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MOLECULAR DEFECTS IN T AND BCELL PRIMARY IMMUNODEFICIENCY DISEASES

Charlotte Cunningham-Rundles and Prashant P. Ponda Abstract | More than 120 inherited primary immunodeficiency diseases have been discovered in the past five decades, and the precise genetic defect in many of these diseases has now been identified. Increasing understanding of these molecular defects has considerably influenced both basic and translational research, and this has extended to many branches of medicine. Recent advances in both diagnosis and therapeutic modalities have allowed these defects to be identified earlier and to be more precisely defined, and they have also resulted in more promising long-term outcomes. The prospect of gene therapy continues to be included in the armamentarium of treatment considerations, because these conditions could be among the first to benefit from gene-therapy trials in humans.

The human immune system is confronted with the chal- cells or complement, whereas opportunistic infections lenge of host defence. This is accomplished through vari- with viruses or fungi are particularly common in ous innate immune responses (which are non-specific) patients with T-cell deficiencies. A subset of primary and adaptive immune responses (which are specific) immunodeficiencies is associated with inflammatory that work synergistically to achieve this goal. Cells of or autoimmune manifestations, and certain subgroups the adaptive immune system include T and B cells, of patients are susceptible to developing malignancies. which are derived from a common multipotent haemato- This Review focuses on the recent advances in the poietic stem cell. Defects involving T and B cells have field, with an emphasis on newly identified genetic been described with respect to their development, effec- deficiencies and therapeutic options for patients. tor function and roles in immunoregulation 1. Although SCID — an immunodeficiency that is character- defined primary natural killer (NK)-cell deficiencies ized by severely reduced numbers or an absence of are rare among primary immuno deficiency diseases, functional T cells, which in turn results in the absence other cells of the innate immune system, which were of an adaptive immune response — is a consequence of previously thought to function independently of adap- a mutation in any one of ten distinct genes that are tive immune responses, are now seen as important inherited in an autosomal recessive or an X-linked Division of Clinical partners in the development of adaptive immunity. manner2–10. Four lymphocyte phenotypes are possible Immunology, Mount Sinai The clinical presentation of patients with a primary on the basis of the influence of the defective gene on School of Medicine, 1425 Madison Avenue, immunodeficiency reflects the complex underpinnings B-cell and NK-cell development TABLE 1. A diag- Box 1089, New York, of the immune system and depends on the underlying nosis is possible at birth, with most affected infants New York 10029, USA. genetic defect. Patients with severe combined immuno- having lymphopaenia (less than 2,000 lymphocytes Correspondence to C.C.R. deficiency (SCID) generally present with opportunistic per mm3 of blood) and their lymphocytes showing e-mail: charlotte. cunningham- infections and fail to thrive within a few months of decreased proliferation in vitro after stimulation with 11 [email protected] life. Recurrent bacterial infections are the hallmark mitogen, antigen or allogeneic cells . Although these doi:10.1038/nri1713 of disease in patients with defects in B cells, phagocytic infants have a severely reduced amount of thymic

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TCELLRECEPTOR EXCISION tissue, accompanied by the absence of normal thymic for thymic insufficiency leading to a T-cell deficiency, CIRCLES architecture, T-cell development is achievable after although it also involves abnormal development of spa- (TRECs). DNA episomes the introduction of normal haematopoietic stem tially related embryological tissues that leads to cardiac, that are normally produced cells12. At present, bone-marrow transplantation using parathyroid and other abnormalities. DiGeorge syn- during the thymic maturation of T cells, specifically during either unfractionated HLA-identical haematopoietic drome is characterized by a decrease in the number of + + recombination of the T-cell- stem cells or T-cell-depleted haploidentical (parental) CD3 cells or an absence of CD3 cells as a consequence receptor genes. haematopoietic stem cells is the standard of care for of hypoplasia or aplasia of the thymus. Depending on these infants, with improved survival in patients who the number of peripheral T cells, the immune pheno- XLINKED receive a transplant within the first 4 weeks of life13. type falls within a range of immunodeficiencies, from LYMPHOPROLIFERATIVE SYNDROME Successful intervention depends on early identifica- full immunocompetence to a SCID-like phenotype. (XLP). A rare, often fatal, tion of infants, before the development of opportunistic Unlike other forms of SCID, severe DiGeorge syndrome primary immunodeficiency infections that contribute to the increased morbidity can be treated effectively by thymic transplantation, disease that is characterized and mortality that is associated with delayed transplan- which allows for the maturation of recipient T cells. by an inability to mount an effective immune response tation. Nevertheless, there is no programme in place Several candidates for the genetic defect in DiGeorge to Epstein–Barr virus. This for screening newborns for SCID; such a programme syndrome have been identified; most recently, a mem- can lead to lymphoma or would allow therapy to be provided within this vital ber of the T-box transcription-factor family, TBX1, has hypogammaglobulinaemia. window of opportunity. In the United States, the been implicated as a cause of most of the main signs of Centers for Disease Control and Prevention has identi- DiGeorge syndrome16,17. fied SCID as a candidate for the development of a new- Genetic defects also affect the signal-transduction born-screening protocol, because SCID meets many of pathways that are essential for T-cell activation. the accepted screening criteria14. Many modalities Components of these pathways include the γ-chain of testing have been explored; most recently, the of CD3 (CD3γ), CD3ε, MHC class I molecules, MHC examination of TCELLRECEPTOR EXCISION CIRCLES (TRECs) class II molecules, LCK, ZAP70 (ζ-chain-associated in DNA isolated from dried blood spots has shown protein kinase of 70 kDa) and CD8α18–25. The result- promise15. TRECs are more abundant in T cells from ing defects are highly variable and range from severe a healthy newborn than from an adult. Their absence cellular dysfunction (from a deficiency in MHC has been confirmed in patients with SCID15, and large- class II molecules) to negligible dysfunction (from a scale implementation of this screening tool might help deficiency in CD8α). to identify affected infants. In addition to genetic defects that reduce or elimi- nate T-cell-based immunity, there is a growing list of Defects that involve T-cell immunity immune defects that result in overactive or abnormal Patients with defects that involve T cells do not have T-cell function that leads to immunodeficiency. An adequate cellular immune responses and are predis- example of a functional mutation is seen in patients posed to developing opportunistic infections. These with XLINKED LYMPHOPROLIFERATIVE SYNDROME (XLP); these T-cell deficiencies are reflected in reduced absolute individuals have a mutation in SH2D1A, which encodes cell numbers, defective activation and function, and SLAM-associated protein (SAP), a cytoplasmic adap- disrupted immunoregulation (FIG. 1; TABLE 2. DiGeorge tor protein that binds signalling lymphocytic activation syndrome has classically been thought of as the model molecule (SLAM) and other SLAM-family molecules26. SLAM is a transmembrane protein that is expressed at low levels at the surface of resting cells and at higher Table 1 | Aetiologies of severe combined immunodeficiency levels after cellular activation; intracytoplasmic binding Type of SCID Chromosomal location Reference of SLAM by SAP has an inhibitory role. For reasons that are unclear, defects in SAP result in uncontrolled prolif- – + + T B NK eration of T cells in individuals who are infected with Interleukin-7 receptor α-chain deficiency 5p13 2 Epstein–Barr virus, as well as ineffective viral elimina- CD3 δ-chain deficiency 11q23 3 tion, lymphoma or hypogammaglobulinaemia. SH2D1A mutations result in fatal infectious mononucleosis in a CD3 ε-chain deficiency 11q23 4 high proportion of cases. T –B+NK– Another emerging role for T cells is that of γ X-linked recessive SCID ( c deficiency) Xq13.1 5 regulation of the immune response to prevent the CD45 deficiency 1q31–1q32 6 recognition of self. Recent studies have outlined aspects of the molecular basis of T-cell defects in JAK3 deficiency 19p13.1 7 three disease states that are characterized by T-cell T –B–NK+ immunodysregulation. Artemis gene-product deficiency 10p13 8 IPEX. Immunodysregulation, polyendocrinopa- RAG1 and RAG2 deficiency 11p13 9 thy and enteropathy, X-linked syndrome (IPEX) T –B–NK– can often be fatal and is a recessive disorder of early Adenosine-deaminase deficiency 20q13.11 10 childhood that involves the classic clinical triad γ γ of endocrinopathy (most commonly in the form of c, common cytokine-receptor -chain; JAK3, Janus kinase 3; NK, natural killer; RAG, recombination-activating gene; SCID, severe combined immunodeficiency. early-onset type 1 diabetes), severe enteropathy with

