Bonnie Dittel - US grants

The influenza virus is a common pathogen of humans with the ability to cause death in the young and immunocompromised. Even though influenza continues to be a great threat to human life, much still needs to be learned about immunity to the virus and how immunity is maintained. To date most studies examining cell responses to influenza have examined peripheral blood and have shown that humans expressing HLA-A2 have a focused T cell response with the majority of T cells using Vbeta17 and recognize residues 58-66 of the influenza protein MI. However, this response is very complex as shown by the number large number of sequences that comprise the CDR3. As a general rule immune responses become more focused and less complex upon repeated antigen exposure. Thus in the lung, at the site of influenza infection, we would expect the influenza-specific T cell repertoire to be more focused and less complex. This hypothesis will be tested in this proposal in three specific aims. In aim 1 we will compare the influenza-specific T cell repertoire in the blood and lung of normal controls and influenza-infected individuals to examine whether the memory T cells in the lung express TCR sequences that are similar or less complex than those in the blood. In the second aim, because certain experiments cannot be performed on humans, we will develop an animal model to study the generation and maintenance of T cell repertoires upon repeated exposure to antigen. In the third aim, we will expand our studies beyond alpha/beta T cells and characterize gamma/delta T cells that reside in the lung in normal and influenza-infected individuals. Gamma/delta T cells are of interest because they are known to regulate alpha/beta T cell responses and can bridge the innate with the adaptive immune response. Little is known about the role of gamma/delta T cells in influenza immunity. These collective studies are designed to further our knowledge of human immunology, by acquiring information on influenza immunity in the lung, the tissue most affected by influenza infection.

DESCRIPTION (provided by applicant): The human disease multiple sclerosis in an autoimmune demyelinating disease of the central nervous system (CNS) and is thought to be initiated by CD4 Th1 cells with specificity for self-antigens present in the myelin sheath. Antigen presentation of the self-antigens is believed to be necessary for the propagation of disease. The cell(s) that present the antigen is currently not known. A candidate cell is the microglial cell that is considered to be a "fixed" macrophage system within the CNS. Resting microglial cells have a distinct ramified morphology and are poor antigen presenting cells (APC) by virtue of low expression levels of MHC class II and costimulatory molecules. Under a variety of pathological conditions microglial cells have shown the capacity to differentiate into "macrophage-like" cells as indicated by a change in morphology and by the upregulation of CD45, MHC class II and costimulatory molecules. Whether microglial cells differentiate and become activated during MS is not known. This proposal will investigate the differentiation and activation of microglial cells in the CNS using the animal model of MS, experimental autoimmune encephalomyelitis (EAE). EAE recapitulates much of the pathology of MS including the presence of an inflammatory infiltrate in the CNS comprised primarily of macrophages. In addition, EAE to a certain extent mimics the paralysis observed in MS patients. By monitoring the extent of the neurological symptoms the severity of disease can be determined. This proposal will examine whether the self-reactive encephalitogenic T cell can signal the differentiation and activation of microglial cells. The following hypothesis will be tested: Antigen presentation by microglial cells in the CNS is necessary for the onset of EAE and the signals required for differentiation of microglial cells into effective APC can be provided exclusively by encephalitogenic T cells. This hypothesis will be tested by two specific aims. Aim 1: The stages and kinetics of microglial cell differentiation during EAE will be determined using BM chimera mice that will allow the distinction between activated microglial cells and peripheral macrophages that currently is not possible by phenotype. Aim 2: The role of encephalitogenic derived signals in the activation of microglial cells in vivo during CNS pathology will be examined using EAE as the model. T cell derived growth factors and costimulation as well as the requirement for antigen presentation will be examined. These studies will determine whether microglial cells become activated in the CNS during EAE and the extent of encephalitogenic T cell involvement. In addition, these studies should be directly applicable to MS pathology and hopefully will lead to improved therapeutics for MS.

