Scientific Resources

It is increasingly evident that neutrophils are able to cross-talk with other leukocytes to shape ongoing inflammatory and immune responses. In this study,we analyzed whether human NK cells may influence the survival and activation of neutrophils under co-culture conditions. We report that NK cells exposed to either IL-15 or IL-18 alone strongly protect the survival of neutrophils via the release of IFNγ and granulocyte macrophage colony-stimulating factor (GM-CSF) plus IFNγ,respectively,and cause a slight up-regulation of neutrophil CD64 and CD11b expression. In comparison,NK cells exposed to both IL-15 and IL-18 show a lesser ability to increase the survival of neutrophils but can more potently up-regulate CD64 and CD11b expression,as well as induce the de novo surface expression of CD69,in neutrophils. Analysis of the events occurring in neutrophil/NK co-cultures exposed to IL-15 plus IL-18 revealed that (i) neutrophil survival is positively affected by NK-derived GM-CSF but negatively influenced by a CD18-dependent neutrophil/NK contact,(ii) NK-derived IFNγ is almost entirely responsible for the induction of CD64,(iii) both soluble factors (primarily GM-CSF) and direct cell-cell contact up-regulate CD11b and CD69 and (iv) NK-derived GM-CSF induces the expression of biologically active heparin-binding EGF-like growth factor (HB-EGF) in neutrophils. Finally,we demonstrate that NK cells can also express HB-EGF when stimulated with either IL-2 or IL-15,yet independently of endogenous GM-CSF. Altogether,our results define a novel interaction within the innate immune system whereby NK cells,by directly modulating neutrophil functions,might contribute to the pathogenesis of inflammatory diseases. View Publication

Killer cell lectin-like receptor F1 (KLRF1) is an activating C-type lectin-like receptor expressed on human NK cells and subsets of T cells. In this study,we show that activation-induced C-type lectin (AICL) is a unique KLRF1 ligand expressed on tumor cell lines of hematopoietic and non-hematopoietic origins. We screened a panel of human tumor cell lines using the KLRF1 reporter cells and found that several tumor lines expressed KLRF1 ligands. We characterized a putative KLRF1 ligand expressed on the U937 cell line. The molecular mass for the deglycosylated ligand was 28 kDa under non-reducing condition and 17 kDa under reducing condition,suggesting that the KLRF1 ligand is a homodimer. By expression cloning from a U937 cDNA library,we identified AICL as a KLRF1 ligand. We generated mAbs against AICL to identify the KLRF1 ligands on non-hematopoietic tumor lines. The anti-AICL mAbs stained the tumor lines that express the KLRF1 ligands and importantly the interaction of KLRF1 with the KLRF1 ligand on non-hematopoietic tumors was completely blocked by the two anti-AICL mAbs. Moreover,NK cell degranulation triggered by AICL-expressing targets was partially inhibited by the anti-AICL mAb. Finally,we demonstrate that AICL is expressed in human primary liver cancers. These results suggest that AICL is expressed on tumor cells of non-hematopoietic origins and raise the possibility that AICL may contribute to NK cell surveillance of tumor cells. View Publication

Although the importance of natural killer (NK) cells in innate immune responses against tumors or viral infections are well documented,their ability to directly recognize pathogens is less well defined. We have recently reported FimH,a bacterial fimbrial protein,as a novel Toll-like receptor (TLR)4 ligand that potently induces antiviral responses. Here,we investigated whether FimH either directly or indirectly can activate human and murine NK cells. We demonstrate that FimH potently activates both human and murine NK cells in vitro to induce cytokines [interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha] and cytotoxicity. Importantly,NK cells directly recognize FimH-expressing pathogens as FimH(+),but not FimH(-),bacteria were able to activate human NK cells. FimH activation of NK cells required TLR4 and MyD88 signaling,as NK cells from both TLR4(-/-) and MyD88(-/-) mice as well as human NK-92 cells,which lack TLR4,were all unresponsive to FimH. In addition,TLR4 neutralization significantly abrogated the response of human NK cells to FimH. Activation of purified NK cells by FimH was independent of lipopolysaccharide (LPS) or other bacterial contaminations. These data demonstrate for the first time that highly purified NK cells directly recognize and respond to FimH via TLR4-MyD88 pathways to aid innate protection against cancer or microbial infections. View Publication

