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 parameters specifying whether autoreactive CD4(+) thymocytes are deleted (recessive tolerance) or differentiate into regulatory T cells (dominant tolerance) remain unresolved. Dendritic cells directly delete thymocytes, partly through cross-presentation of peripheral antigens 'promiscuously' expressed in medullary thymic epithelial cells (mTECs) positive for the autoimmune regulator Aire. It is unclear if and how mTECs themselves act as antigen-presenting cells during tolerance induction. Here we found that an absence of major histocompatibility class II molecules on mTECs resulted in fewer polyclonal regulatory T cells. Furthermore, targeting of a model antigen to Aire(+) mTECs led to the generation of specific regulatory T cells independently of antigen transfer to dendritic cells. Thus, 'routing' of mTEC-derived self antigens may determine whether specific thymocytes are deleted or enter the regulatory T cell lineage.

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.All types of blood cell are generated from a pluripotent haematopoietic stem cell (HSC) through developmentally restricted progenitors which undergo lineage commitment and subsequent differentiation along a single pathway. The lymphoid lineages develop through a common lymphoid progenitor (CLP) which gives rise to natural killer, B and T cells 1 . Early B-cell development can be dissected into different stages according to the rearrangement status of the immunoglobulin heavy-chain (IgH) locus, the expression of stage-specific cell-surface markers and growth factor requirements 2,3 . The earliest B-lineage precursor cells carry the IgH locus still in germline configuration. D H -J H recombination is subsequently initiated in pre-BI cells 3 , which are also known as early pro-B cells (fraction B) 2 . These pro-B cells can be cultured in vitro on stromal cells in the presence of interleukin-7 (IL-7) and express the B-cell surface proteins 5, VpreB, Ig␣ and Ig␤ (refs 3, 4). Completion of a functional V H -DJ H rearrangement results in the expression of the pre-B-cell receptor and subsequent differentiation to small pre-B cells, which are no longer responsive to pro-B-cell growth conditions 5 .The initiation of B-cell development critically depends on two transcription factors; the basic helix-loop-helix proteins encoded by the E2A gene and the early B-cell factor (EBF). In the absence of either protein, B-cell development is aborted at the earliest stage, before D H -J H rearrangement of the IgH gene 6-8 . Moreover, forced expression of E2A and EBF in haematopoietic precursor cells revealed that these regulators cooperatively induce the transcription of several B-lymphoid-specific genes 9,10 . Hence, loss-and gain-offunction experiments have implicated E2A and EBF in the control of B-lineage commitment.A third transcriptional regulator involved in early B-lymphopoiesis is the B-cell-specific activator protein (BSAP), which ...

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. 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.

SummaryWe describe mice that express a transgenic T cell receptor o~/B (TCR-o~/B) specific for peptide 111-119 from influenza hemagglutinin presented by I-E a class II major histocompatibility complex (MHC) molecules. The transgenic TCR is expressed on CD4 +8-as well as CD4-8 + mature T cells even in mice that are deficient in rearrangement or do not express endogenous TCR.-ol genes. The CD4-8 + T cells require I-E a class II MHC molecules for positive selection and can be activated to proliferate and to kill by I-E a molecules presenting the relevant peptide. Full maturation of these cells, however, also requires the presence of class I MHC molecules. The results are compatible with the notion that T cell maturation requires multiple receptor-ligand interactions and establish an exception to the rule that class II-restricted TCRs are exclusively expressed by mature CD4+8-cells.

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.

The B-lymphocyte-specific transcriptional factor called Oct binding factor (OBF)-1, OCA-B or Bob1 (refs 1-3) is thought to be involved in the transcription of immunoglobulin genes through recruitment to the highly conserved octamer site of immunoglobulin promoters, mediated by either Oct-1 or Oct-2. To define the in vivo role of OBF-1 we have used gene targeting in embryonic stem cells to generate mice lacking the coactivator OBF-1. Such OBF-1-/- mice are born normally, are fertile and seem healthy, and surprisingly, rearrangement and transcription of immunoglobulin genes are largely unaffected. However, mice deficient in OBF-1 have reduced numbers of mature B cells and a severe reduction in the number of recirculating B cells, but otherwise show normal B-cell differentiation. Serum IgA and particularly IgG levels are greatly reduced. If mutant mice are immunized with either a thymus-independent or a thymus-dependent antigen, their immune responses are dramatically weakened. Strikingly, germinal centres completely fail to develop after immunization with thymus-dependent antigen. Our results demonstrate that in vivo OBF-1 is not required for initial transcription of immunoglobulin genes or for B cell development, but instead is essential for the response of B cells to antigens, and is required for the formation of germinal centres.

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 (lambda5, VpreB, Igalpha, Igbeta) 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. 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 alpha/beta-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, lambda5, VpreB, Igalpha and Igbeta. Instead, our data implicate Pax5 in the control of B-lineage commitment.