The initiation of an acquired CD4 T cell immune response requires that T cell receptors on antigen-specific T cells recognize and be stimulated by small amounts of foreign antigen expressed on the surface of antigen presenting cells (APCs). These antigens are expressed as polypeptide fragments of foreign proteins immobilized on the surface of the APCs bound to MHC class II (MHC-II) molecules. These polypeptide fragments are generated by proteolysis in endo/lysosomal compartments and to efficiently stimulate T cells MHC-II must traffic to these compartments, bind antigenic peptides, and move out to the plasma membrane of the APC. The goal of my research program is to identify the molecular mechanisms that regulate the expression and stability of peptide-loaded MHC-II (pMHC-II) complexes on the surface of APCs and to elucidate the mechanisms leading to the movement of MHC-II into and out of endo/lysosomal peptide-binding compartments in APCs. Antigen-specific CD4 T cells are stimulated by the binding of their clonotypic T cell receptor (TCR) with specific pMHC-II on the surface of antigen-bearing APCs. DCs are the APCs most responsible for the activation of naive CD4 T cells, a process that requires antigen uptake and processing, antigenic peptide binding to MHC-II, and movement of pMHC-II from intracellular antigen loading compartments to the DC plasma membrane. MHC-II associates with a chaperone protein termed the Invariant chain (Ii) in the endoplasmic reticulum that prevents peptide-binding to endoplasmic reticulum localized MHC-II and assists in MHC-II folding. MHC-II-Ii complexes traverse the secretory pathway, and after arrival at the plasma membrane are internalized by clathrin-mediated endocytosis and travel along the endocytic pathway until they gain access to late endosomal antigen processing and peptide loading compartments. It is in these compartments that MHC-II-associated Ii is degraded by proteinases and the peptide-binding groove blocking fragment of Ii, termed CLIP, is removed by the peptide editor HLA-DM. HLA-DM catalyzes the exchange of CLIP (and other low affinity peptides) from MHC-II in these compartments. Following editing of low kinetic stability pMHC-II complexes by HLA-DM, the pMHC-II complex traffics to the plasma membrane. Surface pMHC-II complexes internalize by clathrin-independent endocytosis, are ubiquitinated in early endosomes, and are rapidly targeted for lysosomal degradation, thereby ensuring pMHC-II turnover in APCs. Activation of DCs by TLR ligands abrogates MHC-II biosynthesis and blocks the turnover of pMHC-II complexes by the E3 ubiquitin ligase March-I, thereby maintaining expression of stimulatory pMHC-II formed at the time of DC activation and increasing the likelihood for effective interaction with pMHC-II-specific CD4 T cells. While delivery of newly-synthesized MHC-II late endosomes for antigenic peptide binding is thought to be the major pathway of MHC-II antigen processing, alternative antigen processing pathways exist in APCs that result in the delivery of antigens into both early and late endosomes. Depending on the activation status of the APC, surface MHC-II can internalize and recycle in APCs, and many studies have shown that recycling MHC-II can bind antigenic peptides in early endosomes. The relative importance of early vs. late endosomes as antigen processing compartments, and understanding the molecular mechanisms regulating MHC-II recycling through them, is a major focus of my research program. Some of our accomplishments in this field over the past year are: -Rab11a regulates pMHC-II recycling in DCs. In our studies of MHC-II endocytosis and recycling we identified Rab11a as a regulator of pMHC-II recycling in DCs. Experimental perturbation of Rab11a expression reduced pMHC-II recycling in activated DCs and also in MHC-II ubiquitination-defective resting APCs, demonstrating that ubiquitination of pMHC-II diverts internalized pMHC-II from an efficient Rab11a-dependent recycling pathway to one of lysosomal degradation. -March-I expression is regulated by an intragenic promoter in DCs. March-I is expressed exclusively in APCs and March-I mRNA expression is rapidly terminated upon APC activation. We have studied the March-I gene to identify regulatory regions controlling March-I expression. We identified an intragenic promoter in March-I that regulates both APC-specific expression of March-I and allows downregulation of March-I upon DC activation. -Ube2D1 regulates ubiquitination of March-I. In our search for E2 ubiquitin conjugating enzymes that regulate March-I function, we discovered that expression of March-I itself is controlled by ubiquitination and we identified the E2 ubiquitin conjugating enzyme Ube2D1 as a regulator of March-I ubiquitination and turnover.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC009404-24
Application #
9779586
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
24
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Bandola-Simon, Joanna; Roche, Paul A (2018) Dysfunction of antigen processing and presentation by dendritic cells in cancer. Mol Immunol :
Cresswell, Peter; Roche, Paul A (2017) Editorial Overview: Antigen Processing and Presentation; many fingers in many pies. Curr Opin Immunol 46:v-vii
Roche, Paul A; Cresswell, Peter (2016) Antigen Processing and Presentation Mechanisms in Myeloid Cells. Microbiol Spectr 4:
Anderson, Howard A; Roche, Paul A (2015) MHC class II association with lipid rafts on the antigen presenting cell surface. Biochim Biophys Acta 1853:775-80
Roche, Paul A; Furuta, Kazuyuki (2015) The ins and outs of MHC class II-mediated antigen processing and presentation. Nat Rev Immunol 15:203-16
Liu, Zhenzhen; Roche, Paul A (2015) Macropinocytosis in phagocytes: regulation of MHC class-II-restricted antigen presentation in dendritic cells. Front Physiol 6:1
Cho, Kyung-Jin; Walseng, Even; Ishido, Satoshi et al. (2015) Ubiquitination by March-I prevents MHC class II recycling and promotes MHC class II turnover in antigen-presenting cells. Proc Natl Acad Sci U S A 112:10449-54
Mittal, Sharad K; Roche, Paul A (2015) Suppression of antigen presentation by IL-10. Curr Opin Immunol 34:22-7
Cresswell, Peter; Roche, Paul A (2014) Invariant chain-MHC class II complexes: always odd and never invariant. Immunol Cell Biol 92:471-2
Bosch, Berta; Heipertz, Erica L; Drake, James R et al. (2013) Major histocompatibility complex (MHC) class II-peptide complexes arrive at the plasma membrane in cholesterol-rich microclusters. J Biol Chem 288:13236-42

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