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γδ T cells are potent anticancer effectors with the potential to target tumours broadly, independent of patient-specific neoantigens or human leukocyte antigen background 1-5 . γδ T cells can sense conserved cell stress signals prevalent in transformed cells 2,3 , although the mechanisms behind the targeting of stressed target cells remain poorly characterized. Vγ9Vδ2 T cells-the most abundant subset of human γδ T cells 4 -recognize a protein complex containing butyrophilin 2A1 (BTN2A1) and BTN3A1 (refs. 6-8 ), a widely expressed cell surface protein that is activated by phosphoantigens abundantly produced by tumour cells. Here we combined genome-wide CRISPR screens in target cancer cells to identify pathways that regulate γδ T cell killing and BTN3A cell surface expression. The screens showed previously unappreciated multilayered regulation of BTN3A abundance on the cell surface and triggering of γδ T cells through transcription, post-translational modifications and membrane trafficking. In addition, diverse genetic perturbations and inhibitors disrupting metabolic pathways in the cancer cells, particularly ATP-producing processes, were found to alter BTN3A levels. This induction of both BTN3A and BTN2A1 during metabolic crises is dependent on AMP-activated protein kinase (AMPK). Finally, small-molecule activation of AMPK in a cell line model and in patient-derived tumour organoids led to increased expression of the BTN2A1-BTN3A complex and increased Vγ9Vδ2 T cell receptor-mediated killing. This AMPK-dependent mechanism of metabolic stress-induced ligand upregulation deepens our understanding of γδ T cell stress surveillance and suggests new avenues available to enhance γδ T cell anticancer activity., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Extending the success of cellular immunotherapies against blood cancers to the realm of solid tumors will require improved in vitro models that reveal therapeutic modes of action at the molecular level. Here we describe a system, called BEHAV3D, developed to study the dynamic interactions of immune cells and patient cancer organoids by means of imaging and transcriptomics. We apply BEHAV3D to live-track >150,000 engineered T cells cultured with patient-derived, solid-tumor organoids, identifying a 'super engager' behavioral cluster comprising T cells with potent serial killing capacity. Among other T cell concepts we also study cancer metabolome-sensing engineered T cells (TEGs) and detect behavior-specific gene signatures that include a group of 27 genes with no previously described T cell function that are expressed by super engager killer TEGs. We further show that type I interferon can prime resistant organoids for TEG-mediated killing. BEHAV3D is a promising tool for the characterization of behavioral-phenotypic heterogeneity of cellular immunotherapies and may support the optimization of personalized solid-tumor-targeting cell therapies., (© 2022. The Author(s).)

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., Competing Interests: JK reports grants from Gadeta, Novartis, and Miltenyi Biotech and is the inventor of patents dealing with γδT cell-related aspects, as well as the co-founder and shareholder of Gadeta. AV, AJ, DB, and ZS are inventors of patents dealing with γδT cell-related aspects. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Vyborova, Janssen, Gatti, Karaiskaki, Yonika, van Dooremalen, Sanders, Beringer, Straetemans, Sebestyen and Kuball.)

γ9δ2T cells play a major role in cancer immune surveillance, yet the clinical translation of their in vitro promise remains challenging. To address limitations of previous clinical attempts using expanded γ9δ2T cells, we explored the clonal diversity of γ9δ2T cell repertoires and characterized their target. We demonstrated that only a fraction of expanded γ9δ2T cells was active against cancer cells and that activity of the parental clone, or functional avidity of selected γ9δ2 T cell receptors (γ9δ2TCRs), was not associated with clonal frequency. Furthermore, we analyzed the target-receptor interface and provided a 2-receptor, 3-ligand model. We found that activation was initiated by binding of the γ9δ2TCR to BTN2A1 through the regions between CDR2 and CDR3 of the TCR γ chain and modulated by the affinity of the CDR3 region of the TCRδ chain, which was phosphoantigen independent (pAg independent) and did not depend on CD277. CD277 was secondary, serving as a mandatory coactivating ligand. We found that binding of CD277 to its putative ligand did not depend on the presence of γ9δ2TCR, did depend on usage of the intracellular CD277, created pAg-dependent proximity to BTN2A1, enhanced cell-cell conjugate formation, and stabilized the immunological synapse (IS). This process critically depended on the affinity of the γ9δ2TCR and required membrane flexibility of the γ9δ2TCR and CD277, facilitating their polarization and high-density recruitment during IS formation.

