Regulation Of NK Cell Function Research Articles

The Circadian ClockPeriod 2Gene Regulates Gamma Interferon Production of NK Cells in Host Response to Lipopolysaccharide-Induced Endotoxic Shock

The Period 2 (Per2) gene is a key molecular component in controlling mammalian circadian rhythms at the levels of gene expression, physiology, and pathogenesis. Although many immune parameters, such as the number of different subtypes of circulating immune cells and the level of cytokine production in response to infection with bacteria and viruses, have been well documented to display a circadian pattern in mammals, the basic features of molecular clock components in the immune system and the role of clock genes in regulating host immune defenses remain uncharacterized. Previously, we have reported that circadian clock genes oscillate in human mononuclear cells. Here we report that Per2-deficient mice were more resistant to lipopolysaccharide (LPS)-induced endotoxic shock than control wild-type mice. We further demonstrate that the levels of the proinflammatory cytokines gamma interferon (IFN-gamma) and interleukin-1beta (IL-1beta) in the serum were dramatically decreased in Per2-/- mice following LPS challenge, while production of tumor necrosis factor alpha, IL-6, and IL-10 was approximately normal, compared to that in control wild-type mice. Flow cytometric analyses confirmed that the cellularity of most of the immune cell subsets in the spleens of LPS-challenged mice was normal and that the impaired IFN-gamma production in Per2-/- mice was attributable to defective NK and NKT cell function. Our data suggest that Per2 is an important regulator of NK cell function, therefore providing the first direct link between the circadian clock system and innate immune responses.

The Inhibitory Receptor NKG2A Determines Lysis of Vaccinia Virus-Infected Autologous Targets by NK Cells

Signals transduced by inhibitory receptors that recognize self-MHC class I molecules prevent NK cells from being activated by autologous healthy target cells. In order for NK cells to be activated upon contact with an infected cell, the balance between the activating and inhibitory signals that regulate NK cell function must be altered in favor of activation. By studying liver-derived NK cells, we show that only a subpopulation of NK cells expressing high levels of the inhibitory receptor NKG2A are able to lyse autologous vaccinia-infected targets, and that this is due to selective down-regulation of HLA-E. These data demonstrate that release from an inhibitory receptor:ligand interaction is one mechanism that permits NK cell recognition of a virally infected target, and that the variegated expression of inhibitory receptors in humans generates a repertoire of NK cells with different antiviral potentials.

Les cellules natural killer : acquisitions récentes et implication en pathologie humaine

Introduction. – Natural killer cells are cytotoxic lymphocytes of innate immunity. These last ten years our knowledge about the mechanisms that regulates NK cell function has greatly improved. Our purpose is to present a review of these new acquisitions and their potential implications in human disease. Current knowledge and key points. – NK cell function is regulated by a repertoire of NK cell receptors and is diversified by recognition of MHC class I by a multigenic and mutiallelic family of NK receptors. Analysis of NK cell repertoire has been used to investigate features that characterize NK cells in pathological situations. Apart from their direct cytotoxic potential to eliminate target cells, recently identification of mechanisms that control NK cell mediated cytokine production and cross talk with dendritic cells emphasize the role of NK cells in the regulation of acquired immune response. Future prospects and projects. – These findings have lead to a better knowledge of the importance of the NK cells in several human diseases. It has been shown that NK cells are actors of the immunosurveillance of tumoral and infectious challenges. Allo or auto reactivity of the NK cell compartment have also been suggested in autoimmune diseases, infertility or foetal loss and transplantation. Ongoing research on NK cells in the fields of human diseases is increasing and will clarify the utility of the evaluation of the NK cell compartment and their receptors in clinical practice.

β-endorphin modulation of interferon-γ, perforin and granzyme B levels in splenic NK cells: Effects of ethanol

The effects of ethanol and beta-endorphin (β-EP) on productions of cytolytic factors granzyme B, perforin and IFN-γ in splenic rat NK cells were determined. Intracranial administration of β-EP increased protein and mRNA levels of cytolytic factors in NK cells. Chronic ethanol feeding reduced the basal and β-EP-induced levels of cytolytic factors in NK cells. In vitro treatment of β-EP on NK cells increased the levels of perforin, granzyme B and IFN-γ and their mRNA transcripts, whereas ethanol pre-treatment prevented β-EP effects on cytolytic factors in these cells. These results suggest that β-EP and ethanol interact to regulate NK cell functions.

