Intracellular Immunoreceptor Tyrosine-based Inhibitory Motifs 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.

Leukocyte immunoglobulin-like receptor subfamily B: therapeutic targets in cancer.

Inhibitory leukocyte immunoglobulin-like receptors (LILRBs 1–5) transduce signals via intracellular immunoreceptor tyrosine-based inhibitory motifs that recruit phosphatases to negatively regulate immune activation. The activation of LILRB signaling in immune cells may contribute to immune evasion. In addition, the expression and signaling of LILRBs in cancer cells especially in certain hematologic malignant cells directly support cancer development. Certain LILRBs thus have dual roles in cancer biology—as immune checkpoint molecules and tumor-supporting factors. Here, we review the expression, ligands, signaling, and functions of LILRBs, as well as therapeutic development targeting them. LILRBs may represent attractive targets for cancer treatment, and antagonizing LILRB signaling may prove to be effective anti-cancer strategies.

Immunosuppressive receptor LILRB1 acts as a potential regulator in hepatocellular carcinoma by integrating with SHP1.

Immunosuppressive receptor LILRB1 regulates tumors progression by transducing immune inhibitory signals via intracellular immunoreceptor tyrosine-based inhibitory motifs. However, its role in Hepatocellular Carcinoma (HCC) remains vague. This study is aimed to disclose the association between LILRB1 and HCC. Immunoblotting and qRT-PCR were employed to evaluate the level of LILRB1 in hepatocarcinoma cells. LILRB1-positive cells in tissue array were measured using immunohistochemistry staining. The relation among LILRB1, SHP1 and SHP2 and survival rates were analyzed using Gene Expression Profiling Interactive Analysis (GEPIA) and Oncomine database. LILRB1 was robustly reduced in hepatocarcinoma cells compared to normal cells. Clinically, LILRB1 was significantly higher in 49 of 75 (65%) paired paracarcinoma tissues than that in paired HCC samples. 48 of 75 (64%) HCC subjects in tissue microarray showed low level of LILRB1, compared to 25 of 75 (33%) in paired-adjacent tissues. Oncomine database and GEPIA analysis confirmed that LILRB1 was lower in HCC than normal tissues. Additionally, lowLILRB1 had a significant association with clinicopathological characteristics and Disease Free Survival, but no association with Overall Survival in HCC patients. Mechanismly, positive correlation between LILRB1 and SHP1, but not SHP2 was observed in HCC. LILRB1 possibly plays an antitumor effect in hepatocarcinoma cells by integrating SHP1, providing evidence that LILRB1 might be involved in the pathologic progression of HCC.

Pivotal role of the carbohydrate recognition domain in self-interaction of CLEC4A to elicit the ITIM-mediated inhibitory function in murine conventional dendritic cells in vitro.

C-type lectin receptors (CLRs), pattern recognition receptors (PRRs) with a characteristic carbohydrate recognition domain (CRD) in the extracellular portion, mediate crucial cellular functions upon recognition of glycosylated pathogens and self-glycoproteins. CLEC4A is the only classical CLR that possesses an intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM), which possibly transduces negative signals. However, how CLEC4A exerts cellular inhibition remains unclear. Here, we report that the self-interaction of CLEC4A through the CRD is required for the ITIM-mediated suppressive function in conventional dendritic cells (cDCs). Human type 2 cDCs (cDC2) and monocytes display a higher expression of CLEC4A than cDC1 and plasmacytoid DCs (pDCs) as well as B cells. The extracellular portion of CLEC4A specifically binds to a murine cDC cell line expressing CLEC4A, while its extracellular portion lacking the N-glycosylation site or the EPS motif within the CRD reduces their association. Furthermore, the deletion of the EPS motif within the CRD or ITIM in CLEC4A almost completely impairs its suppressive effect on the activation of the murine cDC cell line, whereas the absence of the N-glycosylation site within the CRD exhibits partial inhibition on their activation. On the other hand, antagonistic monoclonal antibody (mAb) to CLEC4A, which inhibits the self-interaction of CLEC4A and its downstream signaling in murine transfectants, enhances the activation of monocytes and monocyte-derived immature DCs upon stimulation with a Toll-like receptor (TLR) ligand. Thus, our findings suggest a pivotal role of the CRD in self-interaction of CLEC4A to elicit the ITIM-mediated inhibitory signal for the control of the function of cDCs.

