Primary antibody deficiencies - docvadis.fr fileinformation S1 (table)); however, these conditions...

Our understanding of the physiology of immune responses has been substantially enhanced by the study of natural mutants observed in human primary immu-nodeficiencies (PIDs) — particularly in cases for which an equivalent animal model is not available. Primary antibody deficiencies (PADs) are the most common PIDs in humans. In the past, PADs were mostly attrib-uted to B cell-intrinsic defects (see Supplementary information S1 (table)); however, these conditions can also be caused by functional impairments in other immune cell lineages (including innate immune cells and T cells). Characterization of the genetic features of PADs has revealed new important molecular pathways to be involved in B cell development and antibody pro-duction1–4. The study of PADs has also provided new insights into the genesis of antibody-mediated auto-immunity and checkpoints of B cell reactivity, as well as into defence against infections5. Genetic characteri-zation is also essential for the accurate diagnosis and treatment of patients with these conditions.

However, it seems that the genetic analysis of PADs is more complicated than was previously anticipated: the limited genotype–phenotype correlation observed in many cases suggests that additional genetic and/or environmental factors have a role in PAD pathogenesis. This complexity is further increased by the fact that different mutations in the same gene can lead to dis-tinct phenotypes and can even have different patterns of inheritance, depending on their location in the gene or on as-yet-unknown events related to modifier genes or epigenetic interactions. Last, whereas most PIDs are monogenic, the most prevalent type of PAD — com-mon variable immunodeficiency (CVID) — seems to have a more complex genetic basis6.

Here, we review the different PADs in terms of the main mechanisms that are perturbed in the affected patients. Such defects may be B cell intrinsic or B cell extrinsic, although the aetiology of several PADs is still unknown. In addition, we describe the immune pathologies that have been associated with the various PADs, including increased susceptibility to infections, autoimmunity and cancer.

PADs associated with B cell-intrinsic defectsDefects in B cell development. Genetic defects affecting the expression of the pre‑B cell receptor (pre-BCR) or pre-BCR signalling typically result in the absence of circulat-ing mature B cells and of all immunoglobulin isotypes, accompanied by the accumulation of pre‑B cells in the bone marrow. As a consequence, agammaglobulinaemia occurs early in life (after maternal IgG molecules have disappeared at 6 months of age). As shown in BOX 1 and FIG. 1, several PADs have been associated with defects that affect components of the pre-BCR (the λ5 chain or the μ-chain), the pre-BCR and BCR co-receptors Igα and Igβ, and components of the pre-BCR and BCR sig-nalling pathways, including the p85α subunit of phos-phoinositide 3-kinase (PI3K) and the scaffold protein B cell linker (BLNK)1,7. These defects all lead to rare, autosomal-recessive forms of agammaglobulinaemia (Supplementary information S1 (table)).

The most common PAD that has been associated with defects in B cell development is X-linked agammaglob-ulinaemia (XLA). This condition is caused by mutations in the Bruton’s tyrosine kinase (BTK) gene and accounts for 85% of patients with agammaglobulinaemia8. BTK is a key molecule in pre-B cell activation and differentia-tion. Following pre-BCR engagement in early B cells (and

1National Institute of Health and Medical Research (INSERM) U768, Necker Children’s Hospital, F-75015 Paris, France.2Descartes-Sorbonne Paris Cité University of Paris, Imagine Institute, F-75015 Paris, France.3Center for Primary Immunodeficiencies (CEDI), Assistance Publique-Hopitaux de Paris, Necker Children’s Hospital, F-75015 Paris, France.4Department of Immunology and Haematology, Assistance Publique-Hopitaux de Paris, Necker Children’s Hospital, F-75015 Paris, France.Correspondence to A.D.e-mail: [email protected]:10.1038/nri3466Published online 14 June 2013

Pre‑B cell receptor(Pre‑BCR). A receptor that is formed at the surface of pre‑B cells when rearranged immunoglobulin heavy chains pair with surrogate immunoglobulin light chains; the pre‑BCR is associated with signalling heterodimers of Igα and Igβ. Signalling through the pre‑BCR occurs in the absence of known ligands and is a crucial event in B cell development.

Primary antibody deficienciesAnne Durandy1–4, Sven Kracker1,2 and Alain Fischer1,2,4

Abstract | Primary antibody deficiencies (PADs) are the most common inherited immunodeficiencies in humans. The use of novel approaches, such as whole-exome sequencing and mouse genetic engineering, has helped to identify new genes that are involved in the pathogenesis of PADs and has enabled the characterization of the molecular pathways that are involved in B cell development and function. Here, we review the different PADs in terms of their known or putative mechanisms, which can be B cell intrinsic, B cell extrinsic or not defined so far. We also describe the clinical manifestations (including susceptibility to infections, autoimmunity and cancer) that have been associated with the various PADs.

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Pre‑B cellsHaematopoietic cells that appear in the bone marrow early during B cell development, downstream of the CD34+ pro‑B cell precursor. At the pre‑B1 stage, the cells still express CD34 but acquire the B cell‑specific marker CD19. Pre‑B2 cells are characterized by complete immunoglobulin heavy chain rearrangement in the absence of immunoglobulin light chain rearrangement. They express the pre‑B cell receptor (which comprises a pseudo‑light chain and the μ‑heavy chain), CD19 and cytoplasmic IgM.

V(D)J recombinationA site‑specific recombination process (targeting recombination signal sequences) that takes place in primary lymphoid tissues and stochastically combines the different regions of the T cell and B cell receptors (variable (V), diversity (D) and joining (J) regions) in pre‑T and pre‑B cell precursors. The lymphoid‑specific enzymes RAG1 and RAG2 and the non‑lymphoid‑specific non‑homologous end joining DNA repair complex (which includes DNA protein kinase, Ku70–Ku80, Artemis, XRCC4, DNA ligase 4 and Cernunnos) control this process.

BCR engagement in mature B cells), BTK is phosphoryl-ated by the SRC family kinase LYN and interacts with phosphatidylinositol-3,4,5-trisphosphate (a molecule generated by PI3K). Subsequently, activated BTK directly phosphorylates phospholipase Cγ2 (PLCγ2), which in

turn converts phosphatidylinositol-4,5-bisphosphate into inositol-1,4,5-trisphosphate and diacylglycerol, leading to intracellular Ca2+ release9. Most BTK mutations result in a lack of BTK protein expression, as observed in monocytes (which do not depend on BTK for their development),

Box 1 | B cell development defects in PADs

Primary antibody deficiencies (PADs) result from developmental defects that are either B cell specific or affect several haematopoietic cell lineages (see the figure). B cell development occurs in the bone marrow, where haematopoietic stem cells (HSCs) undergo B cell lineage specification. Some autosomal-recessive forms of severe combined immunodeficiency (SCID) are associated with an early defect in both B cells and T cells and are diagnosed in the first few months or years of life. For example, adenosine deaminase (ADA) deficiency leads to an accumulation of adenosines and thus the death of the lymphocytes113,114. Moreover, adenylate kinase 2 (AK2) deficiency (also known as reticular dysgenesis) is a metabolic defect that mostly affects T cells, natural killer (NK) cells, neutrophils and (in some cases) B cells115,116. It is associated with very-early-onset hypogammaglobulinaemia or even agammaglobulinaemia. Dyskeratosis congenita is caused by mutations in genes encoding components of the telomerase or shelterin complexes117; it is a rare inherited bone-marrow failure syndrome that leads to progressive B cell and T cell lymphopenia and hypogammaglobulinaemia. Finally, mutations in GATA2 (which encodes a transcription factor required for early differentiation of haematopoietic cells in the bone marrow) also lead to B cell, dendritic cell, monocyte and NK cell deficiencies118.

Successive B cell differentiation stages are characterized by ordered gene expression and stochastic immunoglobulin gene rearrangements. The V(D)J recombination of the immunoglobulin locus is achieved by the lymphocyte-specific RAG molecules and the non-lymphocyte-specific non-homologous end joining (NHEJ) complex, and leads to the expression of the pre-B cell receptor (BCR). Defects in V(D)J recombination (as observed in RAG deficiency and NHEJ deficiency) typically result in the absence of mature B cells and T cells119,120. The pre-BCR is affected in these cases, leading to PADs, as V(D)J recombination is required for heavy chain expression.

The dashed lines indicate a block of differentiation. BLNK, B cell linker; BTK, Bruton’s tyrosine kinase; CMP, common myeloid progenitor; ETP, early T cell precursor; GMP, granulocyte/macrophage progenitor; MEP, megakaryocyte/erythrocyte progenitor; MLP, multi-lymphoid progenitor; MPP, multipotent progenitor; PI3K, phosphoinositide 3-kinase.

