Search Results (11)

Natural killer (NK) cells are effector cells of the innate immune system. The cytokine microenvironment influences NK cell function. Dysregulation of NK cell cytotoxicity can manifest in reproductive disorders and is also observed in tumor-transformed tissues. The search for immunotherapies capable of regulating NK cell activity is therefore relevant. This study aimed to evaluate the effect of the TGFβ signaling pathway inhibitor and the cyclin-dependent kinase (CDK) 7/12/13 inhibitor on the transcriptional profile of NK-92 cell line. In the study, the cytokines TGFβ1, IL-12, IL-15, IL-18, and TNFα, and the TGFβ receptor type 1 (TGFβR1) inhibitor LY3200882 and the CDK7/12/13 inhibitor THZ1 were used. The cells were cultured sequentially in the presence of inhibitors and cytokines, followed by assessment of the gene expression of NCR2, NCR3, AHR, NCAM1, B3GAT1, EOMES, GATA3, KLRC1, KLRC2, CCL5, IL10 and TBX21. We observed direct effects of the inhibitors on NK cells. LY3200882 increased the expression of KLRC1 and B3GAT1, and reduced NCAM1. THZ1 increased the expression of KLRC1, KLRC2, AHR and EOMES, while it reduced IL-10 and NCR2. IL-12, IL-15, IL-18, and TNFα modified the gene expression of some phenotypic and cytotoxic receptors and transcription factors. TGFβ1 increased the expression of KLRC1, NCAM1, and B3GAT1. Blocking TGFβ-dependent signaling with LY3200882 abolished TGFβ1 effects. We assessed CD56 presence on NK-92 cell membrane and found its increase in the presence of LY3200882. After LY3200882 treatment, in the presence of TGFβ1 and choriocarcinoma cell line JEG-3, the expression of CD56 receptor on NK cell membrane decreased. Pretreating NK cells with THZ1 decreased the expression of NCAM1, B3GAT1, and EOMES in the presence of TGFβ1. Thus, LY3200882 partially neutralized TGFβ1 effects on the expression of NK cell receptor genes. THZ1 followed by TGFβ1 treatment promoted NK cell transcriptional profile characteristic for CD56dim NK cells. Both LY3200882 and THZ1 affected the NK cell transcription even without cytokine treatment. The independent effects of synthetic inhibitors on NK cells, as well as their influence in the presence of tumor cells, should be considered. Full article

Natural killer (NK) cells are promising candidates for adoptive immunotherapy, but their clinical application requires standardized expansion protocols that yield functional cells in sufficient numbers. This study examined how initial seeding density and donor-intrinsic variability affect NK cell proliferation and receptor phenotype during in vitro expansion in a G-Rex^® 24-well plate under IL-2 stimulation. NK cells from healthy donors were analyzed longitudinally by flow cytometry, and targeted SNP sequencing of selected receptor genes (IL2RA, IL2RB, FCGR3A, NCR1, KLRK1, and ICAM-1) was performed to assess potential genetic contributions. A seeding density of 2.0 × 10⁶ cells/cm² promoted high expansion rates and favorable expression of activating receptors including CD16a, NKp46, and NKG2D. Nonetheless, marked inter-donor differences were observed. Some donors exhibited impaired proliferation and aberrant receptor expression, possibly associated with high-priority SNPs and distinct haplotype structures. Others showed robust proliferation despite the absence of identifiable genetic drivers, suggesting the involvement of variants in other genes or non-genetic mechanisms such as epigenetic priming or adaptive NK-cell differentiation. These results highlight the influence of both culture conditions and donor-intrinsic factors on NK-cell expansion outcomes. Integrating phenotypic and genetic analyses may improve the reproducibility and personalization of NK-cell-based manufacturing protocols for therapeutic use. Full article

