© 2005 Nature Publishing Group http://www.nature.com/naturegenetics
ARTICLES
TACI is mutant in common variable immunodeficiency and IgA deficiency Emanuela Castigli, Stephen A Wilson, Lilit Garibyan, Rima Rachid, Francisco Bonilla, Lynda Schneider & Raif S Geha The tumor necrosis factor receptor family member TACI (transmembrane activator and calcium-modulator and cyclophilin ligand interactor) mediates isotype switching in B cells. We found that 4 of 19 unrelated individuals with common variable immunodeficiency (CVID) and 1 of 16 individuals with IgA deficiency (IgAD) had a missense mutation in one allele of TNFRSF13B (encoding TACI). One of the four individuals with CVID had a single nucleotide insertion in the other TNFRSF13B allele. None of these mutations were present in 50 healthy subjects. TNFRSF13B mutations cosegregated with the phenotype of CVID or IgAD in family members of four index individuals that we studied. B cells from individuals with TACI mutations expressed TACI but did not produce IgG and IgA in response to the TACI ligand APRIL, probably reflecting impaired isotype switching. These results suggest that TACI mutations can result in CVID and IgAD. IgAD is the most common form of primary immunodeficiency, with an incidence of B1 in 600 individuals in the western world1,2. CVID is characterized by a deficiency in all immunoglobulin (Ig) isotypes3–5. Individuals with symptomatic IgAD often have deficiency of IgG subclasses or decreased antibody response to carbohydrate antigens such as pneumococcal polysaccharide vaccine. Individuals with symptomatic IgAD and individuals with CVID suffer from recurrent sinopulmonary and gastrointestinal infections and have an increased incidence of autoimmune disorders and of lymphoid and nonlymphoid malignancies1,6. IgAD and CVID have been known to coexist in families. Some individuals initially present with IgAD and then develop CVID. These observations suggest that some cases of IgAD and CVID may have a common etiology. A small number of individuals diagnosed with CVID have X-linked agammaglobulinemia owing to mutations in BTK (Bruton’s tyrosine kinase; OMIM 300300)7. A subset of individuals with CVID has mutations in SH2D1A (OMIM 308240), the gene mutated in X-linked lymphoproliferative disorder8. A single report has identified mutations in ICOS (OMIM 607594) in individuals with CVID who were probably descended from a common founder9. The molecular basis of IgAD and most cases of CVID remains unknown. In vitro studies have suggested that some individuals with IgAD have impaired isotype class switching to IgA and others may have a post-switch defect10–12. There is also evidence for a global isotype switching defect in some individuals with CVID3,4,13. But CVID is a complex and heterogeneous disease in which defects in B-cell survival, number of circulating CD27+ memory B cells (including IgM+CD27+ B cells), B-cell activation after antigen receptor cross-linking, T-cell signaling and cytokine expression have been observed14.
Isotype switching of human IgM+IgD+ naive B cells to IgG, IgA and IgE involves deletional switch recombination between switch regions that are located upstream of the IgM heavy chain gene (Cm) and the Cg, Ca or Ce heavy chain genes. This class switch recombination requires two signals: one signal is normally delivered by cytokines, and the second signal is normally delivered by select members of the tumor necrosis factor receptor family expressed on B cells that include CD40, TACI and BAFF-R (B cell–activating factor of the tumor necrosis factor family)15–17. The two signals synergize to induce germline transcription of Cg, Ca or Ce and expression of the enzyme activation-induced cytidine deaminase, which is crucial for deletional switch recombination. Mutations in CD40 (OMIM 606843) or its ligand CD40LG (also called CD40L; OMIM 308230), which is expressed on activated T cells, result in failure of T cell–dependent isotype switching and in hyperIgM syndrome18,19. Some individuals with CVID express low levels of CD40L, the product of the gene mutated in X-linked hyperIgM syndrome20. The ligands for TACI are APRIL (a proliferation-inducing ligand) and BAFF21. APRIL and BAFF also bind to BCMA (B-cell maturation antigen), another tumor necrosis factor receptor family member expressed by B cells. In addition, BAFF binds to a unique receptor, BAFF-R. APRIL and BAFF are expressed primarily by monocytes and dendritic cells21,22. Mice deficient in BAFF and BAFF-R have severe failure of B-cell differentiation and very low levels of serum Igs23,24. Mice deficient in TACI and APRIL have IgA deficiency with impaired antibody response to T cell–independent antigens25–27. APRIL and BAFF induce class switch recombination to IgG and IgA in naive human and mouse B cells16,27. We recently showed that TACI and, to a lesser extent, BAFF-R, but not BCMA,
