Activation and growth of colony-stimulating factor-dependent cell lines is cell cycle stage dependent.

Hematopoietic cell development is regulated by a series of growth factors that are progressively restricted in their biological activity. IL-3 is a multi-lineage growth factor that supports the growth and differentiation of progenitor cells belonging to multiple lineages. However, the mechanism by which IL-3 induces proliferation and differentiation of these cells is not completely understood. In this report, we have used two IL-3-dependent cell lines, FDC-P1 (a myeloid progenitor) and F15.12 (a lymphoid progenitor) to investigate IL-3-mediated growth and differentiation. When either FDC-P1 or FL5.12 cells are deprived of IL-3, greater than 90% of all cells accumulate in the G0 phase of the cell cycle. Upon readdition of IL-3, the cells will reenter the active phases of the cell cycle. Therefore, IL-3 can act as both a competence (G0----G1) factor, and a progression (G1----M) factor for hematopoietic precursor clones. FDC-P1 cells can also proliferate in response to granulocyte/macrophage colony-stimulating factor (G/M-CSF) and IL-4 (B cell stimulatory factor 1 [BSF-1]). However, resting (G0) FDC-P1 cells have lost their ability to grow in response to both G/M-CSF and IL-4, even though both factors can induce a G0----G1 transition. Therefore, G/M-CSF or IL-4 behave as progression factors among certain IL-3-responsive clones, and in those cases only in defined points in the cell cycle. Both IL-4 and G/M-CSF can maintain long-term growth of FDC-P1 cells. Upon removal of factor for 24 h, these clones accumulate in the G1 phase of the cell cycle and do not appear to enter G0 even after 36 h of factor deprivation. Therefore, cells maintained in G/M-CSF or IL-4 have altered growth requirements compared with the IL-3-dependent lines from which they were derived. The ability of various hematopoietic growth factors to regulate cell cycle progression in IL-3-dependent cell lines is dependent not only upon the lineage from which these cells were derived, but also the phase of the cell cycle in which those cells reside. The consequences of these interactions dictate the manner by which various clones will respond to CSFs and whether the cells will grow and/or differentiate.

Polymorphism of Fc receptor on murine B cells is Igh-linked.

Analysis of mouse IgG binding to Fc receptors on mouse B cells indicates that the IgG1, IgG2a, and IgGb subclasses bind to the same receptor. No differences in affinity were detected among subclass or between mouse strains. This same receptor bound rat IgG with an affinity that differed between mouse strains. This polymorphism in affinity for rat IgG maps to chromosome 12 distal to the Igh locus.

Deregulated Bcl-2-immunoglobulin transgene expands a resting but responsive immunoglobulin M and D-expressing B-cell population.

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) analysis demonstrated the overproduction of the human 25-kilodalton Bcl-2 protein, which arose from the transgene, in spleen, thymus, and the expanded B-cell subset. Despite the generalized lymphoid pattern of deregulation, two-color flow cytometry and density gradient centrifugation indicated that the expanded lymphocytes were predominantly small, resting B cells coexpressing B220, immunoglobulin M (IgM), IgD, Ia, and kappa. Cell cycle analysis confirmed that about 97% of these expanded B cells reside in G0/G1. An extensive characterization of transgenic lines revealed a fourfold excess of IgM-IgD-expressing B cells in spleen and dramatically increased numbers in bone marrow. While resting, these cells proliferated in response to lipopolysaccharide and anti-IgM and demonstrated normal B-cell colony formation in soft agar. Moreover, these B cells, which demonstrated an extended survival in vitro even in the absence of stroma, were also resting in G0, yet were capable of proliferative responses. These findings provide consistent evidence that the accumulation of B cells after Bcl-2 overproduction is secondary to prolonged cell survival and not increased cell cycling. This suggests a unique role for Bcl-2 as a proto-oncogene that enhances cell survival independent of promoting cell division.

B-cell subsets: functional and structural characteristics.

The review will examine B-cell differentiation with an emphasis on B-cell subpopulations. We will begin with an analysis of current evidence concerning B-cell ontogeny. The development of the B-cell repertoire will be traced from stem cell to effector cells. The CBA/N mouse, which expresses an X-linked immunodeficiency, will serve as a basis for discussing the delineation of B cells into subpopulations. The CBA/N mouse provides evidence for distinct populations of B cells which can be differentiated by cell surface antigens as well as function. We intend to focus on the functional diversity of B-cell subpopulations and how they develop. The CBA/N mouse is not the only evidence for distinct B cell subpopulations and we will attempt to organize these data into a coherent story of B-cell subsets.

