ABSTRACT
The development of T cells is thought to be independent of B cells. However, defects in cell-mediated immunity in individuals with B-cell deficiency suggest the contrary. To test whether B cells affect T-lymphocyte development, we constructed mice with a monoclonal T-cell compartment (MT) and monoclonal B- and T-cell compartments (MBTs). In these mice, the T cells expressed a DO 11.10 transgenic (DO-T) cell receptor restricted to major histocompatibility complex (MHC) class IId. While CD4+ DO-T lymphocytes are rare in transgenic H-2b MT mice, we found that in H-2b MBT mice under the influence of B cells, DO-T lymphocytes mature into large numbers of CD4+ peripheral T cells. H-2b MBT mice have more CD4+ thymocytes than H-2b MT mice. These data are consistent with the view that B cells play some role in thymocyte development.
Subject(s)
B-Lymphocytes/immunology , T-Lymphocytes/immunology , Animals , B-Lymphocyte Subsets/immunology , CD4 Lymphocyte Count , Clonal Deletion/immunology , H-2 Antigens/immunology , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/immunology , Immunity, Cellular , Immunoglobulin lambda-Chains/genetics , Immunoglobulin lambda-Chains/immunology , Immunologic Deficiency Syndromes/genetics , Immunologic Deficiency Syndromes/pathology , Immunophenotyping , Mice , Mice, Knockout , Mice, Transgenic , Models, Immunological , Ovalbumin/immunology , Spleen/cytology , Spleen/immunology , T-Lymphocytes/cytologyABSTRACT
Protein SWAP-70 was initially isolated from nuclei of activated B cells and was implicated in the immunoglobulin class switch process. After B cell activation the protein translocates from the cytoplasm to the nucleus, and may serve to signal nuclear processes. We have generated mice deficient in SWAP-70 and found three main differences when compared to wild-type mice: (i) their B lymphocytes are two- to threefold more sensitive to gamma-irradiation than B cells of wild type; (ii) SWAP-70-deficient mice developed autoantibodies at a much higher frequency; and (iii) the CD40 signaling pathway is compromised in the mutant mice. CD40-dependent switching to the IgE isotype is reduced five- to eightfold in vitro. In SWAP-70-deficient mice, IgE levels prior to immunization were six- to sevenfold lower than in wild-type mice, and after immunization three- to fourfold lower. CD40-induced proliferation was transiently increased in the mutant. LPS-induced switching to other isotypes, however, and LPS-induced proliferation were normal. We propose that SWAP-70 serves a specific role in the CD40 signaling pathway, in particular in the IgE response.
Subject(s)
Autoantibodies/immunology , B-Lymphocytes/immunology , CD40 Antigens/immunology , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/physiology , Guanine Nucleotide Exchange Factors , Immunoglobulin E/immunology , Nuclear Proteins/deficiency , Nuclear Proteins/physiology , Animals , Apoptosis , Autoantibodies/biosynthesis , B-Lymphocytes/cytology , B-Lymphocytes/metabolism , B-Lymphocytes/radiation effects , Cell Division , DNA-Binding Proteins/genetics , Gamma Rays , Gene Targeting , Immunoglobulin Class Switching/genetics , Immunoglobulin Class Switching/immunology , Immunoglobulin E/biosynthesis , Immunoglobulin E/genetics , Immunoglobulin G/biosynthesis , Immunoglobulin G/genetics , Immunoglobulin G/immunology , Lipopolysaccharides/immunology , Lymphocyte Activation , Mice , Mice, Knockout , Minor Histocompatibility Antigens , Mutation/genetics , Nuclear Proteins/genetics , Radiation Tolerance , Signal TransductionABSTRACT
The quasi-monoclonal (QM) mouse has a functionally rearranged H chain gene inserted into its natural position in the IgH locus. In this position, the H chain gene is subject to many of the same activities as normally arranged H chain genes, including somatic hypermutation, V(H) gene replacement, and class switch recombination. Here, we have used this mouse strain to determine some of the rules that govern the V(D)J recombination activity of the IgH locus in thymus. We focused on the requirements for V(H) gene replacement. In normal mice, thymic DJ(H) rearrangements are common, but VDJ(H) rearrangements are not. We found intermediate products of V(H) replacement in double-positive CD4(+)CD8(+) cells of the QM thymus, demonstrating that the inserted V(H) gene was accessible and ruling out the possibility that a V(H) gene per se cannot be rearranged in the thymus. We found transcripts from the knocked-in H chain gene of QM, but no mu H chain protein was detectable in thymocytes. Cloning and sequencing of these transcripts revealed that some had been generated by V(H) gene replacement. Corresponding signal joints could also be identified. These results suggest that neither a B cell-specific signal nor an Ig protein are necessary to activate V(H)-to-VDJ(H) joining in thymocytes. Possible mechanisms remaining to account for overcoming the barrier to V(H) joining in thymocytes include the insertion of a transcriptionally active gene segment and/or the inactivation of a silencer.
