Expression of natural cytotoxicity receptors and a2V-ATPase on peripheral blood NK cell subsets in women with recurrent spontaneous abortions and implantation failures

PROBLEM: Natural cytotoxicity receptors (NCRs) are unique markers, which regulate NK cell cytotoxicity and cytokine production. a2V-ATPase is expressed on subsets of PBMC and regulates the extracellular environment, which facilitates NK cytotoxicity or cytokine secretion. In this study, we aim to investigate the expression of NCRs and a2V-ATPase in peripheral blood NK cells of women with recurrent spontaneous abortions (RSA) or implantation failures. METHOD OF STUDY: Peripheral blood NK cells (CD56(dim) and CD56(bright) were analyzed for the expression of NCRs (NKp46, NKp44 and NKp30) and a2V-ATPase using 3-color flow cytometry in women with RSA (n=24), implantation failures (n=19) or normal healthy women (n=13). RESULTS: CD56+/NKp46+ cells were markedly decreased (P<0.05) and CD56(bright)/a2V-ATPase+ cells were significantly increased (P<0.05) in women with RSA as compared to those of normal controls. In women with RSA or implantation failures, expression of NKp46, NKp44, NKp30, and a2V-ATPase on CD56(bright) NK cells was significantly up-regulated as compared with those of CD56(dim) NK cells. CONCLUSION: The differential expression of NCRs and a2V-ATPase in NK cell subsets may suggest dysregulation of NK cytotoxicity and cytokine production in women with RSA and implantation failures.     

Activating NK cell receptor expression/function (NKp30, NKp46, DNAM-1) during chronic viraemic HCV infection is associated with the outcome of combined treatment

Specific NK cell killer inhibitory receptor (KIR):HLA haplotype combinations have been associated with successful clearance of acute and chronic HCV infection. Whether an imbalance of activating NK cell receptors also contributes to the outcome of treatment of chronic HCV infection, however, is not known. We studied peripheral NK cell phenotype and function in 28 chronically viraemic HCV genotype I treatment‐naïve patients who underwent treatment with pegylated IFN‐α and ribavirin. At baseline, chronically infected patients with sustained virological response (SVR) had reduced CD56^brightCD16^+/? cell populations, increased CD56^dullCD16⁺ NK cell proportions, and lower expression of NKp30, DNAM‐1, and CD85j. Similarly, reduced NK cell IFN‐γ production but increased degranulation was observed among nonresponding (NR) patients. After treatment, CD56^brightCD16^+/? NK cell numbers increased in both SVR and NR patients, with a parallel significant increase in activating NKp30 molecule densities in SVR patients only. In vitro experiments using purified NK cells in the presence of rIL‐2 and IFN‐α confirmed upregulation of NKp30 and also of NKp46 and DNAM‐1 in patients with subsequent SVR. Thus, differences in patient NK cell receptor expression and modulation during chronic HCV‐1 infection are associated with subsequent outcome of standard treatment. Individual activating receptor expression/function integrates with KIR:HLA genotype carriage to determine the clearance of HCV infection upon treatment.     

NKp46 expression discriminates porcine NK cells with different functional properties

So far little is known about natural killer (NK) cells in the pig due to the lack of NK cell-specific markers. In this study, we identified the activating receptor NKp46 (CD335) in swine with newly developed monoclonal antibodies (mAbs) for more detailed studies on NK cells in this species. The NKp46 mAbs showed a specific reactivity with a distinct population of perforin(+) CD2(+) CD3(-) CD8α(+) CD16(+) lymphocytes. In spleen and liver, an additional subset of CD8α(dim/-) lymphocytes with increased NKp46 expression was observed. Surprisingly, we could identify NKp46(-) cells with an NK cell phenotype in all animals analyzed. These lymphocytes showed comparable cytolytic activity against xenogeneic and allogeneic target cells as NKp46(+) NK cells. In contrast, NKp46(+) NK cells produced several fold higher levels of interferon-γ (IFN-γ) than the NKp46(-) cells after cytokine stimulation. Furthermore, an activation-dependent induction of NKp46 expression in formerly NKp46(-) cells after stimulation with interleukin-2 (IL-2), IL-12, and IL-18 could be shown. In summary, our data indicate that NKp46 is not expressed by all porcine NK cells and that NKp46 discriminates porcine NK cells differing in regard to cytokine production, which challenges the paradigm of NKp46 as a comprehensive marker for NK cells across different mammalian species.     

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.     