Division of Immunology, Children’s Hospital and Department of Pediatrics, Harvard Medical School, 300 Longwood Avenue, Boston, Massachusetts 02115, USA. Correspondence should be addressed to R.S.G. (
[email protected]). Published online 10 July 2005; doi:10.1038/ng1601
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ARTICLES Table 1 Characteristics of individuals with IgAD and CVID and mutations in TNFRSF13B Serum level (mg dl
© 2005 Nature Publishing Group http://www.nature.com/naturegenetics
Individual
Diagnosis
Sex
1)a
Percentage of circulating cellsc
Age (y)
Mutation
IgM
IgG
IgA
Antibody response to Pneumovaxb
B cells
T cells
A11 C5
IgAD CVID
M F
17 51
310T-C (C104R) 310T-C (C104R)
105 153
662 497
13 38
Impaired Impaired
11 10
52 80
C12
CVID
F
41
310T-C (C104R) 204insA (frameshift)
82
349
9
NA
16
70
C15 C17
CVID CVID
F F
41 8
542C-A (A181E) 602G-A (R202H)
62 133
381 498
12 32
Impaired NA
1.5 7
81 82
Adults
None
40–240
639–1,344
70–312
Normal
5–21
51–83
Controls
Healthy
M/F
aLaboratory
values were obtained from all affected individuals before they began IVIG therapy. Serum IgG and IgA levels in individual C17 are below the lower limits of the normal range for her age. bImpaired antibody response to immunization with Pneumovax was defined as failure to increase preimmunzation titer by fourfold for at least 7 of 14 serotypes tested. cPercentages of B and T cells in PBMCs as assessed by FACS analysis of CD20+ and CD3+ cells. Abnormal values are shown in bold.
mediate isotype switching induced by APRIL and BAFF17. These findings prompted us to search for TACI mutations in individuals with IgAD and CVID. RESULTS TACI is mutant in individuals with IgAD and CVID We sequenced the five exons of TNFRSF13B in genomic DNA from 19 unrelated individuals with CVID, 16 unrelated individuals with symptomatic IgAD and 50 healthy controls. We found a single missense mutation in one of the TNFRSF13B alleles in each of four individuals with CVID and in one individual with IgAD. In addition, one of the four individuals with CVID also had a mutation in the other TNFRSF13B allele. All five individuals had normal sequences with respect to the genes encoding APRIL, BAFF and BAFF-R (data not shown). The characteristics of the affected individuals are summarized in Table 1. All individuals presented with recurrent sinopulmonary infections that included bronchitis, otitis media and sinusitis and required IVIG replacement therapy. Individual C17 also had a history of osteomyelitis. At presentation, all four individuals with CVID had IgG and IgA serum levels that were below the lower limit of the normal range for their age. Serum IgG levels were within the normal range in the individual with IgAD, but his serum IgA level was severely diminished. Low serum IgG or IgA levels were confirmed on at least two occasions in each individual. The serum antibody response to immunization with pneumococcal antigen (Pneumovax) and the Haemophilus influenzae antigen polyribosyl ribitol phosphate was impaired in all individuals for whom data were available. In contrast, the serum antibody response to immunization with the protein antigen tetanus toxoid was normal (Table 1). Circulating lymphocyte counts were normal in all individuals (data not shown). Except for a diminished percentage of CD20+ B cells in individual C15, all individuals had normal percentages of circulating B and T cells. The percentage of CD20+CD27+ memory B cells was within the normal range in all four individuals studied, except individual C15 (Supplementary Table 1 online). We did not analyze IgM+CD27+ memory B cells. Individual C15 recently developed a non-Hodgkin B-cell lymphoma. Two of the individuals with CVID (C5 and C12) and the individual with IgAD (A11) had the same 310T-C nucleotide substitution in exon 3 of TNFRSF13B (nucleotide position given from the ATG translation initiation site; Supplementary Fig. 1 online). This mutation results in the amino acid substitution C104R in the extracellular domain (Fig. 1). One of the two individuals with CVID with the C104R mutation (C12) also had a single adenine insertion immediately after base pair +204 at the beginning of exon 3 (204insA). This mutation results in a frameshift after amino acid residue 68 and premature
