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The Journal of Immunology, 2001, 166: 5279-5285.
Copyright © 2001 by The American Association of Immunologists

Anergy Induction by Dimeric TCR Ligands1

Heiner Appel*, Nilufer P. Seth*, Laurent Gauthier* and Kai W. Wucherpfennig2,*,{dagger}

* Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute, and {dagger} Department of Neurology, Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
T cells that recognize particular self Ags are thought to be important in the pathogenesis of autoimmune diseases. In multiple sclerosis, susceptibility is associated with HLA-DR2, which can present myelin-derived peptides to CD4+ T cells. To generate molecules that target such T cells based on the specificity of their TCR, we expressed a soluble dimeric DR2-IgG fusion protein with a bound peptide from myelin basic protein (MBP). Soluble, dimeric DR2/MBP peptide complexes activated MBP-specific T cells in the absence of signals from costimulatory or adhesion molecules. This initial signaling through the TCR rendered the T cells unresponsive (anergic) to subsequent activation by peptide-pulsed APCs. Fluorescent labeling demonstrated that anergic T cells were initially viable, but became susceptible to late apoptosis due to insufficient production of cytokines. Dimerization of the TCR with bivalent MHC class II/peptide complexes therefore allows the induction of anergy in human CD4+ T cells with a defined MHC/peptide specificity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD4+ T cells play a central role in the pathogenesis of autoimmune diseases. Transfer experiments with CD4+ T cell clones and transgenic expression of autoreactive TCRs have elegantly shown that CD4+ T cells represent a key effector cell population (1, 2, 3). CD4+ T cells are also important in Ab-mediated autoimmune diseases and provide T cell help forautoantibody-producing B cells. The fact that susceptibility to many human autoimmune diseases is associated with particular alleles of MHC class II genes indicates that CD4+ T cells play an important role in these inflammatory processes (4, 5, 6). Ag-specific CD4+ T cells therefore represent potential targets for selective therapy in human autoimmune diseases.

Two general approaches can be used to induce Ag-specific T cell tolerance. Overstimulation of T cells with large doses of soluble Ag can result in activation-induced cell death, but administration of such large doses of Ag carries the risk of exacerbating an ongoing disease process (7, 8). In contrast, partial stimulation by TCR ligation in the absence of costimulation can result in anergy. Anergic T cells fail to proliferate in response to subsequent stimulation through the TCR, but respond to exogenous IL-2 (9, 10, 11, 12, 13).

T cell anergy has been studied in both human and murine systems (9, 10, 11, 12, 13, 14, 15, 16, 17). In vitro studies with human alloreactive T cell clones demonstrated that T cells become anergic when stimulated with transfectants that express MHC class II, but not B7-1 or B7-2 costimulatory molecules (12). Anergy can also be induced in vitro and in vivo with CTLA4-Ig, which binds to B7-1 and B7-2 and blocks their costimulatory function (14). Anergic T cells are defective in transcription of the IL-2 gene due to an altered ratio of Ras-GTP and Rap1-GTP, a negative regulator of the Ras pathway (15, 16). In vitro experiments have demonstrated that the anergic phenotype can be maintained for long periods of time, as long as exogenous IL-2 is provided to support T cell survival (17). Costimulation has also been shown to enhance T cell survival by enhancing expression of Bcl-xL to levels that prevent T cell death in response to IL-2 withdrawal (18). T cells rendered anergic by costimulation blockade may therefore have a shortened life span in vivo. Two recent studies have shown that long-term tolerance to allografts induced by costimulation blockade requires intact apoptotic pathways (19, 20).

Costimulation blockade impairs all T cell-dependent immune responses, and a more selective approach that targets T cells with defined specificities is desirable in human autoimmune diseases. In this study, we demonstrate that soluble, bivalent HLA-DR2/peptide complexes induce anergy in human autoreactive T cells based on the specificity of their TCR.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Analysis of T cell activation by soluble and immobilized molecules

Ag-specific T cell clones were maintained by weekly restimulation with 1 µg/ml PHA (Murex Diagnostics, Norcross, GA) in RPMI supplemented with 10% human serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES, 2 mM glutamine, and 5 U/ml of human rIL-2 (Roche, Indianapolis, IN) using irradiated human PBMC (mononuclear cells) (MNC)3 as feeder cells. The following T cell clones were used: Ob.1A12 and Ob.2F3, which are specific for the myelin basic protein (MBP) (85–99) peptide bound to HLA-DR2b (DRA, DRB1*1501); Hy.1B11, which is specific for the MBP (85–99) peptide bound to HLA-DQ1 (DQA1*0102, DQB1*0502); KW.TT.1, which is specific for a tetanus toxoid peptide (residues 830–843) bound to HLA-DR2a (DRA, DRB5*0101); and Go.P3.1, which is specific for the desmoglein (190–204) peptide bound to HLA-DR4 (DRA, DRB1*0402) (21, 22).