Chronic liver rejection (CR) represents a complex clinical situation because many patients do not respond to increased immunosuppression. Killer cell immunoglobulin-like receptors/Class I Human Leukocyte Antigens (KIR/HLA-I) interactions allow for predicting Natural Killer (NK) cell alloreactivity and influence the acute rejection of liver allograft. However, its meaning in CR liver graft remains controversial. KIR and HLA genotypes were studied in 513 liver transplants using sequence-specific oligonucleotides (PCR-SSO) methods. KIRs, human leucocyte antigen C (HLA-C) genotypes, KIR gene mismatches, and the KIR/HLA-ligand were analyzed and compared in overall transplants with CR (n = 35) and no-chronic rejection (NCR = 478). Activating KIR (aKIR) genes in recipients (rKIR2DS2⁺ and rKIR2DS3⁺) increased CR compared with NCR groups (p = 0.013 and p = 0.038). The inhibitory KIR (iKIR) genes in recipients rKIR2DL2⁺ significantly increased the CR rate compared with their absence (9.1% vs. 3.7%, p = 0.020). KIR2DL3 significantly increases CR (13.1% vs. 5.2%; p = 0.008). There was no influence on NCR. CR was observed in HLA-I mismatches (MM). The absence of donor (d) HLA-C2 ligand (dC2⁻) ligand increases CR concerning their presence (13.1% vs. 5.6%; p = 0.018). A significant increase of CR was observed in rKIR2DL3⁺/dC1⁻ (p = 0.015), rKIR2DS4/dC1⁻ (p = 0.014) and rKIR2DL3⁺/rKIR2DS4⁺/dC1⁻ (p = 0.006). Long-term patient survival was significantly lower in rKIR2DS1⁺rKIR2DS4⁺/dC1⁻ at 5–10 years post-transplant. This study shows the influence of rKIR/dHLA-C combinations and aKIR gene-gene mismatches in increasing CR and KIR2DS1⁺/C1-ligands and the influence of KIR2DS4⁺/C1-ligands in long-term graft survival. Full article

Innate and adaptive immune systems are evolutionarily divergent. Primary signaling in T and B cells depends on somatically rearranged clonotypic receptors. In contrast, NK cells use germline-encoded non-clonotypic receptors such as NCRs, NKG2D, and Ly49H. Proliferation and effector functions of T and B cells are dictated by unique peptide epitopes presented on MHC or soluble humoral antigens. However, in NK cells, the primary signals are mediated by self or viral proteins. Secondary signaling mediated by various cytokines is involved in metabolic reprogramming, proliferation, terminal maturation, or memory formation in both innate and adaptive lymphocytes. The family of common gamma (γc) cytokine receptors, including IL-2Rα/β/γ, IL-7Rα/γ, IL-15Rα/β/γ, and IL-21Rα/γ are the prime examples of these secondary signals. A distinct set of cytokine receptors mediate a ‘third’ set of signaling. These include IL-12Rβ1/β2, IL-18Rα/β, IL-23R, IL-27R (WSX-1/gp130), IL-35R (IL-12Rβ2/gp130), and IL-39R (IL-23Rα/gp130) that can prime, activate, and mediate effector functions in lymphocytes. The existence of the ‘third’ signal is known in both innate and adaptive lymphocytes. However, the necessity, context, and functional relevance of this ‘third signal’ in NK cells are elusive. Here, we define the current paradigm of the ‘third’ signal in NK cells and enumerate its clinical implications. Full article

NKp30 (Natural Cytotoxicity Receptor 1, NCR1) is a powerful cytotoxicity receptor expressed on natural killer (NK) cells which is involved in tumor cell killing and the regulation of antitumor immune responses. Ligands for NKp30, including BAG6 and B7-H6, are upregulated in virus-infected and tumor cells but rarely detectable on healthy cells. These ligands are released by tumor cells as part of the cellular secretome and interfere with NK cell activity. BAG6 is secreted via the exosomal pathway, and BAG6-positive extracellular vesicles (EV-BAG6) trigger NK cell cytotoxicity and cytokine release, whereas the soluble protein diminishes NK cell activity. However, the extracellular format and activity of B7-H6 remain elusive. Here, we used HEK293 as a model cell line to produce recombinant ligands and to study their impact on NK cell activity. Using this system, we demonstrate that soluble B7-H6 (sB7-H6), like soluble BAG6 (sBAG6), inhibits NK cell-mediated target cell killing. This was associated with a diminished cell surface expression of NKG2D and NCRs (NKp30, NKp40, and NKp46). Strikingly, a reduced NKp30 mRNA expression was observed exclusively in response to sBAG6. Of note, B7-H6 was marginally released in association with EVs, and EVs collected from B7-H6 expressing cells did not stimulate NK cell-mediated killing. The molecular analysis of EVs on a single EV level using nano flow cytometry (NanoFCM) revealed a similar distribution of vesicle-associated tetraspanins within EVs purified from wildtype, BAG6, or B7-H6 overexpressing cells. NKp30 is a promising therapeutic target to overcome NK cell immune evasion in cancer patients, and it is important to unravel how extracellular NKp30 ligands inhibit NK cell functions. Full article

