Constitutive OX40/OX40 Ligand Interaction Induces Autoimmune-Like Diseases Kazuko Murata, Masato Nose, Lishomwa C. Ndhlovu, Takayuki Sato, Kazuo Sugamura and Naoto Ishii This information is current as of October 4, 2021. J Immunol 2002; 169:4628-4636; ; doi: 10.4049/jimmunol.169.8.4628 http://www.jimmunol.org/content/169/8/4628 Downloaded from References This article cites 56 articles, 24 of which you can access for free at: http://www.jimmunol.org/content/169/8/4628.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 © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Constitutive OX40/OX40 Ligand Interaction Induces Autoimmune-Like Diseases1

Kazuko Murata,*† Masato Nose,‡ Lishomwa C. Ndhlovu,* Takayuki Sato,* Kazuo Sugamura,*† and Naoto Ishii2*

The interaction between OX40 and OX40 ligand (OX40L) is suggested to provide T cells with an effective costimulatory signals during T cell-APC interaction. To examine the in vivo effect of constitutive OX40/OX40L interaction during immune regulation, we report the establishment of OX40L-transgenic (OX40L-Tg) mice that constitutively express OX40L on T cells. Markedly elevated numbers of effector memory CD4؉ T cells, but not CD8؉ T cells, were observed in the secondary lymphoid organs of OX40L-Tg mice. Upon immunization with keyhole limpet hemocyanin in the absence of adjuvant, profound T cell proliferative responses and cytokine productions were seen in the OX40L-Tg mice as compared with wild-type mice. Furthermore, in -OX40L-Tg mice administrated with superantigen, this constitutive OX40/OX40L interaction on CD4؉ T cells completely pre vented normal in vivo clonal T cell deletion. Interestingly, OX40L-Tg mice on the C57BL/6 background spontaneously developed Downloaded from interstitial pneumonia and inflammatory bowel disease that was accompanied with a significant production of anti-DNA Ab in the sera. Surprisingly, these diseases were not evident on the OX40L-Tg mice on the BALB/c strain. However, such inflammatory diseases were successfully reproducible in recombination-activating gene (RAG)2-deficient mice upon transfer of OX40L-Tg -CD4؉ T cells. Blockade of OX40/OX40L interaction in the recipient RAG2-deficient mice completely prevented disease develop ment. The present results orchestrated in this study indicate that OX40/OX40L interaction may be a vital link in our under- standing of T cell-mediated organ-specific autoimmunity. The Journal of Immunology, 2002, 169: 4628–4636. http://www.jimmunol.org/

ull activation of naive T cells requires not only the inter- to be a T cell activation marker (18, 19). We and others indepen- action between the TCR and Ag/MHC complexes, but also dently generated OX40L-deficient mice, in which significant im- F costimulation provided by accessory molecules expressed pairment of APC functions, leading to reduction in T cell prolif- on APCs (1–4). In addition to the well-known accessory mole- erative responses and production of both Th1 and Th2 cytokines, cules, CD80 and CD86, both of which bind to CD28 on T cells, were revealed (16, 20). An important avenue in which OX40/ several members of the TNF superfamily, including OX40 ligand OX40L may participate is in regulating the development and sur- (OX40L),3 CD70, 4-1BBL, and LIGHT, have been shown to in- ϩ

