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Clinical Immunology (2010) 135, 193–203

REVIEW

Immunoglobulin class switch recombinationdeficienciesS. Kracker, P. Gardes, F. Mazerolles, A. Durandy⁎

INSERM, U768, Hôpital Necker-Enfants Malades, Université Paris Descartes, Faculté de Médecine Paris V-René Descartes,Paris F-75005, France

Received 27 November 2009; accepted with revision 25 January 2010Available online 18 February 2010

⁎ Corresponding author. Fax: +33 1 4E-mail address: anne.durandy@inse

1521-6616/$ – see front matter © 201doi:10.1016/j.clim.2010.01.012

KEYWORDSInheritedimmunodeficiencies;Class switchrecombination;Somatic hypermutationsCD40 ligand;CD40;NF-kB essential modulator;Activation-inducedcytidine deaminase;Uracil-N-glycosylase;Post-meiotic segregation 2

Abstract Maturation of the secondary antibody repertoire is generated by means of classswitch recombination and somatic hypermutation. The molecular mechanisms underlying theseimportant processes have long remained obscure. Inherited defects in class switch recombinationvariably associated to defects in somatic hypermutation are a group of geneticallyheterogeneous diseases, the characterization of which has allowed recognition that T–B cellinteraction (resulting in CD40-mediated signaling), intrinsic B cell mechanisms, and complex DNArepair machinery are involved in class switch recombination and somatic hypermutation.Elucidation of the molecular defects underlying these disorders has been essential to betterunderstand the molecular basis of immunoglobulin diversification and has offered theopportunity to define the clinical spectrum of these diseases and to prompt more accuratediagnostic and therapeutic approaches.© 2010 Elsevier Inc. All rights reserved.

2 73 06 40.rm.fr (A. Durandy).

0 Elsevier Inc. All rights reserv

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194Class switch recombination and somatic hypermutation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194Ig Class switch recombination deficiencies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194

CSR-D caused by a defect in the CD40 signaling pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194CSR-D caused by an intrinsic B cell defect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196CSR-D with normal in vitro B cell response to CSR activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198

Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199

ed.

194 S. Kracker et al.

Introduction

The study of inherited immunoglobulin class switch recom-bination deficiencies (CSR-Ds) has greatly contributed to ourunderstanding of the normal processes of antibody matura-tion. These syndromes have in common a defect inimmunoglobulin (Ig) class switch recombination (CSR), asdemonstrated by normal or elevated serum IgM levels,contrasting with absent or strongly decreased levels of theother immunoglobulin isotypes. Antibodymaturation leads tothe production of antibodies of different isotypes andformation of B cell receptors (BCR) with high affinity forantigen. This event usually takes place in the secondarylymphoid organs (spleen, lymph nodes, tonsils) in an antigen-and T lymphocyte-dependent manner. Whenmature (but stillnaive) IgM+IgD+ B cells, after emigrating from the bonemarrow (or fetal liver), encounter an antigen that isspecifically recognized by their BCR, they proliferatevigorously and give birth to a unique lymphoid formation,the germinal center. In this location, B cells undergo the twomajor events of maturation: CSR and somatic hypermutation(SHM).

Class switch recombination and somatichypermutation

Class switch recombination is a process of DNA recombinationbetween two different switch (S) regions located upstream ofthe constant (C) regions, while the intervening DNA is deletedby forming an excision circle [1–5]. Replacement of the Cμregion by a downstream Cx region from another class of Igresults in the production of antibodies of different isotypes(IgG, IgA, and IgE) with the same variable (V) region and thusthe same antigen specificity and affinity. The different Igisotypes vary in activities (half-life, binding to Fc receptors,ability to activate the complement system) and tissuelocalization (IgA is secreted by mucosal membranes). ThusCSR is necessary for an optimal humoral response againstpathogens.

Through SHM, mutations and, less frequently, deletions orinsertions are introduced into the V regions of immunoglo-bulins. This process is triggered by activation of BCR andCD40 [6,7]. These mutations occur at a high frequency in theV regions and their proximal flanks (1×10-3 bases/genera-tion). SHM is required as a basis for the selection of B cellsexpressing a BCR with a high affinity for antigen in closeinteraction with follicular dendritic cells [8,9]. CSR and SHMoccur in germinal centers, but neither is a prerequisite forthe other because IgM may be mutated whereas IgG or IgAcan remain unmutated [10,11].

Schematically, three successive steps are required in theprocess of CSR and SHM:

1. Transcription of the targeted DNA. In S regions, this step isinduced by cytokines and leads to the formation of RNA–DNA hybrids, known as stable R-loops, on the templateDNA strand, leaving the single non-template strandaccessible for lesion and cleavage [12–16]. However,the template DNA strand remains accessible in transcrip-tion bubbles [12]. Transcription of V regions is alsorequired for SHM [6] but its induction remains elusive.

2. DNA lesion and cleavage. It is believed that during CSR,the induction of single-stranded DNA lesions is followed bya DNA cleavage step. Since both DNA strands areaccessible to lesions, scattered single-strand breaks(SSBs) occurring on both DNA strands result in bluntdouble-stranded DNA breaks (DSBs) by a mechanismcurrently unknown, possibly involving exonucleases anderror-prone polymerases. This error-prone DNA proces-sing is suggested by the high frequency of mutations foundin Sμ–Sx junctions [17].

3. DNA repair. Although the first steps are shared by CSR andSHM, the following step, DNA repair, differs for bothevents. During CSR, histone H2AX is phosphorylated, andthe repair protein 53BP1 and the complex MRE11/RAD50/NBS1 are recruited to the site of the breaks [18,19].Thereafter, the DNA repair machinery joins Sμ and Sxsequences by means of the widespread, constitutivelyexpressed nonhomologous end-joining (NHEJ) enzymes[20–22]. It has been demonstrated recently that the DNArepair step in CSR can also be accomplished by analternative end-joining pathway (XRCC4/DNA-ligase IV-independent) [23]. In contrast, DNA repair during SHMdoes not require NHEJ [24], but the error-prone poly-merases η and mismatch repair (MMR) enzymes [25]. MMRis also implicated in CSR (see Post-meiotic segregation 2(PMS2) deficiency section), there however in the induc-tion and processing of DNA lesions.

Ig Class switch recombination deficiencies

CSR-Ds lead to a humoral immunodeficiency characterizedby normal or increased production of IgM contrasting with amarked decrease or an absence of other isotypes (IgG, IgA,and IgE), the consequence of which is an increasedsusceptibility to bacterial infections. Depending on thenature of the molecular defect, the CSR-D can be associatedwith a defect in the other process of antibody maturation,the SHM (Table 1).

CSR-D caused by a defect in the CD40 signaling pathwayCSR and SHM are initiated by T and B cell interaction,involving CD40 ligand (CD40L or CD154), a moleculetransiently expressed on activated CD4+ follicular helper Tcells, and CD40, constitutively expressed on B lymphocytesand monocytes/dendritic cells [26] (Fig. 1).

X-linked CSR-D due to CD40L deficiency. The initiallydescribed and most frequent CSR-D is caused by mutationsin the gene encoding the CD40L [27] (Table 1). Patientsexhibit significantly reduced levels of membrane CD40Lexpression on in vitro activated CD4+ T cells, or noexpression at all, making diagnosis of this syndromestraightforward. Due to a CD40 trans-activation defect,the patients' B cells cannot form germinal centers insecondary lymphoid organs in vivo. Impaired production ofIgG and IgA is responsible for specific susceptibility torecurrent bacterial infections as observed in other severeB-cell deficiencies. No antibodies against infectious agentsor vaccines are produced, but iso-hemagglutinins and anti-polysaccharide IgM antibodies are normally detected. Bcells are intrinsically normal, as they can be induced toproliferate and to undergo CSR upon in vitro activation by

Table 1

Gene Relativefrequency+

Transmission Location of theCSR defect

SHM Clinical complications

Cellular and humoral immunodeficienciesCD40L 48% X-L – Diminished Opportunistic infections,

liver damageCD40 2% AR – Diminished Opportunistic infections,

liver damageNEMO# 1% X-L – N or diminished Opportunistic infections

Humoral immunodeficienciesAID 15% AR Upstream from DSB 0 Lymphadenopathies,

auto-immunityUNG 1% AR Upstream from DSB N⁎ B cell lymphomas?PMS2 2% AR Upstream from DSB N CancersAID cofactor? 10% ? Upstream from DSB N Auto-immunityDNA repair? 11% ? Downstream from DSB N Auto-immunity,

B cell lymphomasTFH defect/B cell survival? 10% ? – N Auto-immunity+Relative proportion of the main CSR-D observed in our series, #could be underestimated since only sequenced in patient diagnosed withectodermal dysplasia, DSB: double-stranded DNA breaks, N: normal, ⁎skewed pattern of nucleotide substitution.

