Silva Markovic-Plese - US grants

[unreadable] DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is a chronic autoimmune demyelinating disease of the central nervous system (CNS) affecting primarily young adults in their most productive age, and therefore causing a significant disability. While its etiology remains elusive, most evidence supports the autoimmune pathogenesis of the disease. According to this hypothesis, the activation of autoreactive T-cells is a central event in the development of autoimmune response in MS. The objective of this proposal is to examine molecular events involved in the initiation of autoimmune response in MS. Recent studies have reported an unexpectedly high degree of T-cell receptor (TCR) degeneracy and molecular mimicry as a frequent phenomenon that might play a role in the initiation of autoimmune response in MS. MHC DR2-biased combinatorial peptide libraries are developed as a tool to characterize degenerate T-cells. As auto-antigens are predominantly weak TCR ligands, we propose that myelin-reactive T-cells may be over-represented among the cells with a degenerate TCR. Myelin basic protein (MBP)-specific T-cells exhibit decreased CD28 co-stimulatory requirements in MS patients when compared to healthy controls. We hypothesize that dysregulation of co-stimulatory pathways play a role in the initial activation, prolonged survival, and the expansion of autoreactive cells in MS. Co-stimulation-independent activation might be particularly relevant for the autoantigen recognition within the CNS, as local inflammatory environment enhances autoantigen presentation even in the absence of co-stimulatory molecules on the resident antigen presenting cells. We plan to achieve our objective by pursuing the following Specific Aims: 1) Determine the frequency and TCR repertoire of T-cell clonotypes with a high degree of TCR flexibility. 2) Define co-stimulation requirements for the activation and growth characteristics of T-cells with flexible TCR in RR MS patients and OND controls. 3) Identify mechanisms by which the inflammatory environment induces the autoreactive T-cell activation. The information provided by these studies may yield important insights into the physiologic and pathologic role of the autoreactive T cells, and characterize structurally and functionally the specific targets for the new therapies of MS. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is a chronic autoimmune demyelinating disease of the central nervous system (CNS) affecting primarily young adults in their most productive age, and therefore causing a significant disability. While its etiology remains elusive, most evidence supports the autoimmune pathogenesis of the disease. According to this hypothesis, the activation of autoreactive T-cells is a central event in the development of autoimmune response in MS. The objective of this proposal is to examine molecular events involved in the initiation of autoimmune response in MS. Recent studies have reported an unexpectedly high degree of T-cell receptor (TCR) degeneracy and molecular mimicry as a frequent phenomenon that might play a role in the initiation of autoimmune response in MS. MHC DR2-biased combinatorial peptide libraries are developed as a tool to characterize degenerate T-cells. As auto-antigens are predominantly weak TCR ligands, we propose that myelin-reactive T-cells may be over-represented among the cells with a degenerate TCR. Myelin basic protein (MBP)-specific T-cells exhibit decreased CD28 co-stimulatory requirements in MS patients when compared to healthy controls. We hypothesize that dysregulation of co-stimulatory pathways play a role in the initial activation, prolonged survival, and the expansion of autoreactive cells in MS. Co-stimulation-independent activation might be particularly relevant for the autoantigen recognition within the CNS, as local inflammatory environment enhances autoantigen presentation even in the absence of co-stimulatory molecules on the resident antigen presenting cells. We plan to achieve our objective by pursuing the following Specific Aims: 1) Determine the frequency and TCR repertoire of T-cell clonotypes with a high degree of TCR flexibility. 2) Define co-stimulation requirements for the activation and growth characteristics of T-cells with flexible TCR in RR MS patients and OND controls. 3) Identify mechanisms by which the inflammatory environment induces the autoreactive T-cell activation. The information provided by these studies may yield important insights into the physiologic and pathologic role of the autoreactive T cells, and characterize structurally and functionally the specific targets for the new therapies of MS. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The involvement of Th17-cells in the development of the autoimmune response in relapsing remitting multiple sclerosis (RRMS) is well documented. However, there is still no consensus regarding the cytokines required for the differentiation of human Th17-cells. Our preliminary studies have identified IL- 11 as a new Th17-promoting cytokine. Furthermore, we found that IL-11 is the most significantly increased cytokine in the serum and cerebrospinal fluid (CSF) of patients with clinically isolated syndrome (CIS) suggestive of MS. Although we have also demonstrated an increase in the IL-11 serum and CSF levels in patients with clinically definitive RRMS in comparison to healthy controls (HCs), and significantly higher serum IL-11 levels during the clinical exacerbations in comparison to the remissions, those results still do not confirm a causative role of this cytokine in the development of the inflammatory CNS disease. The rationale for our proposed research is that the in-vivo animal model of the disease will allow us to directly characterize the role of IL-11 in the development of the autoimmune response. Based on preliminary results, our central hypothesis is that IL-11 may play a critical role in the regulation of Th17-cells, which represent a pathogeni cell subset driving the autoimmune response in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Our long-term goal is to characterize the molecular mechanisms through which IL-11 initiates, augments and perpetuates the autoimmune response in RRMS. We will pursue the following specific aims: 1. Characterize the role of IL-11 in the development of the Th17-mediated autoimmune response in EAE. The study will: 1.A. Identify to what extent IL-11 induces the development of RR EAE. 1.B. Determine the effectiveness of anti-IL-11 mAb in the prevention and suppression of the clinical and histopathological parameters of RR EAE. 2. Determine the effect of IL-11-mediated Th17-cell differentiation and expansion on the development of encephalitogenic Th17-cells. More specifically, we will: 2.A. Determine the effect of IL-11-mediated na¿ve CD4+ T-cell differentiation in the Th17-cell passive transfer EAE. 2.B. Identify the effect of IL-11-mediated Th17 memory cell expansion in adoptive transfer EAE. The proposed in-vivo animal studies are expected to characterize the role of IL-11 in the development of the Th17-cell- mediated CNS inflammatory disease, and to provide results that will justify selective targeting of IL-11 in the treatment for RRMS.

  Multiple  sclerosis  (MS),  a  central  nervous  system  (CNS)  inflammatory  demyelinating  disease,  is  a  leading  cause  of  disability  in  the  young  adult  population.    While  dramatic  progress  has  been  made  in  our  understanding  of  the  disease  pathogenesis,  the  initial  disease-­triggering  events  remain  poorly  understood.  Since  immunomodulatory  therapies  are  most  effective  when  administered  early  in  the  course of the disease, we are seeking biomarkers of the clinically isolated syndrome (CIS) suggestive  of MS, the earliest phase of the disease.  Our preliminary studies have identified IL-­11, a new Th17  cell-­polarizing  cytokine,  as  the  most  significantly  increased  cytokine  in  the  serum  and  cerebrospinal  fluid  (CSF)  of  CIS  patients,  whose  serum  levels  are  increased  during  clinical  exacerbations  of  the  disease.    CD4+  cells  are  the  main  source  of  IL-­11  in  the  peripheral  circulation.    The  percentage  of  IL-­ 11+CD4+  cells  is  increased  in  the  peripheral  circulation  of  CIS  patients  in  comparison  to  matched  healthy controls (HCs), and they significantly accumulate in the CSF and the active brain MS lesions in  comparison to matched blood samples.  Animal studies have confirmed the causal role of IL-­11 in the  exacerbation  of  relapsing  remitting  (RR)  experimental  autoimmune  encephalomyelitis  (EAE),  since  IL-­ 11 administration worsened clinical course and induced increased numbers of IL-­17A+CD4+ cells in the  central  nervous  system  (CNS)  inflammatory  infiltrates.    Our  central  hypothesis  is  that  IL-­11  induces  CD4+ cell migratory capacity and encephalitogenicity, mediated via up-­regulation of CCR6, ICAM-­1 and  VLA-­4, which mediate CD4+ cells trans-­endothelial migration.  We propose that IL-­11+CD4+ cells, which  are  also  expanded  in  the  presence  of  IL-­11,  may  represent  a  pathogenic  cell  subset,  whose  transcriptional profiling and T cell receptor (TCR)Vb? repertoire will elucidate their function and antigen  specificity.    a?IL-­11R  mAb  treatment  of  RRAEA  will  provide  pre-­clinical  data  and  identify  markers  of  therapeutic effect for this new therapeutic approach.  The objective of this study is (1) to identify the  molecular  mechanisms  involved  in  IL-­11-­induced  migration  of  CD4+  cell  subsets  to  the  CNS  in  CIS  patients,  (2)  to  characterize  the  phenotype,  transcriptional  profile  and  TCRVb?  repertoire  of  CSF-­ enriched  IL-­11+CD4+  cells  in  CIS  patients,  and  (3)  to  examine  the  potential  of  IL-­11  to  induce  encephalitogenic CD4+ cells capable of passively transferring disease, and to determine the therapeutic  effect of a?IL-­11R mAb in RREAE, an animal model of the disease.  The results are expected to provide  biomarkers  of  early  autoimmune  response  in  CIS  patients  and  to  identify  selective  therapeutic  targets  for this disabling disease.           

