NK Cell Expression Research Articles

Mechanisms Underlying the Antifatigue Effects of the Mycelium Extract of <i>Cordyceps</i> (<i>Paecilomyces hepiali</i>, 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.

NKG2D and DNAM-1 Ligands: Molecular Targets for NK Cell-Mediated Immunotherapeutic Intervention in Multiple Myeloma.

A pivotal strategy to improve NK cell-mediated antitumor activity involves the upregulation of activating ligands on tumor cells. Enhancement of NK cell-mediated recognition of multiple myeloma cells was reported by us and others showing increased surface expression of NKG2D and DNAM-1 ligands on tumor cells following treatment with a number of chemotherapeutic agents, such as genotoxic drugs or inhibitors of proteasome, histone deacetylases, GSK3, and HSP-90. These compounds have the capability to affect tumor survival but also to activate specific transduction pathways associated with the upregulation of different NK cell activating ligands on the tumor cells. Here, we will summarize and discuss the molecular pathways whereby these drugs can regulate the expression of NK cell activating ligands in multiple myeloma cells.

Fas/FasL pathway participates in regulation of antiviral and inflammatory response during mousepox infection of lungs.

Fas receptor-Fas ligand (FasL) signalling is involved in apoptosis of immune cells as well as of the virus infected target cells but increasing evidence accumulates on Fas as a mediator of apoptosis-independent processes such as induction of activating and proinflammatory signals. In this study, we examined the role of Fas/FasL pathway in inflammatory and antiviral response in lungs using a mousepox model applied to C57BL6/J, B6. MRL-Faslpr/J, and B6Smn.C3-Faslgld/J mice. Ectromelia virus (ECTV) infection of Fas- and FasL-deficient mice led to increased virus titers in lungs and decreased migration of IFN-γ expressing NK cells, CD4+ T cells, CD8+ T cells, and decreased IL-15 expression. The lungs of ECTV-infected Fas- and FasL-deficient mice showed significant inflammation during later phases of infection accompanied by decreased expression of anti-inflammatory IL-10 and TGF-β1 cytokines and disturbances in CXCL1 and CXCL9 expression. Experiments in vitro demonstrated that ECTV-infected cultures of epithelial cells, but not macrophages, upregulate Fas and FasL and are susceptible to Fas-induced apoptosis. Our study demonstrates that Fas/FasL pathway during ECTV infection of the lungs plays an important role in controlling local inflammatory response and mounting of antiviral response.

Dasatinib Expands Pre-Existing, CMV-Associated, Highly Differentiated NK Cells in Ph+ Leukemia

[Background] Tyrosine kinase inhibitors (TKIs), imatinib, nilotinib and dasatinib are key drugs for the treatment of Philadelphia chromosome-positive (Ph+) leukemia. Among these TKIs, only dasatinib expands large granular lymphocytes (LGLs) including NK cells in some patients. This dasatinib-induced lymphocytosis is associated with enhanced therapeutic responses. Recently, it has been suggested that dasatinib-associated LGL expansion is linked to cytomegalovirus (CMV) reactivation. CMV infection induces substantial changes in phenotypic and functional profiles of NK cells. However, characteristics of NK cells in dasatinib-treated Ph+ leukemia patients (DA) and mechanisms by which dasatinib expands NK cells remain unknown.[Method] NK-cell marker expression and cytotoxic activity of NK cells were examined in 31 DA, 15 patients treated with other TKIs (OT) and 15 healthy donors (HD). We mapped NK-cell profiles, using principal component analysis (PCA) of 19 NK-cell markers.[Result] NK cells increased in 18 of 26 CMV-seropositive (CMV+) DA, in contrast to no increase in 5 CMV- DA and 13 CMV+ OT. Expression of an activating receptor NKG2C was enhanced on the increased NK cells. PCA mapped NK-cell profiles according to the degree of differentiation, which correlated with higher expression of NKG2C, CD57 and LIR-1, and lower expression of NKp30 and NKp46, revealing that NK cells in CMV+ DA were highly differentiated compared to those in CMV- DA and HD (Figure 1). NK cells in CMV+ OT were mapped between CMV+ DA and HD. In 10 CMV+ DA, recent CMV reactivation was detected by CMV IgM and/or quantitative PCR. Of these, CMV reactivation was symptomatic only in 4 patients, indicating that dasatinib induces subclinical CMV reactivation in the majority of patients. CD107a degranulation assay showed higher cytotoxic activity of NKG2C+ NK cells, compared to NKG2C- NK cells. Interestingly, some of the CMV+ patients with Ph+ leukemia had NKG2C+ highly differentiated NK cells already at diagnosis, suggesting that CMV has been reactivated before starting TKIs at least in some patients. In fact, we detected CMV genome by nested PCR in PBMCs from some of the pretreatment patients. Moreover, the CMV+ patients with highly differentiated NK cells developed NK-LGL expansion earlier after starting dasatinib, compared to those with less differentiated NK cells.[Discussion] Our data indicate that CMV seropositivity underlies the development of NK-LGL expansion in DA patients. Intriguingly, it appears that CMV has been subclinically reactivated at least in some Ph+ leukemia patients even before starting dasatinib, leading to expansion of "ready-to-go" NKG2C+ highly differentiated NK cells. It is likely that dasatinib further expands such NK cells presumably due to chronic stimulation by reactivated CMV. The NKG2C+ NK cells have high cytotoxic activity against leukemic cells, which might contribute to enhanced therapeutic responses in DA patients. The phenotypic profile of NK cells at diagnosis may be a strong predictor of NK-LGL expansion during dasatinib therapy. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Early Disease Relapse after Tyrosine Kinase Inhibitor Treatment Discontinuation in CML Is Related Both to Low Number and Impaired Function of NK-Cells