The influenza virus is a common pathogen of humans with the ability to cause death in the young and immunocompromised. Even though influenza continues to be a great threat to human life, much still needs to be learned about immunity to the virus and how immunity is maintained. To date most studies examining cell responses to influenza have examined peripheral blood and have shown that humans expressing HLA-A2 have a focused T cell response with the majority of T cells using Vbeta17 and recognize residues 58-66 of the influenza protein MI. However, this response is very complex as shown by the number large number of sequences that comprise the CDR3. As a general rule immune responses become more focused and less complex upon repeated antigen exposure. Thus in the lung, at the site of influenza infection, we would expect the influenza-specific T cell repertoire to be more focused and less complex. This hypothesis will be tested in this proposal in three specific aims. In aim 1 we will compare the influenza-specific T cell repertoire in the blood and lung of normal controls and influenza-infected individuals to examine whether the memory T cells in the lung express TCR sequences that are similar or less complex than those in the blood. In the second aim, because certain experiments cannot be performed on humans, we will develop an animal model to study the generation and maintenance of T cell repertoires upon repeated exposure to antigen. In the third aim, we will expand our studies beyond alpha/beta T cells and characterize gamma/delta T cells that reside in the lung in normal and influenza-infected individuals. Gamma/delta T cells are of interest because they are known to regulate alpha/beta T cell responses and can bridge the innate with the adaptive immune response. Little is known about the role of gamma/delta T cells in influenza immunity. These collective studies are designed to further our knowledge of human immunology, by acquiring information on influenza immunity in the lung, the tissue most affected by influenza infection.

DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is a devastating human autoimmune disease that primarily affects females in the third decade of life resulting in a variety of neurological symptoms including problems with motor function, vision, sphincter control and memory. MS is associated with inflammatory lesions in the central nervous system (CNS) that contain large numbers of cells from the immune system that are thought to contribute to the demyelination and axonal loss associated with the disease. Control of the CNS inflammation is critical to the control of MS symptoms and pathogenesis as it is the inflammatory response that is thought to directly cause the neurological symptoms and damage. Thus it is essential to understand how inflammation is controlled in the CNS if new targeted therapies are to be developed for MS treatment. In our studies using an animal model of MS, experimental autoimmune encephalomyelitis (EAE), we have observed that the B lymphocyte, or B cell, is required for recovery from disease symptoms. We have also observed that B cell expression of the co-stimulatory molecules B7 and CD40, and their production of IL-10 are required for recovery. We have also found that B7 expression is required for the timely appearance of Foxp3 T regulatory (Treg) cells and IL-10 in the CNS during EAE. These data demonstrate that B cells are required for the resolution of CNS inflammation. This proposal will investigate these B cell regulatory mechanisms by testing the following hypothesis: CD40-stimulated B cells activate a population of Treg cells via B7/CD28 and IL-10 in the periphery that in turn enter the CNS and suppress inflammation by an IL-10-dependent mechanism. This hypothesis will be tested by three aims: 1) To Identify the IL-10 producing B cell subset(s) required for EAE resolution;2) To identify the B cell-derived immunological signals that promote their production of IL-10 and the presence of IL-10 and Treg in the CNS;and 3) To Identify the T cell subset(s) that interact with the regulatory B cells. We will be identifying the IL-10-producing B cell subset;the regulatory mechanisms induced by B cell derived B7, CD40 and IL-10;the required T cell populations and the IL-10 producing cell in the CNS. Specifically, these studies are designed to determine how B cells either directly or indirectly through interactions with T cells, drive the resolution of inflammation in the CNS. Since little is known about how B cells regulate inflammation, these studies are likely to have an impact on multiple human inflammatory diseases, in addition to MS. It is our hope that the insight gained into how inflammation is regulated in the CNS will aid in the development of therapies for MS and other diseases that are inflammatory in nature.Public Health Relevance Statement In the central nervous system of multiple sclerosis patients the immune system facilitates the development of inflammatory lesions that result in permanent tissue damage resulting in disability if not resolved. B cells have been shown to regulate this autoimmune inflammation in the animal model of multiple sclerosis. This proposal will investigate the cellular mechanisms utilized by regulatory B cells in the resolution of central nervous system inflammation resulting in recovery from disease.

