Regulating NK Cell Activation Research Articles

MTOR Signaling Promotes Rapid m6A mRNA Methylation to Regulate NK-Cell Activation and Effector Functions.

NK cells can be rapidly activated in response to cytokines during host defense against malignant cells or viral infection. However, it remains unclear what mechanisms precisely and rapidly regulate the expression of a large number of genes involved in activating NK cells. In this study, we discovered that NK-cell N6-methyladenosine (m6A) methylation levels were rapidly upregulated upon short-term NK-cell activation and were repressed in the tumor microenvironment (TME). Deficiency of methyltransferase-like 3 (METTL3) or METTL14 moderately influenced NK-cell homeostasis, while double-knockout of METTL3/14 more significantly impacted NK-cell homeostasis, maturation, and antitumor immunity. This suggests a cooperative role of METTL3 and METTL14 in regulating NK-cell development and effector functions. Using methylated RNA immunoprecipitation sequencing, we demonstrated that genes involved in NK-cell effector functions, such as Prf1 and Gzmb, were directly modified by m6A methylation. Furthermore, inhibiting mTOR complex 1 activation prevented m6A methylation levels from increasing when NK cells were activated, and this could be restored by S-adenosylmethionine supplementation. Collectively, we have unraveled crucial roles for rapid m6A mRNA methylation downstream of the mTOR complex 1-S-adenosylmethionine signal axis in regulating NK-cell activation and effector functions.

Inhibition of NK cell cytotoxicity by tubular epithelial cell expression of Clr-b and Clr-f

NK cells participate in ischemia reperfusion injury (IRI) and transplant rejection. Endogenous regulatory systems may exist to attenuate NK cell activation and cytotoxicity in IRI associated with kidney transplantation. A greater understanding of NK regulation will provide insights in transplant outcomes and could direct new therapeutic strategies. Kidney tubular epithelial cells (TECs) may negatively regulate NK cell activation by their surface expression of a complex family of C-type lectin-related proteins (Clrs). We have found that Clr-b and Clr-f were expressed by TECs. Clr-b was upregulated by inflammatory cytokines TNFα and IFNγ in vitro. Silencing of both Clr-b and Clr-f expression using siRNA resulted in increased NK cell killing of TECs compared to silencing of either Clr-b or Clr-f alone (p < 0.01) and when compared to control TECs (p < 0.001). NK cells treated in vitro with soluble Clr-b and Clr-f proteins reduced their capacity to kill TECs (p < 0.05). Hence, NK cell cytotoxicity can be inhibited by Clr proteins on the surface of TECs. Our study suggests a synergistic effect of Clr molecules in regulating NK cell function in renal cells and this may represent an important endogenous regulatory system to limit NK cell-mediated organ injury during inflammation.

Role of NK cells in cord blood transplantation and their enhancement by the missing ligand effect of the killer-immunoglobulin like receptor.

Natural killer (NK) cells are the first lymphocytes reconstituted after allogenic hematopoietic stem cell transplantation (HSCT). Especially, in cord blood transplantation (CBT), the increase in the number of NK cells is sustained for a long period. Although there are conflicting results, many studies show that early reconstitution of NK cells is associated with favorable CBT outcomes, suggesting that maximizing NK cell functions could improve the CBT outcome. Killer immunoglobulin-like receptors (KIRs) include inhibitory and stimulatory receptors, which can regulate NK-cell activity. Because some of the KIRs have HLA class I as their ligand, the KIR-ligand interaction on NK cells can be lost in some cases of CBT, which results in the activation of NK cells and alters HSCT outcome. Thus, effects of KIR-ligand mismatch under various conditions have been widely examined; however, the results have been controversial. Among such studies, those using the largest number of CBTs showed that HLA-C2 (KIR2DL1-ligand) mismatches have a favorable effect on the relapse rate and overall survival only when the CBT used methotrexate for graft-versus-host disease prophylaxis. Another study suggested that KIR-ligand mismatch is involved in reducing the relapse of acute myeloid leukemia, mediated by reactivation of cytomegalovirus. These results indicate that activation of NK cells by KIR-ligand mismatch may have favorable effects on CBT outcomes and could help enhance the NK-cell function.

