Deficiency in TNFRSF13B (TACI) expands T-follicularhelper and germinal center B cells via increasedICOS-ligand expression but impairs plasma cell survivalXijun Oua, Shengli Xua,b, and Kong-Peng Lama,b,c,d,1

aImmunology Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research, Singapore; Departments of bPhysiology,cMicrobiology, and dPediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117599

Edited* by Tasuku Honjo, Graduate School of Medicine, Kyoto University, Kyoto, Japan, and approved August 3, 2012 (received for review January 9, 2012)

Mutations in TNFRSF13B, better known as transmembrane activa-tor and calcium modulator and cyclophilin ligand interactor (TACI),contribute to common variable immunodeficiency and autoimmu-nity in humans. How TACI regulates these two opposing con-ditions is unclear, however. TACI binds the cytokines BAFF andAPRIL, and previous studies using gene KO mice indicated that lossof TACI affected only T-cell–independent antibody responses. Herewe demonstrate that Taci −/− mice have expanded populations of Tfollicular helper (Tfh) and germinal center (GC) B cells in theirspleens when immunized with T-cell–dependent antigen. The in-creased numbers of Tfh and GC B cells in Taci −/− mice are largelya result of up-regulation of inducible costimulator (ICOS) ligand onTACI-deficient B cells, given that ablation of one copy of the Icoslallele restores normal levels of Tfh and GC B cells in Taci −/− mice.Interestingly, despite the presence of increased Tfh and antigen-specific B cells, immunized Taci −/− mice demonstrate defectiveantigen-specific antibody responses resulting from significantly re-duced numbers of antibody-secreting cells (ASCs). This effect isattributed to the failure to down-regulate the proapoptotic mole-cule BIM in Taci −/− plasma cells. Ablation of BIM could rescue ASCformation in Taci −/− mice, suggesting that TACI is more importantfor the survival of plasma cells than for the differentiation of thesecells. Thus, our data reveal dual roles for TACI in B-cell terminaldifferentiation. On one hand, TACI modulates ICOS ligand expres-sion and thereby limits the size of Tfh and GC B-cell compartmentsand prevents autoimmunity. On the other hand, it regulates thesurvival of ASCs and plays an important role in humoral immunity.

costimulation | T-cell–dependent humoral immunity | TNF receptor

Transmembrane activator and calcium modulator and cyclo-philin ligand interactor (TACI/TNFRSF13B), B-cell acti-

vating factor (BAFF) receptor (BAFF-R/TNFRSF13C), andB-cell maturation antigen (BCMA/TNFRSF17) are closely re-lated members of the TNF receptor superfamily and bind B-cellsurvival cytokines BAFF and APRIL (1). Although BAFF-Rand BCMA have been shown to mediate the survival of follic-ular B cells (2) and plasma cells (3), respectively, a similar rolefor TACI in mediating the survival of B lymphocytes at anyparticular stage of B-cell differentiation has not been demonstrateddefinitively.However, mutations in TACI are thought to contribute to

∼10% of common variable immunodeficiency (CVID) in humans(4, 5). This syndrome is characterized by antibody deficiency inlate childhood and early adulthood. Paradoxically, some patientswith TACI-mutated CVID also develop autoimmune diseases(6). How TACI deficiency leads to antibody deficiencies on onehand and autoimmunity on the other hand is not well understood.Mice lacking TACI have been generated (7, 8), and initial

characterizations of these mice revealed modest phenotypes.Taci−/− mice had increased numbers of B cells but exhibiteddecreased antibody responses to T-cell–independent type IIantigens. However, they did not appear to manifest any defectiveantibody responses to T-cell–dependent or protein antigens, as

determined by ELISA measurements of serum antibody titers(7, 8).The host response to T-cell–dependent antigens is known to

involve the generation of germinal centers (GCs), transientstructures found in secondary lymphoid tissues in which T-cell–B-cell interactions occur (9). Here antigen-activated B cells ex-pand and undergo antibody class-switching and affinity matura-tion, and also differentiate into memory B cells and plasma cells(9). GC B-cell differentiation is aided by a subset of CD4+ T cellsknown as T-follicular helper (Tfh) cells, which are characterizedby their surface expression of the chemokine receptor CXCR5and costimulation molecules ICOS and PD-1, as well as theproduction of IL-21 (10). Interestingly, the generation of Tfhcells is also dependent on ICOS ligand (ICOSL, or B7H2) foundon B cells (11), suggesting that GC B cells and Tfh cells mutuallycostimulate their respective differentiation. Whether TACI has arole in the GC reaction is not known; however, TACI expressionis up-regulated on activated B cells and plasma cells (12, 13).In the present study, we reexamined the role of TACI in T-cell–

