Vγ9Vδ2 T-cell Receptor Research Articles

Structures of human γδ T cell receptor–CD3 complex

Gamma delta (γδ) T cells, a unique T cell subgroup, are crucial in various immune responses and immunopathology1–3. The γδ T cell receptor (TCR), which is generated by γδ T cells, recognizes a diverse range of antigens independently of the major histocompatibility complex2. The γδ TCR associates with CD3 subunits, initiating T cell activation and holding great potential in immunotherapy4. Here we report the structures of two prototypical human Vγ9Vδ2 and Vγ5Vδ1 TCR–CD3 complexes5,6, revealing two distinct assembly mechanisms that depend on Vγ usage. The Vγ9Vδ2 TCR–CD3 complex is monomeric, with considerable conformational flexibility in the TCRγ–TCRδ extracellular domain and connecting peptides. The length of the connecting peptides regulates the ligand association and T cell activation. A cholesterol-like molecule wedges into the transmembrane region, exerting an inhibitory role in TCR signalling. The Vγ5Vδ1 TCR–CD3 complex displays a dimeric architecture, whereby two protomers nestle back to back through the Vγ5 domains of the TCR extracellular domains. Our biochemical and biophysical assays further corroborate the dimeric structure. Importantly, the dimeric form of the Vγ5Vδ1 TCR is essential for T cell activation. These findings reveal organizing principles of the γδ TCR–CD3 complex, providing insights into the unique properties of γδ TCR and facilitating immunotherapeutic interventions.

A distinct topology of BTN3A IgV and B30.2 domains controlled by juxtamembrane regions favors optimal human γδ T cell phosphoantigen sensing

Butyrophilin (BTN)–3A and BTN2A1 molecules control the activation of human Vγ9Vδ2 T cells during T cell receptor (TCR)-mediated sensing of phosphoantigens (PAg) derived from microbes and tumors. However, the molecular rules governing PAg sensing remain largely unknown. Here, we establish three mechanistic principles of PAg-mediated γδ T cell activation. First, in humans, following PAg binding to the intracellular BTN3A1-B30.2 domain, Vγ9Vδ2 TCR triggering involves the extracellular V-domain of BTN3A2/BTN3A3. Moreover, the localization of both protein domains on different chains of the BTN3A homo-or heteromers is essential for efficient PAg-mediated activation. Second, the formation of BTN3A homo-or heteromers, which differ in intracellular trafficking and conformation, is controlled by molecular interactions between the juxtamembrane regions of the BTN3A chains. Finally, the ability of PAg not simply to bind BTN3A-B30.2, but to promote its subsequent interaction with the BTN2A1-B30.2 domain, is essential for T-cell activation. Defining these determinants of cooperation and the division of labor in BTN proteins improves our understanding of PAg sensing and elucidates a mode of action that may apply to other BTN family members.

Expression, Purification, and Crystallization of the Vγ9Vδ2 T-cell Receptor Recognizing Protein/Peptide Antigens.

γδ T cells, especially Vγ9Vδ2 T cells, play an important role in mycobacterial infection. We have identified some Vγ9Vδ2 T cells that recognize protein/peptide antigens derived from mycobacteria, which may induce protective immune responses to mycobacterial infection. To clarify the structural basis of the molecular recognition mechanism, we tried many methods to express the Vγ9Vδ2 T-cell receptor (TCR). The Vγ9Vδ2 TCR was not expressed well in a prokaryotic expression system or a baculovirus expression system, even after extensive optimization. In a mammalian cell expression system, the Vγ9Vδ2 TCR was expressed in the form of a soluble heterodimer, which was suitable for crystal screening. Reduced-temperature cultivation (cold shock) increased the yield of the recombinant TCR. The recombinant purified TCR was used for crystal trials, and crystals that could be used for X-ray diffraction were obtained. Although we have not yet determined the crystal structure of the Vγ9Vδ2 TCR, we have established a procedure for Vγ9Vδ2 TCR expression and purification, which is useful for basic research and potentially for clinical application.

