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The Journal of Immunology, 1999, 162: 761-765.
Copyright © 1999 by The American Association of Immunologists

Acquired Thymic Tolerance: Role of CTLA4 in the Initiation and Maintenance of Tolerance in a Clinically Relevant Autoimmune Disease Model1

Shohreh Issazadeh*, ManHua Zhang*, Mohamed H. Sayegh{dagger} and Samia J. Khoury2,*

* Center for Neurologic Diseases, and {dagger} Laboratory of Immunogenetics and Transplantation, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Injection of Ag into the thymus of adult animals induces specific systemic tolerance. The mechanisms of acquired thymic tolerance include anergy and the deletion of Ag-specific T cells. Here, we report that anergy to nominal Ag induced via acquired thymic tolerance requires CTL-associated Ag 4 (CTLA4) engagement. The role of CTLA4 in the induction and maintenance of tolerance was then investigated in the murine experimental autoimmune encephalomyelitis model. CTLA4 blockade abrogated the induction but not the maintenance phase of acquired thymic tolerance induced by intrathymic injection of myelin Ags. In addition, CTLA4 blockade had a restricted window of action after priming with Ag, which is consistent with the expression patterns of CTLA4 in vivo. We conclude that: 1) the induction of acquired thymic tolerance requires signaling through CTLA4 and 2) CTLA4 does not appear to be required for the maintenance of acquired thymic tolerance. This is the first report documenting the role of a CTLA4 negative signaling pathway in the induction of tolerance in an autoimmune disease model.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Injection of Ag into the thymus of adult animals induces Ag-specific tolerance in experimental autoimmune (1, 2, 3, 4, 5) and transplantation models (6, 7, 8). We have shown previously that an injection of guinea pig myelin basic protein (MBP)3 or its encephalitogenic peptide (p71–90) into the thymus induces systemic tolerance and prevents the development of experimental autoimmune encephalomyelitis (EAE) in the Lewis rat model (4). We have also shown that thymic dendritic cells participate in the induction of acquired thymic tolerance (9). In a mouse model, we have recently reported that an intrathymic (i.t.) injection of Ag leads to an Ag-specific suppression of proliferation and cytokine production and to a failure of clonal expansion of Ag-specific T cells in vivo (10). Our data suggest that activated cells circulate to the thymus, where they are inactivated by anergy and/or deletion (10, 11).

Although it is well documented that the activation of mature T lymphocytes requires Ag recognition and costimulatory signals, the requirements for T cell anergy and apoptosis are more controversial. Earlier reports suggested that Ag recognition in the absence of second signals induces a state of unresponsiveness (12, 13). However, recent reports indicate that secondary signaling through costimulatory molecules may be involved in the induction of T cell anergy. CTL-associated Ag 4 (CTLA4) (CD152), a homologue of CD28 that binds with higher affinity to B7 molecules, functions as a negative regulator of immune responses (14, 15). CTLA4 ligation results in an inhibition of IL-2 production and in a consequent arrest in cell cycle progression from G0/G1, rather than in the induction of apoptotic cell death (16, 17). Recent data also indicate that negative T cell signaling through CTLA4 plays an important role in the induction of peripheral tolerance to nominal Ags in vivo (18, 19, 20). The relevance of these findings to in vivo experimental disease models remains unknown.

To investigate the role of CTLA4 negative signaling in the induction and maintenance of tolerance in vivo, we used two models of acquired thymic tolerance. The first model involves an i.t. injection of OVA in BALB/c mice in which the readout was in vitro proliferation and cytokine production; the second model is an in vivo disease model in (SJL/J x PL/J)F1 mice immunized with the immunodominant peptide (Ac1–11) of MBP to induce clinical EAE. Our findings indicate that CTLA4 signaling is necessary for the induction but not the maintenance of tolerance in vivo.