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watery, sometimes bloody, diarrhoea, and eczematous the molecular defect29. FOXP3 is mainly expressed dermatitis27, resulting from a severe deficiency or by, and is a reliable marker of, CD4+CD25hi REGULATORY absence of regulatory T cells. This syndrome is associ- T CELLS30. The role of FOXP3 in the development and ated with autoimmune conditions that affect several function of CD4+CD25hi regulatory T cells, however, organ systems and with moderate to severe recur- is largely an enigma. rent infections with Enterococcus and Staphylococcus At present, immunosuppressive agents that are species28. Mutation of the forkhead box P3 (FOXP3) directed at activated T cells, such as CYCLOSPORIN A OR gene, which encodes a forkhead (also known as winged TACROLIMUS (Prograf; Astellas Pharma US, Inc.) (admini- helix) transcription factor, has been identified as stered together with an optional corticosteroid), are

a Bone marrow Thymic cortex Thymic medulla

DiGeorge syndrome ADA IL-7Rα RAG1, RAG2 γ δ ε c CD3 , CD3 JAK3 artemis CD45 AIRE CD3 NK cells B cells complex αβ-TCR CD8+ T cell pTα CD4 CD8 CD8α MHC class I TCR-β molecules CD3ζ (TAP1, TAP2) LCK Lymphoid ZAP70 lineages Positive MHC class II and molecules CD34+ Pro-T cell: Pre-T cell: DP T cell: negative (CIITA and CD4– CD4– CD4+ selection RFX-family – – + HSC CD8 CD8 CD8 proteins) CD25+ CD25+ CD44+ CD44– CD4+ T cell

Myeloid lineages

b IPEX ALPSCaspase-8 CD95L XLP Caspase-10 SAP SLAM TReg CD4+ cell CD25+ DISC, CD95L caspase-8 and caspase-10 FOXP3 CD95 SAP

CD95 TEff cell APC

Proliferation TCR Peptide – MHC class I or class II inhibited Figure 1 | Protein and gene defects in T-cell development and function. a | Haematopoietic stem cell (HSC)-derived lymphoid progenitor cells migrate from the bone marrow to the thymus and develop into progenitor (pro)-T cells, which then CD4+CD25hi REGULATORY rearrange their T-cell receptor (TCR) genes and differentiate into either γδ or αβ T cells in the cortex. The latter initially co-express T CELLS CD8 and CD4, which interact with MHC class I and class II molecules, respectively, at the surface of medullary thymic stromal A thymus-derived cells. This interaction allows T cells to be ‘educated’ regarding self-antigens and non-self-antigens (enabling the positive or subpopulation of T cells that negative selection of T cells in the thymus) before their migration to the periphery, where they exclusively express CD4 or CD8. expresses the transcription Numerous defects in maturation have been elucidated. Defects in the genes encoding the molecules listed in the yellow boxes factor forkhead box P3 (and the primary immunodeficiency diseases listed) are known to affect the developmental steps indicated. b | Functional (FOXP3) and is involved in defects are also observed after maturation is complete. In patients with IPEX (immunodysregulation, polyendocrinopathy and the suppression of immune enteropathy, X-linked syndrome), self-reactive effector T (T ) cells are not inhibited, because mutations in the forkhead box P3 responses, either by cell–cell Eff gene (FOXP3) result in a loss of CD4+CD25+ regulatory T (T )-cell activity. In patients with autoimmune lymphoproliferative contact or cytokine release. Reg syndrome (ALPS), defects in the CD95, CD95 ligand (CD95L), caspase-8 or caspase-10 genes abrogate formation of the death- inducing signalling complex (DISC), thereby interfering with apoptosis of T cells. In patients with X-linked lymphoproliferative CYCLOSPORIN A AND Eff TACROLIMUS syndrome (XLP), uncontrolled proliferation of T cells occurs as a result of a mutation in SH2D1A, which encodes SAP (signalling Calcineurin inhibitors that lymphocytic activation molecule (SLAM)-associated protein). ADA, adenosine deaminase; AIRE, autoimmune regulator; γ γ are used to prevent transplant APC, antigen-presenting cell; c, common cytokine-receptor -chain; CIITA, MHC class II transactivator; DP, double positive; rejection and that function IL-7Rα, interleukin-7 receptor α-chain; JAK3, Janus kinase 3; NK cell, natural killer cell; pre-T cell, precursor-T cell; pTα, pre-TCR by inhibiting nuclear factor α-chain; RAG, recombination-activating gene; RFX, regulatory factor X; TAP, transporter associated with antigen processing; of activated T cells (NFAT). ZAP70, ζ-chain-associated protein kinase of 70 kDa.