DESCRIPTION (provided by applicant): Multiple sclerosis (MS) a common disease of the central nervous system (CNS) with a direct link to immune system function. MS is characterized as an autoimmune disease, in which the immune system mounts an inappropriate immune response against the CNS. Specifically, self-reactive T lymphocytes are thought to drive disease pathogenesis by inducing an inflammatory response that results in plaque formation characterized by accumulations of immune cells in areas of demyelination. In addition to plaque formation, it has become clear that MS clinical symptoms also result from damage to neurons. Axonal loss has also been reported. A variety of immune-mediated mechanisms have been indicated in neuronal damage, but none have been specifically confirmed in MS. We have recently discovered that self-reactive T cells produce a secreted protein that induces neuronal injury by destabilization of microtubule -tubulin in axons. Axonal function and survival is dependent upon fast axonal transport of neurotransmitters and growth factors in the axons along microtubules. Thus the destablization of microtubules puts the neuron at risk for death. We have named this soluble factor Microtubule Axonal Destablizer or MAD. Using an experimental autoimmune encephalomyelitis (EAE) acute mouse model of MS, we have been able to demonstrate axonal dysfunction consistent with microtubule destablization in the CNS. This neuronal dysfunction was observed early in disease and was reversed upon recovery from EAE clinical disease. In order to further study the mechanisms of how MAD induces neuronal dysfunction, we need to purify and identify the MAD protein. This knowledge is required in order to determine if MAD or its signaling pathways can be targeted as a therapy for MS and other neurodegenerative diseases. Since MAD is an immune-mediated protein that directly affects neuronal function, its identification will facilitate our understanding of functional links between the immune and nervous systems. The hypothesis driving the current proposal is: Activated lymphocytes responding to CNS inflammation secrete a protein (MAD) that directly destabilizes axonal -tubulin causing a transport defect that directly results in MS clinical symptoms. The overall goal of this project is to determine whether MAD is a potential therapeutic target for MS or and other neurodegenerative disease. However, to ultimately achieve this goal, we must purify and identify MAD. This is the single aim of this application. PUBLIC HEALTH RELEVANCE: Many diseases of the nervous system, including multiple sclerosis, are often associated with damage to neurons resulting in disability. We have uncovered a novel mechanism whereby cells of the immune system directly mediate neuronal dysfunction. The goal of this proposal is to purify and identify the protein responsible for the neuronal damage such that it can be investigated as a new therapeutic target for the treatment of neurodegenerative diseases in subsequent studies.

DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is a lifelong debilitating illness with no cures and limited treatment options. Thus new treatments are greatly needed. Using the animal model of MS, experimental autoimmune encephalomyelitis (EAE), we were the first to demonstrate that B cells were required for recovery from disease. These data revealed the potential of harnessing regulatory B cells or Breg for cellular based therapies. However, one major hurdle before this can be accomplished is a better understanding of how Breg control the extent of autoimmune inflammation. We have recently discovered that Breg control both the onset and severity of EAE by the homeostatic maintenance of CD4+Foxp3+ T regulatory cell (Treg) numbers. We found that when B cells were either genetically deficient (?MT) or depleted with anti-CD20 there was a significant reduction in the absolute number of Treg in mice. As for ?MT mice, B cell depletion with anti-CD20 abrogated EAE recovery. Reconstitution of ?MT with WT splenic B cells resulted in the homeostatic expansion of Treg and the subsequent recovery from EAE. These cumulative data solidify a role for Breg in controlling EAE via the maintenance of Treg cell numbers. These data are consistent with the known role of Treg in keeping autoimmune responses in check. When we investigated the mechanism whereby Breg regulate Treg numbers, we discovered that their expression of GITRL and SAP contributed to their ability to promote Treg proliferation and/or promote EAE recovery. The known role for SAP in promoting T cell:B cell interactions in adaptive immune responses suggests that Breg and Treg also directly interact. However, virtually nothing is known regarding the mechanics and biological outcomes of such interactions. When we investigated the phenotype of Breg with the capability of controlling Treg homeostasis we found that a novel population of follicular cells wit IgDlow-neg expression exhibited Breg activity. This population of follicular B cells has never been described indicating that we discovered a novel Breg phenotype. From the above cumulative data, we hypothesize that follicular IgD low-neg Breg control autoimmunity by promoting Treg homeostatic expansion via direct cell-cell interactions facilitated by SAP expression in B cells allowing GITRL:GITR binding promoting Treg proliferation. The testing of this hypothesis will allow us to understand the molecular mechanisms whereby Breg control autoimmunity with the goal of harnessing this knowledge for the development of new therapies. This hypothesis and goal will be achieved in three specific aims: Aim 1: Determine the role of GITRL and SAP in the functional ability of Breg to control Treg homeostasis; Aim 2: Determine the mechanism whereby SAP facilitates Breg:Treg interactions and Aim 3. Characterization and functional analysis of a novel IgDlow-neg follicular Breg.