Subject(s)
Gene Rearrangement, T-Lymphocyte , Genes, Immunoglobulin , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/genetics , Thymus Gland/immunology , Thymus Gland/metabolism , Animals , Base Sequence , CD4 Antigens/biosynthesis , CD8 Antigens/biosynthesis , Immunoglobulin Heavy Chains/isolation & purification , Immunoglobulin Variable Region/isolation & purification , Immunoglobulin mu-Chains/analysis , Immunoglobulin mu-Chains/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Sequence Data , Receptors, Antigen, B-Cell/metabolism , Receptors, Antigen, B-Cell/physiology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombination, Genetic , Thymus Gland/cytology , Transcription, Genetic/immunologyABSTRACT
This study tracks the fate of antigen-reactive B cells through follicular and extrafollicular responses and addresses the function of CD40 in these processes. The unique feature of this system is the use of transgenic B cells in which the heavy chain locus has been altered by site-directed insertion of a rearranged V(H) DJ(H) exon such that they are able to clonally expand, isotype-switch and follow a normal course of differentiation upon immunization. These Ig transgenic B cells when adoptively transferred into non-transgenic (Tg) mice in measured amounts expanded and differentiated distinctively in response to T cell-independent (TI) or T cell-dependent (TD) antigens. The capacity of these Tg B cells to faithfully recapitulate the humoral immune response to TI and TD antigens provides the means to track clonal B cell behavior in vivo. Challenge with TI antigen in the presence of agonistic anti-CD40 mAb resulted in well-defined alterations of the TI response. In vivo triggering of Tg B cells with TI antigen and CD40 caused an increase in the levels IgG produced and a broadening of the Ig isotype profile, characteristics which partially mimic TD responses. Although some TD characteristics were induced by TI antigen and CD40 triggering, the Tg B cells failed to acquire a germinal center phenotype and failed to generate a memory response. Therefore, TD-like immunity can be only partially reconstituted with CD40 agonists and TI antigens, suggesting that there are additional signals required for germinal center formation and development of memory.
Subject(s)
Antibody Formation , B-Lymphocytes/immunology , CD40 Antigens/immunology , Genes, Immunoglobulin , Adoptive Transfer , Animals , Immunoglobulin Class Switching , Mice , Mice, Transgenic , T-Lymphocytes/immunologyABSTRACT
V(H)-gene replacement is a recombination event in which a pre-existing immunoglobulin heavy chain gene can be altered by the replacement of the rearranged V(H) gene segment with another V(H) gene segment. Although this event has been demonstrated in various model systems, its role in generating antibody diversity is still unsettled. We have used a genetically modified mouse strain, QM, with a quasi monoclonal primary B cell repertoire specific for NP to determine whether V(H) gene replacement can generate a new antigen specificity. Hybridomas generated from QM splenocytes after immunization with different antigens, gave rise to antibodies with specificity to the immunizing antigen or with new specificities. We found V(H)-gene replacement was used to change the original heavy chain gene rearrangement specific for NP into a heavy chain gene encoding the new antigen specificity. V(H)-gene replacement intermediates were detected both before and after the immunization, suggesting that the event was selective rather than instructive. These results demonstrate that V(H)-gene replacement can generate a new antibody heavy chain gene with a different functional and selectable antigen specificity.
