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Altered Expression Level of a Systemic Nuclear Autoantigen Determines the Fate of Immune Response to Self This information is current as of June 18, 2017. Kimito Kawahata, Yoshikata Misaki, Yoshinori Komagata, Keigo Setoguchi, Shinji Tsunekawa, Yasuji Yoshikawa, Jun-ichi Miyazaki and Kazuhiko Yamamoto J Immunol 1999; 162:6482-6491; ; http://www.jimmunol.org/content/162/11/6482 Subscription Permissions Email Alerts This article cites 53 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/162/11/6482.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 References Altered Expression Level of a Systemic Nuclear Autoantigen Determines the Fate of Immune Response to Self1 Kimito Kawahata,* Yoshikata Misaki,2* Yoshinori Komagata,*† Keigo Setoguchi,* Shinji Tsunekawa,‡ Yasuji Yoshikawa,§ Jun-ichi Miyazaki,¶ and Kazuhiko Yamamoto* O ne of the prominent features of systemic autoimmune diseases, such as systemic lupus erythematosus (SLE)3 and mixed connective tissue disease (MCTD), is the presence of autoantibodies. The targets of these autoimmune responses are mostly ubiquitous and organ-nonspecific Ags, including nuclear Ags, such as spliceosome components (U1 small nuclear ribonucleoprotein (snRNP)-A, 70K, C, B/B9, etc.) as well as nucleosome components (dsDNA, histones) (1). It has been suggested that these autoantibodies are produced by an Ag-driven mechanism, and that autoantigen-specific T cells are involved in their production. Autoreactive T cells are deleted during development in the thymus. Studies on transgenic (Tg) mice that express a TCR specific for a systemic Ag have confirmed the view that the most efficient way to achieve self tolerance to a systemic self Ag is central deletion (2). However, in recent studies, the autoreactive T cells responding to systemic autoantigens have been demonstrated to be *Department of Allergy and Rheumatology, University of Tokyo Graduate School of Medicine, Tokyo, Japan; †Center for Neurologic Diseases, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02215; ‡Medical and Biological Laboratories, Ina, Japan; §Department of Clinical Laboratory, Medical Institute of Bioregulation, Kyushu University, Beppu, Japan; and ¶Department of Nutrition and Physiological Chemistry, Osaka University Medical School, Suita, Japan Received for publication August 24, 1998. Accepted for publication March 19, 1999. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by grants from the mixed connective tissue disease research group of the Ministry of Health and Welfare, the Ministry of Education of Japan, and Schering-Plough. 2 Address correspondence and reprint requests to Dr. Yoshikata Misaki, Department of Allergy and Rheumatology, University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail address: misaki-tky@ umin.ac.jp 3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; MCTD, mixed connective tissue disease; snRNP, small nuclear ribonucleoprotein; U snRNP, U-type small nuclear ribonucleoprotein complex; HuA, human U1 snRNP-A protein; MuA, murine U1 snRNP-A protein; wt, wild type; B6, C57BL/6; m3G, 2,2,7-trimethylguanosine; LAT, local adoptive transfer; DTH, delayed-type hypersensitivity; Tg, transgenic; LN, lymph node; HEL, hen egg-white lysozyme; MACS, magnetic cell sorting. Copyright © 1999 by The American Association of Immunologists isolated from the peripheral blood not only of patients with autoimmune diseases but also of healthy individuals (3–5). In case of a microbe infection or chronic inflammation, it is possible that these autoreactive T cells expand, leading eventually to autoimmune diseases via proposed mechanisms such as molecular mimicry (6, 7) or superantigen. Therefore, peripheral regulation should be important for the maintenance of tolerance to systemic Ags. Supporting this idea, it was determined that in MRL-lpr/lpr mice, which are a prototype model of human SLE, the pathway of peripheral tolerance to eliminate self-reactive lymphocytes is impaired due to the deficient Fas/Fas ligand system (8). The importance of peripheral tolerance has already been demonstrated using Tg mice in which the extrathymic organs express neo-self Ags under tissue-specific promoters (9 –14). The Tg Ags are not expressed in or are not able to gain access to the thymus. Several mechanisms, including the deletion (15–17) and downregulation of TCRs (18, 19), have been proposed for the establishment and maintenance of peripheral tolerance; it seems that the nature of Tg Ag, such as antigenicity, expression level, and site of expression, is important for the mechanism of the tolerance induction. Peripheral tolerance to systemic Ags was also studied using the adoptive transfer of CD81 T cells from TCR Tg mice into Tg mice expressing the H-Y Ag (20), Ld (21), and the lymphocytic choriomeningitis virus GP epitope (22). A transient activation followed by deletion or unresponsiveness was observed. However, the peripheral regulation of an immune response to intranuclear autoantigens has not been studied. To investigate this point, we employed an autoantigen-Tg mouse system. Because Ag compartmentation and its assembly with other molecules could significantly influence the Ag-specific immune response, we generated Tg mice expressing human U1 snRNP-A protein (HuA) under a class I promoter. U1 snRNP is one of the U-type snRNP complexes (U snRNPs) that constitute a spliceosome. The constituents (U1A, 70K, and U1C polypeptides) are recognized by anti-U1RNP Abs that are found in virtually all patients with MCTD and in ;20 –30% of patients with SLE. The reasons why we selected HuA as a neo-self Ag are as follows: 1) 0022-1767/99/$02.00 Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 One of the hallmarks of systemic autoimmune diseases is immune responses to systemic nuclear autoantigens. We have examined the fate of the immune response against a nuclear autoantigen using human U1 small nuclear ribonucleoprotein-A protein (HuA) transgenic (Tg) mice by adoptive transfer of autoreactive lymphocytes. We obtained two Tg lines that have different expression levels of the transgene. After spleen cells from HuA-immunized wild-type mice were