For T cell proliferation assays with immobilized molecules, DR2/MBP-IgG or Abs (200 ng/well) were bound to a 96-well flat-bottom plate (Maxisorb; Nunc, Naperville, IL) by overnight incubation at 4°C in 50 µl of 100 mM bicarbonate, pH 9.6. An anti-CD3 Ab (UCHT-1) and a mouse IgG2a Ab specific for trinitrophenol (both from PharMingen, San Diego, CA) were used as positive and negative controls, respectively. Wells were washed twice with sterile PBS, and 105 T cells were added per well in 200 µl of RPMI supplemented with 10% human serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES, and 2 mM glutamine. After 48 h of culture, 3H-labeled thymidine was added (1 µCi/well) and cells were harvested 16–18 h later onto glass fiber filters. Incorporated radioactivity was quantitated in a beta scintillation counter (Wallac, Gaithersburg, MD).

T cell proliferation assays with soluble DR2/MBP-IgG or soluble Abs were set up in triplicates in 96-well U-bottom plates with 105 T cells/well in a total volume of 200 µl. T cell proliferation was quantitated after 48 h of culture by [3H]thymidine incorporation, as described above. A mouse IgG2a Ab specific for trinitrophenol (PharMingen) was used as a negative control.

For analysis of the kinetics of T cell proliferation following stimulation with DR2/MBP-IgG or peptide-pulsed MNC, peripheral blood MNC were purified from a DR2+ healthy donor by Ficoll density gradient (Amersham Pharmacia, Piscataway, NJ). MNC were washed in RPMI and pulsed overnight with MBP (85–99) peptide (1 µM) in T cell medium without IL-2. MNC were irradiated with 5000 rad, washed twice in RPMI, resuspended in T cell medium without rIL-2, and plated on 96-well plates (105 cells/well). A total of 105 T cells was added, and T cell proliferation was quantitated at different time points by [3H]thymidine incorporation.

Anergy induction and evaluation

Anergy was induced in T cells (clones Ob.2F3 and Ob.1A12) by treatment with soluble DR2/MBP-IgG (20 µg/ml) as well as immobilized DR2/MBP-IgG or anti-CD3 Ab (200 ng/well). T cells were cultured with these molecules for 4 days in 96-well plates using T cell medium without rIL-2, as described above. Stimulation of T cells with soluble DR2/MBP-IgG in the presence of anti-CD28 was done with 10 µg/ml of soluble anti-CD28 Ab (clone 3D10) (23). As controls, T cells maintained in rIL-2 or previously stimulated with peptide-pulsed B cells were used. Following this pretreatment, T cell reactivity to peptide-pulsed APCs was examined.

T cell proliferation assays with anergic T cells were performed using blood MNC or CD40-activated B cells from a DR2+ normal donor. CD40-activated B cells were obtained by stimulation of MNC with CD40L transfectants and human rIL-4 (PharMingen) in the presence of cyclosporin A (Novartis Pharmaceuticals, East Hanover, NJ), as previously described by Schultze et al. (24). B cells or MNC were pulsed overnight with MBP (85–99) peptide (80 nM-50 µM) in RPMI, 10% human serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES, and 2 mM glutamine. Peptide-pulsed APC were then irradiated with 3200 or 5000 rad, washed twice in RPMI, resuspended in T cell medium without IL-2, and plated on 96-well plates (5 x 104 cells/well). Pretreated T cells were washed twice in RPMI and added at 105 cells/well to a final volume of 200 µl. T cell proliferation was quantitated after 48 h by [3H]thymidine incorporation.