Phosphoantigen sensing combines TCR-dependent recognition of the BTN3A IgV domain and germline interaction with BTN2A1

Vγ9Vδ2 Tcells play critical roles in microbial immunity by detecting target cells exposed to pathogen-derived phosphoantigens (P-Ags). Target cell expression of BTN3A1, the "P-Ag sensor," and BTN2A1, a direct ligand for Tcell receptor (TCR) Vγ9, is essential for this process; however, the molecular mechanisms involved are unclear. Here, we characterize BTN2A1 interactions with Vγ9Vδ2 TCR and BTN3A1. Nuclear magnetic resonance (NMR), modeling, and mutagenesis establish a BTN2A1-immunoglobulin V (IgV)/BTN3A1-IgV structural model compatible with their cell-surface association in cis. However, TCR and BTN3A1-IgV binding to BTN2A1-IgV is mutually exclusive, owing to binding site proximity and overlap. Moreover, mutagenesis indicates that the BTN2A1-IgV/BTN3A1-IgV interaction is non-essential for recognition but instead identifies a molecular surface on BTN3A1-IgV essential to P-Ag sensing. These results establish a critical role for BTN3A-IgV in P-Ag sensing, in mediating direct or indirect interactions with the γδ-TCR. They support a composite-ligand model whereby intracellular P-Ag detection coordinates weak extracellular germline TCR/BTN2A1 and clonotypically influenced TCR/BTN3A-mediated interactions to initiate Vγ9Vδ2 TCR triggering.

Lava-051, a Novel Bispecific Gamma-Delta T-Cell Engager (Gammabody), in Relapsed/Refractory MM and CLL: Pharmacodynamic and Early Clinical Data

Background: LAVA-051 is a Vγ9Vδ2-T cell engaging bispecific antibody of 27 kDa that directly engages tumor expressed CD1d and the Vδ2-TCR chain of Vγ9Vδ2-T cells to kill tumor cells in a tumor target-dependent manner. Binding between LAVA-051 and CD1d also causes the activation of a second effector cell type: invariant natural killer T (iNKT) (Nature Cancer 2020;1:1054-1065). By dual engagement of these unique innate-like T cell subsets with inherent antitumor activity, LAVA-051 has the potential to exhibit high potency antitumor activity. Based on clinical experience with systemic activation of Vγ9Vδ2-T cells through aminobisphosphonates and in vivo studies in non-human primates (NHP) with a surrogate CD1d-γδ T cell engager, a low risk for cytokine release syndrome (CRS) is expected. CD1d is expressed by tumor cells in the majority of patients with CLL (chronic lymphocytic leukemia) and MM (multiple myeloma), while expression in AML (acute myeloid leukemia) is most pronounced on (myelo)monocytic subtypes. The local tolerance of subcutaneous (SC) administration of LAVA-051 was studied in a NHP model with no signs of local intolerance or histopathological abnormalities observed. Methods: An open label, accelerated titration design, phase 1/2a study in patients with relapsed/ refractory CLL and MM (NCT04887259) is ongoing to determine the safety of LAVA-051 and its recommended Phase 2a dose (RP2D). Adverse Events (AEs) are graded per CTCAE V5.0 criteria and CRS is graded per ASTCT criteria. IMWG criteria (MM) and iwCLL guidelines (CLL) are used for disease evaluation. The RP2D will be the optimal biological active dose, supported by a panel of specific pharmacodynamic assays to determine the pattern of change in the binding of LAVA-051 to peripheral blood Vγ9Vδ2-T cells (i.e. Vγ9Vδ2-TCR occupancy), and in the frequency and activation status of Vγ9Vδ2-T and iNKT cells in circulation. LAVA-051 is administered by IV infusion over 2 hours twice a week or by SC administration; observation of changes in PD parameters following SC administration as compared to IV infusion will enhance selection of RP2D administration and schedule. Results: As of July 25, 2022, a dose level of 100 µg has been reached (starting dose 0.45 µg) with no reports of CRS or dose limiting toxicities (DLTs). Eight patients in total have been treated with LAVA-051; 4 patients with MM and 4 patients with CLL with treatment duration range of 1.6 to 18.1 weeks. Treatment emergent AEs (TEAEs) were predominantly of grade 1 and 2 severity, with a minority of AEs observed across more than one dose level: lymphadenopathy, pyrexia (underlying causes different from CRS), epistaxis, dyspnea, and leukopenia. Severity of TEAEs did not increase with escalating doses and no patient discontinued treatment due to an AE. Disease stabilization has been observed in two patients. Despite no observation of formal response, lymph node shrinkage was observed in one CLL patient and decreasing M-protein levels in one MM patient. Pharmacodynamic data reflect changes as expected per LAVA-051 mechanism of action (MoA): Vγ9Vδ2-T cell activation markers (CD25 and CD69) were consistently upregulated in each dose cohort where this could be assessed, maximum measured Vγ9Vδ2-T cell receptor occupancy increased with higher dose cohorts, and iNKT cell activation was observed. Furthermore, no significant increases in IL-6 or other CRS-related cytokines have been observed. Pharmacokinetic data indicate predictable and linear pharmacokinetics. Updated safety, PD, and PK results following IV and SC administration will be presented at the congress. Conclusions: LAVA-051 has been well tolerated early in dose escalation with pharmacodynamics and early clinical signs reflective of its intended MoA. The differentiating importance of these pharmacodynamic parameters with any correlating preliminary antitumor activity will be further elucidated with continued dose escalation by the IV and SC route and in the determination of the RP2D and schedule.

