Inhibition of murine B and T lymphopoiesis in vivo by an anti-interleukin 7 monoclonal antibody.

The effects of interleukin 7 (IL-7) on the growth and differentiation of murine B cell progenitors has been well characterized using in vitro culture methods. We have investigated the role of IL-7 in vivo using a monoclonal antibody that neutralizes IL-7. We find that treatment of mice with this antibody completely inhibits the development of B cell progenitors from the pro-B cell stage forward. We also provide evidence that all peripheral B cells, including those of the B-1 and conventional lineages, are derived from IL-7-dependent precursors. The results are consistent with the rapid turnover of B cell progenitors in the marrow, but a slow turnover of mature B cells in the periphery. In addition to effects on B cell development, anti-IL-7 treatment substantially reduced thymus cellularity, affecting all major thymic subpopulations.

Identification of a source of biologically active CD40 ligand.

We have identified the murine thymoma line EL4 as a source of biologically active CD40 ligand. Using a biotin-labeled soluble CD40.Fc fusion protein, consisting of the extracellular domain of human CD40 and the Fc region of human IgG1, EL4 cells were subjected to repeated flow cytometric cell sorting to select for cells with enhanced biotinylated CD40.Fc binding. After nine rounds of sorting, the number of CD40.Fc binding sites/cell had risen from 450 on the unsorted parental EL4 cells to 15,000 on EL40.9 cells (EL4 cells sorted with biotinylated CD40.Fc for nine rounds). Scatchard analysis of radiolabeled CD40.Fc binding revealed that the surface-expressed CD40 ligand on parental EL4 and EL40.9 cells bound its receptor with a single class of high-affinity sites (Kd = 0.5 nM). Supernatant (SN) from the sorted EL40.9 cells was found to contain human and murine B cell stimulatory activity which could be removed by preclearing with immobilized CD40.Fc, confirming the presence of soluble CD40 ligand in the preparations. EL40.9 supernatant enhanced soluble CD23 (sCD23) release and induced IgE secretion from interleukin 4-stimulated human B cells. In addition, EL40.9 SN contained proliferative activity for anti-IgM-activated murine B cells which could be removed by treatment with immobilized CD40.Fc. However, the same SN had no demonstrable activity on the proliferation of human B cells. The results presented here describe, for the first time, a source of membrane-bound and soluble CD40 ligand. The soluble form of this murine ligand has activity on murine and human B cells and induces some of the functional responses predicted for the ligand based on the action of stimulatory antibodies directed against the human CD40 surface molecule.

The effect of recombinant mast cell growth factor on purified murine hematopoietic stem cells.

Pluripotent hematopoietic stem cells (PHSC) are very rare cells whose functional capabilities can only be analyzed indirectly. For a better understanding and possible manipulation of mechanisms that regulate self-renewal and commitment to differentiation of PHSC, it is necessary to purify these cells and to develop assays for their growth in vitro. In the present study, a rapid and simple, widely applicable procedure to highly purify day 14 spleen colony-forming cells (day 14 CFU-S) is described. Low density bone marrow cells (rho less than or equal to 1.078 g/cm3) were enriched by two successive light-activated cell sorting procedures. In the first sort, cells within a predetermined light scatter (blast cell) window that are wheat germ agglutinin/Texas Red (WGA/TxR) positive and mAb 15-1.4.1/fluorescein isothiocyanate negative (granulocyte-monocyte marker) were selected. In the second sort, cells were selected on the basis of retention of the supravital dye rhodamine 123 (Rh123). Cells that take up little Rh123 (Rh123 dull cells) and those that take up more Rh123 (Rh123 bright cells) were 237-fold and 132-fold enriched, respectively, for day 14 CFU-S. Both Rh123 fractions were cultured for various time periods in vitro in the presence of mast cell growth factor (MGF), with or without interleukin 3 (IL-3) or IL-1 alpha. Both Rh123 fractions proliferated in response to MGF alone as determined by a [3H]TdR assay or by counting nucleated cells present in the cultures over time. MGF also acted synergistically with both IL-3 and IL-1 alpha to promote stem cell proliferation. Stimulation of both Rh123 fractions with MGF alone did not result in a net increase of day 14 CFU-S. Stimulation with MGF + IL-3 or MGF + IL-alpha resulted in a 4.4- or 2.6-fold increase of day 14 CFU-S in the Rh123 dull fraction, and an 11.6-fold or 2.6-fold increase of day 14 CFU-S in the Rh123 bright fraction, respectively. The data presented in this paper indicate that in vitro MGF acts on primitive hematopoietic stem cells by itself and also is a potent synergistic factor in combination with IL-3 or IL-1 alpha.

