Indirect T Cell Allorecognition of the Rt1.A* Mhc Class I Molecule

INDIRECT T CELL ALLORECOGNITION OF THE RT1.A* MHC CLASS I MOLECULE. by Emma Lovegrove A thesis submitted to the University of Glasgow in partial fulfilment for the degree of Doctor of Philosophy with the Faculty of Medicine. Transplantation Laboratories, Department of Surgery, 44, Church Street, Western Infirmary, Glasgow, G il 6NT. October 1999. ProQuest Number: 13818958 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 13818958 Published by ProQuest LLC(2018). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 G! ' 'COW L LftoiTY UE.'iAKY TtieSfV UI2>7 c o p 1-) [ ABSTRACT The extent to which the indirect pathway of T cell recognition contributes to graft rejection remains to be clarified, and this thesis examines its role in the antibody- mediated rejection of allografts in the MHC class I disparate rat strain combination, PVG-R8 (RTl.AaB/CuDu) to PVG-RT1U (RT1.AUB/CUDU). A series of overlapping 15-mer allopeptides (PI-PI8) derived from the donor Aa antigen, were used to map the immunogenic, dominant and sub-dominant epitopes of the Aa molecule. Analysis of the alloantibody response mounted to individual allopeptides following their subcutaneous administration suggested that the 15-mer peptides P7 and P I6, derived from the hypervariable regions of the a l and oc2 domains respectively, and the 24 amino acid a l peptide were immunogenic. The dominant T cell epitope was characterised by examination of the in vitro T cell proliferative responses to individual allopeptides by LNC from RT1U animals immunised with Aa-bearing R8 allografts. Proliferation focused upon those peptides derived from the hypervariable region of the a l domain, an area corresponding to P7 and P8. Analysis of the alloantibody response to the intact Aa molecule following peptide priming demonstrated the presence of two additional sub-dominant T cell epitopes located within PI and P9. These peptides derive from areas of the Aa molecule that are identical in amino acid sequence to the corresponding areas of the RT1U molecule, and are therefore, in effect, self-RTlu peptides. Priming with both dominant and sub-dominant epitopes accelerated the rejection of subsequent R8 cardiac allografts, suggesting that peptide priming is able to indirectly activate recipient T cells. It was examined whether the dominant and sub-dominant T cell epitopes could be used to favourably modulate the immune response to the intact Aa molecule by intravenous administration of high doses of P7 and PI to RT1U animals before challenge with an R8 blood transfusion. Downregulation of the cytotoxic and IgM alloantibody responses were observed and in addition, P7 was able to downregulate the IgG2b response. Slight downregulation of the cytotoxic alloantibody response to an R8 cardiac allograft was seen following immunisation with P7, but the IgM and IgG2b responses were unaltered. Prolonged allograft survival was not observed. These results suggest that the indirect response to an allogeneic MHC molecule may involve additional unexpected epitopes and consequently, that the success of peptide-based tolerogenic protocols requires a fuller understanding of this process. CONTENTS PAGE Title i Abstract ii Contents iv Illustrations and Tables vii Acknowledgements viii Declaration ix Abbreviations x Amino Acid Symbols xv Chapter 1: Introduction 1.1 The Major Histocompatibility Complex 3 1.2 Antigen Processing and Presentation 10 1.3 The T Cell Response 19 1.4 T Cell Development 26 1.5 B Cell Development 30 1.6 Transplantation Immunology 34 1.7 Allorecognition 36 1.8 The Role of Indirectly Activated T Cells in Effecting Allograft Rejection 42 1.9 Tolerance Induction 46 1.10 Aims 52 Chapter 2: Materials and Methods 2.1 Animals 57 2.2 Tissue Culture Media 57 2.3 Cell Preparations 58 2.4 Synthetic Allopeptides 59 - iv - CONTENTS fContinued"} PAGE 2.5 Injections 59 2.6 Skin Transplantation 61 2.7 Cardiac Transplantation 61 2.8 T Cell Proliferation Assays 62 2.9 Detection of Cytotoxic Alloantibody 63 2.10 Detection of Anti-Peptide Antibody 64 2.11 Determination of Class and Subclass of Anti-Aa Alloantibody 65 Chapter 3: The Immune Response to RTl .Aa-Derived Allopeptides in the RT1U Rat Strain 66 3.1 Introduction 67 3.2 The Immune Response Following Immunisation with Allopeptide 70 3.3 T Cell Proliferation Following Immunisation with the Intact RTl .Aa Class I Molecule 76 3.4 Discussion 81 Chapter 4: The Influence of Aa Allopeptide Immunisation on the Immune Response to the Intact Aa Class I Molecule 85 4.1 Introduction 86 4.2 The Immune Response to an R8 Blood Transfusion 91 4.3 The