Phage display mutagenesis of the chimeric dual cytokine receptor agonist myelopoietin.

Myelopoietins comprise a class of chimeric cytokine receptor agonists consisting of an hIL-3 (human interleukin-3) receptor agonist and an hG-CSF (human granulocyte colony-stimulating factor) receptor agonist linked head-to-tail at their respective carboxy and amino termini. The combination of an early acting cytokine (hIL-3) with a late acting one (hG-CSF) allows efficient hematopoeitic reconstruction following myeloablative insult, and drives differentiation of non-myelocytic lineages (ie thrombocytic lineages) that are inaccessible using hG-CSF alone, in both preclinical models and clinical settings. A myelopoietin species was displayed and mutagenized on filamentous bacteriophage: both component agonists of myelopoietin were presented in biologically functional conformations as each recognized its corresponding receptor. Five amino acid positions in a short region of the hG-CSF receptor agonist module of myelopoietin that had been identified as important for proliferative activity were mutagenized. Display was used because it allows very 'deep' mutagenesis at selected residues: >10(5) substitution variants were affinity-screened using the hG-CSF receptor and 130 new, active variants of myelopoietin were identified and characterized. None of the selected variants were significantly more active than the parental myelopoietin species in a hG-CSF-dependent cell proliferation assay, though many were as active. Many of these relatively high-activity variants contained parental amino acids at several positions, suggesting the parental sequence may already be optimal at these positions for the assays used, and potentially accounting for the failure to identify enhanced bioactivity variants. Analysis of substitutions of high-activity variants complements and extends previous alanine scanning, and other genetic and biochemical data for hG-CSF variants.

Enhanced ability of daniplestim and myelopoietin-1 to suppress apoptosis in human hematopoietic cells.

Modified and chimeric cytokines have been developed to aid in the recovery of hematopoietic precursor cells after myeloablative chemotherapy. The interleukin-3 (IL-3) receptor agonist, daniplestim, binds to the IL-3 receptor-alpha subunit with 60-fold greater affinity and induces cell proliferation and colony-forming unit formation 10- to 22-fold better than native IL-3. A chimeric cytokine, myelopoietin-1, composed of daniplestim and a G-CSF receptor agonist binds both the IL-3 and G-CSF receptors. While the in vivo effects of daniplestim and myelopoietin-1 are well described, the mechanisms by which they stimulate growth are not well understood. We have investigated the effects of daniplestim and myelopoietin-1 on the prevention of apoptosis in two human hematopoietic cell lines, OCI-AML.5 and AML 193. Daniplestim and myelopoietin-1 prevented apoptosis to a greater degree than native recombinant IL-3 or G-CSF as determined by annexin V/propidium iodide binding and TUNEL assays. Daniplestim and myelopoietin-1 promoted the maintenance of the mitochondrial membrane potential better than native IL-3 or G-CSF. These cytokines promoted a lower redox potential as higher levels of free radicals were detected after cytokine treatment than in cytokine-deprived cells implying increased respiration. These results indicate that daniplestim and myelopoietin-1 are able to prevent apoptosis in hematopoietic cells more effectively than native IL-3 and G-CSF. These effects of daniplestim and myelopoietin-1 may contribute to their effective ability to repopulate hematopoietic precursor cells after chemotherapy.

The enhanced in vitro hematopoietic activity of leridistim, a chimeric dual G-CSF and IL-3 receptor agonist.

The in vitro activity of leridistim was characterized for cell proliferation, generation of colony-forming units (CFU) and differentiation of CD34+ cells. In AML-193.1.3 cells, leridistim exhibited a significant increase in potency compared to rhG-CSF, SC-65303 (an IL-3 receptor agonist) or an equimolar combination of rhG-CSF and SC-65303. CFU-GM assays demonstrated that at 50% of the maximum response, the relative potency of leridistim was 12-fold greater than the combination of rhG-CSF and rhIL-3 and 44-fold more potent than rhG-CSF alone. In multi-lineage CFU assays, a combination of erythropoietin (rhEPO) and leridistim resulted in greater numbers of BFU-E, CFU-GEMM and CFU-Mk than rhEPO alone. Ex vivo culture of peripheral blood or bone marrow CD34+ cells with leridistim substantially increased total viable cells over cultures stimulated with rhG-CSF, SC-65303, or a combination of rhG-CSF and SC-65303. Culture with leridistim, resulted in a greater increase in myeloid (CD15+/CD11b+), monocytic (CD41-/CD14+) and megakaryocytic (CD41+/CD14-) precursor cells without depleting the progenitor pool (CD34+/CD15-/CD11b-). These results demonstrate that leridistim is a more potent stimulator of hematopoietic proliferation and differentiation than the single receptor agonists (rhG-CSF and SC-65303) either alone or combined. These unique attributes suggest that leridistim may enhance hematopoietic reconstitution following myelosuppressive chemotherapy.

