NK Cell Triggering Research Articles

Deficiency of N-linked glycosylation impairs immune function of B7-H6.

B7-H6 is a novel immune checkpoint molecule that triggers NK cell cytotoxicity, but the role of N-glycosylation in B7-H6 is poorly understood. We here identified the existence of N-glycosylation of B7-H6 in different cell lines and exogenous expression cells by PNGase F digestion and tunicamycin blockage. Subsequently, we demonstrated that B7-H6 contains 6 functional N-linked glycosylation sites by single site mutation and electrophoresis. Phylogenetical and structural analysis revealed that N43 and N208 glycan are conserved in jawed vertebrates and may thus contribute more to the biological functions. We further demonstrated that N43 and N208 glycosylation are essential for B7-H6 to trigger NK cell activation. Mechanistically, we found that N43 and N208 glycan contributed to the stability and membrane expression of B7-H6 protein. Lack of N208 glycosylation led to membrane B7-H6 shedding, while N43 mutation resulted in impaired B7-H6/NKp30 binding affinity. Together, our findings highlight the significance of N-linked glycosylation in B7-H6 biological functions and suggest potential targets for modulating NK cell-mediated immunity.

Impact of HIV-1 Vpu-mediated downregulation of CD48 on NK-cell-mediated antibody-dependent cellular cytotoxicity.

HIV-1 evades antibody-dependent cellular cytotoxicity (ADCC) responses not only by controlling Env conformation and quantity at the cell surface but also by altering NK cell activation via the downmodulation of several ligands of activating and co-activating NK cell receptors. The signaling lymphocyte activation molecule (SLAM) family of receptors, which includes NTB-A and 2B4, act as co-activating receptors to sustain NK cell activation and cytotoxic responses. These receptors cooperate with CD16 (FcγRIII) and other activating receptors to trigger NK cell effector functions. In that context, Vpu-mediated downregulation of NTB-A on HIV-1-infected CD4 T cells was shown to prevent NK cell degranulation via an homophilic interaction, thus contributing to ADCC evasion. However, less is known on the capacity of HIV-1 to evade 2B4-mediated NK cell activation and ADCC. Here, we show that HIV-1 downregulates the ligand of 2B4, CD48, from the surface of infected cells in a Vpu-dependent manner. This activity is conserved among Vpu proteins from the HIV-1/SIVcpz lineage and depends on conserved residues located in its transmembrane domain and dual phosphoserine motif. We show that NTB-A and 2B4 stimulate CD16-mediated NK cell degranulation and contribute to ADCC responses directed to HIV-1-infected cells to the same extent. Our results suggest that HIV-1 has evolved to downmodulate the ligands of both SLAM receptors to evade ADCC. IMPORTANCE Antibody-dependent cellular cytotoxicity (ADCC) can contribute to the elimination of HIV-1-infected cells and HIV-1 reservoirs. An in-depth understanding of the mechanisms used by HIV-1 to evade ADCC might help develop novel approaches to reduce the viral reservoirs. Members of the signaling lymphocyte activation molecule (SLAM) family of receptors, such as NTB-A and 2B4, play a key role in stimulating NK cell effector functions, including ADCC. Here, we show that Vpu downmodulates CD48, the ligand of 2B4, and this contributes to protect HIV-1-infected cells from ADCC. Our results highlight the importance of the virus to prevent the triggering of the SLAM receptors to evade ADCC.

Abstract 3183: Combination of anti-EGFR antibody cetuximab with NKX101, an allogeneic NKG2D-L targeting NK cell therapy, enhances potency and in vitro cytotoxicity against solid tumors

