Immune Evasion by Mycobacterium Tuberculosis: Mannose
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IMMUNE EVASION BY MYCOBACTERIUM TUBERCULOSIS: MANNOSE- CAPPED LIPOARABINOMANNAN INDUCES GRAIL AND CD4+ T CELL ANERGY by OBONDO JAMES SANDE Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Advisor: Dr. W. Henry Boom Department of Pathology: Immunology Training Program CASE WESTERN RESERVE UNIVERSITY May 2016 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Obondo James Sande, candidate for the Ph.D. degree* (Signed) Alan D. Levine (Chair of the committee) Clive Hamlin Clifford V. Harding Roxana E. Rojas W. Henry Boom (Date) 18 January 2016 *We also certify that written approval has been obtained for any proprietary material contained therein. ii Dedication I dedicate this work to my parents Pantaleo O. Wantono (RIP) and Loyce Nandera and to my wife, Hellen Beatrice Anyait. They have provided nothing but support before and throughout my doctoral training. iii Acknowledgements I would first and foremost like to thank Henry, Roxana and Cliff. Their tireless guidance has not only shaped my skills in the laboratory but they have taught me how to effectively communicate and share my ideas, both in text and through regular presentations, which I have come to understand is instrumental in becoming a successful scientist. I would also like to thank all the members of the Boom/Rojas laboratory (Scott Reba, Qing Li, Xuedong Ding, Ahmad Faisal Karim, Sophia Onwuzulike and Keith Chervenak), all have made my graduate student career enjoyable and as fruitful as possible. I would also like to thank all the members of the Harding laboratory (Jaffre Athman, Nancy Nagy, Sukula Supriya, Claire Mazahery and Pamela Wearsch) for their help along the way. Last but not least I would like to thank Robert N. Mahon for initiating this work. Lastly I would like to thank Grace M. Svilar and Marla Manning, the administrators of the Fogarty Scholarship for their kind guidance. I am grateful for your timely assistance throughout my graduate student studies. iv Table of contents Page Dedication iii Acknowledgements iv Table of Contents v List of Figures viii List of Abbreviations x Abstract xi Chapter 1: Introduction Tuberculosis overview 2 Mycobacteria 3 The course of M. tuberculosis infection 4 M. tuberculosis evasion of CD4+ T cells 6 M. tuberculosis cell wall glycolipids 9 Trafficking of M. tuberculosis molecules outside infected cells 11 Mannose-capped Lipoarabinomannan (LAM) and immune regulation 12 LAM and T cell receptor signaling and regulation of T cell activation 14 T cell anergy 17 Models of T cell anergy induction 17 Mechanisms of anergy induction 18 GRAIL (Gene Related to Anergy in Lymphocytes) 19 Other factors that confound anergy 21 Pathogens and anergy induction 22 Reversal of T cell anergy 22 v Chapter 2: Mycobacterium tuberculosis ManLAM inhibits T-cell-receptor signaling by interference with Zap70, Lck and LAT phosphorylation Abstract 24 Introduction 25 Materials and Methods 27 Results 33 Discussion 45 Chapter 3: Mannose-Capped Lipoarabinomannan from Mycobacterium tuberculosis Induces CD4+ T cell Anergy via GRAIL Abstract 50 Introduction 51 Materials and Methods 54 Results 63 Discussion 86 Chapter 4: Discussion and future direction LAM trafficks outside M.tuberculosis-infected macrophages, inhibits T cells 93 LAM and inhibition of TCR signaling 94 A Model of anergy induction by LAM 101 Role and induction of GRAIL by LAM 107 Reversal of LAM-induced anergy by IL-2 111 GRAIL and anergy induction by other pathogens 114 Conclusion and future directions 115 vi Appendix 1: Correlation of figures with experiments 122 Appendix 2: List of Publications and abstracts 123 Works cited 125 vii List of Figures Figure Page Figure 1.1 Potential outcomes of exposure to M. tuberculosis 7 Figure 1.2 Mycobacterial cell envelope 10 Figure 1.3 Proposed structures of LAM, LM, PIM 11 Figure 2.1 ManLAM inhibits antigen-specific CD4+ T cell activation 34 Figure 2.2 ManLAM inhibits the activation of human T cells 36 Figure 2.3 ManLAM inhibits Lck and LAT phosphorylation 38 Figure 2.4 ManLAM does not activate the cAMP/PKA pathway or inhibit Lck-Tyr505 pphosphorylation 40 Figure 2.5 Inhibition of TCR signaling by ManLAM is temperature sensitive 42 Figure 2.6 ManLAM is found in T cell membranes and does not affect localization of Lck in lipid rafts of activated Tcells 44 Figure 3.1 Viability and function of CD4+ T cells with IL-7 treatment 65 Figure 3.2 Presence of LAM on CD4+ T cell membranes is required for inhibition of CD4+ T cell activation after primary stimulation 66 viii Figure 3.3 LAM induces anergy in P25 CD4+ T cells 68 Figure 3.4 LAM-induced P25 CD4+ T cell anergy occurs over a range of Ag85B peptide and LAM concentrations 70 Figure 3.5 LAM does not induce FoxP3-positive regulatory T cells and/or increase activation-Induced cell death (apoptosis) 