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.

The TCR is responsible for the specificity of cytotoxic T lymphocytes (CTL) by recognizing peptides presented in the context of MHC. By producing recombinant soluble TCR, it is possible to study this interaction at the molecular level. We generated single-chain TCR (scTCR) from tumor infiltrating lymphocytes (TIL) and one CTL clone directed against melanoma-associated antigen (MAGE)-1. Sixty-eight day anti-MAGE-1 TIL and one cloned anti-MAGE-1 CTL were analyzed by PCR for their Valpha and Vbeta gene usage. The TIL population showed a restriction in Valpha and Vbeta usage with only Valpha4 and Valpha9 and Vbeta2 and Vbeta7 expressed. The anti-MAGE-1 CTL clone demonstrated absolute restriction with only Valpha12 and Vbeta1 expressed. DNA sequence analysis was performed on all V regions. For the TIL, each possible Valpha-Vbeta combination (i.e. Valpha4-Vbeta2, Valpha9-Vbeta2, Valpha4-Vbeta7 and Valpha9-Vbeta7) was constructed as a distinct scTCR and the recombinant proteins expressed in bacteria. From the anti-MAGE-1 TIL, Valpha4-Vbeta2 scTCR demonstrated binding activity to HLA-A1(+) cells pulsed with MAGE-1 peptide. Results obtained from screening a panel of our scTCR constructs on HLA-A1(+) cells pulsed with MAGE-1 peptide or irrelevant peptide demonstrated that Vbeta2 plays a significant role in binding to the MAGE-1 peptide. Amino acid alignment analysis showed that each Vbeta sequence is distinctly different from the others. These findings demonstrate that soluble TCR in single-chain format have binding activity. Furthermore, the results indicate that in TCR, like antibodies, one chain may contribute a dominant portion of the binding activity.

The combinatorial immune system defined by the presence of antigen specific recognition units from the immunoglobulin family, the genetic machinery necessary for recombination, and cells of the lymphoid series expressing these receptors is fully functional from the earliest extant gnathostomes to mammals; however, the definitive evidence for genes specifying immunoglobulins, T-cell receptors (TCR), or recombination activating genes has not been documented for more primitive agnathan vertebrates, lower deuterostomes, or protostome, or acoelomate invertebrates. This chapter focuses on the appearance and molecular evolution of antibodies and T-cell receptors (TCR), which are the antigen-specific recognition elements of the combinatorial immune system. The combinatorial immune system of jawed vertebrates arose as an evolutionary big bang that involves the generation and duplication of V and C domains from an unknown precursor, and also involves the incorporation of joining segment genes, recombination signal sequences, and transmnembrane/cytoplasmic segments. The most recent major step in the evolution of immune system was the emergence in mammals of germinal centers within the lymph nodes, correlating with the IgM to IgG switch and affinity maturation following from somatic mutation and antigenic selection. The distinct heavy chain isotypes, such as IgY of chickens, reptiles, and amphibians and IgW of sharks have also arisen through gene duplication in evolution, and these may show distant relationships to mammalian immunoglobulins, such as IgD and IgE.

The rearrangement of antibody and T-cell receptor gene segments is indispensable to the vertebrate immune response. All extant jawed vertebrates can rearrange these gene segments. This ability is conferred by the recombination activating genes I and II (RAG I and RAG II). To elucidate their origin and function, the cDNA encoding RAG I from a member of the most ancient class of extant gnathostomes, the Carcharhine sharks, was characterized. Homology domains identified within shark RAG I prompted sequence comparison analyses that suggested similarity of the RAG I and II genes, respectively, to the integrase family genes and integration host factor genes of the bacterial site-specific recombination system. Thus, the apparent explosive evolution (or "big bang") of the ancestral immune system may have been initiated by a transfer of microbial site-specific recombinases.

SummaryWe determined the sequence of 18 DNA clones encoding VH regions of sandbar shark and bull shark. All of these sequences exhibit key structural coding features characteristic of known VH genes of higher vertebrates. These VH sequences disclosed considerable diversity, and can be divided into six families according to the criterion of 80% DNA sequence identity. The overlapping of some VH gene clones to two or more families is a particular feature found in carcharhine sharks, which suggests that VH diversification is a continuing process. The basic sequence patterns of heavy‐chain V regions found in all representative gnathanstomes and in VH of the shark heavy immunoglobulin igW provides evidence for selection of canonical residues in all VH structures, Elasmobranch VH sequences can be divided into two classes or clans, one comprising the ‘classical’ VH set and the other comprising VHs related to those of IgW (Vω). Phylogenetic analyses place the VH cluster as the root of all the classic VHs and indicates that the Vω set is most probably that of the primordial heavy chain.

