Natural Cytotoxicity Receptors Research Articles

Abstract 1319: In vivo programming of natural killer cells and T cells using mRNA delivered cytotoxic chimeric antigen receptors

Abstract Introduction: Current generation of adoptive cell therapy relies on ex vivo engineering of immune cells with tumor-targeting chimeric antigen receptors. In addition to manufacturing challenges, ex vivo engineered cells such as CAR-NK and CAR-T show limited tumor infiltration and persistency within tumor microenvironment. A method leveraging mRNA-based delivery for in-vivo engineering of these cells could address these issues but has not been established. Conventional mRNA/LNP when infused systemically result in significant accumulation in liver tissue and the CAR has no immune cell specificity in their expression or function. Method: Many immune receptors are heterodimeric complexes composed of a ligand binding subunit (e.g. CD16 and TCR) and a signaling adaptor containing Immunoreceptor Tyrosine-based Activation Motif (ITAM) (e.g. FcRγ and CD3ζ). Notably, the cell surface expression of the ligand binding subunit often depends on the expression of the ITAM adaptor. Based on this we fused HER2-targeting scFv to multiple activating receptors whose expression is restricted to NK cells and T cells, and evaluated their expression and anti-tumor function. Results: We developed a series of Natural Cytotoxic Receptor (NCR)-based in vivo CAR constructs by fusing HER2-targeting scFv to the native extracellular and transmembrane sequences of the receptor NKp30, NKp44 and NKp46, These CARs engage signaling adaptor FcRγ or DAP12 expressed in NK cells, and exhibit potent tumoricidal activity against HER2+ ovarian cancer cells as well as mediate tumor-induced cytokine and chemokine response. Using Huh7 liver cell as a model for off-target expression in hepatocytes, we found that NKp44 and NKp46-based CAR requires their cognate signaling adaptors for stable cell surface expression. We also showed that similar strategy can be used to design receptors for selective expression on the surface of T cells utilizing CD3ε chain fused with tumor targeting scFv. Conclusion: These results demonstrated the feasibility of engineering primary NK cells with CARs based on the NCR family of receptors NKp44 and NKp46, whose expression requires the immune-restricted signaling adaptors. These CAR-NK receptors do not contain intracellular signaling motifs but can engage and activate their signaling natural adaptors to initiate tumor killing and cytokine response. An mRNA/LNP based delivery of NKp44-based CAR for cancer treatment is currently under clinical development. Citation Format: Neha Diwanji, Daniel Getts, Yuxiao Wang. In vivo programming of natural killer cells and T cells using mRNA delivered cytotoxic chimeric antigen receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1319.

Abstract 5228: Engineering CD123-targeting vδ1-enriched γδT cells with enhanced preclinical efficacy and safety profile

Abstract Acute myeloid leukemia (AML) is an aggressive hematological malignancy that, despite advances in treatment, has an overall 5-year survival rate of less than 30%. There is an urgent unmet need for new and effective therapies for AML. Chimeric antigen receptor (CAR)-modified cell therapies are emerging as promising options. However, the selection of suitable antigens is challenging, due to the heterogeneous expression of potential antigen targets in AML. Most antigens, such as CD123, are expressed on both leukemic cells and normal hematopoietic stem and progenitor cells. In addition, conventional autologous or allogeneic T cell therapies present many limitations, such as cytokine release syndrome (CRS) and graft-vs-host disease (GvHD). We therefore evaluated CD123-targeting CAR designs in vδ1-enriched γδT cells, an innate immune cell platform that is inherently non-MHC restricted, has low risks of CRS and GvHD, and is capable of discriminating between malignant and healthy cells in preclinical models. We engineered vδ1-enriched γδT cells from peripheral blood of healthy individuals to express various CD123-targeting CAR constructs. The CD123-targeting CAR-engineered vδ1-enriched γδT cells expressed high levels of natural cytotoxicity receptors, suggesting capability to target innate receptor ligands on AML cell surface. CD123-targeting CAR-expressing vδ1-enriched γδT cells demonstrated enhanced cytotoxicity in vitro against multiple AML cell lines, in both short-term co-culture and repeated antigen stimulation assays. CD123-targeting CAR-expressing vδ1-enriched γδT cells also showed enhanced efficacy and survival in vivo against mouse models of human AML. Interestingly, attenuation of CAR activity via novel designs preserved the inherent selectivity of vδ1-enriched γδT cells against AML cells relative to normal hematopoietic stem or progenitor cells, which also express CD123. Together, our results demonstrated through rational CAR designing and engineering in vδ1-enriched γδT cells, an allogeneic cell therapy targeting CD123 can be generated with selective cytotoxicity against AML cells while sparing normal hematopoietic stem cells. Such product designs have the potential to be a transformative therapeutic option for a broad population of AML patients. Citation Format: Mei Rosa Ng, Sarah L. Hayward, Di Zhang, Gary Gu, Bri Paulhus, Donjeta Gjuka, Collin Crowley, Sophie McLaughlin, Anya Lublinsky, Angel Mathew, Evans Berreondo Giron, Doanh Mai, Madisen Lee, Megha Wal, Rebecca Alade, Andre Simoes, Robin Lytle, James Gavin, Shira Cramer, Zhong-hua Yan, Taylor Hickman, Rashmi Choudhary, Cori Abikoff, Yana Wang, Istvan Kovacs, Nandhu M. Sobhana, Lan Cao. Engineering CD123-targeting vδ1-enriched γδT cells with enhanced preclinical efficacy and safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5228.