The NKG2D receptor activates natural killer (NK) cell cytotoxicity and cytokine production on recognition of self-molecules induced by cellular stress under different conditions such as viral infections. The importance of NKG2D in the immune response to human cytomegalovirus (HCMV) is supported by the identification of several viral molecules that prevent the expression of NKG2D ligands by infected cells. In this study we report that,paradoxically,a significant,selective,and transient reduction of NKG2D expression on NK cells is detected during HCMV infection of peripheral blood mononuclear cells if needed. Antagonizing type I interferon (IFN),interleukin-12 (IL-12),and IFNgamma prevented HCMV-induced down-regulation of surface NKG2D. Moreover,treatment of purified NK cells with recombinant IFNbeta1 and IL-12 mimicked the effect,supporting a direct role of these cytokines in regulating NKG2D surface expression in NK cells. The loss of NKG2D expression selectively impaired NK-cell cytotoxicity against cells expressing NKG2D ligands but preserved the response triggered through other activating receptors. These results support that down-regulation of NKG2D expression on NK cells by cytokines with a key role in antiviral immune response may constitute a physiologic mechanism to control NK-cell reactivity against normal cells expressing NKG2D ligands in the context of inflammatory responses to viral infections. View Publication

The ability of NK cells to directly recognize pathogens and be activated via Toll-like receptors (TLR) is increasingly recognized. Nevertheless,controversial results on the NK cell ability to be directly activated by lipopolysaccharide (LPS),the ligand of TLR4,have been recently reported. To start elucidating the reasons explaining the contrasting observations of the literature,we focused on the potential role of currently used NK cell purification procedures to condition putative NK cell responsiveness to LPS. To do so,human NK cells were isolated by negative selection,using three different commercial kits,to be comparatively evaluated for the production of IFNgamma in response to ultra-pure LPS and/or IL-2. Despite the lack of surface TLR4,we found that two out of the three NK cell-enriched populations released IFNgamma (and one of the two,IL-12p70 as well) in response to the LPS plus IL-2 combination,whereas the last one did not. However,the two LPS plus IL-2-responsive NK cell populations were found variably contaminated with 6-sulfo LacNAc(+) dendritic cells (slanDC),demonstrated responsible for triggering,via the production of IL-12p70 in response to LPS,the release of IFNgamma by IL-2-stimulated NK cells. Accordingly,slanDC depletion completely abrogated the capacity to produce both IL-12p70 and IFNgamma in response to LPS plus IL-2 by slanDC-containing NK cells. Taken together,our data uncover that two commercially available kits,specifically designed to isolate NK cells by negative selection,also co-purify variable amounts of slanDC. The latter cells may dramatically affect the outcome of experiments carried on to evaluate NK cell responsiveness to TLR agonists such as LPS. View Publication

Threats of bioterrorism have renewed efforts to better understand poxvirus pathogenesis and to develop a safer vaccine against smallpox. Individuals with atopic dermatitis are excluded from smallpox vaccination because of their propensity to develop eczema vaccinatum,a disseminated vaccinia virus (VACV) infection. To study the underlying mechanism of the vulnerability of atopic dermatitis patients to VACV infection,we developed a mouse model of eczema vaccinatum. Virus infection of eczematous skin induced severe primary erosive skin lesions,but not in the skin of healthy mice. Eczematous mice exhibited lower natural killer (NK) cell activity but similar cytotoxic T lymphocyte activity and humoral immune responses. The role of NK cells in controlling VACV-induced skin lesions was demonstrated by experiments depleting or transferring NK cells. The proinflammatory cytokine interleukin (IL)-17 reduced NK cell activity in mice with preexisting dermatitis. Given low NK cell activities and increased IL-17 expression in atopic dermatitis patients,these results can explain the increased susceptibility of atopic dermatitis patients to eczema vaccinatum. View Publication

