NK Cell Expression Research Articles

A Chimeric Receptor with NKG2D Specificity Enhances Natural Killer Cell Activation and Killing of Tumor Cells

Natural killer (NK) cells rely on surface receptors to distinguish healthy cells from cancer cells. We designed a receptor termed NKG2D-DAP10-CD3ζ that is composed of the NK cell activating molecule NKG2D plus 2 key signaling molecules, DAP10 and CD3ζ, and evaluated its capacity to promote cancer cell killing. Retroviral transduction of NKG2D-DAP10-CD3ζ markedly increased NKG2D surface expression in NK cells, which became consistently more cytotoxic than mock-transduced cells against leukemia and solid tumor cell lines. In contrast, there was no increase in cytotoxicity against nontransformed blood and mesenchymal cells. NKG2D blockade abrogated gains in cytotoxicity to cancer cells. Receptor stimulation triggered signal transduction, secretion of IFN-γ, GM-CSF, IL-13, MIP-1α, MIP-1β, CCL5, and TNF-α, and massive release of cytotoxic granules, which persisted after 48 hours of continuous stimulation. NKG2D-DAP10-CD3ζ-expressing NK cells had considerable antitumor activity in a mouse model of osteosarcoma, whereas activated NK cells were ineffective. Thus, the cytotoxic potential of NK cells against a wide spectrum of tumor subtypes could be markedly enhanced by expression of NKG2D-DAP10-CD3ζ receptors. The development of an electroporation method that permits rapid expression of the receptor in a large number of human NK cells facilitates clinical translation of this NK-based strategy for a generalized cellular therapy that may be useful to treat a wide range of cancers.

Distinct Roles for Neutrophils and Dendritic Cells in Inflammation and Autoimmunity in motheaten Mice

The motheaten mouse has long served as a paradigm for complex autoimmune and inflammatory disease. Null mutations in Ptpn6, which encodes the nonreceptor protein-tyrosine phosphatase Shp1, cause the motheaten phenotype. However, Shp1 regulates multiple signaling pathways in different hematopoietic cell types, so the cellular and molecular mechanism of autoimmunity and inflammation in the motheaten mouse has remained unclear. By using floxed Ptpn6 mice, we dissected the contribution of innate immune cells to the motheaten phenotype. Ptpn6 deletion in neutrophils resulted in cutaneous inflammation, but not autoimmunity, providing an animal model of human neutrophilic dermatoses. By contrast, dendritic cell deletion caused severe autoimmunity, without inflammation. Genetic and biochemical analysis showed that inflammation was caused by enhanced neutrophil integrin signaling through Src-family and Syk kinases, whereas autoimmunity resulted from exaggerated MyD88-dependent signaling in dendritic cells. Our data demonstrate that disruption of distinct Shp1-regulated pathways in different cell types combine to cause motheaten disease.

NAB2 and EGR-1 exert opposite roles in regulating TRAIL expression in human Natural Killer cells

The transcriptional regulator NGFI-A binding protein 2 (NAB2) and the early growth response (EGR) genes are key regulators of effector molecules, such as cell death-inducing genes. We have previously shown that NAB2 modulates the levels of expression of the Tumor Necrosis Factor (TNF) family member TNF-related apoptosis inducing ligand (TRAIL) in T cells and plasmacytoid DCs. Provided that TRAIL plays a key role in NK cell cytotoxicity towards infected and tumor cells, we investigated whether NAB2 also mediates TRAIL expression in human NK cells, and if so through which mechanisms. We show that NAB2 is induced in NK cells upon IL-2 and IL-15 stimulation, and promotes the induction of TRAIL. In addition, we show that the transcription factor EGR-1, which is upregulated by the same stimuli as NAB2, rather acts as a brake on TRAIL expression in NK cells. Overall, these data provide new mechanistic insights in the regulation of TRAIL, and show that the gene regulation through the NAB2/EGR axis allows for a highly controlled expression pattern of this effector molecule in NK cells.

