Egr2 and Egr3 in regulatory T cells cooperatively PNAS PLUS control systemic autoimmunity through Ltbp3-mediated TGF-β3 production

Kaoru 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; and bMax 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 for review July 11, 2016) Systemic lupus erythematosus (SLE) is a prototypical autoimmune treatment, additional approaches for regulating B-cell hyperactivity disease characterized by multiorgan inflammation induced by auto- are needed. antibodies. Early growth response 2 (Egr2), a transcription Regulatory T-cell (Treg) subsets play a major role in the main- factor essential for T-cell anergy induction, controls systemic auto- tenance of immune homeostasis and the prevention of auto- immunity in mice and humans. We have previously identified a immunity (4). The most extensively studied Treg subset is + + − + + + + + + subpopulation of CD4 regulatory T cells, CD4 CD25 LAG3 cells, CD4 CD25 Foxp3 Tregs (CD25 Tregs) (5). The CD25 Tregs that characteristically express both Egr2 and LAG3 and control mice exhibit their suppressive function mainly by expressing CTLA4 β model of lupus via TGF- 3 production. However, due to the mild and CD25 on their cell surface (6). We previously reported phenotype of lymphocyte-specific Egr2-deficient mice, the presence + − − + + CD4 CD25 Foxp3 LAG3 Tregs (LAG3 Tregs), which ex- of an additional regulator has been speculated. Here, we show that ert their suppressive activities via IL-10 production in a Foxp3- Egr2 and Egr3 expressed in T cells cooperatively prevent humoral + immune responses by supporting TGF-β3 secretion. T cell-specific independent manner (7). In contrast to CD25 Tregs, high-affinity interactions with peptide/MHC ligands expressed in the thymus Egr2/Egr3 double-deficient (Egr2/3DKO) mice spontaneously devel- + are not required for the development of LAG3 Tregs. oped an early onset lupus-like disease that was more severe than in + + + T cell-specific Egr2-deficient mice. In accordance with the observation Accumulating evidence has shown that CD4 CD25 Foxp3 + that CD4+CD25−LAG3+ cells from Egr2/3DKO mice completely lost CXCR5 Tregs called follicular regulatory T (Tfr) cells directly the capacity to produce TGF-β3, the excessive germinal center reac- control GC responses and humoral immunity. However, there is tion in Egr2/3DKO mice was suppressed by the adoptive transfer of + − + WT CD4 CD25 LAG3 cells or treatment with a TGF-β3–expressing Significance vector. Intriguingly, latent TGF-β binding (Ltbp)3 expression INFLAMMATION

