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The Journal of Immunology, 2001, 166: 3458-3467.
Copyright © 2001 by The American Association of Immunologists

Fas/Fas Ligand Pathway, Apoptosis, and Clonal Anergy Involved in Systemic Acetylcholine Receptor T Cell Epitope Tolerance1

Caishu Deng2, Elzbieta Goluszko and Premkumar Christadoss3

Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The cellular mechanisms of high dose systemic acetylcholine receptor (AChR) T cell epitope, {alpha}146–162 peptide-induced tolerance in experimental myasthenia gravis were examined. CD4 cells are the prime target for {alpha}146–162 peptide-induced tolerance. The expression of CD69, Fas, and B7.2 molecules on AChR-immune lymphocytes was enhanced within 4–12 h after tolerance induction. A high dose of {alpha}146–162 peptide in IFA failed to suppress T cell proliferation and/or clinical myasthenia gravis in lpr and gld mice deficient in Fas and Fas ligand, respectively. A high dose of {alpha}146–162 peptide in IFA in AChR-immunized mice induced apoptosis of BV6 cells. Further, reconstitution of IL-2 in vitro-recovered {alpha}146–162 peptide tolerized T cell proliferation, IFN-{gamma}, and IL-10 production. The findings implicate the possible role of Fas-/Fas ligand-mediated apoptosis and the resulting clonal anergy as the mechanisms of high dose AChR {alpha}146–162 peptide-induced tolerance on CD4 cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Myasthenia gravis (MG)4 and its animal model, experimental autoimmune myasthenia gravis (EAMG), are Ab-mediated diseases. Acetylcholine receptor (AChR)-specific T cells help B cells to produce anti-AChR Abs (1, 2, 3). Binding of anti-AChR Abs to AChR at the neuromuscular junctions activates the complement cascade and results in AChR loss (4). MHC class II molecules and CD4 T cells play a crucial role in EAMG pathogenesis, because the MHC class II {beta}-chain mutant B6.CH-2bm12 strain and MHC class II- and CD4-deficient mice are resistant to EAMG development (5, 6). Depletion of CD4 cells not only prevented but also treated EAMG (2). Further, CD4 gene knockout (KO) mice are relatively resistant to EAMG development (7).

MG is currently treated by nonspecific immunosuppressive drugs with severe toxic side effects (8, 9, 10, 11, 12). An ideal treatment for MG should be directed at elimination of the AChR-specific T and/or B cells that are involved in the pathogenesis. Torpedo californica AChR {alpha}146–162 peptide is a dominant T cell epitope in the AChR {alpha} subunit and is involved in EAMG pathogenesis in C57BL/6 (B6) mice (13, 14, 15). MG patients’ T cells also respond to human sequence of {alpha}146–162 peptide (16). In a previous study, we demonstrated that high doses of {alpha}146–162 peptide in IFA given s.c. could effectively induce Ag-specific tolerance and prevented EAMG in C57BL/6 (B6) mice (17). High doses of {alpha}146–162 peptide suppressed the AChR-specific T cell responses to AChR and its dominant {alpha}146–162 and subdominant {alpha}182–198 peptides by suppressing IL-2, IFN-{gamma}, and IL-10 production of {alpha}146–162 peptide-specific cells (17). The above tolerance induced by {alpha}146–162 peptide injection is Ag specific because keyhole limpet hemocyanin-specific immune response is not suppressed by {alpha}146–162 peptide injection (17). The precise cellular mechanisms involved in the high dose {alpha}146–162 peptide-induced systemic tolerance in an Ab-mediated disease, EAMG, are not known.

Traditionally, mechanisms of peripheral tolerance have been ascribed to clonal deletion, clonal anergy, or active suppression of self-reactive lymphocytes (18, 19, 20, 21, 22, 23). Other recently postulated mechanisms for tolerance are shift from Th1-mediated cytokines to Th2-mediated cytokines (24, 25) and increased production of immunosuppressive cytokines like TGF-{beta} (26) and IL-10 (27) or by regulatory cells (28). The Fas and Fas ligand (FasL) pathway is involved in activation-induced cell death (AICD) by apoptosis (29), which is one of the main mechanisms in maintaining immunologic homeostasis. Patients with Fas gene mutation developed lymphadenopathy (30, 31). Fas- and FasL-deficient mice develop lymphadenopathy and lupus-like syndrome and die early (32). We hypothesized that Fas-FasL-mediated apoptosis is one of the mechanisms by which high dose AChR T cell epitope {alpha}146–162 peptide induces tolerance in EAMG. In this study, we demonstrate, for the first time, the following findings related to systemic tolerance with AChR {alpha}146–162 peptide in an Ab-mediated autoimmune disease, EAMG: 1) CD4 cells are the prime target for {alpha}146–162 peptide-induced tolerance; 2) Fas-FasL-induced apoptosis does not play a crucial role in the induction of EAMG, because B6.MRL-Faslpr (lpr) and B6.Smn.C3H-gld (gld) mice (Fas and FasL gene-deficient mice, respectively) developed EAMG like the wild-type B6 mice; 3) high doses of {alpha}146–162 peptide in IFA failed to a) suppress T cell proliferation and IL-2 production to AChR and {alpha}146–162 peptide in both lpr and gld mice, b) prevent the development of EAMG, and c) suppress anti-AChR Ab response in lpr mice; 4) the lymphocyte activation molecules, CD69, Fas, and the costimulatory molecule, B7.2, on AChR immune lymph node cells (LNC) were significantly enhanced shortly after high dose {alpha}146–162 peptide injection; 5) IL-2 in vitro could recover {alpha}146–162 peptide-tolerized T cell proliferation. The data most importantly implicate the possible role of Fas-FasL-mediated apoptosis and clonal anergy in high dose {alpha}146–162-induced tolerance in an Ab-mediated disease, EAMG.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Acetylcholine receptor

AChR was purified from the electric organ of T. californica on a neurotoxin affinity column (33). Torpedo AChR {alpha}-chain peptides ({alpha}146–162 and {alpha}182–198) were synthesized in the protein core laboratory, at The University of Texas Medical Branch (Galveston, TX). The sequences are as follow: {alpha}146–162, L-G-I-W-T-Y-D-G-T-K-V-S-I-S-P-E-S; {alpha}182–198, R-G-W-K-H-W-V-Y-Y-T-C-C-P-D-T-P-Y; and {alpha}12–27, L-E-N-Y-N-K-V-I-R-P-V-E-H-H-T-H.

