Summary Human Vγ9Vδ2 T cells respond to tumour cells by sensing elevated levels of phosphorylated intermediates of the dysregulated mevalonate pathway, which is translated into activating signals by the ubiquitously expressed butyrophilin A1 (BTN3A1) through yet unknown mechanisms. Here, we developed an unbiased, genome-wide screening method that identified RhoB as a critical mediator of Vγ9Vδ2 TCR activation in tumour cells. Our results show that Vγ9Vδ2 TCR activation is modulated by the GTPase activity of RhoB and its redistribution to BTN3A1. This is associated with cytoskeletal changes that directly stabilize BTN3A1 in the membrane, and the subsequent dissociation of RhoB from BTN3A1. Furthermore, phosphoantigen accumulation induces a conformational change in BTN3A1, rendering its extracellular domains recognizable by Vγ9Vδ2TCRs. These complementary events provide further evidence for inside-out signaling as an essential step in the recognition of tumor cells by a Vγ9Vδ2TCR.

11393 (to ZS and JK), Marie Curie 749010 (to DXB), NIH grants R01GM100114 (to DSL), P50GM085273 (to the New Mexico Spatiotemporal Modeling Center), and P30CA118100 (to the UNM Comprehensive Cancer Center). DF and AJRH acknowledge financial support from the NWO-funded Netherlands Proteomics Centre through the National Road Map for Large-scale Infrastructures program X-Omics (project 184.034.019). ES and LB are supported by grants from INSERM, CNRS, Université de Nantes, FRM (DEQ20170839118), and Ligue Contre le Cancer AO GO2019 (Côtes d'Armor, Association pour la Recherche contre le Cancer (PJA20191209404). This work was realized in the context of the LabEx IGO program, which is supported by the French National Research Agency Investissements d'Avenir.

Bone marrow niches support multiple myeloma, providing signals and cell-cell interactions essential for disease progression. A 3D bone marrow niche model was developed, in which supportive multipotent mesenchymal stromal cells and their osteogenic derivatives were co-cultured with endothelial progenitor cells. These co-cultured cells formed networks within the 3D culture, facilitating the survival and proliferation of primary CD138+ myeloma cells for up to 28 days. During this culture, no genetic drift was observed within the genomic profile of the primary myeloma cells, indicating a stable outgrowth of the cultured CD138+ population.The 3D bone marrow niche model enabled testing of a novel class of engineered immune cells, so called TEGs (αβT cells engineered to express a defined γδTCR) on primary myeloma cells. TEGs were engineered and tested from both healthy donors and myeloma patients. The added TEGs were capable of migrating through the 3D culture, exerting a killing response towards the primary myeloma cells in 6 out of 8 donor samples after both 24 and 48 hours. Such a killing response was not observed when adding mock transduced T cells. No differences were observed comparing allogeneic and autologous therapy. The supporting stromal microenvironment was unaffected in all conditions after 48 hours. When adding TEG therapy, the 3D model surpassed 2D models in many aspects by enabling analyses of specific homing, and both on- and off-target effects, preparing the ground for the clinical testing of TEGs. The model allows studying novel immunotherapies, therapy resistance mechanisms and possible side-effects for this incurable disease.

Backgroundγ9δ2 T cells hold great promise as cancer therapeutics because of their unique capability of reacting to metabolic changes with tumor cells. However, it has proven very difficult to translate this promise into clinical success.MethodsIn order to better utilize the tumor reactivity of γ9δ2T cells and combine this with the great potential of T cell engager molecules, we developed a novel bispecific molecule by linking the extracellular domains of tumor-reactive γ9δ2TCRs to a CD3-binding moiety, creating gamma delta TCR anti-CD3 bispecific molecules (GABs). GABs were tested in vitro and in vivo for ability to redirect T lymphocytes to a variety of tumor cell lines and primary patient material.ResultsGABs utilizing naturally occurring high affinity γ9δ2TCRs efficiently induced αβT cell mediated phosphoantigen-dependent recognition of tumor cells. Reactivity was substantially modulated by variations in the Vδ2 CDR3-region and the BTN2A1-binding HV4-region between CDR2 and CDR3 of the γ-chain was crucial for functionality. GABs redirected αβT cells against a broad range of hematopoietic and solid tumor cell lines and primary acute myeloid leukemia. Furthermore, they enhanced infiltration of immune cells in a 3D bone marrow niche and left healthy tissues intact, while eradicating primary multiple myeloma cells. Lastly, GABs constructed from natural high affinity γ9δ2TCR sequences significantly reduced tumor growth in vivo in a subcutaneous myeloma xenograft model.ConclusionsWe conclude that GABs allow for the introduction of metabolic targeting of cancer cells to the field of T cell engagers.

In the complex interplay between inflammation and graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (allo-HSCT), viral reactivations are often observed and cause substantial morbidity and mortality. As toxicity after allo-HSCT within the context of viral reactivations is mainly driven by αβ T cells, we describe that by delaying αβ T cell reconstitution through defined transplantation techniques, we can harvest the full potential of early reconstituting γδ T cells to control viral reactivations. We summarize evidence of how the γδ T cell repertoire is shaped by CMV and EBV reactivations after allo-HSCT, and their potential role in controlling the most important, but not all, viral reactivations. As most γδ T cells recognize their targets in an MHC-independent manner, γδ T cells not only have the potential to control viral reactivations but also to impact the underlying hematological malignancies. We also highlight the recently re-discovered ability to recognize classical HLA-molecules through a γδ T cell receptor, which also surprisingly do not associate with GVHD. Finally, we discuss the therapeutic potential of γδ T cells and their receptors within and outside the context of allo-HSCT, as well as the opportunities and challenges for developers and for payers.

γ9δ2T cells fill a distinct niche in human immunity due to the unique physiology of the phosphoantigen-reactive γ9δ2TCR. Here, we highlight reproducible TCRδ complementarity-determining region 3 (CDR3δ) repertoire patterns associated with γ9δ2T cell proliferation and phenotype, thus providing evidence for the role of the CDR3δ in modulating in vivo T-cell responses. Features that determine γ9δ2TCR binding affinity and reactivity to the phosphoantigen-induced ligand in vitro appear to similarly underpin in vivo clonotypic expansion and differentiation. Likewise, we identify a CDR3δ bias in the γ9δ2T cell natural killer receptor (NKR) landscape. While expression of the inhibitory receptor CD94/NKG2A is skewed toward cells bearing putative high-affinity TCRs, the activating receptor NKG2D is expressed independently of the phosphoantigen-sensing determinants, suggesting a higher net NKR activating signal in T cells with TCRs of low affinity. This study establishes consistent repertoire–phenotype associations and justifies stratification for the T-cell phenotype in future research on γ9δ2TCR repertoire dynamics.

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