Characterization of arrangement and expression of the T cell receptor gamma locus in the sandbar shark.

Ig and T cell receptor (TCR) genes consist of separate genomic elements, which must undergo rearrangement and joining before a functional protein can be expressed. Considerable plasticity in the genomic arrangement of these elements has occurred during the evolution of the immune system. In tetrapods, all Ig and TCR chain elements are arranged as translocons. In teleosts, the Ig heavy and TCR chains are translocons, but light chain genes may occur as clusters. However, in chondrichthyes, all of the Ig light and heavy chain genes are arranged as clusters. These clusters vary in number from <10 to several hundred, depending on isotype and species. Here, we report that the germ-line gene for the TCR gamma chain in a chondrichthyan, the sandbar shark (Carcharhinus plumbeus), is present as a single locus arranged in a classic translocon pattern. Thus, the shark utilizes 2 types of genomic arrangements, the unique cluster organization for Ig genes and the "conventional" translocon organization for TCR genes. The TCR gamma translocon contains at least 5 V region genes, 3 J segment genes, and 1 C segment. As expected, the third hypervariable segment (CDR3), formed by the rearrangement of the Vgamma and Jgamma segments, contributed the major variability in the intact V region structure. Our data also suggest that diversity may be generated by mutation in the V regions.

Natural and autoantibodies to human T-cell receptor Vbeta segments: potential roles in immunomodulation.

Although the manifestation of inflammatory autodestructive disease is the result of major immunological dysfunction, recent evidence indicates that the immune system attempts to compensate by the production of immunomodulatory autoantibodies. Healthy humans have low levels of naturally occurring autoantibodies directed against the first complementarity-determining region (CDR1) and third framework region (FR3) of their own T-cell receptor (TCR) Vbeta segments, but individuals suffering from rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE) can have highly elevated levels of these autoantibodies. We cloned and characterized human anti-TCR monoclonal autoantibodies (mAAbs) from RA and SLE patients. Because of the cross-reactions between distinct CDR1 segments of human TCR Vbeta and corresponding murine homologs, it was possible to show that human mAAbs blocked the capacity of a murine TH1 cell line (DO11.10) to produce IL-2 in response to antigenic stimulation in vitro. These results support the hypothesis that autoantibodies against TCR Vbeta can shut down TH1-mediated inflammatory autodestructive reactions.

Rapid clearance of bacteria and their toxins: development of therapeutic proteins.

The emergence of pathogens and toxins with resistance against conventional drugs, vaccines, and host defense peptides and proteins warrants novel countermeasures that can efficiently capture and rapidly clear them. This article examines the utility of chimeric proteins with capture and clearance domains as a novel countermeasure against pathogens and their toxins. The capture and clearance domains are chosen from the large repertoire of host defense peptides and proteins. Although individual capture and clearance domains are rendered ineffective by pathogenic resistance mechanisms, chimeric scaffolds can be designed to retain their antimicrobial activity, even in the face of pathogenic resistance. Here, initial studies on the design of chimeric proteins targeted against (1) intact bacteria such as Xylella fastidiosa (plant pathogens), Salmonella spp. (food-borne pathogens), and Staphylococcus aureus (blood-borne pathogens); and (2) lethal toxins from Bacillus anthracis are described.

The antibody repertoire in evolution: chance, selection, and continuity.

All jawed vertebrates contain the genetic elements essential for the function of the adaptive/combinatorial immune response, have diverse sets of natural antibodies resulting from segmental gene recombination, express comparable functional repertoires and can produce specific antibodies following appropriate immunization. Profound variability occurs in the third hypervariable (CDR3) segments of light and heavy chains even within antibodies of the same ostensible specificity. Germline VH and VL elements, as well as the joining (J) segments are highly conserved among the distinct vertebrate species. Conservation is particularly noted among the VH3-like sequences of all jawed vertebrates in the FR2 and FR3 segments, as well as in the FGXGT(R or K)L J-segment characteristic of light chains and TCRs and the WGXGT(uncharged)VT JH segments. Human VH3-53 and Vlambda6 family orthologs may be present over the entire range of vertebrates. Models of the three-dimensional structures of shark VH/VL combining sites indicate similarity in framework structure and comparable CDR usage to those of man. Although carcharhine shark VH regions show greater than 50% identity to the human VH germline prototype, searches of lower deuterostome and invertebrate databases fail to detect molecules with significant relatedness. Overall, antibodies of jawed vertebrates show tremendous individual diversity, but are constructed incorporating design features that arose with the evolutionary emergence of the jawed vertebrates and have been conserved through at least 450 million years of evolutionary time.

