Interpreting Macrophage Transcripts In Human T Cell-Mediated Rejection by Dina El Sayed Feisal Amin Badr A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Experimental Medicine Department of Medicine University of Alberta © Dina El Sayed Feisal Amin Badr, 2017 ABSTRACT The two types of rejection identified in the Banff histopathologic diagnosis are T cell- mediated rejection (TCMR) and antibody-mediated rejection (ABMR). TCMR is most common early post-transplant while ABMR is the main cause of late graft loss. Kidney transplant TCMR is diagnosed histologically by interstitial inflammation and tubulitis, dominated by T cells and cells of the monocyte-macrophage-dendritic cell (MMDC) lineage. TCMR is mediated by cognate T cell recognition of donor antigen in the allograft. We hypothesized that the transcripts preferentially increased in kidney allografts with TCMR compared to ABMR will reflect MMDC interaction with activated effector T cells. We used gene expression analysis to define the top transcripts preferentially increased in TCMR versus ABMR in 703 clinically-indicated human kidney transplant biopsies. Transcripts for the metalloprotease ADAMDEC1 and chemokines CXCL13 and CCL18 were the top three preferentially increased in TCMR versus ABMR, their expression was also higher when compared to non specific acute kidney injury and non TCMR biopsies, and correlated with the histologic lesions diagnostic for TCMR and with the inflammatory burden in biopsies.. Further analyses identified the chemokine CCL19 as the most strongly increased in TCMR after CXCL13 and CCL18. CCL19 transcript showed similar associations as CXCL13 and CCL18 but to a lesser degree. In vitro studies identified heterogeneous effects on ADAMDEC1, CXCL13, and CCL18 expression in response to macrophage differentiation, and activation following interaction with activated effector T cells but not IFNG alone. Our study suggests that ADAMDEC1, CXCL13, CCL18 are macrophage transcripts capable of differentiating TCMR from ABMR and reflect monocyte to macrophage differentiation, and macrophage interaction with effector T cells. ii DEDICATION To my parents Laila Zada and Feisal Badr, my daughter and my son, Sara and Mostafa Rizk iii ACKNOWLEDGEMENTS I would like to express my deepest respect, appreciation and gratitude to my supervisor Dr. Philip Halloran for giving me the opportunity to participate in this project and to work with his distinguished research team at Alberta Transplant Applied Genomics Centre (ATAGC). I would like to thank Dr. Banu Sis and Dr. Harissios Vliagoftis, members of my graduate committee, who have given time and support for the success of this project and helped my overall scientific progress. The members of ATAGC and Halloran Lab Team, current and previous members: Konrad Famulski, Luis Hidalgo, Anna Hutton, Jeff Reeve, Robert Polakowski, Jeff Venner, Jessica Chang, Pam Publicover, Vido Ramassar, Kara Allanach, Michelle Ryan, Danielle Stewart, Gunilla Einecke, Michael Mengel, Joana Sellares, Gui Rennesto, Sujatha Kamma, Zija Jacaj, Lisa Billesberger, Sakarn Bunnag, Declan De Freitas, Nathalie and Danielle Kaiser, Chatchai Kreepala. They have provided technical and scientific help and have enriched my graduate experience at Halloran Lab. The Department of Medicine particularly graduate program coordinators and advisors, current and previous members, Dr. Sean McMurtry, Dr. Karen Madsen, and Dr. Ross Tsuyuki, Sharon Campbell, Barb Thomson, Eleni Dimos, Aileen Leskow, Maggie Hill for their continuous help, support, and encouragement throughout my graduate program. The Egyptian Ministry of Higher Education for sponsoring my scholarship and giving me such great opportunity to pursue my graduate studies in Canada. The Egyptian Bureau of Cultural and Educational Affairs in Canada for their continuous help and support throughout my graduate studies in Canada. The president of University of Mansoura, Egypt, the Dean of Mansoura Faculty of Medicine, the Chairman of the Department of Microbiology and Medical Immunology, iv University of Mansoura, Egypt, for their patience, understanding, cooperation and the opportunity for extending my stay in Canada to complete my PhD degree. v TABLE OF CONTENTS CHAPTER 1: INTRODUCTION....……………………………………………………1 1.1. Kidney transplant rejection ........................................................................................2 1.2. Generation of alloimmune response during rejection ................................................2 1.3. Effector mechanisms in allograft rejection ................................................................3 a) Antibody-mediated rejection ............................................................................4 b) T cell-mediated rejection ..................................................................................4 1.4. Macrophages as part of the mononuclear phagocyte system .....................................6 1.5. Macrophage classification and heterogeneity ............................................................6 1.6. Dendritic cells ............................................................................................................7 1.7. Overlap between macrophages and dendritic cells ....................................................8 1.8. Chemokines ...............................................................................................................10 1.9. Matrix metalloproteinases .........................................................................................11 1.10. The ADAM Family of metalloptoteases .................................................................12 1.11. Rejection pathology and molecular classification ...................................................13 a) Pathological classification of rejection: The Banff classification ....................13 b) Molecular classification of rejection ................................................................14 1.12. Rationale ..................................................................................................................15 1.13. Hypothesis ...............................................................................................................16 1.14. Aims of the study .................................................................................................... 17 1.15. Research questions ..................................................................................................17 CHAPTER 2: MATERIALS AND METHODS....………………………………...….18 vi 2.1. Human kidney transplants..........................................................................................19 . a) Human patient population and specimens.........................................................19 b) Assessment of human allograft biopsies ..........................................................19 2.2. Human cell isolation and cell cultures........................................................................20 a) Human cell panel ..............................................................................................20 Monocytes and macrophages.................................................................... 