transferred to Tg mice and their non-Tg littermates, these recipients were injected with HuA/IFA to induce a recall memory response. HAB69, which expressed a lower amount of HuA, exhibited a vigorous increase in the autoantibody level and glomerulonephritis. Moreover, the autoreactivity spread to 70K autoantigen. Alternatively, in HAB64, which expressed a higher amount of HuA, the production of autoantibody was markedly suppressed. The immune response to HuA autoantigen was impaired as demonstrated in a both delayed-type hypersensitivity response and proliferation assay. This inhibition was Ag-specific and was mediated by T cells. These data suggest that the expression level of systemic autoantigens influences the outcome of the immune response to self. The Journal of Immunology, 1999, 162: 6482– 6491. The Journal of Immunology Materials and Methods Mice C57BL/6 (B6) mice were obtained from SLC (Shizuoka, Japan); B6-Igha mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were maintained in a temperature- and light-controlled environment with free access to food and water. Female, age-matched mice were used in all experiments were 6 –10 wk old at the start of each experiment. Generation of Tg mice HuA cDNA was isolated from the HeLa cell l gt11 cDNA library (H. Miura et al., unpublished data). The coding nucleotide sequence was identical with the published sequence (27). An expression plasmid (pLG-Em) was constructed by inserting a human Em enhancer into the 59 end of the Ld class I promoter of pLG-2 plasmid (28). HuA cDNA was fused with an additional sequence encoding c-myc tag (AEEQKLISEEDL) at the 39 end. c-myc-tagged HuA cDNA was subcloned into pLG-Em, and the vector sequence was removed by HindIII-SalI digestion. The HuA transgene construct was microinjected into the pronuclei of fertilized eggs from B6 mice. Microinjected eggs were transferred into the oviducts of pseudopregnant females. Mice carrying the transgene were identified by either Southern blot analysis or PCR analysis of tail DNA. Examination of intracellular localization Intracellular localization of the transgene product was studied in the spleen cells of the Tg mice. Nuclear and cytoplasmic extracts were prepared. Both samples were separated by 12.5% SDS-PAGE and were transferred to nitrocellulose membranes (Schleicher and Schuell, Keene, NH). The HuA expression of each extract was detected by Western blotting with anti-HuA mAb (a generous gift of Dr. W. van Venrooij) (29) and anti-c-myc mAb (9E10) (PharMingen, San Diego, CA). Incorporation of HuA into MuA particles Incorporation of the HuA transgene product into MuA particles was confirmed by the immunoprecipitation of thymocytes and splenocytes from the Tg mice using either anti-2,2,7-trimethylguanosine (anti-m3G) mAb (a generous gift of Dr. R. Lührmann) (30) or Y12 (a generous gift of Dr. Joe Craft), as described previously (31). Briefly, the nuclear extract of 2 3 107 thymocytes or splenocytes was probed with anti-m3G mAb or Y12 absorbed to protein A/G-Sepharose (Calbiochem, La Jolla, CA) in 500 ml of IPP150 buffer (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% Nonidet P-40, and 0.1% Tween 20). After 1 h of incubation at room temperature, the Sepharose particles were collected and washed four times with IPP150 buffer. The washed Sepharose particles were resuspended in 20 ml of SDS- gel loading buffer and boiled for 5 min. The supernatant was separated by 12.5% SDS-PAGE and transferred to a nitrocellulose membrane. HuA expression was detected by Western blotting as described above. Preparations of Ags Escherichia coli BL21 that had been lysogenized with phage lDE3 was transformed with pET3c (Novagen, Madison, WI) containing HuA cDNA at NcoI-BamHI sites as described previously (31). After treatment with 0.5 mM isopropylthiogalactose, cells were collected, washed, and disrupted in 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM PMSF, and 10 mM MgCl2 by freeze-thawing and sonication. Because our recombinant HuA protein was insoluble, the insoluble pellets were solubilized in 8 M ureaPBS and were gradually refolded by dialysis against 4 M, 2 M, 1 M ureaPBS sequentially and finally against PBS. The soluble recombinant HuA was purified by gel filtration on a HiLoad16/60 Superdex 200 column (Pharmacia, Uppsala, Sweden) and by cation-exchange chromatography on a MonoS column (Pharmacia) using the fast protein liquid chromatography system (Pharmacia). The protein concentration was measured by a bicinchoninic acid protein assay (Pierce, Rockford, IL). All samples were separated by 12.5% SDS-PAGE and transferred to a nitrocellulose membrane; the purity of recombinant HuA was confirmed by staining with Coomassie brilliant blue. In some experiments, OVA was used as an Ag. Chicken egg OVA (Sigma, St. Louis, MO) was solved in PBS at various concentrations. The endotoxin concentration of these samples was measured by Limulus amebocyte lysate assay (Seikagaku, Tokyo, Japan). All Ags used had ,15 ng endotoxin/mouse at the maximum injected dose. Immunization Mice were immunized s.c. in the base of the tail with 50 mg of Ag emulsified 1:1 (v/v) in CFA (Difco, Detroit, MI). In one part of the experiment, mice were boosted s.c. in the base of the tail 2 wk later with 50 mg of Ag emulsified 1:1 (v/v) in IFA (Difco). Adoptive transfer For adoptive transfer, various numbers of spleen cells or T cell-enriched fractions in 0.5 ml of PBS were injected i.v. into either naive Tg mice or their non-Tg littermates. Cell viability was noted to be .97% as determined by trypan blue exclusion. Preparation of cell populations Mice were sacrificed at 10 days postimmunization, and spleens or inguinal and paraaortic lymph nodes (LNs) were taken. Single-cell suspensions of LN cells or spleen cells were prepared under aseptic conditions by mechanical disaggregation and passed through a sterile nylon mesh, followed by hypotonic shock to remove contaminated erythrocytes. Purified T cells were prepared using the method of Julius et al. (32). Briefly, LN cells (107/ml) suspended in RPMI 1640 complete medium were incubated in a nylon wool column for 1 h at 37°C. After two washings with medium, T cells were collected by