Studying MHC class I peptide loading and exchange in vitro.

In the endoplasmic reticulum (ER), MHC class I molecules associate with several specialized proteins, forming a large macromolecular complex referred to as the "peptide-loading complex" (PLC). In the PLC, antigenic peptides undergo a stringent selection process for binding onto MHC class I molecules. This ensures that the immune system elicits robust CD8+ T-cell responses to viruses and solid tumors. The ability to reconstitute in vitro MHC class I molecules in association with key proteins of the PLC provides a mean for studying at the molecular level how antigenic peptides are selected for presentation to CD8+ T-cells. Here, we describe practical procedures for generating a cell-free system involving MHC class I molecules and tapasin, a critical protein of the PLC, that can be used as a versatile tool for biochemical and mechanistic studies of peptide loading and exchange.