Cellular and Molecular Mechanisms of Regulatory T Cells in EAE

University Of California-Irvine, Irvine CA

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Abstract

? DESCRIPTION (provided by applicant): Our overall goal is to identify cellular and molecular immunoregulatory mechanisms that contribute to disease progression and therapy in multiple sclerosis (MS). Building on our expertise in two-photon (2-P) imaging at the cellular level, and in ion channels and Ca2+ signaling at the molecular level, we will use the myelin oligodendrocyte glycoprotein experimental autoimmune encephalomyelitis (MOG-EAE) mouse model of MS to investigate the cellular interactions and molecular mechanisms underlying disease progression, as well as therapeutic approaches to promote remission. We focus in particular on regulatory T cells (Tregs), which maintain homeostasis and limit autoimmunity. Our central hypothesis is that Tregs limit autoimmune-mediated demyelination in the EAE model at two levels. (i) At the cellular level, Tregs compete with conventional T cells for access to antigen-presenting dendritic cells (DCs), and restrict egress of differentiated, pathogenic Th17 cells from lymph nodes. In Aim 1 we will apply simultaneous 2-P imaging of Tregs, naïve T cells, Th17 cells, and DCs in the intact lymph node (LN) to reveal fundamental cell trafficking and interaction dynamics during EAE induction, progression, and remission. Aim 2 extends those studies to the spinal cord where, by additionally imaging oligodendrocytes and neuronal cells, we will elucidate the cellular dynamics of neuroinflammation and demyelination during disease progression and remission. In both Aims, we further propose to image cellular dynamics during therapies that show great promise for treatment of human MS; including drugs that target S1P1 receptors to cause lymphocyte sequestration within the LN, and stem cell therapy to promote remyelination. (ii) At the molecular level, contact by Tregs inhibits Ca2+ signaling in target lymphocytes, thereby suppressing their activation. Based on our earlier demonstration that that Ca2+ signaling in T cells is mediated by plasma membrane Orai1 channels and triggered by STIM1 in the endoplasmic reticulum, in Aim 3 we propose to investigate the roles of these proteins in contact-induced suppression of Ca2+ signaling. Employing a novel `toolkit' of genetically-encoded Ca2+ indicators and probes of channel function we will monitor cellular Ca2+ signaling in intact LN and cord; test hypothesized mechanisms of Treg-mediated inhibition including dissolution of Orai puncta and transendocytosis of Orai into Tregs; and evaluate Orai as a therapeutic target in MS by visualizing cell dynamics following administration of a specific Orai1 blocker during EAE. Although this proposal is targeted specifically at MS, our findings and novel immunoimaging approaches will contribute in a broader context to a better understanding of how immune responses are initiated, how immunological tolerance is achieved, how regulatory T cells prevent autoimmunity and dampen immune responses, and how autoimmunity and infectious diseases can be effectively treated.