Development 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

摘要

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.