A Strategy for Suppressing Macrophage-mediated Rejection in Xenotransplantation.

Although xenografts are one of the most attractive strategies for overcoming the shortage of organ donors, cellular rejection by macrophages is a substantial impediment to this procedure. It is well known that macrophages mediate robust immune responses in xenografts. Macrophages also express various inhibitory receptors that regulate their immunological function. Recent studies have shown that the overexpression of inhibitory ligands on porcine target cells results in the phosphorylation of tyrosine residues on intracellular immunoreceptor tyrosine-based inhibitory motifs on macrophages, leading to the suppression of xenogenic rejection by macrophages. It has also been reported that myeloid-derived suppressor cells, a heterogeneous population of immature myeloid cells, suppress not only NK and cytotoxic T lymphocyte cytotoxicity but also macrophage-mediated cytotoxicity. This review is focused on the recent findings regarding strategies for inhibiting xenogenic rejection by macrophages.

An agonist TIGIT mab suppresses regulatory T cell activity via an IL-2-mediated mechanism

Abstract The co-inhibitory receptor TIGIT (T cell immunoreceptor with Ig and ITIM domains) is a 26 kDa immunoglobulin domain-containing transmembrane protein that contains both intracellular ITIM (Immunoreceptor Tyrosine-based Inhibitory Motif) and ITT (Immunoreceptor Tail Tyrosine) like domains and is found on the surface of Natural Killer and T cells. CD155 (or the poliovirus receptor) and CD112 (or Nectin-2) are natural ligands for both TIGIT and the activating receptor CD226 (or DNAM-1). In addition to the complexity of the TIGIT/DNAM-1 interplay, TIGIT has been demonstrated to drive both cell intrinsic and extrinsic functions. TIGIT binding to CD155 on Dendritic cells can drive reduction of pro-inflammatory cytokines while inducing tolerogenic cytokine secretion (Yu, 2009). More recently regulatory T cells have been demonstrated to express high levels of TIGIT and upon ligation have been demonstrated to induce effector molecules like FGL2 and reported to promote suppressive function (Joller, 2014). We’ve identified an agonist anti-TIGIT monoclonal antibody, TGTB227, that inhibits T cell responses in vitro in both cellular reporter assays and primary memory T cell assays. TGTB227 is a human IgG1k mab that reduced the expression of the transcription factor, Foxp3, on Tregs. Moreover, TGTB227, but not the related TGTB277, reduced the functional suppressive ability of both natural and induced Tregs. Finally, these data support a TIGIT driven intrinsic CD25/IL-2-dependent mechanism for the modulation of Treg activity.

Chimeric Antigen Receptor T Cells Specific for CLL-1 for Treatment of Acute Myeloid Leukemia

