Development of polychromatic flow cytometry assay to study polyfunctional Epstein-Barr Virus (EBV)-specific CD8+ cytotoxic T-lymphocyte responses

Aim: Development of effective virus-specific CD8+ cytotoxic T lymphocyte (CTL) responses is thought to be crucial in the long term control of Epstein-Barr virus (EBV). We aim to establish a polychromatic flow cytometry assay to study the development of polyfunctional EBV-specific CD8+ CTL responses. Methods: Staphylococcal enterotoxin B (SEB) was used to stimulate cryopreserved peripheral blood mononuclear cells (PBMC) of healthy donors to simulate cytokine and chemokine secretion. Intracellular cytokine assay for three cytokines (IFN-γ, TNF-α and IL2) and one chemokine (MIP1-α) was developed. Cells were stained simultaneously for aqua blue dye, CD3, CD8, CD27, CD45RO and the three cytokines and one chemokine and analyzed by nine-color flow cytometry protocol on BD FACSAria. Setting of PMT voltage of each channel, compensation adjustments and titration of antibodies were performed. Biological (no SEB or peptide stimulation) and FMO (fluorescence minus one) controls served as negative controls. Results: PMT voltage of each channel was determined by unstained PBMC. Compensation beads labeled with antibody of each color showed strong positive signals which were used for compensation adjustments. Aqua blue dye labeled the dead cells. Serial titration of each antibody against the cytokines was essential to determine the optimal concentration for high positive signal to low background. The background of the biological control was as low as 0.001% while the SEB-stimulated positive control population ranged from 1-20% in different samples. FMO control was used to guide the gating of CD45RO and CD27 expression. Conclusion: A sensitive polychromatic flow cytometry assay for the evaluation of polyfunctional EBV-specific CTL is established. Single EBV peptide is used to stimulate cryopreserved PBMC of primary infection subjects with known EBV peptide responses