scholarly journals Innate Cytokine Induced Early Release of IFNγ and CC Chemokines from Hypoxic Human NK Cells Is Independent of Glucose

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 734 ◽  
Author(s):  
Sonia Y. Velásquez ◽  
Bianca S. Himmelhan ◽  
Nina Kassner ◽  
Anna Coulibaly ◽  
Jutta Schulte ◽  
...  
Keyword(s):  
Immune Response ◽  
Nk Cells ◽  
Nk Cell ◽  
Glucose Deprivation ◽  
Innate Immune ◽  
Independent Manner ◽  
Tissue Inflammation ◽  
Metabolic Switching ◽  
Cc Chemokines

Natural killer (NK) cells are among the first innate immune cells to arrive at sites of tissue inflammation and regulate the immune response to infection and tumors by the release of cytokines including interferon (IFN)γ. In vitro exposure to the innate cytokines interleukin 15 (IL-15) and IL-12/IL-18 enhances NK cell IFNγ production which, beyond 16 h of culture, was shown to depend on metabolic switching to glycolysis. NK effector responses are, however, rapid by comparison. Therefore, we sought to evaluate the importance of glycolysis for shorter-term IFNγ production, considering glucose deprivation and hypoxia as adverse tissue inflammation associated conditions. Treatments with IL-15 for 6 and 16 h were equally effective in priming early IFNγ production in human NK cells in response to secondary IL-12/IL-18 stimulation. Short-term priming was not associated with glycolytic switching but induced the release of IFNγ and, additionally, CCL3, CCL4 and CCL5 from both normoxic and hypoxic NK cells in an equally efficient and, unexpectedly, glucose independent manner. We conclude that release of IFNγ and CC chemokines in the early innate immune response is a metabolically autonomous NK effector program.

Human natural killer cells: a unique innate immunoregulatory role for the CD56bright subset

Blood ◽  
2001 ◽  
Vol 97 (10) ◽  
pp. 3146-3151 ◽  
Author(s):  
Megan A. Cooper ◽  
Todd A. Fehniger ◽  
Sarah C. Turner ◽  
Kenneth S. Chen ◽  
Bobak A. Ghaheri ◽  
...  
Keyword(s):  
Immune Response ◽  
Nk Cells ◽  
Natural Killer ◽  
Innate Immune Response ◽  
Nk Cell ◽  
Primary Source ◽  
Interferon Γ ◽  
Innate Immune ◽  
Immunoregulatory Cytokines ◽  
Human Natural Killer Cells

Abstract During the innate immune response to infection, monocyte-derived cytokines (monokines), stimulate natural killer (NK) cells to produce immunoregulatory cytokines that are important to the host's early defense. Human NK cell subsets can be distinguished by CD56 surface density expression (ie, CD56bright and CD56dim). In this report, it is shown that CD56bright NK cells produce significantly greater levels of interferon-γ, tumor necrosis factor-β, granulocyte macrophage–colony-stimulating factor, IL-10, and IL-13 protein in response to monokine stimulation than do CD56dim NK cells, which produce negligible amounts of these cytokines. Further, qualitative differences in CD56bright NK-derived cytokines are shown to be dependent on the specific monokines present. For example, the monokine IL-15 appears to be required for type 2 cytokine production by CD56bright NK cells. It is proposed that human CD56bright NK cells have a unique functional role in the innate immune response as the primary source of NK cell–derived immunoregulatory cytokines, regulated in part by differential monokine production.

Adrenal tumors provide insight into the role of cortisol in NK cell activity

2021 ◽  
Author(s):  
Andrew E. Greenstein ◽  
Mouhammed Amir Habra ◽  
Subhagya A. Wadekar ◽  
Andreas Grauer
Keyword(s):  
Immune Response ◽  
Nk Cells ◽  
Cell Activation ◽  
Nk Cell ◽  
Phase 1 ◽  
Cell Activity ◽  
The Cancer Genome Atlas ◽  
Adrenal Tumors ◽  
Steroid Synthesis