Abstract 5255: A B7-H3-CAR-modified Vδ1 T cells showed potentanti-solid tumor potential

Abstract Background: CAR-αβT cells have made limited advancement in treating solid tumors. Vδ1 T cells, a subtype of γδT cells, are rare in human peripheral blood, but they are the main tissue-resident immune cells found in epithelial and mucosal tissues. This suggests that Vδ1 T cells might be a potential modality for treating solid tumors. To explore this possibility, we hypothesized that Vδ1 T cells modified with B7-H3 CAR (B7-H3-CAR-Vδ1 T) could have great potential of anti-solid tumor activity, making them a promising candidate for further development as a universal cellular therapy product. Methods: A proprietary manufacturing procedure to produce Vδ1 T cells from the leukopak of healthy donors has been established under GMP compliance. The immune checkpoint molecule B7-H3 was selected as the target. The allogeneic B7-H3-CAR-Vδ1 T cells carrying IL-2 (UTAA06), which displayed the most prolonged anti-tumor activity, were chosen for comprehensive functional validation. Single-cell sequencing and mass spectrometry flow cytometry were utilized to characterize the biology of UTAA06. In vivo effectiveness of UTAA06 was confirmed in animal models. Results: A clinical-grade manufacturing process for Vδ1 T cells is developed. This process results in a purity of over 95% of Vδ1 T cells, with an expansion of more than 20,000-fold, and a CAR lentiviral transduction efficiency of over 50%.žSingle-cell sequencing and mass cytometry analysis both demonstrated that the Vδ1 T cells produced by our approach exhibited high expression levels of natural cytotoxic receptors (including NKG2D, DNAM1, and NKp series receptors), chemokine receptors (such as CXCR3 and CXCR6), anti-tumor related molecules (such as CD27 and CD137), and low expression levels of inhibitory molecules (such as PD1 and LAG3).ž UTAA06 cells did not produce IL17A, which is a signal protein with pro-tumor activity. Meanwhile, UTAA06 demonstrated significant and specific inhibitory effects on the growth of various solid tumor cell lines.žUTAA06 cells were found to significantly suppress tumor cell growth in solid tumor mouse models. The complete remission (CR) for each tumor model was 83.3% of neuroblastoma (SK-N-AS, N=6), 66.7% of pancreatic cancer (SW1990, N=6), 100% of lung cancer (A549, N=6), and 70% of colorectal cancer (HCT-15, N=10) respectively. In addition, a potent capacity of UTAA06 to penetrate tumor tissues with a favorable safety profile in mice was observed. Conclusion: UTAA06 exhibited extraordinary anticancer properties both in vitro and in vivo against solid tumors. Additionally, the outstanding manufacturing process of UTAA06 will facilitate the clinical application of this product. Citation Format: Licui Jiang, Changsong Qi, Fengtao You, Shufen Xiang, Ping Zhang, Min Wang, Hai Wu, Kunmin Yan, Jiequn Huang, Huimin Meng, Nan Yang, Shirley Zhang, Lin Shen, Lin Yang. A B7-H3-CAR-modified Vδ1 T cells showed potentanti-solid tumor potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5255.

Angelica gigas polysaccharide induces CR3-mediated macrophage activation and the cytotoxicity of natural killer cells against HCT-116 cells via NF-κB and MAPK signaling pathways

Angelica gigas (A. gigas) is traditional medicinal herb that mainly exists in Korea and northeastern China. There have been relatively few studies conducted thus far on its polysaccharides and their bioactivities. We purified and described a novel water-soluble polysaccharide derived from A. gigas and investigated its immunoenhancing properties. The basic components of crude and purified polysaccharides (F1 and F2) were total sugar (41.07% - 70.55%), protein (1.12–10.33%), sulfate (2.9–5.5%), and uronic acids (0.5–31.05%) in total content. Our results demonstrated that the crude and fractions' molecular weights (Mw) varied from 42.2 to 285.2 × 103 g/mol. As the most effective polysaccharide, F2 significantly stimulated RAW264.7 cells to release nitric oxide (NO) and express several cytokines. Furthermore, F2 increased the expression of tumor necrosis factor-α (TNF-α), interferon-gamma (IFN-ɣ), natural killer cytotoxicity receptors (NKp44), and granzyme-B in NK-92 cells and enhanced the cytotoxicity against HCT-116 cells. In our experiments, we found that F2 stimulated RAW264.7 cells and NK-92 cells via MAPK and NF-κB pathways. The monosaccharide and methylation analysis of the high immunostimulant F2 polysaccharide findings revealed that the polysaccharide was primarily composed of 1 → 4, 1 → 6, 1 → 3, 6, 1 → 3 and 1 → 3, 4, 6 galactopyranose residues, 1 → 3 arabinofuranose residues, 1 → 4 glucopyranose residues. These results demonstrated that the F2 polysaccharide of A. gigas which possesses potential immunostimulatory attributes, could be used to create a novel functional food.

