Early lymphocyte development in bone marrow and thymus.

Haematopoietic stem cells (HSCs), a very rare cell type in the bone marrow, are responsible for the life-long production of all cells of the blood including T and B cells. Until recently, it was thought that the differentiation of HSCs into the various haematopoietic cells was rather hierarchical in that differentiation along a given lineage was associated with a progressive loss of potential to give rise to other blood cell lineages. The recent development of very sensitive and quantitative in vitro assays, together with the identification of new progenitor subpopulations, has challenged this idea. Thus, lymphocyte progenitors can be shown to keep their developmental potential to give rise to myeloid, dendritic and NK cells until just prior to their final commitment stage. Here we review these new findings and concepts.

Early lymphocyte development in bone marrow and thymus.

Haematopoietic stem cells (HSCs), a very rare cell type in the bone marrow, are responsible for the life-long production of all cells of the blood including T and B cells. Until recently, it was thought that the differentiation of HSCs into the various haematopoietic cells was rather hierarchical in that differentiation along a given lineage was associated with a progressive loss of potential to give rise to other blood cell lineages. The recent development of very sensitive and quantitative in vitro assays, together with the identification of new progenitor subpopulations, has challenged this idea. Thus, lymphocyte progenitors can be shown to keep their developmental potential to give rise to myeloid, dendritic and NK cells until just prior to their final commitment stage. Here we review these new findings and concepts.

Critical role for c-kit (CD117) in T cell lineage commitment and early thymocyte development in vitro.

The precise roles played by the transmembrane receptor tyrosine kinase c-kit and its ligand stem cell factor in early T cell development are difficult to study. Using cloned Pax5-deficient progenitor B cells, we show that following Notch signaling, which induces their commitment to the T cell developmental pathway, c-kit expression is rapidly up-regulated at both the transcriptional and cell surface level. Using either an anti-c-kit monoclonal antibody or Gleevec, a pharmacological inhibitor of c-kit signaling, we show that the Notch-induced T cell differentiation of either Pax5-deficient progenitor B cells, or the equivalent cell from the bone marrow of normal mice, is strictly dependent on c-kit signaling, whereas the differentiation of normal progenitors into the B cell lineage is not. Moreover, we show that the Notch and IL-7 signaling-induced proliferation and differentiation of CD44+CD25-c-kit(high) and CD44+CD25+c-kithigh thymocytes along the T cell, but not natural killer cell or macrophage, pathway also requires c-kit signaling, whereas the Notch-induced proliferation and differentiation of CD44-CD25+c-kitint cells along the T cell pathway is independent of c-kit. These results further highlight the complex inter-relationships existing between c-kit, Notch and IL-7 receptor signaling that control the proliferation and differentiation of early T cell progenitors.

A B220+ CD117+ CD19- hematopoietic progenitor with potent lymphoid and myeloid developmental potential.

In this report, we identify in the bone marrow (BM) of normal mice a subpopulation of B220+ CD117+ CD19- NK1.1- cells with potent lymphoid and myeloid developmental potential. These cells represent 0.1-0.2% of nucleated BM cells. By limiting dilution analysis in the presence of the appropriate combination of stromal cells and cytokines, 1 in 5-10 sorted cells formed B cells, 1 in 10-15 formed T cells and 1 in 5-10 generated macrophages. When cultured on a mixture of OP9 stroma and OP9 stromal cells expressing the Notch ligand Delta-like-1, single cells generated both T and B cells. Following intravenous infusion, freshly sorted cells transiently reconstituted both the T and B cell progenitor compartments, generating cohorts of mature T and B lymphocytes. The relationship between B220+ CD117+ CD19- NK1.1- cells of wild-type mice and other multi-lineage BM progenitors is discussed.

The OBF-1 gene locus confers B cell-specific transcription by restricting the ubiquitous activity of its promoter.

Transcription of the gene encoding the transcriptional coactivator Oct-binding factor 1 (OBF-1)/OCA-B/Bob.1 is largely restricted to B cells. During B cell development OBF-1 expression shows two peaks, one in immature B cells in the bone marrow and the other in germinal center B cells. Promoter analysis has identified a cAMP response element (CRE)-binding site present in the OBF-1 proximal promoter that is crucial for activity in B cells and for the induction of OBF-1 expression upon stimulation with CD40 ligand/IL-4. Here we address the question of how transcription of the OBF-1 gene is restricted to B cells. Surprisingly, in transient transfection assays the OBF-1 proximal promoter exhibited an equally strong activity in B and non-B cells. In contrast, upstream promoter regions displayed B cell-specific properties, partly overlapping with DNaseI hypersensitive sites identified in this study. In mice, expression of a neomycin resistance gene under the control of a Polyoma enhancer/TK promoter cassette was restricted to B cells when integrated into the OBF-1 locus, but was ubiquitous when integrated into two other loci, Oct-1 or the large subunit of RNA polymerase II.Therefore, lineage commitment of the OBF-1 gene is promoter independent and is achieved by regulating the entire locus in a B cell-specific manner.

OBF1 enhances transcriptional potential of Oct1.

The POU transcription factors Oct1 and Oct2 bind to DNA in various monomer and dimer configurations. Depending on the DNA sequence to which they bind, the dimers are arranged in configurations that are either accessible (PORE sequence) or inaccessible (MORE sequence) to the B-cell-specific cofactor OBF1 (OcaB, Bob1). As shown previously, the MORE and related sequences (such as the heptamer/octamer motif) are found in immunoglobulin heavy chain promoters. Here we show that the expression of Osteopontin, which contains a PORE sequence in its enhancer region, depends on the presence of OBF1 in B cells. OBF1 alleviates DNA sequence requirements of the Oct1 dimer on PORE-related sequences in vitro. Furthermore, OBF1 stabilizes POU dimer-DNA interactions and overrides Oct1 interface mutations, which abolish PORE-mediated dimerization without OBF1. Our data indicate that the PORE-type Oct1 or Oct2 dimer, rather than the monomer, is the primary target of the cofactor OBF1. Based on our biochemical data, we propose a mode of OBF1-Oct1 dimer interaction, suggesting a novel arrangement of the subdomain connectivities.