Deep mutational scan of tapasin-TM, a variant of the MHC class 1 chaperone tapasin

Tapasin is a member of the peptide loading complex which is responsible for stabilizing empty HLA molecules and facilitating the loading of antigenic peptides. Many neuroblastoma cell lines contain low baseline levels of HLA due to defects in antigen processing and presentation machinery. Supplementing the neuroblastoma cell line EBc1 with tapasin-TM, an altered form of tapasin containing the transmembrane domain of HLA-G, enhances surface HLA expression. Here, we have applied our previously used deep mutational scanning library (GSE159247) to the tapasin-TM construct. We then introduced this library into EBc1 cells and isolated cells with the highest surface HLA-A2 expression. Overall design: EBc1 cells were transduced with lentivirus containing our previously generated site saturation mutagenesis library (GSE159247) encoding 2160 tapasin-TM variants, where 108 tapasin-TM residues were mutated to every other amino acid. Cells were transduced such that the majority of cells contained only one variant, stained for the transduction marker FLAG (DYKDDDDK), and sorted to isolate transduced cells. This population was used as the naïve library. These cells were then expanded, stained for surface HLA-A2 (clone BB7.2) and cells were sorted to isolate those with the highest HLA-A2 expression (top 5%). Both the transduced cell population and the HLA-A2 sorted populations were sequenced. The frequencies of each mutation in the sorted cell library were compared to that of the naïve library.