Mice

B6, lpr, and gld mice (7–8 wk old) and CD4 and CD8 gene KO mice in the B6 background were purchased from The Jackson Laboratory, Bar Harbor, ME. All animals were housed in the viral Ab-free barrier facility at the University of Texas Medical Branch and were maintained according to the Animal Care and Use Committee guidelines.

Induction of EAMG and tolerance

For in vivo studies, all mice were anesthetized and immunized with 20 µg AChR emulsified in CFA (Difco Laboratories, Detroit, MI) s.c. at four sites (two hind footpads and shoulders) on day 0 and boosted with AChR on day 30. To induce tolerance, a high dose (500 µg) of {alpha}146–162 peptide in IFA was administered s.c. on days 10 and 18. For control, mice were injected with PBS in IFA. Mice were screened for clinical EAMG on a daily basis. Clinical muscle weakness was graded as follows: grade 0, mouse with normal muscle strength; grade 1, normal at rest, with muscle weakness characteristically shown by hunchback posture, and inability to raise the head after exercise, consisting of 20–30 paw grips on cage top grid; grade 2, mouse showed above symptoms at rest; and grade 3, dehydrated and moribund. Clinical EAMG was also confirmed by i.p. administration of 50 µl neostigmine bromide (0.015 mg/ml) together with atropine sulfate (0.006 mg/ml) in PBS, and observing improvement in muscle strength.

Induction of T cell tolerance

Mice were immunized with 20 µg AChR in CFA s.c. into footpads and base of tail. Seven days later, they received i.p. injection with 500 µg or other doses of {alpha}l46–162 peptide in IFA or PBS in IFA i.p. (control). Fourteen days after AChR immunization, lymph node cells ((LNC) inguinal and popliteal) were collected for lymphocyte proliferation assay. Supernatants were collected after 4 days of culture and frozen at -20°C for cytokine assay.

Lymphocyte proliferative assay

LNC were collected and suspended in RPMI 1640 supplemented with 25 mM HEPES buffer, penicillin G (100 U/ml), streptomycin (100 µg/ml), 2-ME (3 x 10-5 M), and 10% FBS. Cells (4 x 105) were seeded into triplicate wells of 96-well, flat-bottom microtiter plates (Corning, Corning, NY), and challenged with AChR at 2.5 µg/ml or its {alpha}-chain peptides (0.0025–20 µg/ml). Cells were cultured for 5 days at 37°C in humidified 5% CO2-enriched air and pulsed with 1 µCi/well [3H]thymidine during the last 18–22 h of the culture. 3H incorporation was determined by Beckman scintillation counter (Beckman, Fullerton, CA).

Anti-AChR Ab Assay

A crude extract of mouse muscle AChR was incubated at 4°C in Triton buffer with 125I-labeled {alpha}-bungarotoxin (Amersham, Arlington Heights, IL) for 4 h. To 1 ml labeled AChR, 1 µl serum from an experimental mouse was added. Normal mouse serum served as the control. After overnight reaction at 4°C, rabbit anti-mouse serum Ig (100 µl) was added. After incubation at room temperature for 4 h, samples were centrifuged, and the pellets were washed with 1 ml Triton buffer twice and counted in a gamma counter. The difference in the counts of AChR precipitated in the experimental and the control samples enabled us to calculate the Ab response in nanomols of bungarotoxin binding sites per liter of serum (33). The results are expressed as mean ± SEM.

Flow cytometry

Single-cell suspensions of LNC were incubated for 30 min with one of the following Abs: PE-conjugated CD69; Fas; B7.1; B7.2; and CD40 anti-mouse mAbs (PharMingen, San Diego, CA). PE-conjugated isotypes for CD69, Fas, B7.1, B7.2, and CD40 were used for control. Cells were washed twice, then fixed with 2% paraformaldehyde, and analyzed by FACStation flow cytometry (Becton Dickinson, San Jose, CA). In another experiment, LNC were stained with FITC-labeled CD4 or B220 anti-mouse mAb and costained with PE-labeled CD69, Fas, FasL, B7.2, and CD40 anti-mouse mAb.

Annexin V staining to detect apoptotic cells

Single-cell suspensions of LNC (106) from peptide-injected and control mice were triple-stained with: CD4, CD8, TCR-BV6, or BV8 PE-conjugated Abs; FITC-conjugated annexin V; and 7-amino-actinomycin D (7-AAD) (PharMingen, San Diego, CA). One million cells were washed twice with cold PBS (Mg2+, Ca2+) containing 0.05% sodium azide and 0.5% FBS (FACS buffer). Cells were stained with 1 µg PE-conjugated CD4, CD8, BV6, or BV8 Ab in 100 µl FACS buffer for 30 min on ice. The cells were washed once with FACS buffer and stained in the dark with 5 µg annexin V-FITC in 100 µl binding buffer at room temperature for 15 min. Without any further washing, 20 µl 7-AAD were added 20 min before FACS analysis. Backgrounds were established by staining with standard isotype controls matched by species, fluorochrome, and isotype. All dead cells were excluded, and we calculated only the percentage of apoptotic cells of the indicated live cell population. Analyses were performed on FACScan (Becton Dickinson, Mountain View, CA) using CELLQUEST software.