Monoclonal antibodies block murine IL-4 receptor function.

IL-4 is a cytokine which can induce B-lymphocyte proliferation, increase cell-surface Ia expression, and induce some activated B cells to differentiate and begin to secrete IgE. IL-4 binds specifically to a cell-surface receptor (IL-4R) on cells from a variety of lineages including T and B cells. In general both primary cells and in vitro cell lines express less than 5000 receptors per cell. Utilizing a subclone of the cytotoxic T cell line CTLL-2 expressing a high level of IL-4R, mAb against the murine IL-4R were prepared. Two mAb have been identified which have different properties. These antibodies, designated M1 and M2, recognize sequences specific to the murine IL-4R. Immunoprecipitation studies with M1 and M2 on CTLL-2 cells have identified the receptor as a Mr = 145,000 cell-surface protein. Similar results have been obtained with the recently isolated full length murine IL-4R cDNA expressed in COS-7 cells. In addition the antibodies are capable of inhibiting IL-4 binding. One antibody, M1, is also a potent inhibitor of IL-4-induced proliferation. These antibodies will be useful in dissecting a wide array of activities attributed to IL-4.

Decreased production of interferon-gamma by human neonatal cells. Intrinsic and regulatory deficiencies.

Human neonatal lymphocytes produced little macrophage activation factor in response to mitogens. This correlated with decreased production of interferon-gamma (IFN gamma): adult lymphokines contained 894.2 +/- 177.1 U/ml, whereas neonatal cord and peripheral lymphokines contained 66.9 +/- 17.0 and 116.7 +/- 29.6 U/ml by bioassay. Results by radioimmunoassay (RIA) for IFN gamma were similar. In contrast, the interleukin 2 content of cord lymphokines was greater (P less than 0.01) and that of neonatal peripheral blood lymphokines similar to that of adults. Interleukin 1 production and interleukin 2 receptor expression and affinity were similar for adult and neonatal cells. Interleukins 1 and 2 in amounts comparable to those in adult lymphokines did not increase production of macrophage activation factor or IFN gamma by neonatal cells. Neonatal cells did not contain intracellular IFN or degrade exogenous IFN. Excess suppressor activity was not found in neonatal cultures. Addition of IFN alpha, 10,000-50,000 U/ml of interleukin 2 or phorbol myristate acetate (PMA) to cord mononuclear cells or of adult monocytes or PMA to cord T cells increased IFN gamma production compared to cells stimulated with concanavalin A (ConA) alone. Nevertheless, under optimal conditions (T cells + PMA + Con A), adult cells produced much more IFN gamma (1,360 +/- 261 U/ml by RIA) than cord cells (122 +/- 37 U/ml). Staphylococcal enterotoxin A (SEA) stimulated cord cell IFN gamma production at low cell densities; nevertheless, adult cells produced more IFN in response to SEA 1,341 +/- 350 U/ml) than cord cells (350 +/- 33 U/ml). Decreased production of IFN gamma by neonatal cells appears to be due both to differences in their intrinsic capacity to produce IFN gamma and to differences in regulatory mechanisms.

Quantitative measurement of human interleukin 2 receptor levels with intact and detergent-solubilized human T-cells.

2A3 monoclonal antibody (gamma 1, kappa) is a novel high-affinity reagent for detecting the human interleukin 2 (IL-2) receptor. The antibody inhibits IL-2 binding to its receptor and is an antagonist of IL-2 action. Detailed analysis of the mechanism of binding of the IgG, (Fab')2 and Fab' of 2A3 antibody shows that the bivalent species cross-link on the cell surface when bound. Measurements of IL-2 receptor expression on digitonin-permeabilized cells suggest that the intracellular pool of receptors is small. The antibody will bind to IL-2 receptors on glutaraldehyde-fixed cells in the presence of Triton X-100. This property is used in designing an assay for quantitative measurements of IL-2 receptor concn in solution. This assay can be used to monitor receptor protein during purification to homogeneity.