Hindawi Publishing CorporationISRN ImmunologyVolume 2013 Article ID 365916 10 pageshttpdxdoiorg1011552013365916
Research ArticleSynergy in B-Cell Activation between Toll-Like Receptor 9 andTransmembrane Activator and Calcium-Modulating CyclophilinLigand Interactor (TACI) in A181EC104R CompoundHeterozygous Siblings
Annick A J M van de Ven1 Willemijn J M Janssen1 Lisette van de Corput2
Andries C Bloem2 Joris M van Montfrans1 and Marianne Boes1
1 Department of Pediatric Immunology and Infectious Diseases Wilhelmina Childrenrsquos Hospital University Medical Center UtrechtLundlaan 6 PO Box 85090 KC010690 3584 EA Utrecht The Netherlands
2Department of Medical Immunology University Medical Center Utrecht Heidelberglaan 100 F03821 PO Box 855003508 GA Utrecht The Netherlands
Correspondence should be addressed to Marianne Boes mboesumcutrechtnl
Received 21 April 2013 Accepted 26 May 2013
Academic Editors S-i Fujii F Granucci W W Leitner and S Sanchez-Ramon
Copyright copy 2013 Annick A J M van de Ven et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited
Purpose Approximately 9 of common variable immunodeficiency (CVID) patients harbor variants in the transmembraneactivator and CAML interactor gene TACI which contribute to CVID development We found identical compound heterozygousTACI variants (C104R and A181E) in kindred of which one sibling had severe CVID with refractory auto immunity and a secondsibling remained asymptomatic This study investigated possible differences in B-cell phenotype and function that could explainthis divergent clinical expressionMethodsC104R andA181E TACI variants were identified through Sanger sequencing Phenotypicevaluation of the lymphocyte compartment was performed by flow cytometry analyses Lymphoblastoid cell lines (LCL) from theindex patient asymptomatic sibling and controls were generated Intracellular TACI expression was determined and activation-associated calcium flux capacity was measured In vitro stimulation assays and RT PCR were performed Results Both intracellularlevels and surface expressed TACI protein were higher in the asymptomatic sibling than the CVID patient as were TACI-triggering-induced mRNA expression AID and production of Ig class-switched antibodies In analogy the asymptomatic siblingdisplayed enhanced Toll-like receptor 9 expression and signaling suggesting a compensatory immune mechanism ConclusionsPosttranscriptional regulation of TACI protein and cross-talk with TLR9 signaling may contribute to phenotypic diversity betweenindividuals with TACI variants
1 Introduction
Common variable immunodeficiency (CVID) is a pri-mary immunodeficiency characterized by recurrent bacterialinfections hypogammaglobulinemia and impaired antigen-specific antibody synthesis [1] In approximately 10 ofpatients a genetic defect has been identified In additionfunctional defects have been described for example inToll-like receptor (TLR) signaling [2 3] The most preva-lent genetic alterations are located in the TNFRSF13B gene
encoding transmembrane activator calcium modulator andcyclophilin ligand (CAML) interactor (TACI) [4] TACIis mainly expressed on B-cells and belongs to the tumornecrosis factor receptor (TNFR) family [4]
Two ligands for TACI have been described B-cell acti-vating factor (BAFF) [5] and a proliferation-inducing ligand(APRIL) [6 7] In addition TACI may bind proteoglycansincluding syndecan-2 that stimulate TACI-mediated signal-ing [8 9] BAFF and APRIL also bind B-cell maturation anti-gen (BCMA) [10] and BAFF has a third receptor BAFFR [11]
2 ISRN Immunology
This versatility complicates studying the precise contributionof each molecule to humoral immunity [9]
Following ligand binding the intracellular TACI domainsinteract with TNFR-associated proteins (TRAFs) andCAMLCAML interaction activates nuclear factor of activated Tcells (NFAT) which has a key role in controlling calciumrelease from intracellular stores [4] Additionally nuclearfactor kappa B (NF120581B) is activated [12] probably via TRAFsc-Jun NH2-terminal kinase and transcription factor AP-1[4] TACI has a role in maintenance of B-cell homeostasisimmunoglobulin (Ig) isotype class-switching and antibodyproduction in response to type II T-cell-independent anti-gens Furthermore it may act in synergy with CD40 andTLRs [13ndash15] It was recently shown that TACI induces class-switch recombination via themyeloid differentiation primaryresponse gene 88 (MyD88) adaptor thus employing the samesignaling cascade as multiple TLRs [16]
Currently TACI is considered a CVID susceptibility gene(relative risk 43 95 confidence interval 24ndash76) of whichheterozygous disease-associated variants are present in sim9of CVID patients compared to only monoallelic variantsin lt2 of the healthy controls [17] In healthy or mildlyaffected family members of CVID patients some biallelicpolymorphisms were found suggesting that TACI variantsmay have an incomplete penetrance [17] Furthermore thepresence of TACI polymorphisms in CVID predisposes toautoimmune disease and lymphoid hyperplasia [18]
TwoTACI variants are significantly associatedwithCVIDdisease C104R [17 19] and A181E [19] A characteristicof TNFRs is the presence of two cysteine-rich domain(CRDs) in the extracellular domain which are conservedbetween species [20] TACI monomers engage in a ligand-independent homotypic trimer formation that requires theCRD1 domain [21] The second CRD allows ligation withBAFF or APRIL [22] The amino acid substituting C104Rvariant is located in CRD2 and thus abrogates binding ofAPRIL and BAFF [23 24] as was demonstrated in CVIDpatients heterozygous for C104R Initially the deficiency wasattributed to a dominant-negative interference of the C104Rpolymorphism in trimeric complexes with TACI wildtype[21] Subsequent reports however suggest haploinsufficiencyof the TACI allele [25ndash27]
TheA181E variant is located at the transmembrane regionand permits normal ligand binding [23] Mouse modelswith an A181E equivalent (A144E) show defective preligandand ligand-induced clustering of the intracellular domainand subsequent hampered nuclear factor kappa B (NF120581B)activation possibly this is caused by a conformational changeresulting from the negative charge introduced by the A181Evariant [28] A further consequence was low serum IgAlevels and impaired antibody responses to type II T-cell-independent antigens
To more closely resemble human conditions exhibitingheterozygous alleles A181E and C104R were investigatedin combination with a wildtype TACI allele Neither vari-ant interfered with oligomerization with wildtype TACImolecules nor NF120581B activation was unaffected in 293T cellswith wildtype and mutant TACI [26] Dominant-negative
interference of the variants with wildtype TACI is there-fore improbable but haploinsufficiency was not completelyexcluded It was therefore recommended to study B-cellfunction in patients their healthy relatives and unrelatedsubjects who carry the same mutation [26] Our study hereaddressed this suggestion
We identified a patient compoundheterozygous forA181Eand C104R Her disease course was complicated by cytope-nias interstitial lung disease autoimmune nephritis andeventually mortality [29] In contrast her sibling carried anidentical TACI genotype but was completely healthy whichwas not reported previously Here we describe a clinical andimmunological evaluation of the patient compared to thehealthy sibling and unrelated healthy and CVID controlsWe provide mechanistic support of how individuals withidentical TACI alleles may exhibit differential TACI proteinexpression and downstream signaling function
2 Materials and Methods
21 Patients As part of routine evaluation of CVID patientsin our institute C104R and A181E TACI variants werescreened by polymerase chain reaction (PCR) as describedpreviously [29] and both were found in the index patient Aswe considered stem cell transplantation in the index patientparents and siblings were screened (Figure 2(a)) Informedconsent was obtained to further evaluate the patient andfamily members for clinical indications Further data wereobtained from medical records
22 DNA Sequencing TACI mutations were investigated asdescribed previously [29] Briefly primers were designedfor the pC104R and pA181E variants and their wildtypeswith Primer Express software (Applied Biosystems) To testfor additional mutations or splicing variants [22] primerswere designed for the complete TACI sequence contain-ing 5 exons The following primers were used reverse51015840-TGTAAAACGACGGCCAGTCAGCCCAAGCACTAA-TCAAATC-31015840 and forward 51015840-CAGGAAACAGCTATG-ACCCTCTCTCCCCTCCTCTCCATC-31015840 For splicing vari-ants cDNA was sequenced using a 3730 DNAAnalyzer Priorto sequencing the TACI domain of the cDNA was amplifiedby a PCR starting at 95∘C for 10min followed by 35 cycles of58∘C for 15 s and 60∘C for 1min
23 Flow Cytometry Phenotypic and functional evaluationof the lymphocyte compartment was performed as describedpreviously [29] Next lymphoblastoid cell lines (LCL) weregenerated using Epstein-Barr virus and stained with anti-bodies against CD19 CD20 CD21 (all Becton Dickinson)TLR9 CD40 (both eBioscience) rabbit polyclonal anti-TACI(Abcam) with secondary anti-rabbit Ig-DiLight649 (JacksonImmunoresearch West Grove PA) and the appropriateisotypes Intracellular TACI expression was determined aftertreatment with permeabilization reagents
For calcium assays fresh peripheral blood mononuclearcells (PBMC) were incubated with Fluo-3 Fura Red (bothMolecular Probes) (30min) and CD19 (10min) at 37∘C Cells
ISRN Immunology 3
were washed twice and resuspended in Hankrsquos balanced saltsolution (HBSS) supplemented with 10 fetal calf serum(FCS) Cytosolic calcium levels were measured on FacsCanto II after 1 minute 20120583gmL goat anti-human IgMF(ab1015840)
2fragments (Jackson Immunoresearch) were added
After another 5 minutes 20120583gmL ionomycin (CalbiochemSan Diego CA) was added Area under curves was calculatedusing Facs Diva and Prism software Next LCL were stainedand resuspended in calcium-free phosphate-buffered saline(PBS) Calcium chloridewas added after intracellular calciumstorage depletion with ionomycin or thapsigargin (SantaCruz Biotechnology Santa Cruz CA)
24 In Vitro Stimulation Assays and PCR LCL were culturedin RPMI 1640 containing 10 FCS at the subsequentconditions mouse anti-TACI (05 120583gmL RampD systemsMinneapolis MN USA) and goat anti-mouse IgGmicrobeads (Miltenyi Biotec Carlsbad CA) with or withoutIL-4 (100UmL Immunotools) CpG phosphorothioate-modified oligodeoxynucleotide 2006 (05 120583gmL AlexisBiochemicals) IL-10 (10 ngmL Becton Dickinson) oranti-CD40 (01120583gmL RampD systems) After 48 hours cellswere lysed and total mRNA was isolated using Tripureisolation reagent (Roche Diagnostics Mannheim Germany)according to the manufacturerrsquos instructions Reversetranscriptionwas performed using an iScript cDNA synthesiskit (BioradHercules CA) Primersweremixedwith IQSYBRgreen supermix (BioRad) The detection run started at 95∘Cfor 10min followed by 45 cycles of 95∘C for 15 s and 60∘Cfor 1min Assays were performed in duplicate or triplicateas 15 120583L reactions in 96-well plates using C1000 ThermalCycler (BioRad) Results were normalized to the endogenousGAPDH mRNA (2minusΔCT) The following primers wereused GAPDH forward 51015840-GTCGGAGTCAACGGATT-31015840GAPDH reverse 51015840-AAGCTTCCCGTTCTCAG-31015840 AICDAforward 51015840-TGCTCTTCCTCCGCTACATCTC-31015840 AICDAreverse 51015840-AACCTCATACAGGGGCAAAACC-31015840 NF120581Bforward 51015840-GAAGCACGAATGACAGAGGC-31015840 NF120581B rev-erse 51015840-GCTTGGCGGATTAGCTCTTT-31015840 NFAT forward51015840-GCAGAGCACGGACAGCTATC-31015840 NFAT reverse 51015840-GGGCTTTCTCCACGAAAATGA-31015840 (All Sigma-Aldrich)
3 Results
31 Clinical Evaluation of the CVID Index Patient Theindex patient (Figure 2(a) arrow) was referred at age 11for recurrent upper and lower respiratory tract infectionsand splenomegaly Laboratory investigations showed absentisohemagglutinins and decreased serum titers of total IgGIgA IgM IgG
1 and IgG
2(Table 1) She was lymphopenic
with low numbers of B cells CD4+ and CD8+ T cells andalmost absent class switched memory B cells (Figure 1(a))Functionally in vitro T-cell proliferation assays showed apositive response to mitogens and an incomplete response toparticularly viral antigens A diagnosis of CVID was madeand immunoglobulin replacement therapy was initiated
After some time she developed CVID-related diseasemanifestations including autoimmune hemolytic anemia
(AIHA) idiopathic thrombocytopenic purpura (ITP) sub-mandibular lymphadenopathy and autoimmune nephritisa renal biopsy showed tubulo interstitial nephritis withgranulomatous changes the estimated glomerular filtratedrate was 412mLmin173m2 (as calculated by Schwartzformula) Furthermore she developed exercise-induced dys-pnea a high-resolution computed tomography (HRCT) scanshowed airway and interstitial lung disease with bronchiec-tasis and peribronchovascular abnormalities consisting offocal lesions with ground glass aspect peribronchial con-solidations bronchial wall thickening and air trapping(Figure 2(b)) A broncho alveolar lavage showed no indi-cation for viral bacterial or fungal infections Pulmonaryfindings were suggestive for granulomatous disease and low-dose steroids and tacrolimus were initiated resulting in nor-malization of pulmonary function and glomerular filtrationrate Furthermore viral immunity appeared to be affectedas cytomegalovirus was repeatedly detectable with viral loadsof 100ndash2000 copiesmL in plasma Treatment with tacrolimuswas complicated by tubulopathy and therefore mycopheno-late mofetil was started with good initial response howevercausing pancreatitis Rituximab was administered with pos-itive effect on lymphadenopathy AIHA and ITP Despiteall treatment efforts taken disease manifestations progressedfurther and became refractory The index patient underwentallogeneic stem cell transplantation with matched unrelatedcord blood after myeloablative conditioning but eventuallydied of the complications of graft versus hose disease com-bined with multiple infections
32 Clinical and Immunological Evaluation of Family Mem-bers Further studies were performed to determine the roleof these TACI variants Clinically the parents and sibling2 were healthy sibling 1 developed clinical symptoms ofCVID during the course of investigations Serum Ig levelswere normal in both parents and sibling 2 apart from amodestly decreased serum IgA In contrast sibling 1 washypogammaglobulinemic for IgG IgA and subclasses IgG
1
IgG2 and IgG
4 albeit modestly in comparison to the index
patient (Table 1)To study specific antibody synthesis parents and sibling 1
and 2 were vaccinated with 23-valent Pneumovax (AdventisPasteur MSD) and total IgG antibody titers were measuredafter 1 month Parents and sibling 2 had adequate T cell-independent responses in vivo to pneumococcal polysaccha-rides while the response of sibling 1 was suboptimal (Table 1)
Consequently immunological evaluation was extendedin siblings 1 revealing an increase in relative and absolutenumbers of CD8+ T cells Total B cells were increased(217 of lymphocytes 512 cells120583L) and the compositionof B-cell compartment remained normal Proliferative T-cellresponses in vitro were normal to mitogens but partiallyimpaired to recall antigens similar to the index patientTotal B-cell count and composition of B-cell compartment ofsibling 2 were normal (Figure 1(b))
Sibling 1 slowly developed clinical symptoms consistingof diarrhea fatigue and persistent rhinosinusitis refractory toantibiotic prophylaxisThe spleenwas enlarged on abdominal
4 ISRN Immunology
IgD
13395
01
941 11
01
IgM CD27
IgD
IgD
CD38
CD19+ lymphocytesIndex patient
(a)
94
9
756793
104
IgM CD27
65
CD38
Healthy sibling
IgD
IgD
IgD
(b)
Figure 1 B-cell subpopulations of index patient and healthy sibling (a) B cell subpopulations of index patient showing 941 naıve +transitional B cells (IgM+IgD+CD27minus) 11 Marginal Zone B cells (IgM+IgD+CD27+) 01 class-switched B cells (IgDminusIgMminusCD27+)and 133 transitional B cells (IgD+CD38+) (b) B cell subpopulations of healthy sibling showing 756 naıve + transitional B cells(IgM+IgD+CD27minus) 94Marginal Zone B cells (IgM+IgD+CD27+) 9 class-switched B cells (IgDminusIgMminusCD27+) and 65 transitional Bcells (IgD+CD38+)
echography Based on these clinical and laboratory findingsimmunoglobulin replacement therapy was initiated whichled to significant clinical improvement Further studies werefocused on the index patient and asymptomatic sibling 2
33 TNFRSF13B Compound Heterozygous Variants in theIndex Patient and Asymptomatic Sibling TACI is a CVIDsusceptibility factor and to substantiate the diagnosis CVIDwe performed gene sequencing of the TACI gene Analysisof the patientrsquos TNFRSF13B gene showed two variants C104Rand A181E Subsequently her family was investigated reveal-ing that each parent carried one variant (Figure 2(a)) Thebrother (sibling 3) had two normal TACI alleles and was notfurther investigated The two sisters (siblings 1 and 2) werealso compound heterozygous for A181EC104R (Figure 2(a))Sequencing of the complete TNFRSF13B gene revealed noadditional mutations [22] The index patient and sibling 2exhibited identical alternative splicing patterns of exon 2 thatencode the CRD1 important in trimer formation of TACI[22] This finding however did not help explain the different
clinical and B-cell activation phenotype between our indexpatient and sibling 2
34 TACI Protein Expression and Function in the IndexPatient and Asymptomatic Sibling Lymphoblastoid cell lines(LCL) were generated of the index patient and sibling 2 andcompared to cell lines of healthy controls CVID patients and2 CVID patients with a monoallelic A181E variant LCL of allsubjects exhibited TACI expression on the cell membraneInterestingly the TACI mean fluorescence intensity of theindex patient was lower in comparison with the othersubjects especially with sibling 2 fromwhomLCL repeatedlydisplayed the highest cell surface expression (Figure 2(c))Subsequently intracellular TACI was measured to study thepossibility of impaired transition of TACI to the cell surfaceand ensuing intracellular TACI accumulation IntracellularTACI however correlated with the levels of cell surface TACI(Pearson correlation 070 119875 = 0037) and was also repeatedlylower in the index patient (Figure 2(c)) Basal TACI mRNAlevels varied within a similar range for all LCL including theindex patient and sibling (Figure 2(d))
Figure 2 TACI expression and function are decreased in the CVID patient compared to her asymptomatic sibling (a) Pedigree of the studiedfamily Circles display females squares display males The index patient is indicated with an arrow (b) Chest high-resolution CT scan of theindex patient revealing peribronchial consolidations with ground glass appearance and peribronchial thickening ((c)-(d)) TACI surface (cii) or intracellular (c iii) protein and mRNA expression Light grey line indicated isotype indicated dark grey TACI (c i) Results of 5 healthydonors 3 CVID patients with normal TACI 2 CVID patients withmonoallelic A181E the index patient and sibling 2 For (d) average results of3 independent experiments are shown (e) AID (left) and NFAT (right) mRNA induction after 48 h culture of LCL with the indicated stimuliBars represent means plusmn standard error of the mean
Next we investigated TACI function by measuring itsability to induce activation-induced cytidine deaminase(AICDA) gene transcription The AICDA gene productactivation-induced deaminase (AID) is an enzyme of piv-otal importance for secondary antibody diversification [30]Healthy control and CVID patient LCL with normal bial-lelic TACI showed AID mRNA induction upon stimulation
with agonistic anti-TACI (Figure 2(e)) CVID patients withmonoallelic A181E variants showed minimal AICDA genetranscription The index patient had no AICDA gene tran-scription upon TACI stimulation while AID mRNA levelsof sibling 2 LCL were comparable to B cells with normalTACIThe same pattern was noticed for NFATmRNA induc-tion gene transcription upon TACI triggering was lower in
6 ISRN Immunology
Table 1 Immunological characteristics of patient and family
Patient Sibling 1 Sibling 2 Mother FatherGender C C C C D
the index patient compared to sibling 2 There were nodifferences in NF120581B induction upon TACI stimulation Stim-ulation of the CD40 pathway showed adequate induction ofAID and NFAT mRNA in the index patient IL-10 treatmentwas included as negative control that does not induce AICDAgene expression Taken together TACI protein expressionand function were increased in sibling 2 when compared tothe index patient
35 Appearance of TLR9 Protein Expression and Function inthe Asymptomatic Sibling We considered two possibilitiesthat may explain why the index patient and sibling 2 havingidentical TACI gene mutations exhibit divergent healthconsequences either the index patient had an additionalimmune defect which in combinationwith the TACI variantswould lead to CVID disease or sibling 2 has compensatoryimmune function in the TACI signaling route that preventsCVID disease from occurring First as defects in the B-cell receptor (BCR) and its coreceptor complex have beendescribed [31 32] we screened the BCR signalosome CD19CD21 and CD20 cell surface expression of the index patientwere comparable to expression on control LCL (data notshown) Functionally early B-cell activation seemed intactas measured by calcium mobilization after BCR stimulationof fresh PBMC (Figure 3(a)) The index patient showed
a normal initial but rapidly declining calcium influx aspreviously observed in CVID patients with autoimmunesymptoms (own unpublished observations) and area undercurves were comparable to controls As the index patientrsquosendoplasmic reticulum (ER) calcium storage capacitywas notdecreased compared to sibling 2 an additional defect in BCRsignaling was considered improbable
Second the classical route of BCR isotype class switchingis via CD40 with CD40L on T cells and it was recentlyshown that TACI and CD40 signaling can act in synergy[13 15] However CD40 cell surface expression (Figure 3(b))and CD40-mediated gene transcription (Figures 2(e) and3(b)) were comparable between all LCL suggesting that CD40signaling in the index patient B cells was intact at least in thisassay
Third we explored TLR9 signaling as TLR9 defects havebeen described in CVID [2 3] and TLR9 and TACI signalingemploy common downstream signaling pathways [15 16]Noticeably TLR9 expression was nearly twofold higher insibling 2 compared to other LCL and this increased expres-sion was accompanied by an increased TLR9 expression andfunction (Figure 3(c)) supporting the possibility that com-pensatory TLR9 expression contributes as a mechanism foraltered TACI A181EC104R protein in restoring B-cell overallcapacity to transmit signals towards NFAT and AID Thus
ISRN Immunology 7
CVID patientSibling 1Sibling 2
MotherControl
Anti-IgM Ionomycin
25
20
15
10
5
3
2
1
10000
200 300 400Time (s)
BCR-mediated calcium flux (PBMC)Cy
toso
lic C
a2+
leve
l
(a)
0
5000
10000
15000
Mea
n flu
ores
cenc
e int
ensit
y
Cell surface CD40
Con
trol
(TAC
I nor
mal
)
CVID
(TAC
I nor
mal
)
CVID
(A18
1En
orm
al)
Patie
nt(A
181E
C10
4R)
Sibl
ing
(A18
1EC
104R
)
(b)
IL-10 CpG ODN Anti-CD40
AID mRNA
0AID
mRN
A (r
elat
ive t
o G
APD
H) 20
15
10
5
TLR9
Mea
n flu
ores
cenc
e int
ensit
y
15
10
5
00
Intracellular TLR9times10
2
Con
trol
(TAC
I nor
mal
)CV
ID(T
ACI n
orm
al)
CVID
(A18
1En
orm
al)
Patie
nt(A
181E
C10
4R)
Sibl
ing
(A18
1EC
104R
)
Control (TACI normal)CVID (TACI normal)CVID (A181Enormal)
Patient (A181EC104R)Sibling (A181EC104R)
(c)
Figure 3 Similar BCR signaling but possibly enhanced TLR9 signaling in the healthy sibling (a) Calcium mobilization upon triggering ofthe BCR using IgM F(ab1015840)
2fragments in freshly isolated PBMCs (b) CD40 surface expression asmeasured by flow cytometry (c) TLR9 B-cell
expression (c ii) and function (c iii) Light grey line indicated isotype indicated dark grey TLR9 (c i) Bars represent means plusmn standard errorof the mean
our data supports the notion that compensatory signaling inthe TACI route in the case of sibling 2 via enhanced TLR9signaling may prevent the development of CVID disease
4 Discussion
We here describe differential immunological phenotypesdisplayed by siblings with identical genetic TACI variantsThis family provides the unique opportunity to study the role
and significance of the humanC104R andA181E variants bothin vivo and in vitro
The index patient had severe CVID which graduallyevolved to late onset combined immunodeficiency a phe-nomenon previously described in CVID patients [33] Sibling1 did eventually develop mild CVID but without autoim-mune manifestations To date sibling 2 remains free ofsymptoms and has excellent specific antibody responsesnevertheless this sibling may still develop CVID at a laterage Previous studies have shown that C104R abrogates ligand
8 ISRN Immunology
binding due to haploinsufficiency whereas in the A181Evariant ligand binding remains intact [21 26] At least aproportion of A181EC104R TACI trimers will thus be ableto bind ligand As NF120581B activation was strongly decreasedin both variants we hypothesized TACI function to beaffected AID and NFAT mRNA induction were reduced inthe index patient and seemed also reduced in the CVIDpatients with a heterozygous A181E variant [14] Converselysibling 2 showed AID and NFAT induction comparable tohealthy controls and CVID patients with unaffected TACIalleles NFAT activation induces transcription of cytokinegenes encoding for example IL-4 which is involved in classswitch recombination IL-4 and CD40 ligation induces AIDmRNA and protein expression and depends on STAT6 andNF120581B [34] AID is pivotal for class switch recombination andsomatic hypermutation of theBCR [35 36] bothmechanismsare routinely impaired in CVID