All immunoglobulins and T-cell receptors throughout phylogeny share regions of highly conserved amino acid sequence. To identify possible primitive immunoglobulins and immunoglobulin-like molecules, we utilized 3' RACE (rapid amplification of cDNA ends) and a highly conserved constant region consensus amino acid sequence to isolate a new immunoglobulin class from the sandbar shark Carcharhinus plumbeus. The immunoglobulin, termed IgW, in its secreted form consists of 782 amino acids and is expressed in both the thymus and the spleen. The molecule overall most closely resembles mu chains of the skate and human and a new putative antigen binding molecule isolated from the nurse shark (NAR). The full-length IgW chain has a variable region resembling human and shark heavy-chain (VH) sequences and a novel joining segment containing the WGXGT motif characteristic of H chains. However, unlike any other H-chain-type molecule, it contains six constant (C) domains. The first C domain contains the cysteine residue characteristic of C mu1 that would allow dimerization with a light (L) chain. The fourth and sixth domains also contain comparable cysteines that would enable dimerization with other H chains or homodimerization. Comparison of the sequences of IgW V and C domains shows homology greater than that found in comparisons among VH and C mu or VL, or CL thereby suggesting that IgW may retain features of the primordial immunoglobulin in evolution.

The immune response of jawed vertebrates is an inducible, highly specific defense mechanism that is characterized by an enormous diversity in recognition capacity. This allows responses to potential pathogens that have never been previously encountered in the evolution of the species. The molecules that carry out the specific recognition of antigen are glycoproteins termed immunoglobulins (Igs) or T-cell receptors (TCRs) that occur as heterodimers consisting of pairs of light and heavy chains (Igs) or a/|3 or 7/8 TCR chains (Kabat et al, 1991). These molecules are homologous members of the same family that express variable (V) and constant (C) domains, with the V/V pairs containing the combining site for antigen and the C domains involved in dimerization and effector function. The genes specifying each chain comprise individual multigene families which, in mammals, contain large arrays of variable segments (50-300), 1-10 diversity segments (heavy chains, TCR 3 chains), a set of joining (J) segments, (a few for Igs, but as many as a 100 for TCR a chains), and a few constant domains (1 for K light chains, approximately 10 for the heavy chain translocon). Individual sequence diversity and commitment of a B or T cell to differentiation into an antigen-specific immunocyte (B or T cell) results from the recombination of a V, D, and J element to form a complete variable region for surface expression with an appropriate constant region. Since Igs and TCRs exist as heterodimers, further amplification of recognition capacity is contributed by the selection of the partner chain; for example, if there are 5000 possible complete VH (VDJ) segments and 1500 possible complete VK (VJ) segments, there are 7,500,000 possible VH/VK combining sites that can be formed. This calculation illustrates the magnitude of individual combining sites that can be generated, but is an underestimate because other mechanisms including somatic mutation and junction diversification within the D and J segments contribute additional possibilities. We will summarize recent evidence to illustrate that the fundamental genetic mechanism allowing the combinatorial diversification of antibody occurs in all jawed vertebrates, and is clearly present in representatives of the most primitive of living gnathastomes, the sharks. Early in the course of biochemical studies of Igs, Hill and his associates (Hill et al, 1966) made the seminal discovery that V and C domains of light and heavy chains were homologous to one another and proposed a scheme for the evolution of Igs based upon their derivation from a domain size precursor of approximately 110 residues. Attempts to gain an understanding of the 'big picture' of the evolution of Igs required the application of recombinant DNA technology. We will review recent data showing that clearly defined homologs of light chains (Schluter et al, 1989; Shamblott and Litman, 1989a; Greenberg et al, 1993; Raster al, 1994; Hohman etal, 1995), heavy chains (Kokubu et al, 1988a,b; Vazquez et al, 1992; Shen et al, 1996), and TCRs (Rast and Litman, 1994) are present in the most primitive of jawed vertebrates. In addition, sharks contain an additional class of Ig having many of the properties expected of the primordial Ig (Bernstein et al, 1996b) as proposed by Hill et al. (Hill et al, 1966). Overall, the data obtained on the evolution of Igs, and their close relatives the TCRs, supports the concept that approximately 450 millions years ago (coincident with the origins of ancestral vertebrates) a 'big bang' (Marchalonis and Schluter, 1990a) of gene duplication occurred which, coupled with the addition of DNA processing enzymes facilitating recombination (Greenhalgh et al, 1993; Bernstein et al, 1994, 1996a), generated the combinatorial immune response typical of vertebrates.

A cDNA fragment corresponding to a highly evolutionarily conserved region of the major anion transport protein band 3 was cloned from lamprey mRNA using PCR homology probing. This is the first report providing evidence for a band-3 like transporter in the lowest vertebrates, the agnathostomes. Semi-quantitative PCR showed expression similar to that of higher vertebrates. Lamprey serum contains antibody-like molecules that bind to synthetic peptides of band 3 comprising senescent cell antigen, an aging antigen that terminates the life of cells. The high degree of homology found in nucleic acid and derived proteins sequence and the reaction of "antibodies" in lamprey serum with senescent cell antigen peptides of band 3 suggests that lamprey band 3 plays a role comparable to that in higher vertebrates.

