Peter A. Calabresi - US grants

DESCRIPTION (Adapted from the Investigator's Abstract): Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) of unknown etiology. It is the most common non-traumatic cause of neurologic disability in young adults. Although three drugs have been approved to treat the symptoms of MS, they are not a cure. Development of more effective treatment strategies will be improved by a greater understanding of the pathogenic events in this disease. The pathology of MS is characterized by perivascular infiltration of mononuclear cells. Several lines of evidence suggest that costimulation independent (memory) myelin basic protein (MBP) reactive T cells exist preferentially in MS patients' blood and may be involved in initiating the inflammatory response. T cell activation requires at least two signals: antigen presented in the context of major histocompatibility complex (MHC) class II and a second, costimulatory, signal of binding the CD28 receptor on the T cell. In MS, it is thought that the activated T cell then preferentially produces specific Th1 cytokines that upregulate adhesion molecules on endothelial cells and promote migration into the CNS, where demyelination occurs. Chemokines (CK) are chemoattractant cytokines that mediate tissue specific migration of T cells that express certain chemokine receptors (CKR). In MS, CK and CKR expression are thought to be an important mechanism by which antigen driven T cells migrate to sites of inflammation, but there is little data to support this concept. The investigators will examine CKR expression, function, and correlation with cytokine profiles through the following specific aims: Specific Aim 1. To determine whether MBP reactive memory T cells express more CCR5 and CXCR3 than MBP reactive naive T cells or TT reactive memory cells: a) using primary proliferation assays to isolate the MBP and TT reactive memory T cells that are costimulation independent (do not require CD28 signal) from MS patients, as compared to ONDs and controls; and b) quantifying CKR expression on these cells (by FACS ) and function (by migration studies). Specific Aim 2. To evaluate whether memory MBP reactive T cells that express CCR5 and CXCR3 produce more of the Th1 cytokines: a) isolating T cell subsets with specific CKR profiles by FACS and migration analysis; and b) quantifying cytokine production by intracytoplasmic staining (using flow cytometry). Specific Aim 3. To investigate whether VCAM binding to VLA-4 on T cells alters the expression of CKR on MBP reactive T cells by: co-culturing MBP reactive T cells with VCAM expressing endothelial cells or VCAM-Ig, and analyzing CKR expression by FACS or ELISA. The results from these aims will enhance our understanding of MBP reactive T cells as a model for the pathogenic events in MS.

DESCRIPTION (provided by applicant): Strategies designed to specifically suppress the function of myelin-reactive T cells are an objective of pharmacological therapy in multiple sclerosis (MS). We have focused our studies on identifying markers of costimulation independent memory cells that might be amenable to therapeutic intervention. In the last two years, we have demonstrated that two chemokine receptors, CXCR3 and CXCR6, are highly expressed on Th1 myelin specific effector T cells. We have also determined that the loss of the lymph node homing chemokine receptor, CCR7, is an excellent marker of conversion of myelin reactive T cell lines from an early effector state CD45RA-/CCR7+) to an effector memory state (TEM=CD45RA-/CCR7-), and that these TEM are synonymous with the previously described costimulation independent T cells in MS. In a recent collaboration, we have now identified a voltage-gated potassium (K+) channel, Kv1.3, which is specifically and highly expressed on chronically differentiated TEM. We have demonstrated that the myelin reactive Kv1.3highTEM are present in MS patients and not in controls. We will now characterize the specificity of Kv1.3 expression on effector and regulatory subtypes of CD4 and CD8 T cells, and test the hypothesis that specific Kv1.3 inhibitors will selectively suppress TEM, without compromising immediate immune responses or regulatory T cells. We will examine these hypotheses through the following specific aims: Specific Aim 1. To determine the peptide specificity of the myelin reactive TEM (CCR7-, Kv1.3highlKCa1low). Specific Aim 2. To determine whether CD8 myelin specific T cells from MS patients have the TEM (CCR7-, Kv1.3highlKCa1 low) phenotype as compared to controls using class I tetramers. Specific Aim 3. To evaluate the effects of Kv1.3 antagonists on TEM function. Specific Aim 4. To determine the expression and function of Kv1.3 on myeloid lineage cells in vitro and in the inflammatory infiltrate of MS brain tissue. The results from these K+ channel studies on human immune cells will have broad implications for defining the potential role of K+ channel blockers in specifically targeting effector cells, and could lead to novel treatment strategies for MS.

