Freshly isolated T cells from EBV-seropositive healthy donor IP1 react against bulk antigenic formats prepared from autologous EBV B-LCL cells and presented by autologous DCs.

EBV B-LCL–derived bulk antigens (for preparation, see “Materials and methods”) were pulsed onto autologous immature DCs at a ratio of 100 tumor cell equivalents per DC and were screened for reactivity using CD4⁺ and CD8⁺ T-cell responders purified from the blood of donor IP1 (HLA-A2,32; B7,62; Cw3; DR4,15) in IFN-γ Elispot assays. Protein/peptide yields from 10⁹ EBV B-LCL cells were in the range of the following: freeze–thaw lysates, 30 to 50 mg; TFA lysates, 10 to 30 mg; and eluted naturally presented peptides, 0.5 to 1 mg. Control wells contained T cells with untreated DCs. After a culture period of 20 hours, IFN-γ spots were developed and counted by computer-assisted video image analysis. Each bar represents the mean spot number of triplicates ± SD with 10⁵ CD4⁺ T lymphocytes or CD8⁺ T lymphocytes initially seeded per well. The numbers of antigen-reactive T cells per 10⁵ T lymphocytes are calculated by subtraction of mean spot numbers induced by untreated DCs from mean spot numbers induced by antigen-loaded DCs (asterisks indicate significant results, ie, P < .05). No T-cell responses were observed for freeze–thaw and TFA lysate fractions smaller than 10 kd, acid-eluted HLA-A2 peptide fraction 3 kd or larger, and acid-eluted HLA-DR peptide fraction smaller than 3 kd. Spot production was not detected when T cells were incubated with EBV B-LCL–derived bulk antigens in the absence of DCs. Results were confirmed in 4 independent experiments.