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A simple one-step isolation technique significantly enriched mouse fetal liver cells that respond to interleulkin 3 (IL-3), a multilineage hematopoietic growth factor.The fetal liver cell subpopulation isolated with monoclonal antibody AA4 contained 50-to 100-fold higher frequencies of multipotential (CFU-mix) or restricted (CFU-G/M, BFU-E) erythroid/myeloid precursors as well as precursors that differentiate to become mature B lymphocytes [CFU-mix = erythroid and myeloid colony-forming unit(s); CFU-G/M = CFU-granulocyte/macrophage; BFU-E = burst-forming uniterythroid]. The B-lymphocyte precursors could be cloned in single-cell cultures when IL-3-containing supernatants were present. Growth of these clones was supported by purified IL-3 but not by purified IL-2. Stable growth has been maintained for >6 mo in the presence of IL-3. Such clones express on their cell surface low amounts of class I major histocompatibility complex antigens and high amounts of AA4, GF1, and leukocyte common glycoprotein 200 under an atmosphere of 5% CO2 in air. WEHI-3 supernatant, which contains IL-3 and additional factors (1, 3-10), was stored at -70'C following centrifugation at 500 x g for 15 minand passage through a 0.22-gm filter. This supernatant, added at 10%6 (vol/vol) to IMDM containing 5% fetal calf serum, will be referred to as WEHI-3-conditioned medium (CM). Purified IL-3 and IL-2 (12, 13) were provided by J. Watson (University of Auckland). Monoclonal Antibodies (mAbs) and Quantitation of Cell Surface Determinants. Expression of cell surface determinants recognized by various mAbs was quantitated in a fluorescent antibody binding assay using rat anti-mouse mAbs as described (14,15). mAbs M1/89.18 (16) and D468HLK (17), which recognize leukocyte common glycoprotein (LGP) 200 and rat major histocompatibility complex (MHC) antigens, respectively, were also used.Cell-Separation Techniques. Subpopulations of fetal liver cells were obtained by "panning" on mAb-coated polystyrene dishes (14). Those cells removed after incubation and in the subsequent two rinses were pooled to produce the nonadherent (depleted) population. The adherent (selected) population was composed of cells that remained bound after 8-10 rinses and were recovered by repeatedly pipetting 5-10 ml of medium to gently resuspend cells. 7414The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

We characterized the basis for the follicular lymphoproliferation in transgenic mice bearing a Bcl-2-immunoglobulin (Bcl-2-Ig) minigene representing the t(14;18) of human follicular lymphoma. Discriminatory S1 nuclease protection assays revealed that the Bcl-2-Ig transgene was overexpressed relative to endogenous mouse Bcl-2 in spleen and thymus. Western (immunoblot) Specific types of chromosomal translocations are highly associated with distinct malignant diseases. Previously identified proto-oncogenes were shown to flank several of these breakpoints and were either fused to other genes, e.g., bcr-abl (26), or deregulated as with myc (2,7,27). These models suggested that examination of other recurrent translocations would reveal new proto-oncogenes at their breakpoints. The t(14;18)(q32;q21) of follicular B-cell lymphoma has provided one such opportunity. The molecular cloning of the derivative 14 breakpoint (1, 3, 28) led to the identification of the putative proto-oncogene Bcl-2 at 18q21. The translocation juxtaposed Bcl-2 with the immunoglobulin locus, resulting in inappropriately elevated levels of a Bcl-2-immunoglobulin (Bcl-2-Ig) fusion RNA for the mature stage of these follicular B-cell tumors (4,10,25 S1 nuclease protection assay.

Study of the differentiation of immunoglobulin-producing B lymphocytes has been hampered by the inability to maintain homogeneous populations of precursor cells in vitro. We describe here that interleukin-3 supports the growth of freshly isolated fetal liver pre-B cells and the long-term culture of interleukin-3 dependent pre-B-cell clones that can be induced to mature into antibody secreting cells in vitro.