Expansion of CMV-mediated NKG2C+ NK cells associates with the development of specific de novo malignancies in liver-transplanted patients.

Solid cancers are a major adverse outcome of orthotopic liver transplantation (OLT). Although the use of chronic immunosuppression is known to play a role in T cell impairment, recent insights into the specificities of NK cells led us to reassess the potential modulation of this innate immune cell compartment after transplantation. Our extensive phenotypic and functional study reveals that the development of specific de novo noncutaneous tumors post-OLT is linked to unusual NK cell subsets with maturation defects and to uncommon cytokine production associated with the development of specific cancers. Remarkably, in CMV(+) patients, the development de novo head/neck or colorectal tumors is linked to an aberrant expansion of NK cells expressing NKG2C and a high level of intracellular TNF-α, which impact on their polyfunctional capacities. In contrast, NK cells from patients diagnosed with genitourinary tumors possessed a standard immature signature, including high expression of NKG2A and a robust production of IFN-γ. Taken together, our results suggest that under an immunosuppressive environment, the interplay between the modulation of NK repertoire and CMV status may greatly hamper the spectrum of immune surveillance and thus favor outgrowth and the development of specific de novo tumors after OLT.

A polysaccharide virulence factor of a human fungal pathogen induces neutrophil apoptosis via NK cells.

Aspergillus fumigatus is an opportunistic human fungal pathogen that sheds galactosaminogalactan (GG) into the environment. Polymorphonuclear neutrophils (PMNs) and NK cells are both part of the first line of defense against pathogens. We recently reported that GG induces PMN apoptosis. In this study, we show that PMN apoptosis occurs via a new NK cell-dependent mechanism. Reactive oxygen species, induced by the presence of GG, play an indispensable role in this apoptotic effect by increasing MHC class I chain-related molecule A expression at the PMN surface. This increased expression enables interaction between MHC class I chain-related molecule A and NKG2D, leading to NK cell activation, which in turn generates a Fas-dependent apoptosis-promoting signal in PMNs. Taken together, our results demonstrate that the crosstalk between PMNs and NK cells is essential to GG-induced PMN apoptosis. NK cells might thus play a role in the induction of PMN apoptosis in situations such as unexplained neutropenia or autoimmune diseases.

TNAP, an Essential Player in Membrane Lipid Rafts of Neuronal Cells.

The tissue non-specific alkaline phosphatase (TNAP) is a glycosyl-phosphatidylinositol (GPI) anchored glycoprotein which exists under different forms and is expressed in different tissues. As the other members of the ecto-phosphatase family, TNAP is targeted to membrane lipid rafts. Such micro domains enriched in particular lipids, are involved in cell sorting, are in close contact with the cellular cytoskeleton and play the role of signaling platform. In addition to its location in functional domains, the extracellular orientation of TNAP and the fact this glycoprotein can be shed from plasma membranes, contribute to its different phosphatase activities by acting as a phosphomonoesterase on various soluble substrates (inorganic pyrophosphate -PPi-, pyridoxal phosphate -PLP-, phosphoethanolamine -PEA-), as an ectonucleotidase on nucleotide-phosphate and presumably as a phosphatase able to dephosphorylate phosphoproteins and phospholipids associated to cells or to extra cellular matrix. More and more data accumulate on an involvement of the brain TNAP both in physiological and pathological situations. This review will summarize what is known and expected from the TNAP localization in lipid rafts with a particular emphasis on the role of a neuronal microenvironment on its potential function in the central nervous system.

Identification of a cellular ligand for the natural cytotoxicity receptor NKp44.

