A Dissertation entitled Discovery of a New Dendritic Cell Subset Derived from Immature Granulocytes By Shuo Geng Submitted to the Graduate Fculty as partial fulfillment of the requirement for the Doctor of Philosophy in Biomedical Sciences _______________________________________ Dr. Akira Takashima, Committee Chair _______________________________________ Dr. Kevin Pan, Committee Member _______________________________________ Dr. Stanislaw Stepkowski, Committee Member _______________________________________ Dr. Anthony Quinn, Committee Member _______________________________________ Dr. Randall Worth, Committee Member _______________________________________ Dr. Patricia R. Komuniecki, Dean College of Graduate Studies The University of Toledo June 2011 Copyright 2011, Shuo Geng This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Discovery of a New Dendritic Cell Subset Derived from Immature Granulocytes By Shuo Geng Submitted to the Graduate Fculty as partial fulfillment of the requirement for the Doctor of Philosophy in Biomedical Sciences Compared to other leukocytes, dendritic cells (DCs) are extremely heterogeneous in terms of developmental pathways and immunological properties. More than 10 DC subsets have been identified in mouse, which are distinguishable from each other by surface phenotypes, functions, tissue distributions, and developmental origins. Here we describe a novel subset of DCs that are derived from immature granulocytes, known as band cells. Large numbers of band cells are rapidly recruited to inflammatory sites, where some of them differentiate into this DC subset termed “grDCs.” In addition to showing a typical DC-like morphology, grDCs express many DC markers (MHC class II, CD11c, and CD205) and efficiently present peptide antigens to both CD8 and CD4 T cells. Importantly, grDCs retain the surface expression of Ly6G, which is not detectable on any of the currently known DC subsets, as well as several unique features of granulocytes. GeneChip analyses have revealed a cluster of genes that are selectively expressed by Ly6G+ grDCs, but not Ly6G- conventional DCs – this cluster includes cathelicidin (CRAMP), MMP9 and CD62L. CRAMP-deficient grDC exhibited a diminished bacterial killing activity, indicating a functional contribution of grDC-associated CRAMP. In a peritoneal E. coli infection model, grDC were found to internalize bacteria efficiently and present bacterial antigens to CD4 T cells. Not only have our data unveiled a previously iii unrecognized DC subset and its functions, they also suggest a new concept that granulocytes can directly participate in the induction of adaptive immunity via differentiation into grDCs. iv DEDICATION This work is dedicated to my parents, Wu-qun Geng and You-jie Zhu, as well as my grandparents, Ling-guang Zhu and Jie li, for their endless love. They always have faith in me and encourage me to reach my dreams. This work is also dedicated to my beloved wife, Ran Lu, for her support, understanding and patience. She has been my wonderful cheerleader and makes my life delightful. v ACKNOWLEDGEMENT I would like to thank my major advisor, Dr Akira Takashima, for giving me the opportunity to work on this exciting project. I appreciate his expert training, sagacious guidance, and sustained encouragement during my doctoral education. His enthusiasm in scientific research has always inspired me. He leads me into the field of immunology and I have benefited a lot from him. I would like to thank me committee members: Dr. Kevin Pan, Dr. Stanislaw Stepkowski, Dr. Anthony Quinn and Dr. Randall Worth for their valuable time and constructive suggestions and criticisms during my PhD study. I am grateful to Dr. Robert Blumenthal and Dr. Kristen Williams for providing the E. coli strains used in this study and teaching me how to perform bacterial assays. I thank Dr. William Gunning III for helping us take the EM images. I thank Dr. Richard Gallo for providing anti-CRAMP antibody and BM cells from CRAMP KO mice. I thank Sean Linkes for his service of FACS analysis and sorting. I thank all the current and former members in Takashima’s lab: Dr. Hironori Matsushima, Dr. Ram Veerapaneni, Dr. Claudio Cortes, Dr. Masaaki Miyazawa, Dr. Nobuyasu Mayuzumi, Dr. Taehyung Lee, Ran Lu, Yi Yao, David Leggat, Benjamin Chojnacki, Rachel Mohr, Alan Rupp and Colleen Krout. It is a great pleasure to work with them. vi I would like to thank all the administrative personnel of the Department of Medical Microbiology and Immunology: Diane Ammons, Tamra Chamberlin, Traci McDaniel and Suzanne Payne, who are always ready to help and solve problems. I acknowledge all the faculty, staff and students in the Department of Medical Microbiology and Immunology and the Track of Infection, Immunity and transplantation. They create a very professional and friendly environment. vii Contents ABSTRACT…………………………………………………………….......iii DEDICATION……………………………………………………...