Mary E. Peterson

Controlling Natural Killer Cell Responses: Integration of Signals for Activation and Inhibition

Understanding how signals are integrated to control natural killer (NK) cell responsiveness in the absence of antigen-specific receptors has been a challenge, but recent work has revealed some underlying principles that govern NK cell responses. NK cells use an array of innate receptors to sense their environment and respond to alterations caused by infections, cellular stress, and transformation. No single activation receptor dominates; instead, synergistic signals from combinations of receptors are integrated to activate natural cytotoxicity and cytokine production. Inhibitory receptors for major histocompatibility complex class I (MHC-I) have a critical role in controlling NK cell responses and, paradoxically, in maintaining NK cells in a state of responsiveness to subsequent activation events, a process referred to as licensing. MHC-I-specific inhibitory receptors both block activation signals and trigger signals to phosphorylate and inactivate the small adaptor Crk. These different facets of inhibitory signaling are incorporated into a revocable license model for the reversible tuning of NK cell responsiveness.

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

Summary: 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.

Inhibitory Receptor Signaling via Tyrosine Phosphorylation of the Adaptor Crk

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.

The Adaptor Protein Crk Controls Activation and Inhibition of Natural Killer Cells

Natural killer (NK) cell inhibitory receptors recruit tyrosine phosphatases to prevent activation, induce phosphorylation and dissociation of the small adaptor Crk from cytoskeleton scaffold complexes, and maintain NK cells in a state of responsiveness to subsequent activation events. How Crk contributes to inhibition is unknown. We imaged primary NK cells over lipid bilayers carrying IgG1 Fc to stimulate CD16 and human leukocyte antigen (HLA)-E to inhibit through receptor CD94-NKG2A. HLA-E alone induced Crk phosphorylation in NKG2A(+) NK cells. At activating synapses with Fc alone, Crk was required for the movement of Fc microclusters and their ability to trigger activation signals. At inhibitory synapses, HLA-E promoted central accumulation of both Fc and phosphorylated Crk and blocked the Fc-induced buildup of F-actin. We propose a unified model for inhibitory receptor function: Crk phosphorylation prevents essential Crk-dependent activation signals and blocks F-actin network formation, thereby reducing constraints on subsequent engagement of activation receptors.

Cutting Edge: NK Cell Licensing Modulates Adhesion to Target Cells

Binding of NK cell inhibitory receptors to MHC class I (MHC-I) confers increased responsiveness to NK cells by a process known as NK cell licensing/education. Reduced MHC-I expression or a lack of inhibitory receptors for MHC-I results in diminished NK cell responsiveness. In this study, we evaluated the effect of human and mouse NK cell licensing on early stages of natural cytotoxicity. Unlicensed NK cells did not form as many stable conjugates with target cells. The reduction of NK cell conjugation to target cells was not attributed to altered β2 integrin LFA-1 properties but was instead due to reduced inside-out signaling to LFA-1 by activating receptors. For those unlicensed NK cells that did form conjugates, LFA-1–dependent granule polarization was similar to that in licensed NK cells. Thus, licensing controls signals as proximal as inside-out signaling by activating receptors but not integrin outside-in signaling for granule polarization.

KIR enrichment at the effector-target cell interface is more sensitive than signaling to the strength of ligand binding

Target cell lysis by natural killer cells is inhibited by killer cell immunoglobulin‐like receptors (KIR) that bind major histocompatibility complex class I molecules. Many lymphocyte receptors, including KIR, become enriched at the interface with ligand‐bearing cells. The contribution of the enrichment to inhibitory signaling has not been determined. We now describe a KIR variant with enhanced green fluorescent protein (EGFP) at the N terminus that can mediate inhibitory signaling, but its enrichment is markedly reduced. This receptor is only slightly weaker at inhibiting lysis than the same KIR tagged with EGFP in the cytoplasmic tail, even though the latter enriched as extensively as wild‐type KIR. A slight defect was also detected in the ability of the receptor to reduce adhesion to target cells and for binding of a soluble counterpart to cell surface HLA‐C. Our findings suggest that the strength of the interaction required to readily detect receptor enrichment exceeds that required for signaling.

Natural Killer Cells and Mast Cells from gp49B Null Mutant Mice Are Functional

ABSTRACT Immune responses are controlled by a combination of positive and negative cellular signals. Effector cells in the immune system express inhibitory receptors that serve to limit effector cell expansion and to protect the host from autoreactivity. gp49B is a receptor of unknown function that is expressed on activated mast cells and natural killer (NK) cells and whose cytoplasmic tail endows it with inhibitory potential. To gain insight into the function of gp49B in mice, we disrupted the gp49B gene by homologous recombination. gp49B0 mice were born at expected ratios, were healthy and fertile, and displayed normal long-term survival rates. gp49B0 mice showed no defect in NK or mast cell development. Furthermore, NK and mast cells from the gp49B0mice showed activation properties in vitro similar to those of cells isolated from wild-type mice. Therefore, gp49B is not critical for the development, expansion, and maturation of mast cells and NK cells in vivo. The healthy status of gp49B0 mice makes them suitable for testing the role of gp49B in immune responses to infectious agents.

