Regulation Of NK Cell Function Research Articles

CD8+ and CD8- NK Cells and Immune Checkpoint Networks in Peripheral Blood During Healthy Pregnancy.

Pregnancy involves significant immunological changes to support fetal development while protecting the mother from infections. A growing body of evidence supports the importance of immune checkpoint pathways, especially at the maternal-fetal interface, although limited information is available about the peripheral expression of these molecules by CD8+ and CD8- NK cell subsets during the trimesters of pregnancy. Understanding the dynamics of these immune cells and their checkpoint pathways is crucial for elucidating their roles in pregnancy maintenance and potential complications. This study aims to investigate the peripheral expression and functional characteristics of CD8+ and CD8- NK cell subsets throughout pregnancy, providing insights into their contributions to maternal and fetal health. A total of 34 healthy women were enrolled from the first, 30 from the second and 40 from the third trimester of pregnancy. At the same time, 35 healthy age-matched non-pregnant women formed the control group. From peripheral blood, mononuclear cells were separated and stored at -80 °C. CD8+ and CD8- NK cell subsets were analyzed from freshly thawed samples, and surface and intracellular staining was performed using flow cytometric analyses. The proportions of CD56+ NK cells in peripheral blood were similar across groups. While CD8- NKdim cells increased significantly in all trimesters compared to non-pregnant controls, CD8+ NKdim cells showed no significant changes. CD8- NKbright cells had higher frequencies throughout pregnancy, whereas CD8+ NKbright cells significantly increased only in the first and second trimesters. The expression levels of immune checkpoint molecules, such as PD-1 and PD-L1, and cytotoxic-activity-related molecules were stable, with notable perforin and granzyme B increases in CD8- NKbright cells throughout pregnancy. Our study shows that peripheral NK cell populations, especially CD8- subsets, are predominant during pregnancy. This shift suggests a crucial role for CD8- NK cells in balancing maternal immune tolerance and surveillance. The stable expression of immune checkpoint molecules indicates that other regulatory mechanisms may be at work. These findings enhance our understanding of peripheral immune dynamics in pregnancy and suggest that targeting CD8- NKbright cell functions could help manage pregnancy-related immune complications. This research elucidates the stable distribution and functional characteristics of peripheral NK cells during pregnancy, with CD8- subsets being more prevalent. The increased activity of CD8- NKbright cells suggests their critical role in maintaining immune surveillance. Our findings provide a basis for future studies to uncover the mechanisms regulating NK cell function in pregnancy, potentially leading to new treatments for immune-related pregnancy complications.

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

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

Crosstalk between NFAT and NFκB Signaling Regulates NK Cell Immunosurveillance

One of the major transcriptional regulators in lymphoid cells is NFAT (Nuclear Factor of Activated T Cells), controlling lymphocyte development and activity. The role of NFAT signaling is well defined in T cells, the cytotoxic lymphocytes of the adaptive immunity. However, surprisingly little is known regarding the relevance of this transcription factor family in NK cells as effector cells of the innate immunity. Available data indicate that NFAT activity is dispensable for development of NK cells, whereas effects of the immunosuppressive drugs cyclosporin A and tacrolimus, that inhibit calcineurin and consecutively NFAT, implicate an involvement of the NFAT family in NK cell function. We here employed different genetic mouse models and functional analyses to unravel the role of NFAT1 (NFATc2) and NFAT2 (NFATc1) in NK cell reactivity. In vitro knockout (KO) of NFAT1 (NFATc2) or NFAT2 (NFATc1) enhanced NK cell degranulation and resulted in increased production of granzyme B and perforin upon stimulation of activating receptors like NK1.1 or Nkp46 or upon co-culture with different leukemia and solid tumor cells. In line, cytotoxicity assays revealed increased lysis of YAC-1 and B16F10 tumor cells by both NFAT1- and NFAT2-deficient NK cells as compared to wildtype (WT) controls. The inhibitory effect of NFAT transcription factors on NK cell effector function could also be confirmed in vivo by employing WT and NFAT KO animals in syngeneic B16F10 melanoma, RMA-S lymphoma and LL/2-luc lung carcinoma models, which revealed a significantly reduced tumor burden in NFAT1 and NFAT2 KO mice. Furthermore, we detected higher numbers of tumor infiltrating NK cells in NFAT KO mice, which suggested improved NK cell migration and homing to the tumor side. Comparative analyses with single NFAT as well as NFAT1+NFAT2 double KO and WT animals further confirmed the inhibitory effect of NFAT1 and NFAT2 and pointed to additive effects of NFAT1 and NFAT2 in NK cell tumor immunosurveillance. Proteomics analysis of NK cells from the different NFAT KO strains revealed altered expression of molecules which are regulating chemokine receptor expression as well as a decreased expression of NFκB inhibitory molecules, like NFKBIB (NFKB inhibitor beta) Commd6 (COMM domain containing 6). NFκB signaling was shown to regulate granzyme B and perforin synthesis and expression. Thus, we suggest that NFAT1 and NFAT2 inhibit NFκB signaling in NK cells, and NFAT1 and NFAT2 KO reinforce NFκB induced granzyme B and perforin expression and thereby increase NK cell cytotoxicity. Taken together, our results identify NFAT as a negative regulator of NK cell function. In addition, we provide the first evidence for a direct functional involvement of NFAT1 and NFAT2 in NK cell antitumor reactivity by regulation tumor infiltration and cytotoxicity of NK cell.

