Transcription of a productively rearranged Ig VDJC alpha does not require the presence of HS4 in the IgH 3' regulatory region.

V gene assembly, class switch recombination, and somatic hypermutation are gene-modifying processes essential to the development of an effective Ab response. If inappropriately applied, however, these processes can mediate genetic changes that lead to disease (e.g., lymphoma). A series of control elements within the Ig H chain (Igh) locus has been implicated in regulating these processes as well as in regulating IgH gene transcription. These include the intronic enhancer (Emu) and several elements at the 3' end of the locus (hs1,2, hs3a, hs3b, and hs4) known collectively as the 3' regulatory region. Although it is clear that the Emu plays a unique role in V gene assembly, it has not been established whether there are unique functions for each element within the 3' regulatory region. In earlier studies in mice and in mouse cell lines, pairwise deletion of hs3b and hs4 had a dramatic effect on both class switch recombination and IgH gene transcription; deletion of an element almost identical with hs3b (hs3a), however, yielded no discernible phenotype. To test the resulting hypothesis that hs4 is uniquely required for these processes, we induced the deletion of hs4 within a bacterial artificial chromosome transgene designed to closely approximate the 3' end of the natural Igh locus. When introduced into an Ig-secreting cell line, an Igalpha transcription unit within the bacterial artificial chromosome was expressed efficiently and the subsequent deletion of hs4 only moderately affected Igalpha expression. Thus, hs4 does not play a uniquely essential role in the transcription of a productively rearranged Ig VDJCalpha transcription unit.

Measurement of messenger RNA encoding the alpha-chain, polymeric immunoglobulin receptor, and J-chain in duodenal mucosa from dogs with and without chronic diarrhea by use of quantitative real-time reverse transcription-polymerase chain reaction assays.

OBJECTIVE: To examine the difference in expression of messenger RNA (mRNA) transcripts for polymeric immunoglobulin receptor (plgR), alpha-chain, and J-chain determined by use of quantitative real-time reverse transcription-polymerase chain reaction (QRT-PCR) assays in duodenal biopsy specimens obtained from dogs with and without chronic diarrhea. SAMPLE POPULATION: Biopsy specimens of the proximal portion of the duodenum were obtained endoscopically from 39 dogs evaluated because of chronic diarrhea (12 German Shepherd Dogs and 27 non-German Shepherd Dog breeds); specimens were also obtained from a control group of 7 dogs evaluated because of other gastrointestinal tract diseases and 2 dogs that were euthanatized as a result of nongastrointestinal tract disease. PROCEDURE: Dogs were anesthetized, and multiple mucosal biopsy specimens were obtained endoscopically at the level of the caudal duodenal flexure by use of biopsy forceps; in 2 control dogs, samples were obtained from the descending duodenum within 5 minutes of euthanasia. One-step QRT-PCR was used to quantify the level of expression of transcripts for the housekeeper gene glyceraldehyde-3-phosphate dehydrogenase, plgR, alpha-chain, and J-chain in duodenal mucosal tissue. RESULTS: There was no significant difference in the level of expression of any transcript among non-German Shepherd Dog breeds without diarrhea (control group), non-German Shepherd Dog breeds with chronic diarrhea, and German Shepherd Dogs with chronic diarrhea. Conclusions and Clinical Relevance-Results indicated that the susceptibility of German Shepherd Dogs to chronic diarrhea is not a result of simple failure of transcription of the key genes that encode molecules involved in mucosal IgA secretion.

EBV-encoded latent membrane protein 1 cooperates with BAFF/BLyS and APRIL to induce T cell-independent Ig heavy chain class switching.

By substituting the H chain C region of IgM with that of IgG, IgA, or IgE, class switching enables Abs to acquire new effector functions that are crucial for the neutralization of invading pathogens. Class switching occurs through class switch DNA recombination (CSR) and usually requires engagement of CD40 on B cells by CD40 ligand on Ag-activated CD4(+) T cells. CSR must be tightly regulated because abnormal IgG and IgA production favors the onset of autoimmunity, whereas increased switching to IgE leads to atopy. These inflammatory disorders can be triggered or exacerbated by EBV infection. In this study, we show that EBV induces CD40-independent CSR from C( micro ) to multiple downstream C(gamma), C(alpha), and C(epsilon) genes through latent membrane protein 1 (LMP1), a CD40-like viral protein that signals in a ligand-independent fashion. LMP1-induced CSR is associated with transcriptional activation of germline C(gamma), C(alpha), and C(epsilon) genes and triggers the up-regulation of activation-induced cytidine deaminase, a crucial component of the CSR machinery. In addition, LMP1 induces B cells to express B cell-activating factor of the TNF family and a proliferation-inducing ligand, two molecules that mediate B cell survival and T cell-independent Ab production. B cell-activating factor of the TNF family and a proliferation-inducing ligand cooperate with LMP1 to induce Ig class switching because their neutralization by appropriate soluble decoy receptors attenuates CSR in LMP1-expressing B cells. By showing that LMP1 triggers T cell-independent CSR, our findings suggest that EBV could play an important role in the pathogenesis of disorders with aberrant IgG, IgA, and/or IgE production.