195Immunoglobulin class switch recombination deficiencies

CD40 agonists and appropriate cytokines [28], excluding arole for CD40L/CD40 interaction in B cell differentiation.Most, but not all, patients present reduced numbers of“memory” CD27+ B cells and a low frequency of SHM [29].

The detection of serum IgA and of SHM in some patientssuggest that alternative diversification pathways can occur,for IgA production upon CpG- or proliferating inducible ligand(APRIL) activation of B cells in the gut lamina propria [30],and for SHM in a T-cell-independent manner possibly as aninnate mechanism of defense [31,32].

Impaired CD40L/CD40 interaction leads to defective Tcell interactions with monocytes/dendritic cells with con-sequences as (1) impaired full maturation of dendritic cells,(2) impaired IL-12 production by dendritic cells and macro-phages, and (3) affected T cell priming, resulting in anabnormal cellular immune response [33]. As a consequencepatient suffer from significant susceptibility to opportunistic

Figure 1 T–B cell cooperation in germinal centers. TFH: T folliculacomplex class II, CXCR5: CXC-chemokine receptor-5, ICOSL: ICOS ligaswitch recombination, SHM: somatic hypermutation, AID: activation

infections, which cannot be controlled by Ig substitutiontherapy [34] and thus adversely affect prognosis.

Liver disease is very common and slerosing cholangitis,often associated with Cryptosporidium, is particularly severeand may lead to terminal liver damage.

Intermittent or chronic neutropenia is also a commonfeature of X-linked CD40L deficiency and may result from adefective “stress”-induced CD40-dependent granulopoiesisas myeloid progenitors express CD40 molecules [35]. Othercomplications, such as auto-immune manifestations orcancers, reported in some cases, are not frequent.

Albeit mutations affect the entire CD40L gene, they areirregularly distributed with a majority in exon 5, whichcontains most of the TNF homology domain [36]. No strictrelationship between genotype and phenotype is estab-lished. Although the CD40L gene is located on the Xchromosome, some female patients are rarely affected

r helper, TCR: T cell receptor, MHC II: major histocompatibilitynd, CD40L: CD40 ligand, IL-R: interleukin receptor, CSR: Ig class-induced cytidine deaminase, UNG: uracil-N-glycosylase.

196 S. Kracker et al.

because of a skewed pattern of X inactivation [37] orchromosomal translocation [38].

Autosomal recessive CSR-D due to CD40 deficiency. Thedefect in CD40 has been reported in a very few patients as anautosomal recessive (AR) inherited disease and was diag-nosed based on the lack of expression of CD40 on the surfaceof B lymphocytes and monocytes. The clinical and immuno-logical findings of these patients are identical to thosereported in CD40L deficiency. However, in contrast withwhat is reported in CD40L deficiency, CD40-deficient B cellsare unable to undergo in vitro CSR upon activation with CD40agonists and cytokines [39].

Despite efficient Ig substitution and prophylactic anti-biotics, the long-term prognosis of both CD40L and CD40deficiencies is very severe and death can occur because ofinfections early in life and later on because of severe liverdamage. Hematopoietic stem cell transplantation (HSCT)should be considered if an HLA-identical sibling as a donor isavailable. Results of HSCT with matched unrelated donorsare less satisfactory [40].

X-Linked CSR-D due to defective NF-κB activation. Cross-linking of CD40 results in activation of the NF-κB signalingpathway, the role of which is critical in CSR as shown bythe description of patients affected with ectodermaldysplasia associated with immunodeficiency (EDA-ID) [41–43]. Although heterogeneous, this syndrome can becharacterized by normal to increased IgM levels, lowlevels of serum IgG and IgA, and impaired antibody res-ponses, particularly to polysaccharide antigens. Suscepti-bility to mycobacterial infections is frequent. EDA-ID isinherited as an X-linked trait, and caused by hypomorphicmutations most frequently found in the zinc-finger domainof the NF-κB essential modulator (NEMO, also known asIKKγ) [44], a scaffolding protein that binds to IKKα andIKKβ, two kinase proteins, required for NF-κB activationand nuclear translocation. Likely because of geneticheterogeneity, in vitro CSR and SHM can be either normalor defective [45] (Durandy, unpublished results). However,the defect is not restricted to CD40 activation since NF-κBnuclear translocation is required for many signaling path-ways, including the T and B cell receptors. The ectodermaldysplasia, a key feature of this syndrome, results fromNEMO deficiency as ectodysplasin receptor expressed ontissues derived from the ectoderm activate NF-κB, via theIKKα/β NEMO complex [42].

CSR-D caused by an intrinsic B cell defectOther CSR-Ds are caused by an intrinsic B cell defect,resulting in increased susceptibility to bacterial infections(but not to opportunistic infections) that can be effectivelycontrolled by regular intravenous Ig substitution. SHM can beeither normal or defective, according to the moleculardefect. B cells are intrinsically defective for CSR; althoughthey proliferate normally they are unable to undergo CSRafter activation by CD40L and appropriate cytokines. In suchin vitro activation studies, it is possible to locate the CSRdefect by employing a ligated-mediated PCR technique thatallows detection of DNA-DSBs, which are normally occurringduring CSR in switch regions [46]. The absence of DSBindicates a defect located upstream from the DNA cleavage

step, in contrast normal detection of DSBs suggests a defectin DNA repair.

A CSR defect located upstream from the double-strandedDNA breaks in S regions

Activation-induced cytidine deaminase (AID)deficiency. Activation-induced cytidine deaminase (AID)deficiency is the most frequent of autosomal recessive CSR-D(OMIM #605258). It is caused by mutations in the AICDA gene,which encodes AID, a molecule specifically expressed inactivated B cells. It belongs to the large cytidine deaminasefamily, able to deaminate cytidine into uridine [47]. Its C-terminal domain also contains a leucine-rich region that maybe important for protein–protein interaction [48–50].Recently, a nuclear localization signal (NLS) and a nuclearexport signal (NES) have been described, respectively, in theN and C termini of the molecule, although the function andlocalization of the NLS is still debated [51–53]. A subsequentreport suggested active import of AID and an NLS dependenton the conformation of AID [49]. An APOBEC-1-like domain isalso described but its function remains unknown. Themechanism of action of AID remains open to debate. Becausethe sequence of AID is similar to that of the RNA-editingenzyme APOBEC-1, it was originally proposed that AID editsan mRNA encoding a substrate common to CSR and SHM,probably an endonuclease [54,55]. Conversely, severalpieces of evidence strongly indicate that AID exerts itscytidine deaminase activity directly on DNA [12–15,56].

Whatever its mode of action is, AID plays a crucial role in Bcell terminal differentiation as the inducer of DNA lesion inboth S and V regions and its defect leads to complete lack ofCSR and SHM in AID-deficient patients and AID-/- mice[57,58]. Analysis of the CSR-induced DSB occurrence in Sμregions clearly located the defect upstream from the DNAlesion and cleavage [46].

Apart from bacterial infections of the respiratory anddigestive tracts, lymphoid hyperplasia is a prominent featureof this disease and is due to massive enlargement of germinalcenters, which are filled with actively proliferating B cellsthat coexpress CD38, sIgM, and sIgD, all markers of germinalcenter founder cells. Such lymphoid hyperplasia, affectingPeyer's patches as a consequence of intestinal microbialinfections, is also described in AID knock-out mice [59]. Auto-immunity (hemolytic anemia, thrombocytopenia, hepatitis,SLE) affects about 20% of the patients, with the presence ofauto-antibodies of IgM isotype [60].

AICDAmutations, scattered all along the gene, lead to thesame defect impairing both CSR and SHM, although thepercentage of CD27+ B cells remains normal. Interestingly,several mutations, located in the C-terminal part of theAICDA gene, which retain normal cytidine deaminaseactivity, result in complete lack of CSR, whereas SHM is notaffected [61]. This observation suggests that, in addition toits cytidine deaminase activity, AID acts on CSR by binding aCSR-specific cofactor. Furthermore, a CSR-specific cytoplas-mic AID cofactor has been strongly suggested by recent datausing artificial mutants [48,49]. Because mutations in Sμregions [62] and DSBs [48] are normally found in Sμ regions ofAIDΔC murine B cells stimulated to undergo CSR, a defect insynapse formation of the S regions or in DNA repair is morelikely than an abnormality of AID targeting to S regions.Another unexpected finding reported [63] is that hete-

197Immunoglobulin class switch recombination deficiencies

rozygous nonsense mutations, located in the C-terminaldomain and resulting in the loss of the nine last aminoacids of NES (AIDΔNES) lead to a variable CSR-D transmittedas an autosomal dominant (AD) disease. Haploinsufficiency,although reported in mice with weak consequences on Iglevels [64,65], is highly unlikely because all other humansubjects heterozygous for AID deficiency always exhibitnormal Ig levels. A dominant negative effect exerted bythe mutated allele could be explained if AID acts in ahomomeric complex, which is however still controversiallydiscussed [48,49].

