that CD4⁺ Vγ9Vδ2 T cells comprise 0.1–7% of peripheral blood Vγ9Vδ2 T cells. These cells can be expanded in vitro using zoledronic acid, pamidronic acid or CD3 antibodies combined with IL-2 and feeder cells. Unlike most conventional CD4⁺ αβ T cells, CD4⁺ Vγ9Vδ2 T cells are potently cytotoxic and can kill cancer cells, which is here shown by the killing of cancer cell lines of different histological origins, including breast cancer, prostate cancer and melanoma cell lines, upon treatment with zoledronic acid. Notably, the killing capacity of CD4⁺ Vγ9Vδ2 T cells correlates with co-expression of CD56.     

Identification of a novel proinflammatory human skin-homing Vγ9Vδ2 T cell subset with a potential role in psoriasis

γδ T cells mediate rapid tissue responses in murine skin and participate in cutaneous immune regulation including protection against cancer. The role of human γδ cells in cutaneous homeostasis and pathology is characterized poorly. In this study, we show in vivo evidence that human blood contains a distinct subset of proinflammatory cutaneous lymphocyte Ag and CCR6-positive Vγ9Vδ2 T cells, which is rapidly recruited into perturbed human skin. Vγ9Vδ2 T cells produced an array of proinflammatory mediators including IL-17A and activated keratinocytes in a TNF-α- and IFN-γ-dependent manner. Examination of the common inflammatory skin disease psoriasis revealed a striking reduction of circulating Vγ9Vδ2 T cells in psoriasis patients compared with healthy controls and atopic dermatitis patients. Decreased numbers of circulating Vγ9Vδ2 T cells normalized after successful treatment with psoriasis-targeted therapy. Taken together with the increased presence of Vγ9Vδ2 T cells in psoriatic skin, these data indicate redistribution of Vγ9Vδ2 T cells from the blood to the skin compartment in psoriasis. In summary, we report a novel human proinflammatory γδ T cell involved in skin immune surveillance with immediate response characteristics and with potential clinical relevance in inflammatory skin disease.     

Aminobisphosphonate-pretreated dendritic cells trigger successful Vγ9Vδ2 T cell amplification for immunotherapy in advanced cancer patients

Hepatocellular carcinoma (HCC) and colorectal carcinoma with hepatic metastases (mCRC) are cancers with poor prognosis and limited therapeutic options. New approaches are needed and adoptive immunotherapy with Vγ9Vδ2 T lymphocytes represents an attractive strategy. Indeed, Vγ9Vδ2 T cells were shown to exhibit efficient lytic activity against various human tumor cell lines, and in vitro Vγ9Vδ2 T expansion protocol based on single phosphoantigen stimulation could be easily performed for healthy donors. However, a low proliferative response of Vγ9Vδ2 T cells was observed in about half of the cancer patients, leading to an important limitation in the development of Vγ9Vδ2 T cell-based immunotherapy. Here, for the first time in the context of cancer patients, Vγ9Vδ2 T cell expansions were performed by co-culturing peripheral blood mononuclear cell (PBMCs) with autologous dendritic cells (DCs) pretreated with aminobisphosphonate zoledronate. For patients not responding to the conventional culture protocol, co-culture of PBMC with zoledronate-pretreated DCs induced strong cell expansion and allowed reaching a minimal rate of purity of 70% of Vγ9Vδ2 T cells. The potent immunostimulatory activity of zoledronate-treated DCs was associated with higher amount of isopentenyl pyrophosphate (IPP) in the culture and was correlated with better ability to activate Vγ9Vδ2 T cells as measured by IFN-γ production. Moreover, we demonstrated that the cytotoxic level of Vγ9Vδ2 T cells against freshly autologous tumor cells isolated from patients could be significantly increased by pretreating the tumor cells with zoledronate. Thus, this method of generating Vγ9Vδ2 T cells leads eligible for Vγ9Vδ2 T cell adoptive immunotherapy the HCC and mCRC patients.     

