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The Journal of Immunology, 2000, 164: 1681-1688.
Copyright © 2000 by The American Association of Immunologists

Transgenic Expression of Fas Ligand on Thyroid Follicular Cells Prevents Autoimmune Thyroiditis1

Frédéric Batteux*,{ddagger}, Patrick Lores{dagger}, Danièle Bucchini{dagger} and Gilles Chiocchia2,*

* Institut National de la Santé et de la Recherche Médicale (INSERM) U477 and {dagger} INSERM U257, Université René Descartes, Paris, France; and {ddagger} Laboratoire d’Immunologie, Hôpital Cochin, 5016 Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
"Immune privilege" is defined as tissue resistance to aggression by specifically activated lymphocytes, and involves the interaction between Fas expressed on infiltrating cells and Fas ligand (FasL) constitutively expressed on the target tissue. To test whether ectopic expression of FasL on thyrocytes could prevent autoimmune aggression of the thyroid by activated lymphoid cells, three lines of transgenic mice expressing low, intermediate, and high levels of functional FasL on thyroid follicular cells were generated. Experimental autoimmune thyroiditis was induced by immunization with mouse thyroglobulin. In all of the experiments, the effects were dependent on the level of FasL expression. Low and intermediate expression had no or only weak preventive effects, respectively, whereas high FasL expression strongly inhibited lymphocytic infiltration of the thyroid. Anti-mouse thyroglobulin-proliferative and cytotoxic T cell responses, as well as autoantibody production, were diminished in transgenic mice expressing high levels of FasL relative to controls. Furthermore, in these latter mice Th1 responses to mouse thyroglobulin were profoundly down-regulated, uncovering a new potential role for FasL in peripheral tolerance to organ-specific Ags. In sum, the prevention of experimental autoimmune thyroiditis by FasL on thyrocytes is dependent on the level of FasL expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Adaptive immunity involves specific recognition of Ags by B and TCRs. The diversity of Ag receptors is based on random combinations of gene segments during T and B cell maturation. Thus, in addition to lymphocytes recognizing extrinsic Ags, clones of lymphocytes recognizing self-Ags are constantly generated. These autoreactive lymphocytes specific for ubiquitously expressed self-Ags are inhibited or eliminated in the thymus and bone marrow, resulting in natural T and B cell tolerance. Tolerance to tissue-specific self-Ags by mature lymphocytes is mainly acquired in the periphery.

Natural tolerance of autoreactive cells results from either clonal deletion or anergy. Clonal deletion occurs through apoptotic lymphocyte death, a phenomenon that controls the size of the activated T cell compartment. A breakthrough in our understanding of the regulatory mechanisms of apoptosis in autoimmunity came from studies of mice with lpr (lymphoproliferation) and gld (generalized lymphoproliferative disease) mutations (1, 2, 3, 4). Both mouse strains have expanded lymphocyte compartments with an unusual surface phenotype and a variety of autoimmune reactivity. Lpr and gld are complementary mutations of a receptor, Fas (CD95), a member of the TNF receptor family, and of its ligand FasL (CD95L).3 Mutations of Fas have also been linked to autoimmune lymphoproliferative syndromes in humans (5, 6, 7). Fas appears on the surface of activated lymphocytes and is able to transduce an apoptotic signal through its cytoplasmic "death domain " upon cross-linking with FasL. Fas-induced cell death may occur following cis- or trans-interaction with FasL (8).

The presence of FasL on stromal cells of the eye and on Sertoli cells of the testes confers so-called "immune privilege" on these tissues (9, 10, 11). When activated inflammatory cells enter the eye or testis, they are rapidly killed through the Fas-FasL apoptotic pathway. It has also been proposed that tumor cells expressing FasL use a similar mechanism to eliminate aggressive T cells (12). Thus, the expression of FasL on particular tissues can be responsible for a state of tolerance due to the deletion of specifically activated lymphocytes. This led us to postulate that ectopic tissue expression of FasL might be used experimentally to abrogate an autoimmune response.