vival of memory CD4 T cells (21–23). Recently, we have shown by guest on October 4, 2021 duce costimulatory signals upon binding to their cognate receptors that after adoptive transfer of encephalitogenic wild-type (WT) T on T cells (5, 6), indicating that the TNF/TNFR superfamily mem- cells to OX40L-deficient mice, the mice were apparently unable to bers play major costimulatory roles in the T cell-APC interaction. sustain disease progression despite showing comparable onset and OX40L, a molecule that we originally identified as human gp34 severity of disease to WT recipient mice (24). Furthermore, OX40 whose expression is induced by the tax gene of human T cell stimulation has been reported to prevent peripheral tolerance of leukemia virus (HTLV)-I (7), is expressed on activated T cells ϩ CD4 T cells (25), suggesting a possible involvement of the (7–11), activated B cells (12, 13), dendritic cells (14–16), and OX40/OX40L system in regulating autoimmunity. Indeed, expres- endothelial cells (17), while its receptor, OX40, is primarily known sion of OX40 and/or OX40L has been demonstrated in the tissues of several immune disorders such as experimental allergic enceph- *Department of Microbiology and Immunology, Tohoku University Graduate School alitis (24, 26–28), experimental inflammatory bowel diseases of Medicine, Sendai, Japan; †Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Kanagawa, Japan; and ‡Department of (IBDs) (29, 30), graft-vs-host disease (31–33), human proliferative Pathology, Ehime University School of Medicine, Ehime, Japan lupus nephritis (34), rheumatoid arthritis (35, 36), human IBD (37, Received for publication June 14, 2002. Accepted for publication August 12, 2002. 38), human inflammatory muscle diseases (39), and in thymoma of The costs of publication of this article were defrayed in part by the payment of page patients with myasthenia gravis (40). In vivo blockade of OX40/ charges. This article must therefore be hereby marked advertisement in accordance OX40L interaction has been described not only to suppress ongo- with 18 U.S.C. Section 1734 solely to indicate this fact. ing experimental allergic encephalitis (28) and graft-vs-host dis- 1 This work was supported in part by Core Research for Evolutional Science and Technology of the Japan Science and Technology Corporation (Japan Science and ease (32, 41), but also to ameliorate ongoing colitis in murine Technology Corporation), a grant-in-aid for scientific research on priority areas from models of IBD (29, 30), asthma (42), and collagen-induced arthri- the Ministry of Education, Science, Sports, and Culture of Japan, and a grant-in-aid for scientific research on priority areas from the Japan Society for the Promotion of tis (35). These data suggest that OX40/OX40L interaction may Science. play a definitive role in the immune regulation of various immune- 2 Address correspondence and reprint requests to Dr. Naoto Ishii, Department of related diseases. Microbiology and Immunology, Tohoku University Graduate School of Medicine, To evaluate the influence of constitutive interaction between OX40 2-1 Seiryo-machi, Aobu-ku, Sendai 980-8575, Japan. E-mail address: ishiin@ mail.cc.tohoku.ac.jp and OX40L during immune regulation, we constructed mice stably 3 Abbreviations used in this paper: OX40L, OX40 ligand; OX40L-Tg, OX40L-trans- expressing OX40L on T cells, in which sustained OX40 signaling can genic; IBD, inflammatory bowel disease; RAG, recombination-activating gene; KLH, occur. In this paper, we demonstrate convincing evidence showing the keyhole limpet hemocyanin; SEB, S. aureus enterotoxin B; HTLV, human T cell leukemia virus; CD62L, L-selectin; AICD, activation-induced cell death; WT, wild direct involvement of OX40/OX40L interaction in the development type. of autoimmunity.

Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00 The Journal of Immunology 4629

Materials and Methods or WT mice were added to 96-wells coated with either goat anti-mouse Ig Generation of OX40L-transgenic (OX40L-Tg) mice Ab (Southern Biotechnology Associates, Birmingham, Al), salmon sperm dsDNA (Sigma-Aldrich, St. Louis, MO), or ssDNA and incubated for 2 h The transgene was generated by inserting the cDNA encoding mouse at room temperature. The ssDNA was prepared by boiling salmon sperm OX40L into the T lineage-specific vector p1017 (43) that carries the mouse dsDNA for 5 min. After washing, bound Abs were detected by incubation lck proximal promoter. The vector was released as a NotI fragment en- with either goat anti-mouse IgM, IgG1, IgG2a, IgG2b, IgG3, or IgA con- compassing the tissue-specific transcription unit. The fragment, purified by jugated to alkaline-phosphatase (Southern Biotechnology Associates). standard protocols, was microinjected into the pronuclei of (C57BL/6 ϫ Color reactions using alkaline-phosphatase substrate (Sigma-Aldrich) were

DBA/2)F1 fertilized eggs. We obtained three independent founders, in then evaluated by reading OD450. This was demonstrated to be well within which the presence of the transgene was confirmed by PCR. The founders the linear part of the titration curve. were then mated with C57BL/6 mice to generate OX40L-Tg offspring, and germline transmission was verified either by PCR with DNA from tail Histopathological analysis biopsy specimens or by flow cytometric analysis with peripheral blood Animals were sacrificed at the indicated age. The main organs and tissues from the tail vein. The OX40L-Tg mice were backcrossed at least 12 times that included the heart, lung, aorta, liver, pancreas, submandibular glands, onto the C57BL/6 and 9 times onto the BALB/c backgrounds. stomach, small and large intestines, spleen, thymus, cervical lymph nodes, Mice tibial bones, kidneys, and ankle joints were processed for histopathological examination. Tissues were next fixed in 10% formalin in 0.001 M phos- OX40L-deficient mice were described previously (16) and had been back- phate buffer (pH 7.2) and embedded in paraffin (Wako Pure Chemical, crossed onto the C57BL/6 strains at least 12 times. Age and sex-matched Osaka, Japan). In the case of tibial bones and ankle joints, these tissues WT littermates of the OX40L-Tg mice or OX40L-deficient mice were used were decalcified in 10% formic acid. Two- to 4-␮m thick sections were as control mice. Recombination-activating gene (RAG)2-deficient mice on prepared and stained with H&E using standard techniques and examined the C57BL/6 background, lacking B and T cells, were obtained from the by light microscopy. Central Institute for Experimental Animals (Kawasaki, Japan). The appro- Adoptive transfer of CD4ϩ T cells Downloaded from priate strain combinations were taken into account in all experiments. All the mice were kept under specific pathogen-free conditions. Single-cell suspension was prepared from the spleens of the OX40L-Tg or WT littermates, and incubated with mouse CD4 MicroBeads (Miltenyi FACS analysis Biotec) for 20 min at 4°C and subjected to an autoMACS (Miltenyi Bio- ϩ Ͼ Preincubation with normal rat serum was conducted to prevent labeled tec). The purified WT or OX40L-Tg CD4 T cells (purity 99%) were i.v. ϫ 6 mAbs from nonspecific association. The cells were incubated with labeled injected (1–5 10 ) into the syngenic RAG2-deficient mice. Between 2 mAbs for 30 min at 4°C. The samples were then washed with PBS and and 5 wk after the transfer, some recipient mice were sacrificed, and se- http://www.jimmunol.org/ analyzed with a FACSCalibur flow cytometer (BD Immunocytometry Sys- lected tissues were extracted for histopathological analysis. The body tems, Mountain View, CA). The analyses were conducted using the weights of the recipient mice were monitored after inoculation. CellQuest program (BD Immunocytometry Systems). CD3, CD8, CD4, CD44, and L-selectin (CD62L) were obtained from BD PharMingen (San Results Diego, CA). MGP34 mAb specific for mouse OX40L was described pre- Generation of OX40L-Tg mice viously (16). The cells labeled with the biotinylated mAb were visualized by streptavidin-allophycocyanin (BD PharMingen, San Diego, CA), and Three independent OX40L-Tg mouse lines were generated by us- subjected to flow cytometric analysis. Cells, which were preincubated with ing the T cell-specific p1017 expression vector that carries the unlabeled MGP34 to abolish any specific staining by the biotinylated mouse lck proximal promoter (43). Flow cytometric analysis of MGP34, were used as a negative control for OX40L expression.