The differential TACI function in the index patient andsibling was accompanied by a change in TACI protein levelsshowing increased TACI expression in the sibling both atthe cell surface and intracellular level TACI protein levels inthe healthy sibling were increased in comparison to healthycontrols as well As TNFRSF13B gene transcription wascomparable between all subjects except for the healthy sib-ling these observations suggest that protein turnover in thehealthy sibling may be decreased possibly due to alterationsin posttranslational modification of TACI protein affectingprotein stability and folding transport or degradation
Alternatively altered TACI function in the healthy siblingwas compensated by increasing the TACI protein quantitythrough cross-talk with related immune pathways CVIDis generally assumed to be a polygenetic disorder and itis plausible that alterations in the index patient were notlimited to the TNFRSF13B gene This prompted us to analyzeother essential immune pathways in B lymphocytes andwe investigated CD40 BCR and TLR9 signaling We didnot find any additional defect in BCR or CD40 signalingNevertheless for both pathways defective interaction withother cells or factors such as abrogated CD40L ligation onT cells or CD21- complement C3d fragment interactionscannot be excluded
In contrast TLR9 expression and function were con-siderably dissimilar between index patient and sibling 2Since the TLR9 expression levels and function were compa-rable between the index patient and the other subjects butincreased in sibling 2 we hypothesize that compensatoryTLR9 expression occurs in the sibling TLR9 recognizesunmethylated CpG-motif-containing bacterial or viral DNA[37 38] and has a critical role in the prevention of severalbacterial and viral infections Ligation of TLR9 inducesrecruitment of MyD88 [30 39] initiating several signalingevents which eventually result in activation of NF120581B and AP-1 [40] By the same token increased TACI protein stabilitymay participate in a compensatory mechanism in the healthysibling possibly related to TLR9 signaling TLR9 engagementinduced TACI upregulation and enhanced TACI-mediated Igclass switching of effector B cells resulting in increased IgGsecretion in cells exposed to anti-TACI and IL-10 and inducedsecretion of IgA
This study has some limitations The TLR9 signalingpathway through MyD88 could be studied more extensivelynot only on a functional level but also through whole exomesequencing The effects of TACI malfunctioning on expres-sion and function of several other targets could have beenaddressed including other TNF receptors and their ligandsor signaling proteins downstream of TACI such as TRAFsCAML and AP-1 Additional defects may not be limitedto B-cell lymphocytes TACI expression can be induced inCD4+ and CD8 T+ cells [4 41] study of TACI-related T-cellfunction would be particularly interesting since the patientdemonstrated chronic viral infections in vivo and impairedantigenic T-cells responses to several viruses in vitro
5 Conclusion
We here describe the diverse clinical and immunologicalcharacteristics in siblings with an identical TACI geno-type These clinical differences are supported by differencesin TACI protein expression and function We proposethat compensatory regulatory mechanisms involving post-translational modification of TACI protein and TLR9 existand may overcome the altered function of TACI trimersin asymptomatic TACI A181EC104R individuals Additionalstudies of TACI protein homeostasis the TACI signalingpathway and interactionwithTLR9 and expression and func-tion of TLR9 downstream signaling molecules are thereforeadvisable
Joris M van Montfrans and Marianne Boes contributedequally
Acknowledgments
The authors would like to thank the technicians of theDepartment of Medical Immunology for excellent assistancewith the TACI sequencing setup and Ewoud Compeer andArie Jan Stoppelenburg for help with PCR and data analyses
References
[1] M A Park J T Li J B Hagan D E Maddox and R SAbraham ldquoCommon variable immunodeficiency a new lookat an old diseaserdquo The Lancet vol 372 no 9637 pp 489ndash5022008
[2] C Cunningham-Rundles L Radigan A K Knight L ZhangL Bauer and A Nakazawa ldquoTLR9 activation is defective incommon variable immune deficiencyrdquo Journal of Immunologyvol 176 no 3 pp 1978ndash1987 2006
[3] J E Yu A K Knight L Radigan et al ldquoToll-like receptor 7and 9 defects in common variable immunodeficiencyrdquo Journalof Allergy and Clinical Immunology vol 124 no 2 pp 349ndash3562009
[4] G-U Von Bulow and R J Bram ldquoNF-AT activation inducedby a CAML-interacting member of the tumor necrosis factorreceptor superfamilyrdquo Science vol 278 no 5335 pp 138ndash1411997
[5] P Schneider F Mackay V Steiner et al ldquoBAFF a novel ligandof the tumor necrosis factor family stimulates B cell growthrdquoJournal of Experimental Medicine vol 189 no 11 pp 1747ndash17561999
[6] Y Wu D Bressette J A Carrell et al ldquoTumor necrosis factor(TNF) receptor superfamily member TACI is a high affinityreceptor for TNF family members APRIL and BLySrdquo Journal ofBiological Chemistry vol 275 no 45 pp 35478ndash35485 2000
[7] S A Marsters M Yan R M Pitti P E Haas V M Dixit andA Ashkenazi ldquoInteraction of the TNF homologues BLyS andAPRIL with the TNF receptor homologues BCMA and TACIrdquoCurrent Biology vol 10 no 13 pp 785ndash788 2000
[8] D Bischof S F Elsawa G Mantchev et al ldquoSelective activationof TACI by syndecan-2rdquo Blood vol 107 no 8 pp 3235ndash32422006
[9] F Mackay P Schneider P Rennert and J Browning ldquoBAFFand APRIL a tutorial on B cell survivalrdquo Annual Review ofImmunology vol 21 pp 231ndash264 2003
[10] Y Laabi M P Gras F Carbonnel et al ldquoA new gene BCMon chromosome 16 is fused to the interleukin 2 gene by at(416)(q26p13) translocation in a malignant T cell lymphomardquoEMBO Journal vol 11 no 11 pp 3897ndash3904 1992
[11] J S Thompson S A Bixler F Qian et al ldquoBAFF-R a newlyidentified TNF receptor that specifically interacts with BAFFrdquoScience vol 293 no 5537 pp 2108ndash2111 2001
[12] J Hauer S Puschner P Ramakrishnan et al ldquoTNF receptor(TNFR)-associated factor (TRAF) 3 serves as an inhibitorof TRAF25-mediated activation of the noncanonical NF-120581Bpathway by TRAF-binding TNFRsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 102 no8 pp 2874ndash2879 2005
[13] E Castigli S AWilsonA Elkhal EOzcan LGaribyan andRS Geha ldquoTransmembrane activator and calciummodulator andcyclophilin ligand interactor enhances CD40-driven plasmacell differentiationrdquo Journal of Allergy and Clinical Immunologyvol 120 no 4 pp 885ndash891 2007
[14] E Ozcan L Garibyan J J-Y Lee R J Bram K-P Lamand R S Geha ldquoTransmembrane activator calciummodulatorand cyclophilin ligand interactor drives plasma cell differenti-ation in LPS-activated B cellsrdquo Journal of Allergy and ClinicalImmunology vol 123 no 6 pp 1277ndash1286 2009
[15] E Ozcan I Rauter L Garibyan S R Dillon and R S GehaldquoToll-like receptor 9 transmembrane activator and calcium-modulating cyclophilin ligand interactor and CD40 synergizein causing B-cell activationrdquo Journal of Allergy and ClinicalImmunology vol 128 no 3 pp 601ndash609 2011
[16] B He R Santamaria W Xu et al ldquoThe transmembrane activa-tor TACI triggers immunoglobulin class switching by activatingB cells through the adaptorMyD88rdquoNature Immunology vol 11no 9 pp 836ndash845 2010
[17] U Salzer C Bacchelli S Buckridge et al ldquoRelevance of biallelicversus monoallelic TNFRSF13B mutations in distinguishingdisease-causing from risk-increasing TNFRSF13B variants inantibody deficiency syndromesrdquo Blood vol 113 no 9 pp 1967ndash1976 2009
[18] L Zhang L Radigan U Salzer et al ldquoTransmembrane acti-vator and calcium-modulating cyclophilin ligand interactormutations in common variable immunodeficiency clinical andimmunologic outcomes in heterozygotesrdquo Journal of Allergy andClinical Immunology vol 120 no 5 pp 1178ndash1185 2007
[19] Q Pan-Hammarstrom U Salzer L Du et al ldquoReexamining therole of TACI coding variants in common variable immunode-ficiency and selective IgA deficiencyrdquo Nature Genetics vol 39no 4 pp 429ndash430 2007
[20] R M Locksley N Killeen and M J Lenardo ldquoThe TNF andTNF receptor superfamilies integrating mammalian biologyrdquoCell vol 104 no 4 pp 487ndash501 2001
[21] L Garibyan A A Lobito R M Siegel M E Call K WWucherpfennig and R S Geha ldquoDominant-negative effect ofthe heterozygous C104R TACI mutation in common variableimmunodeficiency (CVID)rdquo Journal of Clinical Investigationvol 117 no 6 pp 1550ndash1557 2007
[22] S GHymowitz D R Patel H J AWallweber et al ldquoStructuresof APRIL-receptor complexes like BCMA TACI employs onlya single cysteine-rich domain for high affinity ligand bindingrdquoJournal of Biological Chemistry vol 280 no 8 pp 7218ndash72272005
[23] E Castigli S A Wilson L Garibyan et al ldquoTACI is mutantin common variable immunodeficiency and IgA deficiencyrdquoNature Genetics vol 37 no 8 pp 829ndash834 2005
[24] U Salzer H M Chapel A D B Webster et al ldquoMutationsin TNFRSF13B encoding TACI are associated with commonvariable immunodeficiency in humansrdquoNature Genetics vol 37no 8 pp 820ndash828 2005
10 ISRN Immunology
[25] C Bacchelli K F Buckland S Buckridge et al ldquoThe C76Rtransmembrane activator and calcium modulator cyclophilinligand interactor mutation disrupts antibody production andB-cell homeostasis in heterozygous and homozygous micerdquoJournal of Allergy and Clinical Immunology vol 127 no 5 pp1253ndash1259 2011
[26] A J Fried I Rauter S R Dillon H H Jabara and R S GehaldquoFunctional analysis of transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) mutationsassociated with common variable immunodeficiencyrdquo Journalof Allergy and Clinical Immunology vol 128 no 1 pp 226ndash2282011
[27] J J Lee H H Jabara L Garibyan et al ldquoThe C104R mutantimpairs the function of transmembrane activator and calciummodulator and cyclophilin ligand interactor (TACI) throughhaploinsufficiencyrdquo Journal of Allergy and Clinical Immunologyvol 126 no 6 pp 1234ndashe2 2010
[28] J J Lee I Rauter L Garibyan et al ldquoThe murine equivalentof the A181E TACI mutation associated with common variableimmunodeficiency severely impairs B-cell functionrdquo Blood vol114 no 11 pp 2254ndash2262 2009
[29] A A JM van deVen L van de Corput CM van Tilburg et alldquoLymphocyte characteristics in children with common variableimmunodeficiencyrdquoClinical Immunology vol 135 no 1 pp 63ndash71 2010
[30] E Latz A Schoenemeyer A Visintin et al ldquoTLR9 signals aftertranslocating from theER toCpGDNA in the lysosomerdquoNatureImmunology vol 5 no 2 pp 190ndash198 2004
[31] C Foerster N Voelxen M Rakhmanov et al ldquoB cell receptor-mediated calcium signaling is impaired in B lymphocytes oftype Ia patients with common variable immunodeficiencyrdquoJournal of Immunology vol 184 no 12 pp 7305ndash7313 2010
[32] M C Van Zelm J Smet B Adams et al ldquoCD81 gene defectin humans disrupts CD19 complex formation and leads toantibody deficiencyrdquo Journal of Clinical Investigation vol 120no 4 pp 1265ndash1274 2010
[33] M Malphettes L Gerard M Carmagnat et al ldquoLate-onsetcombined immune deficiency a subset of common variableimmunodeficiency with severe T cell defectrdquo Clinical InfectiousDiseases vol 49 no 9 pp 1329ndash1338 2009
[34] F Dedeoglu B Horwitz J Chaudhuri F W Alt and R SGeha ldquoInduction of activation-induced cytidine deaminasegene expression by IL-4 and CD40 ligation is dependent onSTAT6 and NF120581Brdquo International Immunology vol 16 no 3 pp395ndash404 2004
[35] M Muramatsu K Kinoshita S Fagarasan S Yamada YShinkai and T Honjo ldquoClass switch recombination andhypermutation require activation-induced cytidine deaminase(AID) a potential RNA editing enzymerdquo Cell vol 102 no 5 pp553ndash563 2000
[36] P Revy T Muto Y Levy et al ldquoActivation-induced cytidinedeaminase (AID) deficiency causes the autosomal recessiveform of the hyper-IgM syndrome (HIGM2)rdquo Cell vol 102 no5 pp 565ndash575 2000
[37] A M Krieg ldquoCpG motifs in bacterial DNA and their immuneeffectsrdquo Annual Review of Immunology vol 20 pp 709ndash7602002
[38] T Kawai and S Akira ldquoInnate immune recognition of viralinfectionrdquo Nature Immunology vol 7 no 2 pp 131ndash137 2006
[39] E Latz A Verma AVisintin et al ldquoLigand-induced conforma-tional changes allosterically activate Toll-like receptor 9rdquoNatureImmunology vol 8 no 7 pp 772ndash779 2007
[40] S Sato H Sanjo K Takeda et al ldquoEssential function for thekinase TAK1 in innate and adaptive immune responsesrdquoNatureImmunology vol 6 no 11 pp 1087ndash1095 2005
[41] F Mackay and H Leung ldquoThe role of the BAFFAPRIL systemon T cell functionrdquo Seminars in Immunology vol 18 no 5 pp284ndash289 2006
This versatility complicates studying the precise contributionof each molecule to humoral immunity [9]
Following ligand binding the intracellular TACI domainsinteract with TNFR-associated proteins (TRAFs) andCAMLCAML interaction activates nuclear factor of activated Tcells (NFAT) which has a key role in controlling calciumrelease from intracellular stores [4] Additionally nuclearfactor kappa B (NF120581B) is activated [12] probably via TRAFsc-Jun NH2-terminal kinase and transcription factor AP-1[4] TACI has a role in maintenance of B-cell homeostasisimmunoglobulin (Ig) isotype class-switching and antibodyproduction in response to type II T-cell-independent anti-gens Furthermore it may act in synergy with CD40 andTLRs [13ndash15] It was recently shown that TACI induces class-switch recombination via themyeloid differentiation primaryresponse gene 88 (MyD88) adaptor thus employing the samesignaling cascade as multiple TLRs [16]
Currently TACI is considered a CVID susceptibility gene(relative risk 43 95 confidence interval 24ndash76) of whichheterozygous disease-associated variants are present in sim9of CVID patients compared to only monoallelic variantsin lt2 of the healthy controls [17] In healthy or mildlyaffected family members of CVID patients some biallelicpolymorphisms were found suggesting that TACI variantsmay have an incomplete penetrance [17] Furthermore thepresence of TACI polymorphisms in CVID predisposes toautoimmune disease and lymphoid hyperplasia [18]
TwoTACI variants are significantly associatedwithCVIDdisease C104R [17 19] and A181E [19] A characteristicof TNFRs is the presence of two cysteine-rich domain(CRDs) in the extracellular domain which are conservedbetween species [20] TACI monomers engage in a ligand-independent homotypic trimer formation that requires theCRD1 domain [21] The second CRD allows ligation withBAFF or APRIL [22] The amino acid substituting C104Rvariant is located in CRD2 and thus abrogates binding ofAPRIL and BAFF [23 24] as was demonstrated in CVIDpatients heterozygous for C104R Initially the deficiency wasattributed to a dominant-negative interference of the C104Rpolymorphism in trimeric complexes with TACI wildtype[21] Subsequent reports however suggest haploinsufficiencyof the TACI allele [25ndash27]
TheA181E variant is located at the transmembrane regionand permits normal ligand binding [23] Mouse modelswith an A181E equivalent (A144E) show defective preligandand ligand-induced clustering of the intracellular domainand subsequent hampered nuclear factor kappa B (NF120581B)activation possibly this is caused by a conformational changeresulting from the negative charge introduced by the A181Evariant [28] A further consequence was low serum IgAlevels and impaired antibody responses to type II T-cell-independent antigens
To more closely resemble human conditions exhibitingheterozygous alleles A181E and C104R were investigatedin combination with a wildtype TACI allele Neither vari-ant interfered with oligomerization with wildtype TACImolecules nor NF120581B activation was unaffected in 293T cellswith wildtype and mutant TACI [26] Dominant-negative
interference of the variants with wildtype TACI is there-fore improbable but haploinsufficiency was not completelyexcluded It was therefore recommended to study B-cellfunction in patients their healthy relatives and unrelatedsubjects who carry the same mutation [26] Our study hereaddressed this suggestion
We identified a patient compoundheterozygous forA181Eand C104R Her disease course was complicated by cytope-nias interstitial lung disease autoimmune nephritis andeventually mortality [29] In contrast her sibling carried anidentical TACI genotype but was completely healthy whichwas not reported previously Here we describe a clinical andimmunological evaluation of the patient compared to thehealthy sibling and unrelated healthy and CVID controlsWe provide mechanistic support of how individuals withidentical TACI alleles may exhibit differential TACI proteinexpression and downstream signaling function
2 Materials and Methods
21 Patients As part of routine evaluation of CVID patientsin our institute C104R and A181E TACI variants werescreened by polymerase chain reaction (PCR) as describedpreviously [29] and both were found in the index patient Aswe considered stem cell transplantation in the index patientparents and siblings were screened (Figure 2(a)) Informedconsent was obtained to further evaluate the patient andfamily members for clinical indications Further data wereobtained from medical records
22 DNA Sequencing TACI mutations were investigated asdescribed previously [29] Briefly primers were designedfor the pC104R and pA181E variants and their wildtypeswith Primer Express software (Applied Biosystems) To testfor additional mutations or splicing variants [22] primerswere designed for the complete TACI sequence contain-ing 5 exons The following primers were used reverse51015840-TGTAAAACGACGGCCAGTCAGCCCAAGCACTAA-TCAAATC-31015840 and forward 51015840-CAGGAAACAGCTATG-ACCCTCTCTCCCCTCCTCTCCATC-31015840 For splicing vari-ants cDNA was sequenced using a 3730 DNAAnalyzer Priorto sequencing the TACI domain of the cDNA was amplifiedby a PCR starting at 95∘C for 10min followed by 35 cycles of58∘C for 15 s and 60∘C for 1min
23 Flow Cytometry Phenotypic and functional evaluationof the lymphocyte compartment was performed as describedpreviously [29] Next lymphoblastoid cell lines (LCL) weregenerated using Epstein-Barr virus and stained with anti-bodies against CD19 CD20 CD21 (all Becton Dickinson)TLR9 CD40 (both eBioscience) rabbit polyclonal anti-TACI(Abcam) with secondary anti-rabbit Ig-DiLight649 (JacksonImmunoresearch West Grove PA) and the appropriateisotypes Intracellular TACI expression was determined aftertreatment with permeabilization reagents
For calcium assays fresh peripheral blood mononuclearcells (PBMC) were incubated with Fluo-3 Fura Red (bothMolecular Probes) (30min) and CD19 (10min) at 37∘C Cells
ISRN Immunology 3
were washed twice and resuspended in Hankrsquos balanced saltsolution (HBSS) supplemented with 10 fetal calf serum(FCS) Cytosolic calcium levels were measured on FacsCanto II after 1 minute 20120583gmL goat anti-human IgMF(ab1015840)
2fragments (Jackson Immunoresearch) were added
After another 5 minutes 20120583gmL ionomycin (CalbiochemSan Diego CA) was added Area under curves was calculatedusing Facs Diva and Prism software Next LCL were stainedand resuspended in calcium-free phosphate-buffered saline(PBS) Calcium chloridewas added after intracellular calciumstorage depletion with ionomycin or thapsigargin (SantaCruz Biotechnology Santa Cruz CA)
24 In Vitro Stimulation Assays and PCR LCL were culturedin RPMI 1640 containing 10 FCS at the subsequentconditions mouse anti-TACI (05 120583gmL RampD systemsMinneapolis MN USA) and goat anti-mouse IgGmicrobeads (Miltenyi Biotec Carlsbad CA) with or withoutIL-4 (100UmL Immunotools) CpG phosphorothioate-modified oligodeoxynucleotide 2006 (05 120583gmL AlexisBiochemicals) IL-10 (10 ngmL Becton Dickinson) oranti-CD40 (01120583gmL RampD systems) After 48 hours cellswere lysed and total mRNA was isolated using Tripureisolation reagent (Roche Diagnostics Mannheim Germany)according to the manufacturerrsquos instructions Reversetranscriptionwas performed using an iScript cDNA synthesiskit (BioradHercules CA) Primersweremixedwith IQSYBRgreen supermix (BioRad) The detection run started at 95∘Cfor 10min followed by 45 cycles of 95∘C for 15 s and 60∘Cfor 1min Assays were performed in duplicate or triplicateas 15 120583L reactions in 96-well plates using C1000 ThermalCycler (BioRad) Results were normalized to the endogenousGAPDH mRNA (2minusΔCT) The following primers wereused GAPDH forward 51015840-GTCGGAGTCAACGGATT-31015840GAPDH reverse 51015840-AAGCTTCCCGTTCTCAG-31015840 AICDAforward 51015840-TGCTCTTCCTCCGCTACATCTC-31015840 AICDAreverse 51015840-AACCTCATACAGGGGCAAAACC-31015840 NF120581Bforward 51015840-GAAGCACGAATGACAGAGGC-31015840 NF120581B rev-erse 51015840-GCTTGGCGGATTAGCTCTTT-31015840 NFAT forward51015840-GCAGAGCACGGACAGCTATC-31015840 NFAT reverse 51015840-GGGCTTTCTCCACGAAAATGA-31015840 (All Sigma-Aldrich)
3 Results
31 Clinical Evaluation of the CVID Index Patient Theindex patient (Figure 2(a) arrow) was referred at age 11for recurrent upper and lower respiratory tract infectionsand splenomegaly Laboratory investigations showed absentisohemagglutinins and decreased serum titers of total IgGIgA IgM IgG
1 and IgG
2(Table 1) She was lymphopenic
with low numbers of B cells CD4+ and CD8+ T cells andalmost absent class switched memory B cells (Figure 1(a))Functionally in vitro T-cell proliferation assays showed apositive response to mitogens and an incomplete response toparticularly viral antigens A diagnosis of CVID was madeand immunoglobulin replacement therapy was initiated
After some time she developed CVID-related diseasemanifestations including autoimmune hemolytic anemia
(AIHA) idiopathic thrombocytopenic purpura (ITP) sub-mandibular lymphadenopathy and autoimmune nephritisa renal biopsy showed tubulo interstitial nephritis withgranulomatous changes the estimated glomerular filtratedrate was 412mLmin173m2 (as calculated by Schwartzformula) Furthermore she developed exercise-induced dys-pnea a high-resolution computed tomography (HRCT) scanshowed airway and interstitial lung disease with bronchiec-tasis and peribronchovascular abnormalities consisting offocal lesions with ground glass aspect peribronchial con-solidations bronchial wall thickening and air trapping(Figure 2(b)) A broncho alveolar lavage showed no indi-cation for viral bacterial or fungal infections Pulmonaryfindings were suggestive for granulomatous disease and low-dose steroids and tacrolimus were initiated resulting in nor-malization of pulmonary function and glomerular filtrationrate Furthermore viral immunity appeared to be affectedas cytomegalovirus was repeatedly detectable with viral loadsof 100ndash2000 copiesmL in plasma Treatment with tacrolimuswas complicated by tubulopathy and therefore mycopheno-late mofetil was started with good initial response howevercausing pancreatitis Rituximab was administered with pos-itive effect on lymphadenopathy AIHA and ITP Despiteall treatment efforts taken disease manifestations progressedfurther and became refractory The index patient underwentallogeneic stem cell transplantation with matched unrelatedcord blood after myeloablative conditioning but eventuallydied of the complications of graft versus hose disease com-bined with multiple infections
32 Clinical and Immunological Evaluation of Family Mem-bers Further studies were performed to determine the roleof these TACI variants Clinically the parents and sibling2 were healthy sibling 1 developed clinical symptoms ofCVID during the course of investigations Serum Ig levelswere normal in both parents and sibling 2 apart from amodestly decreased serum IgA In contrast sibling 1 washypogammaglobulinemic for IgG IgA and subclasses IgG
1
IgG2 and IgG
4 albeit modestly in comparison to the index
patient (Table 1)To study specific antibody synthesis parents and sibling 1
and 2 were vaccinated with 23-valent Pneumovax (AdventisPasteur MSD) and total IgG antibody titers were measuredafter 1 month Parents and sibling 2 had adequate T cell-independent responses in vivo to pneumococcal polysaccha-rides while the response of sibling 1 was suboptimal (Table 1)
Consequently immunological evaluation was extendedin siblings 1 revealing an increase in relative and absolutenumbers of CD8+ T cells Total B cells were increased(217 of lymphocytes 512 cells120583L) and the compositionof B-cell compartment remained normal Proliferative T-cellresponses in vitro were normal to mitogens but partiallyimpaired to recall antigens similar to the index patientTotal B-cell count and composition of B-cell compartment ofsibling 2 were normal (Figure 1(b))
Sibling 1 slowly developed clinical symptoms consistingof diarrhea fatigue and persistent rhinosinusitis refractory toantibiotic prophylaxisThe spleenwas enlarged on abdominal
4 ISRN Immunology
IgD
13395
01
941 11
01
IgM CD27
IgD
IgD
CD38
CD19+ lymphocytesIndex patient
(a)
94
9
756793
104
IgM CD27
65
CD38
Healthy sibling
IgD
IgD
IgD
(b)
Figure 1 B-cell subpopulations of index patient and healthy sibling (a) B cell subpopulations of index patient showing 941 naıve +transitional B cells (IgM+IgD+CD27minus) 11 Marginal Zone B cells (IgM+IgD+CD27+) 01 class-switched B cells (IgDminusIgMminusCD27+)and 133 transitional B cells (IgD+CD38+) (b) B cell subpopulations of healthy sibling showing 756 naıve + transitional B cells(IgM+IgD+CD27minus) 94Marginal Zone B cells (IgM+IgD+CD27+) 9 class-switched B cells (IgDminusIgMminusCD27+) and 65 transitional Bcells (IgD+CD38+)