In this study, we investigated the ability of transgenic FasL expression on thyroid follicular cells (TFC) to prevent autoimmune destruction of the thyroid during experimental autoimmune thyroiditis (EAT). This murine model of Hashimoto’s thyroiditis involves immunization with mouse thyroglobulin (MTg) and adjuvant, and is characterized, like its human counterpart, by autoreactive T and B cell responses and marked lymphocytic infiltration of the thyroid. Splenocytes from mice primed with MTg and adjuvants and restimulated in vitro by MTg can also induce EAT after transfer to irradiated recipients (13). Transgenic mice expressing FasL on their TFC were generated and tested for their susceptibility to experimentally induced thyroiditis. Relative to controls, thyroid infiltration was markedly slowed and attenuated in mice with high expression of FasL on TFC after active induction of EAT. In addition, the anti-Tg cytotoxic T cell response was diminished, IFN-{gamma} production by lymph node cells (LNC) stimulated with MTg was abrogated, and the titers of anti-Tg IgG Abs were significantly reduced.

Thus, FasL expression on TFC confers immune privilege to thyroid and induces tissue-specific peripheral tolerance through the deletion of autoreactive lymphoid clones.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Female CBA/J mice were purchased from Iffa Credo (L’Arbresle, France) and were used at 7–10 wk of age in all experiments. They were maintained in standard environmental conditions with free access to food and water, and were allowed to adapt to their environment for 1 wk before the experiments. Thyroglobulin promoter (pTg)-FasL-transgenic mice were produced by microinjection of FasL constructs into (CBA/J x C57BL/6)F1 fertilized eggs and implantation into pseudopregnant foster mothers.

The construct was obtained by subcloning the EcoRV-KpnI fragment of rat FasL (a gift from S. Nagata, Osaka University, Osaka, Japan) contained in the pBluescript vector (Stratagene, Cambridge, U.K.) into the EcoRV-KpnI sites of the pTg-Gs{alpha} (14) (a gift from D. Christophe, Université Libre de Bruxelles, Brussels, Belgium and J. Feunteun, Centre National de la Recherche Scientifique, Unité de Recherche Associée 1967, Villejuif, France). Three selected transgene-positive founders were crossed with CBA/J mice. Transgenic I-Ak-positive I-Ab-negative mice were then crossed twice with CBA/J mice. Mice were screened for pTg-FasL transgenes by PCR analysis of tail DNA using the following primers: pTg sense 5'-GCCTCCACAAGATTTTCACC-3' and FasL antisense 5'-TGGTAGTGGTGATGGAGGTG-3'. A 450-bp PCR product was obtained in transgenic mice. Only female mice were used for this study because of the female prevalence of experimental autoimmune thyroiditis (EAT).

RT-PCR

Whole thyroids were homogenized, and RNA was prepared by guanidine isothiocyanate-acid phenol extraction. Total RNA was treated with DNase I (Boehringer Mannheim, Mannheim, Germany), and 0.5 µg of RNA was used in a first-strand cDNA synthesis round using oligo(dT) primers. PCR on one-twentieth of the cDNA reaction product generated a 545-bp fragment of rat FasL (sense primer 5'-AAGGACAACATAGAGCTGTG-3', antisense 5'-AAATGGTCAGCAACGGTAAG-3'), a 542-bp fragment of mouse FasL (sense primer 5'-AGGACCACAACACAAATCTG-3', antisense 5'-GGTCAGCACTGGTAAGATTG-3'), or a 348-bp fragment of murine ß actin (sense primer 5'-TGGAATCCTGTGGCATCCATGAAAC-3', antisense 5'-TAAAACGCAGCTCAGTAACAGTCCG-3'). The RT-PCR products were subjected to electrophoresis on a 2% agarose gel and stained with ethidium bromide.

A20 killing assay

The Fas-sensitive lymphoma cell line A20 was used to measure the killing activity of transgenic thyrocytes. Cryostat sections were prepared from thyroids of transgenic and nontransgenic animals. About 15 sections were deposited on a 2-cm in diameter glass slide. Then 2 x 105 A20 target cells were incubated with the thyroid sections. After 12 h at 37°C, the percentage of apoptotic A20 cells was determined by labeling with FITC-conjugated annexin V. Cells were analyzed by flow cytometry using a Coulter XL apparatus (Coulter Pharmaceutical, Margency, France).