Natural killer (NK) cells are part of the innate immunity repertoire, and function in the recognition and destruction of tumorigenic and pathogen-infected cells. Engagement of NK cell activating receptors can lead to functional activation of NK cells, resulting in lysis of target cells. NK cell activating receptors specific for non-major histocompatibility complex ligands are NKp46, NKp44, NKp30, NKG2D, and CD16 (also known as FcγRIII). The natural cytotoxicity receptors (NCRs), NKp46, NKp44, and NKp30, have been implicated in functional activation of NK cells following influenza virus infection via binding with influenza virus hemagglutinin (HA). In this review we describe NK cell and influenza A virus biology, and the interactions of influenza A virus HA and other pathogen lectins with NK cell natural cytotoxicity receptors (NCRs). We review concepts which intersect viral immunology, traditional virology and glycobiology to provide insights into the interactions between influenza virus HA and the NCRs. Furthermore, we provide expert opinion on future directions that would provide insights into currently unanswered questions. Full article

Every year, millions of people worldwide are infected with influenza, causing enormous health and economic problems. The most common type of influenza is influenza A. It is known that Natural Killer (NK) cells play an important role in controlling influenza A infection, mostly through the recognition of the viral protein hemagglutinin (HA) by the activating receptor, NKp46. In contrast, little is known regarding NK cell recognition of influenza B viruses, even though they are responsible for a third of all pediatric influenza deaths and are therefore included in the seasonal vaccine each year. Here we show that NKp46 also recognizes influenza B viruses. We show that NKp46 binds the HA protein of influenza B in a sialic acid-dependent manner, and identified the glycosylated residue in NKp46, which is critical for this interaction. We discovered that this interaction has a binding affinity approximately seven times lower than NKp46 binding of influenza A’s HA. Finally, we demonstrated, using mice deficient for the mouse orthologue of NKp46, named NCR1, that NKp46 is not important for influenza B elimination. These findings enable us to better understand the interactions between the different influenza viruses and NK cells that are known to be crucial for viral elimination. Full article

The role of natural killer (NK) cells in the liver as first-line post infectionem (p.i.) effectors against blood-stage malaria and their responsiveness to protective vaccination is poorly understood. Here, we investigate the effect of vaccination on NK cell-associated genes induced in the liver by blood-stage malaria of Plasmodium chabaudi. Female Balb/c mice were vaccinated at weeks 3 and 1 before being infected with 10⁶P. chabaudi-parasitized erythrocytes. Genes preferentially expressed by NK cells were investigated in livers of vaccination-protected and non-protected mice on days 0, 1, 4, 8, and 11 p.i. using microarrays, qRT-PCR, and chromosome landscape analysis. Blood-stage malaria induces expression of specific genes in the liver at different phases of infection, i.e., Itga1 in expanding liver-resident NK (lrNK) cells, Itga2 in immigrating conventional NK (cNK) cells; Eomes and Tbx21 encoding transcription factors; Ncr1, Tnfsf10, Prf1, Gzma, Gzmb, Gzmc, Gzmm, and Gzmk encoding cytolytic effectors; natural killer gene complex (NKC)-localized genes encoding the NK cell receptors KLRG1, KLRK1, KLRAs1, 2, 5, 7, KLRD1, KLRC1, KLRC3, as well as the three receptors KLRB1A, KLRB1C, KLRB1F and their potential ligands CLEC2D and CLEC2I. Vaccination enhances this malaria-induced expression of genes, but impairs Gzmm expression, accelerates decline of Tnfsf10 and Clec2d expression, whereas it accelerates increased expression of Clec2i, taking a very similar time course as that of genes encoding plasma membrane proteins of erythroblasts, whose malaria-induced extramedullary generation in the liver is known to be accelerated by vaccination. Collectively, vaccination reshapes the response of the liver NK cell compartment to blood-stage malaria. Particularly, the malaria-induced expansion of lrNK cells peaking on day 4 p.i. is highly significantly (p < 0.0001) reduced by enhanced immigration of peripheral cNK cells, and KLRB1F:CLEC2I interactions between NK cells and erythroid cells facilitate extramedullary erythroblastosis in the liver, thus critically contributing to vaccination-induced survival of otherwise lethal blood-stage malaria of P. chabaudi. Full article

