Antibody deficiency associated with an inherited autosomal dominant mutation in TWEAK

Hong-Ying Wanga,1, Chi A. Maa,1, Yongge Zhaoa, Xiying Fana, Qing Zhoub, Pamela Edmondsa, Gulbu Uzelc, Joao Bosco Oliveirad, Jordan Orangee, and Ashish Jaina,2

aLaboratory of Host Defenses, National Institute of Allergy and Infectious Diseases (NIAID), bInflammatory Disease Section, National Research Institute, cImmunopathogenesis Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, and dDepartment of Laboratory Medicine, Clinical Center, National Institutes of Health (NIH), Bethesda, MD 20892; and eDepartment of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104

Edited by Rebecca H. Buckley, Duke University Medical Center, Durham, NC, and approved January 29, 2013 (received for review December 11, 2012)

Mutations in the TNF family of have been associated with In a recent array-based resequencing study, we screened 148 inherited forms of immune deficiency. Using an array-based se- candidate implicated in B-cell development, class switch quencing assay, we identified an autosomal-dominant deficiency in recombination, and NF-κB signaling in 35 patients with defective TNF-like weak inducer of (TWEAK; TNFSF12) in a kindred antibody production (13). In one kindred with impaired humoral with recurrent infection and impaired antibody responses to immunity and recurrent infections we identified a heterozygous and polysaccharide vaccines. This mutation occurs in the sixth exon mutation in the sixth exon of TWEAK (TNF-like weak inducer of of TWEAK and results in the amino acid substitution R145C within apoptosis, APO3L, TNFSF12) that leads to a single amino acid the conserved TNF-homology domain of the full-length protein. substitution of arginine 145 to cysteine (position 52 in the secreted TWEAK mutant protein formed high molecular weight aggregates form). Similar to BAFF and many other TNFSFs, TWEAK can under nonreducing conditions, suggesting an increased propensity function as a type II transmembrane protein or as a secreted for intermolecular interactions. As a result, mutant TWEAK associ- soluble cytokine that is cleaved by furin proteases (14). In general, TWEAK is widely expressed in many tissues and cell types, in- ated with B-cell–activating factor (BAFF) protein and down-regu- κ cluding monocytes/macrophages, dendritic cells, natural killer lated the BAFF-mediated activation of the noncanonical NF- B (NK) cells, and T cells, and its expression is increased during in- pathway through inhibition of p100 processing to p52, resulting flammation (15). Although the precise physiologic function of IMMUNOLOGY in inhibition of BAFF-dependent B-cell survival and proliferation. TWEAK awaits further delineation, it has been proposed that – As BAFF mediates T-cell independent isotype switching and B-cell TWEAK may promote proliferation in endothelial cells (16) and TWEAK survival, our data implicate as a disease-susceptibility modulate innate immunity transition to adaptive Th1 immunity for a humoral immunodeficiency. (17) through its receptor Fn14 (TWEAKR, TNFRSF12A) (18). We found that mutant R145C TWEAK has impaired signaling apoptosis | lymphopenia | cysteine mutation | heteromers function in the Fn14 pathway and may also affect B-cell de- velopment through interaction with BAFF. utations in the TNF superfamily (TNFSF) and their cognate Mreceptors have been identified in humans with inherited Results forms of autoimmune or immune deficiency disorders (1), for Case Report. The proband patient P2 developed pneumococcal example, autoimmune lymphoproliferative syndrome [FAS (CD95 meningitis complicated by multiple infarcts resulting in left-sided or TNFRSF6) mutations], TNF receptor-associated periodic syn- hemiparesis at 3 y of age. A review of the family medical history drome (TNFR1 mutations), hyper IgM syndrome (CD154/CD40 revealed that both siblings (P1 and P2 in the family pedigree; Fig. mutations), and EBV sensitivity (CD27 mutations). Common 1A)andtheirfather(B1)hadahistoryoffrequentearorsino- variable immunodeficiency (CVID) represents a group of heter- pulmonary infections since infancy. The father had been hospital- ogenous primary antibody deficiency disorders characterized by ized several times in infancy with pneumonia and had a prolonged a marked reduction in serum Ig levels with impaired antibody hospitalization for osteomyelitis, although the frequency and se- responses to common bacteria and viruses that result in re- verity of his infections had decreased in adulthood. The father had current infections (2). Single-gene defects in B-cell–activating chronic thrombocytopenia, and both children had intermittent neutropenia in the peripheral blood. The three affected family factor receptor BAFF-R (TNFRSF13C)(3)andTACI (trans- members had multiple warts. Subsequent immunologic evaluation membrane activator and calcium-modulating cyclophilin li- in both siblings and the father revealed the absence of antibody gand interactor, CD267, TNFRSF13B)(4,5)areassociated responses to T-cell–dependent and polysaccharide antigens (Table with some cases of CVID. 1). Serum Ig profiles and lymphocyte phenotypes were further Both BAFF-R and TACI bind to BAFF (B-cell activating fac- analyzed for the father and the two proband patients (Tables 1–3). tor, BLYS, TNFSF13B). BAFF is expressed primarily by mono- – All three affected members had low levels of IgM and IgA. Low cytes and dendritic cells (6) and may induce T-cell independent levels of IgG were revealed in patients P1 and P2, especially P2. isotype switching in naive B cells in the absence of CD40 en- Although the absolute IgG level of the father was considered gagement and exogenous cytokines (7). However, BAFF seems normal, both IgG2 and IgG4 subclasses were far below normal to play a major role in B-cell survival through interaction with BAFF-R (8) based on studies of mice deficient in either BAFF