Immunization of animals

Homemade MTg was emulsified in CFA for immunization on day 0 and in incomplete Freund’s adjuvant for challenge on day 14. The CFA suspension, which contained 1 mg/ml Mycobacterium tuberculosis strain H37Ra (Difco, Detroit, MI), was injected intradermally with 100 µg of MTg. Animals were killed at various times postimmunization, as specified in the text.

Histopathological studies of thyroid specimens

The histological grade of EAT was assessed by blind evaluation of thyroid specimens by three persons. Infiltration indexes were determined on 5-µm-thick sections stained with Masson Goldner’s trichrome solution. EAT was graded as a function of mononuclear cell infiltration of the thyroid as follows: grade 1, interstitial accumulation of inflammatory cells distributed around one or two follicles; grade 2, one or more foci of inflammatory cells reaching at least the size of one follicle; grade 3, 10–40% of the thyroid replaced by inflammatory cells; and grade 4, >40% of the thyroid replaced by inflammatory cells. The results are given as a mean ± SEM of the three individual evaluations of 7–18 mice/group.

In vitro cytotoxic responses to Tg-pulsed syngeneic macrophages

On day 28 postimmunization, spleen cells were suspended at a density of 5 x 106/ml in complete medium and then cultured in 100-mm petri dishes with 40 µg/ml MTg and 1 nM recombinant IL-2 at 37°C for 4 days. At the end of the culture period, the cells were harvested, washed twice in HBSS, and used as effector cells. Peritoneal macrophages from thioglycolate-injected CBA/J mice were collected in HBSS-10% FCS, washed twice, and counted after staining with neutral red. Pelleted cells were then labeled with 100 µCi of 51Cr/106 cells. After a 1-h incubation at 37°C with shaking, cells were washed twice in HBSS-10% FCS, and 104 macrophages were distributed in each well of flat-bottom 96-well plates (model 3799; Costar, Cambridge, MA). A total of 50 µg of MTg was added in a volume of 100 µl for 4 h. The pulsed macrophages were then washed with HBSS, and 100 µl of effector cells at densities of 2.5, 5, 10, and 20 x 105 cells/ml was added. After a 6-h incubation, 100 µl of supernatant was collected and chromium release was measured in a gamma scintillation counter (LKB, Bromma, Sweden). Spontaneous release was always below 21%. Spontaneous and maximal release values were defined by incubation of target cells with culture medium in the absence or presence of Triton X-100 detergent (5% v/v in Tris buffer), respectively.

In vitro proliferative and cytokine responses of LNC to MTg

In vitro proliferative responses to Ag were measured by culturing 4 x 105 LNC with 40 µg/ml MTg in 200 µl for 72 h. At the beginning of the culture, the CD4-CD8 ratios were identical in transgenic and nontransgenic mice as assessed by flow cytometry analysis. Cells were pulsed with 0.5 µCi of [3H]thymidine for the final 12 h and then harvested for liquid scintillation counting. Culture supernatants were collected on days 1–3 for IL-2 assay using the IL-2-dependent CTLL-2 cell line, and for IFN-{gamma} assay by a two-site ELISA using R46-A2 mAb as coating Ab and ß-galactosidase-coupled XMG1-2 mAb as developing Ab. The detection limit for IFN-{gamma} was 250 pg/ml.

Titers and isotypes of Abs to MTg

Mice were bled either by retro-orbital puncture or by cardiac puncture at the time of death. Sera were stored at -20°C until use. Abs to MTg were detected by means of an ELISA method as described previously (15). Briefly, flat-bottom microtiter plates (model 3590; Costar) were coated overnight with 50 µl of MTg (20 µg/ml) at 4°C and then washed twice with PBS-Tween 20. Free protein binding sites were blocked by adding PBS-1% BSA for 2 h at 37°C. Serial 10-fold dilutions (1/102–1/106) of sera from individual mice were incubated overnight at 4°C. After extensive washing of the plates, 1/5000 alkaline phosphatase-conjugated goat anti-mouse IgG (Miles-Yeda, Rehovot, Israel) in PBS-Tween 20 was added as the second Ab, and the colorimetric reaction was revealed by substrate addition. The plates were read at 405 nm with a Titertek multiscan spectrophotometer (Dynatech MR 5000; Dynatech Laboratories, , Guyancourt, France). The isotypes of Abs to MTg were determined in individual sera (serial 10-fold dilutions from 1/103–1/105) using alkaline phosphatase-conjugated goat anti-mouse IgG1, IgG2a, IgG2b, and IgG3 as second Abs (Southern Biotechnology Associates, Birmingham, AL). Dilutions of 1/102–1/104 were used to assay IgG3 Ab. The concentrations of IgG, IgG1, IgG2a, and IgG2b Abs were determined in arbitrary units defined as the amount of anti-Tg Ab providing the same OD as a standard serum diluted 1/106. One unit of IgG3 was defined by the OD provided by 1/104 diluted standard serum. The standard serum was a pool of mouse sera with high concentrations of anti-Tg Abs.