SLAMF7 selectively favors degranulation to promote cytotoxicity in human NK cells.

NK cells play an important role in immunity by recognizing and eliminating cells undergoing infection or malignant transformation. This role is dependent on the ability of NK cells to lyse targets cells in a perforin-dependent mechanism and by secreting inflammatory cytokines. Both effector functions are controlled by several cell surface receptors. The Signaling Lymphocyte Activation Molecule (SLAM) family of receptors plays an essential role in regulating NK cell activation. Several studies have demonstrated that SLAMF7 regulates NK cell activation. However, the molecular and cellular mechanisms by which SLAMF7 influences NK effector functions are unknown. Here, we present evidence that physiological ligation of SLAMF7 in human NK cells enhances the lysis of target cells expressing SLAMF7. This effect was dependent on the ability of SLAMF7 to promote NK cell degranulation rather than cytotoxic granule polarization or cell adhesion. Moreover, SLAMF7-dependent NK cell degranulation was predominantly dependent on PLC-γ when compared to PI3K. These data provide novel information on the cellular mechanism by which SLAMF7 regulates human NK cell activation. Finally, this study supports a model for NK cell activation where activated receptors contribute by regulating specific discrete cellular events rather than multiple cellular processes.

Plasma membrane lipid scrambling causing phosphatidylserine exposure negatively regulates NK cell activation.

One of the hallmarks of live cells is the asymmetric distribution of lipids across their plasma membrane. Changes in this asymmetry due to lipid "scrambling" result in phosphatidylserine exposure at the cell surface thatis detected by annexin V staining. This alteration is observed during cell death processes such as apoptosis, and during physiological responses such as platelet degranulation and membrane repair. Previous studies have shown that activation of NK cells is accompanied by exposure of phosphatidylserine at the cell surface. While this response was thought to be indicative of ongoing NK cell death, it may also reflectthe regulation of NK cell activation in the absence ofcell death. Herein, we found that NK cell activation was accompanied by rapid phosphatidylserine exposure to an extent proportional to the degree of NK cell activation. Through enforced expression of a lipid scramblase, we provided evidence that activation-induced lipid scrambling in NK cells is reversible and does not lead to cell death. In contrast, lipid scrambling attenuates NK cell activation. This response was accompanied by reduced cell surface expression of activating receptors such as 2B4, and by loss of binding ofSrc family protein tyrosine kinases Fyn and Lck to the inner leaflet of the plasma membrane. Hence, lipid scrambling during NK cell activation is, at least in part, a physiological response that reduces the NK cell activation level. This effect is due to the ability of lipid scrambling to alter the distribution of membrane-associated receptors and kinases required for NK cell activation.

Modeling the stimulatory network of CD16, 2B4 and NKG2D in natural killer cell activation

Abstract Natural killer (NK) cells are innate immune effector cells that play an immediate role in host defense by releasing cytotoxic chemicals upon activation. A better understanding of the mechanisms regulating NK cell activation can improve NK cell-based therapies. However, due to the diversity of stimulatory pathways, each with a complex network of signaling species, a systems biology approach is required to understand NK cell activation. We constructed a mathematical model that mechanistically describes the signaling network of CD16, 2B4 and NKG2D. The model was fit to published experimental data and validated with a separate dataset. Baseline model predictions demonstrate that CD16 and NKG2D are qualitatively similar in that they activate the downstream species to a greater degree and at a faster rate when compared to 2B4. Contrastingly, 2B4 activates the signaling species over a longer time interval. In silico pairwise co-stimulation of the receptors, using various ligand concentrations, preferentially impacts the kinetics of species activation: the species are activated faster but the time interval of species activation becomes shorter as more ligands are introduced. Perturbing the stimulatory pathways by altering kinase (activator) and phosphatase (inhibitor) activity, highlights the phosphatases’ predominant control of the network and shows that decreasing their level of activity greatly improves the rate of species activation. Thus, the model predicts (1) qualitative differences between the pathways, (2) increasing receptor stimulation impacts the kinetics of species activation and (3) inhibiting the inhibitor significantly augments signal transduction. This insight can help design NK cell immunotherapies.

