Shannon J. Turley - US grants

Type-1 diabetes (T1D) is an autoimmune disorder characterized by T cell-mediated destruction of pancreatic p-cells. To understand how T cell responses against p cells are provoked is the long-term goal of our work. This proposal is based on the hypothesis that disruptions in the gut epithelium impinge on the initiation of T1D, by exposing dendritic cells (DCs) to enteric factors that boost their capacity to present p cell antigens to potentially diabetogenic T cells. Several findings lead to this hypothesis. First, intestinal permeability and inflammation are exaggerated in diabetes-prone rats before insulitis onset and in patients with T1D. Second, intestinal abnormalities such as hypertrophy or hyperplasia of intestinal villi have been observed in pre- insulitic, diabetes-prone rats. Third, there is a very high prevalence among T1D patients of gluten-induced intestinal inflammation, or celiac disease, and NOD mice exhibit pathogenic hallmarks of celiac disease. Fourth, gluten exhibits diabetogenic potential in genetically susceptible subjects. Finally, our recent studies indicate that perturbations of the gut epithelium modify the course of T1D. Despite an abundance of clinical and epidemiological studies implicating intestinal barrier dysfunction in T1D, the mechanistic underpinnings of this association remain elusive. The objective of this proposal is to elucidate the cellular and molecular mechanisms by which intestinal barrier function regulates the pancreatic autoimmune response. The specific aims are to: 1} Determine whether alterations to the intestinal epithelium affect both MHC class I- and class ll-restricted T cell responses to p cell Ags. These experiments will ascertain the impact of altered intestinal barrier function on the proliferation, activation, survival and effector functions of p cell- specific, CD4+ and CD8+ T cells; 2) Define how changes in intestinal barrier function impact DC function in pancLNs. Here we will determine the impact of mild injury to the intestinal epithelium on the differentiation, maturation, and Ag presentation capacity of specific DC subsets of pancLNs. We will also assess the molecular mechanism by which intestinal injury alters DC function by testing the role of NFicB activation in these processes; 3) Evaluate the role of dietary wheat gluten in provoking the anti-p-cell immune response. These experiments will monitor the impact of alimentary gluten proteins on the priming of p-cell-reactive T cells in pancLNs and the maturation of DCs. The aim of these experiments is to pinpoint the specific mechanism by which gluten provokes pancreatic autoimmunity. The proposed studies will yield insights into the link between environmental provocation and autoimmune pathogenesis.

Peripheral tolerance, or the elimination or functional silencing of potentially auto-reactive T cells, relies on the presentation of self-antigen to T cells. To understand how self-antigen presentation leads to tolerance or autoimmunity is the long-term goal of our work. This proposal is based on the hypothesis that lymph node stromal cells (LNSCs) are highly specialized antigen presenting cells that impose self-tolerance on circulating T cells. Several findings lead to this hypothesis. First, like medullary thymic epithelial cells (mTECs), LNSCs constitutively express peripheral tissue antigens (PTAs), and the autoimmune regulator (Aire) gene, a transcriptional regulator that partially controls PTA expression and central tolerance induction by mTECs. Second, LNSCs are localized in the cortex of the lymph node, which is an ideal location for interacting with circulating T cells. Third, LNSCs can process endogenously expressed antigen into peptide- MHC complexes and induce proliferation in naive, antigen-specific T cells. Fourth, the presentation of endogenously expressed PTA by LNSCs is sufficient to promote deletional tolerance among antigen-specific T cells. The objective of this proposal is to dissect the molecular and cellular mechanisms by which LNSCs present self-antigens to CDS and CD4 T cells and to evaluate their role in imposing tolerance to key autoantigens. The specific aims are to: 1) Investigate the general functional properties of mouse and human LNSCs. Here we will characterize the transcriptional profile of LNSCs and the role of Aire in these novel APCs. We will then assess whether PTA expression by mouse LNSCs is linked to disease susceptibility in experimental autoimmune encephalomyelitis (EAE) and type-1 diabetes (T1D); 2) Define the role of LNSCs in promoting tolerance among MHC class I- and class ll-restricted self-reactive T cells. We will determine whether LNSCs promote tolerance to islet-specific glucose-6-phosphatase, a pancreatic antigen that is targeted in T1D using the 8.3 TCR transgenic mouse model in which pathogenic CD8+ T cells destroy insulin-producing beta cells. Conclusions from these studies will be tested for generality using the 5B6 TCR transgenic mouse model of EAE in which CD4+ T cells recognize the myelin protein, proteolipid protein; 3) Ascertain whether the function of LNSCs is influenced by inflammation. We will examine the impact of inflammation on the function and gene expression profile of LNSCs. Results from these studies will elucidate whether LNSCs are constitutively tolerogenic or whether their capacity for self-antigen presentation is modified under inflammatory conditions.