Identification of distinct functional thymic programming of fetal and pediatric human γδ thymocytes via single-cell analysis

Developmental thymic waves of innate-like and adaptive-like γδ T cells have been described, but the current understanding of γδ T cell development is mainly limited to mouse models. Here, we combine single cell (sc) RNA gene expression and sc γδ T cell receptor (TCR) sequencing on fetal and pediatric γδ thymocytes in order to understand the ontogeny of human γδ T cells. Mature fetal γδ thymocytes (both the Vγ9Vδ2 and nonVγ9Vδ2 subsets) are committed to either a type 1, a type 3 or a type 2-like effector fate displaying a wave-like pattern depending on gestation age, and are enriched for public CDR3 features upon maturation. Strikingly, these effector modules express different CDR3 sequences and follow distinct developmental trajectories. In contrast, the pediatric thymus generates only a small effector subset that is highly biased towards Vγ9Vδ2 TCR usage and shows a mixed type 1/type 3 effector profile. Thus, our combined dataset of gene expression and detailed TCR information at the single-cell level identifies distinct functional thymic programming of γδ T cell immunity in human.

Gamma Delta T-Cell Based Cancer Immunotherapy: Past-Present-Future.

γδ T-cells directly recognize and kill transformed cells independently of HLA-antigen presentation, which makes them a highly promising effector cell compartment for cancer immunotherapy. Novel γδ T-cell-based immunotherapies, primarily focusing on the two major γδ T-cell subtypes that infiltrate tumors (i.e. Vδ1 and Vδ2), are being developed. The Vδ1 T-cell subset is enriched in tissues and contains both effector T-cells as well as regulatory T-cells with tumor-promoting potential. Vδ2 T-cells, in contrast, are enriched in circulation and consist of a large, relatively homogeneous, pro-inflammatory effector T-cell subset. Healthy individuals typically harbor in the order of 50-500 million Vγ9Vδ2 T-cells in the peripheral blood alone (1-10% of the total CD3+ T-cell population), which can rapidly expand upon stimulation. The Vγ9Vδ2 T-cell receptor senses intracellular phosphorylated metabolites, which accumulate in cancer cells as a result of mevalonate pathway dysregulation or upon pharmaceutical intervention. Early clinical studies investigating the therapeutic potential of Vγ9Vδ2 T-cells were based on either ex vivo expansion and adoptive transfer or their systemic activation with aminobisphosphonates or synthetic phosphoantigens, either alone or combined with low dose IL-2. Immune-related adverse events (irAE) were generally \\mild, but the clinical efficacy of these approaches provided overall limited benefit. In recent years, critical advances have renewed the excitement for the potential of Vγ9Vδ2 T-cells in cancer immunotherapy. Here, we review γδ T-cell-based therapeutic strategies and discuss the prospects of those currently evaluated in clinical studies in cancer patients as well as future therapies that might arise from current promising pre-clinical results.