830
termination of the protein 11 amino acids downstream. Analysis of genomic clones and of cDNA clones from individual C12 showed that this insertion and the 310T-C single-base substitution were on different alleles (Supplementary Fig. 1 online and data not shown). Sequencing of genomic DNA from the parents and the children of individual C12 indicated that the two mutated alleles segregated independently (Fig. 2). Therefore, individual C12 with CVID is a compound heterozygote. The third individual with CVID (C15) had a single–base pair mutation, 542C-A, in exon 4 in the sequence encoding the transmembrane domain (Supplementary Fig. 1 online). This mutation results in the amino acid substitution A181E (Fig. 1). The fourth individual with CVID (C17) had a single–base pair substitution, 605G-A, in exon 4 in the sequence encoding the intracellular domain (Supplementary Fig. 1 online). This mutation results in the amino acid substitution R202H (Fig. 1). We found none of the above mutations in 50 control subjects. We detected three single-nucleotide substitutions in both affected individuals and controls. Two of these substitutions reside in exons 2 and 5 but do not result in any amino acid changes. The third substitution is in exon 5 and results in the amino acid substitution P251L; it was found in 6 of the 50 controls. These three nucleotide substitutions probably represent polymorphism. TACI sequence in relatives of individuals with mutant TACI Analysis of genomic DNA from the family members of the four index individuals with CVID and the index individual with IgAD is shown in Figure 2. Individual C5 has a 15-year-old son who is heterozygous with respect to the same C104R mutation. He is affected with CVID and has low serum IgG (420 mg dl 1, normal adult range is 639–1,344 mg dl 1) and IgA (15 mg dl 1, normal adult range is 70–312 mg dl 1) levels. The compound heterozygous individual C12 inherited the mutated TNFRSF13B allele with the 204insA insertion from her mother, who has IgAD (serum IgA levels of 37 mg dl 1) and a history of recurrent sinusitis but normal serum IgG levels. Individual C12 inherited the Frameshift
1
C104R
2
A181E
3
4
EC
R202H
5
TM
IC
Figure 1 Schematic representation of the structure of TACI and its mutations in individuals with CVID and IgAD. Exons 1–5 are color-coded and numbered. EC, extracellular domain; IC, intracellular domain; TM, transmembrane domain.
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C5
C12 C12
C5
IgAD IgAD
IgAD CVID
A11 IgAD IgAD
IgAD
IgAD
CVID
IgAD
C12a CVID
CVID
C104R
C104R
C12b C12c CVID CVID
C104R
C15 C15
C17 C17
CVID CVID
CVID
CVID
A181E
R202H
Figure 2 Analysis of TNFRSF13B mutations in family members of index individuals with CVID and IgAD. White, normal allele; black, missense mutation; green, 204insA (resulting in a frameshift); pale blue, no information available because family members were either unavailable (family of individual C5) or refused consent (family of individual C17). Index cases are indicated by an arrow.
B-cell expression of TACI and serum TACI levels We assessed TACI expression on B cells from the index individuals by FACS analysis on peripheral blood mononuclear cells (PBMCs) by staining them with goat antibody to human TACI. We confirmed the specificity of the antibody by staining 293 cells transfected with a vector expressing wild-type TACI. TACI expression on B cells from all five individuals that we studied was similar in percentage and intensity to that observed in control B cells (Fig. 3). Serum TACI levels were elevated in all four individuals in whom it was tested (43.6 ng ml 1 in individual A11, 48.4 ng ml 1 in individual C5, 44.8 ng ml 1 in individual C12 and 25.3 ng ml 1 in individual C15; normal range was 0.9–4.5 ng ml 1 in eight healthy controls). Serum TACI levels were also high in the two children of individual C12 who were heterozygous with respect to the frameshift mutation (46.5 ng ml 1 and 39.5 ng ml 1). Serum BAFF levels were normal in all individuals, except individual C15 who had B-cell lymphoma. Her serum BAFF level was 16.0 ng ml 1 compared with a normal range of 0.6–1.3 ng ml 1 in eight healthy controls.
