Regulation of hemangioblast development.

The in vitro differentiation of embryonic stem (ES) cells provides a powerful approach for studying the earliest events involved in the commitment of the hematopoietic and endothelial lineages. Using this model system, we have identified a precursor with the potential to generate both primitive and definitive hematopoietic cells as well as cells with endothelial characteristics. The developmental potential of this precursor suggests that it represents the in vitro equivalent of the hemangioblast, a common stem cell for both lineages. ES cells deficient for the transcription factor scl/tal-1 are unable to generate hemangioblasts, while those deficient for Runx1 generate reduced numbers of these precursors. These findings indicate that both genes play pivotal roles at the earliest stages of hematopoietic and endothelial development. In addition, they highlight the strength of this model system in studying the function of genes in embryonic development.

B cell receptor expression level determines the fate of developing B lymphocytes: receptor editing versus selection.

During B lymphocyte development, antibody genes are assembled by DNA recombination. Successful cell surface expression of IgM promotes developmental progression. However, when antigen receptors bind autoantigen, development is blocked and ongoing antibody gene recombination occurs, which often alters antibody specificity in a process called receptor editing. We demonstrate here a significant role of developmental block and receptor editing in B cell receptor quality control. During development a functional, non-self-reactive receptor undergoes receptor editing if its expression is below a certain threshold. Doubling the receptor gene dose promotes development in the absence of autoantigen, but allows editing when autoantigen is present. Thus, both underexpressed and harmful B cell receptors can undergo correction by receptor editing.

T cell-independent rescue of B lymphocytes from peripheral immune tolerance.

Autoimmunity arises when immune tolerance to specific self-antigens is broken. The mechanisms leading to such a failure remain poorly understood. One hypothesis proposes that infectious agents or antigens can break B or T lymphocyte self-tolerance by expressing epitopes that mimic self. Using a transgenic immunoglobulin model, we show that challenge with self-mimicking foreign antigen rescues B cells from peripheral tolerance independent of T cell help, resulting in the accumulation of self-reactive cells in the lymph nodes and secretion of immunoglobulins that bind to a liver-expressed self-antigen. Therefore, our studies reveal a potentially important mechanism by which B lymphocytes can escape self-tolerance.

Antigens varying in affinity for the B cell receptor induce differential B lymphocyte responses.

The B cell receptor (BCR) triggers a variety of biological responses that differ depending upon the properties of the antigen. A panel of M13 phage-displayed peptide ligands with varying affinity for the 3-83 antibody was generated to explore the role of antigen-BCR affinity in cell activation studies using primary 3-83 transgenic mouse B cells. Multiple parameters of activation were measured. T cell-independent B cell proliferation, antibody secretion, induction of germline immunoglobulin gamma1 transcripts, and B cell production of interleukin (IL) 2 and interferon gamma responses were better correlated with antigen-BCR affinity than with receptor occupancy. In contrast, other responses, such as upregulation of major histocompatibility complex class II and B7.2 (CD86), secretion of IL-6, and B cell proliferation in the context of CD40 signaling were only weakly dependent on antigen affinity. Biochemical analysis revealed that at saturating ligand concentrations the ability of phage to stimulate some early signaling responses, such as Ca++ mobilization and tyrosine phosphorylation of syk or Igalpha, was highly affinity dependent, whereas the ability to stimulate Lyn phosphorylation was less so. These data suggest that the BCR is capable of differential signaling. The possibility that differential BCR signaling by antigen determines whether an antibody response will be T independent or dependent is discussed.

Identification of a fetal hematopoietic precursor with B cell, T cell, and macrophage potential.

Despite years of investigation, precursor-progeny relationships within the developing lymphoid lineages of the hematopoietic system remain poorly defined. We have characterized the potential of precursors found within a subpopulation of fetal liver defined by AA4.1 and Fc gammaRII/III expression and predominantly restricted to lymphoid and macrophage development. When cultured in methylcellulose with appropriate cytokines, AA4.1+/Fc gammaR+ precursors generate colonies consisting of various lineages, including the combination of B cell, T cell, and macrophage. Retroviral marking studies showed that the lymphoid cells and macrophages within these colonies arise from a common precursor. These results demonstrate the presence of a common precursor with B cell-, T cell-, and macrophage-restricted potential and as such define an early restriction point within the fetal lymphoid system.

Leptin stimulates fetal and adult erythroid and myeloid development.

The ob gene product, leptin, has been shown in several studies to be involved in weight control and recombinant leptin recently has entered clinical trials to treat obesity. The leptin receptor (OB-R/B219) is expressed in a variety of protein isoforms not only in the central nervous system, but also in reproductive, and hematopoietic tissues. We reported recently that the OB-R/B219 was associated with a variety of hematopoietic lineages as well as the small fraction of cells containing the long-term reconstituting hematopoietic stem cells. Herein we report that leptin significantly stimulates the proliferation and differentiation of yolk sac cells and fetal liver cells and stimulates directly hematopoietic precursors. Leptin alone can increase the number of macrophage and granulocyte colonies, and leptin plus erythropoietin act synergistically to increase erythroid development. These data show that leptin has a significant, direct effect on early hematopoietic development and can stimulate the differentiation of lineage-restricted precursors of the erythrocytic and myelopoietic lineages. These observations along with a recent report strongly support our previous hypothesis that leptin has an unanticipated important role in hematopoietic and immune system development.

Isolation of recombinant partial gag gene product p18 (HIV-1Bru) from Escherichia coli.

The membrane-associated structural protein, p18, of the human immunodeficiency virus (HIV-1), has been expressed in Escherichia coli. The recombinant protein was purified by cation-exchange chromatography on S Sepharose followed by cation-exchange high-performance liquid chromatography (HPLC) on Sulfoethyl Aspartamide. The isolation of 28.7 mg of recombinant p18 from 16.71 of cell culture represents an overall yield of ca. 20%. Recombinant p18 was characterized by sodium dodecyl sulphate polyacrylamide gel electrophoresis, reversed-phase HPLC, amino acid composition and amino acid sequence analysis of the N-terminus. Edman degradation of peptides generated by trypsin or Staphylococcus aureus V8 proteolytic digestion, including the C-terminus, confirmed the amino acid sequence to be that predicted from the cDNA. A C-terminally cleaved form of recombinant p18, p18LM, was separated in the cation-exchange HPLC step and was partially characterized in parallel with the intact molecule. By Western blotting it was shown that recombinant p18 in addition to the cleaved form p18LM is recognized by a monoclonal antibody which was generated against the natural protein from HIV-1.