Analysis of T cell proliferation and survival with CFSE staining

T cells pretreated with DR2/MBP-IgG, DR2/MBP-IgG and anti-CD28 (clone 3D10), or peptide-pulsed APC were harvested at 96 h, washed twice in RPMI, and labeled with CFSE (Molecular Probes, Eugene, OR). Labeling was performed for 1 h at 37°C with 0.5 µM CFSE in serum-free medium (AIM-V; Life Technologies, Gaithersburg, MD) supplemented with 5 U/ml rIL-2. The addition of rIL-2 greatly increased the yield of T cells following CFSE labeling. T cells were washed three times in RPMI and resuspended in T cell medium with or without rIL-2. CFSE-labeled T cells were then restimulated with CD40-activated DR2+ B cells that had been pulsed with the MBP (85–99) peptide. T cells were harvested following 24, 60, or 108 h and counterstained with annexin V for determination of the fraction of apoptotic T cells. T cells were washed twice in RPMI, resuspended in 100 µl annexin V buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4), and stained with 5 µl Alexa 594-labeled annexin V (Molecular Probes) for 20 min at room temperature. Cells were then diluted by addition of 400 µl of annexin V buffer and analyzed in an EPICS XL FACS (Coulter, Miami, FL).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We previously generated a soluble DR2-IgG fusion protein with a covalently linked MBP (85–99) peptide (sequence ENPVVHFFKNIVTPR) (21). Leucine zipper dimerization domains were attached to the 3' end of DR{alpha} and {beta} extracellular domains to facilitate assembly (25, 26). The Fc segment of mouse IgG2a was placed in frame at the 3' end of the DR{alpha}-Fos chain (27). The molecule was expressed in Drosophila Schneider cells and purified by affinity chromatography using a DR-specific mAb (L243) as well as protein A. Experiments using surface plasmon resonance (BIAcore) demonstrated binding of these bivalent DR2/MBP peptide complexes to an immobilized TCR that recognized the DR2/MBP peptide complex. A t1/2 of 2.1–4.6 min was measured, indicating that bivalent binding significantly slowed dissociation from the TCR. No binding was observed with a control TCR that was specific for the HLA-DR1/hemagglutinin peptide complex (27).

Selective induction of T cell activation by soluble, bivalent DR2/peptide complexes

The specificity of TCR stimulation by bivalent DR2/peptide complexes was examined in T cell proliferation assays (Table IGo) using two T cell clones specific for the DR2/MBP peptide complex (clones Ob.1A12 and Ob.2F3) as well as three control clones (clones KW.TT.1, Hy.1B11, and Go.P3.1) (21, 22). The control clones recognized a tetanus toxoid peptide (residues 830–843) bound to HLA-DR2a (DRA, DRB5*0101) (clone KW.TT.1), the MBP (85–99) peptide bound to HLA-DQ1 (DQA1*0102, DQB1*0502) (clone Hy.1B11), and a desmoglein 3 (190–204) peptide bound to HLA-DR4 (DRA, DRB1*0402) (clone Go.P3.1). Mouse IgG2a was used as a negative control because it had the same Fc segment as the DR2/MBP-IgG fusion protein.


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  		Table I. Selective activation of Ag-specific T cell clones with a dimeric DR2/MBP-IgG fusion protein

 
The bivalent DR2/peptide complexes induced TCR-specific activation because proliferation was only observed with T cell clones that recognized the DR2/MBP peptide complex (Table IGo). In contrast, immobilized anti-CD3 Ab induced proliferation by all T cell clones. The functional properties of bivalent DR2/peptide complexes and anti-CD3 were distinct because bivalent DR2/peptide complexes were stimulatory both in soluble and immobilized form. In contrast, the anti-CD3 Ab induced T cell activation only when it was immobilized (Table IGo) or when APC were added that express Fc receptors (data not shown). These results are in agreement with the observation that soluble anti-CD3 inhibits T cell stimulation by immobilized anti-CD3 (28). As an additional control, we also expressed bivalent DR2 molecules without the linked MBP peptide and used these molecules in the T cell proliferation assay. As expected, these molecules did not induce proliferation on any of the T cell clones tested (data not shown).