maintained by Egr2 and Egr3 was required for TGF-β3 production IMMUNOLOGY AND + − + Transcription factors early growth response gene 2 (Egr2) and from CD4 CD25 LAG3 cells. Because Egr2 and Egr3 did not demon- Egr3 have long been regarded as negative regulators of T-cell strate cell intrinsic suppression of the development of follicular activation. Egr2 is also known as a susceptibility gene for sys- helper T cells, Egr2- and Egr3-dependent TGF-β3 production by + − + temic lupus erythematosus characterized by dysregulated CD4 CD25 LAG3 cells is critical for controlling excessive B-cell humoral immune responses to autoantigens. Previously, we responses. The unique attributes of Egr2/Egr3 in T cells may provide reported that Egr2-expressing CD4+CD25-LAG3+ regulatory an opportunity for developing novel therapeutics for autoantibody- T cells regulate lupus pathogenesis via production of TGF-β3. mediated diseases including SLE. However, the role of Egr2 and Egr3 in the regulation of hu- moral immunity is unclear. Here we report that Egr2 and Egr3 β Egr2 | Egr3 | TGF- 3 | systemic lupus erythematosus | regulatory T cell regulate germinal center reactions by promoting TGF-β3 pro- duction from regulatory T cells. Egr2 and Egr3 induce the ex- ntibodies play critical roles in protecting us from infectious pression of latent TGF-β binding protein 3 (Ltbp3), which is Athreats. Effective humoral immune responses depend on required for TGF-β3 secretion. These findings suggest that Egr2 germinal center (GC) reactions. Follicular B cells encounter anti- and Egr3 in T cells may be potential novel therapeutic targets gen in the GC and receive T-cell help to differentiate into memory for autoantibody-mediated autoimmune diseases. B cells and long-lived plasma cells that produce high-affinity anti- bodies (1). However, aberrant humoral immune responses against Author contributions: K.M., T.O., K.Y., and K.F. designed research; K.M., T.O., M.I., T.K., S.T., self-antigens lead to the development of autoimmune diseases. 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., Indeed, suggestive clinical symptoms and the detection of autoan- T.O., K.Y., and K.F. wrote the paper. tibodies in patient sera are essential diagnostic elements. Systemic Conflict of interest statement: K.Y. received financial support or fees from AbbVie, Astellas, lupus erythematosus (SLE) is regarded as a prototypic autoimmune BMS, Daiichi-Sankyo, Mitsubishi Tanabe, Pfizer, Sanofi, Santen, Takeda, Teijin, Boehringer disease with loss of immune tolerance to nucleic acid antigens. Ingelheim, Chugai, Eisai, Ono, Taisho Toyama, UCB, ImmunoFuture, Asahi Kasei, and Antinuclear antibodies (ANAs), which are autoantibodies against 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. nuclear components, including dsDNA, are frequently found in The remaining authors declare no competing financial interests. patients with SLE (2). The importance of B cells in the patho- This article is a PNAS Direct Submission. genesis of SLE is further confirmed by the fact that anti–Bcell– Freely available online through the PNAS open access option. activating factor (BAFF) monoclonal antibody (Belimumab) was Data deposition: The sequence reported in this paper has been deposited in the GenBank approved by the Food and Drug Administration for treatment of database (accession no. NM_008520.2). SLE in 2011 (3). BAFF is a transmembrane protein member of the 1To whom correspondence may be addressed. Email: [email protected] or tomohisa- TNF ligand superfamily, and its overexpression is associated with [email protected]. both murine lupus and human SLE. However, as a sizeable pro- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. portion of patients with SLE remain refractory to Belimumab 1073/pnas.1611286114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1611286114 PNAS | Published online November 30, 2016 | E8131–E8140 Downloaded by guest on September 24, 2021 little evidence regarding the contributions of other Treg pop- development of a mild form of systemic autoimmunity at nearly ulations to humoral immune tolerance. Recently, we have reported 1yofage(8).ToinvestigatetheroleofEgr2andEgr3in + that LAG3 Tregs (7) regulate humoral immunity and lupus dis- T cells, we constructed a mouse strain in which both Egr2 and ease in MRL-Faslpr/lpr (MRL/lpr) mice via TGF-β3 production (8). Egr3 were deleted specifically in T cells. First, we established loxP- Although TGF-β1 is well known for its antiinflammatory effects flanked alleles encoding Egr3 (Egr3fl/fl)mice(Fig. S1A). Egr3fl/fl (9), we have previously revealed the regulatory activity of TGF-β3 mice and Egr2 floxed (Egr2fl/fl) mice (23) were then crossed with + + on humoral immune responses. LAG3 Tregs, which characteris- mice transgenic for Cd4-Cre mice to obtain Egr3fl/flEgr2fl/fl Cd4- + tically express the early growth response gene Cre [Egr2 and Egr3 double conditional knockout (Egr2/3DKO)] 2 (Egr2), were identified as Foxp3-independent Tregs that produce mice. When efficiency of the Cre-mediated recombination in T high amounts of IL-10, and forced expression of Egr2 in naïve cells was evaluated in initial breedings (Fig. S1 B–D), Egr2 and T cells induced IL-10 and LAG3 expression (7). Furthermore, we Egr3 expression was abrogated in naïve T cells, but not in B cells, and our collaborators have also shown that polymorphisms in derived from Egr2/3DKO mice. Egr2/3DKO mice exhibited a EGR2 influence SLE susceptibility (10). Intriguingly, lymphocyte- statistically significant decrease in survival compared with WT specific Egr2-deficient mice develop a mild lupus-like autoimmune and Egr2CKO mice (Fig. 1A). Egr2/3DKO mice generated phenotype (11). These studies suggest that the expression of Egr2 higher concentrations of anti-dsDNA autoantibodies in their se- + in LAG3 Tregs contributes to the control of SLE pathogenesis. rum and progressive proteinuria (Fig. 1 B and C), which are the Egr2, a member of the Egr family, is a C2H2-type hallmark features of SLE in humans (24). Antibodies to dsDNA transcription factor that was first identified as a major regulator of were also detected with the Crithidia luciliae immunofluorescence myelination and hindbrain development (12, 13). Egr2 deficiency tests (Fig. S2A). Egr2/3DKO mice also had inflammatory der- results in perinatal or neonatal death due to respiratory or feeding matitis, which was not observed in WT and Egr2CKO mice. Be- deficits (12). Recent studies have focused on the role of Egr2 in ginning at ∼16 wk of age, Egr2/3DKO mice developed skin immune responses and revealed that Egr2 is essential for full in- inflammation on their backs that was accompanied by hair loss duction of T-cell clonal anergy (14, 15). Egr2 has long been (Fig. 1D). Skin sections from Egr2/3DKO mice at 16 wk of age regarded as a negative regulator of T-cell activation by promoting showed extensive inflammatory cell infiltration, acanthosis, and expression of the E3 ubiquitin ligase Cbl-b and the cyclin-dependent degeneration of the basal layer (Fig. S2B), which are often ob- kinase inhibitor p21cip1 and p27kip, which also contribute to T-cell served in SLE in humans. Consistent with proteinuria progression, anergy induction (11, 15, 16). However, because CD2-Cre–driven kidney sections from Egr2/3DKO mice showed hyperplasia of lymphocyte-specific Egr2-deficient mice demonstrated only a mild mesangial cells at 16 wk of age (Fig. 1 E and F) and massive in- form of systemic autoimmunity with limited anti-dsDNA antibody flammatory cell infiltration and glomerular crescent formation at production (11), the presence of additional regulators that control 36 wk of age (Fig. S2 C and D). When we evaluated glomerular autoimmunity has been speculated. Among the four Egr family deposition of immune complexes in the kidney, one of the dis- members (Egr1–4), it is thought that Egr3 is able to partially tinctive histologic findings in SLE, immunohistochemical analyses compensate for Egr2 (11), although a systemic deletion of Egr3 of kidney sections revealed considerable deposition of IgG in the induces only gait ataxia in mice due to the lack of muscle spindles glomeruli of Egr2/3DKO mice (Fig. 1E). In addition to the lupus- (17). Actually, Egr2 and Egr3 deletion in both T cells and B cells like organ damage, Egr2/3DKO mice also exhibited massive organ causes a more severe early-onset systemic autoimmune syndrome, inflammation, including liver, stomach, salivary gland, lung, and compared with deletion of Egr2 alone using a CD2-Cre–driver (18, pancreas (Fig. S2E). This multiorgan inflammation contrasts 19). The Egr2/Egr3 double-deficient mice showed enhanced ef- sharply with the lung-restricted cellular infiltrate in Egr2CKO fector T-cell differentiation due to the reduction of suppressor of mice. Although Egr2CKO mice also demonstrated a lupus-like cytokines signaling 1 (SOCS1) and SOCS3 and induction of Batf disease, their phenotypes were less severe than those of Egr2/ (18). Whereas there has been no report of T cell- or T cell/B cell- 3DKO mice. Consistent with our previous report (7), the expres- + specific Egr3-deficient mice that develop spontaneous systemic sion of Egr2 mRNA was much higher in LAG3 Tregs than in + − + + autoimmunity (18, 20), these results indicated a compensatory role CD4 CD25 CD44lowCD62Lhigh naïve T cells and CD4 CD25 of Egr3 for Egr2-mediated control of systemic autoimmunity. Nev- Tregs (Fig. 1G). It was reported that, among the Egr family, Egr3 ertheless, both Egr2 and Egr3 expressed in B cells may modulate has a similar role in establishing T-cell anergy as Egr2 (25), and systemic autoimmunity in CD2-Cre–driven lymphocyte-specific potentially compensates for Egr2 in Egr2-deficient conditions (18). + Egr2/Egr3 double-deficient mice, because Egr2 expressed in B cells Intriguingly, LAG3 Tregs from WT mice did not express high regulates the development of B cells (21) and Egr3 is preferentially levels of Egr3; however, Egr2 deficiency heightened the expression + expressed in follicular B cells and marginal zone B cells, among of Egr3 mRNA, especially in LAG3 Tregs, presumably due to a various B-cell populations (22). Therefore, it remains elusive compensatory effect (Fig. 1H). Loss of Egr3 did not change the whether and how Egr2 and Egr3 expression on T cells solely reg- expression of Egr2 mRNA (Fig. 1G). Together, these results sug- ulates humoral immune responses. gested that genetic absence of both Egr2 and Egr3 in T cells, in- + In this report, we identify a previously unknown role of Egr2 and cluding LAG3 Tregs, leads to the spontaneous autoimmune Egr3 in T cells in the regulation of humoral immunity. To elucidate disease resembling SLE and that Egr3 partially compensated for the effect of both Egr2 and Egr3 in T cells, we generated T cell- Egr2 deficiency. specific Egr2/Egr3 double-deficient mice. The double-deficient mice developed an earlier onset lupus-like syndrome compared Excessive Development of Follicular Helper T Cells and GC B Cells in + with T cell-specific Egr2 single-deficient mice. The phenotype in Egr2/3DKO Mice. To investigate the pathogenicity of CD4 T cells + T cell-specific Egr2/Egr3 double-deficient mice is attributed to in- under Egr2 and Egr3 deficiency, we examined CD4 T-cell + sufficient production of TGF-β3fromLAG3 Tregs, which was profiles of WT, Egr2CKO, and Egr2/3DKO mice. Egr2 has been associated with reduced expression of latent TGF-β binding protein found to be involved in the positive selection of thymocytes by (Ltbp)3 required for the assembly and secretion of TGF-β3(9). up-regulating the survival molecule Bcl-2 and IL-7 (26, 27). Consistent with previous reports, we found a small reduction in + Results the frequency of CD4 thymocytes in Egr2/3DKO mice com- Egr2/3 Double Conditional KO Mice Develop a More Severe Lupus-Like pared with WT mice (Fig. S3A). However, the frequency and + Autoimmune Disease than Egr2 Conditional Single KO Mice. We pre- numbers of CD4 thymocytes were not reduced in Egr2/3DKO + viously established Egr2fl/fl Cd4-Cre [Egr2 conditional single knock- mice (Fig. S3B). In contrast, Egr2/3DKO mice had severe out (CKO)] mice, and T cell-specific Egr2-deficiency led to the splenomegaly and increased numbers of lymphocytes compared