echography Based on these clinical and laboratory findingsimmunoglobulin replacement therapy was initiated whichled to significant clinical improvement Further studies werefocused on the index patient and asymptomatic sibling 2
33 TNFRSF13B Compound Heterozygous Variants in theIndex Patient and Asymptomatic Sibling TACI is a CVIDsusceptibility factor and to substantiate the diagnosis CVIDwe performed gene sequencing of the TACI gene Analysisof the patientrsquos TNFRSF13B gene showed two variants C104Rand A181E Subsequently her family was investigated reveal-ing that each parent carried one variant (Figure 2(a)) Thebrother (sibling 3) had two normal TACI alleles and was notfurther investigated The two sisters (siblings 1 and 2) werealso compound heterozygous for A181EC104R (Figure 2(a))Sequencing of the complete TNFRSF13B gene revealed noadditional mutations [22] The index patient and sibling 2exhibited identical alternative splicing patterns of exon 2 thatencode the CRD1 important in trimer formation of TACI[22] This finding however did not help explain the different
clinical and B-cell activation phenotype between our indexpatient and sibling 2
34 TACI Protein Expression and Function in the IndexPatient and Asymptomatic Sibling Lymphoblastoid cell lines(LCL) were generated of the index patient and sibling 2 andcompared to cell lines of healthy controls CVID patients and2 CVID patients with a monoallelic A181E variant LCL of allsubjects exhibited TACI expression on the cell membraneInterestingly the TACI mean fluorescence intensity of theindex patient was lower in comparison with the othersubjects especially with sibling 2 fromwhomLCL repeatedlydisplayed the highest cell surface expression (Figure 2(c))Subsequently intracellular TACI was measured to study thepossibility of impaired transition of TACI to the cell surfaceand ensuing intracellular TACI accumulation IntracellularTACI however correlated with the levels of cell surface TACI(Pearson correlation 070 119875 = 0037) and was also repeatedlylower in the index patient (Figure 2(c)) Basal TACI mRNAlevels varied within a similar range for all LCL including theindex patient and sibling (Figure 2(d))
Figure 2 TACI expression and function are decreased in the CVID patient compared to her asymptomatic sibling (a) Pedigree of the studiedfamily Circles display females squares display males The index patient is indicated with an arrow (b) Chest high-resolution CT scan of theindex patient revealing peribronchial consolidations with ground glass appearance and peribronchial thickening ((c)-(d)) TACI surface (cii) or intracellular (c iii) protein and mRNA expression Light grey line indicated isotype indicated dark grey TACI (c i) Results of 5 healthydonors 3 CVID patients with normal TACI 2 CVID patients withmonoallelic A181E the index patient and sibling 2 For (d) average results of3 independent experiments are shown (e) AID (left) and NFAT (right) mRNA induction after 48 h culture of LCL with the indicated stimuliBars represent means plusmn standard error of the mean
Next we investigated TACI function by measuring itsability to induce activation-induced cytidine deaminase(AICDA) gene transcription The AICDA gene productactivation-induced deaminase (AID) is an enzyme of piv-otal importance for secondary antibody diversification [30]Healthy control and CVID patient LCL with normal bial-lelic TACI showed AID mRNA induction upon stimulation
with agonistic anti-TACI (Figure 2(e)) CVID patients withmonoallelic A181E variants showed minimal AICDA genetranscription The index patient had no AICDA gene tran-scription upon TACI stimulation while AID mRNA levelsof sibling 2 LCL were comparable to B cells with normalTACIThe same pattern was noticed for NFATmRNA induc-tion gene transcription upon TACI triggering was lower in
6 ISRN Immunology
Table 1 Immunological characteristics of patient and family
Patient Sibling 1 Sibling 2 Mother FatherGender C C C C D
the index patient compared to sibling 2 There were nodifferences in NF120581B induction upon TACI stimulation Stim-ulation of the CD40 pathway showed adequate induction ofAID and NFAT mRNA in the index patient IL-10 treatmentwas included as negative control that does not induce AICDAgene expression Taken together TACI protein expressionand function were increased in sibling 2 when compared tothe index patient
35 Appearance of TLR9 Protein Expression and Function inthe Asymptomatic Sibling We considered two possibilitiesthat may explain why the index patient and sibling 2 havingidentical TACI gene mutations exhibit divergent healthconsequences either the index patient had an additionalimmune defect which in combinationwith the TACI variantswould lead to CVID disease or sibling 2 has compensatoryimmune function in the TACI signaling route that preventsCVID disease from occurring First as defects in the B-cell receptor (BCR) and its coreceptor complex have beendescribed [31 32] we screened the BCR signalosome CD19CD21 and CD20 cell surface expression of the index patientwere comparable to expression on control LCL (data notshown) Functionally early B-cell activation seemed intactas measured by calcium mobilization after BCR stimulationof fresh PBMC (Figure 3(a)) The index patient showed
a normal initial but rapidly declining calcium influx aspreviously observed in CVID patients with autoimmunesymptoms (own unpublished observations) and area undercurves were comparable to controls As the index patientrsquosendoplasmic reticulum (ER) calcium storage capacitywas notdecreased compared to sibling 2 an additional defect in BCRsignaling was considered improbable
Second the classical route of BCR isotype class switchingis via CD40 with CD40L on T cells and it was recentlyshown that TACI and CD40 signaling can act in synergy[13 15] However CD40 cell surface expression (Figure 3(b))and CD40-mediated gene transcription (Figures 2(e) and3(b)) were comparable between all LCL suggesting that CD40signaling in the index patient B cells was intact at least in thisassay
Third we explored TLR9 signaling as TLR9 defects havebeen described in CVID [2 3] and TLR9 and TACI signalingemploy common downstream signaling pathways [15 16]Noticeably TLR9 expression was nearly twofold higher insibling 2 compared to other LCL and this increased expres-sion was accompanied by an increased TLR9 expression andfunction (Figure 3(c)) supporting the possibility that com-pensatory TLR9 expression contributes as a mechanism foraltered TACI A181EC104R protein in restoring B-cell overallcapacity to transmit signals towards NFAT and AID Thus
ISRN Immunology 7
CVID patientSibling 1Sibling 2
MotherControl
Anti-IgM Ionomycin
25
20
15
10
5
3
2
1
10000
200 300 400Time (s)
BCR-mediated calcium flux (PBMC)Cy
toso
lic C
a2+
leve
l
(a)
0
5000
10000
15000
Mea
n flu
ores
cenc
e int
ensit
y
Cell surface CD40
Con
trol
(TAC
I nor
mal
)
CVID
(TAC
I nor
mal
)
CVID
(A18
1En
orm
al)
Patie
nt(A
181E
C10
4R)
Sibl
ing
(A18
1EC
104R
)
(b)
IL-10 CpG ODN Anti-CD40
AID mRNA
0AID
mRN
A (r
elat
ive t
o G
APD
H) 20
15
10
5
TLR9
Mea
n flu
ores
cenc
e int
ensit
y
15
10
5
00
Intracellular TLR9times10
2
Con
trol
(TAC
I nor
mal
)CV
ID(T
ACI n
orm
al)
CVID
(A18
1En
orm
al)
Patie
nt(A
181E
C10
4R)
Sibl
ing
(A18
1EC
104R
)
Control (TACI normal)CVID (TACI normal)CVID (A181Enormal)
Patient (A181EC104R)Sibling (A181EC104R)
(c)
Figure 3 Similar BCR signaling but possibly enhanced TLR9 signaling in the healthy sibling (a) Calcium mobilization upon triggering ofthe BCR using IgM F(ab1015840)
2fragments in freshly isolated PBMCs (b) CD40 surface expression asmeasured by flow cytometry (c) TLR9 B-cell
expression (c ii) and function (c iii) Light grey line indicated isotype indicated dark grey TLR9 (c i) Bars represent means plusmn standard errorof the mean
our data supports the notion that compensatory signaling inthe TACI route in the case of sibling 2 via enhanced TLR9signaling may prevent the development of CVID disease
4 Discussion
We here describe differential immunological phenotypesdisplayed by siblings with identical genetic TACI variantsThis family provides the unique opportunity to study the role
and significance of the humanC104R andA181E variants bothin vivo and in vitro
The index patient had severe CVID which graduallyevolved to late onset combined immunodeficiency a phe-nomenon previously described in CVID patients [33] Sibling1 did eventually develop mild CVID but without autoim-mune manifestations To date sibling 2 remains free ofsymptoms and has excellent specific antibody responsesnevertheless this sibling may still develop CVID at a laterage Previous studies have shown that C104R abrogates ligand
8 ISRN Immunology
binding due to haploinsufficiency whereas in the A181Evariant ligand binding remains intact [21 26] At least aproportion of A181EC104R TACI trimers will thus be ableto bind ligand As NF120581B activation was strongly decreasedin both variants we hypothesized TACI function to beaffected AID and NFAT mRNA induction were reduced inthe index patient and seemed also reduced in the CVIDpatients with a heterozygous A181E variant [14] Converselysibling 2 showed AID and NFAT induction comparable tohealthy controls and CVID patients with unaffected TACIalleles NFAT activation induces transcription of cytokinegenes encoding for example IL-4 which is involved in classswitch recombination IL-4 and CD40 ligation induces AIDmRNA and protein expression and depends on STAT6 andNF120581B [34] AID is pivotal for class switch recombination andsomatic hypermutation of theBCR [35 36] bothmechanismsare routinely impaired in CVID
The differential TACI function in the index patient andsibling was accompanied by a change in TACI protein levelsshowing increased TACI expression in the sibling both atthe cell surface and intracellular level TACI protein levels inthe healthy sibling were increased in comparison to healthycontrols as well As TNFRSF13B gene transcription wascomparable between all subjects except for the healthy sib-ling these observations suggest that protein turnover in thehealthy sibling may be decreased possibly due to alterationsin posttranslational modification of TACI protein affectingprotein stability and folding transport or degradation
Alternatively altered TACI function in the healthy siblingwas compensated by increasing the TACI protein quantitythrough cross-talk with related immune pathways CVIDis generally assumed to be a polygenetic disorder and itis plausible that alterations in the index patient were notlimited to the TNFRSF13B gene This prompted us to analyzeother essential immune pathways in B lymphocytes andwe investigated CD40 BCR and TLR9 signaling We didnot find any additional defect in BCR or CD40 signalingNevertheless for both pathways defective interaction withother cells or factors such as abrogated CD40L ligation onT cells or CD21- complement C3d fragment interactionscannot be excluded
In contrast TLR9 expression and function were con-siderably dissimilar between index patient and sibling 2Since the TLR9 expression levels and function were compa-rable between the index patient and the other subjects butincreased in sibling 2 we hypothesize that compensatoryTLR9 expression occurs in the sibling TLR9 recognizesunmethylated CpG-motif-containing bacterial or viral DNA[37 38] and has a critical role in the prevention of severalbacterial and viral infections Ligation of TLR9 inducesrecruitment of MyD88 [30 39] initiating several signalingevents which eventually result in activation of NF120581B and AP-1 [40] By the same token increased TACI protein stabilitymay participate in a compensatory mechanism in the healthysibling possibly related to TLR9 signaling TLR9 engagementinduced TACI upregulation and enhanced TACI-mediated Igclass switching of effector B cells resulting in increased IgGsecretion in cells exposed to anti-TACI and IL-10 and inducedsecretion of IgA
This study has some limitations The TLR9 signalingpathway through MyD88 could be studied more extensivelynot only on a functional level but also through whole exomesequencing The effects of TACI malfunctioning on expres-sion and function of several other targets could have beenaddressed including other TNF receptors and their ligandsor signaling proteins downstream of TACI such as TRAFsCAML and AP-1 Additional defects may not be limitedto B-cell lymphocytes TACI expression can be induced inCD4+ and CD8 T+ cells [4 41] study of TACI-related T-cellfunction would be particularly interesting since the patientdemonstrated chronic viral infections in vivo and impairedantigenic T-cells responses to several viruses in vitro
5 Conclusion
We here describe the diverse clinical and immunologicalcharacteristics in siblings with an identical TACI geno-type These clinical differences are supported by differencesin TACI protein expression and function We proposethat compensatory regulatory mechanisms involving post-translational modification of TACI protein and TLR9 existand may overcome the altered function of TACI trimersin asymptomatic TACI A181EC104R individuals Additionalstudies of TACI protein homeostasis the TACI signalingpathway and interactionwithTLR9 and expression and func-tion of TLR9 downstream signaling molecules are thereforeadvisable
Joris M van Montfrans and Marianne Boes contributedequally
Acknowledgments
The authors would like to thank the technicians of theDepartment of Medical Immunology for excellent assistancewith the TACI sequencing setup and Ewoud Compeer andArie Jan Stoppelenburg for help with PCR and data analyses
References
[1] M A Park J T Li J B Hagan D E Maddox and R SAbraham ldquoCommon variable immunodeficiency a new lookat an old diseaserdquo The Lancet vol 372 no 9637 pp 489ndash5022008
[2] C Cunningham-Rundles L Radigan A K Knight L ZhangL Bauer and A Nakazawa ldquoTLR9 activation is defective incommon variable immune deficiencyrdquo Journal of Immunologyvol 176 no 3 pp 1978ndash1987 2006
[3] J E Yu A K Knight L Radigan et al ldquoToll-like receptor 7and 9 defects in common variable immunodeficiencyrdquo Journalof Allergy and Clinical Immunology vol 124 no 2 pp 349ndash3562009
[4] G-U Von Bulow and R J Bram ldquoNF-AT activation inducedby a CAML-interacting member of the tumor necrosis factorreceptor superfamilyrdquo Science vol 278 no 5335 pp 138ndash1411997
[5] P Schneider F Mackay V Steiner et al ldquoBAFF a novel ligandof the tumor necrosis factor family stimulates B cell growthrdquoJournal of Experimental Medicine vol 189 no 11 pp 1747ndash17561999
[6] Y Wu D Bressette J A Carrell et al ldquoTumor necrosis factor(TNF) receptor superfamily member TACI is a high affinityreceptor for TNF family members APRIL and BLySrdquo Journal ofBiological Chemistry vol 275 no 45 pp 35478ndash35485 2000
[7] S A Marsters M Yan R M Pitti P E Haas V M Dixit andA Ashkenazi ldquoInteraction of the TNF homologues BLyS andAPRIL with the TNF receptor homologues BCMA and TACIrdquoCurrent Biology vol 10 no 13 pp 785ndash788 2000
[8] D Bischof S F Elsawa G Mantchev et al ldquoSelective activationof TACI by syndecan-2rdquo Blood vol 107 no 8 pp 3235ndash32422006
[9] F Mackay P Schneider P Rennert and J Browning ldquoBAFFand APRIL a tutorial on B cell survivalrdquo Annual Review ofImmunology vol 21 pp 231ndash264 2003
[10] Y Laabi M P Gras F Carbonnel et al ldquoA new gene BCMon chromosome 16 is fused to the interleukin 2 gene by at(416)(q26p13) translocation in a malignant T cell lymphomardquoEMBO Journal vol 11 no 11 pp 3897ndash3904 1992
[11] J S Thompson S A Bixler F Qian et al ldquoBAFF-R a newlyidentified TNF receptor that specifically interacts with BAFFrdquoScience vol 293 no 5537 pp 2108ndash2111 2001
[12] J Hauer S Puschner P Ramakrishnan et al ldquoTNF receptor(TNFR)-associated factor (TRAF) 3 serves as an inhibitorof TRAF25-mediated activation of the noncanonical NF-120581Bpathway by TRAF-binding TNFRsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 102 no8 pp 2874ndash2879 2005
[13] E Castigli S AWilsonA Elkhal EOzcan LGaribyan andRS Geha ldquoTransmembrane activator and calciummodulator andcyclophilin ligand interactor enhances CD40-driven plasmacell differentiationrdquo Journal of Allergy and Clinical Immunologyvol 120 no 4 pp 885ndash891 2007
[14] E Ozcan L Garibyan J J-Y Lee R J Bram K-P Lamand R S Geha ldquoTransmembrane activator calciummodulatorand cyclophilin ligand interactor drives plasma cell differenti-ation in LPS-activated B cellsrdquo Journal of Allergy and ClinicalImmunology vol 123 no 6 pp 1277ndash1286 2009
[15] E Ozcan I Rauter L Garibyan S R Dillon and R S GehaldquoToll-like receptor 9 transmembrane activator and calcium-modulating cyclophilin ligand interactor and CD40 synergizein causing B-cell activationrdquo Journal of Allergy and ClinicalImmunology vol 128 no 3 pp 601ndash609 2011
[16] B He R Santamaria W Xu et al ldquoThe transmembrane activa-tor TACI triggers immunoglobulin class switching by activatingB cells through the adaptorMyD88rdquoNature Immunology vol 11no 9 pp 836ndash845 2010
[17] U Salzer C Bacchelli S Buckridge et al ldquoRelevance of biallelicversus monoallelic TNFRSF13B mutations in distinguishingdisease-causing from risk-increasing TNFRSF13B variants inantibody deficiency syndromesrdquo Blood vol 113 no 9 pp 1967ndash1976 2009
[18] L Zhang L Radigan U Salzer et al ldquoTransmembrane acti-vator and calcium-modulating cyclophilin ligand interactormutations in common variable immunodeficiency clinical andimmunologic outcomes in heterozygotesrdquo Journal of Allergy andClinical Immunology vol 120 no 5 pp 1178ndash1185 2007
[19] Q Pan-Hammarstrom U Salzer L Du et al ldquoReexamining therole of TACI coding variants in common variable immunode-ficiency and selective IgA deficiencyrdquo Nature Genetics vol 39no 4 pp 429ndash430 2007
[20] R M Locksley N Killeen and M J Lenardo ldquoThe TNF andTNF receptor superfamilies integrating mammalian biologyrdquoCell vol 104 no 4 pp 487ndash501 2001
[21] L Garibyan A A Lobito R M Siegel M E Call K WWucherpfennig and R S Geha ldquoDominant-negative effect ofthe heterozygous C104R TACI mutation in common variableimmunodeficiency (CVID)rdquo Journal of Clinical Investigationvol 117 no 6 pp 1550ndash1557 2007
[22] S GHymowitz D R Patel H J AWallweber et al ldquoStructuresof APRIL-receptor complexes like BCMA TACI employs onlya single cysteine-rich domain for high affinity ligand bindingrdquoJournal of Biological Chemistry vol 280 no 8 pp 7218ndash72272005
[23] E Castigli S A Wilson L Garibyan et al ldquoTACI is mutantin common variable immunodeficiency and IgA deficiencyrdquoNature Genetics vol 37 no 8 pp 829ndash834 2005
[24] U Salzer H M Chapel A D B Webster et al ldquoMutationsin TNFRSF13B encoding TACI are associated with commonvariable immunodeficiency in humansrdquoNature Genetics vol 37no 8 pp 820ndash828 2005
10 ISRN Immunology
[25] C Bacchelli K F Buckland S Buckridge et al ldquoThe C76Rtransmembrane activator and calcium modulator cyclophilinligand interactor mutation disrupts antibody production andB-cell homeostasis in heterozygous and homozygous micerdquoJournal of Allergy and Clinical Immunology vol 127 no 5 pp1253ndash1259 2011
[26] A J Fried I Rauter S R Dillon H H Jabara and R S GehaldquoFunctional analysis of transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) mutationsassociated with common variable immunodeficiencyrdquo Journalof Allergy and Clinical Immunology vol 128 no 1 pp 226ndash2282011
[27] J J Lee H H Jabara L Garibyan et al ldquoThe C104R mutantimpairs the function of transmembrane activator and calciummodulator and cyclophilin ligand interactor (TACI) throughhaploinsufficiencyrdquo Journal of Allergy and Clinical Immunologyvol 126 no 6 pp 1234ndashe2 2010
[28] J J Lee I Rauter L Garibyan et al ldquoThe murine equivalentof the A181E TACI mutation associated with common variableimmunodeficiency severely impairs B-cell functionrdquo Blood vol114 no 11 pp 2254ndash2262 2009
[29] A A JM van deVen L van de Corput CM van Tilburg et alldquoLymphocyte characteristics in children with common variableimmunodeficiencyrdquoClinical Immunology vol 135 no 1 pp 63ndash71 2010
[30] E Latz A Schoenemeyer A Visintin et al ldquoTLR9 signals aftertranslocating from theER toCpGDNA in the lysosomerdquoNatureImmunology vol 5 no 2 pp 190ndash198 2004
[31] C Foerster N Voelxen M Rakhmanov et al ldquoB cell receptor-mediated calcium signaling is impaired in B lymphocytes oftype Ia patients with common variable immunodeficiencyrdquoJournal of Immunology vol 184 no 12 pp 7305ndash7313 2010
[32] M C Van Zelm J Smet B Adams et al ldquoCD81 gene defectin humans disrupts CD19 complex formation and leads toantibody deficiencyrdquo Journal of Clinical Investigation vol 120no 4 pp 1265ndash1274 2010
[33] M Malphettes L Gerard M Carmagnat et al ldquoLate-onsetcombined immune deficiency a subset of common variableimmunodeficiency with severe T cell defectrdquo Clinical InfectiousDiseases vol 49 no 9 pp 1329ndash1338 2009
[34] F Dedeoglu B Horwitz J Chaudhuri F W Alt and R SGeha ldquoInduction of activation-induced cytidine deaminasegene expression by IL-4 and CD40 ligation is dependent onSTAT6 and NF120581Brdquo International Immunology vol 16 no 3 pp395ndash404 2004
[35] M Muramatsu K Kinoshita S Fagarasan S Yamada YShinkai and T Honjo ldquoClass switch recombination andhypermutation require activation-induced cytidine deaminase(AID) a potential RNA editing enzymerdquo Cell vol 102 no 5 pp553ndash563 2000
[36] P Revy T Muto Y Levy et al ldquoActivation-induced cytidinedeaminase (AID) deficiency causes the autosomal recessiveform of the hyper-IgM syndrome (HIGM2)rdquo Cell vol 102 no5 pp 565ndash575 2000
[37] A M Krieg ldquoCpG motifs in bacterial DNA and their immuneeffectsrdquo Annual Review of Immunology vol 20 pp 709ndash7602002
[38] T Kawai and S Akira ldquoInnate immune recognition of viralinfectionrdquo Nature Immunology vol 7 no 2 pp 131ndash137 2006
[39] E Latz A Verma AVisintin et al ldquoLigand-induced conforma-tional changes allosterically activate Toll-like receptor 9rdquoNatureImmunology vol 8 no 7 pp 772ndash779 2007
[40] S Sato H Sanjo K Takeda et al ldquoEssential function for thekinase TAK1 in innate and adaptive immune responsesrdquoNatureImmunology vol 6 no 11 pp 1087ndash1095 2005
[41] F Mackay and H Leung ldquoThe role of the BAFFAPRIL systemon T cell functionrdquo Seminars in Immunology vol 18 no 5 pp284ndash289 2006
were washed twice and resuspended in Hankrsquos balanced saltsolution (HBSS) supplemented with 10 fetal calf serum(FCS) Cytosolic calcium levels were measured on FacsCanto II after 1 minute 20120583gmL goat anti-human IgMF(ab1015840)
2fragments (Jackson Immunoresearch) were added
After another 5 minutes 20120583gmL ionomycin (CalbiochemSan Diego CA) was added Area under curves was calculatedusing Facs Diva and Prism software Next LCL were stainedand resuspended in calcium-free phosphate-buffered saline(PBS) Calcium chloridewas added after intracellular calciumstorage depletion with ionomycin or thapsigargin (SantaCruz Biotechnology Santa Cruz CA)
24 In Vitro Stimulation Assays and PCR LCL were culturedin RPMI 1640 containing 10 FCS at the subsequentconditions mouse anti-TACI (05 120583gmL RampD systemsMinneapolis MN USA) and goat anti-mouse IgGmicrobeads (Miltenyi Biotec Carlsbad CA) with or withoutIL-4 (100UmL Immunotools) CpG phosphorothioate-modified oligodeoxynucleotide 2006 (05 120583gmL AlexisBiochemicals) IL-10 (10 ngmL Becton Dickinson) oranti-CD40 (01120583gmL RampD systems) After 48 hours cellswere lysed and total mRNA was isolated using Tripureisolation reagent (Roche Diagnostics Mannheim Germany)according to the manufacturerrsquos instructions Reversetranscriptionwas performed using an iScript cDNA synthesiskit (BioradHercules CA) Primersweremixedwith IQSYBRgreen supermix (BioRad) The detection run started at 95∘Cfor 10min followed by 45 cycles of 95∘C for 15 s and 60∘Cfor 1min Assays were performed in duplicate or triplicateas 15 120583L reactions in 96-well plates using C1000 ThermalCycler (BioRad) Results were normalized to the endogenousGAPDH mRNA (2minusΔCT) The following primers wereused GAPDH forward 51015840-GTCGGAGTCAACGGATT-31015840GAPDH reverse 51015840-AAGCTTCCCGTTCTCAG-31015840 AICDAforward 51015840-TGCTCTTCCTCCGCTACATCTC-31015840 AICDAreverse 51015840-AACCTCATACAGGGGCAAAACC-31015840 NF120581Bforward 51015840-GAAGCACGAATGACAGAGGC-31015840 NF120581B rev-erse 51015840-GCTTGGCGGATTAGCTCTTT-31015840 NFAT forward51015840-GCAGAGCACGGACAGCTATC-31015840 NFAT reverse 51015840-GGGCTTTCTCCACGAAAATGA-31015840 (All Sigma-Aldrich)
3 Results
31 Clinical Evaluation of the CVID Index Patient Theindex patient (Figure 2(a) arrow) was referred at age 11for recurrent upper and lower respiratory tract infectionsand splenomegaly Laboratory investigations showed absentisohemagglutinins and decreased serum titers of total IgGIgA IgM IgG
1 and IgG
2(Table 1) She was lymphopenic
with low numbers of B cells CD4+ and CD8+ T cells andalmost absent class switched memory B cells (Figure 1(a))Functionally in vitro T-cell proliferation assays showed apositive response to mitogens and an incomplete response toparticularly viral antigens A diagnosis of CVID was madeand immunoglobulin replacement therapy was initiated
After some time she developed CVID-related diseasemanifestations including autoimmune hemolytic anemia
