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T cell–based cancer immunotherapy has typically relied on membrane-bound cytotoxicity enhancers such as chimeric antigen receptors expressed in autologous αβ T cells. These approaches are limited by tonic signaling of synthetic constructs and costs associated with manufacturing. γδ T cells are an emerging alternative for cellular therapy, having innate antitumor activity, potent antibody-dependent cellular cytotoxicity, and minimal alloreactivity. We present an immunotherapeutic platform technology built around the innate properties of the Vγ9Vδ2 T cell, harnessing specific characteristics of this cell type and offering an allocompatible cellular therapy that recruits bystander immunity. We engineered γδ T cells to secrete synthetic tumor-targeting opsonins in the form of an scFv-Fc fusion protein and a mitogenic IL-15Rα–IL-15 fusion protein (stIL15). Using GD2 as a model antigen, we show that GD2-specific opsonin-secreting Vγ9Vδ2 T cells (stIL15-OPS-γδ T cells) have enhanced cytotoxicity and promote bystander activity of other lymphoid and myeloid cells. Secretion of stIL-15 abrogated the need for exogenous cytokine supplementation and further mediated activation of bystander natural killer cells. Compared with unmodified γδ T cells, stIL15-OPS-γδ T cells exhibited superior in vivo control of subcutaneous tumors and persistence in the blood. Moreover, stIL15-OPS-γδ T cells were efficacious against patient-derived osteosarcomas in animal models and in vitro, where efficacy could be boosted with the addition of zoledronic acid. Together, the data identify stIL15-OPS-γδ T cells as a candidate allogeneic cell therapy platform combining direct cytolysis with bystander activation to promote tumor control. Editor's summary: Both tumor antigen-specific antibodies and cellular therapies based on those antibodies, such as chimeric antigen receptor (CAR) T cells, have shown promise as cancer therapies. Here, Fowler et al. opted to combine the two and added a third feature—the use of γδ T cells instead of their αβ counterparts. The authors engineered γδ T cells, which have the potential to be used as an allogeneic therapy, to secrete opsonins that bound the tumor antigen GD2 and activated Fc receptors on immune cells. They also included an IL-15Rα–IL-15 fusion protein to further improve γδ T cell function and persistence. The opsonins produced by the γδ T cells activated the engineered cells and bystander immune cells in vitro and in vivo. This resulted in efficacy against multiple tumor models in vivo, including orthotopic patient-derived osteosarcomas, particularly when zoledronic acid was used as well. Together, these data support further development of opsonin-secreting γδ T cells as a cancer immunotherapy. —Courtney Malo [ABSTRACT FROM AUTHOR]

Pediatric patients with high-risk neuroblastoma have poor survival rates and urgently need more effective treatment options with less side effects. Since novel and improved immunotherapies may fill this need, we dissect the immunoregulatory interactions in neuroblastoma by single-cell RNA-sequencing of 24 tumors (10 pre- and 14 post-chemotherapy, including 5 pairs) to identify strategies for optimizing immunotherapy efficacy. Neuroblastomas are infiltrated by natural killer (NK), T and B cells, and immunosuppressive myeloid populations. NK cells show reduced cytotoxicity and T cells have a dysfunctional profile. Interaction analysis reveals a vast immunoregulatory network and identifies NECTIN2-TIGIT as a crucial immune checkpoint. Combined blockade of TIGIT and PD-L1 significantly reduces neuroblastoma growth, with complete responses (CR) in vivo. Moreover, addition of TIGIT+PD-L1 blockade to standard relapse treatment in a chemotherapy-resistant Th - ALK F1174L/ MYCN 129/SvJ syngeneic model induces CR. In conclusion, our integrative analysis provides promising targets and a rationale for immunotherapeutic combination strategies. [Display omitted] • Single-cell RNAseq identifies 17 distinct immune subsets infiltrating neuroblastoma • Dysfunctionality of T and NK cells alternates throughout the course of chemotherapy • Interaction analysis identifies the NECTIN2-TIGIT axis as crucial immune checkpoint • TIGIT (+PD-L1) blockade enhances immune responses against neuroblastoma in vivo Wienke et al. analyze the immune landscape of neuroblastoma pre- and post-chemotherapy and identify the NECTIN2-TIGIT axis as a crucial immune checkpoint, which correlates with dysfunction of T/NK cells. TIGIT (+PD-L1) blockade induces numerous complete responses (CR) in vivo, even against chemotherapy-resistant neuroblastoma, highlighting TIGIT blockade as promising immunotherapy for neuroblastoma. [ABSTRACT FROM AUTHOR]

Invariant NKT (iNKT) cell functional subsets are defined by key transcription factors and output of cytokines, such as IL-4, IFN-γ, IL-17, and IL-10. To examine how TCR specificity determines iNKT function, we used somatic cell nuclear transfer to generate three lines of mice cloned from iNKT nuclei. Each line uses the invariant Vα14Jα18 TCRα paired with unique Vβ7 or Vβ8.2 subunits. We examined tissue homing, expression of PLZF, T-bet, and RORγt, and cytokine profiles and found that, although monoclonal iNKT cells differentiated into all functional subsets, the NKT17 lineage was reduced or expanded depending on the TCR expressed. We examined iNKT thymic development in limited-dilution bone marrow chimeras and show that higher TCR avidity correlates with higher PLZF and reduced T-bet expression. iNKT functional subsets showed distinct tissue distribution patterns. Although each individual monoclonal TCR showed an inherent subset distribution preference that was evident across all tissues examined, the iNKT cytokine profile differed more by tissue of origin than by TCR specificity. [ABSTRACT FROM AUTHOR]