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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 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 γ 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 Vγ and Jγ 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.

SUMMARY Immune response (Ir) genes linked to genes encoding histocompatibility antigens affect antibody-formation to synthetic polypeptides, weak native antigens and strong native protein antigens given in limiting doses. It has been proposed that the products of such Ir genes function as receptors for antigen on thymus-derived lymphocytes (T-cells) and these products are not immunoglobulin in nature. Ho wever, recent evidence indicates that (a) Ir genes are probably expressed in both T-cells and in bone-marrow-derived lymphocytes (B-cells); (b) IgM immunoglobulin is present on T-cells and functions in the binding of antigen, and (c) T-cells of nonresponder animals possess the capacity to recognize antigen. These results prompted us to consider alternative hypotheses for the function of Ir genes; namely (1)‘tolerance’hypotheses in which response is dominant over non-response, and (2) the possibility that the Ir gene product can act as an amplifier of T-cell function. These hypotheses are discussed within the context of murine studies and in relation to the association of human diseases, notably immunopathic phenomena, with HL-A.

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.

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 (β) 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.

Infection of people with human immunodeficiency virus (HIV) as well as LP-BM5 infection in mice results in progressive deterioration of the immune system in the majority of untreated hosts. Peptide immunotherapy has been shown to be effective in the stimulation or immunoregulation of T-helper 1 (T(H)1) and T-helper 2 (T(H) 2) response subsets. In murine acquired immunodeficiency syndrome (AIDS), T(H)1 deficiency enables the host to be susceptible to coxsackievirus infection, inducing cardiopathology in a short period. T-cell receptor (TCR) Vbeta8.1 peptide, a 16-mer peptide containing the entire CDR1 segment and part of the FR2 region of human Vbeta8, showed both an immunoregulating and immunostimulating effect in murine AIDS. TCR Vbeta8.1 peptide acts on T cells promoting interleukin-2 production and therefore enhancing a cell-mediated immune response. It retarded development of cardiopathology due to coxsackievirus infection. Retrovirus-infected mice treated with the peptide showed a longer life span than the nontreated, retrovirus-infected animals.

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 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.

Previous studies established that retrovirally infected young mice produced large amounts of autoantibodies to certain T-cell receptor (TCR) peptides whose administration diminished retrovirus-induced immune abnormalities. C57BL/6 young (4 weeks) and old (16 months) female mice were injected with these same synthetic human TCR V beta 8.1 or 5.2 peptides. Administration of these autoantigenic peptides to old mice prevent immunosenescence, such as age-related reduction in splenocyte proliferation and interleukin-2 (IL-2) secretion. TCR V beta peptide injection into young mice had no effect on T- or B-cell mitogenesis and IL-4 production while modifying tumour necrosis factor-alpha (TNF-alpha), IL-6, and interferon-gamma (IFN-gamma) secreted by mitogen-stimulated spleen cells. TCR V beta injection also retarded the excessive production of IL-4, IL-6 and TNF-alpha induced by ageing. These data suggest that immune dysfunction and abnormal cytokine production, induced by the ageing process, were largely prevented by injection of selected TCR V beta CDR1 peptides.

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.

C57BL/6 mice were injected with different doses of human T-cell receptor (TCR) V beta 8.1 CDR1 peptide at different times after murine retrovirus (LP-BM5) infection. Injection with TCR V beta 8.1 CDR1 peptide largely prevented the retrovirus-induced reduction in B- and T-cell proliferation, and T-helper 1 (Th1) cytokines [interleukin-2 (IL-2) and interferon-gamma (IFN-gamma)] secretion. It also suppressed T-helper 2 (Th2) cytokines (IL-6 and IL-10) production, which was stimulated by retrovirus infection. These effects were accomplished using at least 100 micrograms of peptide per mouse and the most effective dose of peptide had to be given within 4 weeks after retrovirus infection. Immunization with doses above 100 micrograms/mouse as long as 4 weeks postinfection maintained natural killer (NK) cell activity during retrovirus infection. Reducing the dose of peptide or delaying it until the disease progressed towards early murine acquired immune deficiency syndrome (AIDS) allowed development of immune dysfunction. These studies provide data suggesting that immune dysfunction, induced by murine retrovirus infection, was largely prevented by TCR V beta CDR1 peptide injection.

