Immunoreceptor Tyrosine-based Switch Motif Research Articles

An immunoglobulin superfamily member (CgIgIT2) functions as immune inhibitory receptor to inhibit the inflammatory cytokine expressions in Crassostrea gigas

Immune inhibitory receptors are increasingly acknowledged as potent regulators of immune response, which inhibit the overactivation of immune system and play an important role in maintaining immune homeostasis. In the present study, a novel immunoglobulin superfamily member (CgIgIT2) was identified from the Pacific oyster, Crassostrea gigas. The protein sequence of CgIgIT2 contained one signal peptide, four Ig domains, one fibronectin type III domain, one transmembrane domain, and a cytoplasmic tail with two intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and one immunoreceptor tyrosine-based switch motif (ITSM). The mRNA transcripts of CgIgIT2 were widely expressed in all the tested tissues, including haemolymph, gill, mantle, adductor muscle, labial palp, gonad and hepatopancreas, with the highest expression in haemolymph. The mRNA expressions of CgIgIT2 in haemocytes increased significantly at 24, 48 and 72 h after Vibrio splendidus stimulation. The positive green signals of CgIgIT2 protein were mainly detected in granulocytes of haemocytes, which were 1.27-fold and 2.15-fold (p < 0.05) higher than that of semi-granulocytes and agranulocytes, respectively. And CgIgIT2 was mainly located in the membrane and cytoplasm of haemocytes. The recombinant protein of CgIgIT2-4 × Ig (rCgIgIT2-4 × Ig) exhibited binding activity towards multiple pathogen-associated molecular patterns (PAMPs), including lipopolysaccharides (LPS), peptidoglycan (PGN), mannose (MAN) and polyinosinic-polycytidylic acid (Poly (I: C)) with the highest affinity for LPS. rCgIgIT2-4 × Ig could also bind Gram-negative bacteria (V. splendidus, V. anguillarum, Escherichia coli), Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis), and fungi (Pichia pastoris). In the blocking assay with anti-CgIgIT2 antibody, the mRNA expressions of interleukins (CgIL17-1, CgIL17-3 and CgIL17-6) and tumor necrosis factors (CgTNF-1 and CgTNF-2) in haemocytes all increased significantly at 12 h after V. splendidus stimulation. These results suggested that CgIgIT2 could function as an inhibitor receptor to bind different PAMPs and microbes, as well as inhibit the mRNA expressions of multiple inflammatory cytokines in oysters.

Expression analysis of Carassius auratus-leukocyte-immune-type receptors (CaLITRs) during goldfish kidney macrophage development and in activated kidney leukocyte cultures.

Carassius auratus leukocyte immune-type receptors (CaLITRs) were recently discovered immunoregulatory receptors in goldfish that have diverse immunoglobulin-like (Ig-like) ectodomains and intracellular signaling motifs. Genomic analysis shows that CaLITR-types are also located as distinct gene clusters across multiple goldfish chromosomes. For example, CaLITR1 (unplaced) is a functionally ambiguous receptor having two Ig-like domains, a transmembrane domain (TM), and a short cytoplasmic tail (CYT) devoid of any recognizable signaling motifs. CaLITR2 (Chr47) is a putative inhibitory receptor containing four Ig-like domains, a TM, and a long CYT with an immunoreceptor tyrosine-based inhibition motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM). A putative activating receptor-type, CaLITR3 (Chr3), has four Ig-like domains, a TM, and a short CYT containing a positively charged histidine residue and CaLITR4 (ChrLG28B) is a receptor with putative multifunctional signaling potential as well as five Ig-like domains, a TM, and a long tyrosine-motif containing CYT region. The variable genomic locations of the CaLITRs suggest that they are likely under the influence of different cis- and/or trans-regulatory elements. To better understand the transcriptional activities of select CaLITRs from variable genomic regions, we used an RT-qPCR-based approach to examine the expression of CaLITR1, CaLITR2, CaLITR3, and CaLITR4 during goldfish primary kidney macrophage (PKM) development and in mixed leukocyte reaction cultures (MLRs) of the goldfish. Our results showed that the select CaLITRs are differentially expressed during PKM development and in goldfish MLRs exposed to T-cell mitogens/immunosuppressive drugs, supporting that the transcription of these CaLITRs is likely regulated by distinct cis- and/or trans-regulatory elements.

Siglec-5 is an inhibitory immune checkpoint molecule for human T cells.

