Figure 2
The spliced gp100mel47–52/40–42 peptide is generated from the gp100mel40–42 peptide by a condensation reaction in cellulo.
(A,B) The spliced peptide gp100mel47–52/40–42 [QLYPEW][RTK] bound the HLA-A*03:01 complex on the cell surface of HeLa (A) and T2 (B) cell lines. In (A) the side / forward scatters charts are reported in the left panel. Cells included in the analysis are marked with a circle and their HLA-A*03:01 amount is shown in the right panel. (B) Shown is the binding affinity of the spliced peptide gp100mel47–52/40–42 compared to a known good HLA-A*03:01 – binder, i.e. [ILRGSVAHK] (NP265–273)35 and to the negative controls HLA-A*32:01-restricted gp100mel40–42/47–52 [RTK][QLYPEW] and HLA-A*02:01-restricted gp100mel209–217 [IMDQVPFSV] epitopes. Cells were transfected with HLA-A*03:01 protein encoding plasmids and pulsed with 100 μM (A) or different peptide concentrations (B). Binding affinity was measured by staining of HLA-A*03:01 and FACS analysis. Values are in mean fluorescence unit (MFU) and are means and SD of 2 replicates in a representative assay of 2–6 independent experiments. (C) Schematic representation of the HA-Ub/gp100mel40–52 fusion construct used in this study. The first amino acid of the gp100mel40–52 [RTKAWNRQLYPEW] sequence was directly fused in frame to the C-terminus of the Ub, which was N-terminally tagged with the YPYDVPDYA HA sequence. When expressed in cells, Ub hydrolases cleaved the fusion-polypeptide directly after the last amino acid (Gly76) of Ub, liberating the gp100mel40–52 peptide from the first residue. (D) CTL response towards HeLa 33/2 cells transfected with HLA-A*03:01 individually or together with HA-Ub/gp100mel40–52 for 24 hrs. Their ability to present gp10047–52/40–42 epitope was measured by using K631 1C CTL. After 16 hrs co-culture, the IFN-γ concentration in supernatants was determined by ELISA. Values are means and SD of 2 replicates in a representative assay of 6 independent experiments. Significant differences in the means of replicates are marked by * (student t-test; E:T ratio: 1:1, p = 0.003; 2:1, p = 0.050). (E) 10 μg of whole HeLa 33/2 cell extracts were resolved on a 15% SDS-PAGE for western blotting with anti-HA antibody to control the expression of the construct. The western blot is representative of 5 independent assays.
