Antibody responses of variable lymphocyte receptors in the lamprey.

Lamprey and hagfish, the living representatives of jawless vertebrates, use genomic leucine-rich-repeat cassettes for the combinatorial assembly of diverse antigen receptor genes encoding variable lymphocyte receptors of two types: VLRA and VLRB. We describe here the VLRB-bearing lineage of lymphocytes in sea lamprey. These cells responded to repetitive carbohydrate or protein determinants on bacteria or mammalian cells with lymphoblastoid transformation, proliferation and differentiation into plasmacytes that secreted multimeric antigen-specific VLRB antibodies. Lacking a thymus and the ability to respond to soluble protein antigens, lampreys seem to have evolved a B cell-like system for adaptive humoral responses.

High-affinity lamprey VLRA and VLRB monoclonal antibodies.

Lamprey are members of the ancestral vertebrate taxon (jawless fish), which evolved rearranging antigen receptors convergently with the jawed vertebrates. But instead of Ig superfamily domains, lamprey variable lymphocyte receptors (VLRs) consist of highly diverse leucine-rich repeats. Although VLRs represent the only known adaptive immune system not based on Ig, little is known about their antigen-binding properties. Here we report robust plasma VLRB responses of lamprey immunized with hen egg lysozyme and beta-galactosidase (beta-gal), demonstrating adaptive immune responses against soluble antigens. To isolate monoclonal VLRs, we constructed large VLR libraries from antigen-stimulated and naïve animals in a novel yeast surface-display vector, with the VLR C-terminally fused to the yeast Flo1p surface anchor. We cloned VLRB binders of lysozyme, beta-gal, cholera toxin subunit B, R-phycoerythrin, and B-trisaccharide antigen, with dissociation constants up to the single-digit picomolar range, equivalent to those of high-affinity IgG antibodies. We also isolated from a single lamprey 13 anti-lysozyme VLRA clones with affinities ranging from low nanomolar to mid-picomolar. All of these VLRA clones were closely related in sequence, differing at only 15 variable codon positions along the 244-residue VLR diversity region, which augmented antigen-binding affinity up to 100-fold. Thus, VLRs can provide a protective humoral antipathogen shield. Furthermore, the broad range of nominal antigens that VLRs can specifically bind, and the affinities achieved, indicate a functional parallelism between LRR-based and Ig-based antibodies. VLRs may be useful natural single-chain alternatives to conventional antibodies for biotechnology applications.

[Cloning and expression of VLRB of Lampetra japonica and generation of the corresponding monoclonal antibodies].

The agnathans (lampreys and hagfishes) are representatives of the jawless vertebrates. The receptor molecules of adaptive immune system in lampreys are different from the antigen receptors in mammal vertebrates. The unique receptor molecules of lampreys are known as variable lymphocyte receptors (VLR). There are three types of VLRs in lampreys, VLRA, VLRB, and VLRC. Multimeric antigen-specific VLRB antibodies are secreted by VLRB+ lymphocytes and constitute the major components of the humoral arm of the lamprey adaptive immune system. Oligomeric VLRB antibodies are composed of four or five disulfide-linked dimeric subunits, which are similar to IgM antibodies in structure and function. In this study, the conservative c-terminal of Lampetra japonica VLRB was cloned and expressed in BL21 E. coli. The recombinant VLRB protein was purified by Ni2+ affinity chromatography column. After Balb/c mice immunity, cell fusion, the positive clones were screened by indirect enzyme-linked immunosorbent assay (ELISA). Finally, the hybridoma cells that produced specific anti-VLRB monoclonal antibodies were obtained. In order to get a large number of antibodies against VLRB, the hybridoma cells were injected into the abdominal cavity of Balb/c mice and the antibodies were purified by protein G sepharose. The results of ELISA indicated that the valence of anti-VLRB antibodies was 1:40000. Western blotting assay showed that the antibodies were able to detect both recombinant VLRB and secreted VLRB in lamprey sera. Flow cytometry analysis also revealed the existence of VLRB on the surface of lymphocytes. In summary, the anti-VLRB monoclonal antibodies provided a major tool for studying lamprey adaptive immune system.

Inside the microcluster: antigen receptor signalling viewed with molecular imaging tools.

