Discovery of a new class of immunoglobulin heavy chain from fugu.

In teleosts, the genomic organization of the immunoglobulin (Ig) heavy (H)-chain locus was thought to follow a typical translocon-type multigene structure; however, recent studies have indicated a variation in the structure and this might be teleost specific. Isotypes of the Ig H-chain, namely IgM, IgD, IgZ and IgT, have been identified. In this study, we report the discovery of a new class of IgH from fugu. This isotype was first identified from the genomic sequence of the fugu IgH locus. This novel IgH gene is composed of two constant (C) domains, a hinge region, and two exons encoding membrane regions. Surprisingly, the new IgH gene is present between the variable (V)H and Cmu regions of the locus. The C domains of the new isotype do not show any significant similarity to mammalian or fish IgH genes. The cloned cDNA from the new isotype has typical Ig H-chain characteristics and is expressed as both secretory and membrane form. Transcript analyses suggest that the new IgH from fugu might only use the joining (J)H segments present in front of the new CH domains and that the usage of DH and JH segments is specific to the isotype expressed. The expression pattern of the gene has been confirmed by in situ hybridization and PCR studies.

The porcine Ig delta gene: unique chimeric splicing of the first constant region domain in its heavy chain transcripts.

The pig delta gene is located approximately 3.4 kb downstream of the second transmembrane exon of the micro gene and shows a similar genomic structure to its counterpart in cow with three exons encoding the CH1, CH2, and CH3 domains. The porcine genomic deltaCH1 exon has been replaced by a recent duplication of the micro CH1 and its flanking sequences, a genetic event that also led to the formation of a short switch delta region, immediately upstream of the delta gene. The deltaCH1 exhibits a 98.7% similarity (314 of 318 bp) to the micro CH1 at the DNA level, whereas the homologies between the deltaCH2 and micro CH3, and the deltaCH3 and micro CH4 are only 33.3 and 35.8%, respectively. Either of the two CH1 exons ( micro and delta) could be observed in the expressed porcine IgD H chain cDNA sequences VDJ- micro CH1-H-deltaCH2-deltaCH3 or VDJ-deltaCH1-H-deltaCH2-deltaCH3, showing a pattern that has not been observed previously in vertebrates. In addition, transfection of a human B cell line, using artificial constructs resembling the porcine C micro -Cdelta locus, also generated both VDJ- micro CH1-deltaCH1-H1-deltaCH2 and VDJ -deltaCH1-H1-deltaCH2 transcripts. An examination of the pig delta genomic sequence shows a putative, second hinge region-encoding exon. Due to the lack of a normal branchpoint sequence for RNA splicing, this exon is not present in the normal pig delta cDNA. However, the exon could be spliced into most of the expressed transcripts in vitro in cell transfection experiments after introduction of a single T nucleotide to restore the branchpoint sequence upstream of the putative H2 exon.

ATM is not required in somatic hypermutation of VH, but is involved in the introduction of mutations in the switch mu region.

Class switch recombination (CSR) and somatic hypermutation (SHM) are mechanistically related processes that share common key factors such as activation-induced cytidine deaminase. We have previously shown a role for ATM (mutated in ataxia-telangiectasia) in CSR. In this paper we show that the frequency, distribution, and nature of base pair substitutions in the Ig variable (V) heavy chain genes in ataxia-telangiectasia patients are largely similar to those in normal donors, suggesting a normal SHM process. Characterization of the third complementarity-determining region in B cells from ataxia-telangiectasia patients also shows a normal V(D)J recombination process. SHM-like mutations could be identified in the switch (S) mu region (up to several hundred base pairs upstream of the S mu -S(alpha) breakpoints) in normal in vivo switched human B cells. In the absence of ATM, mutations can still be found in this region, but at less than half the frequency of that in normal donors. The latter mutations are mainly due to transitions (86% compared with 58% in controls) and are biased to A or T nucleotides. An ATM-dependent mechanism, different from that generating SHM in V genes, is therefore likely to be involved in introducing SHM-like mutations in the S region. ATM may thus be one of the factors that is not shared by the CSR and SHM processes.