Abstract Background: Antibody-dependent cellular cytotoxicity (ADCC) is a mechanism of cell-mediated immune defense against opsonized target cells wherein the Fc-fragment of an antibody binds to the CD16 Fc receptor (FcγRIIIa) on NK cells, triggering NK cell activation and targeted killing. Cetuximab is a human IgG1 antibody targeting epidermal growth factor receptor (EGFR) that is approved for the treatment of colorectal and head and neck cancers. Binding of cetuximab to EGRF+ cell lines has been shown to trigger ADCC mediated by NK cells. NKX101 is an investigational allogeneic NK cell therapy in development for the treatment of cancer. NKX101 is engineered to express an NKG2D-based chimeric antigen receptor (CAR) and membrane-bound IL-15. To investigate the combination of NKX101 and cetuximab, we developed in vitro assays to (i) demonstrate enhanced activity against cancer cells via CD16-mediated ADCC activity, (ii) compare ADCC activity of NKX101 cells generated from high-affinity CD16 and low-affinity CD16 donors, and (iii) evaluate the synergistic interaction between NKX101 and cetuximab. Methods: NKX101 cells were generated from peripheral blood leukopaks from healthy donors. NucRed-labeled EGFR+ NKG2D-L+ human cancer cell lines were pre-incubated with cetuximab or isotype control and co-cultured with NKX101. Specific cell killing of target cells was measured using an Incucyte instrument. For synergy assessments, a dose response matrix of NKX101 cells and cetuximab was used to generate cytotoxicity values. Synergy scores for combinations were determined using the Synergy Finder Plus web application. To determine donor CD16 V/F 158 genotypes, genomic DNA was isolated from each donor and assayed using a TaqMan CD16 genotyping kit. Results: We demonstrate that cetuximab increases the anti-tumor effect of NKX101 in a dose-dependent manner. Assessment of the interaction between NKX101 and cetuximab in vitro revealed that the two agents can combine to kill cancer cells in a synergistic manner. Blocking CD16 on NKX101 cells with a neutralizing antibody significantly decreased potency of the combination, suggesting that the improvement in potency observed is a direct result of CD16-mediated ADCC activity. To determine if common CD16 polymorphisms influence NKX101 ADCC activity, we compared NKX101 cells generated from CD16 158 V/V, V/F, and F/F donors. No significant differences in potency were observed with NKX101 cells expressing the high affinity CD16 158 V/V genotype compared with those derived from CD16 lower affinity 158 V/F or 158 F/F donors. In summary, we show that the combination of an engineered allogeneic NK cell therapy, NKX101, with cetuximab leads to an increase in the specific killing of cancer cells in vitro, supporting further investigation into this combination for the treatment of solid tumors. Citation Format: Cynthia Cho, Kyle Hansen, Max Zhang, Carmel Chan, James Trager. Combination of anti-EGFR antibody cetuximab with NKX101, an allogeneic NKG2D-L targeting NK cell therapy, enhances potency and in vitro cytotoxicity against solid tumors [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 3183.

Blockade of inhibitory killer cell immunoglobulin-like receptors and IL-2 triggering reverses the functional hypoactivity of tumor-derived NK-cells in glioblastomas

Killer cell immunoglobulin-like receptors (KIRs) comprise a group of highly polymorphic inhibitory receptors which are specific for classical HLA class-I molecules. Peripheral blood and freshly prepared tumor cell suspensions (n = 60) as well as control samples (n = 32) were investigated for the distribution, phenotype, and functional relevance of CD158ab/KIR2DL1,-2/3 expressing NK-cells in glioblastoma (GBM) patients. We found that GBM were scarcely infiltrated by NK-cells that preferentially expressed CD158ab/KIR2DL1,-2/3 as inhibitory receptors, displayed reduced levels of the activating receptors CD335/NKp46, CD226/DNAM-1, CD159c/NKG2C, and showed diminished capacity to produce IFN-γ and perforin. Functional hypoactivity of GBM-derived NK-cells persisted despite IL-2 preactivation. Blockade with a specific KIR2DL-1,2/3 monoclonal antibody reversed NK-cell inhibition and significantly enhanced degranulation and IFN-γ production of IL-2 preactivated NK-cells in the presence of primary GBM cells and HLA-C expressing but not HLA class-I deficient K562 cells. Additional analysis revealed that significant amounts of IL-2 could be produced by tumor-derived CD4+ and CD8+CD45RA- memory T-cells after combined anti-CD3/anti-CD28 stimulation. Our data indicate that both blockade of inhibitory KIR and IL-2 triggering of tumor-derived NK-cells are necessary to enhance NK-cell responsiveness in GBM.

