Engagement Of Inhibitory Receptors Research Articles

Mast cell inhibitory receptors

Mast cells play an important role in diverse physiological mechanisms as well as taking part in antimicrobial defense. What is more, these cells are important regulators of a number of pathophysiological processes, involving allergic reactions. Therefore, it seems to be very important to know and understand the factors and receptors influencing mast cell activity. Nowadays it is well established that activating signals are counterbalanced by negative or inhibition signals transmitted by inhibitory receptors containing immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Inhibitory receptor engagement leads to ITIM tyrosine phosphorylation, the recruitment and activation of protein tyrosine phosphatases such as SHP-1, SHP-2 and/or SHIP, and the dephosphorylation of activating receptor associated proteins. There is growing evidence that a number of inhibitory receptors have been identified on mast cells. The scope of this paper is to present the current knowledge on mast cell-associated inhibitory receptors, such as FcγRIIB, paired immunoglobulin-like receptor B (PIR-B), CD300, CD172a, gp49B1, CD200R, sialic acid-binding immunoglobulin-like lectin (Siglec) molecules, CD305, allergin-1, mast cell function-associated antigen (MAFA), and CD72. The role of these inhibitory receptors in regulation mast cell activity is also discussed.

NK Education: Disassociation Between Recovery of Cytoxicity and Cytokine Production In NK Cells After Allogeneic Transplantation.

AbstractAbstract 1462NK cells are the first lymphocyte subset to reconstitute following hematopoietic stem cell transplantation (HSCT) and they play a pivotal role in mediation of the graft versus leukemia (GvL) effect in myeloid leukemia. We hypothesized that for NK cells to mediate GvL, they must be fully functional via a process termed “licensing” or “education”. Although it has been presumed that NK cell functions (cytotoxicity and cytokine production) develop in parallel through interactions with their class I recognizing inhibitory receptors, new data suggests that this may not be the case. To address this issues we developed a 9-color flow cytometric-based assay to simultaneously measure both CD107a expression and IFNy production by CD56+ NK cells in the context of expression of inhibitory receptors for self-class I human leukocyte antigen (HLA). We tested a cohort of 30 patients who received either unmanipulated (T cell replete) or potently T cell depleted (CD34+ selected) grafts from adult unrelated donors. Thawed peripheral blood mononuclear cells (PBMC) were rested overnight in cytokine free media and then incubated with K562 cells to trigger cytotoxicity and cytokine production. PBMC were stained with CD107a (a surrogate for cytotoxicity), IFNy, CD56, CD3, CD45, CD158a, CD158b, CD158e and CD159a simultaneously. The same normal volunteer and the actual transplant donor were used as positive controls in each assay. Cytotoxicity or IFNy production was calculated as a percentage of the normal positive control. Cytotoxicity was intact but modestly suppressed (∼35%) at 3 months after both T cell deplete and T cell replete HSCT with further recovery of killing at 6 months. By contrast, at 3 months after T cell replete HSCT there was potent and sustained suppression of IFNy production by CD56+ cells (57%±11% suppression, p=0.009). The cohort of patients receiving T cell deplete (CD34-selected) grafts also exhibited significant suppression of IFNy at 3 months after HSCT (73%±9.6%, p=0.018), suggesting that the use of post-transplant immune suppression medications did not explain the effect. Suppression of IFNy production when exposed to targets continued through 6 months post-transplant in both cohorts and was partially restored with low concentrations of IL-15. Cells stimulated overnight with IL-12 and IL-18 produced IFNy at 3 and 6 months. Thus the cells were not globally hyporesponsive, suggesting the defect was based on physiologic interactions with the target. NK cells become educated following engagement of inhibitory receptors (eg. Killer-immunoglobulin-like receptors [KIR]) with self class I HLA. Therefore we compared NK cells that expressed at least one KIR with KIR negative NK cells. At 3 months post transplant, KIR expression had no effect on cytotoxicity. In contrast, KIR positive cells produced significantly higher amounts of IFNy than KIR negative cells at 3 (Figure 1) and 6 (data not shown) months post-transplant. Therefore following HSCT, expression of KIR discriminates a population of NK cells that produce IFNy, but does not correlate with cytotoxicity. While NK cell cytotoxicity is only partially suppressed following HSCT, IFNy production is significantly reduced. Consistent with this we found that while all IFNy producing cells degranulate, only a small fraction of CD107a+ cells also make IFNy. This effect is not a result of post-transplant immune suppression or graft versus host disease, as patients receiving CD34+ selected grafts had neither. Perhaps NKG2A, highly expressed on almost all NK cells early after transplant, selectively mediates education for cytotoxcity. In conclusion, our data shows distinct defects in NK cell education for either cytotoxicity or cytokine production. This highlights the importance of analyzing both cytotoxicity and cytokine production when assessing NK cell function post HSCT. Because of their critical anti-tumor and infection protection roles, methods to enhance broad in vivo NK cell function, such as the use of post-transplant IL-15 administration, are warranted. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Natural Killer Cell Killing of Acute Myelogenous Leukemia and Acute Lymphoblastic Leukemia Blasts by Killer Cell Immunoglobulin-Like Receptor–Negative Natural Killer Cells after NKG2A and LIR-1 Blockade

