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1.  Alteration of immunoglobulin phenotype in cell culture-adapted lines of two mouse plasmacytomas 
The Journal of Experimental Medicine  1975;142(4):998-1010.
Murine plasmacytomas can be adapted to continuous in vitro culture by alternate passage between culture and animal. We have found that the kinetics of adaptation reflect a selection for the growth of variant plasmacytoma cells. The inclusion of an altered immunoglobulin phenotype in such variant cells could explain the Ig-producing variants that we observed in two of six transplantable lines of plasmacytomas that were adapted to culture. The first variant, an IgM-producing cell line (104-76), was adapted from a transplanted line of MOPC 104E that had stopped producing IgM with binding specificity for alpha1-3 Dextran. Unlike MOPC 104E, the IgM of 104-76 contains kappa- instead of lambda-light chains and probably contains an altered or different mu- heavy chain. A second variant (352-57) was found in an IgG2b-producing tumor (MOPC 352) which was induced in a BALB/c mouse strain (CB-6) that carried Ig genes of the C57BL/Ka allotype. This cell line apparently switched from producing IgG2b molecules of the C57BL allotype (H9) and of a known idiotype to IgG1 molecules of the BALB/c allotype (F19) without the idiotype marker. The propagation of a biclonal plasmacytoma from the time of original tumor induction does not appear as a likely explanation for these results. Rather, we seem to be dealing with plasmacytoma variants or with the possible induction of secondary tumors of host origin.
PMCID: PMC2189950  PMID: 51902
2.  Nodal and extranodal plasmacytomas expressing immunoglobulin A: an indolent lymphoproliferative disorder with a low risk of clinical progression 
Plasmacytomas expressing immunoglobulin A are rare and not well characterized. In this study, nine cases of IgA-positive plasmacytomas presenting in lymph node and three in extranodal sites were analyzed by morphology, immunohistochemistry, and PCR examination of immunoglobulin heavy and kappa light chain genes. Laboratory features were correlated with clinical findings. There were seven males and five females; age range was 10 to 66 years (median, 32 years). Six of the patients were younger than 30-years-old, five of whom had nodal disease. 67% (6/9) of the patients with nodal disease had evidence of immune system dysfunction, including human immunodeficiency virus (HIV) infection, T-cell deficiency, autoantibodies, arthritis, Sjögren’s syndrome, and decreased B-cells. An IgA M-spike was detected in 6/11 cases, and the M-protein was nearly always less than 30 g/L. All patients had an indolent clinical course without progression to plasma cell myeloma. Histologically, IgA plasmacytomas showed an interfollicular or diffuse pattern of plasma cell infiltration. The plasma cells were generally of mature Marschalko type with little or mild pleomorphism and exclusive expression of monotypic IgA. There was an equal expression of kappa and lambda light chains (ratio 6:6). Clonality was demonstrated in 9 of 12 cases: by PCR in 7 cases, by cytogenetic analysis in 1 case, and by immunofixation in 1 case. Clonality did not correlate with pattern of lymph node infiltration. Our results suggest that IgA plasmacytomas may represent a distinct form of extramedullary plasmacytoma characterized by younger age at presentation, frequent lymph node involvement and low risk of progression to plasma cell myeloma.
doi:10.1097/PAS.0b013e3181f17d0d
PMCID: PMC2947321  PMID: 20871216
Extramedullary plasmacytoma; IgA; lymph node; plasma cell myeloma
3.  The regulated production of mu m and mu s mRNA is dependent on the relative efficiencies of mu s poly(A) site usage and the c mu 4-to-M1 splice. 
Molecular and Cellular Biology  1989;9(2):726-738.
The relative abundance of the mRNAs encoding the membrane (mu m) and secreted (mu s) forms of immunoglobulin mu heavy chain is regulated during B-cell maturation by a change in the mode of RNA processing. Current models to explain this regulation involve either competition between cleavage-polyadenylation at the proximal (mu s) poly(A) site and cleavage-polyadenylation at the distal (mu m) poly(A) site [poly(A) site model] or competition between cleavage-polyadenylation at the mu s poly(A) site and splicing of the C mu 4 and M1 exons, which eliminates the mu s site (mu s site-splice model). To test certain predictions of these models and to determine whether there is a unique structural feature of the mu s poly(A) site that is essential for regulation, we constructed modified mu genes in which the mu s or mu m poly(A) site was replaced by other poly(A) sites and then studied the transient expression of these genes in cells representative of both early- and late-stage lymphocytes. Substitutions at the mu s site dramatically altered the relative usage of this site and caused corresponding reciprocal changes in the usage of the mu m site. Despite these changes, use of the proximal site was still usually higher in plasmacytomas than in pre-B cells, indicating that regulation does not depend on a unique feature of the mu s poly(A) site. Replacement of the distal (mu m) site had no detectable effect on the usage of the mu s site in either plasmacytomas or pre-B cells. These findings are inconsistent with the poly(A) site model. In addition, we noted that in a wide variety of organisms, the sequence at the 5' splice junction of the C mu 4-to-M1 intron is significantly different from the consensus 5' splice junction sequence and is therefore suboptimal with respect to its complementary base pairing with U1 small nuclear RNA. When we mutated this suboptimal sequence into the consensus sequence, the mu mRNA production in plasmacytoma cells was shifted from predominantly mu s to exclusively mu m. This result unequivocally demonstrated that splicing of the C mu 4-to-M1 exon is in competition with usage of the mu s poly(A) site. A key feature of this regulatory phenomenon appears to be the appropriately balanced efficiencies of these two processing reactions. Consistent with predictions of the mu s site-splice model, B cells were found to contain mu m precursor RNA that had undergone the C mu 4-to-M1 splice but had not yet been polyadenylated at the mu m site.
