Strain-Specific Manifestation of -like Systemic Caused by Zap70

This information is current as Takashi Matsuo, Motomu Hashimoto, Shimon Sakaguchi, of September 25, 2021. Noriko Sakaguchi, Yoshinaga Ito, Masaki Hikida, Tatsuaki Tsuruyama, Kaoru Sakai, Hideki Yokoi, Mirei Shirakashi, Masao Tanaka, Hiromu Ito, Hajime Yoshifuji, Koichiro Ohmura, Takao Fujii and Tsuneyo Mimori

J Immunol 2019; 202:3161-3172; Prepublished online 24 Downloaded from April 2019; doi: 10.4049/jimmunol.1801159 http://www.jimmunol.org/content/202/11/3161 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2019/04/23/jimmunol.180115 Material 9.DCSupplemental References This article cites 53 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/202/11/3161.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Strain-Specific Manifestation of Lupus-like Systemic Autoimmunity Caused by Zap70 Mutation

Takashi Matsuo,* Motomu Hashimoto,† Shimon Sakaguchi,‡ Noriko Sakaguchi,‡ Yoshinaga Ito,x Masaki Hikida,{ Tatsuaki Tsuruyama,‖ Kaoru Sakai,# Hideki Yokoi,# Mirei Shirakashi,* Masao Tanaka,† Hiromu Ito,†,** Hajime Yoshifuji,* Koichiro Ohmura,* Takao Fujii,†† and Tsuneyo Mimori*

A defect in TCR-proximal signaling is a major characteristic of CD4 T cells in systemic ; however, it is not fully known how defects in TCR signaling lead to lupus-like systemic autoimmunity characterized by germinal center development and production against nuclear Ags. In this study, we show that SKG mice, which develop autoimmune arthritis in a

BALB/c background due to defective TCR signaling by a Zap70 mutation, develop lupus-like systemic in Downloaded from the C57BL/6 (B6) background (B6SKG mice). B6SKG mice showed multiorgan inflammation with immune complex deposition and anti-dsDNA Ab production. Follicular helper T cells (Tfh), which help germinal center formation, were spontaneously expanded in B6SKG mice. Th cells secreting IFN-g or IL-17 and regulatory T cells were also increased in B6SKG mice compared with wild-type B6 mice, with the regulatory T cell subpopulation losing the expression of CD25. Among the factors related to Tfh differentiation, the number of dendritic cells and the expression levels of the costimulatory CD80, CD86, and ICOSL in

dendritic cells but not in B cells were specifically increased in wild-type B6 mice compared with BALB/c mice. The inhibition of http://www.jimmunol.org/ these costimulatory molecules suppressed Tfh development and lupus-like autoimmunity. Thus, a defect in TCR-proximal sig- naling leads to lupus-like systemic autoimmunity under the specific genetic background that facilitates Tfh development. The Journal of Immunology, 2019, 202: 3161–3172.

ystemic lupus erythematosus (SLE) is a systemic auto- production of B cells (1). A defect in TCR signaling is a major immune disease characterized by the production of auto- characteristic of SLE CD4 T cells (2). Multiple discrete signal- S against nuclear Ags. Although the etiology of ing abnormalities in TCR signaling at the level of the TCR SLE is multifactorial, CD4 T cells have been recognized to play a complex, cytosol, and nucleus have been reported in SLE CD4 fundamental role in its pathogenesis, contributing to the autoantibody T cells (2). Among these, defects of TCR-proximal signaling by guest on September 25, 2021 involving the CD3-TCR complex and its associated ITAMs are the most common abnormalities observed in SLE (3). In fact, *Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, the downregulation of the CD3z chain due to transcriptional or Kyoto University, Kyoto 606-8507, Japan; †Department of Advanced Medicine for posttranscriptional defects has been detected in more than half Rheumatic Diseases, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, ‡ of SLE patients (4). Importantly, decreased expression levels of Japan; Department of Experimental Immunology, Immunology Frontier Research Center, Osaka University, Osaka 565-0871, Japan; xDepartment of Cancer Immunology the TCRz chain in SLE persist regardless of disease activity or { and Virology, Dana-Farber Cancer Institute, Boston, MA 02215; Laboratory for treatments, in contrast with the transient reduction due to inflam- Molecular Cell Physiology, Department of Life Science, Akita University, Akita 010-8502, Japan; ‖Center for Anatomical, Pathological and Forensic Medical Research, matory conditions observed in cancer, infection, or rheumatoid Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan; #Department arthritis (RA) (4, 5). Similar to human studies, abnormalities of of Nephrology, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan; various molecules in TCR signaling, such as CD45, Fyn, and **Department of Orthopedic Surgery, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan; and ††Department of Rheumatology and Clinical Immunology, ZAP70, have been shown to result in the development of systemic Wakayama Medical University, Wakayama 641-8510, Japan autoimmune diseases in mice (6–9). However, the mechanism by ORCIDs: 0000-0003-3805-7224 (T. Matsuo); 0000-0002-3118-2826 (T.T.); 0000- which these defects in TCR signaling lead to systemic autoim- 0003-0803-0397 (K.S.). munity has not been fully addressed. Altered T cell selection in Received for publication August 21, 2018. Accepted for publication March 27, 2019. the thymus (both positive and negative selection) and skewed This work was supported by a Grant-in-Aid for Young Scientists (26860749) and a differentiation of effector T cells and regulatory T cells (Tregs) in Grant-in-Aid for Scientific Research (16K09890) from the Ministry of Education, the periphery have been indicated as potential mechanisms (8, 10). Culture, Sports, Science and Technology, Japan (to M. Hashimoto), and by a research grant from Bristol-Myers Squibb and ONO Pharma (to M. Hashimoto). The SKG mouse strain with a BALB/c background (SKG) has Address correspondence and reprint requests to Dr. Motomu Hashimoto, Department been used as a murine model of RA, which develops autoimmune of Advanced Medicine for Rheumatic Diseases, Graduate School of Medicine, Kyoto arthritis under a nonspecific pathogen-free condition (8). The University, 35 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. E-mail causative factor was identified as a point mutation (W163C) of the address: [email protected] Zap70 gene, located in the SH2 of ZAP70, which alters its The online version of this article contains supplemental material. association with the ITAMs of the CD3z chain, thus reducing, Abbreviations used in this article: ANA, anti-nuclear Ab; DC, dendritic cell; DN, double-negative; DP, double-positive; GC, germinal center; Pdcd1, programmed cell but not abolishing, TCR signal transduction. The decrease in TCR death 1; PTPN22, protein tyrosine phosphatase-22; RA, ; signaling due to the Zap70 mutation alters the thymic selection SLE, systemic lupus erythematosus; SP, single-positive; Tfh, follicular helper T cell; thresholds so that T cells that strongly react with self- Tfr, follicular Treg; Treg, regulatory T cell. fail to be deleted during thymic negative selection and escape Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 to the periphery. These self-reactive T cells then differentiate into www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801159 3162 STRAIN-SPECIFIC LUPUS-LIKE SYSTEMIC AUTOIMMUNITY