Elevated glucocorticoid (GC) activity may limit tumor immune response and immune checkpoint inhibitor (ICI) efficacy. Adrenocortical carcinoma (ACC) provides a unique test case to assess correlates of GC activity, as approximately half of ACC patients exhibit excess GC production (GC+). ACC multi-omics were analyzed to identify molecular consequences of GC+ and assess the rationale for combining the glucocorticoid receptor (GR) antagonist relacorilant with an ICI. GC status, mRNA expression, and DNA mutation and methylation data from 71 adrenal tumors were accessed via The Cancer Genome Atlas. Expression of 858 genes differed significantly between GC- and GC+ ACC cases. KEGG pathway analysis showed higher gene expression of 3 pathways involved in steroid synthesis and secretion in GC+ cases. Fifteen pathways, most related to NK cells and other immune activity, showed lower expression. Hypomethylation was primarily observed in the steroid synthesis pathways. Tumor-infiltrating CD4+ memory (P=.003), CD8+ memory (P=.001), and NKT-cells (P=.014) were depleted in GC+ cases; tumor-associated neutrophils were enriched (P=.001). Given the pronounced differences between GC+ and GC- ACC, the effects of cortisol on NK cells were assessed in vitro (NK cells from human PBMCs stimulated with IL-2 or IL-12/15). Cortisol suppressed, and relacorilant restored, NK cell activation, proliferation, and direct tumor cell killing. Thus, GR antagonism may increase the abundance and function of NK and other immune cells in the tumor microenvironment, promoting immune response in GC+ ACC and other malignancies with GC+. This hypothesis will be tested in a phase 1 trial of relacorilant + ICI.

scholarly journals Stem Cell Factor Enhances Interleukin-2–Mediated Expansion of Murine Natural Killer Cells In Vivo

Blood ◽  
1997 ◽  
Vol 90 (9) ◽  
pp. 3647-3653 ◽  
Author(s):  
Todd A. Fehniger ◽  
William E. Carson ◽  
Ewa Mrózek ◽  
Michael A. Caligiuri
Keyword(s):  
Nk Cells ◽  
Cell Expansion ◽  
Stem Cell Factor ◽  
Low Dose ◽  
Nk Cell ◽  
Interleukin 2 ◽  
Innate Immune ◽  
Ifn Γ

Abstract The administration of low dose interleukin-2 (IL-2) results in a selective expansion of natural killer (NK) cells in vivo, and promotes the differentiation of NK cells from hematopoietic precursor cells in vitro. We have previously shown that stem cell factor (SCF ), the ligand to the c-kit tyrosine kinase receptor, enhances IL-2–induced NK cell proliferation and differentiation in vitro. Here, we investigated the effects of SCF plus IL-2 delivered to mice in vivo. Eight-week-old C57BL/6 mice were treated with a continuous subcutaneous infusion of IL-2 (1 × 104 IU/d) plus a daily intraperitoneal dose of SCF (100 μg/kg/d), IL-2 alone, SCF alone, or vehicle alone for 8 weeks. The in vivo serum concentration of IL-2 ranged between 352 ± 12.0 pg/mL and 606 ± 9.0 pg/mL, achieving selective saturation of the high affinity IL-2 receptor, while the peak SCF serum concentration was 296 ± 13.09 ng/mL. Alone, the daily administration of SCF had no effect on the expansion of NK cells. The continuous infusion of IL-2 alone did result in a significant expansion of NK1.1+CD3− cells compared to mice treated with placebo or SCF. However, mice treated with both SCF and IL-2 showed an increase in the absolute number of NK cells that was more than twofold that seen with IL-2 alone, in the spleen (P ≤ .005), bone marrow (P ≤ .025), and blood (P < .05). NK cytotoxic activity against YAC-1 target cells was significantly higher for mice treated with SCF plus IL-2, compared to mice treated with IL-2 alone (P ≤ .0005). Interferon-γ (IFN-γ) production in cytokine-activated splenocytes was also greater for the SCF plus IL-2 group, over IL-2 treatment alone (P ≤ .01). The effect of SCF plus IL-2 on NK cell expansion was likely mediated via NK cell precursors, rather than mature NK cells. In summary, we provide the first evidence that SCF can significantly enhance expansion of functional NK cells induced by the prolonged administration of low dose IL-2 in vivo. Since the NK cell is a cytotoxic innate immune effector and a potent source of IFN-γ, this therapeutic strategy for NK cell expansion may serve to further enhance innate immune surveillance against malignant transformation and infection in the setting of cancer and/or immunodeficiency.