or BAFF-R, which both display severe defects in peripheral B- Author contributions: H.-Y.W., C.A.M., and A.J. designed research; H.-Y.W., C.A.M., Y.Z., cell maturation and decreased levels of immunoglobulins (9, 10). X.F., Q.Z., P.E., G.U., J.B.O., and A.J. performed research; H.-Y.W. and J.O. contributed new BAFF can form active heterotrimeric molecules when coex- reagents/analytic tools; H.-Y.W., C.A.M., Y.Z., X.F., Q.Z., G.U., J.B.O., J.O., and A.J. analyzed pressed with the TNF ligand APRIL (a proliferation-inducing data; and H.-Y.W. and A.J. wrote the paper. ligand, TNFSF13), and circulating BAFF/APRIL heterotrimers The authors declare no conflict of interest. are present in serum samples from patients with autoimmune This article is a PNAS Direct Submission. disorders (11, 12). These findings raise the possibility that BAFF 1H.-Y.W. and C.A.M. contributed equally to this work. may form heteromeric complexes with other members of the 2To whom correspondence should be addressed. E-mail: [email protected]. TNF ligand superfamily and play a role in other autoimmune or This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. immunodeficient disease states. 1073/pnas.1221211110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1221211110 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 A B1 Cytoplasmic domain D Transmembrane domain Extracellular stalk region TNF Homology domain (THD) P1 P2 R145C 12142 94 249 B –2HN HOOC– A-Fwd/T-Rev G-Fwd/C-Rev aa1 – 249: membrane-bound form C-Fwd/G-Rev T-Fwd/A-Rev aa94 – 249: secreted form C/T E 120 166 TWEAK QDGAQAGVDGTVSGWEEARINSSSPLRYNRQIGEFIVTRAGLYYLYCQ-- Human QDGAQAGVDGTVSGWEETKINSSSPLRYDRQIGEFTVIRAGLYYLYCQ-- Mouse QDGAQAGVDGTVSGWEETKINSSSPLRYDRQIGEFTVIRAGLYYLYCQ-- Rat QDGAQAGVDGTVSGWEEAKINSSNPLRYDCQTGQFTVTRAGLYYLYCQ-- Cattle C C/T QDGAQAGVDGTVSGWEEAKINSSNPLRYDRQSGQFLVTRAGLYYLYCQ-- Wild boar CCTCTG GCTACAACC QDGAQAGVDGTVSGWEEAKINSSNPLRYDRQSGEFIVIRAGLYYLYCQ-- Horse QDGAQAGVDGTVSGWEEARINSSSPLRYNRQIGEFIVTRAGLYYLYCQ-- Chimpanzee QDGAQAGVDGTVSGWEEARINSSSPLRYNRQIGEFIVTRAGLYYLYCQ-- Rhesus Macaque QDGGQAGVDGTVSGWEEAKINSSNPLRYDRQSGEFIVTRAGLYYLYCQ-- Dog ***.*************::****.****: * *:* * **********

