Mice Lpr Disease in MRL- Lymphoproliferation but Not Autoimmune Transgenic Expression of Fas in T Cells Blocks

Transgenic Expression of Fas in T Cells Blocks Lymphoproliferation But Not Autoimmune Disease in MRL-lpr Mice

This information is current as Hidehiro Fukuyama, Masashi Adachi, Sachiko Suematsu, Keiko of September 25, 2021. Miwa, Takashi Suda, Nobuaki Yoshida and Shigekazu Nagata J Immunol 1998; 160:3805-3811; ; http://www.jimmunol.org/content/160/8/3805 Downloaded from

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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 © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Transgenic Expression of Fas in T Cells Blocks Lymphoproliferation But Not Autoimmune Disease in MRL-lpr Mice1

Hidehiro Fukuyama,*† Masashi Adachi,* Sachiko Suematsu,2‡ Keiko Miwa,* Takashi Suda,* Nobuaki Yoshida,‡ and Shigekazu Nagata3*†

Fas is a member of the TNF receptor family. Binding of Fas ligand to Fas induces apoptosis in Fas-bearing cells. Fas is expressed in various cells, including thymocytes, peripheral T cells, and activated B cells. The mouse lpr mutation is a loss of function mutation of Fas. MRL-lpr/lpr mice develop lymphadenopathy and splenomegaly, and produce multiple autoantibodies, which results in autoimmune disease. In this report, we describe the establishment of a line of Fas transgenic MRL-lpr mice in which mouse Fas cDNA was expressed using the T cell-speciﬁc murine lck promoter. The transgenic mice expressed functional Fas in Downloaded from ,(thymocytes and peripheral T cells, but not in B cells. The transgenic mice did not accumulate abnormal T cells (Thy-1؉ B220؉ ,but still accumulated B cells (Thy-1؊ B220؉); they produced a large quantity of Igs (IgG1 and IgG2a), including anti-DNA Abs and developed glomerulonephritis. These results suggest that autoreactive or activated B cells must be killed through Fas expressed in the B cells by the Fas ligand expressed in activated T cells. The Journal of Immunology, 1998, 160: 3805–3811.

omeostasis in the immune system is maintained not only They develop lymphadenopathy and splenomegaly and suffer from http://www.jimmunol.org/ by growth of lymphocytes but also by cell death. Fas autoimmune disease caused by the production of autoantibodies H ligand (FasL)4 is a type II membrane protein belonging (autoimmune lymphoproliferative syndrome) remarkably similar to the TNF family (1, 2). It is predominantly expressed in activated to those found in mouse lpr and gld mutants. T cells and NK cells (3–5). Fas, the receptor for FasL, is a type I Animals carrying the mutation in Fas or FasL accumulate cells that membrane protein, and a member of the TNF receptor family (2, have the surface phenotypes Thy-1ϩ B220ϩ CD4Ϫ CD8Ϫ and are of 6). It is expressed in various cells, including thymocytes, mature T T cell origin (11). The cells also express various activation Ags of T cells, and activated B cells (3, 7–9). Binding of FasL to Fas in- cells, such as CD69 and FasL (20, 21). The lymphoproliferation in the