Missing a "Missing Self" Mechanism: Modeling and Detection of Ly49 Expression in Canine NK Cells.

NK cells are a key focus in immuno-oncology, based on their ability to eliminate malignant cells without prior sensitization. Dogs are valuable models for translational immunotherapy studies, especially for NK cells, where critical species differences exist between mice and humans. Given that the mechanism for recognition of "self" by canine NK cells is currently unknown, we sought to evaluate expression of Ly49 in canine NK cells using in silico and high-throughput techniques. We interrogated the identified polymorphism/mutation in canine Ly49 and assessed the potential impact on structure using computational modeling of three-dimensional protein structure and protein-protein docking of canine Ly49 with MHC class I (MHC-I). Bulk and single-cell RNA-sequencing analysis was performed to detect gene expression of Ly49/KLRA1 in resting and activated NK cells. Tertiary protein structure demonstrated significant structural similarity to the known murine system. Molecular docking of canine Ly49 with MHC-I was favorable, converging at a single low-energy conformation. RNA sequencing revealed expression of Ly49/KLRA1 in both resting and activated NK cells and demonstrated almost exclusive expression of the gene in the NK cluster at the single-cell level. Despite prior reports of a mutated, nonfunctional canine Ly49, our data support that the protein product is predicted to bind to MHC-I in a comparable conformation to the murine system and is expressed in canine NK cells with upregulation following activation. Taken together, these data suggest that Ly49 is capable of recognizing MHC-I and therefore regulating NK cell function in dogs.

Palmitic Acid Upregulates CD96 Expression to Mediate Maternal-Foetal Interface Immune Tolerance by Inhibiting Cytotoxic Activity and Promoting Adhesion Function in Human Decidual Natural Killer Cells.

Decidual natural killer cells (dNK cells) are an essential component of the immune cells present at the maternal-foetal interface during early pregnancy, and they play a vital role in various physiological processes. Abnormalities in the ratio or function of dNK cells have been linked to recurrent miscarriages. CD96 has been previously shown to regulate NK cell function in the tumour microenvironment; however, its role and mechanism at the maternal-foetal interface remains unclear. The present study aimed to investigate the immunomodulatory role of CD96 in dNK cells and its function at the maternal-foetal interface. Immunofluorescence staining and flow cytometry were used to detect the expression of cellular markers such as CD96. Furthermore, the secretory function, adhesion-function-related molecules, and cell proliferation markers of CD96+ and CD96- dNK cells were detected using flow cytometry. In addition, we performed cell culture experiments via the magnetic bead sorting of NK cells to detect changes in the expression of the aforementioned functional molecules in dNK cells after the CD96 blockade. Furthermore, we examined the functional characteristics of dNK cells after palmitic acid treatment at a concentration of 10 μM. We also examined the changes in dNK cell function when subjected to the combined effect of palmitic acid and CD96 antagonists. The results indicated that CD96, TIGIT, CD155, and CD112 were highly expressed at the maternal-foetal interface, with dNK cells predominantly expressing CD96, whereas TIGIT was mainly expressed on T cells, and CD155 and CD112 were mainly present in metaphase stromal and trophoblast cells. CD96+ dNK cells displayed low cytotoxic activity and a high adhesion phenotype, which mediated the immunosuppressive effect on dNK cells at the maternal-foetal interface. Palmitic acid upregulated CD96 expression on the surface of dNK cells in the coculture system, inhibiting dNK cell activity and increasing their adhesion molecule expression. CD96 antagonist treatment blocked the inhibitory effect of trophoblasts on dNK cells, resulting in enhanced cytokine secretion and reduced adhesion. The results of this study provide valuable insight into the immunomodulatory role of CD96 in dNK cells and its mechanism at the maternal-foetal interface, particularly in metaphase NK cells. This study sheds light on the mechanisms of immune regulation at the maternal-foetal interface and their implications for the study of recurrent miscarriages of unknown origin.