Role of NKp46 Expression in Cytokine Production by CD56‐Positive NK Cells in the Peripheral Blood and the Uterine Endometrium

To investigate the role of natural cytotoxicity receptor, NKp46 expression in cytokine-producing NK cells. CD56(+) /NKp46(+) NK cells from the peripheral blood and the uterine endometrium were magnetically separated. IFN-γ, TNF-α, IL-4, IL-10, and TGF-β(1) expressions on NK cells were investigated using multicolor flow cytometry. Peripheral blood and uterine endometrial NK cells were grouped into 4 subpopulations based upon the degree of CD56 and NKp46 expressions. NKp46 expression was associated with higher frequency of cytokine-producing NK cells, including CD56(dim) NK cells. The percentage of TNF-α(+) and IL-10(+) NK cells per total CD56(+) /NKp46(+) NK cells in the uterine endometrium showed a significant correlation with those of the peripheral blood in all subpopulations, but that of IFN-γ(+) , IL-4(+,) and TGF-β(1) (+) NK cells showed partial correlation. Expression of NKp46 is involved in cytokine production of CD56(+) NK cells in the peripheral blood and the uterine endometrium.

Influence of Immunotherapy with Autologous Dendritic Cells on Innate and Adaptive Immune Response in Cancer

The objective of this study was to evaluate some of the mechanisms involved in the activation of the immune system in patients with advanced-stage cancer (n = 7) who received an autologous dendritic cell vaccine. We examined the immune response mediated by macrophages (CD14+), natural killer cells (CD56+), and B lymphocytes (CD19+) by flow cytometry and assessed the expression of Th1 (IFN-γ, TNF-α, IL-2, and IL-12), Th2 (IL-4), and Treg (TGF-β) cytokines by flow cytometry and an enzyme-linked immunosorbent assay. The CD14+ TNF-α+ population was significantly increased (P < 0.04) when patients received the vaccine; IL-2 expression in both NK cells and in B lymphocytes was increased after a transient initial increase showed a nearly significant decrease (P < 0.07 and P < 0.06 respectively), whereas the CD19+ and CD56+ populations did not show significant changes. Dendritic cell-based immunotherapy led to increased secretion of IFN-γ and IL-12 and reduced secretion of TGF-β. In conclusion, it is likely that the autologous dendritic cell vaccine stimulated the immune cells from the peripheral blood of patients with cancer and generally increased the production of Th1 cytokines, which are related to immunomodulatory responses against cancer.

A NOVEL MECHANISM FOR RECOGNITION OF A SMALL MOLECULE CARBOHYDRATE IMMUNOTHERAPEUTIC BY HUMAN MYELOID CELLS: POTENTIAL FOR MODULATION OF EFFICACY IN THE CLINIC

