Regulation of the PD-1/PD-L1 Axis and NK Cell Dysfunction by Exosomal miR-552-5p in Gastric Cancer.

OBJECTIVE: NK cells play a vital role in tumor immune resistance. Various factors affect NK cell activity. While NK cell dysfunction has been observed in numerous malignancies, the underlying mechanisms in gastric cancer remain unclear. METHOD: Flow cytometry was used to identify the phenotypic distribution and expression of activated receptors on NK cells. ELISA was used to determine the expression of cytokines. We examined the expression of NK cell-related genes and explored their association with survival and prognosis. Additionally, we conducted PCR detection of miR-552-5p expression levels in plasma exosomes of patients and investigated its correlation with phenotypic distribution and activated receptors. We used flow cytometry and ELISA to verify the role of miR-552-5p in NK cell dysfunction. Furthermore, we investigated the potential role of PD-1/PD-L1 in regulating NK cell dysfunction in patients' cells. RESULTS: We observed a significant decrease in the percentage of NKG2D and NKp30 and IFN-γ and TNF-α in patients than in healthy volunteers. Patients with low levels of CD56, CD16, NKG2D, and NKP46 exhibited poorer survival prognoses. Moreover, increased expression levels of plasma exosomal miR-552-5p in patients were negatively associated with NK cell phenotypic distribution and activated receptor expression. MiR-552-5p downregulated the secretion of perforin, granzyme, and IFN-γ as well as the expression of NKp30, NKp46, and NKG2D. Additionally, it suppressed the cytotoxicity of NK cells. The inhibitory effect of miR-552-5p, on NK cell function was reversed when anti-PD-L1 antibodies were used. CONCLUSION: Exosomal miR-552-5p targets the PD-1/PD-L1 axis, leading to impaired NK cell function.

Non-fitness status of peripheral NK cells defined by decreased NKp30 and perforin, and increased soluble B7H6, in cervical cancer patients.

The NKp30 receptor is one of the three natural cytotoxic receptors reported in NK cells. This receptor is codified by the NCR3 gene, which encodes three isoforms, a consequence of the alternative splicing of exon 4. A greater expression of the three isoforms (A, B, and C), along with low levels of the NKp30 ligand B7H6, has been reported as a positive prognostic factor in different cancer types. Here, in patients with cervical cancer and precursor lesions, we report an altered immune-phenotype, characterized by non-fitness markers, that correlated with increased disease stage, from CIN 1 to FIGO IV. While overall NK cell numbers increased, loss of NKp30+ NK cells, especially in the CD56dim subpopulation, was found. Perforin levels were decreased in these cells. Decreased expression of the NKp30 C isoform and overexpression of soluble B7H6 was found in cervical cancer patients when compared against healthy subjects. PBMCs from healthy subjects downregulated NKp30 isoforms after co-culture with B7H6-expressing tumour cells. Taken together, these findings describe a unique down-modulation or non-fitness status of the immune response in cervical cancer, the understanding of which will be important for the design of novel immunotherapies against this disease.

Quantitative analysis of human NK cell reactivity using latex beads coated with defined amounts of antibodies.

Natural Killer (NK) cell responses are regulated by a variety of different surface receptors. While we can determine the overall positive or negative effect of a given receptor on NK cell functions, investigating NK cell regulation in a quantitative way is challenging. To quantitatively investigate individual receptors for their effect on NK cell activation, we chose to functionalize latex beads that have approximately the same size as lymphocytes with defined amounts of specific antibodies directed against distinct activating receptors. This enabled us to investigate NK cell reactivity in a defined, clean, and controllable system. Only CD16 and NKp30 could activate the degranulation of resting human NK cells. CD16, NKG2D, NKp30, NKp44, and NKp46 were able to activate cultured NK cells. NK cell activation resulted in the induction of polyfunctional cells that degranulated and produced IFN-γ and MIP-1ß. Interestingly, polyfunctional NK cells were only induced by triggering ITAM-coupled receptors. NKp44 showed a very sensitive response pattern, where a small increase in receptor stimulation caused maximal NK cell activity. In contrast, stimulation of 2B4 induced very little NK cell degranulation, while providing sufficient signal for NK cell adhesion. Our data demonstrate that activating receptors differ in their effectiveness to stimulate NK cells.

