Defects In B-cell Development Research Articles

Primary antibody deficiencies.

Primary antibody deficiencies are characterized by the inability to effectively produce antibodies and may involve defects in B-cell development or maturation. Primary antibody deficiencies can occur at any age, depending on the disease pathology. Certain primary antibody deficiencies affect males and females equally, whereas others affect males more often. Patients typically present with recurrent sinopulmonary and gastrointestinal infections, and some patients can experience an increased risk of opportunistic infections. Multidisciplinary collaboration is important in the management of patients with primary antibody deficiencies because these patients require heightened monitoring for atopic, autoimmune, and malignant comorbidities and complications. The underlying genetic defects associated with many primary antibody deficiencies have been discovered, but, in some diseases, the underlying genetic defect and inheritance are still unknown. The diagnosis of primary antibody deficiencies is often made through the evaluation of immunoglobulin levels, lymphocyte levels, and antibody responses. A definitive diagnosis is obtained through genetic testing, which offers specific management options and may inform future family planning. Treatment varies but generally includes antibiotic prophylaxis, vaccination, and immunoglobulin replacement. Hematopoietic stem cell transplantation is also an option for certain primary antibody deficiencies.

LncRNA00492 is required for marginal zone B-cell development.

B-cell development undergoes a series of steps from the bone marrow to the secondary lymphoid organs. A defect in B-cell development can lead to immunodeficiency or malignant disorders, such as leukaemia or lymphoma. Long non-coding RNAs have been reported to act as important regulators of many pathological processes. However, very little is known regarding the role of lncRNAs during B-cell development and the regulation of their expression. In this study, we explored the expression and role of lncRNA Gme00492 in B-cell development. We observed that lnc00492 was highly expressed in B-cell development and primarily expressed in the nucleus. Lnc00492-deficient mice had fewer marginal zone B cells in the spleen, likely due to a developmental block. Importantly, lnc00492 interacts with CTBP1 and targets it for ubiquitination and degradation during B-cell development, whereas the transcriptional corepressor factor CTBP1 plays a critical role in Notch2signalling. Thus, we identified a novel regulatory axis between lnc00492 and CTBP1 in B cells, suggesting that lnc00492 is essential for marginal zone B-cell development.

Par-4 overexpression impedes leukemogenesis in the Eµ-TCL1 leukemia model through downregulation of NF-κB signaling

AbstractProstate apoptosis response 4 (Par-4) is a tumor suppressor that prevents proliferation and induces cell death in several solid tumors. However, its role in B-cell malignancies has not been elucidated. To describe the role of Par-4 in chronic lymphocytic leukemia (CLL) pathogenesis, we developed a B-cell–specific human Par-4–overexpressing mouse model of CLL using the TCL1 leukemia model. While Par-4 transgenic mice did not display any obvious defects in B-cell development or function, disease burden as evidenced by abundance of CD19+CD5+ B cells in the peripheral blood was significantly reduced in Par-4 × TCL1 mice compared with TCL1 littermates. This conferred a survival advantage on the Par-4–overexpressing mice. In addition, a B-cell–specific knockout model displayed the opposite effect, where lack of Par-4 expression resulted in accelerated disease progression and abbreviated survival in the TCL1 model. Histological and flow cytometry–based analysis of spleen and bone marrow upon euthanasia revealed comparable levels of malignant B-cell infiltration in Par-4 × TCL1 and TCL1 individuals, indicating delayed but pathologically normal disease progression in Par-4 × TCL1 mice. In vivo analysis of splenic B-cell proliferation by 5-ethynyl-2-deoxyuridine incorporation indicated >50% decreased expansion of CD19+CD5+ cells in Par-4 × TCL1 mice compared with TCL1 littermates. Moreover, reduced nuclear p65 levels were observed in Par-4 × TCL1 splenic B cells compared with TCL1, suggesting suppressed NF-κB signaling. These findings have identified an in vivo antileukemic role for Par-4 through an NF-κB–dependent mechanism in TCL1-mediated CLL-like disease progression.