Unique phenotype of human uterine NK cells and their regulation by endogenous TGF-beta.

Natural killer (NK) cells are a major population of lymphocytes in the human endometrium (EM), and NK cells can be a significant source of cytokines that alter local immune responses. The aim of this study was to determine the expression of NK cell receptors in situ and to test whether uterine NK (uNK) cells produce cytokines and how this activity may be regulated by transforming growth factor-beta (TGF-beta). We observed that human uNK cells were CD56+, CD3-, CD57-, CD9+, CD94+, killer inhibitory receptor+, and CD16+/- in situ by confocal microscopy. We examined cytokine production by uNK cells and uNK cell clones derived from human EM. Stimulation of uNK cells with interleukin (IL)-12 and IL-15, both of which are expressed in the human EM, induced interferon-gamma (IFN-gamma) and IL-10 production. IFN-gamma production by uNK cell clones was completely inhibited by TGF-beta1 in a dose-dependent manner with an inhibitory concentration 50% value of 20 pg/ml. IL-10 secretion by uNK cell clones was also inhibited by TGF-beta1 at similar concentrations. Furthermore, blocking endogenous TGF-beta in fresh human endometrial cell cultures increased the production of IFN-gamma by uNK cells. These data indicate that uNK cells have a unique phenotype that is distinct from blood NK cells. Further, data demonstrate that uNK cells can produce immunoregulatory cytokines and that inhibition of uNK cells by locally produced TGF-beta1 is a likely mechanism to regulate NK cell function in the human EM.

IL-4 confers NK stimulatory capacity to murine dendritic cells: a signaling pathway involving KARAP/DAP12-triggering receptor expressed on myeloid cell 2 molecules.

Dendritic cells (DC) regulate NK cell functions, but the signals required for the DC-mediated NK cell activation, i.e., DC-activated NK cell (DAK) activity, remain poorly understood. Upon acute inflammation mimicked by LPS or TNF-alpha, DC undergo a maturation process allowing T and NK cell activation in vitro. Chronic inflammation is controlled in part by Th2 cytokines. In this study, we show that IL-4 selectively confers to DC NK but not T cell stimulatory capacity. IL-4 is mandatory for mouse bone marrow-derived DC grown in GM-CSF (DC(GM/IL-4)) to promote NK cell activation in the draining lymph nodes. IL-4-mediated DAK activity depends on the KARAP/DAP12-triggering receptor expressed on myeloid cell 2 signaling pathway because: 1) gene targeting of the adaptor molecule KARAP/DAP12, a transmembrane polypeptide with an intracytoplasmic immunoreceptor tyrosine-based activation motif, suppresses the DC(GM/IL-4) capacity to activate NK cells, and 2) IL-4-mediated DAK activity is significantly blocked by soluble triggering receptor expressed on myeloid cell 2 Fc molecules. These data outline a novel role for Th2 cytokines in the regulation of innate immune responses through triggering receptors expressed on myeloid cells.

Recognition of CHO cells by inhibitory and activating Ly-49 receptors

Upon ligand recognition, members of the murine Ly-49 receptor family can transmit inhibitory or activating signals that regulate NK cell function. Ly-49A, G, and D have been shown to recognize the murine class I molecule H-2Dd as a potential ligand. Recent studies also have demonstrated also that Ly-49D+ NK cells can lyse CHO cells, although the ligand responsible for this recognition was not identified. Because allorecognition by NK cells may be important in bone-marrow transplantation and because of the overlapping class I recognition by these receptors, recognition of CHO cells by Ly-49G and A was investigated. Our data suggest that Ly-49G and probably A transmit inhibitory signals in response to CHO cells. Receptor inhibition was assessed by examining NK lytic function, IFN-gamma secretion, and DAP12 phosphorylation in response to CHO cells by sorted subsets of Ly-49D vs. G B6 NK cells. Our results suggest that CHO cells may express a common ligand(s) that is capable of engaging Ly-49D, G, and possibly A in C576BL/6 NK cells. In addition to our findings that Ly-49 inhibitory receptors also recognize CHO cells, activating receptors other than Ly-49D are present in B6 mice that can lyse CHO cells.

