Advances in Experimental Medicine and Biology 1254

B Cells in Immunity and Tolerance

Ji-Yang Wang   Editor

Advances in Experimental Medicineand Biology

Volume 1254

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Ji-Yang WangEditor

B Cells in Immunityand Tolerance

123

EditorJi-Yang WangDepartment of ImmunologySchool of Basic Medical SciencesFudan UniversityShanghai, China

ISSN 0065-2598 ISSN 2214-8019 (electronic)Advances in Experimental Medicine and BiologyISBN 978-981-15-3531-4 ISBN 978-981-15-3532-1 (eBook)https://doi.org/10.1007/978-981-15-3532-1

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Preface

B cells produce antibodies that are indispensable for host defence againstinfections, through virus neutralization, opsonization of pathogens for effi-cient phagocytosis by macrophages and antibody-dependent cellular cyto-toxicity. Antibodies are not just effector molecules, but also regulate humoralimmune responses, both positively and negatively, through their cellularreceptors such as Fc receptors. Moreover, B cells may also function tosuppress immune reactions by secreting anti-inflammatory cytokines. Thisbook aims to provide a comprehensive overview of B cell development,maturation, activation and differentiation, and of human diseases caused by Bcell abnormalities.

B cell development in the bone marrow is accompanied byimmunoglobulin (Ig) gene rearrangements and generates a diverse pool ofimmature B cells. Those reactive with self-antigens are deleted or func-tionally inactivated, and the remaining immature B cells migrate to theperiphery to mature. In Chap. 1, Ying Wang et al. discuss the mechanisms ofIg gene rearrangement, regulation of B cell development and maturation, theelimination/inactivation of autoreactive B cells (B cell tolerance), and B-1cell development and function.

Mature B cells in the periphery can be activated by antigen stimulation,toll-like receptor signaling and/or T cell help. Antigen binding to the BCR isa critical step to initiate B cell activation. In Chap. 2, Shinya Tanaka andYoshihiro Baba provide a current update on positive and negative regulationof BCR signaling, focusing on the coordinated function of tyrosine kinases,adaptor molecules and phosphatases. Another important regulator of BCRsignaling is reactive oxygen species (ROS). In Chap. 3, Takeshi Tsubatadescribes the latest findings on the regulation of ROS generation during BCRsignaling and its role in B cell survival and activation.

B cells activated by antigen stimulation and in the presence of T cell helpform germinal centres (GCs). Here, they undergo Ig V gene somatichypermutation (SHM) and class switch recombination (CSR) and finallydifferentiate into memory B or antibody-secreting plasma cells. In Chap. 4,Chuanxin Huang reviews the regulation of the initiation and maintenanceof the GC reaction, mechanisms of Ig gene SHM and CSR, and the selection

v

of high-affinity clones in the GC. In Chap. 5, Saya Moriyama et al. provide acomprehensive overview of the systemic and local memory B cell responsesin humoral protective immunity against pathogens. In addition, Wataru Iseand Tomohiro Kurosaki review the latest progress in the cellular andmolecular mechanisms of plasma cell differentiation from GC B cells inChap. 6.

During a humoral immune response, B cells typically produceantigen-specific IgM first and then IgG later. It is well known that the anti-gen–IgG complex can suppress BCR signaling and B cell activation byco-ligation of the BCR and FccRIIB. However, it is less clear whether theantigen-specific IgM has any role in B cell activation. In Chap. 7, Jun Liuet al. provide a comprehensive overview on the function of IgM, withemphasis on the role of the IgM Fc receptor (FclR). They also discuss therelative contribution of IgM–complement and IgM–FclR pathways in reg-ulating humoral immune responses.

Although there is still some controversy, it is now more appreciated thatthere exists a population of regulatory B cells (Bregs) that function to sup-press immune reactions by secreting anti-inflammatory cytokines such asIL-10. In Chap. 8, Luman Wang et al. summarize the phenotypes andfunctions of Bregs in both mice and humans.

Among the different classes of antibodies, IgA is the most abundantlyproduced antibody in the body (40–60 mg/kg body weight/day in humans)and plays an essential role in mucosal immunity. In Chap. 9, KeiichiroSuzuki gives a cutting-edge overview on the role of diverse IgA–bacteriainteractions in gut homoeostasis. His chapter also provides the latest infor-mation on the dynamics and maintenance of gut IgA, including the regulationof IgA–J chain interaction by a marginal zone B and B-1 cell-specific protein.

