PD-L1 detection on circulating tumor-derived extracellular vesicles (T-EVs) from patients with lung cancer.

BACKGROUND: Recent breakthroughs in therapies with immune checkpoint inhibitors (ICIs) have revolutionized the treatment of lung cancer. However, only 15-25% of patients respond to the ICIs therapy, and methods to identify those responsive patients are currently a hot research topic. PD-L1 expression measured on tumor tissues using immunohistochemistry (IHC) was approved as one of the companion diagnostic methods, but it is invasive and cannot be used to monitor dynamic changes in PD-L1 expression during treatments. METHODS: In this study, we developed an Epcam-PD-L1 extracellular vesicle (EV) detection prototype using the Simoa platform. This assay detected PD-L1 expression levels on tumor-derived exosomes from the lung cancer cell lines A549 and SK-MES1. In addition, 35 plasma samples from patients with lung cancer were tested with this assay and the results were compared to the tissue PD-L1 expression levels represented by the tumor proportion score (TPS). RESULTS: PD-L1 TPS-positive patients (≥1% IHC TPS) had significantly higher Simoa Epcam-PD-L1 signals than TPS-negative patients (<1% IHC TPS, P=0.026). The Simoa Epcam-PD-L1 area under curve (AUC) reached 0.776, with a sensitivity of 92.86% and a specificity of 71.43%. When PD-L1 TPS-positive patients were defined as having an IHC TPS ≥10%, the greatest difference in Epcam-PD-L1 signals was observed between IHC TPS-positive and IHC TPS-negative groups (P=0.0024) and the Simoa Epcam-PD-L1 AUC reached 0.832. Finally, the Spearman's correlation coefficient showed a significant correlation between the TPS and Simoa Epcam-PD-L1 signals (0.428, P=0.0104). CONCLUSIONS: Based on our results, our Simoa Epcam-PD-L1 EV detection assay is a potential liquid biopsy method to predict the PD-L1 expression level in patients with lung cancer.

The Juxtamembrane Domain of Butyrophilin BTN3A1 Controls Phosphoantigen-Mediated Activation of Human Vγ9Vδ2 T Cells.

Vγ9Vδ2 T lymphocytes are the major human peripheral γδ T cell subset, with broad reactivity against stressed human cells, including tumor cells. Vγ9Vδ2 T cells are specifically activated by small phosphorylated metabolites called phosphoantigens (PAg). Stress-induced changes in target cell PAg levels are specifically detected by butyrophilin (BTN)3A1, using its intracellular B30.2 domain. This leads to the activation of Vγ9Vδ2 T cells. In this study, we show that changes in the juxtamembrane domain of BTN3A1, but not its transmembrane domain, induce a markedly enhanced or reduced γδ T cell reactivity. There is thus a specific requirement for BTN3A1's juxtamembrane domain for correct γδ T cell-related function. This work identified, as being of particular importance, a juxtamembrane domain region of BTN3A molecules identified as a possible dimerization interface and that is located close to the start of the B30.2 domain.

Sensing of cell stress by human γδ TCR-dependent recognition of annexin A2.

Human γδ T cells comprise a first line of defense through T-cell receptor (TCR) recognition of stressed cells. However, the molecular determinants and stress pathways involved in this recognition are largely unknown. Here we show that exposure of tumor cells to various stress situations led to tumor cell recognition by a Vγ8Vδ3 TCR. Using a strategy that we previously developed to identify antigenic ligands of γδ TCRs, annexin A2 was identified as the direct ligand of Vγ8Vδ3 TCR, and was found to be expressed on tumor cells upon the stress situations tested in a reactive oxygen species-dependent manner. Moreover, purified annexin A2 was able to stimulate the proliferation of a Vδ2neg γδ T-cell subset within peripheral blood mononuclear cells and other annexin A2-specific Vδ2neg γδ T-cell clones could be derived from peripheral blood mononuclear cells. We thus propose membrane exposure of annexin A2 as an oxidative stress signal for some Vδ2neg γδ T cells that could be involved in an adaptive stress surveillance.

Impact of Graft-Versus-Graft Natural Killer Cell Alloreactivity on Single Unit Dominance After Double Umbilical Cord Blood Transplantation.

