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

Abstract

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.

FIG. 1.

(A) Schematic of aAPCs, followed by peptide loading as described in Materials and Methods. aAPCs were made by immobilizing HLA Ig and anti-CD28 onto magnetic beads. (B) Schematic representation of aAPC-qRT-PCR based assay for detection of CD8⁺ T-cell reactivity. One hundred microliters of whole blood was added to a vial containing 1 × 10⁶ aAPCs in a total volume of 400 μl PBS. After incubation at 37°C for 2.5 h with rotation, RNA was isolated and subsequently reverse transcribed. qRT-PCR was performed to amplify CD8 and IFN-γ cDNA using primers and TaqMan probes specific for CD8 and IFN-γ, respectively. The circled R and Q represent the reporter and quencher dyes that are at the beginning and end of the TaqMan probes.

FIG. 2.

Evaluation of the aAPC-qRT-PCR-based assay using the CMV-specific CD8⁺ T-cell system. Seven donors were tested for CMV-specific CD8⁺ T-cell responses using the aAPC-qRT-PCR assay. (A) One hundred microliters of whole blood per stimulation condition was analyzed with the aAPC-qRT-PCR-based assay for activation and detection of CMV-specific T-cell responses. Asterisks mark all values considered positive. For details, see Materials and Methods. (B) Four-color ICS was performed on whole-blood samples after 6 h of stimulation with CMV aAPCs. The frequencies of CMV-specific CD8⁺ T cells were assessed by gating on CD3, CD8, and IFN-γ triple-positive cells. (C) The CMV specificity for stimulating CD8⁺T cells in whole blood was tested by assessing the frequency of IFN-γ-producing CD4⁺T cells. CD3, CD4, and IFN-γ triple-positive cells are gated.

FIG. 3.

Analysis of measles-reactive cells. Five HLA-A2-positive donors were analyzed for MV peptide-specific memory CD8⁺ T cells using the aAPC-qRT-PCR method. Blood samples were collected and tested using the aAPC-qRT-PCR assay for MV peptide reactivity as indicated. (A) Mean levels of IFN-γ mRNA expression for the three repeat experiments are reported. Asterisks mark all values considered positive; for details, see Materials and Methods. The frequency of MV peptide-specific CD8⁺ T cells was also assessed by staining whole blood with multimers loaded with the MV peptides listed in Table 1. Representative multimer staining results for donor 1 (B) and donor 2 (C) are reported.