ABSTRACT Pregnancy is a complex state that involves immune crosstalks between maternal cells in the decidua and fetal cells. This communication plays an important role in protecting the fetus from rejection. Extravillous trophoblasts (EVTs) are critical fetal cells that shape the immunological microenvironment at the maternal-fetal (M-F) interface. EVTs express a unique set of major histocompatibility complex (MHC I) molecules on their surfaces: the classical HLA-C and non-classical HLA-E, -F, and -G molecules. Quite exceptionally, EVTs do not express HLA-A and HLA-B. HLA-C, -E, and -G molecules have been associated with mechanisms of immune tolerance at the M-F interface, and whether HLA-F functions similarly is less well understood. HLA-F is unique among MHC I molecules in several ways: 1. HLA-F exists in more than one molecular form, with and without associated peptides and b2m; 2. HLA-F binds peptides that are unconventionally long, ranging from 8mers to more than 20mers; and 3. Inhibitory and activating receptors on natural killer (NK) cells can distinguish between peptide-filled and peptide-deficient HLA-F molecules. It is therefore logical to propose that a role for HLA-F in M-F immune crosstalks involves interactions between HLA-F expressed on EVTs and NK receptors on decidual NK cells. This is reinforced by the knowledge that HLA-F expression on EVTs is most abundant in early pregnancy, and that NK cells constitute the largest population of maternal immune cells in the decidua during the first trimester of pregnancy. Thus, to develop an understanding of how HLA-F functions as an immunoregulatory molecule, it is critical that we have a strong understanding of the peptide binding properties of HLA-F and the mechanisms by which peptides modulate interactions with NK cell receptors. This R21 application is an early-stage investigation that aims to fill unresolved gaps in our knowledge of HLA-F immunobiology. In two aims, we propose to examine the unique landscape of HLA-F peptides in relation to specialized aminopeptidases that normally generate MHC I immunopeptidomes inside cells, as well as address key questions centered on why HLA-F presents peptides of extraordinarily long lengths, what biochemical and structural properties of HLA-F support binding of long peptides, and what is the role of peptides in modulating HLA-F interaction with NK cells. For this, we will use a combination of cell-based, biochemical, and structural approaches. Upon completion of this project, we will have generated new knowledge on HLA-F that will help understand how its expression on EVTs and engagement with receptors on decidual NK cells, can support a critical regulatory role in immune crosstalks during the early stages of pregnancy. Given that pregnancy complications are often associated with a loss of immune tolerance mechanisms, a characterization of HLA-F as proposed here is important for stimulating new ideas in managing and decreasing the risks associate...