▪Background: Recent reports suggest that approximately 40% of CML patients who have achieved sustained complete molecular remission are able to stop TKI treatment without disease relapse. However, there are no predictive markers for successful therapy discontinuation. Therefore, we set up an immunological sub-study in the ongoing pan-European EURO-SKI stopping study. Our aim was to identify predictive biomarkers for relapse/non-relapse and to understand more on the mechanisms of immune surveillance in CML.Methods: The EURO-SKI study started in 2012, and patients included were at least three years on TKI and at least one year in MR4 or deeper before the study entry. Basic lymphocyte immunophenotyping (the number of NK-, T- and B-cells) was performed at the time of therapy discontinuation and 1, 6, and 12 months after the TKI stop and in case of relapse (defined as loss of MMR, BCR-ABL1>0.1% IS). In addition, from a proportion of patients more detailed immunophenotypic and functional analyses (cytotoxicity of NK-cells and secretion of Th1 type of cytokines IFN-γ/TNF-α) were done at the same times.Results: Thus far 119 Nordic patients (imatinib n=105, dasatinib n=12, nilotinib n=2) who have discontinued TKI treatment within the EURO-SKI study have been included in the lymphocyte subclass analysis (results are presented from patients who have reached 6 months follow-up). Immunophenotyping analysis demonstrates that imatinib treated patients who were able to maintain remission for 6 months (n=36) had increased NK-cell counts (0.26 vs. 0.15x109cells/L, p=0.01, NK-cell proportion 18.9% vs. 11%, p=0.005) at the time of drug discontinuation compared to patients who relapsed early (before 5 months n=22).Furthermore, the phenotype of NK-cells was more cytotoxic (more CD57+ and CD16+cells and less CD62L+cells), and also their IFN-γ/TNF-α secretion was enhanced (19.2% vs. 13%, p=0.02). Surprisingly, patients who relapsed more slowly (after 5 months, n=16) had similar baseline NK-cell counts (0.37x109cells/L), NK-cell proportion (21.2%), and phenotype and function as patients, who were able to stay in remission. No differences in the NK-cell counts were observed between patients who had detectable or undetectable BCR-ABL1 transcripts at the baseline (0.22 x109cells/L vs. 0.31 x109cells/L, p=0.61). Interestingly, NK-cell count was higher in patients with low Sokal risk score than in patients with intermediate risk (0.33 x109cells/L vs. 0.20 x109cells/L, p=0.04). Furthermore, there was a trend that male patients had a higher proportion of NK-cells than females (21.6% vs. 15.7%, p=0.06). Pretreatment with IFN-α or the duration of imatinib treatment did not have an effect on NK-cell count or proportion. In comparison to the imatinib group, dasatinib treated patients had higher NK-cell counts at the baseline (median 0.52x109cells/L vs. 0.26x109cells/L, p=0.02), and also the proportion of CD27 (median 50% vs. 16%, p=0.01) and CD57 expressing (median 79% vs. 74%, p=0.05) NK-cells was higher. The follow-up time of dasatinib treated patients is not yet long enough to correlate the NK-cell counts with the success of the treatment discontinuation.The absolute number of T-cells or their function did not differ significantly between relapsing and non-relapsing patients at the time of treatment discontinuation. However, both CD4+ and CD8+ T-cells tended to be more mature in patients who stayed in remission compared to patients who relapsed early (CD4+CD57+CD62L- median 5.7% vs. 2.4%, p=0.06, CD8+CD62L+CD45RA+ 13% vs. 26.7%, p=0.05). The analysis of follow-up samples showed that in patients who stayed in remission the Th1 type cytokine (IFN-γ/TNF-α) secretion of CD8+T-cells increased at 6 months compared to baseline (23.6 vs. 18.5%, p=0.07). Same phenomenon was observed in the late relapsing group at relapse compared to baseline (37.9 vs. 13.5%, p=0.03). No similar increase was observed in the early relapsing group.Conclusions: Low NK-cell numbers and poor cytokine secretion may predict early disease relapse after TKI discontinuation. However, patients who relapse later have high numbers of normally functioning NK-cells. Further research (detailed phenotypic analysis of NK- and T-cells including activating and inhibitory receptors and immune checkpoint molecules) and correlation of biomarker data with clinical parameters are ongoing to understand the ultimate determining factors of relapse. DisclosuresSjälander:Novartis: Honoraria. Hjorth-Hansen:Novartis: Honoraria; Bristol-myers Squibb: Honoraria; Ariad: Honoraria; Pfizer: Honoraria. Porkka:BMS: Honoraria; BMS: Research Funding; Novartis: Honoraria; Novartis: Research Funding; Pfizer: Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

