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The Ig superfamily ligand B7H6 subjects T cell responses to NK cell surveillance

ABSTRACT: T cells play a critical role in the immune system by recognizing and responding to foreign and tumor antigens. Dysregulation or dysfunction of T cells can lead to autoimmune diseases, infectious diseases and cancer. Understanding the mechanisms that regulate T cell immunity is therefore critical for the development of effective therapies for diseases associated with T cell dysfunction. Co-inhibitory ‘checkpoint molecules’ such as programmed cell death protein 1 (PD-1) are tightly connected to T cell activation and balance excessive or prolonged immune activation by T cell-intrinsic signaling. Here, by screening for mediators of NK cell recognition on T cells, we identify the Ig superfamily ligand B7H6 to be highly expressed by activated T cells, including patient-infused CD19-targeting chimeric antigen receptor (CAR)-T cells. We show that, unlike other checkpoint molecules, B7H6 mediates NKp30-dependent recognition and subsequent cytolysis of activated T cells by NK cells. B7H6+ T cells are prevalent in the tissue and blood of several diseases, and we find that their abundance in tumor tissue positively correlates with clinical response in a cohort of immune checkpoint inhibitor-treated patients with esophageal cancer. In humanized mouse models, we demonstrate that NK cell surveillance via B7H6 is relevant in limiting the persistence and anti-tumor activity of CAR T cells in vivo and that its genetic depletion enhances T cell proliferation and persistence. In summary, we provide evidence of B7H6 protein expression by activated T cells and suggest the B7H6-NKp30 axis as therapeutically actionable NK cell-dependent immune checkpoint that regulates human T cell function.

ORGANISM(S): Homo sapiens

PROVIDER: GSE253152 | GEO | 2024/01/12

REPOSITORIES: GEO

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Project description:CD19-targeting chimeric antigen receptor (CAR) T cell have become an important therapeutic option for patients with relapsed and refractory B cell malignancies. However, according to the recent clinical data, a significant portion of patients still do not benefit from the therapy, due various resistance mechanisms including the high expression of multiple inhibitory immune checkpoint receptors on activated CAR-T cells. Studies of checkpoint blockade immunotherapy using monoclonal antibodies have shown that simultaneously targeting inhibitory receptors that are functionally non-redundant can synergistically enhance anti-tumor responses. Here we report a lentiviral two-in-one CAR T approach in which two checkpoint receptors can be downregulated simultaneously by a dual short-hairpin RNA (shRNA) cassette integrated into a CAR vector. Using this system, we evaluated CD19-targeting CAR T cells in the context of four different checkpoint combinations, PD-1/TIM-3, PD-1/LAG-3, PD-1/CTLA-4, and PD-1/TIGIT, and found that the CAR T with PD-1/TIGIT downregulation uniquely exhibited synergistic anti-tumor effect in mouse xenograft models compared to the single PD-1 downregulation and maintained cytolytic and proliferative capacity upon repeated antigen exposure. Importantly, functional and phenotypic analysis of CAR T cells as well as analysis of transcriptomic profiles suggests that downregulation of PD-1 enhances short-term effector function while downregulation of TIGIT is primarily responsible for maintaining a less-differentiated/exhausted state, providing a potential mechanism of the synergy. The PD-1/TIGIT downregulated CAR T cells generated from DLBCL patient-derived T cells following clinically applicable manufacturing process also showed a robust anti-tumor activity and significantly improved persistence in vivo compared to conventional CD19-targeting CAR T cells. Overall, our results demonstrate that the cell-intrinsic PD-1/TIGIT dual downregulation strategy may provide an effective way to overcome the immune checkpoint-mediated resistance in CAR T therapy.

Project description:Background: T cell-based immunotherapies including immune checkpoint blockade (ICB) and chimeric antigen receptor (CAR) T cells can induce durable responses in cancer patients. However, clinical efficacy is limited due to the ability of cancer cells to evade immune surveillance. While T cells have been the primary focus of immunotherapy, recent research has highlighted the importance of Natural Killer (NK) cells in directly recognizing and eliminating tumor cells and playing a key role in the set-up of an effective adaptive immune response. The remarkable potential of NK cells for cancer immunotherapy is demonstrated by their ability to broadly identify stressed cells, irrespective of the presence of neoantigens, and their ability to fight tumors that have lost their Major Histocompatibility Complex class I (MHC I) expression due to acquired resistance mechanisms. However, like T cells, NK cells can become dysfunctional within the tumor microenvironment. Strategies to enhance and reinvigorate NK cell activity hold potential for bolstering cancer immunotherapy. Method: In this study, we conducted a high-throughput screen to identify molecules that could enhance primary human NK cell function. After compound validation, we investigated the effect of the top performing compound on dysfunctional NK cells that were generated by a newly developed in vitro platform. Functional activity of NK cells was investigated utilizing compounds alone and in combination with checkpoint inhibitor blockade. The findings were validated on patient-derived intratumoral dysfunctional NK cells from different cancer types. Results: The screening approach led to the identification of a Cbl-b inhibitor enhancing the activity of primary human NK cells. Furthermore, the Cbl-b inhibitor was able to reinvigorate the activity of in vitro generated and patient-derived dysfunctional NK cells. Finally, Cbl-b inhibition combined with TIGIT blockade further increased the cytotoxic potential and reinvigoration of both in vitro generated and patient-derived intratumoral dysfunctional NK cells. Conclusion: These findings underscore the relevance of Cbl-b inhibition in overcoming NK cells dysfunctionality with the potential to complement existing immunotherapies and improve outcomes for cancer patients.

Project description:NK-92 cells and their CAR-modified derivatives exhibit strong cytotoxic activity against tumors and are promising "off-the-shelf" therapeutics compared to CAR-T cells. In order to ensure safety and prevent the occurrence of secondary tumors, (CAR-)NK-92 cells need to be inactivated before transfusion. With increasing clinical use, there is a growing demand for enhanced safety and an efficient method that stops cell proliferation, but better maintains the cytotoxic effector functions of the cells compared to commonly used gamma irradiation. Recently, low-energy electron irradiation (LEEI) was successfully applied for inactivation of bacteria and viruses for vaccine production, and was described as a method for NK-92 inactivation for the first time. In the present publication, data on extensive characterization of LEEI and its comparison with gamma irradiation for the inactivation of parental NK-92 cells as well as CD123-directed CAR-NK-92 cells are provided. Our results show that both irradiation methods cause a progressive decrease in cell viability and are therefore suitable for inhibition of proliferation. Notably, cytotoxicity of the NK cells was significantly reduced by gamma irradiation, but not by LEEI three days after irradiation, compared to non-irradiated cells. Both gamma irradiation as well as LEEI led to substantial DNA-damage and an accumulation of irradiated cells in the G2/M phase. In addition, transcriptomic analysis of cells irradiated with both methods revealed approximately 12-fold more differentially expressed genes after gamma irradiation compared to LEEI. Analysis of surface molecules revealed an irradiation-induced decrease in CD56 expression but no changes in the levels of the activating receptors NKp46, NKG2D, and NKp30. Conclusions: The present data show that LEEI efficiently inactivates (CAR )NK-92 cells and sustains their cytotoxic capacity better than gamma irradiation. Taking into account additional logistic advantages of LEEI proves a potent alternative in the manufacturing of (CAR-)NK-92 cells for clinical application.

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The Ig superfamily ligand B7H6 subjects T cell responses to NK cell surveillance

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