Nature Reviews | Immunology

HSC

MLP

CMP

GMP

MEP

Self-renewal

ADA deficiency

Pre-B cell

Pro-B cell

CD19

ImmatureB cell

B cell

NK cell

Dendriticcell

CD19

Monocyte

Granulocyte

Erythrocyte

Megakaryocyte

Platelets

Hoyeraal-Hreidarsson (shelterin deficiency)

MPP

• RAG (RAG1 and RAG2) deficiency• NHEJ (Artemis) deficiency

ETP T cell

B cell and NK cellprogenitor

GATA2 deficiency

AK2 deficiencyGATA2deficiency

• BTK deficiency• BLNK deficiency• PI3K (p85α) deficiency• λ5 deficiency• Cµ deficiency• Igα or Igβ deficiencies

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CD81CD20


Ca2+

ORAI1

VpreB

CD19 complex deficiency

CD21

Pre-BCR or BCR

λ5

Ca2+

STIM1

λ5deficiency

Cµdeficiency

Igβ deficiency

BLNK deficiency

NF-κBactivation

NFATactivation

PLCγ2 gainof function

p85adeficiency

CD20deficiency

Igα deficiency

ER

IgαIgβ

PP

PPP

SYK

PLCγ2

LYN

RACSOS

ERK

PtdIns(3,4,5)P3 PtdIns(4,5)P2

P

PP

BTK

VAV

BLNK

PI3K

GRB2

HOIL1, CARD11, NEMO and IKKβ deficiencies. IκBα gain of function

p85 p110

Polysaccharide‑specific antibodiesAntibodies that are secreted during T cell‑independent antibody responses (especially by marginal zone B cells).

and a severe block in B cell differentiation. However, the block is less pronounced than that observed in autoso-mal-recessive agammaglobulinaemia. Moreover, some BTK mutations do not affect BTK expression and are generally associated with a milder phenotype (including the presence of low numbers of circulating B cells and residual levels of immunoglobulins)1,10. This genotype–phenotype correlation is not universal, and the observa-tion of intra-family variations11 suggests the influence of as-yet-unknown modifier genes. At one extreme of the phenotypic range, a patient with XLA whose only disease feature was defective production of polysaccharide‑specific antibodies has been reported12.

In addition to the above-mentioned mutations, some genetic defects that affect B cell development also affect non-B cell haematopoietic lineages. These defects are discussed more extensively in BOX 1 and Supplementary information S2 (table).

Overall, the description of the PAD-associated genetic defects that affect B cell development has led to the identification of several discrete steps in this early process. Some of these steps are required for the expan-sion of pro-B cell and pre-B cell populations, and there-fore are B cell specific, whereas other steps are common to B cell and other haematopoietic cell lineages, as they precede lineage specification or are shared by B cell and T cell development.

Defects in B cell migration. B cells migrate from the bone marrow to the blood and then to the spleen (FIG. 2). From there, they recirculate in the blood and home to secondary lymphoid organs. Abnormalities in this migration pattern often lead to the development of PADs (Supplementary information S1 (table)). Warts, hypogammaglobulinaemia, infections and myelokath-exis (WHIM) syndrome is a rare, autosomal-dominant disorder that is caused by gain-of-function mutations in the gene encoding CXC-chemokine receptor 4 (CXCR4). As CXC-chemokine ligand 12 (CXCL12)–CXCR4 interactions retain developing B cells in bone-marrow niches, patients with WHIM syndrome have low circulating B cell counts and hypogammaglobuli-naemia13 (FIG. 2).

In addition to WHIM syndrome, several combined immunodeficiency (CID) syndromes involve PADs, at least in part, as a result of defects in B cell migration. The X-linked disorder Wiskott–Aldrich syndrome (WAS), which is characterized by thrombocytopenia, eczema and immune deficiency, is caused by mutations in the WAS gene and is generally diagnosed during the first few years after birth14. Wiskott–Aldrich syndrome protein (WASP; encoded by WAS) is required for both the adhesion and the migration of haematopoietic cells (including B cells) and for actin polarization dur-ing the formation of the immunological synapse15.

Figure 1 | Pre-BCR and BCR signalling defects in PADs. The pre-B cell receives proliferation and differentiation signals through the pre-B cell receptor (BCR) and the co-receptors Igα and Igβ. Signalling from the pre-BCR involves the immunoreceptor tyrosine-based activation motifs (ITAMs) of the co-receptors Igα and Igβ, which scaffold and activate the tyrosine kinase SYK. SYK either activates the extracellular signal-regulated kinase (ERK) pathway or phosphorylates (P) (together with LYN) the adaptor protein B cell linker (BLNK) and Bruton’s tyrosine kinase (BTK), leading to the activation of phospholipase Cγ2 (PLCγ2) and the phosphoinositide 3-kinase (PI3K) pathway. Defects in this pathway affect the pre-BCR (in Cμ or pseudo light chain λ5), the pre-BCR signal transduction molecules Igα and Igβ, the downstream molecules BTK, BLNK and PI3K, components of the co-stimulatory CD19 complex (CD19, CD21 and CD81) and the B cell marker CD20. The BCR triggers the canonical nuclear factor-κB (NF-κB) pathway through the scaffolding protein CARD11 and activation of the IκB kinase (IKK) complex (comprising IKKα, IKKβ and NEMO). IKK activation leads to the phosphorylation and degradation of NF-κB inhibitor-α (IκBα) and the subsequent release of the p50–p65 NF-κB heterodimer, which then translocates to the nucleus to regulate gene transcription (not shown). Following antigen binding to antigen receptors (such as the BCR), endoplasmic reticulum Ca2+ stores are depleted, STIM1 is activated and ORAI1 Ca2+ release-activated Ca2+ channels open, resulting in store-operated Ca2+ entry. This influx results in activation of the transcription factor NFAT. The dashed arrows indicate downstream signalling events. ER, endoplasmic reticulum; PAD, primary antibody deficiency; PtdIns(4,5)P

2,

phosphatidylinositol-4,5-bisphosphate; PtdIns(3,4,5)P3, phosphatidylinositol-3,4,5-trisphosphate.

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CXCR5

Germinal centre

Follicle

Marginal zone


Immature B cell

CXCR4

IgM

Naive mature B cell

TransitionalB cell

IgMIgDCD19

CD20

CD19

CD20

WHIM syndrome(CXCR4 gain of function)

Bone marrow Blood Spleen

Wiskott–Aldrich syndrome(WASP deficiency)

CID (DOCK8 deficiency and MST1 deficiency)

Marginal zoneAn anatomical site located at the interface between the red pulp and the lymphoid white pulp of the spleen, in which marginal zone B cells are rapidly recruited into early, adaptive immune responses in a T cell‑independent manner. Marginal zone B cells produce IgM as the first line of defence against blood‑borne antigens.

Germinal centreWithin secondary lymphoid tissues, B cells exposed to migration signals (through CXCL13–CXCR5) enter the follicles and, following interaction with cognate T cells, undergo vigorous proliferation and form germinal centres. B cells undergo class‑switch recombination and somatic hypermutation in these germinal centres.

Although WASP is dispensable for B cell development, it has a crucial role in the homeostasis, development and function of peripheral B cells. As a consequence, patients with WAS mutations have low IgM levels, low marginal zone B cell counts and high transitional B cell counts. These features are also observed in Was−/− mice as a consequence of defective BCR-mediated integrin signalling in B cells and as a defective response to the chemokine CXCL12, which is involved in the localiza-tion of B cells to the marginal zone16. This defect in marginal zone B cells probably underlies the inability of patients with WAS to cope with encapsulated, blood-borne pathogens. In addition to defective localization in the marginal zone, other mechanisms (namely, inappropriate B cell development or responses17 and defective T cell help) can lead to the PAD observed in WAS. In addition, autosomal-recessive deficiency in WASP-interacting protein (WIP) leads to a similar phenotype18.

Deficiency in another cytoskeleton regulator — ded-icator of cytokinesis 8 (DOCK8) — underlies a newly described combined PID. This autosomal-recessive syndrome is characterized by susceptibility to viral infections and recurrent sinus and pulmonary bacterial infections, atopy, early-onset malignancies and autoim-munity. Reported immunological abnormalities include a defect in the generation of marginal zone B cells and an impairment in the retention of B cells at the germi‑nal centre and BCR affinity maturation. These defects in combination with T cell lymphopenia and defective T cell proliferation may account for the observed low

IgM levels, variable IgG antibody responses, low num-bers of memory B cells and defective B cell responses to Toll-like receptor (TLR) activation19–21 (FIG. 2). Thus, DOCK8 deficiency results in a CID, and the PAD observed in this syndrome does not result only from defects in B cell migration, but also from defects in other B cell and T cell functions.