Sialic acid-binding immunoglobulin-type lectins (Siglecs) are a family of immunoglobulin-type lectins that mediate protein-carbohydrate interactions via sialic acids attached to glycoproteins or glycolipids. Most of the CD33-related Siglecs (CD33rSiglecs), a major subfamily of rapidly evolving Siglecs, contain a cytoplasmic signaling domain consisting of the immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM) and mediate suppressive signals for lymphoid and myeloid cells. While most CD33rSiglecs are expressed by innate immune cells, such as monocytes and neutrophils, to date, the expression of Siglecs in human T cells has not been well appreciated. In this study, we found that Siglec-5, a member of the CD33rSiglecs, is expressed by most activated T cells upon antigen receptor stimulation. Functionally, Siglec-5suppresses T cell activation. In support of these findings, we found that Siglec-5 overexpression abrogates antigen receptor induced activation of NFAT and AP-1. Furthermore, we show that GBS β-protein, a known bacterial ligand of Siglec-5, reduces the production of cytokines and cytolytic molecules by activated primary T cells in a Siglec-5 dependent manner. Our data also show that some cancer cell lines express a putative Siglec-5ligand(s), and that the presence of soluble Siglec-5 enhances tumor-cell specific T cell activation, suggesting that some tumor cells inhibit T cell activation via Siglec-5. Together, our data demonstrate that Siglec-5 is a previously unrecognized inhibitory T cell immune checkpoint molecule and suggest that blockade of Siglec-5 could serve as a new strategy to enhance anti-tumor T cell functions.

Molecular features underlying differential SHP1/SHP2 binding of immune checkpoint receptors.

A large number of inhibitory receptors recruit SHP1 and/or SHP2, tandem-SH2-containing phosphatases through phosphotyrosine-based motifs immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM). Despite the similarity, these receptors exhibit differential effector binding specificities, as exemplified by the immune checkpoint receptors PD-1 and BTLA, which preferentially recruit SHP2 and SHP1, respectively. The molecular basis by which structurally similar receptors discriminate SHP1 and SHP2 is unclear. Here, we provide evidence that human PD-1 and BTLA optimally bind to SHP1 and SHP2 via a bivalent, parallel mode that involves both SH2 domains of SHP1 or SHP2. PD-1 mainly uses its ITSM to prefer SHP2 over SHP1 via their C-terminal SH2 domains (cSH2): swapping SHP1-cSH2 with SHP2-cSH2 enabled PD-1:SHP1 association in T cells. In contrast, BTLA primarily utilizes its ITIM to prefer SHP1 over SHP2 via their N-terminal SH2 domains (nSH2). The ITIM of PD-1, however, appeared to be de-emphasized due to a glycine at pY+1 position. Substitution of this glycine with alanine, a residue conserved in BTLA and several SHP1-recruiting receptors, was sufficient to induce PD-1:SHP1 interaction in T cells. Finally, structural simulation and mutagenesis screening showed that SHP1 recruitment activity exhibits a bell-shaped dependence on the molecular volume of the pY+1 residue of ITIM. Collectively, we provide a molecular interpretation of the SHP1/SHP2-binding specificities of PD-1 and BTLA, with implications for the mechanisms of a large family of therapeutically relevant receptors.

Siglec-8 Signals Through a Non-Canonical Pathway to Cause Human Eosinophil Death In Vitro.

Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is a glycan-binding receptor bearing immunoreceptor tyrosine-based inhibitory and switch motifs (ITIM and ITSM, respectively) that is selectively expressed on eosinophils, mast cells, and, to a lesser extent, basophils. Previous work has shown that engagement of Siglec-8 on IL-5–primed eosinophils causes cell death via CD11b/CD18 integrin–mediated adhesion and NADPH oxidase activity and identified signaling molecules linking adhesion, reactive oxygen species (ROS) production, and cell death. However, the proximal signaling cascade activated directly by Siglec-8 engagement has remained elusive. Most members of the Siglec family possess similar cytoplasmic signaling motifs and recruit the protein tyrosine phosphatases SHP-1/2, consistent with ITIM-mediated signaling, to dampen cellular activation. However, the dependence of Siglec-8 function in eosinophils on these phosphatases has not been studied. Using Siglec-8 antibody engagement and pharmacological inhibition in conjunction with assays to measure cell-surface upregulation and conformational activation of CD11b integrin, ROS production, and cell death, we sought to identify molecules involved in Siglec-8 signaling and determine the stage of the process in which each molecule plays a role. We demonstrate here that the enzymatic activities of Src family kinases (SFKs), Syk, SHIP1, PAK1, MEK1, ERK1/2, PLC, PKC, acid sphingomyelinase/ceramidase, and Btk are all necessary for Siglec-8–induced eosinophil cell death, with no apparent role for SHP-1/2, SHIP2, or c-Raf. While most of these signaling molecules are necessary for Siglec-8–induced upregulation of CD11b integrin at the eosinophil cell surface, Btk is phosphorylated and activated later in the signaling cascade and is instead necessary for CD11b activation. In contrast, SFKs and ERK1/2 are phosphorylated far earlier in the process, consistent with their role in augmenting cell-surface levels of CD11b. In addition, pretreatment of eosinophils with latrunculin B or jasplakinolide revealed that actin filament disassembly is necessary and sufficient for surface CD11b integrin upregulation and that actin polymerization is necessary for downstream ROS production. These results show that Siglec-8 signals through an unanticipated set of signaling molecules in IL-5–primed eosinophils to induce cell death and challenges the expectation that ITIM-bearing Siglecs signal through inhibitory pathways involving protein tyrosine phosphatases to achieve their downstream functions.