Statistics

Significant differences between groups were identified by using Student’s two-tailed t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of transgenic mice expressing FasL on thyroid follicular cells

To determine how local FasL expression can affect the immune response to thyroid Ags, we generated transgenic (TG) mice expressing rat FasL cDNA driven by the bovine thyroglobulin promoter (pTg-FasL mice) (Fig. 1GoA). Three founder lines were backcrossed once with CBA/J mice, and I-Ak+ I-Ab- (highly susceptible haplotype) transgenic mice were backcrossed twice with CBA/J mice. Using specific primers allowing us to discriminate between mouse and rat FasL, we observed FasL transgene expression in thyroid but not in other organs by means of RT-PCR (Fig. 1Go, B and C). Normal mouse thyroid did not express FasL constitutively. Functional expression of the gene in transgenic mice was shown in a Fas-sensitive A20 cell killing assay (12). The three transgenic lines TG6, TG9, and TG11 expressed low, medium, and high levels of functional FasL, respectively (Fig. 1GoD). This was confirmed by Western blot analysis using two different anti-FasL polyclonal Abs (data not shown). The three lines were named TG6low, TG9med, and TG11high according to FasL expression. pTg-FasL mice were monitored for more than 18 mo, during which they grew normally. None of them developed spontaneous thyroiditis, and examination of thyroid sections showed no abnormalities (Fig. 2GoA).



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  		FIGURE 1. pTg-FasL transgene construction, specificity of expression, and functional activity. A, Rat FasL cDNA was introduced into a construct containing the pTg and was introduced by microinjection into (CBA/J x C57BL/6)F1 fertilized eggs. B, FasL is expressed in transgenic mouse thyroid. Normal and transgenic thyroid and testis were collected and processed for RNA extraction. After DNase treatment, the samples were processed for cDNA synthesis and the presence of FasL-encoding cDNA was tested for by PCR using primers specific for mouse or rat FasL. The rat transgene is expressed only in mouse thyroid. Endogenous mouse FasL is expressed in the testis not in thyroid. C, Transgenic FasL is specifically expressed in thyroid. The presence of FasL was tested for as described in B in various tissue samples. D, The three transgenic pTg-FasL lines selected expressed different levels of functional FasL. The ability of transgenic thyrocytes to kill Fas-sensitive A20 lymphoma cells was used as a measure of their functional FasL expression. The percentage of apoptotic A20 cells was determined by labeling with FITC-conjugated annexin V after 12 h of contact. The sensitivity of A20 cells to Fas-induced apoptosis was measured by incubating the cells with 0.5 µg of anti-Fas Ab (Jo2).

 


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  		FIGURE 2. FasL expression by TFCs does not induce changes in the thyroid and protects mice from thyroiditis. A, Thyroid sections (original magnification, x100) showed no differences between CBA/J and transgenic mice. B, Representative histological aspect of thyroids on day 28 after immunization with MTg in nontransgenic and FasL-transgenic female mice. Note the minimal infiltration in the thyroid of TG11high mice.