dependent humoral immune response. We found that loss ofTACI leads to expansion of GC B and Tfh cells owing to increasedB7H2 expression on Taci−/− B cells. Interestingly, despite the in-creased presence of antigen-specific B cells in Taci−/− mice, thesemice exhibit impaired antibody response to T-cell–dependentantigens as a result of severe reductions in plasma cell numbers.We further demonstrated that TACI can deliver survival signals inplasma cells. Taken together, our data indicate that TACI hasa dual role in B-cell terminal differentiation in limiting the ex-pansion of GC B cells and mediating the survival of plasma cells.

ResultsTACI Deficiency Leads to Expansion of Tfh and GC B cells. TACImutations have been shown to affect Ig class-switching to IgA (7,8, 14–16), an antibody isotype that is enriched in mucosal tissues.Whether TACI deficiency can affect immune cell populations atthese sites is not known, however. To examine this question, weanalyzed the Peyer’s patches (PP) of WT and Taci−/− mice. PPsare sites of chronic immune responses and exhibit sustained GCreactions owing to continuous interactions between gut patho-gens and host immune cells (17, 18). In the GC, one can stain forantigen-activated GC B cells that are CD19+CD38−Fas+GL-7+

and Tfh cells that are CD4+TCRβ+PD-1high(hi)CXCR5+ (10, 19–21). Our flow cytometry analyses indicate that Taci−/− mice havea significantly increased fraction (more than twofold) of Tfh cells

Author contributions: S.X. and K.-P.L. designed research; X.O. performed research; X.O.,S.X., and K.-P.L. analyzed data; and X.O. and K.-P.L. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.1To whom correspondence should be addressed. E-mail: lam_kong_peng@bti.a-star.edu.sg.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1200386109/-/DCSupplemental.

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compared with WT controls (Fig. 1A), along with a significantincrease in the fraction of GC B cells.We next examined whether this phenomenon also occurred in

an acute immune response to T-cell–dependent antigen. Thus, wechallenged WT and Taci−/− mice with 4-hydroxy-3-nitrophenylacetylhapten conjugated to chicken gamma globulin (NP-CGG). At10 d postimmunization, we isolated splenocytes from thesemice and examined their Tfh and GC B-cell compositions.Consistent with the PP data, we found an approximate three-fold increase in Tfh and a twofold increase in GC B-cell fractionsin the spleens of Taci−/− mice compared with WT controls (Fig.1B). Enumeration of total Tfh and GC B cells also confirmedsignificantly increased numbers of these cells in mutant mice(Fig. 1C). Thus, TACI deficiency leads to expansion of Tfh andGC B cells in both acute and chronic immune responses.

B7H2 Expression Is Up-Regulated on Taci−/− B Cells. The de-velopment of GC B and Tfh cells depends on cognate inter-actions involving various receptor–ligand pairs on the two celltypes, including Fas–FasL, CD80/86–CD28, CD40–CD40L,B7H2–ICOS, PDL1/2–PD1, and others (10). Given that TACI isexpressed mainly on B cells, we reasoned that TACI deficiencymight affect the expression of some of these molecules and inturn lead to expansion of Tfh and GC B cells. Thus, we per-formed flow cytometry to examine the expression levels of thekey molecules involved in B-cell–T-cell interactions. Our dataindicate that the expression levels of major histocompatibilityclass II, CD40, Fas (CD95), CD80, CD86, PDL1, and PDL2 onfollicular B cells or GC B cells were comparable in immunizedWT and Taci−/− mice (Fig. S1A). Similarly, there wereno differences in the expression levels of FasL, PD-1, CD28, orCD40L between WT and Taci−/− CD4+ T or Tfh cells (Fig. S1B).Interestingly, the expression of B7H2, previously shown to becritical for GC formation (22), was consistently up-regulated onTaci−/− B cells and GC B cells compared with their WT

