Natural Killer Cell Responses Research Articles

The prognostic impact of NKA dynamics in first-line treatment of metastatic colorectal cancer.

e15514 Background: Metastatic colorectal cancer (mCRC) remains a therapeutic challenge and new markers hold high priority. Natural Killer (NK) cells are immune cells with potent antitumor properties. The association between neoplastic development and level of effector functions of this cell type is well known. Moreover, it is recognized that NK cells are subject to tumor-mediated immune suppression. The present study aimed at elucidating a potential relationship between NK cell activity (NKA) and response to folinic acid, 5-fluorouracil, oxaliplatin, and irinotecan (FOLFOXIRI) in mCRC. Methods: NKA was analyzed in patients enrolled in a randomized trial of mCRC receiving FOLFOXIRI and either tocotrienol or placebo as first-line treatment. Blood (1 mL) was sampled into NK Vue tubes at baseline and before treatment cycles 2-3, placed at 37°C for 24 hours, and after incubation, the level of interferon-γ (IFNγ) in the plasma was a surrogate measure for NKA. A cutoff of 250 pg/mL IFNγ distinguished normal and low NKA. The endpoints were response rate (RR), progression-free survival (PFS), and overall survival (OS). Results: A total of 70 patients were enrolled in the randomized clinical trial. The results showed that tocotrienol had no effect on either RR (p = 0.62), PFS (p = 0.52), or OS (p = 0.80). Patients were classified into two groups according to the dynamics of NKA measured at the available time points. Patients that dropped from a normal to a low level of NKA in samples taken between baseline and before cycle 3 and patients with continuous low NKA (IFNγ < 250 pg/mL) were categorized as NKA-low. The NKA-high group included patients that increased from an abnormal to a normal level of NKA and patients that remained within the normal range (IFNγ > 250pg/mL) in all available samples. The RR in the two groups were 38% (8/21) and 69% (24/35), respectively (p = 0.0496). The median PFS was 186 days (95% confidence interval (CI) 136-225 days, n = 23) for the NKA-low group and 252 days (95% CI 223-305 days, n = 37) for NKA-high (p = 0.0005). The hazard ratio (HR) was 2.65 (95% CI 1.28-5.50). After adjusting for age, performance status (PS), and tumor location, NKA remained an independent prognostic factor for PFS (HR = 3.32, 95% CI 1.67-6.61, p = 0.001). Median OS was 408 days (95% CI 219-679 days, n = 23) and 1051 days (95% CI 645-1225 days, n = 37), respectively (p = 0.0034). For OS, the NKA-low group also showed a poor prognosis, HR 2.23 (95% CI 1.17-4.25). NKA dynamics remained an independent prognostic marker for OS after adjusting for age, PS, and tumor location (HR = 2.74, 95% CI 1.50-5.03, p = 0.001). Conclusions: The results suggest that NKA dynamics within the first two cycles of treatment may hold relevant prognostic information in first-line treatment of mCRC. The possible causative relationship between NKA and treatment effect needs further elucidation, but calls for experimental trials combining chemotherapy with NK cell therapy in mCRC.

Elucidating the role played by the NKG2 family of receptors in natural killer cell responses to JC polyomavirus

Abstract JC polyomavirus (JCPyV) causes progressive multifocal leukoencephalopathy (PML), a potentially fatal complication of severe immune suppression due to monoclonal antibody treatments for cancer, autoimmune diseases and transplantation, or due to uncontrolled HIV infection. There is currently no effective treatment against PML and novel immunotherapies are urgently needed. Natural killer (NK) cells play critical roles in defense against viral infections, yet NK cell responses to JCPyV remain largely unexplored. Here, we first compared NK and T cell responses against JCPyV VP1 peptide pools by intracellular cytokine staining. In about 40% of healthy donors, we detected robust CD107a upregulation and IFNγ production by NK cells, extending beyond T cell responses. Next, using a novel flow cytometry-based killing assay, we showed that co-culture of NK cells and JCPyV-infected SVG-A cells led to a 65% reduction in infection, on average. To identify receptor-ligand interactions modulating NK cell-mediated responses to JCPyV, we performed co-cultures in the presence of select blocking antibodies. We observed enhanced expression of ligands for the activating NKG2D on infected cells and accordingly, NKG2D blockade resulted in decreased NK cell degranulation. In contrast, blockade of inhibitory NKG2A enhanced NK cell response to JCPyV-infected targets. Moreover, we identified JCPyV-derived peptides that elicit dominant NK cell responses and can stabilize HLA-E, the ligand for NKG2A and the activating NKG2C. Altogether, these findings suggest that modulation of NKG2D and NKG2A/C signaling may play key roles in NK cell-mediated control of JCPyV and may be promising immunotherapeutic targets to boost NK cell anti-JCPyV activity. Supported by grant from NIH (R01 NS116278)

The CD16 and CD32b Fc-gamma receptors regulate antibody-mediated responses in mouse natural killer cells

