DEVELOPMENT OF REGULATORY T CELLS CAPABLE OF MAINTAINING IMMUNE HOMEOSTASIS A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY David Lee Owen IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Michael A. Farrar, Advisor September 2020 © David Lee Owen 2020 Acknowledgements I would like to first thank my mentor, Dr. Michael Farrar. Mike has been a fantastic example of the passion, enthusiasm and persistent attitude a scientist must have to succeed. Mike has provided me with a combination of guidance and freedom that allowed me to begin developing my skills as an independent investigator. He has always championed my career and has done whatever possible to promote me throughout my training. I also want to thank my thesis committee, Dr. Yoji Shimizu (chair), Dr. Bruce Blazar, Dr. Scott Dehm, Dr. Kristin Hogquist, and Dr. Daniel Mueller, for contributing their time and effort to supporting my project and progress throughout my thesis. I would also like to thank the other members of the Farrar lab, both past and present, for contributing their time to help me develop the technical skills required in the lab and in theoretical discussion on various projects. The list of collaborators, both inside and outside the University of Minnesota, who contributed to this work is too long to list but I want to express my gratitude for their generosity both of time and resources. Finally, thank you to everyone in the Center for Immunology who create a fun, collaborative and productive environment to develop as a young scientist. i Dedication I want to dedicate this work to my family who have always supported me in any endeavor I have taken on. First, my parents, James and Kathy, and my brother Daniel who have all given me the utmost support throughout the long process of PhD training. I also want to thank the rest of my extended family who have also remained connected and interested in my thesis work. Finally, I want to thank my wife, Wendy, who I met and married during my thesis training in Minnesota. You have always been supportive and patient with me. Thank you to all my family and friends for their interest, support and love. ii Abstract The adaptive immune response, comprised of both T cells and B cells, is essential to control infections and eliminate transformed cancer cells. The success of the adaptive immune system relies on the ability to discriminate self from non-self-antigens. The thymus is the site of selection for T cells, where self-reactive T cells are eliminated, generating a non-self focused T cell compartment. However, this selection process is leaky and potentially pathogenic cells do escape thymic, or central, tolerance. Thus, a population of suppressor cells termed regulatory T cells (Treg cells) co-evolved in order to keep these self-reactive escapees in check. Treg cells that develop in the thymus as part of central tolerance induction are a critical population of T cells that are required to maintain immune homeostasis and prevent autoimmunity. Without intervention, mice or humans that lack the ability to generate Treg cells die shortly after birth from widespread autoimmune-mediated tissue destruction. Further, neonatal thymectomy in mice causes the development of an autoimmune wasting phenotype. These observations highlight the importance of thymic Treg cell selection in immune homeostasis. Thymic Treg cell development occurs via a two-step process. Step one involves developing CD4+ thymocytes receiving strong T cell receptor (TCR) stimulation via engagement of thymic self-antigens, leading to upregulation of CD25, the high affinity subunit of the IL-2 receptor, or FOXP3, the lineage defining transcription factor of Treg cells, generating either + lo CD25 or FOXP3 Treg cell progenitors (TregP). Step two is driven by encounters between TregP cell and intrathymic STAT5 activating cytokines, predominantly IL-2, leading to co- + + expression of CD25 and FOXP3. These CD25 FOXP3 cells represent fully mature Treg cells that disseminate from the thymus to mediate immune tolerance. iii While the framework of this two-step development process is understood, many details of each step remain incompletely understood. This thesis addresses several aspects of thymic Treg cell development. First, we identify that T cells are the critical source of IL-2 + lo required to drive Treg differentiation. Second, we provide evidence that CD25 and FOXP3 TregP arise via distinct selection programs and contribute functionally distinct TCRs to the mature Treg compartment. Third, using single-cell RNA-sequencing analysis of conventional and Treg lineage thymocytes we provide a more detailed analysis of transcriptional signatures and intermediates of thymic Treg development. Finally, we gathered preliminary data to better understand the heterogeneity and function of recirculating or resident thymic Treg