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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), and Egr2fl/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 from serum 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) INFLAMMATION

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 of IMMUNOLOGY AND kidneys 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 in G and H are representative of three independent experiments. The mean ± SD are indicated.

+ + with WT and Egr2CKO mice (Fig. 2A). Analysis of peripheral few splenic CXCR5 PD-1 Tfh cells observed in unimmunized + CD4 T cells from spleens showed that Egr2/3DKO mice had a WT mice, Egr2CKO mice exhibited an increased frequency and + − larger population of CD4 CD25 CD44highCD62Llow effector/ number of Tfh cells from 6 wk of age (Fig. 2 C and D and Fig. memory T cells than WT and Egr2CKO mice (Fig. 2B). S3C). Furthermore, Egr2/3DKO mice displayed an augmented It was demonstrated that Egr2 and Egr3 negatively regulate spontaneous development of Tfh cells compared with Egr2CKO production of Th1 and Th17 cytokines including IFN-γ and IL-17 mice. Excessive development of Tfh cells was also observed in through a SOCS1/3-dependent manner (18). Consistent with these cervical, lumbar, and inguinal lymph nodes of Egr2CKO and previous reports, IFN-γ and IL-17 production was increased in Egr2/3DKO mice compared with those of WT mice (Fig. S3E). Th1 and Th17 condition from aged Egr2CKO mice (Fig. S4A). GC B cells are required for the development of GCs, as well as However, the additional deficiency of Egr3 in Egr2CKO mice did Tfh cells. In GC, GC B cells undergo somatic hypermutation not cause the enhanced differentiation of Th1 and Th17 cells in (SHM) (32, 33) and Ig class switching (34), and differentiate into vitro (Fig. S4 A and B), indicating that Egr3 does not compensate either memory cells or plasma cells that confer lasting humoral the Egr2 role in Th1 and Th17 differentiation intrinsically. Al- immune responses. SHM results in stochastic changes in anti- + though freshly isolated total CD4 T cells from aged Egr2CKO body affinity and specificity and may inadvertently generate mice produced larger amount of IFN-γ and IL-17 compared with autoreactive B cells. It is well known that the development of Tfh those from WT mice, these cytokine levels were not increased in cells and GC B cells are mutually dependent on each other (35). Egr2/3DKO mice compared with Egr2CKO mice (Fig. S4C). Consistent with uncontrolled Tfh cell accumulation in Egr2CKO These findings suggest that the more severe autoimmune syn- and Egr2/3DKO mice, we also observed spontaneous accumu- + + drome in Egr2/3DKO mice than Egr2CKO mice is not explained lation of GL7 Fas GC B cells in the spleens of Egr2CKO and by enhanced response of Th1 and Th17 cells. Egr2/3DKO mice (Fig. 2E). Both the frequency and number of As excessive numbers of follicular helper T (Tfh) cells have GC B cells from Egr2/3DKO mice were higher than those from recently been reported to have harmful effects in autoimmune WT and Egr2CKO mice (Fig. 2F and Fig. S3D). Similar to Tfh diseases both in mice and humans (28–31), we next examined the cells, excessive GC B-cell formation was also observed in the commitment of Egr2 and Egr3 to the differentiation of Tfh cells peripheral lymph nodes of Egr2CKO and Egr2/3DKO mice (Fig. in Egr2CKO and Egr2/3DKO mice. In clear contrast with the S3F). Collectively, these data indicate that the expression of both

