Molecular cloning of IgT from Atlantic salmon, and analysis of the relative expression of τ, µ, and δ in different tissues.

In the present study, IgT genes of Atlantic salmon were cloned and characterised. Analysis of our sequence data as well as ESTs reported to the databases revealed three distinct IgT heavy chain sub-variants in salmon, as opposed to two of IgM and IgD. The IgT sub-variants in salmon are 76-80% identical to each other, and 75-82% identical to the reported rainbow trout sequences, whereas the similarity to the orthologous molecules in zebrafish, grass carp, mandarin fish, and grouper is 25-41%. The heavy chains of both secreted and membrane anchored forms of salmon IgT include four constant Ig domains, τ1-τ4. This parallels the IgM heavy chains in elasmobranch fish and higher vertebrates, but differs from IgM in teleost fish where the membrane anchored form include only three constant Ig domains, µ1-µ3. The similarity between τ1 and µ1 in salmon is relatively high (52%) when compared to the remaining part of the molecules (τ2-τ4 and µ2-µ4 are 13-24% similar). To compare τ, µ and δ expressions in different tissues (head kidney, thymus, spleen, gill, skin, hind gut, brain and muscle) of Atlantic salmon, RT-qPCR assays were designed and evaluated. The analyses revealed that IgM transcripts are most abundant (up to 200 times more than IgD) followed by IgT (up to 20 times more than IgD) in most tissues. Highest expression of IgM, IgT, and IgD was in head kidney and spleen.

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 The porcine genomic deltaCH1 exon has been replaced by a recent duplication of the micro CH1 and its flanking sequences, a genetic event that also led to the formation of a short switch delta region, immediately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.

Aging-dependent exclusion of antigen-inexperienced cells from the peripheral B cell repertoire.

Aging is accompanied by greatly reduced B cell production in the bone marrow, yet peripheral B cell numbers do not decline. We hypothesize that this may reflect filling of the peripheral pool with B cells that are long-lived as a consequence of specificity for, and chronic stimulation by, environmental Ags. To begin to explore this possibility, we analyzed the effects of aging on B cell population dynamics in the anti-H2(k/b) 3-83 mu-delta Ig-transgenic mouse. We predicted that, because they presumably do not bind environmental Ags, B cells bearing the transgenic receptor may be lost in aged animals. As seen in nontransgenic animals, total splenic B cell numbers remained constant with age in the Ig-transgenic animals despite reduced B cell production. Importantly, although the few newly produced B cells in the bone marrow of aged mice are 3-83 positive, the peripheral compartment of these mice is dominated by B cells that express endogenous Ig genes rather than the transgenes. This population includes large numbers of marginal zone-like and CD21(low/-)CD23(low/-)IgM(low) B cells, as well as elevated numbers of CD5+ B cells. Many of these cells express only non-B220 CD45 isoforms, suggesting that they may be memory cells. A significant proportion of aged transgenic animals produce autoantibodies that are reactive with ssDNA, dsDNA, or histones. Results support the hypothesis that, in the face of severely reduced production with age, B cells are selected based on reactivity to environmental Ags, accumulate, and display activated phenotypes. Cells bearing 3-83-transgenic receptors are excluded from this population due to their specificity. Beyond their importance in aging, these findings define a novel form of receptor revision in which B cells are selected rather than deleted based on Ag reactivity.

Natural autoreactive B cells in transgenic mice reproduce an apparent paradox to the clonal tolerance theory.

Naturally occurring autoreactive B cells are thought to be physically eliminated or rendered functionally silent through different mechanisms of tolerance. However, multireactive low affinity natural autoantibody-producing B cells seem to escape these mechanisms in normal adults and could constitute the B cell pool from which pathological autoantibodies can emerge. To analyze this apparent paradox to the clonal tolerance theory, we have made two transgenic mouse lines (mu(k), mudelta(k)) producing a natural low affinity multireactive human autoantibody. These models enable us to test both the central tolerance mechanisms (reactivity with single-stranded DNA) and the peripheral tolerance mechanisms after Ag administration. Not only are the multireactive B cells not deleted in the bone marrow, they circulate and remain in the periphery even after the prolonged administration of Ag, the presence of membrane IgD increasing the number of mature autoreactive B cells. Self-reactive B cells are shown to be autoantigen ignorant both in vivo and in vitro, but they are not anergic because they can be easily activated through both B cell receptor-dependent and -independent pathways. Thus, these mouse lines reproduce an apparent paradox to the clonal tolerance theory meriting further investigation of the biological significance of this phenomenon.