Oligoclonal IgA response in the vascular wall in acute Kawasaki disease.

Kawasaki Disease (KD) is a potentially fatal acute vasculitis of childhood. Although KD is the leading cause of acquired heart disease in children in developed nations, its pathogenesis remains unknown. We previously reported the novel observation that IgA plasma cells infiltrate the vascular wall in acute KD. We have now examined the clonality of this IgA response in vascular tissue from three fatal cases of KD to determine whether it is oligoclonal, suggesting an Ag-driven process, or polyclonal, suggesting nonspecific B cell activation or a response to a superantigen. We first sequenced VDJ junctions of 44 alpha genes isolated from a primary, unamplified KD vascular cDNA library. Five sets of clonally related alpha sequences were identified, comprising 34% (15 of 44) of the isolated alpha sequences. Furthermore, point mutations consistent with somatic mutation were detected in the related sequences. Next, using formalin-fixed coronary arteries from two additional fatal KD cases, we sequenced VDJ junctions of alpha genes isolated by RT-PCR, and a restricted pattern of CDR3 usage was observed in both. We conclude that the vascular IgA response in acute KD is oligoclonal. The identification of an oligoclonal IgA response in KD strongly suggests that the immune response to this important childhood illness is Ag-driven.

Engagement of CD153 (CD30 ligand) by CD30+ T cells inhibits class switch DNA recombination and antibody production in human IgD+ IgM+ B cells.

CD153 (CD30 ligand) is a member of the TNF ligand/cytokine family expressed on the surface of human B cells. Upon exposure to IL-4, a critical Ig class switch-inducing cytokine, Ag-activated T cells express CD30, the CD153 receptor. The observation that dysregulated IgG, IgA, and/or IgE production is often associated with up-regulation of T cell CD30 prompted us to test the hypothesis that engagement of B cell CD153 by T cell CD30 modulates Ig class switching. In this study, we show that IgD+ IgM+ B cells up-regulate CD153 in the presence of CD154 (CD40 ligand), IL-4, and B cell Ag receptor engagement. In these cells, CD153 engagement by an agonistic anti-CD153 mAb or T cell CD30 inhibits S mu-->Sgamma, Smu-->Salpha, and S mu-->Sepsilon class switch DNA recombination (CSR). This inhibition is associated with decreased TNFR-associated factor-2 binding to CD40, decreased NF-kappaB binding to the CD40-responsive element of the Cgamma3 promoter, decreased Igamma3-Cgamma3 germline gene transcription, and decreased expression of Ku70, Ku80, DNA protein kinase, switch-associated protein-70, and Msh2 CSR-associated transcripts. In addition, CD153 engagement inhibits IgG, IgA, and IgE production, and this effect is associated with reduced levels of B lymphocyte maturation protein-1 transcripts, and increased binding of B cell-specific activation protein to the Ig 3' enhancer. These findings suggest that CD30+ T cells modulate CSR as well as IgG, IgA, and IgE production by inducing reverse signaling through B cell CD153.

Correct immunoglobulin alpha mRNA processing depends on specific sequence in the C alpha 3-alpha M intron.

The maturation of IgM-expressing B cells to IgM-secreting plasma cells is associated with both an increase in mu mRNA and the ratio of secreted to membrane forms of mu mRNA which differ at the 3' termini. In contrast, both in vitro and in vivo the secreted form of alpha mRNA is predominant at all stages in the development of a secretory IgA response. Previous studies demonstrated that preferential usage of the alpha s poly(A) site does not result from transcription termination and is independent of either the poly(A) sites or the 3' splice site associated with the exon encoding the membrane exon of IgA (alpha M). The present study demonstrates that a 349-bp region located 774 bp 3' to the alpha s poly(A) site is required for the preferential usage of the alpha s terminus. This region, which is the first isotype-specific cis-acting regulatory sequence not immediately adjacent to a secretory poly(A) site to be identified, contains regulatory elements that increase the efficiency of polyadenylation/cleavage. A ubiquitous, approximately 58-kDa RNA-binding protein interacts specifically with this regulatory region. These studies support the premise that cis-acting elements unique to each CH gene can impinge upon a common mechanism regulating Ig mRNA processing.