scholarly journals CD69 Deficiency Enhances the Host Response to Vaccinia Virus Infection through Altered NK Cell Homeostasis

2016 ◽  
Vol 90 (14) ◽  
pp. 6464-6474 ◽  
Author(s):  
Laura Notario ◽  
Elisenda Alari-Pahissa ◽  
Antonio de Molina ◽  
Pilar Lauzurica
Keyword(s):  
Immune Response ◽  
Infection Control ◽  
Vaccinia Virus ◽  
Nk Cells ◽  
Natural Killer ◽  
Host Response ◽  
Nk Cell ◽  
Innate Immune ◽  
Cell Numbers

ABSTRACTDuring the host response to viral infection, the transmembrane CD69 protein is highly upregulated in all immune cells. We have studied the role of CD69 in the murine immune response to vaccinia virus (VACV) infection, and we report that the absence of CD69 enhances protection against VACV at both short and long times postinfection in immunocompetent and immunodeficient mice. Natural killer (NK) cells were implicated in the increased infection control, since the differences were greatly diminished when NK cells were depleted. This role of NK cells was not based on an altered NK cell reactivity, since CD69 did not affect the NK cell activation threshold in response to major histocompatibility complex class I NK cell targets or protein kinase C activation. Instead, NK cell numbers were increased in the spleen and peritoneum of CD69-deficient infected mice. That was not just secondary to better infection control in CD69-deficient mice, since NK cell numbers in the spleens and the blood of uninfected CD69−/−mice were already augmented. CD69-deficient NK cells from infected mice did not have an altered proliferation capacity. However, a lower spontaneous cell death rate was observed for CD69−/−lymphocytes. Thus, our results suggest that CD69 limits the innate immune response to VACV infection at least in part through cell homeostatic survival.IMPORTANCEWe show that increased natural killer (NK) cell numbers augment the host response and survival after infection with vaccinia virus. This phenotype is found in the absence of CD69 in immunocompetent and immunodeficient hosts. As part of the innate immune system, NK lymphocytes are activated and participate in the defense against infection. Several studies have focused on the contribution of NK cells to protection against infection with vaccinia virus. In this study, it was demonstrated that the augmented early NK cell response in the absence of CD69 is responsible for the increased protection seen during infection with vaccinia virus even at late times of infection. This work indicates that the CD69 molecule may be a target of therapy to augment the response to poxvirus infection.

Elevated CD54 Expression Renders CD4+ T Cells Susceptible to Natural Killer Cell-Mediated Killing

2019 ◽  
Vol 220 (12) ◽  
pp. 1892-1903 ◽  
Author(s):  
Xi Chen ◽  
Huihui Chen ◽  
Zining Zhang ◽  
Yajing Fu ◽  
Xiaoxu Han ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Cd4 T Cells ◽  
Nk Cell ◽  
Killer Cell ◽  
Adaptive Immune Response ◽  
Phenotypic Analysis

Abstract Background Natural killer (NK) cells are an important type of effector cell in the innate immune response, and also have a role in regulation of the adaptive immune response. Several studies have indicated that NK cells may influence CD4+ T cells during HIV infection. Methods In total, 51 HIV-infected individuals and 15 healthy controls participated in this study. We performed the flow cytometry assays and real-time PCR for the phenotypic analysis and the functional assays of NK cell-mediated deletion of CD4+ T cells, phosphorylation of nuclear factor-κB (NF-κB/p65) and the intervention of metformin. Results Here we detected high CD54 expression on CD4+ T cells in HIV-infected individuals, and demonstrate that upregulated CD54 is associated with disease progression in individuals infected with HIV. We also show that CD54 expression leads to the deletion of CD4+ T cells by NK cells in vitro, and that this is modulated by NF-κB/p65 signaling. Further, we demonstrate that metformin can suppress CD54 expression on CD4+ T cells by inhibiting NF-κB/p65 phosphorylation. Conclusions Our data suggest that further studies to evaluate the potential role of metformin as adjunctive therapy to reconstitute immune function in HIV-infected individuals are warranted.

