In Vivo Cytotoxic T Lymphocyte Elimination Assay

To evaluate the overall cellular response elicited in mice injected with our DNA complexes, an in vivo CTL activity assay was carried out according to the procedure outlined in White et al.61

On day 1, mice were primed with the DNA complexes or control formulations. LP/DNA and DSPE-PEG₂₀₀₀/LP/DNA complexes were prepared as described previously using the pCMV-OVA plasmid. The positive control formulation used in this experiment was an injection of 2 × 10⁷ OVA-coated splenocytes with 1 μg of LPS. The OVA-coated splenocytes were prepared by incubating a single-cell suspension of isolated splenocytes (from naive C57BL/6J mice) with OVA. Splenocytes were suspended in red blood cell lysis buffer and incubated at 37°C for 2 min, washed, and passed through a 70-μm cell strainer to filter off any cell debris. Cells were then incubated with OVA (10 mg/mL) at 37°C for 10 min, washed, and then resuspended in PBS. On day 6 post-immunization, mice were injected intravenously with target splenocytes (preparation is in Preparation of Target Splenocytes). The following day, splenocytes were harvested from the mice and analyzed via flow cytometry (preparation is outlined in Flow Cytometry). The percentage of target OVA-pulsed splenocytes eliminated was then calculated for each mouse.

Target splenocytes were prepared by depleting a single-cell suspension of isolated splenocytes of red blood cells and dividing the population of cells equally into two tubes. To one tube, OVA257–264 peptide was added to a concentration of 1 μg/mL. Both tubes were then incubated at 37°C for 1 hr, washed, and resuspended in 10⁷ cells/mL in PBS and 1% fetal bovine serum (FBS). To the OVA_257–264 peptide-pulsed tube of splenocytes, 0.5 μL of 10 mM CFSE was added per 1 mL of cells (i.e., high CFSE concentration). To the unpulsed population of splenocytes, 0.5 μL of 1 mM CFSE was added per 1 mL of cells (i.e., low CFSE concentration). The two tubes were then incubated at 37°C for 10 min, washed, and then resuspended in PBS. The two populations of cells were then mixed at a 1:1 ratio (high CFSE:low CFSE populations), and each mouse received an intravenous injection of 2 × 10⁷ mixed cells in 200 μL of PBS.

Flow cytometry was carried out using the BD FACSCanto II (BD Biosciences, San Jose, CA) to identify the ratio of OVA-pulsed (high CFSE-labeled) population of splenocytes to unpulsed (low CFSE-labeled) splenocytes. For each sample, 2 million events were recorded. The percentage of cells eliminated from OVA-specific T cell activation in each mouse was then calculated using the following formula: