CCL3 AUGMENTS ANTITUMOR RESPONSES IN CT26 BY ENHANCING CELLULAR TRAFFICKING AND INTERFERON-GAMMA EXPRESSION by FREDERICK ALLEN Submitted in Partial Fulfilment of the Requirements for the Degree of Doctor of Philosophy Dissertation Advisor: Alex Y. Huang, M.D., Ph.D. Department of Pathology Immunology Training Program CASE WESTERN RESERVE UNIVERSITY January 2018 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Frederick Allen Candidate for the Ph.D. degree Committee Chair Pamela Wearsch, Ph.D. Committee Member Pushpa Pandiyan, Ph.D. Committee Member Clive Hamlin, Ph.D. Committee Member and Advisor Alex Y. Huang, M.D., Ph.D. Date of Thesis Defense November 13, 2017 *We also certify that written approval has been obtained for any proprietary material contained therein. 2 TABLE OF CONTENTS Acknowlegment ………………………………………………………………………………………. 10 List of major abbreviations ............................................................................................... 12 Abstract ............................................................................................................................. 13 Chapter I - Introduction to the major tissue, cellular and cytokine components involved in the CCL3-expressing CT26 tumor microenvironment ..................................................... 15 A brief history of immunotherapy ................................................................................. 16 The tumor microenvironment – from tumor escape to indirect immunomodulation .... 17 Cytokines ....................................................................................................................... 18 Chemokines ................................................................................................................... 19 Macrophage inflammatory protein 1 �, 1�, and rantes ................................................. 20 CCL3 and CCL4 – Overview .................................................................................... 20 CCL3 and CCL4 ........................................................................................................ 21 CCL3 and CCL5 ........................................................................................................ 23 Lymph node .................................................................................................................. 24 Primary and secondary immune organs – general overview ..................................... 24 Chemokine influence on lymph node cellular trafficking dynamics ......................... 25 Interferon-gamma .......................................................................................................... 25 Natural killer cells ......................................................................................................... 26 Dendritic cells ............................................................................................................... 28 Dendritic cells – Overview ........................................................................................ 28 Dendritic cell maturation ........................................................................................... 29 3 Chapter II - Unique Transcompartmental Bridge: Antigen-Presenting Cells Sampling across Endothelial and Mucosal Barriers (Review) .......................................................... 31 Abstract ......................................................................................................................... 32 Introduction ................................................................................................................... 33 Gut mucosa: apc transepithelial extensions in lamina propria and peyer’s patch ......... 34 Information exchange between LN conduits and follicular DCs (FDCs) ..................... 39 LN-resident and migratory DCs interact directly with lymphatic conduits and HEVs 41 CD169+ Macs extend processes from subcapsular sinus into ln follicular zones to assist in activating the adaptive humoral response .................................................................. 42 Local LN DCs extend dendritic processes into lymphatic fluid to initiate early T cell activation ........................................................................................................................ 43 APC extensions in other anatomical compartments: airway lumen and CNS vessels .. 44 Concluding remarks ...................................................................................................... 46 Chapter III - CCL3 enhances antitumor immune priming in the lymph node via IFN� with dependency on NK cells ................................................................................ 53 Abstract ......................................................................................................................... 54 Introduction ................................................................................................................... 55 Materials and methods .................................................................................................. 57 Results ........................................................................................................................... 61 aCCL3 suppresses tumor growth and promotes tumor rejection .............................. 61 aCCL3 augments T cell activation by enhancing leukocyte migration to the TDLN 62 aCCL3 bolsters the intracellular production of IFN�+ cells in the TDLN ................ 64 L3TU enhanced leukocyte accumulation is dependent on CCL3 but not NK cells .. 65 4 NK cells, but not CD103+ CD11c+ DCs are important for driving the production of IFN�-induced chemokines CXCL9 and CXCL10 in the TDLN ............................... 66 Exposure of BMDCs to rCCL3 enhances Ag-specific T cell proliferation ............... 66 Discussion ..................................................................................................................... 67 Chapter IV - CCL3 augments tumor rejection and enhances CD8+ T cell infiltration through NK and CD103+ dendritic cell recruitment via IFN� .......................................... 89 Abstract ......................................................................................................................... 90 Introduction ................................................................................................................... 91 Material and methods .................................................................................................... 93 Results ........................................................................................................................... 97 CCL3 facilitates tumor rejection via thymic-dependent and thymic-independent mechanisms ............................................................................................................... 97 CCL3 enhances CD4+ and CD8+ T cell infiltration to the primary tumor site .......... 98 CCL3 recruits NK cells to promote CD103+ DCs infiltration and support T cell function within the primary tumor ............................................................................. 99 Subcutaneously administered rCCL3 significantly slowed tumor growth in established tumors ................................................................................................... 101 Discussion ................................................................................................................... 102 Chapter V – Conclusion .................................................................................................. 126 The significance of IFN� in promoting early and late proinflammatory responses with L3TU ............................................................................................................................ 130 Dendritic cell maturation in the tumor microenvironment ......................................... 134 5 The interaction of CCL3, interferon-gamma, and CCL5 in the L3TU tumor microenvironement ...................................................................................................... 137 The dual role of IFN� in tumors .............................................................................. 137 Identifying and targeting tumor intrinsic pathways related to IFN� and WNT/�- catenin, to enhance the constitutive expression CCL3 and tumor rejection ............ 139 Thoughts on the therapeutic application of using high dose recombinant ccl3 .............. 142 Final thoughts ................................................................................................................. 144 Bibliography ................................................................................................................... 145 6 LIST OF FIGURES Figure 1 - Different models of TE and antigen uptake by LP APC .................................. 47 Figure 2 - Overview of LN APC positioning for TE sampling ........................................ 49 Figure 3 - Activated DCs reside near HEV after migrating from the periphery ............... 50 Figure 4 - LN DCs and Macs extend processes into the medullary sinus to directly sample lymph-borne antigen ......................................................................................................... 51 Figure 5 - Lung DCs sample airway pathogens via TEs .................................................. 52 Figure 6 - Autologous or recombinant CCL3 slows tumor growth and promotes tumor rejection ............................................................................................................................