Tyrosine-based Signaling Motifs Research Articles

Counteracting immunotyrosine-based signaling motifs augment zebrafish leukocyte immune-type receptor-mediated phagocytic activity

Leukocyte immune-type receptors (LITRs) represent a polymorphic and polygenic family of immunoregulatory proteins originally discovered in channel catfish (Ictalurus punctatus; IpLITRs). Belonging to the immunoglobulin superfamily (IgSF), IpLITRs are generally classified as stimulatory or inhibitory types based on their utilization of various intracellular tyrosine-based signaling motifs. While research has shown that IpLITRs can activate as well as abrogate different immune cell effector responses including phagocytosis, recent identification of LITRs within the zebrafish genome (Danio rerio; DrLITRs) revealed the existence of fish LITR-types uniquely containing counteracting stimulatory and inhibitory cytoplasmic tail (CYT) region motifs (i.e., an immunoreceptor tyrosine-based activation motif; ITAM, and immunoreceptor tyrosine-based inhibitory motif; ITIM) within the same receptor. This arrangement is unusual as these motifs typically exist on separate stimulatory (i.e., ITAM-containing) or inhibitory (i.e., ITIM-containing) immunoregulatory receptors that then co-engage to fine-tune cellular signaling and effector responses. Using a flow cytometric-based phagocytosis assay, we show here that engagement of DrLITR 1.2-expressing cells with antibody coated 4.5 μm beads causes a robust ITAM-dependent phagocytic response and reveal that its tandem ITIM motif surprisingly enhances the DrLITR 1.2-induced phagocytic activity while simultaneously decreasing the receptors ability to bind the beads. Confocal microscopy studies also revealed that the ITIM-associated inhibitory signaling molecule SHP-2 is localized to the phagocytic synapse during the phagocytic response. Overall, these results provide the first functional characterization of teleost immune receptors containing a tandem ITAM and ITIM and allow for the proposal of an intracytoplasmic tail signaling model for ITIM-mediated enhancement of ITAM-dependent cellular activation.

PD-1 inhibits T cell actin remodeling at the immunological synapse independently of its signaling motifs.

Engagement of the receptor programmed cell death molecule 1 (PD-1) by its ligands PD-L1 and PD-L2 inhibits T cell-mediated immune responses. Blocking such signaling provides the clinical effects of PD-1-targeted immunotherapy. Here, we investigated the mechanisms underlying PD-1-mediated inhibition. Because dynamic actin remodeling is crucial for T cell functions, we characterized the effects of PD-1 engagement on actin remodeling at the immunological synapse, the interface between a T cell and an antigen-presenting cell (APC) or target cell. We used microscopy to analyze the formation of immunological synapses between PD-1+ Jurkat cells or primary human CD8+ cytotoxic T cells and APCs that presented T cell-activating antibodies and were either positive or negative for PD-L1. PD-1 binding to PD-L1 inhibited T cell spreading induced by antibody-mediated activation, which was characterized by the absence of the F-actin-dense distal lamellipodial network at the immunological synapse and the Arp2/3 complex, which mediates branched actin formation. PD-1-induced inhibition of actin remodeling also prevented the characteristic deformation of T cells that contact APCs and the release of cytotoxic granules. We showed that the effects of PD-1 on actin remodeling did not require its tyrosine-based signaling motifs, which are thought to mediate the co-inhibitory effects of PD-1. Our study highlights a previously unappreciated mechanism of PD-1-mediated suppression of T cell activity, which depends on the regulation of actin cytoskeleton dynamics in a signaling motif-independent manner.

