FIGURE 4

Recognition of DB-treated target cells by specific CTL. (a) ELISPOT analysis of HFF, incubated with DB. HLA-A0201 positive MRC-5 fibroblasts (110⁶) (ATCC CCL-171) were either mock treated, incubated for 16 h with 20 g DB, infected with HCMV strain Ad169 over night at a moi of 10, or labeled with synthetic peptide pp65_495-503 at 10^-8 M, respectively, and used as target cells in Interferon- (IFN-) specific ELISPOT analyses. 110⁵ Target cells were seeded per well of a microtiter plate and incubated with 100 effector cells for 20 h. As effectors, a murine polyclonal CTL line (CTLL) specific for the pp65-derived peptide epitope pp65_495-503 presented by human HLA-A0201 (pp65-CTLL) was used. CTLL were generated by immunizing HLA-A2/CD8 double-transgenic mice (a kind gift of L. Sherman, The Scripps Institute, LaJolla, USA) with DB resuspended in incomplete Freund's adjuvant, followed by spleenectonomy 10 days later, similar to procedures described previously.^5,10,11 Splenic cells were restimulated weekly with irradiated, pp65_495-503-loaded feeder cells and TCGF according to established protocols.^10,11 IFN--based ELISPOT analyses were carried out as detailed by others.³² Values given represent the mean number of spots of triplicate wells and the standard deviation. (b) ELISPOT analysis of dendritic cells (DC), incubated with DB. HLA-A0201-positive DC were generated from PBMC of HCMV seronegative donors by adherence to plastic and stimulation with GM-CSF and IL-4 as described previously.^33,34 DC (110⁶) were either mock treated, incubated for 16 h with 20 g DB, infected with HCMV strain TB40/E overnight at a moi of 10, or labeled with synthetic peptide pp65_495-503 at 10^-8 M, respectively, and used as target cells in IFN- specific ELISPOT analyses. pp65-CTLL were used as effector cells. (c) CTL-mediated cytolysis of DB-treated target cells. HLA-A0201-positive MRC-5 cells were either mock treated (MRC5), incubated for 16 h with 20 g DB, infected with HCMV strain Ad169 overnight at a moi of 10, or labeled with synthetic peptide pp65_495-503 at 10^-8 M, respectively, and used as targets in standard 5 h chromium-release assays at the indicated effector/target ratios. A constant number of 10^{3 51}chromium-labeled target cells and graded numbers of effector cells were used in the assay. Throughout, reported cytolytic activities represent the mean percent specific lysis from three replicate microcultures.