counterparts regardless of mouse immunization status (Fig. 2 Aand B). On the other hand, B7H2 expression level was similar onWT and Taci−/− dendritic cells (CD11c+) (Fig. 2B). Moreover,the expression level of its receptor ICOS was also comparable inWT and Taci−/− T and Tfh cells (Fig. 2C). Taken together, thesedata suggest that the expansion of Tfh and GC B cells in Taci−/−

mice possibly could be related to the increased expression of

Fig. 1. TACI deficiency leads to expansion of Tfh andGCB cells. (A andB) Flow cytometry analyses of Tfh andGCB-cell populations in PPs of unchallengedWTandTaci−/− mice (A) and spleens of NP38-CGG–immunized WT and Taci−/− mice (B). Numbers depict PD-1hiCXCR5+ Tfh and CD38−Fas+ or GL-7+Fas+ GC B cells amongCD4+TCRβ+ and CD19+ cells, respectively. (C) Graphical representations of the fraction and total number of Tfh cells (CD4+TCRβ+PD-1hiCXCR5+) and GC B cells(CD19+CD38−Fas+) in the spleens of NP38-CCG–immunized WT and Taci−/− mice. Each data point represents one mouse. ***P < 0.001.

Fig. 2. Up-regulation of B7H2 expression on Taci−/− B cells. (A and B) His-togram depicting B7H2 expression level on GC B cells (CD19+CD38−Fas+) andnon-GC B cells (CD19+CD38+Fas−) from spleens of day 10 NP38-CCG–immu-nized WT and Taci−/− mice (A) and CD19+ B and CD11c+ dendritic cells fromnaïve WT and Taci−/− mice (B). (C) Histogram depicting the level of ICOSexpression on Tfh (CD4+TCRβ+PD-1hiCXCR5+) and CD4+TCRβ+ cells from WTand Taci−/− mice at day 10 postimmunization. Data shown are representativeof more than three independent experiments.

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B7H2 on Taci−/− B cells that enhances Tfh cell development,which in turn further supports GC B-cell differentiation (10, 23).

B7H2 Up-Regulation Is Largely Responsible for the Expansion of Tfhand GC B Cells in Taci−/− Mice. To test the hypothesis that enhancedexpression of B7H2 on Taci−/− B cells drives the expansion ofthe Tfh cell population in Taci−/− mice, we first generated Taci−/−

B7h2−/− and Taci−/−cd28−/− mice and immunized them and vari-ous controls with NP38-CGG. At 10 d postimmunization, we an-alyzed the fractions of Tfh and GC B cells in the spleens of thesemice. Taci−/− mice again displayed increased populations of Tfhand GC B cells compared with WT controls. Taci−/−B7h2−/− andTaci−/−cd28−/−mice could not generate any substantial fraction ofTfh andGCB cells (Fig. S2). These data suggest that the increasednumbers of Tfh and GC B cells in Taci−/− mice arise from cognateinteractions between B cells and T cells. To further examine thecontribution of B7H2 up-regulation in enhancing Tfh and GC B-cell development in Taci−/− mice, we introduced B7h2 heterozy-gosity into these mice to determine whether these populations aresensitive to changes in B7H2 abundance. B7h2 heterozygosity ledto reduced B7H2 expression on Taci−/− B cells (Fig. 3A) to a levelcomparable to that seen on Taci+/+B7h2+/− cells. Interestingly,ablation of one allele of B7h2 was sufficient to modulate the in-creased formation of Tfh cells (Fig. 3B) and GCB cells (Fig. S3) inTaci−/−mice. These data suggest that the increased fraction of Tfhcells in Taci−/− mice is most likely mediated by increased expres-sion of B7H2 on Taci−/− B cells, which provides extra stimulationfor Tfh cell development.It is possible that TACI binding of BAFF or APRIL directly

signals the down-regulation of B7H2 expression on B cells. Totest this, we stimulated WT and Taci −/− B cells in vitro withAPRIL or BAFF. APRIL had no effect on B7H2 expression, butBAFF stimulated equivalent up-regulation of B7H2 on WT andTaci−/− B cells (Fig. S4A). This finding suggests that excessBAFF could stimulate via BAFF-R for the up-regulation ofB7H2 expression. This data led us to hypothesize that TACIbinding of BAFF could perhaps lead to less BAFF for BAFF-Rinduction of B7H2 up-regulation. To test this hypothesis, wegenerated mixed bone marrow (BM) chimeras using CD45.1 WTand CD45.2 WT cells or CD45.1 WT and CD45.2 Taci−/− cells.Reconstituted mice were left for 6 wk before NP38-CGG im-munization. Splenic GC B-cell numbers and B7H2 expression