Clinical responses to checkpoint inhibitors used for cancer immunotherapy seemingly require the presence of αβT cells that recognize tumour neoantigens, and are therefore primarily restricted to tumours with high mutational load. Approaches that could address this limitation by engineering αβT cells, such as chimeric antigen receptor T (CAR T) cells, are being investigated intensively, but these approaches have other issues, such as a scarcity of appropriate targets for CAR T cells in solid tumours. Consequently, there is renewed interest among translational researchers and commercial partners in the therapeutic use of γδT cells and their receptors. Overall, γδT cells display potent cytotoxicity, which usually does not depend on tumour-associated (neo)antigens, towards a large array of haematological and solid tumours, while preserving normal tissues. However, the precise mechanisms of tumour-specific γδT cells, as well as the mechanisms for self-recognition, remain poorly understood. In this Review, we discuss the challenges and opportunities for the clinical implementation of cancer immunotherapies based on γδT cells and their receptors.

Despite extensive usage of gene therapy medicinal products (GTMPs) in clinical studies and recent approval of chimeric antigen receptor (CAR) T cell therapy, little information has been made available on the precise molecular characterization and possible variations in terms of insert integrity and vector copy numbers of different GTMPs during the complete production chain. Within this context, we characterize αβT cells engineered to express a defined γδT cell engineered to express a defined γδT receptor (TEG) currently used in a first-in-human clinical study (NTR6541). Utilizing targeted locus amplification in combination with next generation sequencing for the vector producer clone and TEG001 products, we report on five single-nucleotide variants and nine intact vector copies integrated in the producer clone. The vector copy number in TEG001 cells was on average a factor 0.72 (SD 0.11) below that of the producer cell clone. All nucleotide variants were transferred to TEG001 without having an effect on cellular proliferation during extensive in vitro culture. Based on an environmental risk assessment of the five nucleotide variants present in the non-coding viral region of the TEG001 insert, there was no altered environmental impact of TEG001 cells. We conclude that TEG001 cells do not have an increased risk for malignant transformation in vivo., (Copyright © 2019 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Adoptive transfer of chimeric antigen receptor-transduced T cells is a promising strategy for cancer immunotherapy. The CD38 molecule, with its high expression on multiple myeloma cells, appears a suitable target for antibody therapy. Prompted by this, we used three different CD38 antibody sequences to generate second-generation retroviral CD38-chimeric antigen receptor constructs with which we transduced T cells from healthy donors and multiple myeloma patients. We then evaluated the preclinical efficacy and safety of the transduced T cells. Irrespective of the donor and antibody sequence, CD38-chimeric antigen receptor-transduced T cells proliferated, produced inflammatory cytokines and effectively lysed malignant cell lines and primary malignant cells from patients with acute myeloid leukemia and multi-drug resistant multiple myeloma in a cell-dose, and CD38-dependent manner, despite becoming CD38-negative during culture. CD38-chimeric antigen receptor-transduced T cells also displayed significant anti-tumor effects in a xenotransplant model, in which multiple myeloma tumors were grown in a human bone marrow-like microenvironment. CD38-chimeric antigen receptor-transduced T cells also appeared to lyse the CD38(+) fractions of CD34(+) hematopoietic progenitor cells, monocytes, natural killer cells, and to a lesser extent T and B cells but did not inhibit the outgrowth of progenitor cells into various myeloid lineages and, furthermore, were effectively controllable with a caspase-9-based suicide gene. These results signify the potential importance of CD38-chimeric antigen receptor-transduced T cells as therapeutic tools for CD38(+) malignancies and warrant further efforts to diminish the undesired effects of this immunotherapy using appropriate strategies., (Copyright© Ferrata Storti Foundation.)