IL-17–producing helper CD4 T cells (Th17) driven by homeostatic sections were visualized using an LSM 710 NLO confocal fluorescent mi- proliferation or by innate immune activation and result in the croscope and analyzed with ZEN software (Zeiss). The immunofluorescence development of autoimmune arthritis in SKG mice (11, 12). pattern of the anti-nuclear Ab (ANA) was evaluated by the Hep-2 ANA test (MBL) using mouse anti-IgG (Jackson ImmunoResearch). Although SKG mice have a defect in TCR-proximal signaling, and defects in TCR signaling are major abnormalities in SLE CD4 ELISA T cells, SKG mice do not develop lupus-like systemic autoim- ANA-IgM and IgG levels were evaluated by the MESACUP ANA test munity such as immune complex–mediated nephritis. SKG mice (MBL) using the anti-mouse HRP-IgM Ab (Southern Biotech) and anti- show hypergammaglobulinemia and develop or mouse HRP-IgG Ab (Promega). Anti-dsDNA Ab IgG in the serum was an Ab against type-2 collagen but do not produce anti-dsDNA Ab evaluated by a mouse anti-dsDNA ELISA kit (Shibayagi). Total IgG, IgG2b, (8). A potential explanation for this apparent contradiction may be IgG2c, IgG3, and IgM levels were evaluated by the mouse ELISA Ready- Set-Go kit (eBioscience). IgG1 levels were evaluated by a mouse IgG1 related to the specific genetic background of this mouse strain ELISA kit (Bethyl Laboratories). because BALB/c mice are resistant to the development of lupus in contrast to C57BL/6 (B6) mice in many autoimmune Flow cytometry models. For example, deficiency of FcgRIIb or programmed cell The following Abs were purchased from BioLegend: anti-CD3 (17A2), anti- death protein 1 (Pdcd1) or the transgenic expression of B cell CD4 (RM4-5), anti-CD8 (53-6.7), anti-CXCR5 (L138D7), anti–PD-1 CLL/lymphoma 2 (Bcl2) all result in a lupus-like phenotype in (29F.1A12), anti-ICOS (C398.4A), anti-B220 (RA3-6B2), anti–GL-7 g the B6 background but not in the BALB/c background (13–17). (GL-7), anti–IL-17 (TC11-18H10), anti–IFN- (XMG1.2), anti-CD44 (IM7), anti-CD62L (MEL-14), anti–I-Ad (39-10-8), and anti–I-Ab Although the specific mechanism that renders B6 more (AF6-120.1). The following Abs were purchased from eBioscience: anti- susceptible to the lupus phenotype than BALB/c animals has not CD25 (7D4), anti-Foxp3 (FJK-16s), and anti-AA4.1 (AA4.1). The following Downloaded from been fully defined, several mechanisms have been proposed, such Abs were purchased from BD Bioscience: anti-CD95 (Jo2) and anti–Bcl-6 as a difference in receptor editing of the Ig L chain (18), skewing (K112-91). The factors Foxp3 and Bcl-6 were stained after fixation and permeabilization (eBioscience). For intracellular cytokine staining to Th1 cells (19), or increased type-1 IFN, which are all key of IL-17 and IFN-g, spleen cells were stimulated with 20 ng/ml PMA (Sigma- mediators in the pathogenesis of SLE (20, 21). Aldrich) and 1 mM ionomycin (Sigma-Aldrich) in the presence of GolgiStop In this study, we demonstrate that SKG mice with a B6 back- (BD Biosciences) for 4 h before staining. Costimulatory molecules on splenic DCs were evaluated 24 h after purification by MACS LS columns (Miltenyi ground (B6SKG) develop lupus-like systemic autoimmunity with http://www.jimmunol.org/ marked expansion of follicular helper T cells (Tfh) and germinal Biotec) using CD11c (N418) beads (22). Flow cytometric acquisition was performed on a FACSCalibur system (BD) and analyzed with LSRFortessa center (GC) development. We propose that an increase in the number (BD). Analysis was performed using FlowJo and WinMDI software. of dendritic cells (DCs) with higher expression of costimulatory molecules in the B6 strain compared with the BALB/c strain may Proliferation assay facilitate the strain-specific development of Tfh and lupus in B6SKG CD4 T cells from the spleen were purified in MACS LS columns (Miltenyi mice. Thus, a defect in TCR-proximal signaling may predispose Biotec) using CD4 (RM3-4) beads. Purified cells were loaded with the individuals to autoimmunity, whereas the specific genetic background CFSE Cell Proliferation Kit (Invitrogen), washed, and incubated with plate- m m may provide additional coactivating components necessary for the bound anti-CD3 mAb (10 g/ml) and soluble anti-CD28 mAb (1 g/ml) for 3 d. Cells were harvested and analyzed by flow cytometry. development of systemic autoimmune disease. by guest on September 25, 2021 Real-time PCR Materials and Methods CD4 T cells and CD11chigh DCs isolated from the spleen cells were purified by Mice MACS LS columns (Miltenyi Biotec) using CD4 (RM3-4) and CD11c (N418) beads according to the manufacturer’s instructions. The purity was above 95% BALB/c A (BALB/c), C57BL/6J (B6 wild-type [B6WT]), and SKG mice for total CD4 T cells and above 75% for total CD11chigh DCs. The purified cells were purchased from CLEA Japan. SKG mice were backcrossed to B6WT were lysed, and the RNA was extracted using the RNeasy Micro Kit (Qiagen) mice for eight generations to generate C57BL/6ZAP70 skg/skg (B6SKG) and converted into cDNA with the High-Capacity cDNA Reverse Transcription mice. All mice were maintained in a specific pathogen-free condition in Kit (Applied Biosystems) using Mastercycler (Eppendorf). Cytokine and tran- our animal facility in accordance with the guidelines for animal care ap- scription factor expression levels were assessed by real-time quantitative PCR proved by Kyoto University. Age- and sex-matched mice were used for all using a TaqMan gene expression assay probe and TaqMan Gene Expression experiments. Master Mix run on the 7500 Real-Time PCR system (Applied Biosystems). Expression levels were normalized by the housekeeping gene Hprt. Histology The following primers were purchased from Applied Biosystems: Il-6 Il-21 Hprt The salivary gland, thyroid gland, thymus, lung, heart, stomach, ileum, (Mm00446190_m1), (Mm00517640_m1), and (Mm01545399_m1). intestine, liver, pancreas, kidney, spleen, skin, and ankle joint were fixed in Experimental treatments buffered 10% formalin, and paraffin-embedded sections were stained with H&E. Stained sections were visualized using a system microscope (BX43 For blockade experiments, 8–10-wk-old B6SKG mice were i.p. injected with the and DP26; Olympus) and were evaluated by a single pathologist (T.T.). specific Abs indicated below, and spleen cells were obtained after 2 wk. Anti- OX40L (RM134L, 100 mg; BioLegend) and anti-ICOSL (HK5.3, 100 mg; Immunohistochemistry BioLegend) were injected i.p. every 3 d (a total of five times for 2 wk) in a total m Frozen 5-mm sections of the kidneys from B6WT and B6SKG mice were of 500 g. CTLA4 Ig (Abatacept; Bristol-Myers Squibb) was injected once at m subjected to immunohistochemical staining. The kidney section was 500 g. For evaluating immune complex deposition in the kidney after CTLA4- m stained with IgG (Jackson ImmunoResearch) and C3 (Cappel). The sec- Ig treatment, 125 g of CTLA4-Ig was injected once a week for 8 wk (total m tions were fixed with acetone (Wako) and stained with the indicated 1000 g). For the TLR stimulation experiment, the mice were s.c. implanted for Abs in PBS. Finally, the sections were washed and mounted with Vecta- 14–28 d with an osmotic pump (no. 1002, 1004; DURECT) containing the m m shield (Vector Laboratories), and stained sections were visualized using an following TLR agonists: 700 g of Poly(I:C) (P9582; Sigma-Aldrich), 280 g m all-in-one fluorescence microscope (BZ-700; Keyence Co.). The Crithidia of LPS (L4391; Sigma-Aldrich), 280 g of imiquimod (sc-200385; Santa Cruz m luciliae assay was conducted with the Fluoro nDNA Test kit (MBL) using Biotechnology), and 280 g of CpG-ODN1668 (Hokkaido System Science). mouse anti-IgG (Jackson ImmunoResearch). Zymosan A (Z4250; Sigma-Aldrich) and mannan (M7504; Sigma-Aldrich) Frozen 10-mm sections of the spleens from B6WT and B6SKG mice were injected i.p. twice a week for 4 wk for up to 2 and 20 mg, respectively. were stained with anti-PNA (Vector Laboratories), anti–GL-7 (GL-7; Assessment of arthritis score BD Biosciences), and anti-CD38 (90; eBioscience) Abs. The sections were fixed with acetone (Wako), blocked with Blocking One (Nacalai The arthritis score was assessed as described previously (23). In brief, Tesque), and stained with the indicated Abs in PBS. Finally, the sections swelling of the finger joint received a score of 0.1, and swelling of the wrist were washed and mounted with SlowFade Gold (Invitrogen), and the stained and finger joints received scores of 0.5–1.0. The Journal of Immunology 3163