Abstract Natural killer (NK) cells are innate lymphocytes capable of mediating immune responses without prior sensitization. NK cells express Fc-gamma receptors (FcγR) that engage the Fc region of IgG. Studies investigating the role of FcγR on mouse NK cells have been limited due to lack specific reagents. In this study, we characterize the expression and biological consequences of activating mouse NK cells through their FcγR. We demonstrate that most NK cells express the activating CD16 receptor, and a subset of NK cells also express the inhibitory CD32b receptor. Critically, these FcγRs are functional on mouse NK cells and can modulate antibody-mediated responses. We also characterized mice with conditional knock-out alleles of Fcgr3 (CD16) or Fcgr2b (CD32b) in the NK and innate lymphoid cell (ILC) lineage. NK cells in these mice did not reveal any developmental defects and were responsive to cross-linking activating NK receptors, cytokine stimulation, and killing of YAC-1 targets. Importantly, CD16-deficient NK cells failed to induce antibody-directed cellular cytotoxicity (ADCC) of antibody-coated B cell lymphomas in in vitro assays. In addition, we demonstrate the important role of CD16 on NK cells using an in vivo model of cancer immunotherapy using anti-CD20 antibody treatment of B cell lymphomas. This project was funded by fellowships from Burroughs Wellcome Fund (BWF) and the Cancer Research Institute to O.A.A., and grants from the Parker Institute for Cancer Immunotherapy (PICI), and the NIH (AI068129 and AI146581) to L.L.L.

Association between human genetic variants affecting the host NK cell response and the development of herpes simplex virus type 1 encephalitis.

Herpes simplex virus encephalitis (HSE) is a rare complication of herpes simplex virus type 1 (HSV-1) infection or reactivation. It is so far unclear why only few patients develop HSE. As natural killer (NK) cells provide an important defense against HSV-1, we investigated whether there is an association between distinct human genetic variants associated with the host NK cell response and HSE. Forty-nine adult patients with confirmed HSE and 247 matched controls were analyzed for the distribution of the following genotypes: CD16A (FcγRIIIA) V/F and IGHG1 G1m3/17, both influencing antibody-dependent cellular cytotoxicity; HLA-E*0101/*0103, associated with NK cell activation; and SLFN13 rs9916629C/T associated with NK cell response. Homozygous HLA-E*0101:0101 and HLA-E*0103:0103 variants as well as the rs9916629CC genotype were overrepresented in HSE patients compared to controls (p ≤ 0.001). Notably, cooccurrence of the homozygous HLA-E*0101 and rs9916629CC genotypes was present in 19% of patients but totally absent in controls (p ≤ 0.0001). Distribution of CD16A and IGHG1 variants did not differ between patients and controls. Our data show that the rare combination of HLA-E*0101:0101 and rs9916629CC is significantly associated with HSE. Possibly, these genetic variations could be useful as clinical markers predicting HSE prognosis and helping to adapt the treatment of HSE in the individual patient.

Humanized mouse models with endogenously developed human natural killer cells for in vivo immunogenicity testing of HLA class I-edited iPSC-derived cells

Human induced pluripotent stem cells (hiPSCs) genetically depleted of human leucocyte antigen (HLA) class I expression can bypass T cell alloimmunity and thus serve as a one-for-all source for cell therapies. However, these same therapies may elicit rejection by natural killer (NK) cells, since HLA class I molecules serve as inhibitory ligands of NK cells. Here, we focused on testing the capacity of endogenously developed human NK cells in humanized mice (hu-mice) using MTSRG and NSG-SGM3 strains to assay the tolerance of HLA-edited iPSC-derived cells. High NK cell reconstitution was achieved with the engraftment of cord blood-derived human hematopoietic stem cells (hHSCs) followed by the administration of human interleukin-15 (hIL-15) and IL-15 receptor alpha (hIL-15Rα). Such “hu-NK mice” rejected HLA class I-null hiPSC-derived hematopoietic progenitor cells (HPCs), megakaryocytes and T cells, but not HLA-A/B-knockout, HLA-C expressing HPCs. To our knowledge, this study is the first to recapitulate the potent endogenous NK cell response to non-tumor HLA class I-downregulated cells in vivo. Our hu-NK mouse models are suitable for the non-clinical evaluation of HLA-edited cells and will contribute to the development of universal off-the-shelf regenerative medicine.