Chimeric Antigen Cytotoxic Receptors for In Vivo Engineering of Tumor-Targeting NK Cells.

Chimeric Ag receptor (CAR) NK cells are challenging to manufacture and fail to achieve consistent tumor infiltration and sustained cytolytic function in the tumor microenvironment. In vivo engineering of NK cells using mRNA-based CAR delivery may overcome these issues. In this study, we developed an invivo programming method by designing CARs that leverage the biology of NK cell receptors for cell type-specific expression and function. These CARs were engineered by fusion of a tumor recognition domain with the natural cytotoxic receptor family including NKp30, NKp44, and NKp46. Our results demonstrated that these natural cytotoxic receptor-based CARs can engage endogenous signaling adaptors to effectively activate human NK cells for tumor lysis and cytokine production. Specifically, we discovered that stable expression of an NKp44-based CAR was contingent on the presence of the immune cell-specific signaling adaptor DAP12. This innovative strategy facilitates direct in situ programming of NK cells, enhancing safety and minimizing off-target effects in nontargeted, healthy tissues.

Altered NCR3 Splice Variants May Result in Deficient NK Cell Function in Renal Cell Carcinoma Patients.

The natural killer (NK) cell function of patients with malignant tumours may be suppressed by deficiency, and the poor prognosis of renal cell carcinoma (RCC) patients may be due to escape from NK cell cytotoxicity, especially with respect to natural cytotoxicity receptors (NCRs) on the NK cell surface. However, the specific mechanism remains unclear. Therefore, in this study, we sought to explore the role of NCR, especially NCR3 splice variants, in the process of NK cell deficiency in RCC patients. We used flow cytometry to analyse the phenotype of NK cells from the peripheral blood and kidney tumour tissue of RCC patients. The NKp30-mediated NK cell killing function was measured by antibody-dependent cell-mediated cytotoxicity (ADCC) in NK and RCC cell coincubation. We extracted RNA from the peripheral blood mononuclear cells (PBMCs) of RCC patients and renal carcinoma tissue and carried out real-time quantitative PCR to detect the mRNA levels of NKp30a, NKp30b and NKp30c. mRNA expression levels of cytokines (IL-6, IL-8, IL-10, IL-18 and TGF-β) based on RNA extracted from renal carcinoma tissue and adjacent normal kidney tissues were also measured by real-time quantitative PCR. Regarding the phenotype of NK cells in RCC patients, the proportion of NK cells in tumour tissue was significantly reduced, with changes in the NK cell proportion being most obvious in NKp30+ NK cells. Furthermore, the results of the ADCC function assay showed limited NKp30+ NK cell-mediated cytotoxicity in RCC patients. Through real-time quantitative PCR, we found lower expression of NKp30a and NKp30b, the immunostimulatory splice variants of NCR3 encoding NKp30, in RCC patients. Moreover, expression of activating cytokines (IL-6 and IL-8) in renal cancer tissue was decreased, though inhibitory cytokine (TGF-β) expression remained unchanged, which may result in an immunosuppressive cytokine microenvironment. Decreased expression of immunostimulatory NCR3 splice variants and the inhibitory cytokine microenvironment in RCC patients may contribute to deficient NK cell cytotoxicity and renal carcinoma cell immune escape from NK cell killing, which may provide a theoretical basis for finding new immunotherapeutic targets for RCC.

The Ig Superfamily Ligand B7H6 Subjects Activated T Cells to NK Cell Surveillance and Limits CAR T Cell Persistence

