Mechanisms of tolerance induced by transforming growth factor-ß-treated antigen-presenting cells: CD8 regulatory T cells inhibit the effector phase of the immune response in primed mice through a mechanism involving Fas ligand

Michele M. Kosiewicz1, Pascale Alard1, Shuang Liang1 and Sherry L Clark1

1 Department of Microbiology and Immunology, University of Louisville Health Sciences Center, Louisville, KY 40202, USA

Correspondence to: M. M. Kosiewicz; E-mail: mmkosi01{at}gwise.louisville.edu
Transmitting editor: W. Strober


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Transforming growth factor (TGF)-ß-treated antigen-presenting cells [(APC) adherent peritoneal exudate cells] induce a profound tolerance in primed mice that is thought to be mediated by regulatory T cells induced in the spleen. In the current study, we investigated the mechanism(s) involved in tolerance induced in primed mice by TGF-ß-treated APC. Interestingly, TGF-ß-treated APC from class II knockout mice were unable to mediate tolerance in primed mice and failed to induce not only CD4, but also CD8 regulatory T cells. However, the results of several experiments indicated that it was the CD8 regulatory T cells that were required for tolerance induced in primed mice. Using neutralizing antibody, we found that TGF-ß-treated APC-induced CD8 regulatory T cells did not suppress effector T cell function in vivo through the production of IL-4, TGF-ß or IL-10. On the other hand, our data showed that the Fas–Fas ligand (FasL) pathway was involved in this form of tolerance since TGF-ß-treated APC could not mediate tolerance in primed FasL-deficient mice and CD8 T cells from FasL-deficient mice were unable to suppress effector T cell responses. Moreover, the targets of FasL-mediated suppression were found to be the effector T cells as suggested by the data showing that Fas-deficient effector T cells were not susceptible to suppression mediated by CD8 regulatory T cells induced by TGF-ß-treated APC. In conclusion, our data indicate that TGF-ß-treated APC effect tolerance in primed mice via a Fas–FasL-mediated mechanism that requires CD8 cells.

Keywords: delayed-type hypersensitivity, deletion, Fas


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Tolerogenic antigen-presenting cells (APC) are an ideal modality for the induction of antigen-specific tolerance, and thereby offer great potential as a therapy for the prevention and treatment of T cell-mediated autoimmune diseases. The advantage of using APC pulsed with antigen over a variety of other methods is that the antigen can be targeted exclusively to the tolerogenic APC before injection. This is in comparison to administration of a ‘bolus’ of antigen (e.g. i.v., orally, nasally, etc.) that can result in uncontrolled dissemination of antigen to endogenous APC that could be immunogenic and could, consequently, induce a counterproductive immunogenic, not tolerogenic, response.

A strategy for the generation of effective tolerogenic APC, transforming growth factor (TGF)-ß-treated APC (primarily composed of macrophages), has been developed by Streilein and colleagues (1,2). These cells were originally identified as playing a role in the cascade of events that leads to the development of anterior chamber-associated immune deviation (ACAID). In ACAID, injection of antigen into the anterior chamber of the eye results in the systemic and antigen-specific impairment in the delayed-type hypersensitivity (DTH) response that is mediated by regulatory T cells induced in the spleen. Evidence suggests that F4/80+ cells in the anterior chamber of the eye process the injected antigen under the influence of the ocular environment, which contains high levels of TGF-ß2 as well as other immunomodulatory factors (3,4), then transport the tolerogenic signal through the circulation to the spleen where regulatory T cells are induced (5). A similar phenomenon can be generated by i.v. injection of adherent peritoneal exudate cells (PEC) that have been cultured with TGF-ß2 and antigen (1,2).

A unique feature of TGF-ß-treated APC-induced tolerance is that it can be induced in primed mice and results in the down-regulation of previously activated antigen-specific T cells (6). This characteristic, in particular, makes TGF-ß-treated APC-induced tolerance an especially attractive candidate for development as a therapy for the treatment of established autoimmune diseases. Recent studies using RNA display have begun to elucidate the effect of TGF-ß treatment on APC in TGF-ß-treated APC-induced tolerance (7). Although TGF-ß-treated APC induce regulatory T cells that mediate impairment of the immune response, the precise mechanisms by which the regulatory T cells do this are currently unknown. The goal of the present study was to determine the mechanisms involved in tolerance induced by TGF-ß-treated APC in primed mice. We report here that although class II-restricted CD4 T cells may be required for the development of TGF-ß-treated APC-induced tolerance in primed mice, it is the CD8 regulatory T cell population that very likely mediates impairment of the immune response via a Fas–Fas ligand (FasL) mechanism.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Six- to 8-week-old female BALB/cByJ, C57BL/6, B6.129S2-Cd8atm1Mak (CD8 knockout), B6Smn-C3-Tnfsf6gld (FasL deficient), B6.129S6-Iitm1Liz (class II knockout), B6.129P2-Il4tm1Cgn (IL-4 knockout), BALB/c-Il4ratm1Sz (IL-4 receptor knockout), B6.129P2-Il10tm1Cgn (IL10 knockout) and B6.MRL-Tnfrsf6lpr (Fas deficient) mice were purchased from Jackson Laboratories (Bar Harbor, ME). Tribromoethanol was used as an anesthetic, when necessary i.p.