BACKGROUND: Natural killer (NK) cell alloreactivity is favored after double umbilical cord blood transplantation (dUCBT) in which cord blood (UCB) units and patients are often HLA class I mismatched. Generally, only 1 UCB unit persists after dUCBT. We hypothesize, that NK cell alloreactivity mediated by killer cell immunoglobulin-like receptor (KIR)-HLA interactions may explain the dominance of 1UCB unit over the other after dUCBT. METHODS: We investigated the impact of KIR NK cell alloreactivities on the dominance of 1 full UCB unit in 50 dUCBT. We analyzed the effects of the KIR/HLA genetic incompatibilities and studied cord blood cells at both the phenotypic and functional levels. RESULTS: The genetic combination of KIR3DL1 loser UCB unit/Bw4 winner UCB unit determined both the dominance of 1 UCB unit (hazards ratio, 2.88 [1.32-6.27], P = 0.0077) and correlated with an increased incidence of relapse (hazards ratio, 4.91 [1.39-17.3], P = 0.0134). It is interesting to note that cord blood cells exhibited extremely low HLA class I expression. Moreover, resting cord blood KIR3DL1 NK cells exhibited a basal alloreactivity against Bw4 target cells that increased upon activation, thus triggering death by apoptosis. CONCLUSIONS: Our unicentric study suggests, for the first time, the significant impact of KIR NK cell alloreactivity in the determination of which UCB unit will dominate in dUCBT.

Stereotaxic administrations of allogeneic human Vγ9Vδ2 T cells efficiently control the development of human glioblastoma brain tumors.

Glioblastoma multiforme (GBM) represents the most frequent and deadliest primary brain tumor. Aggressive treatment still fails to eliminate deep brain infiltrative and highly resistant tumor cells. Human Vγ9Vδ2 T cells, the major peripheral blood γδ T cell subset, react against a wide array of tumor cells and represent attractive immune effector T cells for the design of antitumor therapies. This study aims at providing a preclinical rationale for immunotherapies in GBM based on stereotaxic administration of allogeneic human Vγ9Vδ2 T cells. The feasibility and the antitumor efficacy of stereotaxic Vγ9Vδ2 T cell injections have been investigated in orthotopic GBM mice model using selected heterogeneous and invasive primary human GBM cells. Allogeneic human Vγ9Vδ2 T cells survive and patrol for several days within the brain parenchyma following adoptive transfer and can successfully eliminate infiltrative GBM primary cells. These striking observations pave the way for optimized stereotaxic antitumor immunotherapies targeting human allogeneic Vγ9Vδ2 T cells in GBM patients.

Role of the MHC restriction during maturation of antigen-specific human T cells in the thymus.

In the thymus, a T-cell repertoire able to confer protection against infectious and noninfectious agents in a peptide-dependent, self-MHC-restricted manner is selected. Direct detection of Ag-specific thymocytes, and analysis of the impact of the expression of the MHC-restricting allele on their frequency or function has never been studied in humans because of the extremely low precursor frequency. Here, we used a tetramer-based enrichment protocol to analyze the ex vivo frequency and activation-phenotype of human thymocytes specific for self, viral and tumor-antigens presented by HLA-A*0201 (A2) in individuals expressing or not this allele. Ag-specific thymocytes were quantified within both CD4CD8 double or single-positive compartments in every donor. Our data indicate that the maturation efficiency of Ag-specific thymocytes is poorly affected by HLA-A2 expression, in terms of frequencies. Nevertheless, A2-restricted T-cell lines from A2(+) donors reacted to A2(+) cell lines in a highly peptide-specific fashion, whereas their alloreactive counterparts showed off-target activity. This first ex vivo analysis of human antigen-specific thymocytes at different stages of human T-cell development should open new perspectives in the understanding of the human thymic selection process.

Consensus nomenclature for CD8+ T cell phenotypes in cancer.