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PMCID: PMC362650  PMID: 2565533
4.  c-myc Gene rearrangements involving gamma immunoglobulin heavy chain gene switch regions in murine plasmacytomas. 
Nucleic Acids Research  1983;11(23):8303-8315.
In murine plasmacytomas, the c-myc gene has frequently been found to undergo rearrangement by virtue of a T(12;15) chromosome translocation. The immunoglobulin heavy chain gene switch region (S alpha) constitutes the target for most of these recombinations particularly in IgA producing plasmacytomas. We sought to identify non-S alpha myc target sites in several IgG producing tumors. The c-myc target in MPC-11 (a BALB/c IgG2b producing plasmacytoma) has been cloned, localized to the Igh-C locus and identified as the gamma 2a heavy chain gene switch region (S gamma 2a). Furthermore, by Southern blot hybridization, we have determined that the S gamma 2b region is the c-myc target in two NZB IgG2b producing plasmacytomas. The potential relation between Ig class expressed and c-myc translocation target is discussed.
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PMCID: PMC326583  PMID: 6324081
5.  Restricted immunoglobulin VH usage and VDJ combinations in the human response to Haemophilus influenzae type b capsular polysaccharide. Nucleotide sequences of monospecific anti-Haemophilus antibodies and polyspecific antibodies cross-reacting with self antigens. 
Journal of Clinical Investigation  1993;91(6):2734-2743.
To examine the human antibody repertoire generated against a biologically significant antigen we have obtained sequences of heavy chain variable region genes (IgVH) from 15 monoclonal antibodies specific for the capsular polysaccharide of Haemophilus influenzae type b (Hib PS). All VH segments are members of the VH3 family and 9 of 15 are members of the smaller VH3b subfamily. Restriction is evident by the shared use of certain VDJ joints in independent hybridomas from different subjects. Two hybridomas generated from the same subject demonstrate identical heavy chain variable region gene sequences but differ in isotype and rearrange alternative light chain variable region genes (IgVL), suggesting that in a normal immune response, a single pre-B cell clone may use different light chain rearrangements and give rise to progeny capable of reacting with antigen. Using a polymerase chain reaction assay optimized to detect base pair differences among VH genes we demonstrate that at least a portion of expressed anti-Hib PS VH genes have undergone somatic mutation. Anti-Hib PS heavy chain genes are homologous to VH segments encoding autoantibodies and two hybridomas secrete anti-Hib PS antibody that cross-reacts with self antigens (double-stranded DNA and single-stranded DNA). Comparison of VH regions of self-reactive and monospecific anti-Hib PS Ab demonstrates no consistent structural feature correlating with fine antigen specificity. These data demonstrate significant restriction in VH usage and VDJ recombination in the anti-Hib PS response and confirm that autoantibodies may be elicited during normal immune responses.
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PMCID: PMC443339  PMID: 8514881
6.  Plasmacytoma of the Clivus Presenting as Bilateral Sixth Nerve Palsy 
Background and Importance Plasmacytomas are monoclonal proliferations of plasma cells that may arise within soft tissue or bone. The skull base is a rare site for plasmacytomas to occur, and few cases have been reported in the literature. When present in the skull base, plasmacytomas may result in cranial neuropathies and often progress to multiple myeloma more rapidly than other intracranial or skeletal plasmacytomas.
Clinical Presentation A 69-year-old man presented with a primary complaint of diplopia and an examination consistent with bilateral abducens nerve palsy. No other deficits were noted. Magnetic resonance imaging of the skull base demonstrated a large T1 isointense moderately enhancing lesion centered within the clivus. Endoscopic biopsy of the mass revealed sheets and aggregates of mature monoclonal plasma cells. The patient's initial systemic work-up revealed that this was a solitary lesion, and he was treated with radiation therapy to the skull base with a durable local effect at 18-month follow-up. Unfortunately he progressed to multiple myeloma with peripheral osteolytic lesions but has been stabilized on chemotherapeutics.
Conclusion The clivus is an unusual site for intracranial plasmacytomas, and enhancing lesions must be differentiated from chordoma. Characteristic findings on histopathology include an immunoglobulin light-chain restricted clonal proliferation of plasma cells. Treatment is most commonly radiotherapy with surgery reserved for biopsy and palliation. Clinicians should be aware of the increased risk of progression to multiple myeloma in skull base plasmacytomas.
doi:10.1055/s-0035-1554930
PMCID: PMC4520983  PMID: 26251795
plasmacytoma; clivus; skull base; endoscopic sinus surgery
7.  Nonhomologous recombination at sites within the mouse JH-C delta locus accompanies C mu deletion and switch to immunoglobulin D secretion. 
Molecular and Cellular Biology  1991;11(11):5660-5670.
Plasma cells secrete immunoglobulins other than immunoglobulin M (IgM) after a deletion and recombination in which a portion of the immunoglobulin heavy-chain locus (IgH), from the 5'-flanking region of the mu constant-region gene (C mu) to the 5'-flanking region of the secreted heavy-chain constant-region gene (CH), is deleted. The recombination step is believed to be targeted via switch regions, stretches of repetitive DNA which lie in the 5' flank of all CH genes except delta. Although serum levels of IgD are very low, particularly in the mouse, IgD-secreting plasmacytomas of BALB/c and C57BL/6 mice are known. In an earlier study of two BALB/c IgD-secreting hybridomas, we reported that both had deleted the C mu gene, and we concluded that this deletion was common in the normal generation of IgD-secreting cells. To learn how such switch recombinations occur in the absence of a switch region upstream of the C delta 1 exon, we isolated seven more BALB/c and two C57BL/6 IgD-secreting hybridomas. We determined the DNA sequences of the switch recombination junctions in eight of these hybridomas as well as that of the C57BL/6 hybridoma B1-8. delta 1 and of the BALB/c, IgD-secreting plasmacytoma TEPC 1033. All of the lines had deleted the C mu gene, and three had deleted the C delta 1 exon in the switch recombination event. The delta switch recombination junction sequences were similar to those of published productive switch recombinations occurring 5' to other heavy-chain genes, suggesting that nonhomologous, illegitimate recombination is utilized whenever the heavy-chain switch region is involved in recombination.