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100 101 102 103 104
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100 101 102 103 104
TACI
100 101 102 103 104 100 101 102 103 104
Goat TACI
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C104R mutation from her father, who suffers from recurrent sinusitis and bronchitis and has low serum IgA levels (36 mg dl 1) but normal serum IgG levels. Both of her sisters inherited the mutated paternal allele and a normal allele from their mother. Their serum IgG levels are normal, but both have low serum IgA levels (55 and 37 mg dl 1) and one suffers from recurrent sinusitis. Individual C12 transmitted her paternal mutated allele to her son, individual C12a (12 years of age), Surface expression and ligand binding of TACI mutants who received a normal TNFRSF13B allele from his father. Individual To determine whether TACI mutants observed in the affected indiviC12a is affected by CVID, has low serum IgG (384 mg dl 1) and IgA duals could be expressed on the cell surface, we transfected 293 (12 mg dl 1) levels and was placed on IVIG replacement therapy cells with constructs expressing the mutants and analyzed TACI because of recurrent sinopulmonary infections. Individual C12 trans- expression by FACS using goat polyclonal antibody to IgG and mitted her mutated maternal TNFRSF13B allele with the 204insA mouse monoclonal antibody to human TACI. Wild-type TACI, as insertion to her other two children, individuals C12b (9 years of age) well as the A181E and R202H mutants, were readily detectable by both and C12c (5 years of age). Individuals C12b and C12c, like antibodies (Fig. 4). The C104R mutant was detected by the polyclonal their grandmother, have IgAD (serum IgA levels of o7 mg dl 1 and antibody, but not by the monoclonal antibody, suggesting that this 12 mg dl 1, respectively). They also have serum IgG levels (441 mg mutation destroys the epitope recognized by the monoclonal antibody. dl 1 and 404 mg dl 1, respectively) below the lower limit of the normal There was no detectable TACI on the surface of cells transfected with range for their ages. Both individuals C12b and C12c required IVIG the frameshift mutant (data not shown). replacement because of recurrent infections. Individual C15 with the A181E mutation Control 1 Control 2 A11 in the transmembrane domain of TACI devel0.66 2.1 10.0 5.3 oped a B-cell non-Hodgkin lymphoma at the time of the study. Her brother, like her, is heterozygous with respect to the same muta3.6 4.2 6.6 tion. He has low numbers of circulating B 0 101 102 103 104 0 101 102 103 104 0 101 102 103 104 0 101 102 103 104 10 10 10 10 cells (2%), has CVID (serum IgG level of 342 CD20 mg dl 1 and serum IgA level of 15 mg dl 1) C5 C12 C15 C17 and has been on IVIG replacement for several 3.7 12.1 0.5 1.9 years. The sister of individual C15 had a history of CVID (serum IgG level of 362 mg dl 1 and serum IgA level of 29 mg dl 1) and 8.0 1.5 0.4 2.6 died of a gastrointestinal carcinoma. Her 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 mother had a history of recurrent infections CD20 and died of a lymphoma. Her father has two normal TNFRSF13B alleles, normal Ig levels Figure 3 FACS analysis of TACI expression on B cells from index individuals and controls. PBMCs were and is asymptomatic. Taken together, these stained with fluorescein isothiocyanate–conjugated monoclonal antibody to CD20 and with biotinylated data suggests that individual C15 inherited goat antibody to human TACI or biotinylated goat IgG as control, followed by streptavidin-phycoerythrin. the mutated allele from her mother. Numbers in the quadrants represent percentages. 100 101 102 103 104
© 2005 Nature Publishing Group http://www.nature.com/naturegenetics
204insA
Individual A11 inherited the C104R mutated allele from his maternal grandmother; his mother also transmitted this allele to his sister. His grandmother, mother and sister (14 years of age) have IgAD (serum IgA levels of 14 mg dl 1, 36 mg dl 1 and 26 mg dl 1, respectively) and suffer from recurrent sinusitis and upper respiratory infections. Both his mother and grandmother have autoimmune thyroiditis. His father and paternal grandmother have normal TNFRSF13B gene sequence, normal Ig levels and are asymptomatic. The family members of individual C17 did not participate in this study.
