Project Title: Understanding the impacts of HLA-DO in vivo The project described in this R01 application addresses a cellular study aimed at verification of mechanistic understanding of the regulatory role HLA-DO (H2-O in mice) (DO), an accessory molecule of antigen processing and presentation pathway that is dependent on HLA-DM (H2-M in mice) on its cellular expression and has restricted tissue distribution. Despite the discovery of DO for over two decades ago, an understanding of its impact in regulation of antigen processing and epitope selection has been lacking. The current understanding of the DO function is that it inhibits DM. Recently, we have demonstrated that DO does not inhibit DM: we have shown that DO interacts with human MHC class II, HLA-DR1 (DR1), molecules directly and in collaboration with DM optimizes epitope selection. We demonstrated that DO has differential effects on binding of different peptides to DR1 molecules. Those findings need in vivo verification. The key question addressed here is whether DO plays a role in regulation of epitope selection in the thymus leading to changing the expressed T cell repertoire, and possibly regulation of susceptibility to the development of autoimmune diseases.
In aim I, we would examine the role of DO in altering T cell repertoire in DR1 expressing transgenic mice that do, or do not express H2-O. Using unique mouse models expressing a mutant DR1 (DR1bG86Y) that interacts with DO but not with DM, with or without H2-O.
In Aim 2, we would address whether DO inhibits DM, or its function is different from inhibiting DM in vivo.
In Aim 3 a, we would examine precursor frequency for the dominant disease associated epitope of collagen II, the causative antigen in Collagen Induced Arthritis (CIA), in in DR1+ DO-knockout mice.
In Aim3 b we would find out susceptibility of DO-KO mice to CIA, using novel non-invasive fluorescent imaging technology developed by our colleagues at JHU.
In Aim 3 c we would examine samples from Rheumatoid Arthritis patients that are identified as having a single nucleotide polymorphism (SNP) in their HLA-DOA 3'UTR gene for the expression of HLA-DO by intracellular FACS staining. The in vivo verification of HLA-DO contribution to the regulation of antigen processing and epitope selection would be a major leap towards filling the gap in understanding the biological significance of HLA-DO, which can guide the design of effective immunotherapeutics in future.
Helper T cells are stimulated to fight infections or diseases upon recognition of peptides from antigens that are processed and presented by the proteins of Major Histocompatibility Complex (MHC) Class II molecules. Processing of a full protein into small peptide fragments and the events that lead to selection of few short peptides for presentation to T cells is a lengthy process consisting of many steps and chaperones. Malfunctions during any step of antigen processing could lead to the development of self-reactive T cells or defective immune response to pathogens. Although much has been accomplished regarding how antigens are processed and presented to T cells, many questions still remain unanswered, preventing the design of therapeutics for direct intervention with antigen processing. In this proposal focus on a MHC class II molecular chaperone, HLA-DO. While HLA-DO was originally discovered decades ago, elucidating its function has proven challenging. By better understanding of how DO works, we expect to open new ways for design of new therapeutics that may improves many disease conditions.
| Sadegh-Nasseri, Scheherazade; Kim, AeRyon (2018) Selection of immunodominant epitopes during antigen processing is hierarchical. Mol Immunol : |