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

BALB/c and female (SJL x PL/J)F1 mice were obtained from The Jackson Laboratory (Bar Harbor, ME) at 6–8 wk of age. The mice were maintained in our animal facility at the Brigham and Women’s Hospital and Harvard Medical School and were used for all experiments at 8–12 wk of age.

i.t. injection

Mice were injected i.t. with 100 µg of chicken OVA or with Ac1–11 peptide of MBP dissolved in 50 µl of sterile PBS (BioWhittaker, Walkersville, MD), as described previously (10, 11). Briefly, i.t. injections were performed under ether anesthesia by exposing the thymus through a small incision above the sternum. Using a 27-gauge needle, volumes of <=50 µl can be injected into each lobe without evidence of leakage. Either 100 µg of hen egg lysozyme (HEL) in 50 µl of PBS or PBS alone was injected i.t. into control animals. Each experimental group consisted of 5–10 mice.

Immunization

BALB/c mice were immunized in the footpads and axillae with 100 µl of OVA/CFA containing 2 mg/ml of OVA in PBS emulsified with 2 mg/ml of CFA (Difco, Detroit, MI) (total amount of OVA injected is 100 µg/mouse). On day 10 postimmunization, the draining lymph nodes (LNs) and spleen were removed aseptically and mashed carefully to prepare single-cell suspensions. The cells from each group of mice were pooled for the proliferation and cytokine production assays as described previously (10, 11).

EAE induction

(SJL x PL/J)F1 mice were immunized s.c. in the flanks with 1 mg/ml of mouse MBP peptide Ac1–11 emulsified in an equal amount of CFA containing 2 mg/ml CFA (Difco) (total amount of Ac1–11 injected is 100 µg/mouse). The animals received 200 ng of pertussis toxin i.p. (List Biological Laboratories, Campbell, CA) at 24 h postimmunization. Scoring of clinical disease was performed daily as described previously (21). In this model, animals develop acute disease by day 8–12 postimmunization. In our study, the disease incidence is 100%, the day of onset is day 10.78 ± 0.3, and the mean maximal grade is 3.0 ± 0.3 (mean ± SEM from 28 mice in several experiments). The acute disease is followed by a clinical remission, and the mice subsequently have one or more relapses.

Ab treatment

Each mouse received an i.p. injection of 100 µg of hamster anti-mouse CTLA4 Ab from ascites of B cell hybridoma (clone 4F10, kindly provided by Dr. Jeffrey Bluestone, University of Chicago, Chicago, IL (14)); the control group received equal amount of hamster Ig i.p. Ab was administered in single or multiple doses as outlined in Results.

Cell cultures

Cell suspensions were washed twice before resuspension in DMEM (BioWhittaker) and supplemented with 10% (vol/vol) heat-inactivated FCS, 2 mM glutamine, 15 mM HEPES, 1% nonessential amino acids, 1 mM sodium pyruvate (all from BioWhittaker), penicillin (100 U/ml), streptomycin (100 µg/ml), and 20 µM 2-ME (Sigma, St.Louis, MO). For splenocytes, RBCs were lysed with ACK lysing buffer (0.15 M NH4Cl, 10 mM KHCO3, and 0.1 mM Na2 EDTA). Cells (4 x 105 cells/200 µl/well) were cultured in round-bottom microtiter plates (Costar, Cambridge, MA) and stimulated with OVA or Mycobacterium tuberculosis (MT); unstimulated wells served as background response. Proliferation was measured using a standard 72-h lymphocyte proliferation assay. For cytokine production, cell-free supernatants were collected after 48 h for the measurement of IL-2, IFN-{gamma}, IL-4, and IL-10 production. The results of the proliferative and cytokine studies were similar for both splenocytes as well as lymphocytes from draining LN cells; therefore, only data for LN cells are shown.

ELISA of cytokines

A quantitative ELISA for IL-2, IFN-{gamma}, IL-4, and IL-10 was performed using paired mAbs specific for the corresponding cytokine according to the manufacturer’s recommendations (PharMingen, San Diego, CA). Standard curves were generated using known amounts of purified murine rIL-2, rIL-4, rIFN-{gamma}, or rIL-10 (PharMingen).