Antigen presentation genes in gadoid species (haddock: Melanogrammus aeglefinus and Atlantic cod: Gadus morhua) raise questions about cross-presentation pathways and glycosylated beta-2-microglobulin

The Atlantic cod immune system deviates from antigen presentation processes seen in other vertebrates in that it lacks the necessary genes for exogenous antigen presentation (i.e., MHC-II and li) and a key MHC-II interacting molecule necessary for T-helper cell function (i.e., CD4), while possessing an expanded repertoire of MHC-I genes that facilitate endogenous antigen presentation. These observations, combined with the identification of putative endosomal sorting signals in MHC-I cytoplasmic tails, have led to speculation that cod rely on cross-presentation of exogenous antigens to elicit cytotoxic T-lymphocyte responses against extracellular threats. In light of this suggestion, we investigated MHC-I transcriptional profiles and endosomal sorting signals in a closely related gadoid species, the haddock. Analysis of transcripts from one individual identified 13 unique MHC-I molecules, including two non-classical molecules as determined by the level of conservation at their peptide anchoring sites. This suggests that like the cod, the haddock has an expanded MHC-I repertoire. Analysis of haddock MHC-I cytoplasmic tail sequences revealed that the dileucine- and tyrosine-based endosomal signaling motifs, that are suggested to facilitate cross-presentation in cod, were absent. Closer examination of the cod signaling motifs, including their relative position in the cytoplasmic tail region, indicates that these motifs might be non-functional, further supporting the need for functional studies to assess cross-presentation. Finally, in silico analysis and in vitro N-type de-glycosylation experiments demonstrate that haddock and cod beta-2-microglobulin (β2M) are glycosylated at the same NQT sequon. Interestingly, whole genome tBLASTn searches also revealed that putative β2 M glycosylation sites appear frequently within the Gadiformes lineage, as the predictive NQT and other N-X-S/T sequons were located in β2M orthologues from 19 of the 25 additional gadoid genomes analyzed. Though the exact significance of β2M glycosylation has yet to be elucidated, phylogenetic comparisons predict that the same NQT glycosylation sequence occurs in 13 additional species comprising four different orders of Actinopterygii (Gymnotiformes, Esociformes, Beryciformes and Perciformes). This suggests either that this feature has arisen independently in multiple lineages or that it comes from a common ancestor and has been lost or modified in many species. Together, the analysis of gadoid MHC-I genes and β2M molecules highlights the challenges in generalizing immune system paradigms across the most diverse vertebrate lineage (i.e., fish) and between fish and more well-studied mammals.

Identification of goldfish (Carassius auratus L.) leukocyte immune-type receptors shows alternative splicing as a potential mechanism for receptor diversification

Leukocyte immune-type receptors (LITRs) are a multigene family of teleost immunoregulatory proteins that share structural, phylogenetic, and likely functional relationships with several innate immune receptor proteins in other vertebrates, including mammals. Originally discovered in channel catfish (Ictalurus punctatus), representative IpLITR-types have been shown to regulate diverse innate immune cell effector responses including phagocytosis, degranulation, and cytokine secretion. To date, IpLITRs have been primarily characterized using mammalian cell line expression systems, therefore many unanswered questions remain regarding their actual regulatory roles in fish immunity. In the present study, we report on the preliminary molecular characterization of five goldfish (Carassius auratus) CaLITR-types and the identification of several putative splice variants of these receptors cloned from various goldfish tissues and primary myeloid cell cultures. In general, CaLITR mRNA transcripts were detected in all goldfish tissues tested, and also in primary kidney macrophage and neutrophil cultures. Specifically, CaLITR1 is a functionally ambiguous receptor with no charged amino acids in its transmembrane (TM) segment and is devoid of tyrosine-based signaling motifs in its short cytoplasmic tail (CYT) region. CaLITR2 is a putative activating receptor-type that contains immunotyrosine-based activation motifs (ITAMs) within its long CYT region, and CaLITR3 has a positively charged TM segment, suggesting that it may recruit intracellular stimulatory adaptor signaling molecules. CaLITR4 and CaLITR5 appear to have diverse signaling capabilities since they contain various immunoregulatory signaling motifs within their CYT regions including putative Nck and STAT recruitment motifs as well as ITAM-like and ITIM sequences. We also identified putative CaLITR splice variants with altered extracellular Ig-like domain compositions and variable CYT regions. Interestingly, this suggests that alternative splicing-mediated diversification of CaLITRs can generate receptor forms with possible variable binding and/or intracellular signaling abilities. Overall, these findings reveal new information about the teleost LITRs and sets the stage for exploring how alternative splicing leads to the functional diversification of this complex multigene immunoregulatory receptor family.