The dual regulatory role of interleukin 4 is mediated through a direct effect on the target cell.

The mechanisms responsible for regulating the growth and differentiation of pluripotent stem cells involve the complex interaction of a series of specific and nonspecific growth factors and signals. In this report, colony-stimulating factor (CSF)-dependent clonal cell lines, recombinant and/or purified CSFs, and clonal assays were used to investigate the mechanism of interleukin 4 (IL-4)-induced modulation of CSF-dependent cell growth. IL-4 inhibits in a dose-dependent fashion either the interleukin 3 (IL-3)- or granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced growth of the myelomonocytic progenitor cell line, FDC-P1. This inhibitory effect of IL-4- on IL-3- or GM-CSF-induced cell growth was verified using normal bone marrow cells. Our data supports the hypothesis that IL-4 is acting directly on the progenitor cell and not indirectly through the action of accessory cells. Further, because the inhibitory effect of IL-4 is selective and does not affect all CSF-dependent cell lines, other factors, including the maturational state or the lineage of the cell may be important in dictating an effect, if any, mediated by IL-4.

Myelopoietin, a chimeric agonist of human interleukin 3 and granulocyte colony-stimulating factor receptors, mobilizes CD34+ cells that rapidly engraft lethally x-irradiated nonhuman primates.

Myelopoietin (MPO), a multifunctional agonist of interleukin 3 and granulocyte colony-stimulating factor (G-CSF) receptors, was evaluated for its ability to mobilize hematopoietic colony-forming cells (CFC) and CD34+ cells relative to control cytokines in normal nonhuman primates. Additionally, the engraftment potential of MPO-mobilized CD34+ cells was assessed in lethally irradiated rhesus monkeys. Normal rhesus monkeys were administered either MPO (200 microg/kg/day), daniplestim (a high-affinity interleukin 3 receptor agonist) (100 microg/kg/day), G-CSF (100 microg/kg/day), or daniplestim coadministered with G-CSF (100 microg/kg/day each), subcutaneously for 10 consecutive days. The mobilization kinetics were characterized by peripheral blood (PB) complete blood counts, hematopoietic CFC [granulocyte-macrophage CFC (GM-CFC), megakaryocyte CFC (MK-CFC)], and the immunophenotype (CD34+ cells) of PB nucleated cells prior to and on day 3 to days 7, 10, 12, and 14, and at intervals up to day 28 following initiation of cytokine administration. A single large-volume leukapheresis was conducted on day 5 in an additional cohort (n = 10) of MPO-mobilized animals. Eight of these animals were transplanted with two doses of CD34+ cells/kg. A maximum 10-fold increase in PB leukocytes (white blood cells) (from baseline 7.8-12.3 x 10(3)/microL to approximately 90 x 10(3)/microL) was observed over day 7 to day 10 in the MPO, G-CSF, or daniplestim+G-CSF cohorts, whereas daniplestim alone stimulated a less than onefold increase. A sustained, maximal rise in PB-derived GM-CFC/mL was observed over day 4 to day 10 for the MPO-treated cohort, whereas the daniplestim+G-CSF, G-CSF alone, and daniplestim alone treated cohorts were characterized by a mean peak value on days 7, 6, and 18, respectively. Mean peak values for PB-derived GM-CFC/mL were greater for MPO (5,427/mL) than for daniplestim+G-CSF (3,534/mL), G-CSF alone (3,437/mL), or daniplestim alone (155/mL) treated cohorts. Mean peak values for CD34+ cells/mL were noted within day 4 to day 5 of cytokine administration: MPO (255/microL, day 5), daniplestim+G-CSF (47/microL, day 5), G-CSF (182/microL, day 4), and daniplestim (96/microL, day 5). Analysis of the mobilization data as area under the curve indicated that for total CFCs, GM-CFC, MK-CFC, or CD34+ cells, the MPO-treated areas under the curve were greater than those for all other experimental cohorts. A single, large-volume (3.0 x blood volume) leukapheresis at day 5 of MPO administration (PB: CD34+ cell/microL = 438 +/- 140, CFC/mL = 5,170 +/- 140) resulted in collection of sufficient CD34+ cells (4.31 x 10(6)/kg +/- 1.08) and/or total CFCs (33.8 x 10(4)/kg +/- 8.34) for autologous transplantation of the lethally irradiated host. The immunoselected CD34+ cells were transfused into autologous recipients (n = 8) at cell doses of 2 x 10(6)/kg (n = 5), and 4 x 10(6)/kg (n = 3) on the day of apheresis. Successful engraftment occurred with each cell dose. The data demonstrated that MPO is an effective and efficient mobilizer of PB progenitor cells and CD34+ cells, such that a single leukapheresis procedure results in collection of sufficient stem cells for transplantation and long term engraftment of lethally irradiated hosts.