Cloning of shark RAG2 and characterization of the RAG1/RAG2 gene locus.

The recombination-activating genes (RAG) encode a site-specific recombinase that is centrally responsible for the rearrangement of genomic V(D)J exons necessary to form functional immunoglobulin and T-cell receptor genes. To help elucidate the origins of the RAG genes, we have cloned the RAG2 gene from the sandbar shark (Carcharhinus plumbeus) and characterized the entire RAG1/RAG2 gene locus. The shark RAG2 protein consists of 520 amino acids, is approximately 50% identical with RAG2 proteins from other vertebrates, and contains the same three domains identified in mammalian RAG2. Residues critical for RAG2 function are conserved in the shark sequence. In common with other vertebrate species, the shark RAG2 coding region lacks introns and is closely linked in opposite orientation to the RAG1 gene. The intergenic region is 9.4 kb, which is considerably larger than of teleosts (2-3 kb) and is comparable to that of tetrapods. This length is partially explained by the presence of several SINE and LINE fragments. The ancestors of the sharks were apparently the first vertebrates in phylogeny to have RAG genes, and our results confirm that the RAG genes have been highly conserved during evolution both in terms of sequence and gene organization.

Natural recognition repertoire and the evolutionary emergence of the combinatorial immune system.

The primordial combinatorial immune recognition repertoire arose in the evolution of jawed vertebrates approximately 450 million years ago as a rapid genetic process independent of antigenic selection. We propose that it encompassed the entire repertoire of innate immunity involving molecules that had evolved over billions of years. The 'antigen-driven' compartment involving invasive pathogens operates in 'real time' showing inducibility and increases in affinity. Individuals within a species differ in their repertoires because of distinct antigenic challenges, genetics, or local environmental effects. The 'homeostatic' compartment that recognizes invariant cell and serum components should be conserved in all individuals of a species. The potential to recapitulate the entire recognition spectrum must be regenerated during the formation of new species. Evidence for the capacity of the combinatorial response to encompass the entire preexisting repertoire was obtained in studies of natural human IgG antibodies present in intravenous immunoglobulin. Since essential cellular recognition and regulatory elements are conserved throughout evolution, we propose that the natural antibodies of sharks, the most anciently emerged vertebrates to possess the combinatorial immune response, will resemble those of mammals in showing specificity for the conserved recognition/regulatory molecules. If verified, this hypothesis will establish the fundamental importance of natural antibodies not only in defense, but in regulation and functional homeostasis of the individual.

Specificity mapping of human anti-T cell receptor monoclonal natural antibodies: defining the properties of epitope recognition promiscuity.

The classical concept of antibody binding is defined as an exclusive and high-affinity interaction with one epitope. The emerging reality about antibody combing sites, however, is that some can bind unrelated determinants. The studies presented here define this quality as epitope recognition promiscuity by analyzing the capacity of monoclonal human autoantibodies to bind sets of overlapping peptides duplicating the complete structures of T cell receptor (TCR) alpha and beta chains and immunoglobulin lambda chain. We assessed the binding of these monoclonal antibodies (mAbs) to a set of homologous peptides corresponding to the CDR1 segments of human Vbeta gene products, a major epitope used in the selection of the antibodies. We present data on the binding characteristics of four human mAbs selected for the ability to bind TCR epitopes. These mAbs are IgM molecules with VH and VL sequences in germline configuration, but have diverse VH CDR3 regions. These studies aim to characterize the property of epitope promiscuity and show that the relationship between the binding site and its epitope is a complex interaction and unpredictable from antigen sequence alone. Our results support the conclusion that epitope recognition promiscuity is a genuine feature of antibody and TCR recognition.

