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The Journal of Immunology, 2003, 171: 1247-1254.
Copyright © 2003 by The American Association of Immunologists

Regulation of Polyclonal T Cell Responses by an MHC Anchor-Substituted Variant of Myelin Oligodendrocyte Glycoprotein 35-551

Mandy L. Ford and Brian D. Evavold2

Department of Microbiology and Immunology, Emory University, Atlanta, GA 30322


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Analogs of immunogenic peptides containing substitutions at TCR contact residues (altered peptide ligands (APLs)) have been used to manipulate Ag-specific T cell responses in models of autoimmunity, including experimental autoimmune encephalomyelitis. However, recent clinical trials with APL of a myelin basic protein epitope revealed limitations of this therapy. In this study, we demonstrate that individual myelin oligodendrocyte glycoprotein (MOG) 35-55-specific T cell clones responded differentially to a MOG 35-55 APL, raising questions about the ability of peptide analogs containing amino acid substitutions at TCR contact residues to control polyclonal populations of T cells. In contrast, we found that a variant peptide containing a substitution at an MHC anchor residue uniformly affected multiple MOG 35-55-specific clones and polyclonal lines. Stimulation of polyclonal MOG 35-55-specific T cells with an MHC variant peptide resulted in the induction of anergy, as defined by a dramatic reduction in proliferation and IL-2 production upon challenge with wild-type peptide. Furthermore, treatment of T cell lines with this peptide in vitro resulted in a significant reduction in their encephalitogenicity upon adoptive transfer. These results indicate that the use of MHC anchor-substituted peptides may be efficacious in the regulation of polyclonal T cell responses such as those found in EAE.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Although initially considered exquisitely specific for a single peptide:MHC complex, TCRs have recently been characterized as having intrinsic cross-reactivity to many related ligands (1, 2). Over the past decade, several studies have examined the impact of altered TCR ligands on T cell activation and differentiation (3, 4, 5, 6, 7, 8, 9, 10). Most of these studies investigated peptides containing substitutions at TCR contact residues, termed altered peptide ligands (APLs),3 and all of these reports focused on variant peptides that possessed the same affinity for MHC (6). APLs are classified as full agonists, weak agonists, partial agonists, or antagonists, based on the type of response they elicit from the responding T cell (1). Because TCR interaction with variant peptides can lead to an altered state of activation, the use of peptide analogs represents an Ag-specific method of manipulating T cell responses. One area in which specific immunotherapy may prove effective is in the treatment of autoimmune diseases, such as experimental autoimmune encephalomyelitis (EAE).

EAE is an autoimmune attack on myelin-producing cells of the CNS that is initiated by CD4+ T cells and mimics the human disease multiple sclerosis (MS) (11, 12, 13). Myelin oligodendrocyte glycoprotein (MOG) 35-55 is a well-characterized target Ag of encephalitogenic T cells in the C57BL/6 mouse model of EAE (11). As in other models of EAE, the responding CD4+ T cells are polyclonal Th1 cells (11) that proliferate rapidly, secrete IFN-{gamma}, and induce cytolysis of Ag-coated targets in vitro (12). APLs have been used as a method of altering the CNS Ag-specific T cell response in EAE by inducing anergy or skewing the cytokine profile of the responding T cells (14, 15). Although the use of APLs to ameliorate EAE in mouse models proved successful (13, 14, 15, 16, 18), clinical trials assessing the efficacy of APL treatment in MS were less effective (17, 18). In part, these trials were discontinued due to the exacerbation of disease in some of the patients, which was associated with the expansion of both wild-type- and APL-specific T cells. Individual T cell clones may differ in their fine specificity and thus in the spectrum of weak agonists, partial agonists, or antagonists to which they respond. These results highlighted the difficulties in using variant peptide therapy as a treatment for MS and suggested that one explanation for the variable efficacy of APL treatment is the heterogeneity of responding T cell populations.

In this study, we describe a unique approach to regulate polyclonal self-reactive T cell responses. Instead of limiting our analysis to variant peptides containing amino acid substitutions at TCR contact residues, we attempted to manipulate MOG 35-55-specific T cells using a peptide containing an amino acid substitution at an MHC anchor residue. We hypothesized that the lowered affinity of the peptide analog for I-Ab may result in the disruption of the TCR:peptide:MHC complex and consequently alter the activation state of the responding T cell. Because MHC anchor-substituted peptides, unlike classical APLs, do not specifically target amino acids in contact with the TCR, we predict that they more broadly affect polyclonal populations. Results indicate that an MHC anchor-substituted peptide with a 200-fold lower affinity for I-Ab induces anergy in multiple MOG 35-55-specific T cell clones and polyclonal lines, and fails to generate symptoms of EAE when injected into susceptible mice. Furthermore, treatment of MOG 35-55-specific T cells with this peptide reduces their encephalitogenicity upon adoptive transfer. The current study therefore addresses the ability of an MHC anchor-substituted peptide to induce anergy in polyclonal MOG 35-55-specific T cells in vitro, and examines the maintenance of this anergic phenotype in vivo after adoptive transfer.