Event Abstract Back to Event A NOVEL MECHANISM FOR RECOGNITION OF A SMALL MOLECULE CARBOHYDRATE IMMUNOTHERAPEUTIC BY HUMAN MYELOID CELLS: POTENTIAL FOR MODULATION OF EFFICACY IN THE CLINIC Mary A. Antonysamy1*, Mariana I. Nelson1, Richard Walsh1, Nadine C. Ottoson1, Blaine Rathmann1 and William Grossman1 1 Biothera, United States Imprime PGG® (Imprime), is a soluble β-1,3/1,6 immunomodulatory glucan, being developed in combination with monoclonal antibody therapy for multiple oncologic indications. To date, limited studies exist on the recognition and immunomodulatory activities of soluble glucans on the human immune system. Although yeast β-glucans have been shown to be capable of binding human cells through leukocyte complement receptor 3 (CR3, Mac-1, αM β2 integrin), the key components for this event were not elucidated. Here, we demonstrate that Imprime, a soluble carbohydrate pathogen associated molecular pattern (PAMP), is recognized by only certain human cells expressing CR3 (i.e., bind to CR3-expressing myeloid and T cells, but not to CR3 expressing NK cells) and requires opsonization by complement components such as iC3b. Inactivation of serum complement components resulted in loss of binding and function. Surprisingly, not all healthy donor myeloid cells were capable of binding Imprime despite the presence of complement components. Looking into potential reasons for differential binding led to a strong correlation between being a high-Imprime binder and having a high-natural anti-β glucan titer. Both, in vitro and in vivo feasibility studies in humans demonstrate the potential for converting a low-Imprime binder to a high-Imprime binder with anti-β glucan antibodies. Given the preclinical requirement for Imprime binding to myeloid cells for antitumor effects, we propose that assessing Imprime binding to blood immune cells could be a predictive biomarker, and enriching for high-binder patients or converting low-binders to high-binders by treating with high-titer plasma or IVIG, would enhance therapeutic efficacy. Keywords: beta glucan, Immunotherapy, anti-beta glucan antibodies, complement receptor 3, Myeloid Cells, Imprime PGG, IVIg, Cancer, biomarker Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Translational immunology and immune intervention Citation: Antonysamy MA, Nelson MI, Walsh R, Ottoson NC, Rathmann B and Grossman W (2013). A NOVEL MECHANISM FOR RECOGNITION OF A SMALL MOLECULE CARBOHYDRATE IMMUNOTHERAPEUTIC BY HUMAN MYELOID CELLS: POTENTIAL FOR MODULATION OF EFFICACY IN THE CLINIC. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01005 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 28 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Mary A Antonysamy, Biothera, Eagan, United States, mary_antonysamy@hotmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Mary A Antonysamy Mariana I Nelson Richard Walsh Nadine C Ottoson Blaine Rathmann William Grossman Google Mary A Antonysamy Mariana I Nelson Richard Walsh Nadine C Ottoson Blaine Rathmann William Grossman Google Scholar Mary A Antonysamy Mariana I Nelson Richard Walsh Nadine C Ottoson Blaine Rathmann William Grossman PubMed Mary A Antonysamy Mariana I Nelson Richard Walsh Nadine C Ottoson Blaine Rathmann William Grossman Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Natural killer cells and their activation status in normal pregnancy.

Increased peripheral blood-activated NK cell counts are associated with increased risk of miscarriage and failed in vitro fertilization treatment. However, assessment of activated peripheral NK cells in normal and pathological pregnancies beyond implantation and early miscarriage has not been described. Total CD69 expressing NK cells counts were measured by flow cytometry in healthy women with singleton pregnancies, including 45 at 11+6–13+6 weeks' gestation, 46 at 20+0–22+4 weeks, and 42 at 31+6–33+5 weeks. The number of peripheral blood NK cells decreased, whereas the percentage of activated CD69 expressing NK cells increased from the first to the third trimester of pregnancy. This study shows the course of peripheral blood NK cells and activated CD69 expressing NK cells in uncomplicated nulliparous singleton pregnancies. This is a first step in understanding their implication in pathological pregnancies.

Bi(o)communications among peripheral blood fractions: A focus on NK and NKT cell biology in rheumatoid arthritis

Rheumatoid Arthritis (RA) is an autoimmune disease with unknown pathophysiology involving many interwoven signalling cascades. ROS, NK and NKT cells might be crucial in the disease severity of RA of which the role of NK and NKT cells are controversial in literature. However, the role of oxidative stress, its impact on NK and NKT cell immunobiology and disease activity (DAS28) is largely unknown. Therefore, we studied the role of oxidative stress and NK cell subsets in the pathogenesis of RA. The state of oxidative stress in various peripheral blood fractions, percentage NK and NKT cell expression, their altered apoptotic signaling pathways involving mitochondrial membrane potential, FAS associated death domain (FADD) mediated pathways and DNA damage were analyzed. Results indicated a state of profound oxidative stress in the peripheral blood of RA patients where percentage of NK and NKT cell subsets diminished while ROS levels increased. The depolarized mitochondrial membrane potential, FAS, FASL and active caspase-3 positive NK and NKT cell subsets were considerably elevated in patients. The DNA damage, assessed as percentage of DNA in comet tail, was significantly elevated. Findings of the present work indicate increased apoptosis of peripheral NK and NKT cells in the diseased condition. PBMC and RBC are the major sites of enhanced oxidative stress. The state of oxidative stress and altered immunobiology of NK and NKT cells strongly correlated with Disease activity score. The present study strongly supports the protective role of NK cell subsets in the pathogenesis of RA.

