Wild-type Natural Killer Cells Research Articles

Leucine zipper-based SAIM imaging identifies therapeutic agents to disrupt the cancer cell glycocalyx for enhanced immunotherapy.

The abnormally thick glycocalyx of cancer cells can provide a physical barrier to immune cell recognition and effective immunotherapy. Here, we demonstrate an optical method based on Scanning Angle Interference Microscopy (SAIM) for the screening of therapeutic agents that can disrupt the glycocalyx layer as a strategy to improve anti-cancer immune responses. We developed a new membrane labeling strategy utilizing leucine zipper pairs to fluorescently mark the glycocalyx layer boundary for precise and robust measurement of glycocalyx thickness with SAIM. Using this platform, we evaluated the effects of glycosylation inhibitors and targeted enzymatic degraders of the glycocalyx, with particular focus on strategies for cholangiocarcinoma (CCA), a highly lethal malignancy with limited therapeutic options. We found that CCA had the highest mean expression of the cancer-associated mucin, MUC1, across all cancers represented in the cancer cell line encyclopedia. Pharmacological inhibitors of mucin-type O-glycosylation and mucin-specific proteases, such as StcE, could dramatically reduce the glycocalyx layer in the YSCCC model of intrahepatic CCA. Motivated by these findings, we engineered Natural Killer (NK) cells tethered with StcE to enhance NK cell-mediated cytotoxicity against CCA. In a CCA xenograft model, these engineered NK cells demonstrated superior anti-tumor efficacy compared to wild-type NK cells, with no observable adverse effects. Our findings not only provide a reliable imaging-based screening platform for evaluating glycocalyx-targeting pharmacological interventions but also offer mechanistic insights into how CCA may avoid immune elimination through fortification of the glycocalyx layer with mucins. Additionally, this work presents a novel therapeutic strategy for mucin-overexpressing cancers, potentially improving immunotherapy efficacy across various cancer types.

Knockout of the inhibitory receptor TIGIT enhances the antitumor response of ex vivo expanded NK cells and prevents fratricide with therapeutic Fc-active TIGIT antibodies

BackgroundInhibitory receptor T-cell Immunoreceptor with Ig and ITIM domains (TIGIT) expressed by Natural Killer (NK) and T cells regulates cancer immunity and has been touted as the next frontier in...

Human iPSC-Derived NK Cells with Knock-in of the BCL2 G101V Mutation Are Resistant to Venetoclax and Demonstrate Improved Anti-AML Activity In Vivo

Treatment options for acute myeloid leukemia (AML) patients who are unfit for intensive chemotherapy are limited. Both targeted therapy and cellular therapy have been investigated, typically as separate treatments. For patients with AML unfit for intensive induction chemotherapy, venetoclax, a BCL-2 inhibitor, containing regimens have significantly improved treatment outcomes. However, patients treated long-term with venetoclax can develop resistance. Based on the characterization of the BCL2 G101V mutation that mediates resistance to venetoclax, we investigated the ability of homozygous BCL2 G101V knock-in in induced pluripotent stem cell (iPSC)-derived natural killer (NK) cells to promote resistance to venetoclax in these effector cells to enable concurrent treatment with venetoclax and the engineered iPSC-derived NK cells that we hypothesize will provide improved NK cell-mediated killing of AML. Our group has pioneered the production of genetically modified NK cells from human iPSCs. iPSCs provide an advantage of a stable platform for genome engineering and previous studies demonstrate we can engineer iPSCs to express or delete genes of interest to derive genetically modified iPSC-NK cells with improved anti-tumor activity. To generate iPSC-derived NK cells with resistance to venetoclax, we employed CRISPR-Cas9 technology to knock-in the BCL2 G101V mutation (BCL2 G101V) in iPSCs. iPSCs homozygous for BCL2 G101V were selected and differentiated to NK cells. Homozygous BCL2 G101V engineered iPSC-NK cells demonstrated resistance to venetoclax compared to wildtype (WT) iPSC-NK cells in vitro. BCL2 G101V iPSC-NK cells were 94-fold more resistant to venetoclax compared to wildtype iPSC NK cells with an EC50 of 6018 nM, above the serum levels of patients receiving venetoclax. Analysis of cell surface proteins demonstrated that both sets of iPSC-NK cells had a typical NK cell phenotype with no differences in expression of receptors analyzed. There was no difference seen in cytotoxicity against K562 tumor cells between BCL2 G101V iPSC-NK cells and WT-iPSC NK cells. Additional functional analyses demonstrated that activity of the BCL2 G101V iPSC NK cells was preserved upon exposure to venetoclax. Cytotoxic activity, as measured by CD107a expression on WT iPSC-NK cells stimulated by MOLM13 AML tumors cells, was reduced nearly 3-fold upon addition of venetoclax. In contrast, activation of BCL2 G101V iPSC NK cells was not significantly affected by addition of venetoclax. This resistance to venetoclax was confirmed in longer term 36-hour Incucyte cytotoxicity assays where BCL2 G101V iPSC-NK cells demonstrated more than 10-fold increase in anti-AML activity compared to the WT-iPSC-NK cells. We then conducted in vivo studies to test the WT and engineered NK cells with and without addition of venetoclax in an AML xenograft model using NSG mice (Figure Panels A-D). These studies used MOLM13 AML cells made resistant to venetoclax and demonstrated that without venetoclax treatment, both the WT and the BCL2 G101V iPSC NK cells mediate effective anti-AML killing. With addition of venetoclax, the WT NK cells were unable to effectively kill the AML cells while the BCL2 G101V iPSC cells mediated potent anti-AML activity and demonstrated significantly improved survival when the NK cells are given in combination with venetoclax. Specifically, in this system, the mice treated with WT iPSC-NK cells had a median survival of 22.8 days while the median survival of mice treated with the BCL2 G101V iPSC NK cells was not reached (p<0.01) Intriguingly, BCL2 G101V iPSC NK cells also demonstrated improved activity against MOLM13 cells resistant to venetoclax. Together our results demonstrate that iPSC-NK cells can be engineered to generate venetoclax-resistance for use in combination with concurrent venetoclax therapy to markedly improve treatment of AML. Furthermore, this work demonstrates that novel drug resistance mechanisms can be introduced via genome engineering into iPSC-derived NK cells as a new strategy to produce improved cell products for “off-the-shelf” therapy.