whole thymocyte populations derived from the three OX40L-Tg by guest on October 4, 2021 In vivo T cell priming and recall responses to protein Ags lines, but not from the WT mice, expressed OX40L (Fig. 1). Two of the three transgenic lines (OX40L-Tg1 and OX40L-Tg2) ex- OX40L-Tg mice or WT littermates were immunized with 100 ␮g keyhole limpet hemocyanin (KLH) in the presence of CFA to each hind footpad. hibited appreciable expression of OX40L in splenic T cells, Alternatively, these mice were i.p. immunized with 1 mg KLH in PBS in whereas the OX40L-Tg3 line showed comparable OX40L expres- the absence of adjuvant. Fourteen to 30 days afterwards, CD4ϩ T cells sion to WT splenic T cells (Fig. 1). Furthermore, the level of 5 (1 ϫ 10 , purity Ͼ98%) purified from the popliteal lymph node or spleen OX40L expression on OX40L-Tg1 splenic T cells was three times using autoMACS (Miltenyi Biotec, Bergisch Gladbach, Germany) were higher than that on OX40L-Tg2 splenic T cells (Fig. 1). Founders incubated with the indicated dose of KLH in the presence of APCs (5 ϫ 5 ϫ 10 )at37°C for 48 h. The APCs used were irradiated (3000 rad) spleno- of the OX40L-Tg mice on the (C57BL/6 DBA/2)F1 strain de- cytes derived from unimmunized WT littermates. The cultured cells were veloped normally and exhibited no readily detectable abnormali- assayed for [3H]thymidine uptake and cytokine production as described ties. However, when these mice were back-crossed onto the previously (16). For cytokine production, culture supernatants were col- C57BL/6 strain, we noticed weight loss accompanied with diarrhea lected at 48 h for IL-2 and IL-4, or at 72 h for IL-5, IL-10, Il-13, and IFN-␥, and subjected to ELISA. in the OX40L-Tg1 and -Tg2 mice. ϩ ELISA Enhanced number of memory CD4 T cells in OX40L-Tg mice Cytokine levels in tissue culture supernatants or in mice sera were assayed On gross examination of the lymphoid organs of OX40L-Tg1 and using ELISA kits for IL-2, IL-4, IL-5, IL-10, IFN-␥ (BD PharMingen), and OX40L-Tg2 mice, massive splenomegaly and marked lymphade- IL-13 (R&D Systems, Minneapolis, MN) according to the manufacturer’s nopathy were observed. All other organs appeared normal includ- recommendations. ing the thymus when compared with WT littermates. We next de- In vivo clonal T cell deletion induced by Staphylococcus aureus termined by flow cytometry whether the OX40L transgene in mice enterotoxin B (SEB) injection resulted in any intrinsic changes in the overall composition of the lymphocyte populations in the thymus, spleen, and lymph nodes. A total of 100 ␮g of SEB (Toxin Technology, Sarasota, FL) was injected i.p. into 8- to 10-wk-old mice. Peripheral blood from the tail vein was All three OX40L-Tg lines did not display any apparent abnormal- analyzed by flow cytometry on the indicated days. Cells were stained with ity in the numbers or subpopulations of T cells in the thymus as CD4-PE in combination with anti-V␤8-FITC (BD PharMingen) or anti- assessed by CD4ϩ and CD8ϩ expression (data not shown). How- ϩ ϩ ϩ ϩ V␤6-FITC (BD PharMingen). The ratios of V␤8 CD4 and V␤6 CD4 ever, the total number of lymphocytes in the spleen (Fig. 2A) and cell populations to whole CD4ϩ cell populations in the peripheral blood were monitored. lymph nodes (data not shown) were significantly increased in OX40L-Tg1 and OX40L-Tg2 mice as compared with the WT lit- ELISA of serum Igs termates. More precisely, a 2-fold increase of splenic CD4ϩ T cells ϩ The levels of the different Ig subclasses in the various mice sera were but not CD8 T cells was detected in these two OX40L-Tg lines ϩ ϩ assayed as previously described (16). In brief, diluted sera from OX40L-Tg at 7 wk of age (Fig. 2A), while the numbers of splenic IgM B220 4630 OX40L EXPRESSION INDUCES AUTOIMMUNE-LIKE DISEASES