The change of NCR expression and cytokine production of NK cells in peripheral blood and peritoneal fluid by hormonal treatment in patients with endometriosis

Problem: It is considered that NK22 cells might mediate mucosal immune defense in several organ. In response to pathogen, IL-23 stimulates NK22 cells to secrete IL-22. It is only reported that NK22 cells were present in the uterinemucosa. Toexamine theNK22 function,we investigated theproductionand relationof IL-22and theother cytokines using endometrium (EM) and peripheral blood (PB) NK cells of women with recurrent pregnancy loss (RPL). Materials and methods: Cytokines production (IFNgamma, TNF-alpha, IL-4, IL-10, IL-22, TGF-b1) by PB and EMNK cells and the expression of Nkp46were analyzed by triple color flow cytometry inwomenwith RPL (n=43) and controls (n=38). Moreover, we classified into four groups with high or low percentage of IL-22 production (RPL-H, RPL-L, Control-H and Control-L). Results: In EM, the percentage of NK22 cells in women with RPL was significantly higher than that of controls (p<0.05). In PB and EM, the percentage of IFN-gammaproducing NK22 cells in RPL-H was significantly lower than that in Control-L (p<0.05). There were negative correlations between NK22 cells, and IFN-gamma (r=−0.6) or TNF-alpha (r=−0.6) in women with RPL. There was a negative correlation between CD56bright/IL-22+ cells and CD56bright/NKp46+ cells (r=−0.271). Conclusions:NK22 cell might play an important role in women with RPL by regulating cytokine production by NK cells.

The differential characterization of GPR55 receptor in human peripheral blood reveals a distinctive expression in monocytes and NK cells and a proinflammatory role in these innate cells.

G protein-coupled receptor 55 (GPR55) is activated by endogenous, plant-derived and synthetic cannabinoids. Recent studies reported a broad tissue distribution for GPR55 and found prominent roles for this receptor in inflammatory pain, gut and bone physiology, as well as cancer. However, little is known about the expression and function of GPR55 in immune cells. To address this question, we performed a detailed characterization of GPR55 in different human innate and adaptive immune populations using polychromatic flow cytometry and we found that monocytes and NK cells expressed remarkable levels of this receptor compared to several cells of adaptive immunity. GPR55 activation by the specific agonist O-1602 boosted IL-12 and TNF-α production, and decreased endocytic activity, in LPS-activated monocytes. In addition, it increased CD69 activation marker expression, granzyme B and CD107a-dependent cytotoxicity and IFN-γ and TNF-α production in NK cells activated by both IL-2 and IL-12. These over-stimulatory effects of GPR55 were antagonized by its selective antagonist cannabidiol. Altogether, our data thus unveil a proinflammatory role for GPR55 in innate immunity that may be important for the design of new immune therapeutic strategies.

KIR genotype distribution among patients with multiple myeloma: Higher prevalence of KIR 2DS4 and KIR 2DS5 genes

IntroductionNatural killer (NK) cells possess an antitumor activity against multiple myeloma cells proven by the susceptibility of plasmocytes to NK lysis. In the early stage of MM, the killing of MM cells is mediated by natural cytotoxicity receptors (NRC) and NKG2D-dependent pathway, while in the late stage, NK cells lose their killing potential against MM cells due to the high expression of HLA class I molecules on MM cells. AimThe aim of this paper is to study KIR expression of NK cells in MM patients and in healthy controls, to check for any association between KIR genotypes and MM. MethodsKIR genotype was analyzed in 120 healthy Lebanese individuals and 34 MM patients using the KIR Genotyping SSP kit. ResultsKIR 2DS4*001/002 and KIR 2DS5 were found to be significantly more prevalent among MM patients as compared to controls. For MM patients, the AA, AB, and BB genotype frequencies were, respectively, 38.23%, 47.06% and 14.71% with an A:B ratio of 1.62:1. As for the healthy controls, the AA, AB, and BB genotype frequencies were, respectively, 39.17%, 50%, and 10.83% with an A:B ratio of 1.80:1. ConclusionThe interesting observation of the significant presence of KIR2DS4 and KIR2DS5 genes more among multiple myeloma patients than controls is worth further clinical, translational as well as survival research studies in these cases.

