Murine plasma cells secreting more than one class of immunoglobulin heavy chain. II. SAMM 368--a plasmacytoma secreting IgG2b-kappa and IgA-kappa immunoglobulins which do not share idiotypic determinants.

SAMM 368 is a BALB/c plasmacytoma which secretes IgG2b-kappa and IgA-kappa paraproteins. Immunofluorescence studies of ascites cells from the tumor with purified, heavy chain class-specific antiglobulins demonstrate that single cells contain both IgG2 and IgA heavy chains. Idiotypic antisera prepared in mice and rabbits indicate that the two paraproteins produced by the tumor do not share idiotypic determinants. Analysis of the purified paraproteins for allotype markers showed that SAMM 368 IgA bears the BALB/c A12,13,14 determinants. SAMM 368 IgG2b does not carry any detectable allotypic determinants in spite of the fact that heterologous antisera identify the paraprotein as IgG2b.     

Murine plasma cells secreting more than one class of immunoglobulin heavy chain. III. Immunoglobulin production by established cultures and cloned lines of SAMM 368.

SAMM 368, a plasmacytoma which produces IgA-kappa and IgG2b-kappa was established in vitro from primary explants or after animal passage. The 9 lines that were established produced both paraproteins. The production of both immunoglobulins by single cells was demonstrated by immunofluorescent staining and cloning. Continuous culture of 3 parent lines for 18 months and 11 cloned lines for periods from 5 to 7 months demonstrated that double production is a stable characteristic of this plasmacytoma. Two single paraprotein-producing varients (IgG2b or IgA) were derived when cells were cultured in the presence of Fungizone. Chromosomal analysis of SAMM 368 indicates that this double producing tumor has a modal number similar to those observed in myelomas producing a single immunoglobulin class.     

Murine plasma cells secreting more than one class of immunoglobulin. VI. Secretion of completely assembled IgG2b and IgA molecules with segregated heavy chains and free light chains by spontaneous myeloma SAMM 368 in culture.

The antigenic and molecular characteristics of the two immunoglobulins secreted by a single cell line of plasmacytoma SAMM 368 were analyzed by polyacrylamide gel electrophoresis of biosynthesized proteins. Adapted to continuous in vitro cultivation, this BALB/c plasmacytoma secretes at least 98% of its heavy chains as components of fully assembled and isotypically uniform IgG2b and IgA molecules. The IgA is secreted as monomers, dimers, and multimers with chemical properties typical of BALB/c myeloma IgA including disulfide bonded J chain and noncovalently bonded light chains. The noncovalently bonded light chains are monomers rather than dimers. Free light chains are also secreted. The ability to segregate heavy chains is attributed either to chemical, enzymatic, or compartmental regulatory factors operating within these plasma cells.     

Plasmacytoid dendritic cells and the regulation of immunoglobulin heavy chain class switching

By substituting the heavy chain constant region of IgM and IgD with that of IgG, IgA or IgE, immunoglobulin class switching endows antibodies with novel effector functions that enhance the ability of the immune system to effectively clear invading pathogens. Plasmacytoid dendritic cells critically link innate immunity with adaptive immunity by producing massive amounts of type 1 IFN in response to viruses. We have recently found that type 1 IFN triggers class switching by inducing myeloid dendritic cells to upregulate the expression of BAFF and APRIL, two powerful B cell-activating molecules. In this paper, we propose that IFN-producing plasmacytoid dendritic cells modulate class switching by activating B cells through both T cell-dependent and T cell-independent pathways. A better understanding of these pathways may facilitate the development of novel antiviral vaccine strategies and aid in identifying new therapies for antibody-mediated autoimmune disorders, such as lupus.     

Correlation between secreted and membrane-bound IgG in mouse myeloma cells transfected with chimeric immunoglobulin heavy and light chain genes

Mouse myeloma cells were transfected with pSV2-gpt and pSV2-neo based immunoglobulin expression vectors. Double transfectants were selected using the xanthine-guanine phosphoribosyl transferase (gpt) and the neomycin (neo) selection marker genes. A broad distribution in the level of mouse-human chimeric IgG expression was observed with series of independently isolated transfectoma clones. The relative amounts of secreted to membrane-bound antibodies correlated closely, which suggested, that fluorescence-activated cell sorting could be a valuable tool for the selection of high-yielding production cell lines. However, a single cycle of cell sorting did not steer the cloning process significantly toward cells that produce enhanced amounts of recombinant IgG. Only in cases in which the polyclonal transfectoma population contained a large percentage of nonproducing cells, these were successfully separated from the IgG-producing cell population. (c) 1996 John Wiley & Sons, Inc.     

Sequence analysis of non-expressed immunoglobulin heavy chain loci in clonally related, somatically mutated hybridoma cells.

The non-expressed, rearranged JH loci of two hybridoma cells, thought to be derived from a single B cell precursor were cloned and partially sequenced. Identical DJ rearrangements including N sequences at the DJ border were found, proving the common origin of the cells. The non-expressed loci lacked a rearranged VH gene and exhibited a single point mutation in a stretch of 1108 bp. This contrasts with 11 somatic point mutations in the 702 bp of the expressed VDJ regions. We discuss the possibility that the mechanism of somatic hypermutation may require V gene rearrangement.     