OX40L-Tg1 and -Tg2 mice were found (data not shown), indicat- ing the absence of oncogenic features. Because the interaction between OX40 and OX40L is reportedly implicated in the generation or survival of the memory CD4ϩ T cell pool (21–23), we speculated that the elevated CD4ϩ T cell numbers observed in the OX40L-Tg mice mainly reflect an in- crease of memory CD4ϩ T cells. Thus, the phenotypic effector memory CD4ϩ T cell population (CD62LlowCD44high) was next evaluated by flow cytometric analysis in the OX40L-Tg mice. As predicted, a marked increase in the number of splenic effector memory CD4ϩ T cells in the OX40L-Tg1 mice was evident when compared with the WT mice at 4 wk after birth. This increase was age-dependent up to at least 7 wk of age (Fig. 2B). Similarly, this remarkable increase in the memory CD4ϩ T cell population was observed in the OX40L-Tg2 mice; however, this was not evident in the OX40L-Tg3 mice (data not shown). Conversely, a signifi- cant reduction in this population of CD4ϩ T cells was evident in the OX40L-deficient mice as compared with the WT mice (Fig. 2B). The results thus far reveal that OX40L-Tg mice harbor a Downloaded from markedly elevated memory T cell subpopulation, illustrating the importance of OX40/OX40L interaction in the generation or/and maintenance of memory CD4ϩ T cells in vivo.

FIGURE 1. Flow cytometric analysis of three independent lines of Markedly enhanced Ag-specific responses of OX40L-Tg memory http://www.jimmunol.org/ OX40L-Tg mice. Thymocytes from WT or each OX40L-Tg mice were stained CD4ϩ T cells with biotinylated anti-OX40L (MGP34) and visualized by streptavidin-allo- To address the mechanism responsible for the elevated phenotypic phycocyanin. Alternatively, splenocytes from WT or each OX40L-Tg mice ϩ were double-stained with FITC-labeled anti-CD3 mAb and biotinylated anti- memory CD4 T cell population seen of the OX40L-Tg1 and -Tg2 OX40L followed by allophycocyanin-labeled streptavidin. CD3ϩ T cells were mice, we conducted a series of Ag-specific recall proliferation as- separately gated and allophycocyanin staining was analyzed with a FACS- says of CD4ϩ T cells. OX40L-Tg CD4ϩ T cells had a markedly Calibur. Thick lines indicate biotinylated anti-OX40L staining. Dotted lines enhanced proliferative recall response to KLH (Fig. 3A), while indicate background control staining. The individual mouse used in each ge- productions of IL-2, IFN-␥, IL-4, and IL-5 were also significantly notypic group is representative of at least 10 other mice from each genotypic ϩ enhanced when compared with the WT CD4 T cells (Fig. 3B). by guest on October 4, 2021 group in which similar results were obtained. However, secretion of the regulatory cytokine, IL-10, by OX40L-Tg CD4ϩ T cells was reduced in relation to WT CD4ϩ T B cells in all the three OX40L-Tg lines were comparable to WT cells (Fig. 3B). Next, mice were administrated with KLH in the littermates (data not shown). By analyzing the TCR V␤ usage, no absence of adjuvant. As expected, a limited proliferative response skewing of TCR repertoires of the splenic CD4ϩ T cells in both the by the CD4ϩ T cells derived from the WT mice administered with

