Figure 2.

Cross-presentation of the A3-RPL28^{61-91(p76S>F)} SLP relies on a lysosomal endopeptidase. (A) Parallel reaction monitoring (PRM)-analysis shows the detection of the A3-RPL28^{74-84(p76S>F)} epitope (blue peak) in HLA-eluate of HLA-A3+ NBS that were co-incubated with A3-RPL28^{61-91(p76S>F)} SLP for 48 hours. Heavy-isotope labeled A3-RPL28^{74-84(p76S>F)} was spiked-in as a reference and showed the same retention time. (B) Internally quenched A3-RPL28^{61-91(p76S>F)} was loaded on HLA-A3+ NBS fibroblasts and fluorescent spectroscopy was performed to analyze peptide processing. (C) FACS analysis of internally quenched A3-RPL28^{61-91(p76S>F)} variants, chemically protected at either the N- and/or C-termini (orange label), after co-incubation with NBS fibroblasts (18 hours). (D) Spinning confocal microscopy shows colocalization of the internally quenched A3-RPL28^{61-91(p76S>F)} SLP (green) and the lysosome compartment (red). Dashed line indicates the outlines of the fibroblast. Closed line indicates the location of the nucleus. Arrowheads indicate areas of colocalization. Lysotr=lysotracker. NBS, Nijmegen Breakage Syndrome; SLP, synthetic long peptide.