Antonius Rolink

Commitment to the B-lymphoid lineage depends on the transcription factor Pax5

The Pax5 gene encoding the B-cell-specific activator protein (BSAP) is expressed within the haematopoietic system exclusively in the B-lymphoid lineage, where it is required in vivo for progression beyond the pro-B-cell stage. However, Pax5 is not essential for in vitro propagation of pro-B cells in the presence of interleukin-7 and stromal cells. Here we show that pro-B cells lacking Pax5 are also incapable of in vitro B-cell differentiation unless Pax5 expression is restored by retroviral transduction. Pax5-/- pro-B cells are not restricted in their lineage fate, as stimulation with appropriate cytokines induces them to differentiate into functional macrophages, osteoclasts, dendritic cells, granulocytes and natural killer cells. As expected for a clonogenic haematopoietic progenitor with lymphomyeloid developmental potential, the Pax5-/- pro-B cell expresses genes of different lineage-affiliated programmes, and restoration of Pax5 activity represses this lineage-promiscuous transcription. Pax5 therefore plays an essential role in B-lineage commitment by suppressing alternative lineage choices.

The SCID but Not the RAG-2 Gene Product Is Required for Sμ–Sε Heavy Chain Class Switching

We have investigated the capacity of precursor B cells from normal (BDF1) and V(D)J recombinase-deficient (RAG-27) or defective (SCID) mice to be induced by a CD40-specific monoclonal antibody and IL-4 to epsilon H chain gene transcription and to S mu-S epsilon switch recombination. In differentiating precursor B cells from all three strains of mice, the development of similar numbers of CD19+, CD23+, CD40+, and MHC class II+ expressing B lineage cells and similar levels of epsilon H chain gene transcription were induced. Efficient S mu-S epsilon switching occurred in normal and RAG-2-deficient, but not in SCID, precursor B cells. Thus, the transcription of the epsilon H chain is independent of the RAG-2 and the SCID gene product, while the S mu-S epsilon switch recombination requires the SCID gene-encoded DNA-dependent protein kinase, but not the RAG-2 protein.

Long-term in vivo reconstitution of T-cell development by Pax5-deficient B-cell progenitors

The mechanisms controlling the commitment of haematopoietic progenitors to the B-lymphoid lineage are poorly understood. The observations that mice deficient in E2A and EBF lack B-lineage cells have implicated these two transcription factors in the commitment process. Moreover, the expression of genes encoding components of the rearrangement machinery (RAG1, RAG2, TdT) or pre-B-cell receptor (λ5, VpreB, Igα, Igβ) has been considered to indicate B-lineage commitment. All these genes including E2A and EBF are expressed in pro-B cells lacking the transcription factor Pax5 (refs 5,6,7). Here we show that cloned Pax5-deficient pro-B cells transferred into RAG2-deficient mice provide long-term reconstitution of the thymus and give rise to mature T cells expressing α/β-T-cell receptors. The bone marrow of these mice contains a population of cells of Pax5-/- origin with the same phenotype as the donor pro-B cells. When transferred into secondary recipients, these pro-B cells again home to the bone marrow and reconstitute the thymus. Hence, B-lineage commitment is determined neither by immunoglobulin DJ rearrangement nor by the expression of E2A, EBF, λ5, VpreB, Igα and Igβ. Instead, our data implicate Pax5 in the control of B-lineage commitment.

Continued RAG expression in late stages of B cell development and no apparent re-induction after immunization.

Models of B-cell development in the immune system suggest that only those immature B cells in the bone marrow that undergo receptor editing express V (D)J -recombination-activating genes (RAGs). Here we investigate the regulation of RAG expression in transgenic mice carrying a bacterial artificial chromosome that encodes a green fluorescent protein reporter instead of RAG2 (ref. 4). We find that the reporter is expressed in all immature B cells in the bone marrow and spleen. Endogenous RAG messenger RNA is expressed in immature B cells in bone marrow and spleen and decreases by two orders of magnitude as they acquire higher levels of surface immunoglobulin M (IgM). Once RAG expression is stopped it is not re-induced during immune responses. Our findings may help to reconcile a series of apparently contradictory observations, and suggest a new model for the mechanisms that regulate allelic exclusion, receptor editing and tolerance.

Down-regulation of RAG1 and RAG2 gene expression in preB cells after functional immunoglobulin heavy chain rearrangement.

Two waves of immunoglobulin gene rearrangements, first of the heavy, then of the light chain chain gene loci form functional immunoglobulin genes during B cell development. In mouse bone marrow the differential surface expression of B220 (CD45R), c-kit, CD25, and surrogate light chain as well as the cell cycle status allows FACS separation of the cells in which these two waves of rearrangements occur. The gene products of two recombination activating genes, RAG1 and RAG2 are crucial for this rearrangement process. Here, we show that the expression of the RAG genes is twice up- and down-regulated, at the transcriptional level for RAG1 and RAG2, and at the postranscriptional level for RAG2 protein. Expression levels are high in D-->JH and VH-->DJH rearranging proB and preB-I cells, low in preB cells expressing the preB cell receptor on the cell surface, and high again in VL-->JL rearranging small preB-II cells. In immature B cells expressing on the cell surface RAG1 and RAG2 mRNA is down-regulated, whereas RAG2 protein levels are maintained. Down-regulation of RAG1 and RAG2 gene expression after productive rearrangement at one heavy chain allele might be part of the mechanisms that prevent further rearrangements at the other allele.

Identification of BSAP (Pax-5) target genes in early B-cell development by loss- and gain-of-function experiments.

