Ig Superfamily Receptors Research Articles

CD96 targeted antibodies need not block CD96-CD155 interactions to promote NK cell anti-metastatic activity

ABSTRACTCD96 is a transmembrane glycoprotein Ig superfamily receptor, expressed on various T cell subsets and NK cells, that interacts with nectin and nectin-like proteins, including CD155/polio virus receptor (PVR). Here, we have compared three rat anti-mouse CD96 mAbs, including two that block CD96-CD155 (3.3 and 6A6) and one that does not block CD96-CD155 (8B10). Using flow cytometry, we demonstrated that both mAbs 3.3 and 6A6 bind to the first Ig domain of mouse CD96 and compete with CD155 binding, while mAb 8B10 binds to the second Ig domain and does not block CD155. While Fc isotype was irrelevant concerning the anti-metastatic activity of 3.3 mAb, in four different experimental metastases models and one spontaneous metastasis model, the relative order of anti-metastatic potency was 6A6 > 3.3 > 8B10. The metastatic burden control of all of the anti-CD96 clones was highly dependent on NK cells and IFN-γ. Consistent with its inability to block CD96-CD155 interactions, 8B10 retained anti-metastatic activity in CD155-deficient mice, whereas 3.3 and 6A6 lost potency in CD155-deficient mice. Furthermore, 8B10 retained most of its anti-metastatic activity in IL-12p35-deficient mice whereas the activity of 3.3 and 6A6 were partially lost. All three mAbs were inactive in CD226-deficient mice. Altogether, these data demonstrate anti-CD96 need not block CD96-CD155 interactions (ie. immune checkpoint blockade) to promote NK cell anti-metastatic activity.

CD300 family receptors regulate eosinophil survival, chemotaxis, and effector functions.

The CD300 family of receptors is an evolutionary conserved receptor family that belongs to the Ig superfamily and is expressed predominantly by the myeloid lineage. Over the past couple of years, accumulating data have shown that eosinophils express various Ig superfamily receptors that regulate key checkpoints in their biology including their maturation, transition from the bone marrow to the peripheral blood, migration, adhesion, survival, and effector functions in response to numerous activating signals such as IL-4, IL-33, and bacteria. In this review, we will present the emerging roles of CD300 family receptors and specifically CD300a and CD300f in the regulation of these eosinophil activities. The structure and expression pattern of these molecules will be discussed and their involvement in suppressing or co-activating eosinophil functions in health and disease will be illustrated.

CD300f:IL-5 cross-talk inhibits adipose tissue eosinophil homing and subsequent IL-4 production

Eosinophils and their associated cytokines IL-4 and IL-5 are emerging as central orchestrators of the immune-metabolic axis. Herein, we demonstrate that cross-talk between the Ig-superfamily receptor CD300f and IL-5 is a key checkpoint that modifies the ability of eosinophils to regulate metabolic outcomes. Generation of Il5Tg/Cd300f−/− mice revealed marked and distinct increases in eosinophil levels and their production of IL-4 in the white and brown adipose tissues. Consequently, Il5Tg/Cd300f−/− mice had increased alternatively activated macrophage accumulation in the adipose tissue. Cd300f−/− mice displayed age-related accumulation of eosinophils and macrophages in the adipose tissue and decreased adipose tissue weight, which was associated with decreased diet-induced weight gain and insulin resistance. Notably, Il5Tg/CD300f−/− were protected from diet-induced weight gain and glucose intolerance. These findings highlight the cross-talk between IL-5 receptor and CD300f as a novel pathway regulating adipose tissue eosinophils and offer new entry points for therapeutic intervention for obesity and its complications.

Assembly and Expression of Shark Ig Genes.

Sharks are modern descendants of the earliest vertebrates possessing Ig superfamily receptor-based adaptive immunity. They respond to immunogen with Abs that, upon boosting, appear more rapidly and show affinity maturation. Specific Abs and immunological memory imply that Ab diversification and clonal selection exist in cartilaginous fish. Shark Ag receptors are generated through V(D)J recombination, and because it is a mechanism known to generate autoreactive receptors, this implies that shark lymphocytes undergo selection. In the mouse, the ∼2.8-Mb IgH and IgL loci require long-range, differential activation of component parts for V(D)J recombination, allelic exclusion, and receptor editing. These processes, including class switching, evolved with and appear inseparable from the complex locus organization. In contrast, shark Igs are encoded by 100-200 autonomously rearranging miniloci. This review describes how the shark primary Ab repertoire is generated in the absence of structural features considered essential in mammalian Ig gene assembly and expression.