Transcriptional regulation of mu and delta gene expression in bone marrow pre-B and B lymphocytes.

Newly formed B cells first express IgM and subsequently display IgD on the cell surface. This is an ontologically, as well as developmentally, regulated process because IgD is virtually absent on neonatal splenic B cells. In the present studies we have examined, by means of nascent RNA chain labeling, the relative levels of mu to delta gene transcription in bone marrow B cells, pre-B cells, and earlier progenitors of B cells. Pre-B cells were obtained from Whitlock-type long term cultures of bone marrow cells from normal and C.B17 scid mice. Both populations were found to transcribe the delta gene at very low but detectable levels. A similarly low level of delta transcription was found to occur in surface IgM-positive cells from both cultured and freshly isolated bone marrow B cells. In all populations analyzed, termination of the majority of polymerases occurred within a discrete 1-kb region located between the microM and C delta I exons. Analysis of steady state RNA indicated that long term cultured bone marrow cells from normal mice produced both 2.7-kb normal sized microM mRNA as well as 2.9-kb aberrantly spliced I mu-mRNA, whereas those from C.B17 scid mice contained only aberrant sized mu-mRNA. In contrast to these results, our previous findings with spleen cells obtained from both neonatal and adult animals showed that delta gene transcription occurs at a relatively high level. Therefore, it is possible that activation of regulatory signals that allow polymerases to progress beyond the termination site 3' of the microM exons may occur when newly formed B cells migrate from the bone marrow to the splenic environment.

Intracellular processing of membrane and secreted immunoglobulin delta-chains.

Membrane-bound and secreted immunoglobulin delta-chains are synthesized by the mouse hybridoma B1-8 delta.1 as two primary translation products (45,000 and 42,000, respectively) and are converted into three N-glycosylated forms. In addition to N-glycosylation, another modification, reflected in a size increase of 2000 to 3000, occurs within 8 min of synthesis and may be O-glycosylation. After these initial modifications, the N-linked carbohydrates of all three forms are partially trimmed, apparently in the endoplasmic reticulum. The secreted delta-chains acquire galactose and sialic acids less than 10 min before they are secreted. Monensin and CCCP, which are potent inhibitors of IgD secretion, inhibit the terminal glycosylation but not the other modifications of delta-chains. CCCP blocks the intracellular transport of IgD at an earlier stage of trimming than monensin. Within 10 min of the removal of CCCP, some of the accumulated IgD is terminally glycosylated and secreted. Membrane IgD is processed similarly to secreted IgD up to the stage blocked by CCCP. These observations suggest that final trimming and terminal glycosylation of IgD occurs in the Golgi complex, and that the other modifications occur in pre-Golgi compartments, where secreted IgD spends most of its transit time. We suggest that the rate-limiting step in the intracellular transport of both types of IgD is the passage from the ER to the Golgi complex.

Regulation of IgM and IgD synthesis in B lymphocytes. I. Changes in biosynthesis of mRNA for mu- and delta-chains.

Although IgD is expressed on the surface of resting B cells at a higher density than IgM, we determined that both the steady-state level and the biosynthetic rate of mu m-mRNA is higher than that of delta m-mRNA, suggesting that translational or post-translational processing of the Ig heavy chains may modulate the expression of cell surface Ig. After B cell activation by LPS, delta m-mRNA decreases drastically, accounting for the observed decrease in expression of IgD. Concomitantly, a dramatic increase in the level of mu s- and gamma s-mRNA corresponds to the observed increase in secretion of these Ig isotypes in LPS-stimulated cells.

Regulation of IgM and IgD synthesis in B lymphocytes. II. Translational and post-translational events.

Studies investigating the relative rates of biosynthesis of mu- and delta-polypeptide chains in normal resting B lymphocytes have shown that the translation rate of mu m is about sevenfold higher than that of delta m, thus reflecting the relative abundance of mRNA encoding these two chains. The turnover rate of cell surface IgM is faster, however, than that of cell surface IgD, resulting in higher expression of cell surface IgD relative to IgM under steady state conditions. LPS stimulation of B lymphocytes induces the complete cessation of synthesis of the delta-chain, thus accounting for the gradual disappearance of IgD from the cell surface of activated cells.

