Sequence and surface expression CTLA-4 molecules used in these studies. The WT sequence of the intracellular tail of CTLA-4 is shown in full with key residues targeted for mutation depicted in bold. Lysine residues 152, 155 and 156 were changed to alanine residues to generate KLESS CTLA-4. Tyrosine residues located at positions 165 or 182 have been converted to phenylalanine to create Y165F CTLA-4 and Y182F CTLA-4, respectively. The double mutant Y165F/Y182F CTLA-4 contains mutations at both tyrosine residues. PRO- CTLA-4 contains mutations of proline residues 169 and 173 to alanine residues. Stably transfected Jurkat T cell lines have been generated for each of these CTLA-4 variants. Cells were induced overnight with doxycycline (1 μg/ml) and the surface expression of CTLA-4 was measured by flow cytometry (black line, CTLA-4; shaded profile, isotype-matched Ab).