HIV-Differentiated Metabolite N-Acetyl-L-Alanine Dysregulates Human Natural Killer Cell Responses to Mycobacterium tuberculosis Infection

Mycobacterium tuberculosis (Mtb) has latently infected over two billion people worldwide (LTBI) and caused ~1.6 million deaths in 2021. Human immunodeficiency virus (HIV) co-infection with Mtb will affect the Mtb progression and increase the risk of developing active tuberculosis by 10-20 times compared with HIV- LTBI+ patients. It is crucial to understand how HIV can dysregulate immune responses in LTBI+ individuals. Plasma samples collected from healthy and HIV-infected individuals were investigated using liquid chromatography-mass spectrometry (LC-MS), and the metabolic data were analyzed using the online platform Metabo-Analyst. ELISA, surface and intracellular staining, flow cytometry, and quantitative reverse-transcription PCR (qRT-PCR) were performed using standard procedures to determine the surface markers, cytokines, and other signaling molecule expressions. Seahorse extra-cellular flux assays were used to measure mitochondrial oxidative phosphorylation and glycolysis. Six metabolites were significantly less abundant, and two were significantly higher in abundance in HIV+ individuals compared with healthy donors. One of the HIV-upregulated metabolites, N-acetyl-L-alanine (ALA), inhibits pro-inflammatory cytokine IFN-γ production by the NK cells of LTBI+ individuals. ALA inhibits the glycolysis of LTBI+ individuals' NK cells in response to Mtb. Our findings demonstrate that HIV infection enhances plasma ALA levels to inhibit NK-cell-mediated immune responses to Mtb infection, offering a new understanding of the HIV-Mtb interaction and providing insights into the implication of nutrition intervention and therapy for HIV-Mtb co-infected patients.

Abstract 2908: TIGIT knockout enhances anti-tumor response of Natural Killer cells and prevents fratricide when combined with therapeutic ADCC-competent TIGIT antibodies

Abstract In this study we propose knockout of the inhibitory receptor TIGIT on Natural Killer (NK) cells enhances their anti-tumor response and prevents NK cell fratricide when combined with ADCC-competent antibodies targeting TIGIT. T cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) is a major inhibitory receptor of both T cells and NK cells and is an emerging target for immune checkpoint blockade for the treatment of cancer. Although preclinical studies using combinations of anti-TIGIT and anti-PD-(L)1 showed promising results, the phase III SKYSCRAPER-01 trial using the combination of tiragolumab and atezolizumab for NSCLC did not meet its co-primary endpoint of progression free survival (PFS) while the overall survival (OS) at the time of this publication was immature with the study still ongoing. There is critical need to further understand the mechanism of function of TIGIT on immune cells to support its clinical use. Most of the current understanding of TIGIT regulation of immune cell function has been based on T cells, despite comparable or higher TIGIT expression on NK cells. Recent murine studies have shown the therapeutic efficacy of PD-1 and TIGIT blockade depended on the presence of NK cells, indicating they are an important immune population to consider in the efficacy of TIGIT blockade treatments. Furthermore, the majority of TIGIT antibodies have a humanized IgG, which binds to Fc receptors (CD16) and induces antibody-dependent cellular cytotoxicity (ADCC), a major contributor to their efficacy. Since both CD16 and TIGIT are highly expressed on activated NK cells, there is a potential for ADCC-driven fratricide of TIGIT+ NK cells with ADCC-competent anti-TIGIT. In this study, TIGIT knockout was performed in ex vivo-expanded primary human NK cells and anti-tumor response measured. Compared to WT NK cells, TIGIT knockout NK cells had improved in vitro killing of 3D lung cancer spheroids and increased ADCC when combined with cetuximab. TIGIT KO NK cells had upregulated mTORC1 signaling, increased basal glycolytic rate, and increased degranulation. Moreover, fratricide of WT NK cells was observed in the presence of ADCC-competent anti-TIGIT and could be ameliorated by TIGIT knockout. Altogether, this study demonstrated that the highly cytotoxic fratricide resistant TIGIT knockout NK cells have translation potential alone or in combination with ADCC-competent anti-TIGIT antibodies to enhance the efficacy of anti-TIGIT therapeutics. Citation Format: Md Faqrul Hasan, Tayler J. Croom-Perez, Jeremiah L. Oyer, Liza D. Robles-Carrillo, Thomas A. Dieffenthaller, Alicja J. Copik. TIGIT knockout enhances anti-tumor response of Natural Killer cells and prevents fratricide when combined with therapeutic ADCC-competent TIGIT antibodies [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 2908.

Abstract 893: Memory like differentiation enhances in vitro and in vivo NK cell responses against colorectal cancer

Abstract Metastatic (m) colorectal cancer (CRC) is an incurable, frequently lethal disease present in approximately 25% of newly diagnosed CRC patients, and almost 50% of the patients with CRC will develop metastatic disease. For mCRC, while systemic therapies (cytotoxic therapy, targeted therapy, immunotherapy or their combinations) have extended patient’s life expectancy, not all patients are candidates for these treatments. Immunotherapy has achieved significant curative effects in patients with solid tumors; however, a considerable proportion of mCRC patients (~90%) are not responsive to immune checkpoint blockade (ICB) leaving a gap in the CRC immunotherapy options. Natural killer (NK) cells are cytotoxic innate lymphoid cells that display potent effector responses against a wide variety of tumor cells; however, they are frequently dysfunctional in cancer patients. NK cells from CRC patients exhibit decreased expression of activating receptors and reduced cytokine production after stimulation with CRC cells with a more accentuated phenotype in advanced stages of the disease. Memory-like (ML) NK cells differentiated after IL-12, IL-15, and IL-18 activation have been shown to overcome limitations associated with deficient tumor recognition and poor anti-tumor activity. In clinical trials, ML NK cells were safe and active against acute myeloid leukemia (Romee R et al, Sci Transl Med, 2016). Preclinically, ML NK cells exhibited improved responses against melanoma (Marin ND et al, Clin Cancer Res, 2021) and ovarian cancers, compared to conventional NK cells. Here, we hypothesized that memory-like differentiation will enhance multiple aspects of the NK cell response against CRC cells. Allogeneic ML NK cells displayed enhanced IFN-γ production against four CRC cell lines DLD-1 (p=0.015), SW480 (p=0.0005), HT-29 (p=0.0005) and HCT116 (p=0.001), as well as primary patient derived CRC tumoroids (p=0.0156), compared to conventional (c) NK cells. ML NK cells also exhibited superior and sustained killing of CRC cell lines over time compared to cNK cells, as measured by Incucyte assays and using CRC tumoroids. Furthermore, IFN-γ production was significantly reduced after blockade of NKG2D, DNAM-1 and NKp46 (p<0.01) revealing mechanistic insights into how ML NK cells recognize CRC targets. Finally, using a xenograft model of CRC in NSG mice, we demonstrated that ML NK cells exhibited superior control of Luciferase expressing HCT116 cells compared to cNK cells (p=0.01) as measured by bioluminescent imaging (BLI). Collectively, these findings demonstrate that ML NK cells exhibit enhanced responses against CRC cells, and thus warrants further investigation in clinical trials for CRC patients, especially those who are not candidates for standard of care therapy or failed ICB. Citation Format: Nancy D. Marin, Michelle Becker-Hapak, Quazim A. Alayo, Melissa Berrien-Elliot, Lynne Marsala, Naomi Sonnek, Miriam T. Jacobs, Jennifer A. Foltz, Alice Zhou, Jennifer Tran, Pamela Wong, Celia Cubit, Kimberly Hwang, Timothy Schappe, Ryan C. Fields, Matthew A. Ciorba, Todd A. Fehniger. Memory like differentiation enhances in vitro and in vivo NK cell responses against colorectal cancer [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 893.

Abstract 2906: Memory-like differentiation, tumor targeting monoclonal antibodies, and chimeric antigen receptors enhance natural killer cell responses to head and neck cancer

Abstract Background: Head and neck cancer (HNC) is an aggressive tumor with low response rates to frontline PD-1 blockade. Natural killer (NK) cells are a promising cellular therapy for T cell-refractory cancers, but are frequently dysfunctional in patients with HNC. New strategies are needed to enhance NK cell responses against HNC. Activation of NK cells with IL-12, 15 and 18 results in memory-like (ML) differentiation improving NK cell responses in hematological malignancies. We hypothesized that ML NK cell differentiation, tumor targeting with cetuximab, and engineering with α-ephA2 chimeric antigen receptor (CAR) would enhance NK cell responses against HNC. Methods: ML NK and conventional (c) NK cells were generated from healthy donors (HD) and evaluated for their ability to produce IFNγ, TNF, degranulate (CD107a) and kill HNC cell lines and primary HNC cells alone or in combination with cetuximab in vitro and in xenograft models. To identify activating receptors involved in NK cell recognition of HNC, blocking experiments were used. ML and cNK cells were engineered to express α-EphA2 CAR-CD8A-41BB-CD3z and functional responses were assessed in vitro against HNC cell lines and primary HNC targets. Results: ML NK cells generated from HD displayed enhanced IFNγ response compared to cNK cells against HNC cell lines SCC1(11±2% vs. 24±1%, mean ± SEM p<0.001), SCC47 (13±2% vs. 36±2%, p<0.01) and primary HNC targets (17±2% vs. 32±3%, p<0.01) which were further enhanced by cetuximab, SCC1 (cNK + cetuximab vs. ML + cetuximab 16±2% vs. 28±3%, p<0.001), SCC47 (39±3% vs. 56±2%, p<0.001), primary HNC targets (23±2% vs. 40±2%, p<0.0001). The combination of ML NK cells and cetuximab was superior to cNK and cetuximab in 51 Cr release assay and IncuCyte® killing assays for HNC cell lines (p<0.01) and primary cells (p<0.01). Findings were consistent in SCC1 HNC xenograft model, where tumor bearing NSG mice treated with cetuximab and ML NK cells had significantly lower tumor burden at 21 days compared to untreated mice, mice that received cNK cells + cetuximab or cetuximab only (p<0.001). Mechanistically, NK cell IFNγ, CD107a and cytotoxicity against HNC targets is partially dependent on activating receptors NKG2D, CD2 and DNAM-1. ML NK cells expressing α-ephA2 CAR were specific to ephA2+ cell lines and demonstrated increased IFNγ (37±4% vs. 16±3%, p<0.001) and CD107a (36±4% vs. 20±1%, p<0.001) against NK resistant ephA2+ UD-SCC2, compared to CAR negative cells. EphA2 CAR ML NK cells had increased CD107a (26±4% vs. 39±4%, p<0.01) and IFNγ (45±5% vs. 66±3%, p<0.001) against primary ephA2+ HNC targets. Conclusion: These pre-clinical findings show that ML differentiation alone or in concert with cetuximab directed targeting or ephA2 CAR engineering, was effective against HNC and provide the rationale for investigation in early phase clinical trials for HNC patients. Citation Format: Miriam T. Jacobs, Pamela Wong, Alice Zhou, Michelle Becker-Hapak, Nancy Marin, Lynne Marsala, Mark Foster, Jennifer Foltz, Celia Cubitt, Jennifer Tran, Carly Neal, David Russler-Germain, Lily Chang, Timothy Schappe, Ethan McClain, Samantha Kersting-Schadek, Carl DeSelm, Melissa Berrien-Elliott, Sidarth V. Puram, Todd A. Fehniger. Memory-like differentiation, tumor targeting monoclonal antibodies, and chimeric antigen receptors enhance natural killer cell responses to head and neck cancer [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 2906.