The prognosis for the patients is rather good uponregular infusion of Ig for treatment that, however, doesnot control the lymphoid hyperplasia and the auto-immunecomplications.

Uracil-N-glycosylase (UNG) deficiency. The Uracil-N-glycosylase deficiency is a rare cause for CSR-D (OMIM#608106, gene ⁎ 191525) since only three patients have beenreported in the literature [66]. UNG belongs to the family ofuracil-DNA-glycosylases capable of deglycosylating uracilresidues that are misintegrated into DNA. Following removalof uracil residues by UNG, abasic sites are created that canbe attacked by apurinic-apyrimidinic endonucleases leadingto SSB. The processing and repair of the DNA nicks completeboth CSR and SHM [66,67]. In the absence of UNG, thispathway is impaired, resulting in defective CSR and abnormalSHM as shown by the phenotype of both UNG-deficientpatients [66] and ung-/- mice [67]. All three patients had ahistory of frequent bacterial infections of the respiratorytract that are easily controlled by regular IVIG infusions.Lymphadenopathy was observed in two of three patients,with transient enlargement of mediastinal or cervical lymphnodes. The adult patient has developed Sjögren syndrome inrecent years. All 3 patients do suffer from a drastic CSRdefect in vivo and in vitro, the latter being located upstreamfrom the DSB occurrence [66]. Interestingly enough, SHM wasnormal in frequency but characterized by a skewed patternof nucleotide substitution. The excess of transitions occur-ring on C:G residues probably arise from the replication of U:G lesions in the absence of U removal. Mismatch repair (MMR)enzymes may also recognize and repair these mismatches,thereby introducing mutations on neighboring nucleotidesthat result in both transitions and transversions on A:Tresidues [68].

Four different mutations affecting the catalytic domain ofUNG1 and UNG2 have been found. Two patients have smalldeletions leading to a premature stop codon (homozygousmutation in one patient born from a consanguineous family andtwo heterozygous mutations in the other). The third patientcarries a homozygous missense mutation. UNG expression andfunction were defective in Epstein–Barr virus (EBV) B cell lines,providing evidence for the lack of any compensatory UNG–DNAglycosylase activity, at least in B cells.

Patients are well controlled with Ig substitution. Howev-er, UNG is part of the DNA base excision repair involved in therepair of spontaneously occurring base lesions and thereforeconstitutes an anti-mutagenic defense strategy. UNG-defi-cient mice do develop B cell lymphomas when aging [69]. It isthus possible that UNG deficiency predisposes to such tumorsin adulthood. Another consequence of UNG deficiency hasalso been reported in ung-deficient mice since post-ischemicbrain injury is much more severe than in control mice, a

likely consequence of the mitochondrial DNA repair defect[70].

Post-meiotic segregation 2 (PMS2) deficiency. It hasbeen recently shown that homozygous mutations in the PMS2gene, known to be responsible for early occurrence ofcancers, result in a CSR-D, which can be the main symptomfor several years [71]. PMS2 belongs to the MMR pathway thatrecognizes and repairs mismatched nucleotides on DNA. MMRis known to play a role in CSR in mice, as shown by abnormalswitched isotype levels and switch junctions [72,73]. Thereare two main MMR components: the MutS homologue (MSH1–6) and the MutL homologue (PMS2/MLH1/PMS1). The MSH2–MSH6 complex appears to recognize AID-induced DNAmismatches in the absence of UNG, leading to backup CSRand SHM, as shown by the phenotype of a double UNG–MSH2and UNG–MSH6 knockout mutant [68,74]. Recently, it hasalso been reported that MSH5 variants in humans can beassociated with common variable immunodeficiency and IgAdeficiency phenotypes, including abnormal switch junctionsthat are characteristic of DNA repair defects [75], althoughthese results are controversial [76]. The role of the PMS2–MLH1 complex is less clear. It has been proposed that theMMR system can convert DNA SSBs into DSBs in Sμ regionsupon CSR activation [77].

In the SHM process, the repair of V regions requires theMMR and error-prone DNA polymerases. The MSH2–MSH6complex is essential in SHM for recognizing the AID-inducedU/G mismatch, and recruiting exonuclease (EXO1) andpolymerase η [78]. The role of the PMS2–MLH1 complex inSHM remains subject to debate [79,80].

Whereas heterozygous PMS2 mutations are associatedwith colon carcinoma in adulthood [81] homozygousmutations are responsible for high susceptibility to variouscancers (colon adenocarcinoma, T lymphoma, T acutelymphoid leukemia) [82]. Moreover, a variable CSR-D caneven worsen the prognosis, as recently shown in sixpatients carrying homozygous nonsense mutations in PMS2gene, leading to either a truncated protein or a lack ofexpression. Indeed, one of the patients has suffered frombacterial infections from the first years of age, leading tothe diagnosis of CSR-D with high IgM levels, decrease ofIgG, and absence of IgA. Whereas she has been treated byIg substitution for 2 years, she developed a coloncarcinoma that lead to the diagnosis of PMS2 deficiency.The five other patients, already known to suffer from aPMS2 deficiency because of early occurrence of cancers,have no remarkable history of repeated infections;however, they all lack IgG2 and IgG4. IgA and IgG3 werealso decreased in one. SHM was found normal in all with anormal nucleotide substitution pattern. This observationunderlies the role of PMS2 in CSR but not in SHM inhumans, as already shown in mice [72,79]. Interestingly,it was also shown that DNA DSBs do not normally occur inSμ regions in PMS2-deficient B cells upon CSR activation[71].

Expression of MLH1, PMS2 partner in MutLa, was slightlyreduced but still present in the nuclei likely because ofdimerization with PMS1. The decrease occurrence of DSBs inSμ regions upon CSR activation in PMS2-deficient human Bcells strongly suggests a role for PMS2 in DNA cleavage, likelythrough its endonuclease domain [83]. Since UNG-deficientpatients exhibit a drastic CSR defect, it appears that PMS2

198 S. Kracker et al.

does not play a role in CSR as an alternative pathway butacts downstream from UNG in the very same pathway[71].

Indeed, the main symptom of PMS2 deficiency is theoccurrence of cancers during childhood. Nevertheless, theCSR-D, which appears constant in all the studied patients,could be the main feature for several years and thisdiagnosis has to be considered when CSR-D due to anintrinsic B cell defect is not associated with AICDA or UNGmutations.

Ig CSR deficiencies with unknown molecular defect(s). Half of Ig CSR deficiencies due to an intrinsic B celldefect are related neither to AID, UNG nor PMS2 deficiency.Although most of the observed cases are sporadic, the modeof inheritance observed in a fewmultiplex or consanguineousfamilies is compatible with an autosomal recessive (AR)pattern. The clinical phenotype is similar to that of AIDdeficiency, including increased susceptibility to bacterialinfections of the respiratory and gastrointestinal tracts.Lymphoid hyperplasia is milder and less frequent (50%),consisting of moderate follicular hyperplasia without thegiant germinal centers typical of AID deficiency. Auto-immune manifestations are reported [84]. The CSR defectappears to be milder as compared with AID or UNG deficiencysince residual serum levels of IgG can be detected in somepatients. It is located downstream from S-region transcrip-tion and upstream from the S-region DNA cleavage, as nodetectable CSR-induced DSB are detected in Sμ regions ofpatients' B cells. The defect is restricted to CSR, as SHM isnormal in both frequency and pattern in the CD27+ B cellsubset, which is represented in normal numbers.

This type of CSR-D could thus be caused by a direct orindirect impairment of AID targeting on S regions. Althoughtargeting factors of AID are presently unknown, they mightexist because AID deaminates cytosines specifically in the Sand V regions in B cells. AID shares sequence similarity withthe RNA-editing enzyme APOBEC-1, which is only expressedin the gut and requires a cofactor, ACF (APOBEC-1-cofactor),which targets APOBEC-1 on a unique C residue in ApoB mRNA.In addition, specific switch factors have been described inCSR-activated B cells. Although their role remains elusive, ithas been proposed that these cofactors act as dockingproteins for the recruitment of the recombinase complexesto DNA-specific regions [85,86].

A CSR defect located downstream from the double-strandedDNA breaks in S regions. Conversely, a group of CSR-D dueto an intrinsic B cell defect is caused by a defect locateddownstream from the DNA lesion and cleavage step since DSBnormally occur in Sμ regions in CSR-activated B cells. Thus adefect in DNA repair can be suggested.

Ig CSR deficiencies associated to an unknown DNA repairdefect. This CSR deficiency appears to be transmitted asan AR disease since the M:F ratio is around 1 andconsanguinity is observed in 20% of the cases. As otherCSR-D, patients suffer from susceptibility to bacterialinfections, lymphadenopathies, and auto-immune manifes-tations. Some residual IgG or IgA are often observed butthe in vitro CSR upon CD40 activation together withcytokines is strongly reduced. This CSR defect is locateddownstream from the DNA cleavage step, suggesting a DNArepair defect of switching B cells, a hypothesis reinforced

by the observation of tumor occurrence (non-EBV-inducedB cell lymphomas) in 4 out of 45 patients [87]. Moreover, astrong decrease of CD27+ B cells, a defect in switchjunctions repair with a preferential usage of microhomol-ogy, and especially a significant increased radiosensitivityof fibroblasts and EBV B cell lines strongly argue in favor ofthis hypothesis [88].