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A181E
R202H
100
101 102 103 104 FL2-H
100
101 102 103 104 FL2-H
100
50
40
40
IgA (ng/ml)
30 20
Controls C5 (C104R)
30
C12 (C104R) A11 (C104R)
20
C12b (frameshift)
10 0 APRIL
APRIL 60
60
50
50
50
40
40
40
IgA (ng/ml)
60
30 20
IgG (ng/ml)
IgG (ng/ml)
60
50
0
30 20
10
10
0
0
101 102 103 104 FL2-H
We also analyzed the transfectants for binding to APRIL and BAFF. The A181E and R202H mutants bound BAFF to the same extent as wild-type TACI (Fig. 4). In contrast, the C104R mutant had no detectable BAFF binding. The data on APRIL binding were not interpretable because of high background binding to 293 cells, which persisted despite the use of heparin under the same conditions that were recently described to inhibit the binding of APRIL to heparan sulfate proteoglycans on the surface of many cells, including 293 cells28,29. Ig secretion in response to APRIL and BAFF by naive B cells APRIL can mediate class switching to IgA and IgG in B cells16,27. Although APRIL binds both TACI and BCMA, its ability to cause isotype switching is mediated solely by TACI17. To assess the ability of TACI to mediate isotype switching in individuals with TACI mutations, we examined the capacity of highly purified peripheral blood B cells positively selected for IgM and IgD expression to secrete IgA and IgG in response to APRIL and BAFF. Naive control B cells secreted IgM but made little or no detectable IgG and IgA (o2 ng ml 1) when cultured in medium alone (data not shown). APRIL and BAFF induced IgA and IgG synthesis in normal naive B cells, in the absence of exogenous cytokines (Fig. 5), as previously observed in murine B cells17. In contrast, APRIL caused little or no detectable IgA or IgG secretion by B cells from individuals A11, C5, C12 and C12b. BAFF stimulation induced IgG secretion but not IgA secretion in B cells from these individuals. B cells from all individuals that we examined secreted IgG in response to antibody to CD40 plus IL-4 in amounts comparable to those secreted by control B cells, further indicating that their intracellular isotype switching machinery is intact. There was no
30 20 10 0
BAFF
Figure 4 TACI expression and BAFF binding by 293 cells transfected with TACI mutants. 293 cells were transfected with GFP and empty vector, wildtype (WT) TACI or TACI mutants and analyzed by FACS. GFPhi cells were gated and analyzed for surface expression of TACI using goat antibody to human TACI (hTACI) or monoclonal antibody to TACI and for binding FLAGtagged recombinant human BAFF (rBAFF). The dashed lines represent isotype controls; the solid lines, staining for TACI or BAFF binding.
832
60
10
IgG (ng/ml)
C104R
rBAFF-FLAG Counts Counts Counts Counts Counts 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50
© 2005 Nature Publishing Group http://www.nature.com/naturegenetics
WT
mAb α-hTACI Counts Counts Counts Counts Counts 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50
Vector
Counts Counts Counts Counts Counts 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50
Goat α-hTACI
BAFF
anti-CD40 + IL-4
Figure 5 IgG and IgA secretion in response to APRIL and BAFF by B cells from individuals with CVID and IgAD and with TACI mutations. IgG and IgA in supernatants of cultures of naive IgM+IgD+ B cells from affected individuals and four adult controls were stimulated for 14 d with APRIL, BAFF or monoclonal antibody to CD40 plus IL-4. In all cases, IgG and IgA values in supernatants of unstimulated B cells were undetectable or 42 ng ml 1 and were subtracted from the values in stimulated cultures. The lowest net IgG and IgA values in supernatants of control B cells were 23 ng ml 1 and 17 ng ml 1, respectively, after APRIL stimulation and 25 ng ml 1 and 14 ng ml 1, respectively, after BAFF stimulation. Error bars represent s.d.