Although the study of natural killer (NK) cell alloreactivity has been dominated by studies of killer cell immunoglobulin-like receptors (KIRs), we hypothesized that NKG2A and LIR-1, present on 53% +/- 13% and 36% +/- 18% of normal NK cells, respectively, play roles in the NK cell killing of primary leukemia targets. KIR(-) cells, which compose nearly half of the circulating NK cell population, exhibit tolerance to primary leukemia targets, suggesting signaling through other inhibitory receptors. Both acute myelogenous leukemia and acute lymphoblastic leukemia targets were rendered susceptible to lysis by fresh resting KIR(-) NK cells when inhibitory receptor-major histocompatibility class I interactions were blocked by pan-HLA antibodies, demonstrating that these cells are functionally competent. Blockade of a single inhibitory receptor resulted in slightly increased killing, whereas combined LIR-1 and NKG2A blockade consistently resulted in increased NK cell cytotoxicity. Dual blockade of NKG2A and LIR-1 led to significant killing of targets by resting KIR(-) NK cells, demonstrating that this population is not hyporesponsive. Together these results suggest that alloreactivity of a significant fraction of KIR(-) NK cells is mediated by NKG2A and LIR-1. Thus strategies to interrupt NKG2A and LIR-1 in combination with anti-KIR blockade hold promise for exploiting NK cell therapy in acute leukemias.

Education of hyporesponsive NK cells by cytokines

NK-cell tolerance to self is mediated via engagement of inhibitory receptors by cognate MHC molecules. This event is critical for NK-cell education to achieve functional competence. Thus, NK cells expressing self-MHC-specific inhibitory receptors are responsive to activating stimuli while those lacking such receptors are hyporesponsive. Nevertheless, the mechanisms underlying NK-cell education are still poorly understood. Here, we show that after stimulation with cytokines, hyporesponsive NK cells acquire stable expression of killer Ig-like receptors (KIR) as reflected by DNA hypomethylation of their KIR locus. Remarkably, only hyporesponsive NK cells that acquire KIR in the presence of their cognate MHC molecule gain functional competence and this process can occur in the absence of any accessory cells. Acquisition of competence does not result in autoreactivity, since acquired KIR are functional and therefore able to inhibit NK-cell cytotoxicity. Our data demonstrate that competent NK cells can be generated by cytokine stimulation, suggesting that NK-cell education might not only be an early event which takes place during NK-cell development but might also occur in the periphery during an immune response.