Analysis of shark NCR3 family genes reveals primordial features of vertebrate NKp30.

Natural killer (NK) cells play major roles in innate immunity against viruses and cancer. Natural killer receptors (NKR) expressed by NK cells recognize foreign- or self-ligands on infected and transformed cells as well as healthy cells. NKR genes are the most rapidly evolving loci in vertebrates, and it is generally difficult to detect orthologues in different taxa. The unique exception is NKp30, an activating NKR in mammals that binds to the self-ligand B7H6. The NKp30-encoding gene, NCR3, has been found in most vertebrates including sharks, the oldest vertebrates with human-type adaptive immunity. NCR3 has a special, non-rearranging VJ-type immunoglobulin superfamily (IgSF) domain that predates the emergence of the rearranging antigen receptors. Herein we show that NCR3 loci are linked to the shark major histocompatibility complex (MHC), proving NCR3's primordial association with the MHC. We identified eight subtypes of differentially expressed highly divergent shark NCR3 family genes. Using in situ hybridization, we detected one subtype, NS344823, to be expressed by predominantly single cells outside of splenic B cell zones. The expression by non-B cells was also confirmed by PCR in peripheral blood lymphocytes. Surprisingly, high expression of NS344823 was detected in the thymic cortex, demonstrating NS344823 expression in developing T cells. Finally, we show for the first time that shark T cells are found as single cells or in small clusters in the splenic red pulp, also unassociated with the large B cell follicles we previously identified.

Secreted Ligands of the NK Cell Receptor NKp30: B7-H6 Is in Contrast to BAG6 Only Marginally Released via Extracellular Vesicles.

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.

Conserved 33-kb haplotype in the MHC class III region regulates chronic arthritis.

Genome-wide association studies have revealed many genetic loci associated with complex autoimmune diseases. In rheumatoid arthritis (RA), the MHC gene HLA-DRB1 is the strongest candidate predicting disease development. It has been suggested that other immune-regulating genes in the MHC contribute to the disease risk, but this contribution has been difficult to show because of the strong linkage disequilibrium within the MHC. We isolated genomic regions in the form of congenic fragments in rats to test whether there are additional susceptibility loci in the MHC. By both congenic mapping in inbred strains and SNP typing in wild rats, we identified a conserved, 33-kb large haplotype Ltab-Ncr3 in the MHC-III region, which regulates the onset, severity, and chronicity of arthritis. The Ltab-Ncr3 haplotype consists of five polymorphic immunoregulatory genes: Lta (lymphotoxin-α), Tnf, Ltb (lymphotoxin-ß), Lst1 (leukocyte-specific transcript 1), and Ncr3 (natural cytotoxicity-triggering receptor 3). Significant correlation in the expression of the Ltab-Ncr3 genes suggests that interaction of these genes may be important in keeping these genes clustered together as a conserved haplotype. We studied the arthritis association and the spliceo-transcriptome of four different Ltab-Ncr3 haplotypes and showed that higher Ltb and Ncr3 expression, lower Lst1 expression, and the expression of a shorter splice variant of Lst1 correlate with reduced arthritis severity in rats. Interestingly, patients with mild RA also showed higher NCR3 expression and lower LST1 expression than patients with severe RA. These data demonstrate the importance of a conserved haplotype in the regulation of complex diseases such as arthritis.

Coevolution of MHC genes (LMP/TAP/class Ia, NKT-class Ib, NKp30-B7H6): lessons from cold-blooded vertebrates.

