Introduction of an epitope-presenting vector to ES-DCs.
(A) The structure of PCC epitope presentation vector, pCAG-PCC-PI, is shown. CLIP region of human Ii cDNA was replaced with an oligo DNA encoding PCC88-104. The expression of this gene is driven by the chicken β-actin promoter (pCA). The mutant Ii coding sequence is followed by IRES-puromycin N-acetyltransferase gene (Puro-R) and polyadenylation signal sequence of the human growth hormone (GpA).(B-C) Flow cytometric analysis of ES-DCs on the expression of human Ii. ES-DCs without (B) or with (C) expression construct were stained intracellularly with antihuman Ii (CD74) (thick lines) or isotype-matched control mAb (thin dotted lines). (D) Stimulation of PCC-specific T-cell hybridoma, 2B4, by ES-DCs with (○) or without (▪) PCC epitope-presenting vector. As a control, BM-DCs pulsed with PCC peptide (10 μM) for 4 hours (⋄) or left unpulsed (▵) were also used as stimulators. Stimulator cells and hybridomas were cocultured for 24 hours, and IL-2 produced by 2B4 was measured by proliferation of CTLL-20 cells. Results were expressed as mean cpm of triplicate cultures ± standard deviation.
