Deletion, but not anergy, is involved in TGF-ß-treated antigen-presenting cell-induced tolerance

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

1 University of Louisville Health Sciences Center, 319 Abraham Flexner Way, Louisville, KY 40202, USA

Correspondence to: M. M. Kosiewicz; E-mail: mmkosi01{at}gwise.louisville.edu
Transmitting editor: P. S. Ohashi


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Intravenous injection of transforming growth factor (TGF-)-ß-treated antigen-presenting cells (APC) pulsed with antigen induces antigen-specific tolerance in both naive and previously primed mice. Although TGF-ß-treated APC-induced tolerance is associated with induction of regulatory T cells and impaired delayed-type hypersensitivity (DTH) responses, the specific mechanisms that mediate this tolerance are not currently known. The goal of the present report was to study the mechanisms involved in TGF-ß-treated APC-induced tolerance by determining the fate of the antigen-specific effector T cells that are regulated. Using a well-characterized system that allows tracking of small numbers of TCR transgenic T cells, we have found that antigen-specific T cell expansion, either in vivo or in vitro, is inhibited in mice that have been injected with TGF-ß-treated APC. The failure of antigen-specific effector T cells to expand did not appear to be due to the induction of anergy, since carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled cells divided normally in response to antigen and adjuvant in vivo, and addition of exogenous IL-2 was unable to restore T cell expansion in in vitro assays. Interestingly, the percentage of CFSE-labeled cells was decreased after >7–8 divisions following culture in vitro, which correlated with a significant increase in cell death. Cell death was prevented and the ability to expand in vitro was restored by treatment with anti-Fas ligand (FasL) antibody. In conclusion, tolerance induced by TGF-ß-treated APC appears to be associated with deletion of antigen-specific T cells involving the Fas–FasL pathway.

Keywords: anergy, Fas ligand, T cell, tolerance, transforming growth factor-ß


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Transforming growth factor (TGF)-ß is a pluripotent cytokine that is a potent immunosuppressive agent. Adherent peritoneal exudate cells (PEC) cultured overnight with TGF-ß2 and antigen, TGF-ß-treated antigen-presenting cells (APC), induce a profound and long-lasting T cell tolerance in both unsensitized and pre-sensitized mice (15). The latter characteristic makes this form of tolerance unique, since it is far more difficult to down-regulate previously activated T cells than prevent the activation of naive T cells, and, therefore, makes TGF-ß-treated APC-induced tolerance a good candidate for development into a potential therapy for the treatment of inflammatory and autoimmune diseases (5,6). Although TGF-ß-treated APC-induced tolerance is associated with the induction of regulatory T cells, the precise mechanism(s) by which effector T cells are regulated is currently unknown (1,5). In general, a number of mechanisms have been associated with different forms of tolerance and include: (i) the induction of unresponsiveness or anergy which can, for example, occur when T cells are stimulated either in the absence of co-stimulatory signals (7,8) or via the CTLA-4 molecule (9); (ii) the secretion of immunomodulatory or anti-inflammatory cytokines such as IL-4, IL-10 and TGF-ß by a variety of immune cells (1017); and (iii) deletion of effector T cells (18) that can be mediated via effector molecules that are associated with apoptosis such as Fas ligand (FasL) (1922). All of these mechanisms have been associated, either individually or in concert, with a number of different types of tolerance that are either naturally occurring or induced (2326).

The goal of the present report was to study the mechanisms involved in TGF-ß-treated APC-induced tolerance by directly determining the fate of the effector T cells that are being ‘tolerized’. To achieve this, we have used a well-characterized system that allows us to track small numbers of i.v. injected TCR transgenic T cells in vivo (27) after exposure to TGF-ß-treated APC. We have found in the present study that injection of TGF-ß-treated APC inhibited the expansion of antigen-specific T cells in both in vivo and in vitro assays. We further investigated whether one or more mechanisms, including immunosuppressive cytokines, induction of anergy and deletion, was responsible for the inhibition of T cell expansion. Our data strongly suggest that deletion of antigen-specific T cells may be a primary mechanism by which TGF-ß-treated APC mediate tolerance.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Female BALB/cByJ mice (6–8 weeks old) were purchased from Jackson Laboratories (Bar Harbor, ME) and used as recipients. D011.10 (D011) mice transgenic for the TCR for ovalbumin (OVA) peptide (323–339) presented by H-2d were bred and maintained in our colonies.

Reagents
The following reagents were used: phycoerythrin (PE)– or FITC–KJ1-26 antibody (clonotypic antibody specific for D011.10 TCR; Caltag, Burlingame, CA), PE– or PerCP–anti-CD4 antibody, anti-FasL antibody (MFL3), FITC–CD45RB, FITC–CD62L, FITC–CD69, FITC–CD25, FITC–CD44, FITC–CD38, PE–Annexin-V and 7-amino-actinomycin D (7-AAD) (PharMingen, San Diego, CA), OVA (Sigma, St Louis, MO), carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, OR), anti-TGF-ß antibody (1D11.16.8; National Cell Culture Center, Minneapolis, MN), and human IL-2 and porcine TGF-ß2 (R & D Systems, Minneapolis, MN).

