Electroporation of mRNA encoding MART-1 specific TcRs.

<p>(a) SupT1 were electroporated with or without 20 µg mRNA encoding the indicated TcR_2A. Twelve hours later, the cells were stained with HLA-A2/MART-1 multimer-PE and anti-CD3 Alexa Fluor 647. (b) Human PBMC were electroporated with 20 µg mRNA (same constructs as in (a)) and cultured for 4.5 hours. Following the addition of T2 cells pre-loaded or not (grey) with MART-1 peptide (10 µM final concentration), the cells were co-incubated for an additional 5 hours in the presence of anti-CD107a/b Alexa Fluor 647 antibodies, monensin and brefeldin A. Prior to analysis, cells were stained with anti-CD8 PE. The percentage of CD107a/b positive cells from the CD8 positive population is plotted. PHA was used to control for similar maximal degranulation levels regardless of the mRNA used for electroporation. Each bar represents the mean values of duplicates.