Materials and Methods

Cell Lines. The HT2 mouse T cell line (17) was obtained from S. Strober (Stanford University, Stanford, CA), the MC/9 mouse mast cell line (18) and D9 T cell line (19) from G. Nabel and H. Cantor (Harvard University, Boston, MA), the D10. G4. 1 T cell clone (20) from C. Janeway (Yale University, New Haven, CT), and the CDC-25 and CDC-35 mouse T cell clones (21) from D. Parker (University ofMassachusetts, Worcester, MA). The production and maintenance of our antigen-specific mouse T cell clones have been described elsewhere (13, 22). The P3X63Ag myeloma cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD). Antibodies. The S4B6 anti-IL-2 hybridoma has been described previously (13). Purified 11 B11 anti-IL-4 mAb (23) was a kind gift of R. Coffman, DNAX. mAbs recognizing IFN-y were derived from a Lewis rat after immunizing on eight sequential d with soluble recombinant mouse IFN-7 (170 kg/injection). This rat was obtained from M. Kehry (DNAX), and later boosted with IFN-y (100 ug) in CFA (Gibco Laboratories, Grand Island, NY). After 21 d, the rat was boosted with 100 wg IFN-y without adjuvant. 3 d later, the spleen cells were fused with P3X63Ag myeloma cells using 50% polyethylene glycol (Sigma Chemical Co., St. Louis, MO)(24). Antibodies directed against IFN-y were identified by ELISA testing of supernatants. Nine of these were able to block the biological activity of IFN-7 in two different bioassays: the effect of IFN-7 on proliferation of the NFS60 cell line (13) and the inhibition by IFN-y of the enhancement of IgE synthesis by IL-4 (25). XMG1. 2 is a rat IgGl mAb that is effective in both ELISA and blocking assays. Affinity-purified rabbit anti-IFN-7 polyclonal antibodies were obtained by passing serum from rabbits immunized with recombinant mouse IFN-y over a solid-phase immunoadsorbent column containing IFN-y and eluting the anti-IFN-y-specific antibodies. Labeled DNA Probes. The coordinates of the DNA probes used for hybridization were as follows: IL-2, nucleotides 498-570 (26); IL-3, 469-528 (27); IL-4, 445-498 (28); IL-5, 404-461 (29); granulocyte/macrophage colony-stimulating factor (GM-CSF), 549-639 (30); IFN-'y, 900-989 (31); lymphotoxin (LT), 517-618 (32); TNF, 765-863 (33); preproenkephalin (ppENK), 642-730 (34); TY5, 630-732 (Zurawski, G., personal communication); P600, 91-691 (Brown, KD, unpublished observations). With the exception of IL-5 and P600, radioactive probes were prepared by fill-in polymerization essentially following the procedure of Drouin (35). To label a given probe, 0. 4 ug each of a pair of synthetic oligonucleotides were mixed. These oligonucleotides (ranging in length from 30 to 59 nucleotides) were synthesized on a DNA synthesizer (model 380A; Applied Biosystems, Inc., Foster City, CA). The 3'terminal regions of these synthetic strands were complementary for 6-15 bp. Fill-in synthesis was carried out for 60 min at 37 C using 5 U of DNA polymerase (Klenow fragment; IBI, Miami, FL) in the presence of 200, uCi each of a-[s2P] dCTP and a-[" P] dATP (6,000 Ci/mmol, Amersham Corp., Arlington Heights, IL) and 0.01 mM unlabeled dATP and 0.1 mM unlabeled dGTP and dTTP in a total volume of 70 til Klenow reaction buffer (35). The IL-5 probe