TLR3 Mediates Senescence and Immunosurveillance of Hepatic Stellate Cells

Background: Activation of hepatic stellate cells (HSCs) is an important driver of liver fibrosis, which is a health problem of global concern, and there is no effective solution for it at the present. Senescent activated HSCs are preferentially killed by natural killer cells (NK cells) to promote the regression of hepatic fibrosis. Objectives: The purpose of this study was to investigate the effect of polyinosinic-polycytidylic acid (poly I:C) on HSCs’ senescence, a trigger for NK cell-induced cytotoxicity. Methods: The senescence of HSCs was assessed by western blot, qRT-PCR, and flow cytometry, and NK cell cytotoxicity was assessed in a co-culture of NK cells with poly I:C-treated HSCs by measuring CD107a expression. Results: The expression of p16, p21, SA-β-gal, MICA/MICB, and ULBP2 increased in poly I:C-treated HSCs, rendering them significantly susceptible to NK cell cytotoxicity. Conclusions: Poly I:C induces cellular senescence in HSCs and triggers NK cell immunosurveillance, suggesting that the role of poly I:C in HSC senescence may promote fibrosis regression.

Lack of Viral Load Within Chronic Lymphoproliferative Disorder of Natural Killer Cells: What Is Outside the Leukemic Clone?

Large granular lymphocyte leukemias (LGLL) are sustained by proliferating cytotoxic T cells or NK cells, as happens in Chronic Lymphoproliferative Disorder of Natural Killer cells (CLPD-NK), whose etiology is only partly understood. Different hypotheses have been proposed on the original events triggering NK cell hyperactivation and transformation, including a role of viral agents. In this perspective, we revise the lines of evidence that suggested a pathogenetic role in LGLL of the exposure to retroviruses and that identified Epstein Barr Virus (EBV) in other NK cell leukemias and lymphomas and focus on the contrasting data about the importance of viral agents in CLPD-NK. EBV was detected in aggressive NK leukemias but not in the indolent CLPD-NK, where seroreactivity against HTLV-1 retrovirus envelope BA21 protein antigens has been reported in patients, although lacking clear evidence of HTLV infection. We next present original results of whole exome sequencing data analysis that failed to identify viral sequences in CLPD-NK. We recently demonstrated that proliferating NK cells of patients harbor several somatic lesions likely contributing to sustain NK cell proliferation. Thus, we explore whether “neoantigens” similar to the BA21 antigen could be generated by aberrancies present in the leukemic clone. In light of the literature and new data, we evaluated the intriguing hypothesis that NK cell activation can be caused by retroviral agents located outside the hematopoietic compartment and on the possible mechanisms involved with the prospects of immunotherapy-based approaches to limit the growth of NK cells in CLPD-NK disease.

NK cells mediate clearance of CD8+ T cell-resistant tumors in response to STING agonists.

Several immunotherapy approaches that mobilize CD8+ T cell responses stimulate tumor rejection, and some, such as checkpoint blockade, have been approved for several cancer indications and show impressive increases in patient survival. However, tumors may evade CD8+ T cell recognition via loss of MHC molecules or because they contain few or no neoantigens. Therefore, approaches are needed to combat CD8+ T cell-resistant cancers. STING-activating cyclic dinucleotides (CDNs) are a new class of immune-stimulating agents that elicit impressive CD8+ T cell-mediated tumor rejection in preclinical tumor models and are now being tested in clinical trials. Here, we demonstrate powerful CDN-induced, natural killer (NK) cell-mediated tumor rejection in numerous tumor models, independent of CD8+ T cells. CDNs enhanced NK cell activation, cytotoxicity, and antitumor effects in part by inducing type I interferon (IFN). IFN acted in part directly on NK cells in vivo and in part indirectly via the induction of IL-15 and IL-15 receptors, which were important for CDN-induced NK activation and tumor control. After in vivo administration of CDNs, dendritic cells (DCs) up-regulated IL-15Rα in an IFN-dependent manner. Mice lacking the type I IFN receptor specifically on DCs had reduced NK cell activation and tumor control. Therapeutics that activate NK cells, such as CDNs, checkpoint inhibitors, NK cell engagers, and cytokines, may represent next-generation approaches to cancer immunotherapy.

Differential Induction of IFN-α and Modulation of CD112 and CD54 Expression Govern the Magnitude of NK Cell IFN-γ Response to Influenza A Viruses.

