Transfection of DCs with CTLA4-KDEL. Monocytes were transfected with CTLA4-KDEL, mock-KDEL, or left untransfected and then differentiated into DCs with GM-CSF and IL-4. On day 2 G418 was added to select for transfected cells. On day 8, the cells were matured with TNF-α, IL-1β, LPS, IFN-γ, and PGE2. (A) On day 10, the expression of MHC I, MHC II, CD80, CD86, CD83, CD54, CD40, CD11c, and CD14 was analyzed using flow cytometry. (B) Expression of CD80, CD86, CTLA4-KDEL, and β-actin was determined by Western blotting in the presence and absence of 1 μM proteasome inhibitors to determine the degradation pathway of CD80/86. (C) To demonstrate colocalization of CTLA4-KDEL and CD80/86, the cell lysates were immunoprecipitated with anti-CTLA4 (left) and anti–c myc (right) prior to Western blotting and probing with anti-CD80, CD86, or CTLA4 mAb. (D) To determine whether CTLA4-KDEL activated ER stress responses, Western blots were probed with antibodies against phosphorylated PERK and phosphorylated eIF-2α proteins. As a control in these experiments, DCs were also transfected with GFP or with an intrabody (scFv-KDEL) directed against vascular cell adhesion molecule 1 (VCAM-1). (E) The DCs were tested for their ability to stimulate an MLR by allogeneic T cells. The results are expressed as mean ± SD of triplicate wells from a representative experiment. (F) The supernatants from allogeneic T cells cocultured with either untransfected, CTLA4-KDEL, or mock-KDEL transfected DCs were collected on day 4, and the levels of IL-4, IL-12p70, and IFN-γ were measured by ELISA. The results are mean ± SD of 3 cocultures. The data are representative of 3 experiments.