CS1, a novel member of the CD2 family, is homophilic and regulates NK cell function

CS1 is a novel member of the CD2 subset of immunoglobulin superfamily (IgSF) expressed on NK, T and stimulated B cells. The cytoplasmic domain of CS1 contains immunoreceptor tyrosine-based switch motif (ITSM) which is present in 2B4, SLAM and CD84. The signaling adaptor molecule SAP/SH2D1A, the defective gene in X-linked lymphoproliferative disease (XLPD), binds to ITSM and regulates immune cell function. However, recent studies indicate that CS1 may be regulated by a SAP-independent mechanism. In this study, we have examined the ligand specificity of CS1 and the effect of CS1 interaction with its ligand on the cytolytic activity of YT, a human NK cell line. Recombinant fusion protein, CS1-Ig, containing the CS1 extracellular domain and Fc portion of the human IgG bound cells transfected with CS1. CS1-Ig did not show any binding to cells expressing other members of the CD2 family. The cytolytic activity of YT was enhanced in presence of soluble CS1-Ig fusion protein. These results demonstrate that CS1 is a self-ligand and homophilic interaction of CS1 regulates NK cell cytolytic activity.

Regulation of NK cell function in vivo by the dose of tumour transplanted in the peritoneum

Antigen dose is known to regulate T cell activation and anergy. Similarly, dose of antigen also regulates NK cell lytic potential and phenotype development. Resident peritoneal cells of rat contain a small population of NK and NKT cells. Inoculation of AK-5 tumour cells intraperitoneally modulate the cytotoxic function of NK and NKT cells present in the peritoneal exudate cells (PEC) in a dose dependent manner. Low dose of tumour causes activation of NK and NKT cell cytotoxic function and enhanced NK and NKT cell population in PEC, whereas, high doses of tumour cause inactivation of NK and NKT cell cytotoxic function and depletion of the two sub-populations in the peritoneum. Different doses of tumour inoculation in the peritoneal cavity did not suppress the cytotoxic function of NK cells from spleen suggesting that a direct interaction between NK cells and tumour cells is required for the suppression of NK cell cytotoxic function. Tumour inoculation induced secretion of IL-2, IL-12, IFN-γ and TNF-α by tumour infiltrating mononuclear cells (TIM) in ascitic fluid as well as in serum. The levels of IL-2, IL-12, IFN-γ and TNF-α secretion were higher in animals, which rejected tumours as compared with the animals that failed to reject the tumours. Injection of anti IL-12 and anti IFN-γ antibody reduced the survival rate of tumour injected animals, however, anti IL-2 antibody had no effect on the survival of animals. Following incubation with AK-5 tumour cells, activated NK cells upregulated perform expression, whereas, there was upregulation of CD95 expression in inactivated NK cells.

Organization of the mouse macrophage C-type lectin (Mcl) gene and identification of a subgroup of related lectin molecules.

A number of genes encoding C-type lectin molecules have been mapped to the natural killer gene complex (NKC) at the distal region of mouse chromosome 6 and to a syntenic region on human chromosome 12p12-p13. In addition to those receptors which regulate NK cell function, related structures expressed on other cells types have also been localized to this chromosomal region. Among these are a number of recently characterized genes, including macrophage C-type lectin (MCL), macrophage-inducible C-type lectin (Mincle), dendritic cell immunoreceptor (DCIR) and dendritic cell-associated lectin-2 (Dectin-2). The amino acid sequences comprising the single C-type lectin domains of MCL, Mincle, DCIR and Dectin-2 are shown here to be closely related to each other. These molecules show overall similarity to two groups of animal C-type lectins, groups II and V, which demonstrate type II transmembrane topology. In this study, sequence analysis suggests that MCL, Mincle, DCIR and Dectin-2 represent a subset of group II-related C-type lectins which may participate in analogous recognition events on macrophages and dendritic cells. The genomic organization of the MCL gene and the sequence of the promoter region, with putative regulatory elements, were determined from a mouse MCL genomic DNA clone and are described here in detail.

Positive and negative roles of the trans-acting T cell factor-1 for the acquisition of distinct Ly-49 MHC class I receptors by NK cells.