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.

SYNOPSIS. 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.

Although gene segments specifying Igs of all vertebrates show clear homology, their arrangements differ markedly, thereby suggesting that the mechanisms for the generation of diversity and for the regulation of gene expression may be quite distinct. In the sandbar shark, light chain gene segments are distributed as apparently independent clusters consisting of V, J, and C elements that require rearrangement for expression. The usual distance between V and C in the clusters is 3 kb but larger clusters occur. The V, J, and C elements are clearly homologous to those of human λ chains. Shark Igs resemble mammalian IgM in structure and gene similarity. IgM may comprise as much as 50% of serum proteins in the shark. By contrast, IgM in humans comprises less than 5%. Human autoantibodies usually are IgM. These show little dependence on thymic function for expression and tend to increase with age. We have carried out a study of the capacity of Igs of unimmunized sharks and people (normals and patients suffering from autoimmune diseases) to react against a panel of antigens, including those usually considered autoantibodies, such as thyroglobulin and single-stranded DNA. Sharks and humans possess IgM antibodies that react with thyroglobulin and ssDNA. Affinity-purified natural shark antibodies to thyroglobulin or ssDNA constitute small fractions of total IgM. They illustrate extensive cross-reactivity comparable to that shown by polyspecific IgM autoantibodies produced by human B cells (CD5+) that appear early in ontogeny.

A full-length cDNA clone specifying sandbar shark (Carcharhinus plumbeus) immunoglobulin light chain has been isolated and sequenced. By alignment with human lambda chains, the leader, framework, complementarity-determining, joining, and constant regions are clearly identified in the shark light chain. Approximately 40-50% identity is shared between the human and shark sequences in the variable and constant regions. We have performed sequence comparisons of the individual segments and constructed phylogenetic trees for the variable region. These studies identify the shark protein as a lambda chain. In addition, the sandbar shark light chain is only distantly related to that of horned shark (Heterodontus francisci) [Shamblott, M. J. & Litman, G. W. (1989) Proc. Natl. Acad. Sci. USA 86, 4684-4688], demonstrating that the long evolutionary time of divergence among shark species has led to the generation of substantial differences in sequence. The positions of the variable, joining, and constant gene segments in 14 genomic clones have been mapped. The segments are linked in individual clusters (variable, joining, constant) occupying 3-7 kilobases. Cluster arrangement can be grouped into two patterns based upon spacing between the genes in the individual clones. This arrangement is fundamentally different from that observed in higher vertebrates.

Senescent cell antigen (SCA), an aging antigen, is a protein that appears on old cells and marks them for removal by the immune system in mammals. It is derived from band 3, a ubiquitous membrane transport protein found in diverse cell types and tissues. We have used synthetic peptides to identify aging antigenic sites on band 3, using a competitive inhibition assay and immunoblotting with IgG directed against the aging antigen on old cells. Results indicate that: (i) the active antigenic sites of the aging antigen reside on membrane protein band 3 residues that are extracellular regions implicated in anion transport (residues 538-554 and 788-827); (ii) a putative ankyrin-binding-region peptide is not involved in SCA activity; and (iii) carbohydrate moieties are not required for the antigenicity or recognition of SCA because synthetic peptides alone abolish binding of senescent cell IgG to erythrocytes. One of the putative transport sites that contributes to the aging antigen is located toward the carboxyl terminus. A model of band 3 is presented. Localization of the active antigenic site on the band 3 molecule facilitates definition of the molecular changes occurring during aging that initiate molecular as well as cellular degeneration.

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.

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.