Fig. 1. Identification of a rare heterozygous mutation in the TWEAK gene in a family diagnosed with CVID. (A) Pedigree of the affected family members. Highlighted, mutation carrier; square, male; circle, female. (B) Representative image of resequencing array signals showing the heterozygous mutation in the TWEAK coding region. (C)Confirmation of the heterozygous mutation by Sanger sequencing. (D) Schematic representation of TWEAK structure showing full-length and secreted forms and location of the mutation site. (E) Sequence conservation of the TWEAK gene among mammals. Highlighted box indicates the mutation site.

range (Table 1). The father and patient P1 had reduced number of Center for Biotechnology Information Single Nucleotide Poly- B cells, and nearly all the B cells (>94%) in the three affected morphism database. + + patients were IgM IgD naive cells (Table 2, column 6). Fur- thermore, all three affected members had a slight increase in the Mutant TWEAK Fails to Induce Cell Death and Activate NF-κB and + − − percentage of TCRαβ CD4 CD8 T cells [i.e., double-negative T MAPK Pathways. As a TNF-like weak inducer of apoptosis, (DNT) cells] and an increase in total T cell numbers, especially TWEAK is able to induce apoptosis in certain cell lines through + + CD8 T cells, whereas the level and percentage of CD4 T cells induction of TNF-α secretion (21) or through other independent were generally within the normal range (Table 3). The maternal mechanisms (22). We therefore tested whether the R145C mu- medical history was unrevealing. The paternal grandmother died tation affected the ability of TWEAK to induce cell death in fi fi of lymphoma, and the paternal grandfather was not available for TWEAK-sensitive tumor cell lines. We rst puri ed Flag-tagged detailed analysis. soluble WT or mutant TWEAK protein from stable-transfected HEK293 cells, and an equal amount of each protein was used to Identification of Rare Heterozygous Mutation in TWEAK. By using a treat HT-29 cells, an adenocarcinoma cell line that is sensitized fi to TNF-induced cell death. Only WT TWEAK induced signifi- custom resequencing array (13), we identi ed a single-base het- γ erozygous mutation, c.433C>T, in the sixth exon of TWEAK in the cant apoptosis in HT-29 cells in the presence of IFN- ; mutant two siblings P1 and P2 (Fig. 1 B and C). This mutation results in TWEAK showed little apoptotic effect (Fig. 2A, compare col- the amino acid substitution from arginine (R) to cysteine (C) at umns 2 and 3). The proapoptotic effect of TWEAK was clearly blocked by preincubation of the protein with recombinant Fn14: position 145 in full-length TWEAK protein or position 52 in the Fc, the TWEAK receptor (Fig. 2A, column 5), but not by non- secreted protein. The p.R145C mutation is located in the con- specific human IgG (column 4). Moreover, this proapoptotic served TNF-homology domain (THD) of TWEAK and is close to effect of the WT TWEAK protein was dose-dependent, whereas the only N-glycosylation site (N125), which may be responsible for the mutant protein had little effect at any dose tested (Fig. 2B). trimerization of the TWEAK monomer and receptor binding (Fig. The loss of apoptotic function of mutant TWEAK protein may 1 D and E); in addition, PolyPhen prediction (http://genetics.bwh. result from structural changes induced by the R145C mutation. harvard.edu/pph/) indicated that the R-to-C change may be del- As this mutation removes a positive charge from the extracellular eterious (score of 1.992). Other than this mutation, the two sib- domain but leaves a free thiol group in the cysteine residue, an lings had no unusual nucleotide changes in known CVID disease- increase in intermolecular binding to itself or to other proteins is susceptibility genes such as BAFF, BAFF-R,orTACI (19), or in expected. Indeed, SDS/PAGE under nonreducing conditions the related genes FAS, FASLG (, TNFSF6), CASP3 revealed high molecular weight aggregates in lysates of cells (caspase 3, apoptosis-related cysteine peptidase), and CASP8 (cas- expressing the secreted form of mutant TWEAK protein pase 8) (20). The same heterozygous mutation was confirmed in (Fig. 2C). their father, B1, but was not detected in more than 200 internally As previously reported, high molecular weight aggregates of sequenced control samples, or in the latest build of the National TWEAK appear to be nonfunctional under some conditions

Table 1. Serum Ig characterization of three patients carrying the heterozygous mutation in TWEAK Pneumococcal Diphtheria Tetanus Patient Age at diagnosis, y Sex IgM, mg/dL IgG, mg/dL IgA, mg/dL antibodies, μg/mL antibodies, IU/mL antibodies, IU/mL