Immune Functions of Signaling Lymphocytic Activation Molecule Family Molecules in Multiple Myeloma.

Simple SummaryMultiple myeloma (MM) is an incurable hematological malignancy characterized by an increase in abnormal plasma cells. Disease progression, drug resistance, and immunosuppression in MM are associated with immune-related molecules, such as immune checkpoint and co-stimulatory molecules, present in the tumor microenvironment. Novel agents targeting these cell-surface molecules are currently under development, including monoclonal antibodies, bispecific monoclonal antibodies, and chimera antigen receptor T-cell therapies. In this review, we focus on the signaling lymphocytic activation molecule family receptors and provide an overview of their biological functions and novel therapies in MM.The signaling lymphocytic activation molecule (SLAM) family receptors are expressed on various immune cells and malignant plasma cells in multiple myeloma (MM) patients. In immune cells, most SLAM family molecules bind to themselves to transmit co-stimulatory signals through the recruiting adaptor proteins SLAM-associated protein (SAP) or Ewing’s sarcoma-associated transcript 2 (EAT-2), which target immunoreceptor tyrosine-based switch motifs in the cytoplasmic regions of the receptors. Notably, SLAMF2, SLAMF3, SLAMF6, and SLAMF7 are strongly and constitutively expressed on MM cells that do not express the adaptor proteins SAP and EAT-2. This review summarizes recent studies on the expression and biological functions of SLAM family receptors during the malignant progression of MM and the resulting preclinical and clinical research involving four SLAM family receptors. A better understanding of the relationship between SLAM family receptors and MM disease progression may lead to the development of novel immunotherapies for relapse prevention.

Exploring the Allosteric Mechanism of Src Homology-2 Domain-Containing Protein Tyrosine Phosphatase 2 (SHP2) by Molecular Dynamics Simulations.

The Src homology-2 (SH2) domain-containing protein tyrosine phosphatase 2 (SHP2, encoded by PTPN11) is a critical allosteric phosphatase for many signaling pathways. Programmed cell death 1 (PD-1) could be phosphorylated at its immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM) and can bind to SHP2 to initiate T cell inactivation. Although the interaction of SHP2-PD-1 plays an important role in the immune process, the complex structure and the allosteric regulation mechanism remain unknown. In this study, molecular dynamics (MD) simulations were performed to study the binding details of SHP2 and PD-1, and explore the allosteric regulation mechanism of SHP2. The results show that ITIM has a preference to bind to the N-SH2 domain and ITSM has almost the same binding affinity to the N-SH2 and C-SH2 domain. Only when ITIM binds to the N-SH2 domain and ITSM binds to the C-SH2 domain can the full activation of SHP2 be obtained. The binding of ITIM and ITSM could change the motion mode of SHP2 and switch it to the activated state.

Blocking interaction between SHP2 and PD‐1 denotes a novel opportunity for developing PD‐1 inhibitors

Small molecular PD‐1 inhibitors are lacking in current immuno‐oncology clinic. PD‐1/PD‐L1 antibody inhibitors currently approved for clinical usage block interaction between PD‐L1 and PD‐1 to enhance cytotoxicity of CD8+ cytotoxic T lymphocyte (CTL). Whether other steps along the PD‐1 signaling pathway can be targeted remains to be determined. Here, we report that methylene blue (MB), an FDA‐approved chemical for treating methemoglobinemia, potently inhibits PD‐1 signaling. MB enhances the cytotoxicity, activation, cell proliferation, and cytokine‐secreting activity of CTL inhibited by PD‐1. Mechanistically, MB blocks interaction between Y248‐phosphorylated immunoreceptor tyrosine‐based switch motif (ITSM) of human PD‐1 and SHP2. MB enables activated CTL to shrink PD‐L1 expressing tumor allografts and autochthonous lung cancers in a transgenic mouse model. MB also effectively counteracts the PD‐1 signaling on human T cells isolated from peripheral blood of healthy donors. Thus, we identify an FDA‐approved chemical capable of potently inhibiting the function of PD‐1. Equally important, our work sheds light on a novel strategy to develop inhibitors targeting PD‐1 signaling axis.