NKG2A expression and impaired function of NK cells in patients with new onset of Graves' disease.

Graves' disease (GD) is an organ-specific autoimmune disease. A significant decrease of the distribution of NK cells in the peripheral blood in children and adolescents with untreated GD has been observed. However, the role of NK and its subsets in adults with GD remains unclear. A total of 28 adult patients with new onset of GD and 23 healthy controls (HC) were recruited. The number of activated inhibitory NK cells in peripheral blood of individual subjects was determined by flow cytometry. The number of CD3(-)CD56(+) and CD3(-)CD16(+)NK cells in peripheral blood was significantly decreased in the GD patients than the HC. Compared to the HCs, decreased number of NKG2D(+), NKG2C(+), NKp30(+) and NKG2A(+) NK cells and increased number of KIR3DL1(+) NK cells were detected in the GD patients. Moreover, the number of inducible CD107a(+) and IFN-γ-secreting NK cells in GD patients significantly decreased than those in HC. Interestingly, the number of NKG2A(+)NK cells was negatively correlated with the level of serum TRAb in GD patients. Our data indicate that decreased number and impaired function of NK cells may contribute to the pathogenesis of GD.

Phenotypic and Functional Characteristics of Siglec-7, NKG2A and NKG2C Expressing NK Cells in Chronic HIV-1 Clade C Infection

AIDS Research and Human RetrovirusesVol. 30, No. S1 Innate ImmunityPhenotypic and Functional Characteristics of Siglec-7, NKG2A and NKG2C Expressing NK Cells in Chronic HIV-1 Clade C InfectionMichael Z. Zulu, Kewreshini Naidoo, Zenele Mncube, Manjeetha Jaggernath, Philip Goulder, Thumbi Ndung'u, Marcus Altfeld, and Christina ThobakgaleMichael Z. ZuluDoris Duke Medical Research Institute, University of KwaZulu-Natal, HIV Pathogenesis Programme, Durban, South AfricaSearch for more papers by this author, Kewreshini NaidooDoris Duke Medical Research Institute, University of KwaZulu-Natal, HIV Pathogenesis Programme, Durban, South AfricaSearch for more papers by this author, Zenele MncubeDoris Duke Medical Research Institute, University of KwaZulu-Natal, HIV Pathogenesis Programme, Durban, South AfricaSearch for more papers by this author, Manjeetha JaggernathDoris Duke Medical Research Institute, University of KwaZulu-Natal, HIV Pathogenesis Programme, Durban, South AfricaSearch for more papers by this author, Philip GoulderDoris Duke Medical Research Institute, University of KwaZulu-Natal, HIV Pathogenesis Programme, Durban, South AfricaPeter Medawar Building for Pathogen Research, University of Oxford, Department of Paediatrics, London, United KingdomSearch for more papers by this author, Thumbi Ndung'uDoris Duke Medical Research Institute, University of KwaZulu-Natal, HIV Pathogenesis Programme, Durban, South AfricaRagon Institute of MIT, MGH and Harvard, Boston, MA, United StatesSearch for more papers by this author, Marcus AltfeldDoris Duke Medical Research Institute, University of KwaZulu-Natal, HIV Pathogenesis Programme, Durban, South AfricaRagon Institute of MIT, MGH and Harvard, Boston, MA, United StatesHeinrich-Pette Institute, Department of Viral Immunology, Hamburg, GermanySearch for more papers by this author, and Christina ThobakgaleDoris Duke Medical Research Institute, University of KwaZulu-Natal, HIV Pathogenesis Programme, Durban, South AfricaSearch for more papers by this authorPublished Online:30 Oct 2014https://doi.org/10.1089/aid.2014.5256.abstractAboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"Phenotypic and Functional Characteristics of Siglec-7, NKG2A and NKG2C Expressing NK Cells in Chronic HIV-1 Clade C Infection." AIDS Research and Human Retroviruses, 30(S1), p. A130FiguresReferencesRelatedDetails Volume 30Issue S1Oct 2014 InformationCopyright 2014, Mary Ann Liebert, Inc.To cite this article:Michael Z. Zulu, Kewreshini Naidoo, Zenele Mncube, Manjeetha Jaggernath, Philip Goulder, Thumbi Ndung'u, Marcus Altfeld, and Christina Thobakgale.Phenotypic and Functional Characteristics of Siglec-7, NKG2A and NKG2C Expressing NK Cells in Chronic HIV-1 Clade C Infection.AIDS Research and Human Retroviruses.Oct 2014.A130-A130.http://doi.org/10.1089/aid.2014.5256.abstractPublished in Volume: 30 Issue S1: October 30, 2014PDF download

