Identification and initial characterization of a rat monoclonal antibody reactive with the murine interleukin 2 receptor-ligand complex

Thomas Malek, R. J. Robb, E. M. Shevach

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Abstract

Xenogeneic monoclonal antibodies were prepared to the murine interleukin 2 (IL-2)-dependent HT2 cell line. One rat IgM monoclonal antibody (7D4) was identified that inhibited proliferation of the HT2 cells and of IL-2-dependent CTLL cells in the presence of crude rat IL-2 as well as of purified human IL-2. The level of inhibition was dependent on both antibody and IL-2 concentration. Cell distribution studies using a fluorescence-activated cell sorter showed that the antigen identified by 7D4 is expressed at a high density on HT2 cells and on concanavalin A (Con A)-induced T-cell blasts and at a substantially lower density on lipopolysaccharide-induced B-cell blasts; 7D4 binding was not detected on > 95% of nonactivated thymocytes, T cells, or B cells. Competition binding studies indicated that 7D4 fails to inhibit the binding of 3H-labeled human IL-2 to CTLL cells. However, 7D4 specifically immunoprecipitated 3H-labeled human IL-2 from detergent extracts of HT2 cells or Con A-induced T-cell blasts that had been pulsed with [3H]IL-2; in contrast, 7D4 did not react with free [3H]IL-2. Initial biochemical analysis of immunoprecipitates with 7D4 of detergent extracts from surface-iodinated Con A-activated spleen cells showed a major band having apparent molecular weight of 48,000-62,000. Collectively, these results suggest that 7D4 detects an epitope on the IL-2 receptor distal to the ligand binding site or another molecule that physically associates with the receptor.

Original languageEnglish
Pages (from-to)5694-5698
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume80
Issue number181
StatePublished - Dec 1 1983
Externally publishedYes

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Interleukin-2 Receptors
Interleukin-2
Monoclonal Antibodies
Ligands
Concanavalin A
T-Lymphocytes
Detergents
B-Lymphocytes
Heterophile Antibodies
Thymocytes
Cell Extracts
Immunoglobulin M
Epitopes
Spleen
Molecular Weight
Fluorescence
Binding Sites
Cell Proliferation
Antigens
Cell Line

ASJC Scopus subject areas

  • General
  • Genetics

Cite this

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title = "Identification and initial characterization of a rat monoclonal antibody reactive with the murine interleukin 2 receptor-ligand complex",
abstract = "Xenogeneic monoclonal antibodies were prepared to the murine interleukin 2 (IL-2)-dependent HT2 cell line. One rat IgM monoclonal antibody (7D4) was identified that inhibited proliferation of the HT2 cells and of IL-2-dependent CTLL cells in the presence of crude rat IL-2 as well as of purified human IL-2. The level of inhibition was dependent on both antibody and IL-2 concentration. Cell distribution studies using a fluorescence-activated cell sorter showed that the antigen identified by 7D4 is expressed at a high density on HT2 cells and on concanavalin A (Con A)-induced T-cell blasts and at a substantially lower density on lipopolysaccharide-induced B-cell blasts; 7D4 binding was not detected on > 95{\%} of nonactivated thymocytes, T cells, or B cells. Competition binding studies indicated that 7D4 fails to inhibit the binding of 3H-labeled human IL-2 to CTLL cells. However, 7D4 specifically immunoprecipitated 3H-labeled human IL-2 from detergent extracts of HT2 cells or Con A-induced T-cell blasts that had been pulsed with [3H]IL-2; in contrast, 7D4 did not react with free [3H]IL-2. Initial biochemical analysis of immunoprecipitates with 7D4 of detergent extracts from surface-iodinated Con A-activated spleen cells showed a major band having apparent molecular weight of 48,000-62,000. Collectively, these results suggest that 7D4 detects an epitope on the IL-2 receptor distal to the ligand binding site or another molecule that physically associates with the receptor.",
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T1 - Identification and initial characterization of a rat monoclonal antibody reactive with the murine interleukin 2 receptor-ligand complex

AU - Malek, Thomas

AU - Robb, R. J.

AU - Shevach, E. M.

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Y1 - 1983/12/1

N2 - Xenogeneic monoclonal antibodies were prepared to the murine interleukin 2 (IL-2)-dependent HT2 cell line. One rat IgM monoclonal antibody (7D4) was identified that inhibited proliferation of the HT2 cells and of IL-2-dependent CTLL cells in the presence of crude rat IL-2 as well as of purified human IL-2. The level of inhibition was dependent on both antibody and IL-2 concentration. Cell distribution studies using a fluorescence-activated cell sorter showed that the antigen identified by 7D4 is expressed at a high density on HT2 cells and on concanavalin A (Con A)-induced T-cell blasts and at a substantially lower density on lipopolysaccharide-induced B-cell blasts; 7D4 binding was not detected on > 95% of nonactivated thymocytes, T cells, or B cells. Competition binding studies indicated that 7D4 fails to inhibit the binding of 3H-labeled human IL-2 to CTLL cells. However, 7D4 specifically immunoprecipitated 3H-labeled human IL-2 from detergent extracts of HT2 cells or Con A-induced T-cell blasts that had been pulsed with [3H]IL-2; in contrast, 7D4 did not react with free [3H]IL-2. Initial biochemical analysis of immunoprecipitates with 7D4 of detergent extracts from surface-iodinated Con A-activated spleen cells showed a major band having apparent molecular weight of 48,000-62,000. Collectively, these results suggest that 7D4 detects an epitope on the IL-2 receptor distal to the ligand binding site or another molecule that physically associates with the receptor.

AB - Xenogeneic monoclonal antibodies were prepared to the murine interleukin 2 (IL-2)-dependent HT2 cell line. One rat IgM monoclonal antibody (7D4) was identified that inhibited proliferation of the HT2 cells and of IL-2-dependent CTLL cells in the presence of crude rat IL-2 as well as of purified human IL-2. The level of inhibition was dependent on both antibody and IL-2 concentration. Cell distribution studies using a fluorescence-activated cell sorter showed that the antigen identified by 7D4 is expressed at a high density on HT2 cells and on concanavalin A (Con A)-induced T-cell blasts and at a substantially lower density on lipopolysaccharide-induced B-cell blasts; 7D4 binding was not detected on > 95% of nonactivated thymocytes, T cells, or B cells. Competition binding studies indicated that 7D4 fails to inhibit the binding of 3H-labeled human IL-2 to CTLL cells. However, 7D4 specifically immunoprecipitated 3H-labeled human IL-2 from detergent extracts of HT2 cells or Con A-induced T-cell blasts that had been pulsed with [3H]IL-2; in contrast, 7D4 did not react with free [3H]IL-2. Initial biochemical analysis of immunoprecipitates with 7D4 of detergent extracts from surface-iodinated Con A-activated spleen cells showed a major band having apparent molecular weight of 48,000-62,000. Collectively, these results suggest that 7D4 detects an epitope on the IL-2 receptor distal to the ligand binding site or another molecule that physically associates with the receptor.

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