(AIHA) idiopathic thrombocytopenic purpura (ITP) sub-mandibular lymphadenopathy and autoimmune nephritisa renal biopsy showed tubulo interstitial nephritis withgranulomatous changes the estimated glomerular filtratedrate was 412mLmin173m2 (as calculated by Schwartzformula) Furthermore she developed exercise-induced dys-pnea a high-resolution computed tomography (HRCT) scanshowed airway and interstitial lung disease with bronchiec-tasis and peribronchovascular abnormalities consisting offocal lesions with ground glass aspect peribronchial con-solidations bronchial wall thickening and air trapping(Figure 2(b)) A broncho alveolar lavage showed no indi-cation for viral bacterial or fungal infections Pulmonaryfindings were suggestive for granulomatous disease and low-dose steroids and tacrolimus were initiated resulting in nor-malization of pulmonary function and glomerular filtrationrate Furthermore viral immunity appeared to be affectedas cytomegalovirus was repeatedly detectable with viral loadsof 100ndash2000 copiesmL in plasma Treatment with tacrolimuswas complicated by tubulopathy and therefore mycopheno-late mofetil was started with good initial response howevercausing pancreatitis Rituximab was administered with pos-itive effect on lymphadenopathy AIHA and ITP Despiteall treatment efforts taken disease manifestations progressedfurther and became refractory The index patient underwentallogeneic stem cell transplantation with matched unrelatedcord blood after myeloablative conditioning but eventuallydied of the complications of graft versus hose disease com-bined with multiple infections
32 Clinical and Immunological Evaluation of Family Mem-bers Further studies were performed to determine the roleof these TACI variants Clinically the parents and sibling2 were healthy sibling 1 developed clinical symptoms ofCVID during the course of investigations Serum Ig levelswere normal in both parents and sibling 2 apart from amodestly decreased serum IgA In contrast sibling 1 washypogammaglobulinemic for IgG IgA and subclasses IgG
1
IgG2 and IgG
4 albeit modestly in comparison to the index
patient (Table 1)To study specific antibody synthesis parents and sibling 1
and 2 were vaccinated with 23-valent Pneumovax (AdventisPasteur MSD) and total IgG antibody titers were measuredafter 1 month Parents and sibling 2 had adequate T cell-independent responses in vivo to pneumococcal polysaccha-rides while the response of sibling 1 was suboptimal (Table 1)
Consequently immunological evaluation was extendedin siblings 1 revealing an increase in relative and absolutenumbers of CD8+ T cells Total B cells were increased(217 of lymphocytes 512 cells120583L) and the compositionof B-cell compartment remained normal Proliferative T-cellresponses in vitro were normal to mitogens but partiallyimpaired to recall antigens similar to the index patientTotal B-cell count and composition of B-cell compartment ofsibling 2 were normal (Figure 1(b))
Sibling 1 slowly developed clinical symptoms consistingof diarrhea fatigue and persistent rhinosinusitis refractory toantibiotic prophylaxisThe spleenwas enlarged on abdominal
4 ISRN Immunology
IgD
13395
01
941 11
01
IgM CD27
IgD
IgD
CD38
CD19+ lymphocytesIndex patient
(a)
94
9
756793
104
IgM CD27
65
CD38
Healthy sibling
IgD
IgD
IgD
(b)
Figure 1 B-cell subpopulations of index patient and healthy sibling (a) B cell subpopulations of index patient showing 941 naıve +transitional B cells (IgM+IgD+CD27minus) 11 Marginal Zone B cells (IgM+IgD+CD27+) 01 class-switched B cells (IgDminusIgMminusCD27+)and 133 transitional B cells (IgD+CD38+) (b) B cell subpopulations of healthy sibling showing 756 naıve + transitional B cells(IgM+IgD+CD27minus) 94Marginal Zone B cells (IgM+IgD+CD27+) 9 class-switched B cells (IgDminusIgMminusCD27+) and 65 transitional Bcells (IgD+CD38+)
echography Based on these clinical and laboratory findingsimmunoglobulin replacement therapy was initiated whichled to significant clinical improvement Further studies werefocused on the index patient and asymptomatic sibling 2
33 TNFRSF13B Compound Heterozygous Variants in theIndex Patient and Asymptomatic Sibling TACI is a CVIDsusceptibility factor and to substantiate the diagnosis CVIDwe performed gene sequencing of the TACI gene Analysisof the patientrsquos TNFRSF13B gene showed two variants C104Rand A181E Subsequently her family was investigated reveal-ing that each parent carried one variant (Figure 2(a)) Thebrother (sibling 3) had two normal TACI alleles and was notfurther investigated The two sisters (siblings 1 and 2) werealso compound heterozygous for A181EC104R (Figure 2(a))Sequencing of the complete TNFRSF13B gene revealed noadditional mutations [22] The index patient and sibling 2exhibited identical alternative splicing patterns of exon 2 thatencode the CRD1 important in trimer formation of TACI[22] This finding however did not help explain the different
clinical and B-cell activation phenotype between our indexpatient and sibling 2
34 TACI Protein Expression and Function in the IndexPatient and Asymptomatic Sibling Lymphoblastoid cell lines(LCL) were generated of the index patient and sibling 2 andcompared to cell lines of healthy controls CVID patients and2 CVID patients with a monoallelic A181E variant LCL of allsubjects exhibited TACI expression on the cell membraneInterestingly the TACI mean fluorescence intensity of theindex patient was lower in comparison with the othersubjects especially with sibling 2 fromwhomLCL repeatedlydisplayed the highest cell surface expression (Figure 2(c))Subsequently intracellular TACI was measured to study thepossibility of impaired transition of TACI to the cell surfaceand ensuing intracellular TACI accumulation IntracellularTACI however correlated with the levels of cell surface TACI(Pearson correlation 070 119875 = 0037) and was also repeatedlylower in the index patient (Figure 2(c)) Basal TACI mRNAlevels varied within a similar range for all LCL including theindex patient and sibling (Figure 2(d))
Figure 2 TACI expression and function are decreased in the CVID patient compared to her asymptomatic sibling (a) Pedigree of the studiedfamily Circles display females squares display males The index patient is indicated with an arrow (b) Chest high-resolution CT scan of theindex patient revealing peribronchial consolidations with ground glass appearance and peribronchial thickening ((c)-(d)) TACI surface (cii) or intracellular (c iii) protein and mRNA expression Light grey line indicated isotype indicated dark grey TACI (c i) Results of 5 healthydonors 3 CVID patients with normal TACI 2 CVID patients withmonoallelic A181E the index patient and sibling 2 For (d) average results of3 independent experiments are shown (e) AID (left) and NFAT (right) mRNA induction after 48 h culture of LCL with the indicated stimuliBars represent means plusmn standard error of the mean
Next we investigated TACI function by measuring itsability to induce activation-induced cytidine deaminase(AICDA) gene transcription The AICDA gene productactivation-induced deaminase (AID) is an enzyme of piv-otal importance for secondary antibody diversification [30]Healthy control and CVID patient LCL with normal bial-lelic TACI showed AID mRNA induction upon stimulation
with agonistic anti-TACI (Figure 2(e)) CVID patients withmonoallelic A181E variants showed minimal AICDA genetranscription The index patient had no AICDA gene tran-scription upon TACI stimulation while AID mRNA levelsof sibling 2 LCL were comparable to B cells with normalTACIThe same pattern was noticed for NFATmRNA induc-tion gene transcription upon TACI triggering was lower in
6 ISRN Immunology
Table 1 Immunological characteristics of patient and family
Patient Sibling 1 Sibling 2 Mother FatherGender C C C C D
the index patient compared to sibling 2 There were nodifferences in NF120581B induction upon TACI stimulation Stim-ulation of the CD40 pathway showed adequate induction ofAID and NFAT mRNA in the index patient IL-10 treatmentwas included as negative control that does not induce AICDAgene expression Taken together TACI protein expressionand function were increased in sibling 2 when compared tothe index patient
35 Appearance of TLR9 Protein Expression and Function inthe Asymptomatic Sibling We considered two possibilitiesthat may explain why the index patient and sibling 2 havingidentical TACI gene mutations exhibit divergent healthconsequences either the index patient had an additionalimmune defect which in combinationwith the TACI variantswould lead to CVID disease or sibling 2 has compensatoryimmune function in the TACI signaling route that preventsCVID disease from occurring First as defects in the B-cell receptor (BCR) and its coreceptor complex have beendescribed [31 32] we screened the BCR signalosome CD19CD21 and CD20 cell surface expression of the index patientwere comparable to expression on control LCL (data notshown) Functionally early B-cell activation seemed intactas measured by calcium mobilization after BCR stimulationof fresh PBMC (Figure 3(a)) The index patient showed
a normal initial but rapidly declining calcium influx aspreviously observed in CVID patients with autoimmunesymptoms (own unpublished observations) and area undercurves were comparable to controls As the index patientrsquosendoplasmic reticulum (ER) calcium storage capacitywas notdecreased compared to sibling 2 an additional defect in BCRsignaling was considered improbable
Second the classical route of BCR isotype class switchingis via CD40 with CD40L on T cells and it was recentlyshown that TACI and CD40 signaling can act in synergy[13 15] However CD40 cell surface expression (Figure 3(b))and CD40-mediated gene transcription (Figures 2(e) and3(b)) were comparable between all LCL suggesting that CD40signaling in the index patient B cells was intact at least in thisassay
Third we explored TLR9 signaling as TLR9 defects havebeen described in CVID [2 3] and TLR9 and TACI signalingemploy common downstream signaling pathways [15 16]Noticeably TLR9 expression was nearly twofold higher insibling 2 compared to other LCL and this increased expres-sion was accompanied by an increased TLR9 expression andfunction (Figure 3(c)) supporting the possibility that com-pensatory TLR9 expression contributes as a mechanism foraltered TACI A181EC104R protein in restoring B-cell overallcapacity to transmit signals towards NFAT and AID Thus
ISRN Immunology 7
CVID patientSibling 1Sibling 2
MotherControl
Anti-IgM Ionomycin
25
20
15
10
5
3
2
1
10000
200 300 400Time (s)
BCR-mediated calcium flux (PBMC)Cy
toso
lic C
a2+
leve
l
(a)
0
5000
10000
15000
Mea
n flu
ores
cenc
e int
ensit
y
Cell surface CD40
Con
trol
(TAC
I nor
mal
)
CVID
(TAC
I nor
mal
)
CVID
(A18
1En
orm
al)
Patie
nt(A
181E
C10
4R)
Sibl
ing
(A18
1EC
104R
)
(b)
IL-10 CpG ODN Anti-CD40
AID mRNA
0AID
mRN
A (r
elat
ive t
o G
APD
H) 20
15
10
5
TLR9
Mea
n flu
ores
cenc
e int
ensit
y
15
10
5
00
Intracellular TLR9times10
2
Con
trol
(TAC
I nor
mal
)CV
ID(T
ACI n
orm
al)
CVID
(A18
1En
orm
al)
Patie
nt(A
181E
C10
4R)
Sibl
ing
(A18
1EC
104R
)
Control (TACI normal)CVID (TACI normal)CVID (A181Enormal)
Patient (A181EC104R)Sibling (A181EC104R)
(c)
Figure 3 Similar BCR signaling but possibly enhanced TLR9 signaling in the healthy sibling (a) Calcium mobilization upon triggering ofthe BCR using IgM F(ab1015840)
2fragments in freshly isolated PBMCs (b) CD40 surface expression asmeasured by flow cytometry (c) TLR9 B-cell
expression (c ii) and function (c iii) Light grey line indicated isotype indicated dark grey TLR9 (c i) Bars represent means plusmn standard errorof the mean
our data supports the notion that compensatory signaling inthe TACI route in the case of sibling 2 via enhanced TLR9signaling may prevent the development of CVID disease
4 Discussion
We here describe differential immunological phenotypesdisplayed by siblings with identical genetic TACI variantsThis family provides the unique opportunity to study the role
and significance of the humanC104R andA181E variants bothin vivo and in vitro
The index patient had severe CVID which graduallyevolved to late onset combined immunodeficiency a phe-nomenon previously described in CVID patients [33] Sibling1 did eventually develop mild CVID but without autoim-mune manifestations To date sibling 2 remains free ofsymptoms and has excellent specific antibody responsesnevertheless this sibling may still develop CVID at a laterage Previous studies have shown that C104R abrogates ligand
8 ISRN Immunology
binding due to haploinsufficiency whereas in the A181Evariant ligand binding remains intact [21 26] At least aproportion of A181EC104R TACI trimers will thus be ableto bind ligand As NF120581B activation was strongly decreasedin both variants we hypothesized TACI function to beaffected AID and NFAT mRNA induction were reduced inthe index patient and seemed also reduced in the CVIDpatients with a heterozygous A181E variant [14] Converselysibling 2 showed AID and NFAT induction comparable tohealthy controls and CVID patients with unaffected TACIalleles NFAT activation induces transcription of cytokinegenes encoding for example IL-4 which is involved in classswitch recombination IL-4 and CD40 ligation induces AIDmRNA and protein expression and depends on STAT6 andNF120581B [34] AID is pivotal for class switch recombination andsomatic hypermutation of theBCR [35 36] bothmechanismsare routinely impaired in CVID
The differential TACI function in the index patient andsibling was accompanied by a change in TACI protein levelsshowing increased TACI expression in the sibling both atthe cell surface and intracellular level TACI protein levels inthe healthy sibling were increased in comparison to healthycontrols as well As TNFRSF13B gene transcription wascomparable between all subjects except for the healthy sib-ling these observations suggest that protein turnover in thehealthy sibling may be decreased possibly due to alterationsin posttranslational modification of TACI protein affectingprotein stability and folding transport or degradation
Alternatively altered TACI function in the healthy siblingwas compensated by increasing the TACI protein quantitythrough cross-talk with related immune pathways CVIDis generally assumed to be a polygenetic disorder and itis plausible that alterations in the index patient were notlimited to the TNFRSF13B gene This prompted us to analyzeother essential immune pathways in B lymphocytes andwe investigated CD40 BCR and TLR9 signaling We didnot find any additional defect in BCR or CD40 signalingNevertheless for both pathways defective interaction withother cells or factors such as abrogated CD40L ligation onT cells or CD21- complement C3d fragment interactionscannot be excluded
In contrast TLR9 expression and function were con-siderably dissimilar between index patient and sibling 2Since the TLR9 expression levels and function were compa-rable between the index patient and the other subjects butincreased in sibling 2 we hypothesize that compensatoryTLR9 expression occurs in the sibling TLR9 recognizesunmethylated CpG-motif-containing bacterial or viral DNA[37 38] and has a critical role in the prevention of severalbacterial and viral infections Ligation of TLR9 inducesrecruitment of MyD88 [30 39] initiating several signalingevents which eventually result in activation of NF120581B and AP-1 [40] By the same token increased TACI protein stabilitymay participate in a compensatory mechanism in the healthysibling possibly related to TLR9 signaling TLR9 engagementinduced TACI upregulation and enhanced TACI-mediated Igclass switching of effector B cells resulting in increased IgGsecretion in cells exposed to anti-TACI and IL-10 and inducedsecretion of IgA
This study has some limitations The TLR9 signalingpathway through MyD88 could be studied more extensivelynot only on a functional level but also through whole exomesequencing The effects of TACI malfunctioning on expres-sion and function of several other targets could have beenaddressed including other TNF receptors and their ligandsor signaling proteins downstream of TACI such as TRAFsCAML and AP-1 Additional defects may not be limitedto B-cell lymphocytes TACI expression can be induced inCD4+ and CD8 T+ cells [4 41] study of TACI-related T-cellfunction would be particularly interesting since the patientdemonstrated chronic viral infections in vivo and impairedantigenic T-cells responses to several viruses in vitro
5 Conclusion
We here describe the diverse clinical and immunologicalcharacteristics in siblings with an identical TACI geno-type These clinical differences are supported by differencesin TACI protein expression and function We proposethat compensatory regulatory mechanisms involving post-translational modification of TACI protein and TLR9 existand may overcome the altered function of TACI trimersin asymptomatic TACI A181EC104R individuals Additionalstudies of TACI protein homeostasis the TACI signalingpathway and interactionwithTLR9 and expression and func-tion of TLR9 downstream signaling molecules are thereforeadvisable
Joris M van Montfrans and Marianne Boes contributedequally
Acknowledgments
The authors would like to thank the technicians of theDepartment of Medical Immunology for excellent assistancewith the TACI sequencing setup and Ewoud Compeer andArie Jan Stoppelenburg for help with PCR and data analyses
References
[1] M A Park J T Li J B Hagan D E Maddox and R SAbraham ldquoCommon variable immunodeficiency a new lookat an old diseaserdquo The Lancet vol 372 no 9637 pp 489ndash5022008
[2] C Cunningham-Rundles L Radigan A K Knight L ZhangL Bauer and A Nakazawa ldquoTLR9 activation is defective incommon variable immune deficiencyrdquo Journal of Immunologyvol 176 no 3 pp 1978ndash1987 2006
[3] J E Yu A K Knight L Radigan et al ldquoToll-like receptor 7and 9 defects in common variable immunodeficiencyrdquo Journalof Allergy and Clinical Immunology vol 124 no 2 pp 349ndash3562009
[4] G-U Von Bulow and R J Bram ldquoNF-AT activation inducedby a CAML-interacting member of the tumor necrosis factorreceptor superfamilyrdquo Science vol 278 no 5335 pp 138ndash1411997
[5] P Schneider F Mackay V Steiner et al ldquoBAFF a novel ligandof the tumor necrosis factor family stimulates B cell growthrdquoJournal of Experimental Medicine vol 189 no 11 pp 1747ndash17561999
[6] Y Wu D Bressette J A Carrell et al ldquoTumor necrosis factor(TNF) receptor superfamily member TACI is a high affinityreceptor for TNF family members APRIL and BLySrdquo Journal ofBiological Chemistry vol 275 no 45 pp 35478ndash35485 2000
[7] S A Marsters M Yan R M Pitti P E Haas V M Dixit andA Ashkenazi ldquoInteraction of the TNF homologues BLyS andAPRIL with the TNF receptor homologues BCMA and TACIrdquoCurrent Biology vol 10 no 13 pp 785ndash788 2000
[8] D Bischof S F Elsawa G Mantchev et al ldquoSelective activationof TACI by syndecan-2rdquo Blood vol 107 no 8 pp 3235ndash32422006
[9] F Mackay P Schneider P Rennert and J Browning ldquoBAFFand APRIL a tutorial on B cell survivalrdquo Annual Review ofImmunology vol 21 pp 231ndash264 2003
[10] Y Laabi M P Gras F Carbonnel et al ldquoA new gene BCMon chromosome 16 is fused to the interleukin 2 gene by at(416)(q26p13) translocation in a malignant T cell lymphomardquoEMBO Journal vol 11 no 11 pp 3897ndash3904 1992
[11] J S Thompson S A Bixler F Qian et al ldquoBAFF-R a newlyidentified TNF receptor that specifically interacts with BAFFrdquoScience vol 293 no 5537 pp 2108ndash2111 2001
[12] J Hauer S Puschner P Ramakrishnan et al ldquoTNF receptor(TNFR)-associated factor (TRAF) 3 serves as an inhibitorof TRAF25-mediated activation of the noncanonical NF-120581Bpathway by TRAF-binding TNFRsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 102 no8 pp 2874ndash2879 2005
[13] E Castigli S AWilsonA Elkhal EOzcan LGaribyan andRS Geha ldquoTransmembrane activator and calciummodulator andcyclophilin ligand interactor enhances CD40-driven plasmacell differentiationrdquo Journal of Allergy and Clinical Immunologyvol 120 no 4 pp 885ndash891 2007
[14] E Ozcan L Garibyan J J-Y Lee R J Bram K-P Lamand R S Geha ldquoTransmembrane activator calciummodulatorand cyclophilin ligand interactor drives plasma cell differenti-ation in LPS-activated B cellsrdquo Journal of Allergy and ClinicalImmunology vol 123 no 6 pp 1277ndash1286 2009
[15] E Ozcan I Rauter L Garibyan S R Dillon and R S GehaldquoToll-like receptor 9 transmembrane activator and calcium-modulating cyclophilin ligand interactor and CD40 synergizein causing B-cell activationrdquo Journal of Allergy and ClinicalImmunology vol 128 no 3 pp 601ndash609 2011
[16] B He R Santamaria W Xu et al ldquoThe transmembrane activa-tor TACI triggers immunoglobulin class switching by activatingB cells through the adaptorMyD88rdquoNature Immunology vol 11no 9 pp 836ndash845 2010
[17] U Salzer C Bacchelli S Buckridge et al ldquoRelevance of biallelicversus monoallelic TNFRSF13B mutations in distinguishingdisease-causing from risk-increasing TNFRSF13B variants inantibody deficiency syndromesrdquo Blood vol 113 no 9 pp 1967ndash1976 2009
[18] L Zhang L Radigan U Salzer et al ldquoTransmembrane acti-vator and calcium-modulating cyclophilin ligand interactormutations in common variable immunodeficiency clinical andimmunologic outcomes in heterozygotesrdquo Journal of Allergy andClinical Immunology vol 120 no 5 pp 1178ndash1185 2007
[19] Q Pan-Hammarstrom U Salzer L Du et al ldquoReexamining therole of TACI coding variants in common variable immunode-ficiency and selective IgA deficiencyrdquo Nature Genetics vol 39no 4 pp 429ndash430 2007
[20] R M Locksley N Killeen and M J Lenardo ldquoThe TNF andTNF receptor superfamilies integrating mammalian biologyrdquoCell vol 104 no 4 pp 487ndash501 2001
[21] L Garibyan A A Lobito R M Siegel M E Call K WWucherpfennig and R S Geha ldquoDominant-negative effect ofthe heterozygous C104R TACI mutation in common variableimmunodeficiency (CVID)rdquo Journal of Clinical Investigationvol 117 no 6 pp 1550ndash1557 2007
[22] S GHymowitz D R Patel H J AWallweber et al ldquoStructuresof APRIL-receptor complexes like BCMA TACI employs onlya single cysteine-rich domain for high affinity ligand bindingrdquoJournal of Biological Chemistry vol 280 no 8 pp 7218ndash72272005
[23] E Castigli S A Wilson L Garibyan et al ldquoTACI is mutantin common variable immunodeficiency and IgA deficiencyrdquoNature Genetics vol 37 no 8 pp 829ndash834 2005
[24] U Salzer H M Chapel A D B Webster et al ldquoMutationsin TNFRSF13B encoding TACI are associated with commonvariable immunodeficiency in humansrdquoNature Genetics vol 37no 8 pp 820ndash828 2005
10 ISRN Immunology
[25] C Bacchelli K F Buckland S Buckridge et al ldquoThe C76Rtransmembrane activator and calcium modulator cyclophilinligand interactor mutation disrupts antibody production andB-cell homeostasis in heterozygous and homozygous micerdquoJournal of Allergy and Clinical Immunology vol 127 no 5 pp1253ndash1259 2011
[26] A J Fried I Rauter S R Dillon H H Jabara and R S GehaldquoFunctional analysis of transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) mutationsassociated with common variable immunodeficiencyrdquo Journalof Allergy and Clinical Immunology vol 128 no 1 pp 226ndash2282011
[27] J J Lee H H Jabara L Garibyan et al ldquoThe C104R mutantimpairs the function of transmembrane activator and calciummodulator and cyclophilin ligand interactor (TACI) throughhaploinsufficiencyrdquo Journal of Allergy and Clinical Immunologyvol 126 no 6 pp 1234ndashe2 2010
[28] J J Lee I Rauter L Garibyan et al ldquoThe murine equivalentof the A181E TACI mutation associated with common variableimmunodeficiency severely impairs B-cell functionrdquo Blood vol114 no 11 pp 2254ndash2262 2009
[29] A A JM van deVen L van de Corput CM van Tilburg et alldquoLymphocyte characteristics in children with common variableimmunodeficiencyrdquoClinical Immunology vol 135 no 1 pp 63ndash71 2010
[30] E Latz A Schoenemeyer A Visintin et al ldquoTLR9 signals aftertranslocating from theER toCpGDNA in the lysosomerdquoNatureImmunology vol 5 no 2 pp 190ndash198 2004
[31] C Foerster N Voelxen M Rakhmanov et al ldquoB cell receptor-mediated calcium signaling is impaired in B lymphocytes oftype Ia patients with common variable immunodeficiencyrdquoJournal of Immunology vol 184 no 12 pp 7305ndash7313 2010
[32] M C Van Zelm J Smet B Adams et al ldquoCD81 gene defectin humans disrupts CD19 complex formation and leads toantibody deficiencyrdquo Journal of Clinical Investigation vol 120no 4 pp 1265ndash1274 2010
[33] M Malphettes L Gerard M Carmagnat et al ldquoLate-onsetcombined immune deficiency a subset of common variableimmunodeficiency with severe T cell defectrdquo Clinical InfectiousDiseases vol 49 no 9 pp 1329ndash1338 2009
[34] F Dedeoglu B Horwitz J Chaudhuri F W Alt and R SGeha ldquoInduction of activation-induced cytidine deaminasegene expression by IL-4 and CD40 ligation is dependent onSTAT6 and NF120581Brdquo International Immunology vol 16 no 3 pp395ndash404 2004
[35] M Muramatsu K Kinoshita S Fagarasan S Yamada YShinkai and T Honjo ldquoClass switch recombination andhypermutation require activation-induced cytidine deaminase(AID) a potential RNA editing enzymerdquo Cell vol 102 no 5 pp553ndash563 2000
[36] P Revy T Muto Y Levy et al ldquoActivation-induced cytidinedeaminase (AID) deficiency causes the autosomal recessiveform of the hyper-IgM syndrome (HIGM2)rdquo Cell vol 102 no5 pp 565ndash575 2000
[37] A M Krieg ldquoCpG motifs in bacterial DNA and their immuneeffectsrdquo Annual Review of Immunology vol 20 pp 709ndash7602002
[38] T Kawai and S Akira ldquoInnate immune recognition of viralinfectionrdquo Nature Immunology vol 7 no 2 pp 131ndash137 2006
[39] E Latz A Verma AVisintin et al ldquoLigand-induced conforma-tional changes allosterically activate Toll-like receptor 9rdquoNatureImmunology vol 8 no 7 pp 772ndash779 2007
[40] S Sato H Sanjo K Takeda et al ldquoEssential function for thekinase TAK1 in innate and adaptive immune responsesrdquoNatureImmunology vol 6 no 11 pp 1087ndash1095 2005
[41] F Mackay and H Leung ldquoThe role of the BAFFAPRIL systemon T cell functionrdquo Seminars in Immunology vol 18 no 5 pp284ndash289 2006