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the most effective therapy for the treatment of patients IPEX, although the results have mostly been disap- with IPEX31. However, for the most affected patients, pointing32,33. Intriguingly, one patient had improved no acceptable regimen can maintain long-term glucose regulation and reduced diarrhoea during the remission of the disease. Bone-marrow transplanta- conditioning regimen before bone-marrow transplan- tion has been carried out for several patients with tation32, underscoring the role of selectively targeting

Table 2 | Defects that involve T cells Name Clinical phenotype Chromosomal Genetic defect Refs location Development DiGeorge Thymic, cardiac and parathyroid defects, and decreased numbers 22q11.2 Possibly TBX1 16,17 syndrome or absence of CD3+ cells WHN defect Congenital alopecia, and nail dystrophy 17q11–17q12 WHN 113 Activation CD3 deficiency Autoimmune haemolytic anaemia and severe infections 11q23 CD3G 18 Recurrent Haemophilus influenzae pneumonia and otitis media 11q23 CD3E 19 MHC class I Decreased numbers of CD8+ T cells 6p21.3 TAP1 20 deficiency TAP2 21 MHC class II Persistent diarrhoea, bacterial pneumonia, Pneumocystis carinii 1q21.1–1q21.3 RFX5 22 deficiency pneumonia, viral and candidal infections, and low numbers of 13q14 RFXAP 22 CD4+ T cells 19p12 RFXANK 22 16p13 CIITA 22 LCK deficiency Bacterial, viral and fungal infections, lymphopaenia and 1p34.3–1p35 LCK 23 hypogammaglobulinaemia ZAP70 Decreased numbers of CD8+ T cells, normal or decreased 2q12 ZAP70 24 deficiency numbers of CD4+ T cells, and severe recurrent infections CD8 deficiency Absence of CD8+ T cells, and recurrent respiratory infections 2p12 CD8A 25 HIGM1 Pneumocystis carinii pneumonia, pyogenic infections, normal Xq26–Xq27 CD40L 50 or increased level of IgM, and low level or absence of serum IgG, IgA and IgE HIGM3 Pneumocystis carinii pneumonia and Cryptosporidium parvum 20q12–20q13.2 CD40 53,54 infections AD-EDA-ID Lymphocytosis, absence of memory T cells and unresponsive 14q13 NFKBIA 59 naive T cells Function XLP Uncontrolled T-cell proliferation in EBV infection, fatal infectious Xq25 SH2D1A 26,87 mononucleosis in a high proportion of patients, ineffective viral elimination, lymphoma and hypogammaglobulinaemia Regulation IPEX Triad of endocrinopathy, enteropathy and dermatitis, and Xp11.23 FOXP3 27,29 Enterococcus and Staphylococcus species infections APECED Chronic mucocutaneous candidiasis, and parathyroid and 21q22.3 AIRE 34 adrenal autoimmunity ALPS0 Autoimmunity, hypergammaglobulinaemia, lymphoproliferation, 10q24.1 CD95 (homozygous) 41 and excessive numbers of CD3+CD4–CD8–αβ-TCR+ T cells ALPS1a Autoimmunity, hypergammaglobulinaemia, lymphoproliferation, 10q24.1 CD95 (heterozygous, germ line) 42 and excessive numbers of CD3+CD4–CD8–αβ-TCR+ T cells CD95 (heterozygous, somatic) 47 ALPS1b Autoimmunity, hypergammaglobulinaemia, lymphoproliferation, 1q23 CD95L 45 and excessive numbers of CD3+CD4–CD8–αβ-TCR+ T cells ALPS2 Autoimmunity, hypergammaglobulinaemia, lymphoproliferation, 2q33–2q34 CASP8 43 and excessive numbers of CD3+CD4–CD8–αβ-TCR+ T cells CASP10 44 ALPS3 Autoimmunity, hypergammaglobulinaemia, lymphoproliferation, ND ND 46 and excessive numbers of CD3+CD4–CD8–αβ-TCR+ T cells AD-EDA-ID, autosomal dominant ectodermal dysplasia with immunodeficiency; AIRE, autoimmune regulator; ALPS, autoimmune lymphoproliferative syndrome; APECED, autoimmune polyendocrinopathy-candidiasis-ectodermal-dystrophy syndrome; CIITA, MHC class II transactivator; CASP, caspase; CD40L, CD40 ligand; CD95L, CD95 ligand; EBV, Epstein–Barr virus; FOXP3, forkhead box P3; HIGM, hyper-IgM syndrome; IPEX, immunodysregulation, polyendocrinopathy and enteropathy, X-linked syndrome; ND, not determined; NFKBIA, gene encoding IκBα (inhibitor-of-nuclear-factor-κB α); RFX5, regulatory factor X, 5; RFXANK, RFX- associated ankyrin-containing protein; RFXAP, RFX-associated protein; SH2D1A, gene encoding SAP (signalling lymphocytic activation molecule (SLAM)-associated protein); TAP, transporter associated with antigen processing; TBX1, T-box 1; TCR, T-cell receptor; WHN, winged-helix nude (also known as FOXN1); XLP, X-linked lymphoproliferative syndrome; ZAP70, ζ-chain-associated protein kinase of 70 kDa.

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activated T cells. Nevertheless, the usefulness of either that results in the induction of apoptosis. This pathway immunosuppression or bone-marrow transplantation involves formation of the death-inducing signalling would mainly depend on its early implementation, complex in association with caspase-8 and caspase-10. before the onset of permanent organ damage. Further All patients have at least three of the four main features understanding of the mechanisms that are involved in of ALPS: autoimmunity, hypergammaglobulinaemia FOXP3 expression and its influence in patients with (of both IgG and IgA), lymphoproliferation and exces- IPEX will undoubtedly provide therapeutic targets for sive numbers of CD3+CD4–CD8–αβ-TCR+ (double patients with IPEX and potentially for individuals with negative) T cells39. other autoimmune diseases. In vitro study of lymphocyte sensitivity to CD95- induced apoptosis allows for a classification scheme APECED. Autoimmune polyendocrinopathy- that is based on the underlying genetic defect40. candidiasis-ectodermal-dystrophy syndrome (APECED; Defective CD95-induced apoptosis is observed in also known as APS1) results from a defect in the homozygous CD95 deficiency 41 (classified as ALPS0), autoimmune regulator (AIRE) gene34. Patients with heterozygous dominant CD95 mutations42 (classified as APECED usually have chronic mucocutaneous candi- ALPS1a), and signalling pathway defects that involve diasis, as well as autoimmune manifestations that most caspase-8 REF. 43 or caspase-10 REF. 44 (classified commonly affect the parathyroid or adrenal glands and, as ALPS2). CD95-induced apoptosis is intact in two to a lesser extent, the thyroid gland, liver and skin35. additional subtypes of patients: those with a CD95L AIRE is expressed at high levels by purified human mutation45 (classified as ALPS1b), and those with a thymic stromal cells, especially medullary thymic clinical ALPS phenotype but in whom a molecular epithelial cells, and it is thought to regulate the ectopic defect has yet to be identified46 (classified as ALPS3). cell-surface expression of tissue-specific proteins, such A subset of patients who were previously categorized as as insulin and thyroglobulin36. Expression of these having ALPS3 has now been identified to have somatic self-proteins allows the negative selection of autoreac- heterozygous CD95 mutations in unstimulated double- tive T cells during their development. The absence of negative T cells47. Interestingly, all patients in this subset this key regulatory step results in the organ-specific had identical CD95 mutations or mutations that led to autoimmune destruction that is seen in patients with identical structural changes in CD95 to those observed APECED. in patients with ALPS1a. However, mutant CD95 prod- Thymic stromal lymphopoietin, an interleukin-7 ucts could not be detected in T-cell blasts following (IL-7)-like cytokine, has been shown to induce human in vitro activation. It is unclear why the mutations in peripheral-blood CD11c+ dendritic cells (DCs) to these newly identified patients do not lead to defective upregulate AIRE mRNA expression strongly, in con- CD95 expression in activated T cells and, subsequently, junction with cell-surface MHC class II molecules and to defective CD95-induced apoptosis. the co-stimulatory molecules CD80 and CD86 REF. 37. These activated DCs were able to induce a 1,000-fold Hyper-IgM syndromes clonal expansion in an autologous, naive CD4+ T-cell General aspects. Hyper-IgM syndromes (HIGMs) population in culture. This further emphasizes the role constitute a group of molecular defects that is char- of AIRE in the presentation of self-peptides, because acterized by impaired immunoglobulin CLASSSWITCH the CD4+ T-cell proliferation occurred in the absence RECOMBINATION (CSR) and SOMATIC HYPERMUTATION (SHM) of exogenous antigen and was therefore attributed to or by impaired SHM alone. Patients with these syn- the presentation of self-peptide–MHC complexes by dromes typically have recurrent bacterial infections

