Intracellular signaling by the killer immunoglobulin-like receptors and Ly49.

Once thought to be promiscuous killers, it is now known that natural killer (NK) cells possess an elaborate array of receptors that regulate NK cytotoxic and secretory functions upon interaction with target cell MHC class I proteins. These receptors, known as killer cell immunoglobulin-like receptors (KIRs) in humans, and Ly49 receptors in the mouse, have become the focus of intense study in an effort to discern the underlying biology of these large receptor families. These receptor families include both inhibitory and activating receptors. Interrogation of a target expressing KIR ligands leads to coengagement of the inhibitory receptor with as-yet poorly defined activation receptors. Kinases activated during engagement mediate the phosphorylation of the KIR or Ly49 cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). The phosphorylated ITIMs serve as efficient recruitment points for the cytosolic protein tyrosine phosphatases, SHP-1 and SHP-2, resulting in the dephosphorylation of substrates critical for cellular activation. In contrast, some KIRs and Ly49s lack the ITIM and possess a charged residue in their transmembrane domains that mediates interaction with the DAP12 signal transduction chain. DAP12 uses its cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) to mediate cellular activation. Engagement of a DAP12 coupled KIR or Ly49 results in phosphorylation of DAP12, and other key substrates, including the Syk tryosine kinase, phospholipase C, and c-Cbl. DAP12 activation then leads to the Mapk cascade and ultimately to enhanced degranulation, and production of cytokines and chemokines. Although the context in which inhibitory and activating KIR and Ly49s function is not yet known, the dissection of the activating and inhibitory signal transduction pathways should shed light on their method of integration into the activation sequela of NK cells. Ultimately, this work will lead to concrete understanding of the immunobiology of these seemingly antagonistic receptor systems.

Inhibition of natural killer cell activation signals by killer cell immunoglobulin-like receptors (CD158).

The killer cell immunoglobulin-like receptor (KIR) family includes receptors that bind to HLA class I molecules on target cells and inhibit natural killer (NK)-cell cytotoxicity, and receptors such as KIR3DL7 with no known ligand and function. Inhibitory KIR recruit the tyrosine phosphatase SHP-1 to block signals transduced by any one of a number of activation receptors. Inhibition of overall protein tyrosine phosphorylation by SHP-1 during binding of KIR to MHC class I on target cells is selective, suggesting that a limited number of substrates are dephosphorylated by SHP-1. We have chosen to study KIR inhibition as it occurs during binding of KIR to MHC class I on target cells, despite the technical limitations inherent to studies of processes regulated by cell contact. KIR binding to MHC class I on target cells inhibits tyrosine phosphorylation of the activation receptor 2B4 (CD244) and disrupts adhesion of NK cells to target cells. Inhibition of proximal events in NK activation may increase the availability of NK cells by liberating them from non-productive interactions with resistant target cells. As the receptors and the signaling pathways that induce NK cytotoxicity are not fully characterized, elucidation of the inhibitory mechanism employed by KIR may provide insight into NK activation.

Adhesion to target cells is disrupted by the killer cell inhibitory receptor.

Killer cell immunoglobulin-like receptors (KIR) inhibit the cytotoxic activity of natural killer (NK) cells by recruitment of the tyrosine phosphatase SHP-1 to immunoreceptor tyrosine-based inhibition motif (ITIM) sequences in the KIR cytoplasmic tail [1]. The precise steps in the NK activation pathway that are inhibited by KIR are yet to be defined. Here, we have studied whether the initial step of adhesion molecule LFA-1-dependent adhesion to target cells was altered by the inhibitory signal. Using stable expression of an HLA-C-specific KIR in the NK cell line YTS [2] and a two-color flow cytometry assay for conjugate formation, we show that adhesion to a target cell expressing cognate HLA-C was disrupted by KIR engagement. Conjugate formation was abruptly interrupted by KIR within less than 5 minutes. Inhibition of adhesion to target cells was mediated by a chimeric KIR molecule carrying catalytically active SHP-1 in place of its cytoplasmic tail. These results suggest that other ITIM-bearing receptors, many of which have no known function, may regulate adhesion in a wide variety of cell types.

