Phylogenetic origins of antibody structure. 3. Antibodies in the primary immune response of the sea lamprey, Petromyzon marinus.

The sea lamprey, Petromyzon marinus, has been found to produce specific antibodies after immunization with bacteriophage f2. Antibody activity is localized in 6.6S and 14S fractions of lamprey serum. The 6.6S antibodies were purified by a combination of zone electrophoresis, ion exchange chromatography, and gel filtration. Antigenic analysis of the 6.6S antibodies showed them to be free of other serum proteins and antigenically similar or identical to the 14S fraction. Evidence has been obtained which suggests that the 6.6S immunoglobulins consist of light components (molecular weight 25,000) and heavy components (molecular weight 70,000). In the immunoglobulin, these polypeptides appear to be linked via weak interactions but not by interchain disulfide bonds. Molecular weight analyses support the view that the chains can undergo concentration-dependent dissociation in aqueous solutions. Amino acid analyses showed that the compositions of the light and heavy components were similar and that aspartic acid or asparagine was the predominant amino terminal residue. Starch gel electrophoresis indicated that the subunits of lamprey antibodies are diffusely heterogeneous. The heavy chain mobility corresponded to that of micro-chains and resembled that of heavy chains of shark and sting ray immunoglobulins. In the course of the fractionation a 46S natural hemagglutinin composed of lower molecular weight subunits was isolated. This hemagglutinin did not resemble the lamprey immunoglobulin although it had a similar zone electrophoretic mobility in the beta-region. These studies are consistent with the hypothesis that micro-chains were the earliest of the heavy chain classes to emerge and further support the view that the multichain structure of immunoglobulins is a fundamental feature of antibody molecules.

Invertebrate recognition protein cross-reacts with an immunoglobulin idiotype.

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.

Inhibition of a T-cell-dependent immune response in vitro by thymoma cell immunoglobulin.

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.

Isolation and partial characterization of lymphocyte surface immunoglobulins.

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-(125)I 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 micro-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 micro-chain to gamma-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.

Lymphocyte membrane dynamics. Metabolic release of cell surface proteins.

Cell surface proteins of normal and neoplastic lymphocytes were labeled with iodide-(125)I by lactoperoxidase-catalyzed iodination. Incubation of (125)I-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 (125)I 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.

Lymphocyte surface immunoglobulins.

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.

Visualization of a guinea pig T lymphocyte surface component cross-reactive with immunoglobulin.

Thymus-derived lymphocytes (T cells) show exquisite specificity in recognition of antigens, but the nature of the cell surface receptor is controversial. Although antigen recognition mediated by immunoglobulin variable (V) regions remains the minimal hypothesis, it has been extremely difficult to definitely establish the presence of immunoglobulins on these cells. Chicken antibodies, produced against the (Fab')2fragment of mouse immunoglobulin G (IgG) and purified by binding to and elution from IgG-Sepharose 4B, bind to an endogenously synthesized surface component of guinea pig T cells. The binding occurred via a cross-reaction with murine k chain and a heavy chain determinant localized in the Fd region, and was visualized by immunofluorescence and immunoelectronmicroscopy using both transmission and scanning techniques. These data provide direct evidence for the presence of a surface component related to immunoglobulin on T lymphocytes.

Studies on the relationship of the B700 and B50 murine melanoma antigens.

The B50 and B700 proteins of B16 murine melanoma were studied; they were determined to be distinct, unrelated molecules. This was determined by V8 peptide mapping, N-terminal amino acid sequencing, absence of cross-reactivity with specific polyclonal antibodies, and monoclonal antibodies recognizing different epitopes.

Evidence for common and distinct determinants of colon carcinoembryonic antigen, colon carcinoma antigen-III, and molecules with carcinoembryonic antigen activity isolated from breast and ovarian cancer.