With an array of activating and inhibitory receptors, natural killer (NK) cells are involved in the eradication of infected, transformed, and tumor cells. NKp44 is a member of the natural cytotoxicity receptor family, which is exclusively expressed on activated NK cells. Here, we identify natural cytotoxicity receptor NKp44 (NKp44L), a novel isoform of the mixed-lineage leukemia-5 protein, as a cellular ligand for NKp44. Unlike the other MLL family members, NKp44L is excluded from the nucleus, but expressed at the cell-surface level; its subcellular localization is being associated with the presence of a specific C-terminal motif. Strikingly, NKp44L has not been detected on circulating cells isolated from healthy individuals, but it is expressed on a large panel of the tumor and transformed cells. The sharply decreased NK lysis activity induced by anti-NKp44L antibodies directly demonstrates the role of NKp44L in cytotoxicity. Taken together, these results show that NKp44L could be critical for NK cell-mediated innate immunity. The identification and cellular distribution of NKp44L highlight the role of this self-molecule as a danger signal to alert the NK cell network.

Constitutive expression of ligand for natural killer cell NKp44 receptor (NKp44L) by normal human articular chondrocytes.

Normal chondrocytes display susceptibility to lysis by natural killer (NK) cells and this phenomenon may play a role in some inflammatory cartilage disorders. The mechanisms of chondrocyte recognition and killing by NK cells remain unclear. Using flow cytometry and immunohistochemical staining we found that normal human articular chondrocytes constitutively express a ligand for NKp44, one of stimulatory NK cell receptors involved in recognition and killing of target cells. Expression of NKp44 ligand by normal articular chondrocytes is not involved in their killing by unstimulated NK cells; however, it is responsible for anti-chondrocyte cytotoxicity mediated by long-term activated NK cells. Thus, expression of NKp44 ligand may play a role in chondrocyte destruction in course of chronic inflammatory cartilage disorders.

Stat-mediated signaling induced by type I and type II interferons (IFNs) is differentially controlled through lipid microdomain association and clathrin-dependent endocytosis of IFN receptors.

Type I (alpha/beta) and type II (gamma) interferons (IFNs) bind to distinct receptors, although they activate the same signal transducer and activator of transcription, Stat1, raising the question of how signal specificity is maintained. Here, we have characterized the sorting of IFN receptors (IFN-Rs) at the plasma membrane and the role it plays in IFN-dependent signaling and biological activities. We show that both IFN-alpha and IFN-gamma receptors are internalized by a classical clathrin- and dynamin-dependent endocytic pathway. Although inhibition of clathrin-dependent endocytosis blocked the uptake of IFN-alpha and IFN-gamma receptors, this inhibition only affected IFN-alpha-induced Stat1 and Stat2 signaling. Furthermore, the antiviral and antiproliferative activities induced by IFN-alpha but not IFN-gamma were also affected. Finally, we show that, unlike IFN-alpha receptors, activated IFN-gamma receptors rapidly become enriched in plasma membrane lipid microdomains. We conclude that IFN-R compartmentalization at the plasma membrane, through clathrin-dependent endocytosis and lipid-based microdomains, plays a critical role in the signaling and biological responses induced by IFNs and contributes to establishing specificity within the Jak/Stat signaling pathway.

Involvement of the Gab2 scaffolding adapter in type I interferon signalling.

Interferons (IFNs) are pleiotropic cytokines involved in the regulation of physiological and pathological processes. Upon interaction with their specific receptors, IFNs activate the Jak/STAT signalling pathway. Numerous studies suggest, however, that the classical Jak/STAT pathway cannot alone account for the wide range of IFN's biological effects. To better understand the role of alternative signalling pathways in the type I IFNs response, we analyzed novel tyrosine-phosphorylated proteins following IFN-alpha2 stimulation. We showed for the first time that the Grb2-associated binder 2 (Gab2) protein is differentially phosphorylated upon the IFN subtype employed and the cells stimulated. We demonstrated that IFNAR1 physically interacts with Gab2. Moreover, the cellular content of Gab2 varies as a function of IFN receptor chain expression levels, and in particular of the ratio of IFNAR1 to IFNAR2, suggesting that Gab2 and IFNAR2 compete for interaction with IFNAR1. Analysis of Gab2 deletion mutants indicates that IFNAR1 might interact with a Gab2 region containing p85-PI3'kinase binding sites. Our results shed new light on recent data involving both Gab2 and type I IFNs in osteoclastogenesis and oncogenesis.