Measles virus infection and vaccination: potential role in chronic illness and associated adverse events.

Over the last decade, a number of concerns have arisen related to safety issues that have had an adverse effect on the public's trust, particularly among parents whose children are the primary recipient of the vaccine. Historically, the live attenuated measles virus (MV) vaccine and the combination multivalent measles, mumps, and rubella (MMR) vaccine have had a major impact on the health of children worldwide and have been extremely successful at preventing infectious diseases associated with three childhood viral pathogens. In this report, we describe MV infection, replication, pathogenesis, and immunization. MV is a viral pathogen that exhibits a number of complex processes that can effect its replication, pathogenesis, and the induction of an effective antiviral immune response. We describe the published literature as it relates to MV infection and immunization and report adverse events in an attempt to provide a balanced discussion and an historical perspective of the MMR vaccine and autism.

Cloning of a functional vitamin D receptor from the lamprey (Petromyzon marinus), an ancient vertebrate lacking a calcified skeleton and teeth.

The nuclear vitamin D receptor (VDR) mediates the actions of its 1,25-dihydroxyvitamin D(3) ligand to control gene expression in terrestrial vertebrates. Prominent functions of VDR-regulated genes are to promote intestinal absorption of calcium and phosphate for bone mineralization and to potentiate the hair cycle in mammals. We report the cloning of VDR from Petromyzon marinus, an unexpected finding because lampreys lack mineralized tissues and hair. Lamprey VDR (lampVDR) clones were obtained via RT-PCR from larval protospleen tissue and skin and mouth of juveniles. LampVDR expressed in transfected mammalian COS-7 cells bound 1,25-dihydroxyvitamin D(3) with high affinity, and transactivated a reporter gene linked to a vitamin D-responsive element from the human CYP3A4 gene, which encodes a P450 enzyme involved in xenobiotic detoxification. In tests with other vitamin D responsive elements, such as that from the rat osteocalcin gene, lampVDR showed little or no activity. Phylogenetic comparisons with nuclear receptors from other vertebrates revealed that lampVDR is a basal member of the VDR grouping, also closely related to the pregnane X receptors and constitutive androstane receptors. We propose that, in this evolutionarily ancient vertebrate, VDR may function in part, like pregnane X receptors and constitutive androstane receptors, to induce P450 enzymes for xenobiotic detoxification.

Evolution of the acute phase response: iron release by echinoderm (Asterias forbesi) coelomocytes, and cloning of an echinoderm ferritin molecule.

That the plasma concentration of certain divalent cations change during an inflammatory insult provides a major host defense response in vertebrate animals. This study was designed to investigate the involvement of iron sequestration in invertebrate immune responses. A ferritin molecule was cloned from an echinoderm coelomocyte cDNA library. The amino acid sequence showed sequence homology with vertebrate ferritin. The cDNA contained a conserved iron responsive element sequence. Studies showed that stimulated coelomocytes released iron into in vitro culture supernatants. The amount of iron in the supernatants decreased over time when the amebocytes were stimulated with LPS or PMA. Coelomocytes increased expression of ferritin mRNA after stimulation. In vertebrates, cytokines can cause changes in iron levels in macrophages. Similarly, echinoderm macrokines produced decreases in iron levels in coelomocyte supernatant fluids. These results suggest that echinoderm ferritin is an acute phase protein and suggest that sequestration of iron is an ancient host defense response in animals.

The natural antibody repertoire of sharks and humans recognizes the potential universe of antigens.