The first report of siglec-3/CD33 gene in a teleost (rock bream, Oplegnathus fasciatus): An analysis of its spatial expression during stimulation to red seabream iridovirus (RSIV) and two bacterial pathogens

Siglec-3/CD33 is a myeloid-specific inhibitory receptor that is expressed on cells of the immune system, where it is believed to play a regulatory role, modulating the inflammatory and immune responses. We characterized CD33 (RbCD33) in rock bream which is a transmembrane protein with two IG-like domains and a cytoplasmic tail. It has a deduced amino acid sequence of 390 residues and has tyrosine-based signaling motifs in the cytoplasmic tail. The RbCD33 mRNA was highly expressed in peripheral blood leukocytes and was also detected in the muscle, spleen, skin, head kidney, gills, trunk kidney, heart, stomach, brain, intestine and liver by quantitative real-time PCR. A temporal variation in expression of RbCD33 was observed in different tissues after stimulating with E. tarda, S. iniae and red seabream iridovirus (RSIV). In the head kidney tissue, E. tarda and S. iniae induced RbCD33, while a down regulation was observed with RSIV. In addition, in spleen tissue, S. iniae caused a very high induction of RbCD33 in comparison with an E. tarda and RSIV challenge. In the liver and gill tissues, all three pathogens induced a high expression of RbCD33. The expression pattern in various tissues and its high induction after pathogen stimulation suggests that RbCD33 plays an important role in initiating the immune response via the inhibition of signal transduction of the myeloid lineage cells.

Probing Transient Release of Membrane-Sequestered Tyrosine-Based Signaling Motif by Solution NMR Spectroscopy.

Membrane sequestration of tyrosine-based signaling motifs of antigen receptors effectively restricts the signaling activities in resting lymphocytes. However, low level of basal signaling in resting cells is required for lymphocyte survival and antigen responsiveness, of which the molecular mechanism remains obscure. Here we probe the transient release of the cytoplasmic domain of the membrane-bound IgG heavy chain (mIgG-tail) by hydrogen exchange NMR spectroscopy, illustrating a dynamic molecular basis for its basal signaling activity. To solve the severe resonance overlap problem in the 2D spectra of mIgG-tail, a non-uniformly sampled pseudo-4D hydrogen exchange NMR experiment has been exploited to quantitatively measure site-specific hydrogen exchange rates. Our solution NMR study reveals transient solvent exposure of the ITT signaling motif that can be further enhanced by calcium ion, and provides insight into the mechanism of lymphocyte basal signaling.

Chicken TREM-B1, an Inhibitory Ig-Like Receptor Expressed on Chicken Thrombocytes.