This study was designed to answer the question, do molecules with carcinoembryonic antigen (CEA) activity from colon, breast, and ovarian cancer differ? Extracts of two breast and three ovarian cancers with CEA activity were compared to three colon cancer CEA preparations and to the related antigen, colon carcinoma antigen-III, in terms of lectin- and antiserum-binding properties. With the use of Farr-type radioimmunoassays with the lectins, concanavalin A and wheat germ agglutinin, the iodinated colon CEA and CEA-like preparations from breast and ovarian cancer all showed distinctly different patterns of binding. Specificity of binding was confirmed by inhibition studies with the relevant monosaccharides. Similarly, with antisera prepared against colon CEA, colon carcinoma antigen-III, or breast CEA, it was shown that, although all preparations shared some antigens, unique antigenic determinants were also present on all preparations. These data are consistent with the concept of a series of closely related CEA and CEA-like molecules with distinct characteristics for each tissue source of CEA.

Immunomodulation by immunopeptides and autoantibodies in aging, autoimmunity, and infection.

The operation of the immune system is a complex orchestration of specific self and non-self-recognition capacities mediated by cells of the innate system acting in coordination with T and B lymphocytes in a series of processes modulated by cytokines. We provide evidence for a natural immunomodulatory system involving autoantibodies directed against a controlling segment of T cell receptor Vbeta chains that downregulate production of stimulatory cytokines balanced by the peptides which in turn upregulate inflammatory activities mediated by TH1-type helper cells. TCR Vbeta-derived peptides effective in retrovirally induced immunosupression could also reverse the effects of immunosenescence in aged mice by restoring the balance of TH1- and TH2-type immunity and the resistance of the animals to cardiac pathology caused by infection with coxsackievirus. An unexpected finding was an adaptive role of the T cells from peptide-treated mice in remodeling damaged hearts by increasing net collagen synthesis by cardiac fibroblasts.

T-cell receptor-derived peptides in immunoregulation and therapy of retrovirally induced immunosuppression.

Retrovirally infected humans and mice showed progressive acquired immunodeficiency accompanied by the production of elevated levels of autoantibodies directed against T-cell receptor variable-domain epitopes. Epitope mapping analyses indicated that a major determinant recognized was defined by a 16-mer peptide containing the entire CDR1 segment and part of the FR2 region of human Vbeta8, and that both species showed reactivity to the same sequence. Either prophylactic or therapeutic administration of this peptide to retrovirus-infected C57/BL/6 mice normalized the balance of T(H)1- and T(H)2-type helper activity and restored the resistance to infection by the opportunistic parasite Cryptosporidium. Administration of the peptide did not generate significantly increased levels of autoantibody, but had a profound effect on T-cell activity as well as other aspects of inflammation, including NK-cell activity. A 16-mer derived from the Jbeta sequence showed similar functional effects on T cells from retrovirus-infected mice. Direct binding of the VbetaCDR1 peptide to recombinant TCR Valpha/Vbeta constructs, as well as to IgM natural autoantibodies, suggests that the cell surface receptor for the peptide is the alpha/beta TCR on T cells and surface IgM in B cells. The Vbeta CDR1 peptide stimulated division of murine splenocytes in vitro, stimulated the production of the T(H)1 cytokine IL-2, and synergized with the T-cell mitogen concanavalin A in proliferation and IL-2 production. These studies indicate that administration of peptides derived from T-cell receptor variable domains to animals immunosuppressed as a result of retroviral infection has a profound immunomodulatory effect enhancing overall T-cell functional capacity, particularly with respect to the cytokine production characteristic of T(H)1-type cells. Our studies are interpreted in the context of other recent investigations of immunomodulatory peptides.

Binding of human IgG myeloma proteins to autologous T-cell receptor determinants.

IgG myeloma proteins (MPs) produced by monoclonal plasma cells derived from B2 lymphocytes have been reported to bind to various autoantigens but the binding generally has been of low affinity. Moreover, T cells from some multiple myeloma patients can respond specifically to idiotypes of their own paraproteins. We analyzed the capacity of more than 20 human IgG MP to bind, a recombinant single-chain molecule containing complete V beta 8.1 and V alpha 1 structures, sets of synthetic peptide epitopes corresponding to a complete TCR beta chain, and a set of CDR1 epitopes corresponding to 24 human V beta gene products, and intact monoclonal T cells. Two of 20 MPs bound strongly to the recombinant TCR. Five of the same set, including these, bound to a synthetic epitope corresponding to the CDR1 segment. On a mass basis, the binding was approximately 1000-fold greater than that of pooled polyclonal IgG. The binding activity was confined to the Fab fragment and was specifically inhibitable by appropriate peptide determinants. Spectrotypic analysis using a set of CDR1 epitopes indicated that individual proteins showed characteristic binding patterns ranging from highly specific to relatively promiscuous. Highly reactive MPs also bound to TCR on intact cells in immunocytofluorescence by flow cytometry. These results are consistent with the relatively frequent occurrence of autoantibodies to TCR determinants and indicate that MPs can be derived from this autoantibody subset.