72 Figure 3.6 LAM-induced CD4+ T cell anergy is not due to cell death 73 Figure 3.7 LAM does not affect receptor expression on LAM-anergized P25 TCR-Tg CD4+ T cells 76 Figure 3.8 LAM induces increased GRAIL protein expression both during priming and upon re-stimulation 78 Figure 3.9 Knockdown of GRAIL expression by siRNA prevents inhibition of CD4+ T cell activation by LAM 81 Figure 3.10 Exogenous IL-2 down-regulates GRAIL expression and restores T cell proliferation in LAM-anergized CD4+ T cells 83 Figure 3.11 LAM associates with lipid rafts and CD3 on human CD4+ T cells, and up-regulates GRAIL upon activation with anti-CD3/CD28 85 Figure 4.1 Model of anergy induction by LAM 102 ix List of Abbreviations Ag85B, 85B antigen from M. tuberculosis APC, antigen presenting cell BCG, Bacille Calmette Guerin BMM, bone marrow derived macrophages Cbl-b, Castas b-lineage lymphoma proto-oncogene b Csk, C-terminal Src kinase CTLA-4, cytotoxic T lymphocyte antigen-4 Erk, extracellular regulated kinase FITC, fluorescein isothiocyanate Foxp3, forkhead box P3-expressing GRAIL, gene related to anergy in lymphocytes ITAM, Immunoreceptor tyrosine-base activation motif Lag-3, lymphocyte activation gene 3 LAM, Lipoarabinomannan LAT, linker for the activation of T cells Lck, lymphocyte-specific protein tyrosine kinase LM, lipomannan MAPK, mitogen activated protein kinase MHC, Major Histocompatibility Complex PBMC, peripheral blood mononuclear cell PD-1, programmed death-1 PIM, phosphatidylinositol mannosides SLP-76, SH2 domain-containing leuckocyte phosphoprotein of 76 kDa Tim-3, T cell immunoglobulin and mucin domain-containing protein 3 ZAP-70, Zeta-associated protein of 70 kDa x Immune Evasion by Mycobacterium tuberculosis: Mannose-Capped Lipoarabinomannan Induces GRAIL and CD4+ T cell anergy by OBONDO JAMES SANDE Abstract Mycobacterium tuberculosis (Mtb) persists and survives in the host in the face of many T cells subsets recognizing a range of Mtb antigens due to its ability to evade innate and adaptive immune responses. CD4+ T cells and infected antigen presenting cells (APC) are central for control of Mtb but also targets of its immune evasion strategies. The major Mtb cell wall glycolipid, mannose-capped lipoarabinomannan (LAM) is one of the molecules involved in immune evasion. Previous studies determined that LAM can inhibit phagosome maturation and antigen processing in macrophages and thus indirectly affect memory and effector CD4+ T cell function. LAM is trafficked from Mtb-infected macrophages via bacterial vesicles into the microenvironment of the infected site, where it can bind to uninfected APC and T cells. We proposed that this provides a mechanism for direct delivery of LAM to surrounding T cells, thereby further regulating their function. Earlier studies determined that LAM directly inhibits polyclonal murine CD4+ T cell activation by blocking ZAP-70 phosphorylation. In the first part of this thesis we extended our observation of direct inhibition of T cell activation by LAM in two directions. First we determined if LAM inhibition of murine primary CD4+ T cells could be extended to antigen-specific CD4+ T cell xi activation by antigen presenting cells and whether human CD4+ T cells were similarly inhibited. Second, we determined the mechanism of LAM-mediated inhibition of TCR signaling in terms of its effect on Lck and LAT phosphorylation and lipid raft integrity. We found that LAM inhibited antigen-specific murine CD4+ T cells and primary human T cells as well. In addition to ZAP-70, LAM inhibited phosphorylation of Lck and LAT. Inhibition of proximal TCR signaling was temperature sensitive, suggesting that LAM insertion into T cell membranes was required. We established that direct interaction of LAM with T cells inhibits antigen-specific CD4+ T cell activation by interfering with very early events in TCR signaling through LAM’s insertion in T cell membranes. Previous studies show that inhibition of proximal TCR signaling is associated with induction of T cell anergy. In the second focus of this thesis, we tested if LAM-induced inhibition of CD4+ T cell activation results in T cell anergy. We found that LAM induces anergy in P25 TCR transgenic CD4+ T cells (specific for P25 peptide of the 38 kDa antigen 85B of Mtb). Anergy induction required the presence of LAM in the T cell membrane during primary T cell activation. Once anergy was induced, LAM was no longer required in the T cell membrane, as removal of LAM did not affect the T cell anergic state. The induction of anergy was due to up-regulation of GRAIL (Gene Related to Anergy In Lymphocytes) protein in both murine and human CD4+ T cells. We further determined that exogenous IL-2 reversed LAM-induced anergy by downregulating GRAIL expression. We propose that LAM inhibits CD4+ T cell activation and up-regulates GRAIL expression to induce anergy in Mtb-reactive CD4+ T cells.