duces apoptosis in Fas-bearing cells and kills them within hours Fas-deficient animals is explained as follows. The activation of T cells by guest on September 25, 2021 (2). This is one of the effector systems of cytotoxic T cells and NK by foreign Ags activates CD4ϩ-type T cells to produce various lym- cells (10). phokines and cell surface molecules that stimulate the proliferation Mice carrying the mutation of lymphoproliferation (lpr) or gen- and differentiation of lymphocytes. CD8ϩ T cells are also activated by eralized lymphoproliferative disease ( gld) develop lymphadenop- foreign Ags and kill the target cells. These activated T cells must be athy and splenomegaly (11). They also produce large amounts of removed after they have accomplished their tasks. This deletion pro- Igs, including anti-dsDNA or anti-ssDNA Abs, and suffer from cess, called activation-induced suicide of T cells, is mainly mediated autoimmune diseases such as nephritis and vasculitis, particularly by Fas and FasL (22–24). The activated T cells express FasL, which in the mouse strain MRL. The lpr and gld mutations are loss of kills the activated cells by an autocrine or paracrine mechanism. The function mutations of Fas and FasL, respectively (12). Fas-null activated T cells in animals that have defects in Fas or FasL cannot mice, established by gene targeting, show phenotypes that are undergo activation-induced suicide, and they accumulate in the more severe than the leaky lpr mutation (13). Human patients car- periphery (20, 25). rying mutations in Fas or FasL have also been identified (14–19). In contrast to the lymphoproliferation of T cells, it is not clear how animals that are deficient in Fas and FasL produce autoantibodies. Activated B cells express Fas, and they are sensitive to *Department of Genetics, Osaka University Medical School, Yamadaoka, Suita; †Osaka Bioscience Institute, Furuedai, Suita; and ‡Osaka Medical Center for Maternal Fas-induced apoptosis. The number of B cells is increased in lpr and Child Health, Izumi, Osaka, Japan and Fas-null mice (13, 26), and a large number of autoantibody- Received for publication September 8, 1997. Accepted for publication December producing cells was found within the T cell zone of the spleen in 22, 1997. lpr mice (27). From these results, it was suggested that the auto- The costs of publication of this article were defrayed in part by the payment of page reactive B cells are killed through the interaction of FasL-express- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ing T cells with Fas-expressing B cells (28, 29). In this report we have established Fas transgenic MRL-lpr mice 1 This work was supported in part by grants-in-aid from the Ministry of Education, Science, and Culture of Japan, and by Special Coordination Funds from the Science in which Fas was specifically expressed in T cells but not in B and Technology Agency of the Japanese Government. cells. The mice did not accumulate Thy-1ϩ B220ϩ T cells, yet 2 Present address: The Burnham Institute, 10901 N. Torrey Pines Rd., La Jolla, CA produced large amounts of Igs, causing glomerulonephritis. These 92037. results indicate that lymphoproliferation of T cell origin and the 3 Address correspondence and reprint requests to Dr. Shigekazu Nagata, Department production of autoantibodies are independent processes caused by of Genetics, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan. E-mail address: [email protected] defective Fas in T cells and B cells, respectively, and support the 4 Abbreviations used in this paper: FasL, Fas ligand; gld, generalized lymphoprolif- proposal that the autoreactive B cells are removed, at least in part, erative disease; lpr, lymphoproliferation; PE, phycoerythrin; mFas, mouse Fas. by Fas-mediated apoptosis.

Materials and Methods 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide in 10 mM 1-methylimi- ϫ ϫ Transgenic mice dazole, pH 7.0. The plates were extensively washed with 5 SSC (1 SSC is composed of 150 mM NaCl and 15 mM sodium citrate) containing The expression vector, p1017 (30), carrying the proximal murine lck pro- 0.25% SDS at 55°C. Nonspecific binding sites on the DNA-coated plates moter and the human growth hormone gene sequence, was provided by Dr. were blocked by incubation for 60 min at room temperature with blocking R. M. Perlmutter (University of Washington, Seattle, WA). The coding solution (Tris-buffered saline (10 mM Tris-HCl, pH 7.4, and 140 mM sequence of mouse Fas cDNA (EcoRI-AccI fragment of pMF1) (31) was NaCl) containing 1% BSA and 3 mM EDTA). Mouse sera were diluted 100 inserted into the BamHI site of p1017 using a BamHI linker, and the re- times with the blocking solution, and 50-␮l aliquots were applied to the sulting construct was designated p1017Fas. The p1017Fas plasmid DNA DNA-coated plates and incubated for 60 min at room temperature. After was digested with NotI, and a 6.7-kb DNA fragment (5 ng/␮l) carrying washing three times with the washing buffer (Tris-buffered saline contain- mouse Fas cDNA between the lck promoter and human growth hormone ing 0.1% Nonidet P-40, 3 mM EDTA, and 1% gelatin), the plates were sequence was isolated. This fragment was microinjected into the pronuclei incubated for 60 min with peroxidase-conjugated goat anti-mouse Igs of fertilized eggs from MRL-lpr mice. Transgenic mice were screened by (Cappel) at a dilution of 1/4000. The peroxidase activity was detected using Southern blot analysis with mouse Fas cDNA as the probe, using tail DNA o-phenylenediamine as a substrate with a peroxidase-detecting kit (Sumi- prepared as previously described (32). Two transgene-positive lines were tomo, Tokyo, Japan), and the absorbance was measured at 492 nm using a established by backcrossing founder animals with MRL-lpr mice. The MicroElisa reader (Titertek Instruments, Huntsville, AL). presence of the transgene in progenitors was followed by PCR analysis of Immunohistochemistry tail DNA, using Fas cDNA-specific primers, the sense primer P (5Ј- GGAATTCCGCTGTTTTCCCTTGCTGCA-3Ј) and the antisense primer Kidneys from 5- to 7-mo-old mice were fixed with 4% paraformaldehyde ER (5Ј-CACAGTGTTCACAGCAGGA-3Ј). The conditions for PCR were in 0.1 M phosphate buffer, pH 7.2, containing 4% sucrose and embedded described previously (33). MRL/MpJ-lpr/lpr (MRL-lpr) mice were pur- in paraffin. Sections (4 ␮m thick) were prepared with a Microtome (Ya-

