Identification of an interleukin 17F/17A heterodimer in activated human CD4+ T cells

IL-17F and IL-17A are members of the IL-17 pro-inflammatory cytokine family. IL-17A has been implicated in the pathogenesis of autoimmune diseases. IL-17F is a disulfide-linked dimer that contains a cysteine-knot motif. We hypothesized that IL-17F and IL-17A could form a heterodimer due to their sequence homology and overlapping pattern of expression. We evaluated the structure of recombinant IL-17F and IL-17A proteins, as well as that of natural IL-17F and IL-17A derived from activated human CD4+ T cells, by enzyme-linked immunosorbent assay, immunoprecipitation followed by Western blotting, and mass spectrometry. We find that both IL-17F and IL-17A can form both homodimeric and heterodimeric proteins when expressed in a recombinant system, and that all forms of the recombinant proteins have in vitro functional activity. Furthermore, we find that in addition to the homodimers of IL-17F and IL-17A, activated human CD4+ T cells also produce the IL-17F/IL-17A heterodimer. These data suggest that the IL-17F/IL-17A heterodimer may contribute to the T cell-mediated immune responses.     

Functional polymorphism of the KIR3DL1/S1 receptor on human NK cells

NK cells express both inhibitory and activatory receptors that allow them to recognize target cells through HLA class I Ag expression. KIR3DL1 is a receptor that recognizes the HLA-Bw4 public epitope of HLA-B alleles. We demonstrate that polymorphism within the KIR3DL1 receptor has functional consequences in terms of NK cell recognition of target. Inhibitory alleles of KIR3DL1 differ in their ability to recognize HLA-Bw4 ligand, and a consistent hierarchy of ligand reactivity can be defined. KIR3DS1, which segregates as an allele of KIR3DL1, has a short cytoplasmic tail characteristic of activatory receptors. Because it is very similar to KIR3DL1 in the extracellular domains, it has been assumed that KIR3DS1 will recognize a HLA-Bw4 ligand. In this study, we demonstrate that KIR3DS1 is expressed as a protein at the cell surface of NK cells, where it is recognized by the Z27 Ab. Using this Ab, we found that KIR3DS1 is expressed on a higher percentage of NK cells in KIR3DS1 homozygous compared with heterozygous donors. In contrast to the inhibitory KIR3DL1 allotypes, KIR3DS1 did not recognize HLA-Bw4 on EBV-transformed cell lines.     

Identification of novel NK1/NK3 dual antagonists for the potential treatment of schizophrenia

During the lead optimization of NK(1)/NK(3) receptor antagonists program, a focused exploration of molecules bearing a lactam moiety was performed. The aim of the investigation was to identify the optimal position of the carbonyl and hydroxy methyl group in the lactam moiety, in order to maximize the in vitro affinity and the level of insurmountable antagonism at both NK(1) and NK(3) receptors. The synthesis and biological evaluation of these novel lactam derivatives, with potent and balanced NK(1)/NK(3) activity, were reported in this paper.     

Targeting of human dendritic cells by autologous NK cells

NK cells have the capacity to spontaneously kill tumor cell lines, in particular cell lines of hemopoietic origin. In contrast, they do not generally kill nontransformed autologous cells. However, here we demonstrate that short-term activated polyclonal human NK cells, as well as human NK cell lines, efficiently lyse autologous dendritic cells (DC) derived from peripheral blood monocytes as well as Langerhans-like cells derived from CD34+ stem cells isolated from umbilical cord blood. Lysis of autologous DC by short-term activated NK cells and NK cell lines was dependent on granule exocytosis, since total abrogation of lysis was observed in the presence of EGTA. Induction of DC maturation by LPS, monocyte conditioned media (MCM), or stimulation through CD40 ligand (CD40L) rendered the DC less susceptible to lysis by NK cells. Infection of DC with influenza virus was likewise associated with a reduced susceptibility to lysis by NK cells. Thus, susceptibility to lysis by autologous NK cells is a particular property of immature DC. The present results are discussed in relation to the ability of DC to interact with NK cells and to the ability of NK cells to regulate development of specific immunity.     