DNA repair of S regions is achieved through a complexmechanism: it has been shown that upon CSR activation andin an AID-dependent manner, the conformation of the Iglocus is modified, leading the Sμ–Sx regions to be in closeproximity [89]. The maintenance of this synapse requires amultimolecular complex involving the phosphorylated γ-H2AX- and the 53BP1 protein, the complex MRE11/RAD50/NBS1 and ATM [18,19,90–93], leading to (1) cell cycle arrestand (2) DNA repair through the NHEJ pathway [21,22,94].Since the involvement of all these molecules has beenexcluded in these patients, it is likely that (an)other(s)molecule(s), up to now undefined and deficient in thesepatients, plays a role in CSR-induced DNA repair of Sregions.

Although this condition is not molecularly defined, it isimportant to diagnose it in order to prompt an accuratefollow-up of patients because of the risk of lymphomas.

Ig CSR deficiencies as part of a known DNA repair factordefect. As ATM and the MRE11/RAD50/NBS1 (MRN) com-plex are involved in DSB DNA repair, a CSR-D is notunexpected in these syndromes caused by a defect in oneof these molecules. ATM (ataxia–telangiectasia: AT), MRE11(AT-like disease), and NBS1 (Nijmegen breakage syndrome)deficiencies are associated with variable CSR-D, a symptomsometimes preceding the neurological abnormalities in AT[95–97]. Study of recombined switch junctions in the Ig genelocus indicates that the DNA repair does not occur properlyduring CSR [98,99]. Normal SHM generation and pattern in ATconfirms that ATM is not essential for DNA repair of V regions[100]. Conversely, an SHM defect has been reported inNijmegen breakage syndrome [101].

An Ig CSR-D phenotype has also been observed as part ofcombined immunodeficiencies linked to leaky mutations ingenes encoding NHEJ factors, such as Cernunnos, DNA ligaseIV or Artemis [102–105].

CSR-D with normal in vitro B cell response toCSR activationInterestingly, several patients affected with a CSR-Ddisplay normal in vitro CSR. CD40L defect has beenexcluded by normal protein expression and gene sequence.The phenotype of this group of patient is indeed quitedifferent from that of CD40L-deficient patients since thereis no susceptibility to opportunistic infections but only tobacterial infections that are controlled by Ig substitution.Lymphadenopathies with enlarged germinal centers (but nogiant germinal centers that are typical of AID deficiency)are observed and SHM is found normal in frequency andpattern. Several possible causes have been excluded, suchas congenital rubella, in which a defect of T cell activationleads to defective CD40L expression on CD4+ T cells [106],or major histocompatibility complex (MHC) class II defi-ciency in which diminished expression of CD40L byactivated CD4+ T cells can also be responsible for an invivo CSR-D [107]. Deficiencies in ICOS, a T cell activation

199Immunoglobulin class switch recombination deficiencies

molecule, or in transmembrane activator and calciummodulator and cyclophilin ligand interactor (TACI), areceptor expressed on B cells for the activation moleculesBAFF and APRIL, have also been excluded. Althoughreported as responsible for AR or dominant commonvariable immunodeficiencies or IgA deficiencies [108–110], both defects can present with normal serum IgMlevels and decrease of IgG and IgA. In ICOS deficiency, thedefective CSR could be caused by defective TH2 cytokineproduction and/or defective generation of CXC-chemokinereceptor-5 (CXCR5)+ follicular helper T cells as shown inpatients and ICOS-deficient mice [111,112]. In TACIdeficiency, the defective CSR results from impaired TACIactivation as it has been demonstrated that BAFF andAPRIL are inducing CSR to IgG and IgA via TACI activation innaive B cells [113]. A defect in the generation of follicularhelper T cells, their activation, and/ or their interactionwith follicular B cells could be suspected in this CSR-Dcondition.

Conversely, abnormalities in survival signaling ofswitched B cells could underlie this condition. Molecularinteractions are known to be essential for B cell survival,including that of BAFF (B cell activating factor) with its Bcell receptor, BAFF-R [114,115]. However, increased IgMlevels are not reported in patients with a defect in theBAFF-R [116], which might be due to the general decreaseof naive mature B cells in these patients. A response toDNA damage leading to inappropriate cell death should beconsidered. In cells other than germinal center B cells, DSBactivates p53 and p21, resulting in cell cycle arrest andapoptosis. In contrast, in germinal centers, the p53response to DNA damage is directly inhibited by the highlyexpressed transcriptional regulator B cell lymphoma 6(BCL6), while p21-induced cell cycle arrest is suppressedthrough interaction of its transcriptional activator Miz1(protein inhibitor of activated STAT2) with BCL6. Boththese events enable intense proliferation of B cellsundergoing CSR [117,118]. Fitting in with this observation,BCL6-deficient mice are depleted of germinal centersbecause of a strong B cell apoptosis [119]. Such a defectin transcriptional repression of proteins involved in cellcycle arrest induced by DNA damage could also underliethis Ig CSR deficiency. In favor of this last hypothesis is theoccurrence of auto-immunity in 20% of patients. Anotherhypothesis is related to the described role of phosphoinosi-tide-3 kinase (PI3K) acting as a negative regulator of CSR[120]. However, the normal expression of AICDA tran-scripts, AID protein, and in vitro CSR by activated B cellsfrom these patients do not support this hypothesis.

It is likely that this group of patients is quite heteroge-neous, and a precise phenotypical characterization isnecessary for a better understanding of the defectsunderlying this CSR-D with normal in vitro CSR.

Concluding remarks

The ongoing delineation of inherited CSR-D is shedding newlight on the complex molecular mechanisms that T and Bcell interaction govern CSR and SHM. Natural mutantsobserved in human immunodeficiencies have, in somecases, been described before the generation of the

appropriate mouse model. The molecular characterizationof CD40L and of NEMO deficiencies, before the generationof the corresponding deficient mice, provided clearevidence that the CD40 activation pathway, including theactivation and nuclear translocation of NF-κB transcriptioncomplex, was essential in both events of antibodymaturation, CSR and SHM. AID-deficient humans and micewere described concomitantly, demonstrating the key roleof this molecule in the generation of the secondaryantibody repertoire. These examples emphasize the im-portant contribution made by studies of primary immunedeficiencies to improving our understanding of the physi-ology of immune responses. Moreover, a precise diagnosisof the CSR-D aids in assessment of prognosis and promptsto accurate forms of treatment.

Acknowledgments

We acknowledge Mrs. M. Forveille for her excellent technicalassistance.

Thisworkwas supported by INSERM, CEE EUROPADContract7th Framework Program (n201549), and Association Nationalepour la Recherche (MRAR-016-01). S. Kracker is supported bythe EUROPAD contract.

References

[1] T. Iwasato, A. Shimizu, T. Honjo, H. Yamagishi, Circular DNA isexcised by immunoglobulin class switch recombination, Cell62 (1990) 143–149.

[2] M. Matsuoka, K. Yoshida, T. Maeda, S. Usuda, H. Sakano,Switch circular DNA formed in cytokine-treated mousesplenocytes: evidence for intramolecular DNA deletion inimmunoglobulin class switching, Cell 62 (1990) 135–142.

[3] U. von Schwedler, H.M. Jack, M. Wabl, Circular DNA is aproduct of the immunoglobulin class switch rearrangement,Nature 345 (1990) 452–456.

[4] K. Kinoshita, T. Honjo, Unique and unprecedented recombi-nation mechanisms in class switching, Curr. Opin. Immunol. 12(2000) 195–198.

[5] J.P. Manis, M. Tian, F.W. Alt, Mechanism and control of class-switch recombination, Trends Immunol. 23 (2002) 31–39.

[6] U. Storb, A. Peters, E. Klotz, N. Kim, H.M. Shen, J. Hackett, B.Rogerson, T.E. Martin, Cis-acting sequences that affectsomatic hypermutation of Ig genes, Immunol. Rev. 162(1998) 153–160.

[7] H. Jacobs, L. Bross, Towards an understanding of somatichypermutation, Curr. Opin. Immunol. 13 (2001) 208–218.

[8] K. Rajewsky, Clonal selection and learning in the antibodysystem, Nature 381 (1996) 751–758.

[9] J.K. Frazer, J. LeGros, O. de Bouteiller, Y.J. Liu, J.Banchereau, V. Pascual, J.D. Capra, Identification and cloningof genes expressed by human tonsillar B lymphocyte subsets,Ann. N.Y. Acad. Sci. 815 (1997) 316–318.

