Ex vivo screening for immunodominant viral epitopes by quantitative real time polymerase chain reaction (qRT-PCR)
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BioMed Central
Open Access
Methodology
Ex vivo screening for immunodominant viral epitopes by quantitative real time polymerase chain reaction (qRT-PCR) Maurizio Provenzano*1, Simone Mocellin2, Paola Bonginelli1,4, Dirk Nagorsen1, Seog-Woon Kwon3 and David Stroncek1 Address: 1Molecular Immunology section, Department of Transfusion Medicine, Clinical Center, NIH, Bethesda, USA, 2Surgery Branch, Department of Oncological and Surgical Science, University of Padova, Italy, 3Blood Banks, Department of Medicine, Asan Medical Center, Seoul, Korea and 4Current address: Department of Oncology, Division of Medical Oncology, San Filippo Neri, Via Martinotti 20, 00135, Rome, Italy Email: Maurizio Provenzano* - [email protected]; Simone Mocellin - [email protected]; Paola Bonginelli - [email protected]; Dirk Nagorsen - [email protected]; Seog-Woon Kwon - [email protected]; David Stroncek - [email protected] * Corresponding author
Published: 15 December 2003 Journal of Translational Medicine 2003, 1:12
Received: 27 October 2003 Accepted: 15 December 2003
This article is available from: http://www.translational-medicine.com/content/1/1/12 © 2003 Provenzano et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.
qRT-PCRHLAviral peptidememory T lymphocytescytokines
Abstract The identification and characterization of viral epitopes across the Human Leukocyte Antigen (HLA) polymorphism is critical for the development of actives-specific or adoptive immunotherapy of virally-mediated diseases. This work investigates whether cytokine mRNA transcripts could be used to identify epitope-specific HLA-restricted memory T lymphocytes reactivity directly in fresh peripheral blood mononuclear cells (PBMCs) from viral-seropositive individuals in response to ex vivo antigen recall. PBMCs from HLA-A*0201 healthy donors, seropositive for Cytomegalovirus (CMV) and Influenza (Flu), were exposed for different periods and at different cell concentrations to the HLA-A*0201-restricted viral FluM158–66 and CMVpp65495–503 peptides. Quantitative real time PCR (qRT-PCR) was employed to evaluate memory T lymphocyte immune reactivation by measuring the production of mRNA encoding four cytokines: Interferon-γ (IFN-γ), Interleukin-2 (IL-2), Interleukin-4 (IL-4), and Interleukin-10 (IL-10). We could characterize cytokine expression kinetics that illustrated how cytokine mRNA levels could be used as ex vivo indicators of T cell reactivity. Particularly, IFN-γ mRNA transcripts could be consistently detected within 3 to 12 hours of short-term stimulation in levels sufficient to screen for HLA-restricted viral immune responses in seropositive subjects. This strategy will enhance the efficiency of the identification of viral epitopes independently of the individual HLA phenotype and could be used to follow the intensity of immune responses duri
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