Activation of human γδ T cells by cytosolic interactions of BTN3A1 with soluble phosphoantigens and the cytoskeletal adaptor periplakin.

The three butyrophilin BTN3A molecules, BTN3A1, BTN3A2, and BTN3A3, are members of the B7/butyrophilin-like group of Ig superfamily receptors, which modulate the function of T cells. BTN3A1 controls activation of human Vγ9/Vδ2 T cells by direct or indirect presentation of self and nonself phosphoantigens (pAg). We show that the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate binds to the intracellular B30.2 domain of BTN3A1 with an affinity of 1.1 μM, whereas the endogenous pAg isopentenyl pyrophosphate binds with an affinity of 627 μM. Coculture experiments using knockdown cell lines showed that in addition to BTN3A1, BTN3A2 and BTN3A3 transmit activation signals to human γδ T cells in response to (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate and the aminobisphosphonate drug zoledronate that causes intracellular accumulation of isopentenyl pyrophosphate. The plakin family member periplakin, identified in yeast two-hybrid assays, interacted with a membrane-proximal di-leucine motif, located proximal to the B30.2 domain in the BTN3A1 cytoplasmic tail. Periplakin did not interact with BTN3A2 or BTN3A3, which do not contain the di-leucine motif. Re-expression into a BTN3A1 knockdown line of wild-type BTN3A1, but not of a variant lacking the periplakin binding motif, BTN3A1Δexon5, restored γδ T cell responses, demonstrating a functional role for periplakin interaction. These data, together with the widespread expression in epithelial cells, tumor tissues, and macrophages detected using BTN3A antiserum, are consistent with complex functions for BTN3A molecules in tissue immune surveillance and infection, linking the cell cytoskeleton to γδ T cell activation by indirectly presenting pAg to the Vγ9/Vδ2 TCR.

B7–CD28 Costimulatory Signals Control the Survival and Proliferation of Murine and Human γδ T Cells via IL-2 Production

γδ T cells play key nonredundant roles in immunity to infections and tumors. Thus, it is critical to understand the molecular mechanisms responsible for γδ T cell activation and expansion in vivo. In striking contrast to their αβ counterparts, the costimulation requirements of γδ T cells remain poorly understood. Having previously described a role for the TNFR superfamily member CD27, we since screened for other nonredundant costimulatory receptors in γδ T cell activation. We report in this article that the Ig superfamily receptor CD28 (but not its related protein ICOS) is expressed on freshly isolated lymphoid γδ T cells and synergizes with the TCR to induce autocrine IL-2 production that promotes γδ cell survival and proliferation in both mice and humans. Specific gain-of-function and loss-of-function experiments demonstrated a nonredundant function for CD28 interactions with its B7 ligands, B7.1 (CD80) and B7.2 (CD86), both in vitro and in vivo. Thus, γδ cell proliferation was significantly enhanced by CD28 receptor agonists but abrogated by B7 Ab-mediated blockade. Furthermore, γδ cell expansion following Plasmodium infection was severely impaired in mice genetically deficient for CD28. This resulted in the failure to mount both IFN-γ-mediated and IL-17-mediated γδ cell responses, which contrasted with the selective effect of CD27 on IFN-γ-producing γδ cells. Our data collectively show that CD28 signals are required for IL-2-mediated survival and proliferation of both CD27(+) and CD27(-) γδ T cell subsets, thus providing new mechanistic insight for their modulation in disease models.

