David A. Hafler - US grants

Recent advances in immunologic techniques permit the cloning and maintenance of human T-cells that are then available for detailed scientific study. In addition, monoclonal antibodies have been developed that recognize human T-lymphocytes at various stages of differentiation and identify different functional categories of T-cells. Anti-Ta1 is one such monoclonal antibody that reacts with activated T-cells that are more prevalent in the blood of multiple sclerosis patients compared to controls. These approaches will be used for a series of detailed investigations of cellular immunologic studies of central nervous system inflammatory disease, with particular emphasis on multiple sclerosis, a presumed autoimmune disease of the central nervous system associated with abnormalities of cellular immunity. The specific areas of investigation and questions to be asked are as follows: 1. What are the phenotypes and functional characteristics of activation antigen positive cells in the blood and spinal fluid of MS patients and patients with other inflammatory CNS diseases? 2. Using direct single cell clonal analysis of blood and cerebrospinal fluid T-lymphocytes, what is the frequency of different T-cell subpopulations using functional and phenotypic analysis? 3. Are there deficiencies in white matter antigen specific T8 suppressor cells which are induced by T4 suppressor/inducer cells in patients with multiple sclerosis? 4. What are the effects of virus on in vitro immune function and, in particular, the interaction of reovirus type 3 with specific T-cell subpopulations that have a receptor for the viral hemagglutinin?

This grant represents a revised proposal for the study of T cell clonality in multiple sclerosis cerebrospinal fluid by analysing rearrangement patterns of the T cell receptor genes. The studies outlined in the previous grant of peripheral blood and family genetic studies have been deleted from the proposal. New data since submission of the original proposal has been obtained demonstrating oligoclonal T cells in the spinal fluid of an MS patient. In addition, brain tissue has been obtained at an autopsy from a patient in whom T cells were cloned prior to the patient's death. This study will focus on oligoclonal T cells in the spinal fluid of multiple sclerosis patients according to the following specific aims: 1) to what extent are cerebrospinal fluid, oligoclonal T cell populations found in patients with chronic progressive multiple sclerosis? 2) are oligoclonal T cell populations found in early relapsing-remitting patients? 3) do CSF T cell clones reflect the population of T cells in the plaque of MS brain? and 4) what are the functional properties of oligoclonal T cell populations?

DESCRIPTION: (Investigator's abstract): Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system white matter. The similarity of experimental autoimmune encephalomyelitis (EAE) and post viral encephalomyelitis with MS has implicated myelin basic protein (MBP) as a target autoantigen in the disease. The role of MBP has been further supported by the recent demonstration of in vivo activated MBP reactive T-cells in the blood of patients with MS. Other evidence of immune system involvement in the disease pathogenesis has been the association of MS with the MHC class II phenotype HLA-DR2/DQw1. As the ability of immunogenic peptides to bind to polymorphic regions of MHC is one requirement for T-cell receptor (TCR) recognition of antigen, it can be postulated that disease related MHC phenotype selectively present regions of autoantigens that are recognized by T-cells not clonally deleted in the thymus. Thus a major question in the investigation of MS is whether there are DR2 linked immunodominant regions of MBP as has been observed in rodents with EAE. A second major question is whether T-cell recognition of an immunodominant region of MBP is linked to a particular TCR sequence. The applicant's laboratory has demonstrated there is a higher frequency of T-cells reactive with a DR2 linked immunodominant region of MBP between residues 84 and 102 in patients with MS as compared to controls. The investigators have also examined TCR Vbeta gene usage among sixty-four T-cell lines from both MS patients and control subjects which were reactive with this region of human MBP. Vbeta 17 and Vbeta 12 were found to be the major recognition elements for this peptide among different individuals, with 34/64 lines using the Vbeta 17 and 9/64 lines using the Vbeta 12 TCR genes. These data are the first to demonstrate a restricted Vbeta gene usage for an immunodominant region of a human autoantigen. The present grant application has been substantially revised as suggested by the reviewers to reflect these recent observations. There is a series of new questions to be answered to complete these investigations: 1). Which TCR Valpha are used to recognize immunodominant MBP peptides in MS? 2). Are there common sequences among Vbeta chains or among Valpha chains used to recognize peptides of MBP among lines from the same patients or among different patients. 3. What are the MHC restriction elements for recognition of MBP peptides in relationship to TCR usage and are there common DR or DQ sequences used to bind MBP peptides? 4). Are there TCR polymorphisms associated with TCR peptide reactivity? 5). Is there sequestration of T-cells reactive to MBP in the CSF? These questions address the basic hypothesis that an immunodominant region of MBP, between residues 84 and 102, may be an encephalitogenic region in DR2+ individuals and can be one of the target antigens in MS.

This is an application for partial funding of a Conference on AutoImmunity, being held under the auspices of the FASEB, from June 12 - June 18, 1993 at Saxton's River, Vermont. Participation will be limited to 155 scientist applicants, who will be selected on the basis of their expertise and interests as most likely to contribute to a stimulating and productive environment. The meeting will focus on latest developments related to our understanding of autoimmunity. The conference will consist of eight scientific sessions, each with four to five speakers and a discussion leader. In addition there will be two poster sessions during the week and a dinner speaker. The major session topics will be as follows: 1). T cell Activation and Anergy; 2). T cell Recognition of Self Antigen and Thymic Tolerance; 3).MHC structure and relationship to disease; 4). Genetics contribution and genetic manipulation in autoimmunity; 5. What is New in Autoimmune Disease; 6). Autoantibodies and B cell Tolerance 7). Cytokines and autoimmunity; 8). Microbes and autoimmune disease; and 9). New Approaches to Immunotherapy. The conference will present a comprehensive view of both basic immune mechanisms related to autoimmunity, immunopathogenic mechanisms involved in the autoimmune process, and immune intervention. It will provide a forum in which both senior and younger scientists from diverse backgrounds will discuss autoimmunity. Furthermore, we have arranged with Dr. Laurie Glimcher to juxtapose the summer FASEB Conference on Lymphocytes and Antibodies back to back with our more applied meeting so that individuals who wish to attend both meetings can do so. The FASEB Conference on Autoimmunity has been held approximately every two years at Saxton's River and has proven to be an important vehicle for scientific dialogue related to autoimmunity.

Multiple sclerosis (MS) is hypothesized to be an autoimmune disease where the activation of myelin-reactive T cells, possibly by cross-reactive antigens, mediates the initial inflammatory insult leading to the recruitment of effector inflammatory cells. While there are no differences in the frequency of MBP and PLP reactive T cells in MS patients as compared to controls using limiting dilution analysis (LDA), it has become clear that there are both functional differences in the myelin reactive T cells of MS patients and differences in their regulation. We recently developed methods to directly measure the frequency of clonally expanded and activated antigen specific and invariant T cells ex vivo without in vitro manipulations. This analysis uses PCR to measure the number of T cells expressing particular TCR alpha/beta chains. In contrast to frequencies of T cells recognizing the immunodominant MBPp85-99 epitope in MS patients of approximately 1:10/5 as measured by LDA, we found T cell frequencies as high as 1:300 in the same subjects by PCR analysis: This grant will focus on three major issues. First, we will investigate whether the clonal expansion we observed for MBPp85-99 reactive T cells is also observed for T cells recognizing other immunodominant regions of MBP and PLP. This is critical to known in the development of antigen specific immunotherapies. As autoreactive T cells are present in the circulation of normal individuals, it has also become clear that other populations of T cells exist which may regulate the activation of autoreactive T cells, including a recently described subset of T cells expressing the invariant Valpha24JalphaQ sequences. We recently demonstrated that the Valpha24JalphaQ T cells are reduced ion frequency and are Th1 like (lacking IL-4 secretion) in diabetic twins as compared to non-diabetic twins. As the same methods can be used to quantify the frequency of Valpha24JalphaQ in patients with MS. The third aim of the grant will focus on the loss of IL-4 producing CD4+/B7.1+ cells, we recently observed in patients with MS. Using the PCR/TCR technology to identify antigen reactive T cells, we will explore the hypothesis that MS patients have a loss of myelin reactive T cells in the ex vivo IL-4 producing population, while in normal subjects IL-4 producing T cells are autoreactive. These experiments will provide insights that may be important for the design of antigen specific immunotherapies for patients with multiple sclerosis.

The overall goal of this PROGRAM PROJECT is to determine whether B7-1 and B7-2 costimulatory molecules provide the second signal which can induce either the afferent or efferent phases of autoreactive T cell activation leading to pathologic autoimmune disease. To this end, three projects have been constructed. In Project 1, mice lacking B7- and B7-2 molecules will be used to evaluate the potential importance of these costimulatory molecules on the incidence and severity of experimental autoimmune disease. These mice will provide unique models to determine whether costimulatory molecules are capable of providing the minimal second signals to drive autoreactive T cells to induce pathologic autoimmune disease. The central goal of Project 2 is to identify mechanisms by which altered peptide ligands (APLs) and B7 costimulatory molecules affect cytokine balance and suppress antigen-specific proinflammatory activity and enhance anti- inflammatory functions of T cells. The contribution of strength of signal/stimulus to T cell differentiation provided by B7 molecules an APLs will be examined using a panel of CHO transfectants with varying levels of B7 and class II MHC expression and a panel of APLs. Project 3 examines the role of costimulatory pathways in MS. The requirements for expression of costimulatory molecules and il-12 by human glial cells will be examined. The molecular basis for the activated in vivo state of autoreactive T cells in MS patients will be further investigated by comparing myelin antigen- autoreactive peripheral blood T cells in MS patients to controls and determining their capacity to expand in response to antigen presented by CHO cells transfected with MHC class II, DR2, alone or with B7-1 or B7-2 costimulatory molecules and DR2. Additionally, the role of costimulation in negative regulation of T cells will be examined. This Program exemplifies the most fundamental concept of a BASIC/CLINICAL PROGRAM PROJECT since all three projects are highly interdependent. It is through the interrelationship between projects that we wish to rapidly transfer technology from the bench to the understanding and therapy of human autoimmune disease. GRANT-P01AI396710001 Signaling via the B7-CD28/CTLA4 pathway can provide a potent costimulatory signal for T cell activation. For this reason, methods directed toward blocking this pathway have received considerable attention for the treatment of autoimmune disease. B7-CD28/CTLA4 pathway blockade may enable a distinctive treatment strategy, since it would affect only those antigen- specific T cells undergoing activation and not be globally immunosuppressive. We have recently found that murine CTLA4-Ig, which binds to both B7-1 and B7-2, completely inhibits the induction of experimental autoimmune encephalomyelitis (EAE) and induces long-lived protection. We will determine the cellular immunologic basis for the potent therapeutic effects of CTLA-4Ig, and compare it with anti-B7 mAb- mediated immunomodulation. However, the presence of multiple costimulatory molecules in the pathway together with the potential for both positive and negative signaling through the pathway indicate that additional insights are needed in order to successfully manipulate this key immunoregulatory pathway. Using transgenic approaches, we will focus in this project on the function of the B7-CD28/CTLA4 pathway in both the afferent arm of the autoimmune respones where T cells become activated in the periphery and in the effector arm where potentially pathogenic T cells are driven to mediate tissue destruction: Mice lacking B7-1 and/or B7-2 will be used to evaluate the importance of the B7 costimulatory molecules in the induction, reactivation and CNS pathologic stages of EAE. These unique tools will permit us to determine whether the elimination of B7-1 and/or B7-2 will block or modulate the induction and/or effector stages of EAE. These murine models allow us to test the biological significance of the observation of Project 3, that early MS plaques ia the CNS are associated with increased expression of B7-1. We will compare the induction and course of EAE in these knockouts, and in mice given specific B7 antagonists. We will analyze the mechanism of observed effects through the generation and analysis of T cell clones. With Project 2, we will investigate the epitope specificity of T cell clones generated from CTLA- 4Ig treated mice and B7 deficient mice, as a means to determine if there is a contribution of epitope shift to a protective phenotype. Taken together, these studies will define the role of B7-1 and B7-2 in autoimmune disease, enabling rational pharmacological manipulation of this important immunoregulatory pathway.