Immunotherapy with the EGFR-specific mAb cetuximab is clinically effective in 10-20% of patients with squamous cell carcinoma of the head and neck (SCCHN). Little information is available about the mechanism(s) underlying patients' differential clinical response to cetuximab-based immunotherapy,although this information may contribute to optimizing the design of cetuximab-based immunotherapy. Our understanding of these mechanisms would benefit from the characterization of the variables which influence the extent of cell dependent-lysis of SCCHN cells incubated with cetuximab in vitro. Therefore,in this study we have investigated the role of FcgammaR IIIa-158 genotype expressed by effector NK cells,cetuximab concentration,and EGFR expression level by SCCHN cells in the extent of their in vitro lysis and in the degree of NK cell activation. PBMC or purified CD56+ NK cells genotyped at IIIa codon 158 and SCCHN cell lines expressing different levels of EGFR have been used as effectors and targets,respectively,in antibody dependent cellular cytotoxicity (ADCC) assays. Furthermore,supernatants from ADCC assays were analyzed for cytokine and chemokine levels using multiplexed ELISA. We found that the extent of lysis of SCCHN cells was influenced by the EGFR expression level,cetuximab concentration,and FcgammaR polymorphism. Effector cells expressing the FcgammaR IIIa-158 VV allele were significantly (P textless 0.0001) more effective than those expressing FcgammaR IIIa VF and FF [corrected] alleles in mediating lysis of SCCHN cells expressed higher levels of the activation markers CD69 and CD107a,and secreted significantly (P textless 0.05) larger amounts of inflammatory cytokines and chemokines. IL-2 or IL-15 treatment increased cetuximab-mediated ADCC by poor binding FcgammaR IIIa 158 FF expressing NK cells. The importance of the FcgammaR IIIa-158 polymorphism in cytotoxicity of SCCHN cells by NK cells supports a potential role for immune activation and may explain patient variability of cetuximab mediated clinical responses. Cellular and secreted immune profiles and FcgammaR genotypes from patients' lymphocytes may provide clinically useful biomarkers of immune activation in cetuximab treated patients. View Publication

Glatiramer acetate (GA or Copaxone) is a drug used to treat experimental autoimmune encephalomyelitis in mice and multiple sclerosis in human. Here,we describe a new mechanism of action for this drug. GA enhanced the cytolysis of human NK cells against autologous and allogeneic immature and mature monocyte-derived dendritic cells (DCs). This drug reduced the percentages of mature DCs expressing CD80,CD83,HLA-DR or HLA-I. In contrast,it did not modulate the percentages of NK cells expressing NKG2D,NKp30,or NKp44. Nonetheless,anti-NKp30 or anti-CD86 inhibited GA-enhanced human NK cell lysis of immature DCs. Hence,CD86,and NKp30 are important for NK cell lysis of immature DCs,whereas CD80,CD83,HLA-DR and HLA-I are important for the lysis of mature DCs when GA is used as a stimulus. Further,GA inhibited the release of IFN-gamma 24 h but increased the release of TNF-alpha 48 h after incubation with NK cells. View Publication

We analyzed 21 children with leukemia receiving haploidentical hematopoietic stem cell transplantation (haplo-HSCT) from killer immunoglobulin (Ig)-like receptors (KIR) ligand-mismatched donors. We showed that,in most transplantation patients,variable proportions of donor-derived alloreactive natural killer (NK) cells displaying anti-leukemia activity were generated and maintained even late after transplantation. This was assessed through analysis of donor KIR genotype,as well as through phenotypic and functional analyses of NK cells,both at the polyclonal and clonal level. Donor-derived KIR2DL1(+) NK cells isolated from the recipient displayed the expected capability of selectively killing C1/C1 target cells,including patient leukemia blasts. Differently,KIR2DL2/3(+) NK cells displayed poor alloreactivity against leukemia cells carrying human leukocyte antigen (HLA) alleles belonging to C2 group. Unexpectedly,this was due to recognition of C2 by KIR2DL2/3,as revealed by receptor blocking experiments and by binding assays of soluble KIR to HLA-C transfectants. Remarkably,however,C2/C2 leukemia blasts were killed by KIR2DL2/3(+) (or by NKG2A(+)) NK cells that coexpressed KIR2DS1. This could be explained by the ability of KIR2DS1 to directly recognize C2 on leukemia cells. A role of the KIR2DS2 activating receptor in leukemia cell lysis could not be demonstrated. Altogether,these results may have important clinical implications for the selection of optimal donors for haplo-HSCT. View Publication