Culture media and conditions
For the in vitro assays, cells were cultured in complete media (RPMI 1640, 10% heat-inactivated FCS, 2 mM glutamine, 10 mM HEPES, 100 U/ml penicillin G sodium, 100 µg/ml streptomycin sulfate and 1 x 10–5 M 2-mercaptoethanol) at 37°C and 5% CO2.

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/ml) were cultured in 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 OVA in 24-well plates overnight. Non-adherent cells were removed and adherent cells harvested by incubation on ice for 1 h, followed by gentle removal using a cell-lifter. Cells were then washed 3 times in HBSS in preparation for injection.

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

In vivo tracking experiments
Naive BALB/c mice received an i.v. injection of 3–5 x 106 D011 transgenic T cells that had been purified using a T cell-affinity column (R & D Systems). For some experiments, the D011 T cells were labeled with CFSE (20 µM) for 20 min at 37°C and then washed 3 times. Mice were injected i.v. with 4000 TGF-ß-treated or untreated APC and D011 T cells, or D011 T cells alone in 200 µl HBSS. Three days later, injected mice were immunized with OVA/CFA at two sites in the nape of the neck. At 3–4 days after immunization, injected mice were euthanized, and draining lymph nodes (LN) collected and processed for staining or in vitro experiments. All of these experiments were performed using LN cells pooled from three to five mice per group per experiment. To detect D011 TCR transgenic T cells, cells were labeled with anti-KJ1.26 and anti-CD4 antibodies, and analyzed by FACS.

In vitro assays
For the in vitro assays, draining LN were harvested and either cultured unfractionated or purified using T cell affinity columns (R & D Systems) for 24, 48, 72 or 96 h. Both unfractionated LN cells (1 x 106 cells/ml) and purified T cells (1 x 106 cells/ml) plus irradiated spleen cells (2 x 106 cells/ml) were cultured with OVA (0.1–0.2 mg/ml) in 1 ml. In some experiments, either IL-2 (50–100 U/ml), anti-TGF-ß (10 µg/ml) or anti-FasL (10 µg/ml) antibodies were added at the beginning of the cultures. After varying periods of time, cells were harvested for analysis.

Flow cytometry
All flow cytometry was performed using a FACScan and 100,000–200,000 events were acquired for each sample; data were analyzed using CellQuest software (Becton Dickinson, Mountain View, CA). Cells were labeled with the appropriate antibodies for 20 min at 4°C in the dark, washed 3 times and fixed with 2% formalin. Dividing cells were detected by FACS using dilution of CFSE as an indicator of cellular division. Annexin and 7-AAD staining were used to detect cell death, using 5 µl of each per 1 x 106 cells, and cells were acquired within 1 h of staining. Each figure is representative of at least three separate experiments.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Intravenous injection of TGF-ß-treated APC results in a decreased expansion of antigen-specific T cells in vivo
The following experiments were designed to determine whether TGF-ß-treated APC had an effect on the ability of antigen-specific T cells to expand in vivo. In the first series of experiments, BALB/c mice were injected i.v. with both D011 TCR T cells and TGF-ß-treated or untreated APC pulsed with OVA, or D011 T cells alone. Three days later, mice were immunized with OVA and CFA, euthanized after 4 days, and draining LN harvested. As shown by FACS analysis in Fig. 1, the percentage of D011 T cells from mice that had received an injection of TGF-ß-treated APC was decreased >40% in comparison to the controls, D011 T cells alone (Fig. 1A; 42.6% inhibition) or untreated APC pulsed with OVA (Fig. 1B; 45.0% inhibition). This type of experiment was performed >10 times and the decrease ranged from 30 to 50%. In addition, we have found that OVA-pulsed untreated APC in the absence of immunization did not induce an increase in T cell expansion by comparison to TGF-ß-treated APC (Fig. 1C; untreated APC: 1.87% versus TGF-ß-treated APC: 1.85%), suggesting that OVA-pulsed untreated APC do not induce any greater T cell priming in comparison to TGF-ß-treated APC. Taken together, these data indicate that injection of TGF-ß-treated APC results in suppression of T cell expansion in vivo. The D011 TCR T cells from mice injected with TGF-ß-treated APC were also evaluated for changes in CD45RB, CD62L, CD69, CD25, CD44 and CD38 expression by FACS analysis. No differences were found in the expression of these markers (data not shown). We conclude from these data that injection of TGF-ß-treated APC results in suppression of T cell expansion in vivo.