Phase I dose escalation of LAVA-051, a novel bispecific gamma-delta T-cell engager (Gammabody), in relapsed/refractory hematological malignancies.

2577 Background: LAVA-051 is a 27kD humanized bispecific single domain antibody (VHH) that directly engages CD1d and the Vδ2-TCR chain of Vγ9Vδ2-T cells and additionally stabilizes the interaction between CD1d and type 1 NKT cells (Nature Cancer 2020;1:1054-1065) to mediate potent killing of CD1d-expressing tumor cells. By engaging innate-like T cell subsets with inherent antitumor activity, namely Vγ9Vδ2-T and type 1 NKT cells, LAVA-051 has the potential to combine high therapeutic efficacy with limited off-tumor toxicity. CD1d is expressed by tumor cells in the majority of patients with CLL (chronic lymphocytic leukemia) and MM (multiple myeloma), while expression in AML (acute myeloid leukemia) is most pronounced on (myelo)monocytic subtypes. Tolerability studies in non-human primates with cross-reactive surrogate bispecific γδ-T cell engagers showed a good safety profile without signs of CRS (cytokine release syndrome). Methods: This is an open label, accelerated titration design, phase 1/2a study in patients with relapsed/ refractory CLL, MM, and AML (NCT04887259). The phase 1 study starts with single-patient cohorts for the first 3 dose levels followed by a 3+3 design. The primary objectives of the study are to investigate the safety and tolerability of LAVA-051 and to determine its recommended Phase 2a dose (RP2D). Secondary objectives of the study include evaluation of PK, PD, immunogenicity, and preliminary antitumor activity. The (flat) starting dose was determined to be 0.45 µg. LAVA-051 is administered by IV infusion over 2 hours twice a week. Results: As of Feb 14, 2022, a dose level of 45 µg has been reached with no reports of CRS (ASTCT grading) or dose limiting toxicities (DLTs). Six patients in total have been treated with LAVA-051; 4 patients (2 MM, 2 CLL) were evaluable for DLTs during cycle 1 at dose levels 0.45 µg, 3 µg, 15 µg and 45 µg. Treatment emergent AEs (TEAEs) (CTCAEv5.0) were predominantly of grade 1 and 2 severity (e.g. fever, chills, and headache). One patient (45 µg) reported a grade 2 infusion related reaction (IRR) that lasted less than 12 hours following the first administration only. Frequency and severity of TEAEs did not increase with escalating doses and all TEAEs were reversible. One patient with CLL developed symptoms consistent with a tumor flare reaction during cycle 1, and continues on cycle 5 of treatment with stable disease at 15 µg. PK data indicate increasing drug exposure in correlation with increasing doses of LAVA-051, and PD data indicate a dose-dependent increase in LAVA-051 receptor occupancy of the Vγ9Vδ2-T cell receptor. Importantly, no consistent or significant changes in measured cytokines have been observed. Updated results will be presented at the congress. Conclusions: LAVA-051 has been well tolerated early in dose escalation with on-mechanism pharmacodynamics consistent with Vγ9Vδ2-T cell engagement. Clinical trial information: NCT04887259.

A Bispecific Single-Domain Antibody Boosts Autologous Vγ9Vδ2-T Cell Responses Toward CD1d in Chronic Lymphocytic Leukemia.

Although considerable progress has been made with autologous T cell-based therapy in B-cell malignancies, application in chronic lymphocytic leukemia (CLL) lags behind due to disappointing response rates as well as substantial toxicity that is of particular concern in the elderly CLL population. Vγ9Vδ2-T cells form a conserved T-cell subset with strong intrinsic immunotherapeutic potential, largely because of their capacity to be triggered by phosphoantigens that can be overproduced by CLL and other malignant cells. Specific activation of Vγ9Vδ2-T cells by a bispecific antibody may improve the efficacy and toxicity of autologous T-cell-based therapy in CLL. We evaluated CD1d expression in a cohort of 78 untreated patients with CLL and generated and functionally characterized a CD1d-specific Vγ9Vδ2-T cell engager based on single-domain antibodies (VHH). CD1d was expressed by CLL in the majority of patients, particularly in patients with advanced disease. The CD1d-specific Vγ9Vδ2-T cell engager induced robust activation and degranulation of Vγ9Vδ2-T cells, enabling Vγ9Vδ2-T cells from patients with CLL to lyse autologous leukemic cells at low effector-to-target ratios. Expression of CD1d on CLL cells is upregulated by all-trans retinoic acid, and sensitizes the malignant cells to bispecific VHH-induced lysis. Furthermore, we provide evidence that the Vγ9Vδ2-T cell receptor retains responsiveness to phosphoantigens when the bispecific VHH is bound, and aminobisphosphonates can therefore enhance bispecific Vγ9Vδ2-T cell engager-mediated tumor-specific killing. Collectively, our data demonstrate the immunotherapeutic potential of this novel CD1d-specific Vγ9Vδ2-T cell engager in CLL.