Multiple sclerosis (MS) is a chronic inflammatory disease characterized by lymphocyte infiltration and tissue destruction in the central nervous system (CNS). The myelin reactive T cells found in the blood and cerebrospinal fluid of MS patients re in a different state of activation as compared to T cells in normal individuals and thus are thought to be directly involved in the disease pathogenesis. Our long-term goal is to understand the molecular basis for the in vivo activation of autoreactive T cells in MS, providing the basis for rational therapeutic intervention. This project is based on the hypothesis that costimulatory molecules provide the second signal which activates autoreactive T cells which are required to induce autoimmune disease. As we have recently discovered increased expression of B7-1 in acute Ms lesion, the requirement for expression of B7-1 and B7-2 molecules and IL-12 by human glial cells will be examined. In the next aim, the molecular basis for the activated in vivo state of autoreactive T cells in MS patients will be further investigated by comparing myelin antigen-autoreactive peripheral blood T cells in MS patients to controls and determining their capacity to expand in response to antigen presented by CHO cells transfected with MHC class II, DR21, alone or with B7-1 or B7-2 costimulatory molecules and DR2. We will also examine regulatory role of B7-2 expressed on activated T cells, which may be of significance for the disturbed immunoregulation described in MS. Our preliminary data demonstrate that B7-2 expressed on T cells has a lower molecular weight than B7-2 expressed on B cells or CHO cells, and has lost its binding affinity for CD28 but not CTLA4. In our last aim, we will further investigate negative regulatory functions of B7-2 expressed on T cells in a novel CD8 anergy system. These aims will provide basic information on the role of costimulation and anergy induction in CD4 and CD8 T cells as it may apply to understanding immunoregulation of MS, a human autoimmune disease.

DESCRIPTION (adapted from applicant's abstract): The intent of this proposal is to determine what factors prevent pre-diabetic individuals from developing diabetes. This proposal will investigate the regulation of IL-4 in a subset of T cells, which are implicated in the progression of human type I diabetes. In human disease, incomplete concordance of disease progression between identical twins, both with autoreactive T cells and autoantibodies suggests that other factors contribute to the incidence and progression of IDDM. Investigators have recently described a subset of regulatory V-alpha 24J-alphaQ T cells that are reduced in frequency and are Th1-like (lacking IL-4 secretion) in diabetic twins as compared to non-diabetic twins. In addition, the non- or slow-progressors to disease had high levels of the cytokines IL-4 and gamma-IFN in their circulation; this may reflect a more Th0- or Th2-like phenotype for these individuals. Investigator has set up collaborations with laboratories investigating T cell signaling events to address the question, what signaling events lead to the loss of IL-4 secretion in the invariant V-alpha 24J-alphaQ T cells in individuals with diabetes. Expression of the nuclear factors, GATA-3, c-maf, NIP45, and NF-AT species, that have been identified as IL-4 transcription factors, will be quantitated by Northern blot under conditions of activation and conditions of exogenous factors to bias cells toward a Th1 or Th2 phenotype. This approach will involve transfection of c-maf and GATA-3 into non IL-4 secreting V-alpha 24J-alphaQ T cells to determine if there is a dominant negative signal preventing IL-4 secretion. Further, proximal signaling pathways involving phosphorylation of STAT 6 on tyr-641 in the V-alpha 24J-alphaQ T cells will be examined. In addition, as there are defects in Ca(2+) flux in V-alpha 24J-alphaQ T cells from diabetics that do not secrete IL-4, early TCR associated signaling events will be surveyed. Besides examining targeted pathways, broader approaches, to isolate target genes, which may regulate IL-4 expression in V-alpha 24J-alphaQ T cells from subjects with diabetes, will be used. Subtraction methods coupled with PCR (RDA) and DNA microchip array hybridization will be performed. Lastly, whether the cytokine secretion phenotype of V-alpha 24J-alphaQ T cells from the diabetics can be altered to a Th0/Th2 phenotype using the data generated from the first three aims will be examined. The eventual goal of the project is to develop a therapy that can be administered to pre-diabetics that prevent their progression into diabetics.

Oligoclonal IgG banding and the presence of oligoclonal populations of T cells in the central nervous system (CNS) of patients with multiple sclerosis (MS) remains an unexplained hallmark of the disease. Yet, it is still unknown whether there are specific antigen reactivities of clonally expanded T or B cells in the target organ of patients with the disease. In collaboration with Project 1 of this Program Project Grant, we will employ new technologies to examine fundamental issues related to the role of T cells in patients with MS. First, we will use combinatorial peptide libraries to examine the antigen specificity of oligoclonal CSF T cells. These investigations will also be performed on subjects with defined CNS infections, and will allow us to determine whether T cell receptors expressed on CNS T cells in patients with MS are degenerate in their recognition of combinatorial peptide libraries as compared CNS T cells recognizing viral or bacterial antigens. We will then use tetramer/MHC complexes of DR2 (DR2*1501)/MBp85-99 to both define the frequency and phenotypic function of MBPp85-99 reactive T ells in the CSF and blood. In both investigations we will determine whether the cloned T cells are reflective of the original CSF population as defined by TCR CDR3 length analysis and sequencing. Together, these investigations may elucidate whether the T cells in the CSF compartment of patients with MS are clonally expanded by stimulation with a specific antigen, be it a self or microbial antigen. Lastly, in collaboration with Ploegh in Project 3 of this Program Project Grant, we will define immunodominant epitopes and examine the CNS processing of MOG and MBP, CNS proteins of potential importance in the immune response to myelin in patients with MS. Insights into the cell biology of Class II restricted presentation will be applied to the processing of MOG and MBP using APC derived from peripheral blood and from CNS microglial cells. We will determine whether the same epitopes are presented by circulating monocytes as compared to CNS technologies that may allow us to define the importance of CNS T cells in patients with MS, and to develop new surrogate markers for monitoring patients with MS.

The activation of myelin-reactive T cells is postulated to initiate the recruitment of pro-inflammatory Th1 T cells into the CNS of patients with MS. Accumulating evidence suggests that the induction of autoreactive Th2 T cells secreting IL-4 may provide a mechanism for the treatment of autoimmune disease. Altered peptide ligands (APLs) can deliver a lower strength of signal to T cells, resulting in differential cytokine secretion. Investigations by Kuchroo (Project 1) in the EAE model have shown that APLs induce a population of autoreactive T cells with a different fine specificity as compared to the T cells recognized the original self antigen and the T cells generated against APLs of PLP and MBP secrete IL-4 and suppress EAE. In patients with MS, we demonstrated that APLs of MBPp85-99 induce over IFN-gamma secretion in T cell clones. We will examine the mechanism of APL APLs of MBPp85-99 induce IL-4 over IFN-gamma secretion in T cell clones. We will examine the mechanism of APL induced cytokine switching in patients with MS. We hypothesize that APL may expand a small population of low-affinity autoreactive T cells specific for the self-antigen that are different from the pathogenic T cells, and thus generate regulatory effect functions with a different TCR repertoire upon encounter with the self antigen. This would be confirmed by the presence of different TCR sequences between APL and MBPp85-99 reactive T cells. The mechanism of actions of APL in patients with MS will be compared to the mechanism of action in rodents with EAE (Project 1). The ability of APL to differentially signal T cells suggest a mechanism of action for Copaxone (glatiramer acetate which decreases the attack rate in patients with MS. Whether the random amino acid structure of glatiramer acetate may make it a more cross-reactive will be tested in aim 2 using combinatorial peptide libraries. We hypothesize that glatiramer acetate can act as a unive4rsal weak agonist and differentially signal autoreactive T cells to secrete IL-4 rater than IFN-gamma. A prediction of this hypothesis is that the frequency of glatiramer acetate T cells recognizing combinatorial peptide libraries of 13 amino acids will be increased in patients after injections of glatarimer acetate. The use of combinatorial libraries with single fixed amino acid and variable amino acids at the other 13 positions can also be fixed to define the preferential antigens recognized by glatiramer acetate reactive T cells. MBPp85-99 and Copaxone reactive T cell clones will be examined to delineate the biochemical mechanisms for APL and Copaxone-induced alteration in TCR signaling specifically focusing on tintracellular signaling pathways that lead to activation of these nuclear factors, and differentiation of naive cells into those with distinct functions and cytokine profiles.