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Fig. 1. TGF-ß-treated APC inhibit antigen-specific T cell expansion in vivo. Mice were injected i.v. with the following: (A) D011 T cells (KJ1-26+) alone or D011 T cells and 4000 OVA-pulsed TGF-ß-treated APC, (B) D011 T cells (KJ1-26+) and 4000 OVA-pulsed untreated or TGF-ß-treated APC, or (C) D011 T cells (KJ1-26+) and OVA-pulsed untreated or TGF-ß-treated APC. Three days later, mice were immunized (A and B) or not (C) with OVA/CFA. Four days later, draining LN were collected from three to five mice and pooled, cells labeled with PerCP–CD4 and PE–KJ1-26 antibodies, and analyzed by FACS. The percentage of CD4+ cells that are KJ1-26+ was determined. Representative data are shown.

 
The next series of experiments was designed to determine whether antigen-specific T cells from mice that had received TGF-ß-treated APC could expand in vitro after stimulation with antigen. For these experiments, mice received an i.v. injection of D011 T cells and TGF-ß-treated APC, followed by an immunization with OVA and CFA 3 days later. Four days after immunization, LN cells were collected, cultured unfractionated for 24, 48 and 72 h, and analyzed for the percentage of CD4+KJ1-26+ T cells. To determine the rate of expansion (fold-expansion), the ratio between the starting percentage of CD4+KJ1-26+ T cells at 0 h (pre-culture) and the percentage at the designated times after culture initiation was calculated. There was virtually no expansion of cells from either group at 24 h, and an equivalent expansion of cells from both the TGF-ß-treated and untreated APC groups at 48 h (Fig. 2). However, by 72 h culture, cells from untreated APC-injected mice had expanded dramatically, whereas cells from mice injected with TGF-ß-treated APC expanded to a significantly lesser extent (Fig. 2). These data indicate that antigen-specific T cells from mice that have been injected with TGF-ß-treated APC cannot expand to the same extent as cells from mice injected with untreated APC.



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Fig. 2. TGF-ß-treated APC inhibit antigen-specific T cell expansion in vitro. Mice were injected i.v. with (A) D011 T cells or (B) CFSE-D011 T cells and 4000 TGF-ß-treated or untreated APC, and then immunized with OVA/CFA. Four days later, draining LN were collected from three to five mice and pooled, and cells cultured unfractionated for 24, 48 or 72 h with OVA, labeled with PerCP–CD4 and PE–KJ1-26 antibodies, and analyzed by FACS. For (A), fold-expansion was calculated as the percentage of CD4+KJ1-26+ cells after culture divided by the percentage of CD4+KJ1-26+ cells before culture. For (B), the extent of cell division was evaluated by the dilution of CFSE within the CD4+KJ1-26+ gate; the percentage of CD4+ cells that are KJ1-26+ is also included. Representative data are shown.

 
Evaluation of a direct effect of TGF-ß-treated APC on T cell expansion in vivo or in vitro
The next experiments were performed to determine whether the TGF-ß-treated APC themselves directly impair the expansion of antigen-specific T cells in vivo and in vitro. The standard number of TGF-ß-treated APC used for the tolerance experiments was 4000. In the following experiments, mice received either D011 T cells alone or D011 T cells and increasing numbers (4000, 15,000, 50,000 and 100,000) of TGF-ß-treated APC pulsed with antigen, to determine if greater numbers of TGF-ß-treated APC produced greater suppression of T cell expansion. Mice were immunized 3 days later with OVA and CFA, and LN were harvested after 4 days. Cells were analyzed for the percentage of CD4+KJ1-26+ T cells. As shown in Fig. 3, there were no differences in the ability of larger numbers of TGF-ß-treated APC to suppress expansion of T cells.



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Fig. 3. Injection of increasing numbers of TGF-ß-treated APC does not produce a corresponding decrease in antigen-specific T cell expansion in vivo. Mice were injected i.v. with D011 T cells and 0, 4000, 15,000, 50,000 or 100,000 TGF-ß-treated APC, and then immunized with OVA/CFA. Four days later, draining LN were collected from three to five mice and pooled, cells labeled with PerCP–CD4 and PE–KJ1-26 antibodies, and analyzed by FACS. The percentage of CD4+ cells that are KJ1-26+ was determined. Representative data are shown.

 
The next experiments were performed to determine whether the TGF-ß-treated APC directly inhibit the expansion of T cells in vitro. In these experiments, mice were injected with D011 T cells and 4000 TGF-ß-treated APC, and then immunized 3 days later with OVA and CFA. After 3 days, the LN cells were harvested and T cells purified using T cell-affinity columns (>95% purity by FACS analysis). Cells were re-stimulated in the presence of irradiated spleen cells and antigen, and cultured for 24, 48 and 72 h. Similar to the data showing the fate of unfractionated LN cells displayed in Fig. 2, the ability of purified T cells from mice that had received TGF-ß-treated APC to expand after 72 h culture was significantly decreased as compared to the untreated APC-injected controls (Fig. 4).