In ancestral sharks, a rapid emergence in the evolution of the immune system occurred, giving jawed-vertebrates the necessary components for the combinatorial immune response (CIR). To compare the natural antibody (NAb) repertoires of the most divergent vertebrates with the capacity to produce antibodies, we isolated NAbs to the same set of antigens by affinity chromatography from two species of Carcharhine sharks and from human polyclonal IgG and IgM antibody preparations. The activities of the affinity-purified anti-T-cell receptor (anti-TCR) NAbs were compared with those of monoclonal anti-TCR NAbs that were generated from a systemic lupus erythematosus patient. We report that sharks and humans, representing the evolutionary extremes of vertebrate species sharing the CIR, have NAbs to human TCRs, Igs, the human senescent cell antigen, and to numerous retroviral antigens, indicating that essential features of the combinatorial repertoire and the capacity to recognize the potential universe of antigens is shared among all jawed-vertebrates.

Recombinant shark natural antibodies to thyroglobulin.

As cartilaginous fish are the vertebrates most distal from man to produce antibodies, fundamental information regarding conservation and variation of the antigen binding site should be gained by comparing the properties of antibodies directed against the same antigen from the two species. Since monoclonal cell lines cannot be generated using shark B cells, we isolated antigen binding recombinant single chain Fv antibodies (scFv) comprising of the complete variable regions from shark light and heavy chains. Thyroglobulin was used as the selecting antigen as both sharks and humans express natural antibodies to mammalian thyroglobulin in the absence of purposeful immunization. We report that recombinant sandbar shark (Carcharhinus plumbeus) scFvs that bind bovine thyroglobulin consist of heavy chain variable regions (VH) homologous to those of the human VHIII subset and light chain variable regions (VL) homologous to those of the human Vlambda6 subgroup. The homology within the frameworks is sufficient to enable the building of three-dimensional models of the shark VH/VL structure using established human structures as templates. In natural antibodies of both species, the major variability lies in the third complementarity determining region (CDR3) of both VH and VL.

A role for T lymphocytes in mediating cardiac diastolic function.

The induction of T helper (TH) lymphocytes by distinct TH ligands results in a differentiation to TH1/TH2 subsets based on their unique pattern of cytokine secretion and effector functions. We hypothesized that the relative proportion of TH1/TH2 directly relates to cardiac fibroblast (CF) function and thereby cardiac extracellular matrix (ECM) composition and cardiac diastolic function in the absence of injury or altered wall stress. We compared the effect of selective TH1 with TH2 inducers on cardiac gene expression, ECM composition, and diastolic function in C57BL/J mice. Twelve weeks after immune modulation, the left ventricular stiffness (beta) was significantly increased in the TH1 group and decreased in the TH2 group (P < 0.01). The TH2 group also demonstrated significantly increased end-diastolic and end-systolic volumes (P < 0.01). Cardiac gene expression patterns for pro-matrix metalloproteinase (MMP)-9 and -13 were increased by greater than fivefold in the TH2 group and significantly decreased in the TH1 group (P < 0.05). The total cardiac collagen and cross-linked collagen were significantly increased in the TH1 group and decreased in the TH2 group (P < 0.01). Coculturing lymphocytes harvested from the treated mice with naive primary CF demonstrated a direct control of the lymphocytes on CF pro-collagen, pro-MMP gene expression, and MMP activity. These results suggest that the TH phenotype differentially affects diastolic function through modulating CF pro-collagen and pro-MMP gene expression, MMP activity, and cardiac collagen cross-linking, resulting in altered ECM composition. Thus modulation of TH lymphocyte function could promote adaptive remodeling in heart failure and postmyocardial infarction.

Endogenous retroviruses in systemic lupus erythematosus: candidate lupus viruses.

Although the etiology of systemic lupus erythematosus (SLE) remains unclear, there is substantial circumstantial evidence that the development of SLE is dependent on environmental, genetic, and retroviral factors. SLE patients produce high titer antibodies to various retroviral proteins, including Gag, Env, and Nef of HIV and HTLV, in the absence of overt retroviral infection. We review the factors linking HERVs to SLE and consider the various processes utilized by endogenous retroviruses in the etiopathogenesis of SLE. In particular, we consider the role of HTLV-1-related endogenous sequence (HRES-1) in SLE. We propose that molecular mimicry between HRES-1 and the small ribonucleoprotein complex initiates the production of autoantibodies, leading to immune complex formation, complement fixation, and pathological tissue deposition.