Decision making in NK cells

A balance of positive and negative signals that are transmitted by different surface receptors controls the effector functions of NK cells. To date our understanding about the integration of positive and negative signals and the decision-making process inside NK cells remains poor. With the help of bioinformatic modelling we try to understand how NK cells first integrate antagonising signals and then compute a reliable killing decision. Gradual signal input through activating and inhibitory receptors is integrated to come to a yes or no decision by the NK cell to kill an attached target cell. Triggering of activating receptors leads to Src kinase activation and Vav-1 phosphorylation, whereas inhibitory receptors dephosphorylate Vav-1 via the phosphatase SHP-1. Therefore, we proposed in a first hypothesis, that Vav-1 is the decision making point in the signal transduction network. With this hypothesis we created a family of simplified models describing NK cell activation upon various stimuli. The predictions derived from these models were compared with experimental data. Our experiments showed that increased clustering of activating receptors lead to a rapid switch-like increase in Vav-1 phosphorylation. Similarly, titrating the engagement of inhibitory receptors resulted in switch-like dephosphorylation of Vav-1. Testing NK cell activity after various amounts of activating and inhibitory receptor engagement revealed a functional dominance of inhibitory receptors. Our current model is consistent with a central role of Vav-1 in the decision making process of NK cells and enables a novel insight into the integration of positive and negative signals during lymphocyte activation.

Granule polarization is the preferred target for inhibition by NK cell inhibitory receptors.

NK cells kill target cells by polarized release of cytotoxic granules. Granule polarization and degranulation are controlled by different signals in NK cells. The integrin LFA‐1 signals for polarization but not degranulation, whereas the Fc receptor CD16 induces degranulation but not polarization. Lysis of target cells by NK cells is controlled by inhibitory receptors specific for MHC class I. To test the sensitivity to inhibition of polarization and degranulation separately, NK cells were mixed with insect cells expressing ICAM‐1 (ligand of LFA‐1) and MHC class I in the presence or absence of rabbit IgG (ligand of CD16) bound to the insect cells. Engagement of inhibitory receptors by their MHC ligands completely blocked polarization but not degranulation. Similar results were obtained during natural cytotoxicity (independent of CD16) against human K562 cells transfected with MHC class I. Thus, either degranulation is not sensitive to inhibitory signals or inhibition of degranulation requires additional receptor – ligand interactions. Inhibition of degranulation occurred with the human target cell 221 transfected with MHC class I. These data demonstrate that (1) granule polarization is a preferred target of inhibitory signals, and (2) as yet unknown receptor – ligand interactions contribute to inhibition of degranulation.

Tyrosine phosphorylation of an adapter protein induced by NK cell inhibitory receptor contributes to inhibition of cytotoxicity

Many cellular responses, including immune autoreactivity, are under negative control by receptors carrying immunoreceptor tyrosine‐based inhibition motifs (ITIM) in their cytoplasmic tail. Natural killer (NK) cell cytotoxicity is blocked by ITIM‐containing receptors through recruitment of tyrosine phosphatases, which dephosphorylate key components of activation pathways, such as Vav1. Here we show that binding of an NK cell inhibitory receptor to its HLA class I ligand on target cells induces selective phosphorylation of an adapter protein and its dissociation from a signaling complex. Inhibition of NK cell cytotoxicity was impaired by expression of a non‐phosphorylated mutant of the adapter, implying a role in the inhibitory signal. These results show that inhibitory receptor engagement leads to specific phosphorylation as well as dephosphorylation events, and suggest the possibility of other signaling functions for ITIM‐containing receptors. This research was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Allergy and Infectious Diseases.

Mechanisms of Prolongation of Allograft Survival by HLA-G/ILT4-Modified Dendritic Cells

Engagement of inhibitory receptors on dendritic cells (DCs) is a powerful way to modulate their functions to achieve hyporesponsiveness or tolerance induction. Transgenic mice expressing human ILT4 receptor exclusively on DCs and triggered by HLA-G1 developed long-term survival of allogeneic skin transplant. Here we identify the cellular and molecular mechanisms responsible for that induction of hyporesponsiveness to alloantigen in vivo. Engagement of ILT4 receptor by HLA-G1 resulted in down-regulation of expression of MHC class II and costimulatory molecules, and modulation of cytokine production on DCs. HLA-G-modified DCs from ILT4 transgenic mice promote long-term survival of allografts by mechanisms involving both the induction of regulatory T cells and T-cell anergy. A novel feature of our research was to establish a model for the study of the prospective mechanisms of regulation of alloimmune responses by inhibitory receptors in vivo and analysis of the potential of HLA-G and its inhibitory receptors in modulation of DCs and T-cell function.