What Do We Know about How Hantaviruses Interact with Their Different Hosts?

Hantaviruses, like other members of the Bunyaviridae family, are emerging viruses that are able to cause hemorrhagic fevers. Occasional transmission to humans is due to inhalation of contaminated aerosolized excreta from infected rodents. Hantaviruses are asymptomatic in their rodent or insectivore natural hosts with which they have co-evolved for millions of years. In contrast, hantaviruses cause different pathologies in humans with varying mortality rates, depending on the hantavirus species and its geographic origin. Cases of hemorrhagic fever with renal syndrome (HFRS) have been reported in Europe and Asia, while hantavirus cardiopulmonary syndromes (HCPS) are observed in the Americas. In some cases, diseases caused by Old World hantaviruses exhibit HCPS-like symptoms. Although the etiologic agents of HFRS were identified in the early 1980s, the way hantaviruses interact with their different hosts still remains elusive. What are the entry receptors? How do hantaviruses propagate in the organism and how do they cope with the immune system? This review summarizes recent data documenting interactions established by pathogenic and nonpathogenic hantaviruses with their natural or human hosts that could highlight their different outcomes.

Natural killer cell deficiency in patients with non-Hodgkin lymphoma after lung transplantation.

BACKGROUND: Post-transplant non-Hodgkin lymphoma (NHL) is a well-recognized complication of solid-organ transplantation, and pharmacologic suppression of adaptive immunity plays a major role in its development. However, the role of natural killer (NK) cells in post-lung transplant de novo NHL is unknown. METHODS: Extensive phenotypic analyses of NK cells from patients diagnosed with NHL after liver or lung transplantation were conducted with multicolor flow cytometry. Polyfunctionality assays simultaneously assessed NK cell degranulation (CD107a) and intracellular cytokine production (interferon-γ and tumor necrosis factor-α) in the presence of NHL target cells. RESULTS: The development of de novo NHL is linked to NK-cell maturation defects, including overexpression of NKG2A and CD62L and down-modulation of inhibitory killer immunoglobulin-like receptors and CD57 receptors. More importantly, in patients who developed NHL after lung transplantation, we observed a specific down-modulation of the activating receptors (NKp30, NKp46, and NKG2D) and a sharp decrease in perforin expression and degranulation against NHL target cells. CONCLUSIONS: Our results suggest that accumulation of abnormal NK cells could play a role in the outgrowth of NHL after lung transplantation, independently of the immunosuppressive regimen.

NKp44L: A new tool for fighting cancer.

We have recently identified that the ligand of natural cytotoxicity triggering receptor 2 (NCR2, best known as NKp44) is expressed on a large panel of malignant cells. This ligand provides a new tool to investigate how stressed cells are recognized and eliminated by natural killer (NK) cells, and to develop novel immunotherapeutic paradigms against cancer.

Transplantation-induced cancers: Emerging evidence that clonal CMV-specific NK cells are causal immunogenic factors.

Solid cancers are a major adverse outcome of liver transplantation. Recent reassessments have revealed insights into causal factors, primarily centering on modulations of the natural killer (NK) cell compartment in liver transplant recipients. In the presence of cytomegalovirus, the clonal expansion of differentiated NK cells could restrict the diversity of the NK repertoire favoring the development of certain tumors.

The modulation of the cell-cycle: a sentinel to alert the NK cells of dangers.

