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Functional analysis of transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) mutations associated with common variable immunodeficiency

2011; Elsevier BV; Volume: 128; Issue: 1 Linguagem: Inglês

10.1016/j.jaci.2011.01.048

1097-6825

Ari J. Fried, Ingrid Rauter, Stacey R. Dillon, Haifa H. Jabara, Raif S. Geha,

Neurogenetic and Muscular Disorders Research

To the Editor: Mutations in TNFRSF13B, the gene encoding the transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI), have been identified in 5% to 10% of patients with common variable immunodeficiency (CVID).1Castigli E. Wilson S.A. Garibyan L. Rachid R. Bonilla F. Schneider L. et al.TACI is mutant in common variable immunodeficiency and IgA deficiency.Nat Genet. 2005; 37: 829-834Crossref PubMed Scopus (559) Google Scholar, 2Salzer U. Chapel H.M. Webster A.D. Pan-Hammarstrom Q. Schmitt-Graeff A. Schlesier M. et al.Mutations in TNFRSF13B encoding TACI are associated with common variable immunodeficiency in humans.Nat Genet. 2005; 37: 820-828Crossref PubMed Scopus (528) Google Scholar The most common TACI variants in patients with CVID are C104R in the ligand-binding cysteine-rich domain 2 (CRD2) and A181E in the transmembrane domain of TACI. Each is found in the heterozygous state in 2% to 5% of patients with CVID but also in 0.5% to 1% of healthy subjects.1Castigli E. Wilson S.A. Garibyan L. Rachid R. Bonilla F. Schneider L. et al.TACI is mutant in common variable immunodeficiency and IgA deficiency.Nat Genet. 2005; 37: 829-834Crossref PubMed Scopus (559) Google Scholar, 2Salzer U. Chapel H.M. Webster A.D. Pan-Hammarstrom Q. Schmitt-Graeff A. Schlesier M. et al.Mutations in TNFRSF13B encoding TACI are associated with common variable immunodeficiency in humans.Nat Genet. 2005; 37: 820-828Crossref PubMed Scopus (528) Google Scholar, 3Salzer U. Bacchelli C. Buckridge S. Pan-Hammarstrom Q. Jennings S. Lougaris V. et al.Relevance of biallelic versus monoallelic TNFRSF13B mutations in distinguishing disease-causing from risk-increasing TNFRSF13B variants in antibody deficiency syndromes.Blood. 2009; 113: 1967-1976Crossref PubMed Scopus (191) Google Scholar Both destroy the capacity of TACI ligation to activate nuclear factor κB (NF-κB). We have examined the function of TACI coding variants that have been described in patients with CVID and their relatives but thus far not in healthy subjects.3Salzer U. Bacchelli C. Buckridge S. Pan-Hammarstrom Q. Jennings S. Lougaris V. et al.Relevance of biallelic versus monoallelic TNFRSF13B mutations in distinguishing disease-causing from risk-increasing TNFRSF13B variants in antibody deficiency syndromes.Blood. 2009; 113: 1967-1976Crossref PubMed Scopus (191) Google Scholar, 4Pan-Hammarstrom Q. Salzer U. Du L. Bjorkander J. Cunningham-Rundles C. Nelson D.L. et al.Reexamining the role of TACI coding variants in common variable immunodeficiency and selective IgA deficiency.Nat Genet. 2007; 39: 429-430Crossref PubMed Scopus (179) Google Scholar, 5Castigli E. Wilson S. Garibyan L. Rachid R. Bonilla F. Schneider L. et al.Reexamining the role of TACI coding variants in common variable immunodeficiency and selective IgA deficiency.Nat Genet. 2007; 39: 430-431Crossref PubMed Scopus (74) Google Scholar Fig 1, A, shows the location of the missense mutations examined in the TACI protein. The cysteine-rich domain 1 (CRD1) mutants W40R and D41H, the CRD2 mutant I87N, the transmembrane domain mutants C172Yand A181E, and the intracellular domain mutants K188M and V246F were expressed on the surface of 293T cell transfectants at levels comparable with WT TACI (Fig 1, B). Surface expression of the CRD2 mutants Y79C and C104R was modestly decreased to approximately half of the wild-type CRD2, whereas that of the L171R mutant was reduced approximately 8-fold. Protein expression of the L171R mutant in whole-cell lysates was similarly reduced, but its mRNA expression was comparable with that of the wild type (data not shown), suggesting that the mutant protein was unstable. Both of the CRD1 domain mutants, W40R and D41H, showed normal binding of B cell–activating factor of the TNF family (BAFF; Fig 1, C). There was total loss of BAFF binding by the Y79C and C104R mutants. The I87N mutant had