Bivalent DR2/peptide complexes induced T cell proliferation with similar kinetics as peptide-pulsed MNCs from a DR2+ normal donor (Fig. 1Go). Maximum T cell proliferation was observed between 36 and 72 h, and [3H]thymidine incorporation returned close to baseline values at 96 h. Based on these results, T cells treated with soluble DR2/MBP-IgG were restimulated after 96 h in most experiments. FACS analysis demonstrated that CTLA-4 surface expression was up-regulated in T cells stimulated with soluble DR2/MBP-IgG (data not shown).



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  		FIGURE 1. Kinetics of initial T cell proliferation induced by DR2/MBP-IgG. Activation of clone Ob.2F3 by soluble DR2/MBP-IgG (20 µg/ml) was compared with stimulation with rIL-2 or peptide-pulsed MNC from a DR2+ donor. Mouse IgG2a was used as a negative control. Soluble DR2/MBP-IgG induced initial T cell activation with similar kinetics as peptide-pulsed MNC. DR2/MBP-IgG or mouse IgG2a (negative control) was added at 20 µg/ml to 105 T cells/well. For stimulation with MBP (85–99) peptide, MNC from a DR2+ donor were pulsed overnight with MBP (85–99) (1 µM), washed, and cocultured with T cells (105 MNC and 105 T cells/well). rIL-2 was used at a concentration of 5 U/ml. T cell proliferation was quantitated by [3H]thymidine incorporation. The x-axis reflects the total time of T cells in culture, including the thymidine pulse.

 
Bivalent DR2/MBP-IgG induces anergy in MBP-specific T cell clones

Anergy can be induced in both human and murine T cells when costimulation through CD28 is blocked during TCR activation (11, 12). Soluble bivalent DR2/peptide complexes might induce anergy in Ag-specific T cells because they trigger the TCR, but do not deliver a signal through costimulatory or adhesion molecules. This hypothesis was tested by treatment of T cells with soluble DR2/MBP-IgG for 96 h in the absence of rIL-2. T cells that had been stimulated with peptide-pulsed APC or maintained in rIL-2 were used as controls. Following treatment, T cells were washed and tested for the ability to proliferate in response to peptide-pulsed B cells. B cells were obtained from blood MNC of a DR2+ normal donor by stimulation with a CD40L transfectant and rIL-4. CD40-activated B cells express high levels of MHC class II and costimulatory molecules and are efficient APC (24).

Pretreatment of T cell clones with soluble bivalent DR2/peptide complexes greatly reduced their ability to proliferate in response to peptide-pulsed B cells, indicating that the T cells had become anergic (Fig. 2GoA). In contrast, T cells that had been maintained in rIL-2 (Fig. 2GoA) or stimulated with peptide-pulsed B cells or MNC proliferated vigorously. T cells were unresponsive to restimulation at all time points tested, which ranged from 3 to 9 days following treatment with soluble DR2/MBP-IgG. Anergy also resulted when T cells from clone Ob.2F3 were pretreated with immobilized DR2/MBP-IgG (Fig. 2GoB). DR2/MBP-IgG also induced anergy in T cells from clone Ob.1A12, which also is specific for the DR2/MBP peptide complex. With this T cell clone, treatment with immobilized DR2/MBP-IgG was more effective than treatment with soluble DR2/MBP-IgG (Fig. 2Go, C and D). When control clones were initially stimulated with DR2/MBP-IgG, no proliferation was observed. These cells died by apoptosis, due to the absence of rIL-2 and a failure to produce IL-2. Because control clones were not viable following the initial incubation with DR2/MBP-IgG, these clones could not be tested for anergy induction in these experiments.



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  		FIGURE 2. Anergy induction by DR2/MBP-IgG. T cells specific for the DR2/MBP peptide complex become anergic following pretreatment with soluble or immobilized DR2/MBP-IgG. A, T cells from clone Ob.2F3 were treated with either rIL-2 or soluble DR2/MBP-IgG (20 µg/ml in medium without rIL-2) for 96 h. T cells were then washed three times and resuspended in medium without rIL-2. T cells (105) were then assayed by coculture with 5 x 104 peptide-pulsed B cells, and T cell proliferation was measured after 48 h by [3H]thymidine incorporation. B, T cells from clone Ob.2F3 were treated with immobilized DR2/MBP-IgG or anti-CD3 for 96 h. Molecules were immobilized in a 96-well plate by overnight incubation of 200 ng/well in 50 µl of 100 mM bicarbonate, pH 9.6. Wells were washed twice with PBS, and 105 T cells were added to each well. After 96 h, T cells were tested for the ability to proliferate to peptide-pulsed B cells, as described above. The same experiments were performed with T cell clone Ob.1A12, using soluble (C) and immobilized molecules (D).

