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

Figure 1

The effect of glutaraldehyde fixation of Fly/L^d/B7.1/ICAM cells on CD8 T cell activation, proliferation, and CTL function. 1 mM CuSO₄-induced Fly/L^d/B7.1/ICAM cells were fixed in 2.5 ml of PBS containing 1%, 0.3%, 0.1%, and 0% glutaraldehyde at 10 × 10⁶/ml at RT for 30 mins, respectively. After completely washing with Drosophila media, the fixed cells were resuspended in 10 ml of MLR (mixed lymphocyte reaction) culture media, one part of cells was loaded with 10 μM QL9 peptide at RT for 60 mins. (a) For cell activation, 1 × 10⁶ of purified mouse CD8 T cells were incubated with 1 × 10⁶ of QL9 loaded-fixed Fly cells at 37°C, 5% CO₂ for 16 h. Cells were collected and stained with FITC-conjugated anti-mouse CD69 mAb and antibody isotype control at 4°C for 30 minutes. CD69 expression on CD8 cells was analyzed by FACS. (b) For cell proliferation, CD8 cells were labeled with 10 μM CFSE at 37°C for 15 min. After complete washing with MLR media, 1 × 10⁶ of labeled cells were incubated with 1 × 10⁶ of QL9 loaded-fixed Fly cells at 37°C, 5% CO₂ for 48 h. Cells were collected and stained with PE-conjugated anti-mouse CD8 mAb at 4°C for 30 min. CD8-positive cells were gated from FACS and further analyzed for their green florescence intensity. (c) For the measurement of CTL activity, CD8 T cells purified from three mixtures of spleen cells of 2c transgenic mice were stimulated with QL9 loaded-fixed Fly cells at 37°C, 5% CO₂ at day 1, and then propagated in IL-2-supplemented media for 7 days. CTL activity was measured at day 7 by using ⁵¹Cr release assay with peptide-pulsed RmAsL^d target cells (closed dot lines). RmAsL^d cells without QL9 loading in the assay were a negative control (open dot lines).

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