Ex vivo characterization of γδ T-cell repertoire in patients after adoptive transfer of Vγ9Vδ2 T cells expressing the interleukin-2 receptor β-chain and the common γ-chain

Background aimsAdoptive immunotherapy is emerging as a potent anti-tumor treatment modality; Vγ9Vδ2 T cells may represent appropriate agents for such cancer immunotherapy. To improve the currently limited success of Vγ9Vδ2 T-cell–based immunotherapy, we examined the in vivo dynamics of these adoptively-transferred cells and hypothesized that interleukin (IL)-15 is the potential factor for Vγ9δ2 T cell in vivo survival.MethodsWe conducted a clinical trial of adoptive Vγ9Vδ2 T-cell transfer therapy in six colorectal cancer patients who received pulmonary metastasectomy. Patients' peripheral blood mononuclear cells were stimulated with zoledronate (5 μmol/L) and IL-2 (1000 IU/mL) for 14 d. Harvested cells, mostly Vγ9Vδ2 T cells, were given intravenously weekly without additional IL-2 eight times in total. The frequency, phenotype and common γ-chain cytokine receptor expression of Vγ9Vδ2 T cells in peripheral blood was monitored by flow cytometry at each time point during treatment and 4 and 12 weeks after the last administration.ResultsAdoptively transferred Vγ9Vδ2 T cells expanded well without exogenous IL-2 administration or lymphodepleting preconditioning. They maintained effector functions in terms of interferon-γ secretion and prompt release of cytotoxic granules in response to PMA/ionomycin or isopentenyl pyrophosphate–positive cells. Because they are IL-2Rα^?IL-7Rα^?IL-15Rα^?IL-2Rβ⁺γ_c⁺, it is likely that IL-2 or IL-15 is required for their maintenance.ConclusionsThe persistence of large numbers of functionally active adoptively transferred Vγ9Vδ2 T cells in the absence of exogenous IL-2 implies that an endogenous factor, such as IL-15 transpresentation, is adequate to support these cells in vivo.     

Large-scale expansion of Vγ9Vδ2 T cells with engineered K562 feeder cells in G-Rex vessels and their use as chimeric antigen receptor–modified effector cells

Vγ9Vδ2 T cells are a minor subset of lymphocytes in the peripheral blood that has been extensively investigated for their tolerability, safety and anticancer efficacy. A hindrance to the broad application of these cells for adoptive cellular immunotherapy has been attaining clinically appropriate numbers of Vγ9Vδ2 T cells. Furthermore, Vγ9Vδ2 T cells exist at low frequencies among cancer patients. We, therefore, sought to conceive an economical method that allows for a quick and robust large-scale expansion of Vγ9Vδ2 T cells. A two-step protocol was developed, in which peripheral blood mononuclear cells (PBMCs) from healthy donors or cancer patients were activated with Zometa and interleukin (IL)-2, followed by co-culturing with gamma-irradiated, CD64-, CD86- and CD137L-expressing K562 artificial antigen-presenting cells (aAPCs) in the presence of the anti-CD3 antibody OKT3. We optimized the co-culture ratio of K562 aAPCs to immune cells, and migrated this method to a G-Rex cell growth platform to derive clinically relevant cell numbers in a Good Manufacturing Practice (GMP)-compliant manner. We further include a depletion step to selectively remove αβ T lymphocytes. The method exhibited high expansion folds and a specific enrichment of Vγ9Vδ2 T cells. Expanded Vγ9Vδ2 T cells displayed an effector memory phenotype with a concomitant down-regulated expression of inhibitory immune checkpoint receptors. Finally, we ascertained the cytotoxic activity of these expanded cells by using nonmodified and chimeric antigen receptor (CAR)–engrafted Vγ9Vδ2 T cells against a panel of solid tumor cells. Overall, we report an efficient approach to generate highly functional Vγ9Vδ2 T cells in massive numbers suitable for clinical application in an allogeneic setting.     

Phase I study of bromohydrin pyrophosphate (BrHPP, IPH 1101), a Vγ9Vδ2 T lymphocyte agonist in patients with solid tumors

Purpose

Vγ9Vδ2 (γδ) T lymphocytes, a critical peripheral blood lymphocyte subset, are directly cytotoxic against many solid and hematologic tumor types. Vγ9Vδ2 T lymphocytes can be selectively expanded in vivo with BrHPP (IPH1101) and IL-2. The present phase I trial was conducted with the aim of determining the maximum-tolerated dose (MTD) and safety of IPH1101 combined with a low dose of IL-2 in patients with solid tumors.

Experimental design

A 1-h intravenous infusion of IPH11 was administered alone at cycle 1, combined with a low dose of SC IL-2 (1 MIU/M² d1 to d7) in the subsequent cycles (day 1 every 3 weeks). The dose of IPH1101 was escalated from 200 to 1,800 mg/m².