DESCRIPTION (provided by applicant): Strategies designed to specifically suppress the function of myelin-reactive T cells are an objective of pharmacological therapy in multiple sclerosis (MS). We have focused our studies on identifying markers of costimulation independent memory cells that might be amenable to therapeutic intervention. In the last two years, we have demonstrated that two chemokine receptors, CXCR3 and CXCR6, are highly expressed on Th1 myelin specific effector T cells. We have also determined that the loss of the lymph node homing chemokine receptor, CCR7, is an excellent marker of conversion of myelin reactive T cell lines from an early effector state CD45RA-/CCR7+) to an effector memory state (TEM=CD45RA-/CCR7-), and that these TEM are synonymous with the previously described costimulation independent T cells in MS. In a recent collaboration, we have now identified a voltage-gated potassium (K+) channel, Kv1.3, which is specifically and highly expressed on chronically differentiated TEM. We have demonstrated that the myelin reactive Kv1.3highTEM are present in MS patients and not in controls. We will now characterize the specificity of Kv1.3 expression on effector and regulatory subtypes of CD4 and CD8 T cells, and test the hypothesis that specific Kv1.3 inhibitors will selectively suppress TEM, without compromising immediate immune responses or regulatory T cells. We will examine these hypotheses through the following specific aims: Specific Aim 1. To determine the peptide specificity of the myelin reactive TEM (CCR7-, Kv1.3highlKCa1low). Specific Aim 2. To determine whether CD8 myelin specific T cells from MS patients have the TEM (CCR7-, Kv1.3highlKCa1 low) phenotype as compared to controls using class I tetramers. Specific Aim 3. To evaluate the effects of Kv1.3 antagonists on TEM function. Specific Aim 4. To determine the expression and function of Kv1.3 on myeloid lineage cells in vitro and in the inflammatory infiltrate of MS brain tissue. The results from these K+ channel studies on human immune cells will have broad implications for defining the potential role of K+ channel blockers in specifically targeting effector cells, and could lead to novel treatment strategies for MS.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A fundamental objective in multiple sclerosis (MS) research is to elucidate the cellular and molecular pathways that are involved in mediating permanent disability. Clinical, radiographic, and pathological studies suggest that disability can ensue both from acute clinical exacerbations and during the chronic progressive stages of the disease. The underlying hypothesis in our research is that permanent disability in MS, regardless of the stage of the disease, is a result of damage to the axon. Herein, we plan to define the cellular and molecular mechanisms of axonal injury in MS, develop imaging biomarkers of disability, and thereby test rational neuroprotective and neuroreparative strategies. A major goal in translating neuroprotective and neuroreparative strategies for MS from the bench to the bedside is to develop surrogate measures of myelin and axon integrity that can be used in phase II clinical trials to screen for preliminary efficacy. The utility of gadolinium-enhancing lesions as a surrogate marker of inflammation in phase II/ III trials of immunomodulatory drugs is now well accepted. Nonetheless, even in the absence of apparent inflammation, clinical disability progresses. This is thought to occur as a result of axon degeneration mediated by numerous downstream factors. Radiological studies have demonstrated evidence of distant Wallerian degeneration and atrophy that ensue months to years after active inflammatory demyelination. The correlation between disability and conventional measures such as T1 volume (post-gadolinium), T2 volume, and atrophy or T1 black holes is only modest (correlation coefficients between 0.3 and 0.6 in a variety of studies), presumably because all of these measures lack specificity for permanent brain tissue pathology. Recent advances in magnetic resonance imaging (MRI) such as magnetization transfer imaging (MTI), proton magnetic resonance spectroscopy (1H-MRS), and diffusion tensor imaging (DTI) offer promise as more sensitive and specific measures of underlying structural pathology. There is a great need to develop and optimize these measures so as to be able to non-invasively quantify the extent of demyelination and axon degeneration in MS patients. We will focus on developing DTI and MTI to allow quantitative measurement of pathology along white-matter tracts, which can then be used as outcome measures for clinical trials of potential neuroprotective and neuroreparative agents. DTI gives information on the directionality and integrity of white-matter tracts, containing both axonal and myelin information. MTI has been shown to be associated with both axon density and myelin integrity, and therefore may be relevant to tracking local and distant changes in axons that pass through areas of damaged myelin, as well as a measure of myelin repair. Fiber-tracking software allows the 3 dimensional reconstruction of specific pathways distal and proximal to a region of interest (ROI) or between two or more ROIs. In this way, multiple types of quantitative information can then be acquired both locally and at distant sites from an acute inflammatory lesion. Moreover, we can interrogate the reconstructed pathways to measure changes over time in an individual patient. Since all of the MR images are coregistered we can compare DTI and MTI tract specific information, which may improve our ability to discern different pathologies along the trajectories.