KLRC1 knockout overcomes HLA-E-mediated inhibition and improves NK cell antitumor activity against solid tumors.

Natural Killer (NK) cells hold the potential to shift cell therapy from a complex autologous option to a universal off-the-shelf one. Although NK cells have demonstrated efficacy and safety in the treatment of leukemia, the limited efficacy of NK cell-based immunotherapies against solid tumors still represents a major hurdle. In the immunosuppressive tumor microenvironment (TME), inhibitory interactions between cancer and immune cells impair antitumoral immunity. KLRC1 gene encodes the NK cell inhibitory receptor NKG2A, which is a potent NK cell immune checkpoint. NKG2A specifically binds HLA-E, a non-classical HLA class I molecule frequently overexpressed in tumors, leading to the transmission of inhibitory signals that strongly impair NK cell function. To restore NK cell cytotoxicity against HLA-E+ tumors, we have targeted the NKG2A/HLA-E immune checkpoint by using a CRISPR-mediated KLRC1 gene editing. KLRC1 knockout resulted in a reduction of 81% of NKG2A+ cell frequency in ex vivo expanded human NK cells post-cell sorting. In vitro, the overexpression of HLA-E by tumor cells significantly inhibited wild-type (WT) NK cell cytotoxicity with p-values ranging from 0.0071 to 0.0473 depending on tumor cell lines. In contrast, KLRC1 KO NK cells exhibited significantly higher cytotoxicity when compared to WT NK cells against four different HLA-E+ solid tumor cell lines, with p-values ranging from<0.0001 to 0.0154. Interestingly, a proportion of 43.5% to 60.2% of NKG2A- NK cells within the edited NK cell population was sufficient to reverse at its maximum the HLA-E-mediated inhibition of NK cell cytotoxicity. The expression of the activating receptor NKG2C was increased in KLRC1 KO NK cells and contributed to the improved NK cell cytotoxicity against HLA-E+ tumors. In vivo, the adoptive transfer of human KLRC1 KO NK cells significantly delayed tumor progression and increased survival in a xenogeneic mouse model of HLA-E+ metastatic breast cancer, as compared to WT NK cells (p = 0.0015). Our results demonstrate that KLRC1 knockout is an effective strategy to improve NK cell antitumor activity against HLA-E+ tumors and could be applied in the development of NK cell therapy for solid tumors.