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PMCID: PMC361937  PMID: 1922069
8.  A switch region inversion contributes to the aberrant rearrangement of a mu immunoglobulin heavy chain gene in MPC-11 cells. 
Nucleic Acids Research  1982;10(23):7751-7761.
We describe the unique features of an aberrantly rearranged mu immunoglobulin heavy chain gene isolated from MPC-11 cells (a gamma 2b producing Balb/c plasmacytoma). A novel rearrangement has occurred 1.5 Kb 5' of the MPC-11 mu gene (denoted 18b mu) resulting in the deletion of the majority of the repetitive switch region (S mu) and 5' flanking DNA including the Joining (JH) sequences. The remainder (275 bp) of the S mu repeat has undergone a complete sequence inversion. DNA sequences 5' of the inverted S mu sequence do not resemble Variable (VH), Diversity (D), JH or their conserved flanking sequences. A DNA sequence localized 5' of the inverted S mu sequence, (p18b mu-1.4) detects a small family of homologous sequences in Balb/c DNA. The 18b mu-1.4 like sequences lack homology to S mu, exhibit flanking sequence polymorphisms in 5 out of 6 inbred mouse strains and undergo partial or complete deletion in 5 out of 10 plasmacytomas tested. Two 18b mu-1.4 homologous sequences display a higher copy number in C57Bl/6, AL/N and CAL9 mouse strains.
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PMCID: PMC327043  PMID: 6296790
9.  Distinct mechanisms define murine B cell lineage immunoglobulin heavy chain (IgH) repertoires 
eLife  null;4:e09083.
Processes that define immunoglobulin repertoires are commonly presumed to be the same for all murine B cells. However, studies here that couple high-dimensional FACS sorting with large-scale quantitative IgH deep-sequencing demonstrate that B-1a IgH repertoire differs dramatically from the follicular and marginal zone B cells repertoires and is defined by distinct mechanisms. We track B-1a cells from their early appearance in neonatal spleen to their long-term residence in adult peritoneum and spleen. We show that de novo B-1a IgH rearrangement mainly occurs during the first few weeks of life, after which their repertoire continues to evolve profoundly, including convergent selection of certain V(D)J rearrangements encoding specific CDR3 peptides in all adults and progressive introduction of hypermutation and class-switching as animals age. This V(D)J selection and AID-mediated diversification operate comparably in germ-free and conventional mice, indicating these unique B-1a repertoire-defining mechanisms are driven by antigens that are not derived from microbiota.
DOI: http://dx.doi.org/10.7554/eLife.09083.001
eLife digest
Our immune system protects us by recognizing and destroying invading viruses, bacteria and other microbes. B cells are immune cells that produce protective proteins called antibodies to stop infections. These cells are activated by ‘antigens’, which are fragments of molecules from the microbes or from our own cells. When an antigen binds to a B cell, the cell matures, multiplies and produces proteins called antibodies. These antibodies can bind to the antigen, which marks the microbe for attack and removal by other cells in the immune system.
Each antibody consists of two ‘heavy chain’ and two ‘light chain’ proteins. B cells are able to produce a large variety of different antibodies due to the rearrangement of the gene segments that encode the heavy and light chains. In mice, there are two kinds of B cells – known as B-1a and B-2 cells – that play different roles in immune responses. B-1a cells have long been known to produce the ‘natural’ antibodies that are present in the blood prior to an infection. On the other hand, B-2 cells produce antibodies that are specifically stimulated by an infection and are better adapted to fighting it. Previous studies have shown that both types of antibodies are required to allow animals to successfully fight the flu virus.
Here, Yang, Wang et al. used a technique called fluorescence-activated cell sorting (or FACS) and carried out extensive genomic sequencing to study how the B-1a and B-2 populations rearrange their genes to produce heavy chains. This approach made it possible to separate the different types of B cells and then sequence the gene for the heavy chain within the individual cells. The experiments show that the “repertoire” of heavy chains in the antibodies of the B-1a cells is much less random and more repetitive than that of B-2 populations.
Furthermore, Yang, Wang et al. show that B-1a cells produce and maintain their repertoire of heavy chains in a different way to other B-2 populations. B-1a cells develop earlier and the major genetic rearrangements in the gene that encodes the heavy chain occur within the first few weeks of life. Although the gene rearrangements have mostly stopped by adulthood, the B-1a antibody repertoire continues to evolve profoundly as the B-1a cells divide over the life of the animal. On the other hand, the gene rearrangements that make the heavy chains in the B-2 cells continue throughout the life of the animal to produce the wider repertoire of antibodies found in these cells. In addition, the processes that continue to change the antibody reperotire in the B-1a cells during adulthood do not occur in the B-2 populations.
Importantly, the these reperotire-changing processes in B-1a cells also occur in mice that have been raised in germ-free conditions, which demonstrates that – unlike other B cells – the repertoire of heavy chains in B-1a cells is not influenced by antigens from microbes. Instead, it is mainly driven by antigens that are expressed by normal cells in the body. These findings open the way to future work aimed at understanding how B-1a cells help to protect us against infection, and their role in autoimmune diseases, where immune cells attack the body’s own healthy cells.
DOI: http://dx.doi.org/10.7554/eLife.09083.002
doi:10.7554/eLife.09083
PMCID: PMC4714975  PMID: 26422511
V(D)J; diversify; IgH; Mouse
10.  Activation of Aicda gene transcription by Pax5 in plasmacytoma cells 
Immunologic research  2013;55(0):10.1007/s12026-012-8357-8.