Triggering receptors expressed on myeloid cells (TREM) form a multigene family of immunoregulatory Ig-like receptors and play important roles in the regulation of innate and adaptive immunity. In chickens, three members of the TREM family have been identified on chromosome 26. One of them is TREM-B1 which possesses two V-set Ig-domains, an uncharged transmembrane region and a long cytoplasmic tail with one ITSM and two ITIMs indicating an inhibitory function. We generated specific monoclonal antibodies by immunizing a Balb/c mouse with a TREM-B1-FLAG transfected BWZ.36 cell line and tested the hybridoma supernatants on TREM-B1-FLAG transfected 2D8 cells. We obtained two different antibodies specific for TREM-B1, mab 7E8 (mouse IgG1) and mab 1E9 (mouse IgG2a) which were used for cell surface staining. Single and double staining of different tissues, including whole blood preparations, revealed expression on thrombocytes. Next we investigated the biochemical properties of TREM-B1 by using the specific mab 1E9 for immunoprecipitation of either lysates of surface biotinylated peripheral blood cells or stably transfected 2D8 cells. Staining with streptavidin coupled horse radish peroxidase revealed a glycosylated monomeric protein of about 50 kDa. Furthermore we used the stably transfected 2D8 cell line for analyzing the cytoplasmic tyrosine based signaling motifs. After pervanadate treatment, we detected phosphorylation of the tyrosine residues and subsequent recruitment of the tyrosine specific protein phosphatase SHP-2, indicating an inhibitory potential for TREM-B1. We also showed the inhibitory effect of TREM-B1 in chicken thrombocytes using a CD107 degranulation assay. Crosslinking of TREM-B1 on activated primary thrombocytes resulted in decreased CD107 surface expression of about 50–70%.

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.

Molecular and functional characterization of porcine Siglec-3/CD33 and analysis of its expression in blood and tissues

A cDNA clone encoding a 380 a-a type 1 transmembrane protein with homology to human Siglec-3/CD33 was obtained from a swine small intestine library. An analysis of protein sequence identified two immunoglobulin-like domains, a transmembrane region, and a carboxi-terminal tail with two tyrosine-based signalling motifs. Binding assays of Siglec-3 transfected CHO cells to polyacrylamide glycoconjugates showed a preference for α2–6-linked sialic acids. Using mAbs raised against a fragment containing the two Ig-like domains, porcine Siglec-3 was found to be expressed on monocytes and granulocytes, and their bone marrow precursors. It was also detected in lymph node, splenic and alveolar macrophages. MAbs immunoprecipitated, from granulocyte lysates, a protein of 51–60 kDa under both non-reducing and reducing conditions. MAbs were also used to analyse functional activity of Siglec-3 on bone marrow and blood cells. Engagement of Siglec-3 by mAb had no apparent effect on cell proliferation or cytokine production.

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.

Molecular characterization and expression of porcine Siglec-5

In this study we describe the characterization of the porcine orthologue of Siglec-5. A cDNa clone was obtained from a porcine cDNa library derived from swine small intestine which encodes a 555 a-a type 1 transmembrane protein with sequence homology to human Siglec-5. This protein consists of four Ig-like domains, a transmembrane region, and a cytoplasmic tail with two tyrosine-based signalling motifs. When expressed as a recombinant protein fused to the Fc region of human IgG1, porcine Siglec-5 was able to bind porcine red blood cells in a sialic acid-dependent manner. Monoclonal antibodies (mAb) were developed against porcine Siglec-5 and used to analyse its expression in bone marrow and blood cells, and lymphoid tissues. Porcine Siglec-5 expression was mainly restricted to myelomonocytic cells and their precursors, being detected also, although at low levels, on plasmacytoid dendritic cells and B lymphocytes. In lymphoid tissues, ellipsoids of the spleen and subcapsular and medullar sinuses of lymph nodes were positive for Siglec-5. These mAbs were able to precipitate, from granulocyte lysates, a protein of approximately 85kDa under non-reducing conditions, indicating that porcine Siglec-5 is expressed as a monomer in the plasma membrane.

