PROJECT SUMMARY Organ transplantation is a standard-of-care treatment for patients with end-organ failure. Although graft survival has increased significantly over the years, many patients lose their life-saving transplant due to antibody- mediated rejection. Recent work demonstrated that the main target of these antibodies are donor HLA-DQ molecules. This observation is puzzling since the leading convention in the field considered HLA-DR, another type of HLA-class II molecules, as the leading “transplantation antigens”. It is now clear that the frequency and pathogenicity of antibodies against HLA-DQ is higher than HLA-DR (and HLA-DP, the third class II HLA molecules). Currently, there is no mechanistic explanation to these observations. Our central hypothesis is that HLA-DR, HLA-DQ, and HLA-DP were specialized, through evolutionary pressure, to control somewhat different pathways of immune activation. This can be likened to the 3 types of cone photoreceptor cell types that provide similar functionality but for different wavelengths to maximize sensitivity to color. Importantly, the vast majority of mechanistic studies focused on HLA-DR, and the assumption is that HLA-DQ and HLA-DP use identical immune pathways. Our long-term goal is to understand the mechanism leading to the increased immunogenicity and pathogenicity of HLA-DQ mismatches in allo-transplantation (compared with HLA-DR and HLA-DP). The objective is to decipher permissible from non-permissible HLA-DQ mismatches, and further to elucidate immune activation pathways that are preferentially stimulated by HLA-DQ allo-recognition. Specifically, in Aim 1, we will define the immunogenicity of HLA-DQ mismatches that lead to the development of donor-specific HLA-DQ antibodies in transplant recipients. We will use computational and experimental approaches including adsorption/elution and site directed mutagenesis studies to prognosticate qualitative characteristics of HLA-DQ epitopes. In Aim 2, we will compare immune activation pathways triggered by ligation of different HLA class II molecules. We will evaluate the 3 different mechanisms of the immune response: those mediated by qualities of the antibody itself; those transduced inwards, downstream of the ligated HLA class II molecules; and those elicited via the T cell receptor recognizing the different class II molecules. We will use cutting edge proteomic technology as well as CRISPR-Cas9 edited cells expressing only one HLA class II allele as innovative tools to interrogate these specific pathways. Taken together, this project will support two significant advancements in solid organ transplantation: personalized medicine with respect to organ allocation and immunosuppressive management, and identification of novel immune targets for drug development. The shortage of organs for transplantation, the medical and financial ramifications associated with the need to treat allograft rejection, and the impact of immunosuppression and graft dysfun...