Statistics Results We used a two-sided Student t test for all statistical analyses. A p value Development of lupus-like systemic autoimmune disease in ,0.05 was considered statistically significant. B6SKG mice Study approval SKG mice were backcrossed for eight generations with B6WT mice, and their organs were examined histologically. The tissues All experimental procedures were performed in accordance with the ethical guidelines of Kyoto University. This study was approved by the Animal of B6SKG mice were normal or showed only mild abnormality Research Committee, Graduate School of Medicine, Kyoto University at 3 mo of age, whereas obvious abnormalities were found at 12 mo (Medkyo 16106). of age. B6SKG mice showed normal histology of the kidney Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 1. Development of lupus-like systemic autoimmune disease in B6SKG mice. (A–C) H&E staining of tissue sections of B6WT and B6SKG mice at 3 and 12 mo of age. (A) Kidney section. (B) Lung section. (C) Salivary gland section. (D) Major diameter of the glomeruli. Ten glomeruli at the middle cortex area per mouse at original magnification 3400; (n = 3 each) **p , 0.01 versus control mice, Student t test. (E) Immunohistochemistry of glomeruli at 3 mo of age for IgG and complement C3. Scale bars in (A)–(C) and (E), 20 mm. 3164 STRAIN-SPECIFIC LUPUS-LIKE SYSTEMIC AUTOIMMUNITY compared with B6WT mice at 3 mo of age, whereas destruction B6SKG mice compared with age-matched B6WT mice (Fig. 1A, of vascular endothelial cells, a relative increase in the mesangium 1D). In the lungs, there was mild subbronchial accumulation area, and atrophy of the glomeruli were observed in 12-mo-old of lymphocytes detected at 3 mo, which increased at perivascular, Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 2. Serology of B6SKG mice. (A) Immunofluorescent staining of ANA with permeabilized Hep-2 epithelial cells at original magnification 3400. The serum samples were diluted 80 times. The nucleus of Hep-2 cells was stained with the sera of B6SKG mice and B6WT mice. Scale bar, 10 mm. (B) ELISA for ANA-IgM and ANA-IgG. The ELISA plate was coated by a mixture of nuclear Ags, including Sm, RNP, , SS-A, and dsDNA. The serum samples were diluted 100 times (n = 10 each). (C) ELISA for serum IgM (330,000 dilution) and IgG (3100,000 dilution). (D) ELISA for serum IgG1, IgG2b (320,000), IgG2c (310,000), and IgG3 (330,000) levels (n = 10 each). (E) ELISA for anti-dsDNA Ab (351 dilution) (n = 20 each). (F) Crithidia luciliae assay at original magnification 3200. *p , 0.05, **p , 0.01 versus control mice, Student t test. The Journal of Immunology 3165 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 3. Spontaneous development of the GC in B6SKG mice. (A) H&E staining of the spleen. Clear distinction between red pulp and white pulp was observed in B6WT mice but not in B6SKG mice at original magnification 340. (B) Immunohistochemistry of the spleen with staining for PNA (blue), CD38 (green), and GL-7 (red); scale bar, 100 mm. (C) Flow cytometric analysis of GL7 and FAS gated on B220+AA4.12 mature B cells (n =10each).(D) Proportion of B220+ B cells in splenic lymphocytes. (E) Proportion and number of GL7+ FAS+ GC B cells among B220+ AA4.12 Bcells(n = 10 each). (Figure legend continues) 3166 STRAIN-SPECIFIC LUPUS-LIKE SYSTEMIC AUTOIMMUNITY stromal, and intraepithelial lesions, leading to the remodeling of the autoimmune arthritis in SKG mice, only slightly elevated the level bronchus at 12 mo of age in B6SKG mice (Fig. 1B). In the salivary of anti-dsDNA Ab, whereas mannan but not zymosan treatment glands, signs of sialadenitis (accumulation of lymphocytes, edematous triggered mild arthritis in B6SKG mice (12, 23) (Supplemental change, and destruction of the salivary ducts) were observed in Fig. 2B). B6SKG mice at 12 mo of age (Fig. 1C). B6SKG mice also showed Collectively, these results demonstrate that B6SKG mice de- abnormalities in the joints, pancreas, and skin (Supplemental Fig. 1), velop lupus-like systemic autoimmune disease with immune whereas other tissues, including the thyroid gland, heart, liver, complex deposition and anti-dsDNA Ab production, which can be stomach, and intestine, did not show major abnormalities in accelerated by innate immune stimulation. B6SKGmicecomparedwithB6WTmice. Because B6SKG mice showed signs of inflammation in multiple Spontaneous development of the GC and expansion of Tfh in tissues, including the kidney, lung, and salivary glands, we further B6SKG mice investigated whether these changes were mediated by immune Because the production of is highly associated with complexes. Even in the 3-mo-old B6SKG mice that showed the formation of the GC, we analyzed the development of the minimal abnormality in the glomeruli compared with B6WT mice, GC in the spleen of B6SKG mice. B6SKG mice showed mild immunofluorescence analysis of the kidney revealed predominant splenomegaly compared with B6WT mice (data not shown). deposition of IgG in the glomeruli, unlike B6WT mice (Fig. 1E). H&E staining of the spleen further revealed effacement of the Similarly, complement C3 deposition in the glomeruli was observed normal architecture (clearly defined areas of red and white only in B6SKG mice and not in B6WT mice. The deposition of pulp) in the spleen of B6SKG mice, suggesting a hyperreactive