HYPERACTIVATION OF PI3 KINASE CAUSES DEFECTS IN HUMAN B-CELL DEVELOPMENT AND DIFFERENTIATION

Abstract Gain of function (GOF) mutations in PIK3CD, encoding the p110δ subunit of PI3-kinase, cause immune dysregulation characterized by recurrent respiratory infections, increased susceptibility to herpesvirus infection, lymphoma, organomegaly and autoimmunity. Affected individuals display poor Ab responses to protein and polysaccharide Ags suggesting defective humoral immune responses. To better understand humoral immune defects due to hyperactive PI3K, we analysed B cells in these individuals. In peripheral blood, transitional B cells were markedly increased while memory B cells were reduced. The phenotype of naïve B cells with PIK3CD GOF mutations resembled that of normal transitional B cells, indicating an aberrant arrest of developing B cells. Analysis of bone marrow revealed increased proportions of pro/pre-B cells and reduced recirculating mature B cells patients with PIK3CD GOF mutations. Within the memory B cell population there was a reduction in IgG+ cells. Consistent with this, there was a selective impairment in IgG production by transitional of naive B cells following in vitro stimulation. To extend these studies in vivo, we generated a mouse model using CRISPR/Cas9 to introduce the common disease-causing mutation (E1021K) into Pik3cd. Analysis of bone marrow from these mice revealed disrupted B-cell development. Investigation of Ag-specific T-dependent B cell responses using mice expressing a transgenic HEL-specific BCR combined with Pik3cd GOF allowed us to dissect defects in specific Ab generation that are seen in human PIK3CD GOF mutations. These results indicate that intrinsic defects in B-cell development and differentiation underlie poor humoral immune responses in individuals with PIK3CD GOF mutations.

A novel BTK gene mutation, c.82delC (p.Arg28 Alafs*5), in a Korean family with X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) is a hereditary humoral immunodeficiency that results from Bruton’s tyrosine kinase (BTK) gene mutations. These mutations cause defects in B-cell development, resulting in the virtual absence of these lymphocytes from the peripheral circulation. Consequently, this absence leads to a profound deficiency of lg all isotypes, and an increased susceptibility to encapsulated bacterial infections. A 15-month-old Korean boy presented with recurrent sinusitis and otitis media after 6 months of age, and had a family history of 2 maternal uncles with XLA. Laboratory tests revealed a profound deficiency of Ig isotypes, and a decreased count of CD19+ B cells in the peripheral circulation. Based on his family history and our laboratory test results, he was diagnosed with XLA. We performed BTK gene analysis of peripheral blood samples obtained from family members to confirm the diagnosis. Mutational analysis revealed a novel hemizygous frameshift mutation (c.82delC, p.Arg28Alafs*5), in the BTK gene. His mother and maternal grandmother were heterozygous carriers of this mutation and his two maternal uncles were hemizygous at the same position. After XLA diagnosis, intravenous immunoglobulin (400 mg/kg, monthly) treatment was initiated; recurrent sinusitis and otitis media were subsequently brought under control. To our knowledge, this is the first reported case of a Korean pedigree with a novel mutation in the BTK gene.

Atherosclerosis Susceptibility in Mice Is Independent of the V1 Immunoglobulin Heavy Chain Gene.

The V1 (VHS107.1.42) immunoglobulin heavy chain gene is thought to be critical in producing IgM natural antibodies of the T15-idiotype that protect against both atherosclerosis and infection from Streptococcus pneumoniae. Our aim was to determine whether genetic loss of the V1 gene increased atherosclerotic plaque burden in vivo because of a reduction in the T15-idiotype or other atheroprotective antibodies. We crossed VHS107.1.42-deficient mice with the atherosclerosis-prone Apoe(-/-) and Ldlr(-/-) strains. Although these double knockout strains manifested no defects in B-cell development, we did observe a substantial reduction in early immune responses against phosphocholine after immunization. However, the titers of plasma antibodies reacting against defined atherosclerotic antigens such as oxidized low-density lipoprotein, as well as the T15-idiotype, were unaffected by loss of the VHS107.1.42 gene in hypercholesterolemic mice. Furthermore, we observed no increase in atherosclerotic lesion formation, either within the aortic arch or aortic root. Robust deposition of IgM within atherosclerotic plaques could also be readily observed in both control and experimental mice. Our data indicate that IgM-dependent protection against atherosclerosis is unlikely to be dependent on antibodies that use the VHS107.1.42 gene, in contrast to the acute immune response conferred by this heavy chain in the response to phosphocholine and in providing resistance against lethal S pneumoniae infection.

NF-κB-dependent signals control BOB.1/OBF.1 and Oct2 transcriptional activity in B cells.

