Photochemical Treatment of Drosophila APCs Can Eliminate Associated Viruses and Maintain the APC Function for Generating Antigen-Specific CTLs Ex Vivo

Figure 5

The effect of UVADEX-treated APCs on generation of antigen-specific CTLs from both mice and human. (a) CD8 T cells purified from pooled B6 mice spleen and lymph node cells were stimulated in 24-well plates with OVA8-peptide-loaded and UVADEX-treated or nontreated APCs (Fly/K^b/B7.1/ICAM). Cells were prepared as described in Figure 1(c). At day 9, cells were collected and stained with OVA/K^b-specific tetramer-PE or D^b-GagL-specific tetramer and FITC-conjugated anti-mouse CD8 mAb at room temperature for 30 min. FACS analysis was used to determine the number of OVA-specific CTLs. (b) Purified human CD8 T cells from HLA-A2-positive donors were cultured with Mart-1-peptide-loaded APCs or UVADEX-treated Mart-1-peptide-loaded APCs at 37°C, 5% CO₂ for 5 days. Human IL-2 and IL-7 were added for further culture. The activated CD8 T cells were restimulated twice at day 7 and day 15 with non-CD8 adherent cells in PBMC from the same donor in the presence of antigen. The antigen-specific CD8 T cells were identified by tetramer staining to determine the number of antigen-specific CTLs. (c) CTLs generated from human donors described in 5b were assayed for cytolytic activities. ⁵¹Cr-labeled T2 cells were loaded with Mart-1 peptide or without Mart-1 loading and incubated with CTLs generated by UVADEX-treated or nontreated APC stimulation at 37°C, 5% CO₂ for 4 h. The culture supernatant was collected for the determination of ⁵¹Cr release.

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