Comparative immunology provides the long view of what is conserved across all vertebrate taxa versus what is specific to particular organisms or group of organisms. Regarding the major histocompatibility complex (MHC) and coevolution, three striking cases have been revealed in cold-blooded vertebrates: lineages of class Ia antigen-processing and -presenting genes, evolutionary conservation of NKT-class Ib recognition, and the ancient emergence of the natural cytotoxicity receptor NKp30 and its ligand B7H6. While coevolution of transporter associated with antigen processing (TAP) and class Ia has been documented in endothermic birds and two mammals, lineages of LMP7 are restricted to ectotherms. The unambiguous discovery of natural killer T (NKT) cells in Xenopus demonstrated that NKT cells are not restricted to mammals and are likely to have emerged at the same time in evolution as classical α/ß and γ/δ T cells. NK cell receptors evolve at a rapid rate, and orthologues are nearly impossible to identify in different vertebrate classes. By contrast, we have detected NKp30 in all gnathostomes, except in species where it was lost. The recently discovered ligand of NKp30, B7H6, shows strong signs of coevolution with NKp30 throughout evolution, i.e. coincident loss or expansion of both genes in some species. NKp30 also offers an attractive IgSF candidate for the invasion of the RAG transposon, which is believed to have initiated T-cell receptor/immunoglobulin adaptive immunity. Besides reviewing these intriguing features of MHC evolution and coevolution, we offer suggestions for future studies and propose a model for the primordial or proto MHC.

Effect of the in vivo application of granulocyte colony-stimulating factor on NK cells in bone marrow and peripheral blood.

Granulocyte colony-stimulating factor (G-CSF) has been widely used in the field of allogeneic haematopoietic stem cell transplantation (allo-HSCT) for priming donor stem cells from the bone marrow (BM) to peripheral blood (PB) to collect stem cells more conveniently. Donor-derived natural killer (NK) cells have important antitumour functions and immune regulatory roles post-allo-HSCT. The aim of this study was to evaluate the effect of G-CSF on donors' NK cells in BM and PB. The percentage of NK cells among nuclear cells and lymphocyte was significantly decreased and led to increased ratio of T and NK cells in BM and PB post-G-CSF in vivo application. Relative expansion of CD56bri NK cells led to a decreased ratio of CD56dim and CD56bri NK subsets in BM and PB post-G-CSF in vivo application. The expression of CD62L, CD54, CD94, NKP30 and CXCR4 on NK cells was significantly increased in PB after G-CSF treatment. G-CSF treatment decreased the IFN-γ-secreting NK population (NK1) dramatically in BM and PB, but increased the IL-13-secreting NK (NK2), TGF-ß-secreting NK (NK3) and IL-10-secreting NK (NKr) populations significantly in BM. Clinical data demonstrated that higher doses of NK1 infused into the allograft correlated with an increased incidence of chronic graft-vs-host disease post-transplantation. Taken together, our results show that the in vivo application of G-CSF can modulate NK subpopulations, leading to an increased ratio of T and NK cells and decreased ratio of CD56dim and CD56bri NK cells as well as decreased NK1 populations in both PB and BM.

Reconstitution of a ligand-binding competent murine NKp30 receptor.

The activating natural cytotoxicity receptors on natural killer (NK) cells play a fundamental role in immunosurveillance of infections and cancer. Phylogenetic analyses showed that NKp30 is highly conserved in almost all jawed vertebrates and thus, represents one of the most ancient NK cell receptors. However, in contrast to other higher vertebrates, NKp30 is only a pseudogene in mouse, which contains two premature stop codons. To decipher the evolutionary role and biological function of NKp30 in mouse, we removed these premature stop codons and expressed the putative mouse NKp30 (mNKp30) protein as soluble Fc fusion construct and as full-length receptor on A5-GFP reporter cells. Interestingly, even though both NKp30 variants were expressed, maturation and targeting to the plasma membrane were impaired. Previous studies implicated that N-linked glycosylation is crucial for plasma membrane targeting and ligand binding of human NKp30. However, even though present in all other jawed vertebrates analyzed so far, these three N-linked glycosylation sites are missing in mouse NKp30. Interestingly, reconstitution of N-linked glycosylation enabled secretion of a mNKp30-Fc fusion protein which recognized a yet unknown ligand on the plasma membrane of mastocytoma cells. Based on these data, our study is the first to show expression and functional analysis of a mNKp30 protein suggesting that the mouse NKp30 pseudogene is the result of a species-specific loss of function.