[10] M. Kaartinen, G.M. Griffiths, A.F. Markham, C. Milstein, mRNAsequences define an unusually restricted IgG response to 2-phenyloxazolone and its early diversification, Nature 304(1983) 320–324.

[11] J. Jacob, G. Kelsoe, In situ studies of the primary immuneresponse to (4-hydroxy-3-nitrophenyl)acetyl. II. A commonclonal origin for periarteriolar lymphoid sheath-associatedfoci and germinal centers, J. Exp. Med. 176 (1992) 679–687.

[12] R. Bransteitter, P. Pham, M.D. Scharff, M.F. Goodman,Activation-induced cytidine deaminase deaminates deoxycy-

200 S. Kracker et al.

tidine on single-stranded DNA but requires the action of RNase,Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 4102–4107.

[13] J. Chaudhuri, M. Tian, C. Khuong, K. Chua, E. Pinaud, F.W.Alt, Transcription-targeted DNA deamination by the AIDantibody diversification enzyme, Nature 422 (2003) 726–730.

[14] S.K. Dickerson, E. Market, E. Besmer, F.N. Papavasiliou, AIDmediates hypermutation by deaminating single stranded DNA,J. Exp. Med. 197 (2003) 1291–1296.

[15] A.R. Ramiro, P. Stavropoulos, M. Jankovic, M.C. Nussenzweig,Transcription enhances AID-mediated cytidine deamination byexposing single-stranded DNA on the nontemplate strand, Nat.Immunol. 4 (2003) 452–456.

[16] K. Yu, F. Chedin, C.L. Hsieh, T.E. Wilson, M.R. Lieber, R-loopsat immunoglobulin class switch regions in the chromosomes ofstimulated B cells, Nat. Immunol. 4 (2003) 442–451.

[17] X. Chen, K. Kinoshita, T. Honjo, Variable deletion andduplication at recombination junction ends: implication forstaggered double-strand cleavage in class-switch recombina-tion, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 13860–13865.

[18] S. Petersen, R. Casellas, B. Reina-San-Martin, H.T. Chen, M.J.Difilippantonio, P.C. Wilson, L. Hanitsch, A. Celeste, M.Muramatsu, D.R. Pilch, C. Redon, T. Ried, W.M. Bonner, T.Honjo, M.C. Nussenzweig, A. Nussenzweig, AID is required toinitiate Nbs1/gamma-H2AX focus formation and mutations atsites of class switching, Nature 414 (2001) 660–665.

[19] J.P. Manis, J.C. Morales, Z. Xia, J.L. Kutok, F.W. Alt, P.B.Carpenter, 53BP1 links DNA damage-response pathways toimmunoglobulin heavy chain class-switch recombination, Nat.Immunol. 5 (2004) 481–487.

[20] A. Rolink, F. Melchers, J. Andersson, The SCID but not theRAG-2 gene product is required for S mu–S epsilon heavy chainclass switching, Immunity 5 (1996) 319–330.

[21] R. Casellas, A. Nussenzweig, R. Wuerffel, R. Pelanda, A.Reichlin, H. Suh, X.F. Qin, E. Besmer, A. Kenter, K. Rajewsky,M.C. Nussenzweig, Ku80 is required for immunoglobulinisotype switching, EMBO J. 17 (1998) 2404–2411.

[22] J.P. Manis, Y. Gu, R. Lansford, E. Sonoda, R. Ferrini, L.Davidson, K. Rajewsky, F.W. Alt, Ku70 is required for late Bcell development and immunoglobulin heavy chain classswitching, J. Exp. Med. 187 (1998) 2081–2089.

[23] C.T. Yan, C. Boboila, E.K. Souza, S. Franco, T.R. Hickernell,M. Murphy, S. Gumaste, M. Geyer, A.A. Zarrin, J.P. Manis, K.Rajewsky, F.W. Alt, IgH class switching and translocations usea robust non-classical end-joining pathway, Nature 449 (2007)478–482.

[24] M. Bemark, J.E. Sale, H.J. Kim, C. Berek, R.A. Cosgrove, M.S.Neuberger, Somatic hypermutation in the absence of DNA-dependent protein kinase catalytic subunit (DNA-PK(cs)) orrecombination-activating gene (RAG)1 activity [In ProcessCitation], J. Exp. Med. 192 (2000) 1509–1514.

[25] M. Cascalho, J. Wong, C. Steinberg, M. Wabl, Mismatch repairco-opted by hypermutation, Science 279 (1998) 1207–1210.

[26] S. Nonoyama, D. Hollenbaugh, A. Aruffo, J.A. Ledbetter, H.D.Ochs, B cell activation via CD40 is required for specificantibody production by antigen-stimulated human B cells,J. Exp. Med. 178 (1993) 1097–1102.

[27] U. Korthauer, D. Graf, H.W. Mages, F. Briere, M. Padayachee,S. Malcolm, A.G. Ugazio, L.D. Notarangelo, R.J. Levinsky, R.A.Kroczek, Defective expression of T-cell CD40 ligand causes X-linked immunodeficiency with hyper-IgM, Nature 361 (1993)539–541.

[28] A. Durandy, C. Schiff, J.Y. Bonnefoy, M. Forveille, F.Rousset, G. Mazzei, M. Milili, A. Fischer, Induction by anti-CD40 antibody or soluble CD40 ligand and cytokines of IgG,IgA and IgE production by B cells from patients with X-linkedhyper IgM syndrome, Eur. J. Immunol. 23 (1993) 2294–2299.

[29] K. Agematsu, H. Nagumo, K. Shinozaki, S. Hokibara, K. Yasui,K. Terada, N. Kawamura, T. Toba, S. Nonoyama, H.D. Ochs, A.

Komiyama, Absence of IgD-CD27(+) memory B cell populationin X-linked hyper-IgM syndrome, J. Clin. Invest. 102 (1998)853–860.

[30] B. He, W. Xu, P.A. Santini, A.D. Polydorides, A. Chiu, J.Estrella, M. Shan, A. Chadburn, V. Villanacci, A. Plebani, D.M.Knowles, M. Rescigno, A. Cerutti, Intestinal bacteria trigger Tcell-independent immunoglobulin A(2) class switching byinducing epithelial-cell secretion of the cytokine APRIL,Immunity 26 (2007) 812–826.

[31] S. Weller, A. Faili, S. Aoufouchi, Q. Gueranger, M. Braun, C.A.Reynaud, J.C. Weill, Hypermutation in human B cells in vivoand in vitro, Ann. N.Y. Acad. Sci. 987 (2003) 158–165.

[32] F.A. Scheeren, M. Nagasawa, K. Weijer, T. Cupedo, J. Kirberg,N. Legrand, H. Spits, T cell-independent development andinduction of somatic hypermutation in human IgM+IgD+CD27+ Bcells, J. Exp. Med. 205 (2008) 2033–2042.

[33] V. Lougaris, R. Badolato, S. Ferrari, A. Plebani, Hyperimmunoglobulin M syndrome due to CD40 deficiency: clinical,molecular, and immunological features, Immunol. Rev. 203(2005) 48–66.

[34] L.D. Notarangelo, M. Duse, A.G. Ugazio, Immunodeficiencywith hyper-IgM (HIM), Immunodefic. Rev. 3 (1992) 101–121.

[35] J. Banchereau, F. Bazan, D. Blanchard, F. Briere, J.P. Galizzi,C. van Kooten, Y.J. Liu, F. Rousset, S. Saeland, The CD40antigen and its ligand, Annu. Rev. Immunol. 12 (1994)881–922.

[36] D. Hollenbaugh, L.S. Grosmaire, C.D. Kullas, N.J. Chalupny,S. Braesch-Andersen, R.J. Noelle, I. Stamenkovic, J.A.Ledbetter, A. Aruffo, The human T cell antigen gp39, amember of the TNF gene family, is a ligand for the CD40receptor: expression of a soluble form of gp39 with B cell co-stimulatory activity, EMBO J. 11 (1992) 4313–4321.

[37] G. de Saint Basile, M.D. Tabone, A. Durandy, F. Phan, A.Fischer, F. Le Deist, CD40 ligand expression deficiency in afemale carrier of the X-linked hyper-IgM syndrome as a resultof X chromosome lyonization, Eur. J. Immunol. 29 (1999)367–373.

[38] K. Imai, M. Shimadzu, T. Kubota, T. Morio, T. Matsunaga, Y.D.Park, A. Yoshioka, S. Nonoyama, Female hyper IgM syndrometype 1 with a chromosomal translocation disrupting CD40LG,Biochim. Biophys. Acta 1762 (2006) 335–340.

[39] S. Ferrari, S. Giliani, A. Insalaco, A. Al-Ghonaium, A.R.Soresina, M. Loubser, M.A. Avanzini, M. Marconi, R. Badolato,A.G. Ugazio, Y. Levy, N. Catalan, A. Durandy, A. Tbakhi, L.D.Notarangelo, A. Plebani, Mutations of CD40 gene cause anautosomal recessive form of immunodeficiency with hyperIgM, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 12614–12619.