2B4 (CD244) is Involved in Eosinophil Adhesion and Chemotaxis, and its Surface Expression is Increased in Allergic Rhinitis after Challenge

A role for the subtypes of CD2 Ig superfamily receptors has been recently demonstrated in eosinophilic inflammation in experimental asthma and atopic asthmatics. We investigated the functions of 2B4 (CD244) molecules in eosinophil adhesion and chemotaxis, and correlated the results to the pathophysiology of allergic rhinitis (AR). Herein, we show that agonistic stimulation of 2B4 by C1.7, the anti-human 2B4 functional grade purified antibody, resulted in significant increase of eosinophils and eosinophil cell line (Eol-1 cells) adhesion to collagen type IV, and random migration. These functions were associated with tyrosine kinase phosphorylation of several protein residues of low molecular weight. Flow cytometry (FACS) experiments demonstrated that Eol-1 cells, normal peripheral blood eosinophils and eosinophils from AR patients, express surface 2B4 molecules. In vitro AR model demonstrated that the CC-chemokine receptor CCR3 stimulation by eotaxin induced significant increase in the expression of surface 2B4 in eosinophils and Eol-1 cells. Immunofluorescence confocal microscopy images showed that eotaxin induces also redistribution of 2B4 molecules towards the pseudopods in eosinophils and Eol-1 cells, changing their shape. Blocking of 2B4 molecules by the corresponding neutralizing antibody inhibited eotaxin induced Eol-1-adhesion, chemotaxis and the cytoskeleton changes. Pretreatment of Eol-1 cells with 1 microM genistein blocked eotaxin-induced Eol-1 adhesion, chemotaxis and 2B4 up-regulated expression. In vivo correlation demonstrated the expression of 2B4 molecules in eosinophils from AR patients to be significantly increased, after nasal provocation challenge. These results identify a novel role for 2B4 molecules in eosinophil functional migratory response and may point to a novel tyrosine kinase-mediated ligation between CCR3 receptor and 2B4 co-receptor in eosinophil chemotaxis. If so, then 2B4 molecules would be a novel target for therapeutic modalities in diseases characterized by eosinophilia such as AR.

Modulation of the inhibitory receptor leukocyte Ig-like receptor 1 on human natural killer cells

Leukocyte Ig-like receptor 1 (LIR-1) is an inhibitory Ig superfamily receptor with broad specificity for MHC-I expressed on leukocytes including natural killer (NK) and T cells. The extent of LIR-1 expression on NK cells is quite disparate between donors but the regulation of LIR-1 in NK cells is poorly understood. We examined expression profiles of LIR-1 on NK and T lymphocytes in 11 healthy donors over 1 year. Four of the 11 donors demonstrated substantial increases in LIR-1+ NK cells. High levels of LIR-1 expression were not correlated with exposure to human cytomegalovirus or the fraction of CD57+ NK cells in the donor. LIR-1 levels on ex vivo NK and CD56+ T cells were increased in vitro by short term exposure to IL-2 or IL-15 compared to control but not with various other cytokines tested. Sorted CD56bright NK cells also increased LIR-1 expression when cultured in IL-2. Maintenance of LIR-1 on longer term NK cells was also dependent on continuous stimulation by IL-15 or IL-2. While we could not detect increases in total LIR-1 mRNA in response to cytokine treatment by qPCR, we observed a shift in activity of LIR-1 promoter reporter constructs in the presence of IL-2 favoring the more translationally active transcript from the proximal promoter. Together these results show LIR-1 on NK cells is under the control of cytokines known to drive NK cell maturation and activation and suggest availability of such cytokines may alter the NK repertoire in vivo as we observed in several donors with fluctuating levels of LIR-1 on their NK cells.

Functional Diversity of Robo Receptor Immunoglobulin Domains Promotes Distinct Axon Guidance Decisions

Recognition molecules of the immunoglobulin (Ig) superfamily control axon guidance in the developing nervous system. Ig-like domains are among the most widely represented protein domains in the human genome, and the number of Ig superfamily proteins is strongly correlated with cellular complexity. In Drosophila, three Roundabout (Robo) Ig superfamily receptors respond to their common Slit ligand to regulate axon guidance at the midline: Robo and Robo2 mediate midline repulsion, Robo2 and Robo3 control longitudinal pathway selection, and Robo2 can promote midline crossing. How these closely related receptors mediate distinct guidance functions is not understood. We report that the differential functions of Robo2 and Robo3 are specified by their ectodomains and do not reflect differences in cytoplasmic signaling. Functional modularity of Robo2's ectodomain facilitates multiple guidance decisions: Ig1 and Ig3 of Robo2 confer lateral positioning activity, whereas Ig2 confers promidline crossing activity. Robo2's distinct functions are not dependent on greater Slit affinity but are instead due in part to differences in multimerization and receptor-ligand stoichiometry conferred by Robo2's Ig domains. Together, our findings suggest that diverse responses to the Slit guidance cue are imparted by intrinsic structural differences encoded in the extracellular Ig domains of the Robo receptors.