were then analyzed by flow cytometry at 10 d postimmunization.We found that reconstituted CD45.1 WT and CD45.2 Taci−/− Bcells had comparable levels of B7H2 expression (Fig. S4B),which is also similar to that seen on B cells in CD45.1 WT andCD45.2 WT reconstituted mice. This suggests that TACI in-directly controls B7H2 expression on B cells and that as long asTACI is present in the system, even on neighboring WT B cells,there is no increase in B7H2 expression on Taci−/− B cells. Thesedata are consistent with the hypothesized model of TACI se-questering away excess BAFF. Concomitant with the lack ofB7H2 up-regulation in the reconstituted mice, we found no in-crease in the Tfh cell population in the chimeras (Fig. S4C).Interestingly, however, we observed a slight increase of CD45.2Taci−/− GC B cells compared with CD45.1 WT GC B cells (Fig.S4 D and E), suggesting that TACI also may deliver some in-hibitory signals to constrain GC B-cell development, consistentwith previous reports (1).

TACI Deficiency Impairs T-Cell–Dependent Antibody Immune Response.Tfh and GC B cells play critical roles in GC reactions during hu-moral immune responses by regulating antibody class-switchingand memory B-cell and plasma cell differentiation (10). Ourforegoing data indicate that loss of TACI leads to expansion of Tfhand GC B cells via increased B7H2 expression, which in principleshould lead to enhanced T-cell–dependent immune responses.However, it was reported previously that TACI deficiency impairsT-cell–independent responses but does not affect T-cell–depen-dent responses (7, 8). To resolve this discrepancy, we reanalyzedthe antibody responses of NP38-CGG–immunized Taci−/− mice.The high-affinity and total anti-NP antibodies elicited in

C57BL/6 mice were detected using anti-NP2 and anti-NP17ELISA, respectively. Surprisingly, in contrast to previous dataindicating no impairment (7, 8) and our prediction of enhancedresponse, Taci−/− mice had much lower anti-NP antibody titerscompared with WT mice. The ELISA data indicated that anti-NP17 and anti-NP2 IgM and IgG1 antibodies (Fig. 4) and IgG2band IgG3 antibodies (Fig. S5) in the sera of Taci−/− mice weresignificantly reduced across all time points examined comparedwith WT controls, suggesting that TACI is required for optimalT-cell–dependent humoral immune response.The reduced anti-NP antibody response seen in Taci−/− mice

represents an enigma, given that we found expansion of Tfh andGC B cells in these mice during both acute and chronic immuneresponses (Fig. 1). To better understand this conundrum, we

Fig. 3. Ablation of one copy of B7h2 allele modulates surface B7H2expression and Tfh expansion in Taci−/− mice. (A) Histogram depicting B7H2expression level on splenic B cells of WT and Taci−/− mice bearing two copiesor one copy of the B7h2 allele. (B) Flow cytometry analyses of Tfh cell pop-ulation in the spleens of 10 d NP38-CCG–immunized WT, Taci−/−, B7h2+/−, andTaci−/−B7h2+/− mice. Values shown are representative of two independentexperiments.

Fig. 4. Taci−/− mice exhibit a defective T-cell–dependent antibody response.Groups of eight WT and Taci−/− mice were challenged with NP38-CCG, andtheir sera-specific antibody titers at day 7, 14, 28, and 42 were measured byELISA using NP2 and NP17-BSA as coating antigens to detect NP-specific IgMand IgG1 antibodies, respectively. Sera were diluted 1,000-fold for IgM and20,000-fold for IgG1 detection. *P < 0.05; **P < 0.01; ***P < 0.001.

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examined the population of antigen-specific B cells in the GC ofWT and Taci−/− mice by staining for them with anti-B220 andanti-IgG1 antibodies and NIP, an analog of the immunizingantigen, and using a mixture of antibodies (Dump) to gate awayirrelevant cells. We identified a small population of NIP+IgG1+

B cells (∼2.2% of Dump−B220+ cells) in the spleens of WT mice(Fig. 5A). A similar fraction (∼2.5%) of NIP+IgG1+ B cells wasdetected in the spleens of immunized Taci−/− mice, suggestingthat TACI deficiency did not impair the generation of antigen-specific B cells. In fact, enumeration of these antigen-specificIgG1 B cells revealed their presence in much greater abundance(an approximate threefold increase) in Taci−/− mice, owing totheir increased splenocyte numbers.