Cytokine detection

IFN-{gamma}, IL-4, and IL-10 were measured by ELISA. ELISA plates (Immunol 2; Dynatech, Chantilly, VA) were coated with 2 µg/ml (50 µl/well) IFN-{gamma}, IL-4, or IL-10 mAb (PharMingen) in 0.1 M carbonate buffer, pH 8.2, overnight at 4°C. The plates were blocked with 200 µl 10% FBS in PBS for 2 h. Supernatant (100 µl) was added at various dilutions titered to the linear portion of the absorbance/concentration curve in duplicate and incubated overnight at 4°C. After the plates were washed four times with PBS and Tween 20 (0.05%), 100 µl biotinylated anti-cytokine-detecting mAbs (directed to different determinant than the first Ab used to coat ELISA plates) at 1 µg/ml in PBS and 10% FBS were added for 45 min at room temperature. Then 100 µl avidin-peroxidase (2.5 µg/ml) were added and incubated for 30 min. Subsequently, the peroxidase substrate ABTS in 0.1 M citric buffer, pH 4.35, in the presence of H2O2 was added, and the absorbance was measured at 405 nm. IL-2 was measured by IL-2-dependent CTLL-2 cell line (American Type Culture Collection, Manassas, VA) proliferative response. Supernatants (100 µl) were added to 104 CTLL-2 cells/well and cultured for 24 h at 37°C and pulsed with 1 µCi [3H]thymidine 6 h before harvesting. Cells were harvested onto glass fiber filter mats, and the radioactivity incorporated was determined.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The optimal dose of {alpha}146–162 peptide to induce tolerance

To test the optimal T cell tolerance dose of {alpha}146–162 peptide, we tested the effect of five doses of {alpha}146–162 peptide (5 µg, 50 µg, 300 µg, 500 µg, and 1 mg) on AChR- and {alpha}146–162 peptide-specific T cell proliferation and IL-2 production. Twelve B6 mice were immunized with 20 µg AChR in CFA on day 0. Groups of two mice received one of the above doses of {alpha}146–162 peptide in IFA i.p. on day 7. PBS in IFA i.p. was used as control. We have previously demonstrated that {alpha}146–162 peptide-induced tolerance is Ag specific (17); therefore, we did not include an unrelated peptide as control. One week after injection of peptide, all of the mice were euthanized, and the AChR-draining lymph node cells (LNC) were collected. LNC were cultured in the presence of AChR or {alpha}146–162 peptide. AChR and {alpha}146–162 peptide-specific T cell proliferative response and IL-2 production were measured (Fig. 1Go). The results showed that a dose of 500 µg and 1 mg of {alpha}146–162 peptide in IFA i.p. gave the optimal suppression of lymphocyte proliferation to AChR and {alpha}146–162 peptide. At low dose (50 or 5 µg) {alpha}146–162 peptide failed to suppress AChR-specific lymphocyte response. At this dose, {alpha}146–162 peptide markedly enhanced the proliferative response to AChR (Fig. 1GoA). Only a dose of 5 µg {alpha}146–162 peptide enhanced the {alpha}146–162 peptide-specific proliferation (Fig. 1GoB). One milligram of {alpha}146–162 peptide gave the maximum suppression of {alpha}146–162 peptide and AChR-specific IL-2 production (Fig. 1GoC). Therefore, the dose of Ag is crucial to attain the best tolerance effect. It is possible to enhance Ag-primed T cell proliferation with a very low dose of Ag in IFA, leading to augmentation rather than suppression of T cell proliferation, which could lead to detrimental, rather than beneficial effects in autoimmune disease.



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  		FIGURE 1. Optimal tolerance dose of {alpha}146–162 peptide on T cell proliferation and IL-2 production. B6 mice were immunized with 20 µg AChR in CFA on day 0. One of five doses of {alpha}146–162 peptide in IFA was injected i.p. on day 7. Draining LNC were collected on day 14. T cell proliferation to AChR (A), {alpha}146–162 peptide (B), and IL-2 (C) production from supernatant derived from AChR and {alpha}146–162 peptide-exposed culture. Representative of three experiments.

 
Kinetics on the maintenance of tolerance

Next, we tested how long the tolerance effect is maintained at the T cell level after a high dose of {alpha}146–162 peptide in IFA. B6 mice were immunized with 20 µg AChR in CFA, 1 wk later they were injected i.p. with either 500 µg {alpha}146–162 peptide or PBS in IFA; 3, 7, and 28 days after the induction of tolerance, AChR and {alpha}146–162 peptide-specific T cell proliferation, and IFN-{gamma} and IL-2 production were measured. Suppression of AChR and {alpha}146–162 peptide-specific proliferative response could be observed from day 3 to day 28 after a single high dose of {alpha}146–162 peptide in IFA (Fig. 2Goa–f). Suppression of {alpha}146–162 peptide- and AChR-specific IFN-{gamma} and IL-2 production were also observed until day 28. However, very little IL-2 was produced by AChR-specific LNC on days 3 and 28 (Fig. 2Go, g–l). Therefore, a single i.p. injection of 500 µg {alpha}146–162 peptide in IFA could induce T cell low responsiveness to AChR and {alpha}146–162 peptide for at least 28 days, as measured by proliferation, IFN-{gamma}, and IL-2 production.



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  		FIGURE 2. Kinetics on T cell proliferation and cytokine production after single dose of 500 µg {alpha}146–162 peptide tolerance: B6 mice were immunized with 20 µg AChR in CFA on day 0. One dose of 500 µg {alpha}146–162 peptide in IFA was injected i.p. on day 7. Control mice received PBS in IFA i.p.. Draining LNC were collected on day 14. T cell proliferation and IFN-{gamma} and IL-2 production specific for AChR and {alpha}146–162 peptide are evaluated on days 3 (a, b, g, h), 7 (c, d, i, j), and 28 (e, f, k, l) after high dose {alpha}146–162 peptide tolerance. Representative of two experiments.