Understanding the mechanisms that regulate T cell immunity is critical for the development of effective therapies for diseases associated with T cell dysfunction. Co-inhibitory ‘checkpoint molecules’ are tightly connected to T cell activation and balance excessive or prolonged immune activation by suppressing clonal expansion. Previous studies have suggested that NK cells might also suppress lymphocyte expansion, but the specific ligands enabling T cell recognition by natural cytotoxicity receptors (NCRs) remain unknown. In this study, we describe the physiological expression of the immunoglobulin superfamily ligand B7H6 on activated T cells and elucidated the functional and clinical implication of its recognition by autologous NK cells. In a targeted proteomics screen for mediators of NK cell recognition on T cells, we identified the Ig superfamily ligand B7H6 ( NCR3LG1) to be highly expressed by T cells upon activation. We show that, unlike other checkpoint molecules, B7H6 mediates NKp30-dependent recognition and subsequent cytolysis of activated T cells by autologous NK cells. Time-resolved deep RNA-sequencing of anti-CD3/CD28-stimulated peripheral blood-derived T cells from n=24 human donors revealed that NCR3LG1 transcript expression changes dynamically upon stimulation and that B7H6 surface protein recognition by NK cells initiates Granzyme B-mediated ‘fratricide’ as early as 8 hours post T cell stimulation. Genomic knockout of NCR3LG1 rendered activated T cells resistant to NK cell-mediated killing without affecting their viability or proliferative capacity. By analyzing transcriptomic profiles of 313,303 CD8+ and 283.930 CD4+ T cells in datasets of multiple sclerosis (MS, n = 62 patients), inflammatory bowel disease (IBD, n = 4 patients), hepatitis B virus infection (HBV, n = 23 patients) and 9 different cancer entities (n = 47 patients), we found a conserved B7H6+ T cell population that is prevalent in the tissue and blood of most individuals at 1-5% frequency. In line with our experimental data, B7H6 positivity was coupled to a highly activated cell state of T cells. Considering CAR T cell manufacturing involves robust activation and clonal expansion, we hypothesized the B7H6-NKp30 axis is a yet unknown, but relevant factor in limiting CAR T cell persistence. Clinical-grade CD19.CD28.4-1BBzeta-CAR T cell products as well as circulating CAR T cells in lymphoma patients indeed uniformly expressed NCR3LG1 transcripts and B7H6 surface protein and were lysed by autologous NK cells in vitro. We then co-injected CD19.CAR T cells and patient-autologous NK cells in leukemia-engrafted mice and observed reduced CAR T cell persistence in the peripheral blood of mice with NK cell co-injection. Co-injection of NK cells further increased tumor burden and resulted in significantly decreased survival in leukemia-bearing mice treated with CD19-recognizing CAR-T cells, while these effects could be minimized by NKp30 antibody blockade or prior genetic editing of NCR3LG1 on CAR T cells. Given the physiological coupling T cell activation and B7H6 expression, any T cell-directed immunotherapy regimen might trigger the B7H6-NKp30-cytolysis mechanism. We therefore analyzed tissue of patients that were treated with nivolumab and ipilimumab (NCT03416244). In contrast to the previous notion of largely tumor-targeting NK cell function, we observed an increased NK cell abundance in patients that failed to respond to treatment. Notably, we found both B7H6+ CD8+ and CD4+ T cells in situ and that their abundance and a high NK/T ratio was associated with reduced progression-free survival (PFS), even in initial clinical responders, and experimentally deducted a NKp30 dependent cytolysis of these T cells. Together, our data demonstrates a mechanism that couples T cell expansion to eventual clearance by NK cells. This concept differs from the previously known immune checkpoints, as those molecules act through intracellular downstream signaling in T cells. We show that NK cell surveillance via B7H6 counteracts anti-tumor activity of CAR-T cells in vivo and that the genetic or antibody-mediated inhibition of B7H6 enhances T cell persistence. We therefore suggest an alternative immune checkpoint that limits T cell expansion and persistence in physiological and cellular engineering contexts. Targeting the B7H6-NKp30 axis may offer a means to modulate T cell immunity across multiple disease entities.

Analysis of Butyrophilin-Mediated Activation of γδ T Cells from Human Spleen.

There is considerable interest in therapeutically engaging human γδ T cells. However, due to the unique TCRs of human γδ T cells, studies from animal models have provided limited directly applicable insights, and human γδ T cells from key immunological tissues remain poorly characterized. In this study, we investigated γδ T cells from human spleen tissue. Compared to blood, where Vδ2+Vγ9+ T cells are the dominant subset, splenic γδ T cells included a variety of TCR types, with Vδ1+ T cells typically being the most frequent. Intracellular cytokine staining revealed that IFN-γ was produced by a substantial fraction of splenic γδ T cells, IL-17A by a small fraction, and IL-4 was minimal. Primary splenic γδ T cells frequently expressed NKG2D (NK group 2 member D) and CD16, whereas expression of DNAM-1 (DNAX accessory molecule 1), CD28, PD-1, TIGIT, and CD94 varied according to subset, and there was generally little expression of natural cytotoxicity receptors, TIM-3, LAG-3, or killer Ig-like receptors. In vitro expansion was associated with marked changes in expression of these activating and inhibitory receptors. Analysis of functional responses of spleen-derived Vδ2+Vγ9+, Vδ1+Vγ9+, and Vδ1+Vγ9- T cell lines to recombinant butyrophilin BTN2A1 and BTN3A1 demonstrated that both Vδ2+Vγ9+ and Vδ1+Vγ9+ T cells were capable of responding to the extracellular domain of BTN2A1, whereas the addition of BTN3A1 only markedly enhanced the responses of Vδ2+Vγ9+ T cells. Conversely, Vδ1+Vγ9+ T cells appeared more responsive than Vδ2+Vγ9+ T cells to TCR-independent NKG2D stimulation. Thus, despite shared recognition of BTN2A1, differential effects of BTN3A1 and coreceptors may segregate target cell responses of Vδ2+Vγ9+ and Vδ1+Vγ9+ T cells.