Activation Induced Deaminase (AID) is an enzyme responsible for somatic hypermutation (SHM) and immunoglobulin heavy chain class switch recombination (CSR). Because AID causes double-stranded breaks in DNA, its expression is highly regulated and is normally restricted to germinal center B cells. Dysregulated AID expression can lead to cancer as a result of AID-mediated chromosomal translocations. Many transcription factors including Paired box protein 5 (Pax5) have been implicated in regulating the expression of Aicda, the gene encoding AID. In this study, we demonstrate that exogenous expression of Pax5 in a murine plasmacytoma cell line, 558LμM, leads to robust activation of endogenous Aicda transcription. Pax5 is known to initiate transcription both through its N-terminal paired DNA-binding domain (DBD) and C-terminal-activation domain. Through mutational analysis, we demonstrate that Pax5 regulates Aicda transcription through its C-terminal-activation domain. Together, our work describes a novel system that will be useful for determining how Pax5 regulates Aicda transcription.
doi:10.1007/s12026-012-8357-8
PMCID: PMC3844280  PMID: 22956488
Activation induced cytidine deaminase gene; AID; Pax5; transcriptional regulation
11.  Sites That Direct Nuclear Compartmentalization Are near the 5′ End of the Mouse Immunoglobulin Heavy-Chain Locus 
Molecular and Cellular Biology  2005;25(14):6021-6030.
VDJ rearrangement in the mouse immunoglobulin heavy chain (Igh) locus involves a combination of events, including a large change in its nuclear compartmentalization. Prior to rearrangement, Igh moves from its default peripheral location near the nuclear envelope to an interior compartment, and after rearrangement it returns to the periphery. To identify any sites in Igh responsible for its association with the periphery, we systematically analyzed the nuclear positions of the Igh locus in mouse non-B- and B-cell lines and, importantly, in primary splenic lipopolysaccharide-stimulated B cells and plasmablasts. We found that a broad ∼1-Mb region in the 5′ half of the variable-gene region heavy-chain (Vh) locus regularly colocalizes with the nuclear lamina. The 3′ half of the Vh gene region is less frequently colocalized with the periphery, while sequences flanking the Vh gene region are infrequently so. Importantly, in plasmacytomas, VDJ rearrangements that delete most of the Vh locus, including part of the 5′ half of the Vh gene region, result in loss of peripheral compartmentalization, while deletion of only the proximal half of the Vh gene region does not. In addition, when Igh-Myc translocations move the Vh genes to a new chromosome, the distal Vh gene region is still associated with the nuclear periphery. Thus, the Igh region that interacts with the nuclear periphery is localized but is likely comprised of multiple sites that are distributed over ∼1 Mb in the 5′ half of the Vh gene region. This 5′ Vh gene region that produces peripheral compartmentalization is the same region that is distinguished by requirements for interleukin-7, Pax5, and Ezh2 for rearrangement of the Vh genes.
doi:10.1128/MCB.25.14.6021-6030.2005
PMCID: PMC1168801  PMID: 15988016
12.  An incoherent regulatory network architecture that orchestrates B cell diversification in response to antigen signaling 
B cell receptor signaling controls the expression of IRF-4, a transcription factor required for B cell differentiation. This study shows that IRF-4 regulates divergent B cell fates via a ‘kinetic-control' mechanism that determines the duration of a transient developmental state.
The intensity of signaling through the B cell receptor controls the level of expression of IRF-4, a transcription factor required for B cell differentiation. The rate of IRF-4 production dictates the extent of antibody gene diversification that B cells undergo upon antigen encounter before differentiating into antibody-secreting plasma cells.Computational modeling and experimental analyses substantiate a model, whereby IRF-4 regulates B cell fate trajectories via a ‘kinetic-control' mechanism.Kinetic control is a process by which B cells pass through an obligate state of variable duration that sets the degree of cellular diversification prior to their terminal differentiation.An incoherent regulatory network architecture, within which IRF-4 is embedded, is the basis for realization of kinetic control.
The generation of a diverse set of pathogen-specific antibodies, with differing affinities and effector functions, by B lymphocytes is essential for efficient protection from many microorganisms. Antibody gene diversification in B cells is mediated by two molecular processes termed class-switch recombination and somatic hypermutation (CSR/SHM) (F1A). The former enables the generation of antibodies with the same antigen-binding specificity, but different effector domains, whereas the latter results in a repertoire of antibodies with a range of affinities for a given antigen containing the same effector domain. CSR/SHM occurs in antigen-activated B cells before their terminal differentiation into plasma cells. The transcription factor IRF-4 is required for CSR/SHM as well as plasma-cell differentiation, with its highest levels of expression being necessary for the latter. IRF-4 acts in the context of a network of regulators that include Blimp-1, Pax5, Bach2 and Bcl-6 (F1B). Despite extensive characterization of these individual factors, how the network responds to sensing of antigen by the B cell antigen receptor (BCR, antibody molecule expressed on cell surface) to regulate the extent of antibody gene diversification and plasma-cell differentiation remains to be addressed.
To address this issue, we assemble a computational model. The model reveals two contrasting scenarios that can underlie B cell fate dynamics. In one case, the initial rate of IRF-4 production controls a binary cell fate choice that involves either going to the CSR/SHM state or to the plasma-cell state; the time spent in the CSR state is relatively insensitive to the initial rate of IRF-4 production (herein called ‘basic bistability'). In the other case, IRF-4 drives all cells through a transient CSR/SHM state, but the initial rate of IRF-4 production sets its duration (‘kinetic control'). Both scenarios predict that increasing the initial rate of IRF-4 production favors the generation of plasma cells at the expense of CSR/SHM, but they differ fundamentally with respect to the underlying gene expression patterns.