Apart from as a protein involved in cytoskeletal dynamics, mammalian STE20-like protein kinase 1 (MST1; also known as STK4) has also been impli-cated in PAD pathogenesis22. MST1 has a role in lym-phocyte adhesion and migration, and patients with MST1 deficiency present with a CID characterized by normal immunoglobulin levels but defective antigen-specific antibody responses. This autosomal-recessive B cell immunodeficiency may partially result from the defective localization of B cells in the marginal zone or in germinal centres, as MST1 is involved in the Hippo pathway that regulates forkhead box O (FOXO) tran-scription factors and hence chemokine receptor expres-sion. However, MST1 also has a role in B cell and T cell survival, and impaired B cell survival probably underlies the PAD observed in this CID23,24.

The description of several PADs caused by defects in cell migration points to a rather newly perceived disease mechanism. It is nevertheless difficult to fully distinguish B cell defects related to abnormal migration (or retention) of B cells from B cell survival and func-tional defects, as almost all molecules involved in this class of PADs have a role in both types of biological event (further discussed below).

Figure 2 | B cell migration defects in PADs. Immature B cells egress from the bone marrow after attenuation of CXC-chemokine receptor 4 (CXCR4) signalling and differentiate in the periphery into transitional B cells and then mature naive B cells. A gain of function in CXCR4 results in the retention of immature B cells in the bone marrow, leading to warts, hypogammaglobulinaemia, infections and myelokathexis (WHIM) syndrome, which is characterized by low numbers of circulating B cells and hypogammaglobulinaemia. Mature naive B cells undergo two different fates following antigen activation: they either localize in the marginal zone, where they elicit early T cell-independent antibody responses, or enter the follicles in response to migration signals (mediated by CXC-chemokine ligand 13 (CXCL13)–CXCR5) and interact with cognate T cells. Follicular B cells then undergo vigorous proliferation and support germinal centre formation. A deficiency in Wiskott–Aldrich syndrome protein (WASP) or WASP-interacting protein (WIP) leads to defective B cell migration into the marginal zone (owing to an impaired response to CXCL12) but also to additional B cell and T cell activation defects. Dedicator of cytokinesis 8 (DOCK8) and mammalian STE20-like protein kinase 1 (MST1) deficiencies affect both the B cell and the T cell compartment. DOCK8-deficient murine B cells cannot be recruited into the marginal zone and cannot be retained in germinal centres for affinity maturation, probably because of the abnormal polarization of molecules in the immune synapse. MST1 is involved in the HIPPO pathway that regulates forkhead box O (FOXO) transcription factors, and thus MST1 mutations affect B cell migration by preventing chemokine receptor expression. MST1 also has a role in B cell and T cell survival. PAD, primary antibody deficiency.

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TransitionalB cell

IgMIgDCD19

CD20

BAFFR

BAFFBAFFor APRIL

BAFFor APRIL B cell survival defects

• BAFFR deficiency• TWEAK deficiency• TACI deficiency• POLε deficiency

LRBA deficiency

IgM IgDCD19

CD20

Naive mature B cell

IgDIgM

CD27

MarginalzoneB cell

IgM

IgG

IgA

IgE

CD27

MemoryB cell

FollicularB cell

Plasma cell

Plasma cell

IgM

B cell activation defects

CSR deficiencies

TACI

BCMA

T cell‑dependent antibody responseAntibody response to protein antigens that require T cell help. This response mostly occurs in the germinal centre in secondary lymphoid organs, via CD40L–CD40 interactions.

T cell‑independent antibody responseAntibody response to polymeric antigens, such as polysaccharides and lipids, that do not require T cell help.

Defects in B cell survival. The survival and homeosta-sis of B cells at discrete stages of their development are regulated by two cytokines — BAFF (B cell-activating factor) and APRIL (a proliferation-inducing ligand) — which are both produced by stromal and haematopoietic cells (FIG. 3). BAFF binds to three receptors expressed by B cells (the BAFF receptor (BAFFR), transmembrane activator and CAML interactor (TACI) and B cell matu-ration antigen (BCMA)), whereas APRIL only binds to TACI and BCMA (FIG. 3).

Mutations in TACI and BAFFR (but so far no mutations in BCMA) have been reported to cause PADs in humans

(Supplementary information S1 (table)). However, many of the TACI variants described in some patients with CVID are also observed in healthy controls and may well correspond to CVID predisposition genes rather than to disease-causing mutations6. In the few BAFFR-deficient patients reported to date (who have all been diagnosed in adulthood), biallelic mutations in BAFFR seem to be disease causative, despite some phenotypic variability; patients have B cell lymphopenia, with low numbers of marginal zone, follicular and memory B cells. This emphasizes the role of BAFF in the survival of transitional B cells during their differentiation into mature B cells25. In these patients, the T cell‑dependent antibody response to tetanus toxoid is normal but the T cell‑independent response

to polysaccharides is not normal. IgA levels are normal, indicating that the differentiation of mucosal IgA+ human B cells is independent of BAFFR signalling. However, the related immunopathology has low penetrance, indicat-ing that it is very likely that a combination of defects is required to cause clinical and immunological conse-quences. Nonetheless, the association of a heterozygous missense mutation in the gene encoding TNF-like weak inducer of apoptosis (TWEAK) with an autosomal- dominant hypogammaglobulinaemia (Supplementary information S1 (table)) further supports the key role of BAFF signalling in B cell survival, as the TWEAK mutant seems to affect B cell survival by interacting with BAFF to form noneffective BAFF complexes26.

Defects in B cell activation. Antigen recognition by the antigen-specific BCR leads to the activation of naive B cells, which in turn differentiate into either plasmab-lasts or memory B cells. Impaired BCR signalling that results from B cell-intrinsic defects leads to variable pan-hypogammaglobulinaemia, although the numbers of circulating B cells remain normal (FIG. 3). These diseases are typically diagnosed in childhood or early adulthood. BCR-induced signals are regulated by B cell surface mol-ecules that modulate the intensity and threshold of BCR signal transduction. Of these, the CD19 complex, which

Figure 3 | B cell survival defects in PADs. The survival and homeostasis of B cells at discrete stages of their development are regulated by BAFF (B cell-activating factor) and APRIL (a proliferation-inducing ligand). BAFF binds to three receptors expressed by B cells (the BAFF receptor (BAFFR), transmembrane activator and CAML interactor (TACI) and B cell maturation antigen (BCMA)), whereas APRIL only binds to TACI and BCMA. Defects in TACI (although evidence for this is controversial) or BAFFR are responsible for rare forms of common variable immunodeficiency. A mutant of TNF-like weak inducer of apoptosis (TWEAK) also induces a primary antibody deficiency (PAD), probably through competition with BAFF on BAFF receptors. The survival of B cells in the periphery also requires basal stimulation through the B cell receptor (BCR) and co-stimulatory molecules (the CD19 complex and CD20). Following antigen encounter, marginal zone B cells rapidly differentiate into IgM-producing plasma cells, which act as the first line of defence against systemic, blood-borne antigens. Activated follicular B cells receive help from cognate T follicular helper (T

FH) cells to

undergo class-switch recombination (CSR) and somatic hypermutation (SHM) in germinal centres, and then they differentiate into CD27+ long-lived memory B cells and plasma cells. B cell activation defects include B cell-intrinsic defects and combined B cell and T cell defects (see also FIG. 4a). CSR deficiencies are caused by either a B cell-intrinsic defect affecting the CSR machinery or defective T

FH cell–B cell interactions (see also FIG. 4a,b). POLε, DNA polymerase-ε.

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contains CD19, CD21, CD81 and CD225, initiates Ca2+ signalling following BCR stimulation (FIG. 1). Thus, muta-tions in any of the CD19, CD21 and CD81 genes27–29 lead to hypogammaglobulinaemia as a result of defective B cell activation (TABLE 1). As CD21 is the receptor for Epstein–Barr virus (EBV), it would be interesting to investigate whether CD21-deficient patients can be infected by this virus. Biallelic mutations in the gene encoding the co-stimulatory receptor CD20 do not affect BCR-induced Ca2+ signalling, but still lead to partial hypogammaglob-ulinaemia and impaired T cell-independent antibody responses to polysaccharides30. The pathophysiological mechanism of CD20 deficiency is currently unknown.