chased from SLC (Shizuoka, Japan). All mice were maintained under spe- mato Kohki, Saitama, Japan), mounted on silanized slide glasses, and Downloaded from cific pathogen-free conditions in an animal facility at the Osaka Bioscience deparaffinized. For immunohistochemistry, sections were incubated for 60 Institute. min at room temperature in PBS containing 0.1% Triton X-100 and 10% normal goat serum and were stained for 60 min at room temperature with Flow cytometry Ј Cy3-conjugated F(ab )2 of goat anti-mouse IgG (Jackson ImmunoResearch Cell suspensions were prepared from the lymphoid organs of 5-wk-old Laboratories, West Grove, PA) used at a dilution of 1/100. The sections mice as previously described (34). Splenocytes were depleted of erythro- were then washed three times with PBS containing 0.1% Triton X-100 and cytes by treatment for 1 min at room temperature with a lysis buffer (140 observed by the fluorescence microscopy (IX-70, Olympus, Melville, NY). http://www.jimmunol.org/ mM NH4Cl and 17 mM Tris-HCl (pH 7.2) containing 0.5% FCS (Life Technologies, Gaithersburg, MD). To activate B cells, the splenocytes Results were treated at 37°C for 48 h with 50 ␮g/ml LPS (Escherichia coli 026; Generation of transgenic MRL-lpr/lpr mice that express Fas in T B6, Sigma, St. Louis, MO) in RPMI 1640 medium supplemented with 10% cells FCS, 300 ␮g/ml L-glutamine, and 50 ␮M 2-ME. Naive or activated lymphocytes (1–10 ϫ 105 cells) were washed twice The murine lck proximal promoter works specifically in T cells, in in staining solution (PBS containing 2% FCS and 0.02% sodium azide), particular in thymocytes (30). To express Fas specifically in T ␥ incubated for 5 to 10 min at 4°C with anti-Fc II/III receptor Abs (2.4G2, cells, murine Fas cDNA was placed under the control of the lck PharMingen, San Diego, CA), and stained for 30 min at 4°C with Abs in staining solution. The Abs used were phycoerythrin (PE)-conjugated anti- promoter in a p1017 expression vector (30). This vector carries not

