Open AccessDevelopment of an Artificial-Antigen-Presenting-Cell-Based Assay for the Detection of Low-Frequency Virus-Specific CD8+T Cells in Whole Blood, with Application for Measles Virus
Author(s) -
Zaza M. Ndhlovu,
M. Angenendt,
Diana Heckel,
Jonathan P. Schneck,
Diane E. Griffin,
Mathias Oelke
Publication year - 2009
Publication title -
clinical and vaccine immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.649
H-Index - 77
eISSN - 1556-6811
pISSN - 1556-679X
DOI - 10.1128/cvi.00365-08
Subject(s) - biology , antigen , cytotoxic t cell , measles virus , immune system , cd8 , virology , elispot , t cell , microbiology and biotechnology , immunology , measles , in vitro , vaccination , biochemistry
Evaluation of the immune responses induced by childhood vaccines requires measurement of T-cell, as well as antibody, responses. However, cellular immune responses are often not analyzed because of technical hurdles and the volume of blood required. Therefore, a sensitive and specific assay for antigen-specific T cells that utilizes a small volume of blood would facilitate new vaccine evaluation. We developed a novel assay for quantifying virus-specific CD8 + T cells that combines the use of HLA-A2 immunoglobulin-based artificial antigen-presenting cells (aAPCs) for stimulation of antigen-specific CD8 + T cells in whole blood with quantitative real-time reverse transcription-PCR (qRT-PCR) to detect gamma interferon (IFN-γ) mRNA. This assay was optimized using a well-established cytomegalovirus (CMV) CD8 + T-cell system. The aAPC-qRT-PCR assay had comparable sensitivity to intracellular cytokine staining (ICS) in detecting CMV-specific CD8 + T cells with a detection limit of less than 0.004%. The assay was applied to the detection of low-frequency measles virus (MV)-specific CD8 + T cells by stimulating blood from five MV-immune HLA-A*0201 donors with four different MV-specific peptides (MV peptide aAPCs). Stimulation with three of the MV peptide aAPCs resulted in significant increases in IFN-γ mRNA ranging from 3.3- to 13.5-fold. Our results show that the aAPC-qRT-PCR assay is highly sensitive and specific and can be standardized for screening MV-specific CD8 + T cells in vaccine trials. The technology should be transferable to analysis of CD8 + T-cell responses to other antigens.