DESCRIPTION (provided by applicant): Fibroblastic reticular cells (FRCs), one of the major populations of non-hematopoietic stromal cells in lymph node (LNs), secrete extracellular matrix components to form a dense reticular network and lymph-draining conduit system. The T cell zone is delineated by FRCs, forming a scaffold to provide essential guidance cues to immune cells. FRCs orchestrate immune cell migration via expression of CCL19 and CCL21, as well as adhesion molecules, integrins, and glycoproteins. Beyond migration, FRCs maintain na¿ve T cell homeostasis and they have the capacity to impose antigen-specific deletional tolerance, with direct presentation of viral and self-peptides to na¿ve CD8+ T cells. The timing of deletional events in these studies, whether an inevitable outcome of an FRC-mediated activation signal to na¿ve T cells, or a result of subsequent feedback to the FRC from the activated T cell, is unknown, as are its driving molecular mechanisms. Additionally, in the context of immune response, T cells are usually activated by dendritic cells (DCs) while in direct contact with the FRC network, therefore any effect of FRCs on activated T cells is highly relevant. Our recent studies suggest that FRCs can acquire suppressive function at different stages in the T cell response; this newly appreciated function of FRCs appears to control the expansion of potentially pathogenic T cells via a NOS2-dependent mechanism. This project aims to further define the molecular and physical basis of the interactions between activated T cells and FRCs within lymph nodes. Despite recent advances indicating that stromal determinants of secondary lymphoid organs exhibit complex regulatory roles during immune responses, our knowledge of the stromal niche and how it impacts T cell immunity and tolerance is limited. The proposed studies will define the molecular crosstalk that culminates in CD8 T cell suppression by FRCs. They will also establish the influence of FRCs on the positioning and motility of activated T cells Finally, these studies will determine the capacity of FRCs to present lymph-borne antigen to CD4 T cells. The results of these studies will elucidate the molecular and physical interactions between lymph node FRCs and newly activated CD8 and CD4 T cells, and the role of FRCs in patrolling the lymph by sampling antigens from the conduit system, and the impact of inflammation on these processes. PUBLIC HEALTH RELEVANCE: Emerging evidence suggests that stromal determinants of secondary lymphoid organs exhibit complex regulatory roles during immune responses. We recently described a novel in vivo cross-talk between activated T cells and fibroblastic reticular cells (FRCs) that endows these stromal cells with the capacity to constrain the proliferation of activated T cells through regulated nitric oxide (NO) release. The over-arching goal of this proposal is to further elucidate the molecular and physical basis of FRC-T cell cross-talk. This work may provide critical insights for the development of new therapeutic strategies to treat human inflammatory and autoimmune diseases associated with unbridled T cell immunity.

DESCRIPTION (provided by applicant): 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.

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 detection 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: Po patients with MS have increased numbers of myelin-reactive IL- 17/GM-CSF/IFN?-secreting CP4+CCR6+CP45RO+ 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 CP4+ 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 (PPPN) expressed by CSF T cells with CLEC-2 on B cells/dendritic cells, given the importance of PPPN 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 wVo function of myelinreactive T cells, and the impact of distinct APCs on their functional programs.

Genome-wide analyses of MS susceptibility loci have emphasized the importance of MHC class II genes, strongly implicating antigen presentation to CD4 T cells as a key process in the pathogenesis of MS. However, it has been very difficult to study myelin-specific CD4 T cells from MS patients ex vivo, due to low T cell receptor affinities for the relevant peptide-MHC complexes. Therefore, two fundamental questions remain unresolved: First, which molecular properties distinguish myelin-specific T cells in patients with MS from those in healthy individuals? Second, are there distinguishing properties of myelin-specific and virusspecific T cells that could be exploited for therapeutic gain? In Aim 1, we will study TCR recognition by myelin-specific T cells from MS patients. Imaging experiments identified alterations in immunological synapse formation by myelin-specific compared to virus-specific T cells, and structural studies demonstrated unusual binding topologies by some myelin-specific TCRs isolated from MS patients. The Garcia lab recently developed a yeast peptide-MHC display approach to identify entirely novel peptide ligands for a given TCR and showed that one ofthe peptides was recognized with a non-conventional TCR topology and failed to induce signaling. This means that TCR binding topology can have a profound effect on signaling. This approach will be used to examine the irnpact of TCR topology on the function of myelin-specific T cells and determine whether administration of such peptides can prevent spontaneous disease in human TCR/MHC transgenic mice. In Aim 2, we will collaborate with Drs. Love and Hafler to study myelin and virus-specific T cells from MS patients and control subjects identified with the nanowell device. Antigenresponsive T cells will be isolated from nanowells with a robotic device, which enables detailed molecular characterization of myelin-specific T cell populations. The transcriptome of myelin-specific T cells from MS patients and control subjects will be examined, with an emphasis on the expression of genes associated with susceptibility to MS, transcription factors and cytokine signaling molecules. Clonal expansion of myelinspecific will be assessed by sequencing of TCR chains, and the frequency of myelin-reactive TCR sequences in peripheral blood and CSF T cells will be determined by lllumina sequencing ofthe TCR repertoire. These studies will determine whether myelin-specific T cells with particular cytokine proflles and/or gene expression programs are preferentially expanded in MS patients compared to healthy subjects.

