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Blood, Vol. 95 No. 1 (January 1), 2000:
pp. 241-248
Application of the ELISPOT assay to the characterization of
CD8+ responses to Epstein-Barr virus antigens
Jie Yang,
Victor M. Lemas,
Ian W. Flinn,
Chris Krone, and
Richard F. Ambinder
From the Oncology Center, Johns Hopkins University School of
Medicine, Baltimore, MD 21231.
CD8+ cells have an important role in controlling
Epstein-Barr virus (EBV) infection. We adapted the interferon-
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. (Blood. 2000;95:241-248)
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