Subject(s)
Antibody Formation , Antibody Specificity , Gene Rearrangement , Genes, Immunoglobulin , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/genetics , Animals , Base Sequence , Erythrocytes/immunology , Hemocyanins/immunology , Mice , Mice, Inbred C57BL , Molecular Sequence Data , SheepABSTRACT
SWAP-70 is a component of an enzyme complex that recombines Ig switch regions in vitro. We report here the cloning of the human cDNA and its B lymphocyte-specific expression. Although its sequence contains three nuclear localization signals, in small resting B cells, SWAP-70 is mainly found in the cytoplasm. On stimulation, SWAP-70 translocates to the nucleus. In activated, class-switching B cell cultures, it is associated with membrane IgG, but not IgM. The membrane Ig association requires a functional pleckstrin homology domain and is controlled by the C terminus. We suggest that SWAP-70 is involved not only in nuclear events but also in signaling in B cell activation.
Subject(s)
DNA-Binding Proteins/metabolism , Guanine Nucleotide Exchange Factors , Nuclear Proteins/metabolism , Receptors, Antigen, B-Cell/metabolism , Amino Acid Sequence , Animals , Biological Transport , CD40 Antigens/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/immunology , Humans , Immunoglobulin G/immunology , Immunoglobulin M/immunology , Minor Histocompatibility Antigens , Molecular Sequence Data , Mutagenesis , Nuclear Proteins/genetics , Nuclear Proteins/immunology , Rabbits , Tissue Distribution , Tumor Cells, CulturedABSTRACT
The protein SWAP-70 was isolated as part of a DNA recombination complex in B lymphocytes, where it is predominantly expressed. In resting B cells, SWAP-70 is found in the cytoplasm; upon B-cell activation, it is transported both into the nucleus and to the cell membrane, where it is associated with the B-cell receptor complex and may play a role in signal transduction. In the nucleus, its involvement in heavy-chain class switch recombination has been suggested. In this report, using restriction fragment length polymorphism, simple sequence length polymorphism, and fluorescence in situ hybridization, we map the chromosomal localization of the mouse and the human genes to syntenic regions of mouse mid Chromosome (Chr) 7 and human Chr 11p15.
Subject(s)
Chromosomes, Human, Pair 11/genetics , DNA-Binding Proteins/genetics , Guanine Nucleotide Exchange Factors , Nuclear Proteins/genetics , Physical Chromosome Mapping , Animals , Haplotypes , Humans , In Situ Hybridization, Fluorescence , Mice , Mice, Inbred BALB C , Microsatellite Repeats , Minor Histocompatibility Antigens , Polymorphism, Restriction Fragment LengthABSTRACT
Germinal centers are critical for affinity maturation of antibody (Ab) responses. This process allows the production of high-efficiency neutralizing Ab that protects against virus infection and bacterial exotoxins. In germinal centers, responding B cells selectively mutate the genes that encode their receptors for antigen. This process can change Ab affinity and specificity. The mutated cells that produce high-affinity Ab are selected to become Ab-forming or memory B cells, whereas cells that have lost affinity or acquired autoreactivity are eliminated. Normally, T cells are critical for germinal center formation and subsequent B cell selection. Both processes involve engagement of CD40 on B cells by T cells. This report describes how high-affinity B cells can be induced to form large germinal centers in response to (4-hydroxy-3-nitrophenyl) acetyl (NP)-Ficoll in the absence of T cells or signaling through CD40 or CD28. This requires extensive cross-linking of the B cell receptors, and a frequency of antigen-specific B cells of at least 1 in 1,000. These germinal centers abort dramatically at the time when mutated high-affinity B cells are normally selected by T cells. Thus, there is a fail-safe mechanism against autoreactivity, even in the event of thymus-independent germinal center formation.
