IMMUNODEFICIENCY REVIEWEarly B cell defects

The development of B lineage cells from haematopoietic stem cells in the bone marrow to mature antibody-secreting plasma cells in peripheral lymphoid tissues is a complex and tightly regulated process. The proliferation and maturation of these cells occurs in response to numerous signals from the bone marrow microenvironment, soluble cytokines, interaction with antigen and T lymphocytes and the expression of specific gene products. Lymphocyte development, in both T and B lineages, begins with the commitment of multipotent progenitors to the lymphoid lineage. It is likely that these early stages are in part dependent upon extracellular and stromal signals, though the exact mechanisms of early lymphoid commitment are not well defined. The transcription factor Ikaros appears to play an important role in these initial stages, since mice with targeted deletions of the Ikaros gene lack T, B and natural killer cell development [1]. By contrast, the transcription factors E2A and EBF appear to be more B cell-specific, in that mice that are unable to express these genes have normal numbers of T cells and myeloid cells but few if any B lineage cells [2–4]. The earliest stages of normal B cell development are antigen-independent and occur in the bone marrow in response to stromal cell contact and cytokines. From the pre-B cell stage onwards, B cell development is dependent upon the successful assembly, and functional signalling through, pre-B and B cell receptor complexes. Central to this process is expression of the individual components of the receptor and also the molecules that transduce signals from the receptor to initiate cellular events. This is diagrammatically represented in Fig. 1. The understanding of this highly organized developmental pathway comes not only from in vitro data and the generation of transgenic mice but also from the study of naturally occurring human gene abnormalities. In this review we will outline the major gene defects that result in early B lymphocyte maturation arrest and detail the clinical manifestations and the molecular pathogenesis of these conditions. Considerable emphasis will be devoted to X-linked agammaglobulinaemia (XLA), since this is the most common abnormality of B cell development. The stages of B lymphocyte development in the bone marrow and periphery are outlined. The most significant B cell developmental block most commonly seen in individuals with X-linked agammaglobulinaemia (XLA) is shown by a dashed line at the pro B cell to pre-B cell transition. However, there are also blocks at later stages as indicated by the dashed lines at the pre-B to immature B cell stage and at the immature to mature B lymphocyte stage. The knowledge from mouse studies and from individuals with congenital autosomal recessive agammaglobulinaemia has led to an understanding of the role of other genes in B cell development. The maturation arrest arising from abnormalities in these other genes is also demonstrated. A complete block at the pro to pre-B cell transition is seen in patients with μ heavy chain deficiency and in a patient with Igα deficiency. XLA was the first human immunodeficiency described for which the clinical and laboratory findings dictated effective therapy. In 1952 Bruton reported the case of an 8-year-old boy who had suffered from recurrent infections and in whom analysis of serum by protein electrophoresis revealed no demonstrable gammaglobulin fraction [5]. The boy was treated with monthly intramuscular injections of human gamma globulin with significant clinical improvement. Although there was no family history in this initial case, subsequent cases revealed a similar clinical phenotype with an X-linked pedigree [6,7]. The phenotype of XLA is characterized in its classical form by the almost complete absence of immunoglobulin of all isotypes and the profound reduction of B lymphocytes in the peripheral cir-culation. As a result the majority of affected boys are prone to recurrent bacterial infection from the age of approximately 4–12 months following the disappearance of maternal immunoglobulin [8]. However, in a significant number of cases (21% in a survey of 44 patients) onset of infections may occur later at 3–5 years of age [9]. The infections are usually caused by pyogenic bacteria, with Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, and Pseudomonas being the most common species [8], although mycoplasmas are a significant cause of arthritis [10,11]. The site of infection varies considerably, with sinusitis, otitis media and lower respiratory tract infections being the most common [8,9]. Resistance to viral infection remains predominantly intact except for a susceptibility to enteroviral infection which most commonly results in chronic meningoencephalitis or poliomyelitis in a significant number of cases [12]. Indeed, the major cause of mortality in the series reported by Hermaszewski & Webster [9] was as a result of enteroviral meningoencephalitis. The incidence of enteroviral disease in current populations of XLA patients appears to be less frequent, a finding which may be a result of improved clinical monitoring of patients and optimal immunoglobulin substitution therapy. The disease