To distinguish between these two scenarios experimentally, we utilize two different genetic models. The first involves the B1-8i transgenic mouse whose B cells express a rearranged V187.2 VDJ Ig heavy chain gene segment that is specific for the hapten nitrophenol (NP). The second is a newly developed mouse model that allows exogenous control of IRF-4 expression in naive primary B cells using a tet-inducible allele. Using these models, we show that (i) BCR signal strength sets the initial rate of IRF-4 accumulation and (ii) the concentration of IRF-4 is sensed by an incoherent gene regulatory network architecture to regulate the extent of CSR/SHM prior to plasma-cell differentiation. Our results are consistent with the ‘kinetic-control model' in which the levels of BCR-induced IRF-4 expression control the duration of an obligate CSR/SHM state that enables B cell diversification before terminal differentiation into plasma cells. Evidence for the transient CSR/SHM state is corroborated by both patterns of gene expression and the presence of AID-dependent mutations in individual non-switched plasmablasts.
Our results provide a molecular framework for understanding how B cells balance the competing demands for Ig CSR and SHM with the secretion of antibodies during humoral immune responses. The key feature of the network architecture that allows IRF-4 to coordinate the two competing states of gene expression in a temporal manner is that it simultaneously but asymmetrically activates both sides of a bistable mutual repression circuit. Because the two effects of the primary regulator antagonize each other, we describe the circuit as being based on an ‘incoherent' regulatory motif. Other incoherent regulatory motifs in varied biological systems are also associated with the acquisition of transient cell states, and we consider how the kinetic-control mechanism proposed by us could more generally serve to translate developmental cues into elaborate morphogenetic patterns.
The B-lymphocyte lineage is a leading system for analyzing gene regulatory networks (GRNs) that orchestrate distinct cell fate transitions. Upon antigen recognition, B cells can diversify their immunoglobulin (Ig) repertoire via somatic hypermutation (SHM) and/or class switch DNA recombination (CSR) before differentiating into antibody-secreting plasma cells. We construct a mathematical model for a GRN underlying this developmental dynamic. The intensity of signaling through the Ig receptor is shown to control the bimodal expression of a pivotal transcription factor, IRF-4, which dictates B cell fate outcomes. Computational modeling coupled with experimental analysis supports a model of ‘kinetic control', in which B cell developmental trajectories pass through an obligate transient state of variable duration that promotes diversification of the antibody repertoire by SHM/CSR in direct response to antigens. More generally, this network motif could be used to translate a morphogen gradient into developmental inductive events of varying time, thereby enabling the specification of distinct cell fates.
doi:10.1038/msb.2011.25
PMCID: PMC3130558  PMID: 21613984
BCR signal strength; bistability; gene regulatory network; ghost of a fixed point; Irf4
13.  Thymic requirement for cyclical idiotypic and reciprocal anti-idiotypic immune responses to a T-independent antigen 
The role of the thymus in the cyclical appearance of the dominant idiotype of the myeloma protein secreted by the TEPC-15 plasmacytoma (T- 15)-bearing plaque-forming cells (PFC) and anti-idiotypic cells (i.e., cells with receptors for T-15) in the spleen during a primary response to the phosphorylcholine determinant of Streptococcus pneumoniae, strain R36a (Pn) was studied using normal mice, thymus-deficient nude mice, and thymus gland-grafted nude mice (TG-nude). The nude mice and their phenotypically normal littermates (LM) were backcrossed on the BALB/c genetic background. The kinetics of the anti-Pn PFC response of BALB/c inbred mice, littermates of nude mice, and TG-nude mice were essentially the same. There was an initial peak on day 5-6 followed by a decline to near background, and then a second peak on day 12. In nude mice, the first peak of anti-Pn PFC (day 5) was comparable in magnitude to that of mice with an intact thymus; however, there was no second peak. In contrast to the cellular response measured at the level of PFC, the serum antibody response to Pn (assayed by passive hemagglutination of sheep erythrocytes coated with Pn polysaccharide) was comparable in all groups of mice and did not show a measurable oscillation. The anti-idiotypic cellular activity was determined by the ability of spleen cells to bind radiolabeled (125I) TEPC-15 myeloma protein (IgA, kappa) which carries an idiotypic determinant indistinguishable from that of most anti-phosphorylcholine antibodies in BALB/c mice. Binding of radiolabeled McPC-603 (IgA, kappa) and MOPC- 315 (IgA, lambda 2) myeloma proteins (which lack the T-15 idiotypic determinant) served as controls. The changes in T-15 binding by splenic lymphocytes following the Pn immunization differed between normal and athymic mice. BALB/c, LM, and TG-nude mice showed a biphasic pattern with peaks at days 3--4 and 10--11 that was nearly reciprocal to the PFC curve. On the other hand, T-15 binding in nude mice either declined and remained depressed or was not affected by the ongoing anti-Pn response. These observations demonstrate that mature T cells are required for cyclical idiotypic and anti-idiotypic responses to immunization with a T-independent antigen and suggest that the cyclical immune response may result from an interaction between idiotypic and anti-idiotypic cell clones.
PMCID: PMC2185782  PMID: 6965397
14.  EBV-Positive Plasmacytoma of the Submandibular Gland—Report of a Rare Case with Molecular Genetic Characterization 
Head and Neck Pathology  2011;5(4):389-394.