Anti-Siglec-F Antibody Reduces Allergen-Induced Eosinophilic Inflammation and Airway Remodeling

Siglec-F is a sialic acid-binding Ig superfamily receptor that is highly expressed on eosinophils. We have investigated whether administration of an anti-Siglec-F Ab to OVA-challenged wild-type mice would reduce levels of eosinophilic inflammation and levels of airway remodeling. Mice sensitized to OVA and challenged repetitively with OVA for 1 mo who were administered an anti-Siglec-F Ab had significantly reduced levels of peribronchial eosinophilic inflammation and significantly reduced levels of subepithelial fibrosis as assessed by either trichrome staining or lung collagen levels. The anti-Siglec-F Ab reduced the number of bone marrow, blood, and tissue eosinophils, suggesting that the anti-Siglec-F Ab was reducing the production of eosinophils. Administration of a F(ab')(2) fragment of an anti-Siglec-F Ab also significantly reduced levels of eosinophilic inflammation in the lung and blood. FACS analysis demonstrated increased numbers of apoptotic cells (annexin V(+)/CCR3(+) bronchoalveolar lavage and bone marrow cells) in anti-Siglec-F Ab-treated mice challenged with OVA. The anti-Siglec-F Ab significantly reduced the number of peribronchial major basic protein(+)/TGF-beta(+) cells, suggesting that reduced levels of eosinophil-derived TGF-beta in anti-Siglec-F Ab-treated mice contributed to reduced levels of peribronchial fibrosis. Administration of the anti-Siglec-F Ab modestly reduced levels of periodic acid-Schiff-positive mucus cells and the thickness of the smooth muscle layer. Overall, these studies suggest that administration of an anti-Siglec-F Ab can significantly reduce levels of allergen-induced eosinophilic airway inflammation and features of airway remodeling, in particular subepithelial fibrosis, by reducing the production of eosinophils and increasing the number of apoptotic eosinophils in lung and bone marrow.

Neogenin regulates spermatogonia germ cell differentiation

OBJECTIVE: Spermatogonia germ cell differentiation is an essential step for the spermatogenesis. The mechanisms underlying spermatogonial cell differentiation are beginning to be understood. Neogenin, a member of the Ig superfamily receptors, is believed to be a receptor for netrin-1 and RGMs (repulsive guidance molecules). Neogenin is highly expressed in testis. However, its role in spermatogenesis is unknown. To investigate the function of neogenin in the spermatogenesis.

Inhibition of Toll-like receptor and Fc receptor responses in macrophages by TREM-2 and DAP12 (44.7)

Abstract DAP12 is a transmembrane signaling adapter that contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain and is expressed in myeloid cells and natural killer cells. DAP12 associates with receptors that do not have intrinsic signaling ability and therefore use DAP12 to propagate signals. We examined the inflammatory response in macrophages lacking DAP12. Surprisingly, DAP12-deficient macrophages produced increased amounts of inflammatory cytokines in response to Toll-like receptor and Fc receptor stimulation, demonstrating that DAP12 inhibits inflammatory responses in wild-type macrophages. This inhibition was dependent upon the DAP12 ITAM. In vivo, DAP12-deficient mice were more susceptible to LPS/D-galactosamine-induced shock and more resistant to Listeria monocytogenes infection. TREM-2, a DAP12-associated Ig superfamily receptor, was responsible for the DAP12-mediated inhibition of TLR and FcR responses in macrophages. Additionally, a fusion protein containing the extracellular domain of TREM-2 bound to macrophages. These results suggest that the interaction of TREM-2 and its ligand on macrophages results in an inhibitory signal that can reduce the macrophage inflammatory response.