Results

As much as 28 patients with solid tumors underwent a total of 109 treatment cycles. Pharmacodynamics data demonstrate that γδ T lymphocyte amplification in humans requires the co-administration of IL-2 and is dependent on IPH 1101 dose. Dose-limiting toxicity occurred in two patients at a dose of 1,800 mg/m²: one grade 3 fever (1 patient) and one grade 3 hypotension (1 patient) suggesting cytokine release syndrome immediately following the first infusion. At lower doses the treatment was well tolerated; the most frequent adverse events were mild fever, chills and abdominal pain, without exacerbation in the IL-2 combined cycles.

Conclusion

IPH1101 in combination with SC low-dose IL-2 is safe, well tolerated and induces a potent γδ T lymphocyte expansion in patients. Its clinical activity will be evaluated in phase II clinical trials.     

Multieffector-functional immune responses of HMBPP-specific Vγ2Vδ2 T cells in nonhuman primates inoculated with Listeria monocytogenes ΔactA prfA*

Although Listeria monocytogenes can induce systemic infection causing spontaneous abortion, septicemia, and meningitis, studies have not been performed to investigate human anti-L. monocytogenes immune responses, including those of Ag-specific Vγ2Vδ2 T cells, a dominant human γδ T cell subset. L. monocytogenes is the only pathogen known to possess both the mevalonate and non-mevalonate isoprenoid biosynthesis pathways that produce metabolic phosphates or phosphoantigens activating human Vγ2Vδ2 T cells, making it interesting to explore in vivo anti-L. monocytogenes immune responses of Vγ2Vδ2 T cells. In this study, we demonstrated that subclinical systemic L. monocytogenes infection of rhesus macaques via parenteral inoculation or vaccination with an attenuated Listeria strain induced multieffector-functional immune responses of phosphoantigen-specific Vγ2Vδ2 T cells. Subclinical systemic infection and reinfection with attenuated L. monocytogenes uncovered the ability of Vγ2Vδ2 T cells to mount expansion and adaptive or recall-like expansion. Expanded Vγ2Vδ2 T cells could traffic to and accumulate in the pulmonary compartment and intestinal mucosa. Expanded Vγ2Vδ2 T cells could evolve into effector cells producing IFN-γ, TNF-α, IL-4, IL-17, or perforin after L. monocytogenes infection, and some effector Vγ2Vδ2 T cells could coproduce IL-17 and IFN-γ, IL-4 and IFN-γ, or TNF-α and perforin. Surprisingly, in vivo-expanded Vγ2Vδ2 T effector cells in subclinical L. monocytogenes infection could directly lyse L. monocytogenes-infected target cells and inhibit intracellular L. monocytogenes bacteria. Thus, we present the first demonstration, to our knowledge, of multieffector-functional Vγ2Vδ2 T cell responses against L. monocytogenes.     

硼替佐米通过激活Notch/NF-κB通路增强Vγ9Vδ2T细胞对套细胞淋巴瘤的杀伤效应

目的探讨蛋白酶体抑制剂硼替佐米(bortezomib,BZM)增强Vγ9Vδ2T细胞对套细胞淋巴瘤的杀伤作用及其相关机制。方法通过帕米磷酸二钠和IL-2刺激人外周血中单个核细胞得到Vγ9Vδ2T细胞。给予BZM刺激后,溴乙啡锭二聚体-1(ethidium homodimer-1,EthD-1)染色法检测Vγ9Vδ2T细胞对Maver细胞的杀伤效应;CD107a标记法检测Vγ9Vδ2T细胞的脱颗粒效应;CD25与CD69标记法检测Vγ9Vδ2T细胞的活化状况;ELISA法检测Vγ9Vδ2T细胞的TNF-α和IFN-γ的生成水平;Western blot检测Vγ9Vδ2T细胞中Notch1和p-NF-κB水平。结果 BZM增强Vγ9Vδ2T细胞对Maver细胞的杀伤效应和增加Vγ9Vδ2T细胞的脱颗粒能力(CD107a表达增加)。BZM增强Vγ9Vδ2T细胞活化(CD25与CD69的表达增加)以及活化效应功能(TNF-α和IFN-γ的生成水平增加)。BZM能增强Vγ9Vδ2T细胞的Notch1/NF-κB信号(Notch1和p-NF-κB水平增加)。给予Notch1和NF-κB抑制剂处理Vγ9Vδ2T细胞,BZM的以上效应可被部分逆转。结论 BZM可通过Notch/NF-κB通路增强Vγ9Vδ2T细胞的活化,并进一步增强Vγ9Vδ2T细胞对Maver细胞的杀伤效应。