Reagents
The following reagents were used: ovalbumin (OVA; Sigma, St Louis, MO), anti-TGF-ß and isotype control antibody purified (National Cell Culture Center, Minneapolis, MN) from the supernatant of the hybridomas 1D11.16.8 and 1B7.11 (ATCC, Manassas, VA) respectively, anti-IL-4 antibody purified from the supernatant of the hybridoma 11B11 (a kind gift from Dr Rachel Caspi), and porcine TGF-ß2 (R & D Systems, Minneapolis, MN).

TGF-ß-treated APC
PEC were generated by i.p. injection of 2 ml of 3% thioglycolate, 4 days before harvest. Cells (1 x 106 cells/ml) were cultured in either 1% normal mouse serum (Cedarlane, Hornby, Ontario, Canada) or serum-free media [RPMI 1640, 2 mM glutamine, 10 mM HEPES, 100 U/ml penicillin G sodium, 100 µg/ml streptomycin sulfate supplemented with ITS (1 mg/ml iron-free transferrin, 10 ng/ml linoleic acid, 0.3 ng/ml Na2Se and 0.2 mg/ml Fe(NO3)3; Collaborative Biomedical Products, Bedford, MA)], 5 ng TGF-ß2 and 5 mg/ml of lipopolysaccharide (LPS)-free OVA in 24-well plates overnight [OVA solution was passed consecutively though two Acticlean Etox chromatography columns (Sterogene, Carlsbad, CA) to remove LPS (<15 EU/ml) and lyophilized]. Non-adherent cells were removed and adherent cells harvested after incubation on ice for 1 h, using a cell-lifter. Cells were then washed 3 times in HBSS and 4000 antigen-pulsed TGF-ß-treated APC were injected i.v. into primed mice for all experiments. The final APC population that was used for all experiments was >90% F4/80+.

Immunization protocol
Mice received a s.c. immunization with 100 µl of OVA (100 µg) solution emulsified in complete Freund’s adjuvant (CFA) (3.3 mg/ml Mycobacterium tuberculosis H37 RA; Difco, Detroit, MI).

DTH assay
Mice were immunized with OVA and CFA as described above and 7 days later, injected with 4000 TGF-ß-treated APC. Mice were challenged by intradermal injection of OVA-pulsed PEC (1 x 105 PEC/10 µl of HBSS) into the ear pinnae 7 days after injection of TGF-ß-treated APC. The DTH response was determined 24 h later by ear swelling measurements using a micrometer (Mitutoyo, MTI, Paramus, NJ). The negative controls received an ear challenge only, and the positive controls were immunized and received an ear challenge.

Local adoptive transfer (LAT) assay
For detection of regulatory T cells, CD4 or CD8 cell-affinity columns (R & D Systems) were used, respectively, to purify CD4 cells or CD8 cells from the spleens of naive (control) mice or mice that had been injected 7 days earlier with 4000 TGF-ß-treated APC. Either 5 x 105 or 1 x 106 purified CD4 or CD8 spleen cells were injected along with 1 x 106 primed lymph node (LN) cells (from mice that had been immunized with OVA and CFA 10–14 days previously) in 10 µl of HBSS containing 20 mg/ml of OVA into the ear pinnae of naive mice. In the LAT assay that was used to determine whether the T cells or the APC are deleted, T cells from the primed LN were purified by using T cell-affinity columns and injected (0.5 x 106 cells/10 µl) along with PEC that were cultured overnight with OVA then used as APC (0.1 x 106 cells/10 µl) in the mixture of cells that were injected into the ear pinnae. In other experiments, either anti-TGF-ß (100 µg/ml), anti-IL-4 (2 mg/ml) or isotype control antibodies were added to the cell mixture, or mice were injected i.p. with anti-IL-4 or anti-TGF-ß antibody (2 mg/mouse) 1–2 h before injection of cell mixture into the ear pinnae. The ear swelling was measured 24 h later. The negative controls for the LAT received a mixture of naive LN cells, naive spleen T cells and OVA into the ear pinnae. The positive controls received a mixture of primed LN cells, naive spleen CD8 cells and OVA into the ear pinnae.