Members of the Ly-49 gene family code for class I MHC-specific receptors that regulate NK cell function. Due to a combinatorial distribution of Ly-49 receptors, NK cells display considerable clonal heterogeneity. The acquisition of one Ly-49 receptor, Ly-49A is strictly dependent on the transcriptional trans-acting factor T cell-specific factor-1 (TCF-1). Indeed, TCF-1 binds to two sites in the Ly-49a promoter and regulates its activity, suggesting that the Ly-49a gene is a direct TCF-1 target. TCF-1 deficiency resulted in the altered usage of additional Ly-49 receptors. We show in this study, using TCF-1 beta(2)-microglobulin double-deficient mice, that these repertoire alterations are not due to Ly-49/MHC class I interactions. Our findings rather suggest a TCF-1-dependent, cell autonomous effect on the acquisition of multiple Ly-49 receptors. Besides reduced receptor usage (Ly-49A and D), we also observed no effect (Ly-49C) and significantly expanded (Ly-49G and I) receptor usage in the absence of TCF-1. These effects did not in all cases correlate with the presence of TCF binding sites in the respective proximal promoter. Therefore, besides TCF-1 binding to the proximal promoter, Ly-49 acquisition may also be regulated by TCF-1 binding to more distant cis-acting elements and/or by regulating the expression of additional trans-acting factors. Consistent with the observed differential, positive or negative role of TCF-1 for Ly-49 receptor acquisition, reporter gene assays revealed the presence of an inducing as well as a repressing TCF site in certain proximal Ly-49 promoters. These findings reveal an important role of TCF-1 for the formation of the NK cell receptor repertoire.

Differential regulation of Ly49 expression on CD4+ and CD4-CD8- (double negative) NK1.1+ T cells.

The pan-NK cell marker NK1.1, present in some mouse strains, is also found on a subset of TCRalphabeta+ lymphocytes termed NKT cells. These cells are primarily CD4+ or CD4-CD8- (double negative, DN), and both NKT subpopulations contain cells reactive with the MHC class I-like molecule CD1d. Murine NK cells express clonally distributed inhibitory receptors of the Ly49 family that bind to different alleles of MHC class I molecules and transmit negative signals regulating NK cell function. Ly49 receptors are also found on TCRalphabeta+ NK1.1+ T cells. To investigate the potential role of inhibitory Ly49 markers in the regulation of NKT cells, we have done a thorough analysis of their expression on different NKT populations. The CD4+ and DN NK1.1+ T cell subsets have traditionally been dealt with as one NK1.1+ T cell population, but we found dramatic differences between these two NKT cell subsets. We demonstrate here expression of Ly49 receptors on DN, but not on CD4+, NK1.1+ T cells in spleen and liver. Absence of the specific MHC class I ligand in the host was associated with elevated levels of expression and, to a greater extent than has been found for NK cells, increased the frequencies of Ly49-positive cells within the DN subset, while CD4+ NK1.1+ cells remained negative. In the thymus and bone marrow both NK1.1+ T cell subsets contained high frequencies of Ly49-positive cells. Results from in vitro stimulation of DN NKT cells further suggest that activation and expansion of NKT cell subsets are regulated by the Ly49 receptors.

Transgenic expression of Ly49A on T cells impairs a specific antitumor response.

Inhibitory MHC receptors determine the reactivity and specificity of NK cells. These receptors can also regulate T cells by modulating TCR-induced effector functions such as cytotoxicity, cytokine production, and proliferation. Here we have assessed the capacity of mouse T cells expressing the inhibitory MHC class I receptor Ly49A to respond to a well-defined tumor Ag in vivo using Ly49A transgenic mice. We find that the presence of Ly49A on the vast majority of lymphocytes prevents the development of a significant Ag-specific CD8+ T cell response and, consequently, the rejection of the tumor. Despite minor alterations in the TCR repertoire of CD8+ T cells in the transgenic lines, precursors of functional tumor-specific CD8+ T cells exist but could not be activated most likely due to a lack of appropriate CD4+ T cell help. Surprisingly, all of these effects are observed in the absence of a known ligand for the Ly49A receptor as defined by its ability to regulate NK cell function. Indeed, we found that the above effects on T cells may be based on a weak interaction of Ly49A with Kb or Db class I molecules. Thus, our data demonstrate that enforced expression of a Ly49A receptor on conventional T cells prevents a specific immune response in vivo and suggest that the functions of T and NK cells are differentially sensitive to the presence of inhibitory MHC class I receptors.

IL-18 activates STAT3 in the natural killer cell line 92, augments cytotoxic activity, and mediates IFN-gamma production by the stress kinase p38 and by the extracellular regulated kinases p44erk-1 and p42erk-21.