Normal ——34–342 642–1,730 91–499 >0.1 >0.1 >0.6 B1 31 M 61 672* 34† <0.1† 0.04† <0.6† ‡ † † † † † † P1 6F<21 562 <10 <0.03 <0.1 0.02 ‡ † † † † † † P2 4M<21 16 <10 <0.03 <0.1 <0.10

*Low IgG2 (85 mg/dL; normal range, 171–632 mg/dL) and IgG4 (0.3 mg/dL; normal range, 2.4–121 mg/dL). †Abnormal values. ‡Blood samples for subjects P1 and P2 were drawn before IVIG replacement therapy.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1221211110 Wang et al. Downloaded by guest on October 1, 2021 Table 2. B lymphocyte and platelet phenotype report for the three patients + − + + + CD19 IgD CD27 IgM IgD B cells/total + + + Patient Total CD19 B cells CD19 B cells, % CD27 B cells, % cells, % B cells, % Platelets, × 103/μL

Normal 47–409 3.5–7.1 0.7–6.3 0.4–2.3 63–94 161–347 B1 74 2.2* 0.9 0.4* 97* 55* P1 19* 0.9* ND* ND* 95* 226 P2 126 4.6 0.8 0.1* 98* 206

ND, not determined (i.e., too low to be determined accurately). *Abnormal values.

(23), possibly because of changes in protein conformation or loss mutant TWEAK has stronger binding to BAFF than WT of receptor binding activity. As the proapoptotic function of TWEAK (Fig. 3 A and B). In contrast, mutant TWEAK does not TWEAK is dependent on its receptor Fn14, we tested whether bind to CD40L (Fig. S1). the loss of function in this mutant was a result of loss of binding To confirm the in vivo association between BAFF and TWEAK, capability to Fn14. To our surprise, 293T cells transfected with we differentiated monocytes of the two siblings into dendritic mutant TWEAK seemed to have slightly increased binding to cells that express TWEAK and BAFF and performed similar FITC-labeled Fn14 compared with cells transfected with WT coimmunoprecipitation (IP) experiments. Using a monoclonal TWEAK (Fig. 2D). This indicates that the failure of R145C antibody against BAFF, BAFF–TWEAK association was ob- TWEAK to induce cell death in TWEAK-sensitive cell line is not served in activated dendritic cells from patient samples but not in a result of diminished interaction with Fn14. those from normal controls (Fig. 3C). IP experiments with an- TWEAK has been proposed to activate cell death pathways tibody against TWEAK gave a similar result (Fig. S2). We also through multiple pathways involving activation of NF-κB and conducted hybrid ELISA tests on serum samples of the two MAP kinases (22). To determine whether mutant TWEAK patients (P1 and P2) and their father (B1). Serum samples from affects IκB degradation, which leads to NF-κB release and ac- 20 normal individuals served as controls. The ELISA plates were tivation, we treated THP1 cells (a human acute monocytic leu- coated with anti-TWEAK antibody and detected by using a spe- fi fi – kemia cell line) with puri ed Flag-tagged WT or mutant ci c HRP-conjugated anti-BAFF antibody. A low level of BAFF IMMUNOLOGY TWEAK soluble protein. As expected, WT TWEAK induced TWEAK complex was generally detected in normal control serum IκB-α degradation in the presence of cycloheximide (CHX) samples, whereas serum from the two patients displayed signifi- whereas the mutant protein failed to induce IκB-α degradation cantly higher levels of BAFF–TWEAK protein complex, with (Fig. 2E). Similarly, mutant TWEAK lost the ability to induce patient P2 showing the highest levels (Fig. 3D). Interestingly, the MAPK pathways, as evidenced by minimal induction of JNK BAFF–TWEAK complex level was lower in the father, B1, than in phosphorylation in THP1 cells treated with mutant TWEAK the two siblings, which may explain the reduced severity of im- protein (Fig. 2F). Taken together, these data suggest that mutant munodeficient symptoms in the father. The incomplete pene- TWEAK is a partially functional ligand that can still bind to its trance of the phenotype in the kindred could relate to differences receptor but has lost the capability to stimulate NF-κB activation in expression of the mutant protein as well as differences in ge- and induce cell death. netic background of the affected individuals.