 
FasL expression on thyroid protects mice against EAT

To determine whether transgene expression altered the course of autoimmune thyroid disease, transgenic and nontransgenic mice were immunized with MTg and then killed 9, 21, and 28 days later to follow the course of the disease. The results are summarized in Table IGo. The thyroids of all of the transgenic and control mice were devoid of infiltrating cells on day 9 (data not shown). On day 21, a marked lymphocytic infiltrate was observed in the thyroids of CBA/J and TG6low mice, whereas milder infiltrates were detected in the thyroids of TG9med and TG11high animals (p < 0.01, TG11high compared with CBA/J). On day 28 postimmunization, when the disease reaches its peak, nontransgenic mice developed the lymphocytic infiltrates usually observed in this model, whereas TG11high mice had a significantly lower infiltration index (p < 0.02 compared with CBA/J) (Table IGo and Fig. 2GoB). In contrast, the TG6low line generally developed a more acute disease and, on day 28, more granulocytes were found than in nontransgenic animals (Fig. 2GoB). No granulomas were detected in any mice. On day 28, the TG9med line had an infiltration index identical to that of nontransgenic animals. Some immunized TG9med mice had hypertrophic thyroid follicules. All of the nonimmunized animals and those treated with adjuvant alone had normal thyroids (data not shown).


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  		Table I. Effect of TFC FasL expression on EAT

 
Thus, high expression of FasL on thyroid cells prevented lymphoid infiltration of the gland, whereas low expression of FasL had adverse effects.

pTg-FasL-transgenic mice have reduced Tg-specific cytotoxic T cell responses

Since thyroglobulin-specific cytotoxic T cells are effector cells in EAT, their presence was tested for in pTg-FasL mice. The splenic cytotoxic T cell response toward MTg-pulsed syngeneic macrophages was measured on day 28 postimmunization. Cytotoxic T cells were detected in splenocytes from all groups of immunized animals, and TG6low-immunized mice had the same number of cytotoxic T cells as CBA mice (Fig. 3Go). Thus, the exacerbated disease observed in TG6low mice was not linked to an increase in the anti-MTg cytotoxic T cell response. Conversely, high expression of functional FasL (TG11high) induced a lower anti-Tg cytotoxic T cell response than in nontransgenic mice (Fig. 3Go). Intermediate expression of FasL (TG9med) TFC resulted in a less pronounced effect. Thus, transgenic FasL expression in the thyroid induced systemic elimination of Tg-reactive cytotoxic T cells.



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  		FIGURE 3. Cytotoxic T cell responses to MTg-pulsed syngeneic macrophages. Groups of CBA/J and transgenic mice (three to four mice per group) were immunized and boosted on day 14 with MTg. Two weeks later, spleens were harvested and single-cell suspensions were prepared from each mouse. Cells were cultured in vitro as described in Materials and Methods.

 
Abrogation of IFN-{gamma} production by LNC from MTg-immunized pTg-FasL mice

To test whether the systemic CD4+ T cell response was also affected by FasL expression on TFC, proliferative and cytokine responses of LNC from MTg-immunized mice were compared. On day 9 after immunization, LNC from pTg-FasL and CBA mice proliferated equally in response to in vitro stimulation by MTg (Fig. 4GoA) and also by purified protein derivative and Con A as well (data not shown). On day 9, IL-2 production by MTg-stimulated LNC from transgenic and nontransgenic mice mirrored the proliferative response (Fig. 4GoB). Thus, transgenic FasL expression in the thyroid induced neither nonspecific systemic immunosuppression nor anergy of MTg-specific T cells. In contrast, IFN-{gamma} production by MTg-stimulated LNC from TG9med and TG11high but not TG6low mice was almost totally abrogated (Fig. 4GoB). No IL-4, IL-5, or IL-10 was detected in any of the cultures (data not shown).



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  		FIGURE 4. Proliferation and cytokine protein production by MTg-activated LNC from FasL-transgenic and nontransgenic mice. A, On day 9 or 21 after priming, in vitro proliferative responses were measured by culturing 4 x 105 LNC in the presence of 40 µg/ml MTg for 72 h. The CD4/CD8 ratios were identical in transgenic and in nontransgenic mice as assessed by flow cytometry analysis. Cells were pulsed with [3H]thymidine for the final 12 h and then harvested for liquid scintillation counting. B, Culture supernatants were collected on day 1 for IL-2 production and on day 3 for IFN-{gamma} production. IL-2 was assessed using IL-2-dependent CTLL-2 and IFN-{gamma} by ELISA. Results are individual cpm ± SEM from five mice per group.

