Optimization of peptide linker length in production of MHC class II/peptide tetrameric complexes increases yield and stability, and allows identification of antigen-specific CD4+T cells in peripheral blood mononuclear cells.

Journal article

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.

Authors: Cunliffe, S; Wyer, JR; Sutton, J; Lucas, M; Harcourt, G

• Publication status: Published
• Journal: European journal of immunology
• Volume: 32
• Issue: 12
• Pages: 3366-3375
• Publication date: 2002-12-01
• DOI: 10.1002/1521-4141(2002012)32:12<3366::aid-immu3366>3.0.co;2-i
• EISSN: 1521-4141
• ISSN: 0014-2980
• Language: English
• Keywords: HLA-DR1 Antigen; HLA-DR Serological Subtypes; CD4-Positive T-Lymphocytes; Flow Cytometry; Macromolecular Substances; Leukocytes, Mononuclear; Clone Cells; Interferon-gamma; Humans; HLA-DR Antigens; Staining and Labeling