CLASSSWITCH DCs. In vitro studies have elucidated that one role of the and often have lymphoid hyperplasia. They have RECOMBINATION AIRE protein is to function as an E3 ubiquitin ligase, normal numbers of peripheral B cells, albeit with a (CSR). A switch in the DNA indicating its involvement in a ubiquitin–proteasome low proportion of memory B cells (which are CD27+) that encodes the constant pathway38. Furthermore, two known disease-causing and normal or increased levels of serum IgM associ- region of the immunoglobulin heavy chain, from Cµ (which mutations in the AIRE gene abolished this ligase activ- ated with low levels or absent serum IgG, IgA and IgE. 38 48 49 encodes the constant region ity . The precise ubiquitin–proteasome pathway and CSR and SHM occur only after antigen binds B cells of IgM) to DNA that is further ubiquitylation substrates of the AIRE protein have yet displaying cell-surface IgM (that is, the B-cell recep- downstream and encodes the to be identified, and the overall significance of this tor, BCR), and these are two mechanisms by which the constant region of another immunoglobulin class: that is, pathway in the establishment and maintenance of primary antibody repertoire is fine-tuned to generate to Cγ, Cα or Cε, which encode T-cell self-tolerance is not well understood at present. a highly antigen-specific immune response. These the constant region of IgG, events are T-cell dependent and are facilitated through IgA and IgE, respectively. ALPS. There are four known genetic defects that the interaction of CD40L at the surface of activated This is accomplished through have been identified in patients with autoimmune T cells with its receptor CD40, which is constitutively an intrachromosomal deletional rearrangement. lymphoproliferative syndrome (ALPS), an inherited expressed by B cells. B cells that produce high-affinity condition that is associated with dysregulation of specific antibody as a result of SHM have a survival SOMATIC HYPERMUTATION apoptosis mediated by CD95 (also known as FAS). advantage. Two enzymes — activation-induced cyti- (SHM). The introduction CD95 is a cell-surface receptor that is a member of the dine deaminase (AID) and uracil-DNA glycosylase of point mutations at a high frequency in the variable tumour-necrosis factor (TNF)-receptor superfamily, (UNG) — are crucial for this editing process. CSR regions of immunoglobulin and after binding CD95 ligand (CD95L; also known as and SHM work together so that the secondary anti- genes. FAS ligand), it initiates a complex signalling pathway body repertoire has a high affinity. Mutations in the

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components that are involved in these processes result thereby underscoring an inherent T-cell defect. HIGM3 in the inherent defects in patients with HIGM. So far, has been described in four patients from three families seven defects that are known to be involved in HIGM and is characterized by the absence of CD40 expression have been characterized: defects in CD40L, classified at the cell surface of B cells, macrophages and DCs53,54. as HIGM type 1 (HIGM1; also known as X-linked These patients are also susceptible to developing oppor- HIGM, XHIGM); defects in AID, classified as HIGM2; tunistic infections. It is important to note that there defects in CD40, classified as HIGM3; defective CSR might be defects in components of the signalling path- with preserved SHM, classified as HIGM4; defects way that are downstream of CD40–CD40L interactions in UNG; defects in IKKG (IκB (inhibitor of nuclear to account for other patients in the clinical spectrum of factor-κB, NF-κB) kinase-γ; also known as NEMO); HIGM. In addition, other repair mechanisms that work and defects in NFKBIA (which encodes IκBα)50–59 in conjunction with, or independent of, AID and UNG TABLES 2,3. have yet to be clarified. Patients with defects in CD40L, who comprise the Patients with defects in AID or UNG have a HIGM1 subgroup, account for approximately two-thirds similar clinical phenotype51,52. Similar to patients of all patients with HIGM. In these patients, an absence with a CD40L deficiency, the level of serum IgM is of, or a defect in, binding of CD40L to CD40 is caused normal or increased, and this occurs together with by a mutation that affects the extracellular domain of low levels or an absence of IgG and IgA. However, CD40L50. There is no intrinsic B-cell defect observed owing to intact T-cell function, these patients do not in these patients: their B cells generate normal immuno- seem to be susceptible to opportunistic infections and globulin class-switching responses in an appropriate might not be recognized as having an immune defect microenvironment50. Furthermore, in contrast to most until the second or third decade of life60. The exact patients with hypogammaglob ulinaemia, individuals process by which AID and UNG mediate CSR and with HIGM1 are susceptible to opportunistic infections, SHM is not known; however, switch-region double- especially to pneumonia caused by Pneumocystis carinii, stranded-DNA breaks are required for both to occur,