Figure 1 B-cell subpopulations of index patient and healthy sibling (a) B cell subpopulations of index patient showing 941 naıve +transitional B cells (IgM+IgD+CD27minus) 11 Marginal Zone B cells (IgM+IgD+CD27+) 01 class-switched B cells (IgDminusIgMminusCD27+)and 133 transitional B cells (IgD+CD38+) (b) B cell subpopulations of healthy sibling showing 756 naıve + transitional B cells(IgM+IgD+CD27minus) 94Marginal Zone B cells (IgM+IgD+CD27+) 9 class-switched B cells (IgDminusIgMminusCD27+) and 65 transitional Bcells (IgD+CD38+)
echography Based on these clinical and laboratory findingsimmunoglobulin replacement therapy was initiated whichled to significant clinical improvement Further studies werefocused on the index patient and asymptomatic sibling 2
33 TNFRSF13B Compound Heterozygous Variants in theIndex Patient and Asymptomatic Sibling TACI is a CVIDsusceptibility factor and to substantiate the diagnosis CVIDwe performed gene sequencing of the TACI gene Analysisof the patientrsquos TNFRSF13B gene showed two variants C104Rand A181E Subsequently her family was investigated reveal-ing that each parent carried one variant (Figure 2(a)) Thebrother (sibling 3) had two normal TACI alleles and was notfurther investigated The two sisters (siblings 1 and 2) werealso compound heterozygous for A181EC104R (Figure 2(a))Sequencing of the complete TNFRSF13B gene revealed noadditional mutations [22] The index patient and sibling 2exhibited identical alternative splicing patterns of exon 2 thatencode the CRD1 important in trimer formation of TACI[22] This finding however did not help explain the different
clinical and B-cell activation phenotype between our indexpatient and sibling 2
34 TACI Protein Expression and Function in the IndexPatient and Asymptomatic Sibling Lymphoblastoid cell lines(LCL) were generated of the index patient and sibling 2 andcompared to cell lines of healthy controls CVID patients and2 CVID patients with a monoallelic A181E variant LCL of allsubjects exhibited TACI expression on the cell membraneInterestingly the TACI mean fluorescence intensity of theindex patient was lower in comparison with the othersubjects especially with sibling 2 fromwhomLCL repeatedlydisplayed the highest cell surface expression (Figure 2(c))Subsequently intracellular TACI was measured to study thepossibility of impaired transition of TACI to the cell surfaceand ensuing intracellular TACI accumulation IntracellularTACI however correlated with the levels of cell surface TACI(Pearson correlation 070 119875 = 0037) and was also repeatedlylower in the index patient (Figure 2(c)) Basal TACI mRNAlevels varied within a similar range for all LCL including theindex patient and sibling (Figure 2(d))
Figure 2 TACI expression and function are decreased in the CVID patient compared to her asymptomatic sibling (a) Pedigree of the studiedfamily Circles display females squares display males The index patient is indicated with an arrow (b) Chest high-resolution CT scan of theindex patient revealing peribronchial consolidations with ground glass appearance and peribronchial thickening ((c)-(d)) TACI surface (cii) or intracellular (c iii) protein and mRNA expression Light grey line indicated isotype indicated dark grey TACI (c i) Results of 5 healthydonors 3 CVID patients with normal TACI 2 CVID patients withmonoallelic A181E the index patient and sibling 2 For (d) average results of3 independent experiments are shown (e) AID (left) and NFAT (right) mRNA induction after 48 h culture of LCL with the indicated stimuliBars represent means plusmn standard error of the mean
Next we investigated TACI function by measuring itsability to induce activation-induced cytidine deaminase(AICDA) gene transcription The AICDA gene productactivation-induced deaminase (AID) is an enzyme of piv-otal importance for secondary antibody diversification [30]Healthy control and CVID patient LCL with normal bial-lelic TACI showed AID mRNA induction upon stimulation
with agonistic anti-TACI (Figure 2(e)) CVID patients withmonoallelic A181E variants showed minimal AICDA genetranscription The index patient had no AICDA gene tran-scription upon TACI stimulation while AID mRNA levelsof sibling 2 LCL were comparable to B cells with normalTACIThe same pattern was noticed for NFATmRNA induc-tion gene transcription upon TACI triggering was lower in
6 ISRN Immunology
Table 1 Immunological characteristics of patient and family
Patient Sibling 1 Sibling 2 Mother FatherGender C C C C D
the index patient compared to sibling 2 There were nodifferences in NF120581B induction upon TACI stimulation Stim-ulation of the CD40 pathway showed adequate induction ofAID and NFAT mRNA in the index patient IL-10 treatmentwas included as negative control that does not induce AICDAgene expression Taken together TACI protein expressionand function were increased in sibling 2 when compared tothe index patient
35 Appearance of TLR9 Protein Expression and Function inthe Asymptomatic Sibling We considered two possibilitiesthat may explain why the index patient and sibling 2 havingidentical TACI gene mutations exhibit divergent healthconsequences either the index patient had an additionalimmune defect which in combinationwith the TACI variantswould lead to CVID disease or sibling 2 has compensatoryimmune function in the TACI signaling route that preventsCVID disease from occurring First as defects in the B-cell receptor (BCR) and its coreceptor complex have beendescribed [31 32] we screened the BCR signalosome CD19CD21 and CD20 cell surface expression of the index patientwere comparable to expression on control LCL (data notshown) Functionally early B-cell activation seemed intactas measured by calcium mobilization after BCR stimulationof fresh PBMC (Figure 3(a)) The index patient showed
a normal initial but rapidly declining calcium influx aspreviously observed in CVID patients with autoimmunesymptoms (own unpublished observations) and area undercurves were comparable to controls As the index patientrsquosendoplasmic reticulum (ER) calcium storage capacitywas notdecreased compared to sibling 2 an additional defect in BCRsignaling was considered improbable
Second the classical route of BCR isotype class switchingis via CD40 with CD40L on T cells and it was recentlyshown that TACI and CD40 signaling can act in synergy[13 15] However CD40 cell surface expression (Figure 3(b))and CD40-mediated gene transcription (Figures 2(e) and3(b)) were comparable between all LCL suggesting that CD40signaling in the index patient B cells was intact at least in thisassay
Third we explored TLR9 signaling as TLR9 defects havebeen described in CVID [2 3] and TLR9 and TACI signalingemploy common downstream signaling pathways [15 16]Noticeably TLR9 expression was nearly twofold higher insibling 2 compared to other LCL and this increased expres-sion was accompanied by an increased TLR9 expression andfunction (Figure 3(c)) supporting the possibility that com-pensatory TLR9 expression contributes as a mechanism foraltered TACI A181EC104R protein in restoring B-cell overallcapacity to transmit signals towards NFAT and AID Thus
ISRN Immunology 7
CVID patientSibling 1Sibling 2
MotherControl
Anti-IgM Ionomycin
25
20
15
10
5
3
2
1
10000
200 300 400Time (s)
BCR-mediated calcium flux (PBMC)Cy
toso
lic C
a2+
leve
l
(a)
0
5000
10000
15000
Mea
n flu
ores
cenc
e int
ensit
y
Cell surface CD40
Con
trol
(TAC
I nor
mal
)
CVID
(TAC
I nor
mal
)
CVID
(A18
1En
orm
al)
Patie
nt(A
181E
C10
4R)
Sibl
ing
(A18
1EC
104R
)
(b)
IL-10 CpG ODN Anti-CD40
AID mRNA
0AID
mRN
A (r
elat
ive t
o G
APD
H) 20
15
10
5
TLR9
Mea
n flu
ores
cenc
e int
ensit
y
15
10
5
00
Intracellular TLR9times10
2
Con
trol
(TAC
I nor
mal
)CV
ID(T
ACI n
orm
al)
CVID
(A18
1En
orm
al)
Patie
nt(A
181E
C10
4R)
Sibl
ing
(A18
1EC
104R
)
Control (TACI normal)CVID (TACI normal)CVID (A181Enormal)
Patient (A181EC104R)Sibling (A181EC104R)
(c)
Figure 3 Similar BCR signaling but possibly enhanced TLR9 signaling in the healthy sibling (a) Calcium mobilization upon triggering ofthe BCR using IgM F(ab1015840)
2fragments in freshly isolated PBMCs (b) CD40 surface expression asmeasured by flow cytometry (c) TLR9 B-cell
expression (c ii) and function (c iii) Light grey line indicated isotype indicated dark grey TLR9 (c i) Bars represent means plusmn standard errorof the mean
our data supports the notion that compensatory signaling inthe TACI route in the case of sibling 2 via enhanced TLR9signaling may prevent the development of CVID disease
4 Discussion
We here describe differential immunological phenotypesdisplayed by siblings with identical genetic TACI variantsThis family provides the unique opportunity to study the role
and significance of the humanC104R andA181E variants bothin vivo and in vitro
The index patient had severe CVID which graduallyevolved to late onset combined immunodeficiency a phe-nomenon previously described in CVID patients [33] Sibling1 did eventually develop mild CVID but without autoim-mune manifestations To date sibling 2 remains free ofsymptoms and has excellent specific antibody responsesnevertheless this sibling may still develop CVID at a laterage Previous studies have shown that C104R abrogates ligand
8 ISRN Immunology
binding due to haploinsufficiency whereas in the A181Evariant ligand binding remains intact [21 26] At least aproportion of A181EC104R TACI trimers will thus be ableto bind ligand As NF120581B activation was strongly decreasedin both variants we hypothesized TACI function to beaffected AID and NFAT mRNA induction were reduced inthe index patient and seemed also reduced in the CVIDpatients with a heterozygous A181E variant [14] Converselysibling 2 showed AID and NFAT induction comparable tohealthy controls and CVID patients with unaffected TACIalleles NFAT activation induces transcription of cytokinegenes encoding for example IL-4 which is involved in classswitch recombination IL-4 and CD40 ligation induces AIDmRNA and protein expression and depends on STAT6 andNF120581B [34] AID is pivotal for class switch recombination andsomatic hypermutation of theBCR [35 36] bothmechanismsare routinely impaired in CVID
The differential TACI function in the index patient andsibling was accompanied by a change in TACI protein levelsshowing increased TACI expression in the sibling both atthe cell surface and intracellular level TACI protein levels inthe healthy sibling were increased in comparison to healthycontrols as well As TNFRSF13B gene transcription wascomparable between all subjects except for the healthy sib-ling these observations suggest that protein turnover in thehealthy sibling may be decreased possibly due to alterationsin posttranslational modification of TACI protein affectingprotein stability and folding transport or degradation
Alternatively altered TACI function in the healthy siblingwas compensated by increasing the TACI protein quantitythrough cross-talk with related immune pathways CVIDis generally assumed to be a polygenetic disorder and itis plausible that alterations in the index patient were notlimited to the TNFRSF13B gene This prompted us to analyzeother essential immune pathways in B lymphocytes andwe investigated CD40 BCR and TLR9 signaling We didnot find any additional defect in BCR or CD40 signalingNevertheless for both pathways defective interaction withother cells or factors such as abrogated CD40L ligation onT cells or CD21- complement C3d fragment interactionscannot be excluded
In contrast TLR9 expression and function were con-siderably dissimilar between index patient and sibling 2Since the TLR9 expression levels and function were compa-rable between the index patient and the other subjects butincreased in sibling 2 we hypothesize that compensatoryTLR9 expression occurs in the sibling TLR9 recognizesunmethylated CpG-motif-containing bacterial or viral DNA[37 38] and has a critical role in the prevention of severalbacterial and viral infections Ligation of TLR9 inducesrecruitment of MyD88 [30 39] initiating several signalingevents which eventually result in activation of NF120581B and AP-1 [40] By the same token increased TACI protein stabilitymay participate in a compensatory mechanism in the healthysibling possibly related to TLR9 signaling TLR9 engagementinduced TACI upregulation and enhanced TACI-mediated Igclass switching of effector B cells resulting in increased IgGsecretion in cells exposed to anti-TACI and IL-10 and inducedsecretion of IgA
This study has some limitations The TLR9 signalingpathway through MyD88 could be studied more extensivelynot only on a functional level but also through whole exomesequencing The effects of TACI malfunctioning on expres-sion and function of several other targets could have beenaddressed including other TNF receptors and their ligandsor signaling proteins downstream of TACI such as TRAFsCAML and AP-1 Additional defects may not be limitedto B-cell lymphocytes TACI expression can be induced inCD4+ and CD8 T+ cells [4 41] study of TACI-related T-cellfunction would be particularly interesting since the patientdemonstrated chronic viral infections in vivo and impairedantigenic T-cells responses to several viruses in vitro
5 Conclusion
We here describe the diverse clinical and immunologicalcharacteristics in siblings with an identical TACI geno-type These clinical differences are supported by differencesin TACI protein expression and function We proposethat compensatory regulatory mechanisms involving post-translational modification of TACI protein and TLR9 existand may overcome the altered function of TACI trimersin asymptomatic TACI A181EC104R individuals Additionalstudies of TACI protein homeostasis the TACI signalingpathway and interactionwithTLR9 and expression and func-tion of TLR9 downstream signaling molecules are thereforeadvisable
Joris M van Montfrans and Marianne Boes contributedequally
Acknowledgments
The authors would like to thank the technicians of theDepartment of Medical Immunology for excellent assistancewith the TACI sequencing setup and Ewoud Compeer andArie Jan Stoppelenburg for help with PCR and data analyses
References
[1] M A Park J T Li J B Hagan D E Maddox and R SAbraham ldquoCommon variable immunodeficiency a new lookat an old diseaserdquo The Lancet vol 372 no 9637 pp 489ndash5022008
[2] C Cunningham-Rundles L Radigan A K Knight L ZhangL Bauer and A Nakazawa ldquoTLR9 activation is defective incommon variable immune deficiencyrdquo Journal of Immunologyvol 176 no 3 pp 1978ndash1987 2006
[3] J E Yu A K Knight L Radigan et al ldquoToll-like receptor 7and 9 defects in common variable immunodeficiencyrdquo Journalof Allergy and Clinical Immunology vol 124 no 2 pp 349ndash3562009
[4] G-U Von Bulow and R J Bram ldquoNF-AT activation inducedby a CAML-interacting member of the tumor necrosis factorreceptor superfamilyrdquo Science vol 278 no 5335 pp 138ndash1411997
[5] P Schneider F Mackay V Steiner et al ldquoBAFF a novel ligandof the tumor necrosis factor family stimulates B cell growthrdquoJournal of Experimental Medicine vol 189 no 11 pp 1747ndash17561999
[6] Y Wu D Bressette J A Carrell et al ldquoTumor necrosis factor(TNF) receptor superfamily member TACI is a high affinityreceptor for TNF family members APRIL and BLySrdquo Journal ofBiological Chemistry vol 275 no 45 pp 35478ndash35485 2000
[7] S A Marsters M Yan R M Pitti P E Haas V M Dixit andA Ashkenazi ldquoInteraction of the TNF homologues BLyS andAPRIL with the TNF receptor homologues BCMA and TACIrdquoCurrent Biology vol 10 no 13 pp 785ndash788 2000
[8] D Bischof S F Elsawa G Mantchev et al ldquoSelective activationof TACI by syndecan-2rdquo Blood vol 107 no 8 pp 3235ndash32422006
[9] F Mackay P Schneider P Rennert and J Browning ldquoBAFFand APRIL a tutorial on B cell survivalrdquo Annual Review ofImmunology vol 21 pp 231ndash264 2003
[10] Y Laabi M P Gras F Carbonnel et al ldquoA new gene BCMon chromosome 16 is fused to the interleukin 2 gene by at(416)(q26p13) translocation in a malignant T cell lymphomardquoEMBO Journal vol 11 no 11 pp 3897ndash3904 1992
[11] J S Thompson S A Bixler F Qian et al ldquoBAFF-R a newlyidentified TNF receptor that specifically interacts with BAFFrdquoScience vol 293 no 5537 pp 2108ndash2111 2001
[12] J Hauer S Puschner P Ramakrishnan et al ldquoTNF receptor(TNFR)-associated factor (TRAF) 3 serves as an inhibitorof TRAF25-mediated activation of the noncanonical NF-120581Bpathway by TRAF-binding TNFRsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 102 no8 pp 2874ndash2879 2005
[13] E Castigli S AWilsonA Elkhal EOzcan LGaribyan andRS Geha ldquoTransmembrane activator and calciummodulator andcyclophilin ligand interactor enhances CD40-driven plasmacell differentiationrdquo Journal of Allergy and Clinical Immunologyvol 120 no 4 pp 885ndash891 2007
[14] E Ozcan L Garibyan J J-Y Lee R J Bram K-P Lamand R S Geha ldquoTransmembrane activator calciummodulatorand cyclophilin ligand interactor drives plasma cell differenti-ation in LPS-activated B cellsrdquo Journal of Allergy and ClinicalImmunology vol 123 no 6 pp 1277ndash1286 2009
[15] E Ozcan I Rauter L Garibyan S R Dillon and R S GehaldquoToll-like receptor 9 transmembrane activator and calcium-modulating cyclophilin ligand interactor and CD40 synergizein causing B-cell activationrdquo Journal of Allergy and ClinicalImmunology vol 128 no 3 pp 601ndash609 2011
[16] B He R Santamaria W Xu et al ldquoThe transmembrane activa-tor TACI triggers immunoglobulin class switching by activatingB cells through the adaptorMyD88rdquoNature Immunology vol 11no 9 pp 836ndash845 2010
[17] U Salzer C Bacchelli S Buckridge et al ldquoRelevance of biallelicversus monoallelic TNFRSF13B mutations in distinguishingdisease-causing from risk-increasing TNFRSF13B variants inantibody deficiency syndromesrdquo Blood vol 113 no 9 pp 1967ndash1976 2009
[18] L Zhang L Radigan U Salzer et al ldquoTransmembrane acti-vator and calcium-modulating cyclophilin ligand interactormutations in common variable immunodeficiency clinical andimmunologic outcomes in heterozygotesrdquo Journal of Allergy andClinical Immunology vol 120 no 5 pp 1178ndash1185 2007
[19] Q Pan-Hammarstrom U Salzer L Du et al ldquoReexamining therole of TACI coding variants in common variable immunode-ficiency and selective IgA deficiencyrdquo Nature Genetics vol 39no 4 pp 429ndash430 2007
[20] R M Locksley N Killeen and M J Lenardo ldquoThe TNF andTNF receptor superfamilies integrating mammalian biologyrdquoCell vol 104 no 4 pp 487ndash501 2001
[21] L Garibyan A A Lobito R M Siegel M E Call K WWucherpfennig and R S Geha ldquoDominant-negative effect ofthe heterozygous C104R TACI mutation in common variableimmunodeficiency (CVID)rdquo Journal of Clinical Investigationvol 117 no 6 pp 1550ndash1557 2007
[22] S GHymowitz D R Patel H J AWallweber et al ldquoStructuresof APRIL-receptor complexes like BCMA TACI employs onlya single cysteine-rich domain for high affinity ligand bindingrdquoJournal of Biological Chemistry vol 280 no 8 pp 7218ndash72272005
[23] E Castigli S A Wilson L Garibyan et al ldquoTACI is mutantin common variable immunodeficiency and IgA deficiencyrdquoNature Genetics vol 37 no 8 pp 829ndash834 2005
[24] U Salzer H M Chapel A D B Webster et al ldquoMutationsin TNFRSF13B encoding TACI are associated with commonvariable immunodeficiency in humansrdquoNature Genetics vol 37no 8 pp 820ndash828 2005
10 ISRN Immunology
[25] C Bacchelli K F Buckland S Buckridge et al ldquoThe C76Rtransmembrane activator and calcium modulator cyclophilinligand interactor mutation disrupts antibody production andB-cell homeostasis in heterozygous and homozygous micerdquoJournal of Allergy and Clinical Immunology vol 127 no 5 pp1253ndash1259 2011
[26] A J Fried I Rauter S R Dillon H H Jabara and R S GehaldquoFunctional analysis of transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) mutationsassociated with common variable immunodeficiencyrdquo Journalof Allergy and Clinical Immunology vol 128 no 1 pp 226ndash2282011
[27] J J Lee H H Jabara L Garibyan et al ldquoThe C104R mutantimpairs the function of transmembrane activator and calciummodulator and cyclophilin ligand interactor (TACI) throughhaploinsufficiencyrdquo Journal of Allergy and Clinical Immunologyvol 126 no 6 pp 1234ndashe2 2010
[28] J J Lee I Rauter L Garibyan et al ldquoThe murine equivalentof the A181E TACI mutation associated with common variableimmunodeficiency severely impairs B-cell functionrdquo Blood vol114 no 11 pp 2254ndash2262 2009
[29] A A JM van deVen L van de Corput CM van Tilburg et alldquoLymphocyte characteristics in children with common variableimmunodeficiencyrdquoClinical Immunology vol 135 no 1 pp 63ndash71 2010
[30] E Latz A Schoenemeyer A Visintin et al ldquoTLR9 signals aftertranslocating from theER toCpGDNA in the lysosomerdquoNatureImmunology vol 5 no 2 pp 190ndash198 2004
[31] C Foerster N Voelxen M Rakhmanov et al ldquoB cell receptor-mediated calcium signaling is impaired in B lymphocytes oftype Ia patients with common variable immunodeficiencyrdquoJournal of Immunology vol 184 no 12 pp 7305ndash7313 2010
[32] M C Van Zelm J Smet B Adams et al ldquoCD81 gene defectin humans disrupts CD19 complex formation and leads toantibody deficiencyrdquo Journal of Clinical Investigation vol 120no 4 pp 1265ndash1274 2010
[33] M Malphettes L Gerard M Carmagnat et al ldquoLate-onsetcombined immune deficiency a subset of common variableimmunodeficiency with severe T cell defectrdquo Clinical InfectiousDiseases vol 49 no 9 pp 1329ndash1338 2009
[34] F Dedeoglu B Horwitz J Chaudhuri F W Alt and R SGeha ldquoInduction of activation-induced cytidine deaminasegene expression by IL-4 and CD40 ligation is dependent onSTAT6 and NF120581Brdquo International Immunology vol 16 no 3 pp395ndash404 2004
[35] M Muramatsu K Kinoshita S Fagarasan S Yamada YShinkai and T Honjo ldquoClass switch recombination andhypermutation require activation-induced cytidine deaminase(AID) a potential RNA editing enzymerdquo Cell vol 102 no 5 pp553ndash563 2000
[36] P Revy T Muto Y Levy et al ldquoActivation-induced cytidinedeaminase (AID) deficiency causes the autosomal recessiveform of the hyper-IgM syndrome (HIGM2)rdquo Cell vol 102 no5 pp 565ndash575 2000
[37] A M Krieg ldquoCpG motifs in bacterial DNA and their immuneeffectsrdquo Annual Review of Immunology vol 20 pp 709ndash7602002
[38] T Kawai and S Akira ldquoInnate immune recognition of viralinfectionrdquo Nature Immunology vol 7 no 2 pp 131ndash137 2006
[39] E Latz A Verma AVisintin et al ldquoLigand-induced conforma-tional changes allosterically activate Toll-like receptor 9rdquoNatureImmunology vol 8 no 7 pp 772ndash779 2007
[40] S Sato H Sanjo K Takeda et al ldquoEssential function for thekinase TAK1 in innate and adaptive immune responsesrdquoNatureImmunology vol 6 no 11 pp 1087ndash1095 2005
[41] F Mackay and H Leung ldquoThe role of the BAFFAPRIL systemon T cell functionrdquo Seminars in Immunology vol 18 no 5 pp284ndash289 2006
Figure 2 TACI expression and function are decreased in the CVID patient compared to her asymptomatic sibling (a) Pedigree of the studiedfamily Circles display females squares display males The index patient is indicated with an arrow (b) Chest high-resolution CT scan of theindex patient revealing peribronchial consolidations with ground glass appearance and peribronchial thickening ((c)-(d)) TACI surface (cii) or intracellular (c iii) protein and mRNA expression Light grey line indicated isotype indicated dark grey TACI (c i) Results of 5 healthydonors 3 CVID patients with normal TACI 2 CVID patients withmonoallelic A181E the index patient and sibling 2 For (d) average results of3 independent experiments are shown (e) AID (left) and NFAT (right) mRNA induction after 48 h culture of LCL with the indicated stimuliBars represent means plusmn standard error of the mean
Next we investigated TACI function by measuring itsability to induce activation-induced cytidine deaminase(AICDA) gene transcription The AICDA gene productactivation-induced deaminase (AID) is an enzyme of piv-otal importance for secondary antibody diversification [30]Healthy control and CVID patient LCL with normal bial-lelic TACI showed AID mRNA induction upon stimulation
with agonistic anti-TACI (Figure 2(e)) CVID patients withmonoallelic A181E variants showed minimal AICDA genetranscription The index patient had no AICDA gene tran-scription upon TACI stimulation while AID mRNA levelsof sibling 2 LCL were comparable to B cells with normalTACIThe same pattern was noticed for NFATmRNA induc-tion gene transcription upon TACI triggering was lower in
6 ISRN Immunology
Table 1 Immunological characteristics of patient and family
Patient Sibling 1 Sibling 2 Mother FatherGender C C C C D
the index patient compared to sibling 2 There were nodifferences in NF120581B induction upon TACI stimulation Stim-ulation of the CD40 pathway showed adequate induction ofAID and NFAT mRNA in the index patient IL-10 treatmentwas included as negative control that does not induce AICDAgene expression Taken together TACI protein expressionand function were increased in sibling 2 when compared tothe index patient
35 Appearance of TLR9 Protein Expression and Function inthe Asymptomatic Sibling We considered two possibilitiesthat may explain why the index patient and sibling 2 havingidentical TACI gene mutations exhibit divergent healthconsequences either the index patient had an additionalimmune defect which in combinationwith the TACI variantswould lead to CVID disease or sibling 2 has compensatoryimmune function in the TACI signaling route that preventsCVID disease from occurring First as defects in the B-cell receptor (BCR) and its coreceptor complex have beendescribed [31 32] we screened the BCR signalosome CD19CD21 and CD20 cell surface expression of the index patientwere comparable to expression on control LCL (data notshown) Functionally early B-cell activation seemed intactas measured by calcium mobilization after BCR stimulationof fresh PBMC (Figure 3(a)) The index patient showed