In human and murine studies, IFN-γ is a critical mediator immunity to influenza. IFN-γ production is critical for viral clearance and the development of adaptive immune responses, yet excessive production of IFN-γ and other cytokines as part of a cytokine storm is associated with poor outcomes of influenza infection in humans. As NK cells are the main population of lung innate immune cells capable of producing IFN-γ early in infection, we set out to identify the drivers of the human NK cell IFN-γ response to influenza A viruses. We found that influenza triggers NK cells to secrete IFN-γ in the absence of T cells and in a manner dependent upon signaling from both cytokines and receptor-ligand interactions. Further, we discovered that the pandemic A/California/07/2009 (H1N1) strain elicits a seven-fold greater IFN-γ response than other strains tested, including a seasonal A/Victoria/361/2011 (H3N2) strain. These differential responses were independent of memory NK cells. Instead, we discovered that the A/Victoria/361/2011 influenza strain suppresses the NK cell IFN-γ response by downregulating NK-activating ligands CD112 and CD54 and by repressing the type I IFN response in a viral replication-dependent manner. In contrast, the A/California/07/2009 strain fails to repress the type I IFN response or to downregulate CD54 and CD112 to the same extent, which leads to the enhanced NK cell IFN-γ response. Our results indicate that influenza implements a strain-specific mechanism governing NK cell production of IFN-γ and identifies a previously unrecognized influenza innate immune evasion strategy.

Abstract 3699: Activation of NK cells cytotoxicity mediated by alveolar macrophages in the lung against murine melanoma metastases by combined aerosol immunotherapy

Abstract INTRODUCTION: Taking advantage of aerosol for repeated local applications in the lung, we previously demonstrated that combination of Poly(I:C) and CpG-ODN, TLR3 and 9 ligands capable to activate innate effectors, induced a significant antitumor effect against B16 melanoma lung metastases, which correlated with a strong activation of NK cells. We investigated the sequence of events that lead to NK cells activation by aerosolized TLR agonists in the lung. Although NK cells express certain TLRs, controversies exist regarding their direct responsiveness or dependence on macrophages. Alveolar macrophages have several unique features both promoting host defense mechanisms against invading microorganisms, but also establishing an immunosuppressive microenvironment to avoid inflammatory responses to inhaled particles. Our findings, establishing the sequence of events of lung immune cells activation, might represent the basis for the development of combined aerosol therapies that favor NK cell triggering. EXPERIMENTAL PROCEDURE: Alveolar macrophages and NK cells were recovered by bronchoalveolar lavage and spleen of C57BL/6 mice, respectively. Mice i.v. injected with 5×105 B16 cells were treated starting 7 days later every three days with aerosolized Poly(I:C)/CpG-ODN alone or combined to nebulized myeloid derived suppressive cells (MDSC)-depleting RB6-8C5 antibody. Immune cell populations and expression of M1/M2 markers were analysed by flow cytometry and Real Time PCR, respectively, in digested lungs. Cytotoxic activity of NK cells was assessed by 51Cr release assay. RESULTS: In vitro TLR3/TLR9 stimulation induced IFN-γ secretion by naïve NK cells, but an increase in their cytotoxicity was detected only when NK cells were cocultured with alveolar macrophages pretreated with the two agonists. Alveolar macrophages from melanoma lung metastases-bearing mice treated with aerosolized TLR agonists also promoted NK cell cytotoxicity ex vivo. Moreover, naïve alveolar macrophages incubated with activated NK cells from lungs of melanoma metastases-bearing mice aerosolized with TLR9/TLR3 agonists, up-regulated M1- and downregulated M2-related genes, suggesting a bidirectional crosstalk between NK cells and macrophage. Thus, TLR agonists-activated lung macrophages promote NK cell cytotoxicity and, reciprocally, NK cells have the potential to shape the macrophage programming to M1-like phenotype. Our results also indicated that this positive interplay can be further improved by the depletion of myeloid derived suppressive cells that exert a negative effect on macrophage activation. CONCLUSION: This study points to the promise of combinations of immunotherapeutic agents delivered locally by inhalation to promote macrophages-NK cell activation in the lung as a novel strategy to treat lung cancer patients. Citation Format: Michele Sommariva, Valentino Le Noci, Elda Tagliabue, Andrea Balsari, Lucia Sfondrini. Activation of NK cells cytotoxicity mediated by alveolar macrophages in the lung against murine melanoma metastases by combined aerosol immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3699. doi:10.1158/1538-7445.AM2017-3699