Defective natural killer cell anti‐viral capacity in paediatric HBV infection

Natural killer (NK) cells exhibit dysregulated effector function in adult chronic hepatitis B virus (HBV) infection (CHB), which may contribute to virus persistence. The role of NK cells in children infected perinatally with HBV is less studied. Access to a unique cohort enabled the cross‐sectional evaluation of NK cell frequency, phenotype and function in HBV‐infected children relative to uninfected children. We observed a selective defect in NK cell interferon (IFN)‐γ production, with conserved cytolytic function, mirroring the functional dichotomy observed in adult infection. Reduced expression of NKp30 on NK cells suggests a role of impaired NK‐dendritic cell (DC) cellular interactions as a potential mechanism leading to reduced IFN‐γ production. The finding that NK cells are already defective in paediatric CHB, albeit less extensively than in adult CHB, has potential implications for the timing of anti‐viral therapy aiming to restore immune control.     

Expansion of 2B4+ natural killer (NK) cells and decrease in NKp46+ NK cells in response to influenza

Several studies have highlighted the importance of murine natural killer (NK) cells in the control of influenza virus infection, notably through the natural cytotoxicity receptor NKp46. However, little is known about the involvement of NK cells in human influenza infection. Here, we show that upon in vitro exposure to influenza, NKp46 expression on NK cells decreases, whereas expression of 2B4, an activating receptor that can enhance natural cytotoxicity in synergy with NKp46, is up-regulated. Consistent with these observations, NKp46(dull) and 2B4(bright) NK cells had a higher functional activity in response to influenza than NK cells expressing high levels of NKp46 or low levels of 2B4, respectively. Importantly, we assessed whether the expression of these receptors was also modified in vivo in response to influenza antigens and showed that an increase in 2B4-expressing NK cells and a decrease in NKp46(+) NK cells occurred following intramuscular influenza vaccination. Altogether, our results further suggest that NKp46 may play an important role in the innate immune response to human influenza and reveal that exposure to influenza antigens is associated with a previously unrecognized increase in 2B4 expression that can impact NK cell activity against the virus.     

Activation of natural killer cells by hepatitis C virus particles in vitro

Little is known about the ability of hepatitis C virus (HCV) to alter early innate immune responses in infected patients. Previous studies have shown that natural killer (NK) cells are functionally impaired after interaction of recombinant HCV glycoprotein E2 with the co-stimulatory CD81 molecule in vitro; however, the functional consequences of a prolonged contact of NK cells with HCV particles have remained unclear. We have examined the phenotypes of purified, interleukin-2-activated NK cells from healthy donors and HCV genotype 1b patients after culture for 5 days with HCV pseudoparticles (HCVpp) and serum samples containing HCV genotype 1b. NK cells from healthy donors and chronic HCV patients were found to up-regulate receptors associated with activation (NKp46, NKp44, NKp30, NKG2D), while NK receptors from the killer cell immunoglobulin-like receptor family (KIR/CD158), predominantly having an inhibitory function, were significantly down-modulated after culture in the presence of HCV particles compared with control cultures of NK cells. HCV-infected sera and HCVpp elicited significantly higher secretion of the NK effector lymphokines interferon-γ and tumour necrosis factor-α. Furthermore, HCV stimulated the cytotoxic potential of NK cells from normal donors and patients. The enhanced activation of NK cells after prolonged culture with HCVpp or HCV-containing sera for 5 days suggests that these innate effector cells may play an important role in viral control during early phases of HCV infection.     

Hypoxia downregulates the expression of activating receptors involved in NK‐cell‐mediated target cell killing without affecting ADCC

In certain infection sites or tumor tissues, the disruption of homeostasis can give rise to a hypoxic microenvironment, which, in turn, can alter the function of different immune cell types and favor the progression of the disease. Natural killer (NK) cells are directly involved in the elimination of virus‐infected or transformed cells, however it is unknown whether their function is affected by hypoxia or not. In this study, we show that NK cells adapt to a hypoxic environment by upregulating the hypoxia‐inducible factor 1α. However, NK cells lose their ability to upregulate the surface expression of the major activating NK‐cell receptors (NKp46, NKp30, NKp44, and NKG2D) in response to IL‐2 (or other activating cytokines, including IL‐15, IL‐12, and IL‐21). These altered phenotypic features correlate with reduced responses to triggering signals resulting in impaired capability of killing infected or tumor target cells. Remarkably, hypoxia does not significantly alter the surface density and the triggering function of the Fc‐γ receptor CD16, thus allowing NK cells to maintain their capability of killing target cells via antibody‐dependent cellular cytotoxicity. This finding offers an important clue for exploitation of NK cell in antibody‐based immunotherapy of cancer.     

