Figure 3.

Adenovector-Expressed Antigen Is Effective at Recalling T Cell Responses from Immunized Mice

(A) Schema for in vitro antigen discovery. First, A20 APCs are infected with each adenovector in the array. After 24 hr, the specific antigen of interest is expressed and presented on MHC class I molecules. The infected APCs are incubated with splenocytes isolated from immune animals to identify CD8⁺ T cells with specificity for the antigens expressed in the Ad-array. These antigen-specific CD8⁺ T cells are identified by either ICS and flow cytometry or ELISpot assays measuring IFNγ secretion. (B) Ad5 vector effectively transduces APCs. A20 cells were infected with AdGFP at the indicated MOI. The percentage of GFP⁺ cells was determined by flow cytometry. (C) AdPyCSP-infected APCs can recall CD8⁺ T cell responses from immune mice. Target A20 cells were infected with various MOIs of an Ad5 vector expressing PyCSP (AdPyCSP). Control targets were uninfected A20 cells, A20 cells infected with various MOIs of an Ad5 vector that does not express a transgene (AdNull), and A20 cells stimulated with an immunodominant PyCSP peptide (amino acids 280–288, SYVPSAEQI). These targets were used to stimulate splenocytes from BALB/c mice immunized with a PyCSP DNA vaccine. IFNγ-expressing cells were measured by ELISpot. SFC (spot-forming cells); error bars indicate the SEM (n = 3). SFC, spot-forming cell.