Chimeric antigen receptor (CAR) T cells are a novel therapeutic approach which have shown good clinical outcomes in patients receiving CD19 CAR T cells for B cell acute lymphoblastic leukemia. CAR T cells are made to express a CAR that recognizes a specific surface antigen on a cell upon which they can then exert cytotoxic effects. We aim to extend the success of this therapy to acute myeloid leukemia (AML), a disease with generally poor clinical outcomes. However, due to the genetic heterogeneity characteristic of AML and the limited number of distinctive tumor markers, it has been difficult to find effective targets for CAR T cells on AML.C-type lectin like molecule-1 (CLL-1), also known as CD371, is a transmembrane glycoprotein that is expressed on about 90% of AML patient samples. CLL-1 may function as an inhibitory signaling receptor, as it contains an intracellular immunoreceptor tyrosine based inhibitory motif (ITIM). CLL-1 is primarily expressed on myeloid lineage cells in the bone marrow and in peripheral blood. While CLL-1 has been shown to be expressed on some granulocytes in the spleen, it is not reported to be expressed in non-hematopoietic tissues or on hematopoietic stem cells, which make CLL-1 a potential therapeutic target for AML.We generated two types of CLL-1 CARs, termed A and B, by using two different single chain variable fragments (scFvs) recognizing CLL-1. We used second generation CARs containing the scFvs, CD8 hinge and transmembrane domain, 4-1BB co-stimulatory domain, and CD3 zeta signaling domains. Using a lentiviral vector, we transferred the CAR gene into healthy donor human T cells and detected CAR expression by flow cytometry. We then tested the specific cytotoxic effects of CLL-1 CART-A and B on a CLL-1-expressing AML cell line, U937, by conducting a 4-hour chromium release assay. We found that both CAR T cells exhibited a dose-dependent killing of U937 (CLL-1 positive), while the untransduced (UTD) T cells had no cytotoxic effect (Figure 1A). We also found that U937 induces degranulation of CLL-1 CAR T cells as measured by CD107a expression by flow cytometry, while Ramos, a CLL-1 negative cell line, does not (Figure 1B).We then proceeded to investigate the in vivo efficacy of the CAR T cells. We injected NOD/SCID/IL2RG-null (NSG) mice with 1 x 106 THP-1 cells, a CLL-1 positive cell line. We confirmed engraftment by bioluminescent imaging (BLI) after 7 days and then injected 4 x 106 UTD, CLL-1 CART-A or CLL-1 CART-B. Surprisingly, only one of the CAR constructs, CLL-1 CART-A, showed significant activity in vivo, although both CARs had shown comparable activity in vitro. CLL-1 CART-A treated mice had delayed tumor progression and significantly increased length of survival (85 days vs. 63 days, p = 0.0021) compared to mice injected with UTD (Figure 1C and D). While CLL-1 CART-B treated mice also exhibited slower tumor growth and a trend towards better survival (72 days vs. 63 days, p=0.0547) this was not statistically significant. Post-mortem analysis showed that human T cells that continued to express CAR were present in the tumor, bone marrow and spleen of mice treated with CLL-1 CART-A only, while the UTD and CLL-1 CART-B treated mice showed tumor in all organs and no T cells.In summary, we show that CLL-1 CAR T cells can selectively eliminate CLL-1 positive target cells in vitro and in vivo, albeit with different degrees of efficacy modulated by the scFv. Studies are ongoing to investigate the mechanism behind the differential activity of these CAR constructs and to increase the long-term antitumor efficacy. Our results demonstrate that targeting CLL-1 using CAR T cell therapy holds promise for the treatment of AML. [Display omitted] DisclosuresCooper:WUGEN: Consultancy, Equity Ownership.

CD52 glycan binds the proinflammatory B box of HMGB1 to engage the Siglec-10 receptor and suppress human T cell function

CD52, a glycophosphatidylinositol (GPI)-anchored glycoprotein, is released in a soluble form following T cell activation and binds to the Siglec (sialic acid-binding Ig-like lectin)-10 receptor on T cells to suppress their function. We show that binding of CD52-Fc to Siglec-10 and T cell suppression requires the damage-associated molecular pattern (DAMP) protein, high-mobility group box 1 (HMGB1). CD52-Fc bound specifically to the proinflammatory Box B domain of HMGB1, and this in turn promoted binding of the CD52 N-linked glycan, in α-2,3 sialic acid linkage with galactose, to Siglec-10. Suppression of T cell function was blocked by anti-HMGB1 antibody or the antiinflammatory Box A domain of HMGB1. CD52-Fc induced tyrosine phosphorylation of Siglec-10 and was recovered from T cells complexed with HMGB1 and Siglec-10 in association with SHP1 phosphatase and the T cell receptor (TCR). Thus, soluble CD52 exerts a concerted immunosuppressive effect by first sequestering HMGB1 to nullify its proinflammatory Box B, followed by binding to the inhibitory Siglec-10 receptor, triggering recruitment of SHP1 to the intracellular immunoreceptor tyrosine-based inhibitory motif of Siglec-10 and its interaction with the TCR. This mechanism may contribute to immune-inflammatory homeostasis in pathophysiologic states and underscores the potential of soluble CD52 as a therapeutic agent.