Plasmacytomas are differentiated plasma cell tumors that present as a mass lesion in osseous or extraosseous sites. Although the most common site for extramedullary plasmacytomas (EMP) is in the upper respiratory tract, plasmacytomas initially presenting as salivary gland masses are very uncommon. We describe a case of an EBV-positive plasmacytoma presenting as a 7.7 cm submandibular mass in an elderly immunocompetent man which displayed an abundance of “naked nuclei” on fine needle aspiration cytology. The tumor showed lambda light chain restriction and positive expression for CD38, MUM1 and EBER. Subsequent investigation for myeloma revealed absence of M-protein and end-organ damage, except for a lytic lesion in the radial bone. An extensive fluorescent in situ hybridization analysis showed the tumor to be negative for the t(4;14) FGFR3/IGH translocation as well as translocations involving the IGH,IGL, IGK, CCND1, BCL2, BCL6 and C-MYC genes. KRAS genetic analysis did not reveal any mutations of codons 12, 13 and 61.
doi:10.1007/s12105-011-0257-z
PMCID: PMC3210227  PMID: 21442194
Plasmocytoma; Salivary gland; Epstein barr virus; Fluorescence in situ hybridization; CMYC; KRAS
15.  DNA repair defects associated with chromosomal translocation breaksite regions. 
Molecular and Cellular Biology  1994;14(2):1204-1212.
Using an assay that measures the removal of UV-induced pyrimidine dimers in specific DNA sequences, we have found that the Pvt-1, immunoglobulin H-C alpha (IgH-C alpha), and IgL-kappa loci are poorly repaired in normal B lymphoblasts from plasmacytoma-susceptible BALB/cAnPt mice. Breaksites in these genes are associated with the chromosomal translocations that are found in > 95% of BALB/cAnPt plasmacytomas. In contrast to those from BALB/cAnPt mice, B lymphoblasts from plasmacytoma-resistant DBA/2N mice rapidly repair Pvt-1, IgH-C alpha, and IgL-kappa. Further, (BALB/cAnPt x DBA/2N)F1 hybrids, which are resistant to plasmacytoma development, carry an efficient (DBA/2N-like) repair phenotype. Analysis of allele-specific repair in the IgH-C alpha locus indicates that efficient repair is controlled by dominant, trans-acting factors. In the F1 heterozygotes, these factors promote efficient repair of BALB/cAnPt IgH-C alpha gene sequences. The same sequences are poorly repaired in the BALB/cAnPt parental strain. Analysis of the strand specificity of repair indicates that both strand-selective and nonselective forms of repair determine repair efficiency at the gene level in nonimmortalized murine B lymphoblasts.
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PMCID: PMC358476  PMID: 8289801
16.  Immunoglobulin gene rearrangement and cell surface antigen expression in acute lymphocytic leukemias of T cell and B cell precursor origins. 
Journal of Clinical Investigation  1983;71(2):301-313.
We have explored the relationship among immunoglobulin gene rearrangement, cytoplasmic immunoglobulin production, and cell surface antigen expression within 37 cases of acute lymphocytic leukemia. All 12 cases of the T cell type had germ-line kappa and lambda genes and 11 of 12 had germ-line heavy chain genes. In contrast, all 25 cases of the "non-T, non-B" classification, which lacked both definitive T cell markers and surface immunoglobulin, had rearranged immunoglobulin genes, indicating that they represent precursor cells already committed to the B cell lineage at the gene level. 14 had rearranged heavy chain genes, yet retained germ-line light chain genes, whereas 11 cases had both heavy and light chain gene reorganizations. All patterns of immunoglobulin gene rearrangement predicted by a model that proceeds from heavy chain gene recombination to light chain genes were observed. Despite the uniform presence of rearranged immunoglobulin genes, only five cases produced cytoplasmic mu-chain, one exceptional case produced gamma-chain, and another produced only lambda-chain. The cases of B cell precursor type that do not produce immunoglobulin may represent cells that frequently possess ineffectively rearranged immunoglobulin genes. Included in this group may be a set of cells trapped within the B cell precursor series because their ineffective rearrangements have eliminated certain gene subsegments necessary for the assemblage of an effective heavy chain gene. All seven cases of the non-T, non-B subgroup that bore HLA-DR but lacked CALLA (the common acute lymphocytic leukemia-associated antigen) represented the earliest recognizable stage of B cell precursors with rearranged heavy chain genes but germ-line light chain genes. Correlations here suggest that cells entering B cell development express HLA-DR and rearrange heavy chain genes before the expression of a B cell-associated antigen recognized by the antibody BA-1, the antigen CALLA, and any subsequent light chain gene rearrangements.
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PMCID: PMC436868  PMID: 6401769
17.  Receptor editing: an approach by autoreactive B cells to escape tolerance 
The Journal of Experimental Medicine  1993;177(4):999-1008.
To determine the fate of anti-DNA antibody-bearing B cells in normal mice, we generated transgenic mice bearing the heavy (H) and light (L) chain genes of a well-characterized anti-double-stranded DNA antibody. This antibody was originally isolated from a diseased MRL/lpr mouse and has characteristics common to spontaneously arising anti-DNA antibodies. Results show that the H/L transgene (tg) immunoglobulin receptor is not expressed by animals bearing both tgs, although single tg animals (H or L) express their transgenes. Young H/L tg animals express few B cells, whereas adult H/L tg animals maintain almost normal B cell numbers. Analysis of the immunoglobulin receptors used by adult B cells shows that all contain the tg H chain in association with endogenous L chains. These B cells transcribe the L tg as well as the rearranged endogenous L chain gene, and loss of endogenous L chain gene transcription results in resurrection of the 3H9 H/L tg product. Examination of the endogenous L chains used by these cells shows that they represent a highly restricted subset of V genes. Taken together, these data suggest that autoreactive transgenic B cells can rearrange endogenous L chain genes to alter surface receptors. Those L chains that compete successfully with the L tg for H chain binding, and that create a nonautoreactive receptor, allow the B cell to escape deletion. We suggest that this receptor editing is a mechanism used by immature autoreactive B cells to escape tolerance.
PMCID: PMC2190958  PMID: 8459227
18.  The 'zinc knuckle' motif of Early B cell Factor is required for transcriptional activation of B cell-specific genes 
Molecular immunology  2008;45(14):3786-3796.