Rapid structural characterisation of a murine monoclonal IgA alpha chain: heterogeneity in the oligosaccharide structures at a specific site in samples produced in different bioreactor systems.

A murine hybridoma cell line which secretes a monoclonal IgA antibody, directed against the LPS antigen of Vibrio cholerae, was grown in either a continuous stirred tank reactor, a fluidised bed reactor or a hollow fibre reactor. Different methods were used for the structural characterisation of the IgA alpha chains. A classical approach consisted of Edman sequencing and mass determination of peptides separated by reversed phase HPLC. Alternatively, peptides and glycopeptides from a tryptic digest of each alpha chain were identified directly by MALDI-TOF mass spectrometry. A detailed analysis of the oligosaccharide structures at an unique site on the alpha chain was made by labelling the oligosaccharides released by N-glycosidase F with 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone. After separation by HPLC, mass measurements were made using matrix-assisted laser desorption time of flight mass spectrometry before and after digestion with specific exoglycosidases. The primary structure of the alpha chain of IgA was not affected by different cell culture conditions; in contrast, significant variations could be detected in the pattern of N-linked oligosaccharide structures, most prominently in the degree of sialylation. The efficiency of the analytical techniques in providing quality control of the identity, integrity and consistency of the glycoprotein is shown and discussed.

Regulation of the antibody repertoire through control of HCDR3 diversity.

In man, as in mouse, diversification of the antibody repertoire appears to follow a strict developmental program whereby antigen specificities are serially acquired during ontogeny. When compared to the adult repertoire, the fetal antibody repertoire is highly enriched for polyreactive specificities of low affinity. Although the mechanisms governing the development of this fetal repertoire differ between human and mouse, the composition and structure of the fetal antibodies produced by both species are quite homologous. Specifically, both species use similar V gene segments and restrict the sequence and structure of the third complementarity determining region (HCDR3) of the antibody heavy chain. The precise role that this restriction of the HCDR3 might play in the development of immunocompetence in the human remains to be elucidated.

Transgenic mice secreting coronavirus neutralizing antibodies into the milk.

Ten lines of transgenic mice secreting transmissible gastroenteritis coronavirus (TGEV) neutralizing recombinant monoclonal antibodies (rMAbs) into the milk were generated. The rMAb light- and heavy-chain genes were assembled by fusing the genes encoding the variable modules of the murine MAb 6A.C3, which binds an interspecies conserved coronavirus epitope essential for virus infectivity, and a constant module from a porcine myeloma with the immunoglobulin A (IgA) isotype. The chimeric antibody led to dimer formation in the presence of J chain. The neutralization specific activity of the recombinant antibody produced in transiently or stably transformed cells was 50-fold higher than that of a monomeric rMAb with the IgG1 isotype and an identical binding site. This rMAb had titers of up to 10(4) by radioimmunoassay (RIA) and neutralized virus infectivity up to 10(4)-fold. Of 23 transgenic mice, 17 integrated both light and heavy chains, and at least 10 of them transmitted both genes to the progeny, leading to 100% of animals secreting functional TGEV neutralizing antibody during lactation. Selected mice produced milk with TGEV-specific antibody titers higher than 10(6) as determined by RIA, neutralized virus infectivity by 10(6)-fold, and produced up to 6 mg of antibody per ml. Antibody expression levels were transgene copy number independent and integration site dependent. Comicroinjection of the genomic beta-lactoglobulin gene with rMAb light- and heavy-chain genes led to the generation of transgenic mice carrying the three transgenes. The highest antibody titers were produced by transgenic mice that had integrated the antibody and beta-lactoglobulin genes, although the number of transgenic animals generated does not allow a definitive conclusion on the enhancing effect of beta-lactoglobulin cointegration. This approach may lead to the generation of transgenic animals providing lactogenic immunity to their progeny against enteric pathogens.

B cells genetically deficient in the c-Rel transactivation domain have selective defects in germline CH transcription and Ig class switching.

