NK cells that differ in expression of NKp46 might play different roles in endometrium.

NKp46 is a natural cytotoxicity receptor expressed by NK cells and its expression is decreased in reproductive failure patients. NKp46 can be subdivided into NKp46dim and NKp46bright according to different fluorescence staining intensities. We investigated the role of the NKp46 receptor in determining the reproductive outcomes. Uterine endometrium was collected from 34 women with reproductive failure and divided into the pregnant and failed groups based on the results of a pregnancy reaction test during a 1-year follow-up period. NKp46 receptor and other activating or inhibitory receptors expressed on NK cells as well as intracellular cytokine production by NK cells were analyzed by multicolor flow cytometry. In the failed group, the percentage of NKp46dim NK cells (P < 0.05) was significantly higher and percentages of NKp46bright NK cells (P < 0.01) and CD16-/CD56bright NK cells (P < 0.05) were significantly lower than those in the pregnant group. NKp46dim NK cells were significantly and positively correlated with CD16+/NKp46dim NK cells; NKp46bright NK cells were significantly and positively correlated with CD16-/NKp46bright NK cells. CD16+/NKp46dim NK cells were significantly and positively correlated with IFN-γ- and/or TNF-α-producing NK cells; CD16-/NKp46bright NK cells were significantly and positively correlated with TGF-ß1-producing NK cells. We suggest that the NKp46 receptor plays different roles in reproduction based on the different fluorescence intensities associated with NK cells, i.e. NKp46dim NK cells are involved in killing cells, whereas NKp46bright NK cells are involved in cytokine production, indicating that NKp46 could be a predictive marker to see a tolerate condition for embryos.

Cutaneous Squamous Cell Carcinoma Development Is Associated with a Temporal Infiltration of ILC1 and NK Cells with Immune Dysfunctions.

NK cells and tissue-resident innate lymphoid cells (ILCs) are innate effectors found in the skin. To investigate their temporal dynamics and specific functions throughout the development of cutaneous squamous cell carcinoma (cSCC), we combined transcriptomic and immunophenotyping analyses in mouse and human cSCCs. We identified an infiltration of NK cells and ILC1s as well as the presence of a few ILC3s. Adoptive transfer of NK cells in NK cell‒ and ILC-deficient Nfil3-/- mice revealed a role for NK cells in early control of cSCC. During tumor progression, we identified a population skewing with the infiltration of atypical ILC1 secreting inflammatory cytokines but reduced levels of IFN-γ at the papilloma stage. NK cells and ILC1s were functionally impaired, with reduced cytotoxicity and IFN-γ secretion associated with the downregulation of activating receptors. They also showed a high degree of heterogeneity in mouse and human cSCCs with the expression of several markers of exhaustion, including TIGIT on NK cells and PD-1 and TIM-3 on ILC1s. Our data show an enrichment in inflammatory ILC1 at the precancerous stage together with impaired antitumor functions in NK cells and ILC1 that could contribute to the development of cSCC and thus suggest that future immunotherapies should take both ILC populations into account.

Phenotypic and Functional Characteristics of a Novel Influenza Virus Hemagglutinin-Specific Memory NK Cell.

Immune memory represents the most efficient defense against invasion and transmission of infectious pathogens. In contrast to memory T and B cells, the roles of innate immunity in recall responses remain inconclusive. In this study, we identified a novel mouse spleen NK cell subset expressing NKp46 and NKG2A induced by intranasal influenza virus infection. These memory NK cells specifically recognize N-linked glycosylation sites on influenza hemagglutinin (HA) protein. Different from memory-like NK cells reported previously, these NKp46+ NKG2A+ memory NK cells exhibited HA-specific silence of cytotoxicity but increase of gamma interferon (IFN-γ) response against influenza virus-infected cells, which could be reversed by pifithrin-µ, a p53-heat shock protein 70 (HSP70) signaling inhibitor. During recall responses, splenic NKp46+ NKG2A+ NK cells were recruited to infected lung and modulated viral clearance of virus and CD8+ T cell distribution, resulting in improved clinical outcomes. This long-lived NK memory bridges innate and adaptive immune memory response and promotes the homeostasis of local environment during recall response.IMPORTANCE In this study, we demonstrate a novel hemagglutinin (HA)-specific NKp46+ NKG2A+ NK cell subset induced by influenza A virus infection. These memory NK cells show virus-specific decreased cytotoxicity and increased gamma interferon (IFN-γ) on reencountering the same influenza virus antigen. In addition, they modulate host recall responses and CD8 T cell distribution, thus bridging the innate immune and adaptive immune responses during influenza virus infection.