Expression and Activation by Epstein Barr Virus of Human Endogenous Retroviruses-W in Blood Cells and Astrocytes: Inference for Multiple Sclerosis

BackgroundProposed co-factors triggering the pathogenesis of multiple sclerosis (MS) are the Epstein Barr virus (EBV), and the potentially neuropathogenic MSRV (MS-associated retrovirus) and syncytin-1, of the W family of human endogenous retroviruses.Methodology/Principal FindingsIn search of links, the expression of HERV-W/MSRV/syncytin-1, with/without exposure to EBV or to EBV glycoprotein350 (EBVgp350), was studied on peripheral blood mononuclear cells (PBMC) from healthy volunteers and MS patients, and on astrocytes, by discriminatory env-specific RT-PCR assays, and by flow cytometry. Basal expression of HERV-W/MSRV/syncytin-1 occurs in astrocytes and in monocytes, NK, and B, but not in T cells. This uneven expression is amplified in untreated MS patients, and dramatically reduced during therapy. In astrocytes, EBVgp350 stimulates the expression of HERV-W/MSRV/syncytin-1, with requirement of the NF-κB pathway. In EBVgp350-treated PBMC, MSRVenv and syncytin-1 transcription is activated in B cells and monocytes, but not in T cells, nor in the highly expressing NK cells. The latter cells, but not the T cells, are activated by proinflammatory cytokines.Conclusions/Significance In vitro EBV activates the potentially immunopathogenic and neuropathogenic HERV-W/MSRV/syncytin-1, in cells deriving from blood and brain. In vivo, pathogenic outcomes would depend on abnormal situations, as in late EBV primary infection, that is often symptomatic, or/and in the presence of particular host genetic backgrounds. In the blood, HERV-Wenv activation might induce immunopathogenic phenomena linked to its superantigenic properties. In the brain, toxic mechanisms against oligodendrocytes could be established, inducing inflammation, demyelination and axonal damage. Local stimulation by proinflammatory cytokines and other factors might activate further HERV-Ws, contributing to the neuropathogenity. In MS pathogenesis, a possible model could include EBV as initial trigger of future MS, years later, and HERV-W/MSRV/syncytin-1 as actual contributor to MS pathogenicity, in striking parallelism with disease behaviour.

Immune Surveillance Properties of Human NK Cell-Derived Exosomes

Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture supernatant and human biological fluids, such as plasma. Functions of exosomes released by "normal" cells are not well understood. In fact, several studies have been carried out on exosomes derived from hematopoietic cells, but very little is known about NK cell exosomes, despite the importance of these cells in innate and adaptive immunity. In this paper, we report that resting and activated NK cells, freshly isolated from blood of healthy donors, release exosomes expressing typical protein markers of NK cells and containing killer proteins (i.e., Fas ligand and perforin molecules). These nanovesicles display cytotoxic activity against several tumor cell lines and activated, but not resting, immune cells. We also show that NK-derived exosomes undergo uptake by tumor target cells but not by resting PBMC. Exosomes purified from plasma of healthy donors express NK cell markers, including CD56+ and perforin, and exert cytotoxic activity against different human tumor target cells and activated immune cells as well. The results of this study propose an important role of NK cell-derived exosomes in immune surveillance and homeostasis. Moreover, this study supports the use of exosomes as an almost perfect example of biomimetic nanovesicles possibly useful in future therapeutic approaches against various diseases, including tumors.

Molecular definition of the identity and activation of natural killer cells.