Yeast β-glucan modulates macrophages and improves antitumor NK-cell responses in cancer.

As the largest proportion of myeloid immune cells in tumors, macrophages play an important role in tumor growth and regression according to their different phenotypes, thus reprogramming macrophages has become a new research direction for cancer immunotherapy. Yeast-derived whole β-glucan particles (WGPs) can induce M0 macrophages to differentiate into M1 macrophages and convert M2 macrophages and tumor-associated macrophages (TAMs) into M1 macrophages. In vitro, studies have confirmed that WGP-treated macrophages increase the activating receptors in natural killer cells (NK cells) and enhance the cytotoxicity of NK cells. The extracellular regulated protein kinases (ERK) signaling pathway is involved in WGP-mediated regulation of the macrophage phenotype. Further in vivo studies show that oral WGP can significantly delay tumor growth, which is related to the increased proportion of macrophages and NK cells, the macrophage phenotype reversal, and the enhancement of NK cell immune function. NK-cell depletion reduces the therapeutic efficacy of WGP in tumor-bearing mice. These findings revealed that in addition to T cells, NK cells also participate in the antitumor process of WGP. It was confirmed that WGP regulates the macrophage phenotype to regulate NK-cell function.

Bone marrow‐derived mesenchymal stem cells inhibit NK cell function via Tim‐3/galectin‐9 in multiple myeloma patients

Bone marrow‐derived mesenchymal stem cells inhibit NK cell function via Tim‐3/galectin‐9 in multiple myeloma patients

The co-inhibitory receptor TIGIT regulates NK cell function and is upregulated in human intrahepatic CD56bright NK cells.

The crosstalk between NK cells and their surrounding environment is enabled through activating and inhibitory receptors, which tightly control NK cell activity. The co-inhibitory receptor TIGIT decreases NK cell cytotoxicity and is involved in NK cell exhaustion, but has also been associated with liver regeneration, highlighting that the contribution of human intrahepatic CD56bright NK cells in regulating tissue homeostasis remains incompletely understood. A targeted single-cell mRNA analysis revealed distinct transcriptional differences between matched human peripheral blood and intrahepatic CD56bright NK cells. Multiparameter flow cytometry identified a cluster of intrahepatic NK cells with overlapping high expression of CD56, CD69, CXCR6, TIGIT and CD96. Intrahepatic CD56bright NK cells also expressed significantly higher protein surface levels of TIGIT, and significantly lower levels of DNAM-1 compared to matched peripheral blood CD56bright NK cells. TIGIT+ CD56bright NK cells showed diminished degranulation and TNF-α production following stimulation. Co-incubation of peripheral blood CD56bright NK cells with human hepatoma cells or primary human hepatocyte organoids resulted in migration of NK cells into hepatocyte organoids and upregulation of TIGIT and downregulation of DNAM-1 expression, in line with the phenotype of intrahepatic CD56bright NK cells. Intrahepatic CD56bright NK cells represent a transcriptionally, phenotypically, and functionally distinct population of NK cells that expresses higher levels of TIGIT and lower levels of DNAM-1 than matched peripheral blood CD56bright NK cells. Increased expression of inhibitory receptors by NK cells within the liver environment can contribute to tissue homeostasis and reduction of liver inflammation.

