Figure 3: NFκB knock down HEp2 tumor cells acquire increased susceptibility to CD8+ T cell cytotoxicity, and trigger increased secretion of IFN-γ in the presence of decreased IL-6 secretion. (A) T cells were treated with IL-2 (500 units/ml) and cultured with and without HEp2-IκB(S32AS36A) cells for 18 days before they were washed, counted and added to either 51Cr labeled HEp2-vec or 51Cr labeled HEp2-IκB(S32AS36A) cells. T cell cytotoxicity were determined using a standard 4 hour 51Cr release assay, and the lytic units 30/106 were determined using inverse number of effectors required to lyse 30% of the tumor cells X 100. (B) Purified CD8+ T cells were treated with and without IL-2 (500 μ/ml) overnight before their co-culture with and without IFN-γ (200 u/ml) pre-treated vector alone and IκB(S32AS36A) transfected HEp2 cells (E:T ratio 1:1). After two weekly stimulations with freshly supplemented IL2, the supernatants were removed from the CD8+ T /HEp2 cell co-cultures at day 18th and assayed for released IFN- by specific and sensitive ELISA assay. IFN-γ treated HEp2 cell transfectants were washed three times before they were added to CD8+ T cells. The p value for the difference between secretion of IFN- in the co-cultures of CD8+ T cells with vector-alone transfected HEp2 cells vs. IκB(S32AS36A) transfected HEp2 cells were less than 0.05. (C) CD8+ T cells were treated with and without IL-2 (500u/ml) before their co-culture with vector-alone and IκB(S32AS36A) transfected HEp2 cells (E:T ratio 1:1). After two weekly stimulations with freshly supplemented IL2, the supernatants were removed from the CD8+ T /HEp2 cell co-cultures at day 18th and assayed for the released IL-6 and IFN-γ using antibody coated multiplex microbead immunoassay kit obtained from R&D. The p values for the differences in secretion of IL-6 and IFN-γ between the T cells co-cultured with vector-alone vs. IκB(S32AS36A) transfected HEp2 cells were at less than 0.05. (D) The ratio for secreted levels of IL-6 to IFN-γ was calculated for T cells co-cultured with each of HEp2 cell transfectants. 1 of 4 representative experiments is shown in this figure.