Egr2 and Egr3 in regulatory T cells cooperatively …Systemic lupus erythematosus (SLE) is a...

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Egr2 and Egr3 in regulatory T cells cooperatively control systemic autoimmunity through Ltbp3-mediated TGF-β3 production Kaoru Morita a , Tomohisa Okamura a,b,1 , Mariko Inoue a , Toshihiko Komai a , Shuzo Teruya a , Yukiko Iwasaki a , Shuji Sumitomo a , Hirofumi Shoda a , Kazuhiko Yamamoto a,b , and Keishi Fujio a,1 a Department of Allergy and Rheumatology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan; and b Max PlanckUniversity of Tokyo Center for Integrative Inflammology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505 Japan Edited by Shimon Sakaguchi, WPI Immunology Frontier Research Center, Osaka University, Osaka, Japan, and approved November 11, 2016 (received for review July 11, 2016) Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by multiorgan inflammation induced by auto- antibodies. Early growth response gene 2 (Egr2), a transcription factor essential for T-cell anergy induction, controls systemic auto- immunity in mice and humans. We have previously identified a subpopulation of CD4 + regulatory T cells, CD4 + CD25 - LAG3 + cells, that characteristically express both Egr2 and LAG3 and control mice model of lupus via TGF-β3 production. However, due to the mild phenotype of lymphocyte-specific Egr2-deficient mice, the presence of an additional regulator has been speculated. Here, we show that Egr2 and Egr3 expressed in T cells cooperatively prevent humoral immune responses by supporting TGF-β3 secretion. T cell-specific Egr2/Egr3 double-deficient (Egr2/3DKO) mice spontaneously devel- oped an early onset lupus-like disease that was more severe than in T cell-specific Egr2-deficient mice. In accordance with the observation that CD4 + CD25 - LAG3 + cells from Egr2/3DKO mice completely lost the capacity to produce TGF-β3, the excessive germinal center reac- tion in Egr2/3DKO mice was suppressed by the adoptive transfer of WT CD4 + CD25 - LAG3 + cells or treatment with a TGF-β3expressing vector. Intriguingly, latent TGF-β binding protein (Ltbp)3 expression maintained by Egr2 and Egr3 was required for TGF-β3 production from CD4 + CD25 - LAG3 + cells. Because Egr2 and Egr3 did not demon- strate cell intrinsic suppression of the development of follicular helper T cells, Egr2- and Egr3-dependent TGF-β3 production by CD4 + CD25 - LAG3 + cells is critical for controlling excessive B-cell responses. The unique attributes of Egr2/Egr3 in T cells may provide an opportunity for developing novel therapeutics for autoantibody- mediated diseases including SLE. Egr2 | Egr3 | TGF-β3 | systemic lupus erythematosus | regulatory T cell A ntibodies play critical roles in protecting us from infectious threats. Effective humoral immune responses depend on germinal center (GC) reactions. Follicular B cells encounter anti- gen in the GC and receive T-cell help to differentiate into memory B cells and long-lived plasma cells that produce high-affinity anti- bodies (1). However, aberrant humoral immune responses against self-antigens lead to the development of autoimmune diseases. Indeed, suggestive clinical symptoms and the detection of autoan- tibodies in patient sera are essential diagnostic elements. Systemic lupus erythematosus (SLE) is regarded as a prototypic autoimmune disease with loss of immune tolerance to nucleic acid antigens. Antinuclear antibodies (ANAs), which are autoantibodies against nuclear components, including dsDNA, are frequently found in patients with SLE (2). The importance of B cells in the patho- genesis of SLE is further confirmed by the fact that antiB cellactivating factor (BAFF) monoclonal antibody (Belimumab) was approved by the Food and Drug Administration for treatment of SLE in 2011 (3). BAFF is a transmembrane protein member of the TNF ligand superfamily, and its overexpression is associated with both murine lupus and human SLE. However, as a sizeable pro- portion of patients with SLE remain refractory to Belimumab treatment, additional approaches for regulating B-cell hyperactivity are needed. Regulatory T-cell (Treg) subsets play a major role in the main- tenance of immune homeostasis and the prevention of auto- immunity (4). The most extensively studied Treg subset is CD4 + CD25 + Foxp3 + Tregs (CD25 + Tregs) (5). The CD25 + Tregs exhibit their suppressive function mainly by expressing CTLA4 and CD25 on their cell surface (6). We previously reported CD4 + CD25 Foxp3 LAG3 + Tregs (LAG3 + Tregs), which ex- ert their suppressive activities via IL-10 production in a Foxp3- independent manner (7). In contrast to CD25 + Tregs, high-affinity interactions with peptide/MHC ligands expressed in the thymus are not required for the development of LAG3 + Tregs. Accumulating evidence has shown that CD4 + CD25 + Foxp3 + CXCR5 + Tregs called follicular regulatory T (Tfr) cells directly control GC responses and humoral immunity. However, there is Significance Transcription factors early growth response gene 2 (Egr2) and Egr3 have long been regarded as negative regulators of T-cell activation. Egr2 is also known as a susceptibility gene for sys- temic lupus erythematosus characterized by dysregulated humoral immune responses to autoantigens. Previously, we reported that Egr2-expressing CD4 + CD25 - LAG3 + regulatory T cells regulate lupus pathogenesis via production of TGF-β3. However, the role of Egr2 and Egr3 in the regulation of hu- moral immunity is unclear. Here we report that Egr2 and Egr3 regulate germinal center reactions by promoting TGF-β3 pro- duction from regulatory T cells. Egr2 and Egr3 induce the ex- pression of latent TGF-β binding protein 3 (Ltbp3), which is required for TGF-β3 secretion. These findings suggest that Egr2 and Egr3 in T cells may be potential novel therapeutic targets for autoantibody-mediated autoimmune diseases. Author contributions: K.M., T.O., K.Y., and K.F. designed research; K.M., T.O., M.I., T.K., S.T., Y.I., S.S., and H.S. performed research; K.M., T.O., M.I., T.K., S.T., Y.I., S.S., H.S., and K.F. contributed new reagents/analytic tools; K.M., T.O., K.Y., and K.F. analyzed data; and K.M., T.O., K.Y., and K.F. wrote the paper. Conflict of interest statement: K.Y. received financial support or fees from AbbVie, Astellas, BMS, Daiichi-Sankyo, Mitsubishi Tanabe, Pfizer, Sanofi, Santen, Takeda, Teijin, Boehringer Ingelheim, Chugai, Eisai, Ono, Taisho Toyama, UCB, ImmunoFuture, Asahi Kasei, and Janssen. K.F. received financial support or fees from Astellas, BMS, Daiichi-Sankyo, Mitsubishi Tanabe, Pfizer, Santen, Takeda, Chugai, Eisai, Taisho Toyama, UCB, and Janssen. The remaining authors declare no competing financial interests. This article is a PNAS Direct Submission. Freely available online through the PNAS open access option. Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. NM_008520.2). 1 To whom correspondence may be addressed. Email: [email protected] or tomohisa- [email protected]. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1611286114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1611286114 PNAS | Published online November 30, 2016 | E8131E8140 IMMUNOLOGY AND INFLAMMATION PNAS PLUS Downloaded by guest on November 21, 2020

Transcript of Egr2 and Egr3 in regulatory T cells cooperatively …Systemic lupus erythematosus (SLE) is a...

Page 1: Egr2 and Egr3 in regulatory T cells cooperatively …Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by multiorgan inflammation induced by auto-antibodies.

