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Reliable, efficient systems for producing soluble HLA-DR molecules, suitable for multimerization and use as staining reagents, have proved elusive. We found that the addition of a flexible linker between peptide and N terminus of the DRB1*0101-chain (Crawford, F., Kozono, H., White, J., Marrack, P. and Kappler, J., Immunity 1998. 8: 675-682.), results in greater in vitro folding efficiency of Escherichia coli-expressed alpha- and beta-chains, and increases both the yield and stability of the DRA1*0101/DRB1*0101/peptide complexes. Although a 10-amino acid linker functioned efficiently for a 20mer epitope from HIV p24, a longer linker was required to produce a DR1 MHC class II tetramer with the influenza hemagglutinin epitope (HA(306-318)). The DR1-HA tetramer was able to stain positively over 98% of a specific clone (HA 1.7) with only a brief 30-min incubation. The tetrameric complexes detected clone cells diluted into PBMC, with high sensitivity, coupled with low background staining in CD4(+) cells. It was possible to detect antigen-specific CD4(+) T cells within a population of PBMC stimulated with the HA peptide. This demonstrates the potential to monitor CD4(+) T cell responses in peripheral blood in a number of clinical scenarios.

Original publication

DOI

10.1002/1521-4141(200212)32:12<3366::AID-IMMU3366>3.0.CO;2-#

Type

Journal article

Journal

Eur J Immunol

Publication Date

12/2002

Volume

32

Pages

3366 - 3375

Keywords

CD4-Positive T-Lymphocytes, Clone Cells, Flow Cytometry, HLA-DR Antigens, HLA-DR Serological Subtypes, HLA-DR1 Antigen, Humans, Interferon-gamma, Leukocytes, Mononuclear, Macromolecular Substances, Staining and Labeling