DESCRIPTION (provided by applicant): Potassium (K+) channels expressed in immune cells have gained attention recently as promising targets of immunotherapy for multiple sclerosis (MS). We have previously shown that naove and central memory T cells (TCM) predominantly use the calcium-dependent potassium channel KCa3.1 during acute activation, whereas effector memory T cells (TEM), which are likely to be the pathogenic population in MS, utilize the voltage-gated potassium channel Kv1.3. Further, only TEM were inhibited by Kv1.3- specific pharmacological blockade, as naove T cells and TCM bypassed this inhibition via up regulation KCa3.1, indicating the potential for selectivity of this approach. Myelin reactive T cells from MS patients have higher levels of Kv1.3 than either control antigen specific T cells or myelin reactive T cells from healthy volunteers. We have further demonstrated that the inflammatory infiltrate in MS brain tissue consists almost entirely of TEM and that these cells highly express Kv1.3. To better define the specific role of Kv1.3, we have generated a lentivirus construct that expresses a dominant negative (DN) form of Kv1.3 and have recently shown that transduction of CD4+ Tcell subsets with the Kv1.3DN both inhibits differentiation from TCM into TEM and mediates reversion of TEM into TCM. To identify target genes that contribute to these effects, we analyzed gene expression patterns by microarray, which showed significant differences in membrane microdomain, cell cycle, and differentiation genes as a result of Kv1.3 inhibition. The underlying hypothesis of this grant renewal is that Kv1.3 plays a major role in TEM cell derivation and biological function, and that selective blockade of this channel can reversibly inhibit the pathogenic consequences of these cells without compromising the TCM pool that is required for recall immune responses to pathogens. From our preliminary data, we are hypothesizing that Kv1.3 blockade disrupts T cell cycle and differentiation and will test this hypothesis in aim 1. The ultimate goal of this approach is to develop a new therapy for MS, and several pharmaceutical companies are currently generating Kv1.3 peptidyl blockers. Gaining a greater understanding of the in vivo effects will also be necessary for clinical translation. Towards this end, we will examine the effects of Kv1.3 inhibition on T cell infiltration, microglial activation, demyelination, and axon pathology in rat EAE models (Aim 2). The results of these experiments will allow us to better understand the therapeutic potential and safety of Kv1.3 blockade, and may have important implications for the design of upcoming human clinical trials of Kv1.3 blockers. Further, understanding basic mechanisms of T cell function will enhance our knowledge of how the immune system causes disease and allow us to develop novel strategies for correcting abnormal immune responses in diseases like MS. PUBLIC HEALTH RELEVANCE: The relevance of this research is in its ultimate goal to develop a new therapy for Multiple Sclerosis. The results of these experiments will allow us to better understand the therapeutic potential and safety of Kv1.3 blockade, and may have important implications for the design of upcoming human clinical trials of Kv1.3 blockers. Further, understanding basic mechanisms of T cell function will enhance our knowledge of how the immune system causes disease and allow us to develop novel strategies for correcting abnormal immune responses in diseases like MS.

DESCRIPTION (provided by applicant): There are currently no approved therapies for Multiple Sclerosis (MS) that promote remyelination and axon preservation. MS is a chronic immune-mediated disease of the central nervous system (CNS) characterized by demyelinating white matter lesions, glial scar formation and axonal loss. Thyroid hormones (THs) play a critical role in developmental myelination, however their role in remyelination has not yet been clearly defined. During developmental myelination THs act through nuclear hormone receptors to promote oligodendrocyte maturation and myelination. Recent studies in rodent models of demyelination suggest that THs have the capacity to promote remyelination, however non-specific stimulation of TH receptors can also produce significant undesirable side-effects. In this research project, we will investigate if a thyroid hormone receptor ¿ selective thyromimetic, GC-1, can promote recovery in the cuprizone model of demyelination. We will also investigate whether oligodendrocyte progenitor cells (OPCs) in the healthy adult brain are sensitive to exogenous TH administration, an effect that could lead to aberrant OL differentiation, or if they only become reactive following injurious events, such as demyelination. There are two major TH receptors (TRs). TR is predominantly expressed in the heart and is responsible for the known cardiac effects of THs, whereas TR¿ is predominantly expressed in the liver and is