Essential role of LAT in T cell development.

The linker molecule LAT is a substrate of the tyrosine kinases activated following TCR engagement. Phosphorylated LAT binds many critical signaling molecules. The central role of this molecule in TCR-mediated signaling has been demonstrated by experiments in a LAT-deficient cell line. To probe the role of LAT in T cell development, the LAT gene was disrupted by targeting. LAT-deficient mice appeared healthy. Flow cytometric analysis revealed normal B cell populations but the absence of any mature peripheral T cells. Intrathymic development was blocked within the CD4- CD8- stage. No gross abnormality of NK or platelet function was observed. LAT is thus critical to both T cell activation and development.

Conserved residues amino-terminal of cytoplasmic tyrosines contribute to the SHP-1-mediated inhibitory function of killer cell Ig-like receptors.

The sequence I/VxYxxL, often referred to as an immunoreceptor tyrosine-based inhibition motif (ITIM), binds to the C-terminal Src homology 2 domain of the tyrosine phosphatase SHP-1. Conserved residues N-terminal of the tyrosine are not ordinarily found in other Src homology 2 domain binding motifs. The inhibitory forms of killer cell Ig-like receptors (KIR) contain two ITIMs. The role of each ITIM, and of the conserved residues upstream of the tyrosine, in the inhibition of NK cells was tested by vaccinia virus-mediated expression of mutant KIRs. Substitution of the tyrosine in the membrane-proximal ITIM abrogated the ability of KIR to block Ab-dependent cellular cytotoxicity, whereas mutation of the membrane-distal ITIM tyrosine had little effect. Substitution of the conserved hydrophobic amino acid that was located two residues N-terminal to the tyrosine weakened, but did not eliminate, the function of the receptor. In contrast, these substitutions drastically reduced the amount of SHP-1 immunoprecipitated with KIR, suggesting that weak interactions with SHP-1 may be sufficient for inhibition.

Negative signaling pathways of the killer cell inhibitory receptor and Fc gamma RIIb1 require distinct phosphatases.

Inhibition of natural killer (NK) cells by the killer cell inhibitory receptor (KIR) involves recruitment of the tyrosine phosphatase SHP-1 by KIR and is prevented by expression of a dominant negative SHP-1 mutant. Another inhibitory receptor, the low affinity Fc receptor for immunoglobulin G (IgG) (Fc gamma RIIb1), has been shown to bind SHP-1 when cocross-linked with the antigen receptor on B cells (BCR). However, coligation of Fc gamma RIIb1 with BCR and with Fc epsilon RI on mast cells leads to recruitment of the inositol 5' phosphatase SHIP and to inhibition of mast cells from SHP-1-deficient mice. In this study, we evaluated the ability of these two inhibitory receptors to block target cell lysis by NK cells, and the contribution of SHP-1 and SHIP to inhibition. Recombinant vaccinia viruses encoding chimeric receptors and dominant negative mutants of SHP-1 and SHIP were used for expression in mouse and human NK cells. When the KIR cytoplasmic tail was replaced by that of Fc gamma RIIb1, recognition of HLA class I on target cells by the extracellular domain resulted in inhibition. A dominant negative mutant of SHP-1 reverted the inhibition mediated by the KIR cytoplasmic tail but not that mediated by Fc gamma RIIb1. In contrast, a dominant negative mutant of SHIP reverted only the inhibition mediated by the Fc gamma RIIb1 tail, providing functional evidence that SHIP plays a role in the Fc gamma RIIb1-mediated negative signal. These data demonstrate that inhibition of NK cells by KIR involves primarily the tyrosine phosphatase SHP-1, whereas inhibition mediated by Fc gamma RIIb1 requires the inositol phosphatase SHIP.

A novel phosphotyrosine motif with a critical amino acid at position -2 for the SH2 domain-mediated activation of the tyrosine phosphatase SHP-1.