Many cellular responses,such as autoimmunity and cytotoxicity,are controlled by receptors with cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs). Here,we showed that binding of inhibitory natural killer (NK) cell receptors to human leukocyte antigen (HLA) class I on target cells induced tyrosine phosphorylation of the adaptor Crk,concomitant with dephosphorylation of the guanine exchange factor Vav1. Furthermore,Crk dissociated from the guanine exchange factor C3G and bound to the tyrosine kinase c-Abl during inhibition. Membrane targeting of a tyrosine-mutated form of Crk could overcome inhibition of NK cell cytotoxicity,providing functional evidence that Crk phosphorylation contributes to inhibition. The specific phosphorylation of Crk and its dissociation from a signaling complex,observed here with two types of inhibitory receptors,expands the signaling potential of the large ITIM-receptor family and reveals an unsuspected component of the inhibitory mechanism. View Publication

Understanding the cellular mechanisms that ensure an appropriate innate immune response against viral pathogens is an important challenge of biomedical research. In vitro studies have shown that natural killer (NK) cells purified from healthy donors can kill heterologous cell lines or autologous CD4+ T cell blasts exogenously infected with several strains of HIV-1. However,it is not known whether the deleterious effects of high HIV-1 viremia interferes with the NK cell-mediated cytolysis of autologous,endogenously HIV-1-infected CD4+ T cells. Here,we stimulate primary CD4+ T cells,purified ex vivo from HIV-1-infected viremic patients,with PHA and rIL2 (with or without rIL-7). This experimental procedure allows for the significant expansion and isolation of endogenously infected CD4+ T cell blasts detected by intracellular staining of p24 HIV-1 core antigen. We show that,subsequent to the selective down-modulation of MHC class-I (MHC-I) molecules,HIV-1-infected p24(pos) blasts become partially susceptible to lysis by rIL-2-activated NK cells,while uninfected p24(neg) blasts are spared from killing. This NK cell-mediated killing occurs mainly through the NKG2D activation pathway. However,the degree of NK cell cytolytic activity against autologous,endogenously HIV-1-infected CD4+ T cell blasts that down-modulate HLA-A and -B alleles and against heterologous MHC-I(neg) cell lines is particularly low. This phenomenon is associated with the defective surface expression and engagement of natural cytotoxicity receptors (NCRs) and with the high frequency of the anergic CD56(neg)/CD16(pos) subsets of highly dysfunctional NK cells from HIV-1-infected viremic patients. Collectively,our data demonstrate that the chronic viral replication of HIV-1 in infected individuals results in several phenotypic and functional aberrancies that interfere with the NK cell-mediated killing of autologous p24(pos) blasts derived from primary T cells. View Publication

We identify the tumor necrosis factor receptor superfamily 25 (TNFRSF25)/TNFSF15 pair as critical trigger for allergic lung inflammation,which is a cardinal feature of asthma. TNFRSF25 (TNFR25) signals are required to exert T helper cell 2 (Th2) effector function in Th2-polarized CD4 cells and co-stimulate interleukin (IL)-13 production by glycosphingolipid-activated NKT cells. In vivo,antibody blockade of TNFSF15 (TL1A),which is the ligand for TNFR25,inhibits lung inflammation and production of Th2 cytokines such as IL-13,even when administered days after airway antigen exposure. Similarly,blockade of TNFR25 by a dominant-negative (DN) transgene,DN TNFR25,confers resistance to lung inflammation in mice. Allergic lung inflammation-resistant,NKT-deficient mice become susceptible upon adoptive transfer of wild-type NKT cells,but not after transfer of DN TNFR25 transgenic NKT cells. The TNFR25/TL1A pair appears to provide an early signal for Th2 cytokine production in the lung,and therefore may be a drug target in attempts to attenuate lung inflammation in asthmatics. View Publication