Analysis Of CD4+ And CD8+ Epstein-Barr Virus-Specific Polyfunctional T Cell Responses In Chinese Long Term Carriers By A Novel 9-Colour Flow Cytometric Assay

Abstract

Recent evidence shows that polyfunctional CD4+ and CD8+ T cells capable of secreting multiple cytokines simultaneously are the protective immune correlates of different viral infections. Here, we developed a novel 9-color flow cytometric assay for analysis of virusspecific CD4+ and CD8+ T cell responses to EBV infection. Overlapping peptide pools of four EBV latent proteins (EBNA1, EBNA3A, EBNA3B and LMP2) and one lytic protein (BZLF1) were used to stimulate peripheral blood mononuclear cells (PBMCs) of ten healthy Chinese EBV-seropositive long term carriers. After 6-hour stimulation by the peptides, PBMCs were stained sequentially by aqua blue dye (for exclusion of dead cells) and antibodies to surface markers (CD3-APC-Cy7, CD4-PE-Texas Red and CD8-Pacific Blue), cytotoxic marker (CD107a-PE-Cy5) and cytokines (interferon-g [IFN-g]-FITC, interleukin-2 [IL-2]-APC, macrophage inhibitory protein 1-a [MIP1]-a-PE and tumour necrosis factor-a [TNF-a]-PE-Cy7), and analyzed by an optimized 9-colour flow cytometric assay using BDFACS LSR-II. Positive (PBMCs stimulated by Staphylococcal Enterotoxin B), negative (unstimulated and unstained PBMCs) and biological (unstimulated but stained PBMCs) controls were included for each study subject. Polyfunctional CD4+ and CD8+ T cells (defined as those that secrete three or more cytokines) reactive to EBV latent and lytic peptides could be clearly demonstrated in the long term carriers: CD4+ T cells secrete three (IFN-g, TNF-a and MIP1-a) or all four cytokines whereas CD8+ T cells secrete three cytokines (IFN-g, TNF-a and MIP1-a) and have a cytotoxic function (CD107a expression). The new assay can be applied to the study of EBV-specific polyfunctional T cell responses in EBV-related clinical diseases.