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A Natural Killer Cell-Centric Approach Toward New Therapeutics For A natural killer cell-centric approach toward new therapeutics for autoimmune disease A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Ph.D.) in the Immunology Graduate Training Program of the College of Medicine 2019 By Seth D. Reighard B.S., University of Pittsburgh, 2010 Thesis Advisor: Stephen N. Waggoner, Ph.D. Dissertation Committee Chair: William M. Ridgway, M.D. 1 Abstract The debilitating autoimmune disease systemic lupus erythematosus (SLE) is the 5th leading cause of death for African American and Hispanic women between 15 and 24 years of age. Current therapeutics for SLE are blunt weapons that broadly suppress a patient’s immune system, and only one new treatment option has been approved in over 50 years. Standard treatments for SLE potentially lack efficacy because they do not specifically target the immune dysfunction underlying disease. SLE is characterized by the abnormal production of self-targeting autoantibodies that cause widespread organ damage. SLE autoantibodies are generated by rampant germinal center (GC) reactions: immune processes wherein memory B cells and antibody secreting cells (ASC) arise from naïve B lymphocytes following clonal expansion, somatic hypermutation, affinity maturation, and antibody class switching while receiving critical help from follicular helper T (TFH) cells. We hypothesize that inhibition of TFH and GC responses in SLE may be accomplished by harnessing the therapeutic potential of the natural killer (NK) cell. Though classically implicated in anti-tumor and antiviral immunity, NK cells also suppress activated TFH, resulting in reduced GC reactions and decreased antibodies. Of added relevance, NK cells are diminished and defective in SLE patients. Thus, we tested whether increasing endogenous NK cell numbers or artificially enhancing their function could achieve a beneficial result in an autoimmune context like SLE. We treated mice exhibiting an SLE-like disease with drugs that expand the endogenous NK cell (and CD8 T cell) pool and, contrary to our hypothesis, observed worsened autoimmunity. Disease aggravation was attributed to the expansion of CD8 T cells, with expanded NK cells exhibiting negligible effects. Our findings suggest that current cancer treatments that expand these cell populations in vivo should be used with caution in patients with cancer and concurrent SLE. Separately, we utilized chimeric antigen receptor technology, a clinical tool currently being used to safely target tumors, to engineer an NK cell that selectively eliminates TFH cells in hopes of subduing autoreactive GCs. In vitro studies demonstrated that our CAR NK cells not only killed TFH cells but also limited subsequent B cell-mediated generation of ASCs and the downstream production 2 of antibodies. Furthermore, we used RNA sequencing to identify differences in human SLE NK cell gene expression and uncovered several transcriptional alterations with known relevance to both SLE pathogenesis and NK cell function. Gene network analysis predicted the transcription factor PU.1 to underlie several SLE NK cell transcriptional changes, and the upregulation of PU.1 may explain an aberrant and proinflammatory phenotype often observed in disease. Future studies will aim to attenuate the expression of PU.1 in NK cells and determine whether the resulting phenotype is of benefit to disease, thereby identifying PU.1 as a potentially valuable therapeutic target in SLE. Altogether, our innovative NK cell-based therapeutic strategies have the potential to limit the autoantibody-mediated pathology of SLE in a more targeted manner, offering a new hope to thousands of people who suffer from this incurable disease. 3 4 Acknowledgments The thesis work presented herein could not have been accomplished without the help of several people who have made a significant positive impact upon my professional and personal life. I dedicate this work to them. First, I thank my dissertation committee members (Bill Ridgway, George Deepe, Edith Janssen, and Wenhai Shao) for their expertise throughout graduate school. This dissertation would not be nearly as complete without their guidance. Most of them served as members of my qualifying committee as well, so I especially appreciate their willingness to endure my research presentations so many times throughout my tenure as a graduate student. I thank all of my fellow graduate student colleagues and friends, especially Calvin and Rob, for making graduate school bearable and a lot more enjoyable. Similarly, I thank my Waggoner Lab colleagues, past and present, for their endless help and support of my projects and for their enduring friendship. I have extra gratitude for my former