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Fig. 4. Effect of removal of TGF-ß-treated APC on T cell expansion in vitro. Mice were injected i.v. with D011 T cells and TGF-ß-treated or untreated APC, and then immunized with OVA/CFA. Four days later, draining LN were collected from three to five mice and pooled, and T cells purified and cultured for 24, 48 or 72 h with irradiated spleen cells and OVA. After culture, cells were labeled with PerCP–CD4 and PE–KJ1-26 antibodies, and analyzed by FACS. Fold-expansion was calculated as the percentage of CD4+KJ1-26+ cells after culture divided by the percentage of CD4+KJ1-26+ cells before culture. Representative data are shown.

 
The decrease in expansion of antigen-specific T cells induced by TGF-ß-treated APC is not due to the induction of anergy
The induction of anergy is one mechanism by which TGF-ß-treated APC may mediate tolerance. Anergic T cells are, by definition, unresponsive to stimulation with antigen and appropriate co-stimulation (7,8). In the following experiments, both in vivo and in vitro assays were used to determine whether the antigen-specific T cells from mice that had been treated with TGF-ß-treated APC were anergic. We, first, determined whether T cells were recruited into the dividing population at a normal rate in vivo following stimulation with antigen and adjuvant, using CFSE-labeled T cells. The D011 T cells were labeled with CFSE and injected into mice that were subsequently injected with TGF-ß-treated APC, and then immunized with OVA and CFA 3 days later. After 4 days, LN were harvested and cells analyzed by FACS. Although the total percentage of CD4+KJ1-26+ T cells was decreased in mice injected with TGF-ß-treated APC, there were no differences in the pattern of dividing cells and in the percentages of T cells that had either undergone cell division or had not divided (Fig. 5). These data suggest that T cells from TGF-ß-treated APC do not appear to be unresponsive to stimulation and, therefore, do not exhibit characteristics typical of anergized cells.



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Fig. 5. TGF-ß-treated APC do not inhibit antigen-specific T cell division in vivo. Mice were injected i.v. with CFSE-labeled D011 T cells and TGF-ß-treated or untreated APC, and then immunized with OVA/CFA. Four days later, draining LN were collected from three to five mice and pooled, cells labeled with PerCP–CD4 and PE–KJ1-26 antibodies, and analyzed by FACS. The extent of cell division was evaluated by the dilution of CFSE within the CD4+KJ1-26+ gate. Representative data are shown.

 
The next experiment was designed to determine whether the failure of T cells from TGF-ß-treated APC-injected mice to expand in vitro was because the T cells were anergic. An important characteristic of classic anergy is that it can be abrogated by addition of exogenous IL-2 to cultures of T cells stimulated through TCR and co-stimulatory molecules (7). For these experiments, mice were treated as described above and the LN harvested. The T cells were purified, and re-stimulated with irradiated spleen cells and antigen in the presence of high doses of IL-2 (100 U/ml). As shown in Fig. 6, IL-2 was unable to restore the ability of T cells from mice receiving TGF-ß-treated APC to expand to the same extent as the control cells. These data support the conclusion drawn from the data shown in Fig. 5 that T cells from mice that have received TGF-ß-treated APC are not anergic and, thereby, strongly suggest that the mechanism(s) mediating TGF-ß-treated APC-induced tolerance does not involve the induction of anergy.



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Fig. 6. IL-2 does not restore the ability of antigen-specific T cells to expand in vitro. Mice were injected i.v. with D011 T cells and TGF-ß-treated or untreated APC, and then immunized with OVA/CFA. Four days later, draining LN were collected from three to five mice and pooled, and T cells purified and cultured for 24, 48 or 72 h with OVA and irradiated spleen cells in the absence or presence of exogenous IL-2 (100 U), labeled with PerCP–CD4 and PE–KJ1-26 antibodies, and analyzed by FACS. Fold-expansion was calculated as the percentage of CD4+KJ1-26+ cells after culture divided by the percentage of CD4+KJ1-26+ cells before culture. Representative data are shown.