[40] A.R. Gennery, K. Khawaja, P. Veys, R.G. Bredius, L.D.Notarangelo, E. Mazzolari, A. Fischer, P. Landais, M.Cavazzana-Calvo, W. Friedrich, A. Fasth, N.M. Wulffraat, S.Matthes-Martin, D. Bensoussan, P. Bordigoni, A. Lange, A.Pagliuca, M. Andolina, A.J. Cant, E.G. Davies, Treatment ofCD40 ligand deficiency by hematopoietic stem cell transplan-tation: a survey of the European experience, 1993-2002, Blood103 (2004) 1152–1157.

[41] J. Zonana, M.E. Elder, L.C. Schneider, S.J. Orlow, C. Moss, M.Golabi, S.K. Shapira, P.A. Farndon, D.W. Wara, S.A. Emmal,B.M. Ferguson, A novel X-linked disorder of immunedeficiency and hypohidrotic ectodermal dysplasia is allelicto incontinentia pigmenti and due to mutations in IKK-gamma(NEMO), Am. J. Hum. Genet. 67 (2000) 6.

[42] R. Doffinger, A. Smahi, C. Bessia, F. Geissmann, J. Feinberg,A. Durandy, C. Bodemer, S. Kenwrick, S. Dupuis-Girod, S.Blanche, P. Wood, S.H. Rabia, D.J. Headon, P.A. Overbeek, F.Le Deist, S.M. Holland, K. Belani, D.S. Kumararatne, A.Fischer, R. Shapiro, M.E. Conley, E. Reimund, H. Kalhoff, M.Abinun, A. Munnich, A. Israel, G. Courtois, J.L. Casanova, X-linked anhidrotic ectodermal dysplasia with immunodeficien-

201Immunoglobulin class switch recombination deficiencies

cy is caused by impaired NF-kappaB signaling, Nat. Genet. 27(2001) 277–285.

[43] A. Jain, C.A. Ma, S. Liu, M. Brown, J. Cohen, W. Strober,Specific missense mutations in NEMO result in hyper-IgMsyndrome with hypohydrotic ectodermal dysplasia, Nat.Immunol. 2 (2001) 223–228.

[44] E.P. Hanson, L. Monaco-Shawver, L.A. Solt, L.A. Madge, P.P.Banerjee, M.J. May, J.S. Orange, Hypomorphic nuclear factor-kappaB essential modulator mutation database and reconsti-tution system identifies phenotypic and immunologic diversi-ty, J. Allergy Clin. Immunol. 122 (2008) 1169–1177 e1116.

[45] A. Jain, C.A. Ma, E. Lopez-Granados, G. Means, W. Brady,J.S. Orange, S. Liu, S. Holland, J.M. Derry, Specific NEMOmutations impair CD40-mediated c-Rel activation and B cellterminal differentiation, J. Clin. Invest. 114 (2004)1593–1602.

[46] N. Catalan, F. Selz, K. Imai, P. Revy, A. Fischer, A. Durandy,The block in immunoglobulin class switch recombinationcaused by activation-induced cytidine deaminase deficiencyoccurs prior to the generation of DNA double strand breaks inswitch mu region, J. Immunol. 171 (2003) 2504–2509.

[47] M. Muramatsu, V.S. Sankaranand, S. Anant, M. Sugai, K.Kinoshita, N.O. Davidson, T. Honjo, Specific expression ofactivation-induced cytidine deaminase (AID), a novel memberof the RNA-editing deaminase family in germinal center Bcells, J. Biol. Chem. 274 (1999) 18470–18476.

[48] T. Doi, L. Kato, S. Ito, R. Shinkura, M. Wei, H. Nagaoka, J.Wang, T. Honjo, The C-terminal region of activation-inducedcytidine deaminase is responsible for a recombinationfunction other than DNA cleavage in class switch recombina-tion, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 2758–2763.

[49] A.M. Patenaude, A. Orthwein, Y. Hu, V.A. Campo, B. Kavli, A.Buschiazzo, J.M. Di Noia, Active nuclear import and cytoplas-mic retention of activation-induced deaminase, Nat. Struct.Mol. Biol. 16 (2009) 517–527.

[50] R. Geisberger, C. Rada, M.S. Neuberger, The stability of AIDand its function in class-switching are critically sensitive tothe identity of its nuclear-export sequence, Proc. Natl. Acad.Sci. U. S. A. 106 (2009) 6736–6741.

[51] S. Ito, H. Nagaoka, R. Shinkura, N. Begum, M. Muramatsu, M.Nakata, T. Honjo, Activation-induced cytidine deaminaseshuttles between nucleus and cytoplasm like apolipoprotein BmRNA editing catalytic polypeptide 1, Proc. Natl. Acad. Sci.U. S. A. 101 (2004) 1975–1980.

[52] S. Brar, M. Watson, M. Diaz, Activation-induced cytosinedeaminase, AID, is actively exported out of the nucleus butretained by the induction of DNA breaks, J. Biol. Chem. 279(2004) 26935–26401.

[53] K.M. McBride, V. Barreto, A.R. Ramiro, P. Stavropoulos, M.C.Nussenzweig, Somatic hypermutation is limited by CRM1-dependent nuclear export of activation-induced deaminase,J. Exp. Med. 199 (2004) 1235–1244.

[54] K. Kinoshita, T. Honjo, Linking class-switch recombination withsomatic hypermutation, Nat. Rev. Mol. Cell. Biol. 2 (2001)493–503.

[55] T. Honjo, K. Kinoshita, M. Muramatsu, Molecular mechanismof class switch recombination: linkage with somatic hypermu-tation, Annu. Rev. Immunol. 20 (2002) 165–196.

[56] S.K. Petersen-Mahrt, R.S. Harris, M.S. Neuberger, AID mutatesE. coli suggesting a DNA deamination mechanism for antibodydiversification, Nature 418 (2002) 99–104.

[57] M. Muramatsu, K. Kinoshita, S. Fagarasan, S. Yamada, Y.Shinkai, T. Honjo, Class switch recombination and hypermuta-tion require activation-induced cytidine deaminase (AID), apotential RNA editing enzyme, Cell 102 (2000) 553–563.

[58] P. Revy, T. Muto, Y. Levy, F. Geissmann, A. Plebani, O. Sanal,N. Catalan, M. Forveille, R. Dufourcq-Labelouse, A. Gennery,I. Tezcan, F. Ersoy, H. Kayserili, A.G. Ugazio, N. Brousse, M.

Muramatsu, L.D. Notarangelo, K. Kinoshita, T. Honjo, A.Fischer, A. Durandy, Activation-induced cytidine deaminase(AID) deficiency causes the autosomal recessive form of thehyper-IgM syndrome (HIGM2), Cell 102 (2000) 565–575.

[59] S. Fagarasan, M. Muramatsu, K. Suzuki, H. Nagaoka, H. Hiai,T. Honjo, Critical roles of activation-induced cytidinedeaminase in the homeostasis of gut flora, Science 298(2002) 1424–1427.

[60] P. Quartier, J. Bustamante, O. Sanal, A. Plebani, M. Debre, A.Deville, J. Litzman, J. Levy, J.P. Fermand, P. Lane, G.Horneff, G. Aksu, I. Yalcin, G. Davies, I. Tezcan, F. Ersoy, N.Catalan, K. Imai, A. Fischer, A. Durandy, Clinical, immuno-logic and genetic analysis of 29 patients with autosomalrecessive hyper-IgM syndrome due to activation-inducedcytidine deaminase deficiency, Clin. Immunol. 110 (2004)22–29.

[61] V.T. Ta, H. Nagaoka, N. Catalan, A. Durandy, A. Fischer, K.Imai, S. Nonoyama, J. Tashiro, M. Ikegawa, S. Ito, K.Kinoshita, M. Muramatsu, T. Honjo, AID mutant analysesindicate requirement for class-switch-specific cofactors, Nat.Immunol. 4 (2003) 843–848.

[62] V. Barreto, B. Reina-San-Martin, A.R. Ramiro, K.M. McBride,M.C. Nussenzweig, C-terminal deletion of AID uncouples classswitch recombination from somatic hypermutation and geneconversion, Mol. Cell 12 (2003) 501–508.

[63] K. Imai, Y. Zhu, P. Revy, T. Morio, S. Mizutani, A. Fischer, S.Nonoyama, A. Durandy, Analysis of class switch recombinationand somatic hypermutation in patients affected with autoso-mal dominant hyper-IgM syndrome type 2, Clin. Immunol. 115(2005) 277–285.

[64] M. Takizawa, H. Tolarova, Z. Li, W. Dubois, S. Lim, E. Callen,S. Franco, M. Mosaico, L. Feigenbaum, F.W. Alt, A. Nussenz-weig, M. Potter, R. Casellas, AID expression levels determinethe extent of cMyc oncogenic translocations and the incidenceof B cell tumor development, J. Exp. Med. 205 (2008)1949–1957.