Egr2 and Egr3 in regulatory T cells cooperativelycontrol systemic autoimmunity throughLtbp3-mediated TGF-β3 productionKaoru Moritaa, Tomohisa Okamuraa,b,1, Mariko Inouea, Toshihiko Komaia, Shuzo Teruyaa, Yukiko Iwasakia,Shuji Sumitomoa, Hirofumi Shodaa, Kazuhiko Yamamotoa,b, and Keishi Fujioa,1

aDepartment of Allergy and Rheumatology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan; andbMax Planck–University of Tokyo Center for Integrative Inflammology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505 Japan

Edited by Shimon Sakaguchi, WPI Immunology Frontier Research Center, Osaka University, Osaka, Japan, and approved November 11, 2016 (received forreview July 11, 2016)

Systemic lupus erythematosus (SLE) is a prototypical autoimmunedisease characterized by multiorgan inflammation induced by auto-antibodies. Early growth response gene 2 (Egr2), a transcriptionfactor essential for T-cell anergy induction, controls systemic auto-immunity in mice and humans. We have previously identified asubpopulation of CD4+ regulatory T cells, CD4+CD25−LAG3+ cells,that characteristically express both Egr2 and LAG3 and control micemodel of lupus via TGF-β3 production. However, due to the mildphenotype of lymphocyte-specific Egr2-deficient mice, the presenceof an additional regulator has been speculated. Here, we show thatEgr2 and Egr3 expressed in T cells cooperatively prevent humoralimmune responses by supporting TGF-β3 secretion. T cell-specificEgr2/Egr3 double-deficient (Egr2/3DKO) mice spontaneously devel-oped an early onset lupus-like disease that was more severe than inT cell-specific Egr2-deficient mice. In accordance with the observationthat CD4+CD25−LAG3+ cells from Egr2/3DKO mice completely lostthe capacity to produce TGF-β3, the excessive germinal center reac-tion in Egr2/3DKO mice was suppressed by the adoptive transfer ofWT CD4+CD25−LAG3+ cells or treatment with a TGF-β3–expressingvector. Intriguingly, latent TGF-β binding protein (Ltbp)3 expressionmaintained by Egr2 and Egr3 was required for TGF-β3 productionfrom CD4+CD25−LAG3+ cells. Because Egr2 and Egr3 did not demon-strate cell intrinsic suppression of the development of follicularhelper T cells, Egr2- and Egr3-dependent TGF-β3 production byCD4+CD25−LAG3+ cells is critical for controlling excessive B-cellresponses. The unique attributes of Egr2/Egr3 in T cells may providean opportunity for developing novel therapeutics for autoantibody-mediated diseases including SLE.

Egr2 | Egr3 | TGF-β3 | systemic lupus erythematosus | regulatory T cell

Antibodies play critical roles in protecting us from infectiousthreats. Effective humoral immune responses depend on

germinal center (GC) reactions. Follicular B cells encounter anti-gen in the GC and receive T-cell help to differentiate into memoryB cells and long-lived plasma cells that produce high-affinity anti-bodies (1). However, aberrant humoral immune responses againstself-antigens lead to the development of autoimmune diseases.Indeed, suggestive clinical symptoms and the detection of autoan-tibodies in patient sera are essential diagnostic elements. Systemiclupus erythematosus (SLE) is regarded as a prototypic autoimmunedisease with loss of immune tolerance to nucleic acid antigens.Antinuclear antibodies (ANAs), which are autoantibodies againstnuclear components, including dsDNA, are frequently found inpatients with SLE (2). The importance of B cells in the patho-genesis of SLE is further confirmed by the fact that anti–B cell–activating factor (BAFF) monoclonal antibody (Belimumab) wasapproved by the Food and Drug Administration for treatment ofSLE in 2011 (3). BAFF is a transmembrane protein member of theTNF ligand superfamily, and its overexpression is associated withboth murine lupus and human SLE. However, as a sizeable pro-portion of patients with SLE remain refractory to Belimumab

treatment, additional approaches for regulating B-cell hyperactivityare needed.Regulatory T-cell (Treg) subsets play a major role in the main-

tenance of immune homeostasis and the prevention of auto-immunity (4). The most extensively studied Treg subset isCD4+CD25+Foxp3+ Tregs (CD25+ Tregs) (5). The CD25+ Tregsexhibit their suppressive function mainly by expressing CTLA4and CD25 on their cell surface (6). We previously reportedCD4+CD25−Foxp3−LAG3+ Tregs (LAG3+ Tregs), which ex-ert their suppressive activities via IL-10 production in a Foxp3-independent manner (7). In contrast to CD25+ Tregs, high-affinityinteractions with peptide/MHC ligands expressed in the thymusare not required for the development of LAG3+ Tregs.Accumulating evidence has shown that CD4+CD25+Foxp3+

CXCR5+ Tregs called follicular regulatory T (Tfr) cells directlycontrol GC responses and humoral immunity. However, there is

Significance

Transcription factors early growth response gene 2 (Egr2) andEgr3 have long been regarded as negative regulators of T-cellactivation. Egr2 is also known as a susceptibility gene for sys-temic lupus erythematosus characterized by dysregulatedhumoral immune responses to autoantigens. Previously, wereported that Egr2-expressing CD4+CD25-LAG3+ regulatoryT cells regulate lupus pathogenesis via production of TGF-β3.However, the role of Egr2 and Egr3 in the regulation of hu-moral immunity is unclear. Here we report that Egr2 and Egr3regulate germinal center reactions by promoting TGF-β3 pro-duction from regulatory T cells. Egr2 and Egr3 induce the ex-pression of latent TGF-β binding protein 3 (Ltbp3), which isrequired for TGF-β3 secretion. These findings suggest that Egr2and Egr3 in T cells may be potential novel therapeutic targetsfor autoantibody-mediated autoimmune diseases.

Author contributions: K.M., T.O., K.Y., and K.F. designed research; K.M., T.O., M.I., T.K., S.T.,Y.I., S.S., and H.S. performed research; K.M., T.O., M.I., T.K., S.T., Y.I., S.S., H.S., and K.F.contributed new reagents/analytic tools; K.M., T.O., K.Y., and K.F. analyzed data; and K.M.,T.O., K.Y., and K.F. wrote the paper.

Conflict of interest statement: K.Y. received financial support or fees from AbbVie, Astellas,BMS, Daiichi-Sankyo, Mitsubishi Tanabe, Pfizer, Sanofi, Santen, Takeda, Teijin, BoehringerIngelheim, Chugai, Eisai, Ono, Taisho Toyama, UCB, ImmunoFuture, Asahi Kasei, andJanssen. K.F. received financial support or fees from Astellas, BMS, Daiichi-Sankyo,Mitsubishi Tanabe, Pfizer, Santen, Takeda, Chugai, Eisai, Taisho Toyama, UCB, and Janssen.The remaining authors declare no competing financial interests.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.

Data deposition: The sequence reported in this paper has been deposited in the GenBankdatabase (accession no. NM_008520.2).1To whom correspondence may be addressed. Email: [email protected] or [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1611286114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1611286114 PNAS | Published online November 30, 2016 | E8131–E8140