Contribution of inhibitory receptor TIGIT to NK cell education

Engagement of inhibitory receptors by cognate host MHC-I molecules triggers NK cell education, resulting in functional maturation and allowing NK cells to sense missing-self. However, NK cells also express inhibitory receptors for non-MHC-I ligands and their role in NK cell education is poorly understood. TIGIT is a recently identified inhibitory receptor that recognizes a non-MHC-I ligand CD155. Here, we demonstrated that TIGIT+ NK cells from wild-type mice exerted augmented responsiveness to various stimuli, including targets that lacked expression of CD155 ligand. TIGIT+ NK cells derived from CD155-deficient hosts, however, exhibited functional impairment, indicating that the engagement of TIGIT receptor by host CD155 promoted NK cell functional maturation. Furthermore, TIGIT deficiency impaired NK cell-mediated missing-self recognition and rejection of CD155- targets, such as allogenic splenocytes and certain tumor cells, in an MHC-I-independent and CD226-unrelated manner. Thus, TIGIT-CD155 pathway is also involved in the acquisition of optimal NK cell effector function, representing a novel MHC-I-independent education mechanism for NK cell tolerance and activation.

IL-28B Genetic Variants Determine the Extent of Monocyte-Induced Activation of NK Cells in Hepatitis C.

BackgroundImmuno-genetic studies suggest a functional link between NK cells and λ-IFNs. We recently showed that NK cells are negative for the IFN-λ receptor IFN-λR1 and do not respond to IFN-λ, suggesting a rather indirect association between IL-28B genotype and NK cell activity.MethodsA total of 75 HCV(+) patients and 67 healthy controls were enrolled into this study. IL-28B (rs12979860) and IFNL-4 (rs368234815) genotypes were determined by rtPCR. Total PBMC, monocytes, and NK cells were stimulated with IL-29, the TLR-7/8 agonist R848, or a combination of both. NK cell IFN-γ response was analysed by FACS. IL-12 and IL-18 secretion of monocytes was studied by ELISA. In blocking experiments anti-IL-12/anti-IL-18 were used.ResultsFollowing stimulation of total PBMCs with R848 we found NK cell IFN- γ responses to vary with the IL-28B genotype, with carriers of a T/T genotype displaying the lowest frequency of IFN-γ(+)NK cells. When isolated NK cells were studied no such associations were observed, indicating an indirect association between IL-28B genotype and NK cell activity. Accordingly, we found R848-stimulated monocytes of patients with a T/T genotype to be significantly less effective in triggering NK cell IFN- γ production than monocytes from carriers of a non-T/T genotype. In line with these findings we observed monocytes from T/T patients to secrete significantly lower concentrations of IL-12 than monocytes from non-T/T individuals.ConclusionsOur data indicate that monocytes from carriers of an IL-28B T/T genotype display a reduced ability to stimulate NK cell activity and, thus, provide a link between IL-28B genotype and NK functions.

Regulation of Natural Killer cell cytotoxicity by shedding of the Fc receptor CD16

Abstract Natural Killer (NK) cells are cytotoxic lymphocytes that can recognize and kill virally infected or tumour transformed cells by the secretion of cytolytic granules containing perforin. An individual NK cell can kill several target cells sequentially. Each target cell can trigger NK cell activation via different activating ligands and here we report that the order in which ligands are encountered affects the NK cell response. When NK cells are repeatedly activated via their Fc receptor CD16, with the therapeutic antibody rituximab, perforin secretion decreases with each stimulation. However, perforin secretion is restored to its initial level upon subsequent activation by MICA, which ligates NKG2D. Repeated stimulation of NK cells via MICA also decreases the degranulation capacity of NK cells but, strikingly, this effect cannot be rescued by a subsequent stimulation with rituximab. We found that the mechanism underlying these differential outcomes involves shedding of CD16, which occurs upon NK cell activation through both CD16 and NKG2D. Shedding of CD16 renders the cells insensitive to further activation via that receptor but they remain competent for further activation by NKG2D. We also found that NK cells are more motile on rituximab-coated surfaces than on MICA-coated surfaces and their migration speed decreases upon inhibition of CD16 shedding. Thus, although CD16 shedding hinders NK cell responsiveness it can serve to augment NK cell motility to move between target cells. Together these data indicate that manipulation of CD16 shedding might be an important target for NK cell-based therapies including treatments with therapeutic antibodies.