FIGURE 2. Splenic lymphocyte composition in OX40L-Tg mice. A, The total cell number of splenic lymphocytes and subpopulations of splenic CD4ϩ and CD8ϩ T cells of 7-wk-old OX40L-Tg1 (n ϭ 5, f), OX40L-Tg2 (n ϭ 5, u), OX40L-Tg3 (n ϭ 5, o), and WT mice (n ϭ 5, Ⅺ) were counted and stained p Ͻ 0.005; cf WT mice per group as determined by Student’s ,ءء ;p Ͻ 0.05 ,ء .with PE-conjugated anti-CD4-mAb and FITC-conjugated anti-CD8-mAb t test. B, Populations of splenic memory (CD62LlowCD44high) CD4ϩ T cells in OX40L-Tg1 (n ϭ 5, F), OX40L-deficient (n ϭ 4, f), and WT mice (n ϭ 5, E) were evaluated. Splenocytes from each mouse were counted and stained with CD62L-FITC, CD44-CyChrome, and CD4-PE at the indicated days .p Ͻ 0.005; cf WT mice ,ءء ;p Ͻ 0.05 ,ء .after birth The Journal of Immunology 4631

FIGURE 3. Enhanced CD4ϩ T cell responses to protein Ag KLH. A, Eight-week-old OX40L-Tg1 (n ϭ 3, F) and WT (n ϭ 3, E) littermates were immunized with KLH along with CFA in the footpads. Fourteen days after immunization, CD4ϩ T cells from draining popliteal lymph nodes were purified and subjected to an in vitro challenge of KLH in the presence of APCs. After a 48-h culture, their [3H]thymidine uptakes were measured. B, Culture supernatants of OX40L-Tg CD4ϩ T cells (f)orWTCD4ϩ T cells (Ⅺ) were collected at 48 h for IL-2 and IL-4, or at 72 h for IL-5, IL-10, and IFN-␥, and the cytokines were assayed by ELISA. Sim- ilar results were obtained in three independent experi- ments. Alternatively, OX40L-Tg1 (n ϭ 4, F) and WT (n ϭ 4, E) mice were i.p. immunized with 1 mg KLH in PBS. OX40L-Tg1 (n ϭ 3, f) and WT (n ϭ 3, Ⅺ) mice represent unimmunized controls. Eighteen days after immunization, splenic CD4ϩ T cells were ex- tracted and subjected to an in vitro challenge of KLH in the presence of APCs. C, After culturing for 48 h, their Downloaded from [3H]thymidine uptakes were measured. D, Culture su- pernatants of OX40L-Tg1 CD4ϩ T cells (f)orWT CD4ϩ T cells (Ⅺ) were collected at 48 h for IL-2 and IL-4, or at 72 h for IL-5, IL-10, IL-13, and IFN-␥, and the cytokines were assayed by ELISA. Similar results were obtained in four independent experiments. http://www.jimmunol.org/

either no Ag or soluble KLH was seen (Fig. 3C). In contrast, sol- Tg1 mice apparently had markedly elevated serum levels of all Ig uble KLH administration induced an obvious dose-dependent pro- classes except IgG3 when compared with WT littermates (Fig. liferative response by the CD4ϩ T cells derived from the 5A). Notably, anti-DNA Abs to ssDNA and dsDNA were elevated OX40L-Tg mice (Fig. 3C). Furthermore, productions of IL-4, in the sera of OX40L-Tg1 mice (Fig. 5B). These data suggest that IL-5, IL-10, and IL-13, but not IFN-␥, by OX40L-Tg CD4ϩ T the OX40L-Tg1 mice develop polyclonal activation of B cells ac- by guest on October 4, 2021 cells were readily detectable (Fig. 3D), while no marked produc- companied with production of autoantibodies specific for DNA. tion of these cytokines in the WT CD4ϩ T cells were observed We next attempted to determine whether the serum levels of par- (Fig. 3D). These findings indicate that OX40L-Tg mice are able to ticular cytokines that affect Ig secretion of B cells were altered. generate highly functional Ag-specific memory CD4ϩ T cell re- Serum levels of IL-5 and IL-13 were four and seven times higher, sponses even in the absence of adjuvant. respectively, in the OX40L-Tg1 mice in comparison to the WT

