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Clinical and Vaccine Immunology, July 2009, p. 1066-1073, Vol. 16, No. 7
1071-412X/09/$08.00+0     doi:10.1128/CVI.00365-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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{triangledown}

Zaza M. Ndhlovu,1 Monika Angenendt,2 Diana Heckel,2 Jonathan P. Schneck,2 Diane E. Griffin,1 and Mathias Oelke2*

W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, 615 N Wolfe St., Baltimore, Maryland 21205,1 Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland 212052

Received 7 October 2008/ Returned for modification 20 November 2008/ Accepted 21 May 2009

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-{gamma}) 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-{gamma} 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.

* Corresponding author. Mailing address: Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD 21205. Phone: (410) 502-7128. Fax: (410) 614-3548. E-mail: MOELKE1{at}jhmi.edu

{triangledown} Published ahead of print on 3 June 2009.

Clinical and Vaccine Immunology, July 2009, p. 1066-1073, Vol. 16, No. 7
1071-412X/09/$08.00+0     doi:10.1128/CVI.00365-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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