 
Dose-response experiments indicated that anergy was induced with soluble molecules at concentrations ranging from 1.25 to 20 µg/ml (Fig. 3GoA). This dose range corresponded to the concentrationof soluble DR2/MBP-IgG that induced an initial proliferation of these T cells. Anergy was also induced by soluble DR2/MBP-IgG in the presence of MNCs from a DR2+ normal donor, suggesting that these molecules also induced anergy when captured by Fc receptors on the surface of APC (Fig. 3GoB). These data are in agreement with the observation that both soluble and immobilized molecules induced anergy (Fig. 2Go).



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  		FIGURE 3. A, Dose dependence of anergy induction. T cells from clone Ob.2F3 were treated with soluble DR2/MBP-IgG (1.25–20 µg/ml in medium without rIL-2) or with rIL-2 for 96 h. T cells were then washed three times and resuspended in medium without rIL-2. T cells (105) were assayed by coculture with 5 x 104 peptide-pulsed B cells, and T cell proliferation was measured after 48 h by [3H]thymidine incorporation. B, Anergy induction by DR2/MBP-IgG in the presence of MNCs. T cells (5 x 104/well) were pretreated with 20 µg/ml of soluble DR2/MBP-IgG in the absence or presence of irradiated MNC from a DR2+ donor (5 x 104 cells/well). After 96 h, T cells were washed twice in RPMI, resuspended in medium without rIL-2, and cocultured with peptide-pulsed B cells (105 T cells, 5 x 104 B cells/well). After 48 h of culture, T cell proliferation was measured by [3H]thymidine incorporation. These experiments demonstrate that DR2/MBP-IgG can also induce anergy when the molecules can be captured by Fc receptors expressed on APC.

 
Bivalent DR2 molecules without the linked peptide could not be used in these anergy assays because T cells stimulated with these molecules or a control IgG2a Ab died during the initial culture period due to a lack of cytokine production (data not shown). Anergy induction was tested in growth medium without exogenous IL-2, and only T cells that were activated by TCR ligation survived under these conditions.

Anergic T cells are viable and proliferate in the presence of exogenous IL-2

Although anergic T cells are unable to produce IL-2 in response to TCR signaling, anergic T cells proliferate in the presence of exogenous IL-2 (9, 10, 11, 12). When anergic T cells were restimulated with peptide-pulsed B cells in the presence of rIL-2, a strong proliferative response was observed (Fig. 4Go). These experiments indicated that the anergic T cells were viable and that they could respond to exogenous IL-2.



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  		FIGURE 4. Anergic T cells proliferate in response to rIL-2. Anergic T cells are viable and proliferate in response to rIL-2, but not to stimulation via the TCR. T cells (clone Ob.2F3) were pretreated with 20 µg/ml of soluble DR2/MBP-IgG for 96 h in medium without rIL-2. T cells were washed and cocultured with peptide-pulsed B cells in the presence or absence of rIL-2 (5 U/ml). T cell proliferation was measured by [3H]thymidine incorporation after 48 h of culture.

 
To directly visualize anergic T cells and to study their functional properties, T cells were labeled with CFSE before restimulation with peptide-pulsed B cells. Because proliferation results in a stepwise loss of CFSE label with cell division, this method allowed cell viability and the extent of T cell proliferation to be assessed (29). To assess the fraction of apoptotic cells, samples were also stained with Alexa 594-labeled annexin V before FACS analysis. These experiments demonstrated that anergic T cells survived and proliferated in the presence of exogenous rIL-2 (Fig. 5Go).



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  		FIGURE 5. Survival and proliferation of anergic T cells in the presence of rIL-2. T cells from clone Ob.2F3 were pretreated with 20 µg/ml of soluble DR2/MBP-IgG for 96 h and labeled with 0.5 µM CFSE for 1 h at 37°C. T cells were washed three times with PBS, resuspended in medium with rIL-2, and plated on 96-well plates in the presence of peptide-pulsed DR2+ B cells. After 24, 60, or 108 h, T cells were stained with Alexa 594-labeled annexin V and analyzed by FACS. In the presence of IL-2, the anergic T cells were viable at all three time points, indicated by the fact that they remained annexin V negative. Proliferation in response to the rIL-2 was most apparent at 108 h, as indicated by a stepwise loss of CFSE labeling.