A dual positive and negative regulation of monocyte activation by leukocyte Ig-like receptor B4 depends on the position of the tyrosine residues in its ITIMs

The leukocyte Ig-like receptor B4 (LILRB4) is an inhibitory cell surface receptor, primarily expressed on mono-myeloid cells. It contains 2 C-type Ig-like extracellular domains and a long cytoplasmic domain that contains three intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Data suggest that LILRB4 suppresses Fc receptor-dependent monocyte functions via its ITIMs, but relative contributions of the three ITIMs are not characterised. To address this, tyrosine (Tyr) residues at positions 337, 389 and 419 were single, double or triple mutated to phenylalanine and stably transfected into a human monocytic cell line, THP-1. Intact Tyr389 was sufficient to maximally inhibit FcγRI-mediated TNF-α production in THP-1 cells, but, paradoxically, Tyr337 significantly enhanced TNF-α production. In contrast, bactericidal activity was significantly enhanced in mutants containing Tyr419, while Tyr337 markedly inhibited bacteria killing. Taken together, these results indicate that LILRB4 might have dual inhibitory and activating functions, depending on the position of the functional tyrosine residues in its ITIMs and/or the nature of the stimuli.

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.

The middle tyrosine residue of tyrosine-based inhibitory signalling motifs is a key molecule in inhibitory functions of the leukocyte immunoglobulin-like receptor B4 in FcγRI mediated cytokine production and bacterial phagocytosis. (CCR3P.211)

Abstract The leukocyte immunoglobulin-like receptor B4 (LILRB4) is an inhibitory cell surface receptor, primarily expressed on mono-myeloid cells. LILRB4 is a potent inhibitor of FcγRI-mediated pro-inflammatory cytokine production by monocytes and induces tolerogenic dendritic cells in vitro. It is believed that LILRB4 regulates monocyte/macrophage activation through its three intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) by dephosphorylation of non-receptor tyrosine kinases via recruitment of Src homology phosphatase-1 (SHP-1). However, the relative contribution of these ITIMs to LILRB4-mediated inhibition of monocyte/macrophage functions is poorly characterised. To address this, the three functional tyrosine residues of LILRB4 ITIMs at positions 337, 389 and 419 were single, double or triple mutated to non-functional phenylalanine and stably transfected into THP-1 cells. The effects of these mutants on FcγRI-mediated monocyte functions were then assessed. We show that the middle tyrosine residue (Y389) alone maximally inhibited but the proximal tyrosine residue (Y337) enhanced FcγRI-mediated TNF-α production and Fc-dependent bacterial phagocytosis. These results for the first time show that LILRB4 may have variable functions in vitro dependent on the position of the tyrosine residues in its ITIMs. The underlying mechanisms and in vivo implications of these adaptable functions require further investigations.

182: The leukocyte immunoglobulin-like receptor B4 differentially regulates FcγRI-dependent TNF production via its three intracellular immunoreceptor tyrosine-based inhibitory signalling motifs (ITIMs)

