Project description:We developed a novel high-throughput and automated platform for MHC (major histocompatibility complex) peptides purification using AssayMAP with enhanced speed, sensitivity and reproducibility relative to prior studies.
Project description:The central nervous system (CNS), despite the presence of strategically positioned anatomical barriers designed to protect it, is not entirely isolated from the immune system. In fact, it remains physically connected to and can be influenced by the peripheral immune system. How the CNS retains such responsiveness while maintaining “immune privilege” remains an outstanding conundrum. In searching for molecular cues that derive from the CNS and allow its direct communication with the immune system, we discovered a repertoire of CNS-derived endogenous guardian peptides presented on major histocompatibility complex (MHC) II molecules at the CNS borders. During homeostasis, a preponderance of these guardian peptides were found to be bound to MHC II molecules throughout the path of lymphatic drainage from the brain to its surrounding meninges and its draining cervical lymph nodes. With neuroinflammatory disease, however, the presentation of guardian peptides was diminished. Fascinatingly, boosting the presence of these guardian peptides reinforced a population of suppressor CD4+ T cells and significantly reduced CNS autoimmune disease. This unexpected discovery of CNS-derived autoimmune guardian peptides may be the molecular key adapting the CNS to receive information and to maintain continuous dialogue with the immune system while balancing overt autoreactivity. This sheds new light on how we conceptually think about and therapeutically target neuroinflammatory and neurodegenerative diseases.
Project description:We previously reported that a synthetic Nod1 ligand, FK565, induced coronary arteritis in mice similar to Kawasaki disease. However, the molecular mechanisms underlying this site-specific inflammation have remained elusive. In this study, we found that CD11c+MHC class II+ cells accumulated in the heart of FK565-treated mice prior to arteritis development. We used microarray analysis to detail gene expression of CD11c+MHC class II+ cells. To obtain gene expression profile of CD11c+MHC class II+ cells, we isolated these cells from hearts of FK565-treated mice. Briefly, female mice at 8weeks age were administered 500 μg of FK565 subcutaneously at day 0 and day 3. At day6, murine hearts were removed and digested with collagenase. CD11c+MHC II+ cells were sorted as PI–CD45+Ly6G–NK1.1–CD11b+CD11c+MHC II+ using FACS Aria cell sorter (BD Biosciences). Sorted cells were subjected to RNA preparation. Two independent replicates from ten mice were made.
Project description:Hardy’s Fraction D bone marrow pre-B cells and splenic follicular B cells were purified by flow cytometry according to MHC II expression from wild-type and ItgaxcreH2-Ab1c MHC II conditional mice and transcriptionally profiled by RNA-seq. Duplicate or triplicate samples for each genotype and level of MHC II expression were sequenced. This is part of ongoing investigation of a possible cell-intrinsic role of MHC II B cells.
Project description:APCs (A20/ DCs) were stimulated by adjuvants, CpG, MDP and MPLA respectively for 12h. Then MHC-II Immunopeptidome and proteome were perforemd
Project description:Major histocompatibility class II (MHC II) is critical for adaptive immunity. MHC II intracellular trafficking and degradation is regulated by ubiquitination, with poly-ubiquitination (Ub) of MHC II directing it away from the plasma membrane. The MHC II poly-Ub chain that dictates the fate and function of ubiquitinated MHC II has not been characterized. Here, we describe the poly-Ub chain associated with MHC II in primary dendritic cells (DCs) and B cells. Analysis was conducted for endogenous H2-A and H2-E molecules immunoprecipitated from primary cells. We show that the ubiquitination of both I-A and I-E expressed by DCs and B cells is dependent on the E3 Ub ligase MARCH1. Comprehensive Ub chain analysis reveals MARCH1-dependent poly-Ub associated with MHC II is composed of Ub chains with branched K11/K63 linkages.
Project description:The nature of autoantigens that trigger autoimmune diseases has been much discussed but direct biochemical identification is lacking for most. Addressing this question demands unbiased examination of the self-peptides displayed by a defined autoimmune MHC-II molecule. We have examined the immunopeptidome of the pancreatic islets of the NOD mouse that spontaneously develops autoimmune diabetes based on its MHC-II, the I-Ag7 variant. The relevant peptides that induced early CD4 T cells derived from insulin and C-peptide; these were also found in the MHC-II peptidome of the pancreatic lymph node and spleen. The insulin-derived peptides followed a trajectory from internal processing in beta cells to exocytosis, uptake, and presentation in islets and peripheral sites. Such process not only generated conventional epitopes but also resulted in presentation of post-translationally modified peptides, including deamidated and fused sequences. These analyses reveal the key features of a restricted component in the self-MHC-II peptidome causing autoreactivity.