Immunotherapy with innate immune cells has recently evoked broad interest as a novel treatment option for cancer patients. ␥9␦2T cells in particular are emerging as an innate cell population with high frequency and strong antitumor reactivity, which makes them and their receptors promising candidates for immune interventions. However, clinical trials have so far reported only limited tumor control by adoptively transferred ␥9␦2T cells. As a potential explanation for this lack of efficacy, we found unexpectedly high variability in tumor recognition within the physiologic human ␥9␦2T-cell repertoire, which is substantially regulated by the CDR3 domains of individual ␥9␦2TCRs. In the present study, we demonstrate that the reported molecular requirements of CDR3 domains to interact with target cells shape the physiologic ␥9␦2T-cell repertoire and, most likely, limit the protective and thera- IntroductionImmunotherapy with innate immune cells has become widely used because this approach obviates the need to match a cellular product to a defined HLA haplotype, allowing adoptive immunotherapies to be used in virtually any cancer patient without extensive in vitro selection or manipulation of the cellular product. 1-4 ␥9␦2T cells are promising as an innate cell population for this purpose because they are usually observed at high frequencies in the human peripheral blood and provide a strong antitumor reactivity against various solid and hematologic cancers. 5 However, within ␥9␦2T-cell populations, individual clones display great diversity in the repertoire because of the activating or inhibitory receptors expressed. 6 Selecting innate cell products for certain cell types, such as those with a low level of inhibitory receptors, therefore seems plausible, especially considering the limited efficacy of adoptively transferred innate immune cells in clinical trials. 7,8 An alternative proposal is to engineer cells to express defined activating innate receptors that mediate strong antitumor reactivity, such as a defined ␥9␦2TCR, 9 which could pave the way for readily available and more effective cellular products. However, the molecular details of how a ␥9␦2TCR interacts with its target are not fully understood, making it challenging to select defined ␥9␦2T cells or to engineer T cells with defined ␥9␦2TCRs.In "classic" immunoreceptors such as ␣␤TCRs or Igs, the complementary determining regions (CDRs) determine affinity and specificity for a specific (peptide) epitope. V(D)J recombination allows the creation of a highly variable CDR repertoire ensuring recognition of an immense collection of antigens. ␥9␦2T cells also possess a rearranged TCR that mediates recognition. The phosphoantigen isopentenyl pyrophosphate (IPP) has been suggested to be a key player in ␥9␦2TCR-mediated activation, 5,10,11 but no direct interaction between a ␥9␦2TCR and IPP or any other phosphoantigen has ever been demonstrated. It was previously suggested that positively charged residues within the ␥9␦2TCR are crucial for the response to negatively...

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.

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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.

Adoptive transfer of T cells gene-engineered with antigen-specific T cell receptors (TCRs) has proven its feasibility and therapeutic potential in the treatment of malignant tumors. To ensure further clinical development of TCR gene therapy, it is necessary to target immunogenic epitopes that are related to oncogenesis and selectively expressed by tumor tissue, and implement strategies that result in optimal T cell fitness. In addition, in particular for the treatment of solid tumors, it is equally necessary to include strategies that counteract the immune-suppressive nature of the tumor micro-environment. Here, we will provide an overview of the current status of TCR gene therapy, and redefine the following three challenges of improvement: “choice of target antigen”; “fitness of T cells”; and “sensitization of tumor milieu.” We will categorize and discuss potential strategies to address each of these challenges, and argue that advancement of clinical TCR gene therapy critically depends on developments toward each of the three challenges.