Isolation and characterization of a mouse interleukin cDNA clone that expresses B-cell stimulatory factor 1 activities and T-cell-and mast-cell-stimulating activities ( gene cloning / Ia induction / interleukin 2 / interleukin 3 / T-cell lymphokine )

A cDNA sequence coding for a unique mouse interleukin that expresses B-cell-, T-cell-, and mast-cell-stimulating activities has been isolated from a mouse helper T-cell cDNA library. The library, constructed in the pcD expression vector, was screened by transfecting COS monkey cells with DNA pools to express the products encoded by full-length cDNA inserts. By assaying the transfected cell supernatants, we identified clones encoding a factor that stimulates T-cell and mast cell lines. This factor also induces Ia expression on resting B cells and enhances IgGl and IgE production by B cells, two properties of B-cell-stimulatory factor 1. The DNA sequence codes for a polypeptide of 140 amino acid residues including a putative signal peptide. These results demonstrate that a single cDNA clone distinct from interleukin 2 and interleukin 3 encodes a polypeptide with multiple biological activities. The mouse T-cell clone Cl.Lyl+2-/9 was initially shown to produce several biological activities, including (i) stimulation of mast cell proliferation, (ii) stimulation of T-cell proliferation, (iii) activation ofB cells to secrete immunoglobulin, and (iv) formation of hematopoietic colonies of various types (1-3). We previously described the isolation ofcDNA clones coding for interleukin 3 (IL-3) from a cDNA library made with mRNA from activated Cl.Lyl+2-/9 cells (4). These cDNA clones express mast cell growth factor (MCGF) activity in transfected mammalian cells, but even saturating concentrations of IL-3 do not stimulate a cloned mast cell line to the same extent as supernatant derived from the original T-cell clone (5). Recent experiments with Cl.Lyl+2-/9 cell supernatants have demonstrated the existence ofa factor distinct from IL-3 that has MCGF activity and the ability to enhance the MCGF activity of IL-3 (6). Despite multiple biochemical fractionations, the MCGF activity copurifies with a T-cell growth factor (TCGF) activity that is distinct from interleukin 2 (IL-2) (6). These results are consistent with RNA blotting analysis showing that activated Cl.Lyl+2-/9 cells do not produce IL-2 mRNA (unpublished). Furthermore, the TCGF activity in cell supernatants is not blocked by a monoclonal antibody that completely inhibits the activity of mouse IL-2 (unpublished). These results demonstrate that the Cl.Lyl+2-/9 cells produce a factor, which is distinct from IL-3 and IL-2, with both MCGF and TCGF activities (MCGFII/TCGFII). Cl.Lyl+2-/9 cells also produce high levels of three B-cellstimulating activities. These include costimulation of antiIgM-activated B cells (7), induction of Ia antigen on resting B cells (7), and enhancement of IgE and IgG1 production (8). Recent studies show that anti-IgM costimulation (9, 10), Ia induction (11, 12), and IgE (unpublished) and IgG1 (13) enhancement are all properties of partially purified B-cell stimulatory factor 1 (BSF-1). All of these activities in Cl.Lyl+2-/9 supernatants elute following gel filtration with an apparent Mr of -20,000 (ref. 7; unpublished results), the same size reported for BSF-1 from EL-4 cells (10). Together, these results suggest that Cl.Lyl+2-/9 cells produce high levels of a factor functionally identical to BSF-1. Based on our data suggesting that Cl.Lyl+2-/9 cells produce BSF-1 and a factor with MCGF and TCGF activities, we undertook the isolation of cDNA clones for each of these factors. During the course of our work, however, results from other studies suggested that BSF-1 is identical to the factor having MCGFII/TCGFII activity. When the MCGFII/TCGFII activity was highly purified from Cl.Lyl+2-/9 supernatants, it was found to have Ia-inducing activity for B cells (unpublished). Another line of experiments demonstrated that BSF-1 purified from EL-4 cells also possesses MCGFII/TCGFII activity and that anti-BSF-1 antibody (14) can block the MCGFII/TCGFII activity produced by T cells (unpublished). In this paper we describe the isolation and characterization ofcDNA clones that encode a protein with all of these activities. The expression of the functional product in mammalian cells provides final confirmation for the existence of this lymphokine and its ability to stimulate multiple cell types. MATERIALS AND METHODS Cell Lines and Isolation of mRNA. Cl.Lyl+2-/9, a cloned T-cell line derived from a C57BL/6 mouse, was grown as described (1) and stimulated with Con A at 2 ,ug/ml for preparation of induced mRNA. Total cellular RNA was extracted from the cells by using the guanidium thiocyanate method (15), and poly(A)+ RNA was selected by oligo(dT)cellulose chromatography. The HT-2 T-cell line and the MC/9 mast cell line were cultured as described (5). cDNA Library and Screening by Transfection. A pcD cDNA library was constructed with mRNA from Con A-induced Cl.Lyl+2-/9 cells by using a modified pcDV1 plasmid containing an Nsi I site at the previous location of the Kpn I site (16, 17). Transfection of plasmid DNA prepared from pooled cultures of 48 clones into COS monkey cells was performed as described (17). Abbreviations: IL-2, interleukin 2; IL-3, interleukin 3; MCGF, mast cell growth factor; TCGF, T-cell growth factor; BSF, B-cell stimulatory factor; LPS, lipopolysaccharide; GM-CSF, granulocytemacrophage colony-stimulating factor; bp, base pair(s). 2061 The 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. Proc. Natl. Acad. Sci. USA 83 (1986)