Statistical analyses
Data were subjected to statistical analysis by ANOVA and the Tukey–Kramer multiple comparisons test. A value of P < 0.05 was considered significantly different. Each experiment was performed at least twice.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
TGF-ß-treated APC from class II knockout mice fail to induce tolerance in primed mice
We have recently found that TGF-ß-treated APC must express class II for tolerance to be induced in naive mice (8). To determine whether class II expression by TGF-ß-treated APC is also required for tolerance induced in primed mice, we performed a similar DTH experiment where mice were first primed with OVA and CFA, then injected with OVA-pulsed TGF-ß-treated APC from class II knockout mice 7 days later. After an additional 7 days, APC-injected mice were challenged by injection of antigen into the pinnae of the ear. Class II knockout TGF-ß-treated APC were unable to induce tolerance in primed mice (Fig. 1). One interpretation of these data is that it is the CD4 regulatory T cell population that mediates tolerance induced in primed mice. We have already found that CD4 regulatory T cells induced by TGF-ß-treated APC can prevent the induction of naive (DO11.10) LN cells (8). However, in order to mediate tolerance in primed mice, the CD4 regulatory T cells would have to be able to suppress the function of primed effector T cells (and, therefore, the effector phase of the immune response). The following experiment was designed to determine whether CD4 cells from mice injected with TGF-ß-treated APC were capable of suppressing primed LN cells using a LAT assay that was previously described (9). (This LAT assay tests the ability of regulatory T cells to inhibit the effector, rather than the induction, phase of the T cell response.) The CD8 regulatory T cells induced by TGF-ß-treated APC have been shown to be very effective at inhibiting primed LN cells in this LAT assay and were, therefore, used as controls for regulatory T cell function in this experiment (6). For the LAT assay, CD4 and CD8 spleen cells from mice that had been injected with TGF-ß-treated APC were purified, and co-transferred with antigen and LN cells from mice primed 10–14 days earlier with antigen and CFA, into the ear pinnae of naive mice. The CD8, but not CD4, T cells were capable of inhibiting primed effector LN cells from mediating an ear swelling response (Fig. 2). These data indicate that it is only the CD8 regulatory T cells and not the CD4 regulatory T cells that have the potential to inhibit activated T cells in primed mice. The overall conclusion concerning the data presented above is that although class II and, therefore, presumably one or more populations of CD4 T cells are required for tolerance induced by TGF-ß-treated APC in primed mice, the CD4 regulatory T cells themselves appear to be incapable of down-regulating previously activated T cells and would, therefore, be unable to directly suppress a primed response.



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Fig. 1. Class II knockout TGF-ß-treated APC cannot induce tolerance in primed mice. In an DTH assay, naive wild-type (WT) B6 mice were immunized and 7 days later, 4000 wild-type or class II knockout (KO) antigen-pulsed TGF-ß-treated APC were injected i.v. Mice were ear challenged after 7 days and ear swelling measured 24 h later. Positive controls (second column) were immunized 14 days before ear challenge. An asterisk indicates experimental groups (n = 5) significantly different from the positive control (P < 0.05).

 


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Fig. 2. CD8, but not CD4, T cells can inhibit primed LN cells. In an LAT assay, spleen cells from wild-type (WT) B6 mice injected with 4000 antigen-pulsed TGF-ß-treated APC were harvested, and CD4 or CD8 T cells purified by affinity column. Purified CD4 or CD8 cells (regulatory cells) and antigen were co-transferred with LN cells (effector cells) from mice that had been immunized 10–14 days previously into the ear pinnae of naive B6 mice. Ear swelling was measured 24 h later. An asterisk indicates experimental groups (n = 5) significantly different from the positive control (second column in figure; P < 0.05).

 
TGF-ß-treated APC from class II knockout mice fail to induce CD8 regulatory T cells
As shown in Fig. 1, class II knockout TGF-ß-treated APC cannot induce tolerance in primed mice. Paradoxically, the CD4 regulatory T cells induced by wild-type TGF-ß-treated APC are incapable of suppressing primed T cells (Fig. 2). One possible explanation for these results is that CD8 regulatory T cells, which as shown in Fig. 2 can very effectively inhibit primed effector T cells, may not be induced in mice injected with class II knockout TGF-ß-treated APC. To test this possibility, CD8 T cells were harvested from mice that had received class II knockout TGF-ß-treated APC and evaluated for their ability to suppress primed T cell responses in an LAT assay. As shown in Fig. 3, CD8 regulatory T cell regulatory activity could not be detected in mice receiving class II knockout TGF-ß-treated APC as compared to mice receiving wild-type TGF-ß-treated APC. These results indicate that class II knockout TGF-ß-treated APC cannot induce CD8 regulatory T cells and suggest that a class II-restricted CD4 T cell population may be required for the development of CD8 regulatory T cells induced by TGF-ß-treated APC.