2B4 (CD244), NTB-A and CRACC (CS1) stimulate cytotoxicity but no proliferation in human NK cells

The recently described family of SLAM-related receptors plays an important role in the modulation of lymphocyte activity. The members of this family expressed on human NK cells are 2B4 (CD244), NTB-A and CRACC (CS1). The ligands of these surface receptors are also present on all human NK cells, suggesting that 2B4, NTB-A and CRACC are engaged during the contact of neighboring NK cells. Here we investigate the functional consequence of this interaction. We show that blocking the engagement of 2B4, NTB-A and CRACC has no effect on the proliferation or the development of the cytotoxic potential of human NK cells. However, triggering of 2B4, NTB-A or CRACC by their physiological ligands on MHC class I-negative target cells induces potent NK cell cytotoxicity. This suggests that the engagement of inhibitory receptors by MHC class I on neighboring NK cells blocks 2B4-, NTB-A- and CRACC-induced NK cell cytotoxicity, thereby ensuring that NK cells do not kill each other. In support of this, limiting inhibitory receptor engagement by antibodies leads to the autologous killing of NK cells in a 2B4-, NTB-A- and CRACC-dependent manner.

Selective cross-talk among natural cytotoxicity receptors in human natural killer cells.

The cytolytic activity of human natural killer cells is induced by several triggering cell surface receptors upon interaction with specific cellular ligands. These receptors include NKp46, NKp30 and NKp44, collectively termed natural cytotoxicity receptors (NCR). Co-operation among NCR has been shown to occur for optimal recognition and killing of most tumor target cells. In this study, we show that the mAb-mediated engagement and clustering of one or another NCR results in the activation of an identical set of tyrosine kinases. These kinases are included in the signaling cascade leading to tyrosine phosphorylation of different receptor-associated signal transducing molecules i.e. CD3 zeta (associated with NKp46 and NKp30) and KARAP/DAP12 (associated with NKp44). In line with the notion that the engagement of inhibitory receptors prevents NCR-mediated responses, we show that the engagement of CD94/NKG2A virtually abrogates the tyrosine phosphorylation of the NCR-associated signaling molecules, i.e. it acts at the very early steps of the signaling cascade. Importantly, the engagement of a single NCR resulted in the activation of the signaling cascades associated with the other NCR. This "cross-talk" is confined to NKp46, NKp30 and NKp44 since neither CD16-nor KIR2DS4-associated signaling polypeptides were phosphorylated following the NCR engagement. These results suggest that a functional cross-talk specifically occurs among different NCR, possibly resulting in the amplification of the activating signals.

Natural killer cell inhibitory receptors block actin cytoskeleton-dependent recruitment of 2B4 (CD244) to lipid rafts.

A dynamic balance of positive and negative signals regulates target cell lysis by natural killer (NK) cells upon engagement of a variety of different activation receptors and of inhibitory receptors that recruit the tyrosine phosphatase SHP-1. However, the step at which activation signals are blocked by SHP-1 is not known. We have been using activation receptor 2B4 (CD244) to study the influence of inhibitory receptors on NK cell activation. Engagement of inhibitory receptors by HLA class I on target cells blocks phosphorylation of 2B4, placing the inhibitory step at the level, or upstream of 2B4 phosphorylation. Here we show that phosphorylated 2B4, after engagement with either antibodies or target cells that express the 2B4 ligand, is found exclusively in a detergent-resistant membrane fraction that contains lipid rafts. Integrity of lipid rafts was essential for phosphorylation and activating function of 2B4. Coengagement of inhibitory receptors blocked 2B4 phosphorylation and 2B4 association with detergent-resistant membranes, indicating that inhibitory receptors function upstream of raft-dependent signals. Recruitment of 2B4 into detergent-resistant membrane fractions and 2B4 phosphorylation were dependent on actin polymerization. Blocking actin cytoskeleton-dependent raft recruitment of different receptors may be a general mechanism by which inhibitory receptors control NK cell activation.

P46, a novel natural killer cell-specific surface molecule that mediates cell activation.