Progenipoietin-1: a multifunctional agonist of the granulocyte colony-stimulating factor receptor and fetal liver tyrosine kinase-3 is a potent mobilizer of hematopoietic stem cells.

OBJECTIVE: Progenipoietin-1 is an agonist of both the granulocyte colony-stimulating factor and fetal liver tyrosine kinase-3 receptors capable of inducing the proliferation of multiple hematopoietic cell lineages. The potential of progenipoietin-1 to mobilize transplantable hematopoietic stem cells into the peripheral blood was evaluated. METHODS: Cohorts of donor mice were treated with either progenipoietin-1, fetal liver tyrosine kinase-3 ligand, granulocyte colony-stimulating factor, or a vehicle control. Hematopoietic progenitor/stem-cell activity in donor blood was assayed by radioprotection, multilineage reconstitution, secondary transplantation, and competitive repopulation. RESULTS: Only 1 microL of peripheral blood from progenipoietin-1-treated donors was required to protect 80% of lethally irradiated mice, while in contrast 1 microL of peripheral blood from granulocyte colony-stimulating factor-treated donors failed to protect any recipients. The radioprotected recipients of progenipoietin-1-treated donor cells showed donor-derived (Ly5.2) multilineage hematopoietic reconstitution for up to 6 months. Serial transplantation studies using bone marrow from radioprotected, chimeric recipients demonstrated long-term donor-derived hematopoiesis, indicating the successful transplantation of multipotent hematopoietic stem cells. The engraftment potential of progenipoietin-1 donor-derived cells was directly compared with donors treated with granulocyte colony-stimulating factor or fetal liver tyrosine kinase-3 ligand alone or in combination. Both spleen colony-forming activity and competitive repopulating activity was highest in the blood from progenipoietin-1-treated donors. CONCLUSIONS: These studies demonstrate that progenipoietin-1 is a potent mobilizer of transplantable hematopoietic stem cells and indicate that this dual-receptor agonist has greater biologic activity than its constituent molecules.

Progenipoietins: biological characterization of a family of dual agonists of fetal liver tyrosine kinase-3 and the granulocyte colony-stimulating factor receptor.

The progenipoietins, a class of engineered proteins containing both fetal liver tyrosine kinase-3 and granulocyte colony-stimulating factor receptor agonist activities, were functionally characterized in vitro and in vivo. Four representative progenipoietins were evaluated for receptor binding, receptor-dependent cell proliferation, colony-forming unit activity, and their effects on hematopoiesis in the C57BL/6 mouse.The progenipoietins bound to fetal liver tyrosine kinase-3 and the granulocyte colony-stimulating factor receptor with affinities within twofold to threefold of the native ligands, and each progenipoietin bound simultaneously to both fetal liver tyrosine kinase-3 and the granulocyte colony-stimulating factor receptor. The progenipoietins exhibited different levels of activity in receptor-dependent cell proliferation assays. The fetal liver tyrosine kinase-3-dependent cell proliferation activity of three of four progenipoietins was decreased sixfold to 33-fold relative to native fetal liver tyrosine kinase-3 ligand, while granulocyte colony-stimulating factor receptor-dependent activity of the progenipoietins was within twofold to threefold of native granulocyte colony-stimulating factor. At nonsaturating concentrations, the progenipoietins stimulated colony formation to a greater extent than the equimolar combination of fetal liver tyrosine kinase-3 and granulocyte colony-stimulating factor. Treatment of mice with the progenipoietins yielded dramatic increases in peripheral blood and splenic white blood cells, polymorphonuclear leukocytes, and dendritic cells. These preclinical results demonstrate that the progenipoietins are potent hematopoietic growth factors that stimulate cells in a receptor-dependent manner. When administered in vivo, the progenipoietins effectively promote the generation of multiple cell lineages. Thus, in both in vitro and in vivo settings, the progenipoietins as single molecules exhibit the synergistic activity of the combination of fetal liver tyrosine kinase-3 and granulocyte colony-stimulating factor.

