Application of the ELISPOT assay to the characterization of CD8⁺ responses to Epstein-Barr virus antigens

CD8⁺ cells have an important role in controlling Epstein-Barr virus (EBV) infection. We adapted the interferon-γ ELISPOT assay to the quantitative analysis of EBV-specific CD8⁺ cells. Using peripheral blood mononuclear cells (PBMCs) from healthy donors, we measured both the aggregate response to the virus, using EBV-transformed lymphoblastoid cell lines (LCLs) as stimulators, and the specific responses to 2 A2-restricted peptide epitopes: the subdominant latency membrane protein-2 (LMP2) peptide CLGGLLTMV and the early lytic BMLF1 peptide GLCTLVAML. LCL-responsive CD8⁺ cells were detected in all EBV-seropositive donors (range 954 to 37 830 spots/10⁶ CD8⁺ cells). LMP2 peptide-responsive CD8⁺ cells were detected in 10 of 11 healthy seropositive A2 donors (range 11 to 83 spots/10⁶ PBMC). BMLF1 peptide- responsive CD8⁺ cells were detected in all seropositive A2 donors examined (range 13 to 943 spots/10⁶ PBMC). Cytotoxic T-lymphocyte (CTL) lines generated with weekly stimulation of LCLs for therapeutic purposes were also studied. Relative to PBMCs, these CTL lines showed a marked increase in the level of LCL-responsive and LMP2 peptide-responsive CD8⁺ cells and a lesser degree of expansion of BMLF1 peptide-responsive CD8⁺ cells. Finally, we applied the ELISPOT assay to monitor adoptive infusion of EBV CTL lines. In 2 patients examined, a transient increase in LCL-responsive CD8⁺ cells could be detected after infusion. Thus, the ELISPOT assay can be applied to the analysis of CD8⁺ responses to EBV antigens in PBMCs, in ex vivo expanded CTL lines, and in PBMCs from patients treated with ex vivo expanded CTL lines.