A gelatin sponge model of concomitant tumor immunity was employed in order to examine the clonality of T cells associated with progressing and rejected tumor sites. Here we show that freshly isolated T cells bearing TCR V(beta)1, CDR3 RPGTGN, J(beta)1.1 and TCR V(beta)8, CDR3 GD, J(beta)1.6 predominated progressing and rejected tumor sites. Despite the similarity in T cell populations, the T cells from rejected tumor sites were capable of killing the autologous tumor cells, whereas T cells from progressing tumor sites were not able to do so. The differing cytolytic ability could not be attributed to a difference in TCR zeta chain protein expression levels between both T cell populations. After a 5 day mixed lymphocyte tumor culture the T cells from the progressing tumor site were capable of killing autologous tumor cells, which suggested changes took place within the cell population during in vitro culture. Further TCR analysis revealed T cells bearing TCR V(beta)1, CDR3 RPGTGN, J(beta)1.1 and TCR V(beta)8, CDR3 GD, J(beta)1.6 were not expanded following the in vitro culture. These data suggest that the lack of cytotoxicity of freshly isolated tumor-infiltrating lymphocytes (TIL) was not due to abnormal TCR zeta chain expression or major differences in the TCR V(beta) usage. Additionally, the gain of TIL effector function did not correlate with an expansion of the TCR bearing T cells found to predominate the in vivo response. These data suggest that the predominant TCR V(beta) used by lymphocytes infiltrating regressing or rejected tumors may not represent the tumor reactive T cells that grow in culture or respond to the autologous tumor in vitro.

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.

THE WALTER AND ELIZA HALL Institute of Medical Research in Melbourne, Australia is probably the world's best known research center devoted to the study of immunology. This recognition was gained under the leadership of its third director, Sir Frank Macfarlane Burnet, who headed the Institute from 1944 to 1965. Subsequently, the Institute has shown explosive growth under the leadership of its present director, Sir Gustav Nossal (1966 to present). The two individuals differ markedly in personal characteristics, but both made major and unique contributions to the definition of immunology as a modern science. Two recent publications have brought the Hall Institute to general attention: the first is Life among the Scientists: An Anthropological Study of an Australian Scientific Community (Charlesworth et al., 1989). The second is The Seeds of Time (1991), a biography of Sir Macfarlane Burnet by Christopher Sexton. The overall perspectives of the two books are quite distinct. The first has the stated purpose of attempting "to understand how a small group of scientists at a particular research institute, and in a specific scientific field, do science, as distinct from what the received scientific mythology says they do and what philosophers of science and other science watchers suppose they do" (p. 1). For this study a group of anthropologists used interviews and techniques of anthropological analysis to characterize a group of scientists as if they were a separate subculture working within the context of its own historical, sociological and mythological milieu. Charlesworth et al. write from an avowedly Marxist perspective, and address too many issues facing contemporary science to do justice to any single one or to give a coherent picture of the subject. The volume is interesting because it states many of the issues that have currently been brought to the front in various popular criticisms of science and analyses them within the philosophical context of the authors' beliefs and within the framework of interviews with scientists working in a single research institute. It should prove useful to historians of the Hall Institute and Australian Science and to future sociological or anthropological studies of the scientific subculture. Sexton's book is a well-constructed biography of the life of Sir Macfarlane Burnet starting with the immigration of his father, Frank Burnet, to Australia in 1880 and ending with Burnet's death in 1986. The biography makes extensive use of interviews with Burnet, his writings, and interviews of individuals closely associated with him. I found it to be a fascinating book that provided an overall perspective of Burnet's life and times.