The leukocyte immunoglobulin-like receptor (LILR) B4 belongs to a family of activating and inhibitory cell surface receptors that have been increasingly recognised as key regulators of the threshold and amplitude of leukocyte activation. LILRB4 which is highly expressed on monocytes and macrophages transduces inhibitory signals through its three intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Clinically, increased expression is associated with better clinical outcome in renal transplant recipients contrasted with impaired immune effector cell functions in HIV infection. However, mechanisms of how LILRB4 regulates monocyte activation and the exact contribution of the three ITIMs for its immune-regulatory functions are poorly characterised. In this study we show that co-ligation of LILRB4 with FcγRI on THP-1 cells and primary monocytes markedly inhibited the production of TNF, a key pro-inflammatory cytokine. Cross-linking of FcγRI on THP-1 cells increased phosphorylation key protein tyrosine kinase signaling molecules and an adaptor protein that targets protein tyrosine kinases for degradation (c-Cbl). Co-ligation of LILRB4 and FcγRI considerably reduced FcγRI-mediated phosphorylation of these signaling molecules and terminated cellular activation by recruiting Src homology region 2 domain-containing phosphatase-1 (SHP-1)-like molecule. Importantly, using site-targeted mutagenesis we found for the first time that the middle (Y412) and distal (Y442) tyrosine residues in its ITIM motifs were critical for the inhibitory functions of LILRB4 and Y412 alone was sufficient for maximal inhibition. By contrast, the proximal tyrosine residue at Y360 showed unexpectedly enhanced Fc γ RI-mediated TNF production. Collectively, our results suggest that LILRB4 is a potent immune regulatory receptor in which combined contribution of its multiple ITIMs may fine tune innate immune responses.

Inhaled corticosteroids increase siglec-5/14 expression in sputum cells of COPD patients.

Recent studies show that several Siglec receptors, such as Siglec-8 and Siglec-14, may be important therapeutic targets in asthma and COPD. Siglecs are a family of lectins belonging to the immunoglobulin superfamily and recognize sialic acid residues of glycoproteins. Most of Siglecs have intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIM), implicating them in the suppression of immunoreceptor signaling. Siglec-5/14 may be involved in the negative regulation of innate immune responses. The aim of this study was to analyze Siglec-5/14 expression in induced sputum cells of COPD patients in the following treatment combinations: (1) a long-acting beta2-agonist, formoterol; (2) formoterol combined with a long-acting antimuscarinic agent, tiotropium; and (3) formoterol combined with an inhaled corticosteroid or formoterol combined with tiotropium and with an inhaled corticosteroid. Siglec expression was assessed in sputum cells by flow cytometry using a specific monoclonal antibody. Double staining of cells indicated that Siglec-5/14 is expressed in monocyte/macrophages and neutrophils, but not in lymphocytes. Siglec-5/14 expression was significantly higher in patients receiving combined therapy including inhaled corticosteroids compared with patients taking only formoterol or formoterol + tiotropium. Our results suggest that inhaled corticosteroids may exert beneficial or negative effects, depending on the patients' phenotype, through increased immunosuppressive Siglec-5 or immunoactivatory Siglec-14 receptors, respectively.

Ligand Binding and Signaling of Dendritic Cell Immunoreceptor (DCIR) Is Modulated by the Glycosylation of the Carbohydrate Recognition Domain

C-type lectins are innate receptors expressed on antigen-presenting cells that are involved in the recognition of glycosylated pathogens and self-glycoproteins. Upon ligand binding, internalization and/or signaling often occur. Little is known on the glycan specificity and ligands of the Dendritic Cell Immunoreceptor (DCIR), the only classical C-type lectin that contains an intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM). Here we show that purified DCIR binds the glycan structures Lewisb and Man3. Interestingly, binding could not be detected when DCIR was expressed on cells. Since DCIR has an N-glycosylation site inside its carbohydrate recognition domain (CRD), we investigated the effect of this glycan in ligand recognition. Removing or truncating the glycans present on purified DCIR increased the affinity for DCIR-binding glycans. Nevertheless, altering the glycosylation status of the DCIR expressing cell or mutating the N-glycosylation site of DCIR itself did not increase glycan binding. In contrast, cis and trans interactions with glycans induced DCIR mediated signaling, resulting in a decreased phosphorylation of the ITIM sequence. These results show that glycan binding to DCIR is influenced by the glycosylation of the CRD region in DCIR and that interaction with its ligands result in signaling via its ITIM motif.