Abstract 2545: Histamine dihydrochloride and interleukin-2 for relapse prevention in AML: Initial results of the Re:MISSION phase IV trial

Abstract BACKGROUND AND METHODS. Immunotherapy with histamine dihydrochloride and low-dose interleukin-2 (HDC/IL-2) has been approved within the EU for relapse prevention in the post-consolidation phase of acute myeloid leukemia (AML) (Blood 108:88). We have performed a phase IV trial (Re:MISSION) to assess the immunomodulatory properties of this regimen and to correlate potential biomarkers of immunity with outcome. Eighty-nine patients (age 19-80) with AML in first complete remission received HDC (0.5 mg sc bid) and human recombinant IL-2 (1MIU sc bid) in ten repeated three-week cycles for 18 months or until relapse. Mononuclear cells were analyzed for NK and T cell phenotypes by flow cytometry at onset and during cycles 1 and 3. INDUCTION OF NK CELLS AND NCR EXPRESSION DURING THERAPY. Treatment with HDC/IL-2 resulted in a 3-fold expansion of both CD56bright (CD56/CD16-) and CD16+ (CD56+/CD16+) NK cells during cycle 1 (CD56+, P= 10-6, n=47; paired samples t-test). During cycle 1, the median expression intensity of the natural cytotoxicity receptors (NCRs) NKp46 and NKp30 on CD16+ NK cells increased by 50% and 30%, respectively (P=6 x10-8 for NKp46; P=2 x10-7 for NKp30, n=56). For CD56bright cells, the results were discordant for the NCRs with pronounced induction of NKp30 (P=4 x10-13) during cycle 1 but no induction of NKp46 (P=0.51, n=56). ROLE OF NCR EXPRESSION FOR MAINTENANCE OF REMISSION. An interim analysis of immune markers vs. relapse-free survival (RFS) revealed a strong impact of NK cell NKp46 expression. Patients with NKp46 expression above the median after one 21-day cycle of HDC/IL-2 thus showed significantly higher RFS than those with lower expression (CD16+, P=0.003, HR=3.0, CI 1.4-6.4; CD56bright, P=0.002, HR=3.3, CI: 1.5-7.0; logrank test). High NK cell expression of NKp46 after a treatment cycle also identified a population of elderly patients (&amp;gt;60 years old) with favorable outcome (CD16+, P=0.002, HR=4.1, CI: 1.6-10.2; logrank test). We observed a pronounced expansion of CD4+/CD25+/Foxp3+ regulatory T cells during cycles that did not impact on relapse risk. CONCLUSION. These results highlight the role of NK cells and NCR expression for relapse-free survival in AML. Citation Format: Anna Martner, Anna Rydström, Rebecca Riise, Johan Aurelius, Mats Brune, Kristoffer Hellstrand, Fredrik Bergh Thorén. Histamine dihydrochloride and interleukin-2 for relapse prevention in AML: Initial results of the Re:MISSION phase IV trial. [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 2545. doi:10.1158/1538-7445.AM2014-2545

P064 : KIR mRNA EXPRESSION IN NATURAL KILLER CELLS AND PERIPHERAL BLOOD MONONUCLEAR CELLS ASSAYED BY QUANTITATIVE REAL-TIME PCR

Aim To develop assays to quantify the mRNA transcribed from 15 KIR functional genes utilizing a quantitative real-time PCR (qPCR) method. To study the KIR mRNA expression patterns in NK cells and peripheral blood mononuclear cells (PBMC) among 50 healthy volunteers. To correlate the qPCR results from NK cells and PBMC. Methods PBMC were obtained from freshly drawn whole blood (maximum 48 h) from 50 healthy volunteers using a Ficoll method. NK cell enrichment from PBMC was performed by negative selection using a magnetic bead kit. mRNA extraction and cDNA synthesis was followed by qPCR using EvaGreen dye. Relative quantification (RQ) of KIR mRNA expression was quantified by 2-ΔΔCt algorithm with GAPDH as the housekeeping gene and a commercial human cDNA as the universal reference. Results 16 mRNA expression assays for 15 KIR genes, including assays for both the full-length and truncated variants for 2DS4, have been developed and validated. Among the 50 individuals, KIR mRNAs were detected in all samples carrying respective KIR genes and were negative for the KIR gene negative samples tested. In particular, 2DL1, 2DL4 and 3DL2 were positive for all 50 individuals at both genomic DNA and mRNA levels, whereas mRNA was undetected for all 2DL5B and 3DL3 gene positive samples. Among 50 individuals, the most variably expressed KIR mRNA was 2DL1 in NK cells and 3DL1 in PBMC. The strongest correlation between NK cells and PBMC was found for 2DS5 ( R = 0.9582, n = 9, p Conclusions We established qPCR assays to quantify the mRNA expression of 15 KIR genes. KIR mRNA expression varied among healthy individuals carrying the same KIR genotype. Analyzing NK cells and PBMC will allow establishing NK cell specific and overall KIR mRNA expression signatures from an individual. Results from NK cells and PBMC are correlated to a different degree for the 15 KIR genes, confirming that NK cells are the main predictor for KIR mRNA expression in PBMC. However, any KIR mRNA expression in PBMC is only partially reflective of the KIR mRNA expression in NK cells.