 
The {alpha}146–162 peptide-specific CD4 cells are tolerized

It is important to understand the subset of T cells responsible for induction EAMG and in tolerance induction. We have shown that MHC class II-deficient mice with CD4 cell deficiency failed to develop EAMG (5). Also, CD4 cell-depleted B6 mice (2) or CD4 gene KO mice (7) have a reduced incidence of EAMG with suppressed anti-AChR Ab response. MHC class I-deficient mice with CD8 cell deficiency developed EAMG like the wild-type B6 mice (34). Thus, MHC class II-restricted CD4 cells are crucial in EAMG pathogenesis, whereas CD8 cells play an uncertain role in EAMG. Also, LNC from AChR-immunized CD4 KO mice do not undergo AChR-specific proliferation (data not shown), because AChR immune response is primarily influenced by an MHC class II molecule (5, 6). To study the effect of high dose {alpha}146–162 peptide tolerance on CD4 cells, we injected high dose {alpha}146–162 peptide in IFA in CD8 gene KO mice on a B6 background after priming with AChR. CD8 gene KO mice were immunized with AChR in CFA on day 0, and on day 7 they were tolerized with 500 µg {alpha}146–162 peptide in IFA. On day 14, draining LNCs were exposed to AChR, and {alpha}146–162 and {alpha}182–198 peptides and Ag-specific proliferative response and IL-2 production were measured. Tolerance to {alpha}146–162 peptide effectively suppressed AChR and {alpha}146–162 peptide-specific responses in AChR-immunized CD8 gene KO mice (Fig. 3Go). The {alpha}146–162 peptide tolerance also suppressed {alpha}182–198 peptide-specific response by epitope-spread mechanisms, which was discussed earlier (17). Therefore, AChR-activated CD4 cells in CD8-deficient mice could be effectively tolerized, and the suppressed T cell proliferative response correlated with suppressed IL-2 production (Fig. 3Go). Therefore, the {alpha}146–162 peptide tolerance targets the CD4 cells. LNC of AChR-immunized CD4- gene KO mice failed to respond to AChR and {alpha}146–162 peptide in the in vitro lymphocyte assay (data not shown).



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  		FIGURE 3. Effect of high dose {alpha}146–162 peptide tolerance on T cell proliferation and cytokine production in CD8-/- AChR-immunized mice. CD8-/- mice were immunized with 20 µg AChR in CFA on day 0. One dose of 500 µg {alpha}146–162 peptide in IFA was injected i.p. on day 7. Control mice received PBS in IFA i.p. Draining LNC were collected on day 14. T cell proliferation (A) and IL-2 (B) production were measured. Representative of two experiments.

 
Induction of CD69, Fas, and B7.2 molecule expression on AChR-immunized LNC after high dose {alpha}146–162 peptide injection

We hypothesize that AICD by apoptosis is the primary mechanism of AChR {alpha}146–162 peptide-induced tolerance. First, AChR-specific T cells were activated after induction of high dose {alpha}146–162 peptide and then deleted by Fas-FasL-mediated apoptosis. To test whether {alpha}146–162 peptide-reactive cells undergo activation, we first tested the expression of CD69, a very early activation cell surface marker, and Fas on AChR-draining LNC after high dose {alpha}146–162 peptide plus IFA injection. The B7.1/B7.2 costimulatory signals are also required for T cell activation after the engagement of TCR with the MHC class II peptide complex. Therefore, we also tested the expression of B7 molecules on LNC after a high dose {alpha}146–162 peptide injection. The results showed that CD69 was expressed significantly higher on LNC in high dose {alpha}146–162 peptide plus IFA-injected mice than in PBS plus IFA-injected mice between 4 and 12 h after high dose {alpha}146–162 peptide or PBS injection (Fig. 4GoA). Fas expression was higher at 12 and 24 h after administration of high dose of {alpha}146–162 peptide in IFA than in control mice (Fig. 4GoB). The expression of B7.2 molecule on LNC was also enhanced between 4 and 12 h after {alpha}146–162 peptide injection (Fig. 4GoC). A very low level of B7.1 molecule was expressed on AChR-immune LNC (data not shown), suggesting that B7.2, but not B7.1, is up-regulated after AChR immunization. There was no significant difference in the CD40 expression on LNC between {alpha}146–162- and PBS-injected mice at 2–24 h. The in vitro T cell proliferative response to AChR, {alpha}146–162 and {alpha}182–198 peptide was first increased at 2 h and then declined at 12 h in {alpha}146–162 peptide-tolerized mice, when compared with PBS-treated mice (Fig. 4Go, E and G). Therefore, the suppression of lymphocyte proliferative response to AChR and {alpha}146–162 peptide was preceded by augmented expression of CD69 and Fas molecules (initial activation molecules) and B7.2 molecule on AChR-immune LNC.



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  		FIGURE 4. Effect of high dose {alpha}146–162 peptide on T cell proliferation and the expression of CD69, Fas, B7.2, and CD40 molecules on AChR-immune LNC. B6 mice were immunized with 20 µg AChR in CFA on day 0. One dose of 500 µg {alpha}146–162 peptide in IFA was injected i.p. on day 7. Control mice received PBS in IFA i.p. Draining LNC were collected at 2, 4, 6, 12, and 24 h after {alpha}146–162 peptide injection. The expression of CD69, Fas, B7.2, and CD40 molecules on LNC were measured by flow cytometry after staining with specific dye-conjugated mAbs (A–D). LNC from {alpha}146–162 peptide in IFA- and PBS in IFA-injected mice derived after 2, 4, 6, and 12 h were exposed to AChR (E), {alpha}146–162 (F), and {alpha}182–198 (G) peptide and T cell proliferation was measured. Representative of two experiments. *, p < 0.05; #, p < 0.005; +, p < 0.08 (Student’s t test).