ϩ littermates (Fig. 5C). In contrast, the cytokine levels of IL-2, IL-4, Defective clonal CD4 T cell deletion in OX40L-Tg mice IL-10, and IFN-␥ were undetectable in the sera of both the In view of the extraordinary elevated number and functional ca- OX40L-Tg1 mice and WT littermates (data not shown). Similar pacity of the CD4ϩ memory T cells in the OX40L-Tg mice, we results to the OX40L-Tg1 mice were also seen among the OX40L- examined the in vivo clonal T cell deletion of these mice during Tg2 mice (data not shown). activation-induced cell death (AICD), a well-known mechanism of peripheral T cell tolerance. Because clonal T cell deletion induced by superantigen, such as S. aureus enterotoxin A and SEB, mimics Spontaneous development of inflammatory diseases in T cell AICD (44, 45), we examined whether SEB administered to OX40L-Tg mice ϩ OX40L-Tg mice would induce the deletion of the peripheral V␤8 In view of the weight loss, symptoms of diarrhea, and the detection ϩ ϩ T cell repertoire. Interestingly, the V␤8 CD4 T cell population of elevated serum autoantibody levels in both OX40L-Tg1 and in the OX40L-Tg mice remained markedly elevated up to at least OX40L-Tg2 C57BL/6 mice, we conducted histopathological anal- 14 days after the SEB injection (Fig. 4A), while the WT yses of a broad spectrum of tissues in these mice. Cervical lymph ϩ ϩ V␤8 CD4 T cell population, despite peaking on day-2 postin- node sections from the OX40L-Tg1 mice showed increased num- jection, promptly decreased by day 7 (Fig. 4A). Control bers of plasma cells when compared with WT mice (data not ϩ ϩ V␤6 CD4 peripheral T cell populations from OX40L-Tg and shown). Furthermore, numerous Mott cells containing Russell ϩ WT CD4 T cells, TCRs of which are not recognized by SEB, bodies, intracellular acidophilic deposits often found in mice with ϩ remained unchanged (Fig. 4B). V␤8 T cell deletion induced by autoimmune diseases (46), were visible in sections derived from SEB treatment is thus defective in OX40L-Tg mice, suggesting the OX40L-Tg1 mice (data not shown). that constitutive OX40/OX40L interactions prevents clonal dele- A remarkable appearance of inflammatory cells, consisting of ϩ tion of CD4 T cells during AICD. lymphocytes, neutrophils, and macrophages were seen in the al- veolar septa of lung sections obtained from OX40L-Tg1 mice (Fig. Elevated serum levels of Abs and Th2 cytokines in OX40L-Tg mice 6, B and D). These features were consistent with an acute inter- We next determined the levels of all the Ig isotypes, and of various stitial pneumonia presentation. Infiltration of lymphocytes also Th1 and Th2 cytokines in the sera of OX40L-Tg1 mice. OX40L- into the peribronchiolar regions in OX40L-Tg1 lung sections (Fig. 4632 OX40L EXPRESSION INDUCES AUTOIMMUNE-LIKE DISEASES

FIGURE 4. Defective clonal deletion of CD4ϩ T cells induced by SEB administration. OX40L-Tg (n ϭ 5, F) and WT littermates (n ϭ 5, E) were injected i.p. with SEB on day 0. Peripheral blood cells were stained with anti- V␤8-FITC and anti-CD4-PE (A) or with anti- V␤6-FITC and anti-CD4-PE (B), and ana- lyzed by flow cytometry as indicated. Results p Ͻ ,ء .are expressed as the mean Ϯ SEM 0.01. The present results are representative of three independent experiments. Downloaded from

6B) appeared to closely resemble bronchiolitis. These lung histo- All these histopathological findings appeared 3 mo postnatal in logical features were consistently absent in the WT littermates the OX40L-Tg1 and OX40L-Tg2 and not the OX40L-Tg3 line (Fig. 6, A and C). (Table I). This was a significant finding since OX40L-Tg3 mice In colon sections obtained from OX40L-Tg1 mice, marked mu- have marked expression of OX40L only in thymic and not splenic cosal hypertrophy was evident. This was accompanied with severe tissues (Fig. 1). Therefore, it is likely that overexpression of hyperplasia of the lymphoid tissues in the lamina propria (Fig. 6F), OX40L in the periphery may mediate the pathogenesis of these http://www.jimmunol.org/ all of which were visibly absent in the WT colon sections (Fig. inflammatory diseases. Additional results show that the develop- 6E). Further magnification of the OX40L-Tg1 colon sections re- ment of interstitial pneumonia and IBD is age-dependent with the vealed the presence of a mixed inflammatory cell infiltrate in the incidence of the diseases increasing with age (Table I). Other tis- submucosal regions, consisting of lymphocytes, plasma cells, and sue sections obtained from all the three OX40L-Tg lines, including neutrophilic granulocytes (Fig. 6G). In the mucosal epithelium, heart, lung, aorta, liver, pancreas, submandibular glands, stomach, typical crypt abscesses were occasionally identified in the OX40L- small intestines, tibial bones, kidneys, and ankle joints were his- Tg1 colon sections (Fig. 6H). Taken together, this presentation tologically normal (data not shown). Furthermore, all three

appears to be reminiscent of IBD. OX40L-Tg lines back-crossed onto the BALB/c background up to at by guest on October 4, 2021

FIGURE 5. Elevated levels of serum Abs and Th2 cytokines in OX40L-Tg mice. A, Serum levels of Ig isotypes; B, anti-ssDNA and anti-dsDNA Abs in OX40L-Tg1 (F) and WT (E) mice; and C, IL-5 and IL-13 in OX40L Tg1 (n ϭ 11, thick line) and WT (n ϭ 12, dotted line) littermates were measured .p Ͻ 0.005; cf WT mice ,ءء ;p Ͻ 0.05 ,ء .by ELISA. All mice were 8–12 wk old The Journal of Immunology 4633

mice on the C57BL/6 background are mediated by an autoimmune mechanism.