substantial effect of APRIL or BAFF on IgM secretion by B cells from either normal or affected individuals (data not shown). We were unable to study B cells from individuals C15 and C17. Individual C15 was severely B-cell lymphopenic and was started on chemotherapy shortly after we initiated our study. The parents of individual C17 did not give consent for us to draw any more blood in addition to the initial sample used for TNFRSF13B gene sequencing. DISCUSSION Our results suggest that mutations in TNFRSF13B result in CVID and IgAD. This conclusion is based on three observations. First, we found mutations in TNFRSF13B in 4 of 19 individuals with CVID and 1 of 16 individuals with IgAD that were absent in 50 healthy controls. Second, TNFRSF13B mutations cosegregated with the phenotype of CVID or IgAD in family members of four index individuals studied. Third, the capacity of B cells from individuals with TNFRSF13B mutations to secrete IgG and IgA in response to stimulation with the TACI ligand APRIL was severely impaired. We found four mutations in TNFRSF13B in a subgroup of individuals with CVID and IgAD (Table 1 and Fig. 2). Individuals with CVID and TACI mutations may represent a unique subset of CVID, as they all had normal serum IgM levels, which is infrequent in CVID30. The C104R mutation abolishes ligand binding (Fig. 4). The A181E mutation causes a change from a neutral amino acid to a negatively charged amino acid in the transmembrane domain of the protein. The R202H mutation occurs in the membrane proximal region of the intracellular domain of TACI, a region that is implicated in the interaction of TACI with CAML (calcium-modulator and cyclophilin ligand), which positively regulates the activation of the calcium-dependent phosphatase calcineurin, which dephosphorylates and activates NF-AT25,31. The fourth mutation results in a frameshift following amino acid residue 68 in the extracelluar domain and premature termination of the protein 11 amino acids downstream.
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ARTICLES TACI was expressed on B cells from all the affected individuals that we studied (Fig. 3). Transfection experiments showed that all three missense mutants that we identified could be expressed on the cell surface (Fig. 4). In the case of the C104R mutation, this could be predicted by the observation that TACI was expressed on the B cells from the compound heterozygous individual C12, who carries the C104R mutation on one allele and the frameshift mutation on the other allele, the putative product of which would lack a transmembrane anchor. TACI has two cysteine-rich domains (CRDs) in its extracellular domain. The first of these extends from amino acid 32 to amino acid 67, and the second extends from amino acid 68 to amino acid 106 and binds APRIL and BAFF with high affinity32. The CRDs contain a conserved six-residue sequence, (F/Y/W)-D-x-L-(V/T)(R/G), that is required for either APRIL or BAFF binding and is present in the CRDs of BAFF-R and BCMA. Our studies with 293 transfectants showed that BAFF ligand binding was abolished by the C104R mutation but was unaffected by the A181E and R202H mutations. The C104R mutation would disrupt the disulfide bond between Cys93 and Cys104 and would interfere with the formation of the h2 loop in the second CRD of TACI32. In contrast, the A181E transmembrane domain mutation and the R202H intracellular domain mutation would not be expected to affect ligand binding. The frameshift mutation would potentially result in a secreted truncated product that would contain the first CRD of TACI. Serum TACI levels were elevated in all the individuals that we tested, including the two children of individual C12 who were heterozygous with respect to the frameshift mutation. Further studies are needed to determine whether this is a direct consequence of the TACI mutations observed in the individuals. Only individual C15 had an elevated serum BAFF level . This is consistent with the presence of a B-cell lymphoma in this person, as serum BAFF levels are elevated in individuals with B-cell malignancies33. The ability of naive B cells from the three index individuals that we studied and from one relative heterozygous with respect to the frameshift mutation (individual C12b) to secrete IgG and IgA in response to APRIL in vitro was severely impaired (Fig. 5). Although IgG and IgA secretion by normal naive B cells in this system probably represents isotype switching, it may also reflect increased survival of already switched contaminating B cells or increased Ig secretion by these