Some immunodeficiencies that involve impaired B cell activation have been associated with mutations that affect both B cell and T cell signalling. For exam-ple, immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome is caused by muta-tions in the genes that encode DNA methyl transferase DNMT3B31 and ZBTB24 (a transcription factor that is possibly involved in DNA methylation)32. This autoso-mal-recessive syndrome results in a CID mainly charac-terized by pan-hypogammaglobulinaemia. B cells exhibit a naive phenotype, are prone to apoptosis in vitro and are potentially self-reactive33. The mechanistic link between DNA methylation and B cell activation has not yet been described.

Following antigen binding to antigen receptors (such as the BCR), endoplasmic reticulum Ca2+ stores are depleted, STIM1 is activated and ORAI1 Ca2+ release-activated Ca2+ channels open, resulting in store-operated

Ca2+ entry (FIG. 1). This influx results in the activation of the transcription factor NFAT. A defect in this pathway (as observed in patients with biallelic mutations in STIM1 or ORAI1) leads to the abnormal production of specific anti-bodies despite the presence of normal immunoglobulin levels and to autoimmune responses against blood cells. The phenotype is not restricted to B cells, as patients also present with a T cell deficiency, muscle hypotonia and anhydrotic ectodermal dysplasia (EDA)34,35.

Impairment of the canonical nuclear factor-κB (NF-κB) pathway leads to abnormal B cell activation. The BCR and CD40 trigger the canonical NF-κB path-way via activation of the IκB kinase (IKK) complex, which comprises IKKα, IKKβ and NEMO (also known as IKKγ). IKK activation leads to the phosphoryla-tion of IκBα and the subsequent release of the active p50–p65 NF-κB heterodimer, which then translocates to the nucleus to regulate gene transcription (FIG. 4a). The scaffolding protein CARD11 links BCR and T cell receptor (TCR) signalling to the canonical NF-κB path-way (FIG. 4a), so it is not surprising that mutations in CARD11 result in a type of CID. In detail, patients with CARD11 mutations exhibit agammaglobulinaemia and a striking increase in the numbers of transitional B cells36 (FIG. 4a; Supplementary information S1 (table)). It is noteworthy that hemizygous gain-of-function mutations in CARD11 cause a selective deficiency in polysaccharide-specific antibodies and an increased transitional B cell count. This phenotype may be linked to constitutive NF-κB pathway activation, resulting in polyclonal B cell activation and B cell exhaustion37.

Table 1 | The main phenotypes of primary antibody deficiencies

Phenotype Main clinical features Main B cell biological features

Known affected proteins

Pan-agammaglobulinaemia (absence of IgM, IgG and IgA)

Bacterial infections (in the respiratory tract) and enterovirus infections

Absence of CD19+ B cells λ5, BLNK, BTK, Cμ, Igα, Igβ and PI3K

Variable pan-hypogamma-globulinaemia (CVID)

Bacterial infections (in the respiratory tract and gut), autoimmunity, cancer and increased risk of granuloma

Decreased frequency of CD27+ memory B cells; defective plasma cells in tissues

CD19, CD20, CD21, CD27, CD81, DNMT3B, ZBTB24, ICOS, SAP, TACI and BAFFR

CSR deficiencies (absence or decrease in levels of IgG and IgA)

Bacterial and opportunistic infections

Decreased frequency of CD27+ memory B cells

CD40 and CD40L

Bacterial infections, autoimmunity and lymphadenopathies

Normal frequency of CD27+ memory B cells

AID and UNG

Selective IgA deficiency Most often asymptomatic ND ND

Selective IgM deficiency Frequent infections with encapsulated bacteria

No IgM antibody production (absence of allohemaglutinins and polysaccharide-specific antibodies)

Selective IgG2 and/or IgG4 deficiency

Frequent bacterial infections, diagnosis after 2 years of age; sometimes transient in childhood

Defective polysaccharide- specific antibody production

Selective polysaccharide antibody deficiency

Bacterial infections (after 2 years of age)

Normal IgG (including IgG2 and IgG4) levels

NF-κB pathway proteins (CARD11, HOIL1 and NEMO), BTK and CD20

AID, activation-induced cytidine deaminase; BAFFR, BAFF receptor; BTK, Bruton’s tyrosine kinase; BLNK, B cell linker; Cμ, constant region-μ; CD40L, CD40 ligand; CSR, class-switch recombination; CVID, common variable immunodeficiency; ICOS, inducible T cell co-stimulator; ND, not determined; NF-κB, nuclear factor-κB; PI3K, phosphoinositide 3-kinase; SAP, SLAM-associated protein; TACI, transmembrane activator and CAML interactor; UNG, uracil N-glycosylase.

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Class‑switch recombination(CSR). Region‑specific DNA recombination between two different switch regions located upstream of the constant (C) regions in the immunoglobulin locus, with excision of the intervening DNA. Replacement of the Cμ region by a downstream C region from another class of immunoglobulin results in the production of antibodies of different isotypes (IgG, IgA and IgE).

Somatic hypermutation(SHM). SHM introduces mutations into the immunoglobulin variable regions and is a major component of affinity maturation, providing a basis for the selection and proliferation of B cells expressing a B cell receptor with a high affinity for the antigen.

T follicular helper cell(TFH cell). A germinal centre T helper cell that expresses the chemokine receptor CXCR5 and the co‑stimulatory molecules CD40L and ICOS, but only low levels of CCR7. TFH cells are essential for class‑switch recombination and somatic hypermutation in B cells. They secrete cytokines (especially interleukin‑21, which acts in a paracrine and autocrine manner).

Activation‑induced cytidine deaminase(AID). A key enzyme that induces somatic hypermutation and class‑switch recombination by deaminating cytosine bases to uracil bases in single‑stranded DNA in the variable and switch regions of the immunoglobulin locus.

Uracil N‑glycosylase(UNG). A base excision repair enzyme that recognizes and removes uracils (including those induced by AID) from within DNA.

Hypomorphic mutations in either NEMO or IKBKB (which encodes IKKβ) appear in patients with X-linked recessive EDA with immunodeficiency38,39 and in a subset of patients with autosomal-recessive CID, respectively. These patients exhibit variable hypogam-maglobulinaemia as a result of a B cell activation defect40. Moreover, autosomal-dominant EDA with immunodeficiency is caused by a gain-of-function mutation in the gene encoding the inhibitory protein IκBα, resulting in low serum IgG, low or normal IgA and elevated serum IgM levels41,42. Strikingly, specific inactivation of IKBKB in follicular dendritic cells causes impairment of both intercellular adhesion molecules 1 (ICAM1) expression and germinal centre formation43, suggesting that in autosomal-dominant EDA both an extrinsic B cell defect (affecting B cell migration) and an intrinsic B cell defect (affecting B cell activation) are intricately linked and result in the observed PAD.

An autosomal-recessive deficiency in HOIL1, a pro-tein that is required for the ubiquitylation of NF-κB, also results in a defective production of polysaccharide-specific antibodies (and thus an increased susceptibility to invasive bacterial infections) and reduced numbers of memory B cells. Moreover, in some cases, this defi-ciency has been associated with a severe autoinflam-matory syndrome associated with an innate immune defect44 (FIG. 4a). Collectively, the association of defects that affect the NF-κB pathway with PADs highlights the key function of NF-κB in the maturation of B cells (from transitional B cells) and in B cell activation.

Mutations in the gene encoding the LPS-responsive beige-like anchor (LRBA) have recently been reported in several patients affected by an autosomal-recessive CID involving hypogammaglobulinaemia together with either inflammatory bowel disease45 or autoim-munity46. The B cells of these patients are intrinsically defective, as B cell survival, plasmablast generation and immunoglobulin secretion are poor following activation with CD40 ligand (CD40L) and cytokines. However, the exact function of LRBA has not yet been defined.

Defects in B cell proliferation also result in a PAD: we recently reported that a homozygous mis-sense mutation in the POLE1 gene (which encodes the catalytic subunit of DNA polymerase-ε) causes a newly identified disorder characterized by facial dys-morphism, immunodeficiency, livedo and short stat-ure (known as FILS syndrome). Patients suffer from recurrent bacterial infections, low IgM and IgG2 lev-els, a lack of antibodies specific for T cell-independ-ent polysaccharide antigens and low memory B cells counts. B cells fail to proliferate and exhibit impaired progression from G1 phase to S phase of the cell cycle because of partially defective DNA replication, as the capacity for DNA synthesis is limited40. This defect is likely to limit all steps of B cell proliferation regardless of the nature of the trigger, which can be a cytokine and/or an antigen (Supplementary information S1 (table)). The reason why the main phenotype of DNA polymerase-ε deficiency is a B cell deficiency is yet to be determined.

