T cell epitope-mapping by cytokine gene expression assay.
The following method describes the identification of candidate immunogenic peptides through their ability to recall an immune T-cell activation from peripheral blood mononuclear cells (PBMCs) of individuals with defined HLA-peptide restrictions that have been previously exposed to the antigen. Isolated PBMCs are plated out at a concentration of 1 x 10(6) cells/ml in a 200 microl medium and incubated overnight to reduce cytokine gene expression due to cell manipulation. After starving, cells are either directly stimulated with individual peptides or not stimulated and incubated from 3 to 12 h according to experimental conditions. Quantitative real-time PCR (qrt-PCR) is performed on reverse-transcribed complementary DNA (cDNA) from total RNA that is isolated from peptide-cultured PBMCs. To perform high quality qrt-PCR, primers and probes are designed to span exon-intron junctions in order to prevent amplification of genomic DNA and to produce amplicons <150 base pairs (bp). Real-time monitoring of fluorescent emission from the cleavage of sequence specific probe by the nuclease activity of Taq polymerase (TaqMan method) defines threshold cycles during exponential phases of amplification. Standard curves of copy numbers of the gene of interest are normalized using as reference copy numbers of control genes.
Provenzano
Maurizio
M
Department of Urology, University Hospital of Zurich, Zurich, Switzerland.
Spagnoli
Giulio C
GC
D000199:Actins / Q000378:metabolism; D016207:Cytokines / Q000235:genetics*; D004247:DNA / Q000235:genetics; D017931:DNA Primers / Q000378:metabolism; D018604:Epitope Mapping / Q000379:methods*; D018984:Epitopes, T-Lymphocyte / Q000032:analysis* / Q000276:immunology* / Q000494:pharmacology; D018628:Gene Dosage; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects / Q000276:immunology*; D015894:Genome, Human; D006801:Humans; D010455:Peptides; D012313:RNA / Q000235:genetics; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism*
D000199:Actins; D016207:Cytokines; D017931:DNA Primers; D018984:Epitopes, T-Lymphocyte; D010455:Peptides; D012313:RNA; D004247:DNA