Effects of PD1 and CTLA4 Inhibition on Cytotoxicity of CD8+ T-Cells Targeting Ewing’s Sarcoma Cell Line A673

PD1 and CTLA4 inhibitors are often used to improve the effectiveness of TCR immunotherapy. However, their efficacy varies across tumor types. The aim of this research was to determine if PD1 and CTLA4 inhibition improves the cytotoxicity of primary CD8+ T-cells transfected with genes for a TCR targeting the Ewing’s Sarcoma cell line A673. For each experiment that was run, two co-cultures were established: A673 cells co-cultured with non-transfected (NT) CD8+ T-cells and A673 cells co-cultured with transfected CD8+ T-cells. One set of triplicates from each co-culture type had Anti-PDL1 (5ug/ml) and Anti-CTLA4 (5ug/ml) while the other set of triplicates had no added antibodies. The first assay ran was a fluorescence microscopy assay in which PI was added to all wells to identify dead cells and pictures were taken of the co-cultures every hour for 72 hours. The data was quantified using Cell Profiler. However the data had very high standard deviations so a flow cytometry assay was used to verify the results. For this assay the cells were removed from co-culture after 48 hours, stained using PI and Annexin V, then analyzed using flow cytometry. A 4% increase in cytotoxicity was observed. The results showed that Anti-PD1 and Anti-CTLA4 treatments increase cytotoxicity of CD8+ T-cells against A673 cells in vitro over a short period of time. This increase in such a short time frame, although slight, indicates that further research on the effects over a larger time frame and in vivo could be worthwhile.