Eur. J. Immunol. 1991. zyxwvutsrq 21: 447-451 Antibodies against human IFN zyx ap receptor 447 zy Murine tumor cells expressing the gene for the human interferon afl receptor elicit antibodies in syngeneic mice to the active form of the receptor* Gilles UzC, Georges Lutfalla, Pierre Eid, Chantal Maury, Marie-Thirkse Bandu, Ion Gresser and Knud Mogensen Laboratoire d’Oncologie V ile, zyxwvut CNRS UPR 274, Villejuif The cellular receptor for the human a and zyxw p interferons (IFN) was expressed, by gene transfer, in a murine hepatoma-derived cell line, BTG 9A. Injected subcutaneously into the syngeneic mouse (C57BL/6), the parental and the transfected cells grew and formed tumors which later regressed. More than half the mice bearing tumors derived from cells expressing the receptor, developed IgG antibodies capable of blocking the activity, on human cells, of human recombinant IFN-aB, -aA, -aD and of natural human IFN-p, but not of recombinant IFN-y. Cross-reactivity of human IFN-a on murine and bovine cells was unaffected by these antibodies. The binding of human IFN-a to solubilized receptors from human lymphoid cell lines was also blocked and complexes of radiolabeled recombinant IFN-aA or IFN-aB, chemically cross-linked to their human receptor could be immunoprecipitated by the antisera. IFN ap receptor protein, purified by electrophoresis in sodium dodecylsulfate,was not recognized. We conclude that the antibodies are directed against the forms of the IFN ap receptor actually expressed on the membrane. 1 Introduction A large number of polypeptide cytokines appear to act via cellular receptors that are expressed with high affinity but at a low concentration on the surface of cells [l]. It is, therefore, often difficult to obtain a quantity of receptor that is sufficient for immunization and to present it in such a form that useful antibodies are obtained. Antisera of low potency, that block the biological activity of human IFN-a and -p, have been obtained by i.p. injection of mice with mouse-human cell hybrids containing only human chromosome 21 [2]. It has long been recognized that this chromosome contains a gene responsible for the sensitivity of human cells to human IFN-a and IFN-p and it was postulated that this gene encoded the IFN ap receptor [3, 41. We have recently cloned the cDNA coding for the human IFN ap receptor [5].This was achieved by transfection of a murine-derived hepatoma cell line with human DNA followed by a functional selection of cell clones sensitive to human rIFN-aB.The human receptor was thus expressed in an active form and transfected cells were shown to be sensitive to human IFN-aB. However, transfected cells were less sensitive to human IFN-p and rIFN-aA even though the specific activities of both types of IFN were [I 88791 * Supported zyxwvutsrq by grants from Association pour la Recherche sur le Cancer, Fondation pour la Recherche MCdicale, Centre National de la Recherche Scientifique, INSERM 85/2016, DRET 89- 34-132, ANVAR 8906303 QAT. Correspondence: Knud Mogensen, Laboratoire d’Oncologie Virale, CNRS UPR 274, 7 rue Guy Maquet, F-94801 Villejuif a d e x , France 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991 clearly higher than on the parental mouse cells. This apparent discrepancy suggested that a sub-specificity might exist and that the receptor we had cloned might be one of a class. Here we show that the mouse cells, carrying this receptor, form tumors in histocompatible mice and elicit antibodies which block the binding of IFN-aB to human cells. Furthermore, these antibodies are equally effective in blocking the binding and activity of IFN-p as well as other IFN-a. 2 Materials and methods 2.1 Cell lines and transfections BTG 9A [6] an hypoxanthine phosphoribosyl transferase- negative (HPRT-) line derived from the transplantable mouse hepatoma BW 7756 [7], Daudi (ATCC no CCL 213; American Type Culture Collection, Rockville, MD), a line derived from a Burkitt lymphoma, and Ly28, a line from EBV-transformed human PBL, were grown in RPMI 1640 medium. The human amniotic cell line WISH (ATCC no CCL 25) and the bovine kidney MDBK line (ATCC no CCL 22) were grown in Eagle’s MEM. All cell media were supplemented with 10% FCS, 200 U/ml penicillin, zy 60 pg/ml streptomycin and 2 mM, L-glutamine. Isolation of different BTG 9A transfectants was as described [5]. 2.2 IFN Pure human rIFN-aB (as), -aD (al) and the hybrid rIFN-a BDBB, which contains amino acids 1-60 from rIFN-aB, 61-92 from rIFN-aD and 93-166 from rIFN-aB [8] were a gift from Ciba-Geigy Ltd., Basle. Human rIFN-aA (a2) and human rIFN-y were a gift from C. Weissmann, Univer- sity of Zurich. Human IFN-p prepared from diploid fibroblasts was from Rentschler Arzneimittel GmbH, Laupheim, FGR. The preparation, assay and characteriza- 0014-2980/91/0202-0447$3.50 + .25/0