IL-18 is a regulator of NK cell function which utilizes the serine-threonine IL-1R-associated kinase signal transduction pathway and may activate additional not yet characterized signaling pathways. Here we evaluated IL-18-mediated signal transduction using the human NK cell line NK92 as a model. NK92 cells were shown by RT-PCR to express all three IL-18 receptor chains (IL-18R, accessory protein-like chain, IL-18-binding protein). Stimulation by IL-18 strongly enhanced tyrosine phosphorylation of STAT3 and of the mitogen-activated protein kinases (MAPK) p44erk-1and p42erk-2. In contrast, STAT5 was not activated. The cytolytic activity of NK92 against K562 target cells, which was augmented in a dose-dependent manner by IL-18 in the presence of trace amounts of IL-2, was suppressed by the specific inhibitors of MAPK pathways (PD098059 and SB203580). Similarly, the stimulatory effect of IL-18 on IFN-gamma protein production, given in conjunction with IL-2, was counteracted by inhibition of MAPK. IL-18 alone failed to stimulate IFN-gamma protein production despite inducing expression of IFN-gamma mRNA. IL-2 alone stimulated neither IFN-gamma mRNA expression nor IFN-gamma protein production. IL-18 did not stimulate proliferation of NK92 cells, either alone or in combination with IL-2 or IL-12. Inhibition of the MAPK pathway did not significantly alter the IL-2- and IL-12-induced proliferation of NK92 cells, whereas the Janus kinase/STAT pathway inhibitor AG490 strongly suppressed proliferation. MAPK activation appears to play a prominent role in IL-18 signaling, being involved in transcription and translation of IL-18-induced IFN-gamma mRNA and IL-18-induced cytolytic effects. In contrast, proliferation of NK92 cells is not affected by MAPK p44erk-1 and p42erk-2.

NK cell activation: distinct stimulatory pathways counterbalancing inhibitory signals

A delicate balance between positive and negative signals regulates NK cell effector function. Activation of NK cells may be initiated by the triggering of multiple adhesion or costimulatory molecules, and can be counterbalanced by inhibitory signals induced by receptors for MHC class I. A common pathway of inhibitory signaling is provided by immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the cytoplasmic domains of these receptors which mediate the recruitment of SH2 domain-bearing tyrosine phosphate-1 (SHP-1). In contrast to the extensive progress that has been made regarding the negative regulation of NK cell function, our knowledge of the signals that activate NK cells is still poor. Recent studies of the activating receptor complexes have shed new light on the induction of NK cell effector function. Several NK receptors using novel adaptors with immunoreceptor tyrosine-based activation motifs (ITAMs) and with PI 3-kinase recruiting motifs have been implicated in NK cell stimulation.

Mouse NKR-P1B, a Novel NK1.1 Antigen with Inhibitory Function

The mouse NK1.1 Ag originally defined as NK cell receptor (NKR)-P1C (CD161) mediates NK cell activation. Here, we show that another member of the mouse CD161 family, NKR-P1B, represents a novel NK1.1 Ag. In contrast to NKR-P1C, which functions as an activating receptor, NKR-P1B inhibits NK cell activation. Association of NKR-P1B with Src homology 2-containing protein tyrosine phosphatase-1 provides a molecular mechanism for this inhibition. The existence of these two NK1.1 Ags with opposite functions suggests a potential role for NKR-P1 molecules, such as those of the Ly-49 gene family, in regulating NK cell function.

The genomic organization of NKG2C, E, F, and D receptor genes in the human natural killer gene complex.

Interactions of natural killer cell receptors with their cognate ligands play a major role in regulating NK cell function. The NKG2 gene family encodes several highly similar proteins, which are known to form heterodimers with the CD94 receptor. These dimers play a role in the inhibition as well as the activation of NK cells. We have analyzed the gene structures of the NKG2C, D, E, and F genes, and determined their genomic organization. Restriction mapping and sequencing revealed the four genes to be closely linked to one another, and of the same transcriptional orientation. An exon duplication within the NKG2C and E genes was identified, although the duplicated version of this exon has not yet been found in mRNA sequences. The NKG2C, E, and F genes, despite being highly similar, are variable at their 3' ends. We show that NKG2C consists of six exons, whereas NKG2E has seven, and the splice acceptor site for the seventh exon occurs in an Alu repeat. NKG2F consists of only four exons and part of exon IV is in some cases spliced to the 5' end of the NKG2D transcript. NKG2D has only a low similarity to the other NKG2 genes.

The CD94/NKG2-A inhibitory receptor complex is involved in natural killer cell-mediated recognition of cells expressing HLA-G1.