Interaction of SHP-2 SH2 domains with PD-1 ITSM induces PD-1 dimerization and SHP-2 activation

Programmed cell death-1 (PD-1) inhibits T cell responses. This function relies on interaction with SHP-2. PD-1 has one immunoreceptor tyrosine-based inhibitory motif (ITIM) at Y223 and one immunoreceptor tyrosine-based switch motif (ITSM) at Y248. Only ITSM-Y248 is indispensable for PD-1-mediated inhibitory function but how SHP-2 enzymatic activation is mechanistically regulated by one PD-1 phosphotyrosine remains a puzzle. We found that after PD-1 phosphorylation, SHP-2 can bridge phosphorylated ITSM-Y248 residues on two PD-1 molecules via its amino terminal (N)-SH2 and carboxyterminal (C)-SH2 domains forming a PD-1: PD-1 dimer in live cells. The biophysical ability of SHP-2 to interact with two ITSM-pY248 residues was documented by isothermal titration calorimetry. SHP-2 interaction with two ITSM-pY248 phosphopeptides induced robust enzymatic activation. Our results unravel a mechanism of PD-1: SHP-2 interaction that depends only on ITSM-Y248 and explain how a single docking site within the PD-1 cytoplasmic tail can activate SHP-2 and PD-1-mediated inhibitory function.

Phosphorylation of PD-1-Y248 is a marker of PD-1-mediated inhibitory function in human T cells

PD-1 is a target of cancer immunotherapy but responses are limited to a fraction of patients. Identifying patients with T cells subjected to PD-1-mediated inhibition will allow selection of suitable candidates for PD-1-blocking therapy and will improve the therapeutic success. We sought to develop an approach to detect PD-1-mediated inhibitory signaling. The cytoplasmic tail of PD-1 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) encompassing Y223 and an immunoreceptor tyrosine-based switch motif (ITSM) encompassing Y248, which is indispensable for interaction of SHP-2 and delivery of PD-1 inhibitory function. We generated an antibody specific for phosphorylated PD-1-Y248 and examined PD-1pY248+ (pPD-1) expression in human T cells. pPD-1 was upregulated by TCR/CD3 + CD28 stimulation and simultaneous PD-1 ligation. pPD-1+CD8+ T cells were identified in human peripheral blood and had impaired effector function. pPD-1+ T cells were also detected in tumor-draining lymph nodes of tumor bearing mice and in biopsies of patients with glioblastoma multiform. Detection of pPD-1+ T cells might serve as a biomarker for identification of T cells subjected to PD-1-mediated immunosuppression.

Identification of Two Nonrearranging IgSF Genes in Chicken Reveals a Novel Family of Putative Remnants of an Antigen Receptor Precursor.

In this study, we identified a pair of nonrearranging VJ-joined Ig superfamily genes, termed putative remnants of an Ag receptor precursor (PRARP) genes, in chicken. Both genes encode a single V-set Ig domain consisting of a canonical J-like segment and a potential immunoreceptor tyrosine-based inhibitory or switch motif in the cytoplasmic region. In vitro experiments showed that both genes were expressed at the cell surface as membrane proteins, and their recombinant products formed a monomer and a disulfide-linked homodimer or a heterodimer. These two genes were mainly expressed in B and T cells and were upregulated in response to stimulation with poly(I:C) in vitro and vaccination in vivo. Orthologs of PRARP have been identified in bony fish, amphibians, reptiles, and other birds, and a V-C1 structure similar to that of Ig or TCR chains was found in all these genes, with the exception of those in avian species, which appear to contain degenerated C1 domains or divergent Ig domains. Phylogenetic analyses suggested that the newly discovered genes do not belong to any known immune receptor family and appear to be a novel gene family. Further elucidation of the functions of PRARP and their origin might provide significant insights into the evolution of the immune system of jawed vertebrates.

Molecular cloning, expression and characterization of Pekin duck programmed death-1

Programmed death-1 (PD-1) has a pivotal role in the attenuation of adaptive immune responses and peripheral tolerance. Here we describe the identification of the Pekin duck programmed death-1 orthologue (duPD-1). The duPD-1 cDNA encodes a 283-amino acid polypeptide that has an amino acid identity of 70%, 32% and 31% with chicken, murine and human PD-1, respectively. The duck PD-1 gene shares five conserved exons with chicken, murine and human PD-1 genes. A cluster of putative regulatory elements within the conserved region B (CR-B) of the basal promotor is conserved. Homology modeling was most compatible with the two β-sheet IgV domain structure of murine PD-1. Contact residues, shown to be critical for binding of the respective human and murine PD-1 ligands are mostly conserved between avian and mammalian species, whereas residues that define the cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM) are highly conserved across higher vertebrates and frog. Constitutive expression of duPD-1 transcripts was predominantly found in lymphocyte-rich tissues, and mitogen-stimulation of duck peripheral blood mononuclear cells transiently increased duPD-1 mRNA expression. A soluble duPD-1 protein was expressed and shown to engage the identified duck PD-1 ligands. Our observations show considerable evolutionary conservation between mammalian and avian PD-1 orthologues. This work will facilitate further investigation of the role of PD-1 signaling in adaptive immunity in the Pekin duck, a non-mammalian vertebrate and pathogen host with relevance for human and animal health.