 
We also assessed the expression of CD69, Fas, FasL, B7.2, and CD40 molecules on CD4, dendritic (CD11C+), and B (B220+) cells on AChR-immune LNC at 4, 12, and 24 h after {alpha}146–162 peptide injection. The CD69, Fas, FasL, and B7.2 expression on AChR-immune CD4 cells of {alpha}146–162 peptide-injected mice was augmented at 4 h compared with PBS-treated group (Fig. 5Go). Less than 1.6% LNC expressed CD11C molecules at 4, 12, and 24 h after {alpha}146–162 peptide or PBS injection. A double-fold augmented expression of CD69 on B220+ B cells was observed at 4 and 12 h after {alpha}146–162 peptide-injected compared with PBS-injected mice. Expression of Fas and B7.2 molecules on B220+ B cells was also augmented at 4 and 12 h after {alpha}146–162 peptide treatment compared with PBS-treated group (Fig. 5Go). Similar data were obtained on CD19+ B cells (data not shown).



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  		FIGURE 5. Expression of CD69, Fas, FasL, B7.2, and CD40 molecules on CD4 and B cells after {alpha}146–162 peptide injection. Shown are percent of CD4+ and B220+ LNC coexpressing CD69, Fas, FasL, B7.2, and CD40 molecules. Twelve C57BL/6 mice were immunized with 20 µg AChR/CFA. Seven days later, six mice were inoculated (i.p.) with 500 µg {alpha}146–162 peptide in IFA and another six with PBS/IFA. At 4, 12, and 24 h after peptide/IFA or PBS/IFA infection, pooled LNC from two mice from each group were stained with FITC-labeled CD4 or B220 anti-mouse mAb and costained with PE labeled CD69, Fas, FasL, B7.2, and CD40 anti-mouse mAb.

 
The lpr and gld mice developed clinical EAMG on immunization with AChR in CFA

Fas mutation in lpr mice prevented the development of experimental autoimmune encephalomyelitis (EAE), due to defective apoptosis of target cells (35). To study whether Fas-mediated apoptosis is involved in the destruction of AChR in the neuromuscular junctions or in the induction of EAMG, we immunized B6, lpr, and gld mice with AChR in CFA and evaluated for T cell response and clinical manifestation of EAMG. The lpr and gld mice developed EAMG like B6 mice (Table IGo). Further, there is no difference in the anti-AChR Ab response between AChR-immunized lpr and B6 or gld and B6 mice (data not shown). Therefore, Fas- and FasL-mediated apoptosis had minimal influence on the induction of EAMG in lpr and gld mice, implicating the noninvolvement of Fas-FasL in the destruction of AChR at the neuromuscular junction in EAMG.


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  		Table I. lpr andgld mice developed EAMG, and {alpha}146–162 peptide tolerance failed to suppress EAMG

 
Effect of high dose {alpha}146–162 peptide tolerance on T cell proliferation and cytokine production in B6 and lpr mice

Evidence has shown that the Fas-mediated pathway is involved in T cell apoptosis (29, 36). T cells in an lpr mouse that has a Fas mutation cannot undergo apoptosis. To observe whether apoptosis is one of the main mechanisms by which high dose {alpha}146–162 peptide-induces tolerance, we tested the effect of high dose of {alpha}146–162 peptide on AChR and {alpha}146–162 peptide-specific T cell proliferative response and IL-2 and IFN-{gamma} production in lpr and B6 mice. AChR and {alpha}146–162-specific T cell proliferative response and IL-2 production were markedly suppressed by high dose {alpha}146–162 peptide tolerance in B6 mice, but not in lpr mice (Fig. 6Go, A and B). However, the production of IFN-{gamma} by AChR- and {alpha}146–162-specific cells was suppressed by high dose {alpha}146–162 peptide in both B6 and lpr mice (Fig. 6GoC). In gld mice, {alpha}146–162 peptide in IFA injection failed to suppress but did enhance T cell proliferation to AChR and {alpha}146–162 peptide (Fig. 6GoD). Very little suppression (<20%) of AChR- and {alpha}146–162 peptide-specific IL-2 production was observed in gld mice injected with {alpha}146–162 peptide (Fig. 6GoE). However, a moderate suppression of AChR- and {alpha}146–162 peptide-specific IFN-{gamma} production was observed in {alpha}146–162 peptide-injected gld mice (Fig. 6GoF). The data implied that Fas-mediated apoptosis could be one of the possible main mechanisms involved in high dose {alpha}146–162 peptide-induced suppression of AChR- and {alpha}146–162 peptide-specific lymphocyte proliferative response and IL-2 production in B6 mice. IFN-{gamma} production after high dose {alpha}146–162 peptide-induced-tolerance was not influenced by Fas/FasL pathway.



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  		FIGURE 6. Effect of high dose {alpha}146–162 peptide tolerance on Ag-specific T cell proliferation and cytokine production in lpr and gld mice. B6, lpr, and gld mice were immunized with 20 µg AChR in CFA on day 0. One dose of 500 µg {alpha}146–162 peptide in IFA was injected i.p. on day 7. Control mice received PBS in IFA i.p. Draining LNC from B6, lpr, and gld mice were collected on day 14. Percentage of suppression of lymphocyte proliferation (A, D) to AChR and {alpha}146–162 peptide-specific IL-2 (B, E) and IFN-{gamma} (G, F) production were measured. Percent of suppression was calculated using the formula: (1 - (cpm of peptide/cpm of PBS) x 100. Representative of two experiments.

 
High dose {alpha}146–162 peptide failed to suppress clinical EAMG and the anti-AChR Ab response in lpr mice

To determine whether Fas-mediated pathway is involved in the prevention of EAMG after high dose {alpha}146–162 peptide-induced tolerance in B6 mice (17), we studied the effect of high dose {alpha}146–162 peptide tolerance in lpr mice. High dose {alpha}146–162 peptide tolerance did not suppress anti-AChR Ab response (data not shown) and clinical EAMG (Table IGo) in lpr mice deficient in Fas. Thus, the Fas-mediated pathway is involved in the prevention of EAMG by high dose {alpha}146–162 peptide-induced tolerance in B6 mice. The lack of suppression of EAMG in lpr mice by high dose {alpha}146–162 peptide tolerance could be due to defective Fas-mediated apoptosis of AChR- and {alpha}146–162-specific T cells.