Efficient Ex Vivo Expansion of γδ T-Cells from AML Patients Requires Elimination of Circulating Leukemic Blasts

γδ T-cells play an important role in immune surveillance of acute myeloid leukemia (AML). The main circulating subtype expresses a Vγ9Vδ2 T-cell receptor and may be expanded <em>ex vivo</em> following aminobisphosphonate activation. While such protocols operate robustly in healthy donors, they are often inefficient using blood samples from patients with advanced malignancy.

Mistletoe-Extract Drugs Stimulate Anti-Cancer Vγ9Vδ2 T Cells.

Human phosphoantigen-reactive Vγ9Vδ2 T cells possess several characteristics, including MHC-independent recognition of tumor cells and potent killing potential, that make them attractive candidates for cancer immunotherapeutic approaches. Injectable preparations from the hemi-parasite plant Viscum album L. (European mistletoe) are commonly prescribed as complementary cancer therapy in European countries such as Germany, but their mechanism of action remains poorly understood. Here, we investigated in-depth the in vitro response of human T cells towards mistletoe-extract drugs by analyzing their functional and T-cell-receptor (TCR) response using flow cytometry and high-throughput sequencing respectively. Non-fermented mistletoe-extract drugs (AbnobaViscum), but not their fermented counterparts (Iscador), induced specific expansion of Vγ9Vδ2 T cells among T cells. Furthermore, AbnobaViscum rapidly induced the release of cytotoxic granules and the production of the cytokines IFNγ and TNFα in Vγ9Vδ2 T cells. This stimulation of anti-cancer Vγ9Vδ2 T cells was mediated by the butyrophilin BTN3A, did not depend on the accumulation of endogenous phosphoantigens and involved the same Vγ9Vδ2 TCR repertoire as those of phosphoantigen-reactive Vγ9Vδ2 T cells. These insights highlight Vγ9Vδ2 T cells as a potential target for mistletoe-extract drugs and their role in cancer patients receiving these herbal drugs needs to be investigated.

Alpaca (Vicugna pacos), the first nonprimate species with a phosphoantigen-reactive Vγ9Vδ2 T cell subset

Vγ9Vδ2 T cells are a major γδ T cell population in the human blood expressing a characteristic Vγ9JP rearrangement paired with Vδ2. This cell subset is activated in a TCR-dependent and MHC-unrestricted fashion by so-called phosphoantigens (PAgs). PAgs can be microbial [(E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate, HMBPP] or endogenous (isopentenyl pyrophosphate, IPP) and PAg sensing depends on the expression of B7-like butyrophilin (BTN3A, CD277) molecules. IPP increases in some transformed or aminobisphosphonate-treated cells, rendering those cells a target for Vγ9Vδ2 T cells in immunotherapy. Yet, functional Vγ9Vδ2 T cells have only been described in humans and higher primates. Using a genome-based study, we showed in silico translatable genes encoding Vγ9, Vδ2, and BTN3 in a few nonprimate mammalian species. Here, with the help of new monoclonal antibodies, we directly identified a T cell population in the alpaca (Vicugna pacos), which responds to PAgs in a BTN3-dependent fashion and shows typical TRGV9- and TRDV2-like rearrangements. T cell receptor (TCR) transductants and BTN3-deficient human 293T cells reconstituted with alpaca or human BTN3 or alpaca/human BTN3 chimeras showed that alpaca Vγ9Vδ2 TCRs recognize PAg in the context of human and alpaca BTN3. Furthermore, alpaca BTN3 mediates PAg recognition much better than human BTN3A1 alone and this improved functionality mapped to the transmembrane/cytoplasmic part of alpaca BTN3. In summary, we found remarkable similarities but also instructive differences of PAg-recognition by human and alpaca, which help in better understanding the molecular mechanisms controlling the activation of this prominent population of γδ T cells.