Differential recognition of MHC class I molecules of xeno-/allo-endothelial cells by human NK cells

Using human umbilical vein endothelial cells (HUVEC) and porcine aortic endothelial cells (PAEC) as target cells, human peripheral blood NK cells (PBNK) and NK92 cells as effector cells, the differential cytotoxicities of NK cells to allo- and xeno-endothelial cells were studied. The influence of MHC class I molecules on the cytotoxicity of human NK cells was assayed using acid treatment, and blockades of MHC class I antigens, CD94 and KIR (NKB1). The results indicated that the killing of PAEC by the two kinds of NK cells is higher than that of HUVEC. After acid-treatment, the cytotoxicity of the two kinds of NK cells to PAEC and HUVEC is significantly enhanced, but the magnitude of the enhancement is different. The enhancement of NK killing to acid treated HUVEC is much greater than that to PAEC. Blockade of CD94 mAb did not alter the NK cytotoxicity, while blockade of NKB1 mAb enhanced the cytotoxicity of PBNK to HUVEC and PAEC by 95% and 29% respectively. The results above suggested that the different     

Differential recognition of MHC class I molecules of xeno-/allo-endothelial cells by human NK cells

Using human umbilical vein endothelial cells (HUVEC) and porcine aortic endothelial cells (PAEC) as target cells, human peripheral blood NK cells (PBNK) and NK92 cells as effector cells, the differential cytotoxicities of NK cells to allo- and xeno-endothelial cells were studied. The influence of MHC class I molecules on the cytotoxicity of human NK cells was assayed using acid treatment, and blockades of MHC class I antigens, CD94 and KIR (NKB1). The results indicated that the killing of PAEC by the two kinds of NK cells is higher than that of HUVEC. After acid- treatment, the cytotoxicity of the two kinds of NK cells to PAEC and HUVEC is significantly enhanced, but the magnitude of the enhancement is different. The enhancement of NK killing to acid treated HUVEC is much greater than that to PAEC. Blockade of CD94 mAb did not alter the NK cytotoxicity, while blockade of NKB1 mAb enhanced the cytotoxicity of PBNK to HUVEC and PAEC by 95% and 29% respectively. The results above suggested that the differential recognition of MHC I molecules of xeno-endothelial cells by human NK cells could be the major reason for higher NK cytotoxicity to PAEC. KIR might be the primary molecule that transduced inhibitory signals when endothelial cells were injured by NK cells.     

Variable NK cell receptors exemplified by human KIR3DL1/S1

Variegated expression of variable NK cell receptors for polymorphic MHC class I broadens the range of an individual's NK cell response and the capacity for populations and species to survive disease epidemics and population bottlenecks. On evolutionary time scales, this component of immunity is exceptionally dynamic, unstable, and short-lived, being dependent on coevolution of ligands and receptors subject to varying, competing selection pressures. Consequently these systems of variable NK cell receptors are largely species specific and have recruited different classes of glycoprotein, even within the primate order of mammals. Such disparity helps to explain substantial differences in NK cell biology between humans and animal models, for which the population genetics is largely ignored. KIR3DL1/S1, which recognizes the Bw4 epitope of HLA-A and -B and is the most extensively studied of the variable NK cell receptors, exemplifies how variation in all possible parameters of function is recruited to diversify the human NK cell response.     

Adherence of human monocytes and NK cells to human TNF-alpha-stimulated porcine endothelial cells

Discordant xenograft models undergoing delayed rejection response are characterized by xenograft infiltration with host monocytes and NK cells, associated with the release of large quantities of pro-inflammatory cytokines, such as TNF-alpha. In the present study, human monocytes (PBMo)/NK cells (PBNK) isolated from peripheral blood and cultured porcine aortic endothelial cells (PAEC) treated with recombinant human TNF-alpha (rhTNF-alpha) were used to investigate their adhesive interactions and mAbs against porcine E-selectin, human CD11a and CD49d were used to test their relative contributions to such intercellular adhesions. The PBMo exhibited significantly greater adherence to resting (unstimulated) PAEC than PBNK. The rhTNF-alpha upregulated E-selectin and vascular cell adhesion molecule-1 (VCAM-1) expression on PAEC and augmented the adhesiveness of PAEC for PBMo and PBNK in a time- and dose-dependent manner. In mAb blocking assays, anti-E-selectin, anti-CD11a and anti-CD49d mAbs did not inhibit PBMo adherence to rhTNF-alpha-stimulated PAEC when used singly, but resulted in a maximal inhibitory effect when used in combination. Regarding PBNK, anti-E-selectin mAb had no marked influence on PBNK adherence. The combined use of anti-CD11a and anti-CD49d mAbs produced additive reduction in the PBNK binding to rhTNF-alpha-stimulated PAEC, even to far below baseline (unstimulated) levels. Therefore, it is concluded that human TNF-alpha promotes the adhesiveness of PAEC for human monocytes and NK cells and that the mechanism underlying the increased adherence differs for PBMo and PBNK.     