 
Anergic T cells become susceptible to late apoptosis following stimulation with peptide-pulsed APC

The fate of anergic T cells following restimulation with peptide-pulsed APC was examined using CFSE-labeled T cells. CFSE labeling of T cells allowed nonlabeled B cells to be excluded during FACS analysis. Although anergic T cells were viable at early time points, a large fraction of these T cells later became annexin V positive (53.1% at 108 h) (Fig. 6GoA). Similar results were observed when T cells had been treated with immobilized anti-CD3 Ab (data not shown). In contrast, only a small fraction of apoptotic cells was observed among control T cells that had been activated with peptide-pulsed B cells before restimulation. These T cells underwent extensive cell division, as indicated by a striking loss of CFSE label (Fig. 6GoB). A second control T cell population that had been maintained in rIL-2 before restimulation with peptide-pulsed B cells also showed a striking degree of cell division (data not shown).



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  		FIGURE 6. Anergic T cells become susceptible to late apoptosis following stimulation with B cells and peptide. T cells (clone Ob.2F3) were pretreated with 20 µg/ml of soluble DR2/MBP-IgG (A), peptide-pulsed B cells (B), or 20 µg/ml of soluble DR2/MBP-IgG plus 10 µg/ml of soluble anti-CD28 (C). After 96 h, T cells were labeled with CSFE and cocultured with peptide-pulsed B cells. Following 24, 60, and 108 h, T cells were stained with Alexa 594-labeled annexin V, followed by FACS analysis. The gate was set such that cellular debris was excluded from analysis. A, After 24 h of stimulation with peptide-pulsed B cells, the majority of anergic T cells were viable (91.7% annexin V negative). However, after 60 and 108 h, large numbers of anergic T cells were apoptotic. The annexin V-positive population comprised 38% and 53.1% of gated T cells at 60 and 108 h, respectively. B, In contrast, T cells that had been pretreated with peptide-pulsed B cells proliferated vigorously following restimulation. Proliferation was already apparent at 24 h by a loss of CFSE-staining intensity. Significant cell division was also evident at 60 h, and T cells remained viable throughout the experiment. C, T cells pretreated with soluble DR2/MBP-IgG in the presence of soluble anti-CD28 (clone 3D10) were not anergic and showed extensive proliferation and little apoptosis.

 
When a soluble anti-CD28 Ab (clone 3D10) (23) was added during treatment with soluble DR2/MBP-IgG (Fig. 6Goc), T cells proliferated following restimulation with peptide-pulsed B cells. A reduced CFSE-labeling intensity was already evident at 24 h, indicating that the kinetics of proliferation was different from control T cells pretreated with peptide-pulsed B cells. These results demonstrate that the absence of costimulation during treatment with bivalent DR2/peptide complexes resulted in anergy.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Soluble, bivalent DR2/peptide complexes effectively activate Ag-specific T cells, even in the absence of APC. The biological activity of these molecules is distinct from Abs to the TCR-CD3 complex because such Abs require immobilization or capture by APC that express Fc receptors. In fact, soluble Abs to the TCR-CD3 complex block T cell activation by immobilized TCR-CD3 Abs (28). Following initial activation by bivalent DR2/peptide complexes, Ag-specific T cells become anergic and fail to proliferate in response to peptide-pulsed APC. Anergy is not due to the fact that the TCR signal is weak, but rather due to a lack of costimulation in the initial activation phase. CFSE labeling clearly demonstrates that anergic T cells are initially viable and become susceptible to late apoptosis in vitro. Apoptosis can be prevented by addition of rIL-2, indicating that cell death is due to a failure of anergic cells to produce sufficient quantities of cytokines required for survival.