? DESCRIPTION (provided by applicant): Inflammation that accompanies chronic disease if uncontrolled leads to severe tissue damage and even death. B cells have emerged as an important attenuator of inflammation in a variety of diseases including autoimmunity and hypersensitivities. We were the first to show that B cells attenuated disease severity in autoimmunity. This was accomplished using the mouse model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE), where we demonstrated that mice with a deficiency in B cells due to genetic ablation (µMT) were unable to undergo spontaneous recovery. Shortly thereafter, the concept of regulatory B cells or Breg emerged. Subsequently, we discovered a regulatory loop between Breg and CD4+Foxp3+ T regulatory (Treg) cells whereby B cells promote the proliferation of Treg, which are essential for controlling the severity of inflammatory diseases. Of importance, we observed similar results in animal models of contact hypersensitivity and inflammatory bowel disease demonstrating therapeutic potential for Breg for the treatment of chronic inflammatory diseases. In subsequent experiments, we identified a unique Breg phenotype that we will use to uncover the mechanisms whereby Breg impact Treg proliferation, activation and function in inflammatory disease models. In addition, we will determine Breg localization and antigen experience. Finally, we will test the hypothesis that efficacy of B cell depletion by rituximab in MS is due to its inability to deplete Breg.

Inflammation that accompanies chronic disease, if uncontrolled, leads to severe tissue damage resulting in debilitation and even death. It is well established that CD4+Foxp3+ T regulatory cells (Treg) play an essential role in the suppression of autoimmune and inflammatory responses. In addition to Treg, B cells have also emerged as an important attenuator of inflammation in a variety of diseases, including autoimmunity and hypersensitivities. We were the first to show that B cells attenuate disease severity in autoimmunity using the mouse model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). Shortly thereafter the concept of regulatory B cells or Breg emerged. Even though B cells have been shown to regulate inflammation via IL-10 production, they cannot be identified by a definitive cell surface phenotype. In contrast, in our studies investigating IL-10-independent Breg mechanisms, we have successfully identified a cell surface phenotype that we have used to track and purify Breg. Of significance to the translation of our findings is that human Breg promote Treg proliferation. This is the first example of a definitive cell surface phenotype that can be used to identify and purify a specific population of Breg in both mouse and humans. To emphasize their unique function, we termed them B Treg helper cells (BTrh). The goal of this application is to gain a better understanding of the development and function of BTrh and will test the hypothesis that BTrh via their induction of Treg proliferation will have clinical relevance in the attenuation of inflammatory diseases. This hypothesis will be tested in two specific aims: Aim 1. Gain a better understanding of unique BTrh characteristics by studying their developmental status, functional requirements and localization with Treg and Aim 2. Determine the therapeutic potential of GITRL+ BTrh and to test the hypothesis that efficacy of B cell depletion by rituximab in MS is due to its inability to deplete BTrh. Together, the proposed studies represent a tightly focused coordinated series of investigations aimed at broadening the importance of BTrh in a number of clinically important inflammatory disorders in humans.