Interferon-γ production by tubulointerstitial human CD56bright natural killer cells contributes to renal fibrosis and chronic kidney disease progression.

Natural killer (NK) cells are a population of lymphoid cells that play a significant role in mediating innate immune responses. Studies in mice suggest a pathological role for NK cells in models of kidney disease. In this study, we characterized the NK cell subsets present in native kidneys of patients with tubulointerstitial fibrosis, the pathological hallmark of chronic kidney disease. Significantly higher numbers of total NK cells (CD3-CD56+) were detected in renal biopsies with tubulointerstitial fibrosis compared with diseased biopsies without fibrosis and healthy kidney tissue using multi-color flow cytometry. At a subset level, both the CD56dim NK cell subset and particularly the CD56bright NK cell subset were elevated in fibrotic kidney tissue. However, only CD56bright NK cells significantly correlated with the loss of kidney function. Expression of the tissue-retention and -activation molecule CD69 on CD56bright NK cells was significantly increased in fibrotic biopsy specimens compared with non-fibrotic kidney tissue, indicative of a pathogenic phenotype. Further flow cytometric phenotyping revealed selective co-expression of activating receptor CD335 (NKp46) and differentiation marker CD117 (c-kit) on CD56bright NK cells. Multi-color immunofluorescent staining of fibrotic kidney tissue localized the accumulation of NK cells within the tubulointerstitium, with CD56bright NK cells (NKp46+ CD117+) identified as the source of pro-inflammatory cytokine interferon-γ within the NK cell compartment. Thus, activated interferon-γ-producing CD56bright NK cells are positioned to play a key role in the fibrotic process and progression to chronic kidney disease.

Expression and function of NKp46 W32R: the human homologous protein of mouse NKp46 W32R (Noé).

Natural killer (NK) cells eradicate infected cells and tumors following the triggering of activating receptors, like the Natural Cytotoxicity Receptors (NCRs), which include NKp30, NKp44 and NKp46. NKp46 is the only NCR expressed in mice (mNKp46), and except for some Innate Lymphoid Cell (ILC) populations (ILC1/3 subsets), its expression is restricted to NK cells. Previously, a mouse named Noé was generated in which a random point mutation (W32R) impaired the cell surface expression of mNKp46. Interestingly, the Noé mice NK cells expressed twice as much of the transcription factor Helios, and displayed general non-NKp46 specific hyperactivity. We recently showed that the mNKp46 W32R (Noé) protein was expressed on the surface of various cells; albeit slowly and unstably, that it is aberrantly glycosylated and accumulates in the ER. Interestingly, the Tryptophan (Trp) residue in position 32 is conserved between humans and mice. Therefore, we studied here the human orthologue protein of mNKp46 W32R, the human NKp46 W32R. We demonstrated that NKp46 W32R is aberrantly glycosylated, accumulates in the ER, and is unstable on the cell surface. Furthermore, we showed that overexpression of NKp46 W32R or Helios resulted in augmented NK cell activation, which may be applied to boost NK activity for therapeutic applications.

The microenvironment of visceral adipose tissue and liver alter natural killer cell viability and function.

The role of NK cells in visceral adipose tissue (VAT) and liver inflammation in obesity is not fully understood. This study investigated the frequency, cytokine expression, chemokine receptor, and cytotoxicity receptor profile of NK cells in the blood, omentum, and liver of patients with the obesity-associated cancer, oesophageal adenocarcinoma (OAC). The effect of chronically inflamed tissue microenvironments on NK cell viability and function was also examined. We identified significantly lower NK cell frequencies in the liver of OAC patients compared with healthy controls and within the omentum and liver of OAC patients compared with blood, whereas IL-10-producing populations were significantly higher. Interestingly, our data suggest that reduced frequencies of NK cells in omentum and liver of OAC patients are not a result of impaired NK cell chemotaxis to these tissues. In fact, our functional data revealed that secreted factors from omentum and liver of OAC patients induce significant levels of NK cell death and lead to reduced percentages of TNF-α+ and NKP46+ NK cells and higher frequencies of IL-10-producing NK cells. Together, these data suggest that the omental and hepatic microenvironments of OAC patients alter the NK cell phenotype to a more anti-inflammatory homeostatic role.

Increased CD56(bright) NK cells in HIV-HCV co-infection and HCV mono-infection are associated with distinctive alterations of their phenotype.