Augmented apoptosis of TCR-BV6-bearing cells after high dose AChR {alpha}146–162 peptide tolerance

TCR-BV6 cells from Torpedo AChR-immunized B6 mice predominantly respond to {alpha}146–162 peptide (37). BV8 cells also expand in vivo after immunization of B6 mice with AChR in CFA (38, 39). One week after immunization of B6 mice with AChR in CFA, group A was inoculated (i.p.) with 500 µg {alpha}146–162 peptide in IFA, group B was inoculated with peptide {alpha}12–27 (less dominant) in IFA, and group C was inoculated with PBS in IFA. Groups A, B, and C were further subdivided into three subgroups; and at 4, 12, and 24 h after peptide or PBS injection, their LNC were stained with Ab to CD4, CD8, BV6, and BV8, followed by annexin V and 7-AAD. Compared with PBS-inoculated mice, {alpha}146–162 peptide tolerance significantly augmented apoptosis of BV6-bearing cells (Fig. 7Go). Significant numbers of annexin V-positive BV6 cells were detected 24 h after induction of {alpha}146–162 peptide tolerance. CD4 and CD8 cells of the {alpha}146–162 peptide-injected group underwent apoptosis, but not significantly more than PBS control. Although augmented apoptosis is observed among the BV8 population, the data did not attain significance when compared with PBS control. Although {alpha}12–27 peptide-injected mice BV6 and BV8 cells underwent apoptosis, but not significantly more than PBS control. The above data most importantly implicate that high dose {alpha}146–162 peptide tolerance augmented apoptosis of part of the BV6 cells, and it is known that BV6 cells are the predominantly responding cells to {alpha}146–162 peptide (37).



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  		FIGURE 7. Augmented apoptosis of BV6 cells after {alpha}146–162 peptide injection. Shown are percent of CD4+, CD8+, BV6+, and BV8+ LNC undergoing apoptosis after injection of {alpha}146–162, {alpha}12–27, or PBS in IFA. Eighteen C57BL/6 mice were immunized in the footpads and base of tail with 20 µg {alpha}12–27 peptide (less dominant) in IFA and another six with PBS-IFA. At 4, 12, and 24 h after peptide or PBS injection, pooled LNC from two mice from each group were stained with Ab to CD4, CD8, BV6, or BV8, followed by annexin V. Values are mean ± SEM of three experimental data. *, p < 0.00761 (Student’s t test).

 
High dose {alpha}146–162 peptide also induces clonal anergy

Clonal anergy is believed to be due to IL-2 deficiency. If exogenous IL-2 was administered to tolerized T cells, the T cell function could be recovered (19). We tested the effect of IL-2 reconstitution in vitro on T cell proliferative response after high dose {alpha}146–162 peptide in IFA injection. Results showed that IL-2 in vitro (5 ng/ml) did recover the AChR- and {alpha}146–162 peptide-specific T cell proliferative response (Fig. 8GoA), and {alpha}146–162 specific IFN-{gamma} (Fig. 8GoB) and IL-10 (Fig. 8GoC) production. This recovery of AChR and {alpha}146–162 peptide-specific immune response could be due to the activation of nondeleted (nonapoptotic) {alpha}146–162 peptide-reactive cells by IL-2. Thus, clonal anergy is also induced after high dose {alpha}146–162 peptide-induced tolerance. The clonal anergy could be the result of deletion of part of the {alpha}146–162 peptide-reactive cells and the resulting local reduction in IL-2 production.



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  		FIGURE 8. Effect of exogenous IL-2 reconstitution on high dose {alpha}146–162 peptide-induced tolerance. B6 mice were immunized with 20 µg AChR in CFA on day 0 and injected with 500 µg {alpha}146–162 or PBS in IFA i.p. on day 7. On day 14, draining LNC were cultured with AChR, or {alpha}146–162 peptide alone, or in the presence of 1 or 5 ng/ml recombinant mouse IL-2; and T cell proliferation, IFN-{gamma}, and IL-10 production were assessed. Representative of two experiments.

 
Lack of costimulatory signals could result in anergy. To test whether high dose {alpha}146–162 peptide tolerance is due to down-regulation of costimulatory molecule expression, we tested the expression of B7.1, B7.2, and CD40 expression. As mentioned earlier, the expression of B7.1 is very low in AChR-immune lymphocytes. The expression of CD40 on LNC was not significantly different between the {alpha}146–162 peptide-injected and control PBS-injected group (Fig. 4GoD). The B7.2 expression on LNC was significantly higher from 4 to 12 h after high dose {alpha}146–162 peptide-tolerized group compared with PBS-injected group (Fig. 4GoC). The data imply that the up-regulation of B7.2 on APCs (B cells) induce stronger second signal for activation of T cells at a very early stage after high dose tolerance. Therefore, the {alpha}146–162 peptide-induced tolerance is not due to deficiency of costimulatory signals.

Lack of Th1 to Th2 immune deviation after high dose {alpha}146–162 peptide-induced tolerance

A shift of Th1 response to Th2 response is believed to be one of the mechanisms by which high dose Ag tolerance suppresses autoimmune diseases (25, 27, 40, 41). In a previous study, we reported suppression of AChR- and {alpha}146–162 peptide-specific IL-2, IFN-{gamma} (Th1), and IL-10 (Th2) production 1 wk after {alpha}146–162 peptide tolerance (17). Therefore, Th1 to Th2 immune deviation was not observed 1 wk after induction of tolerance. IL-4 cannot be detected in AChR-specific lymphocyte culture supernatants. In this study, we tested the kinetics of cytokine profile after administration of high dose {alpha}146–162 peptide. The results again showed that the production of both Th1 (IFN-{gamma}) and Th2 cytokine (IL-10) specific for {alpha}146–162 peptides was suppressed up to 8 wk after induction of high dose {alpha}146–162 tolerance (Fig. 9Go). Therefore, no Th1 to Th2 shift was observed up to 8 wk after induction of {alpha}146–162 peptide tolerance. Therefore, Th1 to Th2 immune deviation did not play a role in high dose {alpha}146–162 peptide-induced tolerance in EAMG.