Abstract 2909: Genetically engineered Natural Killer cells as an immunotherapeutic approach for the treatment of neuroblastoma

Abstract In this study we test if knockout of DNAM-1 competing inhibitory receptors on Natural Killer (NK) cells improves cytotoxic response against neuroblastoma cell lines. Neuroblastoma and other peripheral nervous cell tumors account for 6% of all pediatric cancer. While the 5-year survival rate has greatly improved, the prognosis of children with high-risk neuroblastoma at diagnosis is less than 50%. Immune checkpoint therapy is a promising new application for pediatric oncology; however, neuroblastoma is often immunologically “cold” and current immune checkpoint targets are not abundant in pediatric solid tumors. To improve response to checkpoint blockade and directly target tumor cells for elimination, this study evaluated the use of NK cells. NK cells prime the immune system for successful response to cancer immunotherapy. NK cells activated with particle technology (PM21-NK cells) are highly cytotoxic, produce IFNγ and induce PD-L1 on cancer cells. Although highly cytotoxic, PM21-NK cells express inhibitory receptors that compete for ligands that trigger NK cell lysis, such as PVR, a ligand highly expressed on neuroblastoma cells. We knocked out expression of multiple PVR inhibitory receptors in PM21-NK cells to further enhance cytotoxicity and prevent exhaustion. Three inhibitory receptors on NK cells that bind PVR - TIGIT, CD96, and PVRIG were knocked out alone or in combination and effector functions of these edited NK cells tested. Cytotoxicity against 3D spheroids of the neuroblastoma cell line SK-NA-S cells was assessed and compared to wild type PM21-NK cells. Knockout (KO) of TIGIT enhanced NK cell killing compared to wildtype NK cells (72%±10 vs 55%±3% killing) as well as PVIRG KO (84±3% vs 46±6% killing) and CD96 KO (62%±9% vs 46±6% killing) at a 1:3 E:T ratio at 72 h. Double or triple KO of these receptors resulted in further increased killing of cancer cells. The knockouts of each of the receptors could affect cytokine production and cytokine responsiveness and this will be examined. Overall, PM21-NK cells show promise for therapeutic use against neuroblastoma and preventing the PVR-mediated inhibitory axis signaling enhances their function. Citation Format: Tayler J. Croom-Perez, Jeremiah L. Oyer, Md Faqrul Hasan, Liza D. Robles-Carrillo, Brian P. Tullius, Alicja J. Copik. Genetically engineered Natural Killer cells as an immunotherapeutic approach for the treatment of neuroblastoma [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 2909.

Inhibition of hepatic natural killer cell function via the TIGIT receptor in schistosomiasis-induced liver fibrosis.

Schistosomiasis is a zoonotic parasitic disease. Schistosoma japonicum eggs deposited in the liver tissue induce egg granuloma formation and liver fibrosis, seriously threatening human health. Natural killer (NK) cells kill activated hepatic stellate cells (HSCs) or induce HSC apoptosis and inhibit the progression of liver fibrosis. However, the function of NK cells in liver fibrosis caused by S. japonicum infection is significantly inhibited. The mechanism of this inhibition remains unclear. Twenty mice were percutaneously infected with S. japonicum cercariae. Before infection and 2, 4, 6, and 8 weeks after infection, five mice were euthanized and dissected at each time point. Hepatic NK cells were isolated and transcriptome sequenced. The sequencing results showed that Tigit expression was high at 4-6 weeks post infection. This phenomenon was verified by reverse transcription quantitative PCR (RT-qPCR) and flow cytometry. NK cells derived from Tigit-/- and wild-type (WT) mice were co-cultured with HSCs. It was found that Tigit-/- NK cells induced apoptosis in a higher proportion of HSCs than WT NK cells. Schistosomiasis infection models of Tigit-/- and WT mice were established. The proportion and killing activity of hepatic NK cells were significantly higher in Tigit-/- mice than in WT mice. The degree of liver fibrosis in Tigit-/- mice was significantly lower than that in WT mice. NK cells were isolated from Tigit-/- and WT mice and injected via the tail vein into WT mice infected with S. japonicum. The degree of liver fibrosis in mice that received NK cell infusion reduced significantly, but there was no significant difference between mice that received NK cells from Tigit-/- and WT mice, respectively. Our findings indicate that Tigit knockout enhanced the function of NK cells and reduced the degree of liver fibrosis in schistosomiasis, thus providing a novel strategy for treating hepatic fibrosis induced by schistosomiasis.

Deletion of Glycogen Synthase Kinase 3 Beta Reprograms NK Cell Metabolism.

