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Clinical and Immunological Diversity Defects of Recombination Hanna IJspeert Clinical and immunological diversity of recombination defects Hanna IJspeert 2014 Clinical and Immunological Diversity of Recombination Defects Hanna IJspeert The research for this thesis was performed within the framework of the Erasmus Postgraduate School Molecular Medicine. The studies described in the thesis were performed at the Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, the Netherlands and collaborating institutions. The studies were financially suported by ‘Sophia Kinderziekhuis Fonds’ (grant 589). The printing of this thesis was supported by Erasmus MC, Stichting Kind & Afweer, CSL Behring and BD Biosciences. ISBN: 978-94-91811-04-3 Illustrations: Sandra de Bruin-Versteeg, Hanna IJspeert Cover: Stephanie van Brandwijk Lay-out: Caroline Linker Printing: Haveka B.V., Alblasserdam, the Netherlands Copyright © 2014 by Hanna IJspeert. All rights reserved. No part of this book may be reproduced, stored in a retrieval system of transmitted in any form or by any means, without prior permission of the author. Clinical and Immunological Diversity of Recombination Defects Klinische en immunologische diversiteit van recombinatie defecten Proefschrift ter verkrijging van de graad van doctor aan de Erasmus Universiteit Rotterdam op gezag van de rector magnificus Prof.dr. H.A.P. Pols en volgens besluit van het College voor Promoties. De openbare verdediging zal plaatsvinden op woensdag 19 maart 2014 om 13.30 uur door Hanna IJspeert geboren te Dordrecht PROMOTIE COMMISSIE Promotoren Prof.dr. J.J.M. van Dongen Prof.dr. A.J. van der Heijden Overige leden Prof.dr. B.H. Gaspar Prof.dr. F.J.T. Staal Dr. D.C. van Gent Copromotoren Dr. M. van der Burg Dr. N.G. Hartwig CONTENTS PART 1 GENERAL INTRODUCTION CHAPTER 1 9 General introduction PART 2 NEW NONHOMOLOGOUS END-JOINING DEFECT CHAPTER 2 43 A DNA-PKcs mutation in a radio sensitive T-B- SCID patient inhibits Artemis activation and nonhomologous end-joining J. Clin. Invest. 2009; 119(1):91-98 PART 3 CLINICAL SPECTRUM OF IMMUNODEFICIENCY WITH RESIDUAL V(D)J RECOMBINATION ACTIVITY CHAPTER 3.1 69 Idiopathic CD4+ T lymphopenia without autoimmunity or granulomatous disease in the slipstream of RAG mutations Blood 2011; 117(22):5892-6 CHAPTER 3.2 83 Similar RAG mutations result in similar immunobiological effects but in different clinical phenotypes J Allergy Clin Immunol. 2014; in press CHAPTER 3.3 105 Artemis splice defects cause atypical SCID and can be restored in vitro by an antisense oligonucleotide Genes Immun. 2011;12(6):434-44 CHAPTER 3.4 131 Clinical spectrum of LIG4 deficiency is broadened with severe dysmaturitas, primordial dwarfism and neurological abnormalities Hum Mutat.2013;34(12):1611-4 PART 4 MECHANISM OF REPERTOIRE DEVELOPMENT CHAPTER 4.1 145 XLF deficiency results in impaired generation of junction diversity by TdT Manuscript in preparation CHAPTER 4.2 163 Antibody deficiency in Ataxia Telangiectasia is caused by disturbed B and T cell homeostasis and reduced immune repertoire diversity J Allergy Clin Immunol. 2013; 131(5):1367-75 PART 5 GENERAL DISCUSSION CHAPTER 5 195 General discussion ADDENDUM Tables with PCR primers and Taqman probes 231 Abbreviations 235 Summary 239 Samenvatting 243 Dankwoord 247 Curriculum Vitae 251 PhD Portfolio 253 Publications 257 Chapter 1 General Introduction General Introduction GENERAL INTRODUCTION 1 The immune system is capable to detect a large variability of pathogens and distinguish them from the body’s own healthy tissue. The immune system consists of the innate- and adaptive immune system. The adaptive immune system consists of B-and T-lymphocytes, which can recognize pathogens with an antigen-specific receptor. B-lymphocytes can adapt their responses during an infection to improve recognition of the pathogen and they generate long-term immunological memory. These antigen-specific receptors are called B-cell receptor (BR) and T-cell receptor (TR), respectively. Since the number of pos- sible antigens is innumerable, an enormous diversity of the antigen-specific receptors is required. To achieve this, the BR and TR contain a variable domain that is unique for each individual T or B cell. This variable domain is generated by recombination of the antigen receptor genes in a process which is called V(D)J recombination. This process requires lymphoid specific proteins to generate DNA double strand breaks (DSB), but is also depen- dent on a common DNA repair pathway to repair the DSB. Defects in V(D)J recombination hamper the production of the antigen-specific receptors, which results in a complete or partial