Human IgG2 can form covalent dimers.

Unlike IgA and IgM, IgG has not yet been shown to form covalent polymers. However in the presence of specific Ag, murine IgG3 has been shown to polymerize through noncovalent interactions. In contrast to the noncovalent oligomers found with murine IgG3, we have detected covalent dimers in three different recombinant human IgG2 Abs produced in myeloma cells. Both IgG2,kappa and IgG2,lambda can form dimers. In addition, analysis of pooled human gamma globulin and several normal sera revealed the presence of IgG2 dimers. The IgG2 dimers are in contrast to the noncovalent IgG dimers found in pooled sera of multiple donors resulting from idiotype/anti-idiotype (Id/anti-Id) interactions. Cyanogen bromide cleavage analysis suggests that one or more Cys residues in the gamma 2 hinge are involved in dimer assembly. The potential role of IgG2 dimers in immunity against carbohydrate Ags is discussed.

Modulation of anti-IgM-induced B cell apoptosis by Bcl-xL and CD40 in WEHI-231 cells. Dissociation from cell cycle arrest and dependence on the avidity of the antibody-IgM receptor interaction.

The demise of B cell progenitors expressing functional IgM receptors for self appears to be the main mechanism by which B cell tolerance is accomplished. The genetic mechanisms that regulate the death process during this critical step of B cell development are still poorly understood. We have studied the regulation of apoptosis in WEHI-231 lymphoma cells after treatment with a panel of anti-IgM mAbs as an in vitro model of clonal B cell deletion. We showed that a product of bcl-x, Bcl-xL, can inhibit anti-IgM-induced apoptosis but not cell cycle arrest in a dose-dependent manner. Bcl-xL was efficient in protecting B cells from low but not high avidity anti-IgM mAbs. In contrast to that observed with Bcl-xL, CD40 stimulation was efficient in inhibiting both cell cycle arrest and apoptosis after IgM cross-linking regardless of the binding avidity of the anti-IgM Ab. Moreover, activation through IgM receptors but not CD40 induced up-regulation followed by rapid down-modulation of Bcl-xL. Thus, the capacity of Bcl-xL to modulate anti-IgM-induced apoptosis in WEHI-231 cells is highly dependent on the avidity of the Ab-IgM receptor interaction.

Primary localized orbital amyloidosis composed of the immunoglobulin gamma heavy chain CH3 domain.

1. Primary orbital amyloidosis is a rare form of localized amyloidosis in which the precursor protein has not previously been identified. We report here the first extraction of amyloid fibrils from a tissue biopsy containing orbital amyloid, and characterization of the fibril protein. 2. The N-terminal nine residues were identical with residues 278-286 and 275-283 of the third constant (CH3) domains of IgG1 and IgG4 gamma heavy chains, respectively. The mass of the fibril subunit protein was 6125Da by time-of-flight mass spectrometry, compared with the expected masses of 6169.9Da and 6214.9Da for the CH3 domains of gamma 1 from residue 278 and gamma 4 from residue 275, respectively. The fibril protein thus appeared to consist exclusively of an immunoglobulin heavy chain constant domain. 3. Only two examples of immunoglobulin heavy chain derived amyloid have been reported previously and both of these, as well as all published cases of the usual immunoglobulin light chain derived amyloid, contained variable domain sequence. The present case therefore represents a form of local, presumably clonal, B-cell/plasma-cell disorder characterized uniquely by deposition of an amyloidogenic immunoglobulin heavy chain constant domain fragment.

Complement and virus-specific antibody-dependent infection of normal B lymphocytes by human immunodeficiency virus type 1.