has always been considered to be confined to the B lymphocyte compartment; however, up to 25% of patients with XLA have profound neutropenia at the time of diagnosis [13]. This may be due to endotoxaemia following severe infections experienced by these patients, or it may be a reflection of the need for the XLA gene product in myeloid cells in situations of stress. In 1993, the gene defective in XLA was identified and termed Btk (Brutons tyrosine kinase) [14,15]. This has had considerable impact on understanding the molecular pathogenesis of the condition (discussed below) but has clinically allowed unambiguous assignment of a molecular defect to individuals with abnormalities in antibody production. Thus, although the majority of Btk-deficient patients display the classical immunophenotype of less than 1% peripheral B lymphocytes and virtual absence of all immunoglobulin isotypes, a significant number of atypical or ‘leaky’ phenotypes have been identified, implying that XLA as a disease entity has considerable clinical and immunological heterogeneity. As many as 20% of patients with mutations in Btk have delayed onset of symptoms or higher concentrations of serum immunoglobulins than expected [16–20]. In addition, there appear to be differences in clinical severity, with some individuals with very low concentrations of serum immunoglobulins and almost no B cells having surprisingly few infective complications [19,21]. The reasons for clinical and immunological heterogeneity in XLA are unclear and may be dependent on factors other than the Btk defect. Btk was identified as the molecular defect in XLA by two groups, one using a positional cloning approach [15] and the other in a search for novel protein kinases expressed in B lymphocytes [14]. The gene is located on the long arm of the X chromosome at Xq21.3 and the human gene encompasses 37·5 kb. Btk is organized into 19 exons [22–25], including a 5′ untranslated region (exon 1), and encodes a 659 amino acid protein that is expressed throughout myeloid and B cell differentiation [26,27]. The Btk protein belongs to a family of cytoplasmic tyrosine kinases that are related to, but distinct from, the Src family kinases. The protein is characterized by its modular structure which includes an amino-terminal pleckstrin homology (PH) domain followed by a tec-homology (TH) domain, a Src homology SH3 domain, an SH2 domain and a carboxy-terminal kinase SH1 domain. Catalytic activity resides in the kinase domain while the other domains are necessary for protein–protein interactions. Other members of this family include Tec [28], Itk [29,30], and Bmx [31], all of which are also expressed in haematopoietic tissues. Identification of the gene has led to mutational analysis being undertaken by a number of groups world wide. Over 500 unique mutations in Btk have now been identified and an international database of mutations and clinical information has now been established (BTKbase: http://http://www.uta.fi/laitokset/imt/bioinfo/BTKbase) [32–34]. Various different types of genetic abnormalities have been found in the Btk gene. One third of mutations are missense mutations and these have been found predominantly in the kinase domain of the gene, although they have been found in all domains except the SH3 domain [32,35]. Premature stop codons, deletions and insertions have also been described throughout the gene. Although the large majority of mutations are found in the coding region, 12% of mutations have been found to affect splice site recognition sequences [36]. Only one mutation has been found in the promoter region and this is thought to affect transcriptional regulation of the gene [37]. About 5–10% of mutations result in gross alterations of the Btk gene [38]. In addition to large deletions, an inversion, a duplication and a retroviral insertion have been identified. Despite the large amount of mutation and phenotypic data available, it has not been possible to make definitive genotype–phenotype correlations. This is emphasized by reports of considerable variation in clinical severity within the same pedigree [17,21] showing that factors other than the genetic mutation may influence the clinical course. It has been suggested that, as in other immunodeficiencies [39], the presence of cytokine gene polymorphisms or defects in innate immunity such as mutations in mannan binding lectin may be important, but as yet there is no evidence to refute or confirm these ideas. It is also possible that environmental factors may play an important role. The consequence of the gene mutation on expression of the Btk protein and its relation to clinical severity has also been explored. However, it has been found that in the majority of individuals, despite the severity of the clinical phenotype and regardless of the nature of the mutation, there is complete lack of Btk expression [18,27,40]. It would appear that even carboxy-terminal amino acid substitutions render the mRNA and/or protein unstable. The consequences of mutations in Btk have also been studied in animal models of the disease. Soon after the identification of the defect in Btk in humans, the X-linked immunodeficiency (xid) mouse was found to have a single amino acid substitution (R28C) in the PH