Promegapoietin, a family of chimeric growth factors, supports megakaryocyte development through activation of IL-3 and c-Mpl ligand signaling pathways.

OBJECTIVE: The signaling pathways induced by promegapoietin (PMP), a family of chimeric growth factors that activate the human IL-3 and c-Mpl receptors, were investigated. METHODS: The biological activity of PMP was examined by receptor binding, cell proliferation, ex vivo expansion of hematopoietic progenitor cells, and in vivo production of platelets. The activation of signaling pathways was examined by Western blot and Northern blot analyses. RESULTS: Two PMP molecules, PMP-1 and PMP-1a, induced proliferation of cells expressing the IL-3 receptor, c-Mpl, or both receptors and bound to the IL-3 receptor and c-Mpl with high affinity. Ex vivo expansion assays using human bone marrow CD34(+) cells suggested that PMP-1 induced greater total cellular expansion as well as expansion of CD41(+) megakaryocytic precursor cells than IL-3 or c-Mpl ligand alone. Subcutaneous administration of 50 microg/kg of PMP-1 for 10 days to rhesus monkeys resulted in increased platelet production in vivo from a baseline of 357 +/- 45 x 10(3) cells/mL to 1376 +/- 151 x 10(3) cell/mL. PMP-1 induced phosphorylation of the beta(c) subunit of IL-3 receptor and c-Mpl, JAK2, and STAT5b, but not STAT3. PMP-1 induced greater expression of Pim-1, c-Myc, and cyclin D2 than did either an IL-3 receptor agonist or c-Mpl receptor agonist alone. The magnitude of induction of early response genes was similar for PMP and the coaddition of IL-3 receptor agonist and c-Mpl receptor agonist. CONCLUSION: PMP combines the biological activities of IL-3 and c-Mpl ligand in a single molecule that can simultaneously activate signaling pathways induced by both these ligands.

Use of combinatorial mutagenesis to select for multiply substituted human interleukin-3 variants with improved pharmacologic properties.

A combinatorial mutagenesis strategy was used to create a collection of nearly 500 variants of human interleukin 3 (IL-3), each with four to nine amino acid substitutions clustered within four linear, nonoverlapping regions of the polypeptide. The variants were secreted into the periplasm of Escherichia coli and supernatants were assayed for IL-3 receptor-dependent cell proliferation activity. Sixteen percent of the variants, containing "region-restricted" substitutions, retained substantial proliferative activity through two rounds of screening. A subset of these was combined to yield variants with substitutions distributed through approximately half of the polypeptide. With one exception, "half-substituted" variants exhibited proliferative activity within 3.5-fold of native IL-3. A subset of the "half-substituted" variants was combined to yield "fully substituted" IL-3 variants having 27 or more substitutions. The combination of the substitutions resulted in a set of polypeptides, some of which exhibit increased proliferative activity relative to native IL-3. The elevated hematopoietic potency was confirmed in a methylcellulose colony-forming unit assay using freshly isolated human bone marrow cells. A subset of the multiply substituted proteins exhibited only a modest increase in inflammatory mediator (leukotriene C4) release. The molecules also exhibited 40- to 100-fold greater affinity for the alpha subunit of the IL-3 receptor and demonstrated a 10-fold faster association rate with the alpha-receptor subunit. The multiply substituted IL-3 variants described in this study provide a unique collection of molecules from which candidates for clinical evaluation may be defined and selected.

Cytokine production by T helper cell subpopulations during prolonged in vitro stimulation.