SHP-1 is a protein-tyrosine phosphatase associated with inhibition of activation pathways in hematopoietic cells. The catalytic activity of SHP-1 is regulated by its two SH2 (Src homology 2) domains; phosphotyrosine peptides that bind to the SH2 domains activate SHP-1. The consensus sequence (I/V)XYXX(L/V) is present in the cytoplasmic tails of several lymphocyte receptors that interact with the second SH2 domain of SHP-1. In several of these receptors, there are two or three occurrences of the motif. Here we show that the conserved hydrophobic amino acid preceding the phosphotyrosine is critical for binding to and activation of SHP-1 by peptides corresponding to sequences from killer cell inhibitory receptors. The interaction of most SH2 domains with phosphopeptides requires only the phosphotyrosine and the three residues downstream of the tyrosine. In contrast, the shortest peptide able to bind or activate SHP-1 also included the two residues upstream of the phosphotyrosine. A biphosphopeptide corresponding to the cytoplasmic tail of a killer cell inhibitory receptor with the potential to interact simultaneously with both SH2 domains of SHP-1 was the most potent activator of SHP-1. The hydrophobic residue upstream of the tyrosine was also critical in the context of the biphosphopeptide. The contribution of a hydrophobic amino acid two residues upstream of the tyrosine in the SHP-1-binding motif may be an important feature that distinguishes inhibitory receptors from those that provide activation signals.

Killer cell inhibitory receptors: diversity, specificity, and function.

NK cells selectively kill target cells that fail to express self-MHC class I molecules. This selective killing results from a balance between inhibitory NK receptors specific for MHC class I molecules and activating receptors that are still largely unknown. Isolation of molecular clones for the human killer cell inhibitory receptors (KIR) revealed that KIR consist of a family of molecules with Ig ectodomains and cytoplasmic tails of varying length. Soluble complexes of KIR and HLA-C molecules established that KIR recognizes and binds to its ligand as an autonomous receptor. A functional expression system in human NK clones demonstrated that a single KIR can provide both recognition of MHC class I and delivery of a dominant negative signal to the NK cell. Functional evidence has been obtained for a role of the tyrosine phosphatase SHP-1 in KIR-mediated inhibition. The presence of a conserved motif used to recruit and activate SHP-1 in the cytoplasmic tail of KIR and of the mouse Ly-49 inhibitory receptor (otherwise structurally unrelated to KIR) represents an interesting case of evolutionary convergence. Furthermore, the motif led to the identification of other receptors with inhibitory potential, including a type I Ig-like receptor shared by mouse mast cells and NK cells.

Type I transmembrane receptor with inhibitory function in mouse mast cells and NK cells.

The MHC class I-specific inhibitory receptors on human and mouse NK cells have surprisingly different structures. The mouse receptors (Ly-49) are type II transmembrane glycoproteins of the C-type lectin family, whereas the human receptors (killer cell inhibitory receptors (KIR)) belong to the Ig superfamily. This difference prompted a search for Ig-like inhibitory receptors in mice. Here we show that gp49, a mouse mast cell protein of unknown function but with sequence similarity to KIR, is expressed in NK cells. The gp49 cytoplasmic tail, containing a sequence related to an inhibitory motif shared by KIR and Ly-49, delivered a strong inhibitory signal in both human and mouse NK cells when substituted for a KIR cytoplasmic tail. These data show that Ig-like receptors with inhibitory properties exist in both species and that mouse mast and NK cells may recognize common inhibitory ligands.

Regulation through inhibitory receptors: Lessons from natural killer cells.

Natural killer (NK) cells employ an unconventional mode of recognition: they kill target cells that lack ligands for inhibitory NK cell receptors. Activation of NK cytotoxicity is tightly controlled by inhibitory receptors that recruit and activate the tyrosine phosphatase SHP-1 through the tyrosine-phosphorylated [I/V]xYxxL amino acid sequence in their cytoplasmic tail. This sequence motif, often referred to as an immunoreceptor tyrosine-based inhibitory motif (ITIM), is found in several other receptors that deliver similar negative signals in diverse types of cells. We suggest that this kind of regulation through inhibition is a widespread mechanism for the control of various cellular responses.