Physiological regulation of immune responses is a fundamental mechanism of limiting autoimmune disease. The cellular basis for a defect underlying autoimmune disease remains under investigation. Recent data from animal models demonstrate that CD4+CD25+ T cells play a major role in regulating autoimmune diseases, particularly diabetes in the NOD mouse. Thus, the CD4+CD25+ T cell may be the long sought-after regulatory T cell, a defect in which underlies human autoimmune disease. The recent identification of CD4+CD25+ Tcell may be the long sought-after regulatory T cell, a defect in which underlies human autoimmune disease. The recent identification of CD4+CD25+ regulatory cells in the circulation of normal humans that exhibit identical in vitro characteristics to the CD4+CD25+ regulatory cells isolated in mice is the basis for the coordinated U-19 Program Project Grant. This U-19 program project is a focused effort of five laboratories: the Fathman Laboratory at Stanford, the Weiner Laboratory at Harvard, the Eisenbarth Laboratory at the Harbara Davis Diabetes Center in Denver, the Khoury and the Hafler laboratories also at Harvard to determine: the mechanism of action of regulatory CD4+CD24+ T cells in models of autoimmune disease, and whether they can be induced by experimental interventions. We will determine whether there are defects in these CD4+CD25+ regulatory T cells in human autoimmune disease, and define the mechanisms of these defects. We will focus on two autoimmune diseases, type 1 diabetes and multiple sclerosis. The fundamental hypothesis for this grant application is that a defect in the number of the CD4+CD25+ regulatory cells or their regulatory function in patients with type I diabetes or multiple sclerosis underlies the dysregulation of the immune system in these diseases. Correction of this potential defect provides a novel approach for prevention of human autoimmune disease. This Program exemplifies the most fundamental concept of a BASIC/CLINICAL PROGRAM PROJECT since all three projects are highly interdependent. It is through the interrelationship between projects that we wish to rapidly transfer technology from the bench to the understanding and development of human autoimmune diseases.

DESCRIPTION: (Adapted from Applicant's Abstract) The Clinical Immunology Society (CIS) has undertaken the task of arranging a federated meeting for clinical immunology to be held yearly commencing in May 2001. This new meeting plans to provide an opportunity for members of each of the individual clinical immunology societies involved to meet together on a yearly basis. The first Federated meeting will be held in Boston, May 4-7, 2001. We believe that this new meeting will consolidate the field of Clinical Immunology as it reaches maturity, particularly as it relates to the major human autoimmune diseases including multiple sclerosis, rheumatoid arthritis, and juvenile diabetes. Resultant cross-fertilization from this meeting among the disease-centric investigators and members of the biotech and pharmaceutical community is of critical importance with the increasing numbers of therapeutics for treatment of these autoimmune diseases. This meeting reflects the new interdisciplinary nature now necessary for the investigation and treatment of human autoimmune diseases. It will also cover additional immune based diseases including, but not limited to, asthma, immuno-oncology, acquired immunodeficiency, primary immunodeficiency, transplantation tolerance and immuno-dermatology. Another key aspect will be the inclusion of the Immune Tolerance Network annual meeting. This will provide an important opportunity to inform industry about the Immune Tolerance Network and to disseminate new information about science and medicine. The format will be three days on topics of mutual interest to all constituent groups. There will be two major plenary lectures to begin each day, followed by concurrent major symposia on topics of interest to constituent groups. Afternoons will be abstract-driven interspersed with breakout meetings for the constituent societies and presentations by biotech and pharmaceutical firms concerning technologies of interest to the assembled groups. There will be two additional meetings held in conjunction with this meeting. The first of these is a "fellows day" in which plenary and abstract driven talks will be held on the day preceding the main scientific sessions. The second parallel session will be on "science for the non-clinician" which targets the lay societies representing the constituent scientific societies.

DESCRIPTION (provided by applicant): The progression towards autoimmune disease is a complex process involving genetic factors and the long term destruction of specific organs by the infiltration of cells of the immune system, including T cells. In a milestone in immunotherapy, Shapiro and colleagues achieved successful islet cell transplantation in patients with Type I diabetes treated with combination therapy including rapamycin where autoimmune destruction of islet cells was prevented. Given this, we are about to embark on a Phase 1/11,open-label, randomized, pilot trial to evaluate the safety and efficacy of Rapamune(r) in patients with MS and this trial has been approved by the Autoimmunity Centers for Excellence. Our laboratory has created an in vitro model of the effects of exposure to rapamycin on CD4+ versus CD8+ T cells. Surprisingly, while rapamycin blocked T cell proliferation at low strengths of signals, strong signals delivered through the immune synapse resulted in higher proliferative response in the presence of rapamycin. Rapamycin-resistant proliferation was associated with bcl-xL induction and p27kip degradation. We propose to extend our in vitro observations to determine the in vivo mechanism of drug action in patients undergoing immunosuppression with rapamycin. We will first examine the in vivo effects of rapamycin therapy in patients with MS to validate our in vitro model of rapamycin exposure. Then, we will examine the array of gene expression induced by in vivo rapamycin therapy. These studies may reveal that only a subset of patients respond to treatment, and these responses are associated with a particular immune response profile. Such data would be critical in determining which immune phenotypes are correlated with response to therapy. Thus, we believe that determining the immunologic effects of rapamycin in patients with MS in relationship to both specific immune abnormalities associated with the disease and therapeutic responses is critical in probing the underlying pathophysiology of the disease. Elucidating the molecular details and in vitro correlates of the effects of in vivo rapamycin treatment may translate into the development of unique combination therapies designed to prevent the development of, or treat, MS.

DESCRIPTION (provided by applicant): During the previous PPG, we showed that the CD28, CTLA4, and ICOS pathways provide critical second signals for the activation of T cells, isotype switching in B cells, and regulation of autoimmunity. During the tenure of the PPG we identified new co-stimulatory molecules (PD-L1/L2 and TIM-3) and delineated new pathways for T cell activation and expansion. However, it has been difficult to establish which of the many co-stimulatory pathways are most crucial in the induction of human autoimmune diseases. This has fundamentally changed because of the major advances that have been made in the genetic analysis of human autoimmune diseases and of congenic mice. Both of these independent investigations converge on two major loci: one on human chromosome 1p12 which contains CD2, CD48/CD58, CD101 and B7H4, and a second locus on chromosome 2 which contains CD28, CTLA4 and ICOS genes. Within the second locus, CTLA4 has emerged to be the most critical because the isoforms of CTLA4 appear to regulate autoimmunity in both mouse and humans. With these new developments and preliminary data, we have refocused this PPG application to study the co-stimulatory molecules/pathways, which based on the genetic data, appear to be critical for the development of autoimmunity in humans. In the renewal application we are proposing to 1) analyze functions of co-stimulatory molecules (CD2, CD58, B7-H4 and CD101) expressed in 1p12 locus and 2) study the mechanism by which CTLA-4 variants regulate tolerance and autoimmunity. Our studies will investigate human diseases (MS and diabetes) and mouse models of these diseases (EAE and diabetes in the NOD mice). Specifically, Wicker and Todd will analyze the genetic variants in mouse chromosome 3 which correspond to the 1p12 locus and in CTLA4 and CD101 in patients with T1D; Hafler and DeJager will analyze the function of the co-stimulatory molecules identified in the 1p12 locus and CTLA4 in relation to human MS; Kuchroo will generate transgenic mice for CTLA4 variants and analyze their effect on the development of autoimmunity in the mouse models; and Sharpe will test the role of B7-H4 and CD48 (mouse ligand for CD2) in the induction of autoimmunity and tolerance. Insights developed by analyzing the co-stimulatory pathways and genetic variants linked to human and mouse autoimmune diseases will enable us to understand the mechanisms by which allelic variants of co-stimulatory molecules regulate activation of autoreactive T and B cells and induce autoimmune disease.

The underlying hypothesis of this grant is that alterations in costimulatory and activation pathways lead to a lower T cell activation threshold and thus to great susceptibility to autoimmune disease. Our efforts will focus on regions identified by the first whole genome admixture association study which led to the discovery of a 4 MB locus on chromosome 1 with an admixture LOD score of 6.3; this admixture locus contains the co-stimulatory molecules: CD2, CD58 (LFA-3), CD101 and B7-h4. Fine association mapping indicated that allelic variation in CD58 is most strongly associated with susceptibility to MS. This finding is of particular interest because of the recently demonstrated role for CD58 engagement of CD2 in the generation of regulatory Tr1 and CD4+CD25high cells. Moreover, genetic variation in CD101 and B7-h4 also exhibit suggestive evidence of association to MS susceptibility. These results parallel those of Wicker & Todd who defined the murine T1D susceptibility loci Idd10, a locus containing the CD101/B7-H4 genes, and Idd18.2 containing the CD2 gene. To examine the hypothesis that allelic variation of costimulatory genes CD58, CD101 & CTLA-4 affect the expression of these and other molecules that are associated with susceptibility to MS, we will in aim 1 identify the risk-associated genetic variation of CD58 in our MS patients by genotyping all publicly available SNPs within this gene followed by resequencing the implicated segment of the CD58. In the second aim, we will determine whether there are additional MS risk alleles of co-stimulation gene variants in the ADMS1 locus and in close collaboration with Todd, determine whether the T1D CTLA4 risk haplotype confers increased risk for MS. The last two aims explore possible functional effects of the risk alleles that are characterized in Aims 1 and 2. These characterizations of CD58 and CTLA4 are driven by the important roles of these molecules in immune regulation in addition to our genetic analyses. In aim 3 we will determine whether soluble CD58 and CTLA-4 levels are altered in MS independently of the effect of the different haplotypes present in the patient and control populations. In close collaboration with Wicker, we will investigate whether the T1D risk haplotype of CTLA4 correlates with decreased soluble CTLA4 and increased CD101 expression on the CD8+CD45RA- cells of subjects with MS. In aim 4 we will determine whether there are differences in the isoform distribution of CD58 between MS cases and control subjects that are independent of the effect of the different haplotypes of CD58. We hypothesize that allelic variants of CD58, CTLA-4 or CD101 dictate subtle but measurable changes in expression and function that are linked to a lower signaling threshold and susceptibility to autoimmune disease.