Inhibitory receptor gp49B regulates eosinophil infiltration during allergic inflammation

gp49B, an Ig‐like receptor, negatively regulates the activity of mast cells and neutrophils through cytoplasmic immunoreceptor tyrosine‐based inhibition motifs. To characterize the role of gp49B further in vivo, gp49B‐deficient mice were tested in two allergic models. Responses to ragweed (RW) challenge in the lung and conjunctiva were assessed in models of allergic inflammation and during an infection with parasitic larvae of the nematode Ascaris suum. Infiltration by inflammatory cells into the lung during allergic responses was under negative control of the inhibitory receptor gp49B. Furthermore, an increase in conjunctival inflammation with a predominance of eosinophils, neutrophils, and degranulated mast cells was observed in RW‐sensitized, gp49B‐deficient mice, which had been challenged in the eye, as compared with C57BL/6 wild‐type (WT) controls. Finally, an increase in allergic inflammation in the lungs of A. suum‐infected, RW‐sensitized mice was observed upon RW challenge, as compared with C57BL/6 WT controls. The observed influx of eosinophils into mucus membranes is characteristic of allergic asthma and allergic conjunctivitis and may contribute to airway hyper‐responsiveness, airway remodeling, and mucus production. Expression of gp49B was detected on peripheral eosinophils of control mice and on eosinophils from lungs of mice treated with RW, suggesting a role for gp49B on eosinophils in dampening allergic inflammatory responses.

Zinc-Induced Polymerization of Killer-Cell Ig-like Receptor into Filaments Promotes Its Inhibitory Function at Cytotoxic Immunological Synapses

The inhibitory function of killer cell immunoglobulin-like receptors (KIR) that bind HLA-C and block activation of human natural killer (NK) cells is dependent on zinc. We report that zinc induced the assembly of soluble KIR into filamentous polymers, as detected by electron microscopy, which depolymerized after zinc chelation. Similar KIR filaments were isolated from lysates of cells treated with zinc, and membrane protrusions enriched in zinc were detected on whole cells by scanning electron microscopy and imaging mass spectrometry. Two independent mutations in the extracellular domain of KIR, away from the HLA-C binding site, impaired zinc-driven polymerization and inhibitory function. KIR filaments formed spontaneously, without the addition of zinc, at functional inhibitory immunological synapses of NK cells with HLA-C(+) cells. Adding to the recent paradigm of signal transduction through higher order molecular assemblies, zinc-induced polymerization of inhibitory KIR represents an unusual mode of signaling by a receptor at the cell surface.

Canonical and cross-reactive binding of NK cell inhibitory receptors to HLA-C allotypes is dictated by peptides bound to HLA-C

Background Human natural killer (NK) cell activity is regulated by a family of killer cell immunoglobulin-like receptors (KIRs) that bind human leukocyte antigen (HLA) class I. Combinations of KIR and HLA genotypes are associated with disease, including susceptibility to viral infection and disorders of pregnancy. KIR2DL1 binds HLA-C alleles of group C2 (Lys80). KIR2DL2 and KIR2DL3 bind HLA-C alleles of group C1 (Asn80). However, this model cannot explain HLA-C allelic effects in disease or the impact of HLA-bound peptides. The goal of this study was to determine the extent to which the endogenous HLA-C peptide repertoire can influence the specific binding of inhibitory KIR to HLA-C allotypes. Results The impact of HLA-C bound peptide on inhibitory KIR binding was investigated taking advantage of the fact that HLA-C*05:01 (HLA-C group 2, C2) and HLA-C*08:02 (HLA-C group 1, C1) have identical sequences apart from the key KIR specificity determining epitope at residues 77 and 80. Endogenous peptides were eluted from HLA-C*05:01 and used to test the peptide dependence of KIR2DL1 and KIR2DL2/3 binding to HLA-C*05:01 and HLA-C*08:02 and subsequent impact on NK cell function. Specific binding of KIR2DL1 to the C2 allotype occurred with the majority of peptides tested. In contrast, KIR2DL2/3 binding to the C1 allotype occurred with only a subset of peptides. Cross-reactive binding of KIR2DL2/3 with the C2 allotype was restricted to even fewer peptides. Unexpectedly, two peptides promoted binding of the C2 allotype-specific KIR2DL1 to the C1 allotype. We showed that presentation of endogenous peptides or HIV Gag peptides by HLA-C can promote KIR cross-reactive binding. Conclusion KIR2DL2/3 binding to C1 is more peptide selective than that of KIR2DL1 binding to C2, providing an explanation for KIR2DL3–C1 interactions appearing weaker than KIR2DL1–C2. In addition, cross-reactive binding of KIR is characterized by even higher peptide selectivity. We demonstrate a hierarchy of functional peptide selectivity of KIR–HLA-C interactions with relevance to NK cell biology and human disease associations. This selective peptide sequence-driven binding of KIR provides a potential mechanism for pathogen as well as self-peptide to modulate NK cell activation through altering levels of inhibition.