 
TGF-ß-treated APC-induced decrease in expansion appears to be due to deletion mediated by FasL
The final series of experiments was designed to determine whether the decrease in expansion of antigen-specific T cells induced by TGF-ß-treated APC was due to a deletion mechanism. For these experiments, we employed the same adoptive transfer system used to generate the data displayed in Fig. 5, in order to follow the CFSE-labeled antigen-specific T cells in vitro after 72 h culture. In these experiments, mice were treated as described above, and LN harvested and T cells purified. The cells were then cultured for 72 h, labeled with anti-CD4 and KJ1-26 antibodies, and then analyzed by FACS. The percentage of CD4+KJ1-26+ T cells from mice receiving TGF-ß-treated APC was markedly decreased by ~60–80% as compared to the controls (Fig. 7A). As found in the in vivo studies using CFSE-labeled cells (Fig. 5), there were no differences in the ability of T cells from mice that had received TGF-ß-treated APC to divide and proliferate in vitro as determined by analysis of CFSE labeling compared to controls (Fig. 7B). However, there was a lower percentage of cells that remained after >7–8 divisions, suggesting that these cells may be undergoing deletion (Fig. 7B). To test this possibility, we determined whether an increase in cell death could be detected in cells from mice receiving TGF-ß-treated APC using Annexin-V and 7-AAD labeling. Mice were treated as described above, the LN cells were cultured for 72 h, and then stained with Annexin-V and 7-AAD to detect dead and dying cells. For these experiments, KJ1-26+ cells were gated, and then analyzed for the presence of Annexin-V+ and 7-AAD+ cells. As shown in Fig. 8, the percentage of KJ1-26+Annexin-V+7-AAD+ from mice receiving TGF-ß-treated APC was twice that found in controls. Moreover, injection of TGF-ß-treated APC resulted in an increase in cell death relative to that found after injection of either D011 cells alone (data not shown), or D011 cells and antigen-pulsed untreated APC (Fig. 8). These data suggest that the decrease in T cell expansion found in mice receiving TGF-ß-treated APC may be due to the induction of cell death, i.e. deletion.



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Fig. 7. TGF-ß-treated APC do not inhibit cell division in vitro, but may delete T cells after 7–8 divisions. Mice were injected i.v. with CFSE-labeled D011 T cells and TGF-ß-treated or untreated APC, and then immunized with OVA/CFA. Four days later, draining LN were collected from three to five mice and pooled, and T cells purified and cultured with irradiated spleen cells and OVA for 72 h. Cells were then labeled with PerCP–CD4 and PE–KJ1-26 antibodies, and analyzed by FACS. (A) The percentage of CD4+ cells that were KJ1-26+ was determined. (B) The extent of cell division was evaluated by the dilution of CFSE within the CD4+KJ1-26+ gate. Representative data are shown.

 


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Fig. 8. TGF-ß-treated APC induce antigen-specific T cell death in vitro. Mice were injected i.v. with D011 T cells and TGF-ß-treated or untreated APC, and then immunized with OVA/CFA. Four days later, draining LN were collected from three to five mice and pooled, and cells cultured for 72 h. Cells were then stained with FITC–KJ1-26 antibody, PE–Annexin-V and 7-AAD, and analyzed by FACS. Percent cell death was determined by the percentage of 7-AAD+ cells within the CD4+KJ1-26+ gate. Representative data are shown.

 
A primary mechanism that is involved in mediating cell death or apoptosis is the Fas–FasL system of apoptosis induction (1922). The following experiments were designed to determine whether FasL is involved in mediating deletion of T cells from mice that had been injected with TGF-ß-treated APC. Mice were treated as described above, LN harvested, and then cultured with antigen and either without antibody, or in the presence of anti-TGF-ß or anti-FasL antibody. Anti-TGF-ß antibody was tested because TGF-ß itself has been implicated in various forms of tolerance (14,16), and alters antigen-presenting abilities and accessory signals of macrophages in vitro (28,29). Therefore, TGF-ß could play a role in TGF-ß-treated APC-induced tolerance. After 72 h, cells were labeled with anti-CD4 and KJ1-26 antibodies, and analyzed by FACS. Treatment with anti-TGF-ß antibody did not affect the expansion of T cells in either the control or TGF-ß-treated APC-injected groups; however, treatment with anti-FasL antibody (a blocking antibody) increased the percentage of T cells in the control group, but more importantly reconstituted the ability of T cells from TGF-ß-treated APC-injected mice to expand at the same rate as cells from control mice (Fig. 9). Furthermore, when cells from each of these groups were stained with 7-AAD (a reagent used to detect dead cells), the percentage of dead T cells (i.e. 7-AAD+) in the untreated cultures from the TGF-ß-treated APC-injected mice was >40% higher than control mice, and this difference did not change significantly with the addition of anti-TGF-ß antibody to the cultures (Fig. 10). However, addition of anti-FasL antibody to the cultures of cells from TGF-ß-treated APC-injected mice decreased cell death to levels similar to those found in cultures of control cells (Fig. 10). Taken together, these data suggest that the decrease in antigen-specific T cell expansion induced by TGF-ß-treated APC is due to FasL-mediated deletion of these T cells.



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Fig. 9. Anti-FasL antibody restores antigen-specific T cell expansion of cells from TGF-ß-treated APC-injected mice in vitro. Mice were injected i.v. with D011 T cells and TGF-ß-treated or untreated APC, and then immunized with OVA/CFA. Four days later, draining LN were collected from three to five mice and pooled, and cells cultured for 72 h with OVA and anti-TGF-ß or anti-FasL antibody, labeled with PerCP–CD4 and PE–KJ1-26 antibodies, and analyzed by FACS. Fold-expansion was calculated as the percentage of CD4+KJ1-26+ cells after culture divided by the percentage of CD4+KJ1-26+ cells before culture. Representative data are shown.

