Fig. 5
Radiation-enhanced sensitivity to T-cell mediated lysis and T-cell activation is reduced in the absence of OX-40L and 4-1BBL. a 4-1BBL was knocked down in tumor cells as described in “Methods” section. Briefly, 1 × 105 SW620 cells were and transfected the following day with 4-1BBL siRNA or a control siRNA. 24 h post-transfection, the cells were irradiated with 10 Gy. Forty-eight hours post-IR, cells were harvested and 4-1BBL mRNA was quantified. *P value <0.05. Data graphed are the mean of two experimental repeats and error bars represent the SEM across the independent experiments. b 2 × 104 SW620 cells were plated in 24-well plates and transfected with 4-1BBL siRNA or a control siRNA. After transfection, the cells were irradiated with 10 Gy. Forty-eight hour post-IR cells were used in a Eu-release cytotoxicity assay using CEA-specific T-cells at an E:T ration of 30:1. In the indicated groups, neutralizing antibody to human OX-40L was added to SW620 cells used in the cytolysis assay. *P value <0.05. Error bars indicate variability in technical replicates. Experiments repeated at least two times with similar results. c Forty-eight hour after cytolysis assay set-up, cells were harvested and stained for markers of T-cell activation. Flow cytometry plots showing the frequency of CD25+ cells within the CD8+ cell population after incubation with irradiated SW620 cells. d Data from three independent experiments is shown graphically and error bars represent the SEM across the independent experiments. *P value <0.05 between untreated versus 10 Gy treated or 10 Gy treated versus dual blockade