Abstract 667: Single-cell functional genomics of natural killer cell evasion by tumor cells

Abstract Background: Natural killer (NK) cells are emerging as a promising therapeutic option in cancer. To better understand how cancer cells evade NK cells, we studied interacting NK and blood cancer cells using single-cell and genome-scale functional genomics screens. Methods: We performed multiplexed single-cell RNA-seq (scRNA-seq) on co-cultures of NK cells and 26 cell lines representing diverse blood cancers. Using screens of pooled DNA-barcoded cell lines (PRISM), we quantified the sensitivity of over 60 blood cancer cell lines and integrated the results with CCLE multi-omics to uncover molecular correlates of tumor cell susceptibility to NK cells. We performed 12 genome-scale CRISPR loss-of-function and gain-of-function screens of cancer-cell intrinsic NK cell resistance mechanisms across 7 blood cancer cell lines. Finally, we investigated the mechanisms-of-action of 65 genome-scale screen hits using CRISPR screens with scRNA-seq readout in both tumor and NK cells. Results: At single-cell resolution, interaction of NK and cancer cells induced distinct activation states in both cell types depending on the cancer cell lineage and molecular phenotype. NK cells transitioned either into an activated state characterized by 4-1BB, GITR, TIM-3, and TIGIT or a state marked by type I interferon signature. Tumor cells responded to NK cell attack by activating interferon gamma (IFNy) signaling, inducing MHC class I. The activation states correlated with sensitivity to NK cells, ranging from more sensitive myeloid to more resistant B-lymphoid cancers. Molecular correlates of increased sensitivity included expression of activating receptor ligands NCR3LG1, PVR, and ULBP1, whereas TRAF3 mutations correlated with resistance. CRISPR screens uncovered cancer cell-intrinsic genes driving sensitivity and resistance, including antigen presentation and death receptor signaling mediators and adhesion molecules. The screens identified new ​​blood cancer-specific NK cell inhibitory regulators (SELPLG, SPN, and MYB ) and genes previously underappreciated in NK cell evasion, including protein fucosylation and transcriptional regulators (e.g. GFI1B). CRISPR screens with scRNA-seq readout identified MHC-I, IFNy, and NF-κB regulation as underlying mechanisms. Cancer cell knockout of positive regulators of NK cell response (CD58, NCR3LG1) induced an inactive NK cell state, providing experimental evidence how cancer cell-intrinsic genetic alterations can shape the molecular profile of attacking immune cells to promote immune evasion. Conclusions: By integrating diverse functional genomics screens and patient genomic profiles, we provide a comprehensive landscape of potential biomarkers and functionally validated genetic mechanisms which influence how NK cells recognize and kill malignant cells. The results offer a roadmap to facilitate development of NK-cell based immunotherapy for blood cancers and beyond. Citation Format: Olli Dufva, Sara Gandolfi, Jani Huuhtanen, Olga Dashevsky, Khalid Saeed, Jay Klievink, Petra Nygren, Jonas Bouhlal, Jenni Lahtela, Anna Näätänen, Bishwa Ghimire, Tiina Hannunen, Pekka Ellonen, Hanna Duàn, Jason Theodoropoulos, Essi Laajala, Jouni Härkönen, Petri Pölönen, Merja Heinäniemi, Shizuka Yamano, Ryosuke Shirasaki, David Barbie, Jennifer Roth, Rizwan Romee, Michal Sheffer, Harri Lähdesmäki, Dean A. Lee, Ricardo De Matos Simoes, Matti Kankainen, Constantine S. Mitsiades, Satu Mustjoki. Single-cell functional genomics of natural killer cell evasion by tumor cells [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 667.

