Defects In B-cell Development Research Articles

The B lymphocyte-specific coactivator BOB.1/OBF.1 is required at multiple stages of B-cell development.

The transcriptional coactivator BOB.1/OBF.1 confers B-cell specificity on the transcription factors Oct1 and Oct2 at octamer site-containing promoters. A hallmark of the BOB.1/OBF.1 mutation in the mouse is the absence of germinal center development in secondary lymphoid organs, demonstrating the requirement for BOB.1/OBF.1 in antigen-dependent stages of B-cell differentiation. Here we analyzed earlier stages of B lymphopoiesis in BOB.1/OBF.1-deficient mice. Examination of B-cell development in the bone marrow revealed that the numbers of transitional immature (B220(+) IgM(hi)) B cells were reduced and that B-cell apoptosis was increased. When in competition with wild-type cells, BOB.1/OBF.1(-/-) bone marrow cells exhibited defects in repopulating the bone marrow B-cell compartment and were unable to establish a presence in the periphery of host mice. The defective bone marrow populations in BOB.1/OBF.1(-/-) mice were rescued by conditional expression of a BOB.1/OBF.1 transgene controlled by the tetracycline gene expression system. However, the restored populations did not restore the numbers of IgD(hi) B cells in the periphery, where the BOB.1/OBF.1 transgene was not expressed. These results show that BOB.1/OBF.1(-/-) B cells exhibit multistage defects in B-cell development, including impaired production of transitional B cells and defective maturation of recirculating B cells.

Fates of human B-cell precursors

Development of mammalian B-lineage cells is characterized by progression through a series of checkpoints defined primarily by rearrangement and expression of immunoglobulin genes. Progression through these checkpoints is also influenced by stromal cells in the microenvironment of the primary tissues wherein B-cell development occurs, ie, fetal liver and bone marrow and adult bone marrow. This review focuses on the developmental biology of human bone marrow B-lineage cells, including perturbations that contribute to the origin and evolution of B-lineage acute lymphoblastic leukemia and primary immunodeficiency diseases characterized by agammaglobulinemia. Recently described in vitro and in vivo models that support development and expansion of human B-lineage cells through multiple checkpoints provide new tools for identifying the bone marrow stromal cell–derived molecules necessary for survival and proliferation. Mutations in genes encoding subunits of the pre-B cell receptor and molecules involved in pre-B cell receptor signaling culminate in X-linked and non–X-linked agammaglobulinemia. A cardinal feature of these immunodeficiencies is an apparent apoptotic sensitivity of B-lineage cells at the pro-B to pre-B transition. On the other end of the spectrum is the apoptotic resistance that accompanies the development of B-lineage acute lymphoblastic leukemia, potentially a reflection of genetic abnormalities that subvert normal apoptotic programs. The triad of laboratory models that mimic the bone marrow microenvironment, immunodeficiency diseases with specific defects in B-cell development, and B-lineage acute lymphoblastic leukemia can now be integrated to deepen our understanding of human B-cell development.

Transcriptional Regulatory Elements Within the First Intron of Bruton's Tyrosine Kinase

AbstractDefects in the gene for Bruton's tyrosine kinase (Btk) result in the disorder X-linked agammaglobulinemia (XLA). Whereas XLA is characterized by a profound defect in B-cell development, Btk is expressed in both the B lymphocyte and myeloid cell lineages. We evaluated a patient with XLA who had reduced amounts of Btk transcript but no abnormalities in his coding sequence. A single base-pair substitution in the first intron of Btk was identified in this patient, suggesting that this region may contain regulatory elements. Using reporter constructs we identified two transcriptional control elements in the first 500 bp of intron 1. A strong positive regulator, active in both pre-B cells and B cells, was identified within the first 43 bp of the intron. Gel-shift assays identified two Sp1 binding sites within this element. The patient's mutation results in an altered binding specificity of the proximal Sp1 binding site. A negative regulator, active in pre-B cells only, was located between base pairs 281 and 491 of the intron. These findings indicate that regulation of Btk transcription is complex and may involve several transcriptional regulatory factors at the different stages of B-cell differentiation.

Transcriptional Regulatory Elements Within the First Intron of Bruton's Tyrosine Kinase

Defects in the gene for Bruton's tyrosine kinase (Btk) result in the disorder X-linked agammaglobulinemia (XLA). Whereas XLA is characterized by a profound defect in B-cell development, Btk is expressed in both the B lymphocyte and myeloid cell lineages. We evaluated a patient with XLA who had reduced amounts of Btk transcript but no abnormalities in his coding sequence. A single base-pair substitution in the first intron of Btk was identified in this patient, suggesting that this region may contain regulatory elements. Using reporter constructs we identified two transcriptional control elements in the first 500 bp of intron 1. A strong positive regulator, active in both pre-B cells and B cells, was identified within the first 43 bp of the intron. Gel-shift assays identified two Sp1 binding sites within this element. The patient's mutation results in an altered binding specificity of the proximal Sp1 binding site. A negative regulator, active in pre-B cells only, was located between base pairs 281 and 491 of the intron. These findings indicate that regulation of Btk transcription is complex and may involve several transcriptional regulatory factors at the different stages of B-cell differentiation.