Human monoclonal antibodies to immunoglobulin superfamily receptor translocation- associated-3, -4 and -5 molecules as potential therapeutic agents for B-cell malignancies

B-cell malignancies, which include various forms of leukaemias and lymphomas as well as multiple myelomas, arise from neoplastic transformation of B-cells at distinct stages of differentiation and result in significant morbidity and mortality. The introduction of therapeutic mAbs against B-cell surface antigens has brought major improvements in disease outcome for several forms of B-cell malignancies. However, a substantial proportion of patients still succumb to these diseases and there is a crucial need for additional reagents directed at novel B-cell surface molecules. Ig superfamily receptor translocation-associated (IRTA) molecules may provide such new targets. IRTA molecules (also called Fc receptor homologues) are cell surface glycoproteins that contain 3 – 9 Ig domains in their extracellular regions and consensus immunoreceptor tyrosine-based activation and/or inhibitory motifs in their cytoplasmic tails. This family of molecules comprises six members, five of which (IRTA-1, -2, -3, -4 and -5) are preferentially expressed during B-cell differentiation. Preliminary evidence indicates that B-cell-associated IRTA molecules are also differentially expressed in various B-cell malignancies. As an approach to exploit the possible therapeutic value of IRTA molecules, Medarex has generated and characterised fully human mAbs with specificity and high affinity for IRTA-3, -4 or -5. These mAbs are claimed to be potentially useful for the treatment of B-cell malignancies, but this remains to be further investigated.

Variable domains in hagfish: NICIR is a polymorphic multigene family expressed preferentially in leukocytes and is related to lamprey TCR-like

The jawless vertebrates, represented by hagfish and lampreys, are the most advanced animals that apparently lack T cell and B cell receptors. As such, they offer unique opportunities for understanding the evolution of antigen receptors and variable (V)-type immunoglobulin (Ig)-like domains. In the present study, we describe four hagfish Ig superfamily (IgSF) members carrying V-type domains. None of them appeared to have direct counterparts in jawed vertebrates, indicating that many IgSF molecules have either evolved independently in jawed and jawless vertebrates or diverged to the extent that clear homology is no longer recognizable. One of the members encoded a molecule closely related to the previously described membrane protein designated novel ITAM (immunoreceptor tyrosine-based activation motif)-containing IgSF receptors (NICIR). We show here that NICIR is a polymorphic multigene family with at least three members and is expressed predominantly in peripheral blood leukocytes. Phylogenetic analysis indicates that among known proteins, NICIR is most closely related to the lamprey molecule recently proposed to be a potential ancestor of T cell receptors.

IRp60 modulates an array of human mast cell functions

RATIONALE: We have previously shown that the Ig-superfamily receptor IRp60 is expressed on human mast cells (MC), and that its cross-linking with FceRI using monoclonal antibodies inhibits mediator release. Our aim was to look for other functions that might be regulated by IRp60 and the underlying mechanism.

2B4 (CD244)-mediated activation of cytotoxicity and IFN-gamma release in human NK cells involves distinct pathways.

2B4 (CD244), a member of the CD2 subset of the Ig superfamily receptors, is expressed on all human NK cells, a subpopulation of T cells, basophils and monocytes. 2B4 activates NK cell mediated cytotoxicity, induces secretion of IFN-gamma and matrix metalloproteinases, and NK cell invasiveness. Although there have been several molecules shown to interact with 2B4, the signaling mechanism of 2B4-mediated activation of NK cells is still unknown. In this study, we found cross-linking of 2B4 on YT cells, a human NK cell line, results in the increased DNA binding activity of activator protein-1 (AP-1), an important regulator of nuclear gene expression in leukocytes. We investigated the possible role of various signaling molecules that may be involved in the activation of lytic function of YT cells via 2B4. Treatment of YT cells with various specific inhibitors indicate that 2B4-stimulation of YT cells in spontaneous and Ab-dependent cytotoxicity is Ras/Raf dependent and involves multiple MAPK signaling pathways (ERK1/2 and p38). However, only inhibitors of transcription and p38 inhibited 2B4-mediated IFN-gamma release indicating distinct pathways are involved in cytotoxicity and cytokine release. In this study we also show that 2B4 constitutively associates with the linker for activation of T cells (LAT) and that 2B4 may mediate NK cell activation via a LAT-dependent signaling pathway. These results indicate that 2B4-mediated activation of NK cells involves complex interactions involving LAT, Ras, Raf, ERK and p38 and that cytolytic function and cytokine production may be regulated by distinct pathways.

CD antigens 2001.

CD antigens 2001.

