Transient transfection of CTLA4-KDEL into artificial APCs reduces their ability to stimulate T cells. (A) M1 cells already transfected with DR1 and CD80 (M1 DR1 CD80) or cells transfected with DR1 alone (M1 DR1) were transiently transfected with mock-KDEL and CTLA4-KDEL constructs or with pCMV/EGFP encoding EGFP. CD80 (FL-4) or EGFP (FL-1) expression was determined by flow cytometry. The number of cells in each quadrant and mean fluorescence intensity (MFI) for CD80 expression are shown below (mean ± SD of 5 independent experiments). (B) The same transfected cells were used as stimulators in a MLR or peptide-specific T-cell response. For the mixed lymphocyte response, various numbers of stimulators were used, and proliferation was determined by ³H-thymidine incorporation after 5 days. The HC3 T-cell clone was used to test a peptide-specific response using the HA peptide at a range of concentrations with ³H-thymidine incorporation being assessed after 3 days. The results are representative of at least 5 independent experiments and show the mean ± SD of triplicate cultures.