This program project has assembled molecular biologist, immunologist, population geneticist and clinical scientist to investigate the mechanism of co-stimulatory responses in patients with MS and type 1 diabetes, and examine the hypothesis that a genetic predisposition for alterations in co-stimulatory signal leads to heightened inflammatory responses that in part underlies the pathophysiology of autoimmune diseases. To accomplish these goals, an administrative core is requested to ensure that financial activities among project participants are in compliance with the appropriate NIH policies and guidelines throughout the project period and to facilitate the report of our findings on an annual basis to the NIH. Additionally, an important function of the administrative core will be the arrangement of monthly conference calls among the PPG participants and arranging the bi-annual meetings of the advisory board. Thus funding the administrative core is important to accomplish the goals of the PPG.

CRISP; Computer Retrieval of Information on Scientific Projects Database; Disease; Disorder; Funding; Gene Combinations; Genes; Genetic; Genetic Condition; Genetic Diseases; Grant; Hereditary; Hereditary Disease; Individual; Inherited; Institution; Investigators; MS (Multiple Sclerosis); Molecular Disease; Multiple Sclerosis; NIH; National Institutes of Health; National Institutes of Health (U.S.); Purpose; Research; Research Personnel; Research Resources; Researchers; Resources; Sclerosis, Disseminated; Source; Time; United States National Institutes of Health; base; disease-causing mutation; disease/disorder; genetic disorder; hereditary disorder; insular sclerosis; response

DESCRIPTION (provided by applicant): Under funding for the original U19, we addressed several hypotheses that have provided new insights into the fundamental, mechanistic understanding of regulatory T cells and how these pathways are altered in animal models of autoimmune disease and in the human diseases type I diabetes and multiple sclerosis. In addition, we have developed novel immunotherapeutic approaches to induce regulatory T cells using oral anti-CD3. The grant will continue to focus on these fundamental aims, reflecting the discoveries we have made over the past five years. In this renewal, the overall goals of this Autoimmunity Prevention Center Project are: 1) To determine, in human autoimmune disease, which of the CD4+CD25+ subsets of DR+ and DR" regulatory T cells are defective; 2) The identification of a "core set" of genes and proteins that are expressed on the "innate", CD4+CD25+ regulatory T cells; 3) To determine which of the "core sets" of genes and proteins are altered in the regulatory T cells found in blood and lymph nodes in patients with diabetes and MS; 4) To understand the mechanism of oral anti-CD3 and GRAIL transfection in T cells that allows the translation into human clinical trials by year five of the grant. The key goal is the development of specific drug targets and methods to induce the function of defective regulatory T cells in patients with autoimmune disease. Lay Summary: Diseases such as type I diabetes and multiple sclerosis are complex genetic diseases thought to be initiated by an autoimmune response directed against self-proteins in the inflamed tissue. Why autoreactive T cells attack the insulin producing islet cells in diabetes or the myelin in multiple sclerosis remains a major question. We recently demonstrated there is a loss of an important "regulatory" immune cell in the circulation of patients with autoimmune disease. This grant assembles a group of investigators to understand why these regulatory cells are dysfunctional, and what can be done to restore their function. PROJECT 1: Mechanism of action of CD4+CD25+ T cells (FATHMAN, GARRISON C.) DESCRIPTION (provided by applicant): This is a competing renewal as a U19 for a previously funded U01 entitled, "CD25+ Regulator CD4+ T Cells." Under funding for the original U01, several specific aims were addressed: initially (1) that expression of GRAIL (a recently identified anergy gene), following peptide administration iv, blocks IL-2 transcription and induces anergy, a form of tolerance, and (2) that GRAIL expression provides a novel and effective screen for the anergic phenotype in mice (and in man), and (3) that CD4+CD25+ suppressor T cells were involved in this form of tolerance induction, and a fourth (4) GVHD will be blocked by adoptive transfer of Tregs, was added in the second year of support. Ten articles were published on these four specific aims and this proposal will extend these studies as follows: Using microarray and RNAi technology for mRNA expression and gene silencing, the "core genes" that define Treg core transcriptome will be identified. In this proposal, these "core Treg" genes, identified by cDNA microarray studies, will be validated by functional genomics (RNAi) and tested in vitro in T cell proliferation assays and in vivo in a model of GVHD, and the "peripheral Treg subset," provisionally defined as CD4+ antigen specific T cells that contact antigen under anergy inducing conditions in the periphery, will be further characterized and studied as proposed in the following four specific aims: * Specific Aim 1: Identification of a "core set" of CD4+CD25+ Treg genes, the Treg core transcriptome. * Specific Aim 2: Characterization of peripherally induced Tregs and core transcriptome identification. * Specific Aim 3: GRAIL transductants as Tregs for immunotherapy. * Specific Aim 4: To evaluate the role of specific genes on Treg function in an in vivo model of graft vs. host disease. The use of adoptive cellular therapy, in particular use of Tregs is rapidly gaining credibility as a useful potential therapy for immunoregulation, in particular in the setting of GVHD, a form of adoptively transferred autoimmune disease. Studies proposed in this project will attempt to identify the "core" set of genes that define Tregs and then using RNA silencing techniques, attempt to identify functionally relevant genes through knock down and loss of function assays in vitro and using a model of GVHD in vivo. All of the techniques required for these studies are currently practiced in our labs.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] Under funding for the original U19, we addressed several hypotheses that have provided new insights into the fundamental, mechanistic understanding of regulatory T cells and how these pathways are altered in animal models of autoimmune disease and in the human diseases type I diabetes and multiple sclerosis. In addition, we have developed novel immunotherapeutic approaches to induce regulatory T cells using oral anti-CD3. The grant will continue to focus on these fundamental aims, reflecting the discoveries we have made over the past five years. In this renewal, the overall goals of this Autoimmunity Prevention Center Project are: [unreadable] 1) To determine, in human autoimmune disease, which of the CD4+CD25+ subsets of DR+ and DR" regulatory T cells are defective; [unreadable] 2) The identification of a "core set" of genes and proteins that are expressed on the "innate", CD4+CD25+ regulatory T cells; [unreadable] 3) To determine which of the "core sets" of genes and proteins are altered in the regulatory T cells found in blood and lymph nodes in patients with diabetes and MS; [unreadable] 4) To understand the mechanism of oral anti-CD3 and GRAIL transfection in T cells that allows the translation into human clinical trials by year five of the grant. The key goal is the development of specific drug targets and methods to induce the function of defective regulatory T cells in patients with autoimmune disease. [unreadable] [unreadable] Lay Summary: Diseases such as type I diabetes and multiple sclerosis are complex genetic diseases thought to be initiated by an autoimmune response directed against self-proteins in the inflamed tissue. Why autoreactive T cells attack the insulin producing islet cells in diabetes or the myelin in multiple sclerosis remains a major question. We recently demonstrated there is a loss of an important "regulatory" immune cell in the circulation of patients with autoimmune disease. This grant assembles a group of investigators to understand why these regulatory cells are dysfunctional, and what can be done to restore their function. [unreadable] [unreadable] PROJECT 1: Mechanism of action of CD4+CD25+ T cells (FATHMAN, GARRISON C.) [unreadable] [unreadable] DESCRIPTION (provided by applicant): [unreadable] This is a competing renewal as a U19 for a previously funded U01 entitled, "CD25+ Regulator CD4+ T Cells." Under funding for the original U01, several specific aims were addressed: initially (1) that expression of GRAIL (a recently identified anergy gene), following peptide administration iv, blocks IL-2 transcription and induces anergy, a form of tolerance, and (2) that GRAIL expression provides a novel and effective screen for the anergic phenotype in mice (and in man), and (3) that CD4+CD25+ suppressor T cells were involved in this form of tolerance induction, and a fourth (4) GVHD will be blocked by adoptive transfer of Tregs, was added in the second year of support. Ten articles were published on these four specific aims and this proposal will extend these studies as follows: Using microarray and RNAi technology for mRNA expression and gene silencing, the "core genes" that define Treg core transcriptome will be identified. In this proposal, these "core Treg" genes, identified by cDNA microarray studies, will be validated by functional genomics (RNAi) and tested in vitro in T cell proliferation assays and in vivo in a model of GVHD, and the "peripheral Treg subset," provisionally defined as CD4+ antigen specific T cells that contact antigen under anergy inducing conditions in the periphery, will be further characterized and studied as proposed in the following four specific aims: [unreadable] * Specific Aim 1: Identification of a "core set" of CD4+CD25+ Treg genes, the Treg core transcriptome. [unreadable] * Specific Aim 2: Characterization of peripherally induced Tregs and core transcriptome identification. [unreadable] * Specific Aim 3: GRAIL transductants as Tregs for immunotherapy. [unreadable] * Specific Aim 4: To evaluate the role of specific genes on Treg function in an in vivo model of graft vs. host disease. [unreadable] [unreadable] The use of adoptive cellular therapy, in particular use of Tregs is rapidly gaining credibility as a useful potential therapy for immunoregulation, in particular in the setting of GVHD, a form of adoptively transferred autoimmune disease. Studies proposed in this project will attempt to identify the "core" set of genes that define Tregs and then using RNA silencing techniques, attempt to identify functionally relevant genes through knock down and loss of function assays in vitro and using a model of GVHD in vivo. All of the techniques required for these studies are currently practiced in our labs. [unreadable] [unreadable] [unreadable] [unreadable]

A number of pathways and newly identified genes appear to be critical for the differentiation and identification of autoreactive Thl T cells. Specifically, T-bet is the master regulator of Thl T cells and TIMS defines Thl T cells in murine systems;in contrast, GATA-3 is a regulator of Th2 differentiation. We have previously cloned T cells from the CSF of patients with MS and demonstrated a significantly higher frequency of IFNy secreting T cells in the CSF of patients with MS as compared to T cells isolated from the CSF of subjects without inflammation. Recent data from Kuchroo (Project 2) demonstrated that engagement of TIMS with TIMS ligand regulated both the function of Thl cells and the ability to induce tolerance. Moreover, TIMS deficient mice were refractory to the induction of high dose tolerance in EAE. We will examine the hypothesis that loss of TIMS expression on T cells in part underlies the loss of tolerance to self- antigens in autoimmune disease and is involved in the pathophysiology of MS. Specifically, in aim 1 we will examine the function of TIMS expression on human T cells, determining the regulation and stability of TIMS expression on differentiated human Thl and Th2 cells from healthy subjects by investigating the relationship between TIMS and T-bet expression with anergy. This aim will directly translate the findings by Glimcher (Project 1) and Kuchroo (Project 2) and will allow us in our second aim to directly examine T cell function in patients with MS, where we will determine whether altered expression of TIMS functionally characterizes the loss of tolerance by T cells from the CNS of patients with MS. This aim is based on new preliminary data suggesting that the high IFNv secreting CD4 T cells in the CSF of patients with MS, but not CD4 T cells from control subjects, exhibit altered levels of TIMS. In the last aim, we will directly interrogate the CNS of patients with MS and ask if there a loss of TIMS expression associated with high IFNy expression of CD4 T cells in the plaque tissue of patients with MS. This aim addresses the hypothesis that loss of TIMS on T cells infiltrating plaque tissue of chronic MS lesions underlies the loss of tolerance and protracted expansion of autoreactive T cells in the brain. These experiments will directly translate the basic immunologic discoveries and findings in the EAE model in attempts to understandthe mechanism for loss of T cell tolerance to myelin antigens in patients with multiple sclerosis.