Whereas preclinical investigations and clinical studies have established that CD8+ T cells can profoundly affect cancer progression, the underlying mechanisms are still elusive. Challenging the prevalent view that the beneficial effect of CD8+ T cells in cancer is solely attributable to their cytotoxic activity, several reports have indicated that the ability of CD8+ T cells to promote tumor regression is dependent on their cytokine secretion profile and their ability to self-renew. Evidence has also shown that the tumor microenvironment can disarm CD8+ T cell immunity, leading to the emergence of dysfunctional CD8+ T cells. The existence of different types of CD8+ T cells in cancer calls for a more precise definition of the CD8+ T cell immune phenotypes in cancer and the abandonment of the generic terms "pro-tumor" and "antitumor." Based on recent studies investigating the functions of CD8+ T cells in cancer, we here propose some guidelines to precisely define the functional states of CD8+ T cells in cancer.

Development of Predictive Value of Urinary Cytokine Profile Induced During Intravesical Bacillus Calmette-Guérin Instillations for Bladder Cancer.

BACKGROUND: We sought to demonstrate a correlation between the response to treatment and the profile of urinary cytokine production during bacillus Calmette-Guérin (BCG) therapy. MATERIAL AND METHODS: From December 2008 to February 2011, 23 patients were included in a prospective study. All patients received 6 instillations of BCG weekly. The mean follow-up period of the population was 16.9 ± 8.4 months. Refractory disease or recurrence was observed in 5 patients. Urine samples were collected and stored at -80°C, before and 4 hours after the first, third, and sixth BCG instillations. The cytokines (interleukin [IL]-2, IL-4, IL-6, IL-10, IL-17, interferon-gamma [IFNγ] and tumor necrosis factor-alpha) were quantified within the collected urine samples using cytometric bead array analysis. The quantitative variables were analyzed using Student's t test, and regression statistical analysis was performed. RESULTS: Urinary cytokine production had increased strongly 4 hours after the sixth instillation but only mildly to moderately after the first and third instillations. IL-2 and IL-6 showed the most dramatic changes after the BCG instillations. Different urinary cytokine production profiles were demonstrated. A trend was observed for the BCG-refractory/recurrence group, with high baseline IL-6 levels, followed by low IL-6 levels before the instillations; low baseline IL-2 levels with only minor changes during treatment; the absence of IFNγ and IL-17 production; and a low peak of cytokine production at the end of treatment. CONCLUSION: Analysis of the urinary cytokine production levels during BCG therapy reflect a specific immune response induced in each patient. Their assessment could allow a more reliable selection of patients eligible for this type of treatment and could help justify the use of maintenance BCG therapy.

Chicago 2014--30 years of γδ T cells.

The international γδ T cell conference takes place every 2 years. After being held in Denver (USA) in 2004, La Jolla (USA) in 2006, Marseille (France) in 2008, Kiel (Germany) in 2010 and Freiburg (Germany) in 2012, the γδ T cell community gathered this time in Chicago (USA). This conference was organized by Zheng Chen from 16 to 18 May 2014 at his home institution, the University of Illinois College of Medicine, and boasted 180 attendants from all over the world and almost 100 submitted abstracts.

Analysis of relationships between peptide/MHC structural features and naive T cell frequency in humans.

The structural rules governing peptide/MHC (pMHC) recognition by T cells remain unclear. To address this question, we performed a structural characterization of several HLA-A2/peptide complexes and assessed in parallel their antigenicity, by analyzing the frequency of the corresponding Ag-specific naive T cells in A2(+) and A2(-) individuals, as well as within CD4(+) and CD8(+) subsets. We were able to find a correlation between specific naive T cell frequency and peptide solvent accessibility and/or mobility for a subset of moderately prominent peptides. However, one single structural parameter of the pMHC complexes could not be identified to explain each peptide antigenicity. Enhanced pMHC antigenicity was associated with both highly biased TRAV usage, possibly reflecting favored interaction between particular pMHC complexes and germline TRAV loops, and peptide structural features allowing interactions with a broad range of permissive CDR3 loops. In this context of constrained TCR docking mode, an optimal peptide solvent exposed surface leading to an optimal complementarity with TCR interface may constitute one of the key features leading to high frequency of specific T cells. Altogether our results suggest that frequency of specific T cells depends on the fine-tuning of several parameters, the structural determinants governing TCR-pMHC interaction being just one of them.