BACKGROUND: HIV-HCV co-infection is associated with accelerated progression to hepatic fibrosis, cirrhosis and hepatocellular carcinoma than HCV mono-infection. The contribution of innate immunity during HIV-HCV co-infection has been a relatively under-investigated area. Natural killer (NK) cells are pivotal sentinels of innate immunity against viruses and tumour cells. In this study we evaluated the effect of HIV-HCV co-infection on peripheral blood NK cell subsets with emphasis on the phenotype of CD56(bright) NK cells. METHODS: Sixty patients were included in the study; HIV mono-infected (n = 12), HCV mono-infected (n = 15), HCV-HIV co-infected (n = 21) and healthy controls (n = 16). PBMCs were isolated and immunophenotyping of NK cells was performed by flowcytometry. RESULTS: We observed an expansion of CD56(bright) NK cell subset in HIV-HCV co-infection as compared to healthy controls and HIV mono-infected group. All the infected groups had an upregulated expression of the activating receptor NKG2D on CD56(bright) NK cells in comparison to healthy controls while not differing amongst themselves. The expression of NKp46 in HIV-HCV co-infected group was significantly upregulated as compared to both HIV as well as HCV mono-infections while NKp30 expression in the HIV-HCV co-infected group significantly differed as compared to HIV mono-infection. The CD56(bright) NK cell subset was activated in HIV-HCV co-infection as assessed by the expression of CD69 as compared to healthy controls but was significantly downregulated in comparison to HIV mono-infection. CD95 expression on CD56(bright) NK cells followed the same pattern where there was an increased expression of CD95 in HIV mono-infection and HIV-HCV co-infection as compared to healthy controls. In contrast to CD69 expression, CD95 expression in HCV mono-infection was decreased when compared to HIV mono-infection and HIV-HCV co-infection. Finally, expression of CXCR3 on CD56(bright) NK cells was increased in HIV-HCV co-infection in comparison to HIV mono-infection while remaining similar to HCV mono-infection. CONCLUSION: Thus, HIV-HCV co-infection is able to modulate the phenotype of CD56(bright) NK cell subset in a unique way such that NKp46 and CXCR3 expressions are distinct for co-infection while both mono-infections have an additive effect on CD56(bright), CD69 with CD95 expressions. HCV mono-infection has a dominant effect on NKp30 expression while NKG2D and CD127 expressions remained same in all the groups.

Unique and redundant functions of NKp46+ ILC3s in models of intestinal inflammation.

Group 3 ILCs (ILC3s) are innate sources of IL-22 and IL-17 and include lymphoid tissue-inducer (LTi)-like and NKp46(+) subsets. Both depend on RORγt and aryl hydrocarbon receptor, but NKp46(+)ILC3s also require Notch and T-bet for their development and are transcriptionally distinct. The extent to which these subsets have unique functions, especially in the context of T cell- and B cell-sufficient mice, remains largely unclear. To investigate the specific function of NKp46(+)ILC3s among other ILC3 subsets and T cells, we generated mice selectively lacking NKp46(+)ILC3s or all ILC3s and crossed them to T cell-deficient mice, thus maintaining B cells in all mice. In mice lacking T cells, NKp46(+)ILC3s were sufficient to promote inflammatory monocyte accumulation in the anti-CD40 innate colitis model through marked production of GM-CSF. In T cell-competent mice, lack of NKp46(+)ILCs had no impact on control of intestinal C. rodentium infection, whereas lack of all ILC3s partially impaired bacterial control. Thus, NKp46(+)ILC3s have a unique capacity to promote inflammation through GM-CSF-induced accumulation of inflammatory monocytes, but are superseded by LTi-like ILC3s and T cells in controlling intestinal bacterial infection.

Profile of natural killer cells after a previous natural Vaccinia virus infection in an in vitro viral re-exposure.

The present study compares the profile of NK cells in an in vitro re-exposure by Vaccinia virus (VACV), in groups that have had a previous vaccination or natural infection. Our data suggests that stimulation with VACV triggers a cytotoxic response by NK cells marked by an increase of NCRs: NKp30, NKp44, and NKp46 in infected (vaccinated and unvaccinated) subjects and in non-infected vaccinated patients, when compared with non-infected unvaccinated individuals. However, the degranulation and secretion processes are inhibited in infected (vaccinated and unvaccinated) subjects and in the non-infected vaccinated patients, when compared with non-infected unvaccinated individuals. We demonstrated that stimulation with VACV downregulates the percentage of expression of Perforin, Granzyme A, and CD107a, but upregulate CD94 in infected (vaccinated and unvaccinated) subjects and in non-infected vaccinated patients, when compared with non-infected unvaccinated individuals. Furthermore, the percentage of IFN-γ(+) NK cells was significantly lower in non-infected unvaccinated subjects, when compared with infected (vaccinated and unvaccinated) and non-infected vaccinated individuals. Our results also show that the percentage of TNF-α(+) NK cells was significantly higher in infected (vaccinated and unvaccinated) subjects and in non-infected vaccinated patients, when compared with non-infected unvaccinated individuals, after in vitro stimulation with UV-inactivated VACV. Our data suggest that the expression of NCRs NKp30, NKp44, NKp46 and cytokines by NK cells are important in the innate response against VACV.