Control of the HIV-1 DNA Reservoir Is Associated In Vivo and In Vitro with NKp46/NKp30 (CD335 CD337) Inducibility and Interferon Gamma Production by Transcriptionally Unique NK Cells.

The size of lentiviral DNA reservoirs reflects the effectiveness of immune responses against lentiviruses. So far, abundant information has been gathered on the control of HIV-1 replication. Understanding the innate mechanisms contributing to containment of the HIV DNA reservoir, however, are only partly clarified and are relevant to guiding interventions for reservoir containment or eradication. We studied the contribution of natural killer (NK) cell functional features in HIV patients controlling replication either spontaneously (HIV controllers [HIC]) or after progression and antiretroviral treatment (progressor patients [PP]). An inverse correlation between HIV DNA copy numbers (either total or integrated) in circulating CD4+ cells and NK cell function was observed. Induced interferon gamma (IFN-γ) production and NKp46/NKp30 activating receptor-induced expression correlated inversely with reservoir size. The correlation was present not only for a homogeneous cohort of HIC patients but also when PP were included in the analysis. Adaptive (NKG2C+ CD57+) NK cell features were not associated with reservoir size. However, a distinct set of 370 differentially expressed transcripts was found to underlie functional differences in NK cells controlling HIV DNA reservoir size. In proof-of-principle in vitro experiments of CD4+ cell infection with HIV-1, purified NK cells with the above-mentioned functional/transcriptional features displayed 10- and 30-fold higher abilities to control HIV replication and DNA burdens in vitro, respectively, than those of other NK cells. Thus, NK cells with a specific functional and transcriptional signature contribute to control of the HIV reservoir in CD4+ cells. Their selection, expansion, and/or adoptive transfer may support strategies to eradicate HIV-1 infection or to safely deescalate antiretroviral treatment.IMPORTANCE The most relevant feature of HIV-1 infection is represented by its DNA reservoir size in the body, which guarantees lifelong infection and resumption of virus replication after antiretroviral treatment interruption. So far, there has been little success in the identification of factors contributing to HIV-1 reservoir containment. In this study, by studying quantitative total and integrated HIV-1 DNA levels and NK cells in HIV-1 patients with either progressive or nonprogressive disease, we observed that inducible IFN-γ and natural cytotoxicity receptor (NCR) expression in a specific subset of NK cells with a characteristic transcriptional signature represents a correlate for HIV-1 reservoir control. This represents an advance in our understanding of the mechanism(s) that controls the lentivirus reservoir. Monitoring, selection, expansion, and adoptive transfer of these NK cells may allow monitoring of treatment efficacy and the likelihood of reservoir control and may support protocols for HIV-1 eradication.

Involvement of NK Cells and NKp30 Pathway in Antisynthetase Syndrome.

Antisynthetase syndrome (aSS) is characterized by the association of interstitial lung disease and myositis with anti-tRNA synthetase autoantibodies. Immune mechanisms leading to aSS could be initiated in the lungs, but the role of NK cells has not yet been studied. Both extensive NK cell phenotype and functions were compared between 33 patients and 26 controls. Direct and redirected polyfunctionality assays (degranulation and intracellular production of TNF-α and IFN-γ) were performed spontaneously or after IL-12 plus IL-18 stimulation in the presence of K562 or P815 target cells, respectively. NK cells from inactive patients showed normal phenotype, whereas active aSS revealed a differentiated NK cell profile, as indicated by increased CD57 and Ig-like transcript 2 and an inability to produce IFN-γ (p = 0.002) compared with controls. Importantly, active aSS was more specifically associated with a significant NKp30 decrease (p = 0.009), although levels of mRNA and intracellular protein were similar in aSS and healthy controls. This NKp30 decrease was strongly correlated with reduced NK cell polyfunctionality in both direct and redirected killing assays with anti-NKp30 Abs (p = 0.009 and p = 0.03, respectively), confirming its important impact in aSS. Histological studies revealed massive infiltrations of NK cells inside the lungs of aSS patients (148 versus 11/mm(2)). Taken together, these data suggest that NK cells and NKp30 could play a role in aSS pathogenesis.

