Reproductive Benefits Conferred by Genetically Foreign Cells That Persist in Mothers and Offspring
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Reproductive benefits conferred by genetically foreign cells that persist in mothers and offspring 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 Program of the University of Cincinnati College of Medicine 2016 By Jeremy M. Kinder B.S., Ball State University, 2010 M.S., Ball State University, 2012 Committee Chair: Sing Sing Way, M.D.,Ph.D. Abstract Our genetic makeup is equally inherited from our mother and father through classical Mendelian genetics. However, each individual is constitutively chimeric, containing genetically foreign cells vertically transferred from their mothers during in utero development. These rare maternal microchimeric maternal cells have been identified to persist many years after birth and across a wide range of tissues demonstrating remarkable immune tolerance to our mothers persists in us long after we are born. Reciprocally during pregnancy, mothers also receive a transfer of fetal cells that persist after parturition. We have shown that transfer of these genetically foreign cells during gestation is not accidental, but instead these cells are intentionally retained to promote reproductive fitness in subsequent pregnancies. Herein, we will explore the teleological and immunological implications of the long-term retention these genetically foreign cells. ii iii Acknowledgements I could not have made it this far without great mentorship. I would like to express my sincere gratitude to my advisor, Dr. Sing Sing Way. Your critical feedback, patience and scientific drive have shaped who I am as a scientist and as an individual. Over the last few years, I have also gained valuable insight observing how you balance a successful scientific career while also maintaining a fulfilling family life. Without your support and guidance, I would not have been able to achieve what I have done so far and what I will do in the future. Before I started my PhD career, Dr. Heather Bruns was another mentor that helped me develop critical scientific skills during my undergraduate and master’s training. There are many intangible skills I have learned from you that I will carry with me throughout my career. These include continuous scientific innovation and organization while maintaining a strong commitment to your family life. Over the last 7 years I have valued not only your scientific mentorship but also your friendship. I would also like to thank the members of my dissertation committee: Drs. Harinder Singh, David Hildeman, Aimen Shaaban, and Jonathan Katz. The time you have devoted during and outside of our meetings to provide important critical feedback on my work is invaluable. Furthermore, I appreciate the investment you have made into not only my time as a graduate student but also advising me in future career decisions as well. I look forward to working with all of you as scientific peers in the future. iv I also owe a lot to the current and past lab members who have provided valuable feedback but also contributed technically to some of the work outlined in this dissertation. I could not have done all of this work without your help and support. I am blessed by the tremendous amount of support my family and friends have given me over the last few years. Pam, Richard and Craig, your encouragement of me and the support you have given to Christina and I is irreplaceable. I have truly felt like a part of your family over the 15 years Christina and I have been together. I owe both my scientific curiosity and tenacity to my mom, Teresa who has always told me I can accomplish whatever I set my mind to and who has forever been right behind cheering me on. To my wife, Christina, your love, dedication and constant support of my career goals has kept me going over the last several years. Importantly, you have kept me grounded during many stressful times and humble over the last 10 years. Finally, to my dad and daughter - while you cannot be here physically you have been and will continue to be my inspiration. v Abbreviations APC antigen-presenting cell BAC bacterial-artificial chromosome CG chorionic gonadotropin CNS1 conserved non-coding sequence 1 CRE Cre recombinase DC dendritic cell DNA deoxyribonucleic acid DT diphtheria toxin DTR diphtheria toxin receptor EAE experimental autoimmune encephelomyelitis FACS fluorescence-activated cell sorting FISH fluorescence in situ hybridization FOXP3 forkhead box P3 Gal1 galectin 1 GFP green fluorescent protein GITR glucocorticoid-induced TNFR-related protein GVHD graft-versus-host disease HLA human leukocyte antigen IDO indoleamine 2,3 dioxygenase IFNg interferon-gamma IL-10 interleukin 10 IPEX immunodysregulation