The transcriptional co-activator BOB.1/OBF.1 is crucial for Octamer-driven transcription in B cells. BOB.1/OBF.1-deficiency leads to tremendous defects in B-cell development and function. Therefore, in the past research focused on the identification of BOB.1/OBF.1 target genes. However, the regulation of BOB.1/OBF.1 expression itself is poorly understood. Here we show that in B cells NF-κB as well as to some extent NFAT proteins are involved in the activation of basal as well as inducible BOB.1/OBF.1 expression by direct binding to the BOB.1/OBF.1 promoter. Moreover, the analysis of different inducers of NF-κB, like several TLR ligands, TNF-α, BAFF, or LTα1β2, revealed that both the canonical and noncanonical NF-κB pathways are involved in the induction of BOB.1/OBF.1 gene. The identification of so far unknown inducers that regulate BOB.1/OBF.1 expression in B cells provides novel insights in the potential function of BOB.1/OBF.1 during different aspects of B-cell development and function.

Hhex is Required at Multiple Stages of Adult Hematopoietic Stem and Progenitor Cell Differentiation.

Hhex encodes a homeodomain transcription factor that is widely expressed in hematopoietic stem and progenitor cell populations. Its enforced expression induces T-cell leukemia and we have implicated it as an important oncogene in early T-cell precursor leukemias where it is immediately downstream of an LMO2-associated protein complex. Conventional Hhex knockouts cause embryonic lethality precluding analysis of adult hematopoiesis. Thus, we induced highly efficient conditional knockout (cKO) using vav-Cre transgenic mice. Hhex cKO mice were viable and born at normal litter sizes. At steady state, we observed a defect in B-cell development that we localized to the earliest B-cell precursor, the pro-B-cell stage. Most remarkably, bone marrow transplantation using Hhex cKO donor cells revealed a more profound defect in all hematopoietic lineages. In contrast, sublethal irradiation resulted in normal myeloid cell repopulation of the bone marrow but markedly impaired repopulation of T- and B-cell compartments. We noted that Hhex cKO stem and progenitor cell populations were skewed in their distribution and showed enhanced proliferation compared to WT cells. Our results implicate Hhex in the maintenance of LT-HSCs and in lineage allocation from multipotent progenitors especially in stress hematopoiesis.

B-cell development and functions and therapeutic options in adenosine deaminase–deficient patients

Adenosine deaminase (ADA) deficiency causes severe cellular and humoral immune defects and dysregulation because of metabolic toxicity. Alterations in B-cell development and function have been poorly studied. Enzyme replacement therapy (ERT) and hematopoietic stem cell (HSC) gene therapy (GT) are therapeutic options for patients lacking a suitable bone marrow (BM) transplant donor. We sought to study alterations in B-cell development in ADA-deficient patients and investigate the ability of ERT and HSC-GT to restore normal B-cell differentiation and function. Flow cytometry was used to characterize B-cell development in BM and the periphery. The percentage of gene-corrected B cells was measured by using quantitative PCR. B cells were assessed for their capacity to proliferate and release IgM after stimulation. Despite the severe peripheral B-cell lymphopenia, patients with ADA-deficient severe combined immunodeficiency showed a partial block in central BM development. Treatment with ERT or HSC-GT reverted most BM alterations, but ERT led to immature B-cell expansion. In the periphery transitional B cells accumulated under ERT, and the defect in maturation persisted long-term. HSC-GT led to a progressive improvement in B-cell numbers and development, along with increased levels of gene correction. The strongest selective advantage for ADA-transduced cells occurred at the transition from immature to naive cells. B-cell proliferative responses and differentiation to immunoglobulin secreting IgM after B-cell receptor and Toll-like receptor triggering were severely impaired after ERT and improved significantly after HSC-GT. ADA-deficient patients show specific defects in B-cell development and functions that are differently corrected after ERT and HSC-GT.

Kynureninase is the conserved self-antigen mimicked by the 2F5 Neutralizing Epitope of HIV-1 gp41 (105.35)

Abstract Accumulating evidence suggests that the generation of neutralizing HIV-1 antibody may be proscribed by immunological tolerance. A significant majority of monoclonal human antibodies, including 2F5, that neutralize multiple clades of HIV-1 are polyreactive and bind avidly to unknown components of mammalian cells. Knock-in mice expressing the VH and VL regions of 2F5 are characterized by a central defect in B-cell development that indicates the presence of a tolerizing autoantigen(s) that is mimicked by the membrane-proximal, 2F5 epitope of HIV-1 gp41. If immunological tolerance does indeed prevent regular expression of 2F5-like neutralizing HIV-1 antibody, then discrete autoantigens that structurally mimic the HIV-1 2F5 epitope must be present in those mammalian species unable to make 2F5-like antibody. We now demonstrate that the 2F5 antibody and its unmutated ancestor avidly bind human kynureninase (KYNU), a conserved enzyme of tryptophan metabolism. The H4 domain of most mammalian KYNU contains the complete 2F5 epitope (ELDKWA) of gp41; a H4 mutation of human KYNU to that (ELEKWA) of laboratory opossums resulted in a fully active enzyme completely unreactive with the 2F5 antibody. Immunization of opossums with HIV-1 gp140 induced extraordinary titers of ELDKWA antibody but little or no response to adjacent gp41 epitopes. Identification of an epitope shared by most mammals and HIV-1 provides direct evidence that immunological tolerance can impair humoral responses to HIV-1.