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  		FIGURE 9. Kinetics of cytokine profile after a single dose of {alpha}146–162 peptide injection. B6 mice were immunized with AChR in CFA on day 0 and injected i.p. with 500 µg {alpha}146–162 or PBS in IFA on day 7. Mice were terminated at 12 h, 24 h, 1 wk, 4 wk, and 8 wk after {alpha}146–162 peptide tolerance induction. Draining LNC were cultured with AChR and {alpha}146–162 peptide. Supernatant IFN-{gamma} and IL-10 were measured. Representative of two experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Traditional mechanisms of periphery tolerance include: 1) clonal deletion by apoptosis through the Fas-FasL pathway; 2) clonal anergy; 3) active suppression by regulatory T lymphocytes secreting suppressive cytokine like TGF-{beta}; and 4) shift in the cytokine profile from the Th1 to the Th2 response. In this study, we present the following original findings on the cellular mechanisms by which high dose AChR {alpha}146–162 peptide induces tolerance in EAMG: 1) CD4 cells are the prime target for {alpha}146–162 peptide-induced tolerance; 2) Fas-FasL-induced apoptosis do not play a crucial role in the induction of EAMG, because lpr and gld mice develop EAMG like the wild-type B6 mice; 3) high doses of {alpha}146–162 peptide in IFA in lpr and gld mice failed to a) suppress T cell response and IL-2 production to AChR and {alpha}146–162 peptide, b) prevent the development of EAMG, and c) suppress anti-AChR Ab response; 4) the lymphocyte activation molecules, CD69, Fas, and costimulatory molecule B7.2 on AChR immune LNC were significantly enhanced shortly after high dose {alpha}146–162 peptide injection; 5) IL-2 in vitro could recover most of the {alpha}146–162 peptide tolerized T cell proliferation. The data most importantly implicate the possible role of Fas-FasL-mediated apoptosis and clonal anergy in high dose {alpha}146–162-induced tolerance in an Ab-mediated disease, EAMG.

Fas-FasL pathway in high dose {alpha}146–162 peptide-induced tolerance

Clonal deletion of Ag-specific T cells by apoptosis is the main mechanism by which the host tolerizes to self or foreign Ags (21, 42, 43, 44, 45, 46). AICD is considered to be the mechanism of peripheral tolerance by high dose Ag (21, 45, 46). Critchfield et al. (21) proposed that AICD-induced by high dose Ag is through the following steps: 1) activation of T cells express growth lymphokines and their receptor; 2) lymphokine stimulates cell-cycle progression; and 3) TCR reengagement leads to programmed cell death. The Fas and FasL pathway provides the key signal to maintain apoptosis. Our data clearly showed that CD69 and B7.2 expression was significantly enhanced before enhancement of Fas expression on LNC shortly after induction of high dose {alpha}146–162 peptide tolerance. Thus, T cells were activated in the early stage after high dose tolerance. Because the frequency of Ag-specific T cells is very low in wild-type animals, almost all of the previous studies used TCR-transgenic mice to trace the fate of Ag-specific T cells after high dose Ag administration (21, 45, 46). Fas- or FasL-deficient mice (lpr and gld mice) are susceptible to EAMG, and their T cells cannot undergo apoptosis due to Fas or FasL mutation. Lack of suppression of T cell proliferative response to AChR and clinical EAMG after high dose {alpha}146–162 peptide-induced tolerance suggests Fas/FasL-mediated AICD as a possible mechanism by which high dose {alpha}146–162 peptide induces tolerance. Our data agree with previous report in which mature CD4+ cells from MRL lpr mice could not be deleted by apoptosis and tolerized after receptor engagement (47). However, the Fas pathway is not involved in the suppression of IFN-{gamma} production by AChR and {alpha}146–162 peptide-reactive T cells after high dose {alpha}146–162 peptide tolerance.

Recent studies implied that Fas and FasL are necessary in the induction of cell-mediated EAE (35). The data suggest that autoreactive FasL+ Th1 cells induce Fas-mediated apoptosis of Fas-expressing CNS tissue (35). In Ab-mediated EAMG, the target site is AChR at the neuromuscular junctions. Successful induction of clinical EAMG in lpr and gld mice suggests that Fas/FasL-induced apoptosis is not involved in the destruction of AChR-expressing muscle membranes, further documenting the lack of cell-mediated pathology during the initial induction of EAMG.

CD4 cells are the target for high dose {alpha}146–162 peptide-induced tolerance

CD4 cells from CD8 gene KO mice responded very well to AChR and its dominant {alpha}146–162 peptide. However, T cells in AChR-immunized CD4 gene KO mice failed to respond to AChR or {alpha}146–162 peptide in vitro. CD8 gene KO mice predominantly expressing CD4 cells could be tolerized (reduced AChR and {alpha}146–162 peptide-specific proliferation and IL-2 production) by high dose {alpha}146–162 peptide injection. Therefore, high dose {alpha}146–162 peptide-induced tolerance target CD4 cells.

Lack of Th1 to Th2 immune deviation during {alpha}146–162 peptide-induced tolerance

Tolerance or autoimmunity has been suggested to be due to the balance between proinflammatory Th1 cytokines and antiinflammatory Th2 cytokines (41). A shift of the destructive Th1 to protective Th2 cytokine response has been observed in a number of studies where Ags were given in tolerogenic doses (25, 27). Also, continuous administration of Ag in tolerogenic doses led to anergy of Ag-specific lymphocytes and up-regulation of Th2 cytokine response (40). More recently, Karachunski et al. (48) showed that nasal administration of synthetic AChR epitope sequences tolerizes Th1 response and prevents EAMG. Evidence has been shown that IFN-{gamma} is crucial in EAMG. IFN-{gamma} gene or receptor KO mice are resistant to EAMG development (49, 50). However, IL-4, a Th2 cytokine, does not play a role in EAMG pathogenesis, because IL-4 gene KO mice developed EAMG like the wild-type mice, despite skewing of the cytokine profile to Th1 (51). Our previous study demonstrated that high dose {alpha}146–162 peptide-induced tolerance suppressed EAMG and is associated with suppression of both Th1 (IL-2 and IFN-{gamma}) and Th2 (IL-10) cytokine production (17), whereas IL-4 was undetectable in tolerized and nontolerized mice. The kinetic analysis in the present study confirmed that Th1 to Th2 immune deviation is not the mechanism of {alpha}146–162 peptide-induced tolerance. Further, recent studies in IL-10 gene KO mice have implicated a facilitative role of IL-10 (a Th2 cytokine) in the development of EAMG (52).