Limited information is available on the surface molecules that are involved in natural killer (NK) cell triggering. In this study, we selected the BAB281 monoclonal antibody (mAb) on the basis of its ability to trigger NK-mediated target cell lysis. BAB281 identified a novel NK cell-specific surface molecule of 46 kD (p46) that is expressed by all resting or activated NK cells. Importantly, unlike the NK cell antigens identified so far, the expression of p46 was strictly confined to NK cells. Upon mAb-mediated cross-linking, p46 molecules induced strong cell triggering leading to [Ca2+]i increases, lymphokine production, and cytolytic activity both in resting NK cells and NK cell clones. The p46-mediated induction of Ca2+ increases or triggering of cytolytic activity was downregulated by the simultaneous engagement of inhibitory receptors including p58, p70, and CD94/NKG2A. Both the unique cellular distribution and functional capability of p46 molecules suggest a possible role in the mechanisms of non-major histocompatibility complex-restricted cytolysis mediated by human NK cells.

Deletion of SHIP or SHP-1 Reveals Two Distinct Pathways for Inhibitory Signaling

Two signaling molecules have been implicated in the modulation of immune receptor activation by inhibitory coreceptors: an inositol polyphosphate 5′-phosphatase, SHIP, and a tyrosine phosphatase, SHP-1. To address the necessity, interaction, or redundancy of these signaling molecules, we have generated SHP-1- or SHIP-deficient B cell lines and determined their ability to mediate inhibitory signaling. Two distinct classes of inhibitory responses are defined, mediated by the selective recruitment of SHP-1 or SHIP. The FcγRIIB class of inhibitory signaling is dependent on SHIP and not SHP-1; conversely, the KIR class requires SHP-1 and not SHIP. The consequence of this selective recruitment by inhibitory receptor engagement is seen in BCR-triggered apoptosis. SHP-1-mediated inhibitory signaling blocks apoptosis, while SHIP recruitment attenuates a proapoptotic signal initiated by FcγRIIB.

Signal minus 1: a key factor in immunological tolerance to tissue-specific self antigens?

Recent data suggest that many autoreactive T cells, particularly to tissue-specific self antigens, can escape thymic deletion. The current dogma is that these autoreactive T cells are silenced by the failure of most tissues to provide co-stimulation (signal 2), antigen alone (signal 1) inducing T cell unresponsiveness. However, I propose that activation of autoreactive T cells frequently occurs but autodestruction by effector T cells is tightly regulated. This phenomenon is most evident with lymph node metastasizing tumour cells where the regional lymph node can mount a vigorous response to the invading tumour cells but tumour growth is unimpaired. I suggest that autodestruction is prevented by inhibitory receptors on T cells which recognize class I MHC structures on target cells. These receptors, which I propose deliver 'signal minus 1' to T cells, were recently described on NK cells and a subpopulation of peripheral T cells. They are also strikingly similar to a family of anti-self receptors that my laboratory described on murine T and B cells 15 years ago. In the 'signal minus 1' model, antigen-activated T cells acquire the inhibitory receptors when they become co-stimulation independent and gain the ability to exit lymphoid organs and enter non-lymphoid tissues. Thus, if autoreactive effector T cells encounter autoantigen in tissues they are functionally silenced by inhibitory receptor engagement and signal minus 1 delivery. In contrast, I propose that in response to intracellular infections, cells down-regulate expression of their ligands for inhibitory receptors. Such a model allows infected cells to be selectively eliminated by effector T cells. If correct, the model predicts that effector T cells, whether foreign-antigen- or autoantigen-specific, can selectively respond to infected cells. This apparent 'usefulness' of autoreactive T cells may explain their observed persistence even after an encounter with autoantigen. It is also suggested that signal minus 1 may silence autoreactive B cells specific for tissue-specific cell surface antigens and lack of signal minus 1 may partially explain the vigorous T cell response to allogeneic MHC. Finally, it is hypothesized that, in evolutionary terms, inhibition of autodestruction by the recognition of a 'self marker' and delivery of signal minus 1 is an ancient process which probably emerged in early metazoans.