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/dimCD16^pos and CD94^lowCD16^pos subsets. By contrast, increased natural cytotoxicity receptor (NCR)‐ mediated degranulation after NKp30 and NKp44 triggering was demonstrated in the CD94^dimCD16^neg subset. Our findings suggest that spontaneous and NCR‐mediated cytotoxicity are effector functions of distinct NK subsets in HCV‐infected chimpanzees.     

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 CD56^dim 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.     

HCV core and NS3 proteins manipulate human blood-derived dendritic cell development and promote Th 17 differentiation

Hepatitis C virus (HCV) chronic infection is characterized by low-level or undetectable cellular immune response against HCV antigens. HCV proteins affect various intracellular events and modulate immune responses, although the mechanisms that mediate these effects are not fully understood. In this study, we examined the effect of HCV proteins on the differentiation of human peripheral blood monocytes to dendritic cells (DCs). The HCV core (HCVc) and non-structural 3 (NS3) proteins inhibited the expression of CD1a, CD1b and DC-SIGN during monocyte differentiation to DCs, while increasing some markers characteristic of macrophages (CD14 and HLA-DR) and also PD-L1 expression. Meanwhile, HCVc and NS3 could induce differentiating monocytes to secrete IL-10. However, anti-IL-10 mAb could not reverse HCVc and NS3 inhibition of monocyte differentiation into DCs. The HCVc and NS3 proteins increased IL-6 secretion both in immature and in fully differentiated DCs and also promoted CD4+ T-cell IL-17 production. Since T(h) 17 cells are active in many examples of immunopathology, these effects may contribute to HCV autoimmune responses in chronically infected patients.     

CD4+ T cell help and innate-derived IL-27 induce Blimp-1-dependent IL-10 production by antiviral CTLs

Interleukin (IL)-10 is an important regulatory cytokine that can modulate excessive immune mediated injury. Several distinct cell types have been demonstrated to produce IL-10, including most recently CD8+ cytotoxic T lymphocytes (CTLs) responding to respiratory virus infection. Here we report that CD4+ T cell help in the form of IL-2 is required for IL-10 production by CTLs, but not for the induction of CTL effector cytokines. We show that IL-2 derived from CD4+ helper T cells cooperates with innate immune cell-derived IL-27 to amplify IL-10 production by CTLs through a Blimp-1-dependent mechanism. These findings reveal a previously unrecognized pathway that coordinates signals derived from innate and helper T cells to control the production of a regulatory cytokine by CTLs during acute viral infection.     

Multiple HLA-class I-specific inhibitory NK receptor expression and IL-4/IL-5 production by CD8+ T-cell clones in HIV-1 infection

Several mechanisms may contribute to the decline in HIV-1 specific CD8+ cytotoxic T-lymphocyte (CTL) activity that is observed in infected patients, including loss of CD4+ cell help, antigenic shift, impaired clonogenicity and functional impairment due to expression of inhibitory NK receptors (iNKRs). In addition to a decrease in HIV-1-specific cytolytic activity, an increased proportion of CD8+ T-cells producing IL-4 and IL-5 has been recently observed in advanced HIV-1 infection. Remarkably, an impaired HIV-1-specific CTL activity was primarily detected among the TC0/Tc2 CD8+ CTLs. A series of CD3+CD8+ T-cell clones expressing inhibitory NK receptors (iNKRs) isolated from HIV-1 infected patients was analyzed in order to determine their cytokine production pattern and to assess the extent of iNKR expression at the single cell level. Our data indicate that iNKR+CD3+CD8+ clones isolated from infected patients frequently express multiple iNKR and may produce IL-4 and IL-5 to a relevant extent.     

Positive selection of cytotoxic T lymphocyte escape variants during acute hepatitis C virus infection

Cellular immune responses are induced during hepatitis C virus (HCV) infection and acute-phase CD8+ T cells are supposed to play an important role in controlling viral replication. In chimpanzees, failure of CD8+ T cells to control HCV replication has been associated with acquisition of mutations in MHC class I-restricted epitopes. In humans, although selection of escape mutations in an immunodominant CTL epitope has been recently described, the overall impact of immune escape during acute HCV infection is unclear. Here, by performing an in depth analysis of the relationship between early cellular immune responses and viral evolution in a chronically evolving HCV acutely infected individual, we demonstrate: (i) the presence of a potent and focused CD8(+ T cell response against a novel epitope in the NS3 protein, (ii) the elimination of the quasi-species harboring the original amino acid sequence within this epitope, and (iii) the selection for a virus population bearing amino acid changes at a single residue within the cytotoxic T cell epitope that strongly diminished T cell recognition. These results support the view that acute-phase CD8+ T cell responses exert a biologically relevant pressure on HCV replication and that viruses escaping this host response could have a significant survival advantage.     