centrifugation; the enrichment of T cells ($90%) was examined with flow cytometry. A T cell-rich population was also prepared by negative selection with magnetic cell sorting (MACS) (Miltenyi Biotech, Bergisch Gladbach, Germany) using anti-B220 mAb (PharMingen) and anti-I-Ab mAb (PharMingen). B cells were prepared by positive selection with MACS using anti-B220 mAb. The enrichment of B cells ($90%) was examined by flow cytometry. Cell viability was noted to be .97% as determined by trypan blue exclusion. Depletion of T cells or B cells was performed by negative selection with MACS using anti-Thy1.2 mAb (PharMingen) or anti-B220 mAb, respectively. The efficiency of depletion ($96%) was examined by flow cytometry. ELISA Anti-HuA Ab production was assayed by ELISA using recombinant protein as described previously (33). Briefly, the microtiter plate (Immulon 4, Dynatech, Chantilly, VA) was coated with recombinant HuA at 5 mg/ml in 0.03 M carbonate buffer at pH 9.6 by overnight incubation at 4°C. After blocking with 1% BSA for 2 h at 37°C, the plates were incubated with mouse serum samples that had been serially diluted in PBS buffer containing 1% BSA and 0.05% Tween 20 for 1 h at 37°C. After washing five times with 0.05% Tween 20 in PBS, the bound Abs were visualized with goat anti-mouse IgG mAb coupled to HRP (Zymed, San Francisco, CA), followed by development with 3,5,39,59-tetramethylbenzidine (Kirkegaard and Perry Laboratories, Gaithersburg, MD). OD was read at 450 nm. All samples were tested in duplicate. A serum of wt mice immunized with HuA exhibiting a high level of anti-HuA Abs was selected as a standard serum, and the level was arbitrarily determined to be 10,000 U/ml. In each experiment, the standard serum was serially diluted and assayed to make a Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 In our previous study, the presence of HuA-reactive T cells was confirmed in patients with SLE or MCTD (3). 2) Although the amino acid sequence of murine U1 snRNP-A protein (MuA) has a 96% homology to that of HuA (23), it has been demonstrated that a considerable immune response to HuA can be induced (24). 3) In murine lupus models, U1A is the immunodominant Ag of U1 snRNP (25), and epitopes spread to other constituents of self U snRNPs through intermolecular/intrastructural help (26). 4) It was shown that tolerance to MuA was broken by the coimmunization of MuA with HuA (26). We obtained two Tg lines that differ in the expression level of the transgene. Because it is not feasible to elicit an immunodominant response to systemic autoantigens, we employed an adoptive transfer strategy: the HuA-specific T cells induced in wild-type (wt) mice were transferred to HuA Tg mice followed by booster immunization to enhance the transferred immune response against HuA. Thus, we were able to observe the fate of the autoimmune response to a systemic intranuclear autoantigen. The experimental results were opposite between the two lines. In one line, which expresses a lower amount of HuA, autoantibody production was vigorously enhanced and the Tg mice showed severe renal involvement. In the other line, which highly expresses HuA, autoantibody production was markedly suppressed. It was demonstrated that this suppression was Ag-specific and mediated by T cells. Therefore, we conclude that the expression level of a systemic autoantigen determines the fate of immune response to self. 6483 6484 REGULATION OF AN AUTOIMMUNE RESPONSE TO A SYSTEMIC NUCLEAR Ag standard curve. The level of anti-HuA Abs in each sample was determined in comparison with this standard curve and was represented as an arbitrary unit. Anti-70K Ab production was assayed by ELISA using the recombinant human 70K protein (MBL, Nagoya, Japan). Measurement of allotypespecific anti-HuA IgG2 Abs was performed as described above, except that IgG2a-specific or IgG2b-specific anti-mouse IgG mAb coupled to HRP (Zymed) was used as second Ab. Proliferation assays Spleen cells and LN cells, obtained as described above, were cultured at 1 3 105 cells/well with various concentrations of HuA in RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, 10% heat-inactivated FCS, and 5 3 1025 M 2-ME for 5 days, followed by a final 16 h of culture in the presence of 1 mCi of [3H]TdR per well. The incorporated radioactivity was counted with a gamma scintillation counter. The proliferative response was expressed as Dcpm (mean cpm of test cultures 2 mean cpm of control cultures without Ag) 6 SD. Cytokine analyses Local adoptive transfer (LAT) assays A LAT assay was employed to detect the regulatory cells that impair the delayed-type hypersensitivity (DTH) response (36). Spleen cells containing a population of presumptive regulatory cells (2 3 105 cells) from Tg mice or their non-Tg littermates (as a control) after adoptive transfer and the injection of HuA/IFA were mixed with HuA (20 mg/ml) and responder cells (DTH-mediating effector cells) (2 3 105 cells) obtained from HuAimmunized wt mice. This mixture (10 ml) was injected into the ear pinnae of wt mice, and ear swelling was measured 24 h and 48 h later. In each experiment, two non-Tg mice and two Tg mice were examined. Evaluation of proteinuria Urine samples were serially collected from Tg mice and non-Tg littermates to which spleen cells from HuA-immunized wt mice were transferred and boosted. The presence of proteinuria was assayed by a murine microalbuminuria ELISA kit (Albuwell, Exocell, Philadelphia, PA). Immunoprecipitations of in vitro-translated protein mRNA was transcribed from human 70K cDNA subcloned into the pGEM3Zf1 vector (Promega, Madison, WI) using the SP6 RNA polymerase promotor followed by translation in rabbit reticulocyte lysates (Promega) in the presence of [35S]methionine. The recombinant human 70K protein labeled in vitro with [35S]methionine was immunoprecipitated with sera as described above and was separated by 7.5% SDS-PAGE. Results Two Tg lines differ in HuA expression level To generate Tg mice with a systemic expression of HuA, we constructed a c-myc-tagged HuA transgene under the control of an Ld MHC class I promoter and Em enhancer. We obtained two Tg lines, HAB64 and HAB69. The successful expression of HuA