The Two SH2 Domains of SHP-2 Bridge Two PD-1 Molecules Resulting in SHP-2 Activation and PD-1-Mediated Inhibition

Programmed cell death-1 (PD-1) is a checkpoint inhibitory receptor member of the B7-CD28 family, which promotes peripheral tolerance and restrains anti-viral and anti-tumor immunity. Although PD-1 blockade leads to durable clinical responses in a fraction of patients, the majority of patients display only transient responses, emphasizing the need for better understanding of the mechanism of PD-1-mediated T cell inhibition. In contrast to other CD28 family family members, which form disulfide-linked homodimers, PD-1 lacks the juxtamembrane cysteine residue responsible for homodimer formation and has been shown to exist as a monomer. The cytoplasmic tail of PD-1 has one immunoreceptor tyrosine-based inhibitory motif (ITIM), centered on Y223 residue, and one immunoreceptor tyrosine-based switch motif (ITSM), centered on Y248 residue. Mutational studies have shown that PD-1-mediated inhibition relies on the interaction of the ITSM with SHP-2 but the mechanism by which PD-1 induces SHP-2 activation is unknown. In this study we sought to determine how PD-1: SHP-2 interaction leads to inhibition of T-cell responses. SHP-2 contains a phosphatase (PTP) domain and two SH2 domains, N-SH2 and C-SH2. To determine whether PD-1 selectively interacts with a specific SH2 domain of SHP-2, we generated five different GST-fusion proteins in which GST was fused with either SHP-2 full length (FL), SHP-2-N-SH2, SHP-2-C-SH2, SHP-2-ΔN-SH2 (lacking the N-terminus SH2 domain) or SHP-2-PTP. Surprisingly, pull-down assays with GST-fusion proteins using lysates from primary human T cells or Jurkat T cell line revealed that PD-1 interacted with SHP-2 via both SH2 domains of SHP-2. Biacore assays confirmed that phosphorylated ITSM Y248 interacted with SHP-2 N-SH2 and C-SH2. To determine whether one of the SHP-2 SH2 domains might preferentially interact with PD-1 in live cells, we mutagenized the functional sites of the N-SH2 or C-SH2 domain at arginine R32 and R138, respectively, and transfected COS cells with cDNA of SHP-2 wild type or each SHP-2 SH2 mutant, together with PD-1 and the TCR proximal kinase Fyn, required for PD-1 phosphorylation and interaction with SHP-2. Immunoprecipitation and immunoblot assays showed that mutagenesis of either SH2 domain abrogated interaction of SHP-2 with PD-1 ITSM Y248, providing evidence that both SH2 domains of SHP-2 are involved in the interaction with PD-1. Because each PD-1 molecule has only one ITSM, these results indicate that SHP-2 interacts via its two SH2 domains with two PD-1 molecules. Assessment of PD-1: PD-1 dimer formation in live cells by split luciferase complementation, using NanoBiT proximity assays, showed that upon PD-1 phosphorylation, PD-1: PD1 interaction occurs only in the presence of SHP-2 with intact N-SH2 and C-SH2 domains. The SH2 domains of SHP-2 have a crucial and distinct role in regulating SHP-2 PTPase activity. In the absence of a tyrosine-phosphorylated binding ligand, N-SH2 is bound to the PTP domain leading to an auto-inhibitory closed conformation that blocks the PTP active site. Phosphorylation of Y542 in the SHP-2 C-terminus tail leads to intramolecular interaction of Y542 with the N-SH2 domain to relieve binding to PTP domain. Similarly, intermolecular interaction of the N-SH2 domain with specific phosphorylated ligands disrupts its PTP recognition surface reversing the auto-inhibitory conformation and activates the phosphatase. We determined that monomeric PD-1 ITIM-pY223 or ITSM-pY248 peptides did not activate the PTP. A dimeric phosphopeptide generated by covalent joining of two PD-1 ITSM-pY248 phosphopeptides with a linker that matches the distance between the binding sites of the two SHP-2 SH2 domains induced rapid activation of SHP-2. In contrast, similarly designed dimeric peptides joining either two PD-1 ITIM-pY223 or ITIM-pY223-and-ITSM-pY248 phosphopeptides did not activate PTP activity of SHP-2. The ability of PD1 ITSM-pY248 to induce SHP-2 activation was correlated with inhibition of antigen-mediated IL-2 production, which was abrogated when Y248 was mutagenized to phenylalanine. Our results reveal the geometry of PD-1: SHP-2 interaction that leads to SHP-2 activation and have implications for the development of PD-1-binding compounds to selectively suppress T cell responses by dimerizing PD-1 or enhance T cell responses by disrupting PD-1 dimerization and SHP-2 activation. DisclosuresFreeman:EMD-Serono: Patents & Royalties; Xios: Membership on an entity's Board of Directors or advisory committees; Origimed: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Patents & Royalties; Bristol-Myers-Squibb: Patents & Royalties; Bristol-Myers-Squibb: Membership on an entity's Board of Directors or advisory committees; Roche: Patents & Royalties; Dako: Patents & Royalties; Boehringer-Ingelheim: Patents & Royalties; Merck: Patents & Royalties; Novartis: Patents & Royalties.

