Abstract
Major histocompatibility complex (MHC) class I heavy chains associate with the light chain beta-2 microglobulin (β2m) to present antigenic peptides to cytotoxic T cells. Upon dissociation of both peptide and β2m, class I free heavy chains (fH) are generated. While the presence of the fH at the cell surface has been reported, their biogenesis, trafficking, and lifetime are insufficiently defined, and their biological functions are largely unknown. Here, we show that class I fH arise via β2m dissociation from peptide-free, β2m-bound class I molecules that are located at the plasma membrane or recycled from intracellular compartments. Using conformation-specific antibodies, peptide rescue assays, brefeldin A blockade, and in silico modeling, we elucidate the short- and long-term kinetics of generation and disappearance of plasma membrane-associated fH. We demonstrate that fH accumulate at the cell surface within minutes and persist for several hours, with rates of endocytic removal and recycling varying substantially between the analyzed allotypes. Notably, a fast-recycling pool of human leukocyte antigen (HLA) class I fH exists in HeLa cells, whereas murine H-2Ld and H-2Db recycle more slowly. These findings elucidate both the early origins of cell surface fH and the kinetics governing their production and disappearance, and they highlight their prolonged surface residency as a potential determinant of novel biological roles.