IgG and C3 in the renal glomeruli strongly suggested that the feature (Fig. 3A). Immunohistochemistry of the spleen showed Downloaded from glomerulonephritis was immune complex mediated (Fig. 1E). the spontaneous development of PNA+ CD382 GL-7+ GC areas in Next, to determine whether the nephritis in B6SKG mice is B6SKG mice but not in B6WT mice (Fig. 3B). driven by lupus-like systemic autoimmunity, we assayed the sera GC development in B6SKG mice was also confirmed by flow for the presence of autoantibodies against nuclear Ags. Immu- cytometry. In B6SKG mice, the proportions of GL7+ FAS+ GC nofluorescent staining of permeabilized Hep-2 epithelial cells B cells were significantly increased among B220+ AA4.12 mature + 2 revealed the presence of ANA, with a peripheral staining pattern B cells, and the numbers of both GC B and FAS GL7 B cells http://www.jimmunol.org/ in the sera of 3-mo-old B6SKG mice, whereas no such reactivity were also increased (Fig. 3C). The proportion of total B cells and was detected in B6WT mice of the same age (Fig. 2A). ELISA both the proportion and total number of GC B cells increased in for ANA (coated with Sm, histone, SS-A, and other Ags) revealed B6SKG mice (Fig. 3D, 3E). an increased titer of ANA-IgG in B6SKG mice compared with Because GC formation is mediated by Tfh, we further examined B6WT mice, whereas the titer of ANA-IgM did not differ be- the proportion of Tfh, characterized by the expression of the tween the two strains (Fig. 2B). Moreover, the total amounts chemokine receptor CXCR5 and the transcription factor BCL6 of serum IgM and IgG were higher in B6SKG mice than in as well as by higher expression levels of PD-1 and ICOS. The B6WT mice (Fig. 2C), suggesting the development of hyper- proportion of CXCR5+ Bcl6+ Tfh was significantly increased in gammaglobulinemia. Among the IgG subsets, the titers of IgG2b, B6SKG mice compared with that in B6WT, BALB/c, or SKG by guest on September 25, 2021 IgG2c, and IgG3, but not IgG1, were significantly elevated in mice (Fig. 3F). Although B6SKG mice had a reduced number B6SKG compared with B6WT mice (Fig. 2D), similar to the of CD4 T cells due to the alteration of thymic selection (Fig. 3G), serological characteristics of Pdcd1 knockout mice (14). ELISA similar to SKG mice, the proportion and total number of Tfh in- revealed that the titer of anti-dsDNA Ab IgG was markedly el- creased in B6SKG mice compared with those of B6WT mice evated in B6SKG mice compared with that in BALB/c, SKG, (Fig. 3H). Splenic CD4 T cells in B6SKG mice expressed high and B6WT mice (Fig. 2E). The specificity for dsDNA was also levels of PD-1 and ICOS (Fig. 3I), and Il-21 mRNA expression confirmed by a Crithidia luciliae assay (9). The sera of anti- was highly elevated in the CD4 T cells of B6SKG mice (Fig. 3J), dsDNA Ab-positive B6SKG mice reacted with the kinetochore consistent with the increased development of Tfh. (which is free of ssDNA and not complexed to a protein), Because the Zap70 gene is primarily expressed in T cells, we thereby confirming the reactivity against dsDNA, whereas only analyzed thymic T cell selection and effector T cell and Treg nonspecific binding was observed in the sera of B6WT, BALB/c, development more precisely in B6SKG mice (Fig. 4). The medulla or SKG mice (Fig. 2F). of the thymus was atrophic in B6SKG mice, suggesting defects in Because systemic autoimmunity can be accelerated by innate thymocyte development (Fig. 4A). Because pre-TCR/TCR sig- immune stimulation (24), we stimulated B6SKG mice with TLR naling via ZAP70 is involved in the passage from the double- agonists, which have been reported to trigger lupus in other negative (DN)3 (CD25+ CD442) to DN4 (CD252 CD442) stage animal models (25–28). We selected 2–3-mo-old B6SKG mice and from the double-positive (DP) to single-positive (SP) stage, for this experiment, which showed a low titer of anti-dsDNA B6SKG mice showed a reduction in the frequency of DN4 cells Ab (,100 mU/ml) by ELISA as well as control B6WT mice, with a corresponding increase in DN3 cells as well as a reduction and both groups were chronically stimulated with various TLR in CD4 SP and CD8 SP cells with a corresponding increase in DP agonists. The anti-dsDNA Ab level significantly increased after cells compared with B6WT mice (Fig. 4B), similar to findings for treatment with imiquimod (TLR7 agonist) or CpG (TLR9 ag- SKG mice with other genetic backgrounds (29). In the periphery, onist) and to a lesser extent after Poly(I:C) (TLR3 agonist) or B6SKG mice showed decreased populations of both CD4 and LPS (TLR4 agonist) treatment in B6SKG mice (Supplemental CD8 T cells (Fig. 4C). CD4 T cells from B6SKG mice were Fig. 2A). Treatment with zymosan or mannan, which triggers hypoproliferative against in vitro anti-CD3/CD28 stimulation