Bovine NK subsets in the afferent lymph and lymph nodes have distinct expression of naïve and activation-associated cell surface expressed molecules, and are differentially stimulated by BCG vaccination

Bovine natural killer (bNK) cells are heterogeneous cell populations defined by constitutive expression of the natural cytotoxicity receptor, NKp46 (CD335). Two major subsets of bNK cells, classified by differential expression of CD2, display divergent functions in innate immunity, and are hypothesised to contribute to adaptive immunity following vaccination. Here we characterised phenotypic variation of bNK cells within afferent lymph and lymph node (LN) tissues and between CD2+ and CD2- bNK subsets, and report phenotypic changes induced by BCG vaccination. CD2- bNK cells, which dominate in the afferent lymph and LN, displayed lower expression of the activation marker CD25 within the LN, with CD25+ cells being less than half as frequent as in afferent lymph. Furthermore, we found bNK cells had a lower expression of CD45RB, associated in cattle with naïve cell status, within LN compared to afferent lymph. Following BCG vaccination, bNK cells in afferent lymph draining the vaccination site showed increased CD2-CD25+ frequencies and increased expression of CD25 on CD2+ bNK cells, although the frequency of these cells remained unchanged. In summary, we provide an overview of the phenotype of bNK cells within bovine lymphatic tissues, and provide an indication of how subsets may diverge following BCG vaccination.

Enhanced expression of natural cytotoxicity receptors on cytokine-induced memory-like natural killer cells correlates with effector function.

Natural killer (NK) cells are a key component of the innate immune system, involved in defending the host against virus-infected cells and tumor immunosurveillance. Under in vitro culture conditions, IL-12/15/18 can induce a memory-like phenotype in NK cells. These cytokine-induced memory-like (CIML) NK cells possess desirable characteristics for immunotherapies, including a longer lifespan and increased cytotoxicity. In this study, NK cells were isolated from peripheral blood of healthy donors and stimulated with IL-12/15/18 to induce a memory-like phenotype or with IL-15 alone as a control. After seven days of culture, multiparametric flow cytometry analysis was performed to evaluate the phenotypic and functional profiles of CIML and control NK cells. Our results showed a significantly higher expression of CD25, CD69, NKG2D, NKp30, NKp44, NKp46, TACTILE, and Granzyme B in CIML NK cells compared to control NK cells. In contrast, KIR2D expression was significantly lower in CIML NK cells than in control NK cells. Moreover, functional experiments demonstrated that CIML NK cells displayed enhanced degranulation capacity and increased intracellular IFN-γ production against the target cell line K562. Interestingly, the degranulation capacity of CIML NK cells was positively correlated with the expression of the activating receptors NKp46 and NKp30, as well as with the inhibitory receptor TACTILE. In conclusion, this study provides a deep phenotypic characterization of in vitro-expanded CIML NK cells. Moreover, the correlations found between NK cell receptors and degranulation capacity of CIML NK cells allowed the identification of several biomarkers that could be useful in clinical settings.

In-depth characterization of NK cell markers from CML patients who discontinued tyrosine kinase inhibitor therapy.

Treatment-free remission (TFR) in patients with chronic myeloid leukemia in chronic phase is considered a safe option if suitable molecular monitoring is available. However, the question arises as to which factors can contribute to the maintenance of TFR, and immunologic surveillance of the remaining leukemic cells is believed to be one of them. Argentina Stop Trial is an open-label, single-arm, multicenter trial assessing TFR after tyrosine kinase inhibitors interruption, that after more than 4 years showed a successful TFR rate of 63%. In this context, we set up an immunological study by flow cytometry in order to analyze specific NK cell subsets from peripheral blood patient samples both at the time of discontinuation as well as during the subsequent months. At the time of discontinuation, patients show a mature NK cell phenotype, probably associated to TKI treatment. However, 3 months after discontinuation, significant changes in several NK cell receptors occurred. Patients with a higher proportion of CD56dim NK and PD-1+ NK cells showed better chances of survival. More interestingly, non-relapsing patients also presented a subpopulation of NK cells with features associated with the expansion after cytomegalovirus infection (expression of CD57+NKG2C+), and higher proportion of NKp30 and NKp46 natural cytotoxicity receptors, which resulted in greater degranulation and associated with better survival (p<0.0001). This NK cell subset could have a protective role in patients who do not relapse, thus further characterization could be useful for patients in sustained deep molecular response.

Identification of natural killer markers associated with fatal outcome in COVID-19 patients.