Broad phylogenetic expression of heavy-chain determinants detected by rabbit antisera to VHa allotypes.

Immunoglobulin molecules from diverse vertebrate species were examined, using an enzyme-linked immunosorbent assay (ELISA), for the expression of determinants detectable by rabbit antisera to VHa allotypes. The data indicate that immunoglobulins of elasmobranchs, teleosts, amphibians and birds express determinants cross-reactive with those specified by the a1, a2 and a3 alleles in the domestic rabbit. We localize VHa cross-reactive specificity to the denatured heavy chain of a primitive vertebrate, the Galapagos shark (Carcharhinus galapagensis). Furthermore, the N-terminal amino acid sequence of the shark heavy chain shows significant homology with rabbit heavy chains of known VHa type at positions where allotype-correlated differences have been implicated. VHa-related determinants are shared by immunoglobulins of a wide range of vertebrates from sharks to man and thus seem to be epitopes which have been conserved during vertebrate evolution. The determinants detected on immunoglobulins of lower vertebrates by rabbit anti-VHa allotype sera most probably are VH-subgroup rather than allotypic markers. Their distribution demonstrates a strong phylogenetic conservation of VH-regions.

Solid-phase antigen binding by purified immunoproteins from antigen-specific monoclonal T cell hybridomas.

We have developed two distinct solid-phase immunoassays for the detection of antigen binding activity by products of antigen binding T cell hybridomas in the absence of MHC. Two suppressor T cell hybridomas studied (34s-18 and 34s-704) are specific for keyhole limpet hemocyanin, a protein antigen, and the other suppressor T cell hybridoma (51H7D) binds specifically to the arsonate hapten. We have adapted these hybridomas to growth in serum-free medium and have isolated molecules with antigen binding activity both from the cell membranes and from the culture fluid in which the cells had been grown. The antigen binding molecules (ABM) produced by the KLH-specific hybridomas bound best to native hemocyanin; binding was decreased when KLH was denatured by reduction and alkylation and no binding was found to an arthropod (Limulus) hemocyanin. The arsonate binding hybridoma, on the other hand, produced molecules specific for this hapten; they showed no capacity to bind KLH. The antigen binding molecules affinity-purified from all three T hybridomas have intact masses of either 145,000, 67,000 or 48,000 when run in SDS-PAGE under non-reducing conditions. Following reduction, ABM resolve in SDS-PAGE into a complex of polypeptide chains having apparent masses of 65,000, 56,000 and 49,000, with either a pair of bands at 26,000 and 22,000, or with a single band at 32,000, which is consistent with the size of translation products of mRNA previously isolated from these hybridomas. Two of the hybridomas, 34s-18 and 34s-704, used for isolation of antigen binding products in this study, were previously reported to lack detectable rearranged gamma or beta genes and therefore to lack expression of the alpha/beta or gamma/delta heterodimers. The antigen binding molecules react in solid-phase immunoassay with some antibodies specific for variable (first framework) region and joining (J) region peptide sequences predicted from T cell receptor gene sequence. Furthermore, the affinity-purified antigen binding molecules from mouse T cell hybridomas cross-react in ELISA with goat anti-rabbit IgG and not with protein G, thus allowing the use of these commercially available reagents in standard laboratory assays. Interestingly, ABM anchored in intact cell membranes, which could be shown to specifically bind antigen, did not cross-react with goat anti-rabbit IgG, indicating that the cross-reactive moiety is not detectable when the ABM are in this situation.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunoglobulin epitopes defined by synthetic peptides corresponding to joining region sequence: conservation of determinants and dependence upon the presence of an arginyl or lysyl residue for cross-reaction between light chains and T-cell receptor chains.

