Immune dysregulation is increasingly being recognized as a prominent feature of genetically determined immunodeficiencies (
Carneiro-Sampaio and Coutinho, 2007;
Liston et al., 2008;
Pessach et al., 2009).
RAG mutations in humans may cause complete or partial defects of V(D)J recombination and, hence, result in SCID or in a leaky phenotype with residual T (and sometimes B) cell development associated with immune dysregulation and skewing of the T lymphocyte repertoire (
Signorini et al., 1999). We and others have previously shown that the reduced thymopoiesis in patients with Omenn syndrome and leaky SCID is accompanied by defects in thymic epithelial cell maturation and/or homeostasis, impaired expression of aire (autoimmune regulator) protein and poor generation and function of CD4
+ CD25
+ Foxp3
+ natural regulatory T cells (nT reg cells;
Cavadini et al., 2005;
Poliani et al., 2009;
Cassani et al., 2010b). However, the possible contribution of defects in B cell tolerance to the immunopathology of Omenn syndrome and leaky SCID has not been previously investigated.
RAG-dependent immunodeficiency is characterized by profound B cell lymphopenia (
Villa et al., 2001), although residual production of antibodies (IgE in particular) and ability to mount low-affinity antibody responses to bacteriophage
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x174 neoantigen have been reported in patients with hypomorphic
RAG mutations (
Ochs et al., 1974). Importantly, autoimmune cytopenias have been observed in several patients with Omenn syndrome or with leaky SCID caused by hypomorphic
RAG defects (
Ehl et al., 2005;
de Villartay et al., 2005), implying that B cell–mediated mechanisms may contribute to the immunopathology of these diseases, The recent availability of suitable animal models has made it possible to study in greater detail B cell development, maturation, and function in conditions associated with hypomorphic
Rag mutations. The Rag1 S723C mutant protein is proficient for DNA cleavage but exhibits severe defects in post-cleavage complex formation and end-joining (
Tsai et al., 2002;
Giblin et al., 2009). Homozygosity for this mutation in
mut/mut mice leads to a severe, although incomplete, block in B cell development but normal or even increased Ig levels. Similar observations had been previously reported in another model of leaky SCID, the MM mouse, with a homozygous
Rag1 R972Q mutation (
Khiong et al., 2007). Using ELISPOT assays, detailed flow cytometric analysis, and molecular assays to track terminal B cell maturation, we have now obtained substantial evidence that the spleens from
mut/mut mice contain an increased proportion of class-switched B cells and ISCs. Consistent with this, significant amounts of Igs of various isotypes are present in the serum of
mut/mut mice. However, both the peripheral B cell repertoire and serum Ig profile from
mut/mut mice show a restricted pattern. This likely reflects a limited number of productive V(D)J rearrangements as a result of the hypomorphic
Rag1 mutation. The stochastic nature of the process may also account for some variability of serum Ig levels in
mut/mut mice, as previously reported for IgE (
Giblin et al., 2009). Several mechanisms may be taken into account to explain the apparent discrepancy between the B cell production bottleneck and significant Ig production in
mut/mut mice. Adoptive bone marrow transfer experiments have shown that when limited numbers of B cells are transferred into immunodeficient mice, a large proportion of the transferred B cells undergo homeostatic proliferation and develop into PCs (
Agenès and Freitas, 1999;
Cabatingan et al., 2002). Furthermore, it has been demonstrated that under in vivo inflammatory conditions, immature/T1 B cells express significant levels of activation-induced cytidine deaminase and Blimp-1 (
Ueda et al., 2007). In vitro activation of immature/T1 B cells by toll-like receptor (TLR) ligands induces these cells to proliferate and secrete Igs in a TI manner (
He et al., 2004). Moreover, innate immune cells are over-reactive in lymphopenic environment (
Cao et al., 2009), and this may lead to increased expression of cytokines (such as BAFF) that may favor B cell survival and/or differentiation (
Ueda et al., 2007). It can be speculated that the profound immunodeficiency and defects in the gut-blood barrier may render
mut/mut mice and patients with Omenn syndrome susceptible to inflammation sustained by endogenous commensal flora, as shown in other human immunodeficiencies (
Brenchley et al., 2006). Interestingly, 4% of
mut/mut mice have developed colitis and/or erythroderma, and their spleens demonstrated very high numbers of ISCs.