MS is an autoimmune disease potentially mediated by activated myelin reactive Th1/Th17 T cells in a genetically susceptible host. Kuchroo recently discovered that Th1 cells selectively express TIM-3, and its ligation regulates tolerance induction. New, complementary data sets generated in the Hafler &Kuchroo labs demonstrate that TIM-3 is also expressed on APCs, including both dendritic cells and monocytes. Contrary to the consequences of TIM-3 engagement on T cells, ligation of TIM-3 on APCs may promote Th1-mediated immunity in that stimulation of monocytes with the TIM-3 ligand galectin-9 (Gal-9) induces TNF-a and IL-6 secretion. Moreover, we have demonstrated that microglia in CNS white matter express high levels of TIM-3, and glial expression of TIM-3 and Gal-9 are up-regulated in MS lesions. This leads us to hypothesize that the altered expression or function of TIM-3 by T cells and circulating APCs and CNS microglia may alter the balance of Th1 differentiation, and perhaps is associated with chronic CNS inflammation due to up-regulation of Gal-9 expression on astrocytes, inducing the activation of TIM-3* microglia. Thus, it is possible that the innate TIM-3/Gal-9 pathway may be involved with transition to secondary progressive disease in patients with MS. Experiments in Aim 1 have been designed to provide us with a detailed mechanistic understanding of how TIM-3 expression on APCs modulates their activity, and how this may subsequently influence the balance of effector versus regulatory T cell differentiation. In Aim 1b, based on our discovery of the presence of TIM-3 on CD11b+ microglia in white matter of normal brain, and the coordinated modulation of TIM-3 &Gal-9 expression depending on the nature of CNS inflammation, we will determine how this pathway modulates glial responses within the CNS. In collaboration with Kuchroo, we propose experiments to examine the kinetics of TIM-3 induction on microglia associated with migration of hematopoietic cells into the CNS. Aim 2 will investigate our recent discovery that 5 distinct TIM- 3 splice variants exist in humans, including membrane &soluble forms with variable potential signaling capacity. Using quantitative RT-PCR analysis in conjunction with techniques to modulate specific TIM-3 isoforms, we will explore how relative differences in the expression of these TIM-3 isoforms may influence T cell vs. APC biology. Our third aim will extend our analysis of TIM-3 regulation of APC function by defining the TIM-3 signaling pathways in T cell vs. monocyte lineage cells. We will perform structure/function experiments to determine which structural elements in the cytoplasmic tail of TIM-3 are responsible for its effects on cytokine production and downstream signaling pathways in TIM-3+ T cells and monocytic cells, and will determine which cytoplasmic signaling proteins are recruited to the cytoplasmic tail of TIM-3 to mediate activation of downstream signaling pathways. These experiments will determine how differences in TIM-3 isoform expression and signaling dictate divergent functional outcomes in adaptive versus innate immunity and how this innate pathway may be involved in the pathoaenesis of autoimmune disease.

DESCRIPTION (provided by applicant): This application, entitled 'The Role of Rare Variants in Multiple Sclerosis Risk', addresses Research and Research Infrastructure 'Grand Opportunities'. Many human autoimmune diseases, including multiple sclerosis (MS) are complex genetic disorders. MS is an inflammatory disease of the central nervous system white matter. The unbiased approach of studying the entire human genome for risk alleles has led to the identification of common allelic variants that are associated with risk to developing MS. A major short-coming of current high-throughput genotyping platforms and study designs is that only genetic variants that have a relatively high frequency in the population have been assessed (and discovered) in MS risk to date. This conclusion is supported by the observation that all of the validated alleles implicated in risk to MS have minor allele frequency (MAF) >0.10 in the populations of European ancestry in which the association studies have been performed. However, recent evidence suggests that rare variants may represent critical components of the allelic architecture of common autoimmune diseases, including Crohn's disease and type 1 diabetes. To assess the role of rare allelic variants in MS risk, we will perform a high-throughput sequencing experiment using Illumina's Solexa technology. Subsequently, we will validate these variants by genotyping a large collection of MS cases and controls. As we have strong evidence that MS shares common risk alleles with Crohn's disease, we will also genotype a set of rare variants discovered in CD cases in our collection of MS cases and controls to assess the commonalities between the two autoimmune diseases. This project will 1) provide a catalogue of rare variants in genomic regions implicated in MS susceptibility, 2) discover novel variants implicated in MS and CD risk, and 3) pin- point towards disease-causing mechanisms. PUBLIC HEALTH RELEVANCE: Title: The Role of Rare Variants in Multiple Sclerosis Risk Narrative - The recent successes of genome wide association scans have provided a growing list of allelic variants implicated in the risk of common human diseases. A major short-coming of current genome-wide genotyping platforms and study designs is that only genetic variants that have a relatively high frequency in the population have been assessed and discovered in multiple sclerosis (MS) risk to date. Using high-throughput sequencing and genotyping technologies to study the largest DNA collection of multiple sclerosis and controls available, this project will 1) provide a catalogue of rare variants in genomic regions implicated in MS susceptibility, 2) discover novel variants implicated in risk of developing MS and other autoimmune diseases, and 3) pin-point towards disease-causing mechanisms.

No abstract provided

Genome wide association (GWA) scans have allowed an assessment of disease pathways in patients with MS and Tl D based on the unbiased identification of gene variants. We will investigate these allelic variants and their pathways that are both common (CD226/TIGIT) and unique (CTLA4-T1D and CD6-MS) to these diseases. The central role of Thi7 and regulatory T cells in MS has recently been elucidated, and as part of this PPG we demonstrated a loss of regulatory T cell function in MS. We also discovered that increasing the strength of signal through the TCR influences the plasticity of CD4+CD25high Tregs to become Thi7 cells. These data have led to the underlying hypothesis that allelic variants in multiple costimulatory pathways lead to a shift from regulatory to inflammatory T cell function. We will explore the costimulatory pathways identified from GWA scans in relationship to Treg and Thi7 function, investigating the CTLA-4/CD28, CD226/TIGIT, and CD6 pathways. Specifically, we will determine: 1) the role of sCTLA4 in the function and plasticity of human Treg populations in close collaboration with Wicker/Todd; 2) how CD6 MS-risk alleles that induce splice variants alter the CD6 costimulatory pathway, investigating the hypothesis that alterations in the CD6 isoforms affect binding to its ligand results in changes influencing the function of Tri and Tregs in patients with MS; and 3) how the CD226/TIGIT costimulatory pathway modulate Treg and Thi 7 function in human autoimmune disease. Our preliminary data demonstrate a lack of TIGIT expression on Thi7 cells while blocking CD226 corrects defects in Treg function in MS leading us to hypothesize that allelic variants in the CD226/TIGIT pathways influence Th17 and Treg function. We will directly examine whether defects in Treg suppression in MS and hyperactivation of monocytes in patients with T1D are corrected by promoting TIGIT signaling. In summary, it is clear that genetic risk to develop autoimmune disease is due to many common and rare allelic variants each with a small effect that together lead to organ specific inflammation in response to undefined environmental challenges. We will apply new tools to examine how genetic variants lead to the heightened immune responses observed in patients with autoimmune diseases.

