Figure 4

Inhibitory function of CTLA-4 molecules with mutation in the cytoplasmic domain. A) Jurkat T cells stably transfected with WT CTLA-4 or mutant CTLA-4 constructs were cultured overnight in the absence or presence of doxycycline (1 μg/ml). Cells were further stimulated with APCs and SEE (1 and 10 ng/ml) for 24 hours at 37°C. IL-2 production was measured by ELISA. Inhibition of IL-2 was determined for each CTLA-4 variant by calculating the percent of IL-2 produced in the presence of CTLA-4 expression normalized to the maximal IL-2 level in the absence of CTLA-4 at each concentration of SEE. The maximal level of inhibition was plotted for each CTLA-4 variant. This graph was generated using triplicate data for each concentration of SEE, from three independent experiments. B) CD28 expression is required for CTLA-4 mediated inhibition. CD28⁺, CD28^- and CD28-reconstituted Jurkat T cells were cultured overnight without (black squares) or with doxycycline (1 μg/ml) (black triangles) to induce the expression of Y165F CTLA-4. Cells were stimulated with SEE:APC and IL-2 was measured as in A). **, p < 0.01; ***, p < 0.001.