Joining or J region sequences of rearranging immunoglobulins and T-cell receptors show considerable sequence homology, particularly in their C-terminal portion corresponding to the fourth framework region of immunoglobulin variable regions. In order to test the question of whether serological cross-reactions between immunoglobulin variable regions and T-cell receptors were due to antigenic similarities in their J regions, we synthesized synthetic peptides corresponding to immunoglobulin J regions and to J regions predicted from gene sequence of the T-cell receptor beta chain. We found that antibodies produced against a synthetic 16-mer J beta sequence reacted with T-cell receptor chains and also with immunoglobulin light chains. The cross-reactivity was dependent upon the J signature sequence FG()GT(R or K)L where the presence of a positively charged lysyl or arginyl residue was essential for cross-reactivity. We were able to classify J region determinants into two distinct antigenic sets; one corresponding to JH and the other corresponding to J kappa, J lambda, J beta and J alpha. Although considerable homology occurs between JH and JL (or J beta) sequences, little cross-reactivity was observed between these two J subsets. Antibodies raised against polyclonal murine IgG immunoglobulins contained antibody subpopulations specifically reactive with either JH or J beta peptides. The serological data derived here using antipeptide antibodies are consistent with computer modeling studies that indicate that the conformations of T-cell receptor variable regions resemble those of classical immunoglobulins. Our data comparing cross-reactivities restricted to the J region indicate that the expression of the J region by intact T-cell receptor beta chains is probably more similar to that of light chains than it is to the corresponding region of heavy chains.

A monoclonal antigen-binding T cell immunoprotein: antigenic relatedness to T cell receptor beta chain FR1 V and J peptide segments: physicochemical distinctiveness from classical immunoglobulins and T cell receptor heterodimers.

The monoclonal murine T cell hybridoma, 51H7D, was previously shown to bind the arsazobenzene hapten and to produce a soluble antigen-binding molecule. In this paper we characterize this antigen-binding immunoprotein for its relationship to known T cell receptors serologically, using antibodies specific for variable region framework, or joining region peptides predicted from gene sequence and by biochemical means. The 51H7D cell expresses a protein with subunit size of approximately 31,000, that reacts antigenically with affinity-purified antibodies directed against synthetic first framework and joining segment peptides, corresponding to the gene sequence of the T cell receptor beta chain, YT35. This molecule does not react with affinity-purified antibodies directed against murine immunoglobulin, framework 1 sequences of alpha and gamma T cell receptors, or with antibodies against synthetic heavy chain joining segments. The subunit of mol. wt. 31,000 can form higher aggregates, notably in the mol. wt range of 60,000-70,000, depending upon extraction conditions. The soluble form of the antigen-binding molecule bears the J beta cross-reactive determinant and occurs predominantly as a charge restricted molecular species of approximate mol. wt 60,000-70,000. The purified molecule has a blocked N-terminus, but quantitative statistical analysis of its amino acid composition indicates a closer relatedness to T cell receptor beta chains and other antigen-binding T cell products, than it has to alpha, gamma or delta TCR chains. No evidence for more than one type of polypeptide chain was found and the polymerization is not dependent upon the formation of disulfide bonds. These studies raise the possibility that antigen-binding soluble T cell molecules might belong to a new family of immunoproteins, that is related to, but distinct from, classical immunoglobulins and alpha beta or gamma delta heterodimers.

Sequence analysis of homogeneous peptides of shark immunoglobulin light chains by tandem mass spectrometry: correlation with gene sequence and homologies among variable and constant region peptides of sharks and mammals.