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Fig. 3. Class II knockout TGF-ß-treated APC cannot induce CD8 regulatory T cells. In an LAT assay, spleen cells from wild-type (WT) B6 mice injected with 4000 wild-type or class II knockout (KO) antigen-pulsed TGF-ß-treated APC were harvested and CD8 T cells purified by affinity column. Purified CD8 cells (regulatory cells) and antigen were co-transferred with LN cells (effector cells) from mice that had been immunized 10–14 days previously into the ear pinnae of naive B6 mice. Ear swelling was measured 24 h later. An asterisk indicates experimental groups (n = 5) significantly different from the positive control (second column in figure; P < 0.05).

 
CD8 cells are required for tolerance induced by TGF-ß-treated APC in primed mice
Next, we determined whether CD8 T cells are required for tolerance induced by TGF-ß-treated APC in primed mice. For this experiment, CD8 knockout mice were first immunized and then injected with TGF-ß-treated APC 7 days later. After 7 days, mice were ear challenged and the DTH response was evaluated. The TGF-ß-treated APC were unable to induce tolerance in primed CD8 knockout mice (Fig. 4), suggesting that CD8 regulatory T cells may be required for tolerance induced in primed mice.



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Fig. 4. CD8 cells are required in TGF-ß-treated APC-induced tolerance in primed mice. In an DTH assay, naive wild-type (WT) B6 or CD8 knockout (KO) mice were immunized and 7 days later, 4000 wild-type antigen-pulsed TGF-ß-treated APC were injected i.v. Mice were ear challenged after 7 days and ear swelling measured 24 h later. Positive controls (second column) were immunized 14 days before ear challenge. An asterisk indicates experimental groups (n = 5) significantly different from the positive control (P < 0.05).

 
CD8 regulatory T cells do not mediate suppression through the production of immunomodulatory cytokines
Since we have recently found that CD4 regulatory T cells mediate suppression of naive effector T cells through the production of TGF-ß (8), the first experiment in this series was performed to determine whether the CD8 regulatory T cells utilize a similar mechanism. Using an LAT assay, splenic CD8 T cells from mice injected with TGF-ß-treated APC were co-transferred with primed LN cells, and anti-TGF-ß antibody or isotype and antigen, into the ear pinnae of naive mice. The ear swelling response was evaluated 24 h later. In the presence of anti-TGF-ß antibody, the CD8 regulatory T cells remained capable of mediating suppression of primed LN cells (Fig. 5A), indicating that TGF-ß is not directly involved in CD8 regulatory T cell-mediated suppression of primed LN cells. To confirm these data, we performed the LAT assay in mice injected i.p. with a high dose of anti-TGF-ß antibody (2 mg/mouse) that has been shown to be effective in vivo (10) and found that the CD8 regulatory T cells were still able to suppress effector T cell responses (Fig. 5A). Previous studies have shown that TGF-ß induces IL-10 production by the APC which is involved in the induction of the tolerogenic response, probably through the induction of regulatory T cells (11). To determine whether IL-10 was also involved directly in the suppression of effector T cell responses, CD8 T cells were purified from IL-10 KO mice that had been injected with TGF-ß-treated APC and used in an LAT assay. CD8 cells from IL-10 KO mice were able to inhibit effector T cell responses (Fig. 5B), indicating that IL-10 is not involved as a direct mediator of effector T cell suppression by CD8 regulatory T cells nor is it involved in regulatory T cell development.



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Fig. 5. CD8 regulatory T cells do not suppress through TGF-ß or IL-10 production. In an LAT assay, spleen cells from (A) wild-type (WT) B6 or (B) wild-type B6 and IL-10 KO mice injected with 4000 wild-type antigen-pulsed TGF-ß-treated APC were harvested and CD8 T cells purified by affinity column. Purified CD8 cells (regulatory cells) and antigen were co-transferred into the ear pinnae of naive mice along with LN cells (effector cells) from mice that had been immunized 10–14 days previously, and (A) anti-TGF-ß antibody or isotype was added to the cell mixture (i.d. injection; left panel) or injected i.p. (right panel), or (B) wild-type or IL-10 KO CD8 cells and antigen were co-transferred with effector cells into the ear pinnae of naive B6 mice. Ear swelling was measured 24 h later. An asterisk indicates experimental groups (n = 5) significantly different from the positive control (second column in figure; P < 0.05).