Statistical analysis

For a comparison of proliferative responses and cytokine production between experimental groups across three to four experiments, we used nonparametric Kruskal-Wallis and Mann-Whitney tests. For a comparison of clinical disease, we used the Mann-Whitney test to compare grade and onset and Fisher’s exact test for incidence rates.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Acquired thymic tolerance to nominal Ag and autoantigen

An injection of OVA into the thymus of adult BALB/c mice induces Ag-specific tolerance. As seen in Fig. 1Go, LN cells from animals injected i.t. with OVA before immunization have decreased proliferation and IL-2 and IFN-{gamma} production after stimulation with OVA in vitro compared with LNs from animals injected with HEL i.t., as described previously (10). MT was used in vitro as a control Ag because the mice were immunized with CFA but not tolerized to MT. Thus, the proliferation and cytokine production by lymphocytes to MT provide evidence of the Ag specificity of tolerance. Similarly, an injection of HEL into the thymus of OVA-immunized mice shows the specificity of the tolerizing Ag (9, 10). For an in vivo model, we used (SJL x PL/J)F1 mice immunized with Ac1–11/CFA. In this model, an i.t. injection of Ac1–11 peptide but not PBS induces protection from clinical EAE (Fig. 2Go). The incidence of disease was six of six for PBS mice and three of four for i.t.-tolerized mice. The mean score was 3.3 ± 0.4 for the PBS group and 1.1 ± 0.6 for the i.t.-tolerized group. Furthermore, this treatment prevents relapsing disease. Interestingly, mice immunized with MBP/CFA and tolerized by an i.t. injection of Ac1–11 peptide are protected from acute disease but not from relapses, suggesting that acquired thymic tolerance is epitope-specific (data not shown) as we have reported in the Lewis rat model.



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  		FIGURE 1. Effects of an i.t. injection of OVA on T cell responses in vitro. BALB/c mice were i.t. injected with OVA ({diamondsuit}) or HEL ({circ}) or were not injected i.t. ({square}); mice were subsequently immunized with OVA/CFA after 48 h. Draining LN cells were obtained on day 10 postimmunization. Cells were pooled from three mice per group, proliferation was measured by [3H]thymidine incorporation, and cytokines were measured by ELISA in the cell-free supernatants. The x-axis represents the dose of OVA or MT used in vitro. The results shown are representative of three experiments.

 


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  		FIGURE 2. Effects of an i.t. injection of Ac1–11 peptide of MBP on the course of EAE. (SJL x PL/J)F1 mice received 100 µg of Ac1–11 ({circ}) or PBS ({blacksquare}) i.t. 48 h before immunization with Ac1–11/CFA. There were six mice in the PBS group and four mice in the i.t.-tolerized group. Incidence of disease was six of six for the PBS group and three of four for the i.t.-tolerized group. The mean score was 3.3 ± 0.4 for the PBS mice and 1.1 ± 0.6 for the i.t.-tolerized mice. Data from this experiment are included in Table IGo. This experiment was repeated five times with similar results. The x-axis represents the days of observation, and the y-axis represents the mean clinical score of each group.