Inhibition of Enhancer of zeste homolog 2 (EZH2) induces natural killer cell-mediated eradication of hepatocellular carcinoma cells.

Natural killer (NK) cell-mediated tumor cell eradication could inhibit tumor initiation and progression. However, the factors that regulate NK cell-mediated cancer cell eradication remain unclear. We determined that hepatocellular carcinoma (HCC) cells exhibit transcriptional down-regulation of NK group 2D (NKG2D) ligands and are largely resistant to NK cell-mediated eradication. Because the down-regulation of NKG2D ligands occurred at the transcriptional level, we tested 32 chemical inhibitors of epigenetic regulators for their ability to re-express NKG2D ligands and enhance HCC cell eradication by NK cells and found that Enhancer of zeste homolog 2 (EZH2) was a transcriptional repressor of NKG2D ligands. The inhibition of EZH2 by small-molecule inhibitors or genetic means enhanced HCC cell eradication by NK cells in a NKG2D ligand-dependent manner. Collectively, these results demonstrate that EZH2 inhibition enhances HCC eradication by NK cells and that EZH2 functions, in part, as an oncogene by inhibiting immune response.

Lymphocytes Negatively Regulate NK Cell Activity via Qa-1b following Viral Infection

NK cells can reduce anti-viral Tcell immunity during chronic viral infections, including infection with the lymphocytic choriomeningitis virus (LCMV). However, regulating factors that maintain the equilibrium between productive Tcell and NK cell immunity are poorly understood. Here, we show that a large viral load resulted in inhibition of NK cell activation, which correlated with increased expression of Qa-1b, a ligand for inhibitory NK cell receptors. Qa-1b was predominantly upregulated on B cells following LCMV infection, and this upregulation was dependent on type I interferons. Absence of Qa-1b resulted in increased NK cell-mediated regulation of anti-viral T cells following viral infection. Consequently, anti-viral Tcell immunity was reduced in Qa-1b- and NKG2A-deficient mice, resulting in increased viral replication and immunopathology. NK cell depletion restored anti-viral immunity and virus control in the absence of Qa-1b. Taken together, our findings indicate that lymphocytes limit NK cell activity during viral infection in order to promote anti-viral Tcell immunity.

Allele-Independent Turnover of Human Leukocyte Antigen (HLA) Class Ia Molecules.

Major histocompatibility complex class I (MHCI) glycoproteins present cytosolic peptides to CD8+ T cells and regulate NK cell activity. Their heavy chains (HC) are expressed from up to three MHC gene loci (human leukocyte antigen [HLA]-A, -B, and -C in humans), whose extensive polymorphism maps predominantly to the antigen-binding groove, diversifying the bound peptide repertoire. Codominant expression of MHCI alleles is thus functionally critical, but how it is regulated is not fully understood. Here, we have examined the effect of polymorphism on the turnover rates of MHCI molecules in cell lines with functional MHCI peptide loading pathways and in monocyte-derived dendritic cells (MoDCs). Proteins were labeled biosynthetically with heavy water (2H2O), folded MHCI molecules immunoprecipitated, and tryptic digests analysed by mass spectrometry. MHCI-derived peptides were assigned to specific alleles and isotypes, and turnover rates quantified by 2H incorporation, after correcting for cell growth. MHCI turnover half-lives ranged from undetectable to a few hours, depending on cell type, activation state, donor, and MHCI isotype. However, in all settings, the turnover half-lives of alleles of the same isotype were similar. Thus, MHCI protein turnover rates appear to be allele-independent in normal human cells. We propose that this is an important feature enabling the normal function and codominant expression of MHCI alleles.

Expression of the inhibitory Ly49E receptor is not critically involved in the immune response against cutaneous, pulmonary or liver tumours.