The Ig heavy chain locus contains a number of binding sites for the transcriptional activator, c-Rel. In this study, we evaluated the capacity of B cells from mice made genetically deficient in the C-terminal, transactivation domain of the c-Rel protein (delta c-Rel) to undergo Ig class switching. Flow-cytometric and digestion circularization PCR analyses revealed that delta c-Rel B cells failed to switch to IgG3 in response to LPS alone, or to IgG1 or IgE in response to LPS + IL-4. This failure to switch to IgG3 or IgG1 was associated with a corresponding loss of germline CH gamma 3 or CH gamma 1 RNA. However, the defective switching to IgE in delta c-Rel B cells was associated with normal levels of germline CH epsilon RNA relative to control B cells. The ability of delta c-Rel B cells to switch to IgG1, in response to LPS + IL-4, could be restored through the action(s) of additional stimuli, and this was associated with induction of normal levels of germline CH gamma 1 RNA relative to controls. In contrast, LPS-activated B cells from delta c-Rel mice underwent normal switching to IgA in the presence of TGF-beta, relative to control B cells. This was associated with equivalent steady state levels of germline CH alpha RNA between the two B cell populations. These data are the first to demonstrate a key and selective role for c-Rel in the regulation of Ig class switching. Furthermore, distinct differences are revealed in the Ig isotype induction profiles of B cells lacking c-Rel activity vs those deficient in p50/nuclear factor-kappa B.

Counterselection against D mu is mediated through immunoglobulin (Ig)alpha-Igbeta.

The pre-B cell receptor is a key checkpoint regulator in developing B cells. Early events that are controlled by the pre-B cell receptor include positive selection for cells express membrane immunoglobulin heavy chains and negative selection against cells expressing truncated immunoglobulins that lack a complete variable region (D mu). Positive selection is known to be mediated by membrane immunoglobulin heavy chains through Ig alpha-Ig beta, whereas the mechanism for counterselection against D mu has not been determined. We have examined the role of the Ig alpha-Ig beta signal transducers in counterselection against D mu using mice that lack Ig beta. We found that D mu expression is not selected against in developing B cells in Ig beta mutant mice. Thus, the molecular mechanism for counterselection against D mu in pre-B cells resembles positive selection in that it requires interaction between mD mu and Ig alpha-Ig beta.

Regulation of transcription of the germ-line Ig alpha constant region gene by an ATF element and by novel transforming growth factor-beta 1-responsive elements.

Inasmuch as transcription of unrearranged, or germ-line, Ig CH genes appears to direct switch recombination, understanding the regulation of this transcription is essential for understanding the regulation of class switching. Transforming growth factor-beta 1 (TGF-beta 1) induces germ-line alpha transcripts and increases class switching to IgA in the I.29 mu B lymphoma and in Peyer's patch and splenic B cells. It has been previously demonstrated that induction of germ-line alpha transcripts by TGF-beta occurs at the transcriptional level in I.29 mu cells. We now demonstrate that the DNA segment located 5' to the initiation sites of germ-line alpha RNA drives expression of a luciferase reporter gene construct in transient transfection experiments. Full constitutive expression requires no more than 106 bp of the 5' flanking segment. By creating a series of deletion and substitution mutations, we have demonstrated that an ATF/CRE site residing within this region is very important for constitutive expression of the germ-line alpha promoter, but mutation of this motif does not diminish TGF-beta induction. Inducibility by TGF-beta requires additional sequences residing between -128 to -106 relative to the first RNA initiation site. Two copies of a tandemly repeated sequence 5' CA-CAG(G)CCAGAC 3' (termed Ig alpha TGF-beta-RE) are located in the region from -127 to -105. An oligonucleotide containing multimers of these repeats confers TGF-beta inducibility to a heterologous promoter. An additional copy of the TGF-beta-RE was identified at -41/-30 and its deletion reduces the TGF-beta response. Thus, we conclude that tandem repeats of a novel TGF-beta-RE are the positive regulatory element for the TGF-beta response. Our study provides further evidence that TGF-beta directs class switching to IgA through induction of transcription of the germ-line C alpha gene and demonstrates that TGF-beta can activate the promoter for the germ-line alpha gene.

[Non-Mediterranean lymphoma producing heavy alpha chains]. / Linfoma no mediterráneo productor de cadenas pesadas alfa.

At the present, immunoproliferative small intestinal disease (IPSID) is considered a fairly homogeneous entity. Isolated heavy chain production is so closely related with IPSID that has been considered as a natural biologic marker for it. By contrast, we report here the case of a 63-year-old female that developed a multinodular small bowel lymphoma without clinical malabsorption symptoms. The main tumour mass was located in proximal jejunum and the neighbour intestinal mucosa did not show neoplastic cell infiltrates. Immunohistochemical methods demonstrated restrictive presence of IgA1 subclass in the neoplastic cells without presence of light chains. Cases as present are extremely infrequent and their potential relationship with IPSID has not been completely outlined, being useful in trying to understand the spectrum of gut lymphomas.

Immunoglobulin synthesis in the human myeloma cell line U-266; expression of two immunoglobulin heavy chain isotypes (epsilon and alpha) after long-term cultivation in vitro.