Fas (Jo2; PharMingen); FITC-, biotin-, or PE-conjugated anti-Thy-1.2 (53- only the lck promoter, but also introns and exons of the human by guest on September 25, 2021 2.1; PharMingen); FITC- or PE-conjugated anti-B220 (RA3-6B2; growth hormone gene. The human growth hormone sequence is in PharMingen); FITC-conjugated anti-CD8a (53-6.7; PharMingen); FITC- the noncoding sequence of the transgene and is used to increase the or PE-conjugated anti-CD4 (RM4-5; PharMingen); and biotin-conjugated Ј ␮ transgene expression (30). Transgenic animals were generated by F(ab )2 of goat anti-mouse IgM Ab ( -chain specific; Cappel Laboratories, Durham, NC). After staining, the cells were washed twice and, if neces- the injection of MRL-lpr mouse embryos with the 6.7-kb NotI sary, were stained with PerCP-streptavidin (Becton Dickinson, San Jose, fragment of p1017Fas. Two independent lines of Fas transgenic CA). The dead cells were stained with FITC-conjugated annexin V (R&D mice were established, and one of them was analyzed in detail. Systems, Minneapolis, MN) or propidium iodide, and flow cytometric analysis was performed on a FACScan flow cytometry (Becton Dickinson). Functional expression of Fas in the T cells of the transgenic The data were analyzed using CellQuest software (Becton Dickinson). mice Preparation of soluble FasL and killing assay Fas is expressed rather ubiquitously, with abundant expression in To produce the soluble mouse FasL (mFasL), BTS1 cells (monkey CV1 thymus, liver, and heart (31). The lpr mice carry a rearrangement cells carrying the temperature-sensitive SV40 T Ag) (35) were transformed in the Fas gene and hardly express Fas (38, 39). When tissues of with a mouse FasL expression plasmid, pEFMFLWX1 (34), as described Fas transgenic mice were analyzed by Northern hybridization with previously (36). The transformants were maintained in DMEM containing mouse Fas cDNA as the probe, the transgenic Fas mRNA of 3.9 kb 10% FCS at the nonpermissive temperature of 39.5°C. To generate culture supernatant fractions containing mFasL, the cells were grown for 4 days at was very strongly detected in the thymus and was weakly detected 33°C in DMEM containing 10% FCS, and mFasL was affinity purified in the spleen (data not shown). No transgenic Fas mRNA was using hamster mAbs against mouse FasL (T. Suda and K. Miwa, unpub- detected in other tissues, such as heart and liver. lished results). The cytotoxic activity of mFasL was determined using To examine which populations of lymphoid cells express Fas, mouse W4 cells as targets as previously described (34). The purified mFasL had a sp. act. of 3 ϫ 106 U/mg protein. the thymocytes, splenocytes, and lymph node cells were analyzed To determine their susceptibility to FasL-induced apoptosis, thymocytes by flow cytometry. As shown in Figure 1A, thymocytes from wild- or splenocytes from 5-wk-old mice were incubated at 37°C with various type mice uniformly expressed Fas, but no significant Fas expres- concentrations of mFasL in RPMI 1640 supplemented with 10% FCS, 300 sion was observed in thymocytes from lpr mice. On the other hand, ␮ ␮ g/ml L-glutamine, and 50 M 2-ME, and the dead cells were quantified most of the thymocytes from the Fas transgenic mice expressed by flow cytometric analysis using FITC-conjugated annexin V (R&D Sys- tems) according to the instructions provided by the manufacturer. Fas, although its expression level was heterogeneous and slightly reduced compared with that in wild-type mice. Similarly, the Serologic studies splenic naive T cells from wild-type, but not lpr, mice expressed Sera were prepared from 5- to 7-mo-old mice, and IgG1 and IgG2a in the Fas (Fig. 1B). The Fas transgenic mice expressed Fas in splenic T sera were quantified by single radial immunodiffusion using an Ig assay kit cells, and its expression level was higher and more heterogeneous (Serotec, Oxford, U.K.). The serum levels of the anti-dsDNA Abs were than that in wild-type mice. There was no significant difference in determined using an ELISA. In brief, pUC19 plasmid DNA was linearized ϩ ϩ with EcoRI. The DNA (5 ␮g/ml) was immobilized on CovaLink plates (37) Fas expression level between CD4 and CD8 thymocytes or ma- (Nunc, Copenhagen, Denmark) by treatment at 50°C for 5 h with 150 mM ture T cells from Fas transgenic mice (data not shown). Resting The Journal of Immunology 3807

FIGURE 1. Expression of Fas in lymphoid cells. Thymocytes and splenocytes were prepared from wild-type (thin line), transgenic (thick line), and lpr (dotted line) mice at age of 5 wk. A, Expression of Fas in thymocytes. Thymocytes were stained with PE-conjugated anti-Fas Abs and were analyzed by ﬂow cytometry as described in Materials and Methods. B, Expression of Fas in naive T cells. Freshly isolated splenocytes were stained with PE-conjugated anti-Fas Abs and biotin-conjugated anti-Thy-1.2 Abs. The cells were then stained with PerCP-conjugated streptavidin and analyzed by ﬂow cytometry. The ϩ