Influence of In Vitro IL-2 or IL-15 Alone or in Combination with Hsp 70 Derived 14-Mer Peptide (TKD) on the Expression of NK Cell Activatory and Inhibitory Receptors on Peripheral Blood T Cells, B Cells and NKT Cells.

Previous studies from Multhoff and colleagues reported that plasma membrane Hsp70 acts as a tumour-specific recognition structure for activated NK cells, and that the incubation of NK cells with Hsp70 and/or a 14-mer peptide derived from the N-terminal sequence of Hsp70 (TKDNNLLGRFELSG, TKD, aa 450–463) plus a low dose of IL-2 triggers NK cell proliferation and migration, and their capacity to kill cancer cells expressing membrane Hsp70. Herein, we have used flow cytometry to determine the influence of in vitro stimulation of peripheral blood mononuclear cells from healthy individuals with IL-2 or IL-15, either alone or in combination with TKD peptide on the cell surface expression of CD94, NK cell activatory receptors (CD16, NK2D, NKG2C, NKp30, NKp44, NKp46, NKp80, KIR2DL4, DNAM-1 and LAMP1) and NK cell inhibitory receptors (NKG2A, KIR2DL2/L3, LIR1/ILT-2 and NKR-P1A) by CD3+CD56+ (NKT), CD3+CD4+, CD3+CD8+ and CD19+ populations. NKG2D, DNAM-1, LAMP1 and NKR-P1A expression was upregulated after the stimulation with IL-2 or IL-15 alone or in combination with TKD in NKT, CD8+ T cells and B cells. CD94 was upregulated in NKT and CD8+ T cells. Concurrently, an increase in a number of CD8+ T cells expressing LIR1/ILT-2 and CD4+ T cells positive for NKR-P1A was observed. The proportion of CD8+ T cells that expressed NKG2D was higher after IL-2/TKD treatment, when compared with IL-2 treatment alone. In comparison with IL-15 alone, IL-15/TKD treatment increased the proportion of NKT cells that were positive for CD94, LAMP1 and NKRP-1A. The more potent effect of IL-15/TKD on cell surface expression of NKG2D, LIR1/ILT-2 and NKRP-1A was observed in B cells compared with IL-15 alone. However, this increase was not of statistical significance. IL-2/TKD induced significant upregulation of LAMP1 in CD8+ T cells compared with IL-2 alone. Besides NK cells, other immunocompetent cells present within the fraction of peripheral blood mononuclear cells were influenced by the treatment with low-dose interleukins themselves or in combination with hsp70 derived (TKD) peptide.

Priming of Human Resting NK Cells by Autologous M1 Macrophages via the Engagement of IL-1β, IFN-β, and IL-15 Pathways.

The cross talk between NK cells and macrophages is emerging as a major line of defense against microbial infections and tumors. This study reveals a complex network of soluble mediators and cell-to-cell interactions allowing human classically activated (M1) macrophages, but not resting (M0) or alternatively activated (M2) macrophages, to prime resting autologous NK cells. In this article, we show that M1 increase NK cell cytotoxicity by IL-23 and IFN-β-dependent upregulation of NKG2D, IL-1β-dependent upregulation of NKp44, and trans-presentation of IL-15. Moreover, both IFN-β-dependent cis-presentation of IL-15 on NK cells and engagement of the 2B4-CD48 pathway are used by M1 to trigger NK cell production of IFN-γ. The disclosure of these synergic cellular mechanisms regulating the M1-NK cell cross talk provides novel insights to better understand the role of innate immune responses in the physiopathology of tumor biology and microbial infections.

Poly-ICLC as an adjuvant for NY-ESO-1 protein vaccination with or without Montanide ISA-51 VG in patients with melanoma.

e14034 Background: Poly-ICLC (Hiltonol, Oncovir Inc) is a synthetic dsRNA complex which directly activates DCs and also triggers NK cells to kill tumor cells. Compared to LPS and R848, Poly-ICLC is...