Targeted activation of human Vγ9Vδ2-T cells controls epstein-barr virus-induced B cell lymphoproliferative disease.

Epstein-Barr virus-induced lymphoproliferative disease (EBV-LPD) after transplantation remains a serious and life-threatening complication. Herein we showed that the aminobisphosphonate pamidronate-expanded human Vγ9Vδ2-T cells efficiently killed EBV-transformed autologous lymphoblastoid B cell lines (EBV-LCL) through γ/δ-TCR and NKG2D receptor triggering and Fas and TRAIL engagement. By inoculation of EBV-LCL in Rag2(-/-)γc(-/-) mice and humanized mice, we established lethal EBV-LPD with characteristics close to those of the human disease. Adoptive transfer of pamidronate-expanded Vγ9Vδ2-T cells alone effectively prevented EBV-LPD in Rag2(-/-)γc(-/-) mice and induced EBV-LPD regression in EBV(+) tumor-bearing Rag2(-/-)γc(-/-) mice. Pamidronate treatment inhibited EBV-LPD development in humanized mice through selective activation and expansion of Vγ9Vδ2-T cells. This study provides proof-of-principle for a therapeutic approach using pamidronate to control EBV-LPD through Vγ9Vδ2-T cell targeting.

CD1d-restricted antigen presentation by Vγ9Vδ2-T cells requires trogocytosis.

CD1d-restricted invariant natural killer T cells (iNKT) constitute an important immunoregulatory T-cell subset that can be activated by the synthetic glycolipid α-galactosylceramide (α-GalCer) and play a dominant role in antitumor immunity. Clinical trials with α-GalCer-pulsed monocyte-derived dendritic cells (moDC) have shown anecdotal antitumor activity in advanced cancer. It was reported that phosphoantigen (pAg)-activated Vγ9Vδ2-T cells can acquire characteristics of professional antigen-presenting cells (APC). Considering the clinical immunotherapeutic applications, Vγ9Vδ2-T APC can offer important advantages over moDC, potentially constituting an attractive novel APC platform. Here, we demonstrate that Vγ9Vδ2-T APC can present antigens to iNKT. However, this does not result from de novo synthesis of CD1d by Vγ9Vδ2-T, but critically depends on trogocytosis of CD1d-containing membrane fragments from pAg-expressing cells. CD1d-expressing Vγ9Vδ2-T cells were able to activate iNKT in a CD1d-restricted and α-GalCer-dependent fashion. Although α-GalCer-loaded moDC outperformed Vγ9Vδ2-T APC on a per cell basis, Vγ9Vδ2-T APC possess unique features with respect to clinical immunotherapeutic application that make them an interesting platform for consideration in future clinical trials.

The intracellular B30.2 domain of butyrophilin 3A1 binds phosphoantigens to mediate activation of human Vγ9Vδ2 T cells.

In humans, Vγ9Vδ2 T cells detect tumor cells and microbial infections, including Mycobacterium tuberculosis, through recognition of small pyrophosphate containing organic molecules known as phosphoantigens (pAgs). Key to pAg-mediated activation of Vγ9Vδ2 T cells is the butyrophilin 3A1 (BTN3A1) protein that contains an intracellular B30.2 domain critical to pAg reactivity. Here, we have demonstrated through structural, biophysical, and functional approaches that the intracellular B30.2 domain of BTN3A1 directly binds pAg through a positively charged surface pocket. Charge reversal of pocket residues abrogates binding and Vγ9Vδ2 T cell activation. We have also identified a gain-of-function mutation within this pocket that, when introduced into the B30.2 domain of the nonstimulatory BTN3A3 isoform, transfers pAg binding ability and Vγ9Vδ2 T cell activation. These studies demonstrate that internal sensing of changes in pAg metabolite concentrations by BTN3A1 molecules is a critical step in Vγ9Vδ2 T cell detection of infection and tumorigenesis.

Characterization of the human CD4(+) T-cell repertoire specific for major histocompatibility class I-restricted antigens.