DESCRIPTION (provided by applicant): PROJECT SUMMARY (See instructions): This renewal application builds upon significant advances in identifying both the genetic basis for autoimmune diseases and discoveries related to the function of costimulatory pathways. As part of this PPG we have defined the immunologic roles of critical costimulatory pathways implicated in the etiology of human autoimmune diseases, focusing on MS and T1D, allowing the identification and molecular characterization of a number of critical costimulatory pathways including B7/CD28, CTLA-4, CD226, ICOS, PD-L1/2 and CD2/CD58 family molecules. The application of genome wide scans to the investigation of these diseases has finally allowed a mechanistic assessment of critical costimulatory pathways based on the unbiased identification of gene variants associated with disease risk. Considering these findings, we will continue to investigate allelic variants and their pathways that are both common (CD226/TIGIT) and unique (CTI_A4-T1D and CDS-MS) to these diseases exploring the underlying hypothesis of this grant that allelic variants in multiple costimulatory pathways lead to a shift from regulatory to inflammatory T cell function. We will explore the costimulatory and signaling pathways identified from genome wide association scans in relationship to regulatory T cell and effector function, investigating the PTPN22, CTLA-4/CD28, CD226/TIGIT, and CDS pathways. This will be accomplished by three approaches: first, we will investigate the immunologic function of these allelic variants in healthy subjects who are not burdened with the inflammatory consequences of autoimmune disease;second, we will directly study patients with MS and T1D, applying our new understanding of disease pathways leading to disease risk in attempts to develop new therapeutic approaches;third;we will study the functional impact of these pathways in mouse models, focusing on how these pathways regulate the functions of effector and regulatory T cells. These goals will be accomplished with four Projects from Harvard and Cambridge Universities with an Administrative Core. RELEVANCE (See instructions): Autoimmune diseases are complex genetic disorders where many common allelic variations lead to disease risk. This understanding is key to developing new therapies. We have elucidated key pathways by performing association scans in patients with MS and Tl D. This grant will translate those genotypic findings to a fundamental understanding of immune phenotypes to identify pathways for future drug targeting. PROJECT 1: Title: - Fuctional Analyses Of Autoimmune Disease Variants Project Leader: Wicker, L PROJECT 1 DESCRIPTION (provided by applicant): PROJECT SUMMARY (See instructions): The project, Functional Analyses of Autoimmune Disease Variants, is focused on three type 1 diabetes (T1D) susceptibility loci, CTLA4, PTPN22 and CD226 in humans and in mice. T1D is a major disease of children with an unexplained steady rise in incidence and increasing numbers of children diagnosed under age 5 years. The research proposed is fully integrated into the PPG activities and goals, and provides a platform to continue the highly productive, interdependent and synergistic collaboration amongst the Pis in Cambridge and Boston aimed at understanding the biological effects of T1D and autoimmune disease gene variants (that we have identified using genetic mapping). T l D gene variants will be studied ex vivo using fresh blood samples from a major resource of genetically-selectable, local healthy volunteers (the Cambridge BioResource) and in vivo, in precisely engineered NOD mouse models of T1D. The Ctla4, Ptpn22 and Cd226 KO alleles will be used to develop NOD strains to model human T1D. A mouse Ptpn22 variant that increases T1D will be a focus of mechanistic studies on the PTPN22 gene, which is part of a molecular pathway that affects multiple human autoimmune diseases. T cells having the susceptibility allele at Ptpn22 have a higher threshold of activation and at a population level fewer of the cells produce IL-2 when stimulated ex vivo. Since both human and mouse gene variants in the IL-2 pathway affect T1D susceptibility, experiments to study gene-gene interactions between the PTPN22 and IL-2 pathways in both species are proposed. Preliminary data indicate that the susceptibility allele at CTLA4, which decreases the expression of soluble CTLA-4, reduces the probability that Tregs will be activated. Overexpression of soluble CTLA-4 in primary T cells and cell lines will be one approach used to study the mechanism by which soluble CTLA-4 affects early events in T cell activation. Since variants at both the PTPN22 and CTLA-4 genes are proposed to alter T cell activation, gene-gene interactions between the PTPN22 and CTLA-4 pathways in both humans and mice will be investigated. RELEVANCE (See instructions): The mortality, morbidity and healthcare costs of type 1 diabetes are enormous. The identification of the genes and pathways that cause type 1 diabetes will allow the identification of early, inherited immunophenotypes or disease precursors that precede autoimmunity and that may be suitable targets or read-outs in ongoing or future clinical trials.

DESCRIPTION (provided by applicant): The genetic variability and Individual variations in immune status dictate responses to vaccinations, infections, and contribute to disease severity. The sequencing of the human genome and generation of the Haplotype Map now enables a mechanistic understanding of how genetic variation influences human immune responses. Yet manifold non-genetic factors also interact to maintain the healthy immune system, and complex analysis will be required to form predictions for its response to perturbations. Here, we will employ systems approaches and novel, high throughput and high-fidelity technologies such as multiplexed gene expression, automated multidimensional flow cytometry, and integrated single-cell assays in nanowells to quantitatively assess leukocyte function to ultimately identify the molecular signatures defining individual immune responses. We will address immune profiles in three related studies. In Research Project 1, we will develop immunologic signatures of influenza vaccine responsiveness and determine the effect of aging and functional status on these signatures. We will identify gene signatures and biological pathways that can distinguish between strong and weak immune responses to vaccination and that predict effective responses. In Research Project 2, we will investigate resistance to flaviviral infections using West Nile virus, and hepatitis C virus; through analysis of responses in patients from stratified cohorts, we will establish correlations between gene expression, immune cell responses and clinical outcome. In Research Project 3, we will generate mathematical models that detect connectivity and predict dynamic functional responses of the immune system. This approach will link data collected on both populations and individuals using multivariate statistical approaches to integrate cohort-wide data including genome wide association studies and novel single-cell analyses to assess immune responsiveness in relationship to genetic variation. Our functional systems immunology approach will allow us to define baseline human immune signatures following viral infection and vaccination along with deviations from this baseline, with the goal of identifying future targets for intervention and establishing sets of biomarkers that predict responses to vaccination.

Immunophenotyping using flow cytometry is in a position similar to that of genomics a decade ago. Highly polychromatic assays with 6 to 18 colors have been demonstrated in academic labs, but have generally not been regularly deployed for clinical studies in a robust, industrialized manner. Immune system monitoring in humans, including the application of sophisticated multi-parameter flow cytometry, would make possible indepth phenotyping that more directly reflects disease pathogenesis and progression. Polychromatic flow cytometry provides a powerful assessment of immune function based on differences in cell numbers, cell types and the expression of cell-associated surface and intracellular molecules related to immune perturbation. Development of a comprehensive platform that introduces: automation to cell processing;highspeed cell interrogation running a 96-well plate in under six minutes;and multidimensional data analysis tools implementing the newly developed "FLAME" program will allow the precise and robust measurement of human immune responses to viral vaccines and infections. The aim of this core is to bring new technologies to the U19 to achieve high throughput but also robust and precise examination of the immune cell status before, during and after immunization or infection. The bulk of all samples collected from Projects 1 and 2 will be processed and analyzed by Flow Cytometry using a state-of-the-art robotics platform. The multidimensional robotic flow core has the capability to use validated robotic methods to process blood samples, resulting in minimal variation in cell preparation. The processed samples will be prepared using the robotic platform to detect the level of immune status markers of different cell populations using polychromatic flow cytometry. The application of robotics, careful quality control of reagents, and automated downstream analysis of flow-data in multi-dimensional space is critical in the U19's challenge to establish this network of human immunology profiling research groups.

DESCRIPTION (provided by applicant): This proposal describes the Clinical Neuroscientist Training Program (CNSTP), the research education program for Neurology residents at the Yale School of Medicine. This program integrates the vast array of innovative and exciting collaborative research opportunities available within the School of Medicine with a select group of outstanding, dedicated mentors in neuroscience. These mentors were selected on the basis of significant research accomplishments, a consistent record of independent funding, and an exceptional track record in the mentoring of clinician scientists. This group of mentors, when combined with the vibrant neuroscience community at Yale, a track record of training distinguished clinicians, rich core facilities and resources, makes the CNSTP at Yale a unique program that develops the careers of physician-scientists. Since 2008, this program already has an established track record of nurturing careers of neurology residents towards career development awards. The present application leverages these existing strengths and institutionalizes them with an executive committee and formal oversight and evaluations, and seeks NINDS funding to buttress these efforts.

DESCRIPTION (provided by applicant): Multiple sclerosis (MS) and type 1 diabetes (T1D) are complex genetic diseases where pathogenic T cell autoimmune responses target and destroy self-tissue. However, the presence of T cells reactive to self alone is not sufficient for disease o occur as autoreactive T cells can be found in healthy control subjects; thus various means are available that control unwanted responses. One of the most important mechanisms is the activity of regulatory T cells (Tregs), which arise both in thymus and in the peripheral immune system as a consequence of exposure to antigens. While it was originally thought that Tregs represented a homogenous, end-stage differentiated population, recent data suggest that Tregs exist in immune response-specific subsets, which parallel the Th subsets that they control, can under certain conditions, be reprogrammed to secrete pro-inflammatory cytokines. Thus, Treg adaptation to the inflammatory milieu and chameleon-like propensity to take on Th-like phenotypes is physiologic and a fundamental attribute observed in mouse models. We have recently found that there is an increased frequency of Tregs from MS or T1D patients that secrete IFN-? compared to healthy controls, suggesting that this reprogramming may be present in humans as well and under certain conditions can be involved in the pathogenesis of autoimmune diseases. Thus, one fundamental question, addressed in this proposal is to determine the mechanisms that generate Th-Tregs in autoimmune diseases and how these cells function in vivo under basal, inflammatory and therapeutic conditions. In addition, the induction and effectiveness of Treg responses are affected by their anatomic location. Factors that have central roles in Treg homeostasis such as TGF-¿, IL-6 and others, are expressed differently at mucosal surfaces, in the CNS, and in the pancreas, and may affect Treg differentiation locally. Thus, in this proposal we will address a second hypothesize that the anatomic location and immune environment of Tregs dictates their pathway of differentiation in humans and that these processes are altered in individuals prone to autoimmunity. The following aims represent a concerted effort between two centers that will allow us to address these questions using the same experimental and disease platforms. 1. To determine what mechanisms are involved in the differentiation and stability of Th-like Treg subsets in healthy individuals and patients with MS and T1D. 2. To determine whether innate factors, driven by TLR ligation modulate Th-like Tregs. 3. To determine the function of human Th-like Tregs in vivo and the effects of immune therapies in humans and humanized mouse models. These studies will identify wide-ranging and disease-specific features of Th-Tregs in autoimmunity, and, hopefully, identify novel mechanisms that are important for prevention of MS, T1D, and potentially other autoimmune diseases. In addition, these studies may identify pathways that may be targeted by existing or novel immune therapeutics. PUBLIC HEALTH RELEVANCE: Multiple sclerosis (MS) and type 1 diabetes (T1D) are complex genetic diseases where pathogenic T cell autoimmune responses target and destroy self-tissue. It is now believed that defects in a population of cells with immunosuppressive properties called regulatory T cells are central to the development of autoimmune diseases, but many facets of the biology of these cells in humans are still unknown. In this project, we will pursue efforts to determine how these cells are generated in health and disease and identify key molecules that could be targeted therapeutically to restore normal processes in MS and T1D.

