RRC ID 50081
Author Watanabe N, Suzuki Y, Yonezu T, Nakagawa Y, Shiina T, Hirayama N, Inokuchi S, Inoue S.
Title A cell-based high-throughput screening assay system for inhibitor compounds of antigen presentation by HLA class II molecule.
Journal Sci Rep
Abstract A number of autoimmune diseases are associated with the genotypes of human leukocyte antigen class II (HLA), some of which present peptides derived from self-proteins, resulting in clonal expansion of self-reactive T cells. Therefore, selective inhibition of self-peptide loading onto such disease-associated HLA could ameliorate the diseases. To effectively identify such compounds, in this study, we established, for the first time, a cell- and 96-well microplate-based high-throughput screening system for inhibitors of antigen presentation. A panel of DRB1 genes plus DRA*01:01 gene were expressed in HEK293T cells and in 3T3 cells, and their binding with biotinylated known self-antigen peptides was measured by flow cytometry. HLA-DR1 (DRB1*01:01) and DR15 (DRB1*15:01) showed a high affinity with myelin basic protein peptide (MBP83-98). Therefore, in 96-well plate wells, MBP83-99 was allowed to bind to DR1 or DR15 on 3T3 cells in competition with a test compound, and the HLA-bound peptide was detected by streptavidin-conjugated β-galactosidase, thereby identifying inhibitor compounds for rheumatoid arthritis or multiple sclerosis. Our assay system has a potential for broad applications, including designing peptide vaccines.
Volume 7(1)
Pages 6798
Published 2017-7-28
DOI 10.1038/s41598-017-07080-4
PII 10.1038/s41598-017-07080-4
PMID 28754892
PMC PMC5533769
MeSH 3T3 Cells Animals Antigen Presentation Autoantigens / immunology* HEK293 Cells HLA-DRB1 Chains / immunology* High-Throughput Screening Assays / methods* Humans Mice Myelin Basic Protein / immunology Peptides / immunology
IF 3.998
Times Cited 2
DNA material pCAG-HIVgp (RDB04394) pCMV-VSV-G-RSV-Rev (RDB04393).