While the general structure of immunoglobulin chains has remained relatively unchanged throughout evolution, the organization of the genes encoding these molecules differs substantially. To understand how the rearranging immunoglobulin system arose, it is necessary to examine living representatives of the most early vertebrate phyla. Elasmo-branches, which include the sharks, skates, and rays, are the most primitive phylogenetic class of vertebrates from which immunoglobulin DNA sequences have been obtained. In the sandbar shark (Carcharhinus plumbeus), the genes are arranged in individual clusters in which a single variable (V), joining (J), and constant (C) region gene, along with upstream regulatory elements, span a distance of approximately 4.4 kb or approximately 5.8 kb. We report the complete sequence of a genomic clone encoding sandbar shark lambda light chain. A unique finding of our study is that the V and J genes are fused in the germ line. Three additional clones have been shown by DNA sequencing to also have fused V and J genes. The four clones have complementarity-determining regions 3 of various lengths and amino acid sequence variability similar to the products of rearranged genes. Furthermore, analysis by polymerase chain reaction technology revealed an additional 26 genomic clones demonstrating fusion of the V and J segments. Therefore, VJ fusion is the prominent organizational feature of sandbar shark immunoglobulin light chain genes. This finding raises questions concerning the necessity of recombination to produce an antibody repertoire capable of reacting against a diverse array of antigens.

We used mapping with synthetic overlapping peptides in combination with molecular modeling to analyze the IgG antibodies that humans naturally produce against human T-cell receptor beta chains and to localize the recognized peptide autoantigens in the three-dimensional structure of the molecule. Healthy individuals produce low levels of antibodies against T-cell receptor peptides, and these can be increased in autoimmune diseases. We characterized the reactivities in detail because IgG molecules reactive with self peptides occur in preparations of intravenous immunoglobulin and can be isolated by immunoaffinity chromatography. Natural IgG antibodies were directed against three major peptides. One corresponds to the first complementarity-determining region of the variable region. A second corresponds to the third framework of the variable region. The third is located in the constant region and is predicted to be a loop that extends out of the beta-barrel structure. This peptide is one that would give a characteristic structural distinction between the beta-chain constant region and the constant regions of immunoglobulin light chains to which beta chains are homologous. The capacity to bind these peptides is found in small fractions of normal polyclonal IgG, which contains both kappa chains and lambda chains. The activity is antibody-like in being confined to the Fab fragment and in its capacity to discriminate among homologous synthetic peptides corresponding to distinct beta-chain variable-region genes. We propose that a recognition and regulatory process naturally occurs that parallels the immune network for the regulation of the production of antibodies.

Isolated light chains of IgM-type immunoglobulins of carcharhine sharks were analyzed by serological and biochemical means. When analyzed by isoelectric focusing analysis, light chains of the tiger shark (Galecerdo cuvieri), the galapagos shark (Carcharhinus galapagensis) and the sandbar shark (Carcharhinus plumbeus) showed a broad, but patterned, spectrum of bands ranging from pI 5.0 to 7.7 in which discrete families were observed. Serologically, light chains of the galapagos shark cross-reacted with rabbit antibodies against mouse immunoglobulin and a synthetic peptide corresponding to the J segment of T cell receptor beta chain. The latter cross-reaction is shared among light chains and T cell receptors. Although there was considerable heterogeneity in isoelectric focusing analysis, the light chains were homogeneous on the basis of apparent mass (23 kDa) and those of tiger shark and galapagos shark had relatively homogeneous dominant N-terminal sequences representing the first framework. The N-terminal sequences of these two shark light chains, were strongly homologous to one another and showed 75% identity to certain V kappa sequences of man and dog. Homology was also shown to V lambda sequences, but the degree of identity was approximately 50%. Following cleavage of the tiger shark light chain with o-iodosobenzoic acid which cleaves at tryptophanyl residues, a constant region peptide was isolated by gel filtration. It was possible to identify the homolog of this peptide within the constant regions of mammalian kappa and lambda chain, but the relationship to C kappa chain was stronger. The degree of identity among the corresponding C region peptides of mammalian, avian and elasmobranch species was much less than that observed for the framework 1 sequence of the light chain variable region. These data support the concept that variable and J region sequence have been conserved in the evolution of placoderm-derived vertebrates, but that constant regions show much greater phylogenetic variation.