We tested the susceptibility of human purified, normal B lymphocytes to human immunodeficiency virus type 1 (HIV-1) infection, in the presence or absence of complement-sufficient serum and of virus-specific antibodies. Virus replication was detected when cells were infected in the presence of both complement and anti-HIV antibodies (C'-ADE conditions), by day 2 postinfection. Similar results were obtained when B lymphocytes were purified either from peripheral blood (three healthy donors) or from tonsils (four individuals with chronic tonsillitis). HIV infection was shown by polymerase chain reaction (PCR) detection of proviral sequences (gag and pol genes), by p24 antigen synthesis, and by cocultivation assay with MT2 cells. The higher p24 production was obtained when B cells were preactivated for 2 days by phorbol 12-myristate 13-acetate (PMA) before infection and then cultured in the presence of low-molecular weight B-cell growth factor (LMW-BCGF). Expression of virus envelope glycoprotein (gp) 120 could also be detected on a subpopulation of B cells (CD19+, CD22+) by flow cytometry. Blocking experiments with monoclonal antibodies (MoAbs) against CD4, CD21 (complement receptor 2 [CR2]), CD35 (CR1), CD19, and CD5 surface molecules indicated that infection of B cells involves CD4, CD21, and CD35 antigens. Indeed, blocking of CD4 receptor inhibited 10% of p24 production, and blocking of both CD21 and CD35 led to extinction of p24 signal. CR-dependent pathway is thus a major route for C'-ADE of HIV infection in normal B cells. Our results emphasize the importance of studying interactions between HIV and the complement system for better understanding infection mechanisms and the major dysfunctions of B cells in HIV-infected individuals.

Role of heavy chain constant domains in antibody-antigen interaction. Apparent specificity differences among streptococcal IgG antibodies expressing identical variable domains.

In this report, we examine the influence of CH domains on antibody specificity, in the context of variable epitope density on bacteria and synthetic glycoconjugates. Hybridomas secreting IgG1 and IgG2b mAb, specific for the N-acetyl-glucosamine (GlcNAc) residues of streptococcal group A carbohydrate, were previously generated from a hybridoma secreting a mouse IgG3 mAb. We show that these three mAb have identical H and L chain V domains, as determined by 1) cDNA sequencing, 2) binding to soluble Ag, and 3) binding to nine monoclonal anti-idiotopes. Nevertheless, the IgG3 mAb binds more effectively than the V region-identical IgG1 or IgG2b mAb to each of three strains of group A streptococci that display different amounts of terminal GlcNAc residues on their cell walls. The magnitude of the subclass-associated differential in binding varies with the target strain, and, whereas the IgG3 mAb binds best to the strain expressing an intermediate amount of GlcNAc, the IgG1 and IgG2b mAb and IgG3-derived F(ab')2 fragments bind best to the strain expressing the highest amount of GlcNAc. The IgG3 mAb also binds better than the IgG1 and IgG2b mAb to solid-phase GlcNAc50-BSA, but the IgG2b mAb binds best to otherwise identical conjugates with lower ratios of GlcNAc to BSA (20:1, 10:1, 5:1, and 1:1). These results suggest that epitope density can significantly influence the magnitude of IgG subclass-associated binding differences, and that structural differences in the CH regions, particularly the CH2 and CH3 domains, can influence the apparent specificities of IgG molecules for multivalent Ag.

Identification of distinct T cell receptor (TCR)-gamma delta heterodimers using an anti-TCR-gamma variable region serum.