Natural killer (NK) lymphocytes are part of the innate immune system and are important in immune protection against tumourigenesis. NK cells display a broad repertoire of activating and inhibitory cell surface receptors that regulate NK cell activity. The Ly49 family of NK receptors is composed of several members that recognize major histocompatibility complex class I (MHC-I) or MHC-I-related molecules. Ly49E is a unique inhibitory member, being triggered by the non-MHC-I-related protein urokinase plasminogen activator (uPA) in contrast to the known MHC-I-triggering of the other inhibitory Ly49 receptors. Ly49E also has an uncommon expression pattern on NK cells, including high expression on liver DX5− NK cells. Furthermore, Ly49E is the only Ly49 member expressed by epidermal γδ T cells. As γδ T cells and/or NK cells have been shown to be involved in the regulation of cutaneous, pulmonary and liver malignancies, and as uPA is involved in tumourigenesis, we investigated the role of the inhibitory Ly49E receptor in the anti-tumour immune response. We demonstrate that, although Ly49E is highly expressed on epidermal γδ T cells and liver NK cells, this receptor does not play a major role in the control of skin tumour formation or in lung and liver tumour development.

Novel Multi-Target Immunosuppressive Approach for Treatment of Severe Aplastic Anemia

Aplastic Anemia (AA) is an immune-mediated form of acquired bone marrow failure (BMF), which is life-threatening in its severe form (SAA). Fundamental pathological features of AA include development and expansion of auto-reactive effector T cells, effector T cell-mediated apoptosis of all hematopoietic cells (including progenitors and hematopoietic stem cells (HSCs)), BM aplasia, pancytopenia, depletion of HSCs, and severe reduction and functional impairment of regulatory T cells (Tregs).Current standard treatments for AA include: (1) immuno-suppressive therapy (IST) with cyclosporine A (CyA) and anti-thymocyte globulin (ATG) which targets all T cells, and (2) allogeneic or matched unrelated donor BM transplant. While IST remains a standard treatment modality, it is not very effective in treating already ongoing and relapsed AA or SAA. There is really no effective therapy for patients with refractory and relapsed AA who are ineligible for BMT.Among different pathophysiological features of AA, IST targets only the effector T cells, and is much more effective in the early than later stages of AA. Moreover, the combination of IST with other immunosuppressive agents (mycophenolate mofetil, sirolimus etc.) or growth factors does not improve the response or survival of AA patients. Since the incidence of AA is on the rise, there is an urgent need for more efficient new therapies that can attenuate the progression and severity of AA in patients with refractory and relapsed AA who are waiting for or are not candidates for BMT.The complex immune and hematological pathophysiology of AA requires new multipurpose treatment approaches. Accumulating evidence shows that β2 integrin CD11b/CD18 (Mac-1) negatively regulates T cell responses and activation, attenuates inflammation, and facilitates the maintenance of tolerance to self-antigens. For example, activated Mac-1 significantly reduces the T cell-activating capacity of dendritic cells (DCs), represses DC cross-priming of cytotoxic T cells, negatively regulates NK cell activation and function, suppresses differentiation of Th17 T cells which are associated with AA and other autoimmune diseases, ameliorates experimental autoimmune hepatitis, and negatively regulates BCR signaling and maintains autoreactive B cell tolerance. For that reason, Mac-1 is an attractive molecular target for new immune-modulating therapies of autoimmune diseases.Using the clinically relevant mouse SAA model we have evaluated the therapeutic efficacy of Leukadherins (LA1-LA3), novel small molecule agonists and activators of Mac-1, as a novel multipurpose immunosuppressive and anti-inflammatory approach to treat AA. The present studies have demonstrated that administration of LA1 safely and significantly (1) suppresses expansion of effector T cells, (2) decreases effector T cell-mediated apoptosis of target BM cells, (2) reduces BM aplasia, (3) minimalizes the loss of HSCs and progenitors, and (4) attenuates the severity of SAA. Furthermore, prolonged treatment of developing SAA with LA1 has therapeutic effects since it not only attenuates the progression and severity of SAA, but also converts otherwise fatal SAA into a survivable disease in mouse SAA models.To begin to address mechanism for these findings we found that LA1 treatment significantly reduces the antigen presenting capacity and T cell activating capacity of DCs. Importantly, in vivo LA1 treatment significantly increases the population of regulatory T cells (Tregs) which may also contribute to the above effects of LA1 on SAA.Simultaneous targeting of multiple pathophysiological features of AA underscores the clinically relevant potential of LA1 treatment as a novel promising multi-target immunosuppressive therapy that can safely and efficiently attenuate the severity of AA and reduce the need for BMT. We are also further evaluating the potential of LA1 treatment combined with IST or in vivo Treg expansion approaches (low dose rIL-2 therapy) to safely and more effectively attenuate the progression and severity of AA in pre-clinical mouse SAA models. These studies will provide an important platform for further translational and clinical testing of LAs as: (1) New therapy to manage ongoing AA in patients who are not responding to IST and are not candidates for BMT, (2) New therapy for relapsed AA, and/or (3) Adjuvant therapy for AA patients who are undergoing IST and are awaiting BM transplant. DisclosuresLevy:Allergan: Consultancy.