Expression of the activating receptor, NKp46 (CD335), in human natural killer and T-cell neoplasia.

OBJECTIVES: To evaluate the expression of CD335 (NKp46), an activation receptor that is selectively expressed on natural killer (NK) cells. METHODS: We assessed CD335's potential utility as a diagnostic marker in 657 cases by flow cytometry and 410 cases by immunohistochemistry. RESULTS: We observed that CD335 was highly specific for NK cells in nonneoplastic tissues. Moreover, 61 (90%) of 68 of NK cell neoplasms demonstrated CD335 expression, whereas B-cell, myelomonocytic, and plasma cell neoplasms lacked expression. Notably, 16 (20%) of 82 mature T-cell neoplasms, particularly T-cell large granular lymphocytic leukemia, mycosis fungoides, and ALK+ anaplastic large cell lymphoma, aberrantly expressed CD335. CONCLUSIONS: Collectively, these data support the diagnostic utility of CD335 in evaluating hematopoietic malignancies and suggest that CD335 could be a useful target for selective immunotherapy in patients with mature NK and T-cell neoplasms.

Flow cytometry in detection of abnormalities of natural killer cell.

The population of natural killer (NK) cells is very heterogeneous and plays a role in the immune system. Several NK cells subpopulations are recognized, differing in phenotype, cytokine release and cytotoxic ability. Different expression of biologically relevant molecules on the surface of NK cells may indicate their multiple functions. The activity of NK cells has mainly to do with their cytotoxic nature. A complete analysis of NK cells function requires application of many tests because a defect may be present at different stages of the cytotoxic process, from signal transduction through lysosome degranulation to target cells destruction. Flow cytometry is actually one of the best methods for the identification of NK cells and tracking their defects.

Altered functions of natural killer cells in response to L-Arginine availability.

L-Arginine (L-Arg) availability is crucial in the regulation of immune response. Indeed, L-Arg deficiency induces T-cell dysfunction and could modulate the properties of natural killer (NK) cells involved in the early host defense against infections and tumors. We explored the impact of L-Arg depletion on NK cell functions using two models - an NK-92 cell line and isolated human blood NK cells. Below 5mg/L of L-Arg, NK-92 cell proliferation was decreased and a total L-Arg depletion reduced NK-92 cell viability. NK cell cytotoxicity was significantly inhibited in presence of low L-Arg concentration (2.5 mg/L). L-Arg depletion reduced the expression of NK-92 activating receptors, NKp46 and NKp30, the expression of NK ζ chain and the NK-92 intracellular production of IFN-γ. Whatever the L-Arg concentrations tested, no significant variation in the gene expression of transporters and enzymes involved in L-Arg metabolism was found. Thus, L-Arg availability modulates the phenotypic and functional properties of NK cells.

Suppressive effect of asbestos on cytotoxicity of human NK cells.

Asbestos, a naturally occurring fibrous mineral, causes malignant mesothelioma (MM). However, it takes a very long time to develop MM, which suggests that effects other than tumorigenicity of asbestos might contribute to the development of MM, and one of the possible targets is anti-tumor immunity. Therefore, we examined the effect of asbestos exposure on human natural killer (NK) cells using the cell line of YT-A1, Peripheral blood mononuclear cells (PBMCs) cultures and specimens from patients with MM. In particular, we focused on expression of NK cell-activating receptors, including NKG2D, 2B4 and NKp46. Analysis of the YT-CB5 subline of YT-A1, cultured with CB for over 5 months, showed a decrease in cytotoxicity with low expressions of NKG2D and 2B4, although there were no decreases after about one month. YT-CB5 showed decreases in phosphorylation of extracellular signal-regulated kinase (ERK) and degranulation stimulated by antibodies to NKG2D. Peripheral blood (PB-) NK cells from MM patients also showed decreased cytotoxicity compared with healthy volunteers (HV), and was accompanied with low expression of NKp46 unlike YT-CB5. PBMCs cultured with CB resulted in decreased expression of NKp46 on NK cells, although this did not occur when using glass wool, an asbestos substitute. These results indicate that asbestos has the potential to suppress cytotoxicity of NK cells. In particular, it is noteworthy that both NK cells from MM patients and those from a culture of PBMCs derived from HVs with asbestos showed the same characteristic of decreased cytotoxicity with low expression of NKp46.