Галектин-9/Tim-3-зависимый механизм регуляции функциональной активности натуральных киллеров при беременности

Galectins are a family of β-galactoside-binding low molecular weight proteins (lectins) homologous in the amino acid sequence of the carbohydrate-binding site. Galectins interact with glycans on the cell surface and are involved in regulating the functional activity of most cells of the human body. Galectin-9 plays a crucial role in controlling the functions of cells of the immune system. Of particular importance are the immunoregulatory effects of galectin-9 during pregnancy, when its production by trophoblast cells increases significantly, which is associated with the formation of peripheral immune tolerance. The main ligand for galectin-9 is the Tim-3 (T-cell immunoglobulin and mucin domain 3) molecule, whose high expression is detected on natural killers (NK cells). During pregnancy, the phenotype and functions of NK cells change significantly, which is necessary to prevent cytotoxic reactions to fetal cells. The review systematizes data on the role of galectin-9 in Tim-3-mediated regulation of NK cell functions in the aspect of pregnancy. The study of the regulation of the functional activity of NK cells is necessary to increase the effectiveness of NK cell therapy.

466 KIR allelic variation is associated with atopic dermatitis

Atopic dermatitis (AD) is a common, pruritic, inflammatory skin disease characterized by life-long periods of acute disease flares and remissions. Predisposition to AD is associated with genetic variation. Killer cell Ig-like receptor family (KIR) receptors interact with HLA ligands and are thought to be a primary regulator of NK cell function. NK cells have also been implicated in the pathophysiology of AD and other skin inflammatory diseases. In this study, we examined allelic variation in KIR genes KIR2DL5, KIR2DS5, and KIR2DS1 with respect to AD.

Abstract 5512: CBLB, CISH and CD70 multiplexed gene knockout with CRISPR/Cas9 enhances cytotoxicity of CD70-CAR NK cells and provides greater resistance to TGF-β for cancer immunotherapy

Abstract Natural killer (NK) cells provide an attractive platform for development of effective cancer immunotherapies. NK cells are known for their ability to kill tumor cells and do not elicit graft-versus-host disease, making them a potential source of ‘off-the-shelf’ allogeneic cell therapy. NK cells are also amenable to CRISPR genomic engineering to enhance the antitumor activity of NK cells by increasing their cytotoxicity, overcoming suppression within the tumor microenvironment, or promoting their persistence and homing to tumor sites. Cytokine inducible SH2-containing protein (CISH) is a NK cell checkpoint for IL-15 mediated NK survival, proliferation, cytotoxicity, and anti-tumor immunity. The E3 ubiquitin ligase CBLB is also a negative regulator of NK cell function and has been shown to mediate TGF-β sensitivity by downregulating inhibitory SMAD7 in primary T cells. We hypothesized that knockout of both CISH and CBLB would not only improve NK cell effector function and but also render NK cells resistant to TGF-β mediated suppression. In this study, we utilized CRISPR-Cas9 ribonucleoproteins (RNPs) to disrupt CISH and CBLB genes in isolated peripheral blood NK cells from healthy donors. Given that CD70 expression is present on activated NK cells, to target CD70 on renal cell carcinoma (RCC) with CAR NK cells, CD70 was knocked out in NK cells to avoid fratricide. Western blotting, FACS and TIDE/Amplicon NGS Sequencing data confirmed all three genes were successfully disrupted. Then we expanded these edited NK cells by using IL-2 and stimulation using NKSTIM, a modified K562 stimulatory cell line expressing membrane-bound form of IL-15 (mbIL-15) and 4-1BBL. IL-12 and IL-18 were added during expansion to drive memory-like NK cell differentiation. Furthermore, we were able to transduce CRISPR/Cas9 edited NK cells to express a CD70-CAR construct and membrane bound IL-15. CAR expression was assessed by flow cytometry. In vitro cytotoxicity was measured using the IncuCyte S3 live cell analysis system. CD70/CISH/CBLB triple knockout CD70-CAR NK cells could be produced efficiently and exhibited similar persistence as CD70/CISH or CD70/CBLB double knockout CD70-CAR NK cells in culture. Cytotoxicity assays demonstrated that CD70/CISH/CBLB triple knockout CD70-CAR NK cells had greater tumor growth control after multiple rechallenges. In the presence of exogenous TGF-β, CD70/CISH/CBLB triple knockout CD70-CAR NK cells showed greater resistance to TGF-β inhibition of cytotoxicity. In summary, we show CD70/CISH/CBLB triple knockout CD70-CAR NK cells demonstrate enhanced anti-tumor activity against relevant solid tumor cell lines and provide greater resistance to tumor microenvironment inhibition. These data support the further exploration of CD70/CISH/CBLB triple gene knockout CD70 CAR NK cells for clinical application. Citation Format: Chao Guo, Yanying Fan, Alexander Aronov, Qi Zhang, Sombeet Sahu, Mary-Lee Dequéant, Changan Guo, Sushant Karnik, Glenn D. Leary, Chandirasegaran Massilamany, Ivan H. Chan, James B. Trager. CBLB, CISH and CD70 multiplexed gene knockout with CRISPR/Cas9 enhances cytotoxicity of CD70-CAR NK cells and provides greater resistance to TGF-β for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5512.