Abstract 2897: Overcoming barriers to the natural killer cell response against multiple myeloma: Manipulating the NKG2A/HLA-E checkpoint and CAR engineering of memory-like natural killer cells

Abstract Recent advances in treatment options for multiple myeloma (MM) have improved patient outcomes, however, MM remains incurable and options are limited for relapsed/refractory disease. Memory-like (ML) differentiation of NK cells using brief IL-12, IL-15, and IL-18 activation generates a safe allogenic NK cellular therapy for acute myeloid leukemia that induce complete remissions. Here, we investigate pre-clinical combinations with ML NK cells for MM, including blockade of the inhibitory NKG2A/HLA-E checkpoint as well as MM-specific targeting via myeloma-targeting monoclonal antibody (mAb) and chimeric antigen receptor (CAR) engineering. Human ML NK cells generated from healthy donors display IFN-γ responses similar to conventional (cNK) cells, and only modestly increased cytotoxicity against myeloma cell lines (MM1.s and OPM2). MM cell lines and primary MM cells express high levels of HLA-E, a major negative checkpoint for ML NK cells that binds to the inhibitory receptor NKG2A expressed on NK cells. Indeed, mass cytometry analysis of MM patients compared to healthy controls demonstrated enrichment of a NK cell population expressing high levels of NKG2A, but also high levels of activation and cytotoxic markers. Disruption of HLA-E binding to NKG2A with the blocking mAb monalizumab (AstraZeneca) significantly increased healthy donor ML NK cell IFN-γ response (P=0.016), degranulation (surface CD107a, P=0.017), and killing (P=0.009) of MM1.s when compared to isotype mAb. NK cells isolated from patients with MM showed similarly enhanced functionality after ML differentiation and NKG2A blockade (IFN-γ P=0.008). In an NSG mouse model of MM using MM1.s cells, healthy donor ML NK cells combined with monalizumab showed improved control of MM tumor growth compared to ML NK cells without monalizumab (P<0.001), conventional NK cells, or control tumors alone (P<0.001). To further improve recognition of MM, ML NK cells responses were assessed in combinationwith a mAb against SLAMF7 (elotuzumab) that both targets MM cells and activates NK cells. Elotuzumab treatment increased the functionality of ML NK cells against MM cell lines (IFN-γ, P=0.004) with an additive effect of both elotuzumab and monalizumab (IFN-γ, P=0.003). In an alternative strategy to enhance MM targeting, we engineered an anti-BCMA CAR (anti-BCMA/4-1bb/z) into ML NK cells. BCMA-CAR+ NK cells exhibited increased IFN-γ (P=0.001) production and degranulation (P<0.001) against MM1.s compared to non-transduced control NK cells. These functional responses were additionally enhanced with ML differentiation. Collectively, these findings suggest that ML NK cell differentiation, combined with strategies to address inhibitory checkpoints (NKG2A) or MM-specific activation (elotuzumab, anti-BMCA CAR), are promising cellular therapy strategies for MM patients. Citation Format: Alice Y. Zhou, Melissa M. Berrien-Elliott, Ravi Vij, Mark Fiala, Michelle Becker-Hapak, Lynne Marsala, Miriam T. Jacobs, Nancy D. Marin, Jennifer Tran, Jennifer Foltz, Pamela Wong, Julie Fortier, Sarah Kelley, Carly Neal, David Russler-Germain, Timothy Schappe, Todd A. Fehniger. Overcoming barriers to the natural killer cell response against multiple myeloma: Manipulating the NKG2A/HLA-E checkpoint and CAR engineering of memory-like natural killer cells [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 2897.

Attenuated Cytokine-Induced Memory-Like Natural Killer Cell Responses to Mycobacterium tuberculosis in Tuberculosis Patients.

This study aimed to investigate the phenotype, proliferation and functional alterations of cytokine-induced memory-like natural killer (CIML NK) cells from healthy subjects and TB patients, and assessed the efficacy of CIML NK cells in response to H37Rv-infected U937 cells in vitro. Fresh peripheral blood mononuclear cells (PBMCs) were isolated from healthy people and tuberculosis patients and activated for 16h using low-dose IL-15, or IL-12, IL-15, IL-18 combination or IL-12, IL-15, IL-18 and MTB H37Rv lysates, respectively, followed by low-dose IL-15 maintenance for another 7 days. Then, the PBMCs were co-cultured with K562 and H37Rv-infected U937, and the purified NK cells were co-cultured with H37Rv infected U937. The phenotype, proliferation and response function of CIML NK cells were assessed using flow cytometry. Finally, colony forming units were enumerated to confirm the survival of intracellular MTB. CIML NK phenotypes from TB patients were similar to healthy controls. CIML NK cells undergo higher rates of proliferation after IL-12/15/18 pre-activation. Moreover, the poor expansion potential of CIML NK cells co-stimulated with MTB lysates. CIML NK cells from healthy individuals showed enhanced IFN-γ functional to H37Rv infected U937 cells, along with significantly enhanced killing of H37Rv. However, the CIML NK cells from TB patients show attenuated IFN-γ production and now enhanced the ability of killing intracellular MTB compared to those from healthy donors after co-cultured with H37Rv infected U937. CIML NK cells from healthy individuals exist the increased ability of IFN-γ secretion and boosted anti-MTB activity in vitro, which from TB patients show impaired IFN-γ production and no enhanced anti-MTB activity compared to those from healthy donors. Additionally, we observe the poor expansion potential of CIML NK cells co-stimulated with antigens from MTB. These results open up new possibilities for NK cell-based anti-tuberculosis immunotherapeutic strategies.