Immune Response to an R8 Cardiac Allograft 97 4.4 Discussion 104 - v - CONTENTS (Continued) PAGE Chapter 5: The Immune Response to the Intact Aa Molecule Following Intravenous Immunisation with Allopeptide 107 5.1 Introduction 108 5.2 The Immune Response to an R8 Blood Transfusion 111 5 .3 The Immune Response to an R8 Cardiac Allograft 116 5.4 Discussion 126 Chapter 6: Discussion 130 Chapter 7: References 141 - vi - ILLUSTRATIONS AND TABLES: PAGE Table 1.1 The Nomenclature of the MHC Class I and Class II Genes in the Human, Mouse and Rat 4 Figure 1.1 Genomic Organisation of the Human, Mouse and Rat MHC Regions 5 Figure 1.2 Schematic Representation of MHC Class I and Class II 7 Figure 1.3 The Crystal Structure of MHC Class I and Bound Peptide 9 Figure 1.4 Schematic Representation of the TCR Complex 20 Figure 1.5 Schematic Representation of the Initiating Events in T Cell Activation 25 Figure 1.6 Schematic Representations of the Pre-BCR and BCR Complexes 32 Figure 1.7 Schematic Representation of Direct and Indirect T Cell Allorecognition 38 Figure 1.8 Mechanisms of T-B Cell Collaboration for Alloantibody Production 44 Table 2.1 The Amino Acid Sequences of Synthetic Aa-Derived Allopeptides 60 Figure 3.1a The Nature of the Amino Acid Disparity Between the PVG-R8 and PVG-RT1U Rat Strains 68 Figure 3.1b The Linear Positions of the Synthetic Aa-Derived Allopeptides Used in This Study 68 Figure 3.5 Cognate T and B Cell Collaboration for the Production of Anti-Peptide Antibodies 74 Figure 4.1 The Mechanism by Which Aa Allopeptide Priming Accelerates the Alloantibody Response to the Intact Aa Molecule 90 - vii - ACKNOWLEDGEMENTS I would like to express my gratitude to the National Kidney Research Fund (NKRF), whose generous funding has allowed this research to take place. For their kind supervision and the opportunity they afforded me to study my Ph.D. in their laboratories, I would also like to thank Dr. Eleanor Bolton and Professor Andrew Bradley. I am very grateful to all the people that helped me with laboratory and animal handling techniques. In particular, I would like to thank Catherine MacDonald, Dr. Hilary Marshall, Dr. Alistair Gracie, Mr. John Casey and the staff of the CRF, Glasgow University, and the CBS, Cambridge University. I must also pass on my thanks to Alan MacIntyre and Dr. Gabsi Brons for their help with radioactive substances. My thanks are also extended to the Medical Illustration Department, Addenbrooke’s Hospital, for their help with photography. I would also like to say to all the lovely people that I have been so fortunate to meet and work with during my period of study, both in the Transplantation Laboratories in the Western Infirmary, Glasgow, and in the Department of Surgery, Addenbrooke’s Hospital, Cambridge, thank you for your kind friendship. In particular, Julie, without whose constant supply of e-mails, I think that I would have gone slowly, but surely, mad.... Thank you also to Vicky Lovegrove for her kind proofreading of this manuscript. Finally, I am indebted to Mr. Gavin Pettigrew, not only for the seemingly endless heart transplants that he performed for me, but also, more importantly, for the continual support, understanding and encouragement that he has given me over the past few months, thank you. DECLARATION I declare that the conceptual ideas and experimental design for the work presented in this thesis were conceived by the author, Miss Emma Lovegrove and Professor JA Bradley. The experimental procedures described in this thesis were undertaken solely by the author, with the exception of the rat cardiac allograft transplants, which were performed by Mr. Gavin Pettigrew. I confirm that all of the work presented in this thesis is original and has not been submitted for another degree at this or any other University. Some of the results described in Chapters 3 and 4 were presented orally at the British Transplantation Society’s Annual meeting in November 1997: “Analysis of Class I MHC Epitopes that Provide Cognate T Cell Help for Alloantibody-Mediated Graft Rejection.” E Lovegrove, G Pettigrew, EM Bolton, JA Bradley. Some of the results described in Chapters 4 and 5 were presented orally at the British Transplantation Society’s Annual meeting in April 1999: “The Manipulation of CD4-Dependant Alloantibody Responses to Class I MHC by Allopeptides.” E Lovegrove, G Pettigrew, EM Bolton, JA Bradley. In addition, some of the results described in Chapter 4 were used for publication: “Indirect T Cell Allorecognition and Alloantibody-Mediated Rejection of MHC Class I-Disparate Heart Grafts.” GJ Pettigrew, E.

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