In summary, except those affecting the CD19 com-plex or CD20 (which are both molecules specifically expressed by B cells), PADs caused by B cell activation defects are part of CID disorders involving both T cells and other cells.

Defects in immunoglobulin class switch recombination. Two key steps in the maturation of B cells, namely class‑switch recombination (CSR) and somatic hypermutation (SHM), are achieved in germinal centres through close B cell–T cell cooperation involving CD40, which is con-stitutively expressed by B cells, and CD40L, which is expressed by activated T helper cells and especially by the T follicular helper cell (TFH cell) subset (FIG. 4a). Following the establishment of B cell–T cell contacts, activation‑induced cytidine deaminase (AID), a B cell-specific molecule, cre-ates DNA lesions by deaminating cytosine to uracil. Uracil N‑glycosylase (UNG) recognizes and processes the AID-induced uracils in DNA. This step is followed (at least dur-ing CSR) by the generation of DNA double-strand breaks (DSBs), which are sensed by ataxia telangiectasia mutated (ATM) and the MRN complex (MRE11–RAD50–NBS1), and repair generally carrried out by the non-homologous end joining (NHEJ) enzymes. This results in immuno-globulin class switching towards IgG, IgA or IgE, and in SHM, which enables the further selection of B cells producing high-affinity antibodies4 (FIG. 4b).

Defects in this pathway result in normal or increased serum IgM levels that contrast with very low levels of IgG, IgA and IgE (TABLE 1). These features may or may not (depending on the molecular defect) be associated with defective SHM, and are generally diagnosed during child-hood. Mutations in CD40 lead to a rare autosomal-reces-sive deficiency that is characterized by a defect in both CSR and SHM47. Other B cell-intrinsic defects can also lead to a CSR deficiency (FIG. 4b). For example, a defect in AID (as observed in autosomal-recessive AID deficiency) results in a complete lack of both CSR and SHM, emphasizing the key role of AID in the two processes48. Notably, muta-tions located in the carboxyl terminus of AID result in a complete CSR defect but do not affect SHM. This feature suggests that AID is not only a cytidine deaminase but also that it has a further role in CSR — possibly as a docking protein that recruits a putative CSR-specific cofactor49,50. Mutations abrogating the nuclear export signal of AID lead to an autosomal-dominant CSR deficiency49, the pheno-type of which is milder than that of autosomal-recessive AID deficiency and very close to that of CVID49.

UNG deficiency causes a very rare autosomal-reces-sive CSR deficiency that is associated with an abnormal pattern of SHM51. Three patients with this disorder have been reported so far, and they displayed susceptibility to bacterial infections, lymphadenopathies and autoim-munity. Moreover, a variable CSR deficiency can develop in patients with ataxia telangiectasia (which is caused by mutations in ATM), in patients with ataxia-telangiecta-sia-like disease associated with mutations in MRE11 and in patients with Nijmegen breakage syndrome (which is due to mutations in NBS1). A CSR deficiency can also be a predominant abnormality in patients with hypo-morphic mutations in genes encoding molecules of the

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SLAM

SLAM

CXCR5

TFH cella

b

Nucleus

ICOSL

ICOSCD40L

CD40BAFFR

IL-21R

IL-21R

IL-21TLR ligand

TLR

Antigen

BCR

TIRAP

MYD88

BAFF

NIK

IKKα IKKα

p100 RELB

CARD11

HOIL1

HOIP

Linear ubiquitylation

Sharpin LUBAC

NEMO

BCL10 MALT1 IRAK4

p52 RELB

p52 RELB p50 p65

p50 p65

Canonical NF-κB

Non-canonical NF-κB

TRAF6

CD40deficiency

CARD11deficiency

IκBα gain of function

AID deficiency and UNG deficiency

NEMOdeficiency

CD40Ldeficiency

IL-21Rdeficiency

ICOSdeficiency

TIRAPdeficiency

IRAK4deficiency

HOIL1 deficiency

IKKβ deficiency

IκBα phosphorylationand degradation

MSH6 deficiency?ATM, MRE11, NBS1, RNF168and NHEJ (LIG4 and Cernunnos) deficiency

MSH6 deficiencyand POLη (XPV)deficiency

Processing ofp100 to p52

PMS2deficiency

MYD88deficiency

V D J II I ICµ Cδ CεCγ3DNA transcription

Somatic hypermutation Class-switch recombination

Double-stranded DNA breaks

DNA repair

Error-prone DNA repair

S S S

I CεS S

CεV D J

V D J

I

I

I

Cµ

Cδ

Cγ3

DNA lesion S

I

S

S

P

P

P

IKKα IKKβ

IκBα IκBα

P P

P

UbUb

UbUb

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◀

Mismatch repair(MMR). A repair pathway that recognizes and corrects mismatched base pairs (typically those that arise from errors of chromosomal DNA replication). There are two main types of MMR components: MutS homologues (MSH1–MSH6) and MutL homologues (PMS2, MLH1 and PMS1).

NHEJ pathway, such as DNA ligase 4 or cernunnos (also known as NHEJ1)52, although these mutations are also associated with B cell and T cell lymphopenia (as NHEJ is required for the expression of the BCR and TCR). A very rare autosomal-recessive CSR deficiency is caused by mutations in the RNF168 gene (which also underlie the radiosensitivity, immunodeficiency, dysmorphic features and learning difficulties (RIDDLE) syndrome), demon-strating the role of RNF168-dependent ubiquitylation of DNA repair proteins53. Defects in the mismatch repair complex (which are also known to lead to the early onset of cancers) have been associated with a variable CSR deficiency that mostly affects IgA and IgG subclasses (as observed in postmeiotic segregation 2 (PMS2) or MutS homologue 6 (MSH6) deficiencies)54,55. Although in vitro CSR is strongly impaired in B cells from patients with these DNA repair deficiencies, one cannot rule out the possibility that an associated T cell immunodeficiency (affecting the TFH subset) is responsible (at least in part) for the CSR deficiency observed in vivo.

The study of CSR deficiencies has been instrumental in deciphering the key steps involved in CSR, from the generation of DNA lesions to the ligation of switch junc-tions, and has contributed to our understanding of how CSR and SHM are tightly but not entirely associated.

Defects in cytokine signalling. In recent years, interleu-kin-21 (IL-21) has emerged as a major inducer of human B cell proliferation and differentiation. IL-21 binds to the IL-21 receptor (IL-21R) on B cells, which then induces the phosphorylation of Janus kinase 3 (JAK3) and the subsequent dimerization of signal transducer and activa-tor of transcription 3 (STAT3). IL-21R signalling results in the generation of high-affinity antibodies and the sur-vival of memory B cells and plasma cells56. An IL-21R deficiency (owing to biallelic mutations in the IL21R gene) has recently been described in two different fami-lies (Supplementary information S1 (table)). Although the phenotype of patients with IL21R mutations is reminis-cent of that of patients with CID (in terms of susceptibility to opportunistic infections), unlike in CID, a combination of hypo-IgM and hyper-IgE syndromes is also noted57.

IL-21 signals through STAT3, and a partial STAT3 deficiency underlies an autosomal-dominant hyper-IgE syndrome with increased susceptibility to recurrent cuta-neous and mucosal bacterial infections (Supplementary information S1 (table)). This susceptibility to bacterial infection partly results from the poor or absent anti-body formation in response to immunization and the reduced frequency of circulating memory B cells that are commonly observed in these patients56,58.

Overall, these data point to the role of IL-21 in main-taining the activation of TFH cells, which are present in reduced numbers in STAT3-deficient patients (discussed below), as well as in the delivery of survival and prolif-eration signals to B cells56. Hyper-IgE syndrome might result from the physiological role of IL-21 in the inhibi-tion of IgE synthesis59.

Severe CIDs (SCIDs) caused by mutations in the genes encoding the common cytokine receptor γ-chain (γc) or JAK3 (Supplementary information S2 (table)) are characterized by normal circulating B cell counts. The γc or JAK3 deficiencies also lead to a T cell defect, but γc- or JAK3-deficient B cells fail to respond to IL-21 and to produce high-affinity antibodies even follow-ing the reversal of the T cell deficiency by allogeneic transplantation or gene therapy60,61. It is noteworthy that an impairment in IL-7 signalling caused by γc (or IL-7Rα) deficiency does not impair B cell development in humans62, highlighting the distinct function of IL-7 in B cell development in humans and mice.