Abstract 2104: NK cell immunosurveillance of tumors is regulated by NFAT1 and NFAT2

Abstract NK cells are lymphoid components of innate immunity and play an important role in tumor immunosurveillance. One of the major transcriptional regulators in lymphoid cells is NFAT (Nuclear Factor of Activated T cells), controlling lymphocyte development and activity. However, while the role of NFAT signaling is well defined in T cells, the cytotoxic lymphocytes of adaptive immunity, surprisingly little is known regarding the relevance of this transcription factor family in NK cells as effector cells of innate immunity. Available data indicate that NFAT activity is dispensable for development of NK cells. However, several lines of evidence including reports on the effects of the immunosuppressive drugs cyclosporin A and tacrolimus, which mediate their effects through inhibition of calcineurin and consecutively NFAT, implicate an involvement of the NFAT family in NK cell function. We here employed different genetic mouse models to unravel the role of NFAT1 (NFATc2) and NFAT2 (NFATc1) in NK cell reactivity. When NK cells with knockout (KO) of NFAT1 or NFAT2 compared to NK cells of control mice (WT) were employed in in vitro analyses, lack of either NFAT was found to surprisingly result in enhanced NK cell degranulation as well as increased production of granzyme B and perforin upon stimulation of activating receptors like NK1.1 or Nkp46 or upon co-culture with different leukemia and solid tumor tumor cells. In line, cytotoxicity assays revealed increased lysis of YAC-1 and B16F10 tumor cells by both NFAT1- and NFAT2-deficient NK cells as compared to WT controls. The inhibitory effect of NFAT transcription factors on NK cell effector function could also be confirmed in vivo by employing WT and NFAT KO animals in syngeneic B16F10 melanoma and RMA-S flank tumor models, which revealed a significantly reduced tumor burden in NFAT1 and NFAT2 KO mice. Comparative analyses with single NFAT as well as NFAT1+NFAT2 double KO and WT animals further confirmed the inhibitory effect of NFAT1 and NFAT2 and pointed to additive effects of NFAT1 and NFAT2 in NK cell tumor immunosurveillance. Taken together, our results provide the first evidence for the direct functional involvement of NFAT1 and NFAT2 in NK cell antitumor reactivity and, in contrast to T and B cells, identify NFAT as a negative regulator of NK cell function. Citation Format: Melanie Märklin, Samuel Holzmayer, Kübra Kaban, Martin R. Müller, Helmut R. Salih. NK cell immunosurveillance of tumors is regulated by NFAT1 and NFAT2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2104.

NK cells require immune checkpoint receptor LILRB4/gp49B to control neurotropic Zika virus infections in mice.