Fas expression in the Thy-1 cells is shown. C, Splenocytes were incubated at 37°C for 52 h with LPS and stained with PE-conjugated anti-Fas Abs and Downloaded from biotin-conjugated anti-mouse IgM Abs as described above. The Fas expression of IgMϩ cells is shown. naive B cells did express a low level of Fas, but its expression the Fas transgenic mice were effectively killed. As previously ob- could be induced by activation (7–9). As shown in Figure 1C, served (8, 9), the activated B cells from wild-type mice were sen- when the splenocytes were treated with LPS, the B cells (IgMϩ sitive to the Fas-induced apoptosis, and Ͼ70% of the cells were cells) from wild-type mice expressed Fas on their surfaces. A sim- killed within 24 h by mFasL (Fig. 2C). On the other hand, the http://www.jimmunol.org/ ilar treatment of splenocytes from the lpr or Fas transgenic mice LPS-activated B cells from lpr or Fas transgenic mice were resis- did not induce Fas expression in B cells. A similar result was tant to mFasL-induced cell death. These results indicate that Fas obtained with lymph node cells. These results indicate that the Fas expressed in the thymocytes and peripheral T cells in Fas trans- transgenic lpr mice speciﬁcally express Fas in thymocytes and genic mice was functional, but the mature B cells in these animals mature peripheral T cells, but not in B cells. could not be killed by the Fas-dependent apoptotic system. We then examined whether the Fas expressed in transgenic mice was functional. Previously, we showed that a recombinant mFasL No development of lymphadenopathy in Fas transgenic mice

(WX1) has a strong cytotoxic activity against thymocytes and na- MRL-lpr mice develop lymphadenopathy and splenomegaly in an by guest on September 25, 2021 ive T cells (34). We used this mFasL-induced cytotoxicity as an age-dependent manner. As shown in Figure 3, the size of the assay for the presence of functional Fas. First, we conﬁrmed our lymph nodes of MRL-lpr mice was more than 40 times larger than previous result with thymocytes and splenic T cells from MRL that of the wild-type mice at 17 wk of age. This lymphadenopathy mice. As shown in Figure 2, A and B, the thymocytes and naive T was greatly diminished in Fas transgenic mice (Fig. 3). The size of cells from spleens of wild-type MRL mice were sensitive to the lymph nodes in the transgenic mice was at most twice that in mFasL-induced apoptosis. In contrast, the thymocytes and naive T the wild-type mice. Similarly, the spleens of MRL-lpr mice were cells from lpr mice were not killed by mFasL; however, those from about 5 times larger than those of the wild-type mice at 17 wk of age. However, the spleens in Fas transgenic mice were, on the average, about 1.7 times larger than those in wild-type mice. The

FIGURE 2. FasL-induced apoptosis of lymphocytes. Thymocytes (A), naive splenocytes (B), and splenocytes activated with LPS (C) from wild- type (ϩ/ϩ), transgenic (Tg), and lpr (lpr) mice at 5 wk of age were incubated at 37°C for3h(A and B)or24h(C) with 5000 U/ml of mFasL. Thymocytes (A) were stained with annexin V alone, while naive and ac- FIGURE 3. No lymphadenopathy in MRL-lpr mice expressing Fas in T tivated splenocytes were stained with annexin V together with anti-Thy-1 cells. The axially and inguinal lymph nodes were excised from wild-type (B) or anti-IgM Abs (C) to represent T cells and B cells, respectively. The (ϩ/ϩ), transgenic (Tg), and lpr mice at the ages of 9 and 17 wk, and the percentages of the annexin V-positive dead cells were then determined average weights of ﬁve or six mice are shown as tissue weights (milli- using a ﬂow cytometer. grams) per body weight (grams). SD values are indicated by bars. 3808 TRANSGENIC EXPRESSION OF FAS IN T CELLS OF lpr MICE

cells (Thy-1ϩ B220Ϫ) was about 24 times higher in lpr mice than in wild-type mice, whereas, the number of T cells in the lymph nodes of Fas transgenic mice was almost comparable to that of the wild-type mice. From these results, we concluded that the loss of function mutation of Fas in lpr mice causes lymphoproliferation not only of T cells but also of B cells. The expression of Fas in T cells in the Fas-transgenic lpr mice prevents the accumulation of T cells but not that of B cells.