Interleukin-7 expression and its effect on natural killer cells in patients with multiple sclerosis

Decreased NK cell numbers and impairment of NK cell function are reported in patients with multiple sclerosis (MS). Interleukin-7 (IL-7) is a member of the common gamma-chain (γc) cytokine superfamily that has well documented roles in lymphocyte development and homeostasis. The interleukin-7 receptor α chain (IL-7Rα) gene was identified as a top non-major histocompatibility complex-linked risk locus for MS. The objective of this study was to test biological function of IL-7/IL-7Rα on NK cells in MS patients. We observed markedly lower IL-7 levels in MS sera, and relatively higher IL-7Rα expression in NK cells of MS. Upon IL-7 stimulation, IL-7Rα on NK cells from MS patients was significantly down-regulated compared with healthy controls (HCs). IL-7 induced a higher increase of IFN-γ production in CD56bright NK cells and a pronounced enhancement of cytotoxicity in NK cells from MS. IL-7 did not impact the proliferation of NK cells differently in MS and HC. In contrast, IL-7 promoted a higher survival of CD56bright NK cells in MS and inhibited their apoptosis by increasing Bcl-2 expression, but had no effect on CD56dim NK cell survival in MS. In conclusion, MS patients have lower serum IL-7 and a higher membrane IL-7Rα expression on CD56bright NK cells. The skew at the IL-7 and IL-7Rα level influences functional responsiveness of NK cells in MS.

Role of Fas/FasL signaling in regulation of anti-viral response during HSV-2 vaginal infection in mice

Fas receptor–Fas ligand (FasL) signaling is involved in apoptosis of virus-infected cells but increasing evidence accumulates on Fas receptor as a mediator of apoptosis-independent processes such as induction of activating and pro-inflammatory signals. In this study, we examined the role of Fas/FasL pathway in regulation of anti-viral response to genital HSV-2 infection using a murine model of HSV-2 infection applied to C57BL6/J, B6. MRL-Faslpr/J and B6Smn.C3-Faslgld/J mice. HSV-2 infection of Fas- and FasL-deficient mice led to decreased migration of IFN-γ expressing NK cells and CD4+ T cells, but not of γδ T cells, into the vaginal tissue. The vaginal tissues of HSV-2 infected Fas- and FasL-deficient mice showed increased production of IL-10, followed by low expression of the early CD69 activation marker on CD4+ and CD8+ T cells and increased numbers of regulatory T cells (Tregs). Experiments in co-cultures of CD4+ T cells and bone marrow derived dendritic cells showed that lack of bilateral Fas–FasL signaling led to expansion of Tregs and increased production of IL-10 and TGF-β1. Our results demonstrate that Fas/FasL can regulate development of tolerogenic dendritic cells and expansion of Tregs early during HSV-2 infection, which further influences effective anti-viral response.

IL-33 promotes IFN-γ production by group 1 innate lymphoid cells in viral hepatitis (IRC8P.490)

Abstract Innate immune response is the first line of the defense against the viral pathogens. The damage associated molecular patterns (DAMP) molecule IL-33 has been proved to be critical for T cell response in viral persistent infection. However, its role in modulating the innate immune response in viral infection is not clear. Herein, we injected the mice with lymphocytic choriomeningitis virus (LCMV) Clone 13 and found that the hepatic gene expression of IL-33 was upregulated at 1 day post-infection (dpi) and reached the peak at 6 dpi. Deficiency of IL-33 not only led to impaired anti-viral T cell responses (low IFN-γ and TNF-α), but also resulted in less numbers of IFN-γ+ innate immune cells, such as NK and γδT cells at 16h postinfection. Treatment of recombinant IL-33 (rIL-33) significantly increased the percentages of IFN-γ +γδT cells. In vitro, rIL-33 inhibited proliferation of IL-17+γδ T cells, but increased IFN-γ+γδ T cells in a dose-dependent manner. rIL-33 treatment also upregulated IFN-γ and granzyme B expression in NK cells in vitro culture. More importantly, hepatic dendritic cells (DC) in IL-33 knockout mice expressed lower level of IL-12 compared with control mice, indicating that deficiency of IL-33 may impair the DC priming. Our data reveal the key role of IL-33 in promoting IFN-γ production by group 1 innate lymphoid cells and subsequently regulate the anti-viral T cell response in viral hepatitis.