in Tg mice was confirmed in the total cell extracts of spleen, thymus, and PBMCs probed by anti-HuA mAb cross-reactive to MuA and anti-c-myc tag mAb (data not shown). To investigate the intracellular localization of HuA, we prepared nuclear and cytoplasmic extracts of spleen cells of Tg mice and non-Tg littermates in each line and performed Western blotting using anti-c-myc mAb. The HuA transgene product was found to be predominantly located in nuclei in both lines, although the ex- FIGURE 1. HuA protein expression in Tg mice. A, The HuA transgene product was predominantly expressed in nuclei. Nuclear and cytoplasmic extracts of splenocytes from wt mice, HAB64 mice, and HAB69 mice were prepared; samples were separated by 12.5% SDS-PAGE, followed by immunoblotting with anti-c-myc mAb. B, Different amounts of HuA were incorporated into MuA between two Tg lines, HAB64 and HAB69. Nuclear extracts of splenocytes and thymocytes from HAB64 and HAB69 mice were immunoprecipitated by anti-m3G mAb. Immunoprecipitants were separated by 12.5% SDS-PAGE, transferred to a nitrocellulose membrane, and probed with anti-c-myc mAb. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 The cytokines involved in the inhibitory effect on T cell proliferation were analyzed. Supernatants were harvested after 40 h of culture and assayed for IL-4, IL-10, and TGF-b by ELISA. Quantitative determinations of IL-4 and IL-10 were performed by commercial assays (Genzyme, Cambridge, MA). Active TGF-b (without acid treatment) was determined by a sandwich ELISA using mouse anti-TGF-b mAb (Genzyme), chicken antiTGF-b mAb (R&D Systems, Minneapolis, MN), and recombinant human TGF-b (R&D Systems) as a standard. In some experiments, to inhibit cytokines in vitro, neutralizing mAbs to IL-4 (10 mg/ml) (R&D Systems), IL-10 (10 mg/ml) (R&D Systems), or TGF-b (1 mg/ml) (R&D Systems) were added in the culture medium at the amounts recommended by the manufacturer (34, 35). pression level of HuA was different between the two Tg lines (Fig. 1A). In a Xenopus oocyte injection study, it was demonstrated that HuA injected into the cytoplasm can be transported into nuclei and that HuA can be incorporated into the U1 snRNP complex (37). Because HuA has a 96% similarity to MuA regarding the amino acid sequence, it would be interesting to know whether HuA can be incorporated into the MuA particle. Therefore, we subsequently examined the expression level in two Tg mouse lines and the exact localization of HuA. The nuclear extracts of the same number of splenocytes and thymocytes of each line were prepared. U snRNP complexes of nuclear extracts were immunoprecipitated with antim3G mAb (30) and were probed with anti-c-myc tag mAb. Fig. 1B showed that HuA was incorporated into mouse U snRNP complexes and that the HAB64 line expressed a higher amount of HuA than did HAB69. We confirmed that the fraction of nuclear extracts that could not be immunoprecipitated by anti-m3G mAb did not contain HuA by Western blot analysis (data not shown). To quantify the expression level of HuA, we analyzed these bands using a densitometer (Table I). The ratio of the expressed transgene product between HAB64 and HAB69 was 2.2 6 0.5 in the thymus and 3.7 6 0.8 in the spleen. We also compared the density of the HuA band with that of the endogenous MuA in the The Journal of Immunology 6485 Table I. Comparison of the HuA expression level between two Tg lines by densitometry analysisa Thymus Spleen HAB64/HAB69 n (HAB64/HAB69) 2.2 6 0.5 3.7 6 0.8 12/12 14/14 a Nuclear extracts of the same number of splenocytes and thymocytes of each line were prepared. U snRNP complexes of nuclear extracts were immunoprecipitated with anti-m3G mAb and probed with anti-c-myc tag mAb. The band densities were analyzed using a densitometer, and the ratio of the expression level was shown. total cell extract of the same number of B cells from the peripheral blood of each line using anti-HuA mAb. In one line HAB64, the amount of HuA was 76% of the endogenous MuA; in the other line HAB69, the amount of HuA was 35%. Therefore, we could confirm both the different expression level of the HuA transgene and the incorporation of HuA into mouse U snRNP. These Tg mice provide us with the advantage of investigating the immune response to an intranuclear autoantigen in physiological conditions. Because the HuA transgene product is expressed in the thymus, Tg mice are expected to be unresponsive to HuA. To confirm this possibility, we immunized Tg mice of both lines and wt mice with 50 mg of HuA emulsified 1:1 (v/v) in CFA in the base of tail. The sera of these mice were serially collected, and the level of antiHuA IgG Abs was measured by ELISA. The proliferative response of spleen cells stimulated with HuA was measured by thymidine incorporation at 10 days postimmunization. Although wt mice responded to HuA, both Tg lines exhibited an unresponsiveness in B cell response and T cell response (data not shown). To exclude the possibility that the Tg mice had a defect in their immune response, we immunized the mice with 50 mg of OVA and examined both T and B cell responses to the Ag. Both lines of the Tg mice demonstrated a good response to OVA, comparable with that of wt mice (data not shown), suggesting that the immune responses in both lines of Tg mice are normal and not significantly different from each other. Therefore, we concluded that both Tg lines are unresponsive to HuA and that the insertion effect of the transgene is negligible. Autoantibody production can be induced in Tg mice by adoptive transfer of autoreactive T cells In systemic autoimmune diseases, expanded autoreactive T cell clones are thought to drive autoantibody production. To create a similar situation in Tg mice, we adoptively transferred the autoreactive lymphocytes that are responsive to HuA into HAB64 Tg mice and observed the immune response to the intranuclear autoantigen. Ten million (1 3 107), 5 3 107, or 2 3 108 viable spleen cells from wt mice immunized with HuA/CFA were injected i.v. into naive Tg mice or their non-Tg littermates. After the adoptive transfer, sera were serially collected and anti-HuA IgG Abs were measured by ELISA. Both Tg mice and their