Negative regulation of leucocyte functions by CD33-related siglecs

The siglecs (sialic acid-binding Ig-like lectins) are a family of transmembrane receptors expressed in the haemopoietic, immune and nervous systems. The CD33-related siglecs are a distinct subset mostly expressed in the innate immune system where they can function as inhibitory receptors by suppressing the signalling mediated by receptors coupled with ITAMs (immunoreceptor tyrosine-based activation motifs). CD33-related siglecs contain ITIMs (immunoreceptor tyrosine-based inhibitory motifs) that recruit and activate SHP-1 [SH2 (Src homology 2) domain-containing phosphatase-1] and SHP-2. In addition, the ITIMs of CD33-related siglecs can suppress siglec-dependent adhesion of sialylated ligands and mediate endocytosis. Siglec-H is a recently characterized murine CD33-related endocytic receptor that lacks intrinsic tyrosine-based signalling motifs and is expressed selectively on PDCs (plasmacytoid dendritic cells). Siglec-H depends on DAP12 (DNAX-activating protein of 12 kDa) for surface expression and cross-linking with anti-siglec-H antibodies can selectively inhibit interferon-alpha production by PDCs following TLR9 (Toll-like receptor 9) ligation. Thus CD33-related siglecs are able to mediate diverse inhibitory functions of leucocytes in the innate immune system via both ITIM-dependent and -independent pathways.

Characterization of Siglec-H as a novel endocytic receptor expressed on murine plasmacytoid dendritic cell precursors

We describe the cloning and characterization of Siglec-H, a novel murine CD33-related siglec-like molecule with 2 immunoglobulin domains. Unlike other CD33-related siglecs, Siglec-H lacks tyrosine-based signaling motifs in its cytoplasmic tail. Although Siglec-H has the typical structural features required for sialic acid binding, no evidence for carbohydrate recognition was obtained. Specific monoclonal and polyclonal antibodies (Abs) were raised to Siglec-H and used to define its cellular expression pattern and functional properties. By flow cytometry, Siglec-H was expressed specifically on plasmacytoid dendritic cell (pDC) precursors in bone marrow, spleen, blood, and lymph nodes. Staining of tissue sections showed that Siglec-H was also expressed in a subset of marginal zone macrophages in the spleen and in medullary macrophages in lymph nodes. Using bone marrow-derived pDC precursors that express Siglec-H, addition of Abs did not influence cytokine production, either in the presence or absence of synthetic oligodeoxynucleotides containing unmethylated cytosine-guanine motifs (CpG). In comparison, Siglec-H functioned as an endocytic receptor and mediated efficient internalization of anti-Siglec-H Abs. By immunizing mice with ovalbumin-conjugated anti-Siglec-H Ab in the presence of CpG, we demonstrate generation of antigen-specific CD8 T cells in vivo. Targeting Siglec-H may therefore be a useful way of delivering antigens to pDC precursors for cross-presentation.

Gene Conversion of Sialic Acid Binding Domains in CD33-Related Siglecs by Adjacent Pseudogenes: A Novel Mechanism To Change Sialic Acid Binding Specificity.

Siglecs (sialic acid-binding immunoglobulin superfamily lectins) are a family of cell surface receptors involved in regulating the immune response. The CD33-Related Siglecs (CD33rSiglecs, namely Siglec-3, -5 through -11 and -XII in humans) are a subgroup of these molecules found primarily on cells of the innate immune system. All are type-1 transmembrane proteins with an N-terminal sialic acid-recognizing V-set domain followed by a variable number of C-2 set domains, a transmembrane region and a cytosolic C-terminal domain that includes two tyrosine-based signaling motifs. Available data suggest an inhibitory signaling role in the innate immune response, mediated by recognition of host sialic acids as “self”. Nine of the 13 known primate Siglec genes along with 14 Siglec pseudogenes comprise the CD33-related Siglec gene cluster on human chromosome 19. Gene conversion is a mechanism for copying part of a genomic sequence into another, contributing to genetic diversity. Pseudogenes are known to play role in generating functional diversity of related genes (e.g., antibody diversity via gene conversion in chickens). We recently analyzed genomic sequences of the CD33-related Siglec gene cluster in three primates (human, chimpanzee and baboon) and found evidence for rapid evolution in this gene family (Angata et al., PNAS, in press). Additional evolutionary studies using distance-based phylogenetic trees shows evidence for three partial gene conversions between Siglec genes and adjacent Siglec pseudogenes. All three involve the coding regions for extracellular domains that mediate sialic acid recognition, and two involve a pseudogene converting a known Siglec gene. Functional analyses using recombinant proteins show marked differences in sialic acid-binding properties between the converted Siglec and its non-converted ortholog. These findings suggest that gene conversion with pseudogenes has contributed to the rapid functional evolution of the Siglecs, and provides a novel mechanism for changing sialic acid binding specificity. We hypothesize that this mechanism allows for rapid evolutionary adjustments in the recognition of endogenous sialic acids as “self”, a potential factor in controlling the innate immune response.