 


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Fig. 10. Anti-FasL antibody prevents death of antigen-specific T cell expansion of cells from TGF-ß-treated APC-injected mice in vitro. Mice were injected i.v. with D011 T cells and TGF-ß-treated or untreated APC, and then immunized with OVA/CFA. Four days later, draining LN were collected from three to five mice and pooled, and cells cultured for 72 h with OVA and anti-TGF-ß or anti-FasL antibody, labeled with PerCP–CD4, PE–KJ1-26 antibodies and 7-AAD, and analyzed by FACS. Percent cell death was determined by the percentage of 7-AAD+ cells within the CD4+KJ1-26+ gate. Representative data are shown.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The goal of the present study was to determine the mechanisms involved in tolerance induced by TGF-ß-treated APC. TGF-ß-treated APC dramatically suppress the DTH response in both naive and pre-primed mice, probably through the induction of regulatory T cells (14). Previous studies have shown that TGF-ß-treated APC do not exhibit changes in expression of class II, B7-1 or B7-2, but express slightly lower levels of CD40 compared to untreated APC, and can directly inhibit IFN-{gamma} production by D011 TCR transgenic T cells in vitro via a mechanism involving TGF-ß (28,29). However, no studies, to our knowledge, have studied the antigen-specific T cells that are being suppressed directly in vivo after treatment with TGF-ß-treated APC. We have used a well-defined adoptive transfer system to track and characterize antigen-specific T cells in mice (27) that have been injected with TGF-ß-treated APC. We have found that injection of TGF-ß-treated APC impairs the ability of antigen-specific T cells to expand after immunization with antigen and adjuvant by comparison to the controls, i.e. D011 T cells alone or D011 T cells plus untreated APC. This impairment in expansion does not seem to be due to a failure to proliferate, since experiments using CFSE-labeled cells indicate that T cells from mice injected with TGF-ß-treated APC appear to be recruited into the dividing population at the same rate as controls. The results of the in vitro studies show that by 72 h of culture, the rate of expansion of T cells from TGF-ß-treated APC-injected mice is dramatically decreased, although again this decrease is not associated with an obvious change in recruitment into the dividing T cell population as determined by CFSE labeling. Furthermore, addition of IL-2 to the cultures did not prevent the impairment in the rate of expansion. Taken together, these data strongly suggest that the tolerance induced by TGF-ß-treated APC does not involve the induction of anergy of the antigen-specific T cells. On the other hand, a decrease in the percentage of CFSE-labeled T cells that had undergone >7–8 divisions was found and this decrease correlated with a corresponding increase in cell death. Interestingly, addition of a blocking anti-FasL antibody resulted in decreased cell death and prevented the impairment in the rate of expansion of T cells from mice receiving TGF-ß-treated APC in vitro. These data strongly suggest that deletion mediated by FasL may be an important mechanism that is involved in the tolerance induced by TGF-ß-treated APC.

A variety of mechanisms have been associated with tolerance including the induction of regulatory T cells, the production of immunosuppressive cytokines, IL-4, IL-10 and TGF-ß, the induction of anergy, and deletion (7,10,11,13,14,18,24). Although previous studies have shown that TGF-ß-treated APC induce populations of regulatory T cells that can adoptively transfer suppression, their mechanism(s) of action has not yet been determined (1,5). We have found no evidence for the production of either IL-4 or IL-10 by either spleen or LN cells from mice that have been injected with TGF-ß-treated APC (data not shown). Although the absence of detectable cytokine production in vitro does not definitively prove that these cytokines are not involved in this form of tolerance, it does make this possibility less likely. The data presented in the present study concerning the inability of anti-TGF-ß antibody to restore expansion of the T cells in vitro strongly suggest that TGF-ß is not involved in that step in the regulation. Furthermore, we believe that our data also strongly suggest that the T cells have not been anergized, thereby ruling out the induction of anergy as a potential mechanism. Deletion of the antigen-specific T cells, on the other hand, appears to be an important mechanism involved in tolerance induced by TGF-ß-treated APC. We have not yet identified the cell or cells that express FasL and are, therefore, responsible for the deletion. There are at least two potential candidates, the TGF-ß-treated APC themselves and one or more populations of T cells. Interestingly, neither removal of the TGF-ß-treated APC by T cell purification in the in vitro studies or injection of greater numbers of TGF-ß-treated APC (100,000 versus 4000) has an impact on the decrease in T cell expansion mediated by TGF-ß-treated APC. Although these data suggest that the TGF-ß-treated APC themselves may not directly suppress the expansion of the antigen-specific T cells, we have not ruled out the possibility that the TGF-ß-treated APC may mediate this effect directly. For example, transient contact of TGF-ß-treated APC with D011 T cells in vivo could possibly initiate a program in the D011 T cells that makes them more prone to apoptosis. However, several studies have shown that TGF-ß-treated APC do not directly suppress proliferation of D011 T cells in vitro and do not induce apoptosis, and, in fact, have been found to rescue D011 T cells from apoptosis in vitro (28,30). On the other hand, there is a possibility that the FasL-expressing perpetrators of deletion are one or more populations of T cells and may be the regulatory T cells that are induced by TGF-ß-treated APC. Experiments are currently underway to test all of these hypotheses.