DESCRIPTION (provided by applicant): Myasthenia gravis (MG) is an autoimmune disorder of neuromuscular transmission with an estimated annual incidence of about 1-2 per 100,000 and prevalence as high as 20-50 per 100,000. Treatment consists of symptomatic therapy with acetylcholinesterase inhibitors and immunotherapy such as corticosteroids, azathioprine, cyclosporine, and plasma exchange (PLEX) and intravenous immunoglobulin (IVIg). Despite current therapies, subset of patients remains medically refractory or has intolerable medication adverse effects. There is need for another agent in the management of MG as there are few effective drugs. Safe, well- tolerated, efficacious and steroid-sparing therapeutics are very desirable. Our proposed research will be instrumental in identifying a novel treatment strategy for MG that may be more effective than current approaches. Several recent studies, including two performed by our group, have demonstrated the benefits of B cell depletion rituximab treatment in MG patients. We completed a small retrospective study to evaluate B cell targeted therapy in medically refractory generalized MG. In this analysis we showed that rituximab led to a sustained clinical improvement in parallel to a reduction or discontinuation of other immunotherapies. We now plan on conducting a multicenter randomized, double-blind, placebo controlled Phase II clinical trial utilizing a futility design. The study would include acetylcholie receptor (AChR) antibody positive generalized MG patients. This study also presents a unique opportunity to study both drug and disease mechanisms because unlike many other autoimmune diseases in which rituximab has been used, MG affords the investigation of antigen-specific components that participate in the immunopathology of the disease. This work will further our understanding of MG immunopathology and it represents the first step toward gaining a more complete understanding of the immune mechanisms underlying treatment of MG with rituximab leading to new ways to treat the disease. The specific primary aim of this study is to determine whether rituximab is safe and shows sufficient promise as a steroid sparing therapeutic for MG to warrant further study in a phase III efficacy trial. Additionally, we plan on collecting specimens to conduct an ancillary exploratory biomarker study, funded by NIAID, focused on identifying how treatment modifies the immunopathology of MG.

PROJECT SUMMARY/ABSTRACT - PROJECT 2 Multiple sclerosis (MS) is an autoimmune inflammatory demyelinating disease of the central nervous system (CNS) associated with increases in activated myelin-reactive T cells. We have previously identified immunoregulatory defects in patients including loss of CD4+Foxp3+ regulatory T cells (Tregs), and a decrease in expression of TIM-3 on CD4 T cells. TIM-3 is selectively expressed on Th1 cells, and engagement of TIM-3 by its ligands galectin-9 or the recently discovered CEACAM-1 regulates Th1 cells and controls tolerance induction. Blocking the TIM-3/CEACAM-1 pathway exacerbates disease in a mouse model for MS. The mechanisms underlying the loss of TIM-3 in MS are not known. We recently undertook a transcriptional profiling study from PBMC of MS patients and identified two subsets of MS patients (MSA and MSB), with MSA patients showing a more active disease. Our preliminary data indicate that lower expression of TIM-3 distinguished the MSA group. Furthermore, a beneficial response to interferon-ß (IFNß) therapy correlated with the degree that TIM-3 was upregulated. Additional experiments identified IFNß and IL-27, which can be induced by IFNß in APCs, as major inducers of TIM-3 on T cells. Taken together with recent reports showing low IFNß serum levels in patients, this suggests that a defect in the IFNß-TIM-3 pathway may play a part in the development of MS. Moreover, our preliminary studies show that TIM-3 is upregulated on human Tregs after activation and that TIM-3+ Tregs are more efficient suppressors than TIM-3- Tregs. Thus, the previously reported loss of Treg function in MS patients that disappeared with IFNß treatment may be linked to defective TIM-3 expression. Our overall hypothesis is that a genetically mediated loss of type I IFN signal in MS drives a loss of TIM-3 expression and of immunoregulation. We propose studies in both humans and mouse models to accomplish the following aims: 1. To determine the mechanism by which IFNß and IL-27 regulate TIM-3 expression and IL-10 secretion by effector T cells. 2. To define the role of TIM-3+ Tregs in regulating autoimmunity in the CNS. These investigations provide a link between the genetic variations underlying autoimmune disease with functional alterations in immune function.

The overall goal of the Human Immunology Project Consortium (HIPC) program is to capitalize on recent advances in immune profiling methods in order to create a novel public resource that characterizes diverse states of the human immune system. We propose to contribute to this program through deep interrogation and a broad systems approach that will identify molecular signatures of divergent human immune responses to infections. The three projects that comprise our U19 each leverage a common experimental infrastructure to focus on a different infectious diseases: the Lyme disease spirochete Borrelia burgdorferi, emerging arthropod-borne West Nile virus, and effects of aging on vaccination against influenza. Our goal is to delineate human immune signatures that are associated with the divergent manifestations in the population, using well-defined patient cohorts and a multidimensional analytical approach to quantitatively assess primary human immune cell function. Our program employs cutting-edge immune profiling such as multidimensional profiling by CyTOF, metabolomics, nanoscale technologies such as MuSIC (MultiSpectral Imaging Cytometry), and RNA-seq on single cells that will inform a systems approach to elucidate the biologic signatures defining immune responsiveness. Commonalities between the responses in different tissues, and to the different infection types, will be determined by quantifying signature enrichments, and by identifying conserved active sub-networks in this immune-specific functional network. This collaborative U19 takes advantage of enormous strengths across our institutions to tackle a challenging issue in human immunology. The investigators in this proposal have established collaborations, regular interactions, and a track record of shared success. Our three research projects are supported by shared cores for Administration, Data Management and Analysis, Single Cell Immunophenotyping, and Clinical Recruitment. The united goal of these varied approaches is to define elements of the immune response that contribute to divergent infection outcomes. This multifactorial, wide-angle view of the immune response will be compiled employing the expertise of each individual approach for Systems Modeling from deep interrogation of three sets of stratified patient cohorts. The output of this functional systems immunology approach will be definitions of human immune signatures following multiple forms of infectious challenges with the ultimate goal of defining future targets for intervention and predicting susceptibility or resistance.

An Administrative Core will coordinate and facilitate all activities within the U19 HIPC Group. This Core will be responsible for the overall organization, management, decision-making, and utilization of institutional resources. The Administrative Core will provide oversight and consultation to each of the Research and Scientific Core Projects to ensure that scientific objectives are met and that there is optimal utilization of resources. Specifically, the Administrative Core will: monitor and assist each group so that their goals are achieved and emergent problems are expeditiously addressed; provide fiscal management and ensure cost-effective utilization of U19 resources; promote the communication and dissemination of research and technology; and organize the presentation and publication of data. This Core will also ensure data sharing, protection of intellectual property, and long-term data storage in coordination with the Data Repository.

Immune function is highly dependent upon costimulatory signals delivered during both the initial process of T cell activation and reactivation with entry into target tissue. There is a key role of costimulatory signals in autoimmune disease with activation of autoreactive T cells which has become evident with the emergence of autoimmmunity after blockade of coinhibitory signals in cancer. Moreover, gene variants in the costimulatory receptor CD226, and its ligand CD155 (shared with TIGIT) are strongly associated with risk of MS, suggesting a central role in MS autoimmunity. We discovered that TIGIT signaling in CD4+ cells induces IL-10, while CD226 is associated with IFN?/IL-17 secretion. Moreover, we identified Foxp3+ T cells expressing TIGIT as a distinct Treg subset with increased suppressor function. New preliminary data revealed that the CD226? TIGIT+CD4+ subpopulation from MS patients did not secrete IL-10 when compared to controls. Additionally, TIGIT was revealed to signal through AKT in MS Tregs with development of dysfunctional IFN? secreting Tregs. These new data have led us to deeply examine the function of TIGIT and CD226 on human T cells. We hypothesize that loss of TIGIT expression or function in MS drives autoimmune responses, while in tumors, increased TIGIT expression or signaling allows tumors to escape immune surveillance. We will determine the role of TIGIT/CD226 in regulating CD4+ T cell activation in human inflammatory disorders. First, we propose to test the hypothesis that the CD226/TIGIT axis regulates balance between IFN?/IL-17 vs. IL-10 production for T cells. Specifically, we propose that loss of signaling through the TIGIT pathway on MS T cells leads to loss of IL-10 while this pathway is hyperactive in glioma T cells. Serial RNAseq, ATACseq, and MINT-ChIP experiments will be performed after stimulation with TIGIT & CD226 mAbs in relationship to cytokine secretion, enabling us to elucidate key regulatory elements and associated epigenetic changes defective in TIGIT signaling in MS as compared to glioma. shRNA/CRISPR/Cas9 knockdowns will validate key regulatory nodes controlling proinflammatory vs. regulatory cytokines. Secondly, we will investigate the role of TIGIT/CD226 on the function of regulatory T cell populations in MS. In this aim, we hypothesize there are defects in the function of TIGIT+ Tregs in MS patients inducing dysfunctional Th1-like Tregs. We will measure frequency and function of TIGIT+ T cells in MS, determining whether TIGIT signaling prevents Th1 reprogramming of Tregs through repression of the AKT pathway. Using agonistic anti-TIGIT mAbs, we will interrogate molecular pathways of TIGIT in Tregs using RNA-seq, ATAC-seq, and MINT-ChIP serially after stimulation as readouts of biologic function allowing the elucidation of key regulatory elements that drive biologic processes which we will confirm by CRISPR/Cas9 and shRNA gene knockdown. In total, these studies will provide insight into the role of costimulatory pathways in the regulation of self- and tumor reactive T cells in autoimmune disease and cancer and potentially direct the development of new therapeutic approaches.

PROJECT 2: PROJECT SUMMARY With the rise in incidence of metastatic melanoma and prolonged survival of patients with this disease, an ever increasing number of patients are living with metastatic melanoma and requiring therapy. Not all patients respond to PD-1 inhibitors, and there is therefore an urgent need for means to predict response to this class of agents, to monitor patients for response, and to understand mechanisms of sensitivity and resistance to these drugs that might be harnessed for designing effective combination regimens. Although tumor cell properties, such as PD- L1 expression, are somewhat associated with response, mounting evidence generated suggests that properties and quantity of tumor infiltrating inflammatory cells might be more informative in terms of predicting and monitoring response and can enable us to determine approaches to overcome resistance. However, melanomas often metastasize to areas that are difficult to biopsy, such as the brain, spleen, small bowel and bone, and we therefore propose approaches to studying properties of tumor infiltrating lymphocytes (TIL) by isolating and profiling T cells that originated in the tumor but can be identified in the circulation. In previous studies we demonstrated that these circulating T cells, which express both PD-1 and TIM-3, are specific to the tumor microenvironment, and are not found in the circulation of healthy individuals or patients with autoimmune disease. We propose to conduct single cell analysis of these TIM3+PD-1+ circulating CD4 and CD8 cells that have been in tumor tissue using state-of-the-art technologies. We have assembled a team of interdisciplinary researchers with the goal of developing liquid biopsy methods to determine mechanisms of resistance to immune checkpoint inhibitors. We will start by conducting single cell CyTOF and functional cytokine analysis of CD4 and CD8 cells from the circulation and tumor tissue to determine similarities and differences at the molecular level. We will then barcode TIM3+PD-1+ cells using TcR chain sequences to identify circulating TIL by the presence of TcR sequences that are identical to TIL isolated from tumors representing sister clones. Using single cell RNA- seq, we will therefore be able to assess TIL function by studying function in this population of circulating TIL. This will be done on prospectively collected matched samples from melanoma patients undergoing resection of metastases, including brain metastases, biopsies from other sites and blood samples. Our liquid biopsy technique then be studied on blood samples from patients treated with PD-1 inhibitors before and on therapy, with complete clinical annotation including response to therapy. If successful, our liquid biopsy approach can be applied to other treatment settings and other tumor types, and thus has the potential to significantly impact patient care by enabling improved patient selection and avoiding the morbidity and expense associated with repeat tumor biopsies. Moreover, the single cell analysis will enable us to better understand mechanisms of resistance, which in turn can facilitate drug development for patients with tumors resistant to PD-1 inhibitors.