Chapters 10–12 focus on human diseases caused by B cell abnormalities.Qing Min et al. provide a detailed overview of different types of primaryantibody deficiencies (PADs) caused by abnormalities in the development,survival, activation or differentiation of B cells. They also review the clinicalmanifestations, the causal genes and the treatments of various PADs.Excessive B cell activation and/or breakdown of B cell tolerance can lead toautoantibody production and autoimmune diseases. In Chap. 11, Xiang Linand Liwei Lu provide an overview of dysregulated B cell responses and themechanisms underlying the development of autoimmunity. They also discussboth autoantibody-dependent and autoantibody-independent B cell functionsin autoimmune diseases and biological therapies targeting B cells. Finally,abnormalities in the genetic alterations experienced by B cells, including Iggene rearrangements, SHM and CSR, can lead to chromosomal transloca-tions, gene mutations and B cell malignancies. In Chap. 12, Xin Meng et al.summarize the morphology, immune phenotypes, clinical features, geneticdefects, treatments and prognosis of human B cell lymphomas.

vi Preface

This book covers as many aspects of B cells as possible in both mice andhumans. We hope that it will become a standard reference for both basicresearchers and clinicians. As the editor, I would like to express my sincerethanks to all of the authors, who have spared their precious time to contributeexcellent chapters, resulting in the timely completion of this exciting book.

Shanghai, China Prof. Ji-Yang Wangwang@fudan.edu.cn

Preface vii

Contents

1 B Cell Development and Maturation . . . . . . . . . . . . . . . . . . . . 1Ying Wang, Jun Liu, Peter D. Burrows, and Ji-Yang Wang

2 B Cell Receptor Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23Shinya Tanaka and Yoshihiro Baba

3 Involvement of Reactive Oxygen Species (ROS) in BCRSignaling as a Second Messenger . . . . . . . . . . . . . . . . . . . . . . . 37Takeshi Tsubata

4 Germinal Center Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47Chuanxin Huang

5 Memory B Cells in Local and Systemic Sites . . . . . . . . . . . . . . 55Saya Moriyama, Yu Adachi, Keisuke Tonouchi,and Yoshimasa Takahashi

6 Regulation of Plasma Cell Differentiation . . . . . . . . . . . . . . . . 63W. Ise and T. Kurosaki

7 Regulation of Humoral Immune Responses and B CellTolerance by the IgM Fc Receptor (FclR) . . . . . . . . . . . . . . . 75Jun Liu, Ying Wang, Qing Min, Ermeng Xiong,Birgitta Heyman, and Ji-Yang Wang

8 Regulatory B Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87Luman Wang, Ying Fu, and Yiwei Chu

9 Diversified IgA–Bacteria Interaction in Gut Homeostasis . . . . 105Keiichiro Suzuki

10 Primary Antibody Deficiencies . . . . . . . . . . . . . . . . . . . . . . . . . 117Qing Min, Xin Meng, and Ji-Yang Wang

11 B Cell-Mediated Autoimmune Diseases . . . . . . . . . . . . . . . . . . 145Xiang Lin and Liwei Lu

12 B Cell Lymphoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161Xin Meng, Qing Min, and Ji-Yang Wang

ix

Contributors

Yu Adachi Department of Immunology, National Institute of InfectiousDiseases, Shinjuku, Tokyo, Japan

Yoshihiro Baba Division of Immunology and Genome Biology, Depart-ment of Molecular Genetics, Medical Institute of Bioregulation, KyushuUniversity, Fukuoka, Japan

Peter D. Burrows Department of Microbiology, University of Alabama atBirmingham, Birmingham, AL, USA

Yiwei Chu Department of Immunology, School of Basic Medical Sciences,and Institutes of Biomedical Sciences, Fudan University, Shanghai, China

Ying Fu Department of Immunology, School of Basic Medical Sciences,and Institutes of Biomedical Sciences, Fudan University, Shanghai, China

Birgitta Heyman Department of Medical Biochemistry and Microbiology,Uppsala University, Uppsala, Sweden

Chuanxin Huang Shanghai Jiao Tong University School of Medicine,Shanghai, China

W. Ise Laboratory of Lymphocyte Differentiation, WPI ImmunologyFrontier Research Center, Osaka University, Osaka, Japan

T. Kurosaki Laboratory of Lymphocyte Differentiation, WPI ImmunologyFrontier Research Center, Osaka University, Osaka, Japan;Laboratory for Lymphocyte Differentiation, RIKEN Center for IntegrativeMedical Sciences (IMS), Yokohama, Kanagawa, Japan

Xiang Lin Department of Pathology and Shenzhen Institute of Research andInnovation, The University of Hong Kong, Hong Kong, China

Jun Liu Department of Immunology, School of Basic Medical Sciences,Fudan University, Shanghai, China

Liwei Lu Department of Pathology, The University of Hong Kong,Pokfulam, Hong Kong, China

Xin Meng Department of Immunology, School of Basic Medical Sciences,Fudan University, Shanghai, China

xi

Qing Min Department of Immunology, School of Basic Medical Sciences,Fudan University, Shanghai, China

Saya Moriyama Department of Immunology, National Institute of Infec-tious Diseases, Shinjuku, Tokyo, Japan

Keiichiro Suzuki Laboratory for Mucosal Immunity, Center for IntegrativeMedical Sciences (IMS), RIKEN, Kanagawa, Japan

Yoshimasa Takahashi Department of Immunology, National Institute ofInfectious Diseases, Shinjuku, Tokyo, Japan

Shinya Tanaka Division of Immunology and Genome Biology, Depart-ment of Molecular Genetics, Medical Institute of Bioregulation, KyushuUniversity, Fukuoka, Japan