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little evidence regarding the contributions of other Treg pop-ulations to humoral immune tolerance. Recently, we have reportedthat LAG3+ Tregs (7) regulate humoral immunity and lupus dis-ease in MRL-Faslpr/lpr (MRL/lpr) mice via TGF-β3 production (8).Although TGF-β1 is well known for its antiinflammatory effects(9), we have previously revealed the regulatory activity of TGF-β3on humoral immune responses. LAG3+ Tregs, which characteris-tically express the transcription factor early growth response gene2 (Egr2), were identified as Foxp3-independent Tregs that producehigh amounts of IL-10, and forced expression of Egr2 in naïveT cells induced IL-10 and LAG3 expression (7). Furthermore, weand our collaborators have also shown that polymorphisms inEGR2 influence SLE susceptibility (10). Intriguingly, lymphocyte-specific Egr2-deficient mice develop a mild lupus-like autoimmunephenotype (11). These studies suggest that the expression of Egr2in LAG3+ Tregs contributes to the control of SLE pathogenesis.Egr2, a member of the Egr family, is a C2H2-type zinc finger

transcription factor that was first identified as a major regulator ofmyelination and hindbrain development (12, 13). Egr2 deficiencyresults in perinatal or neonatal death due to respiratory or feedingdeficits (12). Recent studies have focused on the role of Egr2 inimmune responses and revealed that Egr2 is essential for full in-duction of T-cell clonal anergy (14, 15). Egr2 has long beenregarded as a negative regulator of T-cell activation by promotingexpression of the E3 ubiquitin ligase Cbl-b and the cyclin-dependentkinase inhibitor p21cip1 and p27kip, which also contribute to T-cellanergy induction (11, 15, 16). However, because CD2-Cre–drivenlymphocyte-specific Egr2-deficient mice demonstrated only a mildform of systemic autoimmunity with limited anti-dsDNA antibodyproduction (11), the presence of additional regulators that controlautoimmunity has been speculated. Among the four Egr familymembers (Egr1–4), it is thought that Egr3 is able to partiallycompensate for Egr2 (11), although a systemic deletion of Egr3induces only gait ataxia in mice due to the lack of muscle spindles(17). Actually, Egr2 and Egr3 deletion in both T cells and B cellscauses a more severe early-onset systemic autoimmune syndrome,compared with deletion of Egr2 alone using a CD2-Cre–driver (18,19). The Egr2/Egr3 double-deficient mice showed enhanced ef-fector T-cell differentiation due to the reduction of suppressor ofcytokines signaling 1 (SOCS1) and SOCS3 and induction of Batf(18). Whereas there has been no report of T cell- or T cell/B cell-specific Egr3-deficient mice that develop spontaneous systemicautoimmunity (18, 20), these results indicated a compensatory roleof Egr3 for Egr2-mediated control of systemic autoimmunity. Nev-ertheless, both Egr2 and Egr3 expressed in B cells may modulatesystemic autoimmunity in CD2-Cre–driven lymphocyte-specificEgr2/Egr3 double-deficient mice, because Egr2 expressed in B cellsregulates the development of B cells (21) and Egr3 is preferentiallyexpressed in follicular B cells and marginal zone B cells, amongvarious B-cell populations (22). Therefore, it remains elusivewhether and how Egr2 and Egr3 expression on T cells solely reg-ulates humoral immune responses.In this report, we identify a previously unknown role of Egr2 and

Egr3 in T cells in the regulation of humoral immunity. To elucidatethe effect of both Egr2 and Egr3 in T cells, we generated T cell-specific Egr2/Egr3 double-deficient mice. The double-deficientmice developed an earlier onset lupus-like syndrome comparedwith T cell-specific Egr2 single-deficient mice. The phenotype inT cell-specific Egr2/Egr3 double-deficient mice is attributed to in-sufficient production of TGF-β3 from LAG3+ Tregs, which wasassociated with reduced expression of latent TGF-β binding protein(Ltbp)3 required for the assembly and secretion of TGF-β3 (9).

ResultsEgr2/3 Double Conditional KO Mice Develop a More Severe Lupus-LikeAutoimmune Disease than Egr2 Conditional Single KO Mice. We pre-viously established Egr2fl/fl Cd4-Cre+ [Egr2 conditional single knock-out (CKO)] mice, and T cell-specific Egr2-deficiency led to the

development of a mild form of systemic autoimmunity at nearly1 y of age (8). To investigate the role of Egr2 and Egr3 inT cells, we constructed a mouse strain in which both Egr2 andEgr3 were deleted specifically in T cells. First, we established loxP-flanked alleles encoding Egr3 (Egr3fl/fl) mice (Fig. S1A). Egr3fl/fl

mice and Egr2 floxed (Egr2fl/fl) mice (23) were then crossed withmice transgenic for Cd4-Cre+ mice to obtain Egr3fl/flEgr2fl/fl Cd4-Cre+ [Egr2 and Egr3 double conditional knockout (Egr2/3DKO)]mice. When efficiency of the Cre-mediated recombination in Tcells was evaluated in initial breedings (Fig. S1 B–D), Egr2 andEgr3 expression was abrogated in naïve T cells, but not in B cells,derived from Egr2/3DKO mice. Egr2/3DKO mice exhibited astatistically significant decrease in survival compared with WTand Egr2CKO mice (Fig. 1A). Egr2/3DKO mice generatedhigher concentrations of anti-dsDNA autoantibodies in their se-rum and progressive proteinuria (Fig. 1 B and C), which are thehallmark features of SLE in humans (24). Antibodies to dsDNAwere also detected with the Crithidia luciliae immunofluorescencetests (Fig. S2A). Egr2/3DKO mice also had inflammatory der-matitis, which was not observed in WT and Egr2CKO mice. Be-ginning at ∼16 wk of age, Egr2/3DKO mice developed skininflammation on their backs that was accompanied by hair loss(Fig. 1D). Skin sections from Egr2/3DKO mice at 16 wk of ageshowed extensive inflammatory cell infiltration, acanthosis, anddegeneration of the basal layer (Fig. S2B), which are often ob-served in SLE in humans. Consistent with proteinuria progression,kidney sections from Egr2/3DKO mice showed hyperplasia ofmesangial cells at 16 wk of age (Fig. 1 E and F) and massive in-flammatory cell infiltration and glomerular crescent formation at36 wk of age (Fig. S2 C and D). When we evaluated glomerulardeposition of immune complexes in the kidney, one of the dis-tinctive histologic findings in SLE, immunohistochemical analysesof kidney sections revealed considerable deposition of IgG in theglomeruli of Egr2/3DKO mice (Fig. 1E). In addition to the lupus-like organ damage, Egr2/3DKO mice also exhibited massive organinflammation, including liver, stomach, salivary gland, lung, andpancreas (Fig. S2E). This multiorgan inflammation contrastssharply with the lung-restricted cellular infiltrate in Egr2CKOmice. Although Egr2CKO mice also demonstrated a lupus-likedisease, their phenotypes were less severe than those of Egr2/3DKO mice. Consistent with our previous report (7), the expres-sion of Egr2 mRNA was much higher in LAG3+ Tregs than inCD4+CD25−CD44lowCD62Lhigh naïve T cells and CD4+CD25+

Tregs (Fig. 1G). It was reported that, among the Egr family, Egr3has a similar role in establishing T-cell anergy as Egr2 (25), andpotentially compensates for Egr2 in Egr2-deficient conditions (18).Intriguingly, LAG3+ Tregs from WT mice did not express highlevels of Egr3; however, Egr2 deficiency heightened the expressionof Egr3 mRNA, especially in LAG3+ Tregs, presumably due to acompensatory effect (Fig. 1H). Loss of Egr3 did not change theexpression of Egr2 mRNA (Fig. 1G). Together, these results sug-gested that genetic absence of both Egr2 and Egr3 in T cells, in-cluding LAG3+ Tregs, leads to the spontaneous autoimmunedisease resembling SLE and that Egr3 partially compensated forEgr2 deficiency.

