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Characterizing the Source of Neutrophil Elastase and Proteinase 3 Cross-Presentation in B-Cell Acute Lymphoblastic Leukemia

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Characterizing the Source of Neutrophil Elastase and Proteinase 3 Cross-Presentation in B-Cell Acute Lymphoblastic Leukemia The Texas Medical Center Library DigitalCommons@TMC The University of Texas MD Anderson Cancer Center UTHealth Graduate School of The University of Texas MD Anderson Cancer Biomedical Sciences Dissertations and Theses Center UTHealth Graduate School of (Open Access) Biomedical Sciences 5-2017 Characterizing the source of neutrophil elastase and proteinase 3 cross-presentation in B-cell acute lymphoblastic leukemia Selena Carmona Follow this and additional works at: https://digitalcommons.library.tmc.edu/utgsbs_dissertations Part of the Medicine and Health Sciences Commons Recommended Citation Carmona, Selena, "Characterizing the source of neutrophil elastase and proteinase 3 cross-presentation in B-cell acute lymphoblastic leukemia" (2017). The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access). 751. https://digitalcommons.library.tmc.edu/utgsbs_dissertations/751 This Thesis (MS) is brought to you for free and open access by the The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences at DigitalCommons@TMC. It has been accepted for inclusion in The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access) by an authorized administrator of DigitalCommons@TMC. For more information, please contact [email protected]. CHARACTERIZING THE SOURCE OF NEUTROPHIL ELASTASE AND PROTEINASE-3 CROSS-PRESENTATION IN B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA A THESIS Presented to the Faculty of The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences In Partial Fulfillment Of the Requirements For the Degree of MASTER OF SCIENCE By Selena Nicole Carmona, B.S. Houston, Texas May 2017 iii Dedication This work is dedicated to my loving husband, Sebastian Carmona, who has always supported me in all my hopes and dreams. iii Acknowledgements I would first like to thank my advisors, Dr. Gheath Al-Atrash and Dr. Jeffrey Molldrem, for their scientific and professional guidance throughout my training in their lab. I can truly say that my growth as a scientist is owed to them. I would also like to thank my supervisory committee, Drs. Dean Lee, Joya Chandra, and Greg Lizee, for their insight and input on the progression of my project. All have been crucial in my development as a scientist in these past few years. I would also like to thank all members of the Al-Atrash, Molldrem, and Mittendorf labs. Not only have they made the lab a great place to work, but the numerous scientific discussions, constant advice and patience were always so helpful. Specifically, Drs. Elizabeth Mittendorf, Lisa St. John, Celine Kerros, Anne Phillips, Mao Zhang, Na Qiao, Haley Peters, Haven Garber, and Anna Seergeva along with Pariya Sukhumalchandra, and Alex Perakis. All have helped greatly in the development of protocols, trouble-shooting, advice and analysis of data. Another intricate part of my work is all members of the South Campus Flow Core Facility including Dr. Karen Clise-Dwyer and her entire team. With flow-cytometry being an important aspect of my project, all members of the Flow Core Facility have helped in the planning of experiments and the analysis of data. My work would not have been possible if not for these three groups of scientists. Lastly, I owe a great amount of gratitude to those that have always supported me throughout my graduate school career. My family, including my mom, Cynthia Zaldivar and my sisters, Stefany Hernandez, Christina Spooner and Grace Moon, have always supported my career choices and believed in me. More importantly, my husband, Sebastian Carmona, for always reading my papers, picking up the slack, and being there for me when things did not go as planned. For that, I will always be grateful. iv CHARACTERIZING THE SOURCE OF NEUTROPHIL ELASTASE AND PROTEINASE 3 CROSS PRESENTATION IN ACUTE LYMPHOBLASTIC LEUKEMIA Selena Nicole Carmona, B.S. Advisory Professors: Gheath Al-Atrash, DO, Ph.D./Jeffrey Molldrem, MD Discovery of tumor-associated antigens is an important step in designing effective antigen-targeting immunotherapies. PR1 is a nonameric human leukocyte antigen (HLA)-A2 restricted leukemia-associated antigen derived from serine proteases neutrophil elastase (NE) and proteinase 3 (P3). NE and P3 are primarily expressed in cells of myeloid lineage including granulocytes, bone marrow progenitors and myeloid leukemia. Our lab reported that NE and P3 are cross-presented by antigen presenting cells (APCs) and solid tumors, a mechanism whereby exogenous antigens are endocytosed and presented on HLA class I molecules, inducing a cytotoxic T lymphocyte (CTL)-mediated immune response. Therefore, identifying non-myeloid tumors capable of PR1 cross-presentation broadens the application of PR1-targeted immunotherapies, which to date include PR1 peptide vaccine1, PR1 cellular therapy2,3 and 8F4, a T cell receptor (TCR)-like monoclonal antibody (mAb)4,5. One possible source of NE and P3 in the microenvironment is neutrophil extracellular traps (NETs). NETs are composed of deoxyribonucleic acid (DNA)/histones extruded from polymorphonuclear neutrophils (PMNs) abundant in antimicrobial proteases, including NE and P3. Although a main function of NETs is elimination of pathogens, seminal studies have demonstrated immune-regulatory effects of NETs. Thus there lies a great interest in understanding the role of NETs in modulating adaptive immune system through cross-priming or cross-tolerance in the setting of anti-tumor immunity. We are specifically interested in possible roles of NETs v in facilitating NE and P3 cross-presentation by acute lymphoblastic leukemia (ALL) due to its high abundance in the bone marrow. The major aim of this study was to validate PR1 as a target in B-ALL. This hypothesis is based on strong data from our laboratory showing (1) uptake and cross- presentation of NE and P3 by APCs6,7 including B cells, and (2) susceptibility of non- myeloid tumors to killing by 8F4 and PR1-CTLs (PR1-specific cytotoxic T lymphocytes) following PR1 cross-presentation6. Knowledge gained will define PR1 as a therapeutic target and cross-presentation as a mechanism for antigen expression in B-ALL. My results identify PR1 as a target in B cell-ALL, and identify NETs as a source of NE and P3 in the tumor microenvironment. These findings implicate the use of PR1- targeting immunotherapies as a novel form of treatment in B cell-ALL. vi Table of Contents Approval Page .................................................................................................................. i Title Page ........................................................................................................................ ii Dedication ...................................................................................................................... iii Acknowledgements ........................................................................................................ iv Abstract………………………………...………………………………………………………..v Table of Contents .......................................................................................................... vii List of Illustrations ............................................................................................................ x List of Tables ................................................................................................................. xii Abbreviations ................................................................................................................. xiii Chapter One: Introduction ............................................................................................... 1 1.1 Leukemia ............................................................................................................... 1 1.1.1 B Cell Lymphoblastic Leukemias .................................................................... 1 1.1.2 Allogeneic Stem Cell Transplantation in Leukemia ......................................... 3 1.1.3 NE, P3, and PR1 ............................................................................................. 4 1.1.4 8F4 .................................................................................................................. 7 1.2 Antigen Presentation ............................................................................................. 8 1.3 NETs, abundant in NE and P3, serve as a possible source in the tumor microenvironment ...................................................................................................... 11 1.4 Project Summary and Hypothesis ....................................................................... 12 Chapter 2: Materials and Methods ................................................................................ 14 2.1 Cell Lines ............................................................................................................. 14 vii 2.2 HLA-A2 Transduction .......................................................................................... 16 2.3 Generation of Protein Lysates and Western Blots ............................................... 17 2.4 RNA Purification and RT-PCR ............................................................................. 18 2.5 NE and P3 uptake in B-ALL cell lines .................................................................. 19 2.6 PR1 Cross-Presentation .....................................................................................
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