T cell hybridomas were generated from CD3+, CD4-, CD8- splenocytes and fetal thymocytes. V gamma 1-expressing proteins present on these murine TCR-gamma delta hybridomas were identified by using an anti-TCR V gamma 1 peptide serum. This antiserum specifically immunoprecipitated 41-kDa TCR V gamma-C gamma 4 chains and 31-kDa TCR V gamma-C gamma 1/2 chains from distinct heterodimers expressed on the TCR-gamma delta T cell hybridomas. The RNA from a hybridoma with a 31-kDa TCR-gamma chain hybridized with a V gamma 1 probe but failed to hybridize with a V gamma 2 probe. In contrast, the RNA from a hybridoma with a 32-kDa TCR-gamma chain hybridized with a V gamma 2 probe. This 32-kDa TCR-gamma chain was not immunoprecipitated by the anti-V gamma 1 serum. These data were consistent with the conclusion that the 31-kDa protein was the product of a V gamma 1 to C gamma 2 rearrangement, whereas the 32-kDa protein was the product of a V gamma 2 to C gamma 1 rearrangement. Furthermore, Southern analyses confirmed that the 32-kDa protein was the product of a V gamma 1.2-J gamma 2 rearrangement, and all three of the 41-kDa TCR-gamma chains were the results of V gamma 1.1-J gamma 4 rearrangements. This was the first demonstration at the clonal level of TCR-gamma proteins which use members of the V gamma 1 gene family, as well as the C gamma 2 constant region. Additional biochemical analyses of the TCR-gamma and -delta proteins from three independently derived C gamma 4-bearing T cell hybridomas suggested that most of the molecular mass diversity observed in the bulk subpopulation of peripheral C gamma 4-containing heterodimers may be contributed by the TCR-delta chains.

Isolation of self-recognizing IgG2a monoclonal rheumatoid factors.

Hybridoma cell lines were produced by fusing spleen cells from 11 mice that expressed the lpr mutation to nonsecreting SP2/0 myeloma cells. Five to 20% of the hybridoma cell lines so derived were found to secrete antibodies that recognized autologous IgG2a. Out of 168 anti-IgG2a clones tested, 4 clones produced anti-IgG2a rheumatoid factor (RF) that was itself IgG2a. Two clones recognized self IgG2a while the other two clones each recognized a nonself IgG2a allotypic determinant. These findings indicate that self-recognizing antibodies are a component, albeit a minor one, of the RF repertoire of lpr/lpr mice.

Monoclonal antibodies specific for variable and constant domains of murine lambda chains.

Rat monoclonal antibodies directed against the BALB/c myeloma protein M315 (alpha,lambda 2) are described. 9A8 (IgG1) binds the V domain of lambda 2 and cross-reacts with lambda 1 and lambda 3 chains. 2B6 (IgG2a) is directed to the C domain of lambda 2 and cross-reacts with C lambda 3. The antibodies bind isolated chains as well as complete immunoglobulins. The monoclonals detect soluble immunoglobulin (radioimmunoassay), immunoglobulin immobilized on polystyrene (enzyme-linked immunosorbent assay), immunoglobulin bound to nitrocellulose (immunoblotting), and surface immunoglobulin intercalated in cell membranes (immunofluorescence). The antibodies are easily purified on protein G immunosorbents and may be biotinylated or conjugated with fluorescein isothiocyanate without loss of capacity to bind. In addition to the anti-lambda antibodies, a C alpha 2/C alpha 3-specific monoclonal antibody, 8D2 (IgG2a) is described.

Expression of the human T cell antigen receptor complex in advanced age.

Several studies of T cell-associated surface marker expression in older subjects "with no evidence of diseases known to affect immune function" have suggested that diminished expression of the constant portion (CD3) of the human T cell antigen-receptor complex (CD3/Ti) may be associated with aging. To determine if there is a decrease with age in expression of this complex, we determined the percent of cells positive for WT31, an antibody that recognizes a constant epitope on Ti alpha-beta heterodimers, CD3, CD4 and CD8 by immunofluorescence on peripheral blood lymphocytes from young controls (19-31 years), and 2 well characterized populations of older donors (greater than 69 years). The first group of older donors had no history of chronic or recent acute illness and saw a physician only for routine medical care. The second group was selected from patients seen in a geriatrics clinic for diagnoses that included osteoarthritis and cardiopulmonary disorders. There were no significant differences in the percent of cells positive with WT31, or with antibodies to CD4 and CD8 between young adults and the 2 groups of older donors. The clinic subjects showed a consistent decrease in percent of CD3+ lymphocytes in peripheral blood mononuclear cells compared to young adults (5-10% reduction) with less difference between the healthy older subjects and young adults. In the clinic subjects this reduction appears to be due to a decrease in CD3+, WT31-lymphocytes. These cells represent about 5-6% of the total T cell population in the young and healthy older group and are known to express CD3 in combination with Ti gamma-delta dimers. We show here that these cells are about equally distributed in high and low density lymphocytes after Percoll fractionation. We conclude that significant quantitative reduction in expression of the CD3/Ti antigen-receptor complex on human T cells is not a feature of the primary aging process and cannot account for diminished in vitro proliferative response of healthy subjects with age. However, diminished abundance of T cells expressing products of the Ti gamma-chain in less healthy older subjects may predispose to or result from diseases not usually associated with altered immune function.

