Identification of human antigen-specific T cells using MHC class I and class II tetramers.
Major histocompatibility complex (MHC) tetramers typically consist of a fluorophore-streptavidin complex and biotinylated soluble MHC molecules carrying a peptide of interest. Tetramers bind to T cell receptors (TCR) that recognize the MHC molecule/peptide combination with high specificity. Native MHC molecules are expressed as cell-surface glycoproteins capable of binding a variety of peptides generated from the degradation of self and non-self proteins for display to T cells. The human MHC gene locus is highly polymorphic, with >800 class I and >500 class II alleles currently identified. This heterogeneity contributes to the uniqueness of each person's immune system. This unit describes procedures for labeling CD8(+) T cells with MHC class I tetramers and CD4(+) T cells with MHC class II tetramers. The protocols can be used for detecting and enumerating human antigen-specific T cells. Both CD8(+) and CD4(+) antigen-specific T cells are rare events and require that sufficient numbers of cells be evaluated. To minimize nonspecific tetramer binding contributed by irrelevant cell populations, a cumulative gating strategy using positive selection and/or exclusion gating is described.
Krueger
Lori A
LA
Beckman Coulter Inc., San Diego, California, USA.
Nugent
C Thomas
CT
Hampl
Johannes
J
D015496:CD4-Positive T-Lymphocytes; D018414:CD8-Positive T-Lymphocytes; D019411:Clinical Laboratory Techniques; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D015395:Histocompatibility Antigens Class I; D000949:Histocompatibility Antigens Class II; D006801:Humans; D018655:Lymphocyte Count; D015336:Molecular Probe Techniques; D037182:T-Cell Antigen Receptor Specificity; D013601:T-Lymphocytes / Q000166:cytology* / Q000276:immunology*
D005456:Fluorescent Dyes; D015395:Histocompatibility Antigens Class I; D000949:Histocompatibility Antigens Class II