Accessible surface proteins of thymus-derived lymphocytes (T-cells) of normal CBA mice and bone-marrow-derived lymphocytes (B-cells) of congenitally athymic nu/nu mice were analysed. The surfaces of lymphocytes were radioiodinated by using the enzyme lactoperoxidase (EC 1.11.1.7), then solubilized either in acid-urea or in the non-ionic detergent Nonidet P-40. These lysates were then precipitated with antisera specific to either immunoglobulin or the theta-alloantigen in order to assess the presence of these surface markers. Comparable amounts of radioactivity in proteins specifically precipitable as immunoglobulin were obtained from T-lymphocytes and B-lymphocytes when the cells were disrupted by acid-urea. This immunoglobulin had mol. wt. approx. 180000 and was composed of light chains and mu-type heavy chains. When radioiodinated lymphocytes were solubilized with Nonidet P-40, 3-4% of radioiodinated high-molecular-weight protein of B-cells consisted of immunoglobulin, a result similar to that found with acid-urea extraction. However, with the detergent extraction, only 0.1% of T-cell surface protein was precipitable by anti-globulin reagents. The theta-alloantigen was isolated from CBA T-cells both by acid-urea and by detergent lysis. This protein possessed a mobility on polyacrylamide-gel electrophoresis in sodium dodecyl sulphate which was consistent with a mol. wt. of 60000. An identical component was isolated from the theta-positive thymoma WEHI 105. The theta-antigen was not isolated from B-cells by either of the extraction procedures used. These results provide further evidence that the surface membranes of normal T-cells and B-cells differ in physicochemical properties. In particular, various surface components possess differential solubilities in non-ionic or organic solvents. This observation provides an explanation for discrepant results that have appeared in the literature concerning the isolation of immunoglobulin from T-lymphocytes.

A method is described which allows the isolation of small quantities of DNP-binding antibodies of both high- and low-molecular-weight classes. The only detectable DNP-binding protein in the serum of placoderm-derived vertebrates was immunoglobulin in nature, and of the order of less than or equal to 1% total serum immunoglobulin. Proteins in the serum of the urochordate Pyura stolonifera, which bind to both DNP and erythrocyte antigens, showed charge heterogeneity and an apparent subunit molecular weight of approximately 65,000 to 70,000 daltons.

Proteins unique to B16 melanoma cells grown in culture and as solid tumors have been independently described recently. As both proteins have apparent M r of 65,000-70,000, we have more fully compared their biochemical characteristics. The M r 65,000 protein, which is isolated from B16 cells in culture, has an aggregate molecular weight of >200,000 under nonreducing conditions. This M r 65,000 form is shed into the culture medium exclusively and is the predominant molecular species of serum-free culture supernatants. It was found that B700, a major membrane protein of B16 cells in solid tumors, and the M r 65,000 preparation from cultured cells were electrophoretically identical when compared under four separate sets of conditions. These two proteins also share immunologic determinants. The results suggest the identity of these two proteins isolated by completely different approaches.