Differential Expression and Function of the CD33-Related Siglecs between Humans and Great Apes.

The Siglecs (Sialic acid-binding Immunoglobulin Superfamily Lectins) are a recently discovered family of mammalian glycan-binding proteins that have been shown to recognize the terminal sialic acids of glycoproteins and glycolipids. The CD33-Related Siglecs (CD33rSiglecs, namely Siglec-3, -5 through -11 and -XII in humans) are a subgroup of these molecules, which are thought to be primarily expressed on cells of the innate immune system. All CD33rSiglecs are type-1 transmembrane proteins with an N-terminal sialic acid-recognizing V-set domain followed by a variable number of C-2 set domains, a transmembrane region and a cytosolic C-terminal domain that usually has two tyrosine-based signaling motifs, one of which conforms to a canonical negative regulatory ITIM motif. Although the true function of the CD33rSiglecs has yet to be discovered, available data are most consistent with an inhibitory signaling role in the innate immune response, mediated by recognition of host sialic acids as “self”. CD33rSiglecs also interact with sialic acids on the same cell surface, typically resulting in “masking” of their sialic acid-binding sites. Our recent studies have shown that humans and non-human primates have a similar clustered localization of CD33rSiglec genes, and that true orthologs can generally be identified within each cluster (Angata et al., PNAS, in press). However, humans no longer express CMP-sialic acid hydroxylase (CMAH) the enzyme required to generate one of the potential CD33rSiglec sialic acid ligands called N-glycolylneuraminic acid (Neu5Gc), from its precursor N-acetylneuraminic acid (Neu5Ac). This genetic change occurred after our last common ancestor with the great apes, and dramatically altered the “Sialome” (the sialic acid makeup of a specific species) of humans when compared to that of the great apes. While great ape blood cells express about equal amounts of Neu5Ac and Neu5Gc, human blood cells express almost exclusively Neu5Ac. We also recently discovered that preferential recognition of Neu5Gc is the ancestral condition of most or all of the great ape (chimpanzee and gorilla) CD33rSiglecs (Sonnenburg JL, Altheide TK, Varki A. Glycobiology. 14:339–46, 2004). We therefore reasoned that the sudden and major change in the sialome of our hominid ancestors could have had a significant impact on the evolution, binding specificities and expression patterns of CD33rSiglecs. Indeed, we have found that all human CD33rSiglecs can recognize both Neu5Ac and Neu5Gc. This presumably represents an evolutionarily-selected “relaxation” in binding specificity that was necessary to “remask” the Siglecs that had lost their Neu5Gc ligands. Also, there are differences in CD33rSiglec expression on monocytes and neutrophils between humans and great apes (chimp, bonobo, gorilla and orangutan). Furthermore, while great ape cells often show multiple populations with different signal intensities, humans express a single bright peak for each Siglec in flow cytometry. Surprisingly, while humans showed almost no CD33rSiglec expression on lymphocytes, the great apes show a moderate to high expression of some Siglecs on these cells. Total leukocyte expression of some CD33rSiglecs also shows differences between humans and great apes. Overall, CD33rSiglecs appear to be rapidly evolving in primates, with an apparent further acceleration of changes in humans. Additional studies are needed to define the mechanistic details, as well as the implications for human health and disease.