Organ-specificinflammatory diseases in RAG2-deficient mice reconstituted with OX40L-Tg CD4ϩ T cells Immunohistochemical analysis of the infiltrating lymphocytes in both the peribronchiolar regions of the lung and the intestinal lam- ina propria of the OX40L-Tg1 mice had a CD4ϩ phenotype (data not shown), demonstrating the possible participation of CD4ϩ T cells in the pathogenesis of these organ-specific autoimmune-like diseases. To determine whether OX40L-Tg CD4ϩ T cells mediate the pathogenesis of the diseases seen in the OX40L-Tg mice, we conducted a series of transfer experiments, in which CD4ϩ T cells derived from OX40L-Tg1 or WT mice were transferred i.v. into C57BL/6 RAG2-deficient mice. All the reconstituted RAG2-defi- cient mice displayed signs of sickness that included wasting and diarrhea 8 days after inoculation of OX40L-Tg1 CD4ϩ T cells

(Fig. 7A and data not shown). Between 2 and 5 wk posttransfer, Downloaded from lung histological sections revealed inflammatory features consis- tent with severe interstitial pneumonia (Fig. 7C), while colon sec- tions showed massive infiltration of lymphocytes in the intestinal lamina propria (Fig. 7E). These histological features were repro- duced in RAG2-deficient mice reconstituted with splenic CD4ϩ T

cells derived from OX40L-Tg2 mice (Table I). In contrast, RAG2- http://www.jimmunol.org/ deficient mice inoculated with CD4ϩ T cells from the WT mice had no symptoms of disease (Fig. 7A and data not shown) or his- FIGURE 6. Representative histopathological features of interstitial tological abnormalities in lung or colon tissue sections (Table I and pneumonia and colonic IBD in OX40L-Tg mice. Lung sections (AÐD) and Fig. 7, D and F). Interestingly, administration of an inhibitory ϩ a transverse section of the colon (E) (H&E stain) of a 15-mo-old WT (A, anti-OX40L mAb, MGP34, along with CD4 T cells from C, E) and OX40L-Tg1 (B, D, FÐH) mouse. The arrowhead represents a OX40L-Tg1 mice into RAG2-deficient mice completely prevented crypt abscess (H). Magnifications, approximately ϫ20 (A and B), and the development of the diseases (Table I, Fig. 7, G and H). These ϫ ϫ ϫ ϩ 200 (C and D), 25 (E and F), and 100 (G and H). results not only demonstrate that OX40L-Tg CD4 T cells are the responsible pathogenic effector cells in disease generation, but by guest on October 4, 2021 least 20 mo of age, presented with no symptoms of disease or histo- highlight the critical involvement of OX40/OX40L interaction in logical abnormalities in lung or colonic tissues (Table I), indicating directly mediating the pathogenesis of these diseases. the genetic background specificity in disease development. Although mice were housed under specific pathogen-free con- ditions, to exclude the possibility of pathogenic microorganisms in Discussion the colony as being responsible for the interstitial pneumonia or The present study documents two important findings; first, IBD observed in the OX40L-Tg1 and -Tg2 mice, we conducted OX40L-Tg mice have hyperproliferative CD4ϩ T cells and mark- numerous tests to identify any contagious microorganisms such as edly elevated memory CD4ϩ T cell numbers, probably mediated Pneumocystis carinii and Helicobacter species, among others. No by a disruption in AICD. Secondly, OX40L-Tg mice spontane- known pathogens responsible for interstitial pneumonia or colitis ously develop organ-specific autoimmune-like diseases, namely in the mice were found (data not shown). Taken together, these interstitial pneumonia and colonic IBD, both of which can be re- results strongly suggest that the diseases seen in the OX40L-Tg produced in RAG2-deficient mice reconstituted with pathogenic

Table I. Development of diseases in OX40L-Tg mice

Strain SpecificInflammatory Disease CD4ϩ T Cell Transfer into Recipient RAG2Ϫ/Ϫ Mice

Mice No. of mice with inflammatory diseasea No. of mice with inflammatory diseasea

Age Strain Genotype Interstitial pneumonia IBD Donor mice Treatment Interstitial pneumonia IBD

6–7 wk C57BL/6 WT 0/10 0/10 WT None 0/9 0/9 Tg1 4/10 (40%b) 3/10 (33%b) Tg1 None 15/15 (100%b) 15/15 (100%b) Ͼ3 mo WT 0/10 0/10 Tg2 None 5/5 (100%) 5/5 (100%) Tg1 15/15 (100%) 15/15 (100%) Tg1 Control IgG 13/13 (100%b) 13/13 (100%b) Tg2 9/9 (100%) 9/9 (100%) Tg1 MGP34 (mAb) 0/16 0/16 Tg3 0/9 0/9 BALB/c WT 0/6 0/6 Tg1 0/8 0/8

a No. of mice with histological evidence of disease. b Incidence. 4634 OX40L EXPRESSION INDUCES AUTOIMMUNE-LIKE DISEASES Downloaded from http://www.jimmunol.org/