 
The final series of experiments was designed to determine whether IL-4 is involved in TGF-ß-treated APC-induced tolerance in primed mice. In the first experiment in this series, IL-4 knockout mice were first immunized, then injected with TGF-ß-treated APC and the response determined in an DTH assay. Interestingly, TGF-ß-treated APC could not induce tolerance in primed IL-4 knockout mice (Fig. 6A). These results could be due to one of two possibilities, either (i) regulatory CD8 T cells suppress by directly producing IL-4 or (ii) IL-4 produced by another source is required either directly or indirectly for the development of CD8 regulatory T cells. To determine whether CD8 regulatory T cells suppress primed T cells through the production of IL-4 directly, CD8 T cells from mice injected with TGF-ß-treated APC were co-transferred with primed T cells, anti-IL-4 antibody and antigen into the ear pinnae of naive mice, and the ear swelling was measured 24 h later. The CD8 regulatory T cells were still able to suppress the primed LN response in the presence of anti-IL-4 antibody (Fig. 6B). To confirm that CD8 regulatory T cells do not mediate suppression through the production of IL-4, recipients used in an LAT assay were injected i.p. with a high dose of anti-IL-4 antibody (2 mg/mouse) that has been shown to be effective in vivo (12). Under these circumstances, CD8 T cells were still able to suppress the effector T cell responses (Fig. 6B). These data suggest that the CD8 regulatory T cells do not mediate suppression through the production of IL-4.



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Fig. 6. IL-4 does not directly mediate suppression, but is required for the development of CD8 regulatory T cells. (A) In a DTH assay, naive wild-type (WT) B6 or IL-4 knockout (KO) mice were immunized and 7 days later, 4000 wild-type antigen-pulsed TGF-ß-treated APC were injected i.v. Mice were ear challenged after 7 days and ear swelling measured 24 h later. Positive controls were immunized 14 days before ear challenge. (B) In an LAT assay, spleen cells from wild-type B6 mice injected with 4000 wild-type antigen-pulsed TGF-ß-treated APC were harvested and CD8 T cells purified by affinity column. Purified CD8 cells (regulatory cells) and antigen were co-transferred into the ear pinnae of naive mice along with LN cells (effector cells) from mice that had been immunized 10–14 days previously, and anti-IL-4 antibody or isotype was added to the cell mixture (i.d. injection; left panel) or injected i.p. (right panel). Ear swelling was measured 24 h later. (C) In an LAT assay, spleen cells from wild-type B6 or IL-4 receptor knockout mice injected with 4000 wild-type antigen-pulsed TGF-ß-treated APC were harvested and CD8 T cells purified by affinity column. Purified CD8 cells (regulatory cells) and antigen were co-transferred with LN cells (effector cells) from mice that had been immunized 10–14 days previously into the ear pinnae of naive B6 mice. Ear swelling was measured 24 h later. An asterisk indicates experimental groups (n = 5) significantly different from the positive control (second column in figures; P < 0.05).

 
Since IL-4 does not seem to be directly involved in the suppression of primed LN cells mediated by the CD8 regulatory T cells, the alternative possibility is that IL-4 is somehow involved in the development of the CD8 regulatory T cells. To test this possibility, an LAT assay was performed using CD8 T cells from IL-4 receptor knockout mice. Spleen cells were harvested from IL-4 receptor knockout mice that had received an injection of TGF-ß-treated APC, and purified CD8 cells were co-transferred with primed LN cells and antigen into the ear pinnae of naive mice, and the ear swelling was measured 24 h later. Interestingly, CD8 T cells from IL-4 receptor knockout mice were unable to suppress primed LN cells (Fig. 6C), suggesting that IL-4 is involved in the development of CD8 regulatory T cells. In summary, CD8 regulatory T cells induced by TGF-ß-treated APC do not appear to mediate suppression of primed LN cells through the production of either IL-4, IL-10 or TGF-ß, although the data do suggest that IL-4 may be involved in CD8 regulatory T cell development.