Table 3 | Defects that involve B cells Name Clinical phenotype Chromosomal Genetic References location defect HIGM2 Pyogenic infections, lymphoid hyperplasia, 12p13 AID 51 decreased CD27+ B-cell numbers, normal or 12q23–12q24.1 UNG 52 increased serum IgM level, and low level or no serum IgG, IgA and IgE HIGM4 Pyogenic infections, lymphoid hyperplasia, ND ND 55 decreased CD27+ B-cell numbers, normal or increased serum IgM level, and low level or no serum IgG, IgA and IgE XL-EDA-ID Bacterial and mycobacterial infections, Xq28 IKKG 56–58 and low levels or no antibody specific for carbohydrates Agamma- Low or no levels pre-B-cell and mature B-cell Xq21.3–Xq22 BTK 72 globulinaemia numbers, low serum immunoglobulin levels, and pyogenic infections Normal pro-B-cell numbers, low pre-B-cell 10q23.2 BLNK 114,115 and mature B-cell numbers, low serum immunoglobulin levels, and pyogenic infections Low serum immunoglobulin levels, and t(9; 20) (q33.2; q12) LRRC8 83 pyogenic infections Developmental arrest at the pro-B-cell stage, 22q11.22 IGLL1 76 low serum immunoglobulin levels, and 19q13.2 Iga 77 pyogenic infections 14q32.2 IGHM 78,79 CVID Sinopulmonary infections, low IgG and IgA 16p11.2 CD19 Unpublished* levels, and normal B-cell numbers 22q13.1–22q13.31 BAFFR Unpublished‡ 17p11.2 TACI 116,117 2q33 ICOS 88,89 IgAD Many patients are asymptomatic, although 17p11 TACI 116,117 pyogenic infections are possible Possibly 6p21.3 IGAD1 99,100 (HLA-DQ and HLA-DR) *M. C. van Zelm, personal communication; J. L. Franco, personal communication. ‡V. Salzer, personal communication. AID, activation-induced cytidine deaminase; BAFFR, B-cell-activating-factor receptor; BLNK, B-cell linker; BTK, Bruton’s tyrosine kinase; CVID, common variable immunodeficiency; HIGM, hyper-IgM syndrome; ICOS, inducible T-cell co-stimulator; Iga, gene encoding Igα; IgAD, selective IgA deficiency; IGAD1, IgA-deficiency susceptibility 1; IGHM, gene encoding µ immunoglobulin heavy chain; IGLL1, gene encoding λ5; IKKG, inhibitor-of-nuclear-factor-κB kinase-γ; LRRC8, leucine-rich-repeat-containing 8; ND, not determined; pre-B cell, precursor-B cell; pro-B cell, progenitor-B cell; TACI, transmembrane activator and calcium-modulating cyclophilin-ligand interactor; UNG, uracil-DNA glycosylase; XL-EDA-ID, X-linked ectodermal dysplasia with immunodeficiency.

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and CSR-induced breaks have been shown to occur genes of NF-κB-mediated transcription are AID and much less frequently in AID-deficient B cells than in UNG, so in some patients with IKK-γ deficiency, the AID-sufficient B cells61. processes of CSR and SHM are hindered, leading to Three patients have been described to have a defi- the increased IgM levels that have been observed65. An ciency in UNG52. All three had normal expression of HIGM phenotype similar to IKKG deficiency has been CD40, CD40L and AID, although (similar to AID- described for patients with a mutation in NFKBIA, deficient B cells) their B cells failed to generate switch- which encodes IκBα, an inhibitor of NF-κB. This region double-stranded-DNA breaks after activation hypermorphic gain-of-function mutation prevents through CD40. In addition, a skewed SHM pattern the phosphorylation of IκBα and, in turn, the activa- was observed that showed mutations biased towards tion of NF-κB. Unlike IKKG deficiency, however, this transitions in dG and dC nucleotides, whereas dA and phenotype is associated with a T-cell deficiency that is dT nucleotides showed transitions and transversions characterized by naive T cells that are unresponsive to in similar ratios to control values from normal mem- stimulation through CD3 in vitro and by the absence ory B cells. The current hypothesis to explain these of memory T cells. editing processes involves AID-mediated deamina- Interestingly, an abnormal response to TOLLLIKE tion of C residues into U residues, followed by UNG- RECEPTOR (TLR) signalling has also been noted in mediated removal of U residues. This would create an patients with the IKKG mutation56. On binding and abasic site that could be targeted by an endonuclease activation of the various TLRs by their ligands, a com- to create the required DNA breaks that are crucial for plex signalling cascade is set in motion, the result of CSR and SHM. Lack of either enzyme would therefore which is the activation of NF-κB and the transcription destabilize the development of a secondary antibody of genes encoding several pro-inflammatory cytokines repertoire. Replication protein A, a ubiquitous single- and chemokines66. Mutations in IKKG result in a sub- stranded-DNA-binding protein, has been described as stantially diminished response to lipopolysaccharide, the factor that targets AID to SHM motifs to promote an activator of TLR4. This emphasizes the emerging their deamination62. significance of components of the innate immune Approximately one-quarter of patients with the system in the aetiology of primary immunodeficiency. HIGM phenotype have normal expression and func- Other examples of defects of this type include muta- tion of CD40L, CD40, AID and UNG63. A subset of tions in the IL-1-receptor-associated kinase 4 (IRAK4) these patients has been described to have defective gene and the caspase-12 gene67–69. It is crucial to note CSR with preserved SHM, and these individuals have that the innate and adaptive immune systems, which been categorized into the HIGM4 subtype55. B cells were historically thought of as segregated, do not func- from these patients express AID and show appropri- tion as distinct entities; instead, they are inter dependent ate AID-dependent CSR-induced DNA breaks in and function together to coordinate the host immune the switch region of Cµ (which encodes the constant response. region of IgM), indicating that the molecular defect is downstream of these events: that is, there is a deficiency Defects that involve B-cell immunity in the repair process that occurs after the induction Deficiencies in antibody production and function are of double-stranded-DNA breaks. The precise defect the hallmark of the primary immunodeficiency dis- in individuals with HIGM4 remains undefined, and eases that involve B cells. Patients with these conditions its elucidation should further illuminate the complex are especially vulnerable to recurrent infections with process of CSR and DNA repair. encapsulated pathogens, such as Streptococcus pneumo- HYPOMORPHIC MUTATION A type of mutation that results niae, Haemophilus influenzae and Staphylococcus in either diminished quantity Defects in NF-κB signalling. An increasing number of aureus, and with Gram-negative bacteria, such as of a normal gene product or genetic mutations are being identified that have inap- Pseudomonas species70. A B-cell defect is defined as a diminished function of a gene propriate activation of NF-κB as a common defect64. markedly decreased serum level of at least one of the product. Of these, HYPOMORPHIC MUTATIONS in IKKG are linked to three main immunoglobulin classes: IgG, IgA and HYPODONTIA a clinical phenotype of immunodeficiency, ectodermal IgM; the most marked defects lead to either reduced The partial congenital absence dysplasia, as well as to susceptibility to pyogenic bac- levels or an absence of antibody production. Antibody of one or more teeth. terial infections in early infancy or childhood and to deficiencies are the largest group within the primary mycobacterial infections in early or late childhood58. immunodeficiencies, and multiple molecular defects TOLLLIKE RECEPTORS (TLRs). A family of However, the clinical phenotype is remarkably hetero- have been identified throughout the pathways that are evolutionarily conserved geneous: in some patients it involves only conical involved in B-cell development (FIG. 2; TABLE 3. pattern-recognition receptors. incisors and HYPODONTIA, whereas in others, it involves These molecules are located osteopetrosis with lymphoedema. The range of infec- Forms of agammaglobulinaemia. Several genetic intracellularly and at the cell surface of macrophages, tions and defects in antibody production are similarly defects have been identified that account for the dendritic cells, B cells and diverse, although a severely reduced level, or an absence phenotype of agammaglobulinaemia, which is char- intestinal epithelial cells. Their of, antibody specific for carbohydrate antigens seems acterized by a B-cell defect and intact T-cell func- natural ligands are conserved to be a unifying theme. In addition, whereas NF-κB tion. Of all of the forms of agammaglobulinaemia, molecular patterns, known as κ pathogen-associated molecular was first noted in B cells, active NF- B can be released X-linked agammaglobulinaemia (XLA) provides the patterns, that are found in in the cytoplasm of many cells, indicating that this prototypical clinical description. XLA was the first bacteria, viruses and fungi. defect might affect other tissues. Two important target antibody-deficiency syndrome that was recognized71,