Modulation of Natural Killer Cell Activity in the Setting of Oncolytic Virotherapy and with a Chimeric Antigen Receptor

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 210-210 ◽  
Author(s):  
Chen Xilin ◽  
Jianfeng Han ◽  
Chu Jianhong ◽  
Walter Meisen ◽  
Zhang Jianying ◽  
...  
Keyword(s):  
Brain Tumors ◽  
Nk Cells ◽  
Tumor Cells ◽  
Mouse Models ◽  
Nk Cell ◽  
Cell Activity ◽  
Innate Immune ◽  
The Brain

Abstract Natural killer (NK) cells are innate lymphocytes that can rapidly eradicate tumor cells, especially those lacking MHC Class I molecules. NK cells can also rapidly eradicate herpes virus-infected cells. We designed an oncolytic herpes virus (oHSV) to selectively infect, replicate within, and lyse glioblastoma (GBM), a devastating brain tumor with a median survival of only 15 months following diagnosis. We have shown that the rapid influx of NK cells limits oHSV efficacy in GBM as they impede oHSV replication and spread [Alvarez-Breckenridge et al., Nat Med, 2012, 18(12):1827-34]. In the current study, we developed NK cell-based novel GBM therapies by decreasing the brain influx of NK cells to enhance the efficacy of oHSV, while arming NK cells in the brain with a chimeric antigen receptor (CAR) that targets both the wild-type EGFR and its mutant form EGFRvIII, two GBM tumor-associated antigens. We then investigated the synergistic effects between EGFR-CAR NK cells and oHSV. Transforming growth factor (TGF)-β is a potent immunosuppressive cytokine of NK cells [Yu et al, Immunity, 2006, 24(5):575-90]. We first determined if oHSV efficacy for treatment of GBM would be augmented by inhibiting anti-oHSV activity of NK cells with TGF-β pre-treatment. In vitro, NK cells pre-treated with TGF-β displayed less cytolytic capacity against oHSV-infected GBM cell lines and patient-derived GBM stem-like cells. In viral replication assays, co-culturing oHSV-infected GBM cells with NK cells pre-treated with TGF-β significantly increased virus titers. In an immunocompetent syngeneic GBM mouse model,administration of TGF-β to GBM-bearing mice prior to oHSV injection significantly inhibited intracranial infiltration and activation of NK cells (P < 0.05). In orthotopic human GBM xenograft mouse models and in syngeneic GBM mouse models, TGF-β treatment in vivo prior to oHSV therapy resulted in inhibition of NK cell infiltration, suppression of tumor growth and significantly prolonged survival of GBM-bearing mice (P < 0.05). Furthermore, depletion of NK cells incompletely blocked the positive effects of in vivo treatment of GBM with TGF-β on survival, suggesting that TGF-β may also directly act on other innate immune cells such as macrophages/microglia. These data demonstrate a single dose of TGF-β prior to oHSV administration enhances anti-tumor efficacy for GBM at least in part through the transient inhibition of the innate immune responses to oHSV infection. We next investigated whether NK cell activity could be enhanced to more directly target brain tumors while sparing eradication of oHSV. We therefore infected both human NK-92 cells and primary human NK cells to express the second generation CAR targeting both EGFR and EGFRvIII that we designed. Further, we asked if the treatment with EGFR-CAR NK cells plus oHSV could create a therapeutic synergy for the treatment to brain tumors. In vitro, compared with mock-transduced CAR-NK-cells, EGFR-CAR NK cells exhibited significantly higher cytotoxicity and IFN-γ production when co-cultured with tumor cells, for both NK-92 and primary NK cells (P < 0.01). Further, significantly higher cytolytic activity against tumor cells was obtained when CAR NK cells were combined with oHSV-1 infection of tumor cells, compared to either of the monotherapies alone (P < 0.05). In mice, to avoid oHSV clearance by the EGFR-CAR NK cells following the inoculation of the mouse with tumor cells, we administered these two agents sequentially; administering EGFR-CAR NK cells directly into the tumor first as a single injection of 2 × 106 cells, followed by intracranial infection with 2 × 105 plaque-forming units oHSV five days later, presumably after EGFR-CAR NK survival has diminished. Compared to vehicle controls, intracranial administration of either EGFR-CAR NK cells or oHSV blunted tumor growth. However, the combination of EGFR-CAR NK cells followed by oHSV infection resulted in significantly more efficient killing of tumor cells (P < 0.05) and significantly longer survival for tumor-bearing mice when compared to either monotherapy alone. Collectively, our studies demonstrate that in animal tumor models, we can combine novel NK cell and oHSV therapies to significantly improve survival. Disclosures No relevant conflicts of interest to declare.