The bivalent MHC/peptide complexes only bound to TCRs with the appropriate MHC/peptide specificity based on the following experimental findings: 1) The DR2/MBP-IgG fusion protein only activated T cells specific for the DR2/MBP peptide complex, but not clones with other specificities (Table IGo). The control clones included a HLA-DQ1-restricted clone (Hy.1B11) that was specific for the MBP (85–99) peptide; 2) BIAcore experiments demonstrated specific, dose-dependent binding to a purified DR2/MBP-specific TCR, but not to a control TCR (21); 3) FACS analysis demonstrated staining of DR2/MBP-specific T cell clones, but not of control clones with other specificities (27).

T cell activation was also observed with dimeric MHC class II/peptide complexes in which dimerization was achieved through introduction of a free cysteine at the C terminus and cross-linking (30). This demonstrates that the induction of T cell activation is not an artifact resulting from the attachment of an Ig Fc segment. T cell activation was also reported for other dimeric MHC/peptide-Ig fusion proteins that used different MHC class II molecules and peptides (31). Two distinct mechanisms could account for the initial signaling induced by such molecules: dimerization of two TCR-CD3 complexes or induction of a conformational change in a preassembled TCR-CD3 dimer (32). Crystallographic and functional studies of the erythropoietin receptor demonstrated a preassembled receptor dimer in which the individual membrane-spanning and intracellular domains were too far apart to permit signaling by the receptor-associated Janus kinase 2. Ligand binding induced a major conformational change of the extracellular domain that reduced the distance between the two transmembrane segments from ~73 Å to 39 Å, allowing the associated Janus kinase 2 to come into contact and autophosphorylate (33, 34). Evidence for a preassembled dimer was also provided for glutamate, TNF-R1, IL-2, and epidermal growth factor receptors (35, 36, 37, 38). Because bivalent MHC/peptide complexes also carry the CD4 binding site, which is located in the {beta}2 domain of the DR{beta}-chain, one bivalent MHC/peptide complex could also engage two CD4 molecules (39, 31).

Bivalent, soluble MHC class II/peptide complexes therefore represent an approach for the induction of anergy in defined T cell populations. In contrast to other means of anergy induction that block costimulation of all T cells, these molecules are selective for T cells with a defined MHC/peptide specificity. In addition, treatment with dimeric MHC/peptide complexes delivers a signal only through the TCR and possibly CD4, but not through other cell surface molecules that may still be engaged when particular costimulatory molecules, such as B7-1 and B7-2 or CD40, are blocked. The mechanism of action is also distinct from other approaches for the induction of peptide-specific tolerance. These approaches require administration of Ags and processing/presentation by APC, while the preassembled bivalent MHC/peptide complexes bind directly to the TCR of Ag-specific T cells. Bivalent MHC/peptide complexes have a high molecular weight and may therefore have a long t1/2 in vivo, such as Abs. In contrast, peptides are highly susceptible to proteolytic degradation and are rapidly cleared from the bloodstream through the kidney.

The selective nature of this approach is a potential drawback because it requires knowledge of potentially relevant T cell epitopes. Further characterization of peptide epitopes in human autoimmune diseases with MHC/peptide tetramers and other approaches will therefore be important in the selection of appropriate peptides. Recent studies in celiac disease have demonstrated that two epitopes of gliadin, which are presented by the disease-associated DQ2 molecule, are dominant targets of the T cell-mediated immune response (40, 41). It may also be relevant to interfere relatively early in the disease process, before extensive epitope spreading has occurred (42).

It will be important to further examine the properties of bivalent MHC/peptide complexes in animal models of autoimmunity (43, 44). Particularly relevant may be the nonobese diabetic mouse model because T cells specific for peptides from several islet autoantigens emerge at different stages of the inflammatory process. Studies in such a model may help to elucidate the relative importance of Ag-specific T cell populations in the initiation and progression of disease.


   Acknowledgments
 
We thank Drs. Joachim Schultze, John Gribben, Gordon Freeman, and Lee Nadler for providing the CD40L transfectant and anti-CD28 Abs, as well as Dr. John Daley for valuable advice on FACS analyses.


   Footnotes
 
1 This work was supported by grants from the National Institutes of Health (AI 45757 and NS39096) and the National Multiple Sclerosis Society (to K.W.W.). H.A. was supported by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft. Back

2 Address correspondence and reprint requests to Dr. Kai W. Wucherpfennig, Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA 02115. Back

3 Abbreviations used in this paper: MNC, mononuclear cell; MBP, myelin basic protein. Back

Received for publication July 12, 2000. Accepted for publication February 8, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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