title: ELISPOT and functional T cell analyses using HLA mono-specific target cells.
abstract: Simple T cell assays specific for any chosen HLA class I or class II/peptide combination, are of enormous value in cancer immunotherapy, clinical trials, vaccine and infectious disease research. The reliable measurement of T cell activity can be difficult due to the presence of other alleles on target cells, particularly for the non-HLA-A2 alleles, and the varying baseline characteristics of the different APCs employed. In the absence of pulsing with HLA-A2 restricted peptides, T2 cells are functionally HLA class I and II negative. By coating these cells with recombinant HLA peptide complexes, HLA mono-specific cells are produced that present only a defined single epitope, and generate minimal background immune activation. In ELISPOT, intracellular cytokine staining (ICS) and killing assays using T cells specific for HLA-A2/peptide complexes, the HLA mono-specific cells gave comparable results, to those using standard peptide pulsed HLA-A2 positive T2 cells without significant background. Successful T cell assays for non-HLA-A2 T cells were also performed, with PBMCs recognizing HLA-A24 and HLA-DR15/peptide complexes. The data, obtained with ELISPOT, ICS and FACS-based killing assays, all demonstrate high specificity of T cell activity and low levels of background activity. HLA mono-specific cells are simple to prepare, and can be used with any stable recombinant HLA allele/peptide combination; providing a useful system for improved T cell functional analyses across all HLA allotypes. This represents a significant advance in the generation of reliable functional T cell data.
authors.affiliation: Department of Immunology, Imperial College, London W12 0NN, United Kingdom.
authors.forename: Claire
authors.initials: C
authors.lastname: Horlock
authors.forename: Bryony
authors.initials: B
authors.lastname: Stott
authors.forename: Julian
authors.initials: J
authors.lastname: Dyson
authors.forename: Graham
authors.initials: G
authors.lastname: Ogg
authors.forename: Tess
authors.initials: T
authors.lastname: McPherson
authors.forename: Louise
authors.initials: L
authors.lastname: Jones
authors.forename: Andrew K
authors.initials: AK
authors.lastname: Sewell
authors.forename: Linda
authors.initials: L
authors.lastname: Wooldridge
authors.forename: David K
authors.initials: DK
authors.lastname: Cole
authors.forename: Justin
authors.initials: J
authors.lastname: Stebbing
authors.forename: Philip
authors.initials: P
authors.lastname: Savage
mesh_terms: D015234:HLA-A Antigens; D059871:HLA-A24 Antigen; D006684:HLA-DR Antigens / Q000276:immunology*; D059810:HLA-DR Serological Subtypes; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods; D010455:Peptides / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D015234:HLA-A Antigens; D059871:HLA-A24 Antigen; D006684:HLA-DR Antigens; D059810:HLA-DR Serological Subtypes; C106559:HLA-DR15 antigen; D010455:Peptides