domain of Btk [41,42]. However, the immunophenotype of the xid mouse is very different from the human form. Xid mice have relatively normal serum concentrations of IgG1, IgG2a and IgG2b, but markedly reduced IgG3 and IgM concentrations. In addition, they are unable to make antibody responses to T cell-independent antigens but respond well to stimulation with T cell-dependent antigens [43]. The reduction in the number of B cells is also much less severe in xid mice compared with patients with XLA. While typical XLA patients have < 1% of the normal number of peripheral B cells, xid mice B cell numbers are only reduced to 30–50% of normal. In both species, defects in Btk are associated with an immature B cell phenotype, with the majority of B cells displaying an increased density of surface IgM [43,44]. The differences between the murine and human phenotypes cannot be explained by the nature of the xid genetic defect, since human R28C mutations with severe phenotypes have been described [36], and also Btk-null mice created by homologous recombination have a B cell phenotype that is identical to the xid mouse [45–47]. Tyrosine kinases have been studied in many haematopoietic cell lineages and have been shown to act as signal transduction molecules mediating cell surface receptor activation events to downstream pathways. Cross-linking of a number of cell surface receptors including IL-5, IL-6, CD38, FcRε and perhaps most importantly surface IgM on B cells, results in the recruitment of cytosolic Btk to the plasma membrane and activation of Btk by tyrosine phosphorylation [48]. Migration of Btk to the cell membrane is dependent upon an intact PH domain and its association with PIP3 (phosphatidyl inositol-3,4,5-trisphosphate) [49,50]. The process of Btk activation is initiated by phosphorylation of tyrosine 551 (Tyr551) in the kinase domain of Btk by a Src family kinase, most likely lyn or fyn, followed by Btk autophosphorylation at tyrosine 223 (Tyr223) in the SH3 domain [51–53]. At the cell membrane activated Btk interacts with phospholipase gamma (PLCγ), leading to enhanced tyrosine phosphorylation of PLCγ and resulting in accumulation of inositol 1,4,5-triphosphate (IP3) and mobilization of extracellular calcium. This pathway has been well defined by in vitro models [54,55] and is supported by evidence from Btk-deficient cell lines which show reduced phosphorylation of PLCγ and decreased calcium mobilization [56,57]. Other biochemical consequences of Btk activation include co-immunoprecipitation with, and phosphorylation of a protein termed BAP (Btk associated protein)-135 [58]. This protein has been independently identified as the DNA binding protein TFII and thus links Btk with regulation of transcriptional events [59,60]. Despite the large volume of biochemical and signalling data available, the central question of why the Btk gene defect results in B cell development arrest remains unresolved. There is however evidence that B cell receptor (BCR) expression and signalling is necessary for positive selection of B cells and prevention of death by apoptosis. Xid mice exhibit a progressive loss of splenic and recirculating B cells [61]. Moreover, some of the xid defects can be rescued by over-expression of the anti-apoptotic proteins Bcl-2 and BclXL[62,63]. This, together with evidence for Btk in regulation of BclXL expression, suggests that Btk may be important in mediating anti-apoptotic signals during crucial stages of B cell development [64]. The majority of patients with congenital agammaglobulinaemia and absent B cells have defects in Btk. However, it has been well recognized that there are females who display a similar clinical and immunological profile [65,66], and there are pedigrees in which males and females have early onset hypogammaglobulinaemia and absent B cells. It is therefore likely that autosomally coded genes are sometimes responsible for this disorder. The incidence of these abnormalities is difficult to estimate. In a study of a large cohort of boys with presumed XLA, 90–95% had mutations in Btk [36]. Most of the remaining patients had defects in other genes. When one includes XLA-type females it has been estimated that approximately 10% of agammaglobulinaemic syndromes are autosomally inherited. The molecular defects for three of the autosomally inherited forms have now been identified [67–69]. The first disorder was identified by a combination of a candidate gene approach and It was that the gene would be relatively specific to the B cell lineage and that it would be expressed early in B cell genes that had been to specific in the and proteins in signal transduction the B cell or pre-B cell receptor as candidate B cell signal transduction termed which is an of the EBF transcription factor for and μ immunoglobulin heavy analysis in two that the defect to the μ heavy chain therefore this gene was studied in different mutations from have been identified in the μ heavy chain gene on chromosome In one there was a that and the μ region gene. In two there was a substitution in the splice site of the μ heavy chain gene. This mutation is thought to of the membrane form of the and an amino acid substitution in the form. 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