 
On day 21 postimmunization, MTg-stimulated LNC from TG9med and TG11high mice showed significantly lower MTg-specific proliferation and IL-2 production than those from nontransgenic animals (Fig. 4Go, A and B). IFN-{gamma} production remained abolished in MTg-stimulated LNC from TG9med and TG11high mice (Fig. 4GoB), whereas Con A-induced IFN-{gamma} production was never affected (data not shown).

Thus, FasL expression in the thyroid led to selective down-regulation of a Th1-type response to MTg shown by early abrogation of IFN-{gamma} production. Later, it attenuated proliferation and IL-2 and IFN-{gamma} production in response to MTg stimulation.

Effects of FasL expression on the anti-MTg B cell response

Because humoral tolerance of thyroglobulin is very weak in normal conditions, we examined the impact of TFC-FasL expression on Ab responses in the serum of animals immunized with MTg at various times postimmunization. Two different patterns of Ab response were noted (Table IIGo and Fig. 5Go). CBA and TG6low mice exhibited similar levels of anti-MTg IgG Abs, whereas TG9med and TG11high mice secreted significantly smaller amounts of anti-MTg IgG Abs (p < 0.05 and p < 0.01, respectively, compared with nontransgenic animals on day 21). All of the IgG subclasses were diminished in TG11high mice. In TG9med mice, there was a sharp drop in the anti-Tg IgG2a and IgG2b Ab titers (p < 0.001 and p < 0.02, respectively) compared with CBA mice, whereas the IgG1 titer was not affected. These results were reminiscent of the altered cytokine production by LNC. Four weeks after immunization, some TG6low mice produced large amounts of anti-Tg IgG3 subclass Ab. Although not statistically significant, the latter result might be linked to the slightly higher anti-MTg IgG titers observed in the TG6low group, raising the possibility that these autoantibodies contribute to the pathophysiology of the lesions observed in these mice.


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  		Table II. MTg-specific Abs in sera from animals immunized with MTg on day 21 postimmunization

 


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  		FIGURE 5. Modulation of anti-Tg Ab responses in transgenic FasL mice. Sera were analyzed for Tg-specific Igs by ELISA. The presence of serum Ig was tested for on days 13, 21, and 28 after immunization. The results are means ± SEM of data obtained in one experiment with seven mice per group. Results are expressed in arbitrary units by reference to a standard curve obtained with a pool of sera from Tg-immunized CBA/J mice. Each serum was tested using serial 10-fold dilutions for the IgG titer (1/103–1/107) and 3 dilutions for the different isotypes (1/103–1/105). We used 1/102–1/104 dilutions for IgG3 levels.

 
Thus, FasL expression on thyroid cells had a marked impact on the isotypes and titers of anti-MTg Abs.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We used three transgenic lines of mice expressing increasing levels of FasL to show that FasL expression on mouse thyrocytes can prevent EAT in a "dose-dependent" manner.

On days 21 and 28 after immunization with Tg and adjuvant, thyroid infiltration was minimal in animals expressing high levels of FasL compared with controls and to mice with intermediate or low FasL expression. In mice with low FasL expression, the infiltrates were composed of lymphocytes initially and neutrophils later. Granulocytes have already been observed in tissues expressing FasL, and their presence can be explained by the chemoattractant properties of the molecule (16, 17, 18). However, neutrophils were not observed in the thyroid of mice with intermediate FasL expression, probably because of their susceptibility to apoptosis. Thus, thyroid infiltration correlated strictly with the level of FasL expression on TFC.

Rat insulin promoter-FasL-transgenic nonobese diabetic mice with high levels of FasL are protected against spontaneous diabetes, but transfer of diabetogeneic CD8+ T cell clones into these mice induces intrapancreatic infiltrates, followed by ß cell death and diabetes (19). These effects have been attributed to Fas and FasL coexpression on ß cells that are highly susceptible to Fas-mediated death (16, 19, 20). Similarly, in humans, Giordano et al. (21) proposed that Fas and FasL coexpression on thyrocytes upon IL-1 stimulation could result in the destruction of the thyroid gland. However, recent reports have shown that TFC are resistant to Fas-mediated cell death through expression of antiapoptotic proteins (22, 23). Furthermore, Dayan et al. (24) have suggested that FasL expression on TFC does not threaten the cells but can have a protective effect. This is in keeping with our results showing that a certain level of FasL is necessary to prevent thyroid infiltration.