TACI Is Required for Plasma Cell Survival. Because Taci−/− micehave greater numbers of Tfh and GC B cells and generate greaternumbers of antigen-specific B cells but yet have significantlyreduced specific serum antibody titers, we hypothesize that TACIdeficiency probably affects the generation or survival of plasmacells. To investigate this possibility, we performed a direct ELI-SPOT assay for the presence of NP-specific plasma cells in thespleen and BM of immunized Taci−/− mice. At 14 d and 28d postimmunization with NP38-CGG, we detected a substantialnumber of NP-specific IgG1 antibody-secreting cells (ASCs) inthe spleen and BM of WT mice (Fig. 5B). Interestingly, Taci−/−

mice had greatly reduced numbers of anti-NP IgG1-secretingcells in the spleen at day 14 postimmunization and a furtherdecrease by day 28. The reduction in IgG1 ASCs was even morepronounced in the BM of Taci−/− mice; these cells were hardlydetectable at day 14 or 28 postimmunization. Thus, TACI de-ficiency impairs ASCs.The reduction in ASCs in the spleen and BM of Taci−/− mice

could be due to inefficient generation of plasma cells from ac-tivated antigen-specific GC B cells or impaired survival of plasmacells. Because TACI binds BAFF and APRIL, which are B-cellsurvival factors (1), the latter possibility is more plausible. In

support of this idea, we found that TACI is expressed on WTplasma cells (Fig. 6A). Furthermore, we demonstrated in vitrothat treatment with APRIL or BAFF could increase the sur-vival of WT plasma cells by 1.5- to 2-fold (Fig. 6B and Fig. S6A).However, APRIL treatment did not lead to any increase in thesurvival of Taci−/− plasma cells (Fig. 6B), whereas BAFFtreatment enhanced survival only slightly (Fig. S6A). APRILand BAFF signaling can down-regulate BIM (24), a proa-poptotic molecule whose down-regulation enhances cell sur-vival (25). We next examined the expression level of BIM inAPRIL- and BAFF-treated WT and Taci−/− plasma cells. BimmRNA level was significantly reduced in WT, but not in Taci−/−

plasma cells stimulated with APRIL (Fig. 6C). BAFF treat-ment significantly down-regulated Bim expression in WTplasma cells, but to a much lesser extent in Taci−/− plasmacells (Fig. S6B). Taken together, these data suggest thatAPRIL and BAFF deliver survival signals to plasma cellspredominantly via TACI.To further investigate the role of TACI in plasma cell survival

in vivo, we introduced BIM deficiency into Taci−/− mice. Taci−/−

Bim−/− mice had comparable Tfh cells and more GC B cellscompared with Taci−/−Bim+/+mice (Fig. S6 C,D, and E). A greaternumber of ASCs were found in the spleen and BM of Taci−/−

Bim−/− mice compared with WT and Taci−/− mice at day 14postimmunization (Fig 6 D and E). These data suggest that TACIis required for the survival of plasma cells and not for theirdifferentiation. If the latter were the case, then BIM deficiencywould not restore the ASC compartment in Taci−/− mice.

DiscussionWe report that loss of TACI leads to expansion of Tfh and GC Bcells in mouse PPs and immunized spleens (Fig. 1). This phe-nomenon is attributed to increased B7H2 expression on Taci−/−

B cells (Fig. 2), which enhances cognate interactions between Bcells and T cells and further increases costimulation of Tfh cells.The expanded Tfh cells may provide further positive feedback to

Fig. 5. Presence of antigen-specific B cells and absence of plasma cells in immunized Taci−/− mice. (A) Increased numbers of antigen-specific B cells in Taci−/−

mice. WT and Taci−/− mice on day 10 postimmunization with NP38-CGG were examined by flow cytometry for the presence of antigen-binding (NIP+) IgG1 Bcells in their spleens, and the fraction and total number of these cells were quantified and graphed. (B) Significant reductions in the numbers of plasma cells inthe spleen (SP) and BM of Taci−/− mice. The presence of antigen-specific IgG1 plasma cells in the spleen and BM of WT and Taci−/− mice on days 14 and 28 ofantigenic challenge were examined via ELISPOT to detect NP2- and NP17-specific ASCs. The frequency and number of ASCs in the spleen and BM were graphedas well. Each dot represents one animal tested. **P < 0.01; ***P < 0.001.