HAX1 deficiency: Impact on lymphopoiesis and B‐cell development

HAX1 was originally described as HS1-associated protein with a suggested function in receptor-mediated apoptotic and proliferative responses of lymphoid cells. Recent publications refer to a complex and multifunctional role of this protein. To investigate the in vivo function of HAX1 (HS1-associated protein X1) in B cells, we generated a Hax1-deficient mouse strain. Targeted deletion of Hax1 resulted in premature death around the age of 12 wk accompanied by a severe reduction of lymphocytes in spleen, thymus and bone marrow. In the bone marrow, all B-cell populations were lost comparably. In the spleen, B220(+) cells were reduced by almost 70%. However, as investigated by adoptive transfer experiments, this impairment is not exclusively B-cell intrinsic and we hypothesize that a HAX1-deficient environment cannot sufficiently provide the essential factors for proper lymphocyte development, trafficking and survival. Hax1(-/-) B cells show a significantly reduced expression of CXCR4, which might have an influence on the observed defects in B-cell development.

Clinical consequences of defects in B-cell development

Clinical consequences of defects in B-cell development

Lyl1 and Scl Regulate Bcl2 Expression and Control Apoptosis at Different Stages of Hematopoietic Development.

Abstract 569The Lymphoblastic Leukemia gene 1 (Lyl1) encodes a basic-helix-loop-helix (bHLH) transcription factor that is involved in T cell acute lymphoblastic leukemia via chromosomal translocations. Lyl1 has an almost identical bHLH motif to Scl, a known master regulator of hematopoiesis. In our previous work, we used a double knock-out mouse model to demonstrate functional redundancy between Lyl1 and Scl in adult hematopoietic stem cells (HSCs), where in the absence of both genes in adult mice, HSCs and progenitors undergo rapid apoptosis. Despite their common and overlapping functions, the two genes appear to also function at different aspects of hematopoietic development. Lyl1 knock-out mice exhibit a unique defect in B-cell development. This phenotype is recapitulated in vitro, where Lyl1 progenitors grown on a layer of OP9 cells and allowed to differentiate with the addition of cytokines fail to differentiate to B cells as well as wild type progenitors. Rescue assays, where Lyl1 was transduced using a retrovirus into Lyl1 knock-out progenitors and transplanted into lethally irradiated recipients, showed complete rescue of the B cell lineage. Forced expression of Scl into Lyl1 knock-out progenitors failed to rescue, giving rise to only about 5% of peripheral blood B cells. This demonstrates the distinct functions of the two genes in differentiated cells types, despite their functional redundancy in the HSCs. We further sought to dissect the functions and differences between Lyl1 and Scl based on their target genes. Using bioinformatics analysis and a literature search, we identified a number of genes that were candidates for Lyl1 transcriptional regulation; these interactions with Lyl1 and Scl were confirmed by chromatin immunoprecipitation. We found that Bcl2, and genes belonging to this family, are direct target genes of both Lyl1 and Scl in progenitor cells, and that they are downregulated in Lyl1 and Scl single knock-out mouse models. Since Bcl2 is critical for B cell survival, we hypothesized that the B cell deficiency in Lyl1 knock-out mice could be partly due to a loss of Bcl2. We performed rescue assays where Bcl2 was transduced using a retrovirus into Lyl1 knock-out progenitors which were transplanted into lethally irradiated recipient mice. Bcl2 overexpression rescued the B-cell defect in Lyl1 knock-out bone marrow. Since Bcl2 was identified as a target gene for Scl as well, we tested whether Bcl2 could also rescue the rapid apoptosis phenotype of the Lyl1/Scl double knock-out progenitors. Forced expression of Bcl2 in these cells, followed by deletion of the floxed Scl allele, resulted in a delayed onset of apoptosis, but incomplete rescue of the progenitor cell death. These findings distinguish the activities of Lyl1 and Scl in B cells and demonstrate a possible mechanism for their functions, through in part by their activation of the antiapoptotic gene Bcl2. Disclosures:No relevant conflicts of interest to declare.