FIGURE 4. Flow cytometric analysis of lymphocytes from lymph Serum Ig levels, anti-DNA Ab, and autoimmune disease ϩ ϩ nodes. Lymphocytes from the lymph nodes of wild-type ( / ), transgenic MRL-lpr mice produce a large quantity of pathogenic IgG1 and (Tg), and lpr mice at the age of 17 wk were stained with FITC-conjugated IgG2a autoantibodies and show autoimmune diseases such as glo- anti-Thy-1.2 and PE-conjugated anti-B220 Abs. In each instance, 5000 merulonephritis and vasculitis (11, 40). To examine whether the viable cells were analyzed by ﬂow cytometry. Numbers indicate the percentages of positively stained cells in each quadrant. At least three mice for expression of Fas in T cells, but not that in B cells, affects auto- each genotype were analyzed. immunity in lpr mice, we compared the levels of serum Igs and autoantibodies. As shown in Figure 5, B and C, the serum levels of IgG1 and IgG2a in lpr mice at 5 to 7 mo of age were 2 to 3 times

higher than those in age-matched wild-type mice. This abnormal Downloaded from slight splenomegaly of the Fas transgenic mice was mainly due to production of Igs in lpr mice was more pronounced when the lev- the increased number of B cells (see below). els of the autoantibodies in the serum were compared. As shown in The cells that cause the lymphadenopathy and splenomegaly in Figure 5A, the level of Abs against dsDNA in lpr mice was 5 to 6 MRL-lpr mice are abnormal T cells that express Thy-1 and B220, times higher than that in wild-type mice. This high level of Igs and but neither CD4 nor CD8. As shown in Figure 4, 76% of lympho- autoantibodies was not diminished in the Fas transgenic mice cytes in the lymph nodes of lpr mice at 17 wk of age were these (Fig. 5). abnormal T cells. In contrast, the abnormal T cells in Fas trans- http://www.jimmunol.org/ MRL-lpr mice develop glomerulonephritis, which is caused by genic mice were only 6% of the total. When the total number of the deposit of immune complexes in the glomeruli. In fact, when abnormal lymphocytes was calculated, the prevention of lympho- the kidneys of the lpr mice were immunohistochemically analyzed proliferation in Fas transgenic mice was more apparent. As shown 9 ϩ ϩ with anti-Ig Abs, the glomerulus was strongly positive (Fig. 6). in Table I, a total of 1.1 ϫ 10 Thy-1 B220 abnormal T cells Histologic examination revealed symptoms of renal disease, were found in the lymph nodes of lpr mice. This population de- 5 namely glomerulonephritis with hypercellularity, lobularity, and creased to 6.6 ϫ 10 (1600-fold less) in Fas transgenic mice. focal sclerosis (data not shown). The deposit of Igs in the glomer- Accumulation of apparently normal B cells in Fas transgenic uli was not observed in age-matched wild-type MRL mice, but it mice was observed in glomeruli of the Fas transgenic mice (Fig. 6). by guest on September 25, 2021 ϩ ϩ Furthermore, glomerulonephritis in the Fas transgenic mice was as Although the population of B220 Thy-1 abnormal T cells was ϩ severe as that in lpr mice. From these results, we concluded that greatly reduced in Fas transgenic mice, the population of B220 Ϫ the defect of Fas in B cells leads to the accumulation of B cells Thy-1 cells was still higher in the lymph nodes of the transgenic producing autoantibodies, which causes autoimmune diseases such animals. At 17 wk of age, this population of the cells was 3% in the as glomerulonephritis. lymph nodes of the wild-type mice, and 35% in the transgenic mice (Fig. 4). Accordingly, the absolute number (6.1 ϫ 106 cells) of B220ϩ Thy-1Ϫ cells in the lymph nodes in Fas transgenic mice Discussion was about 40 times higher than that in the wild-type mice (Table In this report we describe the establishment of a line of Fas I). These B220ϩ Thy-1Ϫ cells appear to be normal B cells, since transgenic MRL-lpr mice that express Fas specifically in T they also express IgM on their surface (data not shown). The ac- cells. To express Fas specifically in T cells, we have used the cumulation of these cells was observed even in younger animals; mouse lck proximal promoter. The lck promoter was originally that is, the population of B220ϩ Thy-1Ϫ cells in the lymph nodes reported to operate predominantly in thymocytes (30). In fact, of Fas transgenic mice was 56% at 9 wk of age, while that of the Northern hybridization indicated that while the transgenic Fas wild-type mice was 4% (data not shown). mRNA was very strongly expressed in the thymus, it was very In contrast to the B cells, the accumulation of normal T cells in weakly expressed in spleen and lymph nodes. However, when lpr mice was almost completely rescued by expressing Fas in the Fas protein expressed on cell surfaces was analyzed by FACS, T cells. As shown in Table I, the number of apparently normal T significant expression was found not only in thymocytes but