[65] I.V. Sernandez, V.G. de Yebenes, Y. Dorsett, A.R. Ramiro,Haploinsufficiency of activation-induced deaminase for anti-body diversification and chromosome translocations both invitro and in vivo, PLoS One e3927 (2008) 3.

[66] K. Imai, G. Slupphaug, W.I. Lee, P. Revy, S. Nonoyama, N.Catalan, L. Yel, M. Forveille, B. Kavli, H.E. Krokan, H.D. Ochs,A. Fischer, A. Durandy, Human uracil-DNA glycosylasedeficiency associated with profoundly impaired immunoglob-ulin class-switch recombination, Nat. Immunol. 4 (2003)1023–1028.

[67] C. Rada, G.T. Williams, H. Nilsen, D.E. Barnes, T. Lindahl,M.S. Neuberger, Immunoglobulin isotype switching is inhibitedand somatic hypermutation perturbed in UNG-deficient mice,Curr. Biol. 12 (2002) 1748–1755.

[68] C. Rada, J.M. Di Noia, M.S. Neuberger, Mismatch recognitionand uracil excision provide complementary paths to both Igswitching and the A/T-focused phase of somatic mutation,Mol. Cell. 16 (2004) 163–171.

[69] H. Nilsen, G. Stamp, S. Andersen, G. Hrivnak, H.E. Krokan, T.Lindahl, D.E. Barnes, Gene-targeted mice lacking the Unguracil-DNA glycosylase develop B-cell lymphomas, Oncogene22 (2003) 5381–5386.

[70] M. Endres, D. Biniszkiewicz, R.W. Sobol, C. Harms, M. Ahmadi,A. Lipski, J. Katchanov, P. Mergenthaler, U. Dirnagl, S.H.Wilson, A. Meisel, R. Jaenisch, Increased postischemic braininjury in mice deficient in uracil-DNA glycosylase, J. Clin.Invest. 113 (2004) 1711–1721.

[71] S. Peron, A. Metin, P. Gardes, M.A. Alyanakian, E. Sheridan, C.P. Kratz, A. Fischer, A. Durandy, Human PMS2 deficiency isassociated with impaired immunoglobulin class switch recom-bination, J. Exp. Med. 205 (2008) 2465–2472.

[72] M.R. Ehrenstein, C. Rada, A.M. Jones, C. Milstein, M.S.

202 S. Kracker et al.

Neuberger, Switch junction sequences in PMS2-deficient micereveal a microhomology-mediated mechanism of Ig classswitch recombination, Proc. Natl. Acad. Sci. U. S. A. 98(2001) 14553–14558.

[73] C.E. Schrader, J. Vardo, J. Stavnezer, Role for mismatchrepair proteins Msh2, Mlh1, and Pms2 in immunoglobulin classswitching shown by sequence analysis of recombinationjunctions, J. Exp. Med. 195 (2002) 367–373.

[74] H.M. Shen, A. Tanaka, G. Bozek, D. Nicolae, U. Storb, Somatichypermutation and class switch recombination in Msh6(-/-/-)double-knockout mice, J. Immunol. 177 (2006) 5386–5392.

[75] H. Sekine, R.C. Ferreira, Q. Pan-Hammarstrom, R.R. Graham,B. Ziemba, S.S. de Vries, J. Liu, K. Hippen, T. Koeuth, W.Ortmann, A. Iwahori, M.K. Elliott, S. Offer, C. Skon, L. Du, J.Novitzke, A.T. Lee, N. Zhao, J.D. Tompkins, D. Altshuler, P.K.Gregersen, C. Cunningham-Rundles, R.S. Harris, C. Her, D.L.Nelson, L. Hammarstrom, G.S. Gilkeson, T.W. Behrens, Rolefor Msh5 in the regulation of Ig class switch recombination,Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 7193–7198.

[76] J.E. Guikema, C.E. Schrader, N.G. Leus, A. Ucher, E.K.Linehan, U. Werling, W. Edelmann, J. Stavnezer, Reassess-ment of the role of Mut S homolog 5 in Ig class switchrecombination shows lack of involvement in cis- and trans-switching, J. Immunol. 181 (2008) 8450–8459.

[77] J. Stavnezer, C.E. Schrader, Mismatch repair converts AID-instigated nicks to double-strand breaks for antibody class-switch recombination, Trends Genet. 22 (2006) 23–28.

[78] F. Delbos, S. Aoufouchi, A. Faili, J.C. Weill, C.A. Reynaud,DNA polymerase eta is the sole contributor of A/T modifica-tions during immunoglobulin gene hypermutation in themouse, J. Exp. Med. 204 (2007) 17–23.

[79] N. Kim, G. Bozek, J.C. Lo, U. Storb, Different mismatch repairdeficiencies all have the same effects on somatic hypermuta-tion: intact primary mechanism accompanied by secondarymodifications, J. Exp. Med. 190 (1999) 21–30.

[80] Q.H. Phung, D.B. Winter, R. Alrefai, P.J. Gearhart, Hypermu-tation in Ig V genes from mice deficient in the MLH1 mismatchrepair protein, J. Immunol. 162 (1999) 3121–3124.

[81] Y.M. Hendriks, S. Jagmohan-Changur, H.M. van der Klift, H.Morreau, M. van Puijenbroek, C. Tops, T. van Os, A. Wagner,M.G. Ausems, E. Gomez, M.H. Breuning, A.H. Brocker-Vriends,H.F. Vasen, J.T. Wijnen, Heterozygous mutations in PMS2cause hereditary nonpolyposis colorectal carcinoma (Lynchsyndrome), Gastroenterology 130 (2006) 312–322.

[82] K. Wimmer, J. Etzler, Constitutional mismatch repair-defi-ciency syndrome: have we so far seen only the tip of aniceberg? Hum. Genet. 124 (2008) 105–122.

[83] F.A. Kadyrov, L. Dzantiev, N. Constantin, P. Modrich,Endonucleolytic function of MutLalpha in human mismatchrepair, Cell 126 (2006) 297–308.

[84] A. Durandy, N. Taubenheim, S. Peron, A. Fischer, Pathophys-iology of B-cell intrinsic immunoglobulin class switch recom-bination deficiencies, Adv. Immunol. 94 (2007) 275–306.

[85] A. Shanmugam, M.J. Shi, L. Yauch, J. Stavnezer, A.L. Kenter,Evidence for class-specific factors in immunoglobulin isotypeswitching, J. Exp. Med. 191 (2000) 1365–1380.

[86] L. Ma, H.H. Wortis, A.L. Kenter, Two new isotype-specificswitching activities detected for Ig class switching,J. Immunol. 168 (2002) 2835–2846.

[87] K. Imai, N. Catalan, A. Plebani, L. Marodi, O. Sanal, S. Kumaki,V. Nagendran, P. Wood, C. Glastre, F. Sarrot-Reynauld, O.Hermine, M. Forveille, P. Revy, A. Fischer, A. Durandy, Hyper-IgM syndrome type 4 with a B lymphocyte-intrinsic selectivedeficiency in Ig class-switch recombination, J. Clin. Invest. 112(2003) 136–142.

[88] S. Peron, Q. Pan-Hammarstrom, K. Imai, L. Du, N. Tauben-heim, O. Sanal, L. Marodi, A. Bergelin-Besancon, M. Benker-rou, J.P. de Villartay, A. Fischer, P. Revy, A. Durandy, A

primary immunodeficiency characterized by defective immu-noglobulin class switch recombination and impaired DNArepair, J. Exp. Med. 204 (2007) 1207–1216.

[89] R. Wuerffel, L. Wang, F. Grigera, J. Manis, E. Selsing, T.Perlot, F.W. Alt, M. Cogne, E. Pinaud, A.L. Kenter, S–Ssynapsis during class switch recombination is promoted bydistantly located transcriptional elements and activation-induced deaminase, Immunity 27 (2007) 711–722.

[90] B. Reina-San-Martin, H.T. Chen, A. Nussenzweig, M.C.Nussenzweig, ATM is required for efficient recombinationbetween immunoglobulin switch regions, J. Exp. Med. 200(2004) 1103–1110.

[91] S. Kracker, Y. Bergmann, I. Demuth, P.O. Frappart, G.Hildebrand, R. Christine, Z.Q. Wang, K. Sperling, M. Digweed,A. Radbruch, Nibrin functions in Ig class-switch recombina-tion, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 1584–1589.

[92] C.H. Bassing, F.W. Alt, H2AX may function as an anchor to holdbroken chromosomal DNA ends in close proximity, Cell Cycle 3(2004) 149–153.

[93] B. Reina-San-Martin, M.C. Nussenzweig, A. Nussenzweig, S.Difilippantonio, Genomic instability, endoreduplication, anddiminished Ig class-switch recombination in B cells lackingNbs1, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 1590–1595.