The observation of IL-21-related B cell immunodefi-ciencies points to the role of this cytokine in B cell anti-body production, at least for T cell-dependent responses. The delineation of the precise step or steps at which IL-21 is actually involved will require further work.

PADs associated with B cell-extrinsic defectsDefects in T cell development or activation. Genetic impairments of T cell differentiation also lead to sec-ondary B cell defects (Supplementary information S2 (table)). Mutations in the various chains of the CD3 complex or in IL-7Rα are responsible for SCIDs that are characterized by defective IgG and IgA production and antibody responses62–65 but normal IgM antibody responses. The thymic hypoplasia observed in DiGeorge syndrome is often revealed by the antibody deficiency

Figure 4 | B cell activation defects and CSR defects in PADs. a | In germinal centres, T follicular helper (T

FH) cells and B cells cooperate through CD40 ligand (CD40L)–CD40 and

inducible T cell co-stimulator (ICOS)–ICOS ligand (ICOSL) interactions and cytokine signalling (including through the interleukin-21 receptor (IL-21R)). The defective generation of T

FH cells — as a result of either T cell activation defects (owing to CD40L, ICOS and

SLAM-associated protein (SAP) deficiencies) or IL-21 signalling defects (owing to IL-21R or signal transducer and activator of transcription 3 (STAT3) deficiencies) — indirectly leads to primary antibody deficiencies (PADs). Intrinsic B cell defects, including CD40 deficiency and defects in IL-21R signalling (owing to IL-21R and STAT3 deficiencies), prevent B cell activation. Signalling of CD40 and the BAFF receptor (BAFFR) is mainly mediated through the non-canonical nuclear factor-κB (NF-κB) pathway, whereas Toll-like receptor (TLR) and B cell receptor (BCR) signalling activate the canonical NF-κB pathway. TLR signalling is mediated through TIRAP (also known as MAL), MYD88 and IRAK4, resulting in the activation of TRAF6, and influences the marginal zone B cell subset through an as-yet-undefined mechanism76. BCR signalling activates TRAF6 through the activation of the CARD11–BCL10–MALT1 complex. Deficiencies that affect the canonical NF-κB pathway (CARD 11 and NEMO deficiencies, or NF-κB inhibitor-α (IκBα) gain of function) lead to a combined immunodeficiency that is associated with defective T cell-independent responses. Moreover, MYD88, TIRAP, IRAK4 and HOIL1 deficiencies are all characterized by reduced blood counts of marginal zone B cells and defective T cell-independent responses, an observation that links innate immunity (specifically TLR signalling) and T cell-independent responses. b | Class-switch recombination (CSR) occurs downstream of T cell-dependent B cell activation in germinal centres. CSR involves DNA recombination between two different switch (S) regions located upstream of the constant (C) regions, resulting in the deletion of the intervening DNA. Replacement of the Cμ region by a downstream C region from another class of immunoglobulin results in the production of antibodies of different isotypes (IgG, IgA and IgE). Somatic hypermutation (SHM) introduces mutations into the variable (V) regions of the immunoglobulin locus; these mutations might increase the affinity of the BCR for a given antigen. CSR deficiencies lead to the production of IgM and an absence or markedly decreased levels of other isotypes (IgG, IgA and IgE). Depending on the molecular defect, the CSR deficiencies may also be associated with defective SHM. Defective SHM results in the absence of B cells with high-affinity BCRs. So far, no PAD related to defective SHM has been reported. The dashed arrows represent alternative signalling pathways. AID, activation-induced cytidine deaminase; ATM, ataxia telangiectasia mutated; IKK, IκB kinase; LIG4, DNA ligase 4; MSH6, MutS homologue 6; NHEJ, non-homologous end joining; P, phosphorylation; PMS2, postmeiotic segregation 2; POL, DNA polymerase; Ub, ubiquitylation; UNG, uracil N-glycosylase.

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that leads to recurrent respiratory tract infections and autoimmune conditions66. Genetic defects in the signal-ling components involved in T cell activation (includ-ing the kinases ZAP70, ITK and LCK)67–69 also lead to impaired secondary antibody responses. MHC class II deficiency results in a CD4+ T cell deficiency combined with variable defects in immunoglobulin levels and con-sistently defective T cell-dependent antibody responses against protein antigens70. Overall, these defects provide information about the T cell-independent component of B cell functions, as IgM-mediated antibody responses can still be detected in the absence of cognate T cell interaction.

Defects in TFH cell differentiation and function emphasize the role of this T cell subset in germinal cen-tre reactions. Following antigen-mediated activation, naive CD4+ T cells differentiate into distinct effector T cell subsets, including TFH cells, which provide efficient help to B cells in the germinal centre reaction71. A defect in TFH cells (as shown by low numbers of circulating CXCR5+CD4+ T cells) leads to a PAD in spite of B cells being intrinsically normal. This is illustrated by a very rare autosomal-recessive deficiency of inducible T cell co-stimulator (ICOS), which was one of the first TFH cell-associated immunodeficiencies to be described in humans. ICOS is expressed by activated T cells (includ-ing TFH cells) and interacts with ICOS ligand (ICOSL) that is constitutively expressed on the surface of B cells. Mutations in ICOS are associated with CVID, although some patients have normal IgM levels and low levels of other immunoglobulin isotypes. The low frequency of SHM in patients with ICOS mutations suggests a dys-functional germinal centre reaction, perhaps as a result of the defective production of cytokines (including IL-10)72 by T cells and impaired TFH cell generation73.

The X-linked condition CD40L deficiency is respon-sible for approximately 50% of all CSR deficiencies74 and is diagnosed during childhood. This condition is asso-ciated with a TFH cell defect; in the absence of CD40L, T cells differentiate poorly into TFH cells and B cell–T cell interactions in germinal centres are impaired73. Consequently, patients have a defect in both CSR and SHM (FIG. 4a). However, the fact that a few patients exhibit normal (and sometimes even elevated) IgA levels suggests that compensatory mechanisms operate in the intestinal mucosae, possibly involving the APRIL–TACI pathway75 or B cell activation downstream of TLRs76. The presence of a CD40-independent maturation path-way is also suggested by the observation that certain SHM events take place in marginal zone B cells in some patients with CD40L deficiency77.

X-linked lymphoproliferative disease (XLP) — which is caused by mutations in the gene SH2D1A, encoding SLAM-associated protein (SAP) — is characterized by defective signalling lymphocyte activation molecule (SLAM) homeotypic adhesion between TFH cells and B cells in germinal centres78. As a consequence of defec-tive synapsis, the affected individuals have hypogam-maglobulinaemia, poor antibody responses and low memory B cell numbers, which all lead to susceptibility to bacterial infections. Patients also have an inappropriate

and potentially fatal immune reaction against EBV infec-tion79 (FIG. 4a). A similar susceptibility to EBV infection has recently been reported in autosomal-recessive CD27 defi-ciency, presenting as hypogammaglobulinaemia, impaired T cell-dependent antibody generation and defective T cell responses80,81, although the role of CD27 (a co-stimulatory receptor that binds to CD70) is far from clear.

In autosomal-dominant STAT3 deficiency, the intrin-sic defect in STAT3-mutated B cells (discussed above) is combined with a partial defect not only in the gen-eration but also in the function of TFH cells56,82. Indeed, STAT3-mutated TFH cells are less able to produce IL-21 in response to activation with IL-12 (as STAT3 is also activated downstream of IL-12R) and cannot efficiently activate germinal centre B cells82. However, it should be noted that not all infections suffered by STAT3-mutated patients are due to the TFH cell defect.

Defects in innate immunity. Recently, an unexpected role has been reported for neutrophils in the differentiation of marginal zone B cells. B cell-helper neutrophils (NBH cells) induce CSR and SHM in marginal zone B cells through the production of cytokines such as BAFF, APRIL and IL-21 (REF. 83). Patients with neutropenia or functional neutrophil defects (including chronic granu-lomatous disease (CGD)) have been reported to exhibit not only low numbers of marginal zone B cells, which are hypomutated compared with those of control individu-als, but also low serum levels of IgM and IgG specific for T cell-independent antigens. However, the importance of NBH cells remains to be fully assessed, as patients with neutropenia or CGD are not prone to infections usually associated with B cell deficiencies. Moreover, it has been recently reported that, despite a reduction in the num-ber of CD27+ B cells, patients with CGD retain an intact humoral immunological memory, possibly supported by CD27− memory B cells84.