Recently it has been observed that administration of intravenous immunoglobulin (IVIG) can have profound effects on a wide variety of diseases related to the dysregulation of the immune system. The mechanisms which explain these activities are poorly understood. Human IVIG and various Cohn plasma fractions contain autoantibodies directed against T cell receptors (Tcr). Previous studies have shown that IVIG contains autoantibodies against T cell receptor peptides. In order to further our understanding of autoantibody specificities, a single chain Tcr (scTcr) was constructed by recombinant DNA techniques from the variable alpha and variable beta chains of the Jurkat cell line. Anti-Tcr autoantibodies were isolated from IVIG and Cohn fractions I + III using a scTcr affinity column. This scTcr affinity purified material reacted with the surfaces of T cells at 10 micrograms/ml whereas non-purified IVIG did not. Sera from patients with rheumatoid arthritis (RA) as well as serum from patients with systemic lupus erythematosus (SLE) reacted with the scTcr at levels above that of normals.

With the development of recombinant DNA technology, it has become feasible to clone, construct, and express fully human immunoglobulin molecules. Here we report a novel methodology to make human antitumor single-chain Fv (scFv) antibodies from tumor-infiltrating B lymphocytes. We isolated and expanded tumor-infiltrating B lymphocytes from melanomas in the presence of Epstein-Barr virus. The transformed B cells secreting tumor-specific antibodies were identified and cloned by limiting dilution. From one B cell clone with specific melanoma reactivity, we captured the immunoglobulin variable region genes VH and Vk by PCR, sequenced the genes, and linked them together by PCR assembly with the use of a (Gly4Ser)3 linker. The scFv gene was then cloned into the pET21d vector and expressed. The obtained scFv protein with a M(r), of 29,000 was purified and biotinylated for further characterization. The scFv demonstrated specific tumor reactivity to 21 of 24 different melanoma cell lines and not to 14 nonmelanoma tumor cell lines, such as breast, ovarian, and colon cancer cells lines; normal human melanocytes as well as normal human leukocytes. These results were obtained in (a) a tumor cell ELISA, (b) fixed cell immunofluorescence, and (c) live cell flow cytometry. The immunoprecipitation results indicated that a protein antigen of M(r) 45,000 was recognized by the scFv. Since we reported previously that about 70% of human tumors of different histological types contain tumor-infiltrating B lymphocytes producing specific antitumor antibodies, this approach offers a rapid, effective method by combining in vitro B-cell expansion and PCR gene cloning to elucidate the repertoire of the human antitumor immune response and to make human monoclonal antitumor antibody molecules.

A single chain T cell receptor (scTcr) was constructed from the complete V alpha and V beta regions of Jurkat T-cell receptor alpha/beta chain genes using molecular cloning techniques. The recombinant scTcr reacted with a panel of rabbit antisera generated against synthetic 16-mer peptides duplicating the amino acid sequence of Jurkat V alpha and beta chains but not with antisera directed against peptides from the constant domain. Autoantibodies present in sera from systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) patients bound the scTcr in ELISA assays. The recombinant scTcr described here should prove to be a useful reagent with which to study T-cell receptor activity in serological and functional assays.

Autoimmune reactivity is a consequence of infection with human immunodeficiency virus (HIV). We studied serological cross-reactions of purified pooled IgG from sera of HIV-infected individuals by using nested sets of synthetic overlapping peptides duplicating the covalent structures of T-cell receptors (TCRs) and immunoglobulin light chains and report that two processes of autoantibody production occur. (i) IgG autoantibodies to putative regulatory variable domain CDR1 and FR3 epitopes (where CDR is complementarity-determining region and FR is framework region) are present in pooled IgG from HIV-infected individuals at levels 10-fold greater than that in pooled IgG from healthy humans. (ii) Anti-TCR autoimmunization involves antigenic mimicry between a conserved peptide stretch of the major neutralizing V3 loop determinant of HIV-1 gp120 and the conserved FR4 segment of the TCR V beta. Affinity-purified antibodies to the synthetic V3 loop peptide bound to a recombinant single-chain TCR and to a synthetic TCR joining segment peptide containing the FR4 sequence. Conversely, affinity-purified autoantibodies from pooled IgG from HIV-infected individuals to the TCR peptide bound the V3 loop peptide and a single-chain TCR. Inhibition studies indicated that the cross-reactive immunizing antigen was the V3 loop. These results bear upon the impact of HIV infection on immune regulation and on the selection of peptides for vaccine development.

A 700-bp fragment of the recombinase activating gene 1 (RAG-1) was cloned from several evolutionarily distant (sandbar shark, paddlefish, goldfish, axolotl and pig) species using PCR. The nucleotide and deduced amino acid sequences revealed a highly conserved region that has remained essentially unaltered during 400 million years of evolution; e.g, shark and human sequences were 75% identical at the nucleic acid level and 87% as protein. The RAG-1 mRNA levels in the shark were analyzed using semi-quantitative PCR to reveal expression patterns contrary to normal mammalian expression. These results establish that the genetic mechisms for Ig gene rearrangement are present in all extant gnathanstomes.