Differential expression of leukocyte receptor complex-encoded Ig-like receptors correlates with the transition from effector to memory CTL.

The human leukocyte receptor complex (LRC) on chromosome 19q13.4 encodes Ig superfamily receptors expressed on hemopoietic cells. Killer Ig-like receptors (KIR) are expressed in cytotoxic lymphocytes but other LRC molecules (Ig-like transcript(ILT)/leukocyte Ig-like receptor (LIR)) are more ubiquitous. We investigated expression of the ILT2/LIR1 inhibitory receptor compared with the related KIR. Both ILT2/LIR1 and KIR were expressed by peripheral CD8(+) T cells with a memory/effector phenotype. ILT2/LIR1(+) T cells demonstrated diverse TCRBV repertoires in contrast to KIR(+) T cells, while numbers of peripheral ILT2/LIR1(+) T cells were greater than KIR(+) T cells and the majority of ILT2/LIR1(+) T cells did not coexpress KIR. Analysis of CD8(+) T cells with specific HLA class I tetramers confirmed this pattern of expression, indicating differential regulation of LRC gene expression in T lymphocytes. Only a minor proportion of ILT2/LIR1(+) KIR(-) clones survived in vitro cloning, were more susceptible to anti-CD3 or cognate peptide induced cell death than KIR(+) T cells and exhibited lower levels of the Bcl-2 survival molecule. Our results indicate a sequential program of LRC-encoded receptor expression with initial ILT2/LIR1 expression in effector T cells and KIR gene transcription in the minor proportion of expanded clones which survives activation-induced cell death to become long term memory T cells.

Structural and functional aspects of three major glycoproteins of the human milk fat globule membrane.

The MUC1 mucin, lactadherin, and butyrophilin are 3 major components of the human milk fat globule membrane. The mucin inhibits binding of S-fimbriated Escherichia coli to buccal epithelial cells, and lactadherin prevents symptomatic rotavirus infection in breast-fed infants. Butyrophilin has been suggested to be a structural component of the human milk fat globule (HMFG) membrane and to have receptor functions, but has no known anti-infective activity. These HMFG glycoproteins also are present in skimmed milk, possibly associated with phospholipid micelles, while mucin is also in a soluble form. Mucin and lactadherin resist digestion in the stomach of milk-fed infants, while butyrophilin is rapidly degraded. The MUC1 mucin is an extended rod-like structure forming part of the glycocalyx on the surface of many epithelial cells and membranes of milk, and may act as a decoy for binding of infective agents. The extracellular segment of butyrophilin has homology to Ig superfamily receptors and an intracellular domain with homology to developmentally regulated proteins. Lactadherin is a laterally mobile cell adhesion molecule that interacts with integrins and has a novel means of membrane-association involving specific binding to phosphatidylserine. The structural and functional aspects of these glycoproteins are discussed with regard to their role in human milk for breast-fed infants.

The PTPμ Protein-tyrosine Phosphatase Binds and Recruits the Scaffolding Protein RACK1 to Cell-Cell Contacts

PTPmu, an Ig superfamily receptor protein-tyrosine phosphatase, promotes cell-cell adhesion and interacts with the cadherin-catenin complex. The signaling pathway downstream of PTPmu is unknown; therefore, we used a yeast two-hybrid screen to identify additional PTPmu interacting proteins. The membrane-proximal catalytic domain of PTPmu was used as bait. Sequencing of two positive clones identified the scaffolding protein RACK1 (receptor for activated protein C kinase) as a PTPmu interacting protein. We demonstrate that RACK1 interacts with PTPmu when co-expressed in a recombinant baculovirus expression system. RACK1 is known to bind to the src protein-tyrosine kinase. This study demonstrates that PTPmu association with RACK1 is disrupted by the presence of constituitively active src. RACK1 is thought to be a scaffolding protein that recruits proteins to the plasma membrane via an unknown mechanism. We have shown that the association of endogenous PTPmu and RACK1 in a lung cell line is increased at high cell density. We also demonstrate that the recruitment of RACK1 to both the plasma membrane and cell-cell contact sites is dependent upon the presence of the PTP mu protein in these cells. Therefore, PTPmu may be one of the proteins that recruits RACK1 to points of cell-cell contact, which may be important for PTPmu-dependent signaling in response to cell-cell adhesion.