Mutant TWEAK Associates with BAFF. The free thiol group at the Mutant TWEAK Inhibits BAFF Signaling Function in B Cells. Associa- mutant position C145 may allow TWEAK protein to readily bind tion of mutant TWEAK with BAFF may affect binding of BAFF to other TNFSFs with structurally similar THDs through for- to its receptors. However, neither WT nor mutant TWEAK binds mation of an additional disulfide bridge. BAFF, a critical B-cell BAFF-R (Fig. S3). BAFF has three receptors: a high-affinity activation and survival factor, contains many cysteine residues receptor (BAFF-R), a moderate-affinity receptor (TACI), and and is prone to form homotrimers, oligomers, or heterotrimers alow-affinity receptor (BCMA). Cotransfection of full-length with other proteins such as APRIL (11). In fact, active BAFF/ BAFF with WT or mutant full-length TWEAK slightly enhanced APRIL heterotrimers were detected in serum samples from binding of BAFF to BAFF-R (Fig. 4A) but not to TACI (Fig. 4B); patients with systemic immune-based rheumatic diseases (11). As however, the enhancement effect was indistinguishable between the TWEAK receptor Fn14 resembles BCMA (B cell maturation WT and mutant TWEAK (Fig. 4A). Because an important antigen, TNFRSF17), one of the BAFF receptors, we reasoned function of BAFF is to provide continuous survival signaling to that the THD of TWEAK, which shares structural similarity with antigen-activated B cells through BAFF-R, we reasoned that that of BAFF, might interact with BAFF, and thus the TWEAK mutant TWEAK might affect downstream signaling of BAFF-R. mutation might affect this binding. To test this hypothesis, we Activation of BAFF-R induces the noncanonical NF-κB pathway cotransfected 293T cells with full-length BAFF together with by promoting processing of a precursor protein p100 (NF-κB2) either WT or mutant TWEAK. After 48 h, cells were lysed and to an active p52 subunit (24). Indeed, membrane fraction prep- immunoprecipitated with anti-BAFF or anti-TWEAK antibodies arations from BAFF stably expressing HEK293 cells transfected and then immunoblotted with antibodies against TWEAK or with mutant TWEAK showed decreased BAFF-induced pro- BAFF, respectively. Both experiments clearly demonstrated that cessing of p100 in naive B cells as evidenced by an increase in

Table 3. T lymphocyte phenotype report for the three patients Total α/β α/β DNT + + + + + + Patient Total CD3 T cells CD3 T cells, % Total CD4 T cells CD4 T cells, % Total CD8 T cells CD8 T cells, % DNT cells cells, %

Normal 650–2,108 57.3–86.4 362–1,275 29.0–57.3 344–911 25.2–50.8 2–17 <1.4 B1 3,036* 88.8* 1,294 37.8 1,795* 52.5* 33* 1.4* P1 2,061 92.9* 698 31.5 1,302* 58.7* 31* 1.7* P2 2,235* 82.5 810 30.0 1,346* 49.7 63* 3.2*

*Abnormal values.

Wang et al. PNAS Early Edition | 3of6 Downloaded by guest on October 1, 2021 35 D A B 40 showed a decreased proliferation response (Fig. 4 ). BAFF-me- 35 diated B-cell survival is primarily mediated by BAFF-R (25, 26). 25 ) ) To test whether mutant TWEAK down-regulates BAFF-de- -4 -4 30 15 pendent B-cell survival, human B cells were treated with 25 fi

(LU x 10 * recombinant BAFF with or without puri ed soluble TWEAK * (LU x 10 20 BSA * fi Cell survival index 5 * Cell survival index Twt WT or mutant, or with puri ed BAFF/mutant TWEAK hetero- Tmu 0 15 mers (Fig. S4). As expected, addition of WT or mutant soluble EV None 3 ng/ml 6 ng/ml 9 ng/ml TWEAK did not affect BAFF-induced B-cell survival, whereas Twt Purified TWEAK protein Tmu level in the medium IgG addition of BAFF/mutant TWEAK heteromers decreased cell Fn14:Fc viability to the levels seen in medium alone at all time points C D 16 tested. Finally, to analyze whether mutant TWEAK affects the