Subject(s)
B-Lymphocytes/physiology , Germinal Center/physiology , T-Lymphocytes/physiology , Animals , Antigens, CD/analysis , B7-1 Antigen/analysis , B7-2 Antigen , CD40 Antigens/analysis , Ficoll , Haptens , Membrane Glycoproteins/analysis , Mice , Mice, Nude , Mice, Transgenic , Nitrophenols , Peanut Agglutinin , Phenylacetates , Signal Transduction , Spleen/drug effectsABSTRACT
We describe a new population of non-naive B cells in the peripheral blood of quasimonoclonal (QM) mice. Surface Ig of switched isotypes is expressed, but not B220 nor CD19. These cells are larger and denser than naive B cells but smaller than blasts or plasma cells; they do not stain with syndecan, a marker for plasma cells. Telomerase, which is usually expressed in B cell blasts, was not present in this population. We sorted the switched, idiotype-positive, B220(-) B cells from the peripheral blood of QM mice and sequenced Ig H chain and lambda L chain cDNA. There were many point mutations but no V gene replacements, gene conversions or other type of diversifications. As they express switched isotypes and have mutated their Ig genes, cells in the B220(-), CD19(-) population must have been in an immune response and we suggest that it includes the memory B cell subset.
Subject(s)
Antigens, CD19/analysis , B-Lymphocyte Subsets/immunology , Immunoglobulin Class Switching/genetics , Leukocyte Common Antigens/analysis , Amino Acid Sequence , Animals , Base Sequence , Genes, Immunoglobulin , Genetic Engineering , Hematopoietic Stem Cells , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Idiotypes , Immunoglobulin gamma-Chains/genetics , Membrane Glycoproteins , Mice , Mice, Mutant Strains , Molecular Sequence Data , Peritoneum/cytology , Proteoglycans , Receptors, Antigen, B-Cell , Syndecans , TelomeraseABSTRACT
A subset of myeloid dendritic cells is described which is associated with the ability of splenic and lymph node plasmablasts to survive and differentiate into plasma cells. Plasmablast-associated dendritic cells (PDC) are CD11c(high), DEC-205(-) and unlike conventional dendritic cells do not associate with T cells. The following findings suggest a requirement for PDC if plasmablasts are to differentiate to plasma cells. First, when large numbers of B cells are recruited into antibody responses and plasmablasts outgrow the PDC stroma, only those associated with PDC survive and differentiate into plasma cells. Conversely, if the number of PDC is increased by ligating their CD40, more plasmablasts survive on the expanded PDC stroma and differentiate into plasma cells. Finally, in T cell-deficient mice, the plasma cells that develop atypically in the T zones in response to thymus-independent antigens are associated with ectopic PDC.
Subject(s)
Apoptosis/immunology , Dendritic Cells/immunology , Spleen/cytology , Animals , Cell Differentiation , Cell Division , Cell Survival , Integrin alphaXbeta2/immunology , Lymph Nodes/immunology , Male , Mice , Mice, Inbred C57BLABSTRACT
Variable (V) region gene replacement was recently implicated in B cell repertoire diversification, but the contribution of this mechanism to antibody responses is still unknown. To investigate the role of V gene replacements in the generation of antigen-specific antibodies, we analyzed antiviral immunoglobulin responses of "quasimonoclonal" (QM) mice. The B cells of QM mice are genetically committed to exclusively express the anti-(4-hydroxy-3-nitrophenyl) acetyl specificity. However, approximately 20% of the peripheral B cells of QM mice undergo secondary rearrangements and thereby potentially acquire new specificities. QM mice infected with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus, or poliovirus mounted virus-specific neutralizing antibody responses. In general, kinetics of the antiviral immunoglobulin responses were delayed in QM mice; however, titers similar to control animals were eventually produced that were sufficient to protect against VSV-induced lethal disease. VSV neutralizing single-chain Fv fragments isolated from phage display libraries constructed from QM mice showed VH gene replacements and extensive hypermutation. Thus, our data demonstrate that secondary rearrangements and hypermutation can generate sufficient B cell diversity in QM mice to mount protective antiviral antibody responses, suggesting that these mechanisms might also contribute to the diversification of the B cell repertoire of normal mice.