Over the last decade, live cell imaging has revealed the surprisingly complex orchestration of antigen receptor signalling at the immunological synapse. The imaging studies showed that one of the earliest steps in antigen receptor activation is the formation of submicroscopic clusters, which regulate the early signalling events. However, the molecular mechanisms operating inside these microclusters have remained beyond the resolution of optical microscopy. Recent development of imaging techniques that approach molecular resolution in intact cells offers a first view of the molecular processes inside these structures. Here I review the contributions of molecular imaging of the immunological synapse to our understanding of antigen receptor clustering, binding to antigens, and recruitment of signalling molecules. Finally, I provide an outlook on the future prospects of this rapidly advancing technology.

A dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN)-related protein is highly expressed on human liver sinusoidal endothelial cells and promotes HIV-1 infection.

The discovery of dendritic cell (DC)-specific intercellular adhesion molecule (ICAM)-3-grabbing nonintegrin (DC-SIGN) as a DC-specific ICAM-3 binding receptor that enhances HIV-1 infection of T cells in trans has indicated a potentially important role for adhesion molecules in AIDS pathogenesis. A related molecule called DC-SIGNR exhibits 77% amino acid sequence identity with DC-SIGN. The DC-SIGN and DC-SIGNR genes map within a 30-kb region on chromosome 19p13.2-3. Their strong homology and close physical location indicate a recent duplication of the original gene. Messenger RNA and protein expression patterns demonstrate that the DC-SIGN-related molecule is highly expressed on liver sinusoidal cells and in the lymph node but not on DCs, in contrast to DC-SIGN. Therefore, we suggest that a more appropriate name for the DC-SIGN-related molecule is L-SIGN, liver/lymph node-specific ICAM-3-grabbing nonintegrin. We show that in the liver, L-SIGN is expressed by sinusoidal endothelial cells. Functional studies indicate that L-SIGN behaves similarly to DC-SIGN in that it has a high affinity for ICAM-3, captures HIV-1 through gp120 binding, and enhances HIV-1 infection of T cells in trans. We propose that L-SIGN may play an important role in the interaction between liver sinusoidal endothelium and trafficking lymphocytes, as well as function in the pathogenesis of HIV-1.

New insights emerge as antibody repertoire diversification meets chromosome conformation.

Vast repertoires of unique antigen receptors are created in developing lymphocytes. The antigen receptor loci contain many variable (V), diversity (D), and joining (J) gene segments that are arrayed across very large genomic expanses and are joined to form variable-region exons. This process creates the potential for an organism to respond to large numbers of different pathogens. Here, we consider the underlying molecular mechanisms that favor some V genes for recombination prior to selection of the final antigen receptor repertoire. We discuss chromatin structures that form in antigen receptor loci to permit spatial proximity among the V, D, and J gene segments and how these relate to the generation of antigen receptor diversity.

The integrin receptor alpha 8 beta 1 mediates interactions of embryonic chick motor and sensory neurons with tenascin-C.

This paper identifies a neuronal receptor for tenascin-C (tenascin/cytotactin), an extracellular matrix protein that has previously been detected in developing sensory and motor neuron pathways and has been shown to regulate cell migration in the developing CNS. Antibodies specific for each subunit of the integrin alpha 8 beta 1 are used to demonstrate that alpha 8 beta 1 mediates neurite outgrowth of embryonic sensory and motor neurons on this extracellular matrix protein. In addition, expression of alpha 8 in K562 cells results in surface expression of alpha 8 beta 1 heterodimers that are shown to promote attachment of this cell line to tenascin. The major domain in tenascin that mediates neurite outgrowth is shown to be localized to fibronectin type III repeats 6-8.

Calcium signalling in lymphocytes.

The modulation of intracellular calcium ion concentration, [Ca(2+)](i), is a common signalling mechanism used in many biological systems. B and T lymphocytes rely on Ca(2+) signalling to initiate both developmental and activation programs. Recent data has shed new light on the initiation of this signalling pathway, the connection between the release of intracellular Ca(2+) stores and the influx of extracellular Ca(2+), and the molecular identity of the elusive Ca(2+) release-activated Ca(2+) (CRAC) channel. In addition, recent gene profiling of T lymphocytes has identified the genes that are controlled by [Ca(2+)](i) and the Ca(2+)-dependent phosphatase calcineurin.