Since EAT is a T cell-mediated disease, we investigated whether activated autoreactive T cell functions were affected by FasL expression on TFC. The Tg-specific cytotoxic response was significantly decreased in transgenic animals compared with control mice. The effect was more marked in mice expressing the highest level of functional FasL. This is in agreement with recent data from our laboratory on the role of autoreactive CD8+ T cells in the pathophysiology of EAT and Fas-mediated deletion of these cells in the maintenance of self-tolerance (15). Furthermore, Kurts et al. (25) established the role of Fas in deleting autoreactive CD8+ T cells activated by cross-presentation of exogenous self-Ag. Therefore, it is not surprising that in our model the transgenic FasL expression on TFC can result in the deletion of autoreactive CD8+ T lymphocytes.

The control of CD4+-autoreactive T cell responses through Fas-FasL interaction has also been established. Therefore, we next studied whether FasL expression on TFC altered anti-Tg CD4+ T cell responses. On day 21 after immunization, proliferative responses to Tg, and IL-2 production in the supernatants of Tg-stimulated lymph node (LN) T cells, were significantly decreased relative to controls in transgenic animals expressing intermediate and high levels of FasL. This abrogation of the CD4+ T cell response could be due to the deletion or inactivation of autoreactive T cells before or after immunization, as already established in vivo using TCR-transgenic models (26). Indeed, before immunization, CD4+-autoreactive T cells trafficking through the thyroid could be activated and then deleted if FasL is expressed on TFC. In such conditions, immunization would fail to induce EAT through a lack of responsive clones. In our experiments, the EAT resulting from the transfer of activated splenocytes of transgenic donors into nontransgenic recipients was weaker than when splenocytes from controls were used, but it still occurred, suggesting that full deletion of anti-Tg-autoreactive T cells had not taken place before immunization (data not shown). Another possibility is that FasL expression on TFC may eliminate normally autoreactive T cells, whereas T cells with TCR of lower avidity could evade deletion. The role of functionally hyporesponsive T cells with low-avidity TCR has already been suggested in a number of autoreactive, superantigen-mediated models of tolerance (27, 28, 29, 30, 31, 32). This is improbable in our model, however, because on day 9 after immunization, when no detectable thyroid infiltration had yet occurred, the proliferative responses of LN T cells were the same regardless of the FasL expression level. These results indicate that the early T cell response was not affected by FasL expression on TFC. Therefore, because we cannot rule out the possibility that a decrease in TCR sensitivity or a deletion of autoreactive T cells occurs before immunization, our results rather favor the hypothesis that infiltrating autoreactive lymphocytes are deleted after Tg immunization.

As in other models of T cell-mediated autoimmune diseases, cytokines play a pivotal role in the pathogenesis of EAT. Therefore, we examined whether the protection against EAT in our model was due to a change in cytokine production by Tg-specific T cells. IFN-{gamma} production by Tg-stimulated LN T cells from transgenic mice with the highest level of FasL was almost totally abolished. This could be responsible for the protection against EAT, as Th1 cytokines, and particularly IFN-{gamma}, are highly pathogenic, as shown by the preventive effect of both mAbs to IFN-{gamma} and the Th2 cytokine IL-10 (33, 34, 35, 36). Furthermore, IFN-{gamma} receptor knockout mice are partially resistant to EAT (37). Since IL-4 was undetectable in the supernatants of Tg-stimulated LN T cells or spleen cells (data not shown), we were not able to demonstrate a clear Th2 shift in the response, although it is noteworthy that Th2 lymphocytes are generally more resistant to Fas-mediated apoptosis than are Th1 lymphocytes (38, 39, 40, 41).

IFN-{gamma} production is lower in double transgenic mice expressing both TNF-{alpha} and Leishmania major LACK Ags in the pancreas than in rat insulin promoter LACK single transgenic animals following immunization (42). Thus, in this latter model and our own, autoreactive T cells seem to be pushed away from a Th1 phenotype. Altogether, these results point to a role of proteins of the TNF family in the cytokine pattern produced by autoreactive T cells.