Subject(s)
Antibody Diversity , B-Lymphocytes/immunology , Gene Rearrangement, B-Lymphocyte , Mutation , Animals , Antibodies, Viral/biosynthesis , Antibodies, Viral/genetics , Base Sequence , DNA/genetics , Gene Targeting , Mice , Mice, Inbred C57BL , Neutralization Tests , Sequence Homology, Nucleic Acid , Vesicular stomatitis Indiana virus/immunologyABSTRACT
SWAP-70 is part of a protein complex that catalyzes cell-free DNA recombination between immunoglobulin heavy chain gene switch region substrates. This report studies the expression pattern of SWAP-70 in mouse tissues, sorted cells, and cultured primary cells. SWAP-70 RNA is strongly increased upon switch-induction of spleen cells, and very weakly expressed in thymus and bone marrow. SWAP-70 protein is specifically expressed in B cells, and levels increase rapidly after stimulation. Tissue staining shows strong expression in germinal center B cells, while macrophages and T lymphocytes do not stain. SWAP-70 is not detected in early B cells in the bone marrow. Its expression during mouse ontogeny after birth correlates with the appearance of non-IgM isotypes. While SWAP-70 localizes to the cell nucleus in activated B cells, it is not tightly associated with the chromatin and is found in the cytoplasm as well. SWAP-70 expression is not increased by gamma or UV irradiation of spleen cells, nor does it depend on p53. These characteristics are consistent with the putative role of SWAP-70 in immunoglobulin class switching.
Subject(s)
DNA-Binding Proteins/genetics , Guanine Nucleotide Exchange Factors , Nuclear Proteins/genetics , Animals , B-Lymphocytes/cytology , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Cell Differentiation , Cells, Cultured , DNA-Binding Proteins/biosynthesis , Female , Gene Expression Regulation, Developmental/radiation effects , Male , Mice , Mice, Inbred C57BL , Minor Histocompatibility Antigens , Nuclear Proteins/biosynthesis , Pregnancy , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombination, Genetic , Subcellular Fractions/metabolism , Tissue DistributionABSTRACT
By studying the role of mismatch repair in hypermutation at the immunoglobulin loci, the field of antibody hypermutation has been integrated into the larger area of DNA repair. Trans-acting factors - Ku70, Ku80 and possibly SWAP-70 - have been identified for the temporally related but not mechanistically related immunoglobulin heavy-chain class-switch.
Subject(s)
Antibody Affinity , DNA Repair , Genes, Immunoglobulin , Mutation , Animals , Humans , Immunoglobulin Class Switching , Immunoglobulin Heavy Chains/geneticsABSTRACT
We have generated a monoclonal B-cell mouse by introducing homozygous, nonfunctional RAG-2 alleles and a lambda1 light-chain transgene into the quasi-monoclonal (QM) mouse, which contains a "knocked-in" V(H)DJ(H) rearrangement. Thus, this mouse, which we call MonoB, is devoid of T cells and contains preformed heavy- and light-chain genes encoding immunoglobulin with an anti-NP specificity. The MonoB mouse allows us to examine immunoglobulin diversity in the absence of processes mediated by V(D)J recombination and T cells. Here we report that not only is the MonoB's primary immunoglobulin repertoire monoclonal, but also that its secondary repertoire is not further diversified by V-gene replacement or gene conversion. Among 99 heavy-chain and 41 lambda light-chain genes from peripheral B cells of the MonoB mouse, there were no V-gene replacements. When compared to the QM mouse, which has RAG activity, and for which V-gene replacement is the major diversifying mechanism, these data suggest that V-gene replacement is mediated by V(D)J recombination and not by other recombination systems.