The histone acetyl transferase activity of monocytic leukemia zinc finger is critical for the proliferation of hematopoietic precursors

The monocytic leukemia zinc finger (MOZ) gene encodes a large multidomain protein that contains, besides other domains, 2 coactivation domains for the transcription factor Runx1/acute myeloid leukemia 1 and a histone acetyl transferase (HAT) catalytic domain. Recent studies have demonstrated the critical requirement for the complete MOZ protein in hematopoietic stem cell development and maintenance. However, the specific function of the HAT activity of MOZ remains unknown, as it has been shown that MOZ HAT activity is not required either for its role as Runx1 coactivator or for the leukemic transformation induced by MOZ transcriptional intermediary factor 2 (TIF2). To assess the specific requirement for this HAT activity during hematopoietic development, we have generated embryonic stem cells and mouse lines carrying a point mutation that renders the protein catalytically inactive. We report in this study that mice exclusively lacking the HAT activity of MOZ exhibit significant defects in the number of hematopoietic stem cells and hematopoietic committed precursors as well as a defect in B-cell development. Furthermore, we demonstrate that the failure to maintain a normal number of hematopoietic precursors is caused by the inability of HAT(-/-) cells to expand. These results indicate a specific role of MOZ-driven acetylation in controlling a desirable balance between proliferation and differentiation during hematopoiesis.

The Tec Family Tyrosine Kinase Btk Regulates RANKL-induced Osteoclast Maturation

A spontaneous mutation in Bruton's tyrosine kinase (Btk) induces a defect in B-cell development that results in the immunodeficiency diseases X-linked agammaglobulinemia in humans and X-linked immunodeficiency (Xid) in mice. Here we show an unexpected role of Btk in osteoclast formation. When bone marrow cells derived from Xid mice were stimulated with receptor activator of NF-kappaB ligand, an osteoclast differentiation factor, they did not completely differentiate into mature multinucleated osteoclasts. Moreover, we found that the defects appeared to occur at the stage in which mononuclear preosteoclasts fuse to generate multinucleated cells. Supporting this notion, macrophages from Xid mice also failed to form multinucleated foreign body giant cells. The fusion defect of the Xid mutant osteoclasts was caused by decreased expression of nuclear factor of activated T cells c1 (NFATc1), a master regulator of osteoclast differentiation, as well as reduced expression of various osteoclast fusion-related molecules, such as the d2 isoform of vacuolar H(+)-ATPase V0 domain and the dendritic cell-specific transmembrane protein. This deficiency was completely rescued by the introduction of a constitutively active form of NFATc1 into bone marrow-derived macrophages. Our data provide strong evidence that Btk plays a critical role in osteoclast multinucleation by modulating the activity of NFATc1.

Src-family kinases in B-cell development and signaling.

The Src-family protein tyrosine kinases (SFKs) are known to play key roles in initiating signal transduction by the B-cell antigen receptor (BCR). In addition, numerous studies have shown that this family of molecules also contributes to signaling by BCR surrogates during B-lymphocyte lineage development and maturation. Paradoxically, ablation of SFKs not only results in obvious defects in B-cell development but also in the onset of autoimmunity. Thus SFKs, most notably Lyn, play both activating and inhibitory roles in B-cell function. Confounding analyses of SFK function in B cells is the varied coexpression of family members that mediate redundant as well as unique functions. In this review, we will focus mainly on the role of Lyn in mediating positive and negative roles in B-cell activation and how these affect immune signaling and disease progression.

Id2 Is Dispensable for Myc-Induced Lymphomagenesis

The Emu-Myc transgenic mouse appears to be an accurate model of human Burkitt's lymphoma that bears MYC/Immunoglobulin gene translocations. Id2, a negative regulator of basic helix-loop-helix transcription factors, has also been proposed as a Myc target gene that drives the proliferative response of Myc by binding to and overriding the checkpoint functions of the retinoblastoma tumor suppressor protein. Targeted deletion of Id2 in mice results in defects in B-cell development and prevents the development of peripheral lymphoid nodes. In precancerous B cells and lymphomas that arise in Emu-Myc transgenic mice and in Burkitt's lymphomas, Id2 is overexpressed, suggesting that it plays a regulatory role in lymphoma development. Surprisingly, despite these connections, Emu-Myc mice lacking Id2 succumb to lethal B-cell lymphoma at rates comparable with wild-type Emu-Myc transgenics. Furthermore, precancerous splenic B cells lacking Id2 do not exhibit any significant defects in Myc-induced target gene transactivation and proliferation. However, due to their lack of secondary lymph nodes, Emu-Myc mice lacking Id2 rather succumb to disseminated lymphoma with an associated leukemia, with pronounced infiltrates of the bone marrow and other major organs. Collectively these findings argue that targeting Id2 functions may be ineffective in preventing Myc-associated malignancies.