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enlarge the GC B-cell compartment. Thus, TACI regulates thesize of the Tfh and GC B-cell compartments by modulatingB7H2 expression.Our data indicate that TACI negatively regulates B7H2 ex-

pression on B cells. However, it is not clear whether TACI activelysignals B7H2 down-modulation or sequesters BAFF away fromBAFF-R, which signals B7H2 up-regulation. We favor the latter,because we and others have shown that exogenous BAFF can up-regulate B7H2 expression on B cells (26, 27) (Fig. S4A), andbecause elevated BAFF levels have been found in the sera ofTaci−/− mice (28) and TACI-mutated patients (29). Thus, excessBAFF up-regulates B7H2 expression in Taci−/− mice and humans.In the BM chimera experiments, we found no increase in B7H2expression on Taci−/− B cells compared with WT B cells (Fig.S4B). This is because both WT and Taci−/− B cells are present inthe same organism, and there likely is no excess BAFF in thebody. BAFF could be captured by neighboring TACI+ B cells inthe microenvironment. It is also possible that “soluble TACI” (1)derived from TACI+ B cells can neutralize BAFF, although itsexistence remains questionable. Future experiments with B cellslacking BAFF-R, TACI, or both or expressing truncated TACIwithout its signaling domain would help resolve this issue.Intuitively, it would be expected that with increased Tfh and GC

B cells, Taci−/− mice would mount an enhanced antigen-specificantibody response to a T-cell–dependent antigen, but this was notthe case. We found that Taci−/− mice had reduced antibody titerswhen challenged with NP38-CGG (Fig. 4 and Fig. S4). We furtherdemonstrated that Taci−/− mice had increased numbers of anti-gen-specific B cells but significantly reduced numbers of plasmacells (Fig. 5). Previous studies have shown that TACI is highlyexpressed in plasma cells, whereas BAFF-R expression is de-creased upon B-cell differentiation to plasma cells (13, 30). Thesefindings suggest that TACI could have a role in plasma cells.BAFF is known to down-regulate the proapoptotic molecule BIMin B cells (24). Consistent with this, we found that both APRILand BAFF could significantly down-regulate BIM expression in

WT but not Taci−/− plasma cells, and that the impairment ofASCs in Taci−/− mice could be rectified by ablation of BIM (Fig.6). This suggests that TACI regulates the survival, rather than thedifferentiation, of plasma cells. This finding is significant, giventhat BCMA, a related tumor necrosis factor receptor superfamily(TNFRSF) member, was once thought to regulate the survival oflong-lived plasma cells (3). However, immunization of Bcma−/−

mice with NP-CGG seemed to elicit normal sera antibody titers(3, 31). In contrast, our current data indicate that Taci−/− miceexhibited reductions in both antibody titers and plasma cellnumbers when challenged with NP38-CGG. Given that APRILcould not improve the survival of Taci−/− plasma cells in vitro inwhich BCMA is present (Fig. 6B), TACI might play a more im-portant role in regulating plasma cell survival. While this manu-script was in preparation, Tsuji et al. (32) reported that TACIplays an important role in plasma cells and signals Blimp-1 ex-pression. However, that study was not clear as to whether TACIsignals the survival or differentiation of plasma cells, becauseBlimp1 is a critical transcriptional factor involved in plasma celldevelopment. Our study complements and extends that work byshowing that APRIL engagement of TACI down-regulates BIMexpression and increased plasma cell survival and by generatingTaci−/−Bim−/− mice to demonstrate that TACI is indeed impor-tant for plasma cell survival.TACI mutations in human give rise to CVID and autoimmunity

(4–6). Our findings suggest how these two seemingly opposingconditions could arise in patients. TACI regulates plasma cellsurvival and is thus important for humoral immune response. Onthe other hand, the loss of TACI leads to increased B7H2 ex-pression and expands Tfh and GC B cells. Increased B7H2 ex-pression (33, 34) and expansion of Tfh and GC B cells (35, 36)have been reported to underlie various autoimmune syndromes.