Expression of human CD1d molecules protects target cells from NK cell-mediated cytolysis

The cytotoxic activity of NK cells can be inhibited by classical and nonclassical MHC molecules. The CD1 system is formed by a family of glycoproteins that are related to classical MHC. CD1a, b, and c molecules present lipids or glycolipids to T cells and are involved in defense against microbial infections, especially mycobacteria. It has been shown recently that these molecules can inhibit target cell lysis by human NK cells. It has also been shown that mouse CD1d molecules can protect cells from NK cell-mediated cytotoxicity. In the present study, we describe how human CD1d, orthologous to murine CD1 molecules, can inhibit NK cell-mediated cytolysis. We have expressed CD1d in the HLA class I-deficient cell lines L721.221 and C1R. The inhibitory effect is observed when effector NK cells from different donors are used, as well as in different cell lines with NK activity. The inhibitory effect was reversed by incubating the target cells with a mAb specific for human CD1d. Incubation of target cells with the ligands for CD1d, alpha-galactosylceramide (alpha-GalCer), and beta-GalCer abolishes the protective effect of CD1d in our in vitro killing assays. Staining the effector cells using CD1d tetramers loaded with alpha-GalCer was negative, suggesting that the putative inhibitory receptor does not recognize CD1d molecules loaded with alpha-GalCer.     

IL-18 promotes type 1 cytokine production from NK cells and T cells in human intracellular infection

We investigated the role of IL-18 in leprosy, a disease characterized by polar cytokine responses that correlate with clinical disease. In vivo, IL-18 mRNA expression was higher in lesions from resistant tuberculoid as compared with susceptible lepromatous patients, and, in vitro, monocytes produced IL-18 in response to Mycobacterium leprae. rIL-18 augmented M. leprae-induced IFN-gamma in tuberculoid patients, but not lepromatous patients, while IL-4 production was not induced by IL-18. Anti-IL-12 partially inhibited M. leprae-induced release of IFN-gamma in the presence of IL-18, suggesting a combined effect of IL-12 and IL-18 in promoting M. leprae-specific type 1 responses. IL-18 enhanced M. leprae-induced IFN-gamma production rapidly (24 h) by NK cells and in a more sustained manner (5 days) by T cells. Finally, IL-18 directly induced IFN-gamma production from mycobacteria-reactive T cell clones. These results suggest that IL-18 induces type 1 cytokine responses in the host defense against intracellular infection.     

Tumor-associated neutrophils suppress antitumor immunity of NK cells through the PD-L1/PD-1 axis

Studies have begun to emerge showing the protumor effects of tumor-associated neutrophils (TANs) in tumorigenesis, which may involve dysfunction of NK cells. However, the mechanism through which these rebellious neutrophils modulate NK cell immunity in tumor-bearing state remains unclear. In the present study, we demonstrate that neutrophils can impair the cytotoxicity and infiltration capability of NK cells, and downregulate CCR1 resulting in the weakened infiltration capability of NK cells. Moreover, neutrophils can decrease the responsiveness of NK-activating receptors, NKp46 and NKG2D. Mechanistically, enhanced PD-L1 on neutrophils and PD-1 on NK cells, and subsequent PD-L1/PD-1 interactions were the main mechanisms determining the suppression of neutrophils in NK cell immunity. G-CSF/STAT3 pathway was responsible for PD-L1 upregulation on neutrophils, while IL-18 was essential for PD-1 enhancement on NK cells. The crosstalk between neutrophils and NK cells was cell-cell interaction-dependent. These findings suggest that neutrophils can suppress the antitumor immunity of NK cells in tumor-bearing status through the PD-L1/PD-1 axis, highlighting the importance of PD-L1/PD-1 in the inhibitory effect of neutrophils on NK cells. Targeting G-CSF/STAT3 and IL-18 signaling pathway may be potential strategies to inhibit residual tumor in tumor therapy.     

Quantitative analysis of LacCer/CDw17 in human myelogenous leukaemic cells

LacCer/CDw17 is the most abundant GSL in neutrophils. The cell-surface and intracellular presence of LacCer was determined quantitatively using anti-CDw17 mAbs in a flow cytometry assay. The quantified alterations in the level of CDw17 antigen expression are consistent with alterations in LacCer content, determined chemically. Our results show that CDw17 antigen expression defines successive stages in the maturation of the myeloid cell. The assessment of cell-surface and intracellular CDw17 expression may be useful in evaluating neutrophil physiological status.     

A trivalent anti-erbB2/anti-CD16 bispecific antibody retargeting NK cells against human breast cancer cells

Bispecific antibody (BsAb) can physically cross-link immune cells to tumor cells, circumventing the proper structures for tumor cell-immune cell interactions and activating the cellular cytotoxic mechanisms. The optimal BsAb should target tumor cells with high affinity, but activate trigger molecules on cytotoxic cells by monovalent binding of Fab fragments. In the present study, a trivalent anti-erbB2/anti-CD16 BsAb was produced. This BsAb possesses bivalent arms specifically binding to the extracellular domain of erbB2 and monovalent Fab fragment redirecting NK cells. The recombinant protein could be expressed and purified from Escherichia coli as native proteins without refolding. It was fully functional in bispecific binding to SKBR3 and NK cells. The molecular size of this trivalent BsAb protein is larger than diabody and smaller than whole antibody and expected to have advantages for both high penetration of small antibody fragments and the slow circulation clearance of whole antibody. This novel protein may be an attractive target for further improvement and evaluation.     