Keisuke Tonouchi Department of Immunology, National Institute ofInfectious Diseases, Shinjuku, Tokyo, Japan;Department of Life Science and Medical Bioscience, Waseda University,Shinjuku, Tokyo, Japan

Takeshi Tsubata Department of Immunology, Medical Research Institute,Tokyo Medical and Dental University, Tokyo, Japan

Ji-Yang Wang Department of Immunology, School of Basic MedicalSciences, Fudan University, Shanghai, China

Luman Wang Department of Immunology, School of Basic MedicalSciences, and Institutes of Biomedical Sciences, Fudan University, Shanghai,China

Ying Wang Department of Immunology, School of Basic Medical Sciences,Fudan University, Shanghai, China

Ermeng Xiong Department of Immunology, School of Basic MedicalSciences, Fudan University, Shanghai, China

xii Contributors

1B Cell Development and Maturation

Ying Wang, Jun Liu, Peter D. Burrows, andJi-Yang Wang

Abstract

Since the identification of B cells in 1965(Cooper et al. 1965), three has been tremen-dous progress in our understanding of B celldevelopment, maturation and function. A num-ber of B cell subpopulations, including B-1,B-2 and regulatory B cells, have been identi-fied. B-1 cells mainly originate from the fetalliver and contain B-1a and B-1b subsets. B-2cells are derived from the bone marrow(BM) and can be further classified into follic-ular B (FOB) and marginal zone B(MZB) cells. Regulatory B cells (Bregs) func-tion to suppress immune responses, primarilyby production of the anti-inflammatory cyto-kine IL-10. B cell tolerance is established atseveral checkpoints, during B cell developmentin the BM (central tolerance) as well as duringB cell maturation and activation in the periph-ery (peripheral tolerance). This chapter willfocus on the regulation of important processes

during the development and maturation of B-1and B-2 cells.

Keywords

Ig gene � V(D)J recombination � Follicular B �Marginal zone B � B-1

1.1 Introduction

B cell development proceeds in an orderly fashionand is regulated by intrinsic genetic programs andby external cues such as cytokines that are presentin the specialized microenvironments of fetal liverand BM. One intriguing feature of B cell devel-opment is that it is accompanied byimmunoglobulin (Ig) gene rearrangements. Pro-genitor B cells rearrange their Ig heavy chain(HC) genes to differentiate into precursor B (pre-B) cells that express µ HCs. Pre-B cells thenrearrange their Ig light chain (LC) genes to dif-ferentiate into IgM+ immature B cells and thenbecome IgM+IgD+ mature, resting B cells. Defectsin each stage of the B cell development andmaturation pathway can lead to primary immun-odeficiencies, autoimmune diseases and even Bcell malignancies. In this chapter, we will discussthe mechanism of Ig gene rearrangement, regula-tion of B-2 (FOB and MZB) cell development, theelimination/inactivation of autoreactive B cells

Y. Wang � J. Liu � J.-Y. Wang (&)Department of Immunology, School of BasicMedical Sciences, Fudan University, Shanghai,Chinae-mail: wang@fudan.edu.cn

P. D. BurrowsDepartment of Microbiology, University of Alabamaat Birmingham, Birmingham, AL, USA

© Springer Nature Singapore Pte Ltd. 2020J.-Y. Wang (ed.), B Cells in Immunity and Tolerance, Advances in ExperimentalMedicine and Biology 1254, https://doi.org/10.1007/978-981-15-3532-1_1

1

(central and peripheral tolerance), regulation of B-1 cell development, fate decision of B-1a vs. B-1bsubsets, and the function of B-1 cells.

1.2 Immunoglobulin (Ig) GeneRearrangement

Antigen receptor gene rearrangement, whichis a defining feature of the adaptive immunesystem, is a unique mechanism to generatediversity in both T and B cells from limitednumbers of variable (V) gene segmentsthrough genetic recombination. We willfocus here on Ig gene rearrangement, whichoccurs during the early stages of B celldevelopment to generate a diverse repertoireof antibodies.

1.2.1 Structure of Ig Genes

The genome structure of the mouse Ig heavy(H) and light (L) [kappa (Igj) and lambda (Igk)]loci is shown in Fig. 1.1. An antibody is com-posed of two identical HCs and two identicalLCs (either j or k), consisting of variable(V) and constant (C) regions linked by disulfidebonds. Igh, Igj and Igk genes in mice are locatedon chromosomes 12, 6 and 16 and in humans onchromosomes 14, 2 and 22, respectively. The Vexons of the Ig heavy chains are generated bysomatic recombination (rearrangement) of vari-able (V), diversity (D) and joining (J) gene

segments, whereas the V exons of the Ig lightchains are generated by rearrangement of V and Jbut no D segments. During the early stages of Bcell development, somatic recombination, knownas V(D)J recombination, mediates a physical andpermanent juxtaposition of the Ig gene seg-ments at the DNA level that results in a matureV exon and generation of a functional l HCgene. V(D)J recombination gives rise to alarge, diverse repertoire of Ig proteins. In mice,there are 97 V, 14 D and 4 J segments locatedin the Igh locus, 94–96 V and 4 J segmentslocated in the Igj locus, and 3 (laboratorymice) or 8 (wild mice) V and 3 J segmentslocated in the Igk locus.