We have demonstrated that
mut/mut mice are severely impaired in the antibody response to TI and TD antigens, and in particular are unable to mount high-affinity antibody responses. Adoptive transfer of wild-type CD4
+ T lymphocytes was unable to correct defective antibody response to TNP-KLH, indicating that B cell–intrinsic abnormalities (including restricted B cell repertoire) and/or aberrant architecture of secondary lymphoid organs (characterized by lack of follicles and of germinal centers) account for the impaired antibody response to TD antigens in
mut/mut mice. However, they spontaneously produce high levels of low-affinity antibodies that contain self-reactive specificities. Importantly, we have demonstrated for the first time that hypomorphic
RAG mutations are associated with a high frequency of autoantibodies also in humans. Our data indicate that several mechanisms may be involved in generating and sustaining this B cell–mediated immune dysregulation. Normally, highly polyreactive and self-reactive B cells are counterselected at the primary B cell tolerance checkpoint in the bone marrow, mostly through receptor editing. It has been estimated that both in mice and in humans, ~20–50% of developing B cells undergo receptor editing (
Retter and Nemazee, 1998;
Casellas et al., 2001;
Wardemann et al., 2004). This process is accompanied by a drastic decrease in the proportion of self-reactive antibodies that are detected in early immature versus immature B lymphocytes in humans (
Wardemann et al., 2003). A large fraction of autoantibodies expressed by these developing early immature B cells have low affinity for multiple antigens (i.e., are polyreactive) and contain ANAs (
Wardemann et al., 2003). Using a recently developed assay which is based on quantitation of RS rearrangement at the κ locus (
Panigrahi et al., 2008), we have demonstrated a drastic impairment of receptor editing in the bone marrow of
mut/mut mice. Consistent with this, we have also found that the proportion of peripheral B lymphocytes expressing λ light chain is significantly reduced in
mut/mut mice and that anti-ssDNA antibodies expressed κ, but not λ, light chains. Interestingly, we and others have recently reported increased levels of autoantibodies also in mice with hypomorphic mutations of DNA ligase IV (
Nijnik et al., 2009;
Rucci et al., 2010), another protein which is critically involved in T and B cell development and receptor editing.
Our data suggest that disturbance in peripheral checkpoints of B cell tolerance may also be involved in the pathophysiology of autoimmunity associated with hypomorphic
Rag mutations. BAFF serum levels were 10-fold higher in
rag-mutated mice and humans than in controls. Based on previous studies, this is likely the consequence of the B cell lymphopenic environment (
Lesley et al., 2004;
Lavie et al., 2007). High levels of BAFF-R are expressed by CD21
int T2 B cells, which undergo extensive BAFF-mediated homeostatic proliferation (
Meyer-Bahlburg et al., 2008). Anergic self-reactive B cells in the periphery express low levels of BAFF-R, show reduced BAFF responsiveness, and are normally lost at the T2 stage or beyond (
Lesley et al., 2004;
Thien et al., 2004). However, self-reactive peripheral B cells can be rescued by exogenous administration or overexpression of BAFF (
Mackay et al., 1999;
Lesley et al., 2004;
Thien et al., 2004;
Hondowicz et al., 2007) and produce anti-chromatin and anti-DNA antibodies (
Hondowicz et al., 2007). It has been shown that in vivo BAFF neutralization ameliorates islet-directed autoimmunity in nonobese diabetic mice by increasing competition among transitional B cells for follicular entry (
Zekavat et al., 2008). We did not have access to BAFF-neutralizing antibody and, thus, could not directly test the significance of increased BAFF levels in the autoimmunity of
mut/mut mice. In a companion paper,
Cassani et al. (in this issue) report that i.v. injection of an anti–BAFF-R monoclonal antibody into
Rag2R229Q mice led to disappearance of anti–double-stranded DNA IgG antibodies and significant amelioration of inflammatory infiltrates. In contrast, BAFF-R–deficient A/WySnJ mice develop autoantibodies and severe glomerulonephritis (
Mayne et al., 2008), indicating that balance of BAFF levels may be important to maintain B cell tolerance. Consistent with this, we found that increase of mature B cell number in older
mut/mut mice was associated with decrease of BAFF serum levels and normalization of the titer of anti-ssDNA and anti-chromatin antibodies. Altogether, these data suggest that increased BAFF serum levels play a significant role in autoimmune manifestations associated with hypomorphic
Rag mutations.
