DC priming of <i>S.</i> Typhi-specific T cell responses.

<p>PBMC from volunteer CVD4000#65 were co-cultured with DC alone (media), or pre-mixed with live or heat-killed <i>S.</i> Typhi at a MOI of 10∶1, or uninfected or <i>S.</i> Typhi-infected blasts at a 1∶1 blast∶DC ratio. In some cases, DC were pre-treated with ZVA-D or CCD before exposure to <i>S.</i> Typhi or <i>S.</i> Typhi-infected blasts respectively. In other cases, blasts were treated with ZVA-D before co-culture with DC. After 20 hours of incubation, cells were surface stained with a combination of mAb to CD3, CD4, CD8, CD14 and CD19 as well as ViViD. After fixation and permeabilization, cells were intracellularly stained for IL-2, IFN-γ and TNF-α and analyzed by multichromatic flow cytometry. Lymphocytes were gated based on their scatter characteristics. Single lymphocytes were gated based on forward scatter height vs. forward scatter area. A “dump” channel was used to eliminate dead cells (ViViD⁺) as well as CD14⁺ and CD19⁺ cells from analysis. This was followed by additional gating on CD3, CD4 and CD8, to identify cytokine-producing CD8⁺ T cells. Each cytokine was gated individually. During sample acquisition, routinely 300,000-500,000 events were collected in the forward and side scatter (FS/SS) lymphocyte gate. This large number of gated lymphocyte events was necessary to ensure that a sufficient number of positive cells for a defined subset would be collected for each tube analyzed. Numbers correspond to the percentage of positive cells in the indicated regions in each histogram. These results are representative of 1 of 4 volunteers with similar results.