involved in lipid regulation. Both TR¿ and TR¿ are expressed in the CNS and are known to modulate myelination during development. Despite unequivocal evidence of the important role played by TH in myelination, we do not yet clearly understand which TRs are required to mediate these effects. We have preliminary data showing that GC-1, a TR¿ selective agonist, can induce differentiation of OPCs into myelin forming oligodendrocytes in vitro. We also find that TR¿ expression increases in vivo during remyelination in the cuprizone model of demyelination. We now propose to test the hypothesis that selective agonism of the ¿ thyroid hormone receptor by GC-1 can enhance remyelination following a demyelinating insult in vivo. In aim 1 we will investigate the potential for GC-1 to enhance remyelination in both acute and chronic demyelination scenarios. In aim 2 we will explore the mechanisms by which thyroid hormones and their receptors regulate OPC differentiation in both the healthy adult brain and in the remyelinating state. We hypothesize that in the adult CNS TR expression is one mechanism by which OPC differentiation is tightly regulated, whereas during remyelination TR¿ is up-regulated as part of the endogenous recovery process. The results of the proposed experiments could provide strong preclinical rationale for the further clinical translation of GC-1 in MS. PUBLIC HEALTH RELEVANCE: Multiple Sclerosis (MS) is the most common cause of neurological disability in young adults. It is an immune disorder in which the fatty myelin sheaths around cells of the brain and spinal cord are damaged leading to demyelination and neurological symptoms including numbness, tingling, muscle weakness and visual problems. Myelin can be repaired through the process of remyelination. Thyroid hormones play a critical role in developmental myelination. We will now investigate if a drug related to thyroid hormone can enhance remyelination in an animal model of MS and thus promote recovery from disease. The results of these experiments could provide strong preclinical rationale for the further clinica translation of this drug for MS treatment.

Project Summary One of the significant challenges facing treatment of people with multiple sclerosis (MS) is determining their individual likelihood of progression, as this information would significantly influence the type of therapy selected. Thus, developing specific tools to monitor and predict progression is critical to better manage patient care and to understand mechanisms of disease. We have been developing a multi-faceted approach to more readily monitor (through imaging) and predict (through both imaging and genetic analysis) disease progression in a real-time fashion. In the past cycle of this grant we demonstrated the utility of high resolution spectral domain optical coherence tomography (SD-OCT) and magnetic resonance imaging (3T MRI) in estimating disease burden in different CNS compartments. We showed that retinal degeneration occurs throughout the disease course and mirrors grey matter compartment atrophy in the cerebrum. A critical finding validating the clinical utility of this approach was that in a multicenter analysis of pooled data, a single OCT at baseline predicted risk of disability progression at 5 years of follow up. As MS is thought to have a strong genetic component, we sought to investigate whether there was an underlying genetic predisposition towards progression, which was made possible by the ability to utilize OCT in a real-time fashion to monitor degeneration and correlate with clinical outcome. We thus expanded the imaging study to include a genetic component in which we conducted a gene array to evaluate genetic variation among people with heterogeneous courses of MS and have preliminarily found that several gene variants in network pathways appear to be associated with more rapid rates of retinal neurodegeneration. The large data set that will be generated from these studies will also allow a corollary analysis in which we can begin to develop a risk profile model in which other population characteristics known to be associated with disease such as sex and ethnicity can be incorporated. The central hypotheses of the proposed studies are; that retinal ganglion layer thickness, thalamic and GM volumes predict 10 year disability across MS subtypes, that patients with high genetic load for gene variants in specific network pathways undergo faster neurodegeneration, and that combinations of OCT, MRI and genetic load measures may be used to develop clinically meaningful individual predictive scores for precision medicine. Aim 1: To determine whether baseline retinal ganglion layer thickness and thalamic and GM volumes predict 10 year disease outcomes. Aim 2: To determine whether genetic variation, sex and ethnicity influence rates of GCIP, thalamic, GM atrophy, and disability accumulation. Aim 3: To develop an algorithm disease progression model to predict disease outcome.

