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Cxcr5 Expressing T Helper Cells Mediate Protective Immunity Against Tuberculosis

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Cxcr5 Expressing T Helper Cells Mediate Protective Immunity Against Tuberculosis CXCR5 EXPRESSING T HELPER CELLS MEDIATE PROTECTIVE IMMUNITY AGAINST TUBERCULOSIS by Samantha R. Slight B.S. Microbiology, Brigham Young University, 2008 Submitted to the Graduate Faculty of School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2013 UNIVERSITY OF PITTSBURGH SCHOOL OF MEDICINE This dissertation was presented by Samantha R. Slight It was defended on April 2, 2013 and approved by Tim D. Oury, M.D. Ph.D., Professor of Pathology Jon D. Piganelli, Ph.D., Associate Professor of Pediatrics Doug Reed, Ph.D., Assistant Professor of Immunology Todd A. Reinhart, ScD, Professor of Infectious Diseases and Microbiology Anuradha Ray, Ph.D., Professor of Medicine and Immunology Dissertation Advisor: Shabaana A. Khader, Ph.D., Assistant Professor of Pediatrics ii CXCR5 EXPRESSING T HELPER CELLS MEDIATE PROTECTIVE IMMUNITY AGAINST TUBERCULOSIS Samantha R. Slight, Ph.D. University of Pittsburgh, 2013 COPYRIGHT Copyright permission was granted for the use of parts from: 1. Slight SR, Rangel-Moreno J, Gopal R, Lin Y, Fallert Junecko BA, Mehra S, Selman M, Becerril-Villanueva E, Baquera-Heredia J, Pavon L, Kaushal D, Reinhart TA, Randall TD, and Khader SA. (2013). CXCR5+ T helper cells mediate protective immunity against tuberculosis. J Clin Invest. 123 (2): 712-726. 2. Slight SR and Khader SA. (2012). Chemokines shape the immune response to tuberculosis. Cytokine Crowth Factor Rev. 24 (2): 105-113. Letters are on file with Samantha R. Slight Documentation of permission letters are on file with Samantha R. Slight. iii CXCR5 EXPRESSING T HELPER CELLS MEDIATE PROTECTIVE IMMUNITY AGAINST TUBERCULOSIS Samantha R. Slight, Ph.D. University of Pittsburgh, 2013 One third of the world’s population is infected with Mycobacterium tuberculosis (Mtb). Although most infected people remain asymptomatic, they have a 10% lifetime risk of developing active tuberculosis (TB). Thus, the current challenge is to identify immune parameters that distinguish individuals with latent TB from those with active TB. Using human and experimental models of Mtb infection, we show that organized ectopic lymphoid structures containing CXCR5+ T cells are found in Mtb-infected lungs. In addition, we show that in experimental Mtb infection models, the presence of CXCR5+ T cells inside ectopic lymphoid structures are associated with immune control. Furthermore, in a mouse model of Mtb infection, we show that activated CD4+ CXCR5+ T cells accumulate in Mtb-infected lungs, and produce proinflammatory cytokines. Absence of CXCR5 in mice results in increased susceptibility to TB due to defective T cell localization within the lung parenchyma. We show that CXCR5 expression on T cells mediates correct T cell localization within TB granulomas, efficient macrophage activation, promoting protection against Mtb infection and facilitating lymphoid follicle formation. These data show a novel role for CD4+ CXCR5+ T cells in protective immunity against TB and highlight their potential use for future TB vaccine design and therapy. iv TABLE OF CONTENTS COPYRIGHT .............................................................................................................................. III ACKNOWLEDGEMENTS .................................................................................................... XIII ABBREVIATIONS .................................................................................................................. XVI 1.0 INTRODUCTION ........................................................................................................ 1 1.1 MYCOBACTERIUM TUBERCULOSIS ............................................................. 1 1.1.1 Disease Outcomes ............................................................................................ 1 1.1.2 The TB Granuloma ......................................................................................... 3 1.1.3 Animal Models ................................................................................................. 5 1.2 MTB HOST DEFENSE ....................................................................................... 7 1.2.1 Initiation of Infection....................................................................................... 7 1.2.2 CD4+ T Helper Cells ........................................................................................ 9 1.2.3 CD8+ T cells .................................................................................................... 11 1.2.4 B cells .............................................................................................................. 12 1.3 CYTOKINES IN MTB INFECTION .............................................................. 12 1.3.1 Interleukin-12................................................................................................. 13 1.3.2 Interferon- .................................................................................................... 14 1.3.3 Tumor Necrosis Factor- .............................................................................. 15 1.3.4 Nitric Oxide .................................................................................................... 16 v 1.4 CHEMOKINES IN MTB INFECTION .......................................................... 18 1.4.1 Inflammatory Chemokines in Mtb Infection .............................................. 19 1.4.2 Homeostatic Chemokines in Mtb ................................................................. 21 1.5 ECTOPIC LYMPHOID FOLLICLES ............................................................ 23 1.5.1 Chemokine and Cytokine Induced Formation ........................................... 24 1.5.2 ELOs in Autoimmune Inflammation ........................................................... 26 1.5.3 ELOs in Infectious Disease ........................................................................... 27 2.0 ECTOPIC LYMPHOID FOLLICLES IN M. TUBERCULOSIS INFECTION .. 31 2.1 ABSTRACT........................................................................................................ 31 2.2 INTRODUCTION ............................................................................................. 32 2.3 MATERIALS AND METHODS ...................................................................... 33 2.3.1 Human Tissue Samples and Patient Diagnosis ........................................... 33 2.3.2 Mtb Infection in NHPs .................................................................................. 34 2.3.3 Mtb Infection in Mice .................................................................................... 35 2.3.4 Morphometric Analysis and Immunofluoresence ...................................... 35 2.3.5 CXCL13 In Situ Hybridization .................................................................... 37 2.3.6 Statistics .......................................................................................................... 38 2.4 RESULTS ........................................................................................................... 38 2.4.1 CXCR5+ T cells accumulate within ectopic lymphoid structures of human TB granulomas ........................................................................................................... 38 2.4.2 Ectopic lymphoid structures are associated with immune control during TB in the NHP model of Mtb infection .................................................................... 39 vi 2.4.3 CXCR5+ T cells localize within ectopic lymphoid follicles in murine TB granulomas ................................................................................................................. 42 2.5 DISCUSSION ..................................................................................................... 43 2.6 ACKNOWLEDGEMENTS .............................................................................. 46 3.0 MECHANISMS UNDERLYING ECTOPIC LYMPHOID FOLLICLE FORMATION AND PROTECTION IN M. TUBERCULOSIS INFECTION ...................... 48 3.1 ABSTRACT........................................................................................................ 48 3.2 INTRODUCTION ............................................................................................. 49 3.3 MATERIALS AND METHODS ...................................................................... 51 3.3.1 Mtb Infection in Mice .................................................................................... 51 3.3.2 Mtb Infection In NHPs .................................................................................. 52 3.3.3 Lung and Spleen Single Cell Preparation ................................................... 52 3.3.4 Flow Cytometry ............................................................................................. 53 3.3.5 CXCL13 In Situ Hybridization .................................................................... 53 3.3.6 Morphometric Analysis and Immunofluorescence .................................... 53 3.3.7 Real-Time PCR .............................................................................................. 54 3.3.8 Detection of IFN--Producing Cells by ELISPOT Assay ........................... 54 3.3.9 Protein Estimation by ELISA ....................................................................... 55 3.3.10 Adoptive Transfer of CD4+ Cells ................................................................ 55 3.3.11 CXCL13 Chemotaxis Assay ........................................................................ 55 3.3.12
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