Downstream of these rearranging gene seg-ments are exons to encode the constant (C) re-gions of the antibody heavy and light chains.The C exons are not formed by the rearrange-ment of smaller gene segments and in the Ighlocus encode several antibody classes and sub-classes. Although the Igj locus contains a singleCj exon, the mouse Igk locus contains four Ck

exons. Moreover, the Igh locus contains eight CH

exons, namely Cl, Cd, Cc3, Cc1, Cc2b, Cc2a(BALB/c)/Cc2c (C57BL/6), Ce and Ca. Each CH

exon encodes a particular Ig isotype. For H and Lchains, the most V-proximal C exon is joined tothe mature V exon at the RNA level by con-ventional splicing, which is then translated togenerate the H or L protein.

Because of this specific gene structure andmechanism, B cells can generate about 1011

different BCR or antibodies by V(D)J

VH1 VH2 VH97… DH1 DH2 DH14… JH1 JH2 JH3 JH4 Cμ Cδ Cγ3 Cγ1 Cγ2b Cγ2a/c Cε Cα

Mouse Igh locus

Vκ1 Vκ2 Vκ94-96 … Jκ1 Jκ2 Jκ3 Jκ4 Cκ

Mouse Igκ locus

Jκ5

Ψ

Vλ2 Vλ3 Vλ1Jλ2 Jλ4 Jλ3 Jλ3PCλ2 Cλ3 Cλ1

Mouse Igλ locus

Cλ4

Ψ Ψ Ψ

Jλ1

Fig. 1.1 Immunoglobulin loci of mouse (Notes The organization of mouse Igh locus, Igj locus and Igk locus. W,pseudogene. Not all gene segments and pseudogenes are shown)

2 Y. Wang et al.

recombination (Nussenzweig and Alt 2004). Ig Vgenes also undergo somatic hypermutation dur-ing the germinal center reaction, which furtherincreases the diversity and can change the affinityand sometimes the specificity of antibodies. Sucha vast number of different antibodies, the“repertoire,” are essential for neutralizing a uni-verse of pathogenic bacteria and viruses.

1.2.2 The Mechanism of V(D)JRecombination

V(D)J recombination can be divided into twoprocesses, i.e., DNA cleavage and DNA repair.The DNA cleavage is a site-specific reactioninitiated by the lymphoid-specific proteinsrecombination-activating gene (RAG) 1 andRAG2 and the ubiquitously expressed DNA-binding protein high-mobility group box protein1 or 2 (HMGB1 or HMGB2) (van Gent et al.1997). These proteins bind to the recombinationsignal sequences (RSSs) that flank each genesegment and introduce a double-strand break(DSB) between the RSS and the flanking codingDNA. Then, joining of the DSB is mediated bythe non-homologous end-joining (NHEJ) DNArepair pathway (reviewed by Lieber 2010; Changet al. 2017).

RSSs flank germ line VH segments on their 3ʹsides, JH segments on their 5ʹ sides and DH seg-ments on both sides. Similarly, the VL and JLsegments of both the Igj and Igk loci are flankedby RSSs on their 3ʹ sides and 5ʹ sides, respec-tively. The RSS is a short DNA sequence con-taining a conserved heptamer (consensus sequence5ʹ-CACAGTG-3ʹ) and nonamer (consensussequence 5ʹ-ACAAAAACC-3ʹ) separated byeither 12 or 23 base pairs (±1 bp), namely the 12RSSs and 23 RSSs, respectively (Ramsden et al.1994). The first three positions in the heptamer arethe most conserved and are necessary for guidingthe RAGs to the correct site of cleavage at theborder between the heptamer and the coding seg-ment. Similarly, the tract of adenines is the mostinvariant portion of the nonamer and is known toguide RAG1 binding at the RSS (Yin et al. 2009).While the sequence of the spacer is less well

