Selection of T cell clones with enhanced T-cell activity from a R6C12 CDR3α library
A. Analysis of T cell hybridoma transduced with the R6C12-CDR3α library. hCD8+, 58−/− TCR-negative T cell hybridoma were transduced with retrovirus generated from the R6C12-CDR3α library and populations were dual-stained with anti-cTCR APC mAb (Clone H57-597) and HLA-A2-gp209-2M tetramer using a MoFlo sorter. Cells were sorted once. B. Single cell sorted selected T cell hybridomas expressing mutated TCRs were incubated with 10uM gp209-2M -loaded T2 A2Kb cells for 24 hr and their level of IL-2 cytokine production was evaluated. A subset of clones were compared to the WT (green) and placed in color coded categories characterized by making less cytokine than the WT, 1-2, 2-4, or 4-10 times more cytokine than the WT.