FasL appears to be involved in TGF-ß-treated APC-induced tolerance in primed mice
We have recently found that T cells from TGF-ß-treated APC-injected mice induce FasL-mediated cell death of antigen-specific effector T cells in vitro (13). Based on these data, we next asked whether FasL played a role in TGF-ß-treated APC-induced tolerance in vivo in primed mice. Using an DTH assay, B6gld (FasL-deficient) mice were first immunized, then injected with TGF-ß-treated APC and the response determined. As shown in Fig. 7(A), TGF-ß-treated APC were unable to induce tolerance in primed B6gld mice, suggesting that FasL is an important component of tolerance induced by TGF-ß-treated APC in primed mice. The amplitude of the DTH response was similar in wild-type and B6gld mice. The next experiment was performed to determine whether the CD8 T cells from FasL-deficient mice that have been injected with TGF-ß-treated APC can still mediate suppression of primed LN cells in an LAT assay. Splenic cells were harvested from B6gld mice that had received an injection of TGF-ß-treated APC, and CD8 cells purified and mixed with primed LN cells from wild-type mice and antigen, then injected into the ear pinnae of naive wild-type mice. The DTH response was determined 24 h later. The CD8 T cells from B6gld mice were unable to mediate suppression of primed LN cells (Fig. 7B). These results suggest that FasL is important for tolerance induced by TGF-ß-treated APC in primed mice and CD8 regulatory T cells may mediate suppression of primed LN cells via a mechanism involving FasL.



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Fig. 7. FasL is involved in TGF-ß-treated APC-induced tolerance in primed mice. (A) In an DTH assay, naive wild-type (WT) B6 or gld (FasL-deficient) mice were immunized and 7 days later, 4000 wild-type antigen-pulsed TGF-ß-treated APC were injected i.v. Mice were ear challenged after 7 days and ear swelling measured 24 h later. For negative controls, naive wild-type B6 (left panel) or gld (right panel) mice received an ear challenge only. For positive controls, wild-type B6 (left panel) or gld (right panel) mice were immunized 14 days before ear challenge. (B) In an LAT assay, spleen cells from wild-type B6 or gld mice injected with 4000 wild-type antigen-pulsed TGF-ß-treated APC were harvested and CD8 T cells purified by affinity column. Purified CD8 cells (regulatory cells) and antigen were co-transferred with LN cells (effector cells) from mice that had been immunized 10–14 days previously into the ear pinnae of naive B6 mice. Ear swelling was measured 24 h later. An asterisk indicates experimental groups (n = 5) significantly different from the positive control (second column in figures; P < 0.05).

 
CD8 regulatory T cells appear to mediate Fas-induced deletion of effector T cells
The two goals of the final series of experiments were to confirm that CD8 regulatory T cells suppress primed LN cells through Fas-mediated deletion and, if this is the case, to determine whether the target was the APC or the primed effector T cells. In the first experiment, spleen cells were harvested from wild-type mice that had received an injection of TGF-ß-treated APC, and CD8 cells purified and mixed with primed LN cells from B6lpr (Fas-deficient) mice and antigen, then injected into the ear pinnae of naive wild-type mice. The DTH response was determined 24 h later. The CD8 regulatory T cells were unable to mediate suppression of primed Fas-deficient LN cells (Fig. 8A). These data strongly suggest that CD8 regulatory T cells mediate suppression of primed LN cells through a Fas-mediated mechanism.



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Fig. 8. CD8 regulatory T cells mediate suppression through a FasL-mediated mechanism. (A) In an LAT assay, spleen cells from wild-type (WT) B6 mice injected with 4000 wild-type B6 antigen-pulsed TGF-ß-treated APC were harvested and CD8 T cells purified by affinity column. Purified CD8 cells (regulatory cells) and antigen were co-transferred with LN cells (effector cells) from wild-type or lpr (Fas-deficient) mice that had been immunized 10–14 days previously into the ear pinnae of naive B6 mice. Ear swelling was measured 24 h later. The positive control for WT is represented by the second column; the positive control for lpr is represented by the fourth column. (B) In an LAT assay, spleen cells from wild-type B6 mice injected with 4000 wild-type B6 antigen-pulsed TGF-ß-treated APC were harvested, and CD8 T cells purified by affinity column. To test for APC as targets of Fas-mediated deletion, purified CD8 cells (regulatory cells) and antigen-pulsed APC from wild-type or lpr (Fas-deficient) mice were co-transferred with purified T cells (effector cells) from the LN of wild-type B6 mice that had been immunized 10–14 days previously into the ear pinnae of naive lpr mice. The positive control for the APC deletion experiment is represented by the second column. To test for effector T cells as targets of Fas-mediated deletion, purified CD8 cells (regulatory cells) and antigen-pulsed APC from wild-type B6 mice were co-transferred with purified T cells (effector cells) from the LN of lpr mice that had been immunized 10–14 days previously into the ear pinnae of naive lpr mice. The positive control for the T cell deletion experiment is represented by the fifth column. Ear swelling was measured 24 h later. An asterisk indicates experimental groups (n = 5) that are significantly different from the positive control (P < 0.05).

