Figure 2. Primary human B cells encode plasmid mRNA and mediate expansion of cognate antigen specific CD8 T cells upon plasmid DNA treatment.

a. Different APC subsets were treated with 25μg/mL of plasmid vector alone, or plasmid DNA encoding either EGFP or ovalbumin (OVA) for 1h as indicated, and then in complete medium for 18h. Total RNA was extracted and assayed for expression of EGFP mRNA by qRT-PCR. Shown is one representative agarose gel of an RT-PCR product. Lanes 1-4 in the top segment represent the following: 1: pTVG4 (empty vector), assay for GFP mRNA; 2: pTVG4, assay for OVA mRNA; 3: pEGFPc1, assay for GFP mRNA; 4: psOVA, assay for OVA mRNA. All lanes in the bottom segment represent assay for the actin housekeeping gene mRNA from the corresponding samples. b. Enriched APC subsets from HLA-A2+ donors with known tetramer responses to SSX2 were treated with 25μg/mL empty vector control (pTVG4) or a plasmid encoding SSX2 (pTVG-SSX2) in the presence of autologous T cells. One week later, samples were assayed for p103-specific CD8 T cells by tetramer staining. The dotted line represents baseline ex vivo levels of tetramer staining prior to culture. These data are representative of at least 2 independent experiments from each donor. c. Representative flow cytometry dot-plots of the results obtained from the different conditions, with tetramer-associated fluorescence on the X axis, and CD8a staining on the Y axis. Data are representative of 4 independent experiments, with at least 3 different donors.