Excessive Development of Follicular Helper T Cells and GC B Cells inEgr2/3DKO Mice. To investigate the pathogenicity of CD4+ T cellsunder Egr2 and Egr3 deficiency, we examined CD4+ T-cellprofiles of WT, Egr2CKO, and Egr2/3DKO mice. Egr2 has beenfound to be involved in the positive selection of thymocytes byup-regulating the survival molecule Bcl-2 and IL-7 (26, 27).Consistent with previous reports, we found a small reduction inthe frequency of CD4+ thymocytes in Egr2/3DKO mice com-pared with WT mice (Fig. S3A). However, the frequency andnumbers of CD4+ thymocytes were not reduced in Egr2/3DKOmice (Fig. S3B). In contrast, Egr2/3DKO mice had severesplenomegaly and increased numbers of lymphocytes compared

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with WT and Egr2CKO mice (Fig. 2A). Analysis of peripheralCD4+ T cells from spleens showed that Egr2/3DKO mice had alarger population of CD4+CD25−CD44highCD62Llow effector/memory T cells than WT and Egr2CKO mice (Fig. 2B).It was demonstrated that Egr2 and Egr3 negatively regulate

production of Th1 and Th17 cytokines including IFN-γ and IL-17through a SOCS1/3-dependent manner (18). Consistent with theseprevious reports, IFN-γ and IL-17 production was increased inTh1 and Th17 condition from aged Egr2CKO mice (Fig. S4A).However, the additional deficiency of Egr3 in Egr2CKO mice didnot cause the enhanced differentiation of Th1 and Th17 cells invitro (Fig. S4 A and B), indicating that Egr3 does not compensatethe Egr2 role in Th1 and Th17 differentiation intrinsically. Al-though freshly isolated total CD4+ T cells from aged Egr2CKOmice produced larger amount of IFN-γ and IL-17 compared withthose from WT mice, these cytokine levels were not increased inEgr2/3DKO mice compared with Egr2CKO mice (Fig. S4C).These findings suggest that the more severe autoimmune syn-drome in Egr2/3DKO mice than Egr2CKO mice is not explainedby enhanced response of Th1 and Th17 cells.As excessive numbers of follicular helper T (Tfh) cells have

recently been reported to have harmful effects in autoimmunediseases both in mice and humans (28–31), we next examined thecommitment of Egr2 and Egr3 to the differentiation of Tfh cellsin Egr2CKO and Egr2/3DKO mice. In clear contrast with the

few splenic CXCR5+PD-1+ Tfh cells observed in unimmunizedWT mice, Egr2CKO mice exhibited an increased frequency andnumber of Tfh cells from 6 wk of age (Fig. 2 C and D and Fig.S3C). Furthermore, Egr2/3DKO mice displayed an augmentedspontaneous development of Tfh cells compared with Egr2CKOmice. Excessive development of Tfh cells was also observed incervical, lumbar, and inguinal lymph nodes of Egr2CKO andEgr2/3DKO mice compared with those of WT mice (Fig. S3E).GC B cells are required for the development of GCs, as well as

Tfh cells. In GC, GC B cells undergo somatic hypermutation(SHM) (32, 33) and Ig class switching (34), and differentiate intoeither memory cells or plasma cells that confer lasting humoralimmune responses. SHM results in stochastic changes in anti-body affinity and specificity and may inadvertently generateautoreactive B cells. It is well known that the development of Tfhcells and GC B cells are mutually dependent on each other (35).Consistent with uncontrolled Tfh cell accumulation in Egr2CKOand Egr2/3DKO mice, we also observed spontaneous accumu-lation of GL7+Fas+ GC B cells in the spleens of Egr2CKO andEgr2/3DKO mice (Fig. 2E). Both the frequency and number ofGC B cells from Egr2/3DKO mice were higher than those fromWT and Egr2CKO mice (Fig. 2F and Fig. S3D). Similar to Tfhcells, excessive GC B-cell formation was also observed in theperipheral lymph nodes of Egr2CKO and Egr2/3DKO mice (Fig.S3F). Collectively, these data indicate that the expression of both

Fig. 1. Egr2/3DKO mice develop a more severe lupus-like autoimmune disease than Egr2CKO mice. (A) Survival rates of WT, Egr2fl/fl Cd4-Cre+ (Egr2CKO), andEgr2fl/flEgr3fl/fl Cd4-Cre+ (Egr2/3DKO) mice at the indicated time periods (n = 20 per group). P = 0.001 (log-rank test). (B) Titers of anti-dsDNA antibody fromserum of WT, Egr2CKO, and Egr2/3DKO mice at 16 wk of age (n = 10 per group). *P < 0.05 (Bonferroni posttest). (C) Proteinuria progression of WT, Egr2CKO,and Egr2/3DKO mice (n = 10 per group). *P < 0.05 (Mann–Whitney u test). (D) Skin inflammation in Egr2/3DKO mice. A representative macroscopic view (Top)and H&E staining (Bottom) of the back skin from WT, Egr2CKO, and Egr2/3DKO mice at 16 wk of age. (Scale bars, 500 μm.) (E) Histopathological analysis ofkidneys from WT, Egr2CKO, and Egr2/3DKO mice at 16 wk of age. H&E staining (Top), periodic acid-schiff staining (PAS; Middle), and anti-IgG immunofluo-rescent staining (IgG; Bottom). (Scale bars, 50 μm.) (F ) Histopathological scoring of extent and severity of renal disease from mice as in E (n = 10 per group).(G and H) Quantitative RT-PCR (qRT-PCR) analysis of the expression of Egr2 (G) and Egr3 (H) mRNA in T-cell subsets from WT, Egr2CKO, and Egr2/3DKO mice.Results are presented relative to expression of Actb mRNA encoding β-actin. n.d., not detected (n = 3 per group). *P < 0.05 (Bonferroni posttest). Data inG and H are representative of three independent experiments. The mean ± SD are indicated.

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Egr2 and Egr3 in T cells is necessary for the regulation of Tfhand GC B-cell differentiation.

Dysfunction of LAG3+ Tregs Is the Cause of Aberrant GC Responses inEgr2/3DKO Mice. We have previously reported that LAG3+ Tregsregulate humoral immune responses by suppressing B-cell pro-liferation and antibody production in an Egr2-dependent manner(8). As described above, Egr3 up-regulation in Egr2CKO LAG3+

Tregs suggests a compensatory role of Egr3 for Egr2 in Egr2CKOLAG3+ Tregs (Fig. 1 G and H). Therefore, we addressed whetherthe uncontrollable Tfh and GC B-cell formation in Egr2/3DKO

mice reflects the absence or malfunction of LAG3+ Tregs. Un-expectedly, LAG3+ Tregs were present in both Egr2CKO andEgr2/3DKO mice (Fig. 3A), and the frequency and number ofLAG3+ Tregs were higher in Egr2CKO and Egr2/3DKO mice.Next, we assessed whether deletion of both Egr2 and Egr3 di-minishes the regulatory function of LAG3+ Tregs against B-cellantibody responses. In an in vitro T-cell–B-cell coculture assay,anti–CD3-stimulated LAG3+ Tregs from WT and Egr2/3DKOmice were cultured with WT B cells in the presence of anti-CD40and recombinant IL-4 (rIL-4) for 3 and 7 d. Consistent with ourprevious report (8), LAG3+ Tregs from WT mice effectivelysuppressed B-cell proliferation, survival, and IgG antibody pro-duction. In contrast, LAG3+ Tregs from Egr2/3DKO mice losttheir suppressive capacity (Fig. 3 B–D). We have previously de-fined LAG3+ Tregs as those that do not express CD25 and Foxp3(7). In Egr2CKO and Egr2/3DKO mice, LAG3+ Tregs were stillnegative for both Foxp3 and CD25 expression (Fig. S5 A and B).The ratio of CD25+ Tregs in Egr2CKO and Egr2/3DKO mice wasalso unchanged compared with WT mice (Fig. S5C). UnlikeLAG3+ Tregs from Egr2/3DKO mice, CD25+ Tregs from Egr2/3DKO mice still exhibit suppressive effects on B-cells, which wascomparable to that of WT mice (Fig. S5 D and E), indicating thatEgr3 in concert with Egr2 control the regulatory function ofLAG3+ Tregs, but not CD25+ Tregs, on humoral immune re-sponses. Next, we investigated whether malfunction of Egr2- andEgr3-deficient LAG3+ Tregs leads to excessive accumulation ofTfh and GC B cells in Egr2/3DKO mice. We transferred splenicLAG3+ Tregs from WT mice into Egr2/3DKO mice twice andanalyzed their effect on Tfh and GC B-cell formation. Transfer ofEgr2- and Egr3-sufficient LAG3+ Tregs effectively suppressedexcess Tfh cell and GC B-cell formation (Fig. 3 E and F).As LAG3+ Tregs control humoral immunity via TGF-β3, we