TRAIL-producing NK cells contribute to liver injury and related fibrogenesis in the context of GNMT deficiency

Glycine-N-methyltransferase (GNMT) is essential to preserve liver homeostasis. Cirrhotic patients show low expression of GNMT that is absent in hepatocellular carcinoma (HCC) samples. Accordingly, GNMT deficiency in mice leads to steatohepatitis, fibrosis, cirrhosis, and HCC. Lack of GNMT triggers NK cell activation in GNMT−/− mice and depletion of TRAIL significantly attenuates acute liver injury and inflammation in these animals. Chronic inflammation leads to fibrogenesis, further contributing to the progression of chronic liver injury regardless of the etiology. The aim of our study is to elucidate the implication of TRAIL-producing NK cells in the progression of chronic liver injury and fibrogenesis. For this we generated double TRAIL−/−/GNMT−/− mice in which we found that TRAIL deficiency efficiently protected the liver against chronic liver injury and fibrogenesis in the context of GNMT deficiency. Next, to better delineate the implication of TRAIL-producing NK cells during fibrogenesis we performed bile duct ligation (BDL) to GNMT−/− and TRAIL−/−/GNMT−/− mice. In GNMT−/− mice, exacerbated fibrogenic response after BDL concurred with NK1.1+ cell activation. Importantly, specific inhibition of TRAIL-producing NK cells efficiently protected GNMT−/− mice from BDL-induced liver injury and fibrogenesis. Finally, TRAIL−/−/GNMT−/− mice showed significantly less fibrosis after BDL than GNMT−/− mice further underlining the relevance of the TRAIL/DR5 axis in mediating liver injury and fibrogenesis in GNMT−/− mice. Finally, in vivo silencing of DR5 efficiently protected GNMT−/− mice from BDL-liver injury and fibrogenesis, overall underscoring the key role of the TRAIL/DR5 axis in promoting fibrogenesis in the context of absence of GNMT. Overall, our work demonstrates that TRAIL-producing NK cells actively contribute to liver injury and further fibrogenesis in the pathological context of GNMT deficiency, a molecular scenario characteristic of chronic human liver disease.

Phase I/II study of the TLR3 agonist poly-ICLC as an adjuvant for NY-ESO-1 protein vaccination with or without Montanide ISA-51 vg in patients with melanoma.

TPS9119 Background: Poly-ICLC is a synthetic dsRNA complex which directly activates DCs and also triggers NK cells to kill tumor cells. Compared to LPS and R848, Poly-ICLC is the most potent TLR ad...

Human Cytomegalovirus Infection Promotes Rapid Maturation of NK Cells Expressing Activating Killer Ig–like Receptor in Patients Transplanted with NKG2C−/− Umbilical Cord Blood

NK cells are the first lymphoid population recovering after allogeneic hematopoietic stem cell transplantation and play a crucial role in early immunity after the graft. Recently, it has been shown that human CMV (HCMV) infection/reactivation can deeply influence NK cell reconstitution after umbilical cord blood transplantation by accelerating the differentiation of mature NKG2A(-) killer Ig-like receptor (KIR)(+) NK cells characterized by the expression of the NKG2C-activating receptor. In view of the hypothesis that NKG2C could be directly involved in NK cell maturation driven by HCMV infection, we analyzed the maturation and function of NK cells developing in three patients with hematological malignancies given umbilical cord blood transplantation from donors carrying a homozygous deletion of the NKG2C gene. We show that HCMV infection can drive rapid NK maturation, characterized by the expansion of CD56(dim)NKG2A(-)KIR(+) cells, even in the absence of NKG2C expression. Interestingly, this expanded mature NK cell subset expressed surface-activating KIR that could trigger NK cell cytotoxicity, degranulation, and IFN-γ release. Given the absence of NKG2C, it is conceivable that activating KIRs may play a role in the HCMV-driven NK cell maturation and that NK cells expressing activating KIRs might contribute, at least in part, to the control of infections after transplantation.