HIV post-treatment controllers have distinct immunological and virological features

HIV post-treatment controllers (PTCs) are rare individuals who maintain low levels of viremia after stopping antiretroviral therapy (ART). Understanding the mechanisms of HIV post-treatment control will inform development of strategies aiming at achieving HIV functional cure. In this study, we evaluated 22 PTCs from 8 AIDS Clinical Trials Group (ACTG) analytical treatment interruption (ATI) studies who maintained viral loads ≤400 copies/mL for ≥24 wk. There were no significant differences in demographics or frequency of protective and susceptible human leukocyte antigen (HLA) alleles between PTCs and post-treatment noncontrollers (NCs, n = 37). Unlike NCs, PTCs demonstrated a stable HIV reservoir measured by cell-associated RNA (CA-RNA) and intact proviral DNA assay (IPDA) during analytical treatment interruption (ATI). Immunologically, PTCs demonstrated significantly lower CD4+ and CD8+ T cell activation, lower CD4+ T cell exhaustion, and more robust Gag-specific CD4+ T cell responses and natural killer (NK) cell responses. Sparse partial least squares discriminant analysis (sPLS-DA) identified a set of features enriched in PTCs, including a higher CD4+ T cell% and CD4+/CD8+ ratio, more functional NK cells, and a lower CD4+ T cell exhaustion level. These results provide insights into the key viral reservoir features and immunological profiles for HIV PTCs and have implications for future studies evaluating interventions to achieve an HIV functional cure.

The ion channel CALHM6 controls bacterial infection-induced cellular cross-talk at the immunological synapse.

Membrane ion channels of the calcium homeostasis modulator (CALHM) family promote cell-cell crosstalk at neuronal synapses via ATP release, where ATP acts as a neurotransmitter. CALHM6, the only CALHM highly expressed in immune cells, has been linked tothe induction of natural killer (NK) cell anti-tumour activity. However, its mechanism of action and broader functions in the immune system remain unclear. Here, we generated Calhm6-/- mice and report that CALHM6 is important for theregulation of the early innate control of Listeria monocytogenes infection in vivo. We find that CALHM6 is upregulated in macrophages by pathogen-derived signals and that it relocates from the intracellular compartment to the macrophage-NK cell synapse, facilitating ATP release and controlling the kinetics of NK cell activation. Anti-inflammatory cytokines terminate CALHM6 expression. CALHM6 forms an ion channel when expressed in the plasma membrane of Xenopus oocytes, where channel opening is controlled by a conserved acidic residue, E119. In mammalian cells, CALHM6 is localised to intracellular compartments. Our results contribute to theunderstanding of neurotransmitter-like signal exchange between immune cells that fine-tunes the timing of innate immune responses.

Newborn bacille Calmette-Guérin vaccination induces robust infant interferon-γ-expressing natural killer cell responses to mycobacteria

ObjectivesThe bacille Calmette-Guérin (BCG) vaccine is usually administered at birth to protect against severe forms of tuberculosis in children. BCG also confers some protection against other infections, possibly mediated by innate immune training. We investigated whether newborn BCG vaccination modulates myeloid and natural killer (NK) cell responses to mycobacteria. MethodsBCG vaccination was either administered at birth or delayed to 6 or 10 weeks of age in 130 South African infants. Whole blood was stimulated with BCG and clusters of differentiation (CD)4+ T, myeloid, and NK cell responses were measured by flow cytometry; the levels of secreted cytokines were measured by a multiplex bead array. ResultsNewborn BCG vaccination was associated with significantly higher frequencies of BCG-reactive, cytokine-expressing CD4+ T cells, and interferon (IFN)-γ-expressing NK cells than in unvaccinated infants but no differences in cytokine-expressing CD33+ myeloid cells were observed. The induction of BCG-reactive IFN-γ-expressing NK cells was not associated with the markers of NK cell maturation, differentiation, or cytokine receptor expression. BCG-reactive NK cell responses correlated directly with the levels of secreted interleukin (IL)-2 and IFN-γ and the innate pro-inflammatory cytokines IL-6, IL-1β, and tumor necrosis factor (TNF) in BCG-vaccinated infants only. ConclusionWe showed that BCG-reactive IFN-γ-expressing NK cells are strongly induced by BCG vaccination in infants and are likely amplified through bystander cytokines.

Transcriptional reprogramming of natural killer cells by vaccinia virus shows both distinct and conserved features with mCMV.