Platelet-Derived Ectosomes Reduce NK Cell Function.

Platelet (PLT) transfusions are potentially life saving for individuals with low PLT numbers; however, previous work revealed that PLT transfusions are associated with increased infection risk. During storage, PLT intended for transfusion continuously shed ectosomes (Ecto) from their surface, which express immunomodulatory molecules like phosphatidylserine or TGF-ß1. Recently, PLT-Ecto were shown to reduce proinflammatory cytokine release by macrophages and to favor the differentiation of naive T cells toward regulatory T cells. Whether PLT-Ecto modify NK cells remains unclear. We exposed purified NK cells and full PBMCs from healthy donors to PLT-Ecto. We found a reduced expression of several activating surface receptors (NKG2D, NKp30, and DNAM-1) and decreased NK cell function, as measured by CD107a expression and IFN-γ production. Pretreatment of PLT-Ecto with anti-TGF-ß1 neutralizing Ab restored surface receptor expression and NK cell function. We further observed a TGF-ß1-mediated upregulation of miR-183, which, in turn, reduced DAP12, an important protein for stabilization and downstream signaling of several activating NK cell receptors. Again, these effects could antagonized, in part, when PLT-Ecto were preincubated with anti-TGF-ß1 Ab. Erythrocyte Ecto did not affect NK cells. Polymorphonuclear cell Ecto expressed MHC class I and inhibited NK cell function. In addition, they induced the secretion of TGF-ß1 by NK cells, which participated in an auto/paracrine manner in the suppressive activity of polymorphonuclear cell-derived Ecto. In sum, our study showed that PLT-Ecto could inhibit NK cell effector function in a TGF-ß1-dependent manner, suggesting that recipients of PLT transfusions may experience reduced NK cell function.

The Stalk Domain of NKp30 Contributes to Ligand Binding and Signaling of a Preassembled NKp30-CD3ζ Complex.

The natural cytotoxicity receptor (NCR) NKp30 (CD337) is a key player for NK cell immunosurveillance of infections and cancer. The molecular details of ligand recognition and its connection to CD3ζ signaling remain unsolved. Here, we show that the stalk domain (129KEHPQLGAGTVLLLR143) of NKp30 is very sensitive to sequence alterations, as mutations lead to impaired ligand binding and/or signaling capacity. Surprisingly, the stalk domains of NKp30 and NKp46, another NCR employing CD3ζ for signaling, were not exchangeable without drastic deficiencies in folding, plasma membrane targeting, and/or ligand-induced receptor signaling. Further mutational studies, N-glycosylation mapping, and plasma membrane targeting studies in the absence and presence of CD3ζ suggest two interconvertible types of NCR-CD3ζ assemblies: 1) a signaling incompetent structural NKp30-CD3ζ complex and 2) a ligand-induced signaling competent NKp30-CD3ζ complex. Moreover, we propose that ligand binding triggers translocation of Arg-143 from the membrane interface into the membrane to enable alignment with oppositely charged aspartate residues within CD3ζ and activation of CD3ζ-signaling.

Genetics of Sjögren's syndrome.

The pathogenesis of Sjögren's syndrome has not been elucidated. There has been evidence that genetics play an important role in the development of this disease from earlier studies. However, till now only a number of genes have been identified to be associated with SS, and these have only a weak or moderate effect. In this review we summarize the findings of the genetics studies and emphasize the need of large multicenter projects that will increase the sample sizes to provide more meaningful associations, as is the case in other common autoimmune diseases.