Morita et al. PNAS | Published online November 30, 2016 | E8133 Downloaded by guest on September 24, 2021 + mice reflects the absence or malfunction of LAG3 Tregs. Un- + expectedly, LAG3 Tregs were present in both Egr2CKO and Egr2/3DKO mice (Fig. 3A), and the frequency and number of + LAG3 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-cell antibody responses. In an in vitro T-cell–B-cell coculture assay, + anti–CD3-stimulated LAG3 Tregs from WT and Egr2/3DKO mice were cultured with WT B cells in the presence of anti-CD40 and recombinant IL-4 (rIL-4) for 3 and 7 d. Consistent with our + previous report (8), LAG3 Tregs from WT mice effectively suppressed B-cell proliferation, survival, and IgG antibody pro- + duction. In contrast, LAG3 Tregs from Egr2/3DKO mice lost their 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 still negative for both Foxp3 and CD25 expression (Fig. S5 A and B). + The ratio of CD25 Tregs in Egr2CKO and Egr2/3DKO mice was also unchanged compared with WT mice (Fig. S5C). Unlike + + LAG3 Tregs from Egr2/3DKO mice, CD25 Tregs from Egr2/ 3DKO mice still exhibit suppressive effects on B-cells, which was comparable to that of WT mice (Fig. S5 D and E), indicating that Egr3 in concert with Egr2 control the regulatory function of + + LAG3 Tregs, but not CD25 Tregs, on humoral immune re- sponses. Next, we investigated whether malfunction of Egr2- and + Egr3-deficient LAG3 Tregs leads to excessive accumulation of Tfh and GC B cells in Egr2/3DKO mice. We transferred splenic + LAG3 Tregs from WT mice into Egr2/3DKO mice twice and analyzed their effect on Tfh and GC B-cell formation. Transfer of + Egr2- and Egr3-sufficient LAG3 Tregs effectively suppressed excess 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 production + through LAG3 Tregs. At 3 d after T-cell (TCR) stimu- + lation, LAG3 Tregs from WT mice secreted about 10 ng of TGF- β3protein,whichwas∼50-fold higher than TGF-β1 protein pro- Fig. 2. The deficiency of Egr2 and Egr3 in T cells leads to excessive Tfh and GC + + B-cell formation. (A) Representative macroscopic view (Left), weight (Middle), duced by CD25 Tregs (Fig. 3 G and H). In contrast, LAG3 Tregs and total cellularity (Right) of the spleens from WT, Egr2CKO, and Egr2/3DKO from Egr2CKO mice secreted lower levels of TGF-β3proteinthan + mice at 12 wk of age (n = 6 per group). *P < 0.05 (Bonferroni posttest). (B)Flow WT LAG3 Tregs. Moreover, we did not detect any TGF-β3se- + − + cytometry analysis of the expression of CD44 and CD62L in splenic CD4 CD25 cretion in the supernatants of LAG3 Tregs from Egr2/3DKO TcellsfrommiceasinA (Left). Numbers adjacent to outlined areas indicate + mice. Although it was reported that TGF-β1 could partially com- percentofCD44highCD62Llow memory CD4 T cells. Graph indicates frequency + − + pensate the function of TGF-β3 (36), we did not detect TGF-β1 (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 cytometry production from either WT LAG3 Tregs, Egr2CKO, or Egr2/ + − + analysis of the expression of PD-1 and CXCR5 in splenic CD4 CD25 T cells from 3DKO LAG3 Tregs (Fig. 3H). These results suggest that the WT, Egr2CKO, and Egr2/3DKO mice at 18 wk of age. Numbers adjacent to aberrant development of Tfh and GC B cells in Egr2/3DKO + + outlined areas indicate percent of PD-1 CXCR5 Tfh cells. (D) Frequency (among mice might be caused by the defective regulatory function of + − + total CD4 CD25 T cells) of Tfh cells in the spleens of mice from WT, Egr2CKO, LAG3 Tregs to produce TGF-β3. = < and Egr2/3DKO mice at different weeks of age (n 6pergroup).*P 0.05 As Egr2 and Egr3 have been reported to be transiently up- (Bonferroni posttest). (E) Flow cytometry analysis of the expression of GL7 and + Fas in splenic B220 B cells from mice as in C. Numbers adjacent to outlined regulated in response to TCR stimulation (15, 25), we also ex- + + areas indicate percent of GL7 Fas GCBcells.(F) Frequency (among total plored the possibility that Egr2 and Egr3 intrinsically modulate B220+ B cells) of GC B cells in the spleens of mice as in D (n = 6pergroup).*P < the differentiation of Tfh cells. To evaluate antigen-specific Tfh + 0.05 (Bonferroni posttest). Data are representative of three independent ex- responses, naïve CD4 T cells from WT OT-II, Egr2CKO OT-II, periments. The mean ± SD are indicated. or Egr2/3DKO OT-II mice that express TCR specific for the b ovalbumin (OVA)323–339 peptide in the context of I-A were + adoptively transferred into CD45.1 C57BL/6 (B6) recipient Egr2 and Egr3 in T cells is necessary for the regulation of Tfh mice, followed by immunization of the recipients with OVA con- and GC B-cell differentiation. jugated to 4-hydroxy-2-nitrophenylacetyl (NP-OVA) in complete + Freund’s adjuvant (CFA). At 7 d after the immunization, the fre- Dysfunction of LAG3 Tregs Is the Cause of Aberrant GC Responses in + + + + quency of CD4 CXCR5 PD-1 Tfh cells and the expression of Bcl-6 + Egr2/3DKO Mice. We have previously reported that LAG3 Tregs protein in Egr2-, or Egr2- and Egr3-deficient CD45.2 T cells regulate humoral immune responses by suppressing B-cell pro- were similar to those in their WT counterparts (Fig. 4 A and B). liferation and antibody production in an Egr2-dependent manner + The frequency of GC B cells and the levels of NP-specific IgG (8). As described above, Egr3 up-regulation in Egr2CKO LAG3 were also similar in all groups of recipient mice (Fig. 4 C and D). Tregs suggests a compensatory role of Egr3 for Egr2 in Egr2CKO Thus, Egr2 and Egr3 had little effect on the differentiation of + + LAG3 Tregs (Fig. 1 G and H). Therefore, we addressed whether Tfh cells, supporting that Egr2 and Egr3 expression in LAG3 the uncontrollable Tfh and GC B-cell formation in Egr2/3DKO Tregs controls aberrant GC responses.