Uncoupling ITIM receptor G6b-B from tyrosine phosphatases Shp1 and Shp2 disrupts murine platelet homeostasis

The immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor G6b-B has emerged as a key regulator of platelet homeostasis. However, it remains unclear how it mediates its effects. Tyrosine phosphorylation of ITIM and immunoreceptor tyrosine-based switch motif (ITSM) within the cytoplasmic tail of G6b-B provides a docking site for Src homology 2 domain-containing protein-tyrosine phosphatases Shp1 and Shp2, which are also critical regulators of platelet production and function. In this study, we investigate the physiological consequences of uncoupling G6b-B from Shp1 and Shp2. To address this, we generated a transgenic mouse model expressing a mutant form of G6b-B in which tyrosine residues 212 and 238 within ITIM and ITSM were mutated to phenylalanine. Mice homozygous for the mutation (G6b-B diY/F) were macrothrombocytopenic, as a result of the reduction in platelet production, and had large clusters of megakaryocytes and myelofibrosis at sites of hematopoiesis, similar to those observed in G6b-deficient mice and patients. Platelets from G6b-B diY/F mice were hyporesponsive to collagen, as a result of the significant reduction in the expression of the immunoreceptor tyrosine-based activation motif (ITAM)-containing collagen receptor complex GPVI-FcR γ-chain, as well as thrombin, which could be partially rescued by costimulating the platelets with adenosine diphosphate. In contrast, platelets from G6b-B diY/F, G6b KO, and megakaryocyte-specific Shp2 KO mice were hyperresponsive to antibody-mediated cross-linking of the hemi-ITAM-containing podoplanin receptor CLEC-2, suggesting that G6b-B inhibits CLEC-2-mediated platelet activation through Shp2. Findings from this study demonstrate that G6b-B must engage with Shp1 and Shp2 to mediate its regulatory effects on platelet homeostasis.

Signaling lymphocytic activation molecules Slam and cancers: friends or foes?

Signaling Lymphocytic Activation Molecules (SLAM) family receptors are initially described in immune cells. These receptors recruit both activating and inhibitory SH2 domain containing proteins through their Immunoreceptor Tyrosine based Switch Motifs (ITSMs). Accumulating evidence suggest that the members of this family are intimately involved in different physiological and pathophysiological events such as regulation of immune responses and entry pathways of certain viruses. Recently, other functions of SLAM, principally in the pathophysiology of neoplastic transformations have also been deciphered. These new findings may prompt SLAM to be considered as new tumor markers, diagnostic tools or potential therapeutic targets for controlling the tumor progression. In this review, we summarize the major observations describing the implications and features of SLAM in oncology and discuss the therapeutic potential attributed to these molecules.

Elotuzumab for the Treatment of Relapsed or Refractory Multiple Myeloma, with Special Reference to its Modes of Action and SLAMF7 Signaling.

Elotuzumab, targeting signaling lymphocytic activation molecule family 7 (SLAMF7), has been approved in combination with lenalidomide and dexamethasone (ELd) for relapsed/refractory multiple myeloma (MM) based on the findings of the phase III randomized trial ELOQUENT-2 (NCT01239797). Four-year follow-up analyses of ELOQUENT-2 have demonstrated that progression-free survival was 21% in ELd versus 14% in Ld. Elotuzumab binds a unique epitope on the membrane IgC2 domain of SLAMF7, exhibiting a dual mechanism of action: natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC) and enhancement of NK cell activity. The ADCC is mediated through engagement between Fc portion of elotuzumab and FcgRIIIa/CD16 on NK cells. Enhanced NK cell cytotoxicity results from phosphorylation of the immunoreceptor tyrosine-based switch motif (ITSM) that is induced via elotuzumab binding and recruits the SLAM-associated adaptor protein EAT-2. The coupling of EAT-2 to the phospholipase Cg enzymes SH2 domain leads to enhanced Ca2+ influx and MAPK/Erk pathway activation, resulting in granule polarization and enhanced exocytosis in NK cells. Elotuzumab does not stimulate the proliferation of MM cells due to a lack of EAT-2. The inhibitory effects of elotuzumab on MM cell growth are not induced by the lack of CD45, even though SHP-2, SHP-1, SHIP-1, and Csk may be recruited to phosphorylated ITSM of SLAMF7. ELd improves PFS in patients with high-risk cytogenetics, i.e. t(4;14), del(17p), and 1q21 gain/amplification. Since the immune state is paralytic in advanced MM, the efficacy of ELd with minimal toxicity may bring forward for consideration of its use in the early stages of the disease.

Comparative Analysis of Immune Checkpoint Molecules and Their Potential Role in the Transmissible Tasmanian Devil Facial Tumor Disease.