Increasing evidence has shown that coronavirus disease 19 (COVID-19) severity is driven by a dysregulated immunological response. Previous studies have demonstrated that natural killer (NK) cell dysfunction underpins severe illness in COVID-19 patients, but have lacked an in-depth analysis of NK cell markers as a driver of death in the most critically ill patients. We enrolled 50 non-vaccinated hospitalized patients infected with the initial virus or the alpha variant of SARS-CoV-2 with moderate or severe illness, to evaluate phenotypic and functional features of NK cells. Here, we show that, consistent with previous studies, evolution NK cells from COVID-19 patients are more activated, with the decreased activation of natural cytotoxicity receptors and impaired cytotoxicity and IFN-γ production, in association with disease regardless of the SARS-CoV-2 strain. Fatality was observed in 6 of 17 patients with severe disease; NK cells from all of these patients displayed a peculiar phenotype of an activated memory-like phenotype associated with massive TNF-α production. These data suggest that fatal COVID-19 infection is driven by an uncoordinated inflammatory response in part mediated by a specific subset of activated NK cells.

Abstract 4078: Optimized chimeric antigen receptors (CARs) for CAR-NK cell therapies

Abstract Introduction: CAR-T-cell therapy has shown great success in treating hematopoietic malignancies. Allogenic NK cell-based therapies have also been shown to mount potent responses against hematopoietic malignancies. Unlike T-cell therapies, allogeneic NK cells do not cause toxicities such as serious cytokine release syndrome, neurotoxicity, or graft-vs-host-disease. Several groups have demonstrated the clinical efficacy of allogeneic CAR-expressing NK cells that utilize CARs developed for T-cells. However, activating receptor signaling in NK cells is different from T cells. NK cell-mediated cytotoxicity is regulated by a repertoire of activating and inhibitory receptors. Activating receptors include natural cytotoxic receptors (NCRs) such as NKG2D, CD16, LFA-1, CD244 (2B4), and CD137 (4-1BB) and so on. These activating cell surface receptors have the capacity to trigger cytolytic programs as well as cytokine and chemokine secretion via intra-cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs) such as in 2B4 and 4-1BB and/or via other transmembrane signaling adaptors. Recent studies using NK cell-based activating receptor signaling modules such as NKG2D and 2B4 as CAR co-stimulatory domains proved increased anti-tumor activity compared to T-cell CARs. Methods: The goal of this study is to develop next-generation NK cell-optimized CARs to improve targeting and potency of NK cell treatment. To achieve this, we constructed a library of CARs containing signaling modules from diverse T cell signaling receptors and NK cell specific signaling receptors, and then screened for CAR activity using short-term and long-term NK cell cytotoxicity assays. In comparison with T cell signaling receptors, CAR-NK cells with NK cell signaling receptors showed more potent cytotoxicity. Using a repeated rounds of cancer cell clearance assay, serial re-stimulation by fresh cancer target cells was tested. CAR-NK cells with NK cell signaling receptors showed remarkable persistence and antigen-mediated expansion of CAR-NK cells compared with T cell signaling receptors. Results: We identified a NK cell-CAR containing two NK cell co-stimulatory domain and CD3z signaling domain,showing better persistence than T-cell CAR (CD28-CD28-zeta) and 2 previously described NK cell-CARs (NKG2D-2B4-zeta and 2B4). Conclusions: We have successfully identified an optimized CAR suitable for NK cells that enable increased potency of NK cells compared to other CAR designs. The most potent CARs identified in our screen are being engineered into a highly effective dual targeting BCMA-GPRC5D CAR-NK cell product as a next-generation “off-shelf” cell therapy. Citation Format: Cuiqing Yang, Yifang Wang, Tingting Liu, Fuwei Jiang, Qingyang Wang, Qin Wang, Gang Ye, Renhong Tang, Zhouxiao Cao. Optimized chimeric antigen receptors (CARs) for CAR-NK cell therapies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4078.

NCR negative group 3 innate lymphoid cell (NCR- ILC3) participates in abnormal pathology of lung in cigarette smoking-induced COPD mice.

Natural cytotoxicity receptor negative innate lymphoid cell (NCR- ILC3) involves into mucosal homeostasis, inflammation regulation and tissue remodeling. The proportion of NCR- ILC3 is increased in the lung of smokers with chronic obstructive pulmonary disease (COPD). However, there's still few understandings on the role of NCR- ILC3 in COPD pathogenesis. COPD mice were induced by cigarette smoking. The pathology in lung was detected in histology. The frequency of NCR- ILC3 (CD3-CD45+RORγt+NkP46-) from murine lung was detected using flow cytometry. IL-17+RORγt+ double positive cells in lung were assessed by double immunofluorescence staining. The protein expressions of epithelial-to-mesenchymal transition (EMT) markers, namely E-cadherin and Vimentin, were assessed using immunohistochemistry staining and western blotting. The frequency of NCR- ILC3 in lung was higher in COPD group than controls. The IL-17+RORγt+ cells in lung from COPD mice were more than controls. E-cadherin expression was decreased but Vimentin expression was increased in lung of COPD mice, when compared with controls. The frequency of NCR- ILC3 in lung tissues were positively correlated with mean linear intercept in lung, destructive index in lung and EMT, respectively. NCR- ILC3 could contribute to emphysema and EMT in lung of cigarette smoking-induced COPD, which will provide further understanding on COPD pathogenesis of immune response.