ABSTRACT Not required by RFA

Abstract COVID-19, caused by novel coronavirus SARS-CoV-2, has recently affected over 600,000 people and has caused more than 30,000 deaths worldwide. Dysregulated immune responses against SARS-CoV-2 virus are a critical component of COVID-19 that can lead to severe respiratory failure (SRF). The dysregulated type 1 interferon (IFN-I) production by innate immune cells are likely involved in immunopathogenesis. However, the molecular mechanisms by which the virus causes lethality are not known. It has been found that COVID-19 patients with SRF exhibit a cytokine storm with hyper activated T cells characterized by pro-inflammatory cytokine production of GM-CSF, IFN-?, and TNF-?, though paradoxically, the T cells express high level of co- inhibitory receptors that are thought to limit this aberrant response. These data indicate there are inadequate inhibitory signals on T cells in severe disease. We have identified TIGIT as a critical co-inhibitory receptor expressed on T cells that plays a central role in orchestrating T cell activation and immune homeostasis in autoimmunity, cancer and viral infection, and its expression was found to be coordinated with the PD-1/TIM-3 module in mice. However, our lab recently discovered that while IFN-I drives expression of this module it surprisingly decreases TIGIT expression in humans implicating a unique function of TIGIT during IFN-I responses on human T cells. Moreover, we developed a gene regulatory network using high resolution transcriptional profiling that allows identification of regulatory factors for co-inhibitory receptor expression during IFN-I response. This leads to our overall hypothesis that delayed IFN-I response to SARS-CoV-2 in older individuals disrupts the T cell co-inhibitory response, leading to T cell hyperactivation and severe illness. Specifically, attenuated TIGIT expression on T cells allows aberrant cytokine release which fuels the cytokine storm in severe COVID-19. Moreover, pre-clinical data demonstrated that TIGIT immunopathology . Thus, our goals are to: 1) identify the molecular signaling limits without affecting viral load in vivo mechanism for the dysregulated immune program leading to hyper T cell responses in COVID-19 patients and to identify potential targets. We will probe dynamic T cell responses by incorporating comprehensive multi- omics single cell analysis in patients with mild and severe manifestation of COVID-19 compared to healthy individuals; 2) we will explore the mechanism for driving hyperactivation of T cells in severe COVID-19. Our previously established gene regulatory network for IFN-I response on T cells will be integrated with data acquired from our single cell analysis. This will allow us to identify the key regulatory factors controlling TIGIT expression under IFN-I response and may allow the identification of novel therapeutic targets; 3) Finally, we will determine the therapeutic potential of TIGIT mediated co-inhibitory signaling in COVID-19 by investigating whether agonistic TIGIT antibodies can ameliorate the hyperactivated state of T cells in severe COVID-19 patients. Studying how co-inhibitory signals modulate T cell responses to SARS-CoV-2 may reveal novel molecular targets for COVID-19 immunotherapy.

The overall goal of the Human Immunology Project Consortium (HIPC) program is to capitalize on recent advances in immune profiling methods in order to create a novel public resource that characterizes diverse states of the human immune system. We propose to contribute to this program through deep interrogation and a broad systems approach that will identify molecular signatures of divergent human immune responses to infections. The three projects that comprise our U19 each leverage a common experimental infrastructure to focus on a different infectious diseases: the Lyme disease spirochete Borrelia burgdorferi, emerging arthropod-borne West Nile virus, and effects of aging on vaccination against influenza. Our goal is to delineate human immune signatures that are associated with the divergent manifestations in the population, using well-defined patient cohorts and a multidimensional analytical approach to quantitatively assess primary human immune cell function. Our program employs cutting-edge immune profiling such as multidimensional profiling by CyTOF, metabolomics, nanoscale technologies such as MuSIC (MultiSpectral Imaging Cytometry), and RNA-seq on single cells that will inform a systems approach to elucidate the biologic signatures defining immune responsiveness. Commonalities between the responses in different tissues, and to the different infection types, will be determined by quantifying signature enrichments, and by identifying conserved active sub-networks in this immune-specific functional network. This collaborative U19 takes advantage of enormous strengths across our institutions to tackle a challenging issue in human immunology. The investigators in this proposal have established collaborations, regular interactions, and a track record of shared success. Our three research projects are supported by shared cores for Administration, Data Management and Analysis, Single Cell Immunophenotyping, and Clinical Recruitment. The united goal of these varied approaches is to define elements of the immune response that contribute to divergent infection outcomes. This multifactorial, wide-angle view of the immune response will be compiled employing the expertise of each individual approach for Systems Modeling from deep interrogation of three sets of stratified patient cohorts. The output of this functional systems immunology approach will be definitions of human immune signatures following multiple forms of infectious challenges with the ultimate goal of defining future targets for intervention and predicting susceptibility or resistance.

Abstract A core facility for the IMPACC study group will be formed to conduct analyses of samples collected by the individual study sites. Shared protocols for subject enrollment and sample collection have been established by the IMPACC study group. Collection and shipping to the core site will be overseen by the Data Coordinating Center. Core assays will include multiparameter single cell CyTOF of endotracheal aspirates and DNA sequencing. Core functions will be conducted in close collaboration with the IMPACC study group and include sample tracking and management, design of antibody labeling panel, sample processing and acquisition, DNA sequencing, initial data quality analysis and data sharing.

The overall goal of the Human Immunology Project Consortium (HIPC) program is to capitalize on recent advances in immune profiling methods in order to create a novel public resource that characterizes diverse states of the human immune system. We propose to contribute to this program through deep interrogation and a broad systems approach that will identify molecular signatures of divergent human immune responses to infections. The three projects that comprise our U19 each leverage a common experimental infrastructure to focus on a different infectious diseases: the Lyme disease spirochete Borrelia burgdorferi, emerging arthropod-borne West Nile virus, and effects of aging on vaccination against influenza. Our goal is to delineate human immune signatures that are associated with the divergent manifestations in the population, using well-defined patient cohorts and a multidimensional analytical approach to quantitatively assess primary human immune cell function. Our program employs cutting-edge immune profiling such as multidimensional profiling by CyTOF, metabolomics, nanoscale technologies such as MuSIC (MultiSpectral Imaging Cytometry), and RNA-seq on single cells that will inform a systems approach to elucidate the biologic signatures defining immune responsiveness. Commonalities between the responses in different tissues, and to the different infection types, will be determined by quantifying signature enrichments, and by identifying conserved active sub-networks in this immune-specific functional network. This collaborative U19 takes advantage of enormous strengths across our institutions to tackle a challenging issue in human immunology. The investigators in this proposal have established collaborations, regular interactions, and a track record of shared success. Our three research projects are supported by shared cores for Administration, Data Management and Analysis, Single Cell Immunophenotyping, and Clinical Recruitment. The united goal of these varied approaches is to define elements of the immune response that contribute to divergent infection outcomes. This multifactorial, wide-angle view of the immune response will be compiled employing the expertise of each individual approach for Systems Modeling from deep interrogation of three sets of stratified patient cohorts. The output of this functional systems immunology approach will be definitions of human immune signatures following multiple forms of infectious challenges with the ultimate goal of defining future targets for intervention and predicting susceptibility or resistance.

The overall goal of the Human Immunology Project Consortium (HIPC) program is to capitalize on recent advances in immune profiling methods in order to create a novel public resource that characterizes diverse states of the human immune system. We propose to contribute to this program through deep interrogation and a broad systems approach that will identify molecular signatures of divergent human immune responses to infections. The three projects that comprise our U19 each leverage a common experimental infrastructure to focus on a different infectious diseases: the Lyme disease spirochete Borrelia burgdorferi, emerging arthropod-borne West Nile virus, and effects of aging on vaccination against influenza. Our goal is to delineate human immune signatures that are associated with the divergent manifestations in the population, using well-defined patient cohorts and a multidimensional analytical approach to quantitatively assess primary human immune cell function. Our program employs cutting-edge immune profiling such as multidimensional profiling by CyTOF, metabolomics, nanoscale technologies such as MuSIC (MultiSpectral Imaging Cytometry), and RNA-seq on single cells that will inform a systems approach to elucidate the biologic signatures defining immune responsiveness. Commonalities between the responses in different tissues, and to the different infection types, will be determined by quantifying signature enrichments, and by identifying conserved active sub-networks in this immune-specific functional network. This collaborative U19 takes advantage of enormous strengths across our institutions to tackle a challenging issue in human immunology. The investigators in this proposal have established collaborations, regular interactions, and a track record of shared success. Our three research projects are supported by shared cores for Administration, Data Management and Analysis, Single Cell Immunophenotyping, and Clinical Recruitment. The united goal of these varied approaches is to define elements of the immune response that contribute to divergent infection outcomes. This multifactorial, wide-angle view of the immune response will be compiled employing the expertise of each individual approach for Systems Modeling from deep interrogation of three sets of stratified patient cohorts. The output of this functional systems immunology approach will be definitions of human immune signatures following multiple forms of infectious challenges with the ultimate goal of defining future targets for intervention and predicting susceptibility or resistance.