Mutations in the mu heavy-chain gene in patients with agammaglobulinemia.

Most patients with congenital hypogammaglobulinemia and absent B cells are males with X-linked agammaglobulinemia, which is caused by mutations in the gene for Bruton's tyrosine kinase (Btk); however, there are females with a similar disorder who do not have mutations in this gene. We studied two families with autosomal recessive defects in B-cell development and patients with presumed X-linked agammaglobulinemia who did not have mutations in Btk. A series of candidate genes that encode proteins involved in B-cell signal-transduction pathways were analyzed by linkage studies and mutation screening. Four different mutations were identified in the mu heavy-chain gene on chromosome 14. In one family, there was a homozygous 75-to-100-kb deletion that included D-region genes, J-region genes, and the mu constant-region gene. In a second family, there was a homozygous base-pair substitution in the alternative splice site of the mu heavy-chain gene. This mutation would inhibit production of the membrane form of the mu chain and produce an amino acid substitution in the secreted form. In addition, a patient previously thought to have X-linked agammaglobulinemia was found to have an amino acid substitution on one chromosome at an invariant cysteine that is required for the intrachain disulfide bond and, on the other chromosome, a large deletion that included the immunoglobulin locus. Defects in the mu heavy-chain gene are a cause of agammaglobulinemia in humans. This implies that an intact membrane-bound mu chain is essential for B-cell development.

Mice lacking c-fos have normal hematopoietic stem cells but exhibit altered B-cell differentiation due to an impaired bone marrow environment

Mice lacking c-fos develop severe osteopetrosis with deficiencies in bone remodeling and exhibit extramedullary hematopoiesis, thymic atrophy, and altered B-cell development. In this study, we have used these mice to characterize in detail the developmental potential of hematopoietic stem cells lacking c-fos and to analyze how the lymphoid differentiation is altered. In c-fos -/- mice, B-cell numbers are reduced in the spleen, lymph nodes, and the peripheral blood as a result of a marked reduction (> 90%) in the number of clonogenic B-cell precursors. In contrast, the number and lineage distribution of myeloid progenitor cells are not affected. The thymic defects observed in a large number of these mice correlate with their health status, suggesting that this may be an indirect effect of the c-fos mutation. In vitro differentiation and bone marrow reconstitution experiments demonstrated that hematopoietic stem cells lacking c-fos can give rise to all mature myeloid as well as lymphoid cells, suggesting that the observed B lymphopenia in the mutant mice is due to an altered environment. Transplantation of wild-type bone marrow cells into newborn mutant mice resulted in the establishment of a bone marrow space and subsequent correction of the B-cell defect. These results demonstrate that hematopoietic stem cells lacking Fos have full developmental potential and that the observed defect in B-cell development is most likely due to the impaired bone marrow environment as a consequence of osteopetrosis.

Mice lacking c-fos have normal hematopoietic stem cells but exhibit altered B-cell differentiation due to an impaired bone marrow environment.

Mice lacking c-fos develop severe osteopetrosis with deficiencies in bone remodeling and exhibit extramedullary hematopoiesis, thymic atrophy, and altered B-cell development. In this study, we have used these mice to characterize in detail the developmental potential of hematopoietic stem cells lacking c-fos and to analyze how the lymphoid differentiation is altered. In c-fos -/- mice, B-cell numbers are reduced in the spleen, lymph nodes, and the peripheral blood as a result of a marked reduction (> 90%) in the number of clonogenic B-cell precursors. In contrast, the number and lineage distribution of myeloid progenitor cells are not affected. The thymic defects observed in a large number of these mice correlate with their health status, suggesting that this may be an indirect effect of the c-fos mutation. In vitro differentiation and bone marrow reconstitution experiments demonstrated that hematopoietic stem cells lacking c-fos can give rise to all mature myeloid as well as lymphoid cells, suggesting that the observed B lymphopenia in the mutant mice is due to an altered environment. Transplantation of wild-type bone marrow cells into newborn mutant mice resulted in the establishment of a bone marrow space and subsequent correction of the B-cell defect. These results demonstrate that hematopoietic stem cells lacking Fos have full developmental potential and that the observed defect in B-cell development is most likely due to the impaired bone marrow environment as a consequence of osteopetrosis.

Carrier detection in X-linked agammaglobulinemia by analysis of X-chromosome inactivation.

We used a recently developed strategy to analyze patterns of X-chromosome inactivation in human cell populations in order to study female members of families with X-linked agammaglobulinemia--i.e., to detect the carrier state and to test the hypothesis that the disorder results from a defect intrinsic in the development of B cells. According to this strategy, recombinant-DNA probes simultaneously detect restriction-fragment-length polymorphisms and patterns of methylation of X-chromosome genes. Random X-inactivation patterns were observed in isolated peripheral-blood granulocytes, T lymphocytes, and B lymphocytes of women who were not carriers. In contrast, one of the two X chromosomes was preferentially active in the peripheral B cells, but not the T cells or granulocytes, of three carriers of the disorder. This observation strongly supports the hypothesis that X-linked agammaglobulinemia results from an intrinsic defect in B-cell development. Moreover, the analysis described here can be used for direct identification of carriers in families with this disease.