Immune checkpoint molecules function as a system of checks and balances that enhance or inhibit immune responses to infectious agents, foreign tissues, and cancerous cells. Immunotherapies that target immune checkpoint molecules, particularly the inhibitory molecules programmed cell death 1 and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), have revolutionized human oncology in recent years, yet little is known about these key immune signaling molecules in species other than primates and rodents. The Tasmanian devil facial tumor disease is caused by transmissible cancers that have resulted in a massive decline in the wild Tasmanian devil population. We have recently demonstrated that the inhibitory checkpoint molecule PD-L1 is upregulated on Tasmanian devil (Sarcophilus harrisii) facial tumor cells in response to the interferon-gamma cytokine. As this could play a role in immune evasion by tumor cells, we performed a thorough comparative analysis of checkpoint molecule protein sequences among Tasmanian devils and eight other species. We report that many of the key signaling motifs and ligand-binding sites in the checkpoint molecules are highly conserved across the estimated 162 million years of evolution since the last common ancestor of placental and non-placental mammals. Specifically, we discovered that the CTLA-4 (MYPPPY) ligand-binding motif and the CTLA-4 (GVYVKM) inhibitory domain are completely conserved across all nine species used in our comparative analysis, suggesting that the function of CTLA-4 is likely conserved in these species. We also found that cysteine residues for intra- and intermolecular disulfide bonds were also highly conserved. For instance, all 20 cysteine residues involved in disulfide bonds in the human 4-1BB molecule were also present in devil 4-1BB. Although many key sequences were conserved, we have also identified immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and immunoreceptor tyrosine-based switch motifs (ITSMs) in genes and protein domains that have not been previously reported in any species. This checkpoint molecule analysis and review of salient features for each of the molecules presented here can serve as road map for the development of a Tasmanian devil facial tumor disease immunotherapy. Finally, the strategies can be used as a guide for veterinarians, ecologists, and other researchers willing to venture into the nascent field of wild immunology.

Phosphorylation of Y248 in the ITSM of PD-1 is indicative of PD-1-mediated inhibitory function

Abstract PD-1 is an inhibitory receptor expressed on activated T-cells. SHP-2 tyrosine phosphatase interacts with PD-1 and is critical for PD-1-mediated inhibition. The cytoplasmic tail of PD-1 contains an immunoreceptor tyrosine based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). It has been reported that SHP-2 may interact with either ITIM or ITSM of PD-1. We sought to identify the precise motif of PD-1 that is mandatory for SHP-2 interaction and PD-1 inhibitory function. Using GST-SHP-2 fusion protein we determined that PD-1 interacted with SHP-2 after PD-1 ligation with simultaneous TCR/CD3-mediated activation. This interaction required phosphorylation of the ITSM and was abrogated when the ITSM tyrosine Y248 was mutated to phenylalanine. In contrast, when the ITIM tyrosine Y223 was mutated, interaction of PD-1 with SHP-2 remained unaffected. Based on these findings, we hypothesized that phosphorylation of Y248 might be indicative of PD-1-mediated inhibitory signaling. We generated an antibody specific for Y248 and examined expression and function of PD-1pY248+ T cells in peripheral blood from healthy humans. PD-1pY248+ cells were detected within the CD4 but mostly CD8T cell populations, mainly in central memory and effector memory subsets and in much lower extent in terminally differentiated effectors. Although PD-1high did not correlate with altered ability of CD8 T cells to produce effector cytokines, PD-1pY248+ expression correlated with impaired production of IFN-g and TNFa in response to TCR/CD3+CD28-mediated stimulation. Thus, PD-1pY248 might serve as a biomarker indicative of PD-1 mediated inhibitory signaling in patients with chronic infections and cancer.

Interaction of Both SH2 Domains of SHP-2 with a PD-1 Homodimer Is Required for PD-1-Mediated Inhibition of T Cell Responses

