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1.3 the CRAC Channel

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1.3 the CRAC Channel CRAC channel related proteins in the pathogenesis of inborn errors of immunity Laura Jane Rice Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds School of Medicine and Health Under the supervision of Rashida Anwar PhD and Sinisa Savic MD, PhD June 2021 Publication statement The candidate confirms that the work submitted is her own, except where work which has formed part of jointly-authored publications has been included. The contribution of the candidate and the other authors to this work has been explicitly indicated. The candidate confirms that appropriate credit has been given within the thesis where reference has been made to the work of others. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. The right of Laura Jane Rice to be identified as Author of this work has been asserted by her in accordance with the Copyright, Designs and Patents Act 1988. © 2021 The University of Leeds and Laura Jane Rice ii Acknowledgements This work was funded by University of Leeds and CSL Behring (West Sussex, UK). I would like to thank my supervisors for their continued support. Rashida Anwar for her scientific knowledge, detail driven questions and help during the writing of this thesis. Sinisa Savic for his encouragement and clinical and immunology expertise. The Anwar group and Level 9 WTBB PhD students made this experience enjoyable with coffee and gin. I was fortunate to have collaborators with experience in CRAC channel research. The Gwack lab made the stable cell line work possible and I am so grateful for the opportunely to spend three months in LA. The McKeown group at the University of Leeds helped with the optimisation of calcium flux. The Clinical Immunology and Allergy Department at Leeds Teaching Hospitals provided patient data and expertise. Lastly, I would like to thank Christopher and my greyhound Lucas for their moral support and cuddles. iii Abstract The aim of this project was to identify novel mutations in proteins causing immunodeficiency. This was achieved by confirming whole exome sequencing (WES) results of potential mutations and genes of interest through Sanger sequencing and allelic orientation of mutations. Functional experiments were then designed and performed on patient samples and stable cell lines expressing patient mutants. The experiments examined the known functions of the proteins of interest and cell types that appeared to be affected in the patient immunological profile. The main topic of this thesis is centered around an index case who presented at age 17 with a life-long history of recurrent sinopulmonary infections and established bronchiectasis. Immunological investigations showed severe panhypogammaglobulinaema, marginally reduced CD4 count and reduced T cell proliferative responses. WES identified potentially damaging mutations in Ca2+-release activated Ca2+ regulator 2A (CRACR2A): c.430 A>G (p.144R>G) and c.898 G>T (p.300E>*) which were inherited via the maternal line; c.834 G>T (p.278E>D) in the paternal allele. In primary patient samples, T cells had reduced calcium flux, cytokine production and p-JNK. The two mutant patient alleles were expressed using retroviral transduction in the CRACR2A knock-out Jurkat cell line. Both alleles resulted in reduced calcium flux and IL-2 expression when compared to wild-type CRACR2A. The maternal mutant allele expression produced a truncated protein (resulting from the p.300E>* mutation), with abnormal localisation and significantly reduced p-JNK. This truncated protein was not seen in the primary cells, and so is not likely to be expressed in the patient. The data suggests that biallelic mutations in CRACR2A can lead to primary immunodeficiency, which affects T cell related function. This is the first time CRACR2A has been linked to disease. The identification of novel mutations in patient with primary immunodeficiency and using functional experiments to see their pathogenic effect in patient samples was also done in patients with STIM1 and STING mutations. iv Table of Contents CRAC channel related proteins in the pathogenesis of inborn errors of immunity ........................... i Publication statement ................................................................................................................................... ii Acknowledgements ..................................................................................................................................... iii Abstract ........................................................................................................................................................ iv Table of Contents .......................................................................................................................................... v List of Tables ................................................................................................................................................ ix List of Figures ............................................................................................................................................... x Abbreviations .............................................................................................................................................. xii Chapter 1 Introduction.................................................................................................................................. 1 1.1 Immune cell lineage........................................................................................................................... 1 1.1.1 T cells ....................................................................................................................................... 2 1.1.2 B cells....................................................................................................................................... 4 1.1.3 T and B cell interaction ............................................................................................................. 6 1.1.4 Monocytes and macrophages .................................................................................................. 7 1.2 Primary immunodeficiency ................................................................................................................ 8 1.2.1 Why study PID ......................................................................................................................... 9 1.2.2 Types of PID .......................................................................................................................... 10 1.2.3 Current Therapies for PID ...................................................................................................... 13 1.2.4 Molecular screening of patients .............................................................................................. 14 1.3 The CRAC channel.......................................................................................................................... 15 1.3.1 CRAC channel ....................................................................................................................... 15 1.3.2 Role in the immune system .................................................................................................... 16 1.3.3 CRAC channel deficiencies .................................................................................................... 20 1.4 Aims ................................................................................................................................................ 26 Chapter 2 Materials and Methods .............................................................................................................. 27 2.1 Materials .......................................................................................................................................... 27 2.1.1 Cell lines................................................................................................................................. 33 2.1.2 Patients and Healthy controls ................................................................................................. 35 v 2.2 Methods ........................................................................................................................................... 36 2.2.1 Molecular Biology ................................................................................................................... 36 2.2.2 Clinical Immunology ............................................................................................................... 44 2.2.3 Blood work ............................................................................................................................. 44 2.2.4 Transient transfection methods .............................................................................................. 49 2.2.5 Generation of stable cell lines ................................................................................................ 52 2.2.6 Protein studies ....................................................................................................................... 57 2.2.7 T cell functional studies .......................................................................................................... 60 2.2.8 Calcium flux ...........................................................................................................................
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