non-Tg littermates produced Abs against the HuA protein, and the level thereafter decreased gradually over time for 6 mo. The level of autoantibodies increased according to the number of transferred cells (Fig. 2A). There were no definite differences in the kinetics and the level of anti-HuA Abs between HAB64 Tg mice and their non-Tg littermates. In addition, another line, HAB69, also showed similar results. When a T cell-rich population (.90% purity) was transferred into Tg mice and their non-Tg littermates, anti-HuA IgG Ab production was also observed (data not shown), indicating that HuA-specific T cells could provide help for autoantibody production in recipient-derived B cells. FIGURE 2. Adoptive transfer of HuA-reactive lymphocytes. A, Singlecell suspensions of spleens from wt mice immunized with HuA/CFA were obtained. For adoptive transfer, 1 3 107, 5 3 107, or 2 3 108 viable spleen cells in 0.5 ml of PBS were injected i.v. into naive Tg mice and their littermates. Sera of these mice were serially collected, and anti-HuA IgG Abs of samples were measured by ELISA. The Ab level of samples was calculated as the arbitrary unit according to the standard curve and the mean 6 SD U/ml values are shown. Three mice were tested in each group. B, HuA-specific T cells could provide help for anti-HuA Ab production in recipient-derived B cells. A T cell-rich population was obtained from the spleen cells of HuA-immunized B6-Ighb mice by negative selection with MACS using anti-B220 mAb and anti-I-Ab mAb; a total of 1 3 107 viable T cells were adoptively transferred into two B6-Igha mice, and IgG2a and IgG2b-specific anti-HuA Abs were measured by ELISA. ELISA readings at a 1/100 dilution of sera from recipients were shown as an OD of 450 nm. To confirm this observation, we adoptively transferred the T cell-rich population from HuA-immunized B6-Ighb mice into B6Igha mice and measured allotype-specific anti-HuA IgG Abs by ELISA. As shown in Fig. 2B, the recipients mainly produced antiHuA IgG2a Abs, indicating that recipient-derived B cells were involved in anti-HuA IgG Ab production. Enhancement of autoantibody production in HAB69 resulted in pathogenic renal manifestations Although autoantibody production can be successfully induced in tolerant mice, the autoantibody level was not so high compared with that seen for MRL/lpr lupus-prone mice. This low autoantibody level seems to be due to a low frequency of autoreactive T cells. Therefore, we intended to expand autoreactive lymphocytes by inducing a recall memory response in Tg mice. When 5 3 107 or 1 3 107 viable spleen cells from wt mice immunized with HuA/CFA were adoptively transferred to the Tg mice of both lines and their non-Tg littermates, the recipients were boosted with HuA emulsified in IFA either simultaneously (data not shown) or at 3 mo posttransfer (Figs. 3, A and B). In HAB69, anti-HuA Ab Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 Tg mice are unresponsive to HuA 6486 REGULATION OF AN AUTOIMMUNE RESPONSE TO A SYSTEMIC NUCLEAR Ag FIGURE 3. Enhancement of autoantibody production in HAB64 and HAB69. A single-cell suspension of spleens from HuA-immunized wt mice was prepared. For adoptive transfer, 5 3 107 viable spleen cells were injected i.v. into three Tg mice and three non-Tg littermates. To amplify the recall response, these mice were injected with 50 mg of HuA in IFA at 3 mo posttransfer. The sera of these mice were serially collected, and anti-HuA IgG Abs of samples were measured by ELISA. The Ab level of the samples was calculated as an arbitrary unit according to the standard curve. The mean unit per milliliter values 6 SD are shown. A, Marked suppression of autoantibody production in HAB64. B, Enhancement of autoantibody production in HAB69. The results using 1 3 107 spleen cells were omitted because they gave similar results with the exception of the reduced Ab level due to the reduced number of transferred T cells. the exaggerated autoimmune response in HAB69 led to an autoaggressive disease phenotype. Previous reports have described that the coimmunization of self (murine) snRNPs with HuA could break self tolerance to self snRNPs and epitope spread to other U snRNP constituents. Based on this finding, the model of intermolecular/intrastructural help was proposed to explain autoantibody propagation to the components of an autoantigen complex (26), for example, U1A/70K in the U1 snRNP complex (26) and La/Ro in the RoRNP complex (38). Because the HuA transgene product is incorporated into the MuA complex, HuA Tg mice are a good model to investigate this mechanism. U snRNP complexes of nuclear extracts of Ehrlich ascites tumor cells, a murine lymphoma cell line, were immunoprecipitated with anti-m3G mAb (30) and probed with sera from Tg mice, which FIGURE 4. Renal manifestations in HAB69 suffered from autoaggressive immune response. A, Urine albumin concentration of HAB69 mice and their littermates with an exaggerated HuA Ab response. Urine samples were collected from HAB69 mice and their non-Tg littermates at 5 mo after adoptive transfer. The results from HAB69 mice and their littermates without any treatment were also included as negative controls. The urine albumin concentration was measured by ELISA. In each group of mice, 8 or 10 mice were tested. B, A microscopic picture of a kidney from an HAB69 mouse. Marked proliferation of mesangial cells and matrix was observed (hematoxylin and eosin staining, 3400 magnification). C, Immunofluorescence study of complement 3. There are granular (mesangium) and linear (tubular and Bowman’s capsular basement membranes) deposits of complement 3 (3400 magnification). Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 production was markedly enhanced up to levels that were nearly comparable with those of the wt controls (Fig. 3B). There were no differences in the kinetics of autoantibody production as well. However, HAB69 demonstrated more severe proteinuria than the control mice at 3 mo after the booster (Fig. 4A). The severity is significant because 60% of HAB69 mice (n 5 6/10) exceeded the mean value 1 2 SD of the urine albumin concentration of wt recipient mice. Light microscopic examinations revealed a marked proliferation of mesangial cells, and matrix (Fig. 4B) and immunofluorescence microscopy studies showed granular deposits of complement 3 as well as IgG in the mesangium and in the tubular and capsular basement membranes (Fig. 4C), indicating an immune complex-type glomerulonephritis. Interestingly, some HAB69 exhibited more extensive and severe skin manifestations, including skin ulcers (data not shown). These results suggest that The Journal of Immunology produced a high level of anti-HuA autoantibodies. As shown in Fig. 5A, the Tg mice showing an autoaggressive phenotype developed anti-murine 70K IgG autoantibodies in immunoblots. In vitro-translated, [35S]methionine-labeled recombinant human 70K protein could be immunoprecipitated by sera from Tg mice (Fig. 5B), because anti-murine 70K autoantibodies cross-react to human 70K. These results indicate that anti-70K IgG autoantibodies were produced in HAB69 mice. To compare the level of anti-70K autoantibodies from HuA Tg mice with that of non-Tg mice, sera from individual mice were examined by ELISA using recombinant human 70K (Fig. 5C). A larger number of Tg mice showed a higher level of anti-70K Abs after adoptive transfer of HuA-reactive lymphocytes than did non-Tg mice. These data demonstrate that once an autoimmune response to HuA occurs, self-reactivity spreads to another component of the U1 snRNP complex containing HuA via intermolecular/intrastructural help. By these results, we could again confirm the incorporation of HuA in the mouse U snRNP particle. In contrast to the case of HAB69, in HAB64, anti-HuA autoantibody production was markedly suppressed when the recipients were boosted 3 mo after the successful transfer as shown in Fig. 3A. When we boosted the recipients immediately after the transfer to exclude the possibility that the donor cells were quickly removed by activation-induced cell death (20 –22), we also observed a similar suppression (data not shown). Therefore, we postulated that some regulatory mechanism could appear upon booster immunization. To verify this hypothesis, we performed a LAT assay and a proliferation inhibition assay. The LAT assay has been employed FIGURE 5. Spreading of self-reactivity to 70K autoantigen in HAB69. A, Nuclear extracts of Ehrlich ascites cells were immunoprecipitated by antim3G mAb. The immunoprecipitants were separated by 7.5% SDS-PAGE, transferred to a nitrocellulose membrane, and probed with a representative serum from HAB69 exhibiting an enhanced immune response to HuA (lane 3), from an SLE patient who is known to have a high level of anti-70K autoantibodies as a positive control (lane 2), or from a wt mouse as a negative control (lane 1). Anti-70K Abs could be detected by Western blotting as indicated by the arrow. B, Anti-70K Abs were detected by immunoprecipitation reactions using in vitro-translated and [35S]methionine-labeled human 70K protein. The 70K mRNA was transcribed from the human 70K cDNA that had been previously subcloned into the pGEM-3Zf1 vector followed by translation in rabbit reticulocyte lysates in the presence of [35S]methionine. The human 70K protein labeled in vitro with [35S]methionine was immunoprecipitated with sera from HAB69 mice (lane 2) or with sera from wt mice as a negative control (lane 1). C, The level of anti70K Abs was measured by an ELISA kit using recombinant human 70K. The maximum ELISA readings at a 1/100 dilution of sera from HAB69 Tg mice and their non-Tg littermates were shown as an OD of 450 nm. for the detection of regulatory cells that impair the DTH response (36). Spleen cells from HuA/IFA-immunized HAB64 after adoptive transfer (which presumably contain a population of regulatory cells) were mixed with HuA and DTH-mediating responder cells obtained from HuA/CFA-immunized wt mice. This mixture was injected into the ear pinnae of naive wt mice, and ear swelling was measured after 24 and 48 h. As a positive control, the spleen cells from non-Tg littermates immunized with HuA/IFA after adoptive transfer were employed. The mixture with the spleen cells from HuA/IFA-injected HAB64 exhibited a lower ear swelling response, indicating the presence of regulatory cells that impair the expression of DTH (Fig. 6A). We were also able to confirm the presence of regulatory cells by an in vitro proliferation inhibition assay. Spleen cells or LN cells obtained from HuA/IFA-injected HAB64 after adoptive transfer were mixed with spleen cells or LN cells from HuA/CFA-immunized wt mice at various proportions. Thymidine incorporation of responder cells was reduced by the addition of splenocytes from HuA/IFA-treated HAB64. The inhibitory effect on proliferation was observed when the ratio of regulatory cells to responder cells was either 3:1 or 1:1, whereas this effect was abrogated when the ratio was 1:3 (Fig. 6B), suggesting that the regulatory cell frequency might be important for inhibition. This inhibition was also observed when spleen cells from HAB64 mice, which were only injected with HuA/IFA without the adoptive transfer, were used as regulatory cells. To examine whether this regulation was Ag-specific or not, spleen cells obtained from HuA/IFA-injected HAB64 after the adoptive transfer were mixed with the spleen cells obtained from OVA/CFA-immunized wt mice and were cultured in the presence Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 Regulatory mechanism of autoreactivity in HAB64 6487 6488 REGULATION OF AN AUTOIMMUNE RESPONSE TO A SYSTEMIC NUCLEAR Ag of OVA and HuA. In these experiments, The proliferation response to OVA was not inhibited (data not shown). These data suggest that the regulatory cells in HuA/IFA-injected HAB64 mice are autoantigen specific. Interestingly, when we compared the inhibitory activity between HAB64 and HAB69 using spleen cells from HuA/IFA-injected mice after the adoptive transfer, we found that there was a clear difference between both lines (Fig. 6C). Spleen cells from HAB69, the autoaggressive phenotype line, did not exhibit an impairment of proliferation to HuA, suggesting that the suppression of the immune response to HuA in HAB64 can be attributed to the induction of regulatory cells by HuA/IFA immunization. We