Immune cells express an array of inhibitory checkpoint receptors that are upregulated upon activation and limit tissue damage associated with excessive response to pathogens or allergens. Mouse leukocyte immunoglobulin like receptor B4 (LILRB4), also known as glycoprotein 49B (gp49B), is an inhibitory checkpoint receptor constitutively expressed in myeloid cells and upregulated in B cells, T cells, and NK cells upon activation. Here, we report that expression of LILRB4, which binds Zika virus (ZIKV), was increased in microglia and myeloid cells infiltrating the brains of neonatal mice with ZIKV-associated meningoencephalitis. Importantly, while C57BL/6 mice developed transient neurological symptoms but survived infection, mice lacking LILRB4/gp49B (LILRB4 KO) exhibited more severe signs of neurological disease and succumbed to disease. Their brains showed increased cellular infiltration but reduced control of viral burden. The reduced viral clearance was associated with altered NK cell function in the absence of LILRB4/gp49B. In naive animals, this manifested as reduced granzyme B responses to stimulation, but in ZIKV-infected animals, NK cells showed phenotypic changes that suggested altered maturation, diminished glucose consumption, reduced IFN-γ and granzyme B production, and impaired cytotoxicity. Together, our data reveal LILRB4/gp49B as an important regulator of NK cell function during viral infections.

Abstract 13655: DUSP-1 May Be a Key Regulator of NK Cell Function in Obesity Potentially Accelerating Cardiovascular Disease in African American Women

Background: Obesity disproportionately affects African American (AA) women and contributes to disparate cardiovascular disease (CVD) outcomes. Natural killer cells have been shown to be functionally impaired by obesity. However, underlying mechanisms regulating NK cell function in obesity are largely unknown. Therefore, we sought to elucidate potential signaling pathways involved in obesity-related NK cell function loss in AA women. Methods: All experiments were performed with freshly isolated naïve NK cells. NK cells were isolated from AA women (n=29; 15 BMI&lt;25, 14 BMI&gt;30). NK cell function was measured by flow cytometry by determining the ability of NK cells to degranulate towards K562 cells. NK cell gene expression profile was determined by RNA sequencing to identify important signaling pathways, which were verified using chemical pathway inhibitors. Publicly available datasets were analyzed to determine the importance of identified target genes. Results: NK cells isolated from AA women with obesity displayed diminished capacity to induce K562 cell death as determined by decreased degranulation (decreased surface CD107a expression and decreased intracellular IFNγ and TNFα expression). RNA sequencing analysis of NK cells isolated from lean vs AA women with obesity revealed 1645 significantly regulated genes with DUSP-1 being in the top 50 and displaying a 2.8-fold upregulation in AA women with obesity (p=0.0009). Furthermore, DUSP-1 gene expression was significantly correlated with BMI (R 2 =0.72, p=0.02). In vitro experiments revealed a DUSP-1 inhibitor to enhance NK cell function. Additionally, analysis of publicly available RNA sequencing dataset (GSE20129) in women revealed an increase of DUSP-1 mRNA expression in Black and Latinx women with CVD when compared to women without CVD group; this difference was not observed in White women. Conclusion: Our data demonstrate that NK cells are functionally impaired in NK cells isolated from AA women with obesity. We identified DUSP-1 as a crucial regulator of the observed NK cell dysfunction in obesity, which may be of particular importance in CVD development and progression in AA women, a population disproportionally affected by obesity and CVD.

Purinergic Receptor P2Y6 Is a Negative Regulator of NK Cell Maturation and Function.

NK cells are critical innate immune cells that target the tumor cells and cancer-initiating cells and clear viruses by producing cytokines and cytotoxic granules. However, the role of the purinergic receptor P2Y6 in the NK cells remains largely unknown. In this study, we discovered that the expression of P2Y6 was decreased upon the activation of the NK cells. Moreover, in the P2Y6-deficient mice, we found that the deficiency of P2Y6 promoted the development of the NK precursor cells into immature NK and mature NK cells. We also found that the P2Y6 deficiency increased, but the P2Y6 receptor agonist UDP or UDP analog 5-OMe-UDP decreased the production of IFN-γ in the activated NK cells. Furthermore, we demonstrated that the P2Y6-deficient NK cells exhibited stronger cytotoxicity in vitro and antimetastatic effects in vivo. Mechanistically, P2Y6 deletion promoted the expression of T-bet (encoded by Tbx21), with or without the stimulation of IL-15. In the absence of P2Y6, the levels of phospho-serine/threonine kinase and pS6 in the NK cells were significantly increased upon the stimulation of IL-15. Collectively, we demonstrated that the P2Y6 receptor acted as a negative regulator of the NK cell function and inhibited the maturation and antitumor activities of the NK cells. Therefore, inhibition of the P2Y6 receptor increases the antitumor activities of the NK cells, which may aid in the design of innovative strategies to improve NK cell-based cancer therapy.