(F) Flow cytometric analysis of CXCR5 and Bcl6 gated on CD4 T cells. (G) Proportion of CD4 T cells in splenic lymphocytes (n = 10 each). (H) Proportion and numbers of Tfh (n = 10 each). (I) Flow cytometric analysis of PD-1 and ICOS gated on CD4 T cells. (J) Quantitative real-time PCR for Il-21 mRNA of splenic CD4 T cells (n = 3 per group). Flow cytometric data (C, F, and I) are representative of at least three independent experiments. **p , 0.01 versus control mice, Student t test. The Journal of Immunology 3167 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. Thymic selection: Th1, Th17, and Tregs in B6SKG mice. (A) H&E staining of the thymic gland in 28–35-d-old B6WT and B6SKG mice. (B) Flow cytometric analysis of the maturation of developing T cells in the thymus (n = 5 each). The percentages of DN3 and DN4 were expressed as the percentage of DN3 and DN4 relative to total DNs. The percentages of DNs, DPs, and SPs were assessed on the basis of the percentage of total thymocytes. (C) CFSE dilution assay of CD4 T cells purified from B6WT and B6SKG mice. Proliferation of the CD4 T cells was assessed (Figure legend continues) 3168 STRAIN-SPECIFIC LUPUS-LIKE SYSTEMIC AUTOIMMUNITY due to the Zap70 mutation (Fig. 4D). However, the proportion of strain had a higher number of DCs with higher expression levels of CD62+ CD442 naive CD4 T cells decreased, whereas the pro- costimulatory molecules compared with the BALB/c strain. portions of CD44low CD62L2 effector and CD44high CD62L2 To test whether the expression of these costimulatory molecules memory T cells increased in B6SKG mice compared with B6WT is involved in Tfh differentiation, we treated B6SKG mice with mice (Fig. 4E) because lymphopenia and homeostatic prolif- Abs blocking CD80/86 (CTLA4-Ig), ICOSL, and OX40L. The eration drives their differentiation into the effector/memory proportions of Tfh and GC B cells were significantly decreased phenotype without exogenous Ag exposure. These developmental after CTLA4-Ig or anti-ICOSL treatment (Fig. 5E, 5F). This result defects in thymocytes, CD4 T cell lymphopenia, and spontaneous suggested that interaction with these costimulatory molecules differentiation into effector/memory phenotype were comparable in plays an important role in the expansion of the Tfh population in SKG mice with the BALB/c background (Supplemental Fig. 3). B6SKG mice. Among the CD4 Th cell subsets, the proportion of Th17 cells was Finally, we examined whether the inhibition of CD80/86 by higher in B6SKG than that in B6WT mice (Fig. 4F, 4G), similar to CTLA4-Ig could reduce Tfh and GC development and subse- findings for SKG mice (11). The proportion of Th1 cells was also quently inhibit immune complex deposition in the kidney in B6SKG higher in B6SKG mice than that in B6WT mice, with no difference mice. After repetitive treatment with CTLA4-Ig, IgG and C3 between SKG and BALB/c mice (Fig. 4F, 4G), consistent with the deposition in the glomeruli of kidney sections was significantly known Th1 bias in the B6 strain (19). The proportion of Foxp3+ reduced, suggesting that these costimulatory molecules are indeed Tregs was higher in B6SKG mice than that in B6WT mice (Fig. 4F, involved in Tfh expansion and the development of lupus-like 4G), similar to the previously reported increase in Tregs in SKG nephritis in B6SKG mice (Fig. 5G, 5H). mice compared with BALB/c mice (10). Interestingly, among the Downloaded from Foxp3-positive cells, the proportion of CD252 Foxp3+ CD4 T cells Discussion was markedly increased in B6SKG mice (Fig. 4F). In this study, we demonstrated that a defect in TCR-proximal We also examined the proportion of CXCR5+ Bcl-6+ Foxp3+ signaling caused by a Zap70 mutation specifically predisposes follicular Tregs (Tfr), which suppress Tfh activity in the GC (30–32). mice to the development of both arthritis and lupus, depending on The proportion of Tfr among CD4 T cells was increased in B6SKG the genetic background. In the BALB/c background, the Zap70