A Structural Change in Butyrophilin upon Phosphoantigen Binding Underlies Phosphoantigen-Mediated Vγ9Vδ2 T Cell Activation

Human Vγ9Vδ2 Tcells respond to microbial infections and malignancy by sensing diphosphate-containing metabolites called phosphoantigens, which bind to the intracellular domain of butyrophilin 3A1, triggering extracellular interactions with the Vγ9Vδ2 Tcell receptor (TCR). Here, we examined the molecular basis of this "inside-out" triggering mechanism. Crystal structures of intracellular butyrophilin 3A proteins alone or in complex with the potent microbial phosphoantigen HMBPP or a synthetic analog revealed key features of phosphoantigens and butyrophilins required for γδ Tcell activation. Analyses with chemical probes and molecular dynamic simulations demonstrated that dimerized intracellular proteins cooperate in sensing HMBPP to enhance the efficiency of γδ Tcell activation. HMBPP binding to butyrophilin doubled the binding force between a γδ Tcell and a target cell during "outside" signaling, as measured by single-cell force microscopy. Our findings provide insight into the "inside-out" triggering of Vγ9Vδ2 Tcell activation by phosphoantigen-bound butyrophilin, facilitating immunotherapeutic drug design.

GMP-Grade Manufacturing of T Cells Engineered to Express a Defined γδTCR.

γ9δ2T cells play a critical role in daily cancer immune surveillance by sensing cancer-mediated metabolic changes. However, a major limitation of the therapeutic application of γ9δ2T cells is their diversity and regulation through innate co-receptors. In order to overcome natural obstacles of γ9δ2T cells, we have developed the concept of T cells engineered to express a defined γδT cell receptor (TEGs). This next generation of chimeric antigen receptor engineered T (CAR-T) cells not only allows for targeting of hematological but also of solid tumors and, therefore, overcomes major limitations of many CAR-T and γδT cell strategies. Here, we report on the development of a robust manufacturing procedure of T cells engineered to express the high affinity Vγ9Vδ2T cell receptor (TCR) clone 5 (TEG001). We determined the best concentration of anti-CD3/CD28 activation and expansion beads, optimal virus titer, and cell density for retroviral transduction, and validated a Good Manufacturing Practice (GMP)-grade purification procedure by utilizing the CliniMACS system to deplete non- and poorly-engineered T cells. To the best of our knowledge, we have developed the very first GMP manufacturing procedure in which αβTCR depletion is used as a purification method, thereby delivering untouched clinical grade engineered immune cells. This enrichment method is applicable to any engineered T cell product with a reduced expression of endogenous αβTCRs. We report on release criteria and the stability of TEG001 drug substance and TEG001 drug product. The GMP-grade production procedure is now approved by Dutch authorities and allows TEG001 to be generated in cell numbers sufficient to treat patients within the approved clinical trial NTR6541. NTR6541 will investigate the safety and tolerability of TEG001 in patients with relapsed/refractory acute myeloid leukemia, high-risk myelodysplastic syndrome, and relapsed/refractory multiple myeloma.

Human γδ T-Cells: From Surface Receptors to the Therapy of High-Risk Leukemias.

