Jurkat cells transduced with codon-optimized CN mutants secrete IL-2 in the presence of CN inhibitors. Jurkat cells were retrovirally transduced with CN mutants and FACS sorted. Cultures were stimulated with PMA and ionomycin in the presence of increasing concentrations of FK506 (A) or CsA (B) and secretion of IL-2 assessed by ELISA. Mean and SEM of 3 experiments shown. (A) Jurkat cells transduced with CNa12, CNb30, or a bicistronic construct of these mutants were able to secrete IL-2 throughout the therapeutic range of FK506 (7-12 ng/mL). (B) Jurkat cells transduced with CNa22, CNb30, or the double construct CNa12-CNb30 were able to secrete IL-2 throughout the therapeutic range of CsA (100-250 ng/mL). Selected constructs CNa12, CNa22, CNb30, and CNa12-CNb30 are shown with solid lines; all other constructs are shown with dotted lines. The horizontal line indicates the amount of IL-2 secreted by untransduced Jurkat cells stimulated without CN inhibitors.