Identification and purification of NK cells with lysosomotropic vital stains: correlation of lysosome content with NK activity

The lysosome content of lymphocytes has been analyzed with lysosomotropic vital stains and the fluorescence-activated cell sorter (FACS). Large granular lymphocytes (LGL), which account for virtually all natural killing activity in peripheral blood, are quantitatively different from small lymphocytes (SL) in this respect. LGL obtained by Percoll gradient density centrifugation accumulate more of the lysosomotropic vital dyes than SL do, staining with either neutral red or mepacrine (quinacrine). Furthermore among the LGL-rich, low density lymphocyte population highly, granulated cells can be separated from less granulated ones by mepacrine staining and FACS. Thus, separated highly granulated LGL express very high natural killing, whereas the less granulated low density large lymphocytes do not kill.     

Functional characterization of LFA-1 antigens in the interaction of human NK clones and target cells

In the present studies we analyzed the role of LFA-1 antigens in the interaction between NK clones and target cells. The use of various cloned NK cell lines allowed us to analyze homogeneous populations of NK cells which ordinarily comprise only a small fraction of peripheral blood lymphocytes and are extremely heterogeneous with respect to phenotype and specificity. Indirect immunofluorescence with monoclonal antibodies against the alpha (MHM24) and beta (MHM23) chains of the LFA-1 antigen revealed similar patterns of positive reactivity with all NK clones. Both monoclonal antibodies exerted a significant blocking effect on NK cytotoxicity against target cells such as Molt-4 and CEM, whereas the inhibition was very weak against other targets such as K562 and HSB cells. Additive blocking effects were seen when both monoclonal antibodies MHM23 and MHM24 were added to the cytotoxicity assays. When we compared the inhibitory effect of MHM23 and MHM24 on uncultured peripheral blood NK cells and IL 2-activated NK cells, inhibition of cytotoxicity also was found to be primarily dependent on the individual target cells. Thus, the inhibitory activity of anti-LFA-1 antibody was shown to be independent of the phenotypic and functional heterogeneity of the NK clones, activated NK cells, and unstimulated NK cells utilized in these studies. These blocking effects were found to be independent of the LFA-1 antigen expression on the target cell membrane and inhibition occurred only when antibody was bound to the effector cells. Comparison of the effects of anti-LFA-1, anti-T3, and anti-clonotypic antibodies against a Ti-like structure of different NK clones with a mature T cell phenotype demonstrated that each of these antibodies acts on the effector cells in an independent and additive fashion. However, unlike T3 and NKTa antigen, LFA-1 antigen expression is not modulated by cell surface interaction with antibodies specific for this molecule.     

NK cells stimulate proliferation of T and NK cells through 2B4/CD48 interactions

Few studies have addressed the consequences of physical interactions between NK and T cells, as well as physical interactions among NK cells themselves. We show in this study that NK cells can enhance T cell activation and proliferation in response to CD3 cross-linking and specific Ag through interactions between 2B4 (CD244) on NK cells and CD48 on T cells. Furthermore, 2B4/CD48 interactions between NK cells also enhanced proliferation of NK cells in response to IL-2. Overall, these results suggest that NK cells augment the proliferation of neighboring T and NK cells through direct cell-cell contact. These results provide new insights into NK cell-mediated control of innate and adaptive immunity and demonstrate that receptor/ligand-specific cross talk between lymphocytes may occur in settings other than T-B cell or T-T cell interactions.     

Preferential accumulation of mature NK cells during human immunosenescence.

The major histocompatibility complex-unrestricted, cell-mediated, constitutive anti-tumor cytotoxic function of natural killer cells is highly preserved in healthy elderly. A study of the dynamics of expression of natural killer cell-associated phenotypes during immunosenescence shows that selective, bidirectional, and disproportionate changes in certain natural killer cell subset number and ratio take place during aging. The mean natural killer cell subset ratio (%CD16+CD57+ over %CD56+CD57-) gradually increases from a young adult level of 0.7 to 4.6 with advancing age predominantly due to a tripling of %CD16+57+ cells as opposed to a moderate decrease (-54%) in %CD56+57- phenotype. The parallel increase in natural killer phenotype ratio and cytotoxic activity might represent a shift in the maturity status of these cells. Based on these findings, a model of natural killer cell immunosenescence is proposed. It is concluded that not all immunosenescent changes need be detrimental; some may even improve the potential for survival and represent an adaptational immunosenescent change.