investigated whether Egr2 and Egr3 regulate TGF-β3 productionthrough LAG3+ Tregs. At 3 d after T-cell receptor (TCR) stimu-lation, LAG3+ Tregs from WT mice secreted about 10 ng of TGF-β3 protein, which was ∼50-fold higher than TGF-β1 protein pro-duced by CD25+ Tregs (Fig. 3G andH). In contrast, LAG3+ Tregsfrom Egr2CKO mice secreted lower levels of TGF-β3 protein thanWT LAG3+ Tregs. Moreover, we did not detect any TGF-β3 se-cretion in the supernatants of LAG3+ Tregs from Egr2/3DKOmice. Although it was reported that TGF-β1 could partially com-pensate the function of TGF-β3 (36), we did not detect TGF-β1production from either WT LAG3+ Tregs, Egr2CKO, or Egr2/3DKO LAG3+ Tregs (Fig. 3H). These results suggest that theaberrant development of Tfh and GC B cells in Egr2/3DKOmice might be caused by the defective regulatory function ofLAG3+ Tregs to produce TGF-β3.As Egr2 and Egr3 have been reported to be transiently up-

regulated in response to TCR stimulation (15, 25), we also ex-plored the possibility that Egr2 and Egr3 intrinsically modulatethe differentiation of Tfh cells. To evaluate antigen-specific Tfhresponses, naïve CD4+ T cells from WT OT-II, Egr2CKO OT-II,or Egr2/3DKO OT-II mice that express TCR specific for theovalbumin (OVA)323–339 peptide in the context of I-Ab wereadoptively transferred into CD45.1+ C57BL/6 (B6) recipientmice, followed by immunization of the recipients with OVA con-jugated to 4-hydroxy-2-nitrophenylacetyl (NP-OVA) in completeFreund’s adjuvant (CFA). At 7 d after the immunization, the fre-quency of CD4+CXCR5+PD-1+ Tfh cells and the expression of Bcl-6protein in Egr2-, or Egr2- and Egr3-deficient CD45.2+ T cellswere similar to those in their WT counterparts (Fig. 4 A and B).The frequency of GC B cells and the levels of NP-specific IgGwere also similar in all groups of recipient mice (Fig. 4 C and D).Thus, Egr2 and Egr3 had little effect on the differentiation ofTfh cells, supporting that Egr2 and Egr3 expression in LAG3+

Tregs controls aberrant GC responses.

Fig. 2. The deficiency of Egr2 and Egr3 in T cells leads to excessive Tfh and GCB-cell formation. (A) Representative macroscopic view (Left), weight (Middle),and total cellularity (Right) of the spleens from WT, Egr2CKO, and Egr2/3DKOmice at 12 wk of age (n = 6 per group). *P < 0.05 (Bonferroni posttest). (B) Flowcytometry analysis of the expression of CD44 and CD62L in splenic CD4+CD25−

T cells from mice as in A (Left). Numbers adjacent to outlined areas indicatepercent of CD44highCD62Llow memory CD4+ T cells. Graph indicates frequency(among total CD4+CD25− T cells) of memory CD4+ T cells from mice as in A(Right, n = 6 per group). *P < 0.05 (Bonferroni posttest). (C) Flow cytometryanalysis of the expression of PD-1 and CXCR5 in splenic CD4+CD25− T cells fromWT, Egr2CKO, and Egr2/3DKO mice at 18 wk of age. Numbers adjacent tooutlined areas indicate percent of PD-1+CXCR5+ Tfh cells. (D) Frequency (amongtotal CD4+CD25− T cells) of Tfh cells in the spleens of mice from WT, Egr2CKO,and Egr2/3DKO mice at different weeks of age (n = 6 per group). *P < 0.05(Bonferroni posttest). (E) Flow cytometry analysis of the expression of GL7 andFas in splenic B220+ B cells from mice as in C. Numbers adjacent to outlinedareas indicate percent of GL7+Fas+ GC B cells. (F) Frequency (among totalB220+ B cells) of GC B cells in the spleens of mice as in D (n = 6 per group). *P <0.05 (Bonferroni posttest). Data are representative of three independent ex-periments. The mean ± SD are indicated.

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TGF-β3 Negatively Regulates GC B-Cell Differentiation. Although wehave previously shown that TGF-β3 is responsible for the sup-pressive activity of LAG3+ Tregs on B-cell functions (8), TGF-β1has long been known as the inhibitory cytokine for B-cell responsesthrough inhibiting the activation of Syk and phospholipase C-γ2, aswell as Stat6 phosphorylation (37). TGF-β3 shares 72% amino acididentity with TGF-β1, and TGF-β3 binds to the TGF-β receptor IIin the same way as TGF-β1 does (38, 39). Restrained differentia-tion of Tfh and GC B cells by TGF-β signaling was demonstratedusing TGF-β receptor II KO mice (40), indicating that TGF-β isnecessary for the control of humoral immunity. To investigate therole of TGF-β3 in Tfh and GC B-cell formation, we first analyzedthe suppressive function of TGF-β3 in B cells after in vitro acti-vation under conditions that promote the differentiation of GC-phenotype B cells. Although B cells stimulated with anti-CD40,rIL-4, and rBAFF acquired GL7 and Fas expression, which is thetypical phenotype of GC B cells (41), the addition of TGF-β3 orTGF-β1 suppressed the acquisition of GL7+Fas+ GC phenotypeand proliferation of B cells (Fig. 5 A and B). Moreover, treatmentwith TGF-β3 or TGF-β1 effectively inhibited the expression ofBcl6 mRNA, which is essential for the differentiation of GC B

cells (Fig. 5C) and the production of IgG, IgA, and IgM in theculture supernatants at day 7 (Fig. 5D). Notably, TGF-β3 andTGF-β1 did not suppress the expression of Bcl6 mRNA in in vitroinduced Tfh cells (Fig. 5E). Thus, TGF-β3 acted as a negativeregulator of the differentiation of GC B cells as well as TGF-β1.These results also suggest that B cells are the primary targets ofTGF-β–mediated suppression.We next examined whether excessive Tfh and GC B-cell for-

mation in Egr2/3DKO mice was rescued by exogenous TGF-β3.We injected a TGF-β3 expression plasmid (pCAGGS-Tgfb3) or acontrol pCAGGS plasmid into Egr2/3DKO mice three times andanalyzed Tfh and GC B formation at 4 wk after the final in-jection. Although the excessive Tfh formation was not improvedby the injection of pCAGGS-Tgfb3, the frequency of GC B cells wasmarkedly reduced in Egr2/3DKO mice injected with pCAGGS-Tgfb3, compared with those injected with control pCAGGS (Fig.5 F and G and Fig. S6). This result suggests that loss of TGF-β3production from Egr2- and Egr3-deficient LAG3+ Tregs was re-sponsible for the cause of unrestrained GC B-cell formations inEgr2/3 DKO mice. Moreover, in accordance with suppression ofGC B cells, the injection of pCAGGS-Tgfb3 improved proteinuria

Fig. 3. Dysregulated function of LAG3+ Tregs is responsible for the excessive Tfh and GC B-cell formation in Egr2/3DKO mice. (A) Flow cytometry analysis of splenicCD4+CD25− T cells from WT, Egr2CKO, and Egr2/3DKO mice at 18 wk of age (Left). Numbers adjacent to outlined areas indicate percent of CD4+LAG3+CD45RBlow

T cells (LAG3+ Tregs). Graph indicates frequency (among total CD4+CD25− T cells) of LAG3+ Tregs (Right, n = 8 per group). *P < 0.05 (Bonferroni posttest). (B) Flowcytometry analysis of CFSE-labeled B-cell proliferation. Each anti–CD3-stimulated T-cell subset was cocultured with WT B cells stimulated with anti-CD40 mAb andrIL-4 for 4 d (n = 4 per group). (C) Viability of cocultured B cells as in Bwas assessed by 7-amino-actinomycin D (7-AAD) (n = 4 per group). (D) Quantification of totalIgG production in the culture supernatants of cocultured B cells (as in B) on day 7 (n = 4 per group). *P < 0.05 (Bonferroni posttest). (E and F) Flow cytometry analysisof splenic Tfh and GC B cells in Egr2/3DKO mice. Splenic LAG3+ Tregs from WT mice were transferred into Egr2/3DKO mice at 2 and 4 wk of age. Four weeks afterthe last cell transfer, frequency (among total CD4+CD25− T cells) of Tfh cells (E) and frequency (among total B220+ B cells) of GC B cells (F) in the spleens of Egr2/3DKO mice were examined (n = 6 per group). *P < 0.05 (Bonferroni posttest). (G and H) TGF-β3 (G) and TGF-β1 (H) protein levels in the culture supernatants ofstimulated LAG3+ Tregs fromWT, Egr2CKO, and Egr2/3DKOmice on day 3 (n = 4 per group). *P < 0.05 (Bonferroni posttest). Data are representative of three (A andE–H) or two (B–D) independent experiments. The mean ± SD are indicated.