Evidence That Novel NK-NK Cell Subset Regulation Exists With Regard To Effects In Tumor and Viral Models

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1038-1038
Author(s):  
Ethan G Aguilar ◽  
Can M Sungur ◽  
Anthony E Zamora ◽  
William J Murphy
Keyword(s):  
Immune Response ◽  
Nk Cells ◽  
Mhc Class I ◽  
Viral Infections ◽  
Nk Cell ◽  
Class I ◽  
Inhibitory Receptors ◽  
The Individual

Abstract Natural killer (NK) cells are lymphocytes of the innate immune system and are classically associated with cytotoxic responses to both virally infected as well as neoplastic cells. Activation of NK cells to exhibit their cytotoxicity is dependent on signaling through a number of activating and inhibitory receptors. In mice, one such family of inhibitory receptors is the C-type lectin-like Ly49 family. In humans, the killer immunoglobulin-like receptors (KIRs) serve as the primary family of inhibitory receptors and are functional analogs of the Ly49s. Despite markedly different structures, the Ly49s and KIRs display similar binding capabilities and bind primarily to distinct MHC class I haplotypes, which plays an important role in regulating NK cell function. NK cells that express inhibitory receptors that are specific for the MHC class I haplotype of the individual are termed “licensed” and have been shown to have increased functionality in terms of cytotoxicity and cytokine production. In contrast, NK cells that express inhibitory receptors that are unable to bind to the MHC class I haplotype of the individual are termed “unlicensed” and have been shown to be hyporesponsive. We have recently reported on the role of NK licensing on the immune response to viral infections such as MCMV. In addition, we have previously described how regulatory T cells can regulate NK cell activity in vivo. However, there are limited data examining the interaction and regulation between the different NK subsets based on differences in licensing. We hypothesized that different NK cell subsets, based on licensing, can regulate each other in the context of anti-tumor and anti-viral responses. Here we first provide in vitro data providing evidence to support the hypothesis of NK-NK regulation based on licensing. In vitro killing assays using MCMV infected fibroblasts, or C1498 (murine acute myeloid leukemia) cells as targets and using different combinations of murine NK Ly49 subsets as effectors were used to assess this NK-NK regulation. To further test our hypothesis, in vivo experiments were also performed using a mouse leukemia model as well as an MCMV model. Mice were injected with C1498 cells and then given hematopoietic stem cell transplantation (HSCT). The mice were then depleted of all NK cells or either licensed or unlicensed subsets by antibody depletion once a week, and monitored for survival. Mice that were depleted of the unlicensed population survived significantly longer compared with the other depleted groups, suggesting a negative regulation of the anti-tumor response by the unlicensed population resulting in greater tumor burden and death in the presence of the unlicensed population. This negative regulation by the unlicensed population is further supported by another experiment where mice were infected with MCMC following total NK or subset depletion and monitored for ten days throughout the course of the immune response to MCMV. Mice that were depleted of their unlicensed population displayed a significantly larger expansion of the licensed population of NK cells, without reciprocal greater expansion of the unlicensed population upon licensed NK cell depletion. More specifically, depletion of the unlicensed population resulted in an expansion of the Ly49H+NK cells which have previously been shown to be the primary effector population during MCMV infection. Thus, the unlicensed NK cells are playing a role in down-regulating the anti-viral response by limiting the expansion of the effector licensed population. Our data highlight a role for the murine NK subsets to negatively regulate the immune response of the effector licensed NK population in the context of anti-tumor and anti-viral responses. This new insight into the regulatory role of NK cells may have clinical benefit for patients receiving bone marrow transplants during cancer treatment to enhance graft vs. tumor effects, and to combat opportunistic viral infections that may manifest in the immune compromised environment of the BMT patient. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Cytokine-Induced Apoptosis of Human Natural Killer Cells Identifies a Novel Mechanism to Regulate the Innate Immune Response