 
Role of CTLA4 signaling in acquired thymic tolerance

To investigate the role of the CTLA4 molecule in acquired thymic tolerance, we used a blocking anti-CTLA4 Ab to inhibit signal transduction through the CTLA4-B7 pathway. Studies with this Ab (clone 4F10) and with its Fab fragments have been reported and have shown no functional difference in CTLA4 blockade in vitro or in vivo between the Ab and its Fab fragment (22, 23, 24). Anti-CTLA4 was administered to BALB/c mice at different timepoints after an i.t. injection of OVA and immunization with OVA/CFA. As shown in Fig. 3Go, the administration of anti-CTLA4 on day 3 postimmunization reversed the effects of acquired thymic tolerance and returned the proliferative response and the production of IL-2 and IFN-{gamma} to control levels. The administration of anti-CTLA4 on days -1, 0, or 1 around immunization did not reverse tolerance. Furthermore, anti-CTLA4 administration on days 8, 10, and 12 postimmunization did not reverse tolerance. Interestingly, the administration of anti-CTLA4 on day 6 postimmunization gave intermediate results. Proliferation and IL-2 production remained suppressed, but IFN-{gamma} production returned to control levels, suggesting that some degree of CTLA4-independent tolerance was already established by day 6. Similar results were observed when we stimulated LN cells with higher doses of OVA (50 and 100 µg/ml) in vitro.



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  		FIGURE 3. Effects of blocking CTLA4 signaling on the induction of acquired thymic tolerance. BALB/c mice were i.t. injected with OVA, except for both the control group, which was not injected i.t., and the group that received HEL i.t. All groups were immunized with OVA/CFA after 48 h. Anti-CTLA4 Ab was administered at a dose of 100 µg per mouse on the days noted on the y-axis. Control Ig was administered on days -3, -2, -1, 0, 3, and 6 relative to immunization. The group injected with HEL i.t. received anti-CTLA4 on days -3, -2, and -1. Draining LN cells were obtained on day 10 postimmunization. Cells were pooled from three mice per group and stimulated with OVA at 25 µg/ml, proliferation was measured by [3H]thymidine incorporation, and cytokines were measured by ELISA in the cell-free supernatants. The results shown are representative of three experiments.

 
To address the effect of anti-CTLA4 administration on day 3 in nontolerized animals, we used the LN cells of animals injected i.t. with OVA, immunized with OVA/CFA, and treated with anti-CTLA4 on day 3. In vitro stimulation was conducted using MT as the Ag. Under these conditions, OVA injected into the thymus acts as an irrelevant Ag because the animals are tolerized to OVA but not to MT. Thus, lymphocytes were stimulated in vitro with MT at concentrations of 25, 50, and 100 µg/ml; proliferation, IFN-{gamma} production, and IL-2 production were measured. Fig. 4Go shows the results of stimulation with MT at 25 µg/ml. The data clearly show that anti-CTLA4 administration on day 3 did not enhance proliferation or IFN-{gamma} or IL-2 production compared with controls.



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  		FIGURE 4. Effects of blocking CTLA4 signaling on the response to Ag in the absence of acquired thymic tolerance. BALB/c mice were either injected i.t. with OVA, immunized with OVA/CFA after 48 h, and treated with 100 µg of anti-CTLA4 on day 3 postimmunization (OVA IT, day 3 anti-CTLA4 group) or immunized with OVA/CFA alone (control group). Draining LN cells were obtained on day 10 postimmunization. Cells were pooled from three mice per group and stimulated with MT at 25 µg/ml, proliferation was measured by [3H]thymidine incorporation, and cytokines were measured by ELISA in the cell-free supernatants. The results shown are representative of three experiments.

 
Role of CTLA4 signaling in the induction and maintenance of acquired thymic tolerance in EAE

To investigate the role of CTLA4 signaling in the induction and maintenance of acquired thymic tolerance in a clinically relevant autoimmune disease model, we used the relapsing EAE model. Based on the data obtained in the OVA system, we investigated the effect of anti-CTLA4 administration on day 3 postimmunization (tolerance induction phase) and on later timepoints to evaluate the maintenance phase of tolerance. As shown in Table IGo, administering anti-CTLA4 to i.t. tolerized mice on day 3 postimmunization reversed tolerance, whereas administering anti-CTLA4 on days 10, 12, and 14 postimmunization did not reverse protection (Fig. 5Go and Table IGo). The administration of anti-CTLA4 blocking Ab was reported to worsen EAE (24, 25). However, in our model the reversal of protection seen on day 3 postimmunization cannot be attributed to a worsening of disease. At the doses and protocols used, we observed a slight shortening of disease latency and slightly increased mortality (not statistically significant). The same degree of worsening was observed when anti-CTLA4 was administered on day 3 or on days 10, 12, and 14 postimmunization in the nontolerized animals, but reversal of tolerance was only observed in the day 3 anti-CTLA4-treated thymically injected animals. These data and the observations by Perez et al. (18) in a model of peripheral tolerance to nominal Ag and by Walunas et al. (26) in a model of tolerance to superantigen indicate that CTLA4 blockade is abrogating the induction of tolerance rather than just worsening the immune response.