[94] J.P. Manis, D. Dudley, L. Kaylor, F.W. Alt, IgH class switchrecombination to IgG1 in DNA-PKcs-deficient B cells, Immunity16 (2002) 607–617.

[95] I. Meyts, C. Weemaes, C. De Wolf-Peeters, M. Proesmans, M.Renard, A. Uyttebroeck, K. De Boeck, Unusual and severedisease course in a child with ataxia–telangiectasia, Pediatr.Allergy Immunol. 14 (2003) 330–333.

[96] A. Etzioni, A. Ben-Barak, S. Peron, A. Durandy, Ataxia–telangiectasia in twins presenting as autosomal recessivehyper-immunoglobulin M syndrome, Isr. Med. Assoc. J. 9(2007) 406–407.

[97] A. Soresina, A. Meini, V. Lougaris, G. Cattaneo, S. Pellegrino,M. Piane, F. Darra, A. Plebani, Different clinical andimmunological presentation of ataxia–telangiectasia withinthe same family, Neuropediatrics 39 (2008) 43–45.

[98] A. Lahdesmaki, A.M. Taylor, K.H. Chrzanowska, Q. Pan-Hammarstrom, Delineation of the role of the Mre11 complexin class switch recombination, J. Biol. Chem. 279 (2004)16479–16487.

[99] Q. Pan, C. Petit-Frere, A. Lahdesmaki, H. Gregorek, K.H.Chrzanowska, L. Hammarstrom, Alternative end joiningduring switch recombination in patients with ataxia–telangi-ectasia, Eur. J. Immunol. 32 (2002) 1300–1308.

[100] Q. Pan-Hammarstrom, S. Dai, Y. Zhao, I.F. van Dijk-Hard, R.A.Gatti, A.L. Borresen-Dale, L. Hammarstrom, ATM is notrequired in somatic hypermutation of VH, but is involved inthe introduction of mutations in the switch mu region,J. Immunol. 170 (2003) 3707–3716.

[101] L. Du, D.K. Dunn-Walters, K.H. Chrzanowska, T. Stankovic, A.Kotnis, X. Li, J. Lu, G. Eggertsen, C. Brittain, S.W. Popov, A.R.Gennery, A.M. Taylor, Q. Pan-Hammarstrom, A regulatory rolefor NBS1 in strand-specific mutagenesis during somatichypermutation, PLoS ONE e2482 (2008) 3.

[102] D. Buck, L. Malivert, R. de Chasseval, A. Barraud, M.C.Fondaneche, O. Sanal, A. Plebani, J.L. Stephan, M. Hufnagel,F. le Deist, A. Fischer, A. Durandy, J.P. de Villartay, P. Revy,Cernunnos, a novel nonhomologous end-joining factor, ismutated in human immunodeficiency with microcephaly, Cell124 (2006) 287–299.

[103] Q. Pan-Hammarstrom, A.M. Jones, A. Lahdesmaki, W. Zhou,R.A. Gatti, L. Hammarstrom, A.R. Gennery, M.R. Ehrenstein,Impact of DNA ligase IV on nonhomologous end joiningpathways during class switch recombination in human cells,J. Exp. Med. 201 (2005) 189–194.

[104] L. Du, M. van der Burg, S.W. Popov, A. Kotnis, J.J. van Dongen,

203Immunoglobulin class switch recombination deficiencies

A.R. Gennery, Q. Pan-Hammarstrom, Involvement of Artemisin nonhomologous end-joining during immunoglobulin classswitch recombination, J. Exp. Med. 205 (2008) 3031–3040.

[105] M. O'Driscoll, K.M. Cerosaletti, P.M. Girard, Y. Dai, M. Stumm,B. Kysela, B. Hirsch, A. Gennery, S.E. Palmer, J. Seidel, R.A.Gatti, R. Varon, M.A. Oettinger, H. Neitzel, P.A. Jeggo, P.Concannon, DNA ligase IV mutations identified in patientsexhibiting developmental delay and immunodeficiency, Mol.Cell 8 (2001) 1175–1185.

[106] N. Kawamura, A. Okamura, H. Furuta, S. Katow, M. Yamada, I.Kobayashi, M. Okano, K. Kobayashi, Y. Sakiyama, Improveddysgammaglobulinaemia in congenital rubella syndrome afterimmunoglobulin therapy: correlation with CD154 expression[In Process Citation], Eur. J. Pediatr. 159 (2000) 764–766.

[107] S. Nonoyama, S. Tsukada, T. Yamadori, T. Miyawaki, Y.Z. Jin,C. Watanabe, T. Morio, J. Yata, H.D. Ochs, Functional analysisof peripheral blood B cells in patients with X-linkedagammaglobulinemia, J. Immunol. 161 (1998) 3925–3929.

[108] B. Grimbacher, A. Hutloff, M. Schlesier, E. Glocker, K.Warnatz, R. Drager, H. Eibel, B. Fischer, A.A. Schaffer, H.W.Mages, R.A. Kroczek, H.H. Peter, Homozygous loss of ICOS isassociated with adult-onset common variable immunodefi-ciency, Nat. Immunol. 4 (2003) 261–268.

[109] U. Salzer, H.M. Chapel, A.D. Webster, Q. Pan-Hammarstrom,A. Schmitt-Graeff, M. Schlesier, H.H. Peter, J.K. Rockstroh, P.Schneider, A.A. Schaffer, L. Hammarstrom, B. Grimbacher,Mutations in TNFRSF13B encoding TACI are associated withcommon variable immunodeficiency in humans, Nat. Genet.37 (2005) 820–828.

[110] E. Castigli, S.A. Wilson, L. Garibyan, R. Rachid, F. Bonilla, L.Schneider, R.S. Geha, TACI is mutant in common variableimmunodeficiency and IgA deficiency, Nat. Genet. 37 (2005)829–834.

[111] L. Bossaller, J. Burger, R. Draeger, B. Grimbacher, R. Knoth,A. Plebani, A. Durandy, U. Baumann, M. Schlesier, A.A.Welcher, H.H. Peter, K. Warnatz, ICOS deficiency is associ-ated with a severe reduction of CXCR5+CD4 germinal centerTh cells, J. Immunol. 177 (2006) 4927–4932.

[112] A.T. Bauquet, H. Jin, A.M. Paterson, M. Mitsdoerffer, I.C. Ho,

A.H. Sharpe, V.K. Kuchroo, The costimulatory molecule ICOSregulates the expression of c-Maf and IL-21 in the develop-ment of follicular T helper cells and TH-17 cells, Nat.Immunol. 10 (2009) 167–175.

[113] M.B. Litinskiy, B. Nardelli, D.M. Hilbert, B. He, A. Schaffer, P.Casali, A. Cerutti, DCs induce CD40-independent immuno-globulin class switching through BLyS and APRIL, Nat.Immunol. 3 (2002) 822–829.

[114] F. Mackay, P. Schneider, P. Rennert, J. Browning, BAFF ANDAPRIL: a tutorial on B cell survival, Annu. Rev. Immunol. 21 (2003)231–264.

[115] M. Batten, J. Groom, T.G. Cachero, F. Qian, P. Schneider, J.Tschopp, J.L. Browning, F. Mackay, BAFF mediates survival ofperipheral immature B lymphocytes, J. Exp. Med. 192 (2000)1453–1466.

[116] K. Warnatz, U. Salzer, M. Rizzi, B. Fischer, S. Gutenberger, J.Bohm, A.K. Kienzler, Q. Pan-Hammarstrom, L. Hammar-strom, M. Rakhmanov, M. Schlesier, B. Grimbacher, H.H.Peter, H. Eibel, B-cell activating factor receptor deficiency isassociated with an adult-onset antibody deficiency syndromein humans, Proc. Natl. Acad. Sci. U. S. A. 106 (2009)13945–13950.

[117] B.H. Ye, G. Cattoretti, Q. Shen, J. Zhang, N. Hawe, R. deWaard, C. Leung, M. Nouri-Shirazi, A. Orazi, R.S. Chaganti, P.Rothman, A.M. Stall, P.P. Pandolfi, R. Dalla-Favera, The BCL-6proto-oncogene controls germinal-centre formation and Th2-type inflammation, Nat. Genet. 16 (1997) 161–170.

[118] R.T. Phan, R. Dalla-Favera, The BCL6 proto-oncogene sup-presses p53 expression in germinal-centre B cells, Nature 432(2004) 635–639.

[119] R.T. Phan, M. Saito, K. Basso, H. Niu, R. Dalla-Favera, BCL6interacts with the transcription factor Miz-1 to suppress thecyclin-dependent kinase inhibitor p21 and cell cycle arrestin germinal center B cells, Nat. Immunol. 6 (2005)1054–1060.

[120] S.A. Omori, M.H. Cato, A. Anzelon-Mills, K.D. Puri, M. Shapiro-Shelef, K. Calame, R.C. Rickert, Regulation of class-switchrecombination and plasma cell differentiation by phosphati-dylinositol 3-kinase signaling, Immunity 25 (2006) 545–557.