Figure 1
Generation of surrogate β cell lines and examination of their naturally processed and presented peptide repertoire.
The chronic myelogenous leukemia cell line K562 was variously transfected with the genes for PPI and HLA-A*0201. (A) This yielded cell lines (denoted K562-PPI and K562-PPI-A2) that secrete proinsulin (gray bars) and immunoreactive insulin species (black bars) into cell culture supernatants. No proinsulin or immunoreactive insulin is secreted by single-transfected K562-A2 cells. Bars represent mean levels present in cell supernatants and error bars the SEM. (B) Surface HLA-A2 expression was examined by flow cytometry using the allele-specific mAb BB7.2, showing comparable HLA-A2 levels on the K562-A2 (solid line) and K562-PPI-A2 (dashed line) cells compared with absence of staining on K562-PPI cells (dotted line). Isotype control staining was similar to K562-PPI staining on all cell lines, and similar results were obtained with the pan–HLA-A,B,C–staining mAb W6/32. K562-PPI-A2 and K562-A2 cell lines were grown in large cultures and the natural peptide repertoire extracted and resolved by RP-HPLC, and fractions were compared by MS to identify masses unique to PPI-expressing cells. (C and D) MS analysis of HPLC fractions 55 and 65, respectively, from K562-PPI-A2 cells. Arrows indicate masses unique to these cells (784.37 and 968.48 m/z, respectively) that are not found in the equivalent (or adjacent) fractions from K562-A2 cells (E and F) or K562-PPI cells (data not shown).