IL-10 and IL-24 expression is regulated by distinct pathways in macrophages and NK cells (CCR5P.252)

Abstract Homologous gene in clusters can be coordinately regulated by shared genomic elements to synchronize biologic functions. Within the Il10 locus which includes Il19, Il20 and Il24, only IL-10 and IL-24 are preferentially expressed by Th2 cells. The extent to which IL-24 is regulated in other IL-10-producing cell types and its role in immunity remains unclear. To determine if these genes share regulatory pathways, we examined IL-24 expression in macrophages and NK cells which are key sources of IL-10. In macrophages, maximal LPS-induced IL-24 expression requires IL-4 and Stat6 while IL-10 induction is largely independent of Stat6. Likewise, in NK cells, IL-10 expression is independent of IL-4 but IL-24 is strongly induced by IL-4 in combination with IL-2 and is dependent on Stat6. We identified 4 putative Stat-binding elements in the Il24 gene and found that each site binds IL-4-induced Stat6 in macrophages and NK cells. Interestingly, these sites also bound Stat4 in IL-12-stimulated NK cells but not in macrophages. In NK cells, IL-24 expression required type I IFN signaling regardless of the stimulation while IL-10 expression required type I IFNs only in macrophages. Histone modifications in Il24 were dependent on IL-4/Stat6 in macrophages but not in NK cells. Our results indicate that although IL-10 and IL-24 are co-expressed in macrophages and NK cells, they are governed by distinct signaling pathways suggesting they are unlikely to share genomic regulatory elements.

CCR5 on the NK Cells and its Ligand (RANTES) Expressions are Disrupted in South-Eastern Iranian Patients With Chronic Hepatitis B Infection.

Background:CCR5 is a receptor for CCL3 (MIP-1 α), CCL4 (MIP-1 α) and CCL5 (regulated on activation normal T cell expressed and secreted (RANTES)) and play important roles in recruitment of NK cells to the HBV infected liver.Objectives:The main purpose of this study was to investigate the expression levels of CCR5 on the NK cells and also serum levels of RANTES in chronic HBV infected (CHI) patients.Materials and Methods:In this descriptive study 63 CHI patients and 96 healthy controls were evaluated regarding CCR5 expression on the NK cells and serum levels of RANTES using flow cytometry and ELISA techniques, respectively. Real-Time PCR technique also was used for HBV-DNA quantification.Results:The results revealed that CCR5 expressing NK cells and serum levels of RANTES were decreased significantly in the CHI patients in compare to healthy control.Conclusions:Based on the results it can be concluded that NK cells of Iranian CHI patients are unable to express adequate levels of CCR5 and expression levels of RANTES by immune cells also are defected in CHI patients, hence, the migration of NK cells to the infected hepatocytes and HBV eradication from the cells is interrupted.

Conjunctival scarring in trachoma is associated with the HLA-C ligand of KIR and is exacerbated by heterozygosity at KIR2DL2/KIR2DL3.

Background Chlamydia trachomatis is globally the predominant infectious cause of blindness and one of the most common bacterial causes of sexually transmitted infection. Infections of the conjunctiva cause the blinding disease trachoma, an immuno-pathological disease that is characterised by chronic conjunctival inflammation and fibrosis. The polymorphic Killer-cell Immunoglobulin-like Receptors (KIR) are found on Natural Killer cells and have co-evolved with the Human Leucocyte Antigen (HLA) class I system. Certain genetic constellations of KIR and HLA class I polymorphisms are associated with a number of diseases in which modulation of the innate responses to viral and intracellular bacterial pathogens is central.MethodologyA sample of 134 Gambian pedigrees selected to contain at least one individual with conjunctival scarring in the F1 generation was used. Individuals (n = 830) were genotyped for HLA class I and KIR gene families. Family Based Association Tests and Case Pseudo-control tests were used to extend tests for transmission disequilibrium to take full advantage of the family design, genetic model and phenotype.Principle findingsWe found that the odds of trachomatous scarring increased with the number of genome copies of HLA-C2 (C1/C2 OR = 2.29 BHP-value = 0.006; C2/C2 OR = 3.97 BHP-value = 0.0004) and further increased when both KIR2DL2 and KIR2DL3 (C2/C2 OR = 5.95 BHP-value = 0.006) were present.ConclusionsTo explain the observations in the context of chlamydial infection and trachoma we propose a two-stage model of response and disease that balances the cytolytic response of KIR expressing NK cells with the ability to secrete interferon gamma, a combination that may cause pathology. The data presented indicate that HLA-C genotypes are important determinants of conjunctival scarring in trachoma and that KIR2DL2/KIR2DL3 heterozygosity further increases risk of conjunctival scarring in individuals carrying HLA-C2.

