Antigen presentation is an integral component of every autoimmune disease process, and thus represents an important scientific and clinical problem. The seven investigators who come together in this PPG have highly complementary areas of expertise and have formed a cohesive, multidisciplinary program. The overarching hypothesis is that the development and progression of autoimmune diseases are controlled by specialized populations of antigen-presenting cells (APCs) that serve distinct roles in the induction of different effector and regulatory T cell programs. The team emphasizes direct investigation of APC - T cell interactions in patients with autoimmune diseases, in particular multiple sclerosis (MS), and integrates these human immunological studies with in-depth mechanistic studies in relevant animal models. During the previous funding period, the group developed a novel nanowell-based technology platform for multiplexed investigation of T cell function in autoimmune diseases. The technology enables co-culture of single T cells with mature dendritic cells in wells of subnanoliter volume for multi-dimensional characterization of cytokine secretion and surface markers. Furthermore, responding T cells can be recovered with a robotic device for characterization of transcriptional programs. This technique will be used by all investigators to examine the functional consequences of T cell interactions with distinct populations of APC. The team will address a long-standing challenge in the field and define the functional and molecular differences between self-reactive T cells in patients with MS and healthy subjects. Studies in MS CNS lesions and animal models will examine how the interaction of T cells with different populations of APCs results in the formation of chronic inflammatory microenvironments in the target organ. Of particular interest is the complex interplay between T cells, B cells and stromal cells that results in the formation of ectopic lymphoid follicles in the CNS. Studies during the previous funding period have shown that Th17 cells express podoplanin (PDPN), a surface molecule that interacts with CLEC-2 on B cells and mature dendritic cells. Antibody-based blockade of PDPN function prevents formation of ectopic lymphoid follicles, and the function of these molecules will now be studied in MS lesions and conditional knock-out mice. The program is highly synergistic based on our focus on an important problem in the autoimmunity field, and our highly collaborative approach integrates a unique team of investigators with expertise in molecular and cellular immunology, biophysics, and engineering to investigate disease mechanisms in autoimmunity with cutting-edge technologies.
This PPG investigates one of the central problems in the autoimmunity field, the cellular and molecular mechanisms by which presentation of self-antigens induces autoaggressive T cell populations. Research in this field holds significant promise for the identification of novel therapeutic targets for MS and other autoimmune diseases. The novel technologies that we are developing may be useful for immune monitoring in many autoimmune conditions. Project 1 - Ex vivo Analysis of Autoreactive T Cells from Patients with Multiple Sclerosis Project Leader (PL): J. Christopher Love (Description as provided by applicant): Autoreactive T cells are thought to play a critical role in the pathogenesis of multiple sclerosis (MS), yet the functional T cell programs that promote disease remain unknown. A major challenge hindering the study of self-reactive T cells in MS is the sensitive identification and characterization of these cells. The technologies used to date have insufficient sensitivity for detection of T cells with low TCR affinities and may bias detectin for cells with sufficient in vitro proliferative potential. To overcome these challenges, this collaborative project brings together the Love lab (MIT) with expertise in simultaneous parallel phenotypic and functional analyses of >10 single primary cells in arrays of sub-nanoliter wells (nanowells) and the Hafler lab (Yale School of Medicine) with expertise in characterization of autoreactive T cells in MS as well as the genetics of autoimmune disease. Specifically, this project will address a central question related to understanding the pathology of MS: Do patients with MS have increased numbers of myelin-reactive IL-17/GM-CSF/IFN?-secreting CD4+CCR6CD45RO+ cells compared to age-matched control subjects? To address this critical question, a two-pronged approach will be employed that leverages a new method for polyclonal expansion of CD4+ T cells in small pools, and a novel state-of-the-art nanowell technology for single-cell co-culture assays of individual T cells with autologous mature dendritic cells pulsed with antigen. This technology enables sensitive detection of self-reactive T cells and generates a comprehensive body of data on surface phenotype and cytokine release using a dense, elastomeric array of nanowells. Many different cytokines are captured on a glass slide and quantified on a microarray scanner. Preliminary data show that this approach greatly increases the sensitivity of detection for self-reactive T cells and enables comprehensive assessment of their ex vivo functions. As a second aspect of this aim, we will examine how genotypic variations in HLA haplotypes and cumulative genetic loads in the IL-17 pathway affect the burden of IL-17/GM-CSF/IFN?+ T cells. In Aim 2, we will address how distinct populations of antigen presenting cells (APCs) alter the functional programs of myelin-reactive T cells from blood and cerebrospinal fluid (CSF). Of particular interest is the interaction of podoplanin (PDPN) expressed by CSF T cells with CLEC-2 on B cells/dendritic cells, given the importance of PDPN in the formation of ectopic lymphoid follicles in the CNS of patients with MS. The outcome of these studies will provide new insight into the ex vivo function of myelin-reactive T cells, and th impact of distinct APCs on their functional programs.
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