Roles of Allergin-1 in the pathogenesis of allergic diseases

As a major event in type Ⅰ allergic reaction,degranulation of mast cells is triggered by the interaction between specific IgEs and high affinity IgE receptors (FceRIs) on mast cell membrane,and is followed by the release of a battery of inflammatory mediators,such as histamine,leukotrienes and arachidonic acid,and these inflammatory mediators in turn induce a series of allergic symptoms.Recently,Allergin-1 has been found to be an immunoglobulin-like receptor preferentially expressed on mast cells.The crosslinking of Allergin-1 to FceRIs on mast cells may inhibit the signal transduction from FceRIs to intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM),generate ITIM inhibitory signals,which then participate in the regulation of mast cell degranulation,and finally block the initiation of allergic inflammation. Key words: Allergens; Allergin-1; Mast cells

Synthetic peptides containing ITIM-like sequences of IREM-1 inhibit BAFF-mediated regulation of interleukin-8 expression and phagocytosis through SHP-1 and/or PI3K

B-cell activation factor of the tumour necrosis factor family (BAFF), an important regulator of B-cell survival, has recently been found to be expressed on the surface of murine and human macrophages and engagement with its receptor was shown to trigger induction of pro-inflammatory mediators and block phagocytic activity. In an effort to generate immunomodulatory agents that can regulate BAFF-mediated signal, decapeptides representing the intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) of immune receptor expressed on myeloid cells (IREM)-1, an inhibitory transmembrane protein expressed on myeloid cells, were synthesized in conjugation with HIV-transactivator of transcription (TAT)(48-57,) which facilitates the internalization of peptides into cells. Interestingly, all five of these synthetic peptides, representing the five ITIM-like sequences present in the cytoplasmic tail of IREM-1, exhibited inhibitory action against BAFF-mediated induction of matrix metalloproteinase-9 and interleukin-8 expression. Inhibitor assay and immunoprecipitation assay followed by Western blotting demonstrated that the inhibitory action was mediated by Src homology 2 (SH2)-containing tyrosine phosphatase (SHP)-1 and/or phosphoinositide 3-kinase (PI3K). ELISA-based nuclear factor-κB DNA binding assay observed that the synthetic peptides blocked the activation of nuclear factor-κB in an SHP-1 and phosphoinositide 3-kinase-dependent manner. Three of these synthetic peptides exhibited varying degrees of inhibitory action against BAFF-mediated blockage of phagocytosis in a SHP-1 and PI3K-dependent manner. These data indicate that the synthetic peptides are capable of blocking BAFF-mediated regulation of macrophage activities through the activation of SHP-1 and PI3K as well as inhibition of nuclear factor-κB activation.

Multiple bovine FcγRIIb sub-isoforms generated by alternative splicing

Receptors for the Fc portion of immunoglobulin molecules (FcR) provide an important and vital link between circulating antibody and cellular effector functions. These receptors have been well characterized in human and murine species, however few of these receptors have been investigated in livestock. FcγRII (CD32) is an FcR previously shown in mice and humans to exist in multiple isoforms, both activating (FcγRIIa, FcγRIIc) and inhibitory (FcγRIIb), on a wide variety of cells including B cells, T cells, dendritic cells, monocytes, macrophages and platelets. On B cells, FcγRIIb acts to suppress cell activation and immunoglobulin production by means of an intracellular immunoreceptor tyrosine-based inhibitory motif signaling domain. Two sub-isoforms of FcγRIIb, designated b1 and b2, distinguished by the inclusion of an additional cytoplasmic exon in the b1 form, have been demonstrated in humans and mice, whereas only one sequence corresponding to the human and mouse b2 isoform has been identified in cattle. In this study, the expression profile of FcγRIIb in bovine blood mononuclear cells was characterized by collecting blood samples from mature cattle of dairy and beef breeds, and determining their FcγRIIb mRNA expression profile by RT-PCR. Analysis revealed the presence of two uncharacterized bovine FcγRIIb transcripts in addition to the single previously published transcript. Analysis of the first unknown transcript revealed high homology with published human and murine FcγRIIb1 sequences. This transcript was present in all cell types examined, with little variation in primary sequence between individuals or among breeds. The second unknown sequence was found to be homologous to the murine FcγRIIb3 (IgG-binding protein or soluble FcγR in humans) sequence. This transcript appears to have a much more limited expression profile, which may indicate that expression varies with the cellular activation-state of the cell. These results indicate that cattle, like humans and mice, express multiple sub-isoforms of FcγRIIb. These findings add further complexity to the regulation of IgG-mediated immunity and provide new insight into the role Fc receptors play in antigen acquisition and presentation in cattle.