Structure of immunoglobulin gamma 2b heavy chain gene cloned from mouse embryo gene library.

A mouse DNA clone containing the constant part of the immunoglobulin gamma 2b heavy chain was isolated from a mouse gene library. The library was constructed in Charon 4A from a partial EcoRI digest of mouse embryo DNA and was screened with a plasmid (p gamma (11)7) containing a cDNA insert of the heavy chain constant region of the plasmacytoma MPC-11 (1). The Charon 4A clone contains a 14 kb insert which is cleaved by EcoRI into a 6.8 kb and 7.2 kb fragments, of which only the 6.8 kb contains the sequence for gamma 2b heavy chain. Restriction analysis and partial sequence of the insert in p gamma (11) 7 enabled us to obtain three fragments corresponding to the 5' (amino acid 161-302) middle (amino acid 302-443) and 3' (mostly non coding 107 bp) regions of the constant region. Restriction analysis of the Charon 4A clone and hybridisation to these nick translated fragments revealed that the gamma 2b constant region gene contains about 1.5 kb and has three intervening sequences.     

On immunoglobulin heavy chain gene switching: two gamma 2b genes are rearranged via switch sequences in MPC-11 cells but only one is expressed

During B lymphocytes differentiation, switches in the expression of heavy chain immunoglobulin constant region (CH) genes occur by a novel DNA recombination mechanism. We have investigated the requirements of the CH gene switch by characterizing two rearranged gamma 2b genes from a gamma 2b producing mouse myeloma (MPC-11). One of the two gamma 2b genes is present in 2-3 copies per cell (gamma 2b strong hybridizer) while the other is present in approximately 1 copy per cell (gamma 2b weak hybridizer). Genomic clones of the gamma 2b strongly hybridizing gene indicate that this is an abortive switch event between the S gamma 3 and S gamma 2b regions. However, clones of the gamma 2b weakly hybridizing gene suggest a functional rearrangement due to the presence of VH, JH and S mu sequences. The switch-recombination sites of these rearranged gamma 2b genes and those of other CH genes show a high degree of preference for the sequence AGGTTG 5' of either the S mu donor site or the appropriate CH S acceptor site. AGGTTG and its analogs are rare in the S mu region, are somewhat prevalent in s alpha and in the case of S mu are found 5' of a tandemly repeated DNA sequence (GAGCT, GGGGT) comprising most of S mu.     

Primary structure of a human IgA1 immunoglobulin. IV. Streptococcal IgA1 protease, digestion, Fab and Fc fragments, and the complete amino acid sequence of the alpha 1 heavy chain.

In order to establish the complete amino acid sequence of the human IgA alpha1 chain Bur, IgA1 protease from Streptococcus sanguis was employed to generate Fabalpha and Fcalpha fragments in the final stage of this investigation. Cyanogen bromide cleavage of the Fabalpha fragment followed by reduction and aminoethylation produced the Fd' fragment (residues 84 to 227); this contains part of the variable region (VR), the whole first constant domain (Calpha1), and part of the hinge region of this heavy chain. The tryptic peptides of the Fd' fragment were isolated, characterized, and sequenced. The results together with the data in the preceding papers on chymotryptic, tryptic, and thermolysin peptides permitted the complete amino acid sequence of the human IgA alpha1 chain to be established.     

Three M-components IgA lambda + IgG kappa n + IgG kappa h in one patient (DA): lack of shared idiotypic determinants between IgA and IgG, and the presence of an unusual kappa h chain of 30,000 M.W

Two apparently homogeneous electrophoretic bands were found in the serum of a patient (DA) with multiple myeloma. These M-components were identified as IgA-lambda and IgG-kappa paraproteins bearing different idiotypic determinants. Further analysis of the L chains showed that the lambda-chain was homogeneous but the kappa-chain could be separated by SDS-polyacrylamide gel electrophoresis into two different bands. Both of them were associated with gamma-chains but one (termed kappa n) had normal m.w. (24,500) whereas the other (termed kappa h) was larger (m.w. 30,000). Sugar content of the two DA IgG, as determined by anthrone reaction, was similar in DA IgG kappa n (0.73%) and in DA IgG kappa h (1.1%), clearly demonstrating that the difference in m.w. was not due to a large sugar chain. Furthermore, the peptide map of the kappa h chain included nine peptides absent in those of four other control kappa-chains. Sequence analysis showed that the first 25 N-terminal amino acids of the kappa n differed from those of the kappa h chain at positions 4, 5, 15, 18, and 21. Thus the two kappa-chains had different framework regions.     