Affinity Maturation of the Natural Ligand (B7-H6) for Natural Cytotoxicity Receptor NKp30 by Yeast Surface Display.

In recent years, the development of bispecific antibodies (bsAbs) has experienced tremendous progress for disease treatment, and consequently, a plethora of bsAbs is currently scrutinized in clinical trials. Besides antibody scaffolds, multifunctional molecules referred to as immunoligands have been developed. These molecules typically harbor a natural ligand entity for the engagement of a specific receptor, while binding to the additional antigen is facilitated by an antibody-derived paratope. Immunoligands can be exploited to conditionally activate immune cells, e.g., natural killer (NK) cells, in the presence of tumor cells, ultimately causing target-dependent tumor cell lysis. However, many ligands naturally show only moderate affinities toward their cognate receptor, potentially hampering killing capacities of immunoligands. Herein, we provide protocols for yeast surface display-based affinity maturation of B7-H6, the natural ligand of NK cell-activating receptor NKp30.

Non-fitness status of peripheral NK cells defined by decreased NKp30 and perforin, and increased soluble B7H6, in cervical cancer patients.

The NKp30 receptor is one of the three natural cytotoxic receptors reported in NK cells. This receptor is codified by the NCR3 gene, which encodes three isoforms, a consequence of the alternative splicing of exon 4. A greater expression of the three isoforms (A, B, and C), along with low levels of the NKp30 ligand B7H6, has been reported as a positive prognostic factor in different cancer types. Here, in patients with cervical cancer and precursor lesions, we report an altered immune-phenotype, characterized by non-fitness markers, that correlated with increased disease stage, from CIN 1 to FIGO IV. While overall NK cell numbers increased, loss of NKp30+ NK cells, especially in the CD56dim subpopulation, was found. Perforin levels were decreased in these cells. Decreased expression of the NKp30 C isoform and overexpression of soluble B7H6 was found in cervical cancer patients when compared against healthy subjects. PBMCs from healthy subjects downregulated NKp30 isoforms after co-culture with B7H6-expressing tumour cells. Taken together, these findings describe a unique down-modulation or non-fitness status of the immune response in cervical cancer, the understanding of which will be important for the design of novel immunotherapies against this disease.

Cardiovascular adverse events in chronic myeloid leukemia patients treated with nilotinib or imatinib: A systematic review, meta-analysis and integrative bioinformatics analysis

The aim of this article is to assess the risk and potential mechanisms of cardiovascular adverse events in patients treated with nilotinib or imatinib by conducting a systematic review, meta-analysis and integrative bioinformatics analysis. Three databases were systematically searched for studies published from inception to May 29, 2022. Differential expression analysis and weighted gene coexpression network analysis (WGCNA) were performed to search for modules of genes most associated with cardiotoxicity. Protein-protein interaction (PPI) network analysis was then performed to identify hub genes for the cardiotoxicity of nilotinib. Molecular docking was used to analyze the effects of rosuvastatin and aspirin on these targets. Patients treated with nilotinib as first-line treatment were associated with a higher risk of CAE (OR = 3.43 [95% CI 2.77-4.25]), CAD (OR = 5.30 [95% CI 3.85-7.29]), ACS (OR 2.7 [95% CI 1.60-4.54]), CVA (OR 5.76 [95% CI 2.84-11.28]), PAOD (OR 5.57 [95% CI 3.26-9.50]) and arrhythmia (OR 2.34 [1.17,4.67]) than those treated with imatinib, while no significant difference was found in the risk of HF (OR 1.40 [95% CI 0.42-4.69]) between the two groups. Patients who were treated with more than 600 mg daily dosage of nilotinib or followed up for more than 5 years had a higher risk of ACS and CVA. IL6, CXCL8, CCL2, SOD2, NFKBIA, and BIRC3 were identified as the top 6 hub genes in the magenta module (human cardiomyocyte samples) and were mainly enriched in the NOD-like receptor signaling pathway, IL-17 signaling pathway, TNF signaling pathway, lipid and atherosclerosis signaling pathway. TYROBP and CSF1R were identified as hub genes in the turquoise module (liver samples from Mus musculus). GSEA results showed that type II diabetes mellitus, B-cell receptor, apoptosis, insulin, natural killer cell mediated cytotoxicity,mTOR, chemokine, and T-cell receptor signaling pathways were related to the higher risk of atherosclerosis caused by nilotinib. Rosuvastatin can effectively bind to most of the hub targets and proteins enriched in the inflammatory pathways above. CML patients who start with nilotinib have a higher risk of CAE than those with imatinib. Atherosclerosis caused by the inflammatory response and glycolipid metabolism disorder is the key mechanism of nilotinib cardiotoxicity. Rosuvastatin may be an effective treatment for the cardiotoxicity of nilotinib.