Modulation of lymphocyte function with inhibitory CD2: Loss of NK and NKT cells

Analysis of the NK cell developmental pathway suggests that CD2 expression may be important in regulating NK maturation. To test this hypothesis, we developed mice containing only an inhibitory CD2 molecule by linking the extracellular domain of CD2 to an intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM) motif. Mice containing the CD2 Tg(ITIM) transgene, introduced into a CD2 KO background, have no morphologically detectable lymph nodes, although development of the thymus appears normal. In addition, these mice had major loss of both NK and NKT subsets in peripheral organs, while T and B cell frequencies were intact. Expression of CD2 was low on T cells and lacking on B cells and functional defects were observed in these populations. NKT cells expressing CD4 were absent, while the CD8 + and double negative NKT cells were retained. Small subsets of NK cells were detected but expression of CD2 on these cells was very low or absent, and their maturation was impaired. Based on the phenotype described here, we believe that these mice represent a unique model to study lymphoid organ and lymphocyte development.

Signaling and immune regulatory role of the dendritic cell immunoreceptor (DCIR) family lectins: DCIR, DCAR, dectin-2 and BDCA-2

This review focuses on a distinct family of dendritic cells (DC) expressing C-type lectins that include DC immunoreceptor (DCIR), DC immunoactivating receptor (DCAR), DC-associated C-type lectin (dectin)-2 and blood DC antigen (BDCA)-2. DCIR is a type II C-type lectin expressed on antigen presenting cells and granulocytes and acts as an inhibitory receptor via an intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM). In contrast, DCAR has been identified as a molecule that forms a putative pair with DCIR. While both molecules share the highly homologous extracellular lectin domain, DCAR lacks the ITIM in its short cytoplasmic tail and acts as an activating receptor through association with the Fc receptor γ chain. Two other lectins, dectin-2 and BDCA-2, are highly related to DCAR by similarities of their amino acid sequence, molecular structure and chromosomal localization. Although they also lack the ITIM, they are capable of transducing signal to regulate cellular functions positively or negatively. Here we propose to designate these four highly related molecules as the “DCIR family lectins” and discuss their signaling mechanisms, carbohydrate recognition, and other features that contribute to the function of DC to control immunity.

Genomic analysis of immunity in a Urochordate and the emergence of the vertebrate immune system: "waiting for Godot".

Genome-wide sequence analysis in the invertebrate chordate, Ciona intestinalis, has provided a comprehensive picture of immune-related genes in an organism that occupies a key phylogenetic position in vertebrate evolution. The pivotal genes for adaptive immunity, such as the major histocompatibility complex (MHC) class I and II genes, T-cell receptors, or dimeric immunoglobulin molecules, have not been identified in the Ciona genome. Many genes involved in innate immunity have been identified, including complement components, Toll-like receptors, and the genes involved in intracellular signal transduction of immune responses, and show both expansion and unexpected diversity in comparison with the vertebrates. In addition, a number of genes were identified which predicted integral membrane proteins with extracellular C-type lectin or immunoglobulin domains and intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and immunoreceptor tyrosine-based activation motifs (ITAMs) (plus their associated signal transduction molecules), suggesting that activating and inhibitory receptors have an MHC-independent function and an early evolutionary origin. A crucial component of vertebrate adaptive immunity is somatic diversification, and the recombination activating genes (RAG) and activation-induced cytidine deaminase (AID) genes responsible for the Generation of diversity are not present in Ciona. However, there are key V regions, the essential feature of an immunoglobulin superfamily VC1-like core, and possible proto-MHC regions scattered throughout the genome waiting for Godot.