While CD4(+) T lymphocytes usually recognize antigens in the context of major histocompatibility (MHC) class II alleles, occurrence of MHC class-I restricted CD4(+) T cells has been reported sporadically. Taking advantage of a highly sensitive MHC tetramer-based enrichment approach allowing detection and isolation of scarce Ag-specific T cells, we performed a systematic comparative analysis of HLA-A*0201-restricted CD4(+) and CD8(+) T-cell lines directed against several immunodominant viral or tumoral antigens. CD4(+) T cells directed against every peptide-MHC class I complexes tested were detected in all donors. These cells yielded strong cytotoxic and T helper 1 cytokine responses when incubated with HLA-A2(+) target cells carrying the relevant epitopes. HLA-A2-restricted CD4(+) T cells were seldom expanded in immune HLA-A2(+) donors, suggesting that they are not usually engaged in in vivo immune responses against the corresponding peptide-MHC class I complexes. However, these T cells expressed TCR of very high affinity and were expanded following ex vivo stimulation by relevant tumor cells. Therefore, we describe a versatile and efficient strategy for generation of MHC class-I restricted T helper cells and high affinity TCR that could be used for adoptive T-cell transfer- or TCR gene transfer-based immunotherapies.

Repeated systemic administrations of both aminobisphosphonates and human Vγ9Vδ2 T cells efficiently control tumor development in vivo.

Peripheral Vγ9Vδ2 T lymphocytes compose a major γδ T cell subset in primates with broad reactivity against tumor cells. Vγ9Vδ2 T cells are specifically activated by phosphorylated isoprenoid pathway metabolites called "phosphoagonists." Accordingly, pharmacologic inhibitors of the mevalonate pathway, such as aminobisphosphonates (NBP) that upregulate the intracellular production of phosphoagonists, increase antitumor Vγ9Vδ2 T cell responses. Immunotherapeutic protocols exploiting GMP-grade agonist molecules targeting human Vγ9Vδ2 T lymphocytes have yielded promising, yet limited, signs of antitumor efficacy and therefore need to be improved for next-generation immunotherapies. In this study, we used a model of s.c. human tumor xenografts in severely immunodeficient mice to assess the antitumor efficacy of systemic NBP treatments when combined with the adoptive transfer of human Vγ9Vδ2 T cells. We show that infusion of Vγ9Vδ2 T cells, 24 h after systemic NBP treatment, efficiently delays tumor growth in mice. Importantly, our results indicate efficient but transient in vivo NBP-induced sensitization of tumor cells to human Vγ9Vδ2-T cell recognition. Accordingly, repeated and combined administrations of both NBP and γδ T cells yielded improved antitumor responses in vivo. Because Vγ9Vδ2 T cells show similar responsiveness toward both autologous and allogeneic tumors and are devoid of alloreactivity, these results provide preclinical proof of concept for optimized antitumor immunotherapies combining NBP treatment and adoptive transfer of allogeneic human γδ T cells.

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.

The molecular basis for modulation of human Vγ9Vδ2 T cell responses by CD277/butyrophilin-3 (BTN3A)-specific antibodies.

Human Vγ9Vδ2 T cells are well known for their rapid and potent response to infection and tumorigenesis when in the presence of endogenous or exogenous phosphoisoprenoids. However, the molecular mechanisms behind the activation of this γδ T cell population remains unclear. Evidence pointing to a role for the CD277/butyrophilin-3 (BTN3A) molecules in this response led us to investigate the structures of these molecules and their modifications upon binding to an agonist antibody (20.1) that mimics phosphoisoprenoid-mediated Vγ9Vδ2 activation and an antagonist antibody (103.2) that inhibits this reactivity. We find that the three BTN3A isoforms: BTN3A1, BTN3A2, and BTN3A3, have high structural homology to the B7 superfamily of proteins and exist as V-shaped homodimers in solution, associating through the membrane proximal C-type Ig domain. The 20.1 and 103.2 antibodies bind to separate epitopes on the BTN3A Ig-V domain with high affinity but likely with different valencies based on their binding orientation. These structures directly complement functional studies of this system that demonstrate that BTN3A1 is necessary for Vγ9Vδ2 activation and begin to unravel the extracellular events that occur during stimulation through the Vγ9Vδ2 T cell receptor.

Key implication of CD277/butyrophilin-3 (BTN3A) in cellular stress sensing by a major human γδ T-cell subset.