The Pax‐5 gene codes for the transcription factor BSAP which is essential for the progression of adult B lymphopoiesis beyond an early progenitor (pre‐BI) cell stage. Although several genes have been proposed to be regulated by BSAP, CD19 is to date the only target gene which has been genetically confirmed to depend on this transcription factor for its expression. We have now taken advantage of cultured pre‐BI cells of wild‐type and Pax‐5 mutant bone marrow to screen a large panel of B lymphoid genes for additional BSAP target genes. Four differentially expressed genes were shown to be under the direct control of BSAP, as their expression was rapidly regulated in Pax‐5‐deficient pre‐BI cells by a hormone‐inducible BSAP–estrogen receptor fusion protein. The genes coding for the B‐cell receptor component Ig‐α (mb‐1) and the transcription factors N‐myc and LEF‐1 are positively regulated by BSAP, while the gene coding for the cell surface protein PD‐1 is efficiently repressed. Distinct regulatory mechanisms of BSAP were revealed by reconstituting Pax‐5‐deficient pre‐BI cells with full‐length BSAP or a truncated form containing only the paired domain. IL‐7 signalling was able to efficiently induce the N‐myc gene only in the presence of full‐length BSAP, while complete restoration of CD19 synthesis was critically dependent on the BSAP protein concentration. In contrast, the expression of the mb‐1 and LEF‐1 genes was already reconstituted by the paired domain polypeptide lacking any transactivation function, suggesting that the DNA‐binding domain of BSAP is sufficient to recruit other transcription factors to the regulatory regions of these two genes. In conclusion, these loss‐ and gain‐of‐function experiments demonstrate that BSAP regulates four newly identified target genes as a transcriptional activator, repressor or docking protein depending on the specific regulatory sequence context.

Molecular and cellular origins of B lymphocyte diversity

The diverse repertoire of antibodies or immunoglobulins (Igs) in the immune system is created by B lymphocytes. The specificity of an lg for an antigen is made up by three complementarity-determining regions (CD%) in the variable (V) regions of the heavy(H) and light (L) chains. Each B lymphocyte displays a single combination of H and L chains with a unique set of CDRs, out of millions of possible combinations in the total repertoire of lg molecules. At the molecular level, the diversity of antigen-binding V regions of lg molecules is generated by successive rearrangements and potentially by replacement events at both alleles of the H and L loci, as well as by N and P regions in H but not L chains, which are introduced in the joining reaction. At every step in this generation of diversity, it appears that cells of the B lymphocyte lineage check whether they have rearranged nonproductively or productively by depositing the product of the productively rearranged gene in the surface membrane. The importance of this process for pre-B cells is indicated by the fact that products of certain genes expressed specifically in

The surrogate light chain in B-cell development

The proteins encoded by the VpreB and lambda 5 genes associate with each other to form a light (L) chain-like structure, the surrogate L chain. It can form Ig-like complexes with three partners-the classical heavy (H) chain, the DHJHC mu-protein, or the newly discovered p55 chain; these are expressed on the surface of pre-B cells at different stages of development. Here, Fritz Melchers and colleagues review the structures of the VpreB and lambda 5 genes in mouse and their relatives in humans, describe their pattern of expression, and speculate on their possible evolution and functions.

Characterization of immature B cells by a novel monoclonal antibody, by turnover and by mitogen reactivity

The transit of immature to mature sIgM+ B cells, the life span, maturation kinetics and response to polyclonal activators have been analyzed with the help of a new mAb (493), that distinguishes immature, 493+ from mature, 493− B cells in a variety of mouse strains tested. Analysis of the turnover of immature 493+ B cells by bromodeoxyuridine (BrdU) labeling kinetics indicate that only 10 – 20 % of the cells reach the spleen as immature 493+ cells. The life span of 493+ B cells in bone marrow and spleen is around 4 days. BrdU chase experiments show that most of the immature cells in spleen enter the pool of mature, 493− B cells where they gain a longer life span of 15 – 20 weeks. Immature and mature B cells respond equally well to LPS stimulation; anti‐CD40, however, stimulates mature B cells better than immature B cells. IgM cross‐linking of mature B cells results in proliferation, while it induces apoptosis in immature B cells. This apoptosis of immature cells can be inhibited by co‐stimulation with anti‐CD40 or by overexpression of bcl‐2. We speculate that Ig receptor ligand‐mediated apoptosis (negative selection) plays a major role in the transit of immature B cells from bone marrow to spleen, but only a minor role in the transit from immature B cells to mature B cells in the spleen.

Changes in the VH Gene Repertoire of Developing Precursor B Lymphocytes in Mouse Bone Marrow Mediated by the Pre-B Cell Receptor

The V(H) repertoire on both H chain alleles of normal and lambda5-deficient B lineage cells were analyzed by single-cell PCR. The mu H chains were tested for their capacity to form a pre-B cell receptor. In bone marrow, D-proximal V(H) genes were found preferentially expressed in lambda5-deficient pre-B cells and in a newly identified early c-kit+ cytoplasmic mu H chain+ pre-B cell population of normal mice. Only half of the mu H chains expressed in these cells have the capacity to form a pre-B cell receptor. Representation of the D-proximal V(H) genes was found suppressed on the productive but not on the nonproductive V(H)DJ(H) rearranged alleles of c-kit preB-II cells and splenic lambda5-deficient B cells. More than 95% of the mu H chains expressed in preB-II cells can form a pre-B cell receptor. These results demonstrate that the pre-B cell receptor in normal mice and the B cell receptor in lambda5-deficient mice mediate a shift in the V(H) repertoire.