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+

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 splenic INFLAMMATION + − + + CD4 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 IMMUNOLOGY AND + + − + 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)Flow cytometry 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 and rIL-4for4d(n = 4 per group). (C) Viability of cocultured B cells as in B was assessed by 7-amino-actinomycin D (7-AAD) (n = 4 per group). (D) Quantification of total IgG production in the culture supernatants of cocultured B cells (as in B)onday7(n = 4 per group). *P < 0.05 (Bonferroni posttest). (E and F) Flow cytometry analysis + of 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 after + − + the last cell transfer, frequency (among total CD4 CD25 T cells) of Tfh cells (E) and frequency (among total B220 Bcells)ofGCBcells(F)inthespleensofEgr2/ 3DKO mice were examined (n = 6 per group). *P < 0.05 (Bonferroni posttest). (G and H)TGF-β3(G)andTGF-β1(H) protein levels in the culture supernatants of + stimulated LAG3 Tregs from WT, Egr2CKO, and Egr2/3DKO mice on day 3 (n = 4 per group). *P < 0.05 (Bonferroni posttest). Data are representative of three (A and E–H)ortwo(B–D) independent experiments. The mean ± SD are indicated.

TGF-β3 Negatively Regulates GC B-Cell Differentiation. Although we cells (Fig. 5C) and the production of IgG, IgA, and IgM in the have previously shown that TGF-β3 is responsible for the sup- culture supernatants at day 7 (Fig. 5D). Notably, TGF-β3and + pressive activity of LAG3 Tregs on B-cell functions (8), TGF-β1 TGF-β1 did not suppress the expression of Bcl6 mRNA in in vitro has long been known as the inhibitory cytokine for B-cell responses induced Tfh cells (Fig. 5E). Thus, TGF-β3 acted as a negative through inhibiting the activation of Syk and phospholipase C-γ2, as regulator of the differentiation of GC B cells as well as TGF-β1. well as Stat6 phosphorylation (37). TGF-β3shares72%aminoacid These results also suggest that B cells are the primary targets of identity with TGF-β1, and TGF-β3 binds to the TGF-β receptor II TGF-β–mediated suppression. inthesamewayasTGF-β1 does (38, 39). Restrained differentia- We next examined whether excessive Tfh and GC B-cell for- tion of Tfh and GC B cells by TGF-β signaling was demonstrated mation in Egr2/3DKO mice was rescued by exogenous TGF-β3. using TGF-β receptor II KO mice (40), indicating that TGF-β is We injected a TGF-β3 expression plasmid (pCAGGS-Tgfb3)ora necessary for the control of humoral immunity. To investigate the control pCAGGS plasmid into Egr2/3DKO mice three times and role of TGF-β3 in Tfh and GC B-cell formation, we first analyzed analyzed Tfh and GC B formation at 4 wk after the final in- the suppressive function of TGF-β3 in B cells after in vitro acti- jection. Although the excessive Tfh formation was not improved vation under conditions that promote the differentiation of GC- by the injection of pCAGGS-Tgfb3, the frequency of GC B cells was phenotype B cells. Although B cells stimulated with anti-CD40, markedly reduced in Egr2/3DKO mice injected with pCAGGS- rIL-4, and rBAFF acquired GL7 and Fas expression, which is the Tgfb3, compared with those injected with control pCAGGS (Fig. typical phenotype of GC B cells (41), the addition of TGF-β3or 5 F and G and Fig. S6). This result suggests that loss of TGF-β3 + + + TGF-β1 suppressed the acquisition of GL7 Fas GC phenotype production from Egr2- and Egr3-deficient LAG3 Tregs was re- and proliferation of B cells (Fig. 5 A and B). Moreover, treatment sponsible for the cause of unrestrained GC B-cell formations in with TGF-β3orTGF-β1 effectively inhibited the expression of Egr2/3 DKO mice. Moreover, in accordance with suppression of Bcl6 mRNA, which is essential for the differentiation of GC B GC B cells, the injection of pCAGGS-Tgfb3 improved proteinuria