All vertebrates respond to antigenic challenge by specific cellular reactions and by producing circulating antibodies, and they also contain recognition molecules that are evolutionary relics of primitive non-immune recognition. One such ancient recognition molecule is C-reactive protein (CRP), a member of the pentraxin family, that has homologs occurring in species as diverse as vertebrates, tunicates and the horseshoe crab (an ancient arachnoid). This molecule has lectin-like properties, can act as an opsonin and interact with complement and cells in a manner paralleling immunoglobulins (Igs). The horseshoe crab lectin and CRP, although unrelated to Igs, share functional idiotopes with classical antibodies. This finding reflects either an evolutionary convergence or a mechanism of "mini gene insertion" that allows molecules of distinct evolutionary histories to react to the same ligands. Serum antibodies of all vertebrates are polydisperse in charge and are composed of polypeptide chains comparable in mass to those of mammalian light and heavy chains. Molecular genetic studies of placoderm derived vertebrates are incomplete but are sufficient to allow the conclusion that Igs of these species are specified by variable (V), joining (J) and constant (C) gene segments and that rearrangement are an essential feature for the generation of antibody diversity. Here we present new evidence following from the use of antibodies directed against synthetic joining region peptides as probes in the study of rearranging Igs in evolution, the use of recombinant DNA technology to study T cell receptor V beta genes in a goldfish genomic library and the isolation and characterization of a gene fragment specifying sandbar shark light chain C region. We reached the following conclusions: (1) J region segments are the most conserved in evolution, and this most probably reflects the essential requirements for these gene segments in the formation of intact Ig genes by rearrangement; (2) the framework segments of V regions are highly conserved in vertebrate evolution for both T cell receptors and classical Igs; and (3) although C region segments of light chains of lower vertebrates are homologous to their mammalian counterparts, the degree of conservation of C region structure in phylogeny is apparently less than that for V regions. Essentially, phylogenetic trees can be built using C regions but not V regions. We have identified a molecule of approximate mass of 26 kDa in the hemolymph of the tunicate, Boltenia ovipera, that is serologically cross-reactive with shark heavy chain and with J region peptides.(ABSTRACT TRUNCATED AT 400 WORDS)

Membrane immunoglobulin (Ig) of splenocytes and thymocytes of the goldfish, Carassius auratus, was demonstrated by indirect fluorescent-antibody techniques. Observations on shedding and resynthesis indicated that the thymocyte Ig was endogenously produced. The lymphocyte surface proteins were radioiodinated using the lactoperoxidase-catalyzed reaction, and the labeled Ig molecules were isolated by specific precipitation and analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The IgM-like membrane Igs of splenocytes and thymocytes were shown to differ in their ease of solubilization with nonionic detergent, and in the sodium dodecyl sulfate/electrophoretic mobility of their heavy chains. The significance of these observations for the evolution of T-cell recognition is discussed.

Immunoglobulin light and heavy chains show sequence homology to one another and to the polypeptide chains of putative T-cell receptors in the J (joining) segment of the variable region. Antibodies produced against synthetic peptides corresponding to the entire JH1 region and part of the diversity segment region cross-react serologically with products of normal T cells and monoclonal T-cell lines. In this study we generate immune affinity-purified rabbit antibodies to a synthetic 16-mer peptide consisting of the entire JT sequence and part of the T-cell diversity sequence corresponding to these segments of the human putative T-cell receptor beta gene YT35. Both free peptide and peptide coupled to bovine serum albumin as carrier were found to stimulate the production of antibody. The immune affinity-purified anti-JT peptide antibodies bound to intact immunoglobulin and to light and heavy chain as detected by enzyme-linked immunosorbent assay and by immunoblot transfer. The antibody reacted by these techniques with membrane components of the human monoclonal amplifier T-cell MOLT-3 and the murine suppressor T-cell WEHI-7. The component detected in the MOLT-3 cell corresponded to the beta-chain of the alpha/beta heterodimer putative T-cell receptor; whereas the molecule detected in the WEHI-7 line had properties corresponding to those of antigen-specific T-cell suppressor receptors. The molecular size of this component under reducing conditions was approximately 68 kDa and the intact form had an apparent mass of 140 kDa. These results provide direct proof of serological cross-reaction among products of putative T-cell receptor genes, antigen-binding T-cell receptors, and immunoglobulins, thereby supporting the concept that antigen receptors of T lymphocytes all represent new immunoglobulin translocons.