subsequently examined which cell population is responsible for this inhibition. T cells or B cells were depleted from the spleen cells obtained from either Tg mice or non-Tg littermates after the injection of HuA/IFA by MACS using either anti-Thy-1.2 mAb or anti-B220 mAb. T cell- or B cell-depleted spleen cells were mixed with spleen cells as responder cells obtained from HuA/CFA-immunized wt mice and cultured in the presence of HuA (Fig. 6D). Anti-Thy-1.2 mAb treatment abrogated the inhibitory effect for HuA-reactive T cell proliferation, suggesting that regulatory T cells mediated the inhibition. Several studies have reported that IFA induced Ag-specific tolerance or unresponsiveness. In most of them, the regulatory mechanism is attributed to the induction of Th2 cells by i.p. Ag/IFA injection as demonstrated in a beef insulin Tg model (39) and in neonatal tolerance models (40). Another candidate for the mechanism is Th3 cells secreting TGF-b, which has been shown to play Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 6. Inhibition of the DTH response and the proliferative response in HAB64. A, The DTH response was suppressed by spleen cells from Tg mice injected with HuA/IFA. Spleen cells containing a population of presumptive regulatory cells (2 3 105 cells) obtained from HAB64 mice or their non-Tg littermates (as a control) injected with HuA/IFA after adoptive transfer were mixed with HuA (20 mg/ml) and responder cells (DTH-mediating effector cells) (2 3 105 cells) obtained from HuA/CFA-immunized wt mice. This mixture (10 ml) was injected into the ear pinnae of the wt mice, and ear swelling was measured after 24 h (open bars) and 48 h (striped bars). These data are representative of two separate experiments. In each experiment, two non-Tg mice and two Tg mice were tested. B, Impairment of a proliferative response by the lymphocytes from HAB64. Spleen cells or LN cells were obtained from HAB64 mice or their non-Tg littermates (as a control) that had been injected with HuA/IFA after the adoptive transfer as regulator cells. As responder cells, spleen cells or LN cells were obtained from HuA/CFA-immunized non-Tg mice. Regulatory cells and responder cells were mixed at 3:1, 1:1, and 1:3 and cultured with HuA (50 mg/ml) in RPMI 1640 for 5 days. Proliferation was measured by thymidine incorporation. Results are representative of three independent experiments. C, Proliferative responses of HuA-reactive lymphocytes in the presence of lymphocytes from HuA/IFAinjected HAB64 and HAB69. Proliferation inhibition assays, as described above, were performed. Presumptive regulator cells (105 cells/well) and responder cells (105 cells/well) were mixed and cultured. The percent inhibition was calculated in comparison with the proliferation of responder cells in the absence of regulator cells. Results are representative of three independent experiments. D, Inhibition of the proliferative response in HAB64 was mediated by regulatory T cells. T cells or B cells were depleted from the spleen cells obtained from Tg mice and non-Tg littermates after the injection of HuA/IFA by MACS using anti-Thy-1.2 mAb or anti-B220 mAb, respectively. T cell- or B cell-depleted spleen cells were used as presumptive regulator cells; proliferation inhibition assays, as described above, were performed. The percent inhibition was calculated in comparison with the proliferation of responder cells in the absence of regulator cells. Results are representative of three independent experiments. E, Inhibitory cytokines did not abrogate the proliferative response in HAB64. Proliferation inhibition assays were performed in the presence of neutralizing mAb to IL-4, IL-10, or TGF-b. Proliferation was measured by thymidine incorporation. Results are representative of two independent experiments. The Journal of Immunology Discussion A few studies have examined Ag compartmentation and immunological tolerance (44, 45). Previous studies have revealed that the availability of self Ag depends upon processing and Ag compartmentation (e.g., mitochondria membrane, plasma membrane, and nucleus) and that it could influence the emergence of autoreactive T cells (45). These studies demonstrated that CD4 T cell tolerance via negative selection, a pivotal mechanism for self tolerance, can be achieved by MHC class II Ag presentation of endogenous proteins on thymic epithelial cells, although the contribution of autophagy cannot be excluded. Our Tg model, expressing the neoself Ag in thymocytes, is tolerant of intranuclear neo-autoantigen, and they did not exhibit spontaneous autoimmunity. Although we were not able to define the precise expression site of HuA within the thymus and the amount of circulating Ag, it would be interesting to examine whether bone marrow-derived dendritic cells or thymic epithelial cells contribute to the induction of tolerance. Therefore, the next question would be how the immunological tolerance is induced to intranuclear Ag under physiological conditions, as in the case of our Tg mice, and whether autoantigen-reactive T cells are deleted or anergic in the thymus. By the adoptive transfer of autoreactive (HuA-reactive) T cells from non-Tg mice immunized with HuA/CFA into naive Tg mice or their non-Tg littermates, anti-HuA Abs were successfully induced in both. Therefore, it seems that the autoimmune response is acceptable to some extent in the periphery. Recently, using adoptive transfer of naive autoreactive T cells, it was demonstrated that autoreactive T cells led to an induction of tolerance after a period of transient activation by encountering an autoantigen (22, 46). Because we transferred the autoreactive T cells from immunized mice instead of naive T cells, it is possible that the activated T cells in the transferred T cell population induced Ab production efficiently before being tolerant. However, because the autoantibody level increased until 1 wk after the transfer and there is no difference concerning both the level and the kinetics between Tg and non-Tg mice, it is rather likely that the tolerance induction to the transferred autoreactive T cells in HuA Tg mice might not be so effective as the reported cases. It would be interesting