T cells in Rheumatoid Arthritis By Esther Hidalgo A thesis submitted to The University of Birmingham For the Degree of DOCTOR OF PHILOSOPHY Institute of Biomedical Research, Medical school University of Birmingham June 2010 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. ABSTRACT Identification of the role of T cells and their interaction with other cell types remains a major challenge to our understanding of the pathogenesis of rheumatoid arthritis. In this study we have investigated the regulation of the response of T cells infiltrating the rheumatoid joint to IL-6. Furthermore we have investigated the level of T cell activation in the early stages of rheumatoid arthritis. Interleukin-6 is an important regulator of T cell differentiation and survival. It exerts its biological function by either directly binding to the complete IL-6 receptor consisting of CD126 CD130 or via transsignaling, when sIL6R-IL6 complexes bind to CD130. This study addresses the expression and regulation of these receptor components on the T cells infiltrating the rheumatoid joint. While compared to blood T cells, CD126 expression was found at low levels on synovial fluid and tissue T cells, expression of CD130 on synovial tissue T cells was comparable to that of blood T cells, with lower levels in synovial fluid T cells, both at protein and mRNA level. When exposed to sIL6R-IL6 complexes, tissue derived T cells responded with a higher level of STAT3 phosphorylation compared to cells incubated with IL-6, suggestive of transsignaling. High CD130 expression was demonstrable in T cells in the perivascular cuff area. Among a range of cytokines tested, IL-6 reduced CD126 and CD130 expression while IL-10, which is expressed at high levels in the perivascular infiltrate, induced expression of CD130. Taken together these data suggest that the inflammatory microenvironment maintains responsiveness to IL-6 transsignalling by cytokine driven CD130 expression on CD4 positive T cells. To address the question whether the role of T cells changes during the course of progression of RA, we analysed the expression of T cells activation markers on synovial fluid and peripheral blood T cells from patients at the very early stage of disease (within 3 months of disease onset) compared to patients with established or self resolving arthritis. Expression of CD69, CD71 and HLA-DR was upregulated on synovial fluid T cells compared to peripheral blood but there were no differences between the different groups of patients. Furthermore, we quantified the proportion of T cells expressing the invariant TCR Vα24Jα18 in synovial fluid and blood of the same groups of patients. We found a lower frequency of iNKT cells in the synovial fluid of very early arthritis patients compared to other patients. While this is a preliminary result, it suggests that there may be a role for these cells in the regulation of disease susceptibility. A mi padre, en su memoria AKNOWELEGEMENTS I would like to thank my supervisor Dagmar Scheel-Toellner for her trusting me with the project, for constant help and advice, for teaching me how to work in the lab, teaching me how to do presentations and for her final and extraordinary effort to make me finish writing this PhD thesis. I would like to thank my co-supervisor Karim Raza for his help and advice. I would like to thank Sarah Flavell for letting me benefit from her previous project with Dagmar. For her help around the lab and in the office, especially with reference manager. I would like to thank Professor Lord for making me a “TRIFFID” and Professors Salmon and Buckley for their support and helpful discussions. I would like to thank everyone that has helped me in the lab. Sian Lax introduced me to the world of immunohistochemistry and confocal microscopy, Debbie Hardie also help with the confocal work, especially when the microscope was temperamental. Fionna McConnell taught me how to analyze confocal images. I would like to thank Helen Macgetrick for her help and collaboration in the transmigration experiments. I would like to thank Andrew Thomas and Andy Filer for their help getting human samples. I would also like to thank Katherine Howlett who kindly takes care of the tissue samples and for all her help around the lab. I would like to thank Tony Lammas and Joanne Croudace for their advise and help with iNKT cells and for providing me with α-Gal-Cer. I would like to thank Hema Chahal for general help in and outside the lab, for her help with all chemical calculations and for many coffee breaks. And everybody I share laboratory and office with. I would like to thank Marie Curie Actions for funding my PhD A big thank you to my family, my sisters that are always there for me and Yolanda for taking care of me like if I was her own daugther. And Finally I would like to acknoeledge my father thanks to whom I have grown up to be sufficiently indenpendent and brave to embarq in adventures as big as a PhD. Table of contents Chapter 1 Page 1.1 The immune system…………………………………………………………………....