In man, CD4+ T cells can be divided into phenotypically distinguishable subsets with different function whereas CD4+ T cells with the opposite pheno-CD45RO and low levels of CD45RA antigen provide help for mitogen-induced immunoglobulin production whereas CD4+ cells with the opposite phenotype suppress immunoglobulin production. However, studies examining cytokine production by phenotypically defined CD4+ T cell subsets have led to different conclusions. Further, very few studies have examined cytokine production by freshly isolated CD4+ T cell subsets during extended culture periods. Thus, we examined the production of several cytokines (at various time points) by CD4+ T cell subsets that were isolated in several ways, and stimulated with PWM, Con A, and PHA in a well-defined serum-free culture system. We found that CD4+, CD45RA- (or CD45RO+) T cells consistently produced the most IL-2, IFN-gamma, and TNF-alpha after mitogen stimulation for 2 days. PWM induced the largest quantities of each cytokine, although a similar pattern of production was observed in response to Con A and PHA. We were unable to detect IL-4 production by mononuclear cells and CD4+ T cell subsets suggesting that, if it is produced at all, IL-4 is produced in extremely small quantities. When the culture period of initially CD45RO- T cells was extended beyond 2 days, the culture supernatant contained increased quantities of each cytokine and the cells in the culture had an increased number of cells expressing CD45RO antigen. Together, these data indicate that CD4, CD45RA- (or CD45RO+) T cells in peripheral blood are the major producers of IL-2, IFN-gamma, and TNF-alpha following short-term mitogen stimulation, and that phenotypically defined peripheral blood T cell subsets do not maintain a distinct pattern of cytokines during extended culture periods.

Phage presentation and affinity selection of a deletion mutant of human interleukin-3.

A deletion derivative of the cytokine human interleukin-3 (hIL-3(15-125), comprising amino acids 15-125 of the native protein) was produced as a fusion to the filamentous phage surface protein pIII. The cytokine was detected in association with phage particles by protein immunoblotting. Compared to an equivalent quantity of soluble-cytokine, phage-presented hIL-3(15-125) exhibited reduced biological activity in a hIL-3-dependent cell proliferation assay. The reduction in activity was attributable to presence of phage particles in the assay, rather than directly owing to physical incorporation of the cytokine into the phage particle. Owing to the position of the amber codon in the phagemid vector, the phagemid-produced free hIL-3(15-125) species (designated hIL-3(15-125) epsilon) had 20 amino acids appended to its C-terminus; hIL-3(15-125) epsilon did not exhibit reduced bioactivity. hIL-3(15-125)-presenting phage were affinity-selected with either a hIL-3-reactive polyclonal antibody or with cells expressing the heterodimeric hIL-3 receptor. These data are consistent with the use of phage-display technology for the affinity selection of hIL-3 variants with modified biological properties.

Idiotype-specific tolerance: preferential alteration of idiotype expression after neonatal exposure to tolerogen.

The development of HOPC-8 idiotype-positive and HOPC-8 idiotype-negative B cell subpopulations involved in the immune response to a single PC antigen, has been monitored during normal differentiation and in situations where neonatal tolerance had been induced. It was found that H8 id+ and H8 id- anti-PC antibody-secreting cells appeared at approximately the same time in ontogeny and these late maturing subsets constituted equal fractions of the immune response in CBA/CaJ X BALB/c F1 (JBF1) mice. However, when JBF1 mice were injected at birth with graded doses of PC-FGG, responses obtained in 4-week-old mice were predominantly of the H8 id. This PC-specific, idiotype-restricted, unresponsive state was stable upon adoptive transfer. Specific depletion of Thy-1.2-positive cells and in vitro "mixing" experiments involving combined ratios of tolerant and nontolerant spleen cells suggest that the unresponsive state measured 4 weeks after tolerogen administration does not involve suppressor cells. It would appear that intrinsic differences between these idiotypically defined B cell subsets account for the observed differential susceptibility to tolerance induction among PC-responsive B cells.

Delineation of tolerance-sensitive and tolerance-insensitive B cells in normal and immune defective mice.

B cells responsive to TNP on various thymus-independent carriers acquire immunocompetence at different times during ontogeny and elicit varying levels of responsiveness in immune-defective CBA/N mice. Such B cells have now been found to display differential tolerance susceptibilities in a hapten-specific in vitro tolerance assay. B lymphocytes from normal neonatal or immune-defective adult CBA/N mice responsive to TNP-lipopolysaccharide were shown to be hypersusceptible to tolerogenesis whereas B cell responses to a second TI antigen, TNP-Brucella abortus, were not rendered tolerant until high concentrations of tolerogen had been presented. Therefore, tolerance hypsersusceptibility is characteristic of a fraction of neonatal and immune-defective adult CBA/N antigen-reactive B lymphocytes.