Morita et al. PNAS | Published online November 30, 2016 | E8135 Downloaded by guest on September 24, 2021 is not directly regulated by Egr2 or Egr3. TGF-β3 undergoes com- plex processing steps intracellularly before its secretion from a cell membrane (42, 43). After translation, TGF-β3 precursor protein is cut 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 outside of the membrane as a large latent complex (LLC). Given that Ltbp has been reported to require binding to TGF-β for efficient secretion (44, 45), we hypothesized that the secretion of TGF-β3is also dependent on binding to Ltbp. Among the Ltbp family that + consists of four members from Ltbp1–4 (46), LAG3 Tregs characteristically express Ltbp3 mRNA (Fig. 6C and Fig. S7A). Furthermore, the expression of Ltbp3 mRNA was significantly + down-regulated in Egr2/3DKO LAG3 Tregs compared with + WT and Egr2CKO LAG3 Tregs (Fig. 6C). To investigate whether 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 of TGF-β3 protein after TCR stimulation. Although the expres- sion of Tgfb3 mRNA was unchanged, the secretion of TGF-β3 protein was strongly down-regulated in Ltbp3 siRNA-trans- + fected LAG3 Tregs (Fig. 6D). The expression of Ltbp1, 2, and 4 was not suppressed by Ltbp3 siRNA (Fig. S7B). These results demonstrate that Egr2 and Egr3 positively regulate the secretion of TGF-β3 by inducing Ltbp3 expression. To elucidate whether overexpression of Ltbp3 rescues the loss of TGF-β3 secretion in Egr2/3DKO T cells, we next constructed pMIG-Ltbp3 retroviral vector. Because we previously reported that IL-27–treated T cells express 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 the production of TGF-β3 in the culture supernatants. As shown in Fig. 6E, transduction of pMIG-Ltbp3 significantly increased the production of TGF-β3inIL-27–treated Egr2/3DKO T cells. Fig. 4. Egr2 and Egr3 deficiency do not augment Tfh cell differentiation Moreover, IL-27–treated Egr2/3DKO T cells retrovirally trans- intrinsically. (A) Flow cytometry analysis of the expression of PD-1 and CXCR5 in donor CD45.2+ OT-II CD4+ T cells obtained from draining lymph nodes duced with pMIG-Ltbp3 significantly suppressed B-cell prolifera- + (dLNs) of CD45.1 recipient WT mice given adoptive transfer of naïve WT, tion and antibody production compared with those transduced with + + Egr2CKO, or Egr2/3DKO CD45.2 OT-II CD4 T cells, followed by immuniza- pMIG-mock (Fig. 6 F and G). These results demonstrated that + tion of NP13-OVA in CFA (Left). Numbers adjacent to outlined areas indicate Ltbp3 expression in LAG3 Tregs plays a critical role in controlling + + percent of PD-1 CXCR5 Tfh cells. Graph indicates frequency (among total humoral immunity via TGF-β3 secretion. + − + + + CD4 CD25 CD45.2 T cells) of PD-1 CXCR5 Tfh cells from dLNs (Right, n = 6 per group). (B) Flow cytometry analysis of the expression of Bcl-6 and CXCR5 Discussion in donor CD45.2+ OT-II CD4+ T cells obtained from dLNs as in A (Left). + + Although it is known that dysregulated adaptive immunity is asso- Numbers adjacent to outlined areas indicate percent of Bcl-6 CXCR5 Tfh + − + ciated with the pathogenesis of SLE, the underlying distinct molec- cells. Graph indicates frequency (among total CD4 CD25 CD45.2 T cells) of + + Bcl-6 CXCR5 Tfh cells from dLNs as in A (Right, n = 6 per group). (C) Flow ular mechanisms have been elusive. We demonstrated in this report + cytometry analysis of the expression of GL7 and Fas in B220 B cells obtained that Egr2 and Egr3 in T cells are central molecules for the main- from dLNs as in A (Left). Numbers adjacent to outlined areas indicate per- tenance of humoral immune tolerance in the steady state. In addi- cent of GL7+Fas+ GC B cells. Graph indicates frequency (among total B220+ tion to the previously observed linkage between Egr2 and systemic B cells) of GC B cells from dLNs as in A (Right, n = 6 per group). (D) Quan- autoimmunity in mice and humans (10, 11), our studies indicate that tification of NP-specific IgG in the serum of the recipient mice as in A (n = 6 the combination of Egr2 and Egr3 in T cells is a promising candidate per group). Data are representative of two independent experiments. The target involved in the regulation of humoral immunity. ± mean SD are indicated. The absence of both Egr2 and Egr3 in T cells led to earlier onset of a lupus-like syndrome compared with Egr2CKO mice, progression and titer of anti-dsDNA autoantibodies (Fig. 5 H and indicating that a compensatory function of Egr3 for Egr2 in T cells is necessary for the prevention of lupus pathogenesis. I). Collectively, these results demonstrate that TGF-β3 production + Intriguingly, Egr2/3DKO mice not only developed a lupus-like controlled by Egr2 and Egr3 in LAG3 Tregsisnecessaryforthe phenotype characterized by marked high titers of anti-dsDNA regulation of aberrant GC B-cell differentiation and related to accompanied by severe glomerulonephritis resembling SLE, but disease pathogenesis of Egr2/3DKO mice. also showed a severe systemic autoimmune syndrome with lym- β phocytic infiltration in multiple organs, such as liver and pancreas, Egr2 and Egr3 Regulate the Secretion of TGF- 3 Protein Through an that are rarely impaired in human lupus (48). These findings Ltbp3-Dependent Manner. We next explored the molecular basis suggest that Egr2 and Egr3 expression in T cells is required for the for the requirement of Egr2 and Egr3 for TGF-β3 production from + immune system to avoid attacking self-tissue affected by a wide LAG3 Tregs. Surprisingly, although the secretion of TGF-β3from + range of autoimmune disorders that are not limited to lupus. LAG3 Tregs was strongly regulated by Egr2 and Egr3, the ex- SLE is a B-cell–mediated autoimmune disease characterized by pression of Tgfb3 mRNA was not altered in Egr2- and Egr3-deficient the loss of tolerance to nucleic acid antigens and is regulated by a + LAG3 Tregs, irrespective of the presence of TCR stimulation (Fig. variety of mechanisms including aberrant Tfh cell differentiation + 6 A and B). This result indicates that the expression of Tgfb3 mRNA (49). Our observation that Bcl-6 expression in CD4 TcellsinTfh

E8136 | www.pnas.org/cgi/doi/10.1073/pnas.1611286114 Morita et al. Downloaded by guest on September 24, 2021 PNAS PLUS

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 without cytokines (no stim.) or with rIL-4, anti-CD40 mAb, and BAFF in the presence or absence of rTGF-β1or-β3(Left). Numbers adjacent to outlined areas indicate percent of + + + + GL7 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-labeled INFLAMMATION IMMUNOLOGY AND B-cell proliferation stimulated as in A (Left). Graph indicates frequency of undivided B cells (Right, n = 4 per group). *P < 0.05 (Bonferroni posttest). (C) qRT-PCR analysis of 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 IgM production 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 Bcl6 mRNA in T cells stimulated with rIL-6 and rIL-21 (Tfh cell condition) in the presence or absence of rTGF-β1or-β3for4d(n = 3 per group). *P < 0.05 (Bonferroni posttest). (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+ Bcells)ofGCBcells(G) in the spleens of Egr2/3DKO mice were examined at 4 wk after 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 = 6per group). *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 are representative of two independent experiments. The mean ± SD are indicated.