Transfection: EV Twt TmuTwt Tmu 12 BAFF-induced B-cell isotype switching response, human B cells MW 170 were treated with recombinant BAFF with or without purified WT 8 EVEV fi 95 High- TwtTwt or mutant soluble TWEAK, or with puri ed BAFF/mutant 72 Tmu molecular FITC MFI Tmu weight 4 TWEAK heteromers. BAFF alone promoted secretion of IgG 45 aggregates and IgA, whereas addition of BAFF/mutant TWEAK hetero- 0 25 0.25 0.5 1 1.5 2 mers decreased BAFF-mediated IgG and IgA secretion in cul- TWEAK (soluble) 15 [Fn14:Fc-FITC] (μg/ml) tured B cells (Fig. S5). Reducing Condition Taken together, these data indicate that mutant TWEAK may EFdominantly inhibit B-cell survival and proliferation in addition to Twt Tmu Twt Tmu Ig class switching through association with BAFF and down- Time(h) 010.5 1 2 3 0 0.5 2 3Time(h) 0 0.5 1 2 0 0.5 1 2 regulation of the noncanonical BAFF-induced NF-κB pathway. IKB-α P-JNK

actin actin Discussion

κ The combination of candidate-gene sequencing and protein func- Fig. 2. Mutant TWEAK fails to induce cell death and activate NF- Bpathways. tional analysis used in this study provides an effective strategy for (A) Mutant TWEAK fails to induce cell death in HT-29 cells. Flag-tag fi recombinant WT and mutant TWEAK soluble proteins were purified by M2 identifying genes responsible for rare monogenic immunode - anti-Flag agarose gel and quantified by ELISA. HT-29 cells were cultured in the ciency disorders (27). Here, we describe an autosomal dominant presence of WT TWEAK, mutant soluble TWEAK, or control BSA (3 ng/mL) TWEAK mutation that is associated with impaired antibody γ responses, reduced IgM and IgA levels, and an increased number together with IFN- (10 ng/mL). In some experiments, the WT soluble TWEAK + − − protein was preincubated with human IgG or recombinant Fn14:Fc for 2 h of DNT cells (i.e., TCRαβ CD4 CD8 T cells). The TWEAK p. before addition to HT-29 cells. Cell viability was determined in triplicate after 3 R145C mutation changes a charged arginine residue to a cysteine d. EV, empty vector; Tmu, TWEAK mutant; Twt, TWEAK WT (*P < 0.05 vs. BSA at a position close to the receptor binding sites in the THD. control). (B)Purified mutant TWEAK protein showed minimal capability to induce cell death in HT-29 cells. Equal amounts of WT TWEAK, mutant TWEAK, or control BSA were added in triplicate to HT-29 cell cultures, and cell viability was determined after 3 d (*P < 0.05 vs. BSA control). (C)MutantTWEAKforms A B high molecular weight aggregates. Lysates of Escherichia coli transformed BAFF BAFF with WT or mutant soluble TWEAK were subjected to SDS/PAGE and Western Twt Twt Tmu Tmu blotting under reducing and nonreducing conditions. (D) The R145C mutation IB: IB: does not reduce binding of TWEAK to its receptor Fn14. 293T cells were 13anti-TWEAK IP: anti-BAFF 1 5 anti-BAFF transfected with RFP fusion constructs expressing full-length WT TWEAK or IP: anti-TWEAK IB: IB: mutant TWEAK. Cells transfected with RFP empty vector served as controls. anti-BAFF anti-TWEAK Transfection efficiency of each construct was monitored by RFP expression. IB: IB: fi anti-TWEAK Surface expression of TWEAK was measured by FACS analysis using ve dif- Lysate anti-BAFF + IB: Lysate ferent concentrations of FITC-conjugated Fn14:Fc and gating on RFP cells. anti-BAFF IB: MFI, mean fluorescence intensity. Data in the plots were based on four in- anti-TWEAK dependent experiments. (E) Mutant TWEAK protein was unable to induce NF- κ fi C D B activation in THP1 cells. THP1 cells were treated with 6 ng/mL puri ed WT 200 C1 C2 P1 P2 or mutant TWEAK soluble protein at 37 °C for the indicated time periods. To IB: TWEAK * observe the effect on IκB degradation, 50 μg/mL cycloheximide (CHX) was 150 added to the cell culture. At the indicated times, cells were lysed and subjected IP: BAFF 100 to Western blotting with the indicated antibodies. (F) Western blot analysis of IB: BAFF * phospho-JNK expression was performed as in A but without CHX treatment. 5 BAFF Input 0 p100 levels and a decrease in p52 levels (Fig. 4C, lane 4). In TWEAK-bound BAFF (pg/ml) Normal B1 P1 P2 contrast, BAFF-expressing stable cell lines transfected with WT Fig. 3. Mutant TWEAK associates with BAFF. (A and B) In vitro association of TWEAK showed enhanced p100 processing with increased p52 mutant TWEAK with BAFF. Full-length BAFF and WT (Twt) or mutant TWEAK levels compared with BAFF-expressing stable transfectants alone (Tmu) constructs in pIRES vector were cotransfected into 293T cells. After 48 h, cells (Fig. 4C, lane 3). were lysed and immunoprecipitated with anti-BAFF (A) or anti-TWEAK (B)anti- To further test whether down-regulation of BAFF-R signaling bodies, and the resulting precipitates were subjected to Western blot analysis with by mutant TWEAK is associated with a decreased proliferation the opposing antibodies. Numbers under the bands indicate relative intensity of response in activated B cells, we performed an in vitro B-cell BAFF:TWEAK complex against BAFF-IP or TWEAK-IP input. (C) Association of BAFF proliferation assay using [3H]thymidine incorporation. WT and TWEAK in dendritic cells from patients and normal controls. Monocytes from TWEAK, mutant TWEAK, or empty vector were transfected patients and control subjects were separated and cultured in the presence of GM- CSF and IL-4 for 6 d. The resulting dendritic cells were activated with lipopoly- into BAFF-expressing stable HEK293 cell lines. After 36 h, cells saccharide and subjected to IP and immunoblotting with the indicated antibodies. were irradiated and cocultured with purified human B cells that – ′ (D) Detection of BAFF TWEAK heteromers in serum samples of patients. Serum were stimulated with anti-IgM F(ab )2 fragment. B cells cultured samples from affected family members and normal control subjects were added with WT TWEAK transfectants showed a slightly higher pro- to an ELISA plate coated with anti-TWEAK monoclonal antibodies and detected liferation response than B cells cultured with control transfectants, by HRP-conjugated monoclonal antibody against BAFF. Results from the two whereas B cells cultured with mutant TWEAK transfectants siblings (P1 and P2) and their father (B1) are shown (*P < 0.05 vs. normal controls).