Innate immune signals mediated by TLRs have been recently shown to affect the marginal zone B cell sub-set, as patients with inherited defects in TLR-associated signalling molecules exhibit reduced blood counts of IgM+IgD+CD27+ B cells (Supplementary informa-tion S2 (table)). These results link TLR signalling to T cell-independent B cell immunity76.

The recent description of the role of innate immunity in B cell responses to T cell-independent antigens points to a very new disease mechanism, linking for the first time innate immune cells and B cell immunity.

PADs with unknown aetiologyWhereas most of the early blocks in B cell differentia-tion have been defined in molecular terms, this is not often the case for more distal defects. Indeed, we cur-rently lack a molecular understanding for 25% of CSR deficiencies, and most of the supposed genetic defects in CVID (one of the most common PADs, with an inci-dence of ~1 in 25,000 births) have yet to be identified. In fact, CVID corresponds to a heterogeneous group of dis-orders characterized by pan-hypogammaglobulinaemia with defective antibody responses and, in some cases, is associated with an increased incidence of granuloma,

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autoimmunity and cancer85,86 (TABLE 1). CVID is gener-ally diagnosed in adulthood, although some paediatric cases have been reported. Low numbers of circulating B cells and memory B cells and abnormally high num-bers of transitional B cells are associated with a poor prognosis. Of diagnosed patients, 10–20% have a family history of CVID or IgA deficiency. Although research-ers have attempted to determine the genetic variations associated with CVID, over 90% of these cases have not yet been defined on a molecular basis. Sporadic, autosomal-dominant and autosomal-recessive forms have been reported.

Recently, a genome-wide association approach showed that disease heterogeneity in a large cohort of patients could be related to several variations in the genetic background, including previously reported vari-ations in genes of the MHC locus87. Polymorphisms in genes present in this locus (including those encoding tumour necrosis factor (TNF), MHC class II molecules, complement factors and the mismatch repair protein MSH5) are preferentially associated with CVID. As discussed above, individuals with some TACI variants also have a predisposition to CVID6. As CVID has a late onset, it is probably caused by a combination of several genetic variations rather than by a defect in a single gene.

Similarly, although selective IgA deficiency (SIgAD) is the most common PAD, its genetic basis has not yet been defined (TABLE 1). Most individuals are asympto-matic (suggesting effective compensation by secretory IgM in the mucosae)88, but approximately 25% of symp-tomatic patients have a family history of either SIgAD or CVID. Polymorphisms in TACI89,90 or in MHC genes91 have been reported in some cases, although the involvement of TACI variations has been controversial.

Selective IgG subclass deficiency is defined as a lack of one or more IgG subclasses with normal overall IgG levels (TABLE 1). IgG2 deficiency is most commonly reported and is often associated with IgG4 deficiency and susceptibility to recurrent bacterial infections. Its aetiology remains poorly defined and it can be transient during childhood. IgG3 deficiency, which is less com-mon, leads to susceptibility to recurrent bacterial infec-tions and is often associated with another IgG subclass deficiency. Deletions of constant (C) regions have been reported in a few patients92 but do not account for the majority of cases, as residual amounts of the IgG sub-classes are generally detectable.

Selective IgM deficiency (SIgMD) is a rare disorder that leads to recurrent infections (most frequently with encapsulated pathogens) from infancy onwards. Its pathogenesis remains unclear. Deletion of the Cμ region can be ruled out, as B cells from patients with SIgMD still express surface IgM (TABLE 1).

Last, patients with selective polysaccharide antibody deficiency (SPAD) have normal immunoglobulin levels (including IgG2), but lack polysaccharide-specific anti-bodies, and are therefore susceptible to infections, espe-cially those caused by encapsulated bacteria (TABLE 1). Given that infants are unable to raise polysaccharide-specific responses before the age of 2 years, SPAD can be diagnosed only after this time point. Marginal zone

B cells are especially reactive to bacterial cell wall com-ponents, and splenectomized patients also suffer from SPAD93. Although most SPADs are not molecularly defined, NEMO deficiency38 (which impairs NF-κB signalling downstream of several pathways, including TLR signalling pathways), gain-of-function mutations in CARD11 and HOIL1 deficiency can result in a SPAD (as discussed above). A leaky form of BTK deficiency also reportedly causes SPAD12.

Altogether, B cell functions can be affected in mul-tiple ways in patients with PADs, owing to intrinsic B cell defects in development, migration, activation or survival or as a consequence of extrinsic T cell abnor-malities. Nonetheless, the in vivo immunological conse-quences are all related to defective antibody production, highlighting the roles of antibodies in immunity (as discussed below).

Main clinical features associated with PADsSusceptibility to infections. The production of different immunoglobulin isotypes with high affinities for anti-gens is essential for efficient protection against infec-tions. IgM constitutes the first line of defence against encapsulated bacteria in vascular spaces, IgG has a longer half-life and diffuses in the extracellular compart-ment, IgA is transported to mucosal surfaces (especially in the gut) and IgE is involved in immune responses to helminths. Moreover, these isotypes differentially bind to their respective Fc receptors and some can activate complement components.

Thus, it becomes clear that the imbalanced antibody production observed in PADs results in recurrent bac-terial infections. These infections predominantly affect the respiratory tracts (leading to the severe complica-tions of sinusitis and bronchiectasis, if left untreated). Streptococcus pneumoniae, non-typeable Haemophilus influenzae and Gram-negative bacteria are the most prev-alent microorganisms causing these infections. To a lesser extent, patients with PADs are vulnerable to intestinal tract infections (mainly by Giardia spp., Campylobacter jejuni, Salmonella spp. or Helicobacter pylori) and to bac-terial cutaneous infections (TABLE 1). It is noteworthy that some patients with agammaglobulinaemia also suffer from severe, chronic enteroviral infections, suggesting a major role for antibodies in preventing the dissemination of enteroviruses from the gut. The absence of vulnerabil-ity to fungal infections shows that antibodies are dispen-sable for immunity to fungi. Antibodies also seem to be less important for antiviral responses, with the notable exception of enteroviruses (as mentioned above).

Interestingly, we showed in a prospective study of a cohort of patients with pan-hypogammaglobulinaemia and CSR deficiency who received equal IgG replacement therapy that patients with CSR deficiency had a lower incidence of acute respiratory tract infections and bron-chiectasis and a significantly lower risk of infection with non-typeable H. influenzae compared with agammaglob-ulinemic patients94. These patients carried IgM antibodies specific for non-typeable H. influenzae in their serum and saliva. Interestingly, similar protection has been found in CD40L- and AID-deficient patients (who lack SHM), as

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B cell toleranceB cell tolerance is controlled by two checkpoints: central B cell tolerance is achieved in the bone marrow, through the removal of immature B cells that express polyreactive antibodies, whereas peripheral B cell tolerance mechanisms operate at the transition between immature and mature naive B cells and counterselect self‑reactive B cells that may have encountered peripheral autoantigens that are not expressed in the bone marrow. Disruption of B cell tolerance leads to autoimmunity.

well as in other patients with CSR deficiencies, in whom SHM is normal. Thus, IgM antibodies that have been actively transported to mucosal surfaces88 seem to be clini-cally protective against some microorganisms, irrespective of whether they have undergone SHM. This shows that IgG, which cannot be transported to mucosal surfaces as it fails to bind to the J chain, cannot fully substitute for the immunoglobulin isotypes that can be transported to mucosal surfaces (namely, IgM and IgA).

Autoimmunity and autoinf lammation. Auto-inflammation and/or autoimmunity may be the pre-dominant symptoms of several PADs (TABLE 1). The disease mechanisms are not well known and it is not clear whether B cells always have a direct role in dis-ease pathogenesis. Autoinflammation is characterized by seemingly unprovoked episodes of inflammation in the absence of detectable autoantibodies or auto-reactive T cells. Autoinflammation and granulomas are frequently observed in PADs, especially in CVID and CSR deficiencies.

Antibody-mediated autoimmunity can result from defective checkpoints in the control of self-reactive B cells5. The establishment of central B cell tolerance involves BCR signalling and other complex mechanisms, such as receptor editing, anergy and deletion of immature B cells. Peripheral B cell tolerance occurs during the tran-sition from immature to mature naive B cells and is mostly controlled by B cell-intrinsic factors95. Autoimmunity can also be caused by defective control of self-reactive TFH cells or the impaired development or function of regulatory T cells. Finally, it has been hypothesized (but not proved) that regulatory B cells (that is, IL-10-producing B cells)96 can also have a role in autoimmunity. Several PAD-associated genetic defects are likely to underlie defects in the above-mentioned checkpoints.