Programmed death-1 (PD-1) is a checkpoint receptor expressed on activated T-cells. PD-1 has a key role in maintenance of peripheral tolerance but also restrains anti-viral and anti-tumor immunity. Although PD-1 blockade leads to durable clinical responses in a significant fraction of patients, the majority of patients have only transient responses, emphasizing the need for better understanding of the mechanism of PD-1-mediated T cell inhibition. PD-1 consists of a single N-terminal IgV-like domain, a 20 amino acid stalk separating the IgV domain from the plasma membrane, a transmembrane domain, and a cytoplasmic tail containing two tyrosine-based structural motifs, an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). SHP-2 tyrosine phosphatase interacts with the ITSM and has a critical role in PD-1-mediated inhibition but the precise mechanism is poorly understood. We sought to determine how PD-1: SHP-2 interaction leads to inhibition of T-cell responses. SHP-2 contains two SH2 domains, a phosphatase (PTP) domain and a C-terminus tail (C-tail), forming a structure of N-SH2-C-SH2-PTP-C-tail. We generated five GST-fusion proteins in which GST was fused with either SHP-2 full length, N-SH2, C-SH2 C-SH2-PTP (lacking the N-terminus SH2 domain), or PTP. Pull-down assays using lysates from human T cells revealed that PD-1 interacted with GST-SHP-2 fusion protein only after TCR/CD3-mediated activation with simultaneous PD-1 ligation, and the interaction of PD-1 with SHP-2 was mediated via the SH2 domains of SHP-2. The SH2 domains of SHP-2 have a crucial and distinct role in regulating SHP-2 PTPase activity. In the absence of a tyrosine-phosphorylated ligand, N-SH2 binds the PTP domain leading to an auto-inhibitory closed conformation that blocks the PTP site. Phosphorylation of Y542 in the SHP-2 C-tail leads to an intramolecular interaction of Y542 with the N-SH2 domain that relieves N-SH2 binding to the PTP domain and thereby reverses basal inhibition of the PTPase. Phosphorylation of Y580 in the SHP-2 C-tail relieves the auto-inhibitory closed conformation by interaction with the C-SH2 domain. Subsequent high affinity intermolecular interaction of the N-SH2 with a phosphorylated protein partner completely disrupts its PTP recognition surface, reversing the auto-inhibitory conformation and activating the PTPase activity, whereas the C-SH2 domain contributes to binding energy and specificity. We found that in activated T cells, PD-1-associated SHP-2 was phosphorylated in the tyrosines of the C-tail. To determine whether PD-1 selectively interacts with a specific SH2 domain of SHP-2, we mutagenized the functional sites of N-SH2 and C-SH2 domains at arginines 32 and 138, respectively, to alanine (R32A and R138A) and transfected COS cells with cDNA of SHP-2 wild type or each SH2 mutant together with PD-1 and TCR proximal kinase Fyn, which is required for PD-1 phosphorylation. Immunoprecipitation and immunoblot showed that mutagenesis of either SH2 domain abrogated interaction of SHP-2 with PD-1, indicating that both SH2 domains of SHP-2 are involved in the interaction with PD-1. Surprisingly, each SH2 domain of SHP-2 interacted with tyrosine phosphorylated ITSM of PD-1, as determined by immunoblot with a phopho-PD-1 antibody specific for the phosphorylated tyrosine residue Y245 and by disruption of both N-SH2:PD-1 and C-SH2:PD-1 interaction by mutation of PD-1 ITSM tyrosine residue Y245. These results indicate that SHP-2 brings together two tyrosine phosphorylated PD-1 molecules by interaction with N-SH2 and C-SH2 domains. To determine the functional implications of PD-1 homodimerization, we cultured human T cells in the presence of a soluble dimeric PD-L1 or a monomeric PD-L1. Although dimeric PD-L1 inhibited T cell proliferation and IFN-g production, monomeric PD-L1 had the opposite effect. Our results reveal a previously unidentified mechanism of PD-1: SHP-2 interaction and have implications for the development of PD-1-binding compounds to selectively suppress T cell responses by dimerizing PD-1 or to enhance T cell activation by disrupting PD-1 homodimerization. Our findings open new avenues for the development of selective PD-1-binding compounds in order to augment T cell responses for the induction of antitumor immunity or to suppress aberrant T cell activation in autoimmunity and graft versus host disease. DisclosuresNo relevant conflicts of interest to declare.

Specific subsets of kinases mediate Siglec-8 engagement-induced ROS production and apoptosis in human eosinophils.

Abstract Siglecs are type I transmembrane proteins expressed primarily on leukocytes. Among them is Siglec-8, a CD33 subfamily member that is selectively expressed on the cell surface of human eosinophils. Siglec-8 has an intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM), putatively responsible for signal transduction. In cytokine-activated eosinophils, Siglec-8 binding causes apoptosis with increased mitochondrial damage and ROS production, but exact signaling mechanisms are unknown. Using a mAb (2C4) against Siglec-8 in combination with small molecule inhibitors, we first examined Siglec-8-mediated apoptosis and ROS production by flow cytometry after 24 hr IL-5 priming (30 ng/mL) of human eosinophils. We observed that 2C4-mediated eosinophil apoptosis was inhibited by PP1 and SU6656 (Src kinase inhibitors), ibrutinib (Btk inhibitor), LY294002 (PI3K inhibitor), GF109203x (PKC inhibitor), and sodium orthovanadate (a protein phosphatase inhibitor) at IC50’s of 1.7 μM, 1.8 μM, 0.9 nM, 1.2 μM, 2.4 μM, and 22 μM respectively. Complete inhibition of ROS production occurred at expected IC90’s. Western blot analysis following Siglec-8 cross-linking with 2C4 showed increased phosphorylation of Src530, Csk, PI3Kδ, and Blk that was detectable within 15 min, and c-Abl phosphorylation that was detectable within 60 min. Additionally, co-immunoprecipitation data shows that Siglec-8 associates with SHP-2, a protein tyrosine phosphatase. While the sequence of signaling events is yet to be determined, Siglec-8 mediated apoptosis in eosinophils involves the unexpected recruitment of molecules normally associated with cell survival.