NK cell function triggered by multiple activating receptors is negatively regulated by glycogen synthase kinase-3β

Activation of NK cells is triggered by combined signals from multiple activating receptors that belong to different families. Several NK cell activating receptors have been identified, but their role in the regulation of effector functions is primarily understood in the context of their individual engagement. Therefore, little is known about the signaling pathways broadly implicated by the multiple NK cell activation cues. Here we provide evidence pointing to glycogen synthase kinase (GSK)-3β as a negative regulator of multiple NK cell activating signals. Using an activation model that combines NKG2D and 2B4 and tests different signaling molecules, we found that GSK-3 undergoes inhibitory phosphorylation at regulatory serine residues by the engagement of NKG2D and 2B4, either individually or in combination. The extent of such phosphorylation was closely correlated with the degree of NK cell activation. NK cell functions, such as cytokine production and cytotoxicity, were consistently enhanced by the knockdown of GSK-3β or its inhibition with different pharmacological inhibitors, whereas inhibition of the GSK-3α isoform had no effect. In addition, NK cell function was augmented by the overexpression of a catalytically inactive form of GSK-3β. Importantly, the regulation of NK cell function by GSK-3β was common to diverse activating receptors that signal through both ITAM and non-ITAM pathways. Thus, our results suggest that GSK-3β negatively regulates NK cell activation and that modulation of GSK-3β function could be used to enhance NK cell activation.

Cytokine-Mediated Activation of NK Cells during Viral Infection.

Natural killer (NK) cells provide a first line of defense against infection via the production of antiviral cytokines and direct lysis of target cells. Cytokines such as interleukin 12 (IL-12) and IL-18 are critical regulators of NK cell activation, but much remains to be learned about how cytokines interact to regulate NK cell function. Here, we have examined cytokine-mediated activation of NK cells during infection with two natural mouse pathogens, lymphocytic choriomeningitis virus (LCMV) and murine cytomegalovirus (MCMV). Using a systematic screen of 1,849 cytokine pairs, we identified the most potent combinations capable of eliciting gamma interferon (IFN-γ) production in NK cells. We observed that NK cell responses to cytokine stimulation were reduced 8 days after acute LCMV infection but recovered to preinfection levels by 60 days postinfection. In contrast, during MCMV infection, NK cell responses to cytokines remained robust at all time points examined. Ly49H-positive (Ly49H+) NK cells recognizing viral ligand m157 showed preferential proliferation during early MCMV infection. A population of these cells was still detected beyond 60 days postinfection, but these divided cells did not demonstrate enhanced IFN-γ production in response to innate cytokine stimulation. Instead, the maturation state of the NK cells (as determined by CD11b or CD27 surface phenotype) was predictive of responsiveness to cytokines, regardless of Ly49H expression. These results help define cytokine interactions that regulate NK cell activation and highlight variations in NK cell function during two unrelated viral infections. Natural killer cells play an important role in immunity to many viral infections. From an initial screen of 1,849 cytokine pairs, we identified the most stimulatory cytokine combinations capable of inducing IFN-γ production by NK cells. Ly49H+ NK cells, which can be directly activated by MCMV protein m157, preferentially proliferated during MCMV infection but did not show enhanced IFN-γ production following direct ex vivo cytokine stimulation. Instead, mature CD11b+ and/or CD27+ NK cells responded similarly to innate cytokine stimulation regardless of Ly49H expression. Collectively, our data provide a better foundation for understanding cytokine-mediated NK cell activation during viral infection.