cells in response to APRIL stimulation. Because TACI, but not BCMA, is responsible for IgG and IgA isotype switching and secretion in response to APRIL17, these results suggest that TACI-mediated signaling was impaired in the individuals’ B cells. In the case of individual C12, this is consistent with the observation that the only TACI product expressed on the cell surface, the C104R mutant, is unable to bind BAFF and therefore unlikely to bind APRIL. Given the fact that TACI probably signals after ligand-induced trimerization21, the A181E and R202H mutants may function as dominant negative by disrupting the complex. In the case of the frameshift mutant (in individual C12b), the soluble product of the frameshift mutation may compete with membrane-bound TACI for APRIL binding in vivo and may make complexes with surface TACI that interfere with ligand binding. Alternatively, expression of a single normal allele may not be sufficient to transduce a productive TACI signal. The fact that Tnfrsf13b+/ mice have no phenotypic abnormalities argues against the latter possibility25,26. Naive B cells from the individuals that we studied secreted IgG, but not IgA, in response to BAFF (Fig. 5). These results mirror previous results from TACI-deficient mice, in which BAFF induced IgG secretion but completely failed to induce IgA secretion17. The results suggest that BAFF-R signaling is intact in these individuals and is sufficient for
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IgG secretion, but not for IgA secretion, and that TACI signaling is essential for IgA secretion in response to both APRIL and BAFF. Mice with disruptions of both Tnfrsf13b alleles are IgA-deficient and have impaired response to type II T cell–independent antigens, including pneumococcal polysaccharide antigens, but do not have low serum IgG levels25,26. This phenotype is similar to that of an individual with IgAD and the C104R mutation of TACI. The same C104R mutation was found in individuals with CVID (Table 1 and Fig. 2). In addition, some of the members of the family of individual C12, who carried the allele with the frameshift mutation, suffered from CVID, whereas others suffered from IgAD (Fig. 2). This suggests that the phenotypic expression of TACI mutations is affected by genetic or environmental factors. It is also consistent with the observations that some individuals present with IgAD and then develop CVID and that the two diseases may coexist in the same family. Furthermore, with aging, TACI-deficient mice develop autoantibodies and B-cell lymphoproliferation34, two complications that occur in individuals with IgAD and CVID and that were observed in affected members of the families of individuals A11 and C15, respectively. There was a perfect correlation between the presence of the mutated allele and the occurrence of CVID or IgAD in the families that we studied. In these four families, 12 individuals who carried the mutated allele had CVID or IgAD. Eleven of these twelve had a history of recurrent infections, and four were on IVIG replacement therapy. In contrast, all six individuals who had normal TNFRSF13B genes had normal levels of IgG and IgA and were free of recurrent infections. An association between the MHC locus on chromosome 6 with both IgAD and CVID has been reported1,35–37, suggesting that a gene in that locus may affect the penetrance of the disease. The correlation between the presence of a mutated TNFRSF13B gene, on chromosome 17, and the presence of CVID and IgAD in the family members of our index individuals suggests that the MHC locus exerted little influence on the expression of CVID and IgAD in these individuals with mutations in TACI. HLA typing of members of the families of individuals C12 and A11 showed no evidence of HLA haplotypes that have been reported to be associated with IgAD and CVID (data not shown). Furthermore, one of the three children of individual C12 affected by CVID, individual C12c, shared no MHC haplotypes with his two siblings, who were also affected by CVID, including the sister, individual C12b, who shared the same TNFRSF13B mutation. Also, individual A11 and his sister did not share an MHC haplotype with their maternal grandmother, although all three were affected by IgAD. Nevertheless, we cannot formally rule out the influence of MHC locus genes not previously reported to be associated with CVID and IgAD in these individuals. Because TACI mutations were found in only 5 of the 35 individuals with CVID and IgAD that we examined, the MHC locus may also have a role in the expression of CVID