conserved, the length of the spacer is important asthe 12 and 23 RSSs differ by one turn of the DNAhelix, placing the heptamers and nonamers of thetwo RSSs in the same rotational phase andensuring mutually complementary binding (Ciub-otaru et al. 2015). Therefore, only when one genesegment is flanked on one side by a 12 RSSs andthe other gene segment is flanked by a 23 RSSscan the pair interact and be recognized by theRAG complex and undergo V(D)J recombination.Such a mechanism has been called the 12/23 rule.The V(D)J recombinases first assemble on a single12 or 23 RSSs, creating a signal complex (SC) inwhich nicking can occur (Fig. 1.2). Synapsis witha partner leads to the formation of the pairedcomplex (PC) in which cleavage can occur. Sev-eral lines of evidence suggest that the PC isformed by the “capture model,” in which one SCcaptures the appropriate partner RSS to form thePC, maintaining the same protein content in boththe SC and the PC (Curry et al. 2005; Jones andGellert 2002; Mundy et al. 2002; Swanson 2002).However, the capture model is not universallyaccepted. Some studies support the “associationmode,” in which two preformed SCs associate toform the PC (Shlyakhtenko et al. 2009; Landreeet al. 2001). Under physiological conditions, theDSB is generated only by “coupled cleavage” inthe PC. In a two-step transesterification reaction of“coupled cleavage,” the free 3ʹ hydroxyl attacksthe phosphate on the bottom strand of the DNAdirectly (McBlane et al. 1995). Then, hairpin for-mation in the PC generates two DNA DSBs andyields a pair of blunt DNA ends and two sealedDNA hairpins, all of which are held together bythe RAGs (Hiom and Gellert 1998). Both the bluntends, termed the signal ends, and the DNA hair-pins, termed coding ends, are then handed over tothe NHEJ DNA repair pathway. After repair, theblunt ends are joined to form the discarded pro-duct, a DNA deletion circle. Once opened, thehairpin ends are processed and ligated, yieldingthe coding joint that contains the newly joinedantigen receptor gene segments. In the Igh locus,the recombination of DH and JH segments occursfirst, and then the DHJH recombines with VH

segment to form the complete VHDHJH exon. Inboth the Igj and Igk loci, there is only one

1 B Cell Development and Maturation 3

recombination step between a VL and a JL genesegment. Importantly, the fusion of V, D and Jsegments is not always precise, which leads tojunctional diversity at the site of rearrangement,including the deletion of nucleotides in the jointregion and the addition of the so-called P- or N-nucleotides. In the last steps of V(D)J recombi-nation, if the DNA ends of the rearranging genesegments are digested by an exonuclease prior toligation, there will be deletion of a few nucleotidesin the D to J and/or V to D junctions. If the hairpinis not opened symmetrically, a single-strandedextension is created, resulting in palindromicinsertions (P-nucleotides). Extra nucleotides canalso be created by the terminal dideoxynucleotidyltransferase (TdT), which can randomly add “non-templated” N-nucleotides onto the ends of thesestrands before their final ligation. N-nucleotideaddition occurs almost exclusively in the Igh locusbecause TdT is preferentially expressed in pro-Bcells undergoing VDJH rearrangement. Althoughthis imprecision can introduce diversity in the

CDR3 regions of the antibody heavy and lightchains, it can also lead to frameshift mutations ifdeleted or added nucleotides are not in multiplesof three (one codon). As a result, 2/3 V(D)J rear-rangements are non-functional, aka nonproductive.

The rearrangement of the Ig loci occurs atparticular developmental stages in a temporallyordered and allele-exclusive manner. The Ighlocus is rearranged first, followed by Igj and Igk.After a productive VDJH rearrangement is com-pleted on chromosome 12 in a developing mouseB cell, the now functional VDJ-Cl gene istranscribed and translated. Expression of this lchain marks an important checkpoint in Ig generearrangement and B cell development. Anyfurther V(D)J recombination in the Igh locus onthe other chromosome 12 is blocked. This out-come is called allelic exclusion, because only onechromosome contributes the H chain gene pro-duct (Schatz and Ji 2011; Vettermann and Sch-lissel 2010). Similarly, only one of the L chaingenes, j or k, is expressed during normal B cell

VH1 VH2 VH97… DH1 DH2 DH14… JH1 JH2 JH3 JH4 Cμ Cδ Cγ3 Cγ1 Cγ2b Cγ2a/c Cε CαMouse Igh locus

Rearrangement of DH-JH

VH1 VH2 VH97… DH1 DH2 JH3 JH4 Cμ Cδ Cγ3 Cγ1 Cγ2b Cγ2a/c Cε Cα

Rearrangement of VH-DHJH

VH2 DH2 JH3 Cμ Cδ Cγ3 Cγ1 Cγ2b Cγ2a/c Cε Cα

A

C

Nicking HO

P

Hairpinformation

P HO

B Signal complex (SC)

RAG1RAG2

HMGB1

Paired complex (PC)

Synapsis

Coding joint

CleavageDiscarded DNA product

P or N nucleotides

12

RSS

7 97923

7912

237 9

127 979

12

237 9

127 979

12

237 979

12

JH4

Fig. 1.2 Schematic representation of V(D)J recombina-tion in the mouse lgh locus. a. In this hypotheticalexample, DH2 has joined to JH3, and then VH2 has joinedto DH2JH3. b. A single recombination signal sequence(RSS) can be recognized by a RAG1/RAG2/HMGB1complex and form a signal complex (SC), which thencaptures another RSS to form the paired complex (PC).Nicking can occur in the SC or PC, but hairpin formationcan occur only in the context of the PC. After cleavage,hairpin coding ends and blunt signal ends are processed

by the non-homologous end-joining pathway, creating animperfectly joined coding joint (imperfect joining con-tains P- or N-nucleotides indicated by the yellow sectionbetween coding gene segments) and a discarded DNAproduct. c. Two steps of RAG-mediated DNA cleavageincluding nicking and hairpin formation. The triangledepicts RSS, and the flanking coding gene segment isomitted for clarity. HO, hydroxyl group

4 Y. Wang et al.

development. (Some exceptional situations willbe discussed in the section on B cell centraltolerance.)