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progression and titer of anti-dsDNA autoantibodies (Fig. 5 H andI). Collectively, these results demonstrate that TGF-β3 productioncontrolled by Egr2 and Egr3 in LAG3+ Tregs is necessary for theregulation of aberrant GC B-cell differentiation and related todisease pathogenesis of Egr2/3DKO mice.

Egr2 and Egr3 Regulate the Secretion of TGF-β3 Protein Through anLtbp3-Dependent Manner. We next explored the molecular basisfor the requirement of Egr2 and Egr3 for TGF-β3 production fromLAG3+ Tregs. Surprisingly, although the secretion of TGF-β3 fromLAG3+ Tregs was strongly regulated by Egr2 and Egr3, the ex-pression of Tgfb3mRNAwas not altered in Egr2- and Egr3-deficientLAG3+ Tregs, irrespective of the presence of TCR stimulation (Fig.6 A and B). This result indicates that the expression of Tgfb3mRNA

is not directly regulated by Egr2 or Egr3. TGF-β3 undergoes com-plex processing steps intracellularly before its secretion from a cellmembrane (42, 43). After translation, TGF-β3 precursor protein iscut by furin convertase and forms a small latent complex (SLC)that includes mature TGF-β3 and latency associated peptide(LAP). SLCs are usually associated with Ltbp and secreted outsideof the membrane as a large latent complex (LLC). Given thatLtbp has been reported to require binding to TGF-β for efficientsecretion (44, 45), we hypothesized that the secretion of TGF-β3 isalso dependent on binding to Ltbp. Among the Ltbp family thatconsists of four members from Ltbp1–4 (46), LAG3+ Tregscharacteristically express Ltbp3 mRNA (Fig. 6C and Fig. S7A).Furthermore, the expression of Ltbp3 mRNA was significantlydown-regulated in Egr2/3DKO LAG3+ Tregs compared withWT and Egr2CKO LAG3+ Tregs (Fig. 6C). To investigatewhether Ltbp3 is essential for TGF-β3 to be secreted, we trans-fected LAG3+ Tregs with siRNA specifically designed to down-regulate the expression of Ltbp3 and analyzed the secretion ofTGF-β3 protein after TCR stimulation. Although the expres-sion of Tgfb3 mRNA was unchanged, the secretion of TGF-β3protein was strongly down-regulated in Ltbp3 siRNA-trans-fected LAG3+ Tregs (Fig. 6D). The expression of Ltbp1, 2, and 4was not suppressed by Ltbp3 siRNA (Fig. S7B). These resultsdemonstrate that Egr2 and Egr3 positively regulate the secretionof TGF-β3 by inducing Ltbp3 expression. To elucidate whetheroverexpression of Ltbp3 rescues the loss of TGF-β3 secretion inEgr2/3DKO T cells, we next constructed pMIG-Ltbp3 retroviralvector. Because we previously reported that IL-27–treated T cellsexpress Egr2 and produce TGF-β3 (8, 47), we retrovirally trans-duced WT or Egr2/3DKO T cells with pMIG-mock or pMIG-Ltbp3, followed by treatment with IL-27, and determined theproduction of TGF-β3 in the culture supernatants. As shownin Fig. 6E, transduction of pMIG-Ltbp3 significantly increasedthe production of TGF-β3 in IL-27–treated Egr2/3DKO T cells.Moreover, IL-27–treated Egr2/3DKO T cells retrovirally trans-duced with pMIG-Ltbp3 significantly suppressed B-cell prolifera-tion and antibody production compared with those transduced withpMIG-mock (Fig. 6 F and G). These results demonstrated thatLtbp3 expression in LAG3+ Tregs plays a critical role in controllinghumoral immunity via TGF-β3 secretion.

DiscussionAlthough it is known that dysregulated adaptive immunity is asso-ciated with the pathogenesis of SLE, the underlying distinct molec-ular mechanisms have been elusive. We demonstrated in this reportthat Egr2 and Egr3 in T cells are central molecules for the main-tenance of humoral immune tolerance in the steady state. In addi-tion to the previously observed linkage between Egr2 and systemicautoimmunity in mice and humans (10, 11), our studies indicate thatthe combination of Egr2 and Egr3 in T cells is a promising candidatetarget involved in the regulation of humoral immunity.The absence of both Egr2 and Egr3 in T cells led to earlier

onset of a lupus-like syndrome compared with Egr2CKO mice,indicating that a compensatory function of Egr3 for Egr2 inT cells is necessary for the prevention of lupus pathogenesis.Intriguingly, Egr2/3DKO mice not only developed a lupus-likephenotype characterized by marked high titers of anti-dsDNAaccompanied by severe glomerulonephritis resembling SLE, butalso showed a severe systemic autoimmune syndrome with lym-phocytic infiltration in multiple organs, such as liver and pancreas,that are rarely impaired in human lupus (48). These findingssuggest that Egr2 and Egr3 expression in T cells is required for theimmune system to avoid attacking self-tissue affected by a widerange of autoimmune disorders that are not limited to lupus.SLE is a B-cell–mediated autoimmune disease characterized by

the loss of tolerance to nucleic acid antigens and is regulated by avariety of mechanisms including aberrant Tfh cell differentiation(49). Our observation that Bcl-6 expression in CD4+ T cells in Tfh

Fig. 4. Egr2 and Egr3 deficiency do not augment Tfh cell differentiationintrinsically. (A) Flow cytometry analysis of the expression of PD-1 and CXCR5in donor CD45.2+ OT-II CD4+ T cells obtained from draining lymph nodes(dLNs) of CD45.1+ recipient WT mice given adoptive transfer of naïve WT,Egr2CKO, or Egr2/3DKO CD45.2+ OT-II CD4+ T cells, followed by immuniza-tion of NP13-OVA in CFA (Left). Numbers adjacent to outlined areas indicatepercent of PD-1+CXCR5+ Tfh cells. Graph indicates frequency (among totalCD4+CD25−CD45.2+ T cells) of PD-1+CXCR5+ Tfh cells from dLNs (Right, n = 6per group). (B) Flow cytometry analysis of the expression of Bcl-6 and CXCR5in donor CD45.2+ OT-II CD4+ T cells obtained from dLNs as in A (Left).Numbers adjacent to outlined areas indicate percent of Bcl-6+CXCR5+ Tfhcells. Graph indicates frequency (among total CD4+CD25−CD45.2+ T cells) ofBcl-6+CXCR5+ Tfh cells from dLNs as in A (Right, n = 6 per group). (C) Flowcytometry analysis of the expression of GL7 and Fas in B220+ B cells obtainedfrom dLNs as in A (Left). Numbers adjacent to outlined areas indicate per-cent of GL7+Fas+ GC B cells. Graph indicates frequency (among total B220+

B cells) of GC B cells from dLNs as in A (Right, n = 6 per group). (D) Quan-tification of NP-specific IgG in the serum of the recipient mice as in A (n = 6per group). Data are representative of two independent experiments. Themean ± SD are indicated.