This core will make important resources available to all Projects of this PPG. The nanowell technology represents a powerful tool to study the functional consequences of interactions between T cells and APC. The technology will enable in-depth investigation of the impact of different types of APCs on the functional programs of self-reactive T cells, which is of central importance to this PPG. A novel feature of this technology is that the functional consequences on both T cells and APC can be studied for interacting cell pairs, offering insights into the complex communication that programs self-reactive T cells with autoaggressive behavior. This core will enable integration of datasets from clinical specimens and experimental mouse models (Aim 1). It provides an important link between Projects 1 and 2, which both study myelin-specific T cells from patients with MS. The technology enables robotic isolation of cells of interest, such as myelin-specific T cells that produce combinations of pro-inflammatory cytokines (e.g. IL-17 + GM-CSF), for analysis of single-cell gene expression or clonal expansion. Furthermore, the technology will be made available for analysis of murine T cells, APCs and stromal cells (Projects 3 and 4). The core will also make recombinant proteins available to all Projects of this PPG, including experiments in the nanowell system (Aim 2), This core has provided such reagents to all Projects in the previous funding period and will continue to provide them for several efforts: 1. Studies by Dustin and Wucherpfennig have examined immunological synapse formation by human self-reactive T cells using the planar lipid bilayer system developed by the Dustin lab. Recent collaborative studies by the Love and Wucherpfennig labs have applied this approach to the nanowell system, enabling synapse structure to be related tp T cell function. These experiments require sets of human and murine recombinant proteins, including peptide-MHC complexes, ICAM-1 and CD80 (Projects 1 and 2). 2. Collaborative studies by the Kuchroo and Wucherpfennig labs have shown that tetramers of the MOG extracellular domain can be used to label MOG-specific B cells, and this reagent will be used in Project 3. 3. The turley, Kuchroo and Wucherpfennig labs have utilized Ig fusion proteins of podoplanin and CLEC-2 to study the function of these molecules in T cell - APC communication, which will be provided to Projects 3 and 4. This core will thus enable in-depth investigation of T cell - APC interactions in both humans and animal models to advance our understanding ofthe pathogenesis of MS.

It is well established that leukocyte infiltration of the central nervous system (CNS) is a critical early step in the development of the demyelinating autoimmune disease, multiple sclerosis (MS) however the mechanistic details of this process remain poorly understood. Blocking leukocyte infiltration and destruction of the CNS in a targeted manner remains a significant clinical challenge. The long-term objectives are to develop therapeutics that restore immune tolerance and control autoimmune-mediated destruction of target organs. The obiective of this proposal is to define how stromal cells regulate autoimmune inflammation of the CNS. Stromal cells create inflammatory microenvironments, attract T cells and antigen presenting cells (APCs) into the CNS, create three-dimensional reticular structures that infiltrating leukocytes crawl on, and produce proinflammatory cytokines through interactions with Th17 cells. Moreover, stromal cells can function as APCs and regulate the function of activated T cells in close proximity. The central hvpothesis is that a stromal cell network is essential for the formation of chronic inflammatory lesions in MS. This hypothesis is based on emerging evidence from the literature and preliminary data generated in the applicants' laboratories. The rationale for the proposed research is that elucidating the mechanisms by which stromal cells interact with T cells and APCs at the blood brain barrier and in the CNS parenchyma may illuminate mechanistic insight into this pathogenic process. Two specific aims will be carried out to test this hypothesis: 1) Define the role of PDPN expression by stromal, cells in leukocyte infiltration of the CNS. and 2) Elucidate the impact of stromal cell-leukocyte cross-talk in the inflamed CNS. As part of the first aim, human brain tissue from MS patients and healthy subjects will be studied to evaluate the interactions between leukocytes and PDPN-expressing stromal cells in lesions. In addition, novel conditional knockout mice will be used to evaluate whether PDPN expression by stromal cells and CLEC-2 expression by dendritic ceils and B ceils affects leukocytic infiltration of the CNS during EAE. In the second aim the impact of autoimmune tissue inflammation on stromal cell interactions with T cells and APCs will be evaluated. Furthermore, the mechanism by which the inflammatory milieu converts FRCs from immunosuppressive to proinflammatory cells will be interrogated. The proposed research is innovative because the contribution of stromal cells to the pathogenesis of MS is an understudied field and the role ofthe PDPN-CLEC-2 axis in leukocyte infiltration of inflamed tissues has not been previously addressed. The study is significant because elucidation ofthe mechanisms by which stromal cells influence APC and T cell function may offer new opportunities for intervention.