a normal initial but rapidly declining calcium influx aspreviously observed in CVID patients with autoimmunesymptoms (own unpublished observations) and area undercurves were comparable to controls As the index patientrsquosendoplasmic reticulum (ER) calcium storage capacitywas notdecreased compared to sibling 2 an additional defect in BCRsignaling was considered improbable
Second the classical route of BCR isotype class switchingis via CD40 with CD40L on T cells and it was recentlyshown that TACI and CD40 signaling can act in synergy[13 15] However CD40 cell surface expression (Figure 3(b))and CD40-mediated gene transcription (Figures 2(e) and3(b)) were comparable between all LCL suggesting that CD40signaling in the index patient B cells was intact at least in thisassay
Third we explored TLR9 signaling as TLR9 defects havebeen described in CVID [2 3] and TLR9 and TACI signalingemploy common downstream signaling pathways [15 16]Noticeably TLR9 expression was nearly twofold higher insibling 2 compared to other LCL and this increased expres-sion was accompanied by an increased TLR9 expression andfunction (Figure 3(c)) supporting the possibility that com-pensatory TLR9 expression contributes as a mechanism foraltered TACI A181EC104R protein in restoring B-cell overallcapacity to transmit signals towards NFAT and AID Thus
ISRN Immunology 7
CVID patientSibling 1Sibling 2
MotherControl
Anti-IgM Ionomycin
25
20
15
10
5
3
2
1
10000
200 300 400Time (s)
BCR-mediated calcium flux (PBMC)Cy
toso
lic C
a2+
leve
l
(a)
0
5000
10000
15000
Mea
n flu
ores
cenc
e int
ensit
y
Cell surface CD40
Con
trol
(TAC
I nor
mal
)
CVID
(TAC
I nor
mal
)
CVID
(A18
1En
orm
al)
Patie
nt(A
181E
C10
4R)
Sibl
ing
(A18
1EC
104R
)
(b)
IL-10 CpG ODN Anti-CD40
AID mRNA
0AID
mRN
A (r
elat
ive t
o G
APD
H) 20
15
10
5
TLR9
Mea
n flu
ores
cenc
e int
ensit
y
15
10
5
00
Intracellular TLR9times10
2
Con
trol
(TAC
I nor
mal
)CV
ID(T
ACI n
orm
al)
CVID
(A18
1En
orm
al)
Patie
nt(A
181E
C10
4R)
Sibl
ing
(A18
1EC
104R
)
Control (TACI normal)CVID (TACI normal)CVID (A181Enormal)
Patient (A181EC104R)Sibling (A181EC104R)
(c)
Figure 3 Similar BCR signaling but possibly enhanced TLR9 signaling in the healthy sibling (a) Calcium mobilization upon triggering ofthe BCR using IgM F(ab1015840)
2fragments in freshly isolated PBMCs (b) CD40 surface expression asmeasured by flow cytometry (c) TLR9 B-cell
expression (c ii) and function (c iii) Light grey line indicated isotype indicated dark grey TLR9 (c i) Bars represent means plusmn standard errorof the mean
our data supports the notion that compensatory signaling inthe TACI route in the case of sibling 2 via enhanced TLR9signaling may prevent the development of CVID disease
4 Discussion
We here describe differential immunological phenotypesdisplayed by siblings with identical genetic TACI variantsThis family provides the unique opportunity to study the role
and significance of the humanC104R andA181E variants bothin vivo and in vitro
The index patient had severe CVID which graduallyevolved to late onset combined immunodeficiency a phe-nomenon previously described in CVID patients [33] Sibling1 did eventually develop mild CVID but without autoim-mune manifestations To date sibling 2 remains free ofsymptoms and has excellent specific antibody responsesnevertheless this sibling may still develop CVID at a laterage Previous studies have shown that C104R abrogates ligand
8 ISRN Immunology
binding due to haploinsufficiency whereas in the A181Evariant ligand binding remains intact [21 26] At least aproportion of A181EC104R TACI trimers will thus be ableto bind ligand As NF120581B activation was strongly decreasedin both variants we hypothesized TACI function to beaffected AID and NFAT mRNA induction were reduced inthe index patient and seemed also reduced in the CVIDpatients with a heterozygous A181E variant [14] Converselysibling 2 showed AID and NFAT induction comparable tohealthy controls and CVID patients with unaffected TACIalleles NFAT activation induces transcription of cytokinegenes encoding for example IL-4 which is involved in classswitch recombination IL-4 and CD40 ligation induces AIDmRNA and protein expression and depends on STAT6 andNF120581B [34] AID is pivotal for class switch recombination andsomatic hypermutation of theBCR [35 36] bothmechanismsare routinely impaired in CVID
The differential TACI function in the index patient andsibling was accompanied by a change in TACI protein levelsshowing increased TACI expression in the sibling both atthe cell surface and intracellular level TACI protein levels inthe healthy sibling were increased in comparison to healthycontrols as well As TNFRSF13B gene transcription wascomparable between all subjects except for the healthy sib-ling these observations suggest that protein turnover in thehealthy sibling may be decreased possibly due to alterationsin posttranslational modification of TACI protein affectingprotein stability and folding transport or degradation
Alternatively altered TACI function in the healthy siblingwas compensated by increasing the TACI protein quantitythrough cross-talk with related immune pathways CVIDis generally assumed to be a polygenetic disorder and itis plausible that alterations in the index patient were notlimited to the TNFRSF13B gene This prompted us to analyzeother essential immune pathways in B lymphocytes andwe investigated CD40 BCR and TLR9 signaling We didnot find any additional defect in BCR or CD40 signalingNevertheless for both pathways defective interaction withother cells or factors such as abrogated CD40L ligation onT cells or CD21- complement C3d fragment interactionscannot be excluded
In contrast TLR9 expression and function were con-siderably dissimilar between index patient and sibling 2Since the TLR9 expression levels and function were compa-rable between the index patient and the other subjects butincreased in sibling 2 we hypothesize that compensatoryTLR9 expression occurs in the sibling TLR9 recognizesunmethylated CpG-motif-containing bacterial or viral DNA[37 38] and has a critical role in the prevention of severalbacterial and viral infections Ligation of TLR9 inducesrecruitment of MyD88 [30 39] initiating several signalingevents which eventually result in activation of NF120581B and AP-1 [40] By the same token increased TACI protein stabilitymay participate in a compensatory mechanism in the healthysibling possibly related to TLR9 signaling TLR9 engagementinduced TACI upregulation and enhanced TACI-mediated Igclass switching of effector B cells resulting in increased IgGsecretion in cells exposed to anti-TACI and IL-10 and inducedsecretion of IgA
This study has some limitations The TLR9 signalingpathway through MyD88 could be studied more extensivelynot only on a functional level but also through whole exomesequencing The effects of TACI malfunctioning on expres-sion and function of several other targets could have beenaddressed including other TNF receptors and their ligandsor signaling proteins downstream of TACI such as TRAFsCAML and AP-1 Additional defects may not be limitedto B-cell lymphocytes TACI expression can be induced inCD4+ and CD8 T+ cells [4 41] study of TACI-related T-cellfunction would be particularly interesting since the patientdemonstrated chronic viral infections in vivo and impairedantigenic T-cells responses to several viruses in vitro
5 Conclusion
We here describe the diverse clinical and immunologicalcharacteristics in siblings with an identical TACI geno-type These clinical differences are supported by differencesin TACI protein expression and function We proposethat compensatory regulatory mechanisms involving post-translational modification of TACI protein and TLR9 existand may overcome the altered function of TACI trimersin asymptomatic TACI A181EC104R individuals Additionalstudies of TACI protein homeostasis the TACI signalingpathway and interactionwithTLR9 and expression and func-tion of TLR9 downstream signaling molecules are thereforeadvisable
Joris M van Montfrans and Marianne Boes contributedequally
Acknowledgments
The authors would like to thank the technicians of theDepartment of Medical Immunology for excellent assistancewith the TACI sequencing setup and Ewoud Compeer andArie Jan Stoppelenburg for help with PCR and data analyses
References
[1] M A Park J T Li J B Hagan D E Maddox and R SAbraham ldquoCommon variable immunodeficiency a new lookat an old diseaserdquo The Lancet vol 372 no 9637 pp 489ndash5022008
[2] C Cunningham-Rundles L Radigan A K Knight L ZhangL Bauer and A Nakazawa ldquoTLR9 activation is defective incommon variable immune deficiencyrdquo Journal of Immunologyvol 176 no 3 pp 1978ndash1987 2006
[3] J E Yu A K Knight L Radigan et al ldquoToll-like receptor 7and 9 defects in common variable immunodeficiencyrdquo Journalof Allergy and Clinical Immunology vol 124 no 2 pp 349ndash3562009
[4] G-U Von Bulow and R J Bram ldquoNF-AT activation inducedby a CAML-interacting member of the tumor necrosis factorreceptor superfamilyrdquo Science vol 278 no 5335 pp 138ndash1411997
[5] P Schneider F Mackay V Steiner et al ldquoBAFF a novel ligandof the tumor necrosis factor family stimulates B cell growthrdquoJournal of Experimental Medicine vol 189 no 11 pp 1747ndash17561999
[6] Y Wu D Bressette J A Carrell et al ldquoTumor necrosis factor(TNF) receptor superfamily member TACI is a high affinityreceptor for TNF family members APRIL and BLySrdquo Journal ofBiological Chemistry vol 275 no 45 pp 35478ndash35485 2000
[7] S A Marsters M Yan R M Pitti P E Haas V M Dixit andA Ashkenazi ldquoInteraction of the TNF homologues BLyS andAPRIL with the TNF receptor homologues BCMA and TACIrdquoCurrent Biology vol 10 no 13 pp 785ndash788 2000
[8] D Bischof S F Elsawa G Mantchev et al ldquoSelective activationof TACI by syndecan-2rdquo Blood vol 107 no 8 pp 3235ndash32422006
[9] F Mackay P Schneider P Rennert and J Browning ldquoBAFFand APRIL a tutorial on B cell survivalrdquo Annual Review ofImmunology vol 21 pp 231ndash264 2003
[10] Y Laabi M P Gras F Carbonnel et al ldquoA new gene BCMon chromosome 16 is fused to the interleukin 2 gene by at(416)(q26p13) translocation in a malignant T cell lymphomardquoEMBO Journal vol 11 no 11 pp 3897ndash3904 1992
[11] J S Thompson S A Bixler F Qian et al ldquoBAFF-R a newlyidentified TNF receptor that specifically interacts with BAFFrdquoScience vol 293 no 5537 pp 2108ndash2111 2001
[12] J Hauer S Puschner P Ramakrishnan et al ldquoTNF receptor(TNFR)-associated factor (TRAF) 3 serves as an inhibitorof TRAF25-mediated activation of the noncanonical NF-120581Bpathway by TRAF-binding TNFRsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 102 no8 pp 2874ndash2879 2005
[13] E Castigli S AWilsonA Elkhal EOzcan LGaribyan andRS Geha ldquoTransmembrane activator and calciummodulator andcyclophilin ligand interactor enhances CD40-driven plasmacell differentiationrdquo Journal of Allergy and Clinical Immunologyvol 120 no 4 pp 885ndash891 2007
[14] E Ozcan L Garibyan J J-Y Lee R J Bram K-P Lamand R S Geha ldquoTransmembrane activator calciummodulatorand cyclophilin ligand interactor drives plasma cell differenti-ation in LPS-activated B cellsrdquo Journal of Allergy and ClinicalImmunology vol 123 no 6 pp 1277ndash1286 2009
[15] E Ozcan I Rauter L Garibyan S R Dillon and R S GehaldquoToll-like receptor 9 transmembrane activator and calcium-modulating cyclophilin ligand interactor and CD40 synergizein causing B-cell activationrdquo Journal of Allergy and ClinicalImmunology vol 128 no 3 pp 601ndash609 2011
[16] B He R Santamaria W Xu et al ldquoThe transmembrane activa-tor TACI triggers immunoglobulin class switching by activatingB cells through the adaptorMyD88rdquoNature Immunology vol 11no 9 pp 836ndash845 2010
[17] U Salzer C Bacchelli S Buckridge et al ldquoRelevance of biallelicversus monoallelic TNFRSF13B mutations in distinguishingdisease-causing from risk-increasing TNFRSF13B variants inantibody deficiency syndromesrdquo Blood vol 113 no 9 pp 1967ndash1976 2009
[18] L Zhang L Radigan U Salzer et al ldquoTransmembrane acti-vator and calcium-modulating cyclophilin ligand interactormutations in common variable immunodeficiency clinical andimmunologic outcomes in heterozygotesrdquo Journal of Allergy andClinical Immunology vol 120 no 5 pp 1178ndash1185 2007
[19] Q Pan-Hammarstrom U Salzer L Du et al ldquoReexamining therole of TACI coding variants in common variable immunode-ficiency and selective IgA deficiencyrdquo Nature Genetics vol 39no 4 pp 429ndash430 2007
[20] R M Locksley N Killeen and M J Lenardo ldquoThe TNF andTNF receptor superfamilies integrating mammalian biologyrdquoCell vol 104 no 4 pp 487ndash501 2001
[21] L Garibyan A A Lobito R M Siegel M E Call K WWucherpfennig and R S Geha ldquoDominant-negative effect ofthe heterozygous C104R TACI mutation in common variableimmunodeficiency (CVID)rdquo Journal of Clinical Investigationvol 117 no 6 pp 1550ndash1557 2007
[22] S GHymowitz D R Patel H J AWallweber et al ldquoStructuresof APRIL-receptor complexes like BCMA TACI employs onlya single cysteine-rich domain for high affinity ligand bindingrdquoJournal of Biological Chemistry vol 280 no 8 pp 7218ndash72272005
[23] E Castigli S A Wilson L Garibyan et al ldquoTACI is mutantin common variable immunodeficiency and IgA deficiencyrdquoNature Genetics vol 37 no 8 pp 829ndash834 2005
[24] U Salzer H M Chapel A D B Webster et al ldquoMutationsin TNFRSF13B encoding TACI are associated with commonvariable immunodeficiency in humansrdquoNature Genetics vol 37no 8 pp 820ndash828 2005
10 ISRN Immunology
[25] C Bacchelli K F Buckland S Buckridge et al ldquoThe C76Rtransmembrane activator and calcium modulator cyclophilinligand interactor mutation disrupts antibody production andB-cell homeostasis in heterozygous and homozygous micerdquoJournal of Allergy and Clinical Immunology vol 127 no 5 pp1253ndash1259 2011
[26] A J Fried I Rauter S R Dillon H H Jabara and R S GehaldquoFunctional analysis of transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) mutationsassociated with common variable immunodeficiencyrdquo Journalof Allergy and Clinical Immunology vol 128 no 1 pp 226ndash2282011
[27] J J Lee H H Jabara L Garibyan et al ldquoThe C104R mutantimpairs the function of transmembrane activator and calciummodulator and cyclophilin ligand interactor (TACI) throughhaploinsufficiencyrdquo Journal of Allergy and Clinical Immunologyvol 126 no 6 pp 1234ndashe2 2010
[28] J J Lee I Rauter L Garibyan et al ldquoThe murine equivalentof the A181E TACI mutation associated with common variableimmunodeficiency severely impairs B-cell functionrdquo Blood vol114 no 11 pp 2254ndash2262 2009
[29] A A JM van deVen L van de Corput CM van Tilburg et alldquoLymphocyte characteristics in children with common variableimmunodeficiencyrdquoClinical Immunology vol 135 no 1 pp 63ndash71 2010
[30] E Latz A Schoenemeyer A Visintin et al ldquoTLR9 signals aftertranslocating from theER toCpGDNA in the lysosomerdquoNatureImmunology vol 5 no 2 pp 190ndash198 2004
[31] C Foerster N Voelxen M Rakhmanov et al ldquoB cell receptor-mediated calcium signaling is impaired in B lymphocytes oftype Ia patients with common variable immunodeficiencyrdquoJournal of Immunology vol 184 no 12 pp 7305ndash7313 2010
[32] M C Van Zelm J Smet B Adams et al ldquoCD81 gene defectin humans disrupts CD19 complex formation and leads toantibody deficiencyrdquo Journal of Clinical Investigation vol 120no 4 pp 1265ndash1274 2010
[33] M Malphettes L Gerard M Carmagnat et al ldquoLate-onsetcombined immune deficiency a subset of common variableimmunodeficiency with severe T cell defectrdquo Clinical InfectiousDiseases vol 49 no 9 pp 1329ndash1338 2009
[34] F Dedeoglu B Horwitz J Chaudhuri F W Alt and R SGeha ldquoInduction of activation-induced cytidine deaminasegene expression by IL-4 and CD40 ligation is dependent onSTAT6 and NF120581Brdquo International Immunology vol 16 no 3 pp395ndash404 2004
[35] M Muramatsu K Kinoshita S Fagarasan S Yamada YShinkai and T Honjo ldquoClass switch recombination andhypermutation require activation-induced cytidine deaminase(AID) a potential RNA editing enzymerdquo Cell vol 102 no 5 pp553ndash563 2000
[36] P Revy T Muto Y Levy et al ldquoActivation-induced cytidinedeaminase (AID) deficiency causes the autosomal recessiveform of the hyper-IgM syndrome (HIGM2)rdquo Cell vol 102 no5 pp 565ndash575 2000
[37] A M Krieg ldquoCpG motifs in bacterial DNA and their immuneeffectsrdquo Annual Review of Immunology vol 20 pp 709ndash7602002
[38] T Kawai and S Akira ldquoInnate immune recognition of viralinfectionrdquo Nature Immunology vol 7 no 2 pp 131ndash137 2006
[39] E Latz A Verma AVisintin et al ldquoLigand-induced conforma-tional changes allosterically activate Toll-like receptor 9rdquoNatureImmunology vol 8 no 7 pp 772ndash779 2007
[40] S Sato H Sanjo K Takeda et al ldquoEssential function for thekinase TAK1 in innate and adaptive immune responsesrdquoNatureImmunology vol 6 no 11 pp 1087ndash1095 2005
[41] F Mackay and H Leung ldquoThe role of the BAFFAPRIL systemon T cell functionrdquo Seminars in Immunology vol 18 no 5 pp284ndash289 2006
the index patient compared to sibling 2 There were nodifferences in NF120581B induction upon TACI stimulation Stim-ulation of the CD40 pathway showed adequate induction ofAID and NFAT mRNA in the index patient IL-10 treatmentwas included as negative control that does not induce AICDAgene expression Taken together TACI protein expressionand function were increased in sibling 2 when compared tothe index patient
35 Appearance of TLR9 Protein Expression and Function inthe Asymptomatic Sibling We considered two possibilitiesthat may explain why the index patient and sibling 2 havingidentical TACI gene mutations exhibit divergent healthconsequences either the index patient had an additionalimmune defect which in combinationwith the TACI variantswould lead to CVID disease or sibling 2 has compensatoryimmune function in the TACI signaling route that preventsCVID disease from occurring First as defects in the B-cell receptor (BCR) and its coreceptor complex have beendescribed [31 32] we screened the BCR signalosome CD19CD21 and CD20 cell surface expression of the index patientwere comparable to expression on control LCL (data notshown) Functionally early B-cell activation seemed intactas measured by calcium mobilization after BCR stimulationof fresh PBMC (Figure 3(a)) The index patient showed
a normal initial but rapidly declining calcium influx aspreviously observed in CVID patients with autoimmunesymptoms (own unpublished observations) and area undercurves were comparable to controls As the index patientrsquosendoplasmic reticulum (ER) calcium storage capacitywas notdecreased compared to sibling 2 an additional defect in BCRsignaling was considered improbable
Second the classical route of BCR isotype class switchingis via CD40 with CD40L on T cells and it was recentlyshown that TACI and CD40 signaling can act in synergy[13 15] However CD40 cell surface expression (Figure 3(b))and CD40-mediated gene transcription (Figures 2(e) and3(b)) were comparable between all LCL suggesting that CD40signaling in the index patient B cells was intact at least in thisassay
Third we explored TLR9 signaling as TLR9 defects havebeen described in CVID [2 3] and TLR9 and TACI signalingemploy common downstream signaling pathways [15 16]Noticeably TLR9 expression was nearly twofold higher insibling 2 compared to other LCL and this increased expres-sion was accompanied by an increased TLR9 expression andfunction (Figure 3(c)) supporting the possibility that com-pensatory TLR9 expression contributes as a mechanism foraltered TACI A181EC104R protein in restoring B-cell overallcapacity to transmit signals towards NFAT and AID Thus
ISRN Immunology 7
CVID patientSibling 1Sibling 2
MotherControl
Anti-IgM Ionomycin
25
20
15
10
5
3
2
1
10000
200 300 400Time (s)
BCR-mediated calcium flux (PBMC)Cy
toso
lic C
a2+
leve
l
(a)
0
5000
10000
15000
Mea
n flu
ores
cenc
e int
ensit
y
Cell surface CD40
Con
trol
(TAC
I nor
mal
)
CVID
(TAC
I nor
mal
)
CVID
(A18
1En
orm
al)
Patie
nt(A
181E
C10
4R)
Sibl
ing
(A18
1EC
104R
)
(b)
IL-10 CpG ODN Anti-CD40
AID mRNA
0AID
mRN
A (r
elat
ive t
o G
APD
H) 20
15
10
5
TLR9
Mea
n flu
ores
cenc
e int
ensit
y
15
10
5
00
Intracellular TLR9times10
2
Con
trol
(TAC
I nor
mal
)CV
ID(T
ACI n
orm
al)
CVID
(A18
1En
orm
al)
Patie
nt(A
181E
C10
4R)
Sibl
ing
(A18
1EC
104R
)
Control (TACI normal)CVID (TACI normal)CVID (A181Enormal)
Patient (A181EC104R)Sibling (A181EC104R)
(c)
Figure 3 Similar BCR signaling but possibly enhanced TLR9 signaling in the healthy sibling (a) Calcium mobilization upon triggering ofthe BCR using IgM F(ab1015840)
2fragments in freshly isolated PBMCs (b) CD40 surface expression asmeasured by flow cytometry (c) TLR9 B-cell
expression (c ii) and function (c iii) Light grey line indicated isotype indicated dark grey TLR9 (c i) Bars represent means plusmn standard errorof the mean
our data supports the notion that compensatory signaling inthe TACI route in the case of sibling 2 via enhanced TLR9signaling may prevent the development of CVID disease
4 Discussion
We here describe differential immunological phenotypesdisplayed by siblings with identical genetic TACI variantsThis family provides the unique opportunity to study the role
and significance of the humanC104R andA181E variants bothin vivo and in vitro
The index patient had severe CVID which graduallyevolved to late onset combined immunodeficiency a phe-nomenon previously described in CVID patients [33] Sibling1 did eventually develop mild CVID but without autoim-mune manifestations To date sibling 2 remains free ofsymptoms and has excellent specific antibody responsesnevertheless this sibling may still develop CVID at a laterage Previous studies have shown that C104R abrogates ligand
8 ISRN Immunology
binding due to haploinsufficiency whereas in the A181Evariant ligand binding remains intact [21 26] At least aproportion of A181EC104R TACI trimers will thus be ableto bind ligand As NF120581B activation was strongly decreasedin both variants we hypothesized TACI function to beaffected AID and NFAT mRNA induction were reduced inthe index patient and seemed also reduced in the CVIDpatients with a heterozygous A181E variant [14] Converselysibling 2 showed AID and NFAT induction comparable tohealthy controls and CVID patients with unaffected TACIalleles NFAT activation induces transcription of cytokinegenes encoding for example IL-4 which is involved in classswitch recombination IL-4 and CD40 ligation induces AIDmRNA and protein expression and depends on STAT6 andNF120581B [34] AID is pivotal for class switch recombination andsomatic hypermutation of theBCR [35 36] bothmechanismsare routinely impaired in CVID
The differential TACI function in the index patient andsibling was accompanied by a change in TACI protein levelsshowing increased TACI expression in the sibling both atthe cell surface and intracellular level TACI protein levels inthe healthy sibling were increased in comparison to healthycontrols as well As TNFRSF13B gene transcription wascomparable between all subjects except for the healthy sib-ling these observations suggest that protein turnover in thehealthy sibling may be decreased possibly due to alterationsin posttranslational modification of TACI protein affectingprotein stability and folding transport or degradation
Alternatively altered TACI function in the healthy siblingwas compensated by increasing the TACI protein quantitythrough cross-talk with related immune pathways CVIDis generally assumed to be a polygenetic disorder and itis plausible that alterations in the index patient were notlimited to the TNFRSF13B gene This prompted us to analyzeother essential immune pathways in B lymphocytes andwe investigated CD40 BCR and TLR9 signaling We didnot find any additional defect in BCR or CD40 signalingNevertheless for both pathways defective interaction withother cells or factors such as abrogated CD40L ligation onT cells or CD21- complement C3d fragment interactionscannot be excluded
In contrast TLR9 expression and function were con-siderably dissimilar between index patient and sibling 2Since the TLR9 expression levels and function were compa-rable between the index patient and the other subjects butincreased in sibling 2 we hypothesize that compensatoryTLR9 expression occurs in the sibling TLR9 recognizesunmethylated CpG-motif-containing bacterial or viral DNA[37 38] and has a critical role in the prevention of severalbacterial and viral infections Ligation of TLR9 inducesrecruitment of MyD88 [30 39] initiating several signalingevents which eventually result in activation of NF120581B and AP-1 [40] By the same token increased TACI protein stabilitymay participate in a compensatory mechanism in the healthysibling possibly related to TLR9 signaling TLR9 engagementinduced TACI upregulation and enhanced TACI-mediated Igclass switching of effector B cells resulting in increased IgGsecretion in cells exposed to anti-TACI and IL-10 and inducedsecretion of IgA
This study has some limitations The TLR9 signalingpathway through MyD88 could be studied more extensivelynot only on a functional level but also through whole exomesequencing The effects of TACI malfunctioning on expres-sion and function of several other targets could have beenaddressed including other TNF receptors and their ligandsor signaling proteins downstream of TACI such as TRAFsCAML and AP-1 Additional defects may not be limitedto B-cell lymphocytes TACI expression can be induced inCD4+ and CD8 T+ cells [4 41] study of TACI-related T-cellfunction would be particularly interesting since the patientdemonstrated chronic viral infections in vivo and impairedantigenic T-cells responses to several viruses in vitro