While multiple antiinflammatory therapies are effective in suppressing relapsing-remitting multiple sclerosis (RRMS) disease progression, they have significant side effects and toxicity. We are still seeking a disease specific treatment that would provide an antigen-specific immune tolerance reconstitution. We used microparticle (MP)-encapsulated STING agonist cGAMP to increase its delivery into phagocytic antigen presenting cells (APCs), particularly dendritic cells (DCs). A novel acid-sensitive polymer, acetalated dextran (Ace-DEX) allows regulated MP breakdown due to its pH sensitivity in intracellular acidic compartments. Our preliminary data have demonstrated that the intracellular delivery of MPs encapsulating 2?3?cyclic GMP-AMP dinucleotide (cGAMP) suppressed experimental autoimmune encephalomyelitis (EAE), an animal model of MS, when administered before and at the peak of clinical disease. The therapeutic effect of cGAMP MPs in RR and chronic EAE was mediated via STING-induced IL-27 and IL-10 production, since the therapeutic effect was abrogated in IL-27ra-/- and IL-10-/- mice, while partial disease suppression was maintained in Ifnar-/- mice. Our central hypothesis is that intracellular cGAMP MP delivery will induce IL-27 and IL-10-producing tolerogenic DCs that regulate the differentiation and expansion of IL-10-producing iTregs. We will characterize cGAMP MP tolerogenic DC phenotype and function, as well as suppressive capacity of the induced regulatory CD4+IL-10+ cells, in particular their expression of FoxP3 and type 1 iTreg (Tr1) markers. We propose that presentation of MHC class II DR2-anchored peptide mixtures (DR2-APMs) by cGAMP MP-induced tolerogenic DCs from DR2+ patients with relapsing remitting multiple sclerosis (RRMS) may reconstitute antigen-specific immune tolerance. In order to identify the mechanisms of cAMP MP-mediated disease suppression, we will pursue following specific aims: 1) Demonstrate that cGAMP MP treatment induces iTreg cell expansion in EAE. The proposed studies will determine to what extent cGAMP MP treatment of EAE induces the expansion of CD4+CD25+IL-27R+IL-10+ Tregs in the peripheral immune organs and the CNS inflammatory infiltrates. Studies using aCD25mAb will confirm the role of iTregs in the therapeutic effect. The role of FoxP3+Tregs will be examined using transient inducible FoxP3+Treg depletion and mice with selective FoxP3+Treg IL-27R depletion. 2) In human in-vitro studies, we will characterize cGAMP MP-induced tolerogenic DCs and their capacity to induce iTregs in RRMS patients. Specific experiments will determine the capacity of cGAMP MP-treated monocytes and DCs to induce iTreg expansion, determine the transcriptome of cGAMP MP IL-27- and IL-10- induced Tregs and the reconstitution of their suppressive capacity. Finally, co-administration of cGAMP MPs with DR2-APMs to DCs from RRMS patients will test their capacity to induce antigen-specific tolerogenic DCs and Tregs. The study will provide preclinical data required to translate this therapeutic approach into clinical use in patients with RRMS