Using whole-genome microarray datasets of the Immunological Genome Project, we demonstrate a closer transcriptional relationship between NK and T cells than any other leukocytes, distinguished by their expression of similar signaling functions. While resting NK cells were known to share expression of a few genes with cytotoxic CD8+ T cells, transcriptome-wide analysis demonstrates that the commonalities extend to hundreds of genes, many with unknown functions. The NK cell response to viral infection is dampened relative to cytotoxic CD8+ T cells, in part due to their “pre-primed” state. Collectively, the data provide global context for known and novel molecular aspects of NK cell identity and function by delineating the genome-wide repertoire of gene expression of NK cells in various states.

Rapid early innate control of hepatitis C virus during IFN‐α treatment compromises adaptive CD4+T‐cell immunity

The ability to control HCV with IFN-α-based treatments provides an opportunity in humans to study how the rate of viral clearance in vivo impinges on the development of antiviral responses. Ex vivo (IFN-γ-producing) and cultured antiviral CD4+ T cells, serum cytokines, and viral loads were measured repeatedly in a cohort of chronically HCV-infected subjects (n = 33) receiving IFN-α. Rapid control of virus indicated by an increased calculated rate of virus clearance, occurred in those subjects demonstrating absent/minimal T-cell responses (p < 0.0006). Surprisingly, in subjects who demonstrated the most robust T-cell responses (and reduced serum IL-10), there was actually a reduced rate of early virus clearance. A subsequent analysis of NK-cell function in available subjects (n = 8) revealed an inverse correlation between pretreatment NK-cell expression of NKp46 and the potential to upregulate cytotoxic function on exposure to IFN-α (p < 0.004), as well as the subsequent measured rate of viral clearance (p = 0.045). Thus, the CD4+ T-cell response during IFN-α treatment appears to be shaped by the rate of innate virus suppression. These data suggest that individuals who respond most effectively to immune intervention may be most in need of subsequent vaccination to prevent reinfection.

Monocyte chemoattractant protein-1 secreted by decidual stromal cells inhibits NK cells cytotoxicity by up-regulating expression of SOCS3.

BackgroundDecidual stromal cells (DSCs) are of particular importance due to their pleiotropic functions during pregnancy. Although previous research has demonstrated that DSCs participated in the regulation of immune cells during pregnancy, the crosstalk between DSCs and NK cells has not been fully elucidated. To address this issue, we investigated the effect of DSCs on perforin expression in CD56+ NK cells and explored the underlying mechanism.Methodology/Principal FindingsFlow cytometry analysis showed perforin production in NK cells was attenuated by DSC media, and it was further suppressed by media from DSCs pretreated with lipopolysaccharide (LPS). However, the expression of granzyme A and apoptosis of NK cells were not influenced by DSC media. ELISA assays to detect cytokine production indicated that monocyte chemoattractant protein-1 (MCP-1) in the supernatant of DSCs conditioned culture significantly increased after LPS stimulation. The inhibitory effect of DSC media on perforin was abolished by the administration of anti-MCP-1 neutralizing antibody. Notably, reduced perforin expression attenuated the cytotoxic potential of CD56+NK cells to K562 cells. Moreover, Suppressor of cytokine signaling 3 (SOCS3) expression in NK cells was enhanced by treatment with MCP-1, as measured by RT-PCR and western blot. Interestingly, MCP-1-induced perforin expression was partly abolished by the siRNA induced SOCS3 knockdown. Western blot analysis suggested that both NF-κB and ERK/MAPKs pathway were involved in the LPS-induced upregulation of MCP-1 in DSCs.Conclusions/SignificanceOur results demonstrate that LPS induces upregulation of MCP-1 in DSCs, which may play a critical role in inhibiting the cytotoxicity of NK cells partly by promoting SOCS3 expression. These findings suggest that the crosstalk between DSCs and NK cells may be crucial to maintain pregnancy homeostasis.