Human antibodies to phosphocholine. IgG anti-PC antibodies express restricted numbers of V and C regions.

We examined the IgG subclass composition and isoelectric focusing (IEF) spectrotype pattern of naturally occurring human IgG antibodies that bind phosphocholine (PC) and found direct evidence for restricted expression of both V and C regions among these antibodies. In most individuals, the isotype of these IgG anti-PC antibodies was primarily IgG2. However, serum from some individuals contained significant amounts of IgG1 and IgG3 anti-PC antibodies. We also found that in individual sera, anti-PC antibodies are pauciclonal, as demonstrated by restricted spectrotypic patterns of the anti-PC antibodies. The IEF pattern of these antibodies were for the most part unique for each individual. In some sera, certain anti-PC antibodies with isoelectric points of basic pH bound PC conjugated to bovine serum albumin (PC-BSA) but did not bind pneumococcal C-carbohydrate bearing PC determinants. In two individuals, we found that the spectrotypes that bound only PC-BSA were of the IgG1 subclass. Taken together, these findings demonstrate that within individual sera, human antibodies to PC are quite restricted in both V and C region expression, and furthermore, these V and C regions of human Ig may not randomly associate.

Association of human lambda light chain V/J/C segments: serologic analysis and primary structure of the lambda VI Bence Jones protein THO.

The complete amino acid sequence of the human monoclonal lambda VI light chain Bence Jones protein THO was determined. We have found it to have remarkable similarities to the previously sequenced lambda VI Bence Jones protein SUT. Immunochemical analyses demonstrated that both lambda VI chains belong to a V lambda VI sub-subgroup. The 98-residue V gene-encoded segments of proteins THO and SUT are closely homologous and are distinguished from other lambda VI chains by a one-residue deletion at the V-J recombination site. Proteins THO and SUT have identical 13-residue J segments and therefore are encoded by the same J lambda gene. Further, both proteins have identical 105-residue C regions that by sequence represent products of the C lambda 3 (Kern-, Oz+) gene. The primary structure and serologic properties of proteins THO and SUT imply at the protein level of association between certain types of V lambda, J lambda, and C lambda segments.

Regulatory role of a tripeptide (TKP) from the second constant domain of immunoglobulin G--I. Inhibition of rat and human macrophage activities.

We have previously shown that peptides released after the cleavage of IgG by parasite proteinases were strong inhibitors of the macrophage effector functions against schistosome larvae. The results presented here demonstrate that a single tripeptide set, Thr-Lys-Pro (TKP), inhibits various macrophage functions and can be considered as an immunologically active peptide. Indeed, not only IgE-dependent cytotoxicity but also beta-glucuronidase release, chemiluminescence and ILI production were reduced when rat macrophages were previously incubated with TKP or some analogues. Moreover, chemotaxis and IgE-specific receptor expression were inhibited in both rat and human macrophages after treatment with TKP, without affecting the cell viability. The substitution or acetylation of Thr diminished or suppressed the inhibitory effect of TKP.

Sequence similarities among kappa IIIb chains of monoclonal human IgM kappa autoantibodies.