MHC class II signaling in antigen-presenting cells.

The MHC class II molecules have been recognized as signaling receptors for more than a decade, and recent work has revealed the importance of their signaling for the immune response. Today, we know that the function of MHC class II molecules on antigen-presenting cells (APCs) is not limited to their role as antigen-presenting structures; they are flexible receptors that, by triggering a variety of signaling pathways, can regulate APC activities from proliferation and maturation to apoptosis. Recent advances have provided insights into how these molecules might accommodate such regulation.

Commutators of PAR-1 signaling in cancer cell invasion reveal an essential role of the Rho-Rho kinase axis and tumor microenvironment.

We recently reported that proteinase-activated receptors type I (PAR-1) are coupled to both negative and positive invasion pathways in colonic and kidney cancer cells cultured on collagen type I gels. Here, we found that treatments with the cell-permeant analog 8-Br-cGMP and the soluble guanylate cyclase activator BAY41-2272, and Rho kinase (ROK) inhibition by Y27632 or a dominant negative form of ROK lead to PAR-1-mediated invasion through differential Rac1 and Cdc42 signaling. Hypoxia or the counteradhesive matricellular protein SPARC/BM-40 (SPARC: secreted protein acidic rich in cysteine) overexpressed during cancer progression also commutated PAR-1 to cellular invasion through the cGMP/protein kinase G (PKG) cascade, RhoA inactivation, and Rac1-dependent or -independent signaling. Cultured primary cancer cells isolated from peritoneal and pleural effusions from patients with colon cancer or other malignant tumors harbored PAR-1, as shown by RT-PCR and FACS analyses. These malignant effusions also contained high levels of activated thrombin and fibrin, and induced a proinvasive response in HCT8/S11 human colorectal cancer cells. Our data underline the essential role of the tumor microenvironment and of several commutators targeting cGMP/PKG signaling and the RhoA-ROK axis in the control of PAR-1 proinvasive activity and metastatic potential of cancer cells in distant organs and peritoneal or pleural cavities. We also add new insights into the mechanisms linking the coagulation mediators thrombin and PAR-1 in the context of blood coagulation disorders and venous thrombosis often observed in cancer patients, as described in 1865 by Armand Trousseau.

Mechanisms of signaling by the hematopoietic-specific adaptor proteins, SLP-76 and LAT and their B cell counterpart, BLNK/SLP-65.

Adaptor proteins lack catalytic activity and contain only protein-protein interaction domains. They have been shown to interact with an ever-growing number of signaling proteins and to play essential roles in many signaling pathways. SLP-76 and LAT are cell-type-specific adaptor proteins expressed in T cells, NK cells, platelets, and mast cells. In these cell types, SLP-76 and LAT are required for signaling by immunoreceptor tyrosine-based activation motif(ITAM)-containing receptors, including the T cell receptor (TCR), the pre-TCR, the high-affinity Fc epsilon receptor, and the platelet GPVI collagen receptor. In B cells, an analogous adaptor, BLNK/SLP-65, is required for signaling by the ITAM-containing B cell receptor. This review summarizes recent research on SLP-76, LAT, and BLNK. A major challenge in understanding adaptor protein function has been to sort out the many interactions mediated by adaptor proteins and to define the mechanisms by which adaptors mediate critical signaling events. In the case of LAT, SLP-76, and BLNK, the availability of tractable genetic systems, deficient in expression of each of these adaptor proteins, has facilitated in-depth investigation of their signaling functions and mechanisms of action. The picture that has emerged is one in which multiple adaptor proteins cooperate to bring about the formation of a large signaling complex, localized to specialized lipid microdomains within the cell membrane and known as GEMs. Adaptors not only recruit signaling proteins, but also play an active role in regulating the conformation and activation of many of the proteins recruited to the complex. In particular, recent research has shed light on the mechanisms by which multiple adaptor proteins cooperate to bring about the recruitment and activation of phospholipase C gamma in response to the activation of ITAM-containing receptors.

Therapeutic opportunities in the B7/CD28 family of ligands and receptors.