NK cell therapy in cancer and transplantation

NK cells have unique properties including memory. Although initially described as MHC unrestricted killers, NK cells have several families of receptors that directly recognize MHC Inhibitory receptors to protect “self” expressing normal tissue from being killed by NK cells and protecting against autoimmunity. Therefore, for NK cells to kill and produce cytokines they must encounter activating receptor ligands in the context of “missing self” that occurs with malignant transformation. The second property of inhibitory receptors is to educate or license NK cells to acquire function. This is best demonstrated in the mouse and in humans by enhanced function on self inhibitory receptor expressing NK cells when in a host expressing cognate ligate. In contrast, NK cells without inhibitory receptors or with non‐self inhibitory receptors are relatively hyporesponsive. Understanding the biology of NK cells will translate into strategies to manipulate NK cells for therapeutic purposes in cancer and transplantation.

Influence of Cytokines on HIV-Specific Antibody-Dependent Cellular Cytotoxicity Activation Profile of Natural Killer Cells

There is growing interest in HIV-specific antibody-dependent cellular cytotoxicity (ADCC) as an effective immune response to prevent or control HIV infection. ADCC relies on innate immune effector cells, particularly NK cells, to mediate control of virus-infected cells. The activation of NK cells (i.e., expression of cytokines and/or degranulation) by ADCC antibodies in serum is likely subject to the influence of other factors that are also present. We observed that the HIV-specific ADCC antibodies, within serum samples from a panel of HIV-infected individuals induced divergent activation profiles of NK cells from the same donor. Some serum samples primarily induced NK cell cytokine expression (i.e., IFNγ), some primarily initiated NK cell expression of a degranulation marker (CD107a) and others initiated a similar magnitude of responses across both effector functions. We therefore evaluated a number of HIV-relevant soluble factors for their influence on the activation of NK cells by HIV-specific ADCC antibodies. Key findings were that the cytokines IL-15 and IL-10 consistently enhanced the ability of NK cells to respond to HIV-specific ADCC antibodies. Furthermore, IL-15 was demonstrated to potently activate “educated” KIR3DL1+ NK cells from individuals carrying its HLA-Bw4 ligand. The cytokine was also demonstrated to activate “uneducated” KIR3DL1+ NK cells from HLA-Bw6 homozygotes, but to a lesser extent. Our results show that cytokines influence the ability of NK cells to respond to ADCC antibodies in vitro. Manipulating the immunological environment to enhance the potency of NK cell-mediated HIV-specific ADCC effector functions could be a promising immunotherapy or vaccine strategy.

Costimulatory molecule profiles and NK cell recovery after autologous hematopoietic cell transplantation (HCT) in multiple myeloma (MM) patients.

8089 Background: Increased immune responses post autologous HCT may be of benefit in long term disease control. Responses may be mediated by NK cell function and possibly other alternate pathways, including costimulatory molecule pathways. This pilot study assesses the expression of inhibitory (PD-1 and CTLA-4) and stimulatory (OX-40, ICOS, 4-1BB, and CD28) molecules on NK cells after auto-HCT in MM patients and evaluates the effect of lenalidomide treatment on these pathways. Methods: 17 patients with MM undergoing HCT, median age 56.7 years (36 – 67), were included in the study. Peripheral blood samples were taken 3 days prior to HCT and 14, 30, 60, 90, 180 days after HCT. At d180 post-HCT, 13/17 patients were receiving lenalidomide with d91 as median start date. NK cells and their costimulatory molecules were evaluated by flowcytometry using 2 six color panels of antibodies. One way ANOVA test and Kruskal-Wallis test (non-parametric) were applied to analyze the data using the Graphpad Software. Results: See table below. NK cell number was highest (median: 26% of total lymphocytes) at d14 (p: &lt; 0.0001) compared to pre and post HCT levels. At d180, TNF-R OX40 expression was significantly increased in ≤PR group (n=5) (median: 9.5% of NK cells) compared to ≥VGPR (n=12) (0.8%; p=0.0084). In addition, NK cell number was higher in the lenalidomide group (n=13) (median: 15.15 % of total lymphocytes) compared to the no lenalidomide group (n=4) (6.74%; p=0.0108) at d180 post HCT. Significantly lower level of CTLA-4 expression was also found in the lenalidomide group (0.33% vs. 2.54%; p=0.0362). Conclusions: We observed NK cell recovery to baseline values at 60 days after HCT. At d180 post-HCT, OX-40 expression in NK cells was higher in ≤PR group than ≥VGPR group. Lenalidomide treatment was associated with higher NK cells number and decreased expression of CTLA-4. This observation could be a possible marker of enhanced host NK cell immune response against MM. Future clinical trials will explore therapies that increase NK cell responses. [Table: see text]