The development of autoimmune diseases has long been considered to be exclusively associated with hypermutated IgG antibodies97. Thus, the observation that nearly 30% of AID-deficient patients98 (and ageing Aid−/− mice)99 have autoimmune manifestations came as a surprise. Both central and peripheral B cell toler-ance have been shown to be defective in AID-deficient patients. The mechanism underlying defective central tolerance in AID deficiency remains unclear. In the periphery, AID-deficient self-reactive B cells do not undergo SHM and thus are not fully selected for high affinity to antigens. Therefore, these B cells may accu-mulate and cause autoimmune manifestations follow-ing activation by autoantigens or by microbial antigens (if their self-reactive BCR is crossreactive), as well as in response to non-BCR-mediated signals, such as those activated downstream of innate immune receptors.

The occurrence of autoimmunity in patients with CVID is frequently associated with the presence of circu-lating B cells that express low amounts of CD21 at the cell membrane. This B cell population is clearly distinct from other circulating B cell populations, as these CD21low cells barely express the transitional B cell surface marker CD38, show low levels of Ca2+ influx and decreased pro-liferation downstream of BCR and CD40 stimulation

compared with other B cells, and exhibit polyreactivity and self-reactivity100,101. Moreover, autoimmune manifes-tations and non-infectious granulomas in some patients with CVID are typically associated with hypomorphic mutations in the RAG genes102,103.

The autosomal-dominant condition PLAID (PLCγ2-associated antibody deficiency and immune dysregulation) is caused by deletion of the regulatory domain of PLCγ2 and provides an interesting example of how a mast cell- and B cell-associated immunodeficiency can cause both cold urticaria and autoimmunity104. In patients with PLAID, mutant PLCγ2 is constitutively active at low temperatures in mast cells, thereby caus-ing cold urticaria associated with hyper-IgE syndrome. At 37 °C, PLCγ2 function is reduced, which probably accounts for the mild B cell immunodeficiency104. More recently, a gain-of-function missense mutation in the autoinhibitory domain of PLCγ2 was reported as being associated with mild pan-hypogammaglobulinaemia and inflammatory manifestations105.

Autoimmunity is also a major complication in many patients with CIDs, and can be attributed to B cell-intrin-sic defects, among other causes. For example, B cell-mediated autoimmunity is frequent among individuals with STIM1 deficiency, although this phenotype might also be linked to the low numbers of regulatory T cells in these individuals106. A defect in peripheral B cell tol-erance, leading to antibody-mediated autoimmunity against blood cells, is observed in patients with MHC class II deficiency. WAS syndrome is also frequently asso-ciated with B cell-mediated autoimmunity. Although a regulatory T cell defect may be involved in the patho-genesis of autoimmunity in patients with WAS, the B cell-intrinsic defect conferred by the absence of functional WASP has been shown to perturb B cell tolerance in mice17. Indeed, Was−/− mouse B cells exhibit hyperrespon-siveness to BCR and TLR stimulation17, possibly because of decreased receptor internalization caused by abnormal cytoskeletal rearrangement. Thus, their interactions with cognate wild-type T cells can drive a loss of tolerance in Was−/− B cells and thereby cause severe autoimmune dis-ease. This observation may account for the occurrence of autoimmunity in some patients with WAS following allogeneic bone marrow transplantation that has resulted in mixed B cell chimerism107.

Thus, autoinflammation and autoimmunity appear as severe complications in patients with PADs, and can occur even after immunoglobulin replacement or after haematopoietic stem cell transplantation. Treatment of these manifestations in patients with PADs is associated with morbidity, as it is usually based on immunosuppressive therapies.

Carcinogenesis. Another complication of PADs relates to the occurrence of lymphomas and other types of cancer. The incidence of cancer is high in patients with PADs that are associated with a DNA repair defect, such as ataxia telangiectasia and Nijmegen breakage syndrome. Defective repair of DNA lesions resulting, respectively, from ATM and NBS1 deficiencies probably accounts for this risk. Moreover, malignancies (lymphomas in 50%

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of cases and epithelial carcinomas in the other 50% of cases) occur in 20% of patients with CVID, and cancer is the most severe prognosis factor in these conditions85,86. (TABLE 1). The mechanism underlying the occurrence of B cell lymphomas in CVID remains undefined, and detailed analyses of the tumours are warranted in order to gain insight into this mechanism. Epithelial carci-nomas are thought to result from chronic infections, and are thus indirectly related to the B cell defects that lead to a lack of control over infections. In addition to being associated with CVID, cancers can be observed in patients with other types of PADs, including lymphoma in XLP79 and cholangiocarcinoma or primary neuroecto-dermal tumours in CD40L deficiency108, but the under-lying mechanisms linking these PADs to carcinogenesis still remain to be defined.

Therapeutic approaches. The approaches used for the treatment of PADs greatly depend on disease sever-ity. The morbidity associated with haematopoietic stem cell transplantation (the only curative therapy) means that this therapeutic strategy is restricted to the life-threatening B cell disorders that are generally also associated with a T cell deficiency. However, all anti-body-deficient patients require IgG replacement, with either intravenous or subcutaneous delivery. The use of IgG replacement therapy considerably limits infec-tious complications and chronic lung lesions. However, IgG replacement treatment is not always sufficient, as the other immunoglobulin isotypes are not provided. Administration of antibiotics might also be required, as in the case of patients with SIgMD. As patients with CSR deficiencies are protected from infection with some bac-teria (such as non-typeable H. influenzae), possibly by their IgM antibodies, long-term antimicrobial prophy-laxis could be required for patients with agammaglob-ulinaemia in addition to IgG replacement therapy to compensate for the absence of IgM. Notably, immuno-globulin replacement does not prevent complications such as lymphadenopathies or autoimmunity. However, it could be useful for preventing epithelial cancers by reducing chronic infections.

Recent pathophysiological findings suggest that new therapeutic approaches can be considered. The use of a CXCR4 inhibitor in patients with WHIM syndrome results in a significant improvement in the numbers of circulating leukocytes (including B cells). Moreover, subcutaneous administration of recombinant CD40L

in patients with CD40L deficiency has been reported to restore T helper 1 cell function and thus could be use-ful in cases of severe opportunistic infection. However, B cell responses were not restored following the admin-istration of recombinant CD40L109. Whether the time course or the dosage of CD40L administration was inadequate to provide efficient help to B cells remains undefined.

Finally, gene therapy has been successfully carried out in SCID associated with adenosine deaminase110 and γc deficiencies61 and in WAS111, and it may also be a promising therapeutic strategy for B cell deficiencies in the future. In Btk−/−Tec−/− double-deficient mice (a mouse model of human XLA), transplantation of stem cells transduced with a lentiviral construct encoding BTK under the control of B cell-specific promoters results in long-term B cell rescue and antibody responses112. However, clinical applications of similar gene therapies will require a very strict assessment of safety issues.

ConclusionsIn this Review, we summarize the various disorders influencing antibody responses, emphasizing that PADs are not only caused by B cell-specific defects (from those affecting early B cell development in the bone marrow to those affecting B cell migration, survival and activation in the secondary lymphoid organs) but are also due to other immunological dysfunctions. The characterization of these disorders has revealed unexpected new mecha-nisms that are important for antibody production and maturation, and for B cell-mediated immunity against infections. Moreover, it has emphasized that the coop-eration of B cells with other immune cells, especially the TFH cell subset, is essential for T cell-dependent antibody production. The recently emerging concept that innate immunity is involved in T cell-independent antibody responses also opens up new roads for the investigation and understanding of the genetic defects that under-lie PADs. It is this molecular understanding of PADs that will shed light on the mechanisms that link B cell dysfunction with autoimmunity and lymphomagenesis.

The ongoing genetic delineation of PADs enables accurate diagnosis and contributes to better prognos-tic measures and adequate follow-up. However, several PADs remain undefined, and further advances will be achieved by new molecular approaches and more com-plex genetic analyses of non-monogenic PADs (such as CVIDs) and modifier factors.

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AcknowledgementsThe authors apologize to all colleagues whose work could not be cited owing to length restrictions. The authors thank C. Picard for critical reading of the manuscript. This work was funded by grants from Institut National de la Santé et de la Recherche Médicale, the European Union’s 7th RTD Framework Programme (EURO‑PADnet grant number 201549 and ERC PIDIMMUNE grant number 249816), Association Contre Le Cancer and ANR Blanc 2010‑CSRD. S.K. is a Centre National de la Recherche Scientifique (CNRS) researcher.

Competing interests statementThe authors declare no competing financial interests.

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