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  		Table I. Role of CTLA4 in the induction and maintenance phases of acquired thymic tolerance1

 


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  		FIGURE 5. Effects of blocking CTLA4 signaling on the induction of acquired thymic tolerance in EAE. (SJL x PL/J)F1 mice received 100 µg of Ac1–11 i.t. followed 48 h later by immunization with Ac1–11/CFA. Anti-CTLA4 Ab was administered at a dose of 100 µg per mouse on day 3 postimmunization (single dose) ({blacktriangleup}) and on days 10, 12, and 14 postimmunization ({triangleup}). Some mice received control Ig on days 3, 10, 12, and 14 postimmunization ({diamondsuit}). Control nontolerized animals were immunized at the same time ({square}). The x-axis represents the mean daily clinical score of each group. Each group consisted of three mice; data from this experiment are included in Table IGo. For this experiment, the incidence of disease was three of three in the control and the day 3-treated groups and two of three in the i.t.-tolerized group and the days 10, 12, and 14-treated group. The mean maximal score was 2.3 ± 0.8 for controls, 0.3 ± 0.2 for the i.t.-tolerized group, 2.1 ± 0.2 for the day 3 group, and 0.5 ± 0.3 for the days 10, 12, and 14 group. This experiment was repeated three times with similar results.

 
These results suggest that once the T cell is rendered unresponsive, it is resistant to the effects of CTLA4 blockade. Thus, our data show that CTLA4 negative signaling is not necessary for the maintenance phase of tolerance and are consistent with the findings of Perez et al. for peripheral tolerance to nominal Ag (18).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The induction of specific tolerance with an i.t. injection of Ag was initially described by Waksman and colleagues in the 1960s–1970s (27, 28, 29) and recently applied to several transplantation (8, 30) and autoimmune models (1, 2, 3, 4, 5). In the autoimmune disease model EAE, an i.t. injection of MBP or its major encephalitogenic peptide (p71–90) but not a nonencephalitogenic peptide (p21–40) of MBP prevents disease in the Lewis rat (4); this finding is associated with an Ag-specific suppression of proliferation of the LN T cells of these animals (4). We have shown previously that there is a down-regulation of Th1 cytokines in the brain without evidence of Th2 up-regulation (4). We have also shown that thymic dendritic-enriched cells mediate the induction of acquired thymic tolerance (9), and that the protective effect of thymic dendritic cells is abrogated in thymectomized animals, suggesting that the interaction of dendritic cells and T cells occurs in the thymus (9). More recently, we reported that an i.t. injection of Ag in a mouse model induces Th1 cell unresponsiveness and prevents the peripheral expansion of Ag-specific CD4+ T cells in vivo (10). Injection of OVA into the thymus of OVA TCR-transgenic mice induces apoptosis of immature thymocytes and anergy of more mature thymocytes (11). Thus, our data support the hypothesis that activated cells circulate to the thymus, where they are inactivated by anergy and/or deletion (10, 11). CTLA4 negative signaling reportedly plays a role in peripheral tolerance to nominal Ags (18, 26). In this report, we investigated the role of CTLA4 signaling in acquired thymic tolerance.