 
Next, we determined the target(s) of FasL-induced suppression of the DTH response. The two possibilities include the APC that activate the primed T cells in the LAT assay or the primed effector T cells themselves. To distinguish between these two possibilities, we used a modification of the LAT assay that was described above. To determine whether the target of deletion was the APC, T cells from the LN of primed wild-type mice were purified, and mixed with antigen-pulsed PEC from B6lpr (Fas-deficient) mice and CD8 regulatory T cells from wild-type mice injected with TGF-ß-treated APC. This cell mixture was then injected into the ear pinnae of naive B6lpr mice. In this experimental design, none of the APC (injected as well as endogenous APC in the recipients) expressed the Fas molecule and, therefore, none of the APC should be susceptible to Fas-induced apoptosis. As shown in Fig. 8(B), CD8 regulatory T cells were still capable of suppressing primed LN T cells despite the absence of Fas-expressing APC. These data suggest that the APC that present antigen to the primed LN cells are not the targets of deletion mediated by the CD8 regulatory T cells. In the same experiment, we also determined whether the target of deletion was the primed effector LN T cells themselves. In this experimental design, T cells from the LN of primed B6lpr (Fas-deficient) mice were purified, and mixed with antigen-pulsed PEC from wild-type mice and CD8 regulatory T cells from wild-type mice injected with TGF-ß-treated APC. This cell mixture was then injected into the ear pinnae of naive B6lpr mice. In this case, the primed LN T cells (effector cells) were Fas deficient, whereas the exogenous APC expressed Fas and, therefore, the effector T cells should not be susceptible to Fas-induced apoptosis. Interestingly, the CD8 regulatory T cells were unable to suppress the Fas-deficient effector T cells (Fig. 8B). These data suggest that the CD8 regulatory T cells induced by TGF-ß-treated APC mediate suppression of primed LN cells through a Fas-mediated mechanism that acts directly on the effector T cells and not on the APC that present antigen.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The results of the present study characterize the mechanism of tolerance that is mediated by regulatory T cells induced by TGF-ß-treated APC in primed mice. We have found that although CD4 T cells appear to be required, possibly for the development of CD8 regulatory T cells, the CD8 regulatory T cells appear to directly mediate tolerance induced by TGF-ß-treated APC in primed mice. Furthermore, the CD8 regulatory T cells seem to require IL-4 for their development and mediate suppression via a Fas-mediated mechanism that targets the primed effector T cells.

Since we have shown that the CD4 regulatory T cells induced by TGF-ß-treated APC cannot regulate primed effector T cells, the failure of TGF-ß-treated class II knockout APC to induce tolerance in primed mice is puzzling. The most likely scenario is that one or more populations of CD4 T cells and, possibly, but not necessarily, the CD4 regulatory T cells themselves, may somehow be involved in the development of the CD8 regulatory T cells. For reasons other than simply providing a source of IL-2, CD4 T cells have been found to be important for the development of CD8 T cell responses (14). Alternatively, a less likely possibility is that the CD8 regulatory T cells induced by TGF-ß-treated APC are class II restricted. Although very rare, class II-restricted CD8 T cells (e.g. CD8{alpha}{alpha}) have been shown to exist (15). Moreover, we have reported previously that BALB/c CD8 regulatory T cells can be induced by TGF-ß-treated APC presenting OVA323–339, a peptide traditionally thought to be class II-restricted (16). Although the original interpretation of these data was that the OVA 323–339 peptide also contains an embedded class I-binding motif (e.g. one that could be cleaved and bound, e.g. by H-2Ld) (16), the other possibility is that one or more populations of CD8 regulatory T cells could be class II restricted.