Loss of cytotoxicity and defective metabolism are linked to glycogen synthase kinase 3 beta (GSK3β) overexpression in natural killer (NK) cells from patients with acute myeloid leukemia or from healthy donors after expansion ex vivo with IL-15. Drug inhibition of GSK3β in these NK cells improves their maturation and cytotoxic activity, but the mechanisms of GSK3β-mediated dysfunction have not been well studied. Here, we show that expansion of NK cells with feeder cells expressing membrane-bound IL-21 maintained normal GSK3β levels, allowing us to study GSK3β function using CRISPR gene editing. We deleted GSK3B and expanded paired-donor knockout and wild-type (WT) NK cells and then assessed transcriptional and functional alterations induced by loss of GSK3β. Surprisingly, our data showed that deletion of GSK3B did not alter cytotoxicity, cytokine production, or maturation (as determined by CD57 expression). However, GSK3B-KO cells demonstrated significant changes in expression of genes related to rRNA processing, cell proliferation, and metabolic function, suggesting possible metabolic reprogramming. Next, we found that key genes downregulated in GSK3B-KO NK cells were upregulated in GSK3β-overexpressing NK cells from AML patients, confirming this correlation in a clinical setting. Lastly, we measured cellular energetics and observed that GSK3B-KO NK cells exhibited 150% higher spare respiratory capacity, a marker of metabolic fitness. These findings suggest a role for GSK3β in regulating NK cell metabolism.

The inhibitory NKR-P1B receptor regulates NK cell-mediated mammary tumor immunosurveillance in mice

ABSTRACT Natural killer (NK) cells are an important component of anti-cancer immunity, and their activity is regulated by an array of activating and inhibitory receptors. In mice, the inhibitory NKR-P1B receptor is expressed in NK cells and recognizes the C-type lectin-related protein-b (Clr-b) ligand. NKR-P1B:Clr-b interactions represent a ‘missing-self’ recognition system to monitor cellular levels of Clr-b on healthy and diseased cells. Here, we report an important role for NKR-P1B:Clr-b interactions in tumor immunosurveillance in MMTV-PyVT mice, which develop spontaneous mammary tumors. MMTV-PyVT mice on NKR-P1B-deficient genetic background developed mammary tumors earlier than on wild-type (WT) background. A greater proportion of tumor-infiltrating NK cells downregulate expression of the transcription factor Eomesodermin (EOMES) in NKR-P1B-deficient mice compared to WT mice. Tumor-infiltrating NK cells also downregulated CD49b expression but gain CD49a expression and exhibit effector functions, such as granzyme B upregulation and proliferation in mammary tumors. However, unlike the EOMES+ NK cells, the EOMES‒ NK cell subset is unable to respond to further in vitro stimulation and exhibits phenotypic alterations associated with immune dysfunction. These alterations included increased expression of PD-1, LAG-3, and TIGIT and decreased expression of NKp46, Ly49C/I, CD11b, and KLRG-1. Furthermore, tumor-infiltrating NKR-P1B-deficient NK cells exhibited an elevated dysfunctional immune phenotype compared to WT NK cells. These findings demonstrate that the NKR-P1B receptor plays an important role in mammary tumor surveillance by regulating anti-cancer immune responses and functional homeostasis in NK cells.

BST2, a Novel Inhibitory Receptor, Is Involved in NK Cell Cytotoxicity through Its Cytoplasmic Tail Domain.

Bone Marrow Stromal Cell Antigen 2 (BST2) is a type II transmembrane protein expressed on various cell types that tethers the release of viruses. Natural killer (NK) cells express low levels of BST2 under normal conditions but exhibit increased expression of BST2 upon activation. In this study, we show for the first time that murine BST2 can control the cytotoxicity of NK cells. The cytoplasmic tail of murine BST2 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM). The absence of BST2 on NK cells can enhance their cytotoxicity against tumor cells compared to wild type NK cells. NK cells isolated from NZW mice, which express ITIM-deficient BST2, also showed higher cytotoxicity than wild type NK cells. In addition, we found that galectin-8 and galectin-9 were ligands of BST2, since blocking galectin-8 or -9 with monoclonal antibodies enhanced the cytotoxicity of NK cells. These results suggested that BST2 might be a novel NK cell inhibitory receptor as it was involved in regulating NK cell cytotoxicity through its interaction with galectins.

Altered distribution and function of NK-cell subsets lead to impaired tumor surveillance in JAK2V617F myeloproliferative neoplasms.

