Figure 5. Tumor cell lysis measured by luciferase activity in the surviving target cells.

Luciferase-expressing Lewis lung carcinoma tumor cells (25,000/well) were plated with and without immune effector cells (750,000/well) at an E: T ratio (Effector to Target) of 30:1. A. CD8+ T cells and NK cells were isolated from either wild type or Rad52 knockout mouse spleens, stimulated for 72 h with CD3/CD28 beads with 50 U/ml IL-2 (T-cells) or 1000 U/ml IL-2 (NK cells), and co-cultured with 25,000 luc+ LLC target cells/well. Tumor cell viability was assessed by BLI after 6 h of co-culture (described in Materials and Methods). The % tumor cell survival of Lewis lung cells obtained by the BLI assay is plotted for isolation of both CD8+ T cells and NK cells. Results are represented as mean ± SD of n = 3 independent experiments. Decreased LLC survival was observed in LLC target cells when co-cultured with splenocytes depleted of Rad52. The % survival of Lewis Lung Carcinoma (LLC) cells was calculated using the formula: % survival = (mean BLI test signal/mean BLI maximal signal) × 100. In these assays, LLC cell survival is a direct reflection of T cell-mediated lysis and correlates with classical chromium release assays. B.-C. Levels of granzyme B and IFN-y were calculated using ELISpot kits from R&D Systems. Immune cells were plated either without target cells (CD8+ cells without LLCs and NK cells without LLCs) or with target LLCs at E:T ratios of 1:5 (10,000 effector immune cells: 50,000 target tumor cells). P-value and significance calculated to only compare wild type v. knockout in each individual treatment group. Multiple testing adjustments were performed so that the threshold would be less than the Bonferroni correction using p < 0.05 as threshold. *p < 0.0125 was considered to be statistically significant. ns, Not Significant. Wild-type (n = 3); Rad52^−/− mice (n = 3).