γδ T lymphocytes are potent effector cells, capable of efficiently killing tumor and leukemia cells. Their activation is mediated by γδ T-cell receptor (TCR) and by activating receptors shared with NK cells (e.g., NKG2D and DNAM-1). γδ T-cell triggering occurs upon interaction with specific ligands, including phosphoantigens (for Vγ9Vδ2 TCR), MICA-B and UL16 binding protein (for NKG2D), and PVR and Nectin-2 (for DNAM-1). They also respond to cytokines undergoing proliferation and release of cytokines/chemokines. Although at the genomic level γδ T-cells have the potential of an extraordinary TCR diversification, in tissues they display a restricted repertoire. Recent studies have identified various γδ TCR rearrangements following either hematopoietic stem cell transplantation (HSCT) or cytomegalovirus infection, accounting for their “adaptive” potential. In humans, peripheral blood γδ T-cells are primarily composed of Vγ9Vδ2 chains, while a minor proportion express Vδ1. They do not recognize antigens in the context of MHC molecules, thus bypassing tumor escape based on MHC class I downregulation. In view of their potent antileukemia activity and absence of any relevant graft-versus-host disease-inducing effect, γδ T-cells may play an important role in the successful clinical outcome of patients undergoing HLA-haploidentical HSCT depleted of TCR αβ T/CD19+ B lymphocytes to cure high-risk acute leukemias. In this setting, high numbers of both γδ T-cells (Vδ1 and Vδ2) and NK cells are infused together with CD34+ HSC and may contribute to rapid control of infections and leukemia relapse. Notably, zoledronic acid potentiates the cytolytic activity of γδ T-cells in vitro and its infusion in patients strongly promotes γδ T-cell differentiation and cytolytic activity; thus, treatment with this agent may contribute to further improve the patient clinical outcome after HLA-haploidentical HSCT depleted of TCR αβ T/CD19+ B lymphocytes.

The Armadillo (Dasypus novemcinctus): A Witness but Not a Functional Example for the Emergence of the Butyrophilin 3/Vγ9Vδ2 System in Placental Mammals.

1–5% of human blood T cells are Vγ9Vδ2 T cells whose T cell receptor (TCR) contain a TRGV9/TRGJP rearrangement and a TRDV2 comprising Vδ2-chain. They respond to phosphoantigens (PAgs) like isopentenyl pyrophosphate or (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate (HMBPP) in a butyrophilin 3 (BTN3)-dependent manner and may contribute to the control of mycobacterial infections. These cells were thought to be restricted to primates, but we demonstrated by analysis of genomic databases that TRGV9, TRDV2, and BTN3 genes coevolved and emerged together with placental mammals. Furthermore, we identified alpaca (Vicugna pacos) as species with typical Vγ9Vδ2 TCR rearrangements and currently aim to directly identify Vγ9Vδ2 T cells and BTN3. Other candidates to study this coevolution are the bottlenose dolphin (Tursiops truncatus) and the nine-banded armadillo (Dasypus novemcinctus) with genomic sequences encoding open reading frames for TRGV9, TRDV2, and the extracellular part of BTN3. Dolphins have been shown to express Vγ9- and Vδ2-like TCR chains and possess a predicted BTN3-like gene homologous to human BTN3A3. The other candidate, the armadillo, is of medical interest since it serves as a natural reservoir for Mycobacterium leprae. In this study, we analyzed the armadillo genome and found evidence for multiple non-functional BTN3 genes including genomic context which closely resembles the organization of the human, alpaca, and dolphin BTN3A3 loci. However, no BTN3 transcript could be detected in armadillo cDNA. Additionally, attempts to identify a functional TRGV9/TRGJP rearrangement via PCR failed. In contrast, complete TRDV2 gene segments preferentially rearranged with a TRDJ4 homolog were cloned and co-expressed with a human Vγ9-chain in murine hybridoma cells. These cells could be stimulated by immobilized anti-mouse CD3 antibody but not with human RAJI-RT1Bl cells and HMBPP. So far, the lack of expression of TRGV9 rearrangements and BTN3 renders the armadillo an unlikely candidate species for PAg-reactive Vγ9Vδ2 T cells. This is in line with the postulated coevolution of the three genes, where occurrence of Vγ9Vδ2 TCRs coincides with a functional BTN3 molecule.

A bispecific nanobody approach to leverage the potent and widely applicable tumor cytolytic capacity of Vγ9Vδ2-T cells

ABSTRACTThough Vγ9Vδ2-T cells constitute only a small fraction of the total T cell population in human peripheral blood, they play a vital role in tumor defense and are therefore of major interest to explore for cancer immunotherapy. Vγ9Vδ2-T cell-based cancer immunotherapeutic approaches developed so far have been generally well tolerated and were able to induce significant clinical responses. However, overall results were inconsistent, possibly due to the fact that these strategies induced systemic activation of Vγ9Vδ2-T cells without preferential accumulation and targeted activation in the tumor. Here we show that a novel bispecific nanobody-based construct targeting both Vγ9Vδ2-T cells and EGFR induced potent Vγ9Vδ2-T cell activation and subsequent tumor cell lysis both in vitro and in an in vivo mouse xenograft model. Tumor cell lysis was independent of KRAS and BRAF tumor mutation status and common Vγ9Vδ2-T cell receptor sequence variations. In combination with the conserved monomorphic nature of the Vγ9Vδ2-TCR and the facile replacement of the tumor-specific nanobody, this immunotherapeutic approach can be applied to a large group of cancer patients.

Avoidance of On-Target Off-Tumor Activation Using a Co-stimulation-Only Chimeric Antigen Receptor

Chimeric antigen receptors (CARs) combine Tcell activation with antibody-mediated tumor antigen specificity, bypassing the need for Tcell receptor (TCR) ligation. A limitation of CAR technology is on-target off-tumor toxicity caused by target antigen expression on normal cells. Using GD2 as a model cancer antigen, we hypothesized that this could be minimized by using Tcells expressing Vγ9Vδ2 TCR, which recognizes transformed cells in a major histocompatibility complex (MHC)-unrestricted manner, in combination with a co-stimulatory CAR that would function independently of the TCR. An anti-GD2 CAR containing a solitary endodomain derived from the NKG2D adaptor DAP10 was expressed in Vγ9Vδ2+ Tcells. Differential ligation of the CAR and/or TCR using antibody-coated beads showed that pro-inflammatory cytokine response depended on activation of both receptors. Moreover, in killing assays, GD2-expressing neuroblastoma cells that engaged the Vγ9Vδ2 TCR were efficiently lysed, whereas cells that expressed GD2 equivalently but did not engage the Vγ9Vδ2 TCR were untouched. Differentiation between X-on tumor and X-off tumor offers potential for safer immunotherapy and broader target selection.

Human γδ T cells: From a neglected lymphocyte population to cellular immunotherapy: A personal reflection of 30 years of γδ T cell research

•γδ T cells account for approximately 5% of CD3+ T cells in the blood and frequently express a Vγ9Vδ2 T-cell receptor•Vγ9Vδ2 T cells recognize pyrophosphate molecules that are produced by bacteria but also by many tumor cells•Vγ9Vδ2 T cells kill a broad variety of different tumor cells in an HLA-nonrestricted fashion•Vγ9Vδ2 T cell activation and expansion can be manipulated pharmacologically, e.g. by nitrogen-containing bisphosphonates•Vγ9Vδ2 T cells are attractive candiates for cellular immuotherapy

Construction and identification of a drosophila S2 cell line with stoble expression of Mycobacterium tuberculosis-specific Vγ9Vδ2 T cell receptor monomers

Objective To construct and identify a drosophila S2 cell line that could stably express Mycobacterium tuberculosis-specific Vγ9Vδ2 T cell receptor (TCR) monomers. Methods Four vectors including the TCR γ9-FB-pMT/V5-His B expression vector, TCR δ2-JB-pMT/V5-His A expression vector, pMT/Bip-BirA expression vector and pCoHygro plasmid were transfected into S2 cells by using calcium phosphate transfection. The S2 cells with stable expression of the Vγ9Vδ2 TCR monomers were screened out with Hygromycin-B. CuSO4 and biotin were used together to induce the expression of biotinylated Vγ9Vδ2 TCR monomers in S2 cells after 4 weeks of culturing. The target proteins in the supernatants of cell culture were identified with dot blot, SDS-PAGE and Western blot assays. Results The results of dot blot, SDS-PAGE and Western blot assays showed that the Vγ9Vδ2 TCR monomers could be stably expressed by the screened S2 cell line. Moreover, the expressed Vγ9Vδ2 TCR monomers were successfully biotinylated. Conclusion The drosophila S2 cell line that could stably express Vγ9Vδ2 TCR monomers was successfully established. This study has paved the way for further investigation on the antigen recognition of γδ T cells and its possible mechanism. Key words: γδT cell receptor; Monomer; S2 cell