Identification of NKp80, a novel triggering molecule expressed by human NK cells

The ability of NK cells to kill a wide range of tumor or virally infected target cells as well as normal allogeneic T cell blasts appears to depend upon the concerted action of multiple triggering NK receptors. In this study, using two specific monoclonal antibodies [(mAb) MA152 and LAP171], we identified a triggering NK receptor expressed at the cell surface as a dimer of approximately 80 kDa (NKp80). NKp80 is expressed by virtually all fresh or activated NK cells and by a minor subset of T cells characterized by the CD56 surface antigen. NKp80 surface expression was also detected in all CD3- and in 6 / 10 CD3+ large granular lymphocyte expansions derived from patients with lymphoproliferative disease of granular lymphocytes. In polyclonal NK cells, mAb-mediated cross-linking of NKp80 resulted in induction of cytolytic activity and Ca2+ mobilization. A marked heterogeneity existed in the magnitude of the cytolytic responses of different NK cell clones to anti-NKp80 mAb. This heterogeneity correlated with the surface density of NKp46 molecules expressed by different NK clones. The mAb-mediated masking of NKp80 led to a partial inhibition of the NK-mediated lysis of appropriate allogeneic phytohemagglutinin-induced T cell blasts, while it had no effect on the lysis of different tumor target cells, including T cell leukemia cells. These data suggest that NKp80 recognizes a ligand on normal T cells that may be down-regulated during tumor transformation. Molecular cloning of the cDNA coding for NKp80 revealed a type II transmembrane molecule of 231 amino acids identical to the putative protein encoded by a recently identified cDNA termed KLRF1.     

The impaired NK cell cytolytic function in viremic HIV-1 infection is associated with a reduced surface expression of natural cytotoxicity receptors (NKp46, NKp30 and NKp44)

Signals leading to NK cell triggering are primarily mediated by natural cytotoxicity receptors (NCR) upon binding to as-yet-undefined cell surface ligand(s) on normal hematopoietic cells, pathogen-infected cells or tumor cells. In this study we tried to determine whether the decreased NK cell cytolytic function that is observed in HIV-1-infected patients may be related to a decreased expression of NCR. In HIV-1-infected patients, freshly drawn, purified NK cells expressed significantly decreased surface densities of NKp46 and NKp30 NCR. The low surface density of NKp46, NKp30 and NKp44 was also confirmed in in-vitro-activated NK cell populations and NK cell clones derived from HIV-1 patients compared with uninfected donors. This defective NCR expression in HIV-1 patients was associated with a parallel decrease of NCR-mediated killing of different tumor target cells. Thus, the present study indicates that the defective expression of NCR represents at least one of the possible mechanisms leading to the impaired NK cell function in HIV-1 infection and it can contribute to explain the relatively high frequency of opportunistic tumors reported in cohorts of untreated patients before the occurrence of profound immunosuppression (<200 CD4(+) cells/mm(3)).     

Corrective effects of interleukin-12 on age-related deficiencies in IFN-gamma production and IL-12Rbeta2 expression in virus-specific CD8+ T cells.

Interleukin-12 receptor beta2 (IL-12Rbeta2) has been shown to be selectively expressed on Th1 T cell subsets, and we have previously shown that influenza-specific CD8+ cytotoxic T lymphocyte (CTL) deficiency in old mice was associated with deficient Th1 (interferon-gamma [IFN-gamma]) cytokine production. This study tested whether IL-12Rbeta2 expression was also deficient in CD8+ CTL from old mice and the effect of IL-12 treatment on these responses. Splenic lymphocytes from influenza-primed old and young BALB/c mice were stimulated with influenza virus in vitro with and without IL-12 and then enriched for CD8+ T cells. IFN-gamma was significantly reduced, whereas IL-4 and IL-12p40 (an antagonist of IL-12 function) were evaluated in old when compared with young mice. This was true for secreted protein measured by ELISA and for mRNA levels quantitated by RT-PCR. IL-12Rbeta2 mRNA expression in CD8+ CTL was also significantly reduced in old mice. IL-12 treatment in vitro caused significant upregulation of IFN-gamma and IL-12Rbeta2 and downregulation of IL-4 in CD8+ T cells from old mice and young mice. The present demonstration of an age-related downregulation in IL-12Rbeta2 expression and our previous data showing reduced IFN-gamma and elevated IL-4 production provide strong evidence that CD8+ CTL deficiency in aging results from a Th1/Th2 cytokine production switch. Agents that increase IL-12Rbeta2 expression and redirect Th2 to Thl immune responses are likely to enhance CD8+ CTL-mediated control of viral infections in aging.