Figure 5.

Capacity of CD141⁺, CD1c⁺, and CD16⁺ DCs to cross-present soluble and cell-associated HCMV pp65 antigen. (A) CD8⁺ T cell clone 10, specific for the HLA-A*0201–restricted HCMV pp65 peptide NLVPMVATV (pp65_495–503), was co-cultured with CD141⁺ DCs, CD1c⁺ DCs, CD16⁺ DCs, or pDCs obtained from the buffy coat of one HLA-A*0201⁺ blood donation, with 3 µg/ml of recombinant soluble HCMV pp65 added for the entire culture period. The activation of the T cell clone was determined by measuring the concentration of IFN-γ in the supernatants at the termination of culture after 20 h. Negative controls included addition of irrelevant protein OVA (3 µg/ml) and cultures with only the T cell clone or DCs; addition of 1 µg/ml of pp65_495–503 peptide to the co-cultures served as a positive control. Shown is one representative experiment out of nine; each experiment was performed with cells from a different donor (all experiments included all cDCs subsets; four of them also included pDCs). (B) CD141⁺, CD1c⁺, or CD16⁺ DCs isolated from leukapheresis PBMCs of one HLA-A*0201⁺ donor were co-cultured with CD8⁺ T cell clone 61 at variable ratios (from 1:1 to 1:16) with cell-associated pp65 antigen added for the entire culture period, and IFN-γ was determined in the supernatant after 24 h. Negative controls included irrelevant MART-1_27–35 peptide, and positive controls included HCMV pp65_495–503 peptide (both at 1 µg/ml). Shown are results representative of three experiments with different donors. Error bars represent means ± SEM.