Table I. Accumulation of apparently normal B cells in lymph nodesa

Lymph Nodes (Cell Number ϫ 105)

Genotype Thy-1ϩ B220Ϫ Thy-1Ϫ B220ϩ Thy-1ϩ B220ϩ

ϩ/ϩ 59.8 Ϯ 12.8 1.5 Ϯ 0.7 0.4 Ϯ 0.2 Tg 85.3 Ϯ 44.9* 60.7 Ϯ 31.6* 6.6 Ϯ 3.2* lpr 1,452.2 Ϯ 235.7 1,097.9 Ϯ 151.3 10,531.3 Ϯ 2,301.3

a Proportions of Thy-1ϩ B220Ϫ, Thy-1Ϫ B220ϩ, and Thy-1ϩ B220ϩ cells in the axillary and inguinal lymph nodes of ϩ/ϩ, Tg, and lpr in three 17-wk-old mice were determined by ﬂow cytometry as shown in Figure 4, and the number of each subpopulation was determined based on the total cell number and the percentage of each subpopulation from the dot plot data of ﬂow cytometry, gated out PI positive dead cells. * p Ͻ 0.01 in ϩ/ϩ and Tg. The Journal of Immunology 3809 Downloaded from

FIGURE 5. Production of Igs and autoantibodies. Sera were collected from 17- to 30-wk-old wild-type (ϩ/ϩ), transgenic (Tg), and lpr mice. The concentrations of anti-dsDNA (A), IgG1 (B), and IgG2a Abs (C) were determined as described in Materials and Methods. Results are shown as means of

the values obtained from 12 to 16 mice. SD values are indicated by bars. http://www.jimmunol.org/

also in the mature T cells of the transgenic mice, suggesting that Fas expression is also regulated post-transcriptionally in lymphocytes. The transgenic Fas expressed in thymocytes and peripheral T cells was functional, as the cells were efﬁciently killed by the soluble FasL. On the other hand, in agreement with the tight speciﬁcity of the lck promoter for T cells, the B cells of the transgenic mice did not express Fas. The activated B

cells, which normally are sensitive to Fas-induced apoptosis by guest on September 25, 2021 (9), remained resistant, as found with those from lpr mice. The T cell-specific expression of the transgenic Fas almost completely prevented accumulation of Thy-1ϩ, B220ϩ cells, while the production of autoantibodies and the occurrence of autoimmune diseases such as glomerulonephritis were still observed. Recently, Komano and Shinohara established a line of Fas transgenic MRL-lpr mice that specifically expresses Fas in B cells (N. Shinohara, unpublished observations). These mice developed lymphadenopathy and splenomegaly, but they neither produced autoantibodies nor developed autoimmune disease. Moreover, when Fas was expressed in both T cells and B cells in MRL-lpr mice, neither lymphoproliferation nor autoimmune disease was observed (H. Fukuyama and S. Nagata, unpublished observation). These results indicate that the lymphoproliferation of Thy-1ϩ B220ϩ cells and the production of autoantibodies in MRL-lpr mice are distinct processes. The defect of Fas in T cells causes lymphoproliferation, while the defect of Fas in B cells is responsible for the production of autoantibodies and autoimmune diseases, although the abnormal T cells in lpr mice may further accelerate the disease. This result is in contrast to the previous report by Wu et al. (41) in which they claim that the expression of Fas in the T cells of MRL-lpr mice is sufficient to correct the autoimmune disease. They used the mouse CD2 promoter to express Fas in T cells. Since the expression of CD2 is not strictly restricted to the T cells (42, 43), we think that their transgenic mice expressed Fas FIGURE 6. Development of glomerulonephritis. The kidney sections not only in T cells but also in B cells. Chimeras containing a were prepared from the wild-type (ϩ/ϩ), transgenic (Tg), and lpr mice at mixture of lpr- and normal-derived lymphoid cells were also the age of 6 mo. The sections were stained with Cy3-conjugated anti- constructed. Since the injection of anti-Thy 1 Ab specifically mouse IgG and observed using fluorescence microscopy. Original magni- recognizing the lpr T cells rescued the autoimmune disease, fication, ϫ200. Sobel et al. (44) concluded that lpr T cells are responsible for 3810 TRANSGENIC EXPRESSION OF FAS IN T CELLS OF lpr MICE the production of autoimmune Abs. The transgenic mice ex- 9. Watanabe, D., T. Suda, and S. Nagata. 1995. Expression of Fas in B cells of the pressing Fas in T cells carry 1600-fold less Thy-1ϩ B220ϩ T mouse germinal center and Fas-dependent killing of activated B cells. Int. Im- munol. 7:1949. cells than lpr mice, but this number is still 15 times higher than 10. Ka¨gi, D., F. Vignaux, B. Ledermann, K. Bu¨rki, V. Depraetere, S. Nagata, that found in wild-type mice. Whether these abnormal T cells H. Hengartner, and P. Golstein. 1994. Fas and perforin pathway as major mech- anisms of T cell-mediated cytotoxicity. Science 265:528. contribute to the production of autoantibodies in the transgenic 11. Cohen, P. L., and R. A. Eisenberg. 1991. Lpr and gld: single gene models of mice remains to be studied. systemic autoimmunity and lymphoproliferative disease. Annu. Rev. Immunol. How does the defect of Fas in B cells lead to the production 9:243. 12. Nagata, S., and T. Suda. 1995. Fas and Fas ligand: lpr and gld mutations. Im- of autoantibodies? B cells present Ags to CD4 T cells and ac- munol. Today 16:39. tivate them to express CD40 ligand and FasL. The CD40 ligand 13. Adachi, M., S. Suematsu, T. Suda, D. Watanabe, H. Fukuyama, J. Ogasawara, then activates the B cells to express Fas and makes them sen- T. Tanaka, N. Yoshida, and S. Nagata. 1996. Enhanced and accelerated lymphoproliferation in Fas-null mice. Proc. 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Fas from autoimmune diseases (47–49) similar to those observed in gene mutations in the Canale-Smith syndrome, an inherited lymphoproliferative the MRL-lpr transgenic mice expressing Fas in T cells. The Lyn disorder associated with autoimmunity. N. Engl. J. Med. 335:1643. kinase is involved in signal transduction from CD40, and the 19. Sneller, M. C., J. Wang, J. K. Dale, W. Strober, L. A. Middelton, Y. Choi,

