title: Monitoring antigen-specific T cell responses using real-time PCR.
abstract: Flow cytometry-, ELISA-, and ELISpot-based in vitro assays have played important roles in assessing the frequencies and functional competence of antigen-specific T cells in the setting of infectious disease and cancer. Such methods have helped in the development of antigen-specific vaccines for human disease prevention/treatment and have also served as a foundation for the monitoring of patients' immune responsiveness based on antigen-induced T cell expression of effector molecules (such as cytokines, chemokines, or proteins associated with cytolysis) as a consequence of therapeutic intervention. The following method outlines a protocol employing quantitative real-time PCR (qRT-PCR) with SYBR(®) green technology to examine antigen-specific CD8(+) T cell responses based on their rapid up-regulation of IFN-γ mRNA transcription following in vitro stimulation with peptide (antigen)-loaded, autologous peripheral blood mononuclear cells (PBMCs). The advantages of the current qRT-PCR approach over protein-based detection methods include the sensitivity to distinguish resident CD8(+) T cell responses against multiple antigens without the need to artificially pre-expand T cell numbers ex vivo, as is commonly required for the latter in vitro assay systems. Following qRT-PCR setup and run, the level of human IFN-γ transcript is normalized to CD8 transcript expression level, with data reported as the relative fold change in this index versus a patient-matched PBMC sample stimulated with a negative control peptide (e.g., HIV NEF).
authors.affiliation: Department of Dermatology, University of Pittsburgh School of Medicine, W1003 Biomedical Sciences Tower, 200 Lothrop Street, Pittsburgh, PA, 15213, USA.
authors.forename: Devin B
authors.initials: DB
authors.lastname: Lowe
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Taylor
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Storkus
mesh_terms: D052160:Benzothiazoles; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D002292:Carcinoma, Renal Cell / Q000276:immunology*; D003959:Diamines; D018984:Epitopes, T-Lymphocyte / Q000276:immunology; D015789:HLA-A2 Antigen / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics*; D007680:Kidney Neoplasms / Q000276:immunology*; D007963:Leukocytes, Mononuclear / Q000276:immunology; D009930:Organic Chemicals; D011804:Quinolines; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D018984:Epitopes, T-Lymphocyte; D015789:HLA-A2 Antigen; D009930:Organic Chemicals; D011804:Quinolines; D012333:RNA, Messenger; C098022:SYBR Green I; D007371:Interferon-gamma