Natural killer (NK) cells have an established role in controlling poxvirus infection and there is a growing interest to exploit their capabilities in the context of poxvirus-based oncolytic therapy and vaccination. How NK cells respond to poxvirus-infected cells to become activated is not well established. To address this knowledge gap, we studied the NK cell response to vaccinia virus (VACV) in vivo, using a systemic infection murine model. We found broad alterations in NK cells transcriptional activity in VACV-infected mice, consistent with both direct target cell recognition and cytokine exposure. There were also alterations in the expression levels of specific NK surface receptors (NKRs), including the Ly49 family and SLAM receptors, as well as upregulation of memory-associated NK markers. Despite the latter observation, adoptive transfer of VACV-expercienced NK populations did not confer protection from infection. Comparison with the NK cell response to murine cytomegalovirus (MCMV) infection highlighted common features, but also distinct NK transcriptional programmes initiated by VACV. Finally, there was a clear overlap between the NK transcriptional response in humans vaccinated with an attenuated VACV, modified vaccinia Ankara (MVA), demonstrating conservation between the NK response in these different host species. Overall, this study provides new data about NK cell activation, function, and homeostasis during VACV infection, and may have implication for the design of VACV-based therapeutics.

A tri-specific killer engager against mesothelin targets NK cells towards lung cancer.

New treatments are required to enhance current therapies for lung cancer. Mesothelin is a surface protein overexpressed in non-small cell lung cancer (NSCLC) that shows promise as an immunotherapeutic target in phase I clinical trials. However, the immunosuppressive environment in NSCLC may limit efficacy of these therapies. We applied time-of-flight mass cytometry to examine the state of circulating mononuclear cells in fourteen patients undergoing treatment for unresectable lung cancer. Six patients had earlier stage NSCLC (I-IVA) and eight had highly advanced NSCLC (IVB). The advanced NSCLC patients relapsed with greater frequency than the earlier stage patients. Before treatment, patients with very advanced NSCLC had a greater proportion of CD14- myeloid cells than patients with earlier NSCLC. These patients also had fewer circulating natural killer (NK) cells bearing an Fc receptor, CD16, which is crucial to antibody-dependent cellular cytotoxicity. We designed a high affinity tri-specific killer engager (TriKE®) to enhance NK cytotoxicity against mesothelin+ targets in this environment. The TriKE consisted of CD16 and mesothelin binding elements linked together by IL-15. TriKE enhanced proliferation of lung cancer patient NK cells in vitro. Lung cancer lines are refractory to NK cell killing, but the TriKE enhanced cytotoxicity and cytokine production by patient NK cells when challenged with tumor. Importantly, TriKE triggered NK cell responses from patients at all stages of disease and treatment, suggesting TriKE can enhance current therapies. These pre-clinical studies suggest mesothelin-targeted TriKE has the potential to overcome the immunosuppressive environment of NSCLC to treat disease.

A cytometric survey of immune cell populations reveals an association between allergen-responsive natural killer (NK) cells and human peanut allergy

Food allergies are a leading cause of anaphylaxis, and allergen specific immune responses in both the innate and the adaptive immune system play key roles in its pathogenesis. NK cells can regulate allergic inflammation through cytokine secretion and cell-cell contact-mediated mechanisms in allergic rhinitis, asthma and atopic dermatitis. However, little is known about the response of NK cells in the setting of food allergy.

Natural killercells in COVID-19: from infection, to vaccination and therapy.

Natural killer (NK) cells are among the most important innate immunity members, which are the first cells that fight against infected cells. The function of these cells is impaired in patients with COVID-19 and they are not able to prevent the spread of the disease or destroy the infected cells. Few studies have evaluated the effects of COVID-19 vaccines on NK cells, though it has been demonstrated that DNA vaccines and BNT162b2 can affect NK cell response. In the present paper, the effects of SARS-CoV-2 on the NK cells during infection, the effect of vaccination on NK cells, and the NK cell-based therapies were reviewed.

Human Hematopoietic Stem Cell Engrafted IL-15 Transgenic NSG Mice Support Robust NK Cell Responses and Sustained HIV-1 Infection.

Mice reconstituted with human immune systems are instrumental in the investigation of HIV-1 pathogenesis and therapeutics. Natural killer (NK) cells have long been recognized as a key mediator of innate anti-HIV responses. However, established humanized mouse models do not support robust human NK cell development from engrafted human hematopoietic stem cells (HSCs). A major obstacle to human NK cell reconstitution is the lack of human interleukin-15 (IL-15) signaling, as murine IL-15 is a poor stimulator of the human IL-15 receptor. Here, we demonstrate that immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice expressing a transgene encoding human IL-15 (NSG-Tg(IL-15)) have physiological levels of human IL-15 and support long-term engraftment of human NK cells when transplanted with human umbilical-cord-blood-derived HSCs. These Hu-NSG-Tg(IL-15) mice demonstrate robust and long-term reconstitution with human immune cells, but do not develop graft-versus-host disease (GVHD), allowing for long-term studies of human NK cells. Finally, we show that these HSC engrafted mice can sustain HIV-1 infection, resulting in human NK cell responses in HIV-infected mice. We conclude that Hu-NSG-Tg(IL-15) mice are a robust novel model to study NK cell responses to HIV-1.