Immunodeficiency disorders.

Hematological complications occur frequently in patients with both primary and secondary immunodeficiency disorders. Anemia, thrombocytopenia or leukopenias may bring these individuals to the attention of hematologists. Conversely, evidence suggesting a lymphoproliferative disorder may be the cause for referral. This session will provide an update on the diagnosis and treatment of immunodeficiency diseases ranging from isolated defects in antibody production to the severe combined immunodeficiencies (SCID). Immunodeficiency diseases have traditionally been defined as defects in the development and function of T and B cells, the primary effector cells of specific cellular and humoral immunity. However, it has become increasingly evident that innate immune mechanisms contribute greatly to host defense, either through acting alone or by enhancing specific T and B cell responses. In Section I, Dr. Lewis Lanier reviews the burgeoning information on the extensive families of activating and inhibitory immunoreceptors that are expressed on NK cells, dendritic cells, T and B cells, and phagocytic cells. He provides an overview on the biological functions of these receptors in host defense. In Section II, Dr. Mary Ellen Conley defines the spectrum of antibody deficiency disorders, the most frequently occurring types of primary immunodeficiencies. She covers the different defects in B-cell development and function that lead to antibody deficiencies, and includes diagnosis and therapy of these disorders. In Section III, Dr. Jennifer Puck discusses the diagnosis and treatment of the different types of SCID. She describes the genetic basis for SCID, and the benefits, pitfalls, and complications of gene therapy and bone marrow transplantation in SCID patients.

Variations in the human phospholipase Cgamma2 gene in patients with B-cell defects of unknown etiology.

Our recent studies using targeted gene disruption have shown that defects in phospholipase Cgamma2 (PLCgamma2) result in a B-cell abnormality that is very similar to that seen in Btk-deficient mice. Null mutations in either PLCG2 or BTK are associated with decreased numbers of mature B cells, failure to make antibodies to some T cell-independent antigens and the absence of CD5+ peritoneal B cells. Mutations in BTK in humans cause a more severe defect in B-cell development characterized by almost complete absence of B cells in the peripheral circulation, profound hypogammaglobulinemia and an inability to produce antibodies to any antigens. However, not all patients with severe defects in B-cell development have mutations in BTK or the components of the B-cell signal transduction complex. To explore the possibility that some patients with defects in B-cell development of unknown etiology might have mutations in PLCG2, we determined the genomic structure of this gene and established conditions to analyze the 32 exons of the gene and the flanking sequences by single-strand conformation polymorphism. Although 24 polymorphic variants of this gene were found in 35 patients, we did not identify any alterations that were likely to be the cause of disease.

Regulation of BOB.1/OBF.1 stability by SIAH.

The BOB.1/OBF.1 coactivator is critically involved in mediating octamer-dependent transcriptional activity in B lymphocytes. Mice lacking this coactivator show various defects in B-cell development, most notably they completely lack germinal centers. Consistent with this phenotype, BOB.1/OBF.1 levels are massively upregulated in germinal center B cells as compared with resting B cells. We have addressed the mechanism of upregulation and found that only a minor part of this regulation can be attributed to increased levels of BOB.1/OBF.1-specific mRNA. Apparently, BOB.1/OBF.1 is also regulated at the protein level. In support of this suggestion we have been able to identify two related BOB.1/OBF.1 interacting proteins, SIAH1 and SIAH2, in a yeast two-hybrid screen. SIAH1 and SIAH2 are known regulators of protein stability. Cotransfection experiments revealed that coexpression of SIAH results in a destabilization of BOB.1/OBF.1 protein without affecting mRNA levels. Further more, proteasome inhibitors block the degradation of BOB.1/OBF.1 protein. Finally, B-cell receptor cross-linking also resulted in the degradation of BOB.1/OBF.1 and consequently reduced transcriptional activation of BOB.1/OBF.1-dependent reporters.