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CD56+ T-Cells in Relation to Cytomegalovirus in Healthy Subjects and Kidney Transplant Patients

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CD56+ T-Cells in Relation to Cytomegalovirus in Healthy Subjects and Kidney Transplant Patients CD56+ T-cells in Relation to Cytomegalovirus in Healthy Subjects and Kidney Transplant Patients Institute of Infection and Global Health Department of Clinical Infection, Microbiology and Immunology Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy by Mazen Mohammed Almehmadi December 2014 - 1 - Abstract Human T cells expressing CD56 are capable of tumour cell lysis following activation with interleukin-2 but their role in viral immunity has been less well studied. The work described in this thesis aimed to investigate CD56+ T-cells in relation to cytomegalovirus infection in healthy subjects and kidney transplant patients (KTPs). Proportions of CD56+ T cells were found to be highly significantly increased in healthy cytomegalovirus-seropositive (CMV+) compared to cytomegalovirus-seronegative (CMV-) subjects (8.38% ± 0.33 versus 3.29%± 0.33; P < 0.0001). In donor CMV-/recipient CMV- (D-/R-)- KTPs levels of CD56+ T cells were 1.9% ±0.35 versus 5.42% ±1.01 in D+/R- patients and 5.11% ±0.69 in R+ patients (P 0.0247 and < 0.0001 respectively). CD56+ T cells in both healthy CMV+ subjects and KTPs expressed markers of effector memory- RA T-cells (TEMRA) while in healthy CMV- subjects and D-/R- KTPs the phenotype was predominantly that of naïve T-cells. Other surface markers, CD8, CD4, CD58, CD57, CD94 and NKG2C were expressed by a significantly higher proportion of CD56+ T-cells in healthy CMV+ than CMV- subjects. Functional studies showed levels of pro-inflammatory cytokines IFN-γ and TNF-α, as well as granzyme B and CD107a were significantly higher in CD56+ T-cells from CMV+ than CMV- subjects following stimulation with CMV antigens. This also resulted in higher levels of proliferation in CD56+ T-cells from CMV+ than CMV- subjects. Using class I HLA pentamers, it was found that CD56+ T-cells from CMV+ subjects contained similar proportions of antigen-specific CD8+ T cells to CD56- T cells in healthy donors of several different HLA types. A comprehensive gene expression profile by microarrays of CMV-stimulated sorted CD56+ T-cells was conducted which showed that expression of 106 genes involved in innate and adaptive immune responses was significantly changed, almost all being increased. Some of these changes were validated via RT-PCR and flow cytometry, the latter indicating higher ISG-15 protein expression in response to CMV stimulation in CMV+ than CMV- subjects. Overall, these differences may reflect the expansion and enhanced functional activity of CMV-specific CD56+ memory T cells. In view of the link between CD56 expression and T-cell cytotoxic function, this strongly implicates CD56+ T cells as being an important component of the cytotoxic T-cell response to CMV. - 2 - Dedication I dedicate this work to my mother (Salha) and my father (Janakiram et al.) who pray for me constantly and inspire me. A dedication also goes to my wife (Khadija) and my sons (Aser) and (Moath) who were always inspiring me with their smiles throughout my PhD. Also, special thanks to Taif University and the Saudi Cultural Buereau for their support throughout my scholarship years. Acknowledgment All Thanks to Allah for his countless blessing. I would like to express my sincere appreciation and thanks to my supervisor Dr. Steve Christmas, you have been a remarkable mentor. I would like to thank you for your assisting thoughout my PhD. I would also like to thank my second supervisor Dr Brian Flangan for his invaluable assistance to the study, and thanks to the Department of Women’s and Children’s Health of Alder Hey Children’s Hospital. Sincere gratitude to Sally Heyworth, Mr Abdul Hammad and all renal transplant recipients from Royal Liverpool and Broadgreen University Hospital Transplant Unit for their participation in this study. I am also indebted to Ms Caroline Broughton, Dr Shamsher Ahmed, Dr Suliman Alomar, Dr Waleed Mahallawi, Dr Ali Alejenef, Dr Debbie Howarth and Dr Raju Gautam for their support provided at all levels during this thesis. I am also grateful to all members of the staff of the Department of Clinical Infection, Microbiology and Immunology in IGH, Liverpool University for being cooperative during this course. A special thanks to Dr. Lucille Rainbow, Centre for Genomic Research, IIB for her vital contribution. Thanks also to Ayman Mubarak, Abdullah Aljurayyan, Mustafa Halawi, Wael Alturaiki, and Zaid Al-bayati for their invaluable friendship. Finally, a special thanks to my family. Words cannot express how grateful I am to my mother (Salha), father (Janakiram et al.), wife (Khadija), sons (Aser and Moath), brothers and sisters for all of the help and support provided. Your prayers for me were what sustained me thus far. - 3 - List of Publications Almehmadi M, Flanagan BF, Khan N, Alomar S, Christmas SE. Increased numbers and functional activity of CD56⁺ T cells in healthy cytomegalovirus positive subjects. Immunology. 