In conclusion, our data indicate that tolerance induced by TGF-ß-treated APC is associated with deletion of antigen-specific T cells involving the Fas–FasL pathway. Since one of the unique features of TGF-ß-treated APC-induced tolerance is that it can be effectively induced in pre-primed mice, i.e. mice with an established immune response, it is very possible that deletion of these activated antigen-specific T cells may be the primary mechanism by which TGF-ß-treated APC mediate their tolerogenic effect. The TGF-ß-treated APC may be very effective in the treatment of ongoing autoimmune disease through the deletion of activated pathogenic autoreactive T cells. This approach has already been used successfully to prevent and treat autoimmune uveitis in mice and may, therefore, be very useful in the development of therapies for the treatment of a variety of other types of T cell-mediated autoimmune diseases (5,6).


    Acknowledgements
 
This work was supported by a grant from the National Institutes of Health (R01DK56206).


    Abbreviations
 
7-AAD—7-amino-actinomycin D

APC—antigen-presenting cell

CFA—complete Freund’s adjuvant

CFSE—carboxyfluorescein diacetate succinimidyl ester

DTH—delayed-type hypersensitivity

LN—lymph node

OVA—ovalbumin

PEC—peritoneal exudate cell

TGF—transforming growth factor


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Wilbanks, G. A., Mammolenti, M. and Streilein, J. W. 1992. Studies on the induction of anterior chamber-associated immune deviation (ACAID). III. Induction of ACAID depends upon intraocular transforming growth factor-beta. Eur. J. Immunol. 22:165.[ISI][Medline]
  2. Hara, Y., Okamoto, S., Rouse, B. and Streilein, J. W. 1993. Evidence that peritoneal exudate cells cultured with eye-derived fluids are the proximate antigen-presenting cells in immune deviation of the ocular type. J. Immunol. 151:5162.[Abstract/Free Full Text]
  3. Kosiewicz, M. M., Okamoto, S., Miki, S., Ksander, B. R., Shimizu, T. and Streilein, J. W. 1994. Imposing deviant immunity on the presensitized state. J. Immunol. 153:2962.[Abstract/Free Full Text]
  4. Kosiewicz, M. M. and Streilein, J. W. 1996. Intraocular injection of class II-restricted peptide induces an unexpected population of CD8 regulatory cells. J. Immunol. 157:1905.[Abstract]
  5. Hara, Y., Caspi, R. R., Wiggert, B., Dorf, M. and Streilein, J. W. 1992. Analysis of an in vitro-generated signal that induces systemic immune deviation similar to that elicited by antigen injected into the anterior chamber of the eye. J. Immunol. 149:1531.[Abstract/Free Full Text]
  6. Okamoto, S., Kosiewicz, M. M., Caspi, R. R. and Streilein, J. W. 1994. ACAID as a potential therapy of established experimental autoimmune uveitis. In Nussenblatt, S. M., Caspi, R. R. and Gery, I., eds, Proceedings of the 6th International Symposium on the Immunology and Immunopathology of the Eye, p. 195. Elsevier Science, Bethesda, MD.
  7. Schwartz, R. H., Mueller, D. L., Jenkins, M. K. and Quill, H. 1989. T-cell clonal anergy. Cold Spring Harbor Symp. Quant. Biol. 54 :605.
  8. Schwartz, R. H. 1996. Models of T cell anergy: is there a common molecular mechanism? [Comment]. J. Exp. Med. 184:1.[ISI][Medline]
  9. Greenwald, R. J., Boussiotis, V. A., Lorsbach, R. B., Abbas, A. K. and Sharpe, A. H. 2001. CTLA-4 regulates induction of anergy in vivo. Immunity 14:145.[ISI][Medline]
  10. von Herrath, M. G., Dyrberg, T. and Oldstone, M. B. A. 1996. Oral insulin treatment suppresses virus-induced antigen-specific destruction of beta cells and prevents autoimmune diabetes in transgenic mice. J. Clin. Invest. 98:1324.[Abstract/Free Full Text]
  11. Asseman, C., Mauze, S., Leach, M. W., Coffman, R. L. and Powrie, F. 1999. An essential role for interleukin 10 in the function of regulatory T cells that inhibit intestinal inflammation. J. Exp. Med. 190:995.[Abstract/Free Full Text]
  12. Groux, H., O‘Garra, A., Bigler, M., Rouleau, M., Antonenko, S., de Vries, J. E. and Roncarolo, M. G. 1997. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 389:737.[CrossRef][ISI][Medline]