The primary structure of skeletal muscle myosin heavy chain: IV. Sequence of the rod, and the complete 1,938-residue sequence of the heavy chain

In the preceding paper [Maita, T., Miyanishi, T., Matsuzono, K., Tanioka, Y., & Matsuda, G. (1991) J. Biochem. 110, 68-74], we reported the amino-terminal 837-residue sequence of the heavy chain of adult chicken pectoralis muscle myosin. This paper describes the carboxyl terminal 1,097-residue sequence and the linkage of the two sequences. Rod obtained by digesting myosin filaments with alpha-chymotrypsin was redigested with the protease at high KCl concentration, and two fragments, subfragment-2 and light meromyosin, were isolated and sequenced by conventional methods. The linkage of the two fragments was deduced from the sequence of an overlapping peptide obtained by cleaving the rod with cyanogen bromide. The rod contained 1,039 amino acid residues, but lacked the carboxyl-terminal 58 residues of the heavy chain. A carboxyl-terminal 63-residue peptide obtained by cleaving the whole heavy chain with cyanogen bromide was sequenced. Thus, the carboxyl terminal 1,097-residue sequence of the heavy chain was completed. The linkage of subfragment-1 and the rod was deduced from the sequence of an overlapping peptide between the two which was obtained by cleaving heavy meromyosin with cyanogen bromide. Comparing the sequence of the adult myosin thus determined with that of chicken embryonic myosin reported by Molina et al. [Molina, M.I., Kropp, K.E., Gulick, J., & Robbins, J. (1987) J. Biol. Chem. 262, 6478-6488], we found that the sequence homology is 94%.     

Identification of a stimulus-dependent DNase I hypersensitive site between the Ialpha and Calpha exons during immunoglobulin heavy chain class switch recombination

The complete humoral response to foreign antigen depends upon two distinct recombination events within the heavy chain locus of immunoglobulin. The first recombination event takes place in what will become the antigen combining site of the antibody molecule, encoded by V, D and J segments. The second recombination event involves the looping-out of large spans of DNA which separate the various clusters of heavy chain exons which define the different immunoglobulin isotypes, or classes. While a great deal has been learned about the nature of the VDJ recombinase, very little is known about the nature of the class-switch recombinase. Using a cell system where class-switch recombination occurs primarily to the IgA locus, we have looked for stimulus-dependent changes in the chromatin structure of the IgA locus which might result from interactions between components of the recombinase and cis-elements within the region. We present evidence that strongly suggests that the class-switch recombinase interacts between the Ialpha and Calpha exons of IgA, just upstream of the highly reiterated DR1 and DR2 elements. However, although multiple potential SMAD-4 sites are located precisely within the DNase I hypersensitive site and 160 bp upstream of that site, we failed to detect any evidence of DNA/protein interactions near the hypersensitive site. Moreover, recombinant SMAD-3/4 proteins fail to interact with these sites with appreciable affinity in vitro. These data suggest that some other structural alteration at this site (e.g. RNA/DNA hybrid) may mediate the nuclease sensitivity.     

Conservation and continuity of periodic bent DNA in genomic rearrangements between the c-myc and immunoglobulin heavy chain mu loci.

Periodic bent DNA was mapped in the human c- myc and immunoglobulin heavy chain mu (Ig mu) loci. A total of 12 DNA bend sites in the c- myc gene and 11 sites in the Ig mu locus were aligned at average intervals of 694.2 +/- 281.4 and 654.5 +/- 222.7 bp respectively. Although some of the bend sites retained the distance of 700 bp, their periodicity was disturbed at several locations, including the exons of the c- myc gene and the enhancer element present in the Ig mu locus. Analysis of rearrangements that resulted in tumorigenesis of lymphocytes showed that the continuity of DNA bend sites was conserved in three lymphoma cell lines, Manca, BL22 and Ramos, suggesting that the genomic rearrangements gain stability by retaining their periodicity. This adds further evidence that the periodic bent DNA plays a crucial role in genomic structure.     

Abnormalities in the glycosylation of immunoglobulin heavy chain and an h-2 transplantation antigen in a mouse myeloma mutant.

Two mutant cell lines derived from the MPC-11 mouse myeloma synthesize immunoglobulin with abnormal heavy chains and normal light chains. The defective heavy chains have molecular weights of 38,000-42,000 (M3.11) and 50,000 daltons (ICR 11.19) as compared to 55,000 daltons of the wild-type. The glycosylation of the defective heavy chains demostrated several unusual features: first, 30-50% of the M3.11 heavy chain contained no carbonydrate, while 100% of the wildtype and ICR 11.19 heavy chains were glycosylated; second, the glycopeptides of the M3.11 heavy chains revealed an altered gel filtration pattern when compared with the wild-type; and third, digestion with an endoglycosidase indicated that the heterogeneity of the wild-type and M3.11 glycopeptides involved structural changes in the core region of the oligosaccharide. Examination of two other glycoproteins (the major histocompatibility complex antigens) in these cell lines showed that in M3.11, the H-2D but not the H-2K product was abnormally glycosylated and contained a smaller glycopeptide. However, in a subclone of M3.11 that had lost the ability to produce immunoglobulin heavy chains, the H-2D glycopeptide had returned to wild-type size. We concluded from these studies that the defective M3.11 immunoglobulin heavy chain interfered both with its own glycosylation and the glycosylation of another protein, H-2D.