Abstract The major histocompatibility complex (MHC) class I gene locus in humans (human leukocyte antigens (HLA) class I) is the most polymorphic of human genes. Individual variants have profound influences on disease outcomes in infectious and inflammatory diseases and in cancer. HLA class I variants display distinct peptide binding preferences which allows a diverse set of peptide antigens to be presented to T cell receptors (TCRs) of CD8+ T cells. MHC class I molecules also play critical roles in the natural killer (NK) cell response via the engagement of activating and inhibitory receptors of NK cells. In the textbook defined MHC class I assembly pathway, peptides are derived from the cytosol of cells, transported into the endoplasmic reticulum (ER) via the transporter associated with antigen presentation (TAP), and bound to MHC class I molecules in the ER in a process edited by tapasin. This pathway is expected to culminate in the presentation of a stable repertoire of high affinity peptide-MHC class I complexes on the cell surface. However, there is also a need to maintain surveillance of endosomal compartments, as antigens derived from these compartments initiate CD8+ T cell responses. Additionally, many HLA class I variants can assemble independently of components the classical pathway. Some HLA class I allotypes are also found in complex with peptides that have broad predicted affinity ranges, and present as imperfect and peptide-receptive forms in cells. There are fundamental gaps in our knowledge about the non-canonical pathways that govern the formation of such molecules and their functions in the immune response. It is our central hypothesis that a balance of canonical and non- canonical HLA class assembly pathways is needed to enable both cytosolic and endosomal surveillance, immunity in the face of pathogen evasion of the canonical pathway, and immunity under conditions of inflammatory and nutritional stress. To address this hypothesis, the endosomal HLA class I assembly pathway will be interrogated, to examine how specific factors affect the expression, localization, and antigen presentation functions of different HLA class I allotypes. Additionally, assembly under tapasin and TAP deficient conditions will also be examined, with a focus on their intersections with the endolysosomal pathway, the resulting immunopeptidomes, and glycosylation changes to HLA class I molecules. Together, these studies will unravel how HLA class I molecules maintain multicompartmental immune surveillance and elucidate how the divergent assembly features of HLA class I allotypes ensure the maintenance of immunity under pathogenic conditions. This knowledge will be important towards the development of future immunotherapeutic strategies, by identifying features of HLA class I molecules that can be exploited for enhanced immune protection.