residual BAFF binding, which increased with increasing concentrations of ligand (see Fig E1, A, in this article’s Online Repository at www.jacionline.org). The poorly expressed L171R mutant had negligible ligand binding. The transmembrane domain mutants C172Y and A181E and the intracellular domain mutants K188M and V246F had intact BAFF binding. TACI signaling was examined by measuring NF-κB and nuclear factor of activated T cells (NFAT) activation with a dual luciferase reporter system.6Garibyan L. Lobito A.A. Siegel R.M. Call M.E. Wucherpfennig K.W. Geha R.S. Dominant-negative effect of the heterozygous C104R TACI mutation in common variable immunodeficiency (CVID).J Clin Invest. 2007; 117: 1550-1557Crossref PubMed Scopus (74) Google Scholar Mutant or WT TACI was transfected into 293T cells with either NF-κB luciferase or NFAT luciferase reporter plasmids together with Renilla luciferase, and the capacity of a proliferation-inducing ligand (APRIL; 50 ng/mL) to induce reporter gene activity was determined. The CRD1 domain mutants (W40R and D41H) activated NF-κB and NFAT at levels comparable with those seen with WT TACI (Fig 2, A and B). The Y79C and C104R mutants failed to activate either NF-κB or NFAT, which is consistent with their failure to bind BAFF. The I87N mutant demonstrated residual reporter gene activation. Increasing the concentration of APRIL resulted in increased NF-κB activation in I87N transfected cells (see Fig E1, B), which is consistent with the decreased ligand-binding affinity of this mutant. Ligand-induced activation of NF-κB and NFAT was abrogated in the transmembrane domain mutant L171R, likely a consequence of its markedly diminished surface expression. Despite normal ability to bind ligand, the transmembrane mutants C172Y and A181E failed to signal. Signaling by the intracellular domain mutants (K188M and V246F) was intact. Because TACI assembles as a trimer or higher-order oligomer,6Garibyan L. Lobito A.A. Siegel R.M. Call M.E. Wucherpfennig K.W. Geha R.S. Dominant-negative effect of the heterozygous C104R TACI mutation in common variable immunodeficiency (CVID).J Clin Invest. 2007; 117: 1550-1557Crossref PubMed Scopus (74) Google Scholar TACI mutants with disrupted function might potentially exert a dominant-negative (DN) effect on signaling by WT TACI in heterozygotes. 293T cells were cotransfected with equal amounts of WT TACI and either empty vector or TACI mutant cDNA, and ligand-driven NF-κB activity was measured. Results were calculated as the percentage of the net increase in NF-κB activity in cells transfected with WT TACI and empty vector. None of the mutants analyzed exhibited a significant inhibition of NF-κB activation by WT TACI (Fig 2, C). The absence of a DN effect by the C104R mutant is consistent with our recent demonstration that introduction of a murine TACI transgene, which carries the C76R TACI mutation equivalent to the human C104R mutation, into the TACI+/− background does not exert a DN effect on TACI function.7Lee J.J. Jabara H.H. Garibyan L. Rauter I. Sannikova T. Dillon S.R. et al.The C104R mutant impairs the function of transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) through haploinsufficiency.J Allergy Clin Immunol. 2010; 126 (1234-41 e2)Abstract Full Text Full Text PDF Scopus (37) Google Scholar The data obtained on the mutants included in our study are summarized in Table I. The mutations that impaired TACI function did not exert a DN effect but could cause B-cell dysfunction in heterozygotes because of haploinsufficiency. This is supported by the observation that patients with CVID who are heterozygous for C104R have poor in vitro responses to APRIL.1Castigli E. Wilson S.A. Garibyan L. Rachid R. Bonilla F. Schneider L. et al.TACI is mutant in common variable immunodeficiency and IgA deficiency.Nat Genet. 2005; 37: 829-834Crossref PubMed Scopus (559) Google Scholar, 2Salzer U. Chapel H.M. Webster A.D. Pan-Hammarstrom Q. Schmitt-Graeff A. Schlesier M. et al.Mutations in TNFRSF13B encoding TACI are associated with common variable immunodeficiency in humans.Nat Genet. 