Abstract Human NK cells bear surface receptors that inhibit their cytolytic activity upon specific recognition of MHC class Ia Ags; little is known about the capacity of class Ib molecules to regulate NK cell function. We have studied the roles of different NK inhibitory receptors in recognition of the class Ib HLA-G. To this end, we analyzed the ability of an HLA-defective tumor cell line (721.221) transfected with the membrane form of HLA-G1, which contains the three external domains, to inhibit the cytolytic activity mediated by a panel of NK clones from several donors. A substantial proportion of peripheral blood NK clones appeared to be significantly inhibited by the HLA-G1-transfected cell line (referred to as .221-G1); nevertheless, no relation was observed between the expression and the function of serologically identifiable Ig-SF receptors (p58/p70) and specific recognition of .221-G1 cells. Moreover, p58 killer cell inhibitory receptor-IgG soluble fusion proteins specifically bound to 721.221 transfectants bearing their corresponding HLA-C ligands, but only a weak reactivity with .221-G1 cells was detectable. By contrast, most NK clones blocked by HLA-G1 expressed the CD94/NKG2-A inhibitory receptor, and moreover, CD94-specific mAbs reconstituted their cytolytic activity comparably to anti-HLA class I mAbs. These data support the idea that the CD94/NKG2 receptor complex is involved in the recognition of cells expressing HLA-G1.

Regulation of NK cell functions by TGF-beta 1.

The contribution of exogenous and endogenous TGF-beta 1 to human peripheral blood NK cell proliferation and activity in vitro was investigated. Exogenous bioactive TGF-beta 1 inhibited NK cell DNA synthesis and production of IFN-gamma, TNF-alpha, and granulocyte-macrophage CSF (GM-CSF) by NK cultures consisting of > 98% CD56+ cells. The cytotoxic activity of NK cells was also weakly inhibited by exogenous TGF-beta 1. All TGF-beta 1-induced inhibitory effects occurred in the absence and presence of the NK cell-activating cytokines IFN-alpha, IL-2, and IL-12. Unstimulated NK cell cultures expressed steady state TGF-beta 1 mRNA detected by Northern blot analysis and produced TGF-beta protein (1.6 ng/ml), as determined by ELISA. When NK cell proliferation was induced by IL-2, IL-12, IFN-alpha, or a combination of IL-2 and IL-12, expression of TGF-beta 1 mRNA and protein was moderately and consistently reduced by approximately 20%, as compared with unstimulated control cultures. Unstimulated and rapidly proliferating NK cell cultures secreted primarily latent TGF-beta into their culture medium, as determined by the Mv1Lu bioassay. These results indicate that, in vitro, endogenous NK cell-derived TGF-beta 1 has no negative autocrine effect upon activation of NK cells by various cytokines.

Stimulation of protein tyrosine phosphorylation by interaction of NK cells with fibronectin via alpha 4 beta 1 and alpha 5 beta 1.

Recent evidence indicates that integrins do transduce signals that are involved in the regulation of a number of cellular processes. We have shown previously that human NK cells express alpha 4 beta 1 and alpha 5 beta 1 integrins, which mediate their adhesion to fibronectin (FN). Here we investigate whether cross-linking of beta 1 FN receptors on human NK cells stimulates tyrosine kinase activation. Our results indicate that cross-linking of beta 1 integrins on NK cells, either freshly isolated from PBL or generated from 10-day coculture of nonadherent PBMC with an irradiated EVB+ lymphoblastoid B cell line (long-term activated NK cells), altered the tyrosine phosphorylation pattern. In particular, we found stimulation of tyrosine phosphorylation of two proteins migrating with an apparent mass of 105 and 115 kDa, which was not observed after CD16 engagement. Phosphorylation of pp105-115 was already observed at 1 min, raised maximal values at 3 to 5 min, and persisted until 20 min after stimulation. Tyrosine phosphorylation of pp105-115 was also observed upon engagement of alpha 4 beta 1 and alpha 5 beta 1 FN receptors either with mAb directed against the alpha subunits or after NK cell adhesion to FN or its 120- and 40-kDa fragments. Pretreatment of NK cells with the tyrosine kinase inhibitor herbimycin A resulted in marked decrease of phosphorylation stimulated through alpha 4 beta 1 and alpha 5 beta 1 integrins, indicating that ligation of FN receptors on NK cells activates tyrosine kinase(s). Overall our results suggest that beta 1 integrins on NK cells play a major role as signaling molecules in the regulation of NK cell functions.