2014 Jun;142(Varnum et al.):258-68. Al Omar S, Flanagan BF, Almehmadi M, Christmas SE. The effects of IL-17 upon human natural killer cells. Cytokine. 2013 Apr;62(1) 123-30. Manuscript under preparation Almehmadi M, Hammad A, Heyworth S, Christmas SE. CD56+ T cells are increased in kidney transplant patients following cytomegalovirus infection. - 4 - Table of Contents Abstract 2 Dedication 3 Acknowledgements 3 List of publications 4 Table of contents 5 List of figures 13 List of tables 18 Abbreviations complete list 19 1. Introduction 22 1. Summary of the immune system 23 1.1 Antigen presentation 24 1.2 T-lymphocytes 25 1.2.1 CD4 + T-cells 25 1.2.2 CD8+ T-cells 28 1.2.3 γδ-T-cells 28 1.2.4 Natural Killer cells (NK cells) 29 1.3 B-Lymphocytes 29 1.4 CD56+ T cells (CD56+CD3+ cells) 29 1.4.1 History of CD56+ T-cells 29 1.4.2 CD56+ T-cell functional capability combines that of T-cells 34 and NK cells 1.4.3 CD56 neural adhesion molecule (NCAM) 34 - 5 - 1.4.4 CD56+ T cells functions in infections 34 1.5 Human Cytomegalovirus (HCMV) 40 1.5.1 Cytomegalovirus Transmission, Latency and Pathogenicity 42 1.5.1.1 Transmission 42 1.5.1.2 Latency 44 1.5.1.3 HCMV Pathogenicity 45 1.5.1.3.a Solid Organ Transplantation (SOT) 45 1.5.2 Immune response to HCMV 49 1.5.2.1 Innate Immunity 49 1.5.2.2 Natural Killer cells (NK) 49 1.5.2.2.a Missing self-hypothesis (Reduction in MHC-I expression) 52 1.5.2.2.b Self-cells expression of self-induced Ligands 52 1.5.2.3 Adaptive Immune response 53 1.5.2.3.1 Humoral Response to CMV 53 1.5.2.3.2 T-cell-mediated immune responses 54 1.5.2.3.2.a γδT-cells in response to CMV 54 1.5.2.3.2.b CD8+T-cells in response to CMV 54 1.5.2.3.2.c CD4+ T cells in response to CMV 57 1.5.3 CMV immune-evasion 59 1.5.3.1 CMV genes homologue to the host genes 60 1.5.3.2 CMV evading detection by Cytotoxic T cells 61 1.6 Kidney Transplant Rejection 61 1.6.1 T cells and B cells interface and generation of antibodies 63 1.6.2 T-cell role response in graft rejection and graft tolerance 64 1.6.2.1 The allorecognition process 65 - 6 - 1.6.2.1.a Direct allorecognition pathway 66 1.6.2.1.b Indirect allorecognition pathway 67 1.6.2.1.c Semi-direct allorecognition pathway 68 1.6.3 NK cell allorecognition 69 1.6.4 Development of Alloresponses 70 1.6.4.1 The alloresponse of CD4+ T-cells 71 1.6.4.2 The alloresponse of CD8+ T -cells 71 1.6.5 CD56+ T-cells immunological role to Graft 72 1.6.6 The role of CMV in allograft rejection 73 1.7 Aims and objectives 74 2. Materials and Methods 76 2.1 CMV IgG ELISA 76 2.1.1 CMV IgG ELISA assay Principle 76 2.1.2 Reagents and materials supplied and preparation 76 2.1.3 ELISA assay procedure and measurement 77 2.2 CMV lysate preparation 78 2.2.1 Fibroblast Cell Culture 78 2.2.2 CMV lysate preparation protocol 79 2.3 Peripheral blood 80 2.3.1 PBMC Donors 80 2.3.2 PBMC Isolation 81 2.3.3 Counting of PBMCs 82 2.4 Flow cytometry (FACSCalibur™ BD Biosciences) 82 2.4.1 Surface staining for phenotypic analysis 83 - 7 - 2.4.2 Functional analysis of CD56+ T-cells 85 2.4.2.1 Intracellular staining of CD56+ T-cells 85 2.4.2.2 Degranulation assay of CD56+ T-cells; detection by LAMP-1 87 (CD107a) expression 2.4.2.2.1 MHC negative targets 87 2.4.2.3.2 PBMC stimulation by K562 cells and CMV lysate 88 2.4.2.4 Proliferation assay of CD56+ T-cells 89 2.4.2.4.1 Carboxy-Fluorescein Succinimidyl Ester (CFSE) assay 89 2.4.2.4.2 PBMC CFSE labelling 90 2.4.2.4.3 PBMC stimulation by CMV lysate and SEB 90 2.4.2.5 CD56+ T-cell CMV specificity detection 92 2.4.2.5.1 HLA tissue type 93 2.4.2.5.2 CD56+ T-cells Pro5 Pentamer staining 96 2.4.2.6 Identifying CD56+ T-cell gene expression profile in response to 96 CMV 2.4.2.6.1 CD56+ T-cell sorting 97 2.4.2.6.2 RNA extraction protocol 98 2.4.2.6.3 DNase-treatment of RNA 99 2.4.2.6.4 One-Colour Microarray-Based Gene Expression 100 2.4.2.6.5 Data Analysis of microarrays using GeneSpring GX9.0 101 2.4.2.6.6 Validation of Microarrays results 102 2.4.2.6.7 Synthesis of cDNA (Reverse Transcription) 103 2.4.2.6.8 RT-PCR gene expression analysis 103 2.4.2.6.9 Protein expression analysis 105 2.4.2.6.9a CD38 surface staining of CD56+ T-cells 106 - 8 - 2.4.2.6.9b ISG-15 intracellular staining of CD56+ T-cells 106 3.
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