1.2.3 B Cell Receptor Expressionand Tonic Signaling

After VHDHJH recombination of the Igh locus issuccessfully completed, an intact l HC proteincan be synthesized. Such a free l HC wouldusually be retained in the endoplasmic reticulum(ER) as part of the ER quality control system.However, the l HC in pre-B cells can beexpressed at low levels on the cell surfacebecause it associates with a surrogate light chain(SLC), which is composed of two proteins,VpreB and k5. Two l HCs and two SLCs plusthe Iga/Igb signaling heterodimer form the pre-BCR, which is expressed transiently to test thefunctionality of this particular VHDHJH-Clcombination. The pre-BCR triggers an intra-cellular signal to inform the cell that the l HCprotein is successfully expressed and associatedwith the SLCs. If the Ig HC gene rearrangementis unsuccessful or the l HC fails to associatewith the SLC, VHDHJH recombination willensue on the other Igh allele. This newly gen-erated l HC protein undergoes the same processof quality testing through the pre-BCR. Failureof Ig HC rearrangement on both chromosomesoccurs in about half of the pre-B cells, whichwill be eliminated by apoptotic death. If the pre-B cell generates a functional pre-BCR, it willpromote proliferation and Igj or Igk rear-rangement; typically, the Igj locus rearrangesfirst. Only B cells with fully functional H and Lchains can survive to become immature B cells.A BCR complex on the B cell surface, whichcontains two functional H and L chains plus theIga/Igb signaling heterodimer, delivers anintracellular signal that terminates all Ig locusrearrangements and mediates central toleranceat the same time. Moreover, both pre-BCR andmature BCR can also generate low-level tonicsurvival signals through a ligand-independentpathway, termed tonic (pre-)BCR signaling, tosupport (pre-)B cell survival and further

development. Furthermore, the tonic (pre-)BCRsignal also plays an important role in B cellcarcinogenesis. However, the mechanisms forthe initiation of tonic (pre-)BCR signaling arestill not fully understood. Based on the existingdata, three models have been suggested, namelythe homotypic pre-BCR-binding model, lipidraft compartmentalization model and equilib-rium model (reviewed in Monroe 2004).

1.3 B-2 Cell Developmentand Maturation

The majority of B-2 cells originate from themultipotent hematopoietic stem cells (HSCs) inthe BM; therefore, the BM microenvironmenthas a major influence on their development.When an immature B cell starts migrating intothe periphery via the bloodstream, it firstbecomes a transitional B cell and finally differ-entiates into a FOB or MZB. Only a fraction ofBM transitional B cells differentiate into B-1cells.

1.3.1 BM Microenvironment

BM stromal cells create distinct microenviron-ments, known as niches, which provide supportfor self-renewal and differentiation of HSCs intomature blood cells. Although B cell developmentis known to occur in the niches, their functionalorganization remains unclear. Some importantenvironmental factors supplied by the cellularniches maintain B cell development.

The adult BM niches contain several celltypes associated with HSC development. Osteo-blasts generate bone and control the differentia-tion of HSCs, endothelial cells line the bloodvessels and also regulate HSC differentiation,reticular cells mediate processes connecting cellsto bone and blood vessels, and sympatheticneurons control the release of hematopoietic cellsfrom the BM. The BM is densely packed withstromal cells and hematopoietic cells of everyBM lineage and at every stage of differentiation.With age, however, fat cells gradually replace

1 B Cell Development and Maturation 5

50% or more of the BM compartment, and theefficiency of hematopoiesis decreases. The lin-eage choices that an HSC makes depend largelyon the environmental signals it receives. Severalmicroenvironmental components that act on Bcell precursors have been identified. CXC che-mokine ligand 12 (CXCL12), FLT3 ligand(FLT3L), interleukin-7 (IL-7), stem cell factor(SCF) and receptor activator of nuclear factor-jBligand (RANKL) have each been shown to beessential for B cell development in vivo.

The main physiological receptor for CXCL12is CXC chemokine receptor 4 (CXCR4). TheCXCL12–CXCR4 axis is essential for the earli-est stages of B cell development in the fetus andadult (Nagasawa 2006; Nagasawa et al. 1996;Tokoyoda et al. 2004). FLT3L is a ligand forFTL3, which is essential for the development ofpre-pro-B cells (Sitnicka et al. 2002; Sitnickaet al. 2003; Hunte et al. 1996). The receptor forIL-7 is comprised of two chains: IL-7Ra and thecytokine receptor common c-chain. IL-7 caninduce the expression of MCL1 to mediate thesurvival of B cell precursors (Opferman et al.2003). IL-7Ra has been shown to deliver a signalthat specifically induces VDJ recombination atthe Igh locus (Corcoran et al. 1998). SCF is aligand for the class III receptor tyrosine kinaseKIT and is encoded by a gene that maps to thesteel locus of mice. Cells expressing membrane-bound SCF might function as a niche for pro-Band pre-B cells (Driessen et al. 2003). Thetransmembrane protein RANKL is a tumor

necrosis factor (TNF) family member that isessential for the development of osteoclasts andfor bone remodeling. The receptor for RANKL isRANK, which belongs to TNF receptor (TNFR)family and is expressed by DCs, T cells andosteoclast precursors. RANKL expression bylymphoid cells is important for the developmentof pre-B cells and immature B cells (Kong et al.1999; Anderson et al. 1997).