5 Conclusion
We here describe the diverse clinical and immunologicalcharacteristics in siblings with an identical TACI geno-type These clinical differences are supported by differencesin TACI protein expression and function We proposethat compensatory regulatory mechanisms involving post-translational modification of TACI protein and TLR9 existand may overcome the altered function of TACI trimersin asymptomatic TACI A181EC104R individuals Additionalstudies of TACI protein homeostasis the TACI signalingpathway and interactionwithTLR9 and expression and func-tion of TLR9 downstream signaling molecules are thereforeadvisable
Joris M van Montfrans and Marianne Boes contributedequally
Acknowledgments
The authors would like to thank the technicians of theDepartment of Medical Immunology for excellent assistancewith the TACI sequencing setup and Ewoud Compeer andArie Jan Stoppelenburg for help with PCR and data analyses
References
[1] M A Park J T Li J B Hagan D E Maddox and R SAbraham ldquoCommon variable immunodeficiency a new lookat an old diseaserdquo The Lancet vol 372 no 9637 pp 489ndash5022008
[2] C Cunningham-Rundles L Radigan A K Knight L ZhangL Bauer and A Nakazawa ldquoTLR9 activation is defective incommon variable immune deficiencyrdquo Journal of Immunologyvol 176 no 3 pp 1978ndash1987 2006
[3] J E Yu A K Knight L Radigan et al ldquoToll-like receptor 7and 9 defects in common variable immunodeficiencyrdquo Journalof Allergy and Clinical Immunology vol 124 no 2 pp 349ndash3562009
[4] G-U Von Bulow and R J Bram ldquoNF-AT activation inducedby a CAML-interacting member of the tumor necrosis factorreceptor superfamilyrdquo Science vol 278 no 5335 pp 138ndash1411997
[5] P Schneider F Mackay V Steiner et al ldquoBAFF a novel ligandof the tumor necrosis factor family stimulates B cell growthrdquoJournal of Experimental Medicine vol 189 no 11 pp 1747ndash17561999
[6] Y Wu D Bressette J A Carrell et al ldquoTumor necrosis factor(TNF) receptor superfamily member TACI is a high affinityreceptor for TNF family members APRIL and BLySrdquo Journal ofBiological Chemistry vol 275 no 45 pp 35478ndash35485 2000
[7] S A Marsters M Yan R M Pitti P E Haas V M Dixit andA Ashkenazi ldquoInteraction of the TNF homologues BLyS andAPRIL with the TNF receptor homologues BCMA and TACIrdquoCurrent Biology vol 10 no 13 pp 785ndash788 2000
[8] D Bischof S F Elsawa G Mantchev et al ldquoSelective activationof TACI by syndecan-2rdquo Blood vol 107 no 8 pp 3235ndash32422006
[9] F Mackay P Schneider P Rennert and J Browning ldquoBAFFand APRIL a tutorial on B cell survivalrdquo Annual Review ofImmunology vol 21 pp 231ndash264 2003
[10] Y Laabi M P Gras F Carbonnel et al ldquoA new gene BCMon chromosome 16 is fused to the interleukin 2 gene by at(416)(q26p13) translocation in a malignant T cell lymphomardquoEMBO Journal vol 11 no 11 pp 3897ndash3904 1992
[11] J S Thompson S A Bixler F Qian et al ldquoBAFF-R a newlyidentified TNF receptor that specifically interacts with BAFFrdquoScience vol 293 no 5537 pp 2108ndash2111 2001
[12] J Hauer S Puschner P Ramakrishnan et al ldquoTNF receptor(TNFR)-associated factor (TRAF) 3 serves as an inhibitorof TRAF25-mediated activation of the noncanonical NF-120581Bpathway by TRAF-binding TNFRsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 102 no8 pp 2874ndash2879 2005
[13] E Castigli S AWilsonA Elkhal EOzcan LGaribyan andRS Geha ldquoTransmembrane activator and calciummodulator andcyclophilin ligand interactor enhances CD40-driven plasmacell differentiationrdquo Journal of Allergy and Clinical Immunologyvol 120 no 4 pp 885ndash891 2007
[14] E Ozcan L Garibyan J J-Y Lee R J Bram K-P Lamand R S Geha ldquoTransmembrane activator calciummodulatorand cyclophilin ligand interactor drives plasma cell differenti-ation in LPS-activated B cellsrdquo Journal of Allergy and ClinicalImmunology vol 123 no 6 pp 1277ndash1286 2009
[15] E Ozcan I Rauter L Garibyan S R Dillon and R S GehaldquoToll-like receptor 9 transmembrane activator and calcium-modulating cyclophilin ligand interactor and CD40 synergizein causing B-cell activationrdquo Journal of Allergy and ClinicalImmunology vol 128 no 3 pp 601ndash609 2011
[16] B He R Santamaria W Xu et al ldquoThe transmembrane activa-tor TACI triggers immunoglobulin class switching by activatingB cells through the adaptorMyD88rdquoNature Immunology vol 11no 9 pp 836ndash845 2010
[17] U Salzer C Bacchelli S Buckridge et al ldquoRelevance of biallelicversus monoallelic TNFRSF13B mutations in distinguishingdisease-causing from risk-increasing TNFRSF13B variants inantibody deficiency syndromesrdquo Blood vol 113 no 9 pp 1967ndash1976 2009
[18] L Zhang L Radigan U Salzer et al ldquoTransmembrane acti-vator and calcium-modulating cyclophilin ligand interactormutations in common variable immunodeficiency clinical andimmunologic outcomes in heterozygotesrdquo Journal of Allergy andClinical Immunology vol 120 no 5 pp 1178ndash1185 2007
[19] Q Pan-Hammarstrom U Salzer L Du et al ldquoReexamining therole of TACI coding variants in common variable immunode-ficiency and selective IgA deficiencyrdquo Nature Genetics vol 39no 4 pp 429ndash430 2007
[20] R M Locksley N Killeen and M J Lenardo ldquoThe TNF andTNF receptor superfamilies integrating mammalian biologyrdquoCell vol 104 no 4 pp 487ndash501 2001
[21] L Garibyan A A Lobito R M Siegel M E Call K WWucherpfennig and R S Geha ldquoDominant-negative effect ofthe heterozygous C104R TACI mutation in common variableimmunodeficiency (CVID)rdquo Journal of Clinical Investigationvol 117 no 6 pp 1550ndash1557 2007
[22] S GHymowitz D R Patel H J AWallweber et al ldquoStructuresof APRIL-receptor complexes like BCMA TACI employs onlya single cysteine-rich domain for high affinity ligand bindingrdquoJournal of Biological Chemistry vol 280 no 8 pp 7218ndash72272005
[23] E Castigli S A Wilson L Garibyan et al ldquoTACI is mutantin common variable immunodeficiency and IgA deficiencyrdquoNature Genetics vol 37 no 8 pp 829ndash834 2005
[24] U Salzer H M Chapel A D B Webster et al ldquoMutationsin TNFRSF13B encoding TACI are associated with commonvariable immunodeficiency in humansrdquoNature Genetics vol 37no 8 pp 820ndash828 2005
10 ISRN Immunology
[25] C Bacchelli K F Buckland S Buckridge et al ldquoThe C76Rtransmembrane activator and calcium modulator cyclophilinligand interactor mutation disrupts antibody production andB-cell homeostasis in heterozygous and homozygous micerdquoJournal of Allergy and Clinical Immunology vol 127 no 5 pp1253ndash1259 2011
[26] A J Fried I Rauter S R Dillon H H Jabara and R S GehaldquoFunctional analysis of transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) mutationsassociated with common variable immunodeficiencyrdquo Journalof Allergy and Clinical Immunology vol 128 no 1 pp 226ndash2282011
[27] J J Lee H H Jabara L Garibyan et al ldquoThe C104R mutantimpairs the function of transmembrane activator and calciummodulator and cyclophilin ligand interactor (TACI) throughhaploinsufficiencyrdquo Journal of Allergy and Clinical Immunologyvol 126 no 6 pp 1234ndashe2 2010
[28] J J Lee I Rauter L Garibyan et al ldquoThe murine equivalentof the A181E TACI mutation associated with common variableimmunodeficiency severely impairs B-cell functionrdquo Blood vol114 no 11 pp 2254ndash2262 2009
[29] A A JM van deVen L van de Corput CM van Tilburg et alldquoLymphocyte characteristics in children with common variableimmunodeficiencyrdquoClinical Immunology vol 135 no 1 pp 63ndash71 2010
[30] E Latz A Schoenemeyer A Visintin et al ldquoTLR9 signals aftertranslocating from theER toCpGDNA in the lysosomerdquoNatureImmunology vol 5 no 2 pp 190ndash198 2004
[31] C Foerster N Voelxen M Rakhmanov et al ldquoB cell receptor-mediated calcium signaling is impaired in B lymphocytes oftype Ia patients with common variable immunodeficiencyrdquoJournal of Immunology vol 184 no 12 pp 7305ndash7313 2010
[32] M C Van Zelm J Smet B Adams et al ldquoCD81 gene defectin humans disrupts CD19 complex formation and leads toantibody deficiencyrdquo Journal of Clinical Investigation vol 120no 4 pp 1265ndash1274 2010
[33] M Malphettes L Gerard M Carmagnat et al ldquoLate-onsetcombined immune deficiency a subset of common variableimmunodeficiency with severe T cell defectrdquo Clinical InfectiousDiseases vol 49 no 9 pp 1329ndash1338 2009
[34] F Dedeoglu B Horwitz J Chaudhuri F W Alt and R SGeha ldquoInduction of activation-induced cytidine deaminasegene expression by IL-4 and CD40 ligation is dependent onSTAT6 and NF120581Brdquo International Immunology vol 16 no 3 pp395ndash404 2004
[35] M Muramatsu K Kinoshita S Fagarasan S Yamada YShinkai and T Honjo ldquoClass switch recombination andhypermutation require activation-induced cytidine deaminase(AID) a potential RNA editing enzymerdquo Cell vol 102 no 5 pp553ndash563 2000
[36] P Revy T Muto Y Levy et al ldquoActivation-induced cytidinedeaminase (AID) deficiency causes the autosomal recessiveform of the hyper-IgM syndrome (HIGM2)rdquo Cell vol 102 no5 pp 565ndash575 2000
[37] A M Krieg ldquoCpG motifs in bacterial DNA and their immuneeffectsrdquo Annual Review of Immunology vol 20 pp 709ndash7602002
[38] T Kawai and S Akira ldquoInnate immune recognition of viralinfectionrdquo Nature Immunology vol 7 no 2 pp 131ndash137 2006
[39] E Latz A Verma AVisintin et al ldquoLigand-induced conforma-tional changes allosterically activate Toll-like receptor 9rdquoNatureImmunology vol 8 no 7 pp 772ndash779 2007
[40] S Sato H Sanjo K Takeda et al ldquoEssential function for thekinase TAK1 in innate and adaptive immune responsesrdquoNatureImmunology vol 6 no 11 pp 1087ndash1095 2005
[41] F Mackay and H Leung ldquoThe role of the BAFFAPRIL systemon T cell functionrdquo Seminars in Immunology vol 18 no 5 pp284ndash289 2006
Control (TACI normal)CVID (TACI normal)CVID (A181Enormal)
Patient (A181EC104R)Sibling (A181EC104R)
(c)
Figure 3 Similar BCR signaling but possibly enhanced TLR9 signaling in the healthy sibling (a) Calcium mobilization upon triggering ofthe BCR using IgM F(ab1015840)
2fragments in freshly isolated PBMCs (b) CD40 surface expression asmeasured by flow cytometry (c) TLR9 B-cell
expression (c ii) and function (c iii) Light grey line indicated isotype indicated dark grey TLR9 (c i) Bars represent means plusmn standard errorof the mean
our data supports the notion that compensatory signaling inthe TACI route in the case of sibling 2 via enhanced TLR9signaling may prevent the development of CVID disease
4 Discussion
We here describe differential immunological phenotypesdisplayed by siblings with identical genetic TACI variantsThis family provides the unique opportunity to study the role
and significance of the humanC104R andA181E variants bothin vivo and in vitro
The index patient had severe CVID which graduallyevolved to late onset combined immunodeficiency a phe-nomenon previously described in CVID patients [33] Sibling1 did eventually develop mild CVID but without autoim-mune manifestations To date sibling 2 remains free ofsymptoms and has excellent specific antibody responsesnevertheless this sibling may still develop CVID at a laterage Previous studies have shown that C104R abrogates ligand
8 ISRN Immunology
binding due to haploinsufficiency whereas in the A181Evariant ligand binding remains intact [21 26] At least aproportion of A181EC104R TACI trimers will thus be ableto bind ligand As NF120581B activation was strongly decreasedin both variants we hypothesized TACI function to beaffected AID and NFAT mRNA induction were reduced inthe index patient and seemed also reduced in the CVIDpatients with a heterozygous A181E variant [14] Converselysibling 2 showed AID and NFAT induction comparable tohealthy controls and CVID patients with unaffected TACIalleles NFAT activation induces transcription of cytokinegenes encoding for example IL-4 which is involved in classswitch recombination IL-4 and CD40 ligation induces AIDmRNA and protein expression and depends on STAT6 andNF120581B [34] AID is pivotal for class switch recombination andsomatic hypermutation of theBCR [35 36] bothmechanismsare routinely impaired in CVID
The differential TACI function in the index patient andsibling was accompanied by a change in TACI protein levelsshowing increased TACI expression in the sibling both atthe cell surface and intracellular level TACI protein levels inthe healthy sibling were increased in comparison to healthycontrols as well As TNFRSF13B gene transcription wascomparable between all subjects except for the healthy sib-ling these observations suggest that protein turnover in thehealthy sibling may be decreased possibly due to alterationsin posttranslational modification of TACI protein affectingprotein stability and folding transport or degradation
Alternatively altered TACI function in the healthy siblingwas compensated by increasing the TACI protein quantitythrough cross-talk with related immune pathways CVIDis generally assumed to be a polygenetic disorder and itis plausible that alterations in the index patient were notlimited to the TNFRSF13B gene This prompted us to analyzeother essential immune pathways in B lymphocytes andwe investigated CD40 BCR and TLR9 signaling We didnot find any additional defect in BCR or CD40 signalingNevertheless for both pathways defective interaction withother cells or factors such as abrogated CD40L ligation onT cells or CD21- complement C3d fragment interactionscannot be excluded
In contrast TLR9 expression and function were con-siderably dissimilar between index patient and sibling 2Since the TLR9 expression levels and function were compa-rable between the index patient and the other subjects butincreased in sibling 2 we hypothesize that compensatoryTLR9 expression occurs in the sibling TLR9 recognizesunmethylated CpG-motif-containing bacterial or viral DNA[37 38] and has a critical role in the prevention of severalbacterial and viral infections Ligation of TLR9 inducesrecruitment of MyD88 [30 39] initiating several signalingevents which eventually result in activation of NF120581B and AP-1 [40] By the same token increased TACI protein stabilitymay participate in a compensatory mechanism in the healthysibling possibly related to TLR9 signaling TLR9 engagementinduced TACI upregulation and enhanced TACI-mediated Igclass switching of effector B cells resulting in increased IgGsecretion in cells exposed to anti-TACI and IL-10 and inducedsecretion of IgA
This study has some limitations The TLR9 signalingpathway through MyD88 could be studied more extensivelynot only on a functional level but also through whole exomesequencing The effects of TACI malfunctioning on expres-sion and function of several other targets could have beenaddressed including other TNF receptors and their ligandsor signaling proteins downstream of TACI such as TRAFsCAML and AP-1 Additional defects may not be limitedto B-cell lymphocytes TACI expression can be induced inCD4+ and CD8 T+ cells [4 41] study of TACI-related T-cellfunction would be particularly interesting since the patientdemonstrated chronic viral infections in vivo and impairedantigenic T-cells responses to several viruses in vitro
5 Conclusion
We here describe the diverse clinical and immunologicalcharacteristics in siblings with an identical TACI geno-type These clinical differences are supported by differencesin TACI protein expression and function We proposethat compensatory regulatory mechanisms involving post-translational modification of TACI protein and TLR9 existand may overcome the altered function of TACI trimersin asymptomatic TACI A181EC104R individuals Additionalstudies of TACI protein homeostasis the TACI signalingpathway and interactionwithTLR9 and expression and func-tion of TLR9 downstream signaling molecules are thereforeadvisable
Joris M van Montfrans and Marianne Boes contributedequally
Acknowledgments
The authors would like to thank the technicians of theDepartment of Medical Immunology for excellent assistancewith the TACI sequencing setup and Ewoud Compeer andArie Jan Stoppelenburg for help with PCR and data analyses
References
[1] M A Park J T Li J B Hagan D E Maddox and R SAbraham ldquoCommon variable immunodeficiency a new lookat an old diseaserdquo The Lancet vol 372 no 9637 pp 489ndash5022008
[2] C Cunningham-Rundles L Radigan A K Knight L ZhangL Bauer and A Nakazawa ldquoTLR9 activation is defective incommon variable immune deficiencyrdquo Journal of Immunologyvol 176 no 3 pp 1978ndash1987 2006
[3] J E Yu A K Knight L Radigan et al ldquoToll-like receptor 7and 9 defects in common variable immunodeficiencyrdquo Journalof Allergy and Clinical Immunology vol 124 no 2 pp 349ndash3562009
[4] G-U Von Bulow and R J Bram ldquoNF-AT activation inducedby a CAML-interacting member of the tumor necrosis factorreceptor superfamilyrdquo Science vol 278 no 5335 pp 138ndash1411997
[5] P Schneider F Mackay V Steiner et al ldquoBAFF a novel ligandof the tumor necrosis factor family stimulates B cell growthrdquoJournal of Experimental Medicine vol 189 no 11 pp 1747ndash17561999
[6] Y Wu D Bressette J A Carrell et al ldquoTumor necrosis factor(TNF) receptor superfamily member TACI is a high affinityreceptor for TNF family members APRIL and BLySrdquo Journal ofBiological Chemistry vol 275 no 45 pp 35478ndash35485 2000
[7] S A Marsters M Yan R M Pitti P E Haas V M Dixit andA Ashkenazi ldquoInteraction of the TNF homologues BLyS andAPRIL with the TNF receptor homologues BCMA and TACIrdquoCurrent Biology vol 10 no 13 pp 785ndash788 2000
[8] D Bischof S F Elsawa G Mantchev et al ldquoSelective activationof TACI by syndecan-2rdquo Blood vol 107 no 8 pp 3235ndash32422006
[9] F Mackay P Schneider P Rennert and J Browning ldquoBAFFand APRIL a tutorial on B cell survivalrdquo Annual Review ofImmunology vol 21 pp 231ndash264 2003
[10] Y Laabi M P Gras F Carbonnel et al ldquoA new gene BCMon chromosome 16 is fused to the interleukin 2 gene by at(416)(q26p13) translocation in a malignant T cell lymphomardquoEMBO Journal vol 11 no 11 pp 3897ndash3904 1992
[11] J S Thompson S A Bixler F Qian et al ldquoBAFF-R a newlyidentified TNF receptor that specifically interacts with BAFFrdquoScience vol 293 no 5537 pp 2108ndash2111 2001
[12] J Hauer S Puschner P Ramakrishnan et al ldquoTNF receptor(TNFR)-associated factor (TRAF) 3 serves as an inhibitorof TRAF25-mediated activation of the noncanonical NF-120581Bpathway by TRAF-binding TNFRsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 102 no8 pp 2874ndash2879 2005
[13] E Castigli S AWilsonA Elkhal EOzcan LGaribyan andRS Geha ldquoTransmembrane activator and calciummodulator andcyclophilin ligand interactor enhances CD40-driven plasmacell differentiationrdquo Journal of Allergy and Clinical Immunologyvol 120 no 4 pp 885ndash891 2007
[14] E Ozcan L Garibyan J J-Y Lee R J Bram K-P Lamand R S Geha ldquoTransmembrane activator calciummodulatorand cyclophilin ligand interactor drives plasma cell differenti-ation in LPS-activated B cellsrdquo Journal of Allergy and ClinicalImmunology vol 123 no 6 pp 1277ndash1286 2009
[15] E Ozcan I Rauter L Garibyan S R Dillon and R S GehaldquoToll-like receptor 9 transmembrane activator and calcium-modulating cyclophilin ligand interactor and CD40 synergizein causing B-cell activationrdquo Journal of Allergy and ClinicalImmunology vol 128 no 3 pp 601ndash609 2011
[16] B He R Santamaria W Xu et al ldquoThe transmembrane activa-tor TACI triggers immunoglobulin class switching by activatingB cells through the adaptorMyD88rdquoNature Immunology vol 11no 9 pp 836ndash845 2010
[17] U Salzer C Bacchelli S Buckridge et al ldquoRelevance of biallelicversus monoallelic TNFRSF13B mutations in distinguishingdisease-causing from risk-increasing TNFRSF13B variants inantibody deficiency syndromesrdquo Blood vol 113 no 9 pp 1967ndash1976 2009
[18] L Zhang L Radigan U Salzer et al ldquoTransmembrane acti-vator and calcium-modulating cyclophilin ligand interactormutations in common variable immunodeficiency clinical andimmunologic outcomes in heterozygotesrdquo Journal of Allergy andClinical Immunology vol 120 no 5 pp 1178ndash1185 2007
[19] Q Pan-Hammarstrom U Salzer L Du et al ldquoReexamining therole of TACI coding variants in common variable immunode-ficiency and selective IgA deficiencyrdquo Nature Genetics vol 39no 4 pp 429ndash430 2007
[20] R M Locksley N Killeen and M J Lenardo ldquoThe TNF andTNF receptor superfamilies integrating mammalian biologyrdquoCell vol 104 no 4 pp 487ndash501 2001
[21] L Garibyan A A Lobito R M Siegel M E Call K WWucherpfennig and R S Geha ldquoDominant-negative effect ofthe heterozygous C104R TACI mutation in common variableimmunodeficiency (CVID)rdquo Journal of Clinical Investigationvol 117 no 6 pp 1550ndash1557 2007
[22] S GHymowitz D R Patel H J AWallweber et al ldquoStructuresof APRIL-receptor complexes like BCMA TACI employs onlya single cysteine-rich domain for high affinity ligand bindingrdquoJournal of Biological Chemistry vol 280 no 8 pp 7218ndash72272005
[23] E Castigli S A Wilson L Garibyan et al ldquoTACI is mutantin common variable immunodeficiency and IgA deficiencyrdquoNature Genetics vol 37 no 8 pp 829ndash834 2005
[24] U Salzer H M Chapel A D B Webster et al ldquoMutationsin TNFRSF13B encoding TACI are associated with commonvariable immunodeficiency in humansrdquoNature Genetics vol 37no 8 pp 820ndash828 2005
10 ISRN Immunology
[25] C Bacchelli K F Buckland S Buckridge et al ldquoThe C76Rtransmembrane activator and calcium modulator cyclophilinligand interactor mutation disrupts antibody production andB-cell homeostasis in heterozygous and homozygous micerdquoJournal of Allergy and Clinical Immunology vol 127 no 5 pp1253ndash1259 2011
[26] A J Fried I Rauter S R Dillon H H Jabara and R S GehaldquoFunctional analysis of transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) mutationsassociated with common variable immunodeficiencyrdquo Journalof Allergy and Clinical Immunology vol 128 no 1 pp 226ndash2282011
[27] J J Lee H H Jabara L Garibyan et al ldquoThe C104R mutantimpairs the function of transmembrane activator and calciummodulator and cyclophilin ligand interactor (TACI) throughhaploinsufficiencyrdquo Journal of Allergy and Clinical Immunologyvol 126 no 6 pp 1234ndashe2 2010
[28] J J Lee I Rauter L Garibyan et al ldquoThe murine equivalentof the A181E TACI mutation associated with common variableimmunodeficiency severely impairs B-cell functionrdquo Blood vol114 no 11 pp 2254ndash2262 2009
[29] A A JM van deVen L van de Corput CM van Tilburg et alldquoLymphocyte characteristics in children with common variableimmunodeficiencyrdquoClinical Immunology vol 135 no 1 pp 63ndash71 2010
[30] E Latz A Schoenemeyer A Visintin et al ldquoTLR9 signals aftertranslocating from theER toCpGDNA in the lysosomerdquoNatureImmunology vol 5 no 2 pp 190ndash198 2004
[31] C Foerster N Voelxen M Rakhmanov et al ldquoB cell receptor-mediated calcium signaling is impaired in B lymphocytes oftype Ia patients with common variable immunodeficiencyrdquoJournal of Immunology vol 184 no 12 pp 7305ndash7313 2010
[32] M C Van Zelm J Smet B Adams et al ldquoCD81 gene defectin humans disrupts CD19 complex formation and leads toantibody deficiencyrdquo Journal of Clinical Investigation vol 120no 4 pp 1265ndash1274 2010
[33] M Malphettes L Gerard M Carmagnat et al ldquoLate-onsetcombined immune deficiency a subset of common variableimmunodeficiency with severe T cell defectrdquo Clinical InfectiousDiseases vol 49 no 9 pp 1329ndash1338 2009
[34] F Dedeoglu B Horwitz J Chaudhuri F W Alt and R SGeha ldquoInduction of activation-induced cytidine deaminasegene expression by IL-4 and CD40 ligation is dependent onSTAT6 and NF120581Brdquo International Immunology vol 16 no 3 pp395ndash404 2004
[35] M Muramatsu K Kinoshita S Fagarasan S Yamada YShinkai and T Honjo ldquoClass switch recombination andhypermutation require activation-induced cytidine deaminase(AID) a potential RNA editing enzymerdquo Cell vol 102 no 5 pp553ndash563 2000
[36] P Revy T Muto Y Levy et al ldquoActivation-induced cytidinedeaminase (AID) deficiency causes the autosomal recessiveform of the hyper-IgM syndrome (HIGM2)rdquo Cell vol 102 no5 pp 565ndash575 2000
[37] A M Krieg ldquoCpG motifs in bacterial DNA and their immuneeffectsrdquo Annual Review of Immunology vol 20 pp 709ndash7602002
[38] T Kawai and S Akira ldquoInnate immune recognition of viralinfectionrdquo Nature Immunology vol 7 no 2 pp 131ndash137 2006
[39] E Latz A Verma AVisintin et al ldquoLigand-induced conforma-tional changes allosterically activate Toll-like receptor 9rdquoNatureImmunology vol 8 no 7 pp 772ndash779 2007
[40] S Sato H Sanjo K Takeda et al ldquoEssential function for thekinase TAK1 in innate and adaptive immune responsesrdquoNatureImmunology vol 6 no 11 pp 1087ndash1095 2005
[41] F Mackay and H Leung ldquoThe role of the BAFFAPRIL systemon T cell functionrdquo Seminars in Immunology vol 18 no 5 pp284ndash289 2006
binding due to haploinsufficiency whereas in the A181Evariant ligand binding remains intact [21 26] At least aproportion of A181EC104R TACI trimers will thus be ableto bind ligand As NF120581B activation was strongly decreasedin both variants we hypothesized TACI function to beaffected AID and NFAT mRNA induction were reduced inthe index patient and seemed also reduced in the CVIDpatients with a heterozygous A181E variant [14] Converselysibling 2 showed AID and NFAT induction comparable tohealthy controls and CVID patients with unaffected TACIalleles NFAT activation induces transcription of cytokinegenes encoding for example IL-4 which is involved in classswitch recombination IL-4 and CD40 ligation induces AIDmRNA and protein expression and depends on STAT6 andNF120581B [34] AID is pivotal for class switch recombination andsomatic hypermutation of theBCR [35 36] bothmechanismsare routinely impaired in CVID