In cancer, tumor cells and their neoplastic microenvironment can sculpt the immunogenic phenotype of a developing tumor. In this context, natural killer (NK) cells are subtypes of lymphocytes of the innate immune system recognized for their potential to eliminate neoplastic cells, not only through direct cytolytic activity but also by favoring the development of an adaptive antitumor immune response. Even though the protective effect against leukemia due to NK-cell alloreactivity mediated by the absence of the KIR-ligand has already been shown, and some data on the role of NK cells in myeloproliferative neoplasms (MPN) has been explored, their mechanisms of immune escape have not been fully investigated. It is still unclear whether NK cells can affect the biology of BCR-ABL1-negative MPN and which mechanisms are involved in the control of leukemic stem cell expansion. Aiming to investigate the potential contribution of NK cells to the pathogenesis of MPN, we characterized the frequency, receptor expression, maturation profile, and function of NK cells from a conditional Jak2V617F murine transgenic model, which faithfully resembles the main clinical and laboratory characteristics of human polycythemia vera, and MPN patients. Immunophenotypic analysis was performed to characterize NK frequency, their subtypes, and receptor expression in both mutated and wild-type samples. We observed a higher frequency of total NK cells in JAK2V617F mutated MPN and a maturation arrest that resulted in low-numbered mature CD11b+ NK cells and increased immature secretory CD27+ cells in both human and murine mutated samples. In agreement, inhibitory receptors were more expressed in MPN. NK cells from Jak2V617F mice presented a lower potential for proliferation and activation than wild-type NK cells. Colonies generated by murine hematopoietic stem cells (HSC) after mutated or wild-type NK co-culture exposure demonstrated that NK cells from Jak2V617F mice were deficient in regulating differentiation and clonogenic capacity. In conclusion, our findings suggest that NK cells have an immature profile with deficient cytotoxicity that may lead to impaired tumor surveillance in MPN. These data provide a new perspective on the behavior of NK cells in the context of myeloid malignancies and can contribute to the development of new therapeutic strategies, targeting onco-inflammatory pathways that can potentially control transformed HSCs.

Mitochondrial cyclophilin D promotes disease tolerance by licensing NK cell development and IL-22 production against influenza virus.

Severity of pulmonary viral infections, including influenza A virus (IAV), is linked to excessive immunopathology, which impairs lung function. Thus, the same immune responses that limit viral replication can concomitantly cause lung damage that must be countered by largely uncharacterized disease tolerance mechanisms. Here, we show that mitochondrial cyclophilin D (CypD) protects against IAV via disease tolerance. CypD-/- mice are significantly more susceptible to IAV infection despite comparable antiviral immunity. This susceptibility results from damage to the lung epithelial barrier caused by a reduction in interleukin-22 (IL-22)-producing natural killer (NK) cells. Transcriptomic and functional data reveal that CypD-/- NK cells are immature and have altered cellular metabolism and impaired IL-22 production, correlating with dysregulated bone marrow lymphopoiesis. Administration of recombinant IL-22 or transfer of wild-type (WT) NK cells abrogates pulmonary damage and protects CypD-/- mice after IAV infection. Collectively, these results demonstrate a key role for CypD in NK cell-mediated disease tolerance.

Reprogramming NK cells and macrophages via combined antibody and cytokine therapy primes tumors for elimination by checkpoint blockade.

SUMMARYTreatments aiming to augment immune checkpoint blockade (ICB) in cancer often focus on T cell immunity, but innate immune cells may have important roles to play. Here, we demonstrate a single-dose combination treatment (termed AIP) using a pan-tumor-targeting antibody surrogate, half-life-extended interleukin-2 (IL-2), and anti-programmed cell death 1 (PD-1), which primes tumors to respond to subsequent ICB and promotes rejection of large established tumors in mice. Natural killer (NK) cells and macrophages activated by AIP treatment underwent transcriptional reprogramming; rapidly killed cancer cells; governed the recruitment of cross-presenting dendritic cells (DCs) and other leukocytes; and induced normalization of the tumor vasculature, facilitating further immune infiltration. Thus, innate cell-activating therapies can initiate critical steps leading to a self-sustaining cycle of T cell priming driven by ICB.

ADAM17-Deficient Pluripotent Stem Cell-Derived Natural Killer Cells Possess Improved Antibody-Dependent Cellular Cytotoxicity and Antitumor Activity