2005; 37: 820-828Crossref PubMed Scopus (528) Google Scholar It remains to be addressed whether healthy subjects with this and other TACI mutations that affect TACI function have TACI haploinsufficiency that remains clinically silent. Deficiency in the TACI homologue BAFF receptor has been reported to result in poor antibody responses to pneumococcal cell-wall polysaccharides in 2 siblings who carry the same BAFFR mutation but caused CVID in only one of the siblings.8Warnatz K. Salzer U. Rizzi M. Fischer B. Gutenberger S. Bohm J. et al.B-cell activating factor receptor deficiency is associated with an adult-onset antibody deficiency syndrome in humans.Proc Natl Acad Sci U S A. 2009; 106: 13945-13950Crossref PubMed Scopus (280) Google Scholar Potential synergy of TACI haploinsufficiency with other defects, such as defective B-cell responsiveness to Toll-like receptor 9,9Yu J.E. Knight A.K. Radigan L. Marron T.U. Zhang L. Sanchez-Ramon S. et al.Toll-like receptor 7 and 9 defects in common variable immunodeficiency.J Allergy Clin Immunol. 2009; 124 (349-56, e1-3)Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 10Cunningham-Rundles C. Radigan L. Knight A.K. Zhang L. Bauer L. Nakazawa A. TLR9 activation is defective in common variable immune deficiency.J Immunol. 2006; 176: 1978-1987PubMed Google Scholar which is important for memory B-cell formation, might determine its penetrance and needs to be investigated in patients with CVID with TACI mutations.Table ISummary of the effect of TACI mutations on TACI expression and functionMutationDomainExpressionLigand bindingNF-κB/NFATsignalingDN effectW40RCRD1NormalNormalNormalNAD41HCRD1NormalNormalNormalNAY79CCRD2Modestly decreasedAbsentAbsentNoI87NCRD2NormalImpairedImpairedNoC104RCRD2Modestly decreasedAbsentAbsentNoL171RTMStrongly decreasedAbsentAbsentNoC172YTMNormalNormalAbsentNoA181ETMNormalNormalAbsentNoK188MICNormalNormalNormalNAV246FICNormalNormalNormalNAIC, Intracellular; NA, Not applicable (these mutations do not affect TACI function); TM, transmembrane. Open table in a new tab IC, Intracellular; NA, Not applicable (these mutations do not affect TACI function); TM, transmembrane. The absence of a DN effect of deleterious TACI mutants on signaling by WT TACI is unlikely the result of their failure to assemble with WT TACI because the C104R and A181E mutants assemble normally with WT TACI (data not shown).6Garibyan L. Lobito A.A. Siegel R.M. Call M.E. Wucherpfennig K.W. Geha R.S. Dominant-negative effect of the heterozygous C104R TACI mutation in common variable immunodeficiency (CVID).J Clin Invest. 2007; 117: 1550-1557Crossref PubMed Scopus (74) Google Scholar We previously suggested that the C104R mutant might exert a DN effect based on calculations of fold induction of NF-κB activity in cells cotransfected with WT and mutant TACI compared with cells cotransfected with WT TACI and empty vector.6Garibyan L. Lobito A.A. Siegel R.M. Call M.E. Wucherpfennig K.W. Geha R.S. Dominant-negative effect of the heterozygous C104R TACI mutation in common variable immunodeficiency (CVID).J Clin Invest. 2007; 117: 1550-1557Crossref PubMed Scopus (74) Google Scholar However, re-evaluation of the data indicates that the absolute increase in NF-κB activity was comparable in both cotransfectants but that background NF-κB activity in the cells cotransfected with WT and mutant TACI was approximately double that of cells transfected with WT TACI and empty vector. This increased background was responsible for the observed reduction in fold induction and is likely due to the fact that the mutant preassembles and constitutively signals comparably with WT TACI.6Garibyan L. Lobito A.A. Siegel R.M. Call M.E. Wucherpfennig K.W. Geha R.S. Dominant-negative effect of the heterozygous C104R TACI mutation in common variable immunodeficiency (CVID).J Clin Invest. 2007; 117: 1550-1557Crossref PubMed Scopus (74) Google Scholar The use of transfectants to examine the expression and function of mutant proteins has limitations because the unphysiologic levels of expression of the mutant protein in this system might mask subtle alterations of protein expression and because it does not reveal the effect of the mutants on B-cell function in a physiologic setting. This is best achieved by examining B-cell function in patients, their healthy relatives, and unrelated subjects who carry the same mutations and by the study of transgenic mice that carry the mutant allele on a homogeneous background.