The CD28 and B7 protein families are critical regulators of immune responses and, in the past few years, several new family members have been identified. Preclinical studies exploring the role of members of the CD28 and B7 families support the targeting of these pathways for new therapeutic approaches. Indeed, recent Phase I clinical studies using agonists and antagonists of the CD28/CTLA-4/B7 pathway have shown promise in inflammatory diseases and cancer.

CAR T Cells Targeting Podoplanin Reduce Orthotopic Glioblastomas in Mouse Brains.

Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor in adults with a 5-year overall survival rate of less than 10%. Podoplanin (PDPN) is a type I transmembrane mucin-like glycoprotein, expressed in the lymphatic endothelium. Several solid tumors overexpress PDPN, including the mesenchymal type of GBM, which has been reported to present the worst prognosis among GBM subtypes. Chimeric antigen receptor (CAR)-transduced T cells can recognize predefined tumor surface antigens independent of MHC restriction, which is often downregulated in gliomas. We constructed a lentiviral vector expressing a third-generation CAR comprising a PDPN-specific antibody (NZ-1-based single-chain variable fragment) with CD28, 4-1BB, and CD3ζ intracellular domains. CAR-transduced peripheral blood monocytes were immunologically evaluated by calcein-mediated cytotoxic assay, ELISA, tumor size, and overall survival. The generated CAR T cells were specific and effective against PDPN-positive GBM cells in vitro. Systemic injection of the CAR T cells into an immunodeficient mouse model inhibited the growth of intracranial glioma xenografts in vivo. CAR T-cell therapy that targets PDPN would be a promising adoptive immunotherapy to treat mesenchymal GBM.

DC-SIGN, a C-type lectin on dendritic cells that unveils many aspects of dendritic cell biology.

Dendritic cells (DC) are present in essentially every tissue where they operate at the interface of innate and acquired immunity by recognizing pathogens and presenting pathogen-derived peptides to T cells. It is becoming clear that not all C-type lectins on DC serve as antigen receptors recognizing pathogens through carbohydrate structures. The C-type lectin DC-SIGN is unique in that it regulates adhesion processes, such as DC trafficking and T-cell synapse formation, as well as antigen capture. Moreover, even though several C-type lectins have been shown to bind HIV-1, DC-SIGN does not only capture HIV-1 but also protects it in early endosomes allowing HIV-1 transport by DC to lymphoid tissues, where it enhances trans infection of T cells. Here we discuss the carbohydrate/protein recognition profile and other features of DC-SIGN that contribute to the potency of DC to control immunity.

Recognition and function in a degenerate immune system.

Degeneracy in the immune system not only refers to the recognition by its specific receptors, but also at the level of functional degeneracy of reactivity by its lymphocytes. Features of the immune system such as dominance and competition occur at each of these two levels. Another characteristic of the immune system as a fuzzy system, is its continuum of states, where difficult choices lead to the emergence of unpredictable phenotypes.

Biochemical events in naturally occurring forms of cell death.

Several molecular elements of programmed cell death and apoptosis have recently been revealed. The function of gene products which deliver the lethal 'hit' is still not known. Well-characterized and newly discovered cell surface structures (e.g. antigen receptors, FAS/APO-1), as well as transcriptional factors (steroid receptor, c-myc, P53, retinoblastoma protein and others), have been implicated in the initiation of the death pathway. Negative regulators of the process (ced-9 gene product in programmed death of cells in Caenorhabditis elegans and bcl-2 protein in apoptosis) have been described. Biochemical mechanisms responsible for the silent nature of natural deaths of cells include their rapid engulfment (mainly through integrin receptors), transglutaminase-catalyzed cross-linking of cellular proteins, and fragmentation of DNA. Several lines of evidence suggest that distinct molecular mechanisms may operate in various forms of natural cell death.

Multisubunit receptors in the immune system and their association with the cytoskeleton: in search of functional significance.

Various multisubunit receptors of the immune system share similarities in structure and induce closely related signal transduction pathways upon ligand binding. Examples include the T cell antigen receptor (TCR), the B cell antigen receptor (BCR), and the high-affinity receptor for immunoglobulin E (Fc epsilon RI). Although these receptors are devoid of intrinsic kinase activity, they can associate with a similar array of intracellular kinases, phosphatases and other signaling molecules. Furthermore, these receptor complexes all form an association with the cytoskeletal matrix. In this review, we compare the structural and functional characteristics of the TCR, BCR and Fc epsilon RI. We examine the role of the cytoskeleton in regulating receptor-mediated signal transduction, as analyzed in other well-characterized receptors, including the epidermal growth factor receptor and integrin receptors. On the basis of this evidence, we review the current data depicting a cytoskeletal association for multisubunit immune system receptors and explore the potential bearing of this interaction on signaling function.