Antibody-dependent cellular cytotoxicity (ADCC) is a key pathway that mediates natural killer (NK) cell cytotoxicity against antibody-opsonized target cells. This process helps mediate the therapeutic efficacy of anti-tumor antibodies. On NK cells, ADCC occurs via engagement of antibody-coated target cells with activating receptor FcγRIIIa, or CD16a, leading to proinflammatory cytokine upregulation, degranulation, and target cell death. Upon cellular activation, the CD16a ectodomain is cleaved from the NK cell surface by A Disintegrin and Metalloprotease-17 (ADAM17). Cleavage of the ectodomain prevents further antibody binding and signaling through CD16a, which dampens NK cell activity. Blocking activation-induced ADAM17-mediated CD16a cleavage has been previously demonstrated to augment ADCC activity and provides a novel strategy to improve efficacy of therapeutic antibodies in combination with adoptive transfer of engineered NK cells. To further define the ability of ADAM17 to regulate NK cell activity, we have generated and characterized ADAM17-deficient (ADAM17-KO) NK cells derived from CRISPR/Cas9-modified human induced pluripotent stem cells (iPSCs). ADAM17-KO iPSCs successfully differentiate into hematopoietic progenitor cells, then to NK cells that uniformly express typical NK cell surface markers including CD56, CD94, NKG2D, NKp44, and NKp46. ADAM17-KO iPSC-NKs are functional and kill K562 erythroleukemia cells comparable to wildtype iPSC-derived NK cells (WT iPSC-NK cells) and healthy donor-derived peripheral blood NK cells (PB-NK cells) in vitro. Surprisingly, upon differentiation, ADAM17-KO iPSC-NK cells express ~20% lower CD16a surface expression compared to WT iPSC-NK cells, but stably retain CD16a expression after enrichment for CD16a+ cells and over 6 weeks of expansion in culture. WT iPSC-NKs and PB-NKs rapidly lose CD16a surface expression upon stimulation with phorbol esters, while ADAM17 KO iPSC-NK cells maintain over 90% CD16a expression after this stimulation. Additionally, a significantly higher proportion of ADAM17-KO iPSCs express TNF-α (71%) and CD62L (L-Selectin) (36%) - two other known ADAM17 substrates, on the cell surface after stimulation with phorbol esters for 4 hours compared to WT iPSC-NK (7% TNF-α+, 2% L-Selectin+) and PB-NK (2% TNF-α+, 1% L-Selectin+). CD16a+ ADAM17-KO iPSC-NK cells mediate increased CD107a (45%) and IFNγ (39%) expression when co-incubated with RAJI B-lymphoma cells in the presence of the anti-CD20 antibody rituximab, compared to CD16a+ WT iPSC-NK (32% CD107a+, 11% IFNγ) and PB-NK (37% CD107a+, 7% IFNγ) cells. Similarly, CD16a+ ADAM17-KO iPSC-NK cells upregulate increased CD107a (29%) and IFNγ (42%) expression when co-incubated with CAL27 squamous cell carcinoma cells in the presence of the anti-EGFR antibody cetuximab, compared to CD16a+ WT iPSC-NK (12% CD107a+, 8% IFNγ) and PB-NK (14% CD107a+, 6% IFNγ). Long-term (24 hour) cytotoxicity assay against RAJI cells in the presence of rituximab demonstrates higher cytotoxicity in CD16a+ ADAM17-KO iPSC-NK cells compared to CD16a+ WT iPSC-NK and CD16a+ PB-NK cells over time (see associated figure). In vivo studies to determine the therapeutic efficacy of ADAM17-KO iPSC-NK cells compared to WT iPSC-NK and PB-NK cells are ongoing. Together, these studies demonstrate ADAM17-KO iPSC-NK cells derived from a renewable source of gene-edited iPSCs possess enhanced ADCC potential, and provide a promising candidate to be used for standardized, off-the-shelf NK cell-based therapies in conjunction with therapeutic antibodies. Figure Disclosures Blum: Fate Therapeutics: Current Employment. Kaufman:Fate Therapeutics: Consultancy.

IL-10-producing NK cells exacerbate sublethal Streptococcus pneumoniae infection in the lung

Lung inflammation is tightly controlled to balance microbial clearance with the tissue damage that accompanies this response. Bacterial pathogens including Streptococcus pneumoniae (S. pneumoniae) modulate immune regulation by promoting secretion of the anti-inflammatory cytokine IL-10. The important cellular sources of IL-10 that impact protection against different bacterial infections are not well characterized. We find that S. pneumoniaeactivates IL-10 secretion from natural killer (NK) cells in the lung, which restrict host protection in a mouse model of sublethal infection. Direct transfer of wild-type NK cells into the lungs of IL-10-deficient mice drives bacterial expansion, identifying NK cells as a critical source of IL-10 promoting S. pneumoniae infection. The S. pneumoniae virulence protein Spr1875 was found to elicit NK cell IL-10 production in purified cells and in the lungs of live animals. These findings reveal therapeutic targets to combat bacterial-driven immune regulation in the lung.