Transmembrane adapters: structure, biochemistry and biology

Transmembrane adapter proteins (TRAPs) represent a relatively new and unique group of signalling molecules in hematopoetic cells. They differ from other signalling proteins as they lack any enzymatic or transcriptional activity, instead they possesses multiple tyrosine-based signalling motifs (TBSMs). Triggering of immunoreceptors induces tyrosine phosphorylation of these motifs by members of the Src-, Syk- or Tec-family of protein tyrosine kinases thus enabling the TRAPs to recruit cytosolic adapter and/or effector molecules via their SH2-domains into close proximity to the immunoreceptors, a position from which they can coordinate and modulate signal transduction pathways important for lymphocyte function.

Erratum: Transmembrane adaptor proteins: organizers of immunoreceptor signalling

Nature Reviews Immunology 4, 603–616 (2004). When published, in Figure 2 , the tyrosine-based signalling motifs of the molecule LIME were misaligned. See the corrected version below.

Large-scale sequencing of the CD33-related Siglec gene cluster in five mammalian species reveals rapid evolution by multiple mechanisms.

Siglecs are a recently discovered family of animal lectins that belong to the Ig superfamily and recognize sialic acids (Sias). CD33-related Siglecs (CD33rSiglecs) are a subgroup with as-yet-unknown functions, characterized by sequence homology, expression on innate immune cells, conserved cytosolic tyrosine-based signaling motifs, and a clustered localization of their genes. To better understand the biology and evolution of CD33rSiglecs, we sequenced and compared the CD33rSiglec gene cluster from multiple mammalian species. Within the sequenced region, the segments containing CD33rSiglec genes showed a lower degree of sequence conservation. In contrast to the adjacent conserved kallikrein-like genes, the CD33rSiglec genes showed extensive species differences, including expansions of gene subsets; gene deletions, including one human-specific loss of a novel functional primate Siglec (Siglec-13); exon shuffling, generating hybrid genes; accelerated accumulation of nonsynonymous substitutions in the Sia-recognition domain; and multiple instances of mutations of an arginine residue essential for Sia recognition in otherwise intact Siglecs. Nonsynonymous differences between human and chimpanzee orthologs showed uneven distribution between the two beta sheets of the Sia-recognition domain, suggesting biased mutation accumulation. These data indicate that CD33rSiglec genes are undergoing rapid evolution via multiple genetic mechanisms, possibly due to an evolutionary "arms race" between hosts and pathogens involving Sia recognition. These studies, which reflect one of the most complete comparative sequence analyses of a rapidly evolving gene cluster, provide a clearer picture of the ortholog status of CD33rSiglecs among primates and rodents and also facilitate rational recommendations regarding their nomenclature.

An inhibitory Ig superfamily protein expressed by lymphocytes and APCs is also an early marker of thymocyte positive selection.

Positive selection of developing thymocytes is associated with changes in cell function, at least in part caused by alterations in expression of cell surface proteins. Surprisingly, however, few such proteins have been identified. We have analyzed the pattern of gene expression during the early stages of murine thymocyte differentiation. These studies led to identification of a cell surface protein that is a useful marker of positive selection and is a likely regulator of mature lymphocyte and APC function. The protein is a member of the Ig superfamily and contains conserved tyrosine-based signaling motifs. The gene encoding this protein was independently isolated recently and termed B and T lymphocyte attenuator (Btla). We describe in this study anti-BTLA mAbs that demonstrate that the protein is expressed in the bone marrow and thymus on developing B and T cells, respectively. BTLA is also expressed by all mature lymphocytes, splenic macrophages, and mature, but not immature bone marrow-derived dendritic cells. Although mice deficient in BTLA do not show lymphocyte developmental defects, T cells from these animals are hyperresponsive to anti-CD3 Ab stimulation. Conversely, anti-BTLA Ab can inhibit T cell activation. These results implicate BTLA as a negative regulator of the activation and/or function of various hemopoietic cell types.

Positive and negative adaptors in T-cell signalling.

Positive and negative adaptors in T-cell signalling.