mice, whereas the Tfr/Tfh ratio was not significantly different mutation causes a Th17-dependent form of autoimmune arthritis http://www.jimmunol.org/ between B6SKG and B6WT mice (Fig. 4H). Interestingly, (11, 12), whereas in the B6 background, the same mutation causes B6SKG mice had increased more CD252 Tfr and fewer CD25+ lupus-like systemic autoimmune disease with an expansion of the Tfr compared with B6WT mice (Fig. 4I). Tfh population. Although the mechanism that leads to the pref- Collectively, these findings show that B6SKG mice exhibit erential development of lupus in the B6 background may be high abnormal thymic T cell development, CD4 T cell lymphopenia, and multifactorial, the increased number of CD11c DCs with spontaneous differentiation into effector and memory CD4 T cells, higher expression of costimulatory molecules in the B6 strain including Th1 and Th17 cells, along with an increase in Tregs. In might be one factor that has facilitated the differentiation of Tfh addition, the loss of CD25 in the subpopulations of Tregs and Tfr is and the development of lupus-like systemic autoimmune disease a characteristic feature of B6SKG mice. in B6SKG mice. These results suggest that the pathogenic effect by guest on September 25, 2021 of a defect in TCR-proximal signaling depends on the complex Factors facilitating the strain-specific development of Tfh interaction with genetic and environmental factors, which may To gain insights into the molecular mechanisms that facilitated the determine the ultimate manifestation of the disease. strain-specific expansion of the Tfh population in B6SKG mice, we Although the disease in B6SKG mice is less severe than that compared the total number, cytokine production, and costimulatory observed in the classical models such as NZB/W F1 or MRL/lpr expression in DCs and B cells between wild-type BALB/c mice, it is more akin to the lupus-like diseases that occur in several andB6mice,whichhavebeenreportedtoregulateTfhde- other genetically engineered mice, such as Pdcd1 knockout mice velopment (33). The proportion of CD11chigh DCs in the spleen or Bcl2 transgenic mice, in which significant early mortality or was significantly higher in B6WT mice than in BALB/c mice overt proteinuria has not been reported (14, 16). One of the main (Fig. 5A), whereas Il-6 mRNA expression in DCs was not sig- characteristics of B6SKG mice is the spontaneous development nificantly different between B6WT and BALB/c mice (Fig. 5B). of Tfh and the GC, which has also been reported in other lupus Notably, the expression levels of the costimulatory molecules animal models (34, 35). An increase in the number of Tfh-like CD80/86 and IOCSL but not OX40L were significantly higher in cells in the peripheral blood has also been reported in human the DCs of B6WT mice than in those of BALB/c mice (Fig. 5C, SLE patients. Because defects in TCR-proximal signaling and Supplemental Fig. 4), consistent with the reported upregulation of Tfh expansion are also common findings in human SLE patients CD86 in B6 DCs (22). In contrast, the expression levels of these (36), B6SKG is a suitable model to study how TCR signaling costimulatory molecules in B cells were not different between defects contribute to the development of lupus. BALB/c and B6WT mice (Fig. 5D). The number of DCs, Il-6 A critical question remaining is why lupus-like systemic auto- mRNA expression levels in DCs, and costimulatory molecule immune disease developed only in B6SKG mice and not in SKG expression levels in DCs and B cells were also compared be- mice with the BALB/c background. The strain-specific develop- tween mice with wild-type Zap70 and mutated Zap70 mice in a ment of lupus in the B6 strain but not in the BALB/c strain has BALB/c and B6 background (Supplemental Fig. 4). The B6 mouse been observed in other lupus animal models (13–17). Thus, the B6