Biogenesis of membrane-bound and secreted immunoglobulins: two primary translation products of the human delta chain, differentially N-glycosylated to four discrete forms in vivo and in vitro.

Structural differences between the heavy chain of membrane IgD (delta m) and the heavy chain of secreted IgD (delta s) were investigated by using a human lymphoblastoid cell line that expresses idiotypically identical IgM and IgD. In a wheat germ cell-free system, mRNA from this cell line was shown to encode two distinct delta chains that differed in molecular weight. When translated in vitro in the presence of dog pancreatic microsomal membranes or when synthesized in vivo, these two delta chains were processed to four discrete glycosylated forms, all of which shared idiotypic determinants, C region determinants, and light chain linkage. As shown by digestion with endo-beta-N-acetylglucosaminidase H, these four delta forms represent two delta polypeptide chains that are differentially N-glycosylated. Pulse-chase experiments demonstrated that, after endo-beta-N-acetylglucosaminidase H treatment, delta m has a higher molecular weight than delta s. After integration into dog pancreatic microsomal membranes in vitro, delta m was found not to have a large cytoplasmic domain exposed to proteolytic digestion. The finding that delta m and delta s differ in primary structure is analogous to previous work with the corresponding heavy chains of IgM (mu m and mu s) from the same cell line. Thus, this cell line produces four Ig heavy chains (mu m, mu s, delta m, and delta s), with the same idiotype. The observation of differential N-glycosylation, apparently unique for the delta class, is discussed.

Rat IgD myeloma protein: cell-free translation of the delta mRNA and biochemical analysis of intracellular and membrane delta chain.

The analysis of IgD molecules produced by the tumor-derived IR 731 cell line showed that these molecules are not secreted and are only present at the cell surface as covalent dimers of 48 000 dalton delta chains. The original IR 731 tumor secreted complete IgD; thus, the disppearance of secretion in the cell line was correlated with the absence of light chain synthesis. The mRNA coding for the cell line delta chain was isolated and translated. The analysis of the translated delta chain (mol. wt.: 40 000) confirmed the absence of a region having the size of a domain as previously shown by partial amino acid sequence. The observation that the absence in delta chain of this region, which comprises most of the CH2 domain, could be a general feature of murine species is discussed.

In vitro immune response of human peripheral lymphocytes. VII. Effect of anti-mu and anti-delta antibodies on B colony formation and detection of abnormal B cells in patients with juvenile rheumatoid arthritis.

Effect of anti-mu and anti-delta antibodies on PHA- and protein A-induced B colony formation was studied. Anti-mu antibody at any concentrations tested did not show inhibitory or enhancing effect on colony formation. On the other hand, anti-delta antibody enhanced both PHA- and protein A-induced colony formation. Optimum concentration of anti-delta antibody for maximum enhancement was 0.1 microgram/ml. and F(ab')2 fragment of anti-delta antibody also showed comparable enhancing effect. Simultaneous addition of IgD with anti-delta antibody abrogated anti-delta-induced enhancement, and anti-delta antibody did not show any facilitation of colony formation in IgM+ IgD- cell population. In marked contrast with normal B cells, anti-mu antibody showed a remarkable enhancing effect on protein A-induced colony formation of B cells from JRA patients. F(ab')2 fragment of anti-mu antibody also showed comparable enhancing effect. Anti-mu antibody did not show any enhancement of colony formation of B cells from several other autoimmune diseases. The result indicated the presence of abnormal B cells in JRA patients.

Asynchronous development of two monoclonal proteins (IgM lambda and delta 1 chains) in a patient with abdominal lymphoma.

An IgM paraprotein was detected in a patient with abdominal lymphoma. Due to some doubts about the malignant nature of the situation, no cytostatic drugs were administered. When seen two years later the patient had considerably deteriorated in clinical status, with evidence of extra-abdominal involvement. At this time he presented a second paraprotein of low of molecular weight (74,000 to 80,000), consisting of two covalently linked delta1-chain fragments.