20 Effector T cells .........................................................................................20 B cells .......................................................................................................20 NK cells.....................................................................................................21 Endothelial and epithelial cells .................................................................21 Interferon gamma treatment......................................................................21 b) THP-1 cell culture and stimulation ..................................................................21 THP-1 cell culture......................................................................................21 THP-1 cell stimulation ..............................................................................21 c) Monocyte-derived macrophage culture ............................................................22 Monocyte isolation ...................................................................................22 Monocyte-derived macrophage differentiation ........................................22 Monocyte-derived M-CSF macrophage differentiation ...........................22 Macrophage stimulation ...........................................................................23 M-CSF macrophage stimulation ...............................................................23 M-CSF- macrophage time course..............................................................23 d) Monocyte-derived dendritic cell culture...........................................................24 Monocyte-derived dendritic cell differentiation .......................................24 Monocyte-derived dendritic cell stimulation ............................................24 vii e) Co-cultures of macrophages and activated T cells ...........................................24 Transwell (no contact) co-culture .............................................................25 Contact co-culture .....................................................................................25 2.3. Flow cytometry ..........................................................................................................25 2.4. Enzyme-linked immunosorbent assay (ELISA).........................................................26 2.5. V-PLEX cytokine assay .............................................................................................27 2.6. RNA preparation and microarray ..............................................................................27 2.7. Real time RT-PCR..................................................................................................... 28 2.8. Mouse kidney transplants ..........................................................................................28 RNA preparation and microarray .............................................................29 2.9. Statistical analyses .....................................................................................................29 2.10. Tables .......................................................................................................................31 2.11. Figures .....................................................................................................................34 CHAPTER 3: DEFINING TRANSCRIPTS PREFERENTIALLY INCREASED IN TCMR VERSUS ABMR....……………………………………………………………..41 3.1. Overview ....................................................................................................................42 3.2. Human population demographics and biopsy diagnoses ...........................................43 3.3. Algorithm for defining transcripts preferentially increased in TCMR versus ABMR ...................................................................................................................44 3.4. Defining the transcripts increased in TCMR versus normal kidneys ........................44 3.5. Defining the transcripts preferentially increased in TCMR versus ABMR............... 45 3.6. The top transcripts with the highest association with TCMR ....................................46 3.7. The significance of the top transcripts preferentially increased in TCMR versus ABMR in differentiating TCMR from AKI and non TCMR biopsies ................. 46 viii 3.8. Chemokines and chemokine receptors preferentially increased in TCMR versus ABMR ....................................................................................................................48 3.9. Metalloproteases preferentially increased in TCMR versus ABMR ......................... 49 3.10. Selection of ADAMDEC1, CXCL13, CCL18 and CCL19 for further study ..........50 3.11. Expression of ADAMDEC1, CXCL13, CCL18 and CCL19 in different histologic diagnoses ..........................................................................................................50 3.12. The relationship between ADAMDEC1, CXCL13, CCL18 and CCL19 expression and histologic lesions ......................................................................................51 3.13. Pathogenesis-based transcript sets ...........................................................................52 3.14. The relationship between ADAMDEC1, CXCL13, CCL18 and CCL19 expression and inflammatory cell burden .........................................................................53 3.15. Selectivity of ADAMDEC1, CXCL13, CCL18 and CCL19 expression for TCMR .........................................................................................................................55 3.16. Establishing Real-Time RT-PCR method for assessment of ADAMDEC1, CXCL13, CCL18, and CCL19 expression........................................................................55 3.17. Tables ...................................................................................................................... 57 3.18. Figures .....................................................................................................................68 CHAPTER 4: CHARACTERIZATION OF ADAMDEC1, CXCL13, CCL18, AND CCL19 EXPRESSION IN HUMAN MMDC….………………………………...…….76 4.1. Overview ....................................................................................................................77 4.2. THP-1 monocytic cell line as in vitro model to study ADAMDEC1 and CXCL13.............................................................................................................................78 a) ADAMDEC1 and CXCL13 expression in THP-1 cells versus primary monocytes ............................................................................................................79 ix b) Effect of THP-1 stimulation on expression of ADAMDEC1 and CXCL13 …79 4.3. ADAMDEC1, CXCL13, CCL18 and CCL19 expression in human cells .................80 4.4. Effect of IFNG on ADAMDEC1, CXCL13, CCL18 and CCL19 expression ...........81 Heterogeneity of expression of ADAMDEC1, CXCL13, CCL18 and CCL19 in MMDC ..........................................................................................................................82 4.5. ADAMDEC1, CXCL13, CCL18 and CCL19 expression during differentiation of monocyte-derived macrophages and dendritic cells ....................................................82 4.6. ADAMDEC1, CXCL13, CCL18 and CCL19 expression during macrophage stimulation .........................................................................................................................83 4.7. ADAMDEC1, CXCL13, CCL18 and CCL19 expression during dendritic cell stimulation ..................................................................................................................85 4.8. ADAMDEC1, CXCL13, CCL18 and CCL19 expression in M-CSF- differentiated macrophages ...............................................................................................86 4.9. ADAMDEC1, CXCL13, CCL18 and CCL19 expression during M-CSF- macrophage stimulation ....................................................................................................87 4.10. Time course for ADAMDEC1, CXCL13, CCL18 and CCL19 expression in M-CSF- macrophages ...................................................................................................88 ADAMDEC1, CXCL13, CCL18 and CCL19 expression during T cell – Macrophage interaction ....................................................................................................90 4.11. Overview .................................................................................................................90 4.12. Effect of soluble factors released from activated T cells......................................... 90 4.13. Effect of contact interaction between activated T cells and macrophages ..............93 4.14. Cytokine profiling ....................................................................................................94 4-15. Figures .....................................................................................................................96 x