and IgAD in cases that do not involve mutations in TNFRSF13B. METHODS Blood samples. We obtained blood by venipuncture from affected individuals, their relatives and healthy adult controls after obtaining informed consent. The study was approved by the Ethics Committee/Internal Review Board of Children’s Hospital, Boston. DNA and cDNA sequencing. We extracted genomic DNA and RNA using standard procedures from PBMCs prepared by Ficoll-Hypaque density gradient centrifugation. We prepared cDNA by reverse transcription. We amplified the five exons of TNFRSF13B from genomic DNA by PCR using five sets of primers that hybridized to intronic sequences that flanked the exons (Supplementary Table 2 online). We used three sets of primers that yielded overlapping
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sequences to amplify the coding sequence of TNFRSF13B cDNA (Supplementary Table 2 online). We sequenced PCR products in both directions on all subjects. Sequencing was done at the Children’s Hospital Genomics Facility on an ABI 3730 DNA analyzer. For individual C12 and a normal control, the PCR product that amplified the third exon of TNFRSF13B from genomic DNA and the PCR product that spanned the sequence encoded by third exon from TNFRSF13B cDNA were cloned in pCR-TOPO vector (Invitrogen). We sequenced ten colonies from each cloning reaction. FACS analysis. We stained PBMCs with fluorescein isothiocyanate–conjugated monoclonal antibody to CD20 (BD-Pharmingen) and biotin-conjugated goat antibody to human TACI (2 mg ml 1, PeproTech, Inc.) and then with streptavidin-phycoerythrin (BD-Pharmingen) and analyzed them on a FACSCalibur (BD) flow cytometer. TACI expression and BAFF binding by 293 cells transfected with TACI mutants. We cloned TNFRSF13B cDNAs from affected (mutants) and control (wild-type) individuals into pcDNA3.1 plasmid and then transfected 293 cells (a gift from L. Glimcher, Harvard University) with TNFRSF13B plasmids at a 10:1 ratio with pcDNA3.1-GFP plasmid using the Fugene transfection method (Roche) in accordance with the manufacturer’s instruction. After 48 h we collected the cells without trypsin and stained them with a biotin-conjugated polyclonal goat antibody to human TACI , with a phycoerythrin-conjugated monoclonal antibody to TACI (R&D) or with recombinant human BAFF-FLAG (Alexis) followed first by biotinylated monoclonal antibody to FLAG (Sigma-Aldrich) and then by streptavidin-phycoerythrin. We analyzed TACI expression by FACS on cells gated for high GFP expression. Serum TACI and BAFF levels. We assayed serum TACI by ELISA using maxisorb plates (Nunc) and goat antibody to human TACI as a capture antibody. We used the same antibody in the biotinylated form with streptavidin –horseradish peroxidase (BD-Pharmingen) and ABTS-peroxide (KPL) as developing reagent. We used recombinant human TACI (PeproTech, Inc.) as standard. We detected BAFF in serum by ELISA using a commercial kit (R&D). In vitro immunoglobulin synthesis. We prepared naive B cells from PBMCs by positive selection. We incubated PBMCs with biotin-conjugated monoclonal antibodies to human IgM and human IgD (BD-Pharmingen) and positively sorted them with magnetic beads coated with antibody to biotin (Milltenyi) in accordance with the manufacturer’s instructions. The resulting cell population was 498% IgM+IgD+. We cultured 1 106 cells ml 1 in 0.5 ml of RPMI 1640 medium with 10% fetal calf serum, 100 U ml 1 penicillin, 100 mg ml 1 streptomycin and 2 mM L-glutamine and stimulated them with recombinant APRIL (1 mg ml 1, R&D), recombinant BAFF (1 mg ml 1, Alexis) or monoclonal antibody to CD40 626.1 (5 mg ml 1, a gift from S.-M. Fu, University of Virginia) plus IL-4 (5 ng ml 1, R&D). We assayed supernatants at day 14 for IgA and IgG by ELISA. We used goat antibodies to human IgG and IgA (Southern Biotech.) for capture and the same antibodies conjugated to alkaline phosphatase as developing Abs. Note: Supplementary information is available on the Nature Genetics website. ACKNOWLEDGMENTS We thank the affected individuals and their families for participation in the study and H. Jabara, J. Manis, H. Oettgen, N. Ramesh and L. Kunkel for discussions. This work was supported by grants from the US Public Health Service, the Jeffrey Modell Foundation, the New England Primary Immunodeficiency Network, the March of Dimes and the Wallace Fund. COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests. Received 24 March; accepted 10 June 2005 Published online at http://www.nature.com/naturegenetics/
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