Stable transfection of CTLA4-KDEL into B cells inhibits their ability to stimulate T cells. (A) HLA-DR1–expressing B cells were stably transfected with CTLA4-KDEL and mock-KDEL. The expression of CD40, CD54, and MHC II (i-iii), CD80 and CD86 (iv-vi), IgG and CD19 (vii-ix) was analyzed by flow cytometry. Staining with isotype matched control antibodies is shown in the solid profile. (B) Expression of the 29-kDa CTLA4-KDEL in cell lysates and supernatants of HLA DR1- or HLA DR11-expressing B cells transfected with either CTLA4-KDEL or mock-KDEL was determined by Western blotting using Ab against the c-myc protein tag incorporated in the construct. β-Actin was used as a housekeeping control. (C) HLA-DR1–expressing-B-cell lines that were either untransfected or transfected with the mock-KDEL or the CTLA4-KDEL plasmid were used as stimulators in a MLR and for a peptide-specific response. In the MLR, 10 μg/mL CTLA4-Ig was used as a control, and ³H-thymidine incorporation was determined after 5 days. The HC3 T-cell clone was used to test the peptide-specific response, and the B cells were incubated with different concentrations of HA peptide. ³H-thymidine incorporation was determined after 3 days. The results are representative of at least 3 experiments. (D) Culture supernatants from the HC3 clone and allogeneic T cells were collected on day 2 and 4, respectively, after coculture with HLA-DR1–expressing B cells that were either untransfected or stably transfected with CTLA4-KDEL or mock-KDEL. The levels of IL-4, IFN-γ, and IL-10 were measured using ELISA. The results are mean ± SD of 3 independent experiments.