cell-polarizing conditions was not suppressed by TGF-β3 does not Tregs (52), play a major role in the regulation of humoral immunity. exclude the suppressive activity of TGF-β in longer duration be- However, contradictory results have been reported for the num- cause Tfh cells overly accumulate in the absence of TGF-β signaling bers and function of Treg in SLE patients, and the precise roles of + in T cells (40). Ogbe et al. reported that Egr2 and Egr3 directly CD25 Tregs in SLE remain elusive (53), because the clinical regulate Bcl-6 expression and are essential for the late stage of Tfh manifestations of immunodysregulation, polyendocrinopathy, en- cell differentiation during viral infection (50). They also revealed teropathy, X-linked (IPEX) syndrome caused by a mutation in the that the key molecules involved in the regulation of Tfh cell che- FOXP3 gene and SLE differ considerably (54, 55). In addition to + + motaxis, such as expression levels of Cxcr5, Icos, and Sh2d1a, are CD25 Treg, Egr2-expressing LAG3 Tregs may also contribute not affected by Egr2/Egr3 deficiency in lymphocytes. Similarly, we to the control of systemic autoimmunity. Egr2 and Egr3 appeared + revealed that absence of Egr2/Egr3 does not alter Tfh cell devel- to exhibit regulatory activity in LAG3 Tregs, because both Egr2- + opment. These results suggested that Tfh cell intrinsic expression of and Egr3-deficient CD25 Tregs retain in vitro suppressive activity + Egr2/Egr3 does not explain the enhanced humoral immunity ob- (18) and the adoptive transfer of WT LAG3 Tregs into Egr2/ served in Egr2/3DKO mice. The fact that treatment with a TGF-β3 3DKO mice effectively suppressed the aberrant development of expression vector ameliorated only GC B development in Egr2/ both Tfh and GC B cells. 3DKO mice indicated that a primary direct target of suppression In the present study, we observed that TGF-β3 suppressed mediated by Egr2/Egr3–TGF-β3 axis may be B cells. B-cell activation and differentiation as efficaciously as TGF-β1, To date, a number of studies have demonstrated that Treg sub- suggesting that TGF-β3 is a potent regulator of humoral immu- + + sets maintain immunological self-tolerance. Foxp3 CD25 Tregs nity. It was recently identified that TGF-β1 plays an integral role in have been the most intensively studied based on the accumu- maintaining immune tolerance (9). As for regulation of humoral lating evidence for their therapeutic effects to prevent various immunity, TGF-β1 controls B-cell activation by inhibiting both Ig autoimmune diseases. It has been reported that some specialized synthesis and the switch from the membrane form to the secreted + + + − + + − CD25 Treg subsets, such as Tfr cells (51) and CD4 CD25 CD69 forms of μ-andγ-mRNA (56). CD4 CD25 CD69 Treg-mediated

Morita et al. PNAS | Published online November 30, 2016 | E8137 Downloaded by guest on September 24, 2021 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-cell subsets (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-CD28 mAb (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 (Bonferroni posttest). (D) qRT-PCR analysis of the expression of Ltbp3 (Left) and Tgfb3 (Middle) mRNA, and quantification of TGF-β3 protein levels (Right) in the culture + supernatants 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 in + the 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 were retrovirally 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 = 4 per 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 were cocultured with pMIG-mock or pMIG-Ltbp3 vector transduced CD4+ T cells that were treated with IL-27 after transduction. B-cell proliferation was assessed by CFSE 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 = 4 per group). *P < 0.05 (Bonferroni posttest). Data are representative of three (A–C)ortwo(D–G) independent experiments. The mean ± SD are indicated.

suppression of B-cell antibody production is mediated at least Ltbp3 as a potential therapeutic target. Further investigation is partially through expression of TGF-β1 (52). On the other hand, the needed to confirm whether Ltbp3 is a direct or indirect target of immune suppressive role of TGF-β3 has not been extensively Egr2 and Egr3. evaluated, and a mechanism for the production of TGF-β3hasnot As described above, our study suggests the importance of Egr2 been clarified. We found that double deficiency of both Egr2 and and Egr3 in T cells for the maintenance of humoral immune tol- Egr3 in T cells affects TGF-β3 production and Ltbp3 expression, + erance. Although Li et al. reported that mice in which Egr2 and and not Tgfb3 mRNA expression, in LAG3 Tregs. Among the four Egr3 are deleted specifically in both T and B cells die within 8 mo isoforms of Ltbp, Ltbp1, and Ltbp3 can associate efficiently with (18), three-quarters of T-cell–specific Egr2 and Egr3 double- – β β β pro TGF- 1, - 2, and - 3, whereas Ltbp2 reportedly does not (57). deficient (Egr2/3DKO) mice were still alive after 1 y of age (Fig. β – On the other hand, Ltbp4 binds to TGF- 1 LAP more weakly than 1A), suggesting the importance of Egr2 and Egr3 in B cells in the Ltbp1 and Ltbp3, indicating that Ltbp1 and Ltbp3 might be the immune system. However, there have been no reports of B-cell– primary responsible for binding to the TGF-β–LAP com- specific Egr2/Egr3 double-deficient mice. Detailed analyses of the plex SLC (58). Several lines of evidence indicate that the Ltbps, which are not necessary for latency (59), play a major role in the role of Egr2 and Egr3 in B cells may provide novel therapeutic secretion (44), extracellular membrane localization (17), and acti- targets on B cells for autoantibody-mediated autoimmune diseases. In summary, our findings have provided insight into the mecha- vation (60, 61) of latent TGF-β. However, it was reported that, + unlike Ltbp1, Ltbp3 is not suitable for integrin-mediated latent nism of how Egr2 and Egr3 in CD4 T cells regulate humoral im- β munity and established the combination of Egr2 and Egr3 as a TGF- activation, and this isoform-specific function is most likely + related to the great sequence divergence of their hinge domains pivotal regulator of TGF-β3 secretion from LAG3 Tregs. Un- (62). In this study, we revealed a previously unrecognized function derstanding the molecular basis of TGF-β3secretionwillbekeyto for Ltbp3 as an efficient regulator of TGF-β3 secretion from understanding lupus pathogenesis and will provide new therapeutic + LAG3 Tregs. These results suggest that Ltbp3 may play a critical avenues for manipulating the excessive humoral immune responses + role in regulating TGF-β3–mediated immune tolerance and define using LAG3 Tregs.

