Figure 1

EL4 stable transfectants express the SIINFEKL peptide derived from HEL targeted to the ER or to the cytosol. (A) Schematic representation of the constructs used to generate EL4 stable transfectants. Modified coding sequences of HEL [34, 35] in frame with the region coding for the ovalbumin-derived peptide SIINFEKL and its flanking region were cloned into the pIRES-EGFP2 vector. HEL-ER-SIINFEKL possesses HEL N-terminal signal sequence (ss) and the ER-retention signal KDEL and targets HEL to the ER; HEL-Cyto-SIINFEKL lacks the N-terminal signal sequence and the ER-retention signal and targets HEL to the cytoplasm (see materials and methods). (B) EL4 stable transfectants express K^b-SIINFEKL at the cell surface. EL4 cells were transfected with the pIRES-EGFP2 vector encoding HEL-ER-SIINFEKL or HEL-Cyto-SIINFEKL. Stable transfectants were selected by repeated cycles of FACS of EGFP-positive cells combined with drug resistance (1000 μg/ml of G418). Cells were stained with 25-D1.16 monoclonal antibody, recognizing the K^b-SIINFEKL complex, followed by staining with APC-conjugated anti-mouse IgG₁ as secondary antibody. Depicted in the graphs are EGFP and K^b-SIINFEKL MFI values of untransfected EL4 cells (upper left), EGFP-transfected cells (lower left) and the two representative clones that were used in further studies: EL4/HEL-ER-SIINFEKL (upper right) and EL4/HEL-Cyto-SIINFEKL (lower right). Percentages represent the proportion of cells expressing EGFP and K^b-SIINFEKL.