Even if the lesional mechanism of EAT mainly involves T cells, a hallmark of the disease is the emergence of serum anti-Tg Abs (43). Because Fas-FasL interaction is involved in B cell tolerance (44, 45, 46, 47), we studied changes in anti-Tg production in FasL-transgenic mice. FasL expression on TFC resulted in a decrease in the titers of anti-Tg Abs. Levels of all the subclasses of anti-Tg IgG Abs fell in TG11 mice expressing the highest levels of FasL, whereas only Th1-associated isotypes were affected in TG9 animals with intermediate FasL expression. The decrease in autoantibody production could be explained by the deletion of autoreactive B cells before immunization. However, Akkaraju et al. (48), using transgenic mice expressing hen egg lysozyme (HEL) in the thyroid, showed that B cells reactive to HEL were neither deleted nor inactivated but, contrary to T cells, kept separate from the neo-self-autoantigen by the basement membrane and vascular endothelial cells of the thyroid. In addition, transgenic mice expressing HEL on TFC produced autoantibodies to HEL after immunization provided that T cell tolerance to HEL had been bypassed. Thus, the production of autoantibodies to a thyroid autoantigen is clearly dependent on T cell-specific tolerance. Our transgenic mice with intermediate levels of FasL on TFC showed a decrease in the Tg-specific CD4+ T cell response associated with a decrease in IFN-{gamma} production, suggesting a shift away from a Th1-type response, as confirmed by decreased anti-Tg IgG2a and constant anti-Tg IgG1 Ab levels. Moreover, in the transgenic mice with the highest levels of FasL on TFC, the profound decrease in the anti-Tg T cell response was also associated with a sharp decrease in the anti-Tg B cell response affecting all of the anti-Tg IgG subclasses. Thus, the variations observed in anti-Tg B cell responses resulted from the decrease in T cell help, which was itself related to the level of FasL expression on TFC. Intermediate levels of FasL led to the deletion of Th1 lymphocytes, favoring a Th2-B cell response, whereas high levels of FasL affected both Th1 and Th2 lymphocytes and resulted in a decrease in all anti-Tg IgG subclasses.

In conclusion, our work demonstrates that FasL expression on thyrocytes avoids thyroid autoimmunity. We show for the first time that this protection is clearly dependent on the level of FasL expression on TFC. Furthermore, the dose-dependent effect of FasL is not restricted to autoimmunity, as a similar effect is observed on allograft rejection (L. Tourneur, F. Batteux, P. Lores, D. Bucchini, and G. Chiocchia, manuscript in preparation). A high level of FasL protects the thyroid against autoreactive lymphocytes in a two-step process involving both clonal deletion of activated effector cells and a deviation of the immune response. In contrast, low FasL expression is ineffective and could even worsen the disease by attracting inflammatory cells. These findings form the groundwork for FasL immunotherapy of organ-specific autoimmune diseases.


   Acknowledgments
 
We thank Drs. Shigekazu Nagata (Osaka University, Osaka, Japan), Pierre Golstein (Institut National de la Santé et de la Recherche Médicale Unité U136, Luminy, France), Nicolas Glaischenhaus (Centre National de la Recherche Scientifique Unité Propre de Recherche 411, Valbonne, France), Jean Feunteun (Centre National de la Recherche Scientifique Unité de Recherche Associée 1967, Villejuif, France), Daniel Christophe (Université Libre de Bruxelles, Brussels, Belgium), and Gunther Richter (Max Delbrück-Centrum, Berlin, Germany) for generously providing invaluable reagents. We are also indebted to S. Mistou, E. Lallemand, and A. Gaston for their excellent technical assistance, F. Lager for help with animal care, Dr. M. Fabre for photographs and comments, and Drs. J. Charreire, C. Fournier, and B. Weill for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by INSERM. Back

2 Address correspondence and reprint requests to Dr. Gilles Chiocchia, INSERM U477, Hôpital Cochin, 27 rue du Faubourg Saint-Jacques 75679 Paris, Cedex 14, France. E-mail address: Back

3 Abbreviations used in this paper: L, ligand; Tg, thyroglobulin; LN, lymph node; LNC, lymph node cell; MTg, mouse thyroglobulin; pTg, thyroglobulin promoter; EAT, experimental autoimmune thyroiditis; TG, transgenic; TFC, thyroid follicular cell; HEL, hen egg lysozyme. Back

Received for publication August 24, 1999. Accepted for publication November 17, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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