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cell-polarizing conditions was not suppressed by TGF-β3 does notexclude the suppressive activity of TGF-β in longer duration be-cause Tfh cells overly accumulate in the absence of TGF-β signalingin T cells (40). Ogbe et al. reported that Egr2 and Egr3 directlyregulate Bcl-6 expression and are essential for the late stage of Tfhcell differentiation during viral infection (50). They also revealedthat the key molecules involved in the regulation of Tfh cell che-motaxis, such as expression levels of Cxcr5, Icos, and Sh2d1a, arenot affected by Egr2/Egr3 deficiency in lymphocytes. Similarly, werevealed that absence of Egr2/Egr3 does not alter Tfh cell devel-opment. These results suggested that Tfh cell intrinsic expression ofEgr2/Egr3 does not explain the enhanced humoral immunity ob-served in Egr2/3DKO mice. The fact that treatment with a TGF-β3expression vector ameliorated only GC B development in Egr2/3DKO mice indicated that a primary direct target of suppressionmediated by Egr2/Egr3–TGF-β3 axis may be B cells.To date, a number of studies have demonstrated that Treg sub-

sets maintain immunological self-tolerance. Foxp3+CD25+ Tregshave been the most intensively studied based on the accumu-lating evidence for their therapeutic effects to prevent variousautoimmune diseases. It has been reported that some specializedCD25+ Treg subsets, such as Tfr cells (51) and CD4+CD25+CD69−

Tregs (52), play a major role in the regulation of humoral immunity.However, contradictory results have been reported for the num-bers and function of Treg in SLE patients, and the precise roles ofCD25+ Tregs in SLE remain elusive (53), because the clinicalmanifestations of immunodysregulation, polyendocrinopathy, en-teropathy, X-linked (IPEX) syndrome caused by a mutation in theFOXP3 gene and SLE differ considerably (54, 55). In addition toCD25+ Treg, Egr2-expressing LAG3+ Tregs may also contributeto the control of systemic autoimmunity. Egr2 and Egr3 appearedto exhibit regulatory activity in LAG3+ Tregs, because both Egr2-and Egr3-deficient CD25+ Tregs retain in vitro suppressive activity(18) and the adoptive transfer of WT LAG3+ Tregs into Egr2/3DKO mice effectively suppressed the aberrant development ofboth Tfh and GC B cells.In the present study, we observed that TGF-β3 suppressed

B-cell activation and differentiation as efficaciously as TGF-β1,suggesting that TGF-β3 is a potent regulator of humoral immu-nity. It was recently identified that TGF-β1 plays an integral role inmaintaining immune tolerance (9). As for regulation of humoralimmunity, TGF-β1 controls B-cell activation by inhibiting both Igsynthesis and the switch from the membrane form to the secretedforms of μ- and γ-mRNA (56). CD4+CD25+CD69− Treg-mediated

Fig. 5. TGF-β3–mediated suppression of B-cell differentiation. (A) Flow cytometry analysis of the expression of GL7 and Fas in CFSE-labeled B cells stimulated withoutcytokines (no stim.) or with rIL-4, anti-CD40 mAb, and BAFF in the presence or absence of rTGF-β1 or -β3 (Left). Numbers adjacent to outlined areas indicate percent ofGL7+Fas+ B cells. Graph indicates frequency of GL7+Fas+ B cells (Right, n = 4 per group). *P < 0.05 (Bonferroni posttest). (B) Flow cytometry analysis of CFSE-labeledB-cell proliferation stimulated as inA (Left). Graph indicates frequency of undivided B cells (Right, n = 4 per group). *P < 0.05 (Bonferroni posttest). (C) qRT-PCR analysisof the expression of Bcl6 and Aicda mRNA in B cells stimulated as in A (n = 4 per group). *P < 0.05 (Bonferroni posttest). (D) Quantification of IgG, IgA, and IgMproduction in the culture supernatants of B cells stimulated as in A (n = 4 per group). *P < 0.05 (Bonferroni posttest). (E) qRT-PCR analysis of the expression of Bcl6mRNA in T cells stimulated with rIL-6 and rIL-21 (Tfh cell condition) in the presence or absence of rTGF-β1 or -β3 for 4 d (n = 3 per group). *P < 0.05 (Bonferroniposttest). (F and G) Analysis of splenic Tfh and GC B cells in Egr2/3DKO mice after i.v. injection with pCAGGS-control or pCAGGS-Tgfb3 plasmid vector. Frequency(among total CD4+CD25− T cells) of Tfh cells (F) and frequency (among total B220+ B cells) of GC B cells (G) in the spleens of Egr2/3DKO mice were examined at 4 wkafter last injection (n = 6 per group). *P < 0.05 (Bonferroni posttest). (H) Quantification of serum anti-dsDNA antibodies in Egr2/3DKO mice treated as in F (n = 6 pergroup). *P < 0.05 (Student’s t test). (I) Proteinuria progression in Egr2/3DKO mice treated as in F (n = 6 per group). *P < 0.05 (Mann–Whitney u test). Data arerepresentative of two independent experiments. The mean ± SD are indicated.

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suppression of B-cell antibody production is mediated at leastpartially through expression of TGF-β1 (52). On the other hand, theimmune suppressive role of TGF-β3 has not been extensivelyevaluated, and a mechanism for the production of TGF-β3 has notbeen clarified. We found that double deficiency of both Egr2 andEgr3 in T cells affects TGF-β3 production and Ltbp3 expression,and not Tgfb3mRNA expression, in LAG3+ Tregs. Among the fourisoforms of Ltbp, Ltbp1, and Ltbp3 can associate efficiently withpro–TGF-β1, -β2, and -β3, whereas Ltbp2 reportedly does not (57).On the other hand, Ltbp4 binds to TGF-β1–LAP more weakly thanLtbp1 and Ltbp3, indicating that Ltbp1 and Ltbp3 might be theprimary proteins responsible for binding to the TGF-β–LAP com-plex SLC (58). Several lines of evidence indicate that the Ltbps,which are not necessary for latency (59), play a major role in thesecretion (44), extracellular membrane localization (17), and acti-vation (60, 61) of latent TGF-β. However, it was reported that,unlike Ltbp1, Ltbp3 is not suitable for integrin-mediated latentTGF-β activation, and this isoform-specific function is most likelyrelated to the great sequence divergence of their hinge domains(62). In this study, we revealed a previously unrecognized functionfor Ltbp3 as an efficient regulator of TGF-β3 secretion fromLAG3+ Tregs. These results suggest that Ltbp3 may play a criticalrole in regulating TGF-β3–mediated immune tolerance and define

Ltbp3 as a potential therapeutic target. Further investigation isneeded to confirm whether Ltbp3 is a direct or indirect target ofEgr2 and Egr3.As described above, our study suggests the importance of Egr2

and Egr3 in T cells for the maintenance of humoral immune tol-erance. Although Li et al. reported that mice in which Egr2 andEgr3 are deleted specifically in both T and B cells die within 8 mo(18), three-quarters of T-cell–specific Egr2 and Egr3 double-deficient (Egr2/3DKO) mice were still alive after 1 y of age (Fig.1A), suggesting the importance of Egr2 and Egr3 in B cells in theimmune system. However, there have been no reports of B-cell–specific Egr2/Egr3 double-deficient mice. Detailed analyses of therole of Egr2 and Egr3 in B cells may provide novel therapeutictargets on B cells for autoantibody-mediated autoimmune diseases.In summary, our findings have provided insight into the mecha-

nism of how Egr2 and Egr3 in CD4+ T cells regulate humoral im-munity and established the combination of Egr2 and Egr3 as apivotal regulator of TGF-β3 secretion from LAG3+ Tregs. Un-derstanding the molecular basis of TGF-β3 secretion will be key tounderstanding lupus pathogenesis and will provide new therapeuticavenues for manipulating the excessive humoral immune responsesusing LAG3+ Tregs.