cells. Developing a holistic understanding of Treg development is essential to discern the etiology of immune disorders and properly modulate Treg cells to treat autoimmune disease, infections and cancer. iv Table of Contents Section Page Acknowledgements i Dedication ii Abstract iii List of Tables vi List of Figures vii Chapter 1 – Introduction 1 Chapter 2 – Defining the important sources of IL-2 required 27 for regulatory T cell development and homeostasis Chapter 3 – Development of regulatory T cells in the thymus 48 from two distinct developmental pathways Chapter 4 – Visualizing thymic Treg cell differentiation using 90 single-cell sequencing Chapter 5 – Role of recirculating Treg cells in thymus function 120 Chapter 6 – Summary and Future Directions 139 Chapter 7 – Materials and Methods 157 References 166 v List of Tables Table Title Page + Table 3.1. List of genes differentially regulated between CD25 TregP 62 lo and FOXP3 TregP in both scRNAseq data sets. Table 4.1. Selected genes associated with immature or mature CD4SP 95 clusters. Table 4.2. Selection gene associated with the “Interferon Signature” 99 thymocyte subset. + Table 4.3. Selected genes differentially regulated between CD25 TregP 101 lo and FOXP3 TregP agonist selection clusters. + Table 4.4. Selected genes differentially regulated between CD25 TregP 107 agonist and post-agonist clusters. Table 4.5. Selected genes differentially regulated between post-agonist 109 and mature Treg cell clusters. lo Table 4.6. Selected genes differentially regulated between FOXP3 TregP 112 lo and FOXP3 TregP agonist clusters. Table 4.7. Genes differentially regulated between mature Treg cells and 114 lo FOXP3 TregP. Table 5.1. Table of selected differentially regulated genes in clusters from 129 + thymic CD73 Treg cell scRNAseq analysis. vi List of Figures Figure Title Page Figure 1.1 Model of thymic Treg cell development 6 Figure 1.2 Foxp3iDTR schematic 23 Figure 2.1 Thymocytes and thymic DCs produce IL-2. 30 Figure 2.2 T cell-derived IL-2 is important for Treg cell homeostasis 32 in IL-15 sufficient mice. Figure 2.3 Dendritic cell- and B cell-derived IL2 is dispensable for 33 Treg cell development and homeostasis. Figure 2.4 T cell-derived IL2 is critical for thymic Treg differentiation. 36 Figure 2.5 Cell intrinsic IL2 is not required for thymic Treg differentiation. 39 + Figure 2.6 CD25 TregP cells undergoing agonist selection are a 40 major source of T cell derived intrathymic IL-2. Figure 2.7 T cell-derived IL2 is critical for Treg cell homeostasis. 42 Figure 2.8 T cell-derived IL2 is important in maintaining resting Treg cells. 44 Figure 3.1. Two thymic Treg progenitor cell populations exist. 50 + lo Figure 3.2. CD25 TregP cells and FOXP3 TregP cells have distinct 53 TCR repertoires. Figure 3.3. Germ-free and NME mice show no defect in either thymic 56 TregP cell pathway. + lo Figure 3.4. CD25 TregP and FOXP3 TregP cells are distinct TregP 58 populations. Figure 3.5. Single-cell RNA-seq of thymic Treg cell lineage. 60 Figure 3.6. Maturation analysis of thymic Treg cell populations. 63 vii + lo Figure 3.7. CD25 and FOXP3 TregP cells are in discrete selection stages. 65 Figure 3.8. Frequency of contaminating recirculating cells in thymic Treg 67 cell lineage subsets. lo Figure 3.9. Treg cells and FOXP3 TregP cells show different localization 69 in the thymus. lo Figure 3.10. FOXP3 TregP cells are dependent on NFκB1 activation. 71 + lo Figure 3.11. CD25 TregP cell and FOXP3 TregP cell development is 73 regulated by distinct enhancers. Figure 3.12. Enhancer deletions do not cause reduced levels of FOXP3 75 or CD25. –/– Figure 3.13. Itk mice show increased Treg cell production from both 77 TregP cell pathways via distinct molecular mechanisms. lo + Figure 3.14. FOXP3 and CD25 TregP respond distinctly to IL-4 stimulation. 80 lo Figure 3.15. FOXP3 TregP cells depend on Tuft cells and iNKT cells. 83 + lo Figure 3.16. Treg cells derived from CD25 and FOXP3 TregP cells are 85 functionally distinct. + lo Figure 3.17. CD25 but not FOXP3 TregP cells react with the 87 self-antigen MOG. + Figure 4.1. Identification of CD4 thymocyte and Treg cell lineage 92 developmental stages. Figure 5.1. RT-Treg cells are activated. 124 Figure 5.2. scRNAseq identification of RT-Treg cell subsets. 126 Figure 5.3. Low dose intrathymic DT treatment in Foxp3DTR mice 132 preferentially depletes RT-Treg cells. viii + Figure 5.4. RT-Treg cell depletion causes an increase in AIRE mTEC 134 density. ix Chapter 1: Introduction *Portions of the introductory text are derived from a review article written by the author1 Adaptive immunity evolved as a powerful defense mechanism to eliminate foreign pathogens and eradicate transformed cells. This system relies on two chief capabilities- extensive repertoire diversity and the ability to discriminate “self” versus “non-self” 2.