Mechanisms Regulating NK Cell Activation During Viral Infection

ABSTRACT NK cells constitute a population of lymphocytes involved in innate immune functions. They play a critical role in antiviral immune surveillance. Viruses have evolved with their host species for millions of years, each exerting a selective pressure upon the other. As a corollary, the pathways used by the immune system that are critical to control viral infection can be revealed by defining the role of viral gene products that are nonessential for virus replication. We relate here the battery of resources available to NK cells to recognize and eliminate viruses and reciprocally the immune evasion mechanisms developed by viruses to prevent NK cell activation.

Mechanisms Underlying the Antifatigue Effects of the Mycelium Extract of &amp;lt;i&amp;gt;Cordyceps&amp;lt;/i&amp;gt; (&amp;lt;i&amp;gt;Paecilomyces hepiali&amp;lt;/i&amp;gt;, CBG-CS-2) in Mice in the Forced Swimming Test

Cordyceps (CS) is used as an alternative medicine and functional food. We examined in vivo mechanisms underlying the antifatigue effects of the cultured mycelium extract of CS (CS extract) in forced swimming mice, a fatigue model that is induced by muscle exercise. Animals orally administered with CS extract significantly extended the loaded forced-swimming time, indicating its antifatigue effects. CS extract modulated the increased levels of blood IL-6 that was induced by forced swimming. CS extract protected the forced swimming-induced increase in NKp46 expression of splenic NK cells, suggesting regulation of fatigue-elicited hyper-reactivity by activated NK cells. By DNA microarray analysis of the quadriceps femoris muscle, it was uncovered that CS extract prevented the forced swimming-mediated upregulation of the expression of 5 genes associating with muscular inflammation (Ccl6, Ccl8, and Wfdc17) and muscle regeneration (Sfrp4 and Nfil3), whereas it regulated the downregulation in the expression of Svs5 participating in actin binding. CS extract exhibits the antifatigue effects through preventing IL-6 accumulation in blood, regulating NK cell activation in the spleen, and alleviating altered expression of genes related to inflammation, regeneration, and actin binding in the local muscle. Thus, CS extract is an effective functional food for preventing fatigue.

Abstract 651: Ex vivo and in vivo characterization of IPH4102, a humanized anti-KIR3DL2 antibody for the treatment of cutaneous T-cell lymphomas