Structural, antigenic and evolutionary analyses of immunoglobulins and T cell receptors.

We have had the pleasure of collaborating with Allen Edmundson for the past 15 years on the structure, binding properties and evolution of immunoglobulins and T cell receptors. Among the most significant contributions of our joint efforts were: (1) the predictive use of structural features of immunoglobulin domains to model the three-dimensional structures of the immunoglobulin domains of human T-cell receptor alpha and beta chains as well as shark light chains and V(H) domains; (2) the finding that normal humans and other vertebrates express autoantibodies against combining site epitopes of their own T cell receptors; (3) the mapping of the peptide autoepitopes recognized in health, autoimmunity and retroviral infection; and (4) the determination that epitope recognition promiscuity is a characteristic property of the combining sites of IgM immunoglobulins ranging from those of sharks to those of humans. We briefly review the salient findings and status of these studies and indicate the future directions that we will pursue in their continuation.

Rapid evolutionary emergence of the combinatorial recognition repertoire.

Although the capacity of cells to respond to environmental challenges such as oxidative damage are ancient evolutionary developments that have been carried through to modern higher vertebrates as "innate" immunity, the characteristic immune response of vertebrates is a relatively recent evolutionary development that is present only in jawed vertebrates. The vertebrate "combinatorial" response is defined by the presence of lymphocytes as specific antigen recognition cells and by the complete panel of antibodies, T cell receptors, and major histocompatibility complex molecules all of which are members of the immunoglobulin family. Its emergence in evolution was an extremely rapid event (approximately 10 million years) that was catalyzed by the horizontal transfer of recombinase activator genes (RAG) from microbes to an ancestral jawed vertebrate. RAGs occur in jawed vertebrates, but have not been found in invertebrates and other intermediate species. We propose that antigen recognition capacity contributed by this novel combinatorial mechanism gave jawed vertebrates the ability to recognize the entire range of potential antigenic molecular structures, including self components and molecules of infectious microbes not shared with vertebrates. The contrast within the vertebrates is striking because the most ancient extant jawed vertebrates, sharks and their kin, have the complete panoply of T-cell receptors, antibodies, MHC products and RAG genes, whereas agnathans possess cells resembling lymphocytes but ostensibly lack all of the molecules definitive of combinatorial immunity. Another vertebrate innovation may have been the utilization of nuclear receptor superfamily, in the regulation of lymphocytes and other cells of the immune lineage. Unlike, RAG, however, this superfamily occurs in all metazoans with the exception of sponges.

Human monoclonal natural autoantibodies against the T-cell receptor inhibit interleukin-2 production in murine T cells.

Natural autoantibodies (NAAbs) specific for the T-cell receptor (TCR) are present in all human sera, but individuals with rheumatoid arthritis (RA) generally produce higher titres of immunoglobulin M (IgM) isotype autoantibodies (AAbs) against Vbeta TCR epitopes. To investigate possible correlations between the specificity of such AAbs and their role in immunomodulation, we generated seven B-cell hetero-hybridomas, secreting monoclonal IgM NAAbs, from the synovial tissue and peripheral blood of patients with RA. Here we report three anti-TCR monoclonal autoantibodies (mAAbs)--OR2, OR5 and Syn 2H-11--with the ability to bind subsets of murine T cells, including the ovalbumin-specific DO-11.10 clone. These antibodies did not induce apoptosis in vitro, but prevented interleukin-2 (IL-2) production by antigen-specific T cells. These findings suggest an immunomodulatory function for NAAbs to TCR V-region epitopes and serve as the foundation for testing human anti-TCR mAAbs in animal models with the eventual goal of using them as therapeutic agents in human disease.