Subject(s)
B-Lymphocytes/immunology , Gene Rearrangement, B-Lymphocyte , Immunoglobulin Isotypes/genetics , Immunoglobulin Isotypes/immunology , Immunoglobulin Variable Region/genetics , Animals , Genetic Variation , Mice , Mice, Knockout , Mice, TransgenicABSTRACT
We have purified and biochemically characterized a multiprotein complex designated SWAP. In a DNA transfer assay, SWAP preferentially recombines ("swaps") sequences derived from Ig heavy chain switch regions. We identified four of the proteins in the SWAP complex: B23 (nucleophosmin), C23 (nucleolin), poly(ADP-ribose) polymerase (PARP), and SWAP-70. The first three are proteins known to be present in most cells. B23 promotes single-strand DNA reannealing and the formation of joint molecules in a D-loop assay between homologous, but also between Smu and Sgamma sequences. SWAP-70 is a novel protein of 70 kDa. Its cDNA was cloned and sequenced, and the protein was overexpressed in Escherichia coli. SWAP-70 protein expression was found only in B lymphocytes that had been induced to switch to various Ig isotypes and in switching B-cell lines. SWAP-70 is a nuclear protein, has a weak affinity for DNA, binds ATP, and forms specific, high affinity complexes with B23, C23, and poly(ADP-ribose) polymerase. These findings are consistent with SWAP being the long elusive "switch recombinase" and with SWAP-70 being the specific recruiting element that assembles the switch recombinase from universal components.
Subject(s)
B-Lymphocytes/metabolism , DNA, Recombinant/genetics , DNA-Binding Proteins/genetics , Guanine Nucleotide Exchange Factors , Nuclear Proteins/genetics , Recombination, Genetic , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA-Binding Proteins/metabolism , Escherichia coli/genetics , Hot Temperature , Kinetics , Mice , Mice, Inbred C57BL , Minor Histocompatibility Antigens , Molecular Sequence Data , Nuclear Proteins/metabolism , Nucleophosmin , Phosphoproteins/metabolism , Poly(ADP-ribose) Polymerases/metabolism , Protein BindingABSTRACT
Hypermutation at the Ig loci is confined to the area between the promoter and the intronic enhancer, which includes the rearranged variable region gene segment. We identified factors that contribute to the site-specificity at the heavy chain locus. We found that distance from both the promoter and the intronic enhancer is crucial in hypermutation. The presence of the enhancer is required, and, in contrast to its definition for transcriptional activity, its effect is orientation-sensitive.
Subject(s)
Enhancer Elements, Genetic , Genes, Immunoglobulin , Introns , Animals , Base Composition , Base Sequence , Cell Line , Cytosine , Guanine , Humans , Mice , Mutagenesis , Restriction Mapping , TransfectionABSTRACT
Mice homozygous for a disrupted allele of the mismatch repair gene Pms2 have a mutator phenotype. When this allele is crossed into quasi-monoclonal (QM) mice, which have a very limited B cell repertoire, homozygotes have fewer somatic mutations at the immunoglobulin heavy chain and lambda chain loci than do heterozygotes or wild-type QM mice. That is, mismatch repair seems to contribute to somatic hypermutation rather than stifling it. It is suggested that at immunoglobulin loci in hypermutable B cells, mismatched base pairs are "corrected" according to the newly synthesized DNA strand, thereby fixing incipient mutations instead of eliminating them.