There is supporting evidence that elevated BAFF serum levels may also associate with immune dysregulation in humans (
Moisini and Davidson, 2009). Very elevated serum BAFF concentrations were detected in our series of 19 patients with
RAG mutations, including 14 patients with Omenn syndrome or leaky SCID, 9 of which had evidence of autoantibodies. Levels of BAFF were higher in patients with
RAG mutations than in those with SCIDX1, none of which developed signs of autoimmunity. It is possible that a certain threshold of BAFF concentration must be reached to rescue self-reactive B cells in the periphery. However, other non–mutually exclusive mechanisms may also account for the different incidence of autoimmunity between patients with hypomorphic
RAG defects and those with SCIDX1. Impaired receptor editing is anticipated in
RAG deficiency but not in SCIDX1. Furthermore, patients with SCIDX1 typically lack mature T lymphocytes, whereas both in humans and in mice hypomorphic
RAG mutations are often associated with a variable number of oligoclonal and in vivo activated T cells that infiltrate target organs and may promote and/or sustain B cell–mediated autoimmunity. In the companion manuscript,
Cassani et al. (2010a) show that in vivo depletion of CD4
+ cells in
Rag2R229Q mice results in the decrease of ISCs and normalization of serum IgE. In contrast, autoimmunity develops to the same extent in both T cell–sufficient and in T cell–deficient BAFF-transgenic mice (
Groom et al., 2002), indicating that BAFF-mediated rescue of self-reactive B cells may be T cell independent. In this regard, it is noteworthy that adoptive transfer of wild-type CD4
+ T cells did not ameliorate autoimmunity in
mut/mut mice. Nonetheless, it is likely that the severe defects in T cell development and function observed in
mut/mut mice and in patients with Omenn syndrome and leaky SCID may contribute to B cell–mediated immune dysregulation. In particular, we and others have shown that hypomorphic
RAG mutations are associated with a severe defect in the ability to support nT reg cells development and function both in mice (
Marrella et al., 2007) and in humans (
Poliani et al., 2009;
Cassani et al., 2010b).
We have shown that
mut/mut mice produce polyreactive low-affinity antibodies with self-reactive specificities. Usually, such antibodies are not associated with organ-specific autoimmunity and tissue damage. Consistent with this,
mut/mut mice did not show significant signs of disease, such as weight loss, proteinuria, or reduced life span, under SPF conditions, and only a minority (4%) of them developed alopecia or colitis. In contrast, a significant proportion of
Rag2R229Q mice develop features of Omenn syndrome by 8–10 wk of age (
Marrella et al., 2007). Furthermore, >10% of
Rag2R229Q mice produce high-affinity anti–double-stranded DNA antibodies and show prominent T and B cell infiltrates in the kidney (leading to proteinuria), the gut, and the skin, as reported by
Cassani et al. (2010a) in the companion paper. Several mechanisms may account for the phenotypic heterogeneity both between and within these
Rag-mutated mouse models. It is possible that the distinct
Rag mutations carried by these models exert a different effect on the efficiency of V(D)J recombination, generation of T and B lymphocytes, and receptor editing and that the stochastic nature of the process contributes to the heterogeneity of the phenotype, even within the same model. Moreover, it is possible that differences in housing conditions and in the microbial gut flora may modulate B cell–mediated autoimmunity through TLR-mediated signaling. Consistent with this,
Cassani et al. (2010a) have shown that B lymphocytes from
Rag2R229Q mice respond efficiently to TLR agonists undergoing terminal differentiation, Ig production, and class switch recombination. Finally, T lymphocytes may also play a role in precipitating the disease phenotype. In this regard,
Cassani et al. (2010a) have shown that depletion of CD4
+ T cells in
Rag2R229Q mice leads to reduction of ISCs and normalization of serum IgE, associated with a decrease of IL-21 and IFN-γ serum levels. These factors may contribute to the significant variability of the clinical phenotype that has also been observed in patients with hypomorphic
RAG mutations, even within the same family (
Villa et al., 2001).
In conclusion, we have established that hypomorphic RAG mutations in mice and humans are associated with autoantibody production. We have determined that this B cell–mediated immune dysregulation reflects profound abnormalities in the mechanisms that govern central and peripheral B cell tolerance. Experiments based on adoptive transfer of T reg cells, in vivo injection of TLR agonists, and generation of mut/mut mice carrying additional defects in TLR signaling may permit to define the relative contribution of impaired T cell–mediated control of B cell immune dysreactivity and TLR-mediated B cell activation to the autoimmune phenomena of leaky SCID and Omenn syndrome.
3 and Luigi D. Notarangelo
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