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Bone marrow RAG1, RAG2 Periphery Igα IL-7Rα Igµ CVID γ λ c 5 IgAD AID HIGM4 ICOS BAFFR JAK3 BLNK BTK Immature CD40 UNG IKK-γ CD19 TACI B cell Igβ Pre-BCR IgG, IgM Mature IgA IgM B cell or IgE Pro-B cell Igα Plasma cell Lymphoid lineages Pre-B cell + CD34+ CD10 • Negative • Class-switch CD19+ B220low IgD + selection recombination CD34 • Receptor • Somatic editing hypermutation HSC Memory B cell CD34+ T cells

Myeloid NK cells lineages

Figure 2 | Protein and gene defects in B-cell development and function. Haematopoietic stem cells (HSCs) give rise to progenitor (pro)-B cells, which then rearrange their immunoglobulin heavy-chain gene segments to generate precursor (pre)- B cells. Pre-B cells subsequently rearrange their immunoglobulin light-chain gene segments to produce a functional cell-surface receptor (IgM). This protein is composed of heavy and light chains that are derived from these gene rearrangements, and it functions as a receptor for responding to stimulation with antigen, resulting in the induction of proliferation and differentiation of the B cell. In the periphery, after stimulation with antigen, mature B cells further develop following class-switch recombination and somatic hypermutation and, ultimately, differentiate into memory B cells or plasma cells. Developmental blocks throughout B-cell maturation and differentiation occur as a result of defects in genes encoding the molecules listed in the yellow boxes. Blocks in the function of mature B cells can also occur. Primary immunodeficiency syndromes that cause these blocks are also listed. AID, activation-induced cytidine deaminase; BAFFR, B-cell-activating-factor receptor; BCR, B-cell receptor; BLNK, B-cell γ γ linker; BTK, Bruton’s tyrosine kinase; c, common cytokine-receptor -chain; CVID, common variable immunodeficiency; HIGM4, hyper-IgM syndrome 4; ICOS, inducible T-cell co-stimulator; IgAD, selective IgA deficiency; Igµ, µ immunoglobulin heavy chain; IKK-γ, inhibitor-of-nuclear-factor-κB kinase-γ; IL-7Rα, interleukin-7 receptor α-chain; JAK3, Janus kinase 3; NK cell, natural killer cell; RAG, recombination-activating gene; TACI, transmembrane activator and calcium-modulating cyclophilin-ligand interactor; UNG, uracil-DNA glycosylase.

and it results from a mutation in the gene encoding Various autosomal recessive mutations and one Bruton’s tyrosine kinase (BTK), which has a crucial role translocation have also been described in patients in B-cell development. This gene is a member of the with agammaglobulinaemia. Defects in the indi- SRC family of proto-oncogenes, which encodes protein vidual pre-BCR components λ5 (also known as tyrosine kinases72. These patients have precursor (pre)- 14.1, in humans) and Igα have been identified in B cells in their bone marrow; however, the absence single patients76,77. The surrogate immunoglobulin of BTK prevents these cells from differentiating into light chain is composed of λ5 and VpreB, and it is circulating, mature B cells and plasma cells65. Afflicted normally expressed only by progenitor (pro)-B cells patients have a low number of circulating B cells and and pre-B cells. It escorts the µ immunoglobulin extremely low levels of serum immunoglobulin of all heavy chain (Igµ) to the cell surface and might also classes. During the first few months of life, patients assess the capacity of Igµ to bind immunoglobulin light with XLA are protected by circulating maternal IgG, chains. Igα and Igβ form a complex with the surrogate which crossed the placenta during gestation. Because light chain and Igµ and then migrate to the cell surface, the concentration of this serum IgG diminishes, the where both Igα and Igβ function in transmembrane clinical presentation is one of recurrent pyogenic bac- signal transduction through their immunoreceptor terial infections, especially sinopulmonary infections73. tyrosine-based activation motifs (ITAMs). Lack of BRONCHIECTASIS A permanent dilation of the BRONCHIECTASIS is the most concerning complication these crucial pre-BCR components results in the arrest bronchi, owing to chronic of these recurrent infections and is most commonly of B-cell development at the pro-B-cell stage. inflammation, that increases found in the middle or lower lobes of the lungs, with Defective cell-surface expression of Igµ also results susceptibility to recurrent the upper lobes being spared74. Germinal-centre for- in arrest of B-cell differentiation at the CD19+CD34+ infections. mation in these patients is defective, and this leads TERMINAL DEOXYNUCLEOTIDYLTRANSFERASE (TdT)+ pro-B-cell TERMINAL DEOXY to the underdevelopment of lymphoid tissues, such as stage. In contrast to patients with XLA, patients with NUCLEOTIDYLTRANSFERASE the lymph nodes, Peyer’s patches, spleen, tonsils and this defect might have an earlier onset of disease that (TdT). An enzyme that inserts adenoids. The standard treatment for patients with is associated with more severe complications and no nucleotides into the variable 78 regions of T-cell receptor and XLA is monthly immunoglobulin-replacement therapy detectable B cells . In one study, bone-marrow-derived immunoglobulin genes, thereby to prevent chronic lung disease and to protect against pro-B cells from two patients with distinct mutations creating junctional diversity. enteroviral meningoencephalitis75. in the gene encoding Igµ were used to investigate the

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influence of Igµ on BCR development79. Igµ expression antibody73. One approach to classifying the B-cell pheno- did not have an effect on usage of the gene segments type in patients with CVID involves characterizing the encoding the immunoglobulin heavy chain — the population of class-switched memory B cells (which + – – variable-region segment (VH), the diversity segment have the phenotype CD27 IgM IgD ) in these patients.