Morphologically, sharks are living fossils that are remarkably similar to their Devonian ancestors of ca. 400 million years ago. If a parallel conservation in biochemical properties characterizes shark evolution, knowledge of the properties of shark immunoglobulins should provide information on the structure of primordial immunoglobulins and their genes. The problem of polyclonality of shark immunoglobulins has precluded detailed analysis of shark immunoglobulin light polypeptide chains. Here, we approach the problem of obtaining direct sequence information on polyclonal light chains of shark immunoglobulins by isolating homogeneous peptides from tryptic digests of shark light chains and sequencing these by tandem mass spectrometry. To confirm the location of the peptides, we isolated a complementary DNA (cDNA) clone from a sandbar shark cDNA library in the expression vector lambda gt11, identifying the clone by its ability to produce a peptide serologically detectable using rabbit antibody to purified shark light chain. The correspondence between peptide sequence and that derived from gene sequence provided direct proof that the gene studied was that of a major expressed serum light chain. Using this combined approach, we isolated homogeneous peptides from both constant and variable regions. The variable region peptides showed homology to corresponding sequences of mammalian V lambda and V kappa sequences. The constant region gene sequence we obtained was homologous to mammalian C lambda sequence. The four constant region tryptic peptides we sequenced corresponded exactly to stretches of the C lambda sequence derived from the DNA sequence. The combined approach described here shows that shark light chains exhibit heterogeneity at both the protein and gene level, but that the constant regions of these chains can be identified as homologs of mammalian lambda chains and that evolutionary conservation has occurred in V region sequences ranging from elasmobranchs to man.

Evolutionary perspectives on amyloid and inflammatory features of Alzheimer disease.

We propose that the amyloid deposits in senile plaques of Alzheimer's Disease (AD) result from ancient mechanisms in wound-healing and inflammatory processes that preceded the evolution of the inducible combinatorial immune responses characteristic of jawed vertebrates. AD plaques are unlike active plaques in MS, because antibodies, T-cells and, B cells are not conspicuous components of senile plaques or other loci of degeneration. However, senile plaques contain amyloids and other inflammatory proteins of ancient origin that appear to be made by local brain cells, including neurons, astrocytes, and microglia. We describe a highly conserved 16-mer found in pentrakins from mammals and from the horseshoe crab. The senile plaque thus provides a novel opportunity to study primitive features of complement-mediated inflammatory responses in the absence of immunoglobulins.

Lack of preferential V beta usage in synovial T cells of rheumatoid arthritis patients.

The T-cell receptor V beta subfamily repertoires of synovial and peripheral T cells of 8 rheumatoid arthritis (RA) patients were determined using the polymerase chain reaction. Three normal controls were included. Some of the rheumatoid synovial samples did not express the complete range of V beta families and lacked as many as 6 gene families. However, these patients showed considerable individual variation in expression. Overall, the data do not support preferential T-cell receptor V beta usage in synovial T cells of RA patients either in comparison to their autochthonous peripheral T cells or to peripheral T cells of normal subjects.

Antibodies to synthetic peptides corresponding to variable-region first-framework segments of T cell receptor. Detection of T cell products and cross-reactions with classical immunoglobulins.

Recent studies at the gene level have shown that T cells express rearranged genes for four types of T cell receptors that are strongly homologous to classical immunoglobulins in the joining region and in the framework 1 (Fr1) and 3 segments of the variable region. Based upon the homologies in gene sequence, it follows that the gene products would show similarities in amino acid sequence and in the folding of the proteins so that cross-reactivities in antigenic determinants would be expected between variable regions of the T cell receptors and classical immunoglobulins. We have synthesized peptides corresponding to predicted protein sequences of the Fr1 residues of T cell receptor alpha, beta- and gamma-chains and have produced antibodies in rabbits against these synthetic peptides. Use of antisera and affinity-purified antipeptide antibodies indicated that high-titer antibodies could be raised that were specific for individual Fr1 peptides. Cross-reactions among Fr1 peptides of T cell receptors and immunoglobulin light chains were observed. In addition, some rabbit antisera raised against classical polyclonal immunoglobulins or affinity-purified immunoglobulin-like T cell receptors were found to exhibit binding activity against Fr1 peptides of T cell receptor beta- and gamma-chains. The sequence homology, although real among the Fr1 of T cell receptors and immunoglobulin light chains, is moderate and the antigenic cross-reaction must reflect the configuration and types of amino acids present. The development of antipeptide antibodies holds promise for the characterization of T cell receptors of various T cell sources and also offers a new means for the identification of molecules related to rearranging immunoglobulins.