Induction of BV6 cell apoptosis and anergy after high dose {alpha}146–162 peptide injection

Clonal deletion has been suggested to be a mechanism of high dose myelin basic protein (MBP) tolerance in autoimmune encephalomyelitis (21). Multiple doses of MBP injected i.v. induced deletion of transgenic AV2.3+, BV8.2+ cells reactive to Ac1–11 peptide of MBP. Also, high doses of MBP induced deletion of CD4 cells in nontransgenic mice transferred with MBP-reactive lymph node T cells. We have used the wild-type B6 mice having an intact immune system and a full constellation of TCR genes to study the cellular mechanisms of high dose AChR T cell epitope tolerance. Because the frequency of {alpha}146–162 peptide-reactive T cells should be very low in AChR-immunized B6 mice, we tested the effect of tolerance in BV6 cells that are reactive to AChR and {alpha}146–162 peptide (37). For the first time, we show augmented apoptosis of BV6 cells 24 h after {alpha}146–162 peptide injection into wild-type AChR-immunized B6 mice. Although BV8 cells underwent apoptosis after {alpha}146–162 peptide injection, but the apoptotic index was less than that of BV6 cells.

Anergy of Ag-specific T cells has been proposed by other investigators (41) to be involved in high dose Ag-induced tolerance (18, 19, 53). The anergized T cells are functionally hyporesponsive due to lack of IL-2. Exogenous IL-2 can reverse their function. Falb et al. (54) reported that injection of soluble antigenic peptide into transgenic mice could induce the Ag-specific T cell hyporesponsiveness, whereas the T cell number is similar to that of the control. High dose synthetic immunodominant peptides of MBP prevented MBP-induced EAE, blocked the progression, and decreased the severity of EAE by anergy of MBP-reactive cells (19). Anergized T cells could be activated by costimulation through the CD28/B7 pathway. More recent studies demonstrated that high dose Ag-induced tolerance is through the negative signal through the CTLA4/B7 pathway (55, 56). Anergy could also take place after apoptosis (57). In our study, the low response of T cells to AChR and {alpha}146–162 peptide by high dose {alpha}146–162 peptide tolerance could be reversed by exogenous supplementation of IL-2. Thus, anergy also contributes to high dose {alpha}146–162-induced tolerance in EAMG. Anergy of {alpha}146–162 peptide-reactive cells could be due to the deficiency of locally produced IL-2, resulting possibly from Fas/FasL-mediated apoptosis of part of the {alpha}146–162 peptide-reactive T cells. Thus, we show for the first time that both clonal deletion by apoptosis and clonal anergy participate in {alpha}146–162 peptide-induced tolerance. Interestingly, B220+ or CD19+ cells (B cells) are activated after {alpha}146–162 peptide injection. Future studies could suggest whether or not B220+ or CD19+ B cells also undergo apoptosis after {alpha}146–162 peptide injections. It is likely that {alpha}146–162 sequence contains both T and B cell epitopes or that activation of CD4 cells (e.g., BV6) by AChR or {alpha}146–162 peptide augments the activation of B220+ cells by production of cytokines (e.g., IL-2, IFN-{gamma}, or IL-10). The latter event is unlikely, because B220 expression was augmented within 4 h after injection of {alpha}146–162 peptide.

The following is the scenario of cellular events that could take place after systemic injection of a high dose of {alpha}146–162 peptide in IFA into AChR-immunized B6 mice to induce tolerance: 1) activation of CD4 cells (B cells?) specific for {alpha}146–162 peptide, and augmented expression of CD69, Fas, and B7.2 molecules; 2) Fas-FasL-mediated apoptosis of part of {alpha}146–162 peptide-specific (e.g., BV6) cells; 3) reduction of IL-2 production due to deletion of IL-2-producing {alpha}146–162 peptide-reactive (e.g., BV6) cells; 4) clonal anergy of part of {alpha}146–162 peptide-specific cells that had escaped apoptosis; and 5) clonal anergy of other AChR ({alpha}182–198 peptide)-specific cells due to local IL-2 deficiency (infectious tolerance or determinant spread (17)). Therefore, Ag-specific therapy of EAMG and MG could be achieved by Fas-mediated apoptosis and anergy of AChR dominant peptide-reactive T cells by high dose dominant T cell epitope tolerance by activating Fas on the AChR pathogenic epitope-reactive CD4 cells.


   Footnotes
 
1 This work was supported by grants from the Muscular Dystrophy Association and Association Française Center les Myopathies to P.C. C.D. is a J. W. McLaughlin Foundation postdoctoral fellow. Back

2 Current address: Department of Neurology, University of Texas Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75235-9036. Back

3 Address correspondence and reprint requests to Dr. Premkumar Christadoss, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1070. Back

4 Abbreviations used in this paper: MG, myasthenia gravis; EAMG, experimental autoimmune myasthenia gravis; AChR, acetylcholine receptor; KO, knockout; B6, C57BL/6; lpr, B6.MRL-Faslpr; gld, B6.Smn.C3H-gld; FasL, Fas ligand; LNC, lymph node cells; MBP, myelin basic protein; EAE, experimental autoimmune encephalomyelitis; AICD, activation-induced cell death; 7-AAD, 7-amino-actinomycin D. Back

Received for publication December 21, 1999. Accepted for publication December 8, 2000.


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