Christopher J. Paige

Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities.

PU.1 is a member of the ets family of transcription factors and is expressed exclusively in cells of the hematopoietic lineage. Mice homozygous for a disruption in the PU.1 DNA binding domain are born alive but die of severe septicemia within 48 h. The analysis of these neonates revealed a lack of mature macrophages, neutrophils, B cells and T cells, although erythrocytes and megakaryocytes were present. The absence of lymphoid commitment and development in null mice was not absolute, since mice maintained on antibiotics began to develop normal appearing T cells 3–5 days after birth. In contrast, mature B cells remained undetectable in these older mice. Within the myeloid lineage, despite a lack of macrophages in the older antibiotic‐treated animals, a few cells with the characteristics of neutrophils began to appear by day 3. While the PU.1 protein appears not to be essential for myeloid and lymphoid lineage commitment, it is absolutely required for the normal differentiation of B cells and macrophages.

Normal B lymphocyte development but impaired T cell maturation in CD45-Exon6 protein tyrosine phosphatase-deficient mice

The transmembrane tyrosine phosphatase CD45 is expressed in multiple isoforms on all nucleated hematopoietic cells, resulting from alternative splicing of variable exons. We generated mice with a mutation in the variable CD45 exon 6, using homologous recombination. In mice homozygous for the CD45-exon6 mutation, B cells and most T cells did not express CD45. Development of B cells appeared normal, although Ig mu-induced proliferation was completely abrogated. Thymocyte maturation was blocked at the transitional stage from immature CD4+CD8+ to mature CD4+ or CD8+ cells, and only a few T cells could be detected in peripheral lymphoid organs. Clonal deletion of superantigen-reactive T cells still occurred. Cytotoxic T cell responses to lymphocytic choriomeningitis virus were absent in CD45-exon6-/- mice. These data imply that CD45 is differentially required for the development and function of B and T lymphocytes.

Development of B lymphocytes from lymphoid committed and uncommitted progenitors

Rapid experimental advances in B-cell biology are yielding an increasingly clear image of B-cell progenitors as they develop from multipotential stem cells to immunoglobulin (Ig)-secreting plasmacytes. Intermediate cells have been identified, growth and difTerentiation factors have been purified, stromal elements have been cloned, regulatory mechanisms of gene expression have been described. Not surprisingly, a wide consensus has been reached regarding many key elements of the developmental process. Despite this progress, other critical issues remain either obscure or controversial. These include fundamental questions such as the origins of lymphopoiesis, the genetic basis for lymphoid commitment, and the role of Ig in lymphoid progression, as well as detailed issues such as the role of particular growth factors or adhesion molecules. In this review, we will summarize the findings we have accumulated using in vitro and in vivo approaches to B-ceil development, and note the current issues which arise from this work. Particular emphasis is placed on the emergence of B-cell progenitors during fetal development.

Detection of RNA transcripts in normal lymphoid and myeloid colonies.

A procedure is described for the routine detection of RNA transcripts in small numbers of hematopoietic cells growing in semi-solid agar. It is suggested that hybridization depends upon RNA expression and that as few as 2500 mRNA molecules per colony are easily detected. Applications of this technique are described in three diverse experimental systems; immunoglobulin gene expression in B cell colonies; neo expression in normal and transformed B cell clones derived from multipotent stem cells infected with a neo-containing retrovirus; and c-myc expression in factor-dependent myeloid colonies.

A sensitive dot blot procedure for detecting mRNA in lymphoid cells grown in liquid culture

A sensitive dot blot procedure for detecting RNA transcripts with radiolabelled cDNA probes in small numbers of cells in suspension culture is described. For a frequent mRNA species such as that for immunoglobulin in an IgM secretor hybridoma, as few as 30 cells can be detected using a C mu probe; for a less frequent mRNA species such as transcripts coding for alpha, beta or gamma genes of the T cell receptor in T cell tumors or CTL clones, as few as 3 X 10(3) to 10(4) cells can be detected using C alpha, C beta or C gamma probes. High sensitivity is achieved by confining the cells to a very small area on the filter on which they are probed, and by treating the filter with formaldehyde.

DIFFERENTIATION OF B CELL TUMORS BY PRODUCTS OF MONOCLONAL T CELL IMMUNE REACTIONS

ABSTRACT When cloned T helper cells encounter antigen presented by macrophages of the proper I-A type, soluble mediators are produced which affect the differentiation and immunoglobulin metabolism of normal B lymphocytes and cell lines of the B lineage. Such culture supernatants cause the 70Z/3 pre-B cell line to begin to synthesize Ig L chains and gain membrane Ig detectable by immunofluorescence. Similar treatment of the WEHI-279.1 cell line, which represents a mature, Ig(+) B cell, causes a shift in the ratio of μ chains produced from mostly membrane to mostly secretory type, secretion of large amounts of IgM and massive cell death in culture. Products from T cell immune reactions thus exert multiple effects on B cell development and activation, at several stages of the B cell developmental pathway. The molecules responsible are distinct from the previously recognized lymphokines TCGF and LAF on the basis of molecular weight.

PLAQUE FORMATION BY B CELL COLONIES GROWING IN SOFT AGAR

ABSTRACT Aproximately 20% of sIg + murine B cells are able to form clones in semi-solid agar when cultured in the presence of lipopolysaccharide and sheep red blood cells. The method for growing these cells has been modified to allow the detection of secreted antibody. Thus both the proliferation and maturation of single B cells can be routinely assessed without the need for filler cells or other accessory cells. Protein A-conjugated SRBC as well as hapten-conjugated SRBC are suitable targets for this analysis. The frequency of B cell clones which secrete IgM, IgG, or specifie antibody has been determined. Using this information the clonable B cell population is being examined.

Hematopoietic Growth Factors Involved in B-Cell Development

B lymphocytes are continuously generated throughout life. Like all other members of the hematopoietic system, B cells are derived from multipotent hematopoietic stem cells (Wu et al., 1967). The developmental pathway that leads from multipotent stem cells to committed B lymphocytes is characterized by a series of differentiation steps. Many of these steps can be recognized based on the appearance of proteins such as growth factor receptors or the B-lineage-specific molecules that allow or promote the further development of progenitor cells. These stages of differentiation can also be recognized based on functional assays that have been developed over the last 30 years. Initially, such assay systems relied on the ability of progenitor cells to repopulate the hematopoietic system of a mouse that had previously been subjected to high doses of ionizing radiation. This approach has been crucial for the identification of multipotent stem cells and defining cells, which have long-term reconstituting potential (Dick et al., 1985; Keller et al.,1985; Lemischka et al., 1986). Irradiation/reconstitution experiments have been less useful for identifying intermediate stages in the developmental process. This method is also inadequate for studying either the essential cellular interactions or the growth and differentiation factors that promote hematopoiesis.