It is worth noting that most of the precedinginformation about the BM microenvironment isderived from studies done in mice. Although itsgeneral features are likely also true for humans,the requirements for B cell development are notcompletely identical in the two species. Forexample, IL-7 is essential for both T and B celldevelopments in mice, but only for T celldevelopment in humans (Prieyl and LeBien1996).

1.3.2 Stages of B Cell Developmentand ImportantTranscription Factors

The major stages of B cell development in theBM include the HSC, the multipotent progenitor(MPP), the common lymphoid progenitor (CLP),and then the progenitor B cell (pro-B cell), theprecursor B cell (pre-B cell) and the immature Bcell. Hardy et al. have elegantly shown that B celldevelopmental stages can be divided into frac-tions A, B, C, Cʹ, D and E. Ig gene

Cell stage Status of Ig genes

Surface Ig AA4.1 B220 CD43 HSA BP-1 C-Kit IL-7Ra CD19 CD25

Pre-pro B Fraction A Germ line arrangement None + + + + - + + - -

Early pro B Fraction B DHJH None + + + ++ - ++ ++ + -

Late pro B Fraction C Some VHDHJH None + + + ++ - ++ ++ + -

Large pre B Fraction C’ VHDHJH Pre-BCR + + + +++ + ++ ++ + +

Small pre B Fraction D VHDHJH & VLJL arrangement

Decreased Pre-BCR + + - +++ + - ++ + +

Immature B Fraction E VHDHJH & VLJL IgM-BCR + + - +++ - - - + -

Mature B Fraction F VHDHJH & VLJL IgM-BCRIgD-BCR - + - + - - - + -

Fig. 1.3 Ig gene rearrangements and expression of marker proteins during B cell development

6 Y. Wang et al.

rearrangement status and the expression of sev-eral defining cell surface proteins during thesestages of B cell development are shown inFig. 1.3.

With the expression of the B cell lineage-specific marker B220 (CD45R) and the increasedexpression of the transcription factor EBF1, thedeveloping cell enters pre-pro-B cell stage orfraction A. EBF1, along with E2A, binds to theIgh locus, promoting DHJH recombination.Moreover, EBF1 is also essential for theexpression of many B cell-associated proteins,including Iga (CD79a), Igb (CD79b), and thegenes encoding the pre-BCR. Pre-pro-B cellsremain in contact with CXCL12-secreting stro-mal cells in the BM. But, after the successfulDHJH recombination, which means the cell entersthe early pro-B cell stage or fraction B, thedeveloping cell moves within the BM in searchof IL-7-secreting stromal cells. Meanwhile, theexpression of PAX5, which is one of the EBF1transcriptional targets, blocks the expression ofnon-B lineage genes. In addition, many impor-tant B cell genes are turned on at this stage, underthe control of PAX5 and other transcriptionfactors. During this stage, one of the definingmarkers of B lineage cells and an importantcomponent of the (pre-)B cell co-receptor, CD19,is expressed. In addition, PAX5 promotes VH toDH recombination by contracting the Igh locus,bringing the VH gene segments and the DHJHregion into closer proximity. Then, the develop-ing B cell enters the late pro-B cell stage orfraction C and most cells have initiated VH toDHJH Ig gene segment recombination, which iscompleted by the onset of the early pre-B cellstage. During the pro-B-cell stage or fractions A-C, Iga and Igb, which are signaling componentsof the BCR, begin to be expressed, and theexpression of c-Kit enables the cell to receivesignals from stem cell factor. However, by thebeginning of the pre-B cell stage, the expressionof c-Kit is irreversibly turned off.

After successful VHDHJH recombination, thecell expresses a pre-BCR, which is composed ofthe rearranged µ heavy chain, complexed withVpreB and k5 plus Iga and Igb. The developingB cell enters the large proliferating pre-B cell

stage or fraction Cʹ. This is an importantcheckpoint, and only cells with a productivelyrearranged heavy chain can be selected into thenext stage. The pre-BCR is then lost from thesurface, and this signals entry into the small/latepre-B cell stage or fraction D. At this stage, lightchain rearrangement is initiated with the re-expression of the Rag1/2 genes. In the mouse,light chain rearrangement begins on one of the jchain chromosomes, followed by the other. Ifneither j chain rearrangement is successful,rearrangement is then attempted on each of thek chain chromosomes. At the same time, there isvery little expression of TdT and therefore Nregion addition occurs less frequently in lightchains than in heavy chains. After successfullight chain gene rearrangement and expression,the integrated IgM receptor (BCR) is expressedon the cell surface, which means the developingB cell enters the immature B cell stage or frac-tion E.