Immunoglobulins have been isolated from the surface of B (bone marrow-derived) and T (thymus-derived) lymphocytes. Two types of membrane immunoglobulin occur on B lymphocytes; one type resembles the 200,000-dalton subunit of IgM, the second possesses a heavy chain electrophoretically distinct from mu chain and does not correspond to any of the known classes of mouse immunoglobulins. It might correspond to human sigma chain. T lymphocytes possess only one type of surface immunoglobulin. This molecule has a mass of approximately 200,000 daltons and contains light chains and heavy chains similar to, but not identical to, mu chains. Evidence now exists that surface IgM-like immunoglobulins of B lymphocytes and T lymphocytes activated to certain antigens can bind specifically to antigen. These observations suggest that surface immunoglobulin functions as a receptor for antigen on B cells and at least on some T cells. The mechanisms by which combination of antigen with surface immunoglobulin initiate differentiation remain to be determined.

In this review, we analyze data pertinent to the origins of specific immune recognition by lymphocytes. The phenomena of immunity in invertebrates and the cells that might be involved in these processes are considered. We conclude that the existence of vertebrate-type immunocompetent lymphocytes in invertebrates is not yet proven. All vertebrates apparently possess immunologically competent lymphocytes, and the following conclusions may be drawn: (1) Specific antigen recognition by lymphocytes of all vertebrates appears to be mediated by membrane-bound immunoglobulins. These immunoglobulins are, in all probability, distinct from secreted, serum immunoglobulins (antibodies), although serum and surface immunoglobulins share combining sites for antigen which are formed of variable regions. (2) There is evidence for similar functional divisions among the immune systems of all vertebrates, as reflected in the results of anatomical, functional, and physicochemical investigations of lymphocytes from animals as diverse as fish and mammals.

We have detected and partially characterized multiple lectins present in the serum of the freshwater prawn Macrobrachium rosenbergii. Since agglutination of erythrocytes (RBC) is not abolished by treatment with Vibrio cholerae neuraminidase (VCN), Macrobrachium shows an agglutination pattern different from that of other sialic acid-specific lectins such as Limulus polyphemus lectin. However, after absorption with primate and bird VCN-treated RBC, Macrobrachium serum exhibits high titers with untreated and pronase-treated RBC and no agglutination of VCN-treated RBC, suggesting a typical sialic acid specific lectin agglutination profile. Hemagglutination-inhibition tests indicate that sialic acid containing compounds are the best inhibitors for Macrobrachium lectins. Subterminal sugars and type of linkage are probably important for the lectin binding since bovine submaxillary mucin (containing mainly terminal NANA-alpha-2 6-GalNAc-) is a better inhibitor than fetuin (containing mainly terminal NANA-alpha-2 leads to 3-Gal-) and colominic acid (-NANA-alpha-2 leads to 8-NANA-) is a weak inhibitor.

To estimate the minimal structural requirements for cross-reaction of idiotypic determinants, we determined the capacity of monoclonal antibodies specific for the idiotype of the phosphorylcholine (PC)-binding myeloma protein TEPC-15 for cross-reactivities with the PC-binding, acute-phase protein C-reactive protein (CRP) and the hemagglutinin from the horseshoe crab Limulus polyphemus (limulin), which binds sialic acid and PC. Certain monoclonal antibodies (MAb) to the TEPC-15 idiotype showed strong cross-reactions with CRP and limulin when tested by enzyme-linked immunoadsorbent assays. The specificity of the cross-reactivities was confirmed by testing the binding of the reactive anti-TEPC-15 MAb to both CRP and limulin in the presence of p-nitrophenylphosphorylcholine (pNPPC), N-acetylneuraminic acid, and bovine submaxillary mucin. The binding of the MAb to both CRP and limulin was strongly decreased by pNPPC, partially decreased by free PC, and not affected by N-acetylneuraminic acid or bovine submaxillary mucin. Neither CRP nor limulin showed significant overall sequence homology to vertebrate immunoglobulins. However, CRP, limulin, and TEPC-15 variable region heavy chain (VH) shared short stretches of homology (8-10 amino acids) that mapped to a stretch comprised of the second complementarity determining region and third framework region of the TEPC-15 VH. These results might reflect either evolutionary convergence forced upon molecules of diverse evolutionary histories because of steric requirements of binding the same ligand, or a conservation of primitive combining site gene segments in evolution.