Natural killer (NK) cells are an essential component of innate immunity that provides a rapid response to detect stressed, infected, or transformed target cells. This system is controlled by a balance of inhibitory and activating signals transmitted by a myriad of receptors and their specific ligands. Inhibitory receptors mainly recognize self-MHC class-I molecules, whereas activating receptors, such as natural cytotoxic receptors, NKG2D, and DNAM-1, interact with self-proteins, normally not expressed on the cell surface of healthy cells, but up-regulated by cellular stress or infections and are frequently expressed on tumor cells. In these circumstances, regulatory controls ensure that specific ligands are induced mainly in diseased cells and not in normal cells. Each ligand seems to exhibit some distinct specializations providing broad "coverage" for numerous stresses associated with various diseases. Deregulated cell proliferation is a hallmark of these abnormal situations, and may serve as a sentinel for the elimination of the targets by NK cells. The purpose of this review is to discuss recent implications of cell-cycle to create a warning control system that relays various danger signals via specific ligands to the NK receptor system.

NKp44L expression on CD4+ T cells is associated with impaired immunological recovery in HIV-infected patients under highly active antiretroviral therapy.

OBJECTIVE: HIV-infected immunological nonresponders (InRs) patients fail to show satisfactory CD4+ T-cell recovery despite virologically effective HAART. We propose that NKp44L, the cellular ligand of an activating natural killer (NK) receptor, expressed only on uninfected bystander CD4+ T cells from HIV-1 infected patients, could play a major role in this phenomenon by sensitizing these cells to NK killing. DESIGN: Phenotype and multifunctional status of CD4+ T cells, especially the subsets expressing and not expressing NKp44L, were characterized for HIV-infected patients receiving HAART for at least 2 years, during which their viral load remained less than 40 copies/ml; 53 were InRs (CD4 cell count always <350 cells/µl), and 82 immunological responders (CD4 cell count always ≥350 cells/µl). Flow cytometry determined NKp44L expression in association with specific markers of proliferation, maturation, activation, homeostasis, and intracellular cytokine production. Degranulation of NKp44+ determined the functional capacity of NK cells. RESULTS: InRs exhibited high levels of NKp44L+CD4+ T cells. Compared with NKp44L negative cells, the frequency of naive CD45RA+CCR7+ T cells expressing NKp44L fell (P < 0.001) and their proliferative capacity grew. Moreover, apoptosis and a unique ability to produce multiple cytokines (IL-2, IFN-γ, and TNF-α) without or after phytohemagglutinin or anti-CD3/CD28 stimulation distinguished NKp44L+ T cells. CONCLUSION: InR status is associated to a significant expansion of highly differentiated, multifunctional and apoptotic CD4+ T cells expressing NKp44L. This could explain a rapid CD4+ T-cell turnover in InR preventing immune recovery. These data suggest a new target for developing therapeutic strategies to prevent NKp44L expression and then stimulating immune recovery in InRs.

The cellular prion protein interacts with the tissue non-specific alkaline phosphatase in membrane microdomains of bioaminergic neuronal cells.

BACKGROUND: The cellular prion protein, PrP(C), is GPI anchored and abundant in lipid rafts. The absolute requirement of PrP(C) in neurodegeneration associated to prion diseases is well established. However, the function of this ubiquitous protein is still puzzling. Our previous work using the 1C11 neuronal model, provided evidence that PrP(C) acts as a cell surface receptor. Besides a ubiquitous signaling function of PrP(C), we have described a neuronal specificity pointing to a role of PrP(C) in neuronal homeostasis. 1C11 cells, upon appropriate induction, engage into neuronal differentiation programs, giving rise either to serotonergic (1C11(5-HT)) or noradrenergic (1C11(NE)) derivatives. METHODOLOGY/PRINCIPAL FINDINGS: The neuronal specificity of PrP(C) signaling prompted us to search for PrP(C) partners in 1C11-derived bioaminergic neuronal cells. We show here by immunoprecipitation an association of PrP(C) with an 80 kDa protein identified by mass spectrometry as the tissue non-specific alkaline phosphatase (TNAP). This interaction occurs in lipid rafts and is restricted to 1C11-derived neuronal progenies. Our data indicate that TNAP is implemented during the differentiation programs of 1C11(5-HT) and 1C11(NE) cells and is active at their cell surface. Noteworthy, TNAP may contribute to the regulation of serotonin or catecholamine synthesis in 1C11(5-HT) and 1C11(NE) bioaminergic cells by controlling pyridoxal phosphate levels. Finally, TNAP activity is shown to modulate the phosphorylation status of laminin and thereby its interaction with PrP. CONCLUSION/SIGNIFICANCE: The identification of a novel PrP(C) partner in lipid rafts of neuronal cells favors the idea of a role of PrP in multiple functions. Because PrP(C) and laminin functionally interact to support neuronal differentiation and memory consolidation, our findings introduce TNAP as a functional protagonist in the PrP(C)-laminin interplay. The partnership between TNAP and PrP(C) in neuronal cells may provide new clues as to the neurospecificity of PrP(C) function.