Fig. 6. Egr2 and Egr3 regulate TGF-β3 secretion through induction of Ltbp3. (A) qRT-PCR analysis of the expression of Tgfb3 mRNA in freshly isolated T-cellsubsets (n = 3 per group). (B) qRT-PCR analysis of the expression of Tgfb3 mRNA in T-cell subsets stimulated for 3 d with or without anti-CD3 and anti-CD28mAb (n = 3 per group). (C) qRT-PCR analysis of the expression of Ltbp3 mRNA in T-cell subsets stimulated as in B (n = 3 per group). *P < 0.05 (Bonferroniposttest). (D) qRT-PCR analysis of the expression of Ltbp3 (Left) and Tgfb3 (Middle) mRNA, and quantification of TGF-β3 protein levels (Right) in the culturesupernatants of LAG3+ Tregs transfected with control siRNA or Ltbp3 siRNA (n = 4 per group). *P < 0.05 (Student’s t test). (E) Analysis of TGF-β3 production inthe culture supernatants of IL-27–treated T cells from WT and Egr2/3DKO mice, transduced with pMIG-mock or pMIG-Ltbp3 vector. After CD4+ T cells wereretrovirally transduced with pMIG-mock or pMIG-Ltbp3 vector, the cells were treated with IL-27 for 2 d and TGF-β3 production was determined by ELISA (n = 4per group). *P < 0.05 (Student’s t test). (F) In vitro suppression of B cells by Ltbp3-transduced Egr2 and Egr3-deficient T cells. CFSE-labeled B cells werecocultured with pMIG-mock or pMIG-Ltbp3 vector transduced CD4+ T cells that were treated with IL-27 after transduction. B-cell proliferation was assessed byCFSE after 4 d of culture (n = 4 per group). (G) Quantification of total IgG production in the culture supernatants of cocultured B cells as in F on day 7 (n = 4per group). *P < 0.05 (Bonferroni posttest). Data are representative of three (A–C) or two (D–G) independent experiments. The mean ± SD are indicated.

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Materials and MethodsMice. C57BL/6 (B6) mice were purchased from Japan SLC. B6mice congenic forthe CD45 locus (B6-CD45.1+) were purchased from Sankyo Lab Service. TCRtransgenic OT-II mice (specific for the chicken ovalbumin peptide (amino acidresidues 323–339) in the context of MHC class II I-Ab) were purchased from TheJackson Laboratory. Egr2fl/fl mice were provided by Patrick Charnay (INSERM)(23). CD4-Cre transgenic mice (line 4196), originally generated by C. B. Wilsonand colleagues, were purchased from Taconic. Egr2fl/fl mice were crossedwith CD4-Cre transgenic mice to generate Egr2CKO mice (Egr2fl/fl Cd4-Cre+).Egr2/3DKO mice (Egr2fl/fl Egr3fl/fl Cd4-Cre+) were generated by crossingEgr2CKO mice with Egr3fl/fl mice. All animal experiments were approved bythe ethics committee of the University of Tokyo Institutional Animal Care andUse Committee.

Generation of Egr3 Floxed Mice. The targeting vector was constructed byinserting a 1.4-kb fragment containing exon 2 of the Egr3 gene flanked byloxP sites, 3.0 kb of a 3′ sequence, 3.9 kb of a 5′ sequence, and a neomycin-resistance gene flanked by Frt sites into a pBluescriptII SK (+) vector. Thetargeting vector was linearized and transfected into B6 embryonic stemcells by electroporation. Recombinant ES clones were selected in mediumsupplemented with G418. Thymidine kinase was used as a counter-selection. G418-resistant clones were screened for homologous recombi-nation by PCR and Southern blot analysis. The positive clones weremicroinjected into blastocytes derived from BALB/c mice and transferredto surrogate mothers. Mating of chimeric male mice to B6 female miceresulted in the transmission of the floxed allele to the germline. Theneomycin selection cassette flanked by Frt sites was excised in vivo bycrossing the C57BL/6-Tg (CAG-FLP) mice. For detection of floxed alleles,genomic DNA obtained from B cells and T cells was assessed by PCR. PCRprimer pairs were as follows: forward CGAGGACAAAAGCGTCGAAGCTCand reverse GATCAAGGCGATCCTAACTGAAC.

Reagents, Antibodies, and Media. Purified and conjugated antibodies werepurchased from BD Bioscience, eBiosciences, or Biolegend and recombinantcytokines were purchased from Miltenyi Biotec, R&D, and Biolegend. See SIMaterials and Methods for details.

Flow Cytometry and Cell Sorting. These procedures are described in SI Ma-terials and Methods.

Histopathological Examination.Histopathologic examination ofWT, Egr2CKO,and Egr2/3DKO mice was done at 16 or 36 wk of age. See SI Materials andMethods for details. Renal pathology was graded as described in SI Materialsand Methods.

B- and T-Cell Isolation and Proliferation. These procedures are described in SIMaterials and Methods.

T Cell–B Cell Coculture Assay. These procedures are described in SI Materialsand Methods.

Transfer of LAG3+ Tregs into Egr2/3DKO Mice. FACS-sorted LAG3+ Tregs (3 ×105 cells) from WT mice were i.v. transfused into Egr2/3DKO mice at 2 and4 wk of age. At 4 wk after the transfer, the mice were killed and splenic Tfhand GC B-cell formation was analyzed by flow cytometry.

Adoptive Transfer of Naïve T Cells from OT-II Mice. These procedures are de-scribed in SI Materials and Methods.

Injection of TGF-β3 Expressing Plasmid Vector into Egr2/3DKO Mice. Construc-tion of TGF-β3 expressing pCAGGs vector (pCAGGS-Tgfb3) was previouslydescribed (8). Egr2/3DKO mice were injected i.v. with 100 μg of pCAGGS-Tgfb3 or control pCAGGS in sterile PBS at 4, 6, and 8 wk of age. At 4 wk afterthe last injection, the mice were killed and splenic Tfh and GC B-cell for-mation was analyzed by flow cytometry.

Transfection of siRNA. LAG3+ Tregs (4 × 105 cells per well) were transfectedwith siRNA in Accell siRNA delivery media (GE Healthcare) according to themanufacturer’s protocol. siRNA targeting Ltbp3 and control siRNA wereused at a concentration of 1 μM. At 48 h after the transfections, the cellswere transferred to 96-well flat-bottomed plates coated with anti-CD3 mAb(2 μg/mL) and anti-CD28 mAb (2 μg/mL) in RPMI-1640 medium as describedabove. The cells were incubated for 72 h and then gene expression and TGF-β3 protein production were analyzed.

Retroviral Transduction into IL-27–Treated T cells. These procedures are de-scribed in SI Materials and Methods.

RNA Isolation, cDNA Synthesis, and Quantitative Real-Time PCR. These proce-dures are described in SI Materials and Methods.

Statistical Analysis. Survival rates were analyzed with the log-rank test.Quantitative proteinuria progression was analyzed with the Mann–Whitneyu test. For the comparison of more than three groups, a one-way analysis ofvariance followed by a Bonferroni multiple comparison test was performed.All other statistical differences were determined using the two-tailed Stu-dent’s t test. All data are presented as mean and SD. Statistical significancewas defined as a P value of <0.05 for all tests.

ACKNOWLEDGMENTS. We thank K. Watada, J. Takezawa, and R. Yamagatafor excellent technical assistance; Dr. P. Charnay (INSERM) for providing Egr2floxed mice; and Dr. J. Miyazaki (Osaka University Medical School) for kindlyproviding the pCAGGS vector. This work was supported by Grant-in-Aid forJapan Society for the Promotion of Science Fellows Grant 254186 andScientific Research Grant 16K09918.

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