A human IgE-producing myeloma has been cultivated in vitro as a continuous cell line (U-266) since 1968. Analysis of immunoglobulin production during early passages of the cell line demonstrated a high synthesis rate of monoclonal IgE. Analysis of late passages, cultivated after 1980, revealed a 3-6-fold lower rate of IgE secretion. This decrease was accompanied by the appearance of small amounts of IgA in the culture medium together with IgE. RNA was extracted from a late passage of U-266 and analyzed by Northern blotting, using epsilon and alpha-specific oligonucleotides as hybridization probes. The results showed the presence of epsilon as well as alpha-specific mRNA. Moreover the results demonstrated that the latter mRNA was derived from the alpha 2 locus and that the epsilon and the alpha 2-specific mRNA contained the same V region sequences. Southern blot analysis of DNA from the late passage of the U-266 cell line failed to reveal a recombinatory switch from the epsilon locus to the alpha 2 locus. The expression of alpha 2 is thus likely to be caused by differential splicing rather than by an isotype switch at the DNA level.

The regulation of membrane-bound and secreted alpha-chain biosynthesis during the differentiation of the B cell lymphoma I.29.

The regulation of the synthesis of membrane-bound and secreted IgA was investigated in the murine B lymphoma I.29 during the differentiation from IgA-bearing lymphocytes to IgA-secreting cells, as caused by treatment with lipopolysaccharide (LPS). LPS induced a threefold to fivefold increase in the amount of IgA synthesized, and induced a shift from the synthesis of the membrane form of alpha-chain (alpha m) to the synthesis of the secreted form of alpha-chain (alpha s), resulting in a 60-fold increase in the amount of IgA secreted. In vitro translation of sucrose gradient-fractionated RNA indicated that two mRNA molecules, 3.1 and 2.1 kilobase pairs (kb), encode alpha m-chains, whereas a smaller RNA molecule, 1.7 kb, encodes alpha s. Analyses by RNA blotting showed that the relative amounts of the three alpha mRNA changed rapidly during LPS-induced differentiation. The amount of the 3.1 and 2.1 kb alpha mRNA decreased, and the amount of the 1.7 kb alpha s mRNA increased in LPS-stimulated cells as compared with controls. These observations suggest that the regulation of alpha m/alpha s synthesis is controlled mostly at the pretranslational level.

IgA polymorphism in mice: individual light chains can pair covalently with some alpha heavy chains and non-covalently with others.

The molecular basis for the two different forms of IgA in mice, distinguished by the covalent or non-covalent association of light (L) and heavy (H) chains, is unknown. In this communication, we show, using somatic cell hybridization to construct cells producing new combinations of alpha and L chains, that individual L chains probably can pair both covalently and non-covalently depending on the alpha chain.

Hairy cell leukaemia occurring with an unrelated paraproteinaemia. A biochemical and immuno-electron microscopic study.

A detailed study is described of a case of hairy cell leukaemia, presenting with a serum paraprotein of an immunoglobulin (Ig) class different from that synthesised by the neoplastic cells. The case was unusual in its association with leukaemic arthropathy but ultrastructurally the hairy cells were typical. By immunofluorenscence and immuno-electron microscopy the neoplastic cells expressed IgA lambda both on the cell surface and intracellularly in the rough endoplasmic reticulum, perinuclear space and Golgi apparatus. No Ig was observed in the ribosomal-lamellae complexes. These cells also synthesised and secreted Ig of class A lambda in culture. However the serum paraprotein was of class IgA chi and could not be attributed to an abnormal population of plasma cells in the bone marrow. There was no other evidence for myeloma and the IgA chi paraproteinaemia appeared to be benign, apparently unrelated to the neoplastic proliferation of hairy cells.

Synthesis and processing of the alpha heavy chains of secreted and membrane-bound IgA.

We have compared the synthesis and processing of immunoglobulin alpha chains in two murine cell lines, a B cell lymphoma that expresses membrane-bound IgA and a hybridoma that secretes IgA. Results of biosynthetic labeling experiments demonstrated that membrane-bound and secreted alpha chains have two distinct intracellular precursors, of different molecular weights and isoelectric points. RNAs from both of these cell lines direct the synthesis in vitro of two alpha polypeptides of Mr 59,000 and 62,000, the larger one being the precursor for membrane-bound alpha chain and the smaller one being the precursor for secreted alpha chain. These cell lines each contain three RNAs, 1.7, 2.1, and 3.1 kilobases in length, which hybridize with cDNA for the alpha constant region and which are present in different concentrations. Our results suggest that the smallest RNA encodes the secreted alpha chain and one or both of the larger RNAs encode(s) the membrane-bound alpha chain.