colleague, (Dr.) Erik, for helping me to proofread sections of this thesis in a time of dire need. I thank my past mentors, Drs. M. Brian Traw and Thomas Cherpes, for giving me a chance to pursue scientific research early in my career, and for developing in me a strong foundation of skills, knowledge, and perseverance that is required for earning a PhD. I especially thank my PhD mentor, Dr. Stephen Waggoner, for being the best scientist and teacher that I could have chosen for my graduate school research. Steve is the true embodiment of everything it takes to be a successful scientist: grit, optimism, creativity, sociability, salesmanship, foresight, intelligence, and determination. As I continue my journey along the research career path, I will forever use lessons learned from Steve to guide my way. 5 I thank the family I was born into: my mom, dad, sister and brother, for their relentless love and support of all of my academic pursuits throughout the years. I thank the family I married into: my mother, father, and siblings-in-law, for their support and frequent visits to Cincinnati that lessened the homesickness within our household. I also thank the family I chose: my closest friends from childhood to adulthood (Justin, Mike, Matt, Sam, & Kayla) for always being there when I need them to forget about my professional life for a while. Words cannot express the amount of love and gratitude that I feel for my wife, Brittany, who uprooted everything she knew to move with me to Cincinnati and start a new life together while I pursued an additional 8 years of school. She is truly my rock, my cornerstone, and my utmost source of motivation throughout the trials and tribulations of graduate school. I fully believe that I could not have done this without her. Finally, I thank and dedicate this thesis to my newborn son Forrest who, at the time of finishing this thesis, is just over a month old. I never imagined I could love anything more in my life until I met him. I can’t predict what the future holds for Forrest, but I wish a few things for him. I hope that science and logic can survive and prevail in this time of “alternative facts” that he was born into. I hope that technology, reason, and unity can help humanity to heal the environmental mess he has inherited from the generations that came before him. Lastly, I hope that, no matter the passion he pursues, he will be inspired by the power of science and have a deep admiration for the phenomena that underlie the natural world. 6 Table of Contents Abstract…………………………………………………………………………………………………………………………………..……….2 Acknowledgments……………………………………………………………………………………………………………….………..…5 Table of Contents……………………………………………………………………………………………………………………………..7 Abbreviations frequently used in this dissertation………………………………………………………………………….12 Chapter 1. Introduction to critical concepts underlying this dissertation………………………………………….15 o B lymphocytes, the germinal center reaction, and follicular helper T cells…………….……..….…..16 o Systemic lupus erythematosus: disease variety and pathogenesis………………………………….…..19 o Current treatment approaches for systemic lupus erythematosus..…………………………………….25 o A brief overview of modeling systemic lupus erythematosus in mice……………………………………29 o Natural killer cell development, maturation, and function…………………………………………………..31 o Natural killer cells in systemic lupus erythematosus…………………………………………………….……..35 o The therapeutic potential of natural killer cells in human disease…………..…………………..……..38 o Summary & overall hypotheses…………………………………………………………………………………....…….42 Chapter 2. Immunomodulatory effects of IL-2Rβ/γc-mediated expansion of natural killer cells and CD8 T cells in a mouse model of SLE-like disease………………………………………………………………………………………44 o Rationale & Hypothesis……………………………………………………………………………………………………….45 o Chapter 2 Graphical Abstract………………………………………………………………………………………………47 o Results………………………………………………………………………………………………………………………………..48 o Acknowledgements…………………………………………………………………………………………………………….52 o Figures………………………………………………………………………………………………………………………………..53 . Figure 1: Bm12-induced lupus-like disease is associated with phenotypic alteration of NK cells, as well as a contraction in the NK cell pool that is reversed via targeted cytokine treatment…………………………………………………………………………………..…………...53 7 . Figure 2: IL-2/αIL-2 (S4B6) complexes and the IL-15 superagonist N-803 selectively expand peripheral CD8 T and NK cell populations……………………………………………………55 . Figure 3: Pretreatment with IL-2 complexes or N-803 transiently exacerbates cGvHD……………………………………………………………………………………………………………..…….56 . Figure 4: Therapeutic application of IL-2 complexes or N-803 exacerbates lupus-like disease…………………………………………………………………………………………………………………..58 . Figure 5: N-803 expansion
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