Speculations on the origin of the vertebrate immune system.

As one studies more and more the immune systems of invertebrates their multiplicity and diversity increase. On one hand, the number of different local and systemic innate mechanisms (or cascades of mechanisms) increases with every phylum studied, on the other hand the diversity increases within each system as multiple loci and polymorphisms are discovered. Even somatic variation is not restricted to vertebrates. Yet no immune system similar to that of vertebrates characterized by the usage of RAG enzymes, has been found below jawed vertebrates. The availability of genome projects in early chordates allows understanding better the different causal lines that led to the generation of the vertebrate system with its diverse repertoire of antigen receptors generated somatically. This paper singles out a linkage group in human, gathering genes of the Ig superfamily with structural relationships to the antigen receptor JAM/CTX, nectin, or to some haematopoietic cells CD that have homologues in protochordates such as Branchiostoma and Ciona or in more primitive protostomes. The role of virus receptor for many of these clustered genes products provides perhaps one way of envisaging the recruitment of this family in the adaptive immune system from an ancient form of innate antiviral immunity.

The non-specific cytotoxic cell receptor (NCCRP-1): molecular organization and signaling properties.

The evolutionary precursor to mammalian natural killer cells in teleost fish is called non-specific cytotoxic cells (NCC). NCC collaborate with other non-specific effector mechanisms to provide innate resistance during acute stress responses. The NCC receptor protein (NCCRP-1) contains 238 amino acid residues and is believed to be a type III membrane protein with three distinct functional domains. The antigen-binding domain has been mapped to amino acids nos. 104-119. The intracellular C-terminus contains a high concentration of potential phosphorylation sites (Y, S, T). Indeed, we have shown that activation of NCC by crosslinking of NCCRP-1 leads to receptor tyrosine and serine phosphorylation. The N-terminus of the molecule is also inside the cells and has as well signature amino acids, proline-rich motifs (PRM), that are indicative of functional relevance. The cytokine/hormone receptor-like PRMs are known docking sites for JAK kinases. We have evidence that following activation, NCCRP-1 comes in contact with JAK kinase and as a result of this interaction, STAT 6 is translocated into the nucleus. These results suggest that NCCRP-1 may play a dual role in the activation of NCC: first, as an antigen recognition molecule necessary for target cell lysis, and second, as an initiator of cytokine release from NCC. Both of these processes are required for a competent innate immune response.

Function associated molecules on nonspecific cytotoxic cells: role in calcium signaling, redirected lysis, and modulation of cytotoxicity.

Monoclonal antibodies (mAbs) specific for a putative antigen receptor (function associated molecule, FAM) on fish NCC were tested for their ability to initiate signal transduction responses. Anti-FAM hybridoma target cells were significantly lysed by NCC when tested by redirected lysis (RL). Redirected lysis was enhanced by 1-h pretreatment of the NCC with anti-FAM mAb; however, 18-h pretreatment produced significant inhibition. The effects of the calcium ionophore A23187 on RL were also examined: 10(-4) M but not 10(-5) M A23187 significantly increased RL. Purified NCC were treated with mAb and cellular DNA synthesis was determined. Significant increases in NCC incorporation of tritiated thymidine were measured 72 h after treatment with mAb. Experiments were also conducted to determine levels of free cytosolic calcium in NCC following mAb binding. Anti-FAM mAb binding produced a rapid rise in cytosolic calcium (200-500 nM) determined by Fura-2/AM fluorescence. Calcium ionophore A23187, PHA, and Con-A treatment of NCC also produced significantly increased [Ca2+]i. Target cell binding by NCC caused a three-fold increase in [Ca2+]i. These data suggest that triggering of the FAM may initiate a signal(s) that activates cytotoxicity, increases cytosolic free calcium concentrations, and initiates DNA synthesis.