…1 1.2 T cells…………………………………………………………………………...................3 1.2.1 T cell development, positive and negative selection…………………………………….4 1.2.2 T regs…………………………………………………………………………………….8 1.2.3 iNKT cells…………………………………………………………………………...….9 1.2.3.1 iNKT cell development………………………………………………………………10 1.2.3.2 iNKT cell activation and function……………………………………………..…..11 1.2.4 T cell activation markers……………………………………………………………….13 1.3 Cytokines………………………………………………………………………………...13 1.3.1 IL-6 family of cytokines………………………………………………………….……14 1.3.2. IL-6…………………………………………………………………………………….14 1.3.2.1 IL-6, functions………………………………………………………………….….…15 1.3.2.2 IL-6 signalling………………………………………………………………..……...15 1.3.2.3 Regulation of IL-6R expression……………………………………..……………..17 1.3.2.4 IL-6 and signaling regulation………………………………………..…………….18 1.3.1.5 IL-6, chronic inflammation ………………………………………….………….....19 1.3.2 Interleukin 10 (IL-10)……………………………………………………………..……20 1.3.2.1 The IL-10 Receptor…………………………………………………….…………....21 1.3.2.2 IL-10, Biological activity…………………………………………………….…......21 1.4 Inflammation…………………………………………………………………….….23 1.4.1 Acute Inflammation……………………………………………………………………23 1.4.2 Chronic Inflammation………………………………………………………………….24 1.4.3 Resolution of Inflammation……………………………………………………...….…26 1.5 Rheumatoid arthritis (RA) and classification criteria …………........27 1.5.1 Immunobiology and origin of Rheumatoid arthritis…………………………………...29 1.5.2 The synovium, in health and in RA……………………………………………………31 1.5.3 RA, a two phase disease ………………………………………………………………33 1.5.4 T cells in RA……………………………………………………………………...……34 1.5.4.1 T cell distribution and phenotype in the rheumatoid synovium……………..…34 1.5.4.2 Potential T cell antigens in the synovium……………………………………..…35 1.5.4.3 Interactions of T cells with other cells in the rheumatoid joint……………..…35 1.5.4.4 Role of the newly identified Th17 T cell subset in RA…………..................….36 1.5.4.5 Regulatory T cells in RA…………………………………………………….……...37 1.5.5 iNKT cells and autoimmune disease……………………………………………….….38 1.5.6 NKT cells and RA……………………………………………………….................….39 1.5.7 IL-6 and RA…………………………………………………………...………….....…41 1.5.8 IL-10 and RA……………………………………………………………………….….42 1.6 Cytokine targeting therapies of RA…………………………………………….....…...43 1.6.1 B cell depletion ……………………………………………………….....…………......44 1.6.2 TNF targeting therapies……………………………………………….....................…..45 1.6.3 IL-10 as a therapeutic agent…………………………………………….…………...…46 1.6.3 IL-6 as a therapeutic target for RA……………………………………………………..46 1.7 RA therapies targeting T cells……………………………………………………....….47 1.8 Project rationale………………………………………………………….…………..…48 1.9. Project aims……………………………………………………………….…………....49 Chapter 2…………………………………………………………………..…..50 2. MATERIALS AND METHODS………………………………………………………..50 2.1 Patients………………………………………………………………………………......50 2.2 Isolation of PBMC’s by density gradient centrifugation…………………………......50 2.3 Isolation of CD4 T cells by cell-sorting from PBMCs and STMCs……………….....52 2.4 Isolation of CD45RO CD4 positive T cells………………………………………...…..52 2.5 Processing of ST samples to obtain STMC’s…………………………………...…......53 2.6 Flow cytometry…………………………………………………………………...……..55 2.6.1. Three-color flow cytometry to quantify iNKT cells and determine T cells activation status………………………………………………………………………………………….55 2.6.2. Three-color flow cytometry to determine the expression of CD130 and CD126 on freshly isolated CD4 T cells…………………………………………………………...……..56 2.6.3 Four colour flow cytometry to investigate the expression of CD126 and CD130 on CD45RO positive CD4 T cells from PB, SF and ST………………………………….....…..58 2.7 Cytokine CD4 T cell treatments…………………………………………………...…...58 2.8 PBMC, SFMC and STMC treatment………………………………………………….59 2.9 Tissue sectioning………………………………………………………………………...59 2.10 Immunohistochemistry Staining…………………………………………………...…60 2.11 Quantification of CD130 signal on CD4 T cells within and outside the perivascular cuffs in ST sections of RA patients…………………………………………………….…..61 2.12 FACS staining for phosphorylated STAT3…………………………..……………....61 2.13 RNA extraction. …………………………………………………………………...…..62 1.14 Reverse transcription of RNA to produce cDNA……………………………………63 2.15 cDNA purification………………………………………………………..................…63 2.16 Real Time Polymerase Chain Reaction (RT-PCR)………………………………….63 2.16.1 RT-PCR Analysis………………………………………………………………...…...64