……….v ACKNOWLEDGEMENT…………………………….…….……..…....….vi CONTENTS……………………................……………….…………....…viii LIST OF ABBREVIATIONS…………………………………………...….ix INTRODUCTION…………………………………………………………...1 LITERATURE REVIEW……………………………………………………5 MATERIALS AND METHODS…………………………………………..34 RESULTS…………………………………………………………………..46 DISCUSSION…..………………………………………………………….79 SUMMARY………………………………………………………………..90 REFERENCES……………………………………………………………..91 viii LIST OF ABBREVIATIONS APC antigen presenting cell BM bone marrow BMDC bone marrow-derived dendritic cell cDC conventional dendritic cell CDP common Dendritic cell precursor CFU colony forming unit CHS contact hypersensitivity CLP common lymphoid precursor CMP common myeloid precursor CRAMP cathelicidin CTL cytotoxic T lymphocyte DC dendritic cell DT diphtheria toxin EM electron microscopy Ep-CAM epithelial cell adhesion molecule ER endoplasmic reticulum FDC follicular cendritic cell Flt3 fms-related tyrosin kinase 3 GC germinal center GCDC germinal center dendritic cell G-CSF granulocyte colony-stimulating factor GM-CSF granulocyte macrophage colony-stimulating factor grDC granulocyte-derived dendritic cell IFN interferon IKDC interferon-producing killer dendritic cell i.p. intraperitoneal i.v. intravenous KO knock out LC Langerhans cell LN lymph node M-CSF macrophage colony-stimulating factor MFI mean fluorescence intensity MHC major histocompatibility complex MMR macrophage mannose receptor NK natural killer moDC monocyte-derived dendritic cell OVA ovalbumin OX oxazolone pDC plasmacytoid dendritic cell ix PEC peritoneal exudate cell PMNp precursor of polymorphonuclear granulocyte RA rheumatoid arthritis ROS reactive oxygen species SP spleen SR scavenger receptor TG transgenic Tip-DC TNF-α/iNOS producing dendritic cell TLR toll like receptor UV ultraviolet WT wild type x INTRODUCTION Dendritic cells (DCs) are specialized antigen presenting cells (APCs), which initiate and modulate the immune response as well as maintain the immune tolerance to self-antigens. Although Langerhans cells (LCs), a DC subset, were observed as early as 1868, the characterization of DC only began about 25 years ago (Banchereau & Steinman, 1998; Paul, 2007). Immature DCs, abundant in the body surface, have a potent capacity to uptake antigens and subsequently migrate to lymphoid tissues, where they mature. Then the processed antigen peptides will be loaded onto major histocompatibility complex (MHC) molecules and presented to CD4 or CD8 T cells, resulting in a dramatic expansion of T cells. Activated CD8 cytotoxic T cells migrate back to the inflamed sites to eliminate the infected cells, while CD4 helper T cells secret cytokines to facilitate the activation of macrophages, natural killer (NK) cells and eosinophils or the antibody production of B cells. B cells also can be activated by direct contact with DCs, and produce neutralizing antibodies to the initial pathogen (Banchereau et al., 2000). Compared to other bone marrow (BM) derived leukocytes, DC represent a highly heterogeneous population, since various DC subsets have been reported in the literature. Generally speaking, DC can be divided into two families. One is conventional DC (cDC), which has a dendritic morphology and potent antigen processing and presentation capacity. The other one is plasmacytoid DC (pDC), which is a round, non-dendritic shape, and relatively long-lived cell (Facchetti & Vergoni, 2000; Liu 2005). There are at least 1 seven cDC subsets identified in mouse. Recently, a novel DC subset, interferon- producing killer DC (IKDC), was identified from mouse spleen (Bonmort et al., 2008). Although they share many common features, different DC subsets have been demonstrated to have distinct phenotype, tissue distribution and biological functions. The diversity of DCs permits the immune system to mount various types of responses to different pathogens and stimulus (Shortman & Liu, 2002; Merad & Manz, 2009). Most of these DC subsets have a short lifespan, based on the evidence that only less than 5% of DCs are dividing and the half-life is 1.5-7 days in the steady state. DCs must be renewed continually, perhaps by the cells originating from hematopoietic stem cells or BM or some tissue resident precursor cells. The differentiation of DC is crucial not only to the establishment of DC network, but also to the dynamic response against pathogens, self-antigens or tumor cells. The research on DC differentiation and identification of DC precursors is also important to DC-based immunotherapy, which