Early B cell factor (EBF) is a critical regulator of B lymphocyte-specific gene transcription. EBF functions, in part, by binding to regulatory sites of genes required for the pre-B- and mature B cell receptors. These DNA targets include the promoters of the mb-1 and Vpreb1 genes that encode Ig-α and one of the components of surrogate light chain, respectively. The biochemical basis of DNA binding and gene activation by EBF is poorly understood. The DNA-binding domain (DBD) of EBF includes a putative zinc-binding motif (HX3CX2CX5C), which we have designated the 'Zn-knuckle'. The Zn-knuckle is required for binding of the mb-1 promoter site in EMSA, but it has not been demonstrated to be important for functional activities of EBF in B cells. Therefore, we expressed EBF with mutations in the Zn-knuckle motif or flanking sequences in plasmacytoma cells in which activation of endogenous mb-1 and Vpreb1 genes is dependent on EBF. EBF with mutations that prevent zinc coordination by the Zn-knuckle did not activate transcription of either target gene. Other mutations affected the sequence preference of DNA binding and differentially inhibited activation of these genes. Our results demonstrate the importance of the Zn-knuckle motif in EBF. These experiments also confirm that EBF can re-activate multiple genes of the early B cell program in plasmacytoma cells, which provide a useful cell-based assay for dissecting mechanisms involving EBF.
doi:10.1016/j.molimm.2008.05.018
PMCID: PMC2593158  PMID: 18606452
Early B cell Factor; EBF; mb-1 promoter; DNA-binding specificity; Vpreb1 promoter; zinc-binding motif
19.  The coupling between enhancer activity and hypomethylation of kappa immunoglobulin genes is developmentally regulated. 
Molecular and Cellular Biology  1988;8(2):930-937.
Previous studies have indicated that immunoglobulin enhancers are essential for establishing transcriptional competence but not for maintaining the activity of constitutively transcribed genes. To understand the basis for this developmental shift away from dependence on enhancer function, we have investigated the relationship between transcriptional activity and methylation status of the immunoglobulin kappa light-chain genes (kappa genes) in mouse cell lines representing different stages of B-cell maturation. Using pre-B-cell lines in which the level of a critical kappa enhancer-binding factor, NF-kappa B, was controlled by the administration or withdrawal of lipopolysaccharide and plasmacytoma lines that either contain or lack this factor, we studied the properties of endogenous kappa genes and of transfected kappa genes which were stably integrated into the genomes of these cells. In the pre-B cells, the exogenous (originally unmethylated) kappa genes, as well as endogenous kappa genes, were fully methylated and persistently dependent on enhancer function, even after more than 30 generations in a transcriptionally active state. In plasmacytoma cells, the endogenous kappa genes were invariably hypomethylated, whereas exogenous kappa genes were hypomethylated only in cells that contain NF-kappa B and are thus permissive for kappa enhancer function. These results indicate that the linkage of hypomethylation to enhancer-dependent activation of kappa transcription occurs after the pre-B-cell stage of development. The change in methylation status, together with associated changes in chromatin structure, may suffice to eliminate or lessen the importance of the enhancer for the maintenance of the transcriptionally active state.
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PMCID: PMC363225  PMID: 3127693
20.  The Immunoglobulin Heavy Chain Locus Control Region Increases Histone Acetylation along Linked c-myc Genes 
Molecular and Cellular Biology  1998;18(11):6281-6292.
In chromosome translocations characteristic of Burkitt lymphomas (BL) and murine plasmacytomas, c-myc genes become juxtaposed to immunoglobulin heavy-chain (IgH) sequences, resulting in aberrant c-myc transcription. Translocated c-myc alleles that retain the first exon exhibit increased transcription from the normally minor c-myc promoter, P1, and increased transcriptional elongation through inherent pause sites proximal to the major c-myc promoter, P2. We recently demonstrated that a cassette derived from four DNase I-hypersensitive sites (HS1234) in the 3′Cα region of the IgH locus functions as an enhancer-locus control region (LCR) and directs a similar pattern of deregulated expression of linked c-myc genes in BL and plasmacytoma cell lines. Here, we report that the HS1234 enhancer-LCR mediates a widespread increase in histone acetylation along linked c-myc genes in Raji BL cells. Significantly, the increase in acetylation was not restricted to nucleosomes within the promoter region but also was apparent upstream and downstream of the transcription start sites as well as along vector sequences. Histone hyperacetylation of control c-myc genes, which was induced by the deacetylase inhibitor trichostatin A, mimics the effect of the HS1234 enhancer on expression from the c-myc P2 promoter, but not that from the P1 promoter. These results suggest that the HS1234 enhancer stimulates transcription of c-myc by a combination of mechanisms. Whereas HS1234 activates expression from the P2 promoter through a mechanism that includes increased histone acetylation, a general increase in histone acetylation is not sufficient to explain the HS1234-mediated activation of transcription from P1.
PMCID: PMC109215  PMID: 9774645
21.  Changes in Locus-specific V(D)J Recombinase Activity Induced by Immunoglobulin Gene Products during B Cell Development 
The process of V(D)J recombination is crucial for regulating the development of B cells and for determining their eventual antigen specificity. Here we assess the developmental regulation of the V(D)J recombinase directly, by monitoring the double-stranded DNA breaks produced in the process of V(D)J recombination. This analysis provides a measure of recombinase activity at immunoglobulin heavy and light chain loci across defined developmental stages spanning the process of B cell development. We find that expression of a complete immunoglobulin heavy chain protein is accompanied by a drastic change in the targeting of V(D)J recombinase activity, from being predominantly active at the heavy chain locus in pro-B cells to being exclusively restricted to the light chain loci in pre-B cells. This switch in locus-specific recombinase activity results in allelic exclusion at the immunoglobulin heavy chain locus. Allelic exclusion is maintained by a different mechanism at the light chain locus. We find that immature, but not mature, B cells that already express a functional light chain protein can undergo continued light chain gene rearrangement, by replacement of the original rearrangement on the same allele. Finally, we find that the developmentally regulated targeting of V(D)J recombination is unaffected by enforced rapid transit through the cell cycle induced by an Eμ-myc transgene.
