title: 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.
abstract: 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.affiliation: MRC Human Immunology Unit, W.I.M.M., J.R. Hospital, Oxford, GB.
authors.forename: Sharon L
authors.initials: SL
authors.lastname: Cunliffe
authors.forename: Jessica R
authors.initials: JR
authors.lastname: Wyer
authors.forename: Julian K
authors.initials: JK
authors.lastname: Sutton
authors.forename: Michaela
authors.initials: M
authors.lastname: Lucas
authors.forename: Gillian
authors.initials: G
authors.lastname: Harcourt
authors.forename: Paul
authors.initials: P
authors.lastname: Klenerman
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: McMichael
authors.forename: Anthony D
authors.initials: AD
authors.lastname: Kelleher
mesh_terms: D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D002999:Clone Cells; D005434:Flow Cytometry; D006684:HLA-DR Antigens / Q000737:chemistry* / Q000302:isolation & purification; D059810:HLA-DR Serological Subtypes; D015798:HLA-DR1 Antigen / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000276:immunology; D046911:Macromolecular Substances; D013194:Staining and Labeling
chemical_list: D006684:HLA-DR Antigens; D059810:HLA-DR Serological Subtypes; D015798:HLA-DR1 Antigen; C080766:HLA-DR11 antigen; D046911:Macromolecular Substances; D007371:Interferon-gamma