T. A. Fleisher, M. S. Lim, E. S. Jaffe, J. M. Puck, M. J. Lenardo, and S. E. Straus. http://www.jimmunol.org/ inability of B cells to express Fas in lyn-null mice has recently 1997. Clinical, immunologic, and genetic features of an autoimmune lympho- been demonstrated (50). In addition, the autoimmune diseases proliferative syndrome associated with abnormal lymphocyte apoptosis. Blood ␣␤ 89:1341. in MRL-lpr mice were shown to occur by non- T cells by a 20. Watanabe, D., T. Suda, H. Hashimoto, and S. Nagata. 1995. Constitutive activation CD40 ligand-dependent or -independent mechanism (51). It of the fas ligand gene in mouse lymphoproliferative disorders. EMBO J. 14:12. will be interesting to examine whether the autoimmune disease 21. Giese, T., and W. F. Davidson. 1992. Evidence for early onset, polyclonal acti- ␣␤ vation of T cell subsets in mice homozygous for lpr. J. Immunol. 149:3097. (renal failure) in the Fas transgenic mice is caused by T cells 22. Dhein, J., H. Walczak, C. Baümler, K.-M. Debatin, and P. H. Krammer. 1995. or non-␣␤ T cells, and whether it is CD40 ligand dependent. In Autocrine T-cell suicide mediated by APO-1/(Fas/CD95). Nature 373:438. any case, our results agree with the proposal that T cells not 23. Brunner, T., R. J. Mogil, D. LaFace, N. J. Yoo, A. Mahboubi, F. Echeverri, S. J. Martin, W. R. Force, D. H. Lynch, C. F. Ware, and D. R. Green. 1995. only help B cells to proliferate and differentiate, but also kill Cell-autonomous Fas (CD95)/Fas-ligand interaction mediates activation-induced by guest on September 25, 2021 them through the Fas/FasL system (45). In addition to autore- apoptosis in T-cell hybridomas. Nature 373:441. active B cells, B cells reactive to foreign Ags undergo apoptosis 24. Ju, S.-T., D. J. Panka, H. Cui, R. Ettinger, M. El-Khatib, D. H. Sherr, B. Z. Stanger, and A. Marshak-Rothstein. 1995. Fas (CD95)/FasL interaction upon activation in the germinal centers (52). 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