FIGURE 7. Interstitial pneumonia and IBD in RAG2-deficient mice reconstituted with OX40L-Tg CD4ϩ T cells. Purified splenic CD4ϩ T cells (purity Ͼ99%, 3 ϫ 106) from OX40L-Tg1 or WT littermates on the C57BL/6 background were injected into the tail vein of syngenic RAG2-deficient C57BL/6 mice. Alternatively, 500 ␮g of anti-OX40L (MGP34) mAb or control rat IgG was i.p. injected to the recipient RAG2-deficient mice at day 0, 1, and 4 after T cell transfer. A, Body weights of the RAG2-deficient mice injected with OX40L-Tg1 CD4ϩ T cells (n ϭ 7, F) and WT CD4ϩ T cells (n ϭ 7, E) were monitored and are represented as the average body weight index: (body weight at the indicated days)/(body weight at day 0) Ϯ SD. Similar results were obtained in three independent experiments. B, Similarly, average body weight indices of RAG2-deficient mice injected with OX40L-Tg1 CD4ϩ T cells plus MGP34 (n ϭ 7, f), RAG2-deficient mice injected with OX40L-Tg1 CD4ϩ T cells plus control rat IgG (n ϭ 4, F), and WT CD4ϩ T cells (n ϭ 3, E) were represented. The present results are representative of three independent experiments. H&E-stained sections of the lung (C and D) and colon (E and F)of by guest on October 4, 2021 the reconstituted RAG2-deficient mice, 27 days after transfer of OX40L-Tg1 CD4ϩ T cells (C and E)orWTCD4ϩ T cells (D and F). Lung histological sections of RAG2-deficient mice treated with either control rat Ig (G) or MGP34 (H), 27 days after reconstitution with OX40L-Tg CD4ϩ T cells. Magnifications, approximately ϫ25 (C and D), ϫ60 (E and F), and ϫ50 (G and H).

OX40L-Tg CD4ϩ T cells. Furthermore, administration of an in- nity, defective AICD seen in OX40L-Tg mice may cause a marked hibitory anti-OX40L mAb prevented the development of these dis- elevation of effector memory CD4ϩ T cells and lead to autoim- eases in the reconstituted RAG2-deficient mice. This report dem- mune-like disease development. In addition, a recent report sug- onstrates the first direct evidence linking the OX40/OX40L signaling gested a possible involvement of OX40 signaling in breaking pe- pathway to the development of autoimmune-like diseases. Many ripheral CD4ϩ T cell tolerance (25). However, to further clarify previous reports have suggested that T cell costimulatory molecules the plausible association between the defective AICD, probably (CD28, inducible costimulator, LFA-1, TNFR family molecules in- resulting in a break in peripheral tolerance and the autoimmune- cluding OX40, etc.) may be involved in the development of autoim- like diseases seen in the OX40L-Tg mice, studies using Ag-spe- mune diseases (6, 47–49). However, to our knowledge, no report has cific T cells derived from a TCR transgenic mouse intercrossed to revealed that constitutive costimulatory signaling in T cells lead to an OX40L-Tg mouse are required. autoimmune/allergic diseases. In this context, our present observa- The spontaneous development of autoimmune-like diseases of tions should provide us with important information to understand T the lung and colon in OX40L-Tg mice may provide us with the cell-mediated organ-specific autoimmunity. first evidence demonstrating the direct link of OX40 signaling to OX40 has been reported to be expressed on autoreactive or ef- the development of immune disorder-associated diseases. This as- fector T cells in patients with autoimmune diseases (34, 36, 40) and inflammatory diseases linked to immune disorders (37–39). sociation is strongly supported by our results showing the effec- The OX40/OX40L system may thus play a potential key role in the tiveness of an inhibitory anti-OX40L mAb in completely prevent- ing these diseases in syngenic RAG2-deficient mice reconstituted immune regulation of various autoimmune diseases. One mecha- ϩ nism to limit autoimmunity is by AICD, which has been consid- with pathogenic OX40L-Tg CD4 T cells. Although elevated se- ered to limit accumulation of autoantigen-specific T cells (50). rum autoantibody levels were seen in the OX40L-Tg mice, it is Although a previous report demonstrated that treatment with an clear that B cells are not essential in the pathogenesis of interstitial agonistic anti-OX40 mAb alone was not able to prevent S. aureus pneumonia or IBD, since the reconstituted RAG2-deficient mice enterotoxin A-induced AICD (21), AICD triggered by SEB ad- which lack B cells developed these diseases. However, this finding ministration was markedly impaired in OX40L-Tg mice (Fig. 4A). does not rule out the possibility that autoantibodies might lead to Because AICD plays an important role in regulating autoimmu- autoimmune manifestations in OX40L-Tg mice. The Journal of Immunology 4635

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