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1221211110 Wang et al. Downloaded by guest on October 1, 2021 A EV BAFF B underlying apoptotic mechanism awaits further study. Because BAFF + Tmu BAFF + Twt the patients have papillomatosis, we were intrigued by the fact 100 100 that TWEAK protein can be up-regulated by IFN-γ or phorbol myristate acetate in cultured human peripheral NK cells (17, 28). 80 80 Although further investigation is warranted, TWEAK expression 60 60 by NK cells and its subsequent engagement of Fn14 on the surface of epithelial cells may be important for controlling local 40 40 immune responses to papilloma virus. Cell number 20 20 Mutations in BAFF-R in humans have been associated with reduced antibody production. The findings in our patients of 0 0 absent antibody responses to T-cell–dependent and poly- BAFF-R binding (APC) TACI binding (APC) saccharide antigens, as well as reduced B cell numbers, have also been reported in BAFF-R–deficient patients (3). However, in contrast to the patients presented here, BAFF-R–deficient CD) -3 3 patients have normal or even elevated IgA serum concentrations. CBB + TwtB + Tmu Differences in genetic background among patient groups may fl 2 in uence the development of some of these phenotypes. It is p100 possible that R145C TWEAK/BAFF heterotrimers or oligomers also bind to TACI in a dominant-negative manner and limit the 1 receptor’s association with another TNF ligand, APRIL. Con-