Homeostasis of Human NK Cells Is Not IL-15 Dependent

IL-15 is a proinflammatory cytokine that plays an important role in the development and activation of NK cells and is a potential target for inflammatory disease therapy. Studies conducted in IL-15- and IL-15R knockout mice identified IL-15 as an important cytokine for NK cell homeostasis. Consistent with this information derived from genetically modified mice, we demonstrated that neutralizing IL-15 with a mouse anti-mouse IL-15 mAb (M96) depletes C57BL/6 mouse NK cells. An mAb directed against macaque IL-15 (Hu714MuXHu) was manufactured and demonstrated to block IL-15-induced activation of nonhuman primate (NHP) NK cells in vitro. Neutralization of macaque IL-15 by parenteral administration of Hu714MuXHu reduces (>95%) circulating NK cell counts in NHPs. A blocking mAb directed against human IL-15 (huIL-15; AMG 714) was manufactured. Unexpectedly, when human subjects were treated with the blocking anti-IL-15 Ab AMG 714 in clinical trials, no reductions in circulating NK cell counts were observed despite achieving significantly higher exposures than the levels of Hu714MuXHu needed to cause NK cell count reductions in NHPs in vivo. Both AMG 714 and Hu714MuXHu are able to block huIL-15 activity in a human T cell blast proliferation and IFN-γ production assay. Both Abs block huIL-15-mediated Stat5 activation and CD69 expression in human NK cells. Collectively, these results demonstrate that NK cell homeostasis is obligatorily dependent upon IL-15 in both mice and NHPs, but that IL-15 is dispensable for maintenance of circulating human NK cells.

Menstrual blood closely resembles the uterine immune micro-environment and is clearly distinct from peripheral blood

Is menstrual blood a suitable source of endometrial derived lymphocytes? Mononuclear cells isolated from menstrual samples (menstrual blood mononuclear cells (MMC)) are clearly distinct from peripheral blood mononuclear cells (PBMC) and show a strong resemblance with biopsy-derived endometrial mononuclear cells. A critical event in the onset of pregnancy is the implantation of the embryo in the uterine wall. The immune cell composition in the endometrium at the time of implantation is considered pivotal for success. Despite advancing knowledge on the composition of the immune cell population in the uterus, the role of endometrial immune cells in reproductive disorders is still not fully resolved, mainly due to the fact that this type of research requires invasive techniques. Here, we collected menstrual fluid and validated this unique non-invasive technique to obtain and study the endometrium-derived immune cells which would be present around the time of implantation. Five healthy non-pregnant females with regular menstruation cycles and not using oral contraceptives collected their menstrual blood using a menstrual cup in five consecutive cycles. Sampling took place over the first 3 days of menses, with 12 h intervals. Peripheral blood samples, taken before and after each menstruation, were obtained for comparative analysis. MMC and PBMC samples were characterized for the different lymphocyte subsets by flow cytometry, with emphasis on NK cells and T cells. Next, the functional capacity of the MMC-derived NK cells was determined by measuring intracellular production of IFN-γ, granzyme B and perforin after culture in the presence of IL-2 and IL-15. In support of their endometrial origin, MMC samples contained the typical composition of mononuclear cells expected of endometrial tissue, were phenotypically similar to the reported phenotype for biopsy-derived endometrial cells, and were distinct from PBMC. Increased percentages of NK cells and decreased percentages of T cells were found in MMC when compared with PBMC from the same female. The MMC-derived NK cells were pre-dominantly CD56(bright)/CD16(-), in contrast to the primarily CD56(dim)/CD16(+) peripheral blood NK cells. MMC-derived NK cells expressed CD103, indicating their mucosal origin. In addition, the pattern of natural cytotoxicity receptor (NCR) expression in MMC-derived NK cells was comparable with that in endometrial biopsy-derived NK cells. Compared with PBMC, the NKp30 expression was decreased, while the percentage of NKp44 positive cells was increased in MMC samples. CXCR3 and CXCR4 were hardly expressed by MMC-derived NK cells, indicating that these cells are not of PBMC origin. NK cells from MMC samples were functional as shown by their capacity to produce IFN-γ, granzyme B and perforin, upon stimulation with IL-2 and IL-15. MMC-derived T cells revealed an increased expression of CD103, CD69 and CXCR4 compared with PBMC-derived T cells. Importantly, MMC collection using a menstrual cup proved highly reliable and reproducible between women and between cycles. Based on the parameters we studied, MMC appear similar to biopsy-derived endometrial mononuclear cells. However, sampling is not done at the exact same time in the menstrual cycle, and thus we cannot exclude some, as yet undetected, differences. Also, it should be considered that for some women, the use of the menstrual cup may be unpleasant. Menstrual blood may be a source of endometrial cells and may create new opportunities to study uterine immunological cells in fertility issues. No external funding was obtained for the present study. None of the authors have any conflict of interest to declare. NA.