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UNDERSTANDING & OPTIMIZING HUMAN T REGULATORY CELL FUNCTION IN PATIENTS WITH AUTOIMMUNITY AND/OR UNDERGOING TRANSPLANTATION by Katherine Grace MacDonald B.Sc., The University of Victoria, 2010 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Experimental Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2015 © Katherine Grace MacDonald, 2015 Abstract CD4+FOXP3+ T regulatory cells (Tregs) are potent suppressors of inflammatory immune activity. Cellular therapy with Tregs is a promising way to induce antigen specific tolerance in transplantation and autoimmunity, as it would allow the reduction of nonspecific immunosuppression. Currently, Tregs are being tested in clinical trials; however, outstanding questions regarding stability, specificity, and longevity of transferred cells remain. The aim of this research was to better understand the potential plasticity of Tregs, develop novel methods of creating antigen specific Tregs, and determine the optimal signals for Tregs to persist after transfer. To better understand the pathological conversion of Tregs to inflammatory cells, I examined the phenotype of Tregs in systemic sclerosis, a Th2-biased disease. I found that Tregs in patient skin and blood had acquired Th2-cytokine and homing marker expression, respectively, and that both tissue-localized and homing cells express the receptor for IL-33, which was expressed in patient skin. This work suggests that sub-populations of Tregs have the capacity to become pathogenic upon encountering tissue-specific inflammatory signals. Next, in order to create antigen specific Tregs, I developed a novel chimeric antigen receptor (CAR) against HLA-A2 and tested its function. A2CAR-Tregs were highly activated and proliferative in response to HLA-A2, but they retained their suppressive capacity and expression of the transcription factors FOXP3 and Helios, and the effector molecules CD25 and CTLA-4. A2CAR-Tregs were also superior to polyclonal Tregs at preventing xenoGVHD in mice, even at low doses. Thus, A2CAR-Treg cell therapy is a promising new technology to create potent Tregs for antigen-specific transplant tolerance. Finally, to optimize Treg activity and persistence in patients, I developed a series of CARs containing different co-stimulatory domains. Four TNFR ii superfamily domains were cloned, from 4-1BB, OX40, GITR and TNFR2, and three signalling domains were cloned from the B7/CD28 superfamily, from ICOS, PD-1, and CTLA-4. 4-1BB, OX40, ICOS, and PD-1 containing CARs were expressed on the surface of cells. These tools will provide valuable information in future research on Treg survival in vivo. Collectively, these studies have provided insights to improve both the safety and efficacy of Treg cell therapy. iii Preface Chapter 1 Versions of chapter 1 have been published: MacDonald KG, Orban PC, Levings MK. T regulatory cell therapy in transplantation: stability, localization and functional specialization. Curr Opin Organ Transplant. 2012 Aug;17(4):343-8. and: Vent-Schmidt J, Han JM, MacDonald KG, Levings MK. The role of FOXP3 in regulating immune responses. Int Rev Immunol. 2014 Mar;33(2):110-28. I performed literature review and wrote the majority of the text represented in my thesis. Levings MK reviewed, edited, and oversaw manuscript preparation. Chapter 3 A version of chapter 3 has been published: MacDonald KG, Dawson NA, Huang Q, Dunne JV, Levings MK, Broady R. (2015). Regulatory T cells produce profibrotic cytokines in the skin of patients with systemic sclerosis. J Allergy Clin Immunol. 135(4):946-955. I conducted the experiments on fibroblasts, sorted Tregs, and expanded T cells, analyzed all data, and wrote most of the manuscript. Patient skin and data were collected by Dunne, JV, and cultured and stained by Huang, Q. Chemokine staining and immunofluorescence were performed by Huang Q and Dawson NA. Levings MK and Broady R supervised the project and reviewed the manuscript. iv Chapter 4 I was the primary contributor to Chapter 4. I planned, executed, and analyzed in vitro experiments. Orban PC planned and assisted with cloning lentivirus constructs. Hoppli R and Huang Q monitored and sacrificed mice with my supervision. All work was supervised by Borady R, Orban PC, and Levings MK This work has been submitted as: MacDonald KG, Orban PC, Hoppli RE, Huang Q, Broady R and Levings MK. Generation of alloantigen-specific T regulatory cells by expression of a novel chimeric antigen receptor targeting HLA-A2. Manuscript submitted to JCI. Chapter 5 I conceptualized, planned, and executed chapter 5. I designed the lentiviral constructs. I cloned the co-stimulatory domains with the assistance of Huang Q. Orban PC planned and created co- inhibitory constructs. Huang Q performed transient transfection of co-inhibitory domains. All work was supervised by Broady R, Orban PC, and Levings MK. Ethics approval for all chapters was obtained from the Clinical Research Ethics Board for “C03- 0657 Elucidation of the Mechanisms by Which the Human Immune System Responds to Infection & Organ Transplantation” (H03-70657) and “C03-0062 - Human T Regulatory Cells” (H03-70062). Approval for in vivo models (chapter 4) was obtained from the Animal Care Committee for “Xeno GVHD Mouse” (A14-0110). v Table of contents Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iv Table of contents .......................................................................................................................... vi List of tables................................................................................................................................ viii List of figures ................................................................................................................................ ix List of abbreviations ......................................................................................................................x Acknowledgements ..................................................................................................................... xii Dedication ................................................................................................................................... xiii Chapter 1: Introduction ................................................................................................................1 1.1 T helper cells and the adaptive immune response .......................................................... 1 1.2 T regulatory cells ............................................................................................................ 3 1.2.1 FOXP3 driven mechanisms of suppression ................................................................ 4 1.2.2 Epigenetic maintenance of the Treg lineage ............................................................... 5 1.2.3 Treg cell therapy ......................................................................................................... 7 1.2.4 Aspects of Treg biology to consider for successful cell therapy ................................ 8 1.3 Th-like Treg subsets ...................................................................................................... 12 1.3.1 The role of TBET in Tregs........................................................................................ 12 1.3.2 The relationship between Th2 cells and Tregs ......................................................... 13 1.3.3 Cross-talk between FOXP3 and ROR-γt .................................................................. 15 1.3.4 FOXP3-independent transcriptional regulation of Tregs .......................................... 16 1.3.5 Implications of Th-like Treg subsets for cell therapy ............................................... 17 1.4 Antigen specific Tregs and chimeric antigen receptors ................................................ 18 1.4.1 Antigen choice and targets of current CARs ............................................................ 20 1.4.2 T cell activating domains in CARs ........................................................................... 22 1.4.3 Considerations for additional CAR components ...................................................... 23 1.5 Conclusion .................................................................................................................... 24 1.6 Synopsis of research questions ..................................................................................... 25 Chapter 2: Material and methods ..............................................................................................27 2.1 Patient samples (chapter 3) ........................................................................................... 27 2.2 CAR generation and testing (chapters 4 and 5) ............................................................ 30 Chapter 3: T regulatory cells produce pro-fibrotic cytokines in the skin of patients with systemic sclerosis ..........................................................................................................................34 3.1 Introduction ................................................................................................................... 34 3.2 SSc patient characteristics............................................................................................

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