Interestingly, although IL-4 (protein or mRNA) could not be detected in cultures of LN or spleen cells (data not shown), this cytokine appears to play an important role in tolerance induced by TGF-ß-treated APC in primed mice. Although the results of our study indicate that CD8 regulatory T cells do not directly mediate suppression of primed LN cells through the production of either IL-4, IL-10 or TGF-ß, the data from the IL-4 receptor knockout experiments strongly suggest that IL-4 is involved in the development of the CD8 regulatory T cells at some level (i.e. either directly with the development of CD8 cells themselves or with cells that are involved in CD8 cell development). IL-4 has been reported to be required for CD8 cell development in other systems and to influence CD8 cell function (17,18). The source of IL-4 is currently unknown, but there are two possible candidates. One possible source of IL-4 is NK T cells, since they have the potential to produce IL-4 rapidly after stimulation (19), and have been shown to be important for the development of ACAID and TGF-ß-treated APC-induced tolerance (20,21). TGF-ß-treated APC must express CD1d in order to mediate tolerance. Furthermore, ACAID cannot be induced in mice that do not have functional NKT cells and although NKT cells do not directly mediate tolerance (i.e. do not function as the regulatory T cells themselves), they are somehow involved in the development of the response (i.e. possibly in the development of the regulatory T cells). Another potential source is the CD4 T cells, possibly including a population(s) that may be required for the development of CD8 regulatory cells and that is induced by injection with wild-type, but not class II knockout, TGF-ß-treated APC. These CD4 cells may also be the CD4 regulatory T cells found to mediate tolerance induced by TGF-ß-treated APC in naive mice or they may be an entirely unrelated population. We are interested in how and whether CD4 T cells and IL-4 are involved in the development of TGF-ß-treated APC-induced CD8 regulatory T cells. These issues are currently under investigation in our laboratory.

The Fas–FasL system of apoptosis induction plays important roles in both homeostasis and the maintenance of immune privilege (2225). FasL has also been used in strategies designed to treat autoimmune diseases and prevent transplant rejection (2631). Since FasL is very effective at down-regulating activated T cells, we tested the possibility that it may be involved in TGF-ß-treated APC-induced tolerance. The results of our previous study in which TCR transgenic T cells co-transferred with TGF-ß-treated APC into recipient mice were evaluated in vitro and found to undergo cell death, and thereby deletion, through a mechanism that involves FasL, strongly suggested that FasL may be involved in TGF-ß-treated APC-induced tolerance (13). The current study extends these findings to determine whether FasL plays a role in vivo in primed mice. We have found that the Fas–FasL system seems to be critical for tolerance mediated by TGF-ß-treated APC-induced CD8 regulatory T cells in primed mice and may act through the deletion of the effector T cells, but not the APC. Induction of apoptosis of antigen-activated CD4 cells by FasL-expressing CD8 cells has been reported previously (32,33) and FasL-induced apoptosis has also been associated with suppressor T cell activity in other models of immune suppression (34). Interestingly, IL-4 has been shown to decrease perforin activity and increase FasL-mediated activity by CD8 T cells in vivo (35). It is tempting to speculate that perhaps IL-4 participates in TGF-ß-treated APC-induced tolerance in primed mice by up-regulating FasL-mediated activity in the CD8 regulatory T cells.

Our data concerning the role that FasL plays in regulatory T cell-mediated inhibition of primed effector T cells differ from that previously reported in ACAID (36). In the current study, we have found that CD8 regulatory T cells from mice injected with TGF-ß-treated APC cannot suppress primed effector T cells from Fas-deficient mice, whereas Kezuka and Streilein have found that intraocular injection of soluble protein antigen induces splenocytes capable of suppressing primed effector T cells from Fas-deficient mice (36). One reason for this disparity may be that the role that Fas–FasL plays in the function of regulatory T cells induced by intraocular injection of antigen may differ from those induced by antigen-pulsed TGF-ß-treated APC. Therefore, it is possible that the mechanisms involved in the impairment in the DTH response that is induced by intraocular injection versus TGF-ß-treated APC are not identical. This possibility is further supported by two studies that suggest that FasL may play different roles in tolerance induced by intraocular injection versus TGF-ß-treated APC in naive mice (36,37).

In conclusion, our data support the hypothesis that CD8 regulatory T cells induced by TGF-ß-treated APC suppress activated effector T cells (in primed mice) through a mechanism that involves the Fas–FasL pathway. The ultimate goal of these studies is to develop this tolerance-inducing strategy into a therapy to prevent and treat established autoimmune and inflammatory diseases. The feasibility of this approach has already been demonstrated in ‘Proof of Principle’ experiments showing that autoimmune uveitis can be prevented and inflammatory damage limited in established disease by injection of TGF-ß-treated APC before or after onset of disease, respectively (38,39).


    Acknowledgements
 
We would like to thank Jean N. Manirarora and Michael J. Myers for technical support and help in the preparation of this manuscript. This work was supported by a grant from the National Institutes of Health (DK-56206).


    Abbreviations
 
ACAID—anterior chamber-associated immune deviation

APC—antigen-presenting cell

CFA—complete Freund’s adjuvant

DTH—delayed-type hypersensitivity

FasL—Fas ligand

LAT—local adoptive transfer

LN—lymph nodes

OVA—ovalbumin

PEC—peritoneal exudate cell

TGF—transforming growth factor


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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