Abstract LB-374: A novel method for the detection of circulating NK cells in archived blood reveals a decrease in NK cells in head and neck squamous cell carcinoma

Abstract Patients with head and neck squamous cell carcinoma (HNSCC) frequently exhibit abnormal immune cell levels and activities, leading many to believe that progression of the disease is linked to evasion of immune surveillance. A more complete understanding of immune cell alterations could be obtained from analysis of archival samples, but this type of study is not possible using current immunodiagnostic methods. Combining information on cell lineage specific differentially methylated regions (DMRs) with methylation specific quantitative PCR (MS-qPCR) enables highly sensitive and accurate counts of individual cell types in archival blood and tissues. DNA methylation and mRNA expression in magnetic microbead (MACS) isolated, flow cytometry (FACS) validated normal human peripheral blood leukocytes were assessed using the Infinium HumanMethylation27 and Whole-Genome DASL HT (Illumina) microarrays. Cell-specific DMRs were identified by comparative analysis of DNA methylation and mRNA expression patterns between different cell lineages, and validated by MS-qPCR and pyrosequencing. Analysis of the microarray data revealed CpG loci significantly demethylated in NK cells compared to all other leukocyte lineages (q &amp;lt; 0.1) within genes exhibiting significantly increased mRNA expression in NK cells (q &amp;lt; 0.1). Pyrosequencing and MS-qPCR assays confirmed a NK cell specific demethylated region. The MS-qPCR assay was calibrated using known quantities of NK cell DNA to facilitate quantification of these cells in biological samples, and was then applied to archival peripheral blood DNA samples from 122 control donors and 122 HNSCC patients. Demethylation of the candidate region was significantly lower in HNSCC patient bloods compared with control bloods (p &amp;lt; 0.001), but was not significantly associated with age, gender, HPV16 (E6 and/or E7) serology, cigarette smoking, alcohol consumption, BMI, or disease treatment. Measurements from control samples were used to establish normal demethylation tertiles. Proportion of total HNSCC cases decreased significantly with increasing tertile (p &amp;lt; 0.001). Multivariate logistic regression controlling for age, gender, cigarette smoking, alcohol consumption, BMI, and HPV16 serology confirmed increased HNSCC risk for individuals in the lowest tertile (OR = 5.6, 95% CI: 2.0, 17.4) and second lowest tertile (OR = 4.9, 95% CI: 1.8, 16.1). The advent of DNA methylation based immunodiagnostic methods will make assessment of immune cell profiles in archived samples that have not undergone rigorous special handling requirements possible. Using a novel NK cell specific demethylated region to detect and quantify NK cells in the blood, we have detected a HNSCC associated decrease in circulating NK cells that is independent of disease treatments, known exposures and other putative risk factors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-374. doi:1538-7445.AM2012-LB-374

TLR3 and RIG-I gene variants: Associations with functional effects on receptor expression and responses to measles virus and vaccine in vaccinated infants