E8138 | www.pnas.org/cgi/doi/10.1073/pnas.1611286114 Morita et al. Downloaded by guest on September 24, 2021 Materials and Methods T Cell–B Cell Coculture Assay. These procedures are described in SI Materials PNAS PLUS Mice. C57BL/6 (B6) mice were purchased from Japan SLC. B6 mice congenic for and Methods. the CD45 locus (B6-CD45.1+) were purchased from Sankyo Lab Service. TCR + + × transgenic OT-II mice (specific for the chicken ovalbumin peptide (amino acid Transfer of LAG3 Tregs into Egr2/3DKO Mice. FACS-sorted LAG3 Tregs (3 5 residues 323–339) in the context of MHC class II I-Ab) were purchased from The 10 cells) from WT mice were i.v. transfused into Egr2/3DKO mice at 2 and Jackson Laboratory. Egr2fl/fl mice were provided by Patrick Charnay (INSERM) 4 wk of age. At 4 wk after the transfer, the mice were killed and splenic Tfh (23). CD4-Cre transgenic mice (line 4196), originally generated by C. B. Wilson and GC B-cell formation was analyzed by flow cytometry. and colleagues, were purchased from Taconic. Egr2fl/fl mice were crossed with CD4-Cre transgenic mice to generate Egr2CKO mice (Egr2fl/fl Cd4-Cre+). Adoptive Transfer of Naïve T Cells from OT-II Mice. These procedures are de- Egr2/3DKO mice (Egr2fl/fl Egr3fl/fl Cd4-Cre+) were generated by crossing scribed in SI Materials and Methods. Egr2CKO mice with Egr3fl/fl mice. All animal experiments were approved by the ethics committee of the University of Tokyo Institutional Animal Care and Injection of TGF-β3 Expressing Plasmid Vector into Egr2/3DKO Mice. Construc- Use Committee. tion of TGF-β3 expressing pCAGGs vector (pCAGGS-Tgfb3) was previously described (8). Egr2/3DKO mice were injected i.v. with 100 μg of pCAGGS- Generation of Egr3 Floxed Mice. The targeting vector was constructed by Tgfb3 or control pCAGGS in sterile PBS at 4, 6, and 8 wk of age. At 4 wk after inserting a 1.4-kb fragment containing exon 2 of the Egr3 gene flanked by the last injection, the mice were killed and splenic Tfh and GC B-cell for- loxP sites, 3.0 kb of a 3′ sequence, 3.9 kb of a 5′ sequence, and a neomycin- mation was analyzed by flow cytometry. resistance gene flanked by Frt sites into a pBluescriptII SK (+) vector. The targeting vector was linearized and transfected into B6 embryonic stem Transfection of siRNA. LAG3+ Tregs (4 × 105 cells per well) were transfected cells by electroporation. Recombinant ES clones were selected in medium with siRNA in Accell siRNA delivery media (GE Healthcare) according to the supplemented with G418. Thymidine kinase was used as a counter- manufacturer’s protocol. siRNA targeting Ltbp3 and control siRNA were selection. G418-resistant clones were screened for homologous recombi- used at a concentration of 1 μM. At 48 h after the transfections, the cells nation by PCR and Southern blot analysis. The positive clones were were transferred to 96-well flat-bottomed plates coated with anti-CD3 mAb microinjected into blastocytes derived from BALB/c mice and transferred (2 μg/mL) and anti-CD28 mAb (2 μg/mL) in RPMI-1640 medium as described to surrogate mothers. Mating of chimeric male mice to B6 female mice above. The cells were incubated for 72 h and then and TGF- resulted in the transmission of the floxed allele to the germline. The β3 protein production were analyzed. neomycin selection cassette flanked by Frt sites was excised in vivo by crossing the C57BL/6-Tg (CAG-FLP) mice. For detection of floxed alleles, Retroviral Transduction into IL-27–Treated T cells. These procedures are de- genomic DNA obtained from B cells and T cells was assessed by PCR. PCR scribed in SI Materials and Methods. primer pairs were as follows: forward CGAGGACAAAAGCGTCGAAGCTC and reverse GATCAAGGCGATCCTAACTGAAC. RNA Isolation, cDNA Synthesis, and Quantitative Real-Time PCR. These proce- dures are described in SI Materials and Methods. Reagents, Antibodies, and Media. Purified and conjugated antibodies were purchased from BD Bioscience, eBiosciences, or Biolegend and recombinant Statistical Analysis. Survival rates were analyzed with the log-rank test. cytokines were purchased from Miltenyi Biotec, R&D, and Biolegend. See SI Quantitative proteinuria progression was analyzed with the Mann–Whitney Materials and Methods for details. u test. For the comparison of more than three groups, a one-way analysis of variance followed by a Bonferroni multiple comparison test was performed. Flow Cytometry and Cell Sorting. These procedures are described in SI Ma- All other statistical differences were determined using the two-tailed Stu- terials and Methods. ’ INFLAMMATION

dent s t test. All data are presented as mean and SD. Statistical significance IMMUNOLOGY AND was defined as a P value of <0.05 for all tests. Histopathological Examination. Histopathologic examination of WT, Egr2CKO, and Egr2/3DKO mice was done at 16 or 36 wk of age. See SI Materials and ACKNOWLEDGMENTS. We thank K. Watada, J. Takezawa, and R. Yamagata Methods for details. Renal pathology was graded as described in SI Materials for excellent technical assistance; Dr. P. Charnay (INSERM) for providing Egr2 and Methods. floxed mice; and Dr. J. Miyazaki (Osaka University Medical School) for kindly providing the pCAGGS vector. This work was supported by Grant-in-Aid for B- and T-Cell Isolation and Proliferation. These procedures are described in SI Japan Society for the Promotion of Science Fellows Grant 254186 and Materials and Methods. Scientific Research Grant 16K09918.

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