Fc gamma receptors (FcγRs) are widely expressed on a variety of immune cells and play a myriad of regulatory roles in the immune system because of their structural diversity. Apart from their indispensable role in specific binding to the Fc portion of antibody subsets, FcγRs manifest diverse biological functions upon binding to their putative ligands. Examples of such manifestation include phagocytosis, presentation of antigens, mediation of antibody-dependent cellular cytotoxicity, anaphylactic reactions, and the promotion of apoptosis of T cells and natural killer cells. Functionally, the equilibrium between activating and inhibiting FcγR maintains the balance between afferent and efferent immunity. The γ subunit of the immunoglobulin Fc receptor (FcRγ) is a key component of discrete immune receptors and Fc receptors including the FcγR family. Furthermore, FcγRs exert a key role in terms of crosslinking the innate and adaptive workhorses of immunity. Ablation of one of these receptors might positively or negatively influence the immune response. Very recently, we discovered that FcRγ derived from natural cytotoxicity triggering receptor 1 (NCR1) curtails CD8⁺ T cell expansion and thereby turns an acute viral infection into a chronic one. Such a finding opens a new avenue for targeting the FcγRs as one of the therapeutic regimens to boost the immune response. This review highlights the structural heterogeneity and functional diversity of the ubiquitous FcγRs along with their featured subunit, FcRγ. Full article

Interleukin (IL)-22-producing Natural Killer (NK) cells protect the gut epithelial cell barrier from pathogens. A strain of probiotics, Lactobacillus plantarum (L. plantarum, LP), was previously found by our laboratory to significantly improve the mucosal barrier integrity and function of the small intestine in pigs. However, it was unclear whether LP benefited the intestinal mucosal barrier via interactions with the intestinal NK cells. The present study, therefore, was focused on the therapeutic effect of NK cells that were stimulated by LP on attenuating enterotoxigenic Escherichia coli (ETEC)-induced the damage to the integrity of the epithelial cell barrier. The results showed that LP can efficiently increase protein levels of the natural cytotoxicity receptor (NCR) family, and the expression levels of IL-22 mRNA and protein in NK cells. Transfer of NK cells stimulated by LP conferred protection against ETEC K88-induced intestinal epithelial barrier damage in NCM460 cells. We found that NK cells stimulated by LP could partially offset the reduction in NCM460 cell monolayers transepithelial electrical resistance (TEER) caused by ETEC K88, and increase ZO-1 and occludin mRNA and protein expressions by ETEC K88-infected NCM460 cells. Furthermore, adding NK cells that were stimulated by LP to ETEC K88-infected NCM460cells, IL-22R1, p-Stat3, and p-Tyk2 expression by NCM460 cells was increased. Mechanistic experiment showed that NK cells stimulated by LP lost the function of maintaining TEER of NCM460 cells challenged with ETEC K88, when polyclonal anti-IL-22 antibody was used to block IL-22 production. Collectively, our results suggested that LP stimulation of NK could enhance IL-22 production, which might be able to provide defense against ETEC-induced damage to the integrity of intestinal epithelial barrier. Full article

Natural-killer receptor group 2, member D (NKG2D) is a well characterized natural killer (NK) cell activating receptor that recognizes several ligands poorly expressed on healthy cells but up-regulated upon stressing stimuli in the context of cancer or viral infection. Although NKG2D ligands represent danger signals that render target cells more susceptible to NK cell lysis, accumulating evidence demonstrates that persistent exposure to ligand-expressing cells causes the decrease of NKG2D surface expression leading to a functional impairment of NKG2D-dependent NK cell functions. Upon ligand binding, NKG2D is internalized from the plasma membrane and sorted to lysosomes for degradation. However, receptor endocytosis is not only a mechanism of receptor clearance from the cell surface, but is also required for the proper activation of signalling events leading to the functional program of NK cells. This review is aimed at providing a summary of current literature relevant to the molecular mechanisms leading to NKG2D down-modulation with particular emphasis given to the role of NKG2D endocytosis in both receptor degradation and signal propagation. Examples of chronic ligand-induced down-regulation of NK cell activating receptors other than NKG2D, including natural cytotoxicity receptors (NCRs), DNAX accessory molecule-1 (DNAM1) and CD16, will be also discussed. Full article