Measles virus causes severe morbidity and mortality, despite the availability of measles vaccines. Successful defence against viral pathogens requires early recognition of virus-specific patterns by innate receptors like Toll-like receptor (TLR)3 and the RNA helicase, retinoic acid inducible gene-I (RIG-I). Genetic differences in these receptors may influence the primary immune responses to measles and the efficacy of measles vaccine. In 1-year-old Australian infants after their first measles vaccine dose, we investigated functional effects of TLR3 and RIG-I polymorphisms on intracellular protein expression using flow cytometry, cytokine responses to receptor ligands and measles lysate, and post-vaccination measles IgG levels. We found that TLR3 Leu412Phe was significantly associated with IFN-α/β response after stimulation with TLR3 ligand, poly(I:C) (P=0.024). Downregulation of TLR3 protein expression in NK cells after poly(I:C) was also associated with this variant (P=0.011). In contrast, measles-specific expression, cytokine responses and antibody responses were not associated with TLR3 polymorphisms. No associations were found with RIG-I variants. These results suggest that a TLR3 polymorphism has functional effects on receptor expression and cytokine response. However, this did not translate to an effect on specific responses to measles virus or vaccine. We found no evidence that RIG-I polymorphisms were involved in measles immune responses.

Extracellular heat‐shock protein 90 down‐regulates NK cell immune responses

90‐Kda heat‐shock proteins (Hsp90s) are expressed on various cells. The function of Hsp90 is different depending on the intracellular and extracellular locations. Intracellular Hsp90 has protective function regulating programmed cell death in lethal condition and thereby blocks the apoptotic cell death. Cancer cells abnormally express a high level of Hsp90 and intracellularly overexpressed Hsp90 facilitates oncogenesis and chemotherapy resistance. In contrast, extracellular Hsp90 or membrane‐bound Hsp90 is related to immune responses but the function of extracellular Hsp90 is not known well. NK cells are cytotoxic lymphocytes that play an important role in the rejection of tumor and virus infected cells. Here we showed the effects of the extracellular Hsp90 to NK cells. We investigated interferon‐γ (IFN‐γ) secretion, cytotoxic effects, and the changes of various receptors and ligands expression of NK cells when treated with recombinant Hsp90 with/without IL‐2. As a result, the recombinant Hsp90 was not effective in IFN‐γ secretion but it inhibited IFN‐γ secretion of IL‐2 stimulated NK cells. In addition, NK cell cytotoxicity to breast and ovarian cancer cell lines, MCF7 and CAOV3, was inhibited when NK cells were treated with Hsp90. Consistently with the above results, the recombinant Hsp90 down regulated TRAIL, NKG2D, and HLA‐DR and up regulated inhibitory receptor KIR. These results demonstrated that the extracellular Hsp90 down‐regulates NK cell immune responses, therefore, blocking the intracellular and extracellular Hsp90 can be helpful in preventing cancer progression and in enhancing immune responses against the cancer cells.

Glucocorticoid Receptor Mediates the Effect of Progesterone on Uterine Natural Killer Cells

Uterine natural killer cells (uNK) do not express progesterone receptor, but express glucocorticoid receptor (GR). So, we speculate that progesterone may regulate uNK cells through a GR-mediated process. After mouse NK cells were stimulated with CpG with or without IL-12 in the presence or absence pre-treatment of progesterone, the effects of progesterone on NK via GR were investigated by using RU486 (progesterone receptor and GR antagonist) and CDB-2914 (progesterone receptor antagonist). The expressions of CD69 and IFN-γ were determined by flow cytometry and qPCR. Phosphorylation of IκB and STAT4 was determined by Western blot. Furthermore, we purified uNK cells from human decidual tissues using anti-CD56 microbeads to verify the effect of progesterone on uNK via GR. Progesterone suppressed CD69 and IFN-γ expression of mouse spleen NK cells and human uNK cells induced by CpG combined with IL-12. CDB-2914 had no effect on IFN-γ expression suppressed by progesterone, while RU486 could abolish the inhibitory effect of progesterone. In addition, progesterone could decrease the phosphorylation of both STAT4 and IκB. In the present study, we first prove that progesterone can regulate NK cells via GR. It is valuable for further understanding the role of uNK in progesterone regulated gestation process.