Different endurance exercises modulate NK cell cytotoxic and inhibiting receptors

PurposeInduction of IDO depends on the activation of AhR forming the AhR/IDO axis. Activated AhR can transcribe various target genes including cytotoxic and inhibiting receptors of NK cells. We investigated whether AhR and IDO levels as well as activating (NKG2D) and inhibiting (KIR2DL1) NK cell receptors are influenced by acute exercise and different chronic endurance exercise programs.Methods21 adult breast and prostate cancer patients of the TOP study (NCT02883699) were randomized to intervention programs of 12 weeks of (1) endurance standard training or (2) endurance polarized training after a cardiopulmonary exercise test (CPET). Serum was collected pre-CPET, immediately post-CPET, 1 h post-CPET and after 12 weeks post-intervention. Flow cytometry analysis was performed on autologous serum incubated NK-92 cells for: AhR, IDO, KIR2DL1 and NKG2D. Differences were investigated using analysis-of-variance for acute and analysis-of-covariance for chronic effects.ResultsAcute exercise: IDO levels changed over time with a significant increase from post-CPET to 1 h post-CPET (p = 0.03). KIR2DL1 levels significantly decreased over time (p < 0.01). NKG2D levels remained constant (p = 0.31). Chronic exercise: for both IDO and NKG2D a significant group × time interaction, a significant time effect and a significant difference after 12 weeks of intervention were observed (IDO: all p < 0.01, NKG2D: all p > 0.05).ConclusionBoth acute and chronic endurance training may regulate NK cell function via the AhR/IDO axis. This is clinically relevant, as exercise emerges to be a key player in immune regulation.

Influence of Galectin-9 Treatment on the Phenotype and Function of NK-92MI Cells in the Presence of Different Serum Supplements.

Galectins are one of the critical players in the tumor microenvironment–tumor crosstalk and the regulation of local immunity. Galectin-9 has been in the limelight in tumor immunology. Galectin-9 possesses its multiplex biological functions both extracellularly and intracellularly, plays a pivotal role in the modulation of adaptive and innate immunity, and induces immune tolerance. NK-92MI cell lines against different malignancies were extensively studied, and recently published trials used genetically chimeric antigen receptor-transfected NK-92MI cells in tumor immunotherapy. Besides the intensive research in tumor immunotherapy, limited information is available on their immune-checkpoint expression and the impact of checkpoint ligands on their effector functions. To uncover the therapeutic potential of modulating Galectin-9-related immunological pathways in NK-cell-based therapy, we investigated the dose-dependent effect of soluble Galectin-9 on the TIM-3 checkpoint receptor and NKG2D, CD69, FasL, and perforin expression of NK-92MI cells. We also examined how their cytotoxicity and cytokine production was altered after Gal-9 treatment and in the presence of different serum supplements using flow cytometric analysis. Our study provides evidence that the Galectin-9/TIM-3 pathway plays an important role in the regulation of NK cell function, and about the modulatory role of Galectin-9 on the cytotoxicity and cytokine production of NK-92MI cells in the presence of different serum supplements. We hope that our results will aid the development of novel NK-cell-based strategies that target Galectin-9/TIM-3 checkpoint in tumors resistant to T-cell-based immunotherapy.

KIR2DL2/S2 and KIR2DS5 in alcoholic cirrhotic patients undergoing liver transplantation

IntroductionThe molecular mechanisms underlying alcoholic liver fibrosis and cirrhosis are not completely understood. Hepatic fibrosis involves the interplay of diverse cells and factors, including hepatic stellate cells (HSCs), Kupffer, NK cells, and T-lymphocyte subsets. Killer-cell immunoglobulin-like receptors (KIR) are membrane receptors involved in mediation between NK and activated HSCs, regulating NK cell function through their interaction with HLA-I molecules. The aim of this study was to analyse the genetic association between KIR genes and the susceptibility to or protection from alcoholic cirrhosis (AC) in a cohort of male AC patients undergoing liver transplantation (LT) with and without concomitant viral infections.Material and methodsKIR genotyping was performed in nuclear DNA extracted from 281 AC patients and compared with 319 male controls.ResultsSignificant differences between total AC patients and healthy controls were only found in the case of KIR2DL2 and KIR2DS5. KIR2DL2 was significantly underrepresented in non-viral AC patients (52.6% vs. 63.3%; p = 0.015), while patients heterozygous for KIR2DL2 were also underrepresented in the non-viral AC group compared with controls (p = 0.034). KIR2DS5 was overrepresented in this group compared with healthy controls (p = 0.002). All these observations were only evident in AC patients older than 54 years old.ConclusionsOur data suggest a contrary effect of KIR2DL2 and KIR2DS5 in AC patients older than 54 years, in whom the presence of KIR2DL2 appears to be protective against AC, whereas the presence of KIR2DS5 seems to promote the fibrotic process, particularly in patients with no associated viral infection.

Dysregulation of Metabolic Pathways in Circulating Natural Killer Cells Isolated from Inflammatory Bowel Disease Patients.

Background and AimsInflammatory bowel diseases [IBD], comprising Crohn’s disease [CD] and ulcerative colitis [UC], are chronic conditions characterized by severe dysregulation of innate and adaptive immunity resulting in the destruction of the intestinal mucosa. Natural killer [NK] cells play a pivotal role in the dynamic interaction between the innate and adaptive immune response. There is an increasing appreciation for the key role immunometabolism plays in the regulation of NK cell function, yet little remains known about the metabolic profile, cytokine secretion, and killing capacity of human NK cells during active IBD.MethodsPeripheral blood mononuclear cells were isolated from peripheral blood of patients with moderate to severely active IBD and healthy controls. NK cells were stained with a combination of cell surface receptors, intracellular cytokines, and proteins and analyzed by flow cytometry. For measurements of NK cell cytotoxicity, the calcein-AM release assay was performed. The metabolic profile was analyzed by an extracellular flux analyzer.ResultsNK cells from IBD patients produce large quantities of pro-inflammatory cytokines, IL-17A and TNF-α ex vivo, but have limited killing capability. Furthermore, patient NK cells have reduced mitochondrial mass and oxidative phosphorylation. mTORC1, an important cell and metabolic regulator, demonstrated limited activity in both freshly isolated cells and cytokine-stimulated cells.ConclusionsOur results demonstrate that circulating NK cells of IBD patients have an unbalanced metabolic profile, with faulty mitochondria and reduced capacity to kill. These aberrations in NK cell metabolism may contribute to defective killing and thus the secondary infections and increased risk of cancer observed in IBD patients.

KIR/HLA ligands immunogenetics markers associated with outcome of hepatitis B virus infection in the Bulgarian population.

Hepatitis B virus (HBV) infection is one of the most common infections worldwide, having negative impact on world health due to the tendency for chronification with late complications such as liver cirrhosis and hepatocellular carcinoma. Natural killer (NK) cells as part of innate antiviral defense influence the clinical course of HBV infection: elimination of the virus or chronic disease. Therefore, we investigated the polymorphisms of the main gene systems, regulating NK-cell function: killer cell immunoglobulin-like receptors (KIRs) and their appropriate HLA class I ligands in 144 HBV infected patients (124 chronic carriers and 20 spontaneously recoved) and 126 ethnically matched healthy controls from the Bulgarian population in a case-control study. KIRs and HLA ligands were determined by PCR-SSP or PCR high-resolution typing methods. KIR2DL5B allele variant was significantly less frequent in spontaneously recovered (SR) patients compared to healthy controls (10.0% vs. 45.5%, Pcorr=0.006). The presence of KIR3DL1*004 allele was higher in chronic HBV carriers (CH) than in controls (33.1% vs. 17.6%, Pcorr=0.036). Additionally, SR patients differed from healthy individuals by the lower frequency of HLA-Bw4Ile80 group ligands (30.0% vs 63.7%, P=0.015). Three KIR genotypes were found more frequent in healthy in comparison with HBV infected individuals: ID2 (13.5% vs 5.6%, P=0.025), KIR genotype containing 6 activating KIRs (18.0% vs 7.6%, P=0.017), and KIR genotype composed of 4 activating and 5 inhibitory KIRs (23.8% vs 5.6%, P=0.001). These data suggest that inherited KIR and HLA class I ligand polymorphisms may influence the clinical course of HBV infection.