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Fig. 6 | BMC Research Notes

Fig. 6

From: Effector function of CTLs is increased by irradiated colorectal tumor cells that modulate OX-40L and 4-1BBL and is reversed following dual blockade

Fig. 6

Radiation-enhanced T-cell lytic activity and radiation-enhanced T cell survival 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 HCT 116 cells were and transfected the following day with 4-1BBL siRNA or a control siRNA. Twenty-four hour post-transfection, the cells were irradiated with 10 Gy. Twenty-four hour post-IR, cells were harvested and 4-1BBL mRNA was quantified. *P value <0.05 and ** <0.0001. Data graphed are the mean of two experimental repeats and error bars represent the SEM across the independent experiments. b 2 × 104 HCT 116 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 and used the next day in a Eu-release cytotoxicity assay as described in the “Methods” section. In the indicated group, neutralizing antibody to human OX-40L was added to tumor cells used in the cytolysis assay. Error bars indicate variability in technical replicates. Experiments repeated at least two times with similar results. c Flow cytometry histograms showing active Caspase-3 expression in T-cells incubated alone or with treated HCT116 cells for 5 h. d The frequency of Caspase 3+ cells within the gated CD8+ population is shown. *P value <0.05 between incubation with 10 Gy-treated tumor cells versus untreated or 10 Gy-treated cells verus 10 Gy-treated cells plus dual blockade. Line indicates the mean and error bars represent the SD across four independent experiments

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