Asymptomatic CMV infections in long-term renal transplant recipients are associated with the loss of FcRγ from LIR-1+ NK cells.

While it is established that cytomegalovirus (CMV) disease affects NK-cell profiles, the functional consequences of asymptomatic CMV replication are unclear. Here, we characterize NK cells in clinically stable renal transplant recipients (RTRs; n = 48) >2 years after transplantation. RTRs and age-matched controls (n = 32) were stratified by their CMV serostatus and the presence of measurable CMV DNA. CMV antibody or CMV DNA influenced expression of NKG2C, LIR-1, NKp30, NKp46, and FcRγ, a signaling adaptor molecule, on CD56dim NK cells. Phenotypic changes ascribed to CMV were clearer in RTRs than in control subjects and affected NK-cell function as assessed by TNF-α and CD107a expression. The most active NK cells were FcRγ- LIR-1+ NKG2C- and displayed high antibody-dependent cell cytotoxicity responses in the presence of immobilized CMV glycoprotein B reactive antibody. However, perforin levels in supernatants from RTRs with active CMV replication were low. Overall we demonstrate that CMV can be reactivated in symptom-free renal transplant recipients, affecting the phenotypic, and functional profiles of NK cells. Continuous exposure to CMV may maintain and expand NK cells that lack FcRγ but express LIR-1.

Spontaneous and natural cytotoxicity receptor-mediated cytotoxicity are effector functions of distinct natural killer subsets in hepatitis C virus-infected chimpanzees.

In humans, CD16 and CD56 are used to identify functionally distinct natural killer (NK) subsets. Due to ubiquitous CD56 expression, this marker cannot be used to distinguish between NK cell subsets in chimpanzees. Therefore, functional analysis of distinct NK subsets during hepatitis C virus (HCV) infection has never been performed in these animals. In the present study an alternative strategy was used to identify four distinct NK subsets on the basis of the expression of CD16 and CD94. The expression of activating and inhibiting surface receptors showed that these subsets resemble human NK subsets. CD107 expression was used to determine degranulation of the different subsets in naive and HCV-infected chimpanzees. In HCV-infected chimpanzees increased spontaneous cytotoxicity was observed in CD94(high/dim) CD16(pos) and CD94(low) CD16(pos) subsets. By contrast, increased natural cytotoxicity receptor (NCR)- mediated degranulation after NKp30 and NKp44 triggering was demonstrated in the CD94(dim) CD16(neg) subset. Our findings suggest that spontaneous and NCR-mediated cytotoxicity are effector functions of distinct NK subsets in HCV-infected chimpanzees.

Siglec-7 Defines a Highly Functional Natural Killer Cell Subset and Inhibits Cell-Mediated Activities.

Sialic acid-binding immunoglobulin-like lectin-7 (Siglec-7) is an inhibitory receptor expressed on natural killer (NK) cells. In this study, we investigated the relationship between Siglec-7 expression and NK cell functions. Siglec-7 was highly expressed on NK cells and was preferentially expressed by mature NK cells from peripheral blood of healthy adults. Siglec-7(+) NK cells displayed higher levels of activating receptors CD38, CD16, DNAM1, NKp30 and NKp46, but lower levels of inhibitory receptors such as NKG2A and CD158b, compared with Siglec-7(-) NK cells. Functional tests showed that Siglec-7(+) NK cells displayed more CD107a degranulation and IFN-γ production than Siglec-7(-) NK cells. Siglec-7 inhibited NK cell functions when interacting with specific antibodies. These data suggest that Siglec-7 defines a highly functional NK cell subset and suppresses NK cell-mediated functions when cross-linked with specific antibodies.

Homo-oligomerization of the activating natural killer cell receptor NKp30 ectodomain increases its binding affinity for cellular ligands.