p52 [3H] - Thymidine sistent with this hypothesis are the observations of reduced se- 3.9 1.3 1.2 2.7 rum IgA levels in TACI-deficient humans and mice deficient in incorporation (cpm x10 0 APRIL (4, 5, 33). Thus, the phenotype that results from the β-actin R145C TWEAK mutation may not only be influenced by its EV+293 EV+293 Twt+293 -BAFF Tmu+293 interaction with BAFF, but also by association of the mutant -BAFF -BAFF heteromeric complex with multiple TNF family receptors. Fig. 4. Mutant TWEAK affects BAFF downstream signaling. (A) Association TWEAK is located on human 17p13.1 approxi- of TWEAK with BAFF slightly increases binding of BAFF to BAFF-R. 293T cell mately 878 bp upstream of APRIL, and these two genes are lines were cotransfected with full-length DsRed2–BAFF fusion vector to- tightly linked in human and mouse with similar intron–exon IMMUNOLOGY gether with empty vector (EV) or WT or mutant TWEAK in pEGFP-C1 vector. organization. APRIL is also an important regulator of T-cell– Surface expression of BAFF was measured by FACS analysis using recombi- independent class-switch pathways in B cells. An intergene nant Fc-BAFF-R and APC-conjugated secondary IgG. (B) TWEAK does not splicing transcript TWE-PRIL, which combines the first six exons affect binding of BAFF to TACI. Experiments were conducted as in A except of TWEAK and the last five exons of APRIL, was identified in that surface expression of BAFF was measured by FACS analysis using activated T cells and monocytes (34). TWE-PRIL is a membrane- recombinant TACI:Fc. (C) Membrane fraction of HEK293 stable BAFF- expressing cell line cotransfected with mutant TWEAK showed decreased anchored protein that possesses the intracellular transmembrane p100 processing in human B cells. Membrane fractions were prepared from stalk region of TWEAK but the receptor-binding domain of HEK293 BAFF stable cells transfected with WT or mutant TWEAK or empty APRIL, thus signaling through the APRIL binding domain may vectors and used to stimulate purified human B cells. After 42 h, B cells were be linked to downstream pathways shared with the TWEAK lysed and subjected to Western blotting using anti-p52 antibodies. B, BAFF; intracellular domain. As the R145 residue is also present in Tmu, TWEAK mutant; Twt, TWEAK WT. Numbers under the bands indicate TWE-PRIL, the possibility that the R145C mutation may in- relative intensity of p100 and p52 bands. (D) Mutant TWEAK down-regulates terfere with the signaling pathways of APRIL cannot be ruled the BAFF-induced B-cell proliferation response. B cells were purified from out. However, we were not able to detect a band corresponding PBMCs and cocultured in the presence of anti-human IgM F(ab′)2 fragment to the TWE-PRIL protein in activated monocytes or T cells and irradiated HEK293 cells that stably expressed full-length BAFF and were using monoclonal antibodies against APRIL or TWEAK in transfected with WT or mutant TWEAK construct or empty vector. B-cell normal subjects. Furthermore, we prepared single-cell suspen- proliferation response was measured as described in SI Materials and Methods < sions from lymph nodes of normal subjects and did not detect (*P 0.05 vs. 293-BAFF plus EV control). TWE-PRIL expression on the surface of T cells and monocytes. These results call into question whether TWE-PRIL exists as a functional protein in human leukocytes. Although this mutation does not affect binding of TWEAK to its fi receptor, it appears to impair its ability to induce apoptosis in These ndings lead us to consider the physiologic function of TWEAK-sensitive cell lines by decreasing activation of NF-κB naturally occurring heteromeric TNF ligands. Further study of these complexes could lead to the rational design of immunosuppressive and MAPK pathways. The demonstration that mutant TWEAK fi associates with BAFF indicates that the mutant protein might agents that target speci c aspects of immunity. In addition, further also dominantly inhibit B-cell function by forming noneffective analysis of cysteine mutations in other TNF family members and ligand trimers or oligomers, thereby blocking effective receptor their contribution to the formation of heteromeric complexes binding and downstream signaling. might provide a mechanism for some of the phenotypic variability fi Of particular interest among the observations in these patients associated with immune de ciency disorders. is the increased number of DNT cells and presence of cutaneous papillomatosis. Previous reports suggest that TWEAK works Materials and Methods with other proapoptotic TNFSF ligands such as FASLG, TRAIL Patients and Protocols. All patients and healthy controls were studied with (TNF-related apoptosis inducing ligand, TNFSF10), and TNF-α informed consent at the NIH clinical center under clinical protocol (00-I-006), to facilitate cytotoxicity in many cell types, including activated which was approved by the NIAID institutional review board. A detailed of monocytes (28), dendritic cells (29), NK cells (30), and T cells summary of research methods (7, 13, 23, 35), reagents, and statistical analysis (31). Autoimmune lymphoproliferative syndrome caused by im- is available in the SI Materials and Methods. paired FAS-mediated cell death is characterized by an accumu- ’ lation of DNT cells and autoimmunity (32). It seems that the loss ACKNOWLEDGMENTS. We thank the patients families for their participa- tion in this study; Ron Hornung and Margaret Brown for immunologic eval- of apoptotic function of TWEAK protein is correlated to the + uations; Mary Derry for editoral assistance; and Ivona Aksentijevich, Linda increase in peripheral DNT cells and CD8 T cells in patients Burkly, and Warren Strober for helpful discussions. This research was sup- carrying the mutant R145C allele; however, the exact link and ported by the Intramural Research Program of the NIH/NIAID.

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