Light chains of the serologically and chemically defined V region sub-subgroup kappa IIIb are preferentially associated with several types of human IgM kappa (monoclonal) autoantibodies and are remarkably homologous in primary structure, as evidenced by partial amino acid sequence data. To establish the extent of homology among such proteins, we have determined the complete variable region (V) sequence of the light chains of four monoclonal IgM kappa autoantibodies, of which two (GAR and GOT) are rheumatoid factors (RFs), the third (SON) has anti-apo beta lipoprotein specificity, and the fourth (PIE) binds specifically to intermediate filaments. The region encoded by the V kappa segment gene (positions 1-95) in all four light (L) chains is virtually identical in sequence, differing by only one residue in the FR3 of protein SON and in the first CDR of protein GOT. Further, the CDR3 of kappa chain SON contains an additional residue (prolyl) located at the carboxyl-terminus of the V segment. The region encoded by the J gene (positions 96-108) is identical after position 96 for the two RFs GAR and GOT (J kappa 2), but different in proteins SON (J kappa 4) and PIE (J kappa 1). The amino acid residue at position 96, located in CDR3 at the site of combinatoriaL joining of the V kappa and J kappa gene segments and involved as a contacting residue in the hapten binding site, is different in all four light chains. These results demonstrate the extensive homology in sequence among light chains of IgM kappa autoantibodies and indicate that a particular V kappa germ line gene, kappa IIIb, is expressed as a phylogenetic response to certain self antigens or as part of a selection process by which these autoimmune responses are regulated.

Nucleotide sequence of a cDNA encoding the constant region of a rabbit immunoglobulin light chain of the lambda type.

A cDNA library has been constructed in the plasmid pBR322 using as template 12 S poly(A)-RNA isolated from spleen cells of a hyperimmunized Basilea rabbit. One of these cDNA-containing clones was used to determine the nucleotide sequence coding for the lambda light chain constant (C) region. The deduced amino acid sequence of this cDNA was found in good agreement with a Basilea rabbit C lambda region amino acid sequence previously determined. The nucleotide sequence of the rabbit C lambda-coding region was compared with man, mouse and chicken C lambda sequences and showed 78%, 72% and 66% homology, respectively. Southern blot hybridization analyses of liver DNA from various rabbits were carried out. The comparison of the restriction patterns suggests that a few C lambda-related genes occur in the rabbit genome. In addition, discrete differences in the restriction patterns may exist between rabbits of different genetic backgrounds.

Isoelectric focusing of human anti-diphtheria toxoid antibodies: identical spectrotypes of anti-fragment A antibodies with the same IgG subclass and light chain constant regions are expressed in multiple individuals.

The serum antibodies from humans booster-immunized with diphtheria toxoid were analyzed by isoelectric focusing. To restrict the antibody response, we visualized those antibodies that reacted to the Fragment A moiety of diphtheria toxin. Of the 100 normal human donors examined, 20 were estimated to be nonresponders to Fragment A. Sixty-three of the 80 donors who responded to Fragment A had restricted IgG-anti-Fragment A spectrotypes that consisted of three to eight distinct bands. Six series of repeat or shared spectrotypes were delineated among our donor population with repeat frequencies ranging from 0.025 to 0.312. We determined that the repeat spectrotypes were IgG1-kappa anti-Fragment A antibodies. Three percent of our donors had complex IgG-anti Fragment A spectrotypes consisting of greater than 10 bands. In some instances, the complex spectrotypes were composed of simple spectrotypes found among other donors. The complex IgG-anti Fragment A spectrotypes of these donors consisted of both IgG1 and IgG4-anti-Fragment A antibodies. From a statistical analysis, we calculate that only a limited number of IgG-anti-Fragment A spectrotypes are expressed in vivo after booster immunization. Our analysis suggests two groups of IgG-anti-Fragment A spectrotypes exist in our donor population. One IgG-anti-Fragment A spectrotype group is expressed randomly, and a second group is preferentially expressed among individuals in our donor population.