HYBRIDOMA Volume 11, Number 5, 1992 Mary Ann Liebert, Inc., Publishers
Production of a Monoclonal Antibody That Defines the a-Subunit of the Feline IL-2 KOICHI ATSUHIKO
Receptor
OHNO,1'2 RYO GOITSUKA,2 KATSUHIKO KIT AMURA,1 HASEGAWA,2 TOHRU TOKUNAGA,1 and MITSUO HONDA"
'Laboratory of Immunology, AIDS Research Center,
National Institute
of Health, Tokyo 141, Japan
2Department of Veterinary Internal Medicine, Faculty of Agriculture, University of Tokyo, Tokyo 113
ABSTRACT A mAb, termed 9F23, to feline Con A-stimulated PBMC was prepared to characterize feline IL-2R. 9F23 was identified by FACS studies, which showed that the antigen was expressed at a high density on Con A-induced feline T cell blasts while 9F23 binding was not detected on nonactivated PBMC or the Crandell feline kidney cell line CRFK. Chemical crosslinking of 125I-IL-2 to membrane IL-2Rs on Con A-stimulated feline PBMC under the low-affinity condition resulted in detection of a major 65-kDa band. 9F23 specifically immunoprecipitated the lL-2-IL-2Ra complex in a cell extract; in contrast, neither anti-human IL-2Ra H48 nor anti-mouse IL-2Ra 7D4 reacted with the complex. Moreover, immunoprecipitation with 9F23 of the extract from surface-iodinated Con A-stimulated PBMC showed a major 50-55 kDa band. Furthermore, 9F23 had no effect on either IL-2-driven proliferation of the Con Astimulated PBMC or IL-2 binding. Finally, the expression of feline IL-2Ra on Con Astimulated PBMC was up-regulated by addition of exogenous IL-2. Thus, 9F23 defines an epitope different from the IL-2 binding site on the a-subunit of feline IL-2R.
antibody; HTLV, human T-lymphotropic virus; FIV, feline feline acquired immunodeficiency syndrome; FeLV, feline leukemia virus; CRFK, Crandell feline kidney cell line. Abbreviations: mAb, monoclonal
Immunodeficiency virus; FAIDS,
INTRODUCTION Activation of mature T lymphocytes by an antigen or a mitogen induces the induction of highaffinity receptors for IL-2 (1). The interaction of IL-2 with its receptor drives the clonal expansion of antigen-reactive T cells and consequently determines the magnitude and duration of a T cell response (1-3). Moreover, three classes of IL-2 receptors that differ in their affinity for IL-2 have been defined by ligand binding studies: high-affinity receptors (kd=10"nM, 2, 4), intermediateaffinity receptors (kd=109M, 5,6) and low-affinity IL-2 receptors (kd=10"8M, 2,4). The availability of mAbs to human (7-10), mouse (11) and rat (12) IL-2 receptors has provided a better understanding of the structure and function of this receptor, which is composed of at least two different IL-2 binding glycoproteins, a 55- to 60-kDa (p58) protein referred to the a-chain (IL-2R
Production of a Monoclonal Antibody That Defines the a-Subunit of the Feline IL-2 KOICHI ATSUHIKO
Receptor
OHNO,1'2 RYO GOITSUKA,2 KATSUHIKO KIT AMURA,1 HASEGAWA,2 TOHRU TOKUNAGA,1 and MITSUO HONDA"
'Laboratory of Immunology, AIDS Research Center,
National Institute
of Health, Tokyo 141, Japan
2Department of Veterinary Internal Medicine, Faculty of Agriculture, University of Tokyo, Tokyo 113
ABSTRACT A mAb, termed 9F23, to feline Con A-stimulated PBMC was prepared to characterize feline IL-2R. 9F23 was identified by FACS studies, which showed that the antigen was expressed at a high density on Con A-induced feline T cell blasts while 9F23 binding was not detected on nonactivated PBMC or the Crandell feline kidney cell line CRFK. Chemical crosslinking of 125I-IL-2 to membrane IL-2Rs on Con A-stimulated feline PBMC under the low-affinity condition resulted in detection of a major 65-kDa band. 9F23 specifically immunoprecipitated the lL-2-IL-2Ra complex in a cell extract; in contrast, neither anti-human IL-2Ra H48 nor anti-mouse IL-2Ra 7D4 reacted with the complex. Moreover, immunoprecipitation with 9F23 of the extract from surface-iodinated Con A-stimulated PBMC showed a major 50-55 kDa band. Furthermore, 9F23 had no effect on either IL-2-driven proliferation of the Con Astimulated PBMC or IL-2 binding. Finally, the expression of feline IL-2Ra on Con Astimulated PBMC was up-regulated by addition of exogenous IL-2. Thus, 9F23 defines an epitope different from the IL-2 binding site on the a-subunit of feline IL-2R.
antibody; HTLV, human T-lymphotropic virus; FIV, feline feline acquired immunodeficiency syndrome; FeLV, feline leukemia virus; CRFK, Crandell feline kidney cell line. Abbreviations: mAb, monoclonal
Immunodeficiency virus; FAIDS,
INTRODUCTION Activation of mature T lymphocytes by an antigen or a mitogen induces the induction of highaffinity receptors for IL-2 (1). The interaction of IL-2 with its receptor drives the clonal expansion of antigen-reactive T cells and consequently determines the magnitude and duration of a T cell response (1-3). Moreover, three classes of IL-2 receptors that differ in their affinity for IL-2 have been defined by ligand binding studies: high-affinity receptors (kd=10"nM, 2, 4), intermediateaffinity receptors (kd=109M, 5,6) and low-affinity IL-2 receptors (kd=10"8M, 2,4). The availability of mAbs to human (7-10), mouse (11) and rat (12) IL-2 receptors has provided a better understanding of the structure and function of this receptor, which is composed of at least two different IL-2 binding glycoproteins, a 55- to 60-kDa (p58) protein referred to the a-chain (IL-2R