Ratios Of FcγRIIa and FcγRIII Binding Affinities Govern The Efficacy Of PMN-Mediated Cytotoxicity – Implications For Fc-Engineering Approaches Of Therapeutic Antibodies

IntroductionAntibody-dependent cell-mediated cytotoxicity (ADCC) has been suggested to be an essential effector mechanism for the in vivo activity of tumor-targeting therapeutic monoclonal antibodies (mAbs). Thus, enhancing the affinity of human IgG1 mAbs to NK cell-expressed FcγRIIIa by glyco- or protein-engineering of their Fc moiety has been demonstrated to improve NK cell-mediated ADCC and represents a promising strategy to improve antibody therapy. However, human polymorphonuclear (PMN) cells express the homologous FcγRIIIb isoform, which does not trigger ADCC. The aim of the present study was to analyze a panel of distinct IgG1 mAbs, displaying different affinities for FcγRIIa and FcγRIII, with respect to PMN recruitment for tumor cell destruction. MethodsAffinities of analyzed mAbs to distinct FcγR were determined by surface plasmon resonance technology. Induction of ADCC was assessed by 51Cr release experiments in the absence or presence of FcγRIII- or FcγRII-blocking agents to unravel the contribution of both receptors in these assays. ResultsNon-fucosylated or protein-engineered IgG1 variants with optimized FcγRIII binding capacities demonstrated the expected benefit in triggering NK cell- mediated ADCC but did not mediate ADCC by PMN, which could be restored by FcγRIIIb blockade. Additionally, eosinophils as well as PMN from paroxysmal nocturnal hemoglobinuria (PNH) patients – expressing no or low levels of FcgRIIIb – mediated effective ADCC with FcgRIII-optimized mAbs. Additional experiments with Fc variants displaying enhanced FcγRIIa binding or with double FcγRIIa/FcγRIII-optimized constructs demonstrated enhanced PMN-mediated ADCC compared to control mAbs. Statistical analyses revealed that FcγRIIa/FcγRIIIb affinity ratios correlated with the extent of human PMN-mediated ADCC. ConclusionsTo conclude, the present study represents novel findings concerning recruitment of PMN for tumor cell destruction by Fc-engineered antibodies. While PMN-mediated ADCC was completely abolished by FcγRIII-optimized antibodies through predominant FcγRIIIb binding, it was potently enhanced by optimization of FcγRIIa binding affinity. Importantly, functional analyses unraveled the ratio between FcγRIIa and FcγRIII binding affinities to control PMN-mediated ADCC activity and therefore opened new alleys in engineering of therapeutic antibodies. Disclosures:Muchhal:Xencor Inc.: Employment. Desjarlais:Xencor Inc.: Employment.

Inhibition of Glycogen Synthase Kinase-3 Increases NKG2D Ligand MICA Expression and Sensitivity to NK Cell–Mediated Cytotoxicity in Multiple Myeloma Cells: Role of STAT3

Engagement of NKG2D and DNAX accessory molecule-1 (DNAM-1) receptors on lymphocytes plays an important role for anticancer response and represents an interesting therapeutic target for pharmacological modulation. In this study, we investigated the effect of inhibitors targeting the glycogen synthase kinase-3 (GSK3) on the expression of NKG2D and DNAM-1 ligands in multiple myeloma (MM) cells. GSK3 is a pleiotropic serine-threonine kinase point of convergence of numerous cell-signaling pathways, able to regulate the proliferation and survival of cancer cells, including MM. We found that inhibition of GSK3 upregulates both MICA protein surface and mRNA expression in MM cells, with little or no effects on the basal expression of the MICB and DNAM-1 ligand poliovirus receptor/CD155. Moreover, exposure to GSK3 inhibitors renders myeloma cells more efficient to activate NK cell degranulation and to enhance the ability of myeloma cells to trigger NK cell-mediated cytotoxicity. We could exclude that increased expression of β-catenin or activation of the heat shock factor-1 (transcription factors inhibited by active GSK3) is involved in the upregulation of MICA expression, by using RNA interference or viral transduction of constitutive active forms. On the contrary, inhibition of GSK3 correlated with a downregulation of STAT3 activation, a negative regulator of MICA transcription. Both Tyr(705) phosphorylation and binding of STAT3 on MICA promoter are reduced by GSK3 inhibitors; in addition, overexpression of a constitutively active form of STAT3 significantly inhibits MICA upregulation. Thus, we provide evidence that regulation of the NKG2D-ligand MICA expression may represent an additional immune-mediated mechanism supporting the antimyeloma activity of GSK3 inhibitors.