to know whether this inefficiency is related to the accessibility and availability of the autoantigen. HAB69, a low expresser, exhibited enhanced production of antiHuA and anti-70K autoantibody, resulting in glomerulonephritis. However, because anti-HuA Ab and proteinuria could be also induced in wt mice to some extent, glomerulonephritis might be induced by the deposition of immune complexes. Nevertheless, the significant difference in the severity of proteinuria suggests that the HuA transgene product plays some role in the pathogenesis of the kidney in these mice. In SLE patients, proteinuria was usually not associated with anti-U1RNP autoantibody but with antidsDNA autoantibody. Ab to native DNA in sera from SLE patients and BWF1 lupus-prone mice has been shown to have cross-reactivity with U1A and Sm D proteins frequently, and this crossreactivity was suggested to be part of the original immunogenic drive in the production of anti-native DNA autoantibody (47). We could also detect anti-dsDNA reactivity in HAB69 (our unpublished observations). This implies the possibility that enhanced immune responses to HuA led to the production of nephritogenic Abs such as anti-dsDNA autoantibody and that these nephritogenic Abs were involved in the severity of glomerulonephritis in HAB69. HAB64, a high expresser, demonstrated a regulatory phenotype. This suppression was Ag-specific. Thus far, a few reports have described the relationship between the amount of tolerogen and T cell tolerance (19, 48). It has been shown that peripheral tolerance has multiple levels that are significantly influenced by the amount of tolerogens in organ-specific autoantigens (19). This view has also been proposed in studies that investigated the immune response to a systemic Ag (20, 21). According to the previously reported two Tg lines producing different amounts of secreted hen egg-white lysozyme (HEL), T cells specific for the minimal immunodominant epitope of HEL were deleted or inactivated in both lines, whereas T cell clones of lower affinity reacting with epitopes on longer peptides persist only in the line that produced a lower amount of HEL (48). Therefore, we can postulate that in our Tg mice, the different expression levels of the nuclear autoantigen led to the difference of either the amount or affinity of T cell clones that were deleted or inactivated, and that the tolerized T cells compete for IL-2 (49), resulting in the inhibitory effect on Ag-specific T cell proliferation. Because our system does not use TCR Tg mice, and we could not examine the phenotype of single T cell clones (e.g., TCR down-regulation), we can not completely exclude the possibility that the tolerance level of a single T cell clone is more profound in HAB64. However, this mechanism is less likely, because an addition of IL-2 could not restore the inhibitory effect (our unpublished observations). We prefer to postulate the presence of regulatory T cells, which appear after HuA/IFA immunization. It has been proposed that CD41 T cells are the executors of Ag-specific surveillance of autoantibody production (50). This mechanism depends upon the Fas/Fas ligand system (51, 52). Based on this idea, an alternative explanation might be possible. That is, the memory HuA-specific T cells transferred and activated by HuA/IFA immunization could exhibit cytolytic activity against the Tg mice B cells, leading to a loss of APC. However, when we introduced the HuA transgene into B6/lpr/lpr to investigate the involvement of the Fas/Fas ligand system, a suppression of autoantibody production was observed. Because the spleen cells with the suppressive activity did not show Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 an important role in oral tolerance (41). Therefore, we examined the possibility that these humoral factors are involved in the inhibition. At first, we collected culture supernatants at 48 h in the proliferation inhibition assays and analyzed the cytokines. We were unable to detect any significant increase in Th2-type cytokines (IL-4 and IL-10) (42, 43) and TGF-b in the supernatant of responder cells mixed with the regulatory spleen cells from HAB64 or in the supernatant of HAB64 spleen cells stimulated with the Ag. We could observe only lower amounts of IL-2 in the supernatant of responder cells mixed with spleen cells from HAB64 mice (131.3 6 1.1 pg/ml) compared with that seen for wt mice (177.7 6 2.1 pg/ml). Because it is possible that the frequency of the Ag-specific T cells was not sufficient to be distinguished by their lymphokine secretion, we employed neutralizing Abs to the inhibitory cytokines. However, the addition of Abs to IL-4, IL-10, and TGF-b did not abrogate the inhibition, suggesting that the inhibitory effect is not mediated by these cytokines (Fig. 6E). Taken together, the two HuA autoantigen Tg lines, HAB64 and HAB69, which show different expression levels of the autoantigen, demonstrated different outcomes when autoreactive lymphocytes were expanded. Pathogenic lesions (manifestations of autoimmune diseases) appeared in one line, whereas a regulatory mechanism was seen in the other. 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Interestingly, Marusic and Tonegawa reported that a mechanism other than Th2 deviation or activation-induced cell death can be induced by peritoneal IFA injection; they observed an unresponsive induction using either Fas-deficient or IL-4 deficient anti-myelin basic protein TCR Tg mice (53). In conclusion, these data suggest that the immune system to intranuclear autoantigens has a regulatory mechanism that appears according to the intensity and the context of the immune response to self. This peripheral regulation might be a fail-safe mechanism against the disturbance of self tolerance by several mechanisms including cryptic epitope (54), molecular mimicry (6, 7) and bystander activation (55). Our Tg mouse models can be expected to present the key for the understanding and treatment of systemic autoimmune diseases such as SLE and MCTD, because the location and assembly with other molecules of the introduced Ag are physiological rather than artificial. 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