The natural cytotoxicity receptors, comprised of three type I membrane proteins NKp30, NKp44, and NKp46, are a unique set of activating proteins expressed mainly on the surface of natural killer (NK) cells. Among these, NKp30 is a major receptor targeting virus-infected cells, malignantly transformed cells, and immature dendritic cells. To date, only few cellular ligands of NKp30 have been discovered, and the molecular details of ligand recognition by NKp30 are poorly understood. Within the current study, we found that the ectodomain of NKp30 forms functional homo-oligomers that mediate high affinity binding to its corresponding cellular ligand B7-H6. Notably, this homo-oligomerization is strongly promoted by the stalk domain of NKp30. Based on these data, we suggest that homo-oligomerization of NKp30 in the plasma membrane of NK cells, which might be favored by IL-2-dependent up-regulation of NKp30 expression, provides a way to improve recognition and lysis of target cells by NK cells.

Tumor-released Galectin-3, a soluble inhibitory ligand of human NKp30, plays an important role in tumor escape from NK cell attack.

Human Galectin-3 (Gal-3), a ß-galactoside-binding protein expressed by tumor cells, has been reported to act as an immune regulator in antitumor T cells. However, its effect on natural killer (NK) cells is elusive. Using a recombinant human NK cell-activating receptor, NKp30 fusion protein (NKp30-Fc), we found that soluble NKp30-Fc could immunoprecipitate Galectin-3. The direct interaction between NKp30 and Galectin-3 was further confirmed using surface plasmon resonance experiments. Because Galectin-3 was mainly released from tumor cells in a soluble form in our study, the binding assay was performed to show that soluble Galectin-3 specifically bound to NK cells and NKp30 on the surface of the NK cells. Functionally, when soluble Galectin-3 was added to the NK-tumor cell coculture system, the NKp30-mediated, but not NKG2D-mediated, cytolysis and CD107a expression in the NK cells were inhibited, and these phenotypes could be restored by preincubation of soluble Galectin-3 with NKp30-Fc fusion protein or the addition of anti-Gal-3 antibody alone. Moreover, genetic down-regulation of Galectin-3 (shGal-3) resulted in tumor cells being more sensitive to NK cell lysis, and, reversely, Galectin-3-overexpressing HeLa cells (exGal-3) became less sensitive to NK cell killing. The results of these in vitro experiments were supported by studies in shGal-3-HeLa or exGal-3-HeLa xenograft non-obese diabetic/severe combined immunodeficiency mice after NK cell adoptive immunotherapy, indicating that Galectin-3 strongly antagonizes human NK cell attack against tumors in vivo. These findings indicate that Galectin-3 may function as an immune regulator to inhibit NK cell function against tumors, therefore providing a new therapeutic target for tumor treatment.

NKp30 isoforms in patients with chronic hepatitis C virus infection.

Natural killer (NK) cells play an important role in virus infection, their action being regulated by several activating and inhibitory receptors. The NKp30 activating receptor and its isoforms have recently emerged as important determinants of efficient NK cell responses. We determined the relative proportions of NKp30 isoforms in patients with chronic hepatitis C virus (HCV) infection and healthy donors (HD). NK cell function (degranulation and cytokine production) and correlations with clinical parameters were assessed following unsupervised hierarchical clustering of patients according to isoform expression. NKp30 receptor expression on NK cells and all isoforms were reduced in HCV-infected patients. Patients were clustered into two groups: the HCV-1 group had similar isoform expression to the HD group, whereas the HCV-2 group had lower expression. The latter showed a better functional activity, and a higher proportion of the activating a isoform and of the NKp30 isoform a/c ratio compared with the HCV-1 cluster. There was a positive correlation between the activating a isoform and liver stiffness and an inverse relationship between the immunosuppressive c isoform and the fibrosis 4 score, suggesting a potentially important role of NKp30 isoforms in influencing liver damage and ensuing fibrosis.