DESCRIPTION (provided by applicant): Progressive MS remains an untreatable form of the disease, and in order to intervene, a better understanding of the mechanisms by which infiltrating TEM cells of the adaptive immune system interface with glial cells is needed. Lymphoid follicles within the meninges and compartmentalized inflammation may be a major effector population for grey matter injury and incomplete remyelination, which renders axons susceptible to mitochondrial stress and calcium mediated degeneration. Alternatively, a low level of inflammation may be beneficial for promoting remyelination. To date, there is little information regarding how CNS infiltrating TEM subsets may differentially impact oligodendrocyte progenitor cells (OPCs) and remyelination, and the molecular pathways through which this occurs. Our preliminary data show that in vitro, IFN? suppresses OPC differentiation whereas, remarkably, IL-4 promotes OPC differentiation. In order to investigate the effects of T cells on remyelination in an in vivo model in which there is extensive remyelination, we have developed a novel model in which we adoptively transfer MOG reactive effector T cells (TEff) into mice with cuprizone induced demyelination (AT-cuprizone). In this system, adoptively transferred MOG reactive TEff markedly inhibited the endogenous remyelination that occurs after cessation of cuprizone feeding, without causing EAE. In order to probe the molecular signals that mediate the downstream effects of these cytokines, we have investigated several nuclear hormone receptor (NHR) family members including; thyroid hormone receptor (TR) and peroxisome proliferator activated receptor (PPAR). Ligation of PPAR? and TR?/ß is known to enhance differentiation of OPC and induce (re)myelination, but how these NHRs are coordinately signaled and whether they can overcome the inhibitory effects of IFN? is unclear. We now show that IL-4 enhances PPAR? in OPCs, and this occurs in association with increased pSTAT6 and expression of the coactivator PGC1a in a heteromeric complex with TRb1. IFN? suppresses this pathway. We thus hypothesize that TEff cytokines mediate direct effects on OPCs by regulation of PPAR? through the canonical cytokine STAT signaling pathways known to modulate immune cells.

Multiple sclerosis (MS) is an immune-mediated, inflammatory disorder of the central nervous system. Despite being classically considered a demyelinating disorder, it has been demonstrated that neuro-axonal injury occurs early in the disease course and represents the pathologic substrate for permanent neurological disability in people with MS (PwMS). In clinical practice, disease monitoring in PwMS is performed by clinical evaluation and use of conventional magnetic resonance imaging (MRI) measures, including new T2 lesions and/or presence of T1 post-gadolinium (Gd) enhancing lesions. Notably, these conventional MRI measures assess for the presence of inflammatory disease activity rather than neuro-axonal loss and are only modestly associated with clinical measures of disability in MS, a phenomenon known as the ?clinico-radiological paradox?. There is an unmet need in MS for a biomarker that may identify PwMS with ongoing neuro-axonal damage prior to the accrual of permanent clinical disability, in order to allow for timely intervention. Neurofilaments are neuron-specific cytoskeletal proteins that are released following neuro-axonal damage. Increased neurofilament light chain (NfL) levels have been found in the blood and cerebrospinal fluid (CSF) in several neurological disorders, including MS. In MS, there is evidence that serum NfL (sNfL) levels correlate closely with CSF NfL levels, are associated with clinico-radiological measures of disease activity, are modulated by disease modifying therapies (DMTs), and predict disability worsening and brain atrophy. However these data are derived mainly from small, single-center studies, and the influence of factors including demographics, disease subtype, and co-morbid conditions on sNfL levels in MS remains poorly characterized. It is also remains unclear if sNfL levels may be used to monitor response to therapy, guide decision-making regarding potency of immunotherapy, and predict long-term outcomes. While, given this emerging evidence, there is great interest in sNfL for use as a prognostic and monitoring biomarker of neuro-axonal injury in MS, further clinical validation is necessary in larger, demographically diverse, clinically heterogeneous, multi-center cohorts. Furthermore, it is of utmost importance that validation is performed utilizing a robust, scalable assay that may be rapidly implemented in the clinical realm. We plan to measure sNfL utilizing a novel automated immunoassay (Siemens Healthineers; performed on an existing clinically available platform) in serum samples from two large multi-center studies: 1) MS PATHS (Multiple Sclerosis Partners Advancing Technology and Health Solutions) is a network of 10 healthcare institutions in the United States and Europe, merging research with ongoing patient care by collecting standardized clinical/imaging data and biospecimens during routine medical visits. As of February 1st, 2019, >15,000 patients have opted to participate in MS PATHS with biospecimens already available for >5,000 patients. 2) TREAT-MS (Traditional versus Early Aggressive Therapy for Multiple Sclerosis) is an ongoing, pragmatic, randomized controlled trial, designed to evaluate in treatment naïve MS patients whether an ?early aggressive? therapy approach, versus starting with a traditional, first-line therapy approach, influences the longer-term risk of disability (at 48 months). TREAT-MS is prospectively recruiting 900 subjects across ~45 sites in the US (83 enrolled as of February 1st, 2019) with anticipated enrollment of 700 subjects in the bio-banking sub-study. We anticipate that large-scale clinical validation of sNfL in MS as a prognostic and monitoring tool in this study will lead to submission to the FDA of a full qualification package and will lead to availability of the first blood-based biomarker of MS.