NCAM Expression Regulates Integrin and Actin Cytoskeletal Function in Human Natural Killer Cells

Human natural killer (NK) cells are innate immune cells that are broadly defined as being CD56+CD3−, however a functional role for CD56 (neural cell adhesion molecule, NCAM) on NK cells has not been described. NCAM is a member of the Ig superfamily that engages in homotypic and heterotypic interactions in cis and in trans. These include NCAM-NCAM interactions and binding to heparin sulfate proteoglycans. In neuronal cells, NCAM plays a role in neurite outgrowth, cell migration and adhesion, in part through direct interactions with focal adhesion kinase (FAK). We have previously shown that CD56 is required for human NK cell migration on stromal cells, and that deletion of CD56 in primary human NK cells significantly impairs this migration. We deleted CD56 by CRISPR-Cas9 mediated gene editing in a human NK cell line and measured the effect of CD56 deletion on adhesion and migration. Using super-resolution (STED) nanoscopy, we found that activation through NK cell activating receptors and/or αLβ2 integrin leads to an increase in the frequency of actin foci >0.05 um2 in the cortical actin of CD56-knockout (KO) NK cells. This was accompanied by increased colocalization of actin and vinculin in actin asters. In wild-type NK cells, we found that CD56 co-localized with β1 integrin in the uropod of migrating cells. Using live-cell interference reflection microscopy, we showed that while adhesion to integrin ligands was not impaired in CD56-KO NK cells, integrin detachment, specifically in the uropod of migrating cells was dysfunctional. Impaired phosphorylation of the FAK homologue Pyk2 in CD56-KO NK cells in response to activation and integrin ligation further supports a novel role for CD56 in regulating integrin detachment. In summary, we have defined NCAM/CD56 as a novel regulator of human immune cell migration.

Natural killer cells induce neutrophil extracellular trap formation in venous thrombosis

Essentials Neutrophil extracellular traps (NETs) are generated during deep vein thrombosis (DVT). The role of interferon γ (IFNγ) and natural killer (NK) cells in NET formation was studied. IFNγ promote venous thrombosis through NET formation. NK cell depletion reduces DVT. SUMMARY: Background Neutrophils contribute to venous thrombosis through the release of neutrophil extracellular traps (NETs), but the mechanism triggering their formation remains unclear. Invitro data show that interferon (IFN)-γ induces the formation of NETs. Objectives To determine whether IFN-γ and the transcription factor T-box expressed on T cells (Tbet) promote venous thrombosis through neutrophil activation. Methods Venous thrombosis was induced by flow restriction in the inferior vena cava in IFN-γ-/- , Tbet-/- or wild-type (WT) mice. After 48h, thrombus size was measured by the use of high-frequency ultrasound. NET formation was determined by immunofluorescence. Results and Conclusions Thrombus formation was reduced in Tbet-/- and IFN-γ-/- mice, suggesting that Tbet/IFN-γ-expressing cells are required for venous thrombosis. The number of NETs formed during thrombosis was significantly lower in Tbet-/- and IFN-γ-/- mice. NET formation was also decreased in WT mice treated with an IFN-γ-blocking antibody. Injection of recombinant IFN-γ into IFN-γ-/- mice rescued the phenotype. Natural killer (NK) cells were specifically depleted prior to venous thrombosis induction. NK cell depletion results in decreased NET formation and smaller thrombi, suggesting that NK cells are required for thrombus development. In depleted mice, adoptive transfer of WT NK cells induced a similar thrombosis burden as in WT mice. In contrast, adoptive transfer of IFN-γ -/- NK cells resulted in thrombi similar in size to those in depleted mice. Invitro, we showed that WT neutrophils released fewer NETs when they were cocultured with IFN-γ-/- NK cells. This study demonstrates that NK cell-dependent IFN-γ production is crucial for thrombus development by promoting the formation of NETs by neutrophils.

Unexpected role of natural killer cell‐derived interferon‐γ as a driver of NETosis and DVT

Unexpected role of natural killer cell‐derived interferon‐γ as a driver of NETosis and DVT

CXCR3-deficient natural killer cells fail to migrate to B16F10 melanoma cells

Natural killer (NK) cells eliminate cancer cells in a contact-dependent manner. However, how NK cells find cancer cells remain unclear. Here, using time-lapse imaging, we investigated how individual NK cells migrate toward cancer cells. Although naïve B16F10 cancer cells produce low levels of chemokines, IFN-γ-treated B16F10 cells secreted high levels of CXCL10, low levels of CCL5, but did not secrete CCL2, CCL7, or CXCL12. Wild-type NK cells migrated well toward cancer cells and killed them, whereas NK cells deficient in CXCR3 did not. CXCR3-deficient NK cells also showed slower migration speed than did wild-type NK cells. Taken together, our data show that NK cells find cancer cells, at least in part, by sensing CXCL10 produced by cancer cells and suggest that a strategy to increase CXCL10 secretion by cancer cells may improve the efficacy of NK cell–based immunotherapy.