Subject(s)
Adenosine Triphosphatases , DNA Repair Enzymes , DNA Repair , DNA-Binding Proteins , Genes, Immunoglobulin , Immunoglobulin Variable Region/genetics , Mutation , Proteins/genetics , Alleles , Amino Acid Sequence , Animals , B-Lymphocytes/immunology , Base Composition , Base Sequence , Cloning, Molecular , Crosses, Genetic , Female , Gene Rearrangement , Heterozygote , Immunoglobulin Heavy Chains/chemistry , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/chemistry , Immunoglobulin lambda-Chains/chemistry , Immunoglobulin lambda-Chains/genetics , Male , Mice , Mice, Knockout , Mismatch Repair Endonuclease PMS2 , Molecular Sequence Data , Proteins/physiologyABSTRACT
The Na,K-ATPase, or sodium pump, a ubiquitous transmembrane enzyme in higher eukaryotes, consists of an alpha and a beta subunit. Here we investigate the expression pattern of the two beta isotypes in mouse B cell lines. Neither primary cells nor cell lines express beta 2. Abelson virus-transformed pre-B cells express beta 1, while B lymphomas and plasmacytomas do not. Thus, beta 1 expression in transformed cells follows that of their untransformed counterparts. Some subclones of pre-B cell line 70Z/3 express beta 1, and others do not, but lipopolysaccharide induces the beta 1-negative cells to become beta 1-positive.
Subject(s)
B-Lymphocytes/enzymology , Sodium-Potassium-Exchanging ATPase/biosynthesis , Abelson murine leukemia virus , Animals , B-Lymphocytes/drug effects , Brain/cytology , Cell Line , Cell Transformation, Viral , Enzyme Induction , Gene Expression Regulation , Isoenzymes/biosynthesis , Isoenzymes/genetics , Lymphoma, B-Cell/enzymology , Lymphoma, B-Cell/genetics , Mice , Molecular Sequence Data , Plasmacytoma/enzymology , Plasmacytoma/genetics , Sodium-Potassium-Exchanging ATPase/geneticsABSTRACT
The Abelson-virus-transformed mouse pre-B-cell line 18-81 switches almost exclusively from mu to gamma 2b. From nuclear extracts of this cell line, we have isolated a factor that specifically binds to S gamma 2b. After an eight-step purification scheme, in which different types of DNA-affinity chromatography were used as key elements, we obtained a preparation with two narrowly spaced bands at approximately 69 kD on a silver-stained SDS gel. Binding specificity of main-peak fractions of affinity-purified proteins was analyzed by gel shift assays, in which S gamma 2b, but not S mu, competes. The results are consistent with this factor being part of the switch recombinase.
Subject(s)
DNA-Binding Proteins/metabolism , Immunoglobulin G/genetics , Immunoglobulin Switch Region , Animals , Antibody Specificity , Autoradiography , Base Sequence , Cell Line, Transformed , Cell Transformation, Viral , Chromatography, Affinity , DNA Footprinting , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/isolation & purification , Mice , Molecular Sequence Data , Molecular Weight , Recombination, Genetic , Repetitive Sequences, Nucleic AcidABSTRACT
The primary repertoire of the quasimonoclonal mouse is monospecific. However, among peripheral B cells, there is a high frequency of variant cells with V(H) replacements, which are also hypermutated. We show in this work that these hyperselected cells expand in numbers as the animals increase in age, switch their isotypes, and with increasing age become the almost exclusive contributors to the pool of serum Ig. The fraction of such cells is higher in the peritoneum than in peripheral blood, supporting the view that the peritoneum is a site of production of nonspecific serum Ig. We have also isolated and partially sequenced the replacing V(H) gene segments from B220-positive, Id-negative cells, and mu-negative, lambda-positive (i.e., switched) cells, and matched them with their germline counterparts. V(H) families are represented proportional to the number of members in the germline, a finding that is consistent with the idea that environmental Ag pressure maintains the germline repertoire of V gene segment.