The differential TACI function in the index patient andsibling was accompanied by a change in TACI protein levelsshowing increased TACI expression in the sibling both atthe cell surface and intracellular level TACI protein levels inthe healthy sibling were increased in comparison to healthycontrols as well As TNFRSF13B gene transcription wascomparable between all subjects except for the healthy sib-ling these observations suggest that protein turnover in thehealthy sibling may be decreased possibly due to alterationsin posttranslational modification of TACI protein affectingprotein stability and folding transport or degradation
Alternatively altered TACI function in the healthy siblingwas compensated by increasing the TACI protein quantitythrough cross-talk with related immune pathways CVIDis generally assumed to be a polygenetic disorder and itis plausible that alterations in the index patient were notlimited to the TNFRSF13B gene This prompted us to analyzeother essential immune pathways in B lymphocytes andwe investigated CD40 BCR and TLR9 signaling We didnot find any additional defect in BCR or CD40 signalingNevertheless for both pathways defective interaction withother cells or factors such as abrogated CD40L ligation onT cells or CD21- complement C3d fragment interactionscannot be excluded
In contrast TLR9 expression and function were con-siderably dissimilar between index patient and sibling 2Since the TLR9 expression levels and function were compa-rable between the index patient and the other subjects butincreased in sibling 2 we hypothesize that compensatoryTLR9 expression occurs in the sibling TLR9 recognizesunmethylated CpG-motif-containing bacterial or viral DNA[37 38] and has a critical role in the prevention of severalbacterial and viral infections Ligation of TLR9 inducesrecruitment of MyD88 [30 39] initiating several signalingevents which eventually result in activation of NF120581B and AP-1 [40] By the same token increased TACI protein stabilitymay participate in a compensatory mechanism in the healthysibling possibly related to TLR9 signaling TLR9 engagementinduced TACI upregulation and enhanced TACI-mediated Igclass switching of effector B cells resulting in increased IgGsecretion in cells exposed to anti-TACI and IL-10 and inducedsecretion of IgA
This study has some limitations The TLR9 signalingpathway through MyD88 could be studied more extensivelynot only on a functional level but also through whole exomesequencing The effects of TACI malfunctioning on expres-sion and function of several other targets could have beenaddressed including other TNF receptors and their ligandsor signaling proteins downstream of TACI such as TRAFsCAML and AP-1 Additional defects may not be limitedto B-cell lymphocytes TACI expression can be induced inCD4+ and CD8 T+ cells [4 41] study of TACI-related T-cellfunction would be particularly interesting since the patientdemonstrated chronic viral infections in vivo and impairedantigenic T-cells responses to several viruses in vitro
5 Conclusion
We here describe the diverse clinical and immunologicalcharacteristics in siblings with an identical TACI geno-type These clinical differences are supported by differencesin TACI protein expression and function We proposethat compensatory regulatory mechanisms involving post-translational modification of TACI protein and TLR9 existand may overcome the altered function of TACI trimersin asymptomatic TACI A181EC104R individuals Additionalstudies of TACI protein homeostasis the TACI signalingpathway and interactionwithTLR9 and expression and func-tion of TLR9 downstream signaling molecules are thereforeadvisable
Joris M van Montfrans and Marianne Boes contributedequally
Acknowledgments
The authors would like to thank the technicians of theDepartment of Medical Immunology for excellent assistancewith the TACI sequencing setup and Ewoud Compeer andArie Jan Stoppelenburg for help with PCR and data analyses
References
[1] M A Park J T Li J B Hagan D E Maddox and R SAbraham ldquoCommon variable immunodeficiency a new lookat an old diseaserdquo The Lancet vol 372 no 9637 pp 489ndash5022008
[2] C Cunningham-Rundles L Radigan A K Knight L ZhangL Bauer and A Nakazawa ldquoTLR9 activation is defective incommon variable immune deficiencyrdquo Journal of Immunologyvol 176 no 3 pp 1978ndash1987 2006
[3] J E Yu A K Knight L Radigan et al ldquoToll-like receptor 7and 9 defects in common variable immunodeficiencyrdquo Journalof Allergy and Clinical Immunology vol 124 no 2 pp 349ndash3562009
[4] G-U Von Bulow and R J Bram ldquoNF-AT activation inducedby a CAML-interacting member of the tumor necrosis factorreceptor superfamilyrdquo Science vol 278 no 5335 pp 138ndash1411997
[5] P Schneider F Mackay V Steiner et al ldquoBAFF a novel ligandof the tumor necrosis factor family stimulates B cell growthrdquoJournal of Experimental Medicine vol 189 no 11 pp 1747ndash17561999
[6] Y Wu D Bressette J A Carrell et al ldquoTumor necrosis factor(TNF) receptor superfamily member TACI is a high affinityreceptor for TNF family members APRIL and BLySrdquo Journal ofBiological Chemistry vol 275 no 45 pp 35478ndash35485 2000
[7] S A Marsters M Yan R M Pitti P E Haas V M Dixit andA Ashkenazi ldquoInteraction of the TNF homologues BLyS andAPRIL with the TNF receptor homologues BCMA and TACIrdquoCurrent Biology vol 10 no 13 pp 785ndash788 2000
[8] D Bischof S F Elsawa G Mantchev et al ldquoSelective activationof TACI by syndecan-2rdquo Blood vol 107 no 8 pp 3235ndash32422006
[9] F Mackay P Schneider P Rennert and J Browning ldquoBAFFand APRIL a tutorial on B cell survivalrdquo Annual Review ofImmunology vol 21 pp 231ndash264 2003
[10] Y Laabi M P Gras F Carbonnel et al ldquoA new gene BCMon chromosome 16 is fused to the interleukin 2 gene by at(416)(q26p13) translocation in a malignant T cell lymphomardquoEMBO Journal vol 11 no 11 pp 3897ndash3904 1992
[11] J S Thompson S A Bixler F Qian et al ldquoBAFF-R a newlyidentified TNF receptor that specifically interacts with BAFFrdquoScience vol 293 no 5537 pp 2108ndash2111 2001
[12] J Hauer S Puschner P Ramakrishnan et al ldquoTNF receptor(TNFR)-associated factor (TRAF) 3 serves as an inhibitorof TRAF25-mediated activation of the noncanonical NF-120581Bpathway by TRAF-binding TNFRsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 102 no8 pp 2874ndash2879 2005
[13] E Castigli S AWilsonA Elkhal EOzcan LGaribyan andRS Geha ldquoTransmembrane activator and calciummodulator andcyclophilin ligand interactor enhances CD40-driven plasmacell differentiationrdquo Journal of Allergy and Clinical Immunologyvol 120 no 4 pp 885ndash891 2007
[14] E Ozcan L Garibyan J J-Y Lee R J Bram K-P Lamand R S Geha ldquoTransmembrane activator calciummodulatorand cyclophilin ligand interactor drives plasma cell differenti-ation in LPS-activated B cellsrdquo Journal of Allergy and ClinicalImmunology vol 123 no 6 pp 1277ndash1286 2009
[15] E Ozcan I Rauter L Garibyan S R Dillon and R S GehaldquoToll-like receptor 9 transmembrane activator and calcium-modulating cyclophilin ligand interactor and CD40 synergizein causing B-cell activationrdquo Journal of Allergy and ClinicalImmunology vol 128 no 3 pp 601ndash609 2011
[16] B He R Santamaria W Xu et al ldquoThe transmembrane activa-tor TACI triggers immunoglobulin class switching by activatingB cells through the adaptorMyD88rdquoNature Immunology vol 11no 9 pp 836ndash845 2010
[17] U Salzer C Bacchelli S Buckridge et al ldquoRelevance of biallelicversus monoallelic TNFRSF13B mutations in distinguishingdisease-causing from risk-increasing TNFRSF13B variants inantibody deficiency syndromesrdquo Blood vol 113 no 9 pp 1967ndash1976 2009
[18] L Zhang L Radigan U Salzer et al ldquoTransmembrane acti-vator and calcium-modulating cyclophilin ligand interactormutations in common variable immunodeficiency clinical andimmunologic outcomes in heterozygotesrdquo Journal of Allergy andClinical Immunology vol 120 no 5 pp 1178ndash1185 2007
[19] Q Pan-Hammarstrom U Salzer L Du et al ldquoReexamining therole of TACI coding variants in common variable immunode-ficiency and selective IgA deficiencyrdquo Nature Genetics vol 39no 4 pp 429ndash430 2007
[20] R M Locksley N Killeen and M J Lenardo ldquoThe TNF andTNF receptor superfamilies integrating mammalian biologyrdquoCell vol 104 no 4 pp 487ndash501 2001
[21] L Garibyan A A Lobito R M Siegel M E Call K WWucherpfennig and R S Geha ldquoDominant-negative effect ofthe heterozygous C104R TACI mutation in common variableimmunodeficiency (CVID)rdquo Journal of Clinical Investigationvol 117 no 6 pp 1550ndash1557 2007
[22] S GHymowitz D R Patel H J AWallweber et al ldquoStructuresof APRIL-receptor complexes like BCMA TACI employs onlya single cysteine-rich domain for high affinity ligand bindingrdquoJournal of Biological Chemistry vol 280 no 8 pp 7218ndash72272005
[23] E Castigli S A Wilson L Garibyan et al ldquoTACI is mutantin common variable immunodeficiency and IgA deficiencyrdquoNature Genetics vol 37 no 8 pp 829ndash834 2005
[24] U Salzer H M Chapel A D B Webster et al ldquoMutationsin TNFRSF13B encoding TACI are associated with commonvariable immunodeficiency in humansrdquoNature Genetics vol 37no 8 pp 820ndash828 2005
10 ISRN Immunology
[25] C Bacchelli K F Buckland S Buckridge et al ldquoThe C76Rtransmembrane activator and calcium modulator cyclophilinligand interactor mutation disrupts antibody production andB-cell homeostasis in heterozygous and homozygous micerdquoJournal of Allergy and Clinical Immunology vol 127 no 5 pp1253ndash1259 2011
[26] A J Fried I Rauter S R Dillon H H Jabara and R S GehaldquoFunctional analysis of transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) mutationsassociated with common variable immunodeficiencyrdquo Journalof Allergy and Clinical Immunology vol 128 no 1 pp 226ndash2282011
[27] J J Lee H H Jabara L Garibyan et al ldquoThe C104R mutantimpairs the function of transmembrane activator and calciummodulator and cyclophilin ligand interactor (TACI) throughhaploinsufficiencyrdquo Journal of Allergy and Clinical Immunologyvol 126 no 6 pp 1234ndashe2 2010
[28] J J Lee I Rauter L Garibyan et al ldquoThe murine equivalentof the A181E TACI mutation associated with common variableimmunodeficiency severely impairs B-cell functionrdquo Blood vol114 no 11 pp 2254ndash2262 2009
[29] A A JM van deVen L van de Corput CM van Tilburg et alldquoLymphocyte characteristics in children with common variableimmunodeficiencyrdquoClinical Immunology vol 135 no 1 pp 63ndash71 2010
[30] E Latz A Schoenemeyer A Visintin et al ldquoTLR9 signals aftertranslocating from theER toCpGDNA in the lysosomerdquoNatureImmunology vol 5 no 2 pp 190ndash198 2004
[31] C Foerster N Voelxen M Rakhmanov et al ldquoB cell receptor-mediated calcium signaling is impaired in B lymphocytes oftype Ia patients with common variable immunodeficiencyrdquoJournal of Immunology vol 184 no 12 pp 7305ndash7313 2010
[32] M C Van Zelm J Smet B Adams et al ldquoCD81 gene defectin humans disrupts CD19 complex formation and leads toantibody deficiencyrdquo Journal of Clinical Investigation vol 120no 4 pp 1265ndash1274 2010
[33] M Malphettes L Gerard M Carmagnat et al ldquoLate-onsetcombined immune deficiency a subset of common variableimmunodeficiency with severe T cell defectrdquo Clinical InfectiousDiseases vol 49 no 9 pp 1329ndash1338 2009
[34] F Dedeoglu B Horwitz J Chaudhuri F W Alt and R SGeha ldquoInduction of activation-induced cytidine deaminasegene expression by IL-4 and CD40 ligation is dependent onSTAT6 and NF120581Brdquo International Immunology vol 16 no 3 pp395ndash404 2004
[35] M Muramatsu K Kinoshita S Fagarasan S Yamada YShinkai and T Honjo ldquoClass switch recombination andhypermutation require activation-induced cytidine deaminase(AID) a potential RNA editing enzymerdquo Cell vol 102 no 5 pp553ndash563 2000
[36] P Revy T Muto Y Levy et al ldquoActivation-induced cytidinedeaminase (AID) deficiency causes the autosomal recessiveform of the hyper-IgM syndrome (HIGM2)rdquo Cell vol 102 no5 pp 565ndash575 2000
[37] A M Krieg ldquoCpG motifs in bacterial DNA and their immuneeffectsrdquo Annual Review of Immunology vol 20 pp 709ndash7602002
[38] T Kawai and S Akira ldquoInnate immune recognition of viralinfectionrdquo Nature Immunology vol 7 no 2 pp 131ndash137 2006
[39] E Latz A Verma AVisintin et al ldquoLigand-induced conforma-tional changes allosterically activate Toll-like receptor 9rdquoNatureImmunology vol 8 no 7 pp 772ndash779 2007
[40] S Sato H Sanjo K Takeda et al ldquoEssential function for thekinase TAK1 in innate and adaptive immune responsesrdquoNatureImmunology vol 6 no 11 pp 1087ndash1095 2005
[41] F Mackay and H Leung ldquoThe role of the BAFFAPRIL systemon T cell functionrdquo Seminars in Immunology vol 18 no 5 pp284ndash289 2006
Joris M van Montfrans and Marianne Boes contributedequally
Acknowledgments
The authors would like to thank the technicians of theDepartment of Medical Immunology for excellent assistancewith the TACI sequencing setup and Ewoud Compeer andArie Jan Stoppelenburg for help with PCR and data analyses
References
[1] M A Park J T Li J B Hagan D E Maddox and R SAbraham ldquoCommon variable immunodeficiency a new lookat an old diseaserdquo The Lancet vol 372 no 9637 pp 489ndash5022008
[2] C Cunningham-Rundles L Radigan A K Knight L ZhangL Bauer and A Nakazawa ldquoTLR9 activation is defective incommon variable immune deficiencyrdquo Journal of Immunologyvol 176 no 3 pp 1978ndash1987 2006
[3] J E Yu A K Knight L Radigan et al ldquoToll-like receptor 7and 9 defects in common variable immunodeficiencyrdquo Journalof Allergy and Clinical Immunology vol 124 no 2 pp 349ndash3562009
[4] G-U Von Bulow and R J Bram ldquoNF-AT activation inducedby a CAML-interacting member of the tumor necrosis factorreceptor superfamilyrdquo Science vol 278 no 5335 pp 138ndash1411997
[5] P Schneider F Mackay V Steiner et al ldquoBAFF a novel ligandof the tumor necrosis factor family stimulates B cell growthrdquoJournal of Experimental Medicine vol 189 no 11 pp 1747ndash17561999
[6] Y Wu D Bressette J A Carrell et al ldquoTumor necrosis factor(TNF) receptor superfamily member TACI is a high affinityreceptor for TNF family members APRIL and BLySrdquo Journal ofBiological Chemistry vol 275 no 45 pp 35478ndash35485 2000
[7] S A Marsters M Yan R M Pitti P E Haas V M Dixit andA Ashkenazi ldquoInteraction of the TNF homologues BLyS andAPRIL with the TNF receptor homologues BCMA and TACIrdquoCurrent Biology vol 10 no 13 pp 785ndash788 2000
[8] D Bischof S F Elsawa G Mantchev et al ldquoSelective activationof TACI by syndecan-2rdquo Blood vol 107 no 8 pp 3235ndash32422006
[9] F Mackay P Schneider P Rennert and J Browning ldquoBAFFand APRIL a tutorial on B cell survivalrdquo Annual Review ofImmunology vol 21 pp 231ndash264 2003
[10] Y Laabi M P Gras F Carbonnel et al ldquoA new gene BCMon chromosome 16 is fused to the interleukin 2 gene by at(416)(q26p13) translocation in a malignant T cell lymphomardquoEMBO Journal vol 11 no 11 pp 3897ndash3904 1992
[11] J S Thompson S A Bixler F Qian et al ldquoBAFF-R a newlyidentified TNF receptor that specifically interacts with BAFFrdquoScience vol 293 no 5537 pp 2108ndash2111 2001
[12] J Hauer S Puschner P Ramakrishnan et al ldquoTNF receptor(TNFR)-associated factor (TRAF) 3 serves as an inhibitorof TRAF25-mediated activation of the noncanonical NF-120581Bpathway by TRAF-binding TNFRsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 102 no8 pp 2874ndash2879 2005
[13] E Castigli S AWilsonA Elkhal EOzcan LGaribyan andRS Geha ldquoTransmembrane activator and calciummodulator andcyclophilin ligand interactor enhances CD40-driven plasmacell differentiationrdquo Journal of Allergy and Clinical Immunologyvol 120 no 4 pp 885ndash891 2007
[14] E Ozcan L Garibyan J J-Y Lee R J Bram K-P Lamand R S Geha ldquoTransmembrane activator calciummodulatorand cyclophilin ligand interactor drives plasma cell differenti-ation in LPS-activated B cellsrdquo Journal of Allergy and ClinicalImmunology vol 123 no 6 pp 1277ndash1286 2009
[15] E Ozcan I Rauter L Garibyan S R Dillon and R S GehaldquoToll-like receptor 9 transmembrane activator and calcium-modulating cyclophilin ligand interactor and CD40 synergizein causing B-cell activationrdquo Journal of Allergy and ClinicalImmunology vol 128 no 3 pp 601ndash609 2011
[16] B He R Santamaria W Xu et al ldquoThe transmembrane activa-tor TACI triggers immunoglobulin class switching by activatingB cells through the adaptorMyD88rdquoNature Immunology vol 11no 9 pp 836ndash845 2010
[17] U Salzer C Bacchelli S Buckridge et al ldquoRelevance of biallelicversus monoallelic TNFRSF13B mutations in distinguishingdisease-causing from risk-increasing TNFRSF13B variants inantibody deficiency syndromesrdquo Blood vol 113 no 9 pp 1967ndash1976 2009
[18] L Zhang L Radigan U Salzer et al ldquoTransmembrane acti-vator and calcium-modulating cyclophilin ligand interactormutations in common variable immunodeficiency clinical andimmunologic outcomes in heterozygotesrdquo Journal of Allergy andClinical Immunology vol 120 no 5 pp 1178ndash1185 2007
[19] Q Pan-Hammarstrom U Salzer L Du et al ldquoReexamining therole of TACI coding variants in common variable immunode-ficiency and selective IgA deficiencyrdquo Nature Genetics vol 39no 4 pp 429ndash430 2007
[20] R M Locksley N Killeen and M J Lenardo ldquoThe TNF andTNF receptor superfamilies integrating mammalian biologyrdquoCell vol 104 no 4 pp 487ndash501 2001
[21] L Garibyan A A Lobito R M Siegel M E Call K WWucherpfennig and R S Geha ldquoDominant-negative effect ofthe heterozygous C104R TACI mutation in common variableimmunodeficiency (CVID)rdquo Journal of Clinical Investigationvol 117 no 6 pp 1550ndash1557 2007
[22] S GHymowitz D R Patel H J AWallweber et al ldquoStructuresof APRIL-receptor complexes like BCMA TACI employs onlya single cysteine-rich domain for high affinity ligand bindingrdquoJournal of Biological Chemistry vol 280 no 8 pp 7218ndash72272005
[23] E Castigli S A Wilson L Garibyan et al ldquoTACI is mutantin common variable immunodeficiency and IgA deficiencyrdquoNature Genetics vol 37 no 8 pp 829ndash834 2005
[24] U Salzer H M Chapel A D B Webster et al ldquoMutationsin TNFRSF13B encoding TACI are associated with commonvariable immunodeficiency in humansrdquoNature Genetics vol 37no 8 pp 820ndash828 2005
10 ISRN Immunology
[25] C Bacchelli K F Buckland S Buckridge et al ldquoThe C76Rtransmembrane activator and calcium modulator cyclophilinligand interactor mutation disrupts antibody production andB-cell homeostasis in heterozygous and homozygous micerdquoJournal of Allergy and Clinical Immunology vol 127 no 5 pp1253ndash1259 2011
[26] A J Fried I Rauter S R Dillon H H Jabara and R S GehaldquoFunctional analysis of transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) mutationsassociated with common variable immunodeficiencyrdquo Journalof Allergy and Clinical Immunology vol 128 no 1 pp 226ndash2282011
[27] J J Lee H H Jabara L Garibyan et al ldquoThe C104R mutantimpairs the function of transmembrane activator and calciummodulator and cyclophilin ligand interactor (TACI) throughhaploinsufficiencyrdquo Journal of Allergy and Clinical Immunologyvol 126 no 6 pp 1234ndashe2 2010
[28] J J Lee I Rauter L Garibyan et al ldquoThe murine equivalentof the A181E TACI mutation associated with common variableimmunodeficiency severely impairs B-cell functionrdquo Blood vol114 no 11 pp 2254ndash2262 2009
[29] A A JM van deVen L van de Corput CM van Tilburg et alldquoLymphocyte characteristics in children with common variableimmunodeficiencyrdquoClinical Immunology vol 135 no 1 pp 63ndash71 2010
[30] E Latz A Schoenemeyer A Visintin et al ldquoTLR9 signals aftertranslocating from theER toCpGDNA in the lysosomerdquoNatureImmunology vol 5 no 2 pp 190ndash198 2004
[31] C Foerster N Voelxen M Rakhmanov et al ldquoB cell receptor-mediated calcium signaling is impaired in B lymphocytes oftype Ia patients with common variable immunodeficiencyrdquoJournal of Immunology vol 184 no 12 pp 7305ndash7313 2010
[32] M C Van Zelm J Smet B Adams et al ldquoCD81 gene defectin humans disrupts CD19 complex formation and leads toantibody deficiencyrdquo Journal of Clinical Investigation vol 120no 4 pp 1265ndash1274 2010
[33] M Malphettes L Gerard M Carmagnat et al ldquoLate-onsetcombined immune deficiency a subset of common variableimmunodeficiency with severe T cell defectrdquo Clinical InfectiousDiseases vol 49 no 9 pp 1329ndash1338 2009
[34] F Dedeoglu B Horwitz J Chaudhuri F W Alt and R SGeha ldquoInduction of activation-induced cytidine deaminasegene expression by IL-4 and CD40 ligation is dependent onSTAT6 and NF120581Brdquo International Immunology vol 16 no 3 pp395ndash404 2004
[35] M Muramatsu K Kinoshita S Fagarasan S Yamada YShinkai and T Honjo ldquoClass switch recombination andhypermutation require activation-induced cytidine deaminase(AID) a potential RNA editing enzymerdquo Cell vol 102 no 5 pp553ndash563 2000
[36] P Revy T Muto Y Levy et al ldquoActivation-induced cytidinedeaminase (AID) deficiency causes the autosomal recessiveform of the hyper-IgM syndrome (HIGM2)rdquo Cell vol 102 no5 pp 565ndash575 2000
[37] A M Krieg ldquoCpG motifs in bacterial DNA and their immuneeffectsrdquo Annual Review of Immunology vol 20 pp 709ndash7602002
[38] T Kawai and S Akira ldquoInnate immune recognition of viralinfectionrdquo Nature Immunology vol 7 no 2 pp 131ndash137 2006
[39] E Latz A Verma AVisintin et al ldquoLigand-induced conforma-tional changes allosterically activate Toll-like receptor 9rdquoNatureImmunology vol 8 no 7 pp 772ndash779 2007
[40] S Sato H Sanjo K Takeda et al ldquoEssential function for thekinase TAK1 in innate and adaptive immune responsesrdquoNatureImmunology vol 6 no 11 pp 1087ndash1095 2005
[41] F Mackay and H Leung ldquoThe role of the BAFFAPRIL systemon T cell functionrdquo Seminars in Immunology vol 18 no 5 pp284ndash289 2006
[25] C Bacchelli K F Buckland S Buckridge et al ldquoThe C76Rtransmembrane activator and calcium modulator cyclophilinligand interactor mutation disrupts antibody production andB-cell homeostasis in heterozygous and homozygous micerdquoJournal of Allergy and Clinical Immunology vol 127 no 5 pp1253ndash1259 2011
[26] A J Fried I Rauter S R Dillon H H Jabara and R S GehaldquoFunctional analysis of transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) mutationsassociated with common variable immunodeficiencyrdquo Journalof Allergy and Clinical Immunology vol 128 no 1 pp 226ndash2282011
[27] J J Lee H H Jabara L Garibyan et al ldquoThe C104R mutantimpairs the function of transmembrane activator and calciummodulator and cyclophilin ligand interactor (TACI) throughhaploinsufficiencyrdquo Journal of Allergy and Clinical Immunologyvol 126 no 6 pp 1234ndashe2 2010
[28] J J Lee I Rauter L Garibyan et al ldquoThe murine equivalentof the A181E TACI mutation associated with common variableimmunodeficiency severely impairs B-cell functionrdquo Blood vol114 no 11 pp 2254ndash2262 2009
[29] A A JM van deVen L van de Corput CM van Tilburg et alldquoLymphocyte characteristics in children with common variableimmunodeficiencyrdquoClinical Immunology vol 135 no 1 pp 63ndash71 2010
[30] E Latz A Schoenemeyer A Visintin et al ldquoTLR9 signals aftertranslocating from theER toCpGDNA in the lysosomerdquoNatureImmunology vol 5 no 2 pp 190ndash198 2004
[31] C Foerster N Voelxen M Rakhmanov et al ldquoB cell receptor-mediated calcium signaling is impaired in B lymphocytes oftype Ia patients with common variable immunodeficiencyrdquoJournal of Immunology vol 184 no 12 pp 7305ndash7313 2010
[32] M C Van Zelm J Smet B Adams et al ldquoCD81 gene defectin humans disrupts CD19 complex formation and leads toantibody deficiencyrdquo Journal of Clinical Investigation vol 120no 4 pp 1265ndash1274 2010
[33] M Malphettes L Gerard M Carmagnat et al ldquoLate-onsetcombined immune deficiency a subset of common variableimmunodeficiency with severe T cell defectrdquo Clinical InfectiousDiseases vol 49 no 9 pp 1329ndash1338 2009
[34] F Dedeoglu B Horwitz J Chaudhuri F W Alt and R SGeha ldquoInduction of activation-induced cytidine deaminasegene expression by IL-4 and CD40 ligation is dependent onSTAT6 and NF120581Brdquo International Immunology vol 16 no 3 pp395ndash404 2004
[35] M Muramatsu K Kinoshita S Fagarasan S Yamada YShinkai and T Honjo ldquoClass switch recombination andhypermutation require activation-induced cytidine deaminase(AID) a potential RNA editing enzymerdquo Cell vol 102 no 5 pp553ndash563 2000
[36] P Revy T Muto Y Levy et al ldquoActivation-induced cytidinedeaminase (AID) deficiency causes the autosomal recessiveform of the hyper-IgM syndrome (HIGM2)rdquo Cell vol 102 no5 pp 565ndash575 2000
[37] A M Krieg ldquoCpG motifs in bacterial DNA and their immuneeffectsrdquo Annual Review of Immunology vol 20 pp 709ndash7602002
[38] T Kawai and S Akira ldquoInnate immune recognition of viralinfectionrdquo Nature Immunology vol 7 no 2 pp 131ndash137 2006
[39] E Latz A Verma AVisintin et al ldquoLigand-induced conforma-tional changes allosterically activate Toll-like receptor 9rdquoNatureImmunology vol 8 no 7 pp 772ndash779 2007
[40] S Sato H Sanjo K Takeda et al ldquoEssential function for thekinase TAK1 in innate and adaptive immune responsesrdquoNatureImmunology vol 6 no 11 pp 1087ndash1095 2005
[41] F Mackay and H Leung ldquoThe role of the BAFFAPRIL systemon T cell functionrdquo Seminars in Immunology vol 18 no 5 pp284ndash289 2006