PMCID: PMC2196138  PMID: 9034140
22.  IGHV1-69-Encoded Antibodies Expressed in Chronic Lymphocytic Leukemia React with Malondialdehyde–Acetaldehyde Adduct, an Immunodominant Oxidation-Specific Epitope 
PLoS ONE  2013;8(6):e65203.
The immunoglobulins expressed by chronic lymphocytic leukemia (CLL) B cells are highly restricted, suggesting they are selected for binding either self or foreign antigen. Of the immunoglobulin heavy-chain variable (IGHV) genes expressed in CLL, IGHV1-69 is the most common, and often is expressed with little or no somatic mutation, and restricted IGHD and IGHJ gene usage. We found that antibodies encoded by one particular IGHV1-69 subset, designated CLL69C, with the HCDR3 encoded by the IGHD3-3 gene in reading frame 2 and IGHJ6, specifically bound to oxidation-specific epitopes (OSE), which are products of enhanced lipid peroxidation and a major target of innate natural antibodies. Specifically, CLL69C bound immunodominant OSE adducts termed MAA (malondialdehyde–acetaldehyde-adducts), which are found on apoptotic cells, inflammatory tissues, and atherosclerotic lesions. It also reacted specifically with MAA-specific peptide mimotopes. Light chain shuffling indicated that non-stochastically paired L chain of IGLV3-9 contributes to the antigen binding of CLL69C. A nearly identical CLL69C Ig heavy chain was identified from an MAA-enriched umbilical cord phage displayed Fab library, and a derived Fab with the same HCDR3 rearrangement displayed identical MAA-binding properties. These data support the concept that OSE (MAA-epitopes), which are ubiquitous products of inflammation, may play a role in clonal selection and expansion of CLL B cells.
doi:10.1371/journal.pone.0065203
PMCID: PMC3688726  PMID: 23840319
23.  Lymphocyte Defect in Plasmacytoma-bearing Mice 
British Journal of Cancer  1978;37(4):545-552.
Multiple myeloma is often associated with humoral immunodepression in both man and mouse. When mice bearing the humorally immunodepressive plasmacytomas TEPC-183 and SPQC-11 were injected with SRBC, the rise of serum haemolysins was significantly less than that of non-tumour-bearing mice. Mice with the plasmacytomas MPC-11 and MOPC-315 have an antibody response similar to normal mice when injected with SRBC. Following immunization, normal mice and those bearing MPC-11 showed a 2- to 3-fold increase in total spleen lymphocytes. Mice bearing TEPC-183 or SPQC-11, the plasmacytomas causing an impaired antibody response, has significant increase in spleen lymphocytes under the same conditions. Mice bearing MOPC-315 had a very high initial count of spleen lymphocytes, which did not further increase upon immune stimulation.
Incubation of lymphocytes from plasmacytoma-bearing mice with PHA did not produce an increase in TdR incorporation and in some cases even caused a decrease in TdR incorporation.
Lymphocytes from mice bearing TEPC-183, SPQC-11, and MOPC-315 incorporated less TdR in response to LPS than did normal mice. On the other hand, mice bearing MPC-11 incorporated about as much TdR as did normal mice following LPS stimulation. Thus, the defect in the ability to respond to LPS in vitro correlated with the lack of an increase of spleen lymphocytes in mice bearing these tumours following antigenic stimulation in vivo.
No immunodepressive properties of serum from mice with plasmacytoma could be detected.
PMCID: PMC2009570  PMID: 646925
24.  Cleaved variant of plasmacytoma with myelomonocytic differentiation--immunohistochemical and ultrastructural studies. 
Journal of Korean Medical Science  1997;12(5):443-446.
Although plasma cells are terminally differentiated B cells, neoplastic plasma cells frequently express not only pre-B cell antigen, but also megakaryocytic, myelomonocytic, or erythroid markers. Since morphologic diagnosis of plasmacytoma is based on the recognition of neoplastic cells closely resembling normal plasma cells, unusual morphologic variants of neoplastic cells associated with these aberrant immunohistochemical features frequently cause diagnostic difficulty. The authors report a case of plasmacytoma with cleaved nuclei and myelomonocytic features occurring in the clavicle. The tumor was composed of immature plasma cells showing irregular, cleaved, and multilobated nuclei and abundant cytoplasm with prominent eosinophilic granules. A few tumor cells showing recognizable plasmacytic differentiation were admixed within the tumor. Immunohistochemically, the tumor cells expressed CD45RB, CD68, lysozyme, myeloperoxidase and kappa light chain with focal positivity for lambda chain. Ultrastructurally, the tumor cells contained numerous membrane bound electron dense lysosomal granules, some of them resembling Auer rods, as well as rough endoplasmic reticula arranged in lamellated stacks. Small biopsied nasal mucosal tissue in same patient revealed well differentiated plasmacytoma composed of tumor cells showing round, eccentric nuclei devoid of marked nuclear cleavage and cytoplasmic granularity. Immunohistochemically, these cells were kappa(+), lambda(-), myeloperoxidase(-), lysozyme(-) and CD68(-).
PMCID: PMC3054300  PMID: 9364303
25.  Structure of immunoglobulin gamma 2b heavy chain gene cloned from mouse embryo gene library. 
Nucleic Acids Research  1980;8(3):453-466.
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.
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PMCID: PMC327283  PMID: 6255443

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