Dr. Wucherpfennig has provided scientific and administrative leadership for this PPG since 1999, and this effort has been productive as evidenced by the large number of collaborative publications, many of which have been in high-impact journals. He will continue to closely interact with all members ofthe team to foster collaboration and identify new scientific opportunities. Dr. Wucherpfennig will organize regular formal scientific meetings at which recent progress will be presented and new opportunities for collaboration will be discussed. All project leaders work in the Boston area, Drs. Wucherpfennig, Turtey and Kuchroo on the Harvard Medical School (HMS) campus, and Dr. Love at the Koch Institute of MIT. Drs. Turley and Wucherpfennig are neighbors at the Dana-Farber Cancer Institute, and Drs. Kuchroo, Turtey and Wucherpfennig frequently meet at seminars and as part of the Program for Immunology at HMS. Drs. Kuchroo and Wucherpfennig also jointly teach an annual quarter course on Autoimmunity' for graduate students (since 1999). These frequent interactions foster dialogue and collaboration. In addition. Dr. Wucherpfennig will organize annual meetings with the Scientific Advisory Board (SAB) at which each project leader will present recent progress and discuss directions for the coming year. All members of our SAB work at HMS, and we will therefore also have many opportunities to consult them on an informal basis. Dr. Wucherpfennig will also continue to be in charge of all administrative aspects and will be assisted by this administrator and Dana-Farber grants and financial management specialists.

Experimental autoimmune encephalomyelitis (EAE) is an animal model that reproduces most of the clinical and pathological features of multiple sclerosis (MS). The development and the progression of EAE, like other autoimmune diseases, result from the pathogenicity of effector T cells and the negative regulation imposed by regulatory T cells (Tregs). However, the role of B cells in MS pathogenesis and the interplay of myelin-specific B, T effector, and regulatory T cells is not well understood. Therefore, we have established a trangenic mouse model (2D2xTH mice) in which both T and B cells are specific for the myelin oligodendrocyte glycoprotein (MOG). The majority of the 2D2xTH mice (about 59%) develop a very severe form of spontaneous EAE characterized by the presence of ectopic lymphoid follicle like structures in the spinal cord of affected mice. By gene expression profiling we have identified Th17 cytokines differentially upregulated in the spinal cord of sick 2D2xTH vs. 2D2 mice. 2D2 TCR transgenic Thi7 cells were specifically found to induce autoimmune tissue inflammation characterized by formation of ectopic lymphoid follicles under the meninges of the recipients. The effector cytokine IL-17 and a cell surface molecule Podoplanin (PDPN) specifically expressed on Th17 cells were involved in the induction of ectopic follicles in the affected animals, since anti-PDPN antibody treatment suppressed development of ectopic lymphoid follicles in the CNS. CLEC-2, the ligand for PDPN, is predominantly expressed on DCs and B cells. We have recently generated/obtained PDPN and CLEC-2-deficient mice to address the role of this pathway in T:B ceir collaboration and induction of ectopic lymphoid follicles in autoimmune disease. Based on our results, we propose that antigen presentation by MOG-specific B cells results in the generation of highly pathogenic T cells and limits the generation and function of MOG specific CD4+CD25+ regulatory T cells. We will: 1) Study the mechanism by which B cells induce pathogenic Thi7 cells in vivo; 2) Study the mechanism by which Th17 cells induce ectopic lymphoid follicles and germinal center formation and 3) Study the role of Foxp3+ Tregs in regulating ectopic lymphoid follicles and germinal center formation in the CNS. A better characterization of the role of auto-reactive T and B cells, pathogenic cytokines and their interactions during disease progression, as described in our proposal, will provide invaluable insight into the role of autoantigen-specific B cells in the generation of autopathogenic T cells.