(D) and the joining segment (JH) — or on immuno- Two groups can be identified on this basis, using a classi- globulin light-chain gene recombination or expression. fication system that was proposed by Warnatz et al.85 However, persistent secondary VJ rearrangements of Patients in group 1 have a low percentage (less than the gene encoding Igκ were noted in Igµ-deficient 0.4%) of class-switched memory B cells, and patients in pro-B cells compared with control pro-B cells. This group 2 have a normal percentage (greater than 0.4%). is probably caused by the lack of signalling through The former can be subdivided into those patients with the BCR complex, which is required during normal an increased proportion of CD19+CD21– peripheral differentiation of these cells into pre-B cells. Although B cells (group 1a) and those with a normal proportion the substrates for BTK have not been elucidated, (group 1b). Many patients with splenomegaly and IgM at the surface of B cells is thought to be one of its autoimmune cytopaenias were found to segregate into key activators80,81. Collectively, these data underscore group 1a. the important role of transmembrane IgM in B-cell Unlike patients with XLA, T-cell proliferation to signalling and in normal B-cell development. mitogen is impaired in 40% of patients with CVID, BTK has also been implicated in the regulation of and it is directly associated with the serum level of B-cell tolerance thresholds82. In these experiments, anti- IgG86. Patients with CVID are at an increased risk bodies were cloned from isolated CD10+CD19+CD27– of developing numerous associated diseases or con- IgM+ B cells (which are newly emigrated from the ditions, including infections, autoimmune diseases, bone marrow) from four patients with XLA. These hepatitis, granulomatous infiltrations, gastrointestinal antibodies were found to have a repertoire consisting and pulmonary diseases, and malignancies73,86. The

of specific VH and D gene segments and to undergo development of structural damage to the lungs that extensive secondary recombination on both immuno- leads to bronchiectasis is also of concern and occurs globulin light-chain loci compared with antibodies with a similar distribution to that of patients with from normal, control B cells. In addition, B cells from XLA, although at a later age of onset74. patients with XLA were found to produce a consider- The mutated genes that produce the CVID ably higher frequency of self-reactive and polyreactive phenotype are known only for a minority of patients, antibodies than normal, control B cells. Taken together, and they are diverse in their influence on immune these data indicate an essential role for BTK in BCR function. They include INDUCIBLE TCELL COSTIMULATOR signalling that involves subsequent deletion of cells that (ICOS), SH2D1A26,87 (which is involved in XLP), and produce autoreactive antibodies. three genes that have recently been described to be Last, karyotypic analysis of the leukocytes of one involved: CD19, B-cell-activating factor (BAFF) recep- patient with a novel form of agammaglobulinaemia tor (BAFFR) and TACI (transmembrane activator and showed a balanced chromosomal translocation calcium-modulating cyclophilin-ligand interactor). — 46,XX,t(9; 20) (q33.2; q12) — that resulted in a Homozygous loss of ICOS as a result of a large truncated product being encoded by the affected genomic deletion has been characterized in nine gene, leucine-rich-repeat-containing 8 (LRRC8)83. In a patients from four families that are not known to mouse model, retroviral transfection of bone-marrow be related88,89. Binding of ICOS to its ligand induces cells with this mutant gene followed by bone-marrow a marked increase in T-cell proliferation and cyto- transplantation led to developmental arrest of B cells at kine production, especially of IL-10, which has been the pro-B cell stage, together with a marked deficiency implicated in the differentiation of B cells into plasma in pre-B cells. Intriguingly, the unaffected allele in this cells90. Further genetic testing of these nine individu- patient could produce normal LRRC8 protein, indicat- als showed that they all had identical homozygous ing that the mutant protein has a dominant-suppressor haplotypes in the ICOS locus, indicating that the effect on B-cell development. mutation was most probably inherited from the same ancestor. ICOS deficiency has also been investigated CVID. Common variable immunodeficiency (CVID) as a potential aetiology in patients with HIGM that is is characterized by a defect in antibody production. caused by an unknown genetic defect91. In this study,

INDUCIBLE TCELL Males and females are equally affected, with an inci- 33 patients from 30 families were examined; however, COSTIMULATOR dence between 1 in 10,000 and 1 in 50,000. It is usually none showed a defect in ICOS expression by acti- (ICOS). A homodimeric diagnosed in the second or third decade of life after vated T cells or a defect in the sequence of the coding transmembrane protein that a history of recurrent pyogenic sinopulmonary infec- region or intron–exon boundaries of ICOS. Therefore, is selectively expressed at the 84 surface of activated T cells. tions . Serum levels of IgG and IgA are lower than in although it is probably involved in B-cell activation It specifically interacts with unaffected individuals; however, approximately one- and class switching, ICOS could not be identified as ICOS ligand (also known as half of patients have a normal serum level of IgM. The the genetic defect that is responsible for the clinical B7-H2), which is expressed number of circulating B cells is reduced or normal, and presentation of this subset of patients. by many cell types, including professional antigen-presenting these cells can respond and proliferate appropriately Patients with a clinical presentation of CVID have cells, fibroblasts, epithelial cells to stimulation with antigen; however, they fail to ter- also been identified to have mutations in intermediate and endothelial cells. minally differentiate into plasma cells, which secrete components in B-cell signalling and B-cell development