on day 3. (D) Flow cytometric analysis of CD4 and CD8 on lymphocytes in the spleen. (E) Flow cytometric analysis of CD44 and CD62L gated on CD3+ CD4+ T cells in the spleen. (F) Flow cytometric analysis of IL-17 and IFN-g gated on CD4 T cells (upper). Flow cytometric analysis of CD25 and Foxp3 gated on CD4 T cells (lower). (G) Proportion of Th1, Th17, and Tregs (n = 5 each). (H) Proportion of CXCR5 and Bcl6 coexpressing Foxp3+ cells (Tfr) in the spleen. The proportion of Tfr (CXCR5+ Bcl6+ Foxp3+) among CD4 T cells and ratio of Tfr (CD4+ CXCR5+ Bcl6+ Foxp3+) to Tfh (CD4+ CXCR5+ Bcl6+) are shown (n = 5 each). (I) Proportion of CD25+ Tfr and CD252 Tfr (n = 5 each). Flow cytometric data (D, E, and F) are representative of at least three independent experiments. **p , 0.01 versus control mice, Student t test. The Journal of Immunology 3169 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 5. Factors facilitating the strain-specific expansion of Tfh. (A) Proportion of CD11chigh DCs in the spleen (n = 5 each). (B) Il-6 mRNA expression of splenic CD11c DCs (n = 3 each). (C) Histograms of CD80, CD86, ICOSL, and OX40L expression in CD11chigh DCs. BALB/c (blue-filled histogram), B6WT (pink- filled histogram), and isotype control (gray line). (D) Histograms of CD80, CD86, ICOSL, and OX40L expression in B cells. BALB/c (blue-filled histogram), B6WT (pink-filled histogram), and isotype control (gray line). (E) Flow cytometric analysis of CXCR5+ Bcl6+ Tfh and GL7+ FAS+ GC (Figure legend continues) 3170 STRAIN-SPECIFIC LUPUS-LIKE SYSTEMIC AUTOIMMUNITY strain, which is normally considered to be disease-free and in- may facilitate the development of Tfh by enhancing TCR sig- nocuous in genetic analyses, may nevertheless harbor certain loci naling and compensating for defective TCR signaling caused by that predispose mice to the development of systemic autoimmune the Zap70 mutation. disease. The causes for these strain differences in the suscepti- Alteration of TCR signaling by a Zap70 mutation may not only bility to lupus have been studied, and some candidates have been affect Tfh but also the differentiation and function of Tregs. reported, including receptor editing of the Ig L chains (18), bias Agonistic TCR signaling is required for Treg differentiation in to a Th1 response (19), or increased type-1 IFN in B6 mice (20). the thymus and their suppressive function in the periphery (45). One major reason for the difference in the disease manifestation In SKG mice, the number of Tregs is increased irrespective of between B6SKG and SKG mice might be the difference in MHC genetic background, although their suppressive function is im- class II molecules and repertoire selection because SKG mice paired due to defective TCR signaling (10). Interestingly, sub- with a C57BL/6 MHC H2-Aq (B6.Q) background develop ar- populations of Tregs and Tfrs in B6SKG mice lost the expression thritis but not lupus, although the production of anti-DNA Abs or of the typical Treg marker CD25. We previously reported that a immune complex deposition in tissues was not examined (29). In subpopulation of Tfr localized in the GC lost CD25 expression but addition to these factors, we suggest that the increased number retained similar suppressive function to CD25+ Tfr (32), whereas of CD11chigh DCs with higher expression of the costimulatory another report suggested that CD252 Tregs tended to lose Foxp3 molecules CD80, CD86, and ICOSL in the B6 strain might be expression and trans-differentiated into Tfh (46). It remains to be one of the causes that facilitated the differentiation of Tfh and determined how these alterations in Treg phenotypes in B6SKG the development of lupus-like systemic autoimmune disease in mice contribute to the development of lupus-like autoimmune

B6SKG mice. disease. Downloaded from Differentiation of Tfh is a multistage, multifactorial process In addition to SKG mice, other of TCR-proximal characterized by initial priming by the interaction with DCs and signaling such as mutations of Zap70 or the CD3z chain lead to further commitment by the interaction with B cells (33). Tfh a variety of phenotypes (11, 47, 48). Partial attenuation of TCR differentiation is determined by the strength of TCR signaling, signaling by Zap70 mutations leads to autoimmune disease de- the specific cytokines milieu (including IL-6 and IL-21), and the velopment or autoantibody production, depending on the mutation