Human peripheral Vγ9Vδ2 T cells are activated by phosphorylated metabolites (phosphoagonists [PAg]) of the mammalian mevalonate or the microbial desoxyxylulose-phosphate pathways accumulated by infected or metabolically distressed cells. The underlying mechanisms are unknown. We show that treatment of nonsusceptible target cells with antibody 20.1 against CD277, a member of the extended B7 superfamily related to butyrophilin, mimics PAg-induced Vγ9Vδ2 T-cell activation and that the Vγ9Vδ2 T-cell receptor is implicated in this effect. Vγ9Vδ2 T-cell activation can be abrogated by exposing susceptible cells (tumor and mycobacteria-infected cells, or aminobisphosphonate-treated cells with up-regulated PAg levels) to antibody 103.2 against CD277. CD277 knockdown and domain-shuffling approaches confirm the key implication of the CD277 isoform BTN3A1 in PAg sensing by Vγ9Vδ2 T cells. Fluorescence recovery after photobleaching (FRAP) experiments support a causal link between intracellular PAg accumulation, decreased BTN3A1 membrane mobility, and ensuing Vγ9Vδ2 T-cell activation. This study demonstrates a novel role played by B7-like molecules in human γδ T-cell antigenic activation and paves the way for new strategies to improve the efficiency of immunotherapies using Vγ9Vδ2 T cells.

Ex vivo measurement of the cytotoxic capacity of human primary antigen-specific CD8 T cells.

The major function of CD8 T cells is to kill specifically target cells. Moreover in certain incurable diseases, antigen-specific human CD8 T cells are impaired, and assessment of their cytolytic activity could bring insights into their physiopathological role and ways to restore immune dysfunctions for immunotherapeutic purposes. Despite this, T cell cytolytic function has been seldom analyzed thoroughly in humans, due to the lack of approaches well suited for ex vivo assessment of T cell cytotoxicity. Current techniques require prior in vitro expansion of antigen-specific CD8 T cell populations and the use of immortalized cells as targets to measure the cell-mediated killing. Furthermore, bulk cytotoxic activity is frequently measured using percentage of specific lysis calculations that do not quantify actual target cell death and effector numbers at the single cell level. Here we established a new flow cytometry-based assay that allows accurate single-cell analysis of cytotoxic capacity of primary antigen-specific CD8 T cells generated in vivo in humans after antigenic exposure without in vitro amplification that can be used for specificities restricted by different HLAs as target cells are autologous cells. We show that this assay is robust, highly sensitive irrespective of the frequency of antigen-specific CD8 T cells, and allows accurate calculation of the index of cytotoxic capacity in lytic units. This new assay provides a sensitive method to measure the intrinsic cytotoxic activity of antigen-specific CD8 T cells directly ex vivo on human primary cells.

Activated iNKT cells promote Vγ9Vδ2-T cell anti-tumor effector functions through the production of TNF-α.

Vγ9Vδ2-T cells constitute a proinflammatory lymphocyte subpopulation with established antitumor activity. Phosphoantigens activate Vγ9Vδ2-T cells in vivo and in vitro. We studied whether the antitumor activity of Vγ9Vδ2-T cells can be potentiated by invariant NKT cells (iNKT), an important immunoregulatory T cell subset. When activated by the glycolipid α-galactosylceramide (α-GalCer), iNKT produce large amounts of cytokines involved in antitumor immune responses. Monocyte-derived dendritic cells were loaded with both phosphoantigens (using aminobisphosphonates) and α-GalCer during maturation and subsequently co-cultured with Vγ9Vδ2-T and iNKT cells. Aminobisphosphonates dose-dependently enhanced Vγ9Vδ2-T cell activation, and this was potentiated by α-GalCer-induced iNKT co-activation. iNKT co-activation also enhanced the IFN-γ production and cytolytic potential of Vγ9Vδ2-T cells against tumor cells. Using transwell experiments and neutralizing antibodies cross-talk between iNKT and Vγ9Vδ2-T cells was found to be mediated by TNF-α. Our data provide a rationale for combining both activating ligands to improve Vγ9Vδ2-T cell based approaches in cancer-immunotherapy.