Abstract 4743: Inhibition of the Hif1a-mediated checkpoint refuels NK activation in cancer

Abstract Solid tumors remain a major challenge for immunotherapy of cancer due to their immunosuppressive microenvironment. Hypoxia is a common feature of solid tumors and might affect immune cell activation within the tumor tissue. Upon hypoxia the transcription factor Hypoxia inducible factor 1a (HIF-1α) that affects many cellular processes including cell proliferation, survival and effector function becomes induced. Its impact on innate immune cells such as Natural Killer (NK) cells, however, is still incompletely understood. Natural Killer (NK) cells are considered as highly potent effector cells against tumors. Within the tumor tissue, however, NK effector function is often impaired. Our microarray analyses of tumor-infiltrated NK cells revealed upregulated levels of Hif1a. To determine the role of the HIF-1α in NK cell function, we generated the mouse strain Hif1aΔ/ΔNcr1-iCreTg in which HIF-1α is deficient in the NKp46+ lineage. Hif1a deficient NK cells showed greatly enhanced anti-tumor activity compared to wild-type NK cells, leading to a pronounced reduction of tumor growth. Tumor-infiltrated Hif1a deficient NK cells expressed lower levels of checkpoint molecules and show enhanced responses to tumor cells indicating a less exhausted phenotype. Hif1a deficient NK cells preferentially utilize oxidative phosphorylation (OXPHOS) than glycolysis to fuel their energy resulting in enhanced IFN-γ production in the hypoxic environment. In addition, using HIF-1α inhibitors we were able to greatly enhance human NK cell anti-tumor activity. Accordingly, our data reveal HIF-1α as a negative metabolic checkpoint for NK cells. Moreover, inhibition of HIF-1α holds high promise to refuel NK cell activation in cancer with a high potential for improving current strategies of NK cell-based cancer immunotherapy. Citation Format: Jing Ni, Lea Bühler, Ana Stojanovic, Annette Arnold, Veronika Sexl, Adelheid Cerwenka. Inhibition of the Hif1a-mediated checkpoint refuels NK activation in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4743.

Interleukin (IL)-15 superagonist mediates immunotoxicity by inducing hyperproliferation of activated NK cells and production of IFNγ

Abstract Interleukin (IL)-15 is currently undergoing clinical trials to assess its anti-tumor efficacy. IL-15 is essential for the generation, function and survival of natural killer (NK), natural killer T (NKT) and memory phenotype (MP) CD8+T cells. The combination of IL-15 with its soluble IL-15 receptor α (IL-15Rα) generates a complex termed IL-15 superagonist (IL-15 SA) that exhibits more profound biological activities on NK, NKT and MP CD8+T cells than IL-15 alone. Thus, IL-15 SA is a potentially more attractive therapeutic agent for use against advanced tumors. However, the safety of IL-15 SA administration in vivo is not clearly elucidated. This study was designed to evaluate toxic consequences of IL-15 SA in mice. Administration of 2 μg IL-15 SA for 4 days caused a time- and dose-dependent immune-toxicity, characterized by hypothermia, weight loss, liver damage and death. At 2ug, IL-15 SA expanded NK cells in spleens and livers compared to vehicle and significantly increased NK cell expression of CD69, NKG2D and NKp46 as well as production of IFNγ, granzyme B and perforin. IL-15 SA also prompted NK cell maturity with a preferential expansion of the pro-inflammatory (CD27+ CD11b+) NK subset. Mice depleted of NK cells by anti-asialoGM1or anti-NK1.1 were resistant to IL-15 SA-induced toxicity, whereas adoptive transfer of wild type NK cells to Rag2−/−γc−/− mice re-established IL-15 SA toxicity. While IL-15 SA expanded NKT and mCD8+ T cells, mice depleted of NKT or CD8+ T cells remained sensitive to IL-15 SA-mediated toxicity. In addition, IFNγ KO, but not TNF-α KO or perforin KO, mice were resistant to IL-15 SA-induced toxicity. In conclusion, high-dose IL-15 SA causes systemic immunotoxicity via induction of NK cell proliferation and activation.