Our results clearly show that CTLA4 signaling is required during the induction of but not the maintenance of acquired thymic tolerance. Administration of anti-CTLA4 on day 3 postimmunization reverses acquired thymic tolerance. One could argue that blocking CTLA4 signaling enhances immunity rather than abrogates tolerance. Several observations in our study counter this hypothesis. First, our in vivo protocol of anti-CTLA4 administration to control (nontolerized) animals did not worsen disease. The differential effect of anti-CTLA4 administration on day 3 vs days 10–14 is another argument against this hypothesis. Second, we demonstrate in our in vitro experiments that the administration of anti-CTLA4 on day 3 postimmunization did not augment the immune response to MT (an Ag to which the mice were not tolerized). Thus, there is no evidence that administration of anti-CTLA4 on day 3 postimmunization augments the immune response or worsens disease. Our data demonstrate that anti-CTLA4 administered on day 3 postimmunization prevents the anergy normally induced by acquired thymic tolerance.

We found that there is a narrow window for CTLA4 signaling in acquired thymic tolerance. Our in vitro data suggest that this window is between days 3 and 6 postimmunization. Interestingly, administration of anti-CTLA4 around the day of immunization did not abrogate the induction of tolerance. One would have expected that an IgG Ab might persist in the circulation so that some effect can be seen on day 3. However, several factors determine the efficacy of Ab blockade in vivo, including t1/2 (shorter with a xenogenic Ab), peak serum concentration, tissue distribution, and the level of expression of the target molecule. In our experiments with nominal Ag, the administration of anti-CTLA4 on day 0 may not provide an optimal tissue level at the time of peak CTLA4 expression. This is supported by data from Bluestone (20) showing the delayed expression of CTLA4 after Ag priming in vivo. Interestingly, a similar narrow window for anti-CTLA4 action has been reported in a model of autoimmune diabetes in which the administration of anti-CTLA4 during a critical period before the onset of insulitis leads to a more rapid onset of diabetes (31).

Our findings are different from those reported by Perez et al. (18) in a model of peripheral tolerance in vivo in which the administration of anti-CTLA4 at the time of Ag administration before priming abrogated the induction of tolerance. Alternatively, our observations are in keeping with our hypothesis of the mechanisms of acquired thymic tolerance; peripheral activation of Ag-specific T cells is followed by a migration of activated T cells to the thymus (10), where they interact with thymic dendritic cells and either become anergic or are deleted (11). Anergy induced via acquired thymic tolerance is not reversed by the addition of IL-2 in vitro (10), suggesting that this anergy is not a form of clonal ignorance but specific inactivation (18). This possibility is supported by the reversal of tolerance seen after blocking B7-CTLA4 interaction. Consequently, we hypothesize that blocking CTLA4 signaling can reverse acquired thymic tolerance only at the time at which the tolerizing signal is given to the T cell (i.e., after the initial activation through immunization). Therefore, the role of CTLA4 signaling in the induction of tolerance in different models is dependent upon the exact mechanisms mediating the tolerant state in vivo.

Our findings are the first to demonstrate an important role for CTLA4 negative signaling in the induction of tolerance in a clinically relevant autoimmune disease model in vivo. Our data have relevant clinical implications, because they suggest that the development of novel strategies in humans should take into account the importance of keeping T cell signaling through CTLA4 intact if true tolerance is to be achieved.


   Footnotes
 
1 This work was supported by research grants from the National Multiple Sclerosis Society (RG-2589-A-2, to S.J.K.) and the National Institutes of Health (AI-40945, to S.J.K.; AI-34965, to M.H.S.; and PO1 AI-41521). Back

2 Address correspondence and reprint requests to Dr. Samia J. Khoury, Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, 77 Louis Pasteur Avenue, Boston, MA. E-mail address: Back

3 Abbreviations used in this paper: MBP, myelin basic protein; CTLA4, CTL-associated Ag 4; i.t., intrathymic(ally); EAE, experimental autoimmune encephalomyelitis; HEL, hen egg lysozyme; LN, lymph node; MT, Mycobacterium tuberculosis. Back

Received for publication July 17, 1998. Accepted for publication October 6, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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