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

Author:

Ndhlovu Zaza M.12,Angenendt Monika12,Heckel Diana12,Schneck Jonathan P.12,Griffin Diane E.12,Oelke Mathias12

Affiliation:

1. W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, 615 N Wolfe St., Baltimore, Maryland 21205

2. Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Abstract

ABSTRACTEvaluation 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.

Publisher

American Society for Microbiology

Subject

Microbiology (medical),Clinical Biochemistry,Immunology,Immunology and Allergy

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