  13. Roncarolo, M. G., Bacchetta, R., Bordignon, C., Narula, S. and Levings, M. K. 2001. Type 1 T regulatory cells. Immunol. Rev. 182:68.[CrossRef][ISI][Medline]
  14. Powrie, F., Carlino, J., Leach, M. W., Mauze, S. and Coffman, R. L. 1996. A critical role for transforming growth factor-beta but not interleukin 4 in the suppression of T helper type 1-mediated colitis by CD45RBlow CD4+ T cells. J. Exp. Med. 183:2669.[Abstract]
  15. Seddon, B. and Mason, D. 1999. Regulatory T cells in the control of autoimmunity: the essential role of transforming growth factor beta and interleukin 4 in the prevention of autoimmune thyroiditis in rats by peripheral CD4+CD45RC cells and CD4+CD8 thymocytes. J. Exp. Med. 189:279.[Abstract/Free Full Text]
  16. Chen, Y., Kuchroo, V. K., Inobe, J., Hafler, D. A. and Weiner, H. L. 1994. Regulatory T cell clones induced by oral tolerance: suppression of autoimmune encephalomyelitis. Science 265:1237.[ISI][Medline]
  17. Miller, A., Lider, O., Roberts, A. B., Sporn, M. B. and Weiner, H. L. 1992. Suppressor T cells generated by oral tolerization to myelin basic protein suppress both in vitro and in vivo immune responses by the release of transforming growth factor beta after antigen-specific triggering. Proc. Natl Acad. Sci. USA 89:421.[Abstract]
  18. Chen, Y., Inobe, J., Marks, R., Gonnella, P., Kuchroo, V. K. and Weiner, H. L. 1995. Peripheral deletion of antigen-reactive T cells in oral tolerance. Nature 376:177.[CrossRef][ISI][Medline]
  19. Lynch, D. H., Ramsdell, F. and Alderson, M. R. 1995. Fas and FasL in the homeostatic regulation of immune responses. Immunol. Today 16:569.[CrossRef][ISI][Medline]
  20. Martin, D. A., Zheng, L., Siegel, R. M., Huang, B., Fisher, G. H., Wang, Jackson, C. E., Puck, J. M., Dale, J., Strauss, S. E., Peter, M. E., Krammer, P. H., Fesik, S. and Lenardo, M. J. 1999. Defective CD95/APO-1/Fas signal complex formation in the human autoimmune lymphoproliferative syndrome, type Ia. Proc. Natl Acad. Sci. USA 96:4552.[Abstract/Free Full Text]
  21. Takahashi, T., Tanaka, M., Brannan, C. I., Jenkins, N. A., Copeland, N. G., Suda, T. and Nagata, S. 1994. Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand. Cell 76:969.[ISI][Medline]
  22. Watanabe-Fukunaga, R., Brannan, C. I., Copeland, N. G., Jenkins, N. A. and Nagata, S. 1992. Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 356:314.[CrossRef][ISI][Medline]
  23. Egan, R. M., Yorkey, C., Black, R., Loh, W. K., Stevens, J. L., Storozynsky, E., Lord, E. M., Frelinger, J. G. and Woodward, J. G. 2000. In vivo behavior of peptide-specific T cells during mucosal tolerance induction: antigen introduced through the mucosa of the conjunctiva elicits prolonged antigen-specific T cell priming followed by anergy. J. Immunol. 164:4543.[Abstract/Free Full Text]
  24. Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, M. and Toda, M. 1995. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 155:1151.[Abstract]
  25. Weiner, H. L. 1997. Oral tolerance: immune mechanisms and treatment of autoimmune diseases. Immunol. Today 18:335.[CrossRef][ISI][Medline]
  26. Chen, Z. K., Cobbold, S. P., Waldman, H. and Metcalfe, S. 1996. Amplification of natural regulatory immune mechanisms for transplantation tolerance. Transplantation 62:1200.[ISI][Medline]
  27. Kearney, E. R., Pape, K. A., Loh, D. Y. and Jenkins, M. K. 1994. Visualization of peptide-specific T cell immunity and peripheral tolerance induction in vivo. Immunity 1:327.[ISI][Medline]
  28. Takeuchi, M., Kosiewicz, M. M., Alard, P. and Streilein, J. W. 1997. On the mechanisms by which transforming growth factor-ß2 alters antigen-presenting abilities of macrophages on T cell activation. Eur. J. Immunol. 27:1648.[ISI][Medline]
  29. Takeuchi, M., Alard, P. and Streilein, J. W. 1998. TGF-ß promotes immune deviation by altering accessory signals of antigen-presenting cells. J. Immunol. 160:1589.[Abstract/Free Full Text]
  30. Takeuchi, M., Alard, P., Verbik, D., Ksander, B. and Streilein, J. W. 1999. Anterior chamber-associated immune deviation-inducing cells activate T cells, and rescue from antigen-induced apoptosis. Immunology 98:576.[CrossRef][ISI][Medline]