Membrane-bound and soluble IL-15/IL-15Ralpha complexes display differential signaling and functions on human hematopoietic progenitors.

Membrane-bound and soluble interleukin-15 (IL-15)/IL-15 receptor alpha (Ralpha) complexes trigger differential transcription factor activation and functions on human hematopoietic progenitors. Indeed, human spleen myofibroblasts (SMFs) are characterized by a novel mechanism of IL-15 trans-presentation (SMFmb [membrane-bound]-IL-15), based on the association of an endogenous IL-15/IL-15Ralpha complex with the IL-15Rbetagamma c chains. SMFmb-IL-15 (1) induces lineage-specific signaling pathways that differ from those controlled by soluble IL-15 in unprimed and committed normal progenitors; (2) triggers survival and proliferation of leukemic progenitors expressing low-affinity IL-15R (M07Sb cells); (3) causes only an antiapoptotic effect on leukemic cells expressing high-affinity receptors (TF1beta cells). This behavior is likely due to the IL-15Ralpha chain present on these cells that interact with the SMFmb-IL-15, inhibiting signal transducer and transcriptional activator 5 (STAT5) activation. On the other hand, the soluble IL-15/IL-15Ralpha complex (hyper IL-15) displays a dominant pattern of action, activating only those cells expressing low-affinity IL-15R (IL-15Rbetagamma c). Thus, hyper IL-15 induces antiapoptotic effects on M075b cells and the up-regulation of STAT6 activation on adult peripheral blood (PB) pre-natural killer (NK) committed progenitors. The latter effect using 100-fold concentrations of recombinant (r)-IL-15. In conclusion, SMFmb-IL-15 and soluble IL-15Ralpha/IL-15 complexes seem to play a pivotal role in the control of the survival, proliferation and differentiation of both normal and leukemic circulating progenitors, highlighting new functions of IL-15 and of IL-15Ralpha.

Identification of residues of the IFNAR1 chain of the type I human interferon receptor critical for ligand binding and biological activity.

The antiviral and antiproliferative activities of human type I interferons (IFNs) are mediated by two transmembrane receptor subunits, IFNAR1 and IFNAR2. To elucidate the role of IFNAR1 in IFN binding and the establishment of biological activity, specific residues of IFNAR1 were mutated. Residues (62)FSSLKLNVY(70) of the S5-S6 loop of the N-terminal subdomain of IFNAR1 and tryptophan-129 of the second subdomain of IFNAR1 were shown to be crucial for IFN-alpha binding and signaling and establishment of biological activity. Mutagenesis of peptide (278)LRV in the third subdomain shows that these residues are critical for IFN-alpha-induced biological activity but not for ligand binding. These data, together with the sequence homology of IFNAR1 with cytokine receptors of known structure and the recently resolved NMR structure of IFNAR2, led to the establishment of a three-dimensional model of the human IFN-alpha/IFNAR1/IFNAR2 complex. This model predicts that following binding of IFN to IFNAR1 and IFNAR2 the receptor complex assumes a "closed form", in which the N-terminal domain of IFNAR1 acts as a lid, resulting in the activation of intracellular kinases. Differences in the primary sequence of individual IFN-alpha subtypes and resulting differences in binding affinity, duration of ligand/receptor association, or both would explain differences in intracellular signal intensities and biological activity observed for individual IFN-alpha subtypes.