Mapping cell surface antigens on mouse pre-B cell lines.

Characterization of cell surface molecules expressed by B cell precursors has been hampered by the inability to routinely assay such cells from freshly explanted normal lymphoid tissues. However, a large number of transformed pre-B cell clones have been isolated which presumably include those precursors recently entering the B cell lineage (obtained from fetal liver and more rarely from adult bone marrow) and extending to those clones which are poised to become surface Ig-positive B cells. In this report, we have quantitated expression of 10 different cell surface molecules, 5 of which bear special interest as B cell differentiation antigens, among a large panel of pre-B cell clones.

Interleukin-3 and phorbol esters induce different patterns of immediate-early gene expression in an interleukin-3 dependent cell line.

The myeloid interleukin-3 (IL-3) dependent cell line, FDC-P1, enters the G0 stage of the cell cycle after IL-3 deprivation for 24 hr. The expression of 13 protooncogenes and immediate-early genes was compared with 4 "control" genes after the addition of either IL-3 or phorbol myristate acetate (PMA) to IL-3-deprived cells. mRNA transcripts encoding c-myc and the T-cell receptor c-gamma gene were induced to high levels only after IL-3 addition, whereas c-fos, fos-B, c-jun, jun-B, Krox-20, and Krox-24 were induced transiently only after PMA addition. The remaining genes (fra-1, p53, jun-D, c-Ha-ras, c-Ki-ras, c-raf, beta-actin, ornithine decarboxylase, and histone 2B) were detected after culture with either IL-3 or PMA. When cells were serum- and IL-3-deprived, c-fos, fos-B, c-jun, jun-B, Krox-20, and Krox-24 were detected after exposure to either serum or PMA. Moreover, culture with cycloheximide and PMA resulted in superinduction of these genes, whereas cycloheximide and IL-3 addition did not. mRNAs encoding these genes had half-lives of 10-20 min after PMA treatment, whereas that of beta-actin was longer (greater than 2 hr), suggesting that short mRNA half-lives contributed to the transient nature of these genes. Although c-fos, fos-B, c-jun, jun-B, Krox-20, and Krox-24 expression can be detected in IL-3-dependent cells after exposure to either PMA or serum, these genes were not detected after IL-3 addition, which allows cell-cycle progression. These results document the existence of IL-3 and PMA-responsive genes and demonstrate that IL-3 and protein kinase C agonists can induce distinct patterns of gene expression.

Transformation of B and non-B cell lines with the 2,4,6,-trinitrophenyl (TNP)-specific immunoglobulin genes.

The rearranged mu and kappa genes from the 2,4,6-trinitrophenyl (TNP)-specific hybridoma Sp6 have been introduced into B cells from three different stages of differentiation as well as 5 non-B cell lines to determine the levels and modes of immunoglobulin (Ig) gene expression. In pre-B cells transformed with the mu and kappa genes, low levels of Sp6-specific mu RNA were produced and approximately 210-fold less mu and 800-fold less kappa proteins were produced than in the hybridoma Sp6. The Ig proteins were present intracellularly, but were not detected on the cell membrane. In mature surface sIg+ B cell transformants, higher levels of mu Sp6 and kappa Sp6 proteins and RNA were produced than in the pre-B cell transformants (12 X mu, 70 X kappa). These transformants displayed the mu Sp6 and kappa Sp6 proteins on the cell membrane and also secreted the transfected Ig product. Plasma cell transformants produced the highest amounts of mu Sp6 and kappa Sp6 proteins. These transformants secreted pentameric IgM but did not display detectable amounts of these proteins on the cell membrane. T cell and one fibroblast transformant produced Ig as normal sized mu Sp6 and kappa Sp6 proteins. All other mu Sp6 and kappa Sp6 non-B cell transformants (melanoma, teratoma and macrophage) failed to produce enough Ig to determine whether the Ig proteins were of the correct molecular weights. The T cell and fibroblast transformants that produced Ig proteins did not secrete or display detectable Ig on the cell membrane. The expression of Ig did not inhibit the expression of the T cell antigen Thy-1 in the T cell transformants.