expression level of costimulatory molecules that interact with type and the genetic background of the mice (47, 48), whereas http://www.jimmunol.org/ TCR signaling (33). Although IL-6 and IL-21 contribute to Tfh Zap70 deficiency leads to a lack of mature CD4 T cells and im- differentiation, their roles are redundant because both Il6 and munodeficiency. In CD3z knockout mice (Cd2472/2), TCR sig- Il21 knockout mice show normal Tfh or GC development (37). naling is severely impaired but not completely inhibited because By contrast, both Cd28 and Icos knockout mice fail to develop the signal can be transmitted via a CD3 complex other than that Tfh or the GC (38, 39), suggesting the critical importance of of the z family (49). Interestingly, CD3z knockout mice develop costimulatory molecules for the development of Tfh. The inter- multiorgan inflammation accompanied by Th1 infiltration but fail action of CD80/86 with DCs is required for the initial priming to develop autoantibody production (50). Thus, there appears to be of Tfh as well as other helper CD4 T cell subsets (38, 40). The a distinct threshold for the attenuation of TCR signaling to de- ICOS2ICOSL interaction at the time of DC priming is required velop a systemic autoimmune disease characterized by autoanti- by guest on September 25, 2021 for the expression of Bcl6 on CD4 T cells, and the ICOS2ICOSL body production. Although TCR signaling should be defective interaction with B cells further upregulates Bcl6 expression (39, within a certain range for self-reactive T cells to escape thymic 41, 42). Although Ox40 knockout mice show normal Tfh devel- negative selection, a certain strength of TCR signaling is required opment, the OX40–OX40L interaction induces CXCR5 expression for their differentiation into Tfh in the periphery. Partially and is involved in CD4 T cell migration to the T–B cell border impaired TCR signal transduction that meets these central and after priming (43). Thus, costimulatory molecules are critically peripheral activation thresholds may provoke the final mani- important for the development of Tfh, and multiple signals acting festation of systemic autoimmune disease characterized by in concert, including CD80/86, ICOSL, and OX40L, at the time of Tfh and GC development. DC priming are required for the initiation of Tfh differentiation, Recent -wide linkage analyses revealed that multiple consistent with previous reports (33). genes in the TCR signaling pathway are associated with a wide In SKG mice, a TCR signaling defect alters the thymic selection variety of autoimmune diseases, including lupus. Among them, thresholds, and T cells that strongly react with self-antigens are not a polymorphism in protein tyrosine phosphatase-22 (PTPN22) eliminated in the thymus and thus escape into the periphery (8). is known to be one of the strongest risk factors associated with However, the activation and proliferation of these self-reactive various autoimmune diseases, including SLE, RA, type 1 diabetes, T cells upon TCR stimulation is severely impaired because of and autoimmune thyroiditis (51). PTPN22 encodes the TCR- the equal decrease in TCR signaling due to the Zap70 mutation. associated protein tyrosine kinases and potently suppresses Unlike other Th cell subsets, Tfh differentiation requires the TCR signaling through the dephosphorylation of Lck, Fyn, or strongest level of TCR signaling and continuous interaction with ZAP70. However, it remains controversial as to whether the DCs (44). Therefore, T cells with a defective TCR signaling disease-associated PTPN22 polymorphisms reduce or enhance molecule due to a Zap70 mutation are less likely to differentiate TCR signaling (51). Several reports suggest that the PTPN22 into Tfh than other Th cell subsets. In this regard, increased ex- W620 mutation is a gain-of-function mutation that reduces pression levels of costimulatory molecules in a B6 background TCR-proximal signaling, whereas other reports suggest that this is

B cells after the inhibition of costimulatory molecules after 2-wk treatment with anti-OX40L, anti-ICOSL, and CTLA-4 Ig (up to 500 mg/body for 2 wk). (F) Proportion of Tfh among CD4 T cells and proportion of GC B cells among B220+ AA4.12 mature B cells after 2-wk treatment with anti-OX40L, anti-ICOSL, andCTLA-4Ig(upto500mg/body, for 2 wk) (n = 5 each). (G) Immunohistochemistry of IgG and C3. Kidney sections of B6SKG mice with or without CTLA4-Ig treatment were stained with IgG and C3. Scale bar, 20 mm(n = 4 each). (H) Area intensity of IgG and C3 fluorescence in the kidney of B6SKG mice. The integrated brightness of IgG and C3 deposition was analyzed by an all-in-one fluorescence microscope (BZ-700; KEYENCE). Five glomeruli per mouse were analyzed. Flow cytometric data (C, D,andE) are representative of at least three independent experiments. **p , 0.01 versus control mice, Student t test. The Journal of Immunology 3171 a loss-of-function mutation that causes hyperresponsive TCR sig- responses and elicits autoimmune disease. Proc. Natl. Acad. Sci. USA naling (51–53). Defective TCR signaling in the thymus may help 88: 8661–8665. 17. Kuo, P., M. Bynoe, and B. Diamond. 1999. Crossreactive B cells are present the survival of self-reactive T cells in the periphery (8), whereas during a primary but not secondary response in BALB/c mice expressing a bcl-2 enhanced TCR signaling in the periphery may help their differ- transgene. Mol. Immunol. 36: 471–479. 18. Fukuyama, H., F. Nimmerjahn, and J. V. Ravetch. 2005. The inhibitory entiation into Tfh (53). Thus, the effect of the alteration in TCR Fcgamma receptor modulates autoimmunity by limiting the accumulation of signaling for the development of systemic autoimmunity should immunoglobulin G+ anti-DNA plasma cells. Nat. Immunol. 6: 99–106. be studied in further detail, considering both its influence in the 19. Segal, R., B. L. Bermas, M. Dayan, F. Kalush, G. M. Shearer, and E. Mozes. 1997. Kinetics of cytokine production in experimental systemic lupus eryth- thymus and in the periphery. ematosus: involvement of T helper cell 1/T helper cell 2-type cytokines in dis- In conclusion, we established a novel animal model of SLE with ease. J. Immunol. 158: 3009–3016. a defect in TCR-proximal signaling, which develops lupus-like 20. De Maeyer-Guignard, J., F. Dandoy, D. W. Bailey, and E. De Maeyer. 1986. Interferon structural genes do not participate in quantitative regulation of in- systemic autoimmune disease in a strain-specific manner. The terferon production by if loci as shown in C57BL/6 mice that are congenic with model will be useful to study the pathogenesis of SLE and to BALB/c mice at the alpha interferon gene cluster. J. Virol. 58: 743–747. determine how alterations in TCR-proximal signaling contribute 21. Lauwerys, B. R., J. Ducreux, and F. A. Houssiau. 2014. Type I interferon blockade in systemic lupus erythematosus: where do we stand? Rheumatology to the development of systemic autoimmune disease under the (Oxford) 53: 1369–1376. influence of genetic and environmental factors. 22. Liu, T., T. Matsuguchi, N. Tsuboi, T. Yajima, and Y. Yoshikai. 2002. Differences in expression of toll-like receptors and their reactivities in dendritic cells in BALB/c and C57BL/6 mice. Infect. Immun. 70: 6638–6645. Acknowledgments 23. Yoshitomi, H., N. Sakaguchi, K. Kobayashi, G. D. Brown, T. Tagami, We thank Atsuko Tamamoto and Sumie Nakagawa for technical assistance. T. Sakihama, K. Hirota, S. Tanaka, T. Nomura, I. Miki, et al. 2005. A role for

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