Abstract KIR3DL2 belongs to the killer immunoglobulin (Ig)-like receptors (KIRs) family and is composed of three extracellular Ig-like domains. KIR3DL2 is naturally expressed on some NK cells and minor subpopulations of CD8+ and CD4+ T cells. Physiologically, KIRD3L2 is an inhibitory receptor for human leukocyte antigen (HLA) class I molécules regulating NK cell activation. Interestingly, KIR3DL2 is also expressed on several subtypes of T lymphomas/leukemias, and in particular on Advanced cutaneous T cell lymphomas (CTCL), making it a unique therapeutic target in cancer. From a wide collection of anti-KIR3DL2 monoclonal antibodies (mAbs) with distinct properties related to several epitopes, we have selected IPH4102 as the lead therapeutic candidate. IPH4102 is a humanized IgG1 that selectively kills KIR3DL2-positive cells in vitro, mainly through antibody-dependent cell cytotoxicity (ADCC). IPH4102 also improves survival of mice engrafted with KIR3DL2-positive tumors. More importantly, IPH4102 exerts remarkable anti-tumor activity against primary KIR3DL2-positive tumor cells ex vivo: using blood taken from Sézary Syndrome patients as experimental model, we demonstrated the efficient killing of primary leukemic cells by autologous NK cells engaged by IPH4102. IPH4102 IND-enabling studies are ongoing. In parallel, other anti-KIR3DL2 mAbs were also developed as unique and sensitive tools for the detection by immunohistochemistry of KIR3DL2 on tumor biopsies. Owing to its efficacy profile and to the highly restricted expression pattern of its target KIR3DL2, IPH4102 stands as a very promising therapeutic option for the treatment of advanced cutaneous T cell lymphomas. Citation Format: Anne Marie-Cardine, Nicolas Viaud, Arnaud Dujardin, Rachel Joly, Laurent Gauthier, Cecile Bonnafous, Mathieu Blery, Carine Paturel, Armand Bensussan, Martine Bagot, Helene Sicard. Ex vivo and in vivo characterization of IPH4102, a humanized anti-KIR3DL2 antibody for the treatment of cutaneous T-cell lymphomas . [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 651. doi:10.1158/1538-7445.AM2014-651

Effect of tumor cells and tumor microenvironment on NK-cell function.

The ability of tumors to manage an immune-mediated attack has been recently included in the "next generation" of cancer hallmarks. In solid tumors, the microenvironment that is generated during the first steps of tumor development has a pivotal role in immune regulation. An intricate net of cross-interactions occurring between tumor components, stromal cells, and resident or recruited immune cells skews the possible acute inflammatory response toward an aberrant ineffective chronic inflammatory status that favors the evasion from the host's defenses. Natural killer (NK) cells have powerful cytotoxic activity, but their activity may be eluded by the tumor microenvironment. Immunosubversion, immunoediting or immunoselection of poorly immunogenic tumor cells and interference with tumor infiltration play a major role in evading NK-cell responses to tumors. Tumor cells, tumor-associated fibroblasts and tumor-induced aberrant immune cells (i.e. tolerogenic or suppressive macrophages, dendritic cells (DCs) and T cells) can interfere with NK-cell activation pathways or the complex receptor array that regulate NK-cell activation and antitumor activity. Thus, the definition of tumor microenvironment-related immunosuppressive factors, along with the identification of new classes of tissue-residing NK-like innate lymphoid cells, represent key issues to design effective NK-cell-based therapies of solid tumors.

Gap Junction Intercellular Communications Regulate NK Cell Activation and Modulate NK Cytotoxic Capacity

Gap junctions (GJs) mediate intercellular communication between adjacent cells. Previously, we showed that connexin 43 (Cx43), the main GJ protein in the immune system, mediates Ag transfer between human dendritic cells (DCs) and is recruited to the immunological synapse during T cell priming. This crosstalk contributed to T cell activation, intracellular Ca(2+) responses, and cytokine release. However, the role of GJs in NK cell activation by DCs and NK cell-mediated cytotoxicity against tumor cells remains unknown. In this study, we found polarization of Cx43 at the NK/DC and NK/tumor cell-contact sites, accompanied by the formation of functional GJs between NK/DCs and NK/tumor cells, respectively. Cx43-GJ-mediated intercellular communication (GJIC) between human NK and DCs was bidirectional. Blockage of Cx43-GJIC inhibited NK cell activation, though it affected neither the phenotype nor the function of DCs. Cx43 knockdown or inhibition using mimetic peptides greatly reduced CD69 and CD25 expression and IFN-γ release by DC-stimulated NK cells. Moreover, blocking Cx43 strongly inhibited the NK cell-mediated tumor cell lysis associated with inhibition of granzyme B activity and Ca(2+) influx. Our data identify a novel and active role for Cx43-GJIC in human NK cell activation and antitumor effector functions that may be important for the design of new immune therapeutic strategies.