Korean Red Ginseng modulates immune function by upregulating CD4+CD8+ T cells and NK cell activities on porcine

In the present study, we investigated whether treatment with KRG improve the parameters of immune activity such as the cytotoxicity, populations of CD4+ CD8+T cell, CD3−CD172−CD8+ NK cell and CD172+ monocyte as well as natural cytotoxicity receptors such as Nkp46, Nkp44, Nkp30. In results, KRG significantly increased these immune activities. These results indicate that KRG has distinct immune-enhancing effects by increasing the roles of T cells and NK cell in porcine.

Critical roles of Parkin and PINK1 in antiviral immunity and protection from cell death in Enterovirus-induced viral myocarditis

Abstract Background Myocarditis is an inflammatory disease of the heart muscle often resulting in cardiac dysfunction and death. One of the most common causes is a cardiac infection with viruses, especially with enteroviruses of the Coxsackievirus B (CVB) family. CVB3-induced viral myocarditis is a well-established model to elucidate viral myocarditis in mice. It is assumed that mitochondrial damage control and mitochondria-associated innate immunity is essential for defence against viral infection of the heart muscle. And Parkinson's disease genes Parkin and PINK1 are well known for their critical function in mitochondrial homeostasis and damage control. We investigated the roles of Parkin and PINK1 in CVB3-induced viral myocarditis mouse model. Material and methods Male C57BL/6J WT, Parkin KO and PINK1 KO mice were used in an age of 8 to 12 weeks. The animals were injected intraperitoneally with 1x104 plaque forming units of CVB3 diluted in phosphate-buffered saline. Control and survived virus-infected mice were sacrificed for tissue sampling at day 7 to investigate imaging and molecular workup. Result After virus inoculation, the Parkin KO and PINK1 KO groups showed a significantly lower 28-day survival rate compared with the WT group (p&amp;lt;0.001 in Parkin KO; p&amp;lt;0.001 in PINK1 KO). Evans-Blue Dye (EBD) staining image of day 7 after virus-infection showed that the hearts of Parkin KO and PINK1 KO mice were more severely damaged than the hearts of WT mice (3.2-fold, p&amp;lt;0.01 in Parkin KO; 2.6-fold, p&amp;lt;0.05 in PINK1 KO). H&amp;E staining showed that severe inflammatory reaction occurred in the hearts of WT mice, although the inflammatory reaction was much lower compared to WT in the hearts of Parkin KO and PINK1 KO mice (p&amp;lt;0.001 in Parkin KO; p&amp;lt;0.001 in PINK1 KO). In immunohistochemistry, EBD staining positive area colocalized well with CVB3 in Parkin KO and PINK1 KO mice hearts, although EBD staining positive area was rare and didn't colocalized well CVB3 in WT mice hearts. mRNA levels of cell markers (CD3, natural cytotoxicity receptors, F4/80), inflammatory cytokines (IL-1β, IL-6, TNF-α), type II interferon, macrophage-derived protective factors, and CXCL10 family cytokines were lower in virus-infected Parkin/PINK1 KO mice than WT mice. Western blots showed that phospho-p65 increased less than WT in the heart of Parkin KO (0.7-fold, p&amp;lt;0.05) and PINK1 KO mice (0.58-fold, p&amp;lt;0.05). Conclusion The Parkin KO and PINK1 KO mice showed a higher mortality rate and more severe cardiomyocyte damage than the WT mice in the viral myocarditis mouse model. Parkin and PINK1 is critical to the type II IFN pathway that inhibits virus replication. And Parkin and PINK1 play an important role in the recruitment of IFN-γ-producing lymphocytes through NF-κB pathway and CXCL10 family chemokines. Funding Acknowledgement Type of funding sources: None.

The natural cytotoxicity receptor genes in the family Felidae.

Natural killer (NK) cells belong to the innate immune system. The germline-encoded natural killer cell receptors represent activating and inhibitory receptors regulating multiple NK cell activities. The natural cytotoxicity receptors (NCRs) are activating natural cytotoxicity triggering receptors 1, 2, and 3 (NKp46, NKp44, and NKp30), encoded by the genes NCR1, NCR2, and NCR3, respectively. NCRs may be expressed in different cell types engaged in mechanisms of innate and adaptive immunity. The family Felidae, comprising the domestic cat and a wide variety of free-ranging species represents a well-suited model for biomedical and evolutionary studies. We characterized the NCR1, NCR2, and NCR3 genes in a panel of felid species. We confirmed the presence of potentially functional genes NCR1, NCR2, and NCR3 in all species. All three genes are conserved within the family and are similar to other phylogenetically related mammalian families. The NCR1 and NCR2 phylogenetic trees based on both nucleotide and protein sequences corresponded to the current zoological taxonomy, with some exceptions suggesting effects of different selection pressures in some species. Highly conserved NCR3 sequences did not allow a robust phylogenetic analysis. Most interspecific differences both at the nucleotide and protein level were found in NCR2. Within species, the most polymorphic CDS was detected in NCR1. Selection analyses indicated the effects of purifying selection on individual amino acid sites in all three genes. In stray cats, a rather high intraspecific diversity was observed.