Recruitment of tyrosine phosphatase HCP by the killer cell inhibitor receptor.

Cytolysis of target cells by natural killer (NK) cells and by some cytotoxic T cells occurs unless prevented by inhibitory receptors that recognize MHC class I on target cells. Human NK cells express a p58 inhibitory receptor specific for HLA-C. We report association of the tyrosine phosphatase HCP with the p58 receptor in NK cells. HCP association was dependent on tyrosine phosphorylation of p58. Phosphotyrosyl peptides corresponding to the p58 tail bound and activated HCP in vitro. Furthermore, introduction of an inactive mutant HCP into an NK cell line prevented the p58-mediated inhibition of target cell lysis. These data imply that the inhibitory function of p58 is dependent on its tyrosine phosphorylation and on recruitment and activation of HCP.

Dynamics of peptide binding to purified antibody-bound H-2Db and H-2Db beta 2m complexes.

Although it is clear that each component of the class I MHC trimolecular complex (heavy chain, beta 2m, and antigenic peptide) contributes to its formation and stability, the specific interaction governing assembly and disassembly remain to be clarified. In an effort to address these issues using purified H-2Db molecules, we used a solid-phase binding assay recently developed in our laboratory to quantify kinetic parameters for class I assembly and disassembly. It was found that the influenza NP peptide Y367-374 associated with preformed empty complexes of 28-14-8S- (i.e., anti-alpha 3) bound Db beta 2m dimers much more quickly (t 1/2 < 0.2 h at 22 degrees C) than it did when coincubated with an anti-alpha 3 bound Db and human beta 2m (t1/2 3.5 h at 22 degrees C). The previously reported potential for the NP peptide Y367-374 to interact directly with free Db heavy chains and configure the conventionally beta 2m-dependent B22 epitope in the absence of beta 2m, was confirmed using our assay system. However, the rate of B22 epitope formation induced in the Db heavy chain by NP Y367-374 was considerably slower (t1/2 13 h, at 22 degrees C) and much less efficient on a molar basis than that resulting from the addition of beta2m (t1/2, 0.75 h, at 22 degrees C). In contrast, the Db heavy chain with NP-Y367-374 was more resistant to thermal disassembly (as measured by loss of the B22 epitope, t1/2 2h, 37 degrees C) than the Db beta 2m empty dimer (t1/2 0.2 h). Finally, stability of the preformed trimolecular complex of heavy chain, beta 2m, and peptide was found to diminish in accordance with deviation of the peptide from the optimal length and with increasing temperature from 4 to 37 degrees C.

High occupancy binding of antigenic peptides to purified, immunoadsorbed H-2Db beta 2m molecules.

In an effort to examine the peptide binding properties of purified class I MHC molecules, we have developed a solid phase, radiolabeled peptide binding assay based on the use of H-2Db molecules bound to agarose beads via heavy chain-specific mAb. Using purified Db beta 2m, recovered from RMA-S cells and bound to immunoadsorbent beads through either alpha 1 or alpha 3 region specific antibodies, complete occupancy of these molecules could be achieved with 125I-Y366-374 influenza nucleoprotein peptide (Kd 10(-7) M). Approximately 12% of the Db beta 2m dimers recovered from RMA cells could be occupied by this influenza nucleoprotein peptide under the same conditions. When free Db heavy chains were isolated from beta 2m negative R1E.Db cells by bead-bound alpha 3-region specific antibody (28-14-8S) and were incubated with human beta 2m, high affinity (Kd 10(-8) M) binding sites were created for the 125I-Y367-374 influenza nucleoprotein peptide. In addition to demonstrating that a significant fraction of the heavy chains present in R1E.Db cells are in a beta 2m-reactive form, the R1E.Db cells provide an alternate approach to that of RMA-S derived Db beta 2m empties for the creation of homogeneous complexes of Db, beta 2m, and antigenic peptide. We anticipate that these bead-bound empty and defined peptide-class I complexes may be useful in the further study of class I MHC target structure formation and recognition.