Figure 1.

MHC class I antigen presentation from OVA protein and SIINFEKL-containing oligopeptides. (A) E36.12.4 APCs were infected with vTF7–3 and then transfected with the indicated concentrations of plasmid encoding intact OVA cDNA. Assays for presentation of SIINFEKL on K^b were performed as described (7). (B) Similar to A, except that cells were treated with (▪) or without (□) lactacystin (2 μM) for 30′ before transfection and during the subsequent incubation, and 5 μg of a plasmid encoding p.5+SIINFEKL+5 was transfected. (C) Same as B except that the plasmid encoded p.SIINFEKL (0.7 μg transfected). (D) Similar to B except that 8+SIINFEKL+8 synthetic peptide (300 μg/ml) was introduced into the cytosol of LB27.4 cells (17) (APCs) by electroporation (7) (instead of vaccinia infection and plasmid transfection) and lactacystin was used at 40 μM. (E) Same as D except that SIINFEKL (0.5 μg/ml) was used instead of 8+SIINFEKL+8 peptide. (F) Same as D except that LLnL (40 μM) was used instead of lactacystin. (G) Same as E except LLnL (40 μM) was used instead of lactacystin. Some APCs were fixed immediately after electroporation of antigen (○) to rule out peptide binding directly to cell surface MHC molecules (7). All results in Figs. 1, 2, 3, 4 are representative of data obtained in repeated experiments. OVA constructs expressed from plasmids are indicated with the prefix p.