polyendocrinopathy enteropathy X-linked syndrome LCMV lymphocytic choriomeningitis virus Lm Listeria monocytogenes MHC major histocompatibility complex MS multiple sclerosis NFAT nuclear factor of activated-Tcells NIMA non-inherited maternal antigen NIPA non-inherited paternal antigen OVA chicken egg ovalbumin PCR polymerase chain reaction RA rheumatoid arthritis vi Rh rhesus factor SCID severe combined immunodeficiency SLE systemic lupus erythematosus Smad3 mothers against decapentaplegic homolog 3 TCR T-cell receptor TGF-B transforming growth factor beta Th T helper cell Treg regulatory T cell vii Table of Contents Abstract ii Acknowledgements iv List of Abbreviations vi Table of Contents viii CHAPTER 1: General Introduction 1 1.1 Introduction 2 1.2 Evolution of the placenta 2 1.3 Immunological conundrum of pregnancy 4 1.4 Maternal-fetal tolerance during pregnancy 6 1.5 Systemic immunological shifts during pregnancy 9 1.6 Fetal tolerance to non-inherited maternal antigen 17 1.7 Genetically foreign microchimeric cells 18 1.8 Dissertation Aims 23 1.9 References 26 CHAPTER 2: Pregnancy-induced maternal regulatory T cells, 36 bona fide memory or maintenance by antigenic reminder from fetal cell microchimerism? CHAPTER 3: Tolerance to noninherited maternal antigens, 48 reproductive microchimerism and regulatory T cell memory: 60 years after ‘Evidence for actively acquired tolerance to Rh antigens’ 3.1 Abstract 49 3.2 Introduction, pioneering observations on immunological 50 tolerance by Dr. Ray Owen 3.3 Human immunological tolerance to noninherited 51 maternal antigens 3.4. Animal models of immune tolerance with early 57 developmental antigen exposure 3.5 Teleological benefits and immunological consequences 62 of NIMA-specific tolerance 3.6 Concluding perspectives 68 viii 3.7 References 70 CHAPTER 4: Offspring’s tolerance of mother goes viral 79 CHAPTER 5: Cross-generational reproductive fitness 87 enforced by microchimeric maternal cells 5.1 Summary 88 5.2 Introduction 89 5.3 Results 91 5.4 Discussion 100 5.5 Experimental procedures 104 5.6 References 108 5.7 Main figures 113 5.8 Supplementary figures 121 Chapter 6: Sustained protection against fetal wastage 127 conferred by prior pregnancy despite numerical loss of maternal regulatory CD4+ T cell memory 6.1 Abstract 129 6.2 Body 130 6.3 Materials and methods 138 6.4 Main figures 141 6.5 Extended data figures 145 6.6 References 149 Chapter 7: Summary and Discussion 152 7.1 Overall summary 153 7.2 Discussion 157 7.3 References 169 Appendices 172 Appendix I: Reuse Licenses 173 ix Appendix II: pdf of Chapter 2 185 Appendix III: pdf of Chapter 3 189 Appendix IV: pdf of Chapter 4 203 Appendix V: pdf of Chapter 5 206 x Chapter 1: General Introduction 1 Introduction Reproductive success is essential for the survival of all species. In turn, this immense selective pressure drives continuous refinement and evolutionary conservation of methods for procreation that promote reproductive fitness. One such advantageous adaptation allowed the development of the embryo from within the maternal body known as viviparity. In contrast to other methods of reproduction such as laying of eggs (oviparity), viviparity allows prolonged maturation of offspring with minimal threat of destruction from predation and permits long-term regulated transfer of parental nutrients throughout development1. Broadly speaking, despite reduced numbers of offspring, increased investment of time and resources devoted to the developing fetus with viviparity drastically increases their chances of survival and reduces the wasted allocation of resources to offspring that will likely not survive. Evolution of the placenta Emergence of viviparity simultaneously required additional physiological adaptations to facilitate development of offspring within the mother. Formation of a specialized organ known as the placenta allows attachment of the fetus to maternal uterine tissue and regulates exchange of nutrients, gas and waste between mother and fetus2,3. Although the placenta has evolved independently amongst multiple different animal groups (cartilaginous fish, reptiles, amphibians, etc.) all viviparous mammalian species require a placenta for successful reproduction3,4. 2 In mammals the placenta is formed early in gestation by a specialized lineage of fetal cells known as trophoblasts that continuously expand to form a multilayered organ3,5. Early in pregnancy, trophoblasts initiate systemic physiological changes through release of hormones such as chorionic gonadotropin (CG) that halts shedding or resorption of the uterine decidua and instead triggers its expansion. In turn, fetal trophoblasts continuously expand and invade maternal uterine tissue to form the placenta. This process mediates anchoring of the developing