Regulatory dendritic cells suppress NK cell activation by promoting IL-10 secretion and activating an NKp46-mediated inhibitory circuit. (P1072)

Abstract Regulatory dendritic cells (regDC) constrain the immune response in physiological and pathological conditions including tumor settings. Cross-talk between mature dendritic cells (mDC) and NK cells leads to reciprocal activation of both cells, but the impact of regDC on NK cells remains unknown. Our aim was to investigate the functional outcome and mechanisms underlying the interactions between regDC and NK cells. Human regDC generated from monocyte-derived DC treated with LPS and dexamethasone, opposed to mDC, triggered very little amounts of IFN-γ secretion by NK cells. This effect was partially due to insufficient amounts of IL-18. Blocking experiments demonstrated that regDC have the potential ability to stimulate IFN-γ secretion by NK cells through NKp30, but that this effect was overridden by a suppressive circuit that involves IL-10, NK cell inhibitory receptors and, unexpectedly, engagement of the activating receptor NKp46. Surprisingly, the opposing role of NKp30 and NKp46 on IFN-γ secretion was also observed during the cross-talk between mDC and NK cells. Accordingly, similar amounts of NKp46 ligands were found to be expressed on mDC and regDC. Our findings unveil a previously unrecognized regulatory mechanism through which regDC regulate NK cell activation and identify a novel inhibitory role for NKp46 which counterbalances the stimulatory activity of NKp30. These results might be relevant in shutting-down NK cell-mediated anti-tumor immune responses.

Abstract 493: Antitumoral efficacy of therapeutic human anti-KIR antibody (BMS-986015/IPH2102) in a preclinical xenograft tumor model.

Abstract Natural Killer cells (NK cells) are lymphocytes able to recognize and kill tumors for which the expression of Major Histocompatibility Complex (MHC) class I molecules is altered. This “missing self” recognition is mediated in humans by the lack of engagement of MHC class I i.e. Human Leucocyte Antigens (HLA) molecules with NK cell inhibitory receptors that include Killer Immunoglobulin-like Receptors (KIR). Some tumors escape NK cell immune surveillance by increasing the expression of HLA molecules on their surface. Consequently, blocking interactions between KIR and HLA molecules constitutes an interesting therapeutic strategy. The anti-KIR2DL1/2/3-specific monoclonal antibody, BMS-986015/IPH2102, is a human IgG4 that is being developed for treating both hematologic malignancies and solid tumors. In rodents, the MHC class I inhibitory system regulating NK cell activation is based on lectin-like family Ly49 but the KIR molecules are not expressed. The objective of this study was to develop a preclinical model to assess the efficacy of the drug candidate used in clinical trials, BMS-986015/IPH2102. Mice expressing the human NK inhibitory KIR2DL3, on the surface of NK cells were generated on a RAG-1deficient background (KIRtgRAG mice). The human B cell lymphoma cell line, 721.221, transduced with HLA-Cw3 molecule, a ligand of KIR2DL3, was intra-venously engrafted in these mice. The expression of HLA-C by tumor cells was sufficient to allow them to escape control of NK cells, leading to mice death in around 30 days. IPH2102 treatment increased mice survival in a dose dependent manner when injected at the same time as the tumor challenge. This protective effect was NK cell mediated and directly correlated with the duration of KIR saturation. Interestingly, BMS-986015/IPH2102 treatment also improved survival in therapeutic conditions i.e. when the antibody was injected 5 days after the tumor, also in a NK cell dependent manner. In conclusion, this study showed efficacy of BMS-986015/IPH2102 as single agent in a HLA-Cw3-expressing tumor model and this xenogenic pre-clinical model will be an excellent tool to investigate the therapeutic benefits of combination treatments. Citation Format: Caroline Sola, Fabien Chanuc, Ariane Thielens, Nicolas Fuseri, Isabelle Palacios, Mathieu Blery, Pascale André, Eric Vivier, Sophie Ugolini, Robert Graziano, François Romagne, Cécile Bonnafous. Antitumoral efficacy of therapeutic human anti-KIR antibody (BMS-986015/IPH2102) in a preclinical xenograft tumor model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 493. doi:10.1158/1538-7445.AM2013-493