Blood ◽  
1997 ◽  
Vol 89 (3) ◽  
pp. 910-918 ◽  
Author(s):  
Mary E. Ross ◽  
Michael A. Caligiuri
Keyword(s):  
Immune Response ◽  
Nk Cells ◽  
Natural Killer ◽  
Innate Immune Response ◽  
Cell Apoptosis ◽  
Nk Cell ◽  
Innate Immune ◽  
Tnf Α ◽  
Ifn Γ ◽  
Induced Apoptosis

Abstract Interferon-γ (IFN-γ) is critical for an effective innate immune response against infection. A combination of interleukins (ILs) derived from activated T cells (IL-2) and monocytes (IL-12), or monocytes alone (IL-15 and IL-12), induces optimal production of IFN-γ from natural killer (NK) cells. The mechanism by which human NK cells downregulate their production of IFN-γ is unknown. Here we show that the same cytokines that induce human NK cell IFN-γ production subsequently induce apoptosis of the NK cells. Fas, bcl-2, or bax do not appear to be involved in this process. The mechanism of cytokine-induced apoptosis of human NK cells appears to involve NK cell production of tumor necrosis factor-α (TNF-α). Neutralization of TNF-α or inhibition of TNF-α binding to the p80 TNF-α receptor partially inhibited apoptosis. Transforming growth factor-β, which inhibits cytokine-induced NK cell production of IFN-γ and TNF-α, also decreased cytokine-induced NK cell apoptosis. Costimulation of a CD3−CD56+ NK leukemia cell line with IL-2 and IL-12 or IL-15 and IL-12 induced apoptosis in vitro, which increased when combined with a chemotherapeutic agent. In summary, costimulation of human NK cells via the IL-2 receptor and the IL-12 receptor induces significant IFN-γ production, followed by NK cell apoptosis and a decline in IFN-γ production. Hence, cytokines that activate this innate immune response may also serve to limit it via apoptosis. This novel observation may have implications for the regulation of the innate immune response during infection, the toxicity of combination cytokine therapy, and the treatment of NK cell leukemia.

scholarly journals Aneuploid cells activate NF-κB to promote their immune clearance by NK cells

2020 ◽  
Author(s):  
Ruoxi W. Wang ◽  
Sonia Viganò ◽  
Uri Ben-David ◽  
Angelika Amon ◽  
Stefano Santaguida
Keyword(s):  
Immune Response ◽  
Immune System ◽  
Nk Cells ◽  
Cancer Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Immune Surveillance ◽  
Immune Recognition ◽  
Immune Clearance

SummaryThe immune system plays a major role in the protection against cancer. Identifying and characterizing the pathways mediating this immune surveillance is thus critical for understanding how cancer cells are recognized and eliminated. We previously found that untransformed cells that had undergone senescence due to highly abnormal karyotypes are eliminated by Natural Killer (NK) cells in vitro. Here we show that this is also true for aneuploid untransformed cells that had not lost their ability to proliferate. Their elimination by NK cells, like that of aneuploid senescent cells, is predominantly mediated by non-cell autonomous mechanisms. Our data further indicate that NF-κB signaling in aneuploid cells is central to eliciting this immune response. Inactivating NF-κB abolishes NK-cell mediated clearance in aneuploid cells in vitro. In cancer cell lines, NF-κB signaling correlates with degree of aneuploidy, raising the possibility that aneuploidy-induced immune recognition is partially retained in cancer.