block in B- and T-cell differentiation, leading to a combined B- and T-cell deficiency. In this General Introduction the B and T-cell development will be described, with spe- cial focus on the generation of the antigen-specific receptor repertoire. The role of DNA repair during the V(D)J recombination process and during further maturation of the B cells will be highlighted. Subsequently, clinical and immunological aspects of V(D)J recombina- tion and DNA repair defects resulting in immunodeficiency will be addressed. Finally, the aims of this thesis will be outlined. IMMUNOGLOBULIN AND T-CELL RECEPTOR MOLECULES B-cell receptor The BR, also called immunoglobulin (IG) is composed of two identical heavy chains (IGH) and two identical light chains, either IGκ or IGλ (Figure 1A). The heavy and light chains all consist of a variable and a constant domain. The variable domain of the IG is encoded by a combination of one the V, D and J genes (heavy chain) (Figure 1B), or by a combination of the available V, and J genes (IGK or IGL). The IGH locus contains 9 constant genes (Cμ, Cδ, Cγ3, Cγ1, Cα1, Cγ2, Cγ4, Cε and Cα2), which have different effector functions. Naive B cells express Cμ and Cδ, but upon encountering an antigen the effector function can be changed, by replacing the constant gene during a process called class switch recombina- tion (CSR). 11 Chapter 1 T-cell receptor A TR molecule consists of two chains, either a TRα and a TRβ chain (TRαβ) or a TRγ and a TRδ chain (TRγδ) (Figure 1C). The vast majority of mature T lymphocytes (85-95%) expresses TRαβ; a minority expresses TRγδ (5-15%).1 The variable domain of the TR is encoded by a combination of one of the many variable (V), diversity (D) and joining (J) genes (TRB en TRD loci), or by a combination of the available V and J genes (TRA and TRG loci).1, 2 The constant domains of the TR chains are encoded by constant (C) genes. B-CELL DEVELOPMENT Precursor B cells derive from multipotent hematopoietic progenitors and develop in the bone marrow into immature B cells (Figure 2). This differentiation occurs in a stepwise manner and the main objective is to create a unique IG molecule. At the immature B-cell stage, the IG molecule is tested for functionality without high affinity for auto-antigens, after which they go to the periphery and become transitional B cells. The transitional B cells are immature in their migration capacity and response to antigen, but develop rapidly into naive mature B cells. After B cells have encountered antigen, they can further mature V V A D IgH IgH D C J J V V C C TR α TRβ TR γ TRδ J J IgL IgL C C C C V V V V D D C C J J J J b C C C C 9 a 7 9 7 C C D D CD3 C CD3 C ε CD3 CD3 ε CD3 CD3 γ δ γ δ CD3 CD3 CD3 CD3 ξ ξ ξ ξ B-lymphocyte T-lymphocyte T-lymphocyte B VH DH JH Cµ Cδ Cγ3 Cγ1 Cα1 Cγ2 Cγ4 Cε Cα2 ψε ψγ 1 70 1 27 1 2 3 4 5 6 sµ Sγ1 Sγ3 Sα1 Sγ2 Sγ4 Sα2 Figure 1. Composition and generation of T- and B-cell receptors. A) The B-cell receptor (BR) consist of two heavy (IGH) and two light chains (IGL). B) Schematic overview of IGH locus including the constant genes. C) The T-cell receptor (TR) consist of either a TRα and TRβ chain or a TRγ and TRδ 12 General Introduction in a T-cell dependent or T-cell independent manner and become an antibody secreting 1 plasma cell or a memory B cell. IG rearrangements During precursor B-cell differentiation, V(D)J recombination starts with the incomplete DH-JH rearrangements on both IGH loci (Figure 3),3, 4 which occur at the pro-B cell stage (Figure 2).5-7 Subsequently, in the pre-B-I cell stage only one allele continues with the V to DJ rearrangement, and the second allele only rearranges when the first is not productive (e.g. out of frame or with a stop codon). Once a VDJ exon is formed, RNA transcripts are produced and the exon is spliced to the Cμ exon. If the resulting Igμ heavy chain is capable of pairing with the surrogate light chain proteins λ14.1 and VpreB, this pre-B-cell receptor complex (pre-BcR) is expressed on the plasma membrane of the cell at the large pre-B-II cell stage. Expression of a functional pre-BcR is an important checkpoint in precursor B-cell dif- ferentiation.8, 9 Expression of the pre-BcR receptor induces several processes, which include allelic exclusion and induction of proliferation. As soon as the pre-BcR complex is down regulated, proliferation will be limited and rearrangement of the IG light chains is initiated, and cells will progress to the small pre-B-II cell stage. First, a Vκ will rearrange to a Jκ, and if this junction is not productive, subsequent Vκ-Jκ rearrangements can take place until a functional Vκ-Jκ junction is formed.
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