Materials and MethodsMice. Taci−/− mice were obtained from Vishva Dixit (Genentech); C57BL/6CD45.1, Cd28−/−, and Bim−/− mice were obtained from Jackson Laboratory;

Fig. 6. TACI is critical for the down-regulation of BIM and plasma cell survival. (A) Plasma cells express TACI. Purified WT and Taci−/− B cells were treated with20 μg/mL of LPS for 3 d, and TACI expression on plasma cells (B220lowCD138hi) was analyzed by flow cytometry. (B) APRIL stimulation of TACI enhanced plasmacell survival. WT and Taci−/− plasma cells generated via LPS treatment were purified using anti-CD138 microbeads, and their survival was monitored afterovernight culture with or without treatment with APRIL, 400 ng/mL. Cell viability was analyzed by annexin-V staining. Numbers indicate the percentage ofannexin-V–negative live cells. (C) Quantitative real-time PCR analyses of Bim mRNA expression in WT and Taci−/− plasma cells stimulated as in B. Results arepresented relative to the expression of Gapdh mRNA. *P < 0.05. NS, not significant. (D and E) ELISPOT analyses of NP17- and NP2-specific ASCs (D) andquantification of the antigen-specific numbers (E) in the spleen and BM of WT, Taci−/−, Bim−/−, and Taci−/−Bim−/− mice on day 14 postimmunization. Data arerepresentative of two or three independent experiments.

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and B7h2−/− mice were generated in the laboratory as described previously(22). Mice were bred to a C57BL/6 background and maintained under spe-cific pathogen-free conditions. The mouse experiments were approved bythe A*STAR Biological Resource Centre (BRC) Institutional Animal Care andUse Committee. For analyses of T-cell–dependent antibody responses,mice were immunized i.p. with 100 μg of alum-precipitated NP38-CGG(Biosearch Technologies).

Flow Cytometry. Single-cell suspensions from spleens, BM, and PPs wereprepared and stained with various combinations of fluorochrome-conju-gated antibodies to CD4, CD19, CD38, CD45.1, CD45.2, and B220 (BioLegend)and CD40, TCR-β, ICOS, PD-1, CXCR5, Fas, GL7, and B7H2 (BD Biosciences).Antigen-specific B cells were detected as described previously (21) with anti-B220 and anti-IgG1 antibodies and NIP and using a mixture of antibodies asDump. Samples were acquired on an LSRII cytometer (BD Biosciences) andanalyzed with FlowJo software (TreeStar).

BM Chimeras. BM reconstitution was performed as described previously withminor modifications (37). In brief, 3 × 106 mixed BM cells of CD45.1 WT andCD45.2 WT (1:1) or CD45.1 WT and CD45.2 Taci−/− (1:1) origins were injectedi.v. into lethally irradiated C57BL/6 CD45.1 mice (1,000 rads). Recipient micewere left for 6 wk before NP38-CGG immunization.

ELISA and ELISPOT. NP-specific antibodies and ASCs were detected via ELISAand ELISPOT, respectively, as described previously (21).

Cell Isolation and Culture. Total B cells were isolated from the spleens usinganti-CD43 antibody-conjugated microbeads (Miltenyi Biotec) and culturedwith 20 μg/mL of LPS for 3 d. Plasma cells were purified with anti-CD138microbeads and cultured with or without 400 ng/mL of APRIL or BAFF (R&DSystems) overnight for the subsequent survival and RT-PCR analysis.

Quantitative Real-Time PCR. Total RNA was isolated using isopropanol pre-cipitation, and the cDNA was prepared with the RevertAid H Minus First-Strand cDNA Synthesis Kit (Fermentas). SYBR Green Master Mix (AppliedBiosystems) was used for real-time PCR. The primer sequences were as fol-lows: Gapdh forward, 5′-TGTGTCCGTCGTGGATCTGA-3′, Gapdh reverse, 5′-TTGCTGTTGAAGTCGCAGGAG-3′; Bim forward, 5′-CGACAGTCTCAGGAGG-AACC-3′, Bim reverse, 5′-CAATGCCTTCTCCATACCAGA-3′.

Statistical Analysis Two-tailed unpaired t tests were performed usingGraphPadPrism software.

ACKNOWLEDGMENTS. We thank members of the K.-P.L. laboratory for in-sightful discussion and the A*STAR Biomedical Research Council forgrant support.

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