Human Natural Killer Cells Are Able to Kill Aspergillus Fumigatus but Not Via the Perforin - Granzyme Pathway.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1640-1640 ◽  
Author(s):  
Maria Bouzani ◽  
Michael Ok ◽  
Oliver Kurzai ◽  
Hermann Einsele ◽  
Juergen Loeffler
Keyword(s):  
Gene Expression ◽  
Immune System ◽  
Aspergillus Fumigatus ◽  
Nk Cells ◽  
Innate Immune System ◽  
Nk Cell ◽  
Surface Expression ◽  
Innate Immune ◽  
Cytokine Release

Abstract Abstract 1640 Poster Board I-666 Introduction Natural killer (NK) cells are CD3- CD56+ lymphocytes demonstrating confirmed cytotoxicity against neoplastic and virus infected host cells. Increasing data provide evidence of a direct NK cell effect against extracellular pathogens, such as bacteria, parasites and yeasts, but there is a relative lack of data on their interaction with filamentous fungus and especially with Aspergillus fumigatus. Aspergillus is an omnipresent mold, living in close vicinity with humans, being constantly inhaled in the lungs and thereafter cleared by the innate immune system. Otherwise harmless for healthy people, it is at the origin of invasive Aspergillosis (IA), an extremely devastating disease for immunocompromised subjects. Host's innate immune system controls Aspergillus growth through a complex system of potent effector cells, mediating their antifungal activity mainly by phagocytosis. Our study aims to shed light for the first time on the direct interaction between human NK cells, mediators of extracellular cytotoxicity, and Aspergillus. Methods NK cells were isolated after magnetic depletion of the peripheral blood of healthy volunteers and they were used after 24h priming with 500 U/ml recombinant interleukin – 2 rhIL-2. To determine gene expression and cytokine release of interferon gamma (IFNg) and Tumor Necrosis Factor- a (TNF-a), NK cells were stimulated for 0, 3, 6 and 12h with different morphologies of Aspergillus: conidia and germlings. To evaluate the lethal impact of NK cells on Aspergillus, plate killing assays were performed at 0, 3 and 6h time points. To illustrate the role of antibody dependent cellular cytotoxicity, ADCC a monoclonal IgG antibody, against germlings, was tested. Transwell permeable membranes, with pores of 0,4 μm, prohibiting the direct contact of cells placed on their opposite sides, but allowing the free circulation of molecules, were used to estimate the effect of cell-fungal contact. To investigate the cytotoxic mechanism involved, NK cells were depleted from perforin and granzymes by treatment with strontium chloride and they had their death ligands, TNF- related apoptosis- inducing ligand (TRAIL) and FasL, neutralised by means of blocking antibodies. The release of cytotoxic granules was estimated by the NK cell surface expression of the marker of degranulation CD107a/b. Results Observing the in vitro interaction of NK cells with Aspergillus, fungal germinated morphologies (germlings) showed to be highly immunogenic towards NK cells, compared to conidia, inducing the gene expression and cytokine release of Th1 immune mediators such as IFN-g (p <0,05) and TNF-a.(p <0,1). NK cells demonstrated also a strong lethal impact against germlings (p <0,05). Moreover, the presence of antifungal antibody further potentiated both immunoregulatory and cytotoxic activities. Investigating the means engaged by NK cells to perceive and kill Aspergillus, direct effector–pathogen cell to cell contact was revealed as prerequisite; when this condition was not present there was neither cytokine induction, nor fungal damage (p <0,05). This finding was confirmed by the lack of surface expression of CD107a/b, after NK cell- Aspergillus co-incubation. Investigating the killing pathway we compared the effectiveness of perforin – granzymes depleted NK cells to this of intact cells against germlings and it was found equivalent (p =NS). In a similar way, neutralisation of TRAIL and FasL ligands did not alter the cytotoxic ability of NK cells towards Aspergillus. Conclusion Our data show that human NK cells are stimulated in vitro by Aspergillus germlings, which triggers an immunoregulatory Th1 orientated response and causes important fungal killing. NK cells are not aware of conidia, they are not stimulated by them and par consequence they do not kill them. Finally, we showed that NK cells do not mediate their cytotoxic effect via perforin – granzymes pathway, neither through the engagement of TRAIL, FasL death receptors, suggesting that another pathway is involved in NK cell – Aspergillus fumigatus interplay. We suggest that further investigation of these striking findings might offer a potent immunotherapeutic tool against IA. Disclosures No relevant conflicts of interest to declare.

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