SYNOPSIS. Recent evidence suggests Ihat amphibians represent a key transition stage in the evolution of immunological competence. In particular, anuran amphibians exhibit a significant advance relative to more primitive vertebrates in their capacity to synthesize two distinct classes of immunoglobulins in response to antigenic stimulation.These two classes of antibody molecule resemble the γM and γG antibodies of man in size and polypeptide chain structure. Furthermore, cellular aspects of antibodyproduction in anura resemble those of mammals in the method of retention of antigen and the range of antibody-forming cells (lymphocytes to plasma cells). In addition to these parameters of antibody-formation, a state of specific nonresponsiveness (high zone tolerance) can be induced in adult anura by pretreatment with large doses of protein antigen. Studies of larval anurans establish that these forms become immunologically competent early in development, but possess only γM immunoglobulins. These larval stages provide feasible model systems for the study of the activation of genes which specify immunoglobulins. Antibody-formation in urodeles is discussed in relation to the emergence of immunological competence in amphibians.

Cell surface proteins of normal and neoplastic lymphocytes were labeled with iodide-125I by lactoperoxidase-catalyzed iodination. Incubation of 125I-labeled iodide cells in vitro resulted in the release of iodinated surface proteins at a rapid rate which was dependent on cellular respiration and protein synthesis. Comparisons by disc electrophoresis showed a marked similarity between urea-soluble surface proteins extracted from iodinated cells and iodinated material released by the cells during in vitro incubation. The rate of release of cell surface proteins from thymus cells was three times faster than that of spleen cells or bone marrow-derived thoracic duct lymphocytes. In addition, different proteins were released at different rates as evidenced by the rate of release of 125I of rabbit anti-mouse immunoglobulin specifically bound to mouse spleen cells and comparisons by disc electrophoresis of urea-soluble iodinated surface proteins extracted from cells before and after incubation. The results suggest that a dynamic state exists at the cell surface. The possible role of the release of cell surface proteins in cell regulation and communication is discussed.

Concentrated medium obtained from cultures of a continuous thymus-derived mouse lymphoma cell line (WEHI-22.1) was found to inhibit a T-cell-dependent (antidonkey red blood cell), but not a T-cell-independent (anti-DNP) immune response in vitro. Passage of such a concentrate through an anti-mouse Ig immunoadsorbent column removed its inhibitory activity. It is suggested that the tumor cell Ig can compete with specific normal T-cell Ig in its collaborative function in immune responses. A similar mechanism may account for anergy associated with some human lymphoid neoplasms.

Immunoglobulins were isolated from the surfaces of lymphocytes from a variety of lymphocyte populations including murine and human thymus lymphocytes and murine spleen and thoracic duct lymphocytes. Cell surface proteins were labeled with iodide-125I by lactoperoxidase-catalyzed iodination, and recovered in solution either by solubilization in dissociating solvents or active metabolic release. Immunoglobulins were identified and isolated by immunological coprecipitation. The polypeptide chain structure of immunoglobulins isolated from lymphocyte surfaces was analyzed by polyacrylamide gel electrophoresis of reduced, alkylated samples in acid urea. Human and murine thymus lymphocytes possessed only IgM immunoglobulin on their surfaces. This protein contained light chains and µ-type heavy chains and was characterized by a molecular weight of approximately 200,000. Murine splenic lymphocytes from CBA x C57 animals and congenitally athymic (nu/nu) mice possessed both IgM and IgG on their surfaces. The ratio of µ-chain to γ-chain was about 3/1. The presence of IgM on thymus lymphocytes probably does not reflect trace contamination by B lymphocytes because comparable quantities of IgM were isolated from both cell populations. Metabolic turnover data suggest that this immunoglobulin is synthesized by the cell population studied. These results provide direct evidence for the presence of immunoglobulins composed of light and heavy polypeptide chains on the surfaces of lymphocytes of all classes.