Throughout B cell development, PU.1 sets thestage for lymphoid and myeloid development,and lineage priming in lymphoid progenitors ismediated by Ikaros. In addition, E2A regulatesthe chromatin landscape to promote geneexpression during B cell development and EBF1plays an important role as a central coordinator ofB cell development, collaborating with FOXO1.

1.3.3 B Cell Central Tolerance

There are three mechanisms of central B celltolerance, clonal deletion, receptor editing andanergy. If an immature B cell recognizes a self-antigen that is present at high concentration inthe BM, its BCRs are cross-linked, delivering astrong signal to the cell. In this case, the B cellmay undergo apoptosis, a process called clonaldeletion. Alternatively, such a B cell may reac-tivate RAG1 and RAG2 expression and initiate anew round of VLJL recombination. A Vj seg-ment upstream of the originally rearranged VjJjunit is joined to a downstream Jj. As a result, theformer rearranged VjJj exon in the self-reactiveimmature B cell is deleted and a new Ig lightchain is expressed, thus creating a B cell receptor

1 B Cell Development and Maturation 7

with a new, potentially no longer autoreactive,specificity. This process is called receptor edit-ing. If the edited light chain rearrangement isnonproductive, rearrangement may proceed atthe other j locus, and if that is also nonproduc-tive, rearrangements at the k light chain loci mayfollow. Finally, if the receptors on the developingB cell recognize self-antigens with low affinity,the cells may become functionally unresponsiveor anergic, due to downregulation of BCRexpression and BCR signaling. In principle,receptor editing at the Igh locus is not possible,since there are no germ line DH gene segmentsavailable for further VDJ rearrangement; all weredeleted during the original V!DJ rearrangementstep. However, a “cryptic” RSS heptamer ispresent in many VH genes and can be used in aprocess called VH gene replacement, in which anupstream VH gene segment recombines with thecryptic heptamer, replacing most of the V regionin a preexisting VDJ exon. Whether this processis more important for diversification than forcentral B cell tolerance is somewhat controver-sial, since it appears to occur mainly in pre-Bcells that have not yet undergone light chain generearrangement and thus their antigen specificityhas not yet been defined (Kumar et al. 2015; Sunet al. 2015; Kelsoe 2015).

David Nemazee and colleagues generatedmice transgenic for both a heavy and a lightchain specific for the H-2Kk MHC molecule. Inmice with H-2Kd but no H-2Kk MHC molecules,the transgenic BCR was expressed on B cells anda high concentration of transgenic antibody couldbe measured in the serum. However, in mice withH-2Kk but no H-2Kd MHC molecule, no anti-H-2Kk B cells or secreted antibodies could bedetected, suggesting that all immature B cellsbearing the potentially autoimmune BCR hadbeen deleted in the BM. Interestingly, in micewith both H-2Kd and H-2Kk MHC, not all B cellsbearing the autoimmune transgenes were deleted,and some of them underwent light chain receptorediting and no longer bound the H-2Kk antigen(Nemazee and Bürki 1989; Tiegs et al. 1993).

Goodnow and colleagues developed double-transgenic mice carrying transgenes encodingmembrane hen egg white lysozyme (mHEL)

driven by a class I MHC promoter and anti-HELBCR, which mimics the situation of a self-antigen and a corresponding autoreactive BCR.B cells in double-transgenic mice were arrestedat the pre-B stage and underwent efficient clonaldeletion, with few B cells reaching the periphery(Hartley et al. 1993). However, when the mHELtransgene in double-transgenic mice wasreplaced by a soluble HEL (sHEL) transgenelinked to a metallothionein promoter and sHELwas expressed in the periphery, but not in theBM, the double-transgenic mice were able togenerate mature, peripheral B cells bearing theanti-HEL BCR. However, these B cells werefunctionally nonresponsive, or anergic, a mech-anism called B cell peripheral tolerance (Cysteret al. 1994). Based on these results, it appearsthat in normal animals, not all self-reactive Bcells are deleted in the BM. Some are released tothe periphery but are inactivated. It has beensuggested that such cells could be abnormallyreactivated by non-BCR-mediated signals,resulting in their differentiation into plasma cellsand resultant antibody-mediated autoimmunediseases.

1.3.4 B-2 Cell Maturationin the Periphery

1.3.4.1 Transitional B CellsImmature B cells have a short half-life and areready for export to the peripheral lymphoidorgans, usually the spleen, where they completetheir developmental program. During B-2 cellmaturation in the periphery, transitional B cellsplay an important role in linking BM immatureand peripheral mature B cells. Moreover, transi-tional B cells express similar defining cell surfacemarkers, such as AA4.1, HSA and IgM, asimmature B cells, and are still susceptible tonegative selection.

Newly generated immature B cells that haveyet to acquire the ability to recirculate throughoutthe body are known as T1 B cells. They arefound in the BM and the periarteriolar lymphoidsheath (PALS) of spleen and are AA4.1+ IgMhigh

IgD− CD21− CD23−. After entering spleen

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