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pathways. Specifically, defects in CD19 (J. L. Franco, that is maintained in the serum. Enzyme replacement personal communication, and M. C. van Zelm, personal with bovine adenosine deaminase (ADA) modified communication), BAFFR (U. Salzer, personal commu- by polyethylene glycol can be used to treat patients nication) and TACI 116,117 have recently been identified with ADA-deficient SCID who are not candidates for in patients who have a B-cell-defect phenotype. BAFF haematopoietic stem-cell or bone-marrow transplan- is a ligand for BAFFR, TACI and B-cell maturation tation101. Both modalities of replacement therapy are antigen (BCMA)92,93. Another TNF-family member, generally well tolerated and provide these patients with a proliferation-inducing ligand (APRIL), also binds a much improved quality of life. BCMA and, with lower affinity, TACI; however, it does Haematopoietic stem-cell transplantation has been not bind BAFFR94. The expression of BAFF and APRIL attempted for patients with multiple types of primary has been shown to be upregulated by human DCs and immunodeficiency, most successfully to treat patients monocytes after exposure to interferon-α, interferon-γ with SCID102. The largest study of patients with SCID or CD40L95. In the presence of IL-10 or transforming who had received a haematopoietic stem-cell transplant growth factor-β, BAFF and APRIL have been shown to was reported by a multinational registry, the European induce CSR from Cµ to Cγ and/or Cα gene segments Group for Blood and Marrow Transplantation and the in B cells. Therefore, patients with mutations in BAFFR European Society for Immunodeficiencies103. There or TACI probably do not have the B-cell signalling that were 475 patients who were studied, and each had is provided through interaction with BAFF and APRIL received a transplant in the previous three decades, and is required to promote maturation of B cells and including 205 (43%) who did not receive a chemo- generation of a diverse antibody repertoire. ablative preconditioning regimen. Factors that were associated with poorer outcomes among patients IgAD. Selective IgA deficiency (IgAD) is the most receiving non-HLA-identical transplants included a common primary immunodeficiency, with a preva- B– SCID phenotype, absence of a protective environ- lence of between 1 in 400 and 1 in 3,000 in healthy ment and/or presence of a pulmonary infection before blood donors96. Ethnicity-specific differences are transplantation. Although the use of a preconditioning more disparate, ranging in prevalence from 1 in 500 regimen was shown to promote functional engraft- (in Caucasians) to 1 in 18,000 (in Japanese)97. Although ment in the group of patients with B– SCID, this was most patients are asymptomatic, recurrent pyogenic not statistically significant compared with the results sinopulmonary infections are the most frequent achieved for other groups of patients. Among patients illnesses that are associated with IgAD. Several auto- receiving HLA-identical transplants, survival rates immune diseases that involve multiple organ systems were improved when transplantation occurred at are also associated with IgAD96. The molecular defect less than 6 months of age and when prophylaxis with that accounts for the absence of class switching to the antibiotics trimethoprim and sulphamethoxazole IgA is unknown in most cases, although mutations in was used. TACI can lead to an absence of class switching116,117. One study of 132 patients with SCID who had Familial studies have implicated the existence of an received a haematopoietic stem-cell transplant(s) allelic relationship between IgAD and CVID, indicat- within the previous two decades has also been ing that these disorders reflect differential expression reported11. Most of these patients received neither of the same molecular aetiology98. In one study of pre-transplantation chemotherapeutic condition- 83 multiply-affected families with IgAD and CVID, ing (to aid engraftment) nor post-transplantation increased allele sharing at chromosome 6p21, which is graft-versus-host-disease prophylaxis. Normal T-cell in the proximal region of the MHC, was observed, and function was seen within 2 weeks of transplanta- this susceptibility locus was designated IGAD1 REF. 99. tion of unfractionated HLA-identical bone marrow More sensitive genetic analysis was later carried out but was delayed by up to 4 months in patients who in 101 multiple-case families (in which more than received T-cell-depleted bone marrow, owing to the one family member is affected) and 110 single-case fact that mature T cells are not transferred to these families, and this further localized the defect to the patients104. The survival rate for these 132 patients HLA-DQ and HLA-DR loci100. was positively correlated with Caucasian race, female gender and younger age at the time of transplanta- Therapeutic options tion. It is important to note that, although patients Replacement therapy, haematopoietic stem-cell in this study who had not received myeloablative transplantation (using bone marrow, cord blood or pre-transplantation chemotherapy had higher rates peripheral blood) and gene therapy are the avail- of survival, long-term studies that were carried out able treatments for patients with primary immuno- more than a decade after transplantation have shown deficiency. Immunoglobulin-replacement therapy is an accelerated rate of decline in the TRECs in T cells the main treatment for antibody-deficiency disorders from such patients and in the development of oligo- and is usually given every 3–4 weeks. It can be given by clonal populations of T cells12,105. Similar longitudinal either an intravenous or a subcutaneous route, and the studies of T-cell function and thymic output have not dose and frequency of administration can be adjusted been carried out in patients with SCID who received on the basis of the clinical response of the patient pre-transplantation conditioning. Further long-term and on the adequacy of the concentration of serum IgG follow-up studies are needed to assess the efficacy

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Box 1 | Retroviral insertional mutagenesis of ten treated patients developing normal T-cell function and not requiring gammaglobulin-replacement therapy. The clinical trials of gene therapy for the treatment of X-linked severe combined However, ∼3 years after gene therapy, the 2 youngest + immunodeficiency (SCID) used a retroviral vector to transduce CD34 patients developed clonal proliferation of mature T-cell haematopoietic stem cells with the gene encoding the common cytokine-receptor populations109. A third child also developed an abnormal γ-chain (γ ). Retroviral vectors can stably integrate themselves into the DNA of c clonal phenotype. In the first two cases, leukaemia-like the host cell; however, the precise location of their insertion cannot be predicted. proliferation was caused by retroviral-vector insertion This property, combined with the propensity of these vectors to insert themselves in a locus that contains the LIM domain only 2 (LMO2) into transcriptionally active genes, is the reason why the unfavourable side-effect oncogene, and the expression of LMO2 in mature of insertional mutagenesis is a distinct possibility, and it could result in oncogene 110 activation by the inserted target gene. T cells led to their uncontrolled proliferation . This Two patients in the French clinical trial have developed T-cell acute lymphoblastic phenomenon is known as retroviral insertional muta- leukaemia, which is probably a consequence of retroviral insertion near the LIM genesis BOX 1. Clearly, there is tremendous potential domain only 2 (LMO2) oncogene109,110. A third patient developed lymphatic cancer. for the use of gene therapy to treat patients with SCID Increased expression of LMO2 is postulated to block T-cell differentiation, and or other selective immune defects, and advances in the γ selection of additional vectors and the elucidation of expression of c by these cells might then facilitate signalling to induce their division, resulting in clonal proliferation110. The exact mechanism by which the LMO2 locus the mechanisms of gene transduction might allow its was selectively targeted for pro-virus integration in these patients is uncertain. re-implementation for the treatment of these patients. Before this trial, the risk of retroviral insertional mutagenesis was thought to be mainly theoretical, because a single round of transduction would not account for Looking forward the multiple mutations that are generally required for clonal proliferation. This idea As we look to the future of the field that encompasses was further supported by the absence of this phenomenon in preclinical and clinical primary immunodeficiency diseases, emphasis needs trials using replication-incompetent retroviral vectors112. Nevertheless, insertional to be placed on the early detection of life-threatening mutagenesis proved to be a serious adverse event in these patients, and further insight SCID, the elucidation of the unknown molecular is needed for the design and delivery of retroviral vectors before this life-saving defects that underlie immune dysfunction and the therapy can be provided in the future. implementation of new modalities of therapy that aim to correct these genetic defects. Adoption of SCID of various approaches to haematopoietic stem-cell into newborn-screening programmes will aid in the transplantation and to assist in the identification of diagnosis, treatment and long-term management of patient subgroups that are likely to benefit from one these patients. Advances in haematopoietic stem-cell form of therapy over another. isolation protocols allow transplantation of far greater Clinical trials have been carried out using gene numbers of highly purified haematopoietic stem therapy for the treatment of patients with X-linked, cells, and in conjunction with more efficient negative recessive SCID (which is caused by a deficiency in the selection of T cells, this will undoubtedly improve γ γ common cytokine-receptor -chain, c) and for patients the outcomes of haematopoietic stem-cell transplan- with ADA deficiency106–108. For patients with X-linked, tation111. Gene therapy, although in its infancy, has recessive SCID, those without an HLA-identical sibling shown remarkable success in some patients, and a were infused with autologous CD34+ haematopoietic greater understanding of the mechanisms that are stem cells (enriched from bone marrow) that had been involved and development of safer methods of gene γ incubated with cytokines and exposed to a c-encoding correction might allow this promising therapy to be retroviral vector. Initial results were promising, with nine used in clinical practice.

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