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

Female C57BL/6 mice (H-2b;B6) were purchased from the National Cancer Institute (Frederick, MD) and were housed in the Emory University Department of Animal Resources facility. Mice were used at 5–8 wk of age.

Peptides

Peptides were synthesized using standard 9-fluorenylmethyloxycarbonyl chemistry on a Symphony/Multiplex Peptide Synthesizer and were analyzed by HPLC (Rainin Instruments, Boston, MA) and mass spectrometry at the Emory University Department of Chemistry core facility. The sequences of the peptides used are as follows: MOG 35-55 (MEVGWYRSPFSRVVHLYRNGK), 45D (MEVGWYRSPFDRVVHLYRNGK), and 47A (MEVGWYRSPFSRAVHLYRNGK).

Competition-based affinity ELISA

Membrane-bound I-Ab was purified from T2BB cells by detergent lysis and subsequent purification on an affinity column coupled to anti-I-Ab mAb (YP3), as described (19). Purified class II molecules (0.5 µM), biotinylated MOG 35-55 reference peptide (1 µM), and various concentrations of unlabeled competitor peptides were incubated at 37°C in 0.1 M citrate phosphate buffer (pH 4.5) with 0.2% Nonidet P-40 containing protease inhibitors, as described (20). After 48 h of incubation, peptide:MHC complexes were captured on ELISA microtiter plates using anti-I-Ab mAb (YP3). MHC molecules loaded with biotinylated reference peptide were detected using avidin-alkaline phosphatase (Sigma-Aldrich, St. Louis, MO) and p-nitrophenylphosphate substrate (Bio-Rad, Hercules, CA). The data are expressed as the relative IC50, the amount of peptide required to inhibit the binding of the reference peptide by 50%. Results were normalized to a value of one for wild-type peptide.

Cells and reagents

MOG 35-55-specific T cell lines and clones were generated by priming 6-wk-old C57BL/6 mice with 200 µg MOG 35-55 emulsified in CFA containing 1 mg/ml heat-inactivated Mycobacterium tuberculosis (H37 RA; Difco, Detroit, MI) in the hind footpad and base of tail. Popliteal and inguinal lymph nodes were harvested on day 10. For the generation of MOG 35-55-specific polyclonal T cell lines, 3 x 106 lymph node cells were incubated with irradiated syngeneic splenocytes (2000 rad), 1 µM MOG 35-55, and 10 pg/ml IL-2 in a 24-well plate for 7 days. To generate MOG 35-55-specific T cell clones, serial dilutions of MOG 35-55-primed lymph node cells were cultured in a 96-well plate with irradiated syngeneic splenocytes, 1 µM MOG 35-55, and 10 pg/ml IL-2. Culture medium consisted of RPMI medium 1640 supplemented with 10% FBS (Mediatech, Herndon, VA), 2 mM L-glutamine, 0.01 M HEPES buffer, 100 µg/ml gentamicin (Mediatech), and 2 x 10-5 M 2-ME (Sigma-Aldrich). TCR V{beta} usage was determined by flow cytometry using FITC-conjugated mAbs to V{beta}2, 3, 4, 5.1/5.2, 6, 7, 8.1/8.2, 8.3, 9, 10b, 11, 12, 13, 14, and 17a (BD PharMingen, San Diego, CA). Flow cytometry was performed on a BD FACSCalibur, and data were processed using FlowJo software (Tree Star, San Carlos, CA).

Proliferation assay

MOG 35-55-specific T cell lines or clones (5 x 104 per well) were incubated in a 96-well plate with irradiated syngeneic splenocytes (5 x 105 per well) and the indicated concentration of peptide, as described (21). After 48 h in culture, cells were labeled with 0.4 µCi/well of [3H]thymidine. Eighteen hours later, the plates were harvested on a FilterMate harvester (Packard, Meriden, CT) and analyzed on a Matrix 96 Direct beta counter (Packard).

Induction of hypoproliferation/anergy

MOG 35-55-specific T cell lines or clones (2 x 105 per well) were incubated with irradiated syngeneic splenocytes (5 x 106 per well), 10 pg/ml IL-2, and 10 µM 45D (or 1 µM MOG 35-55 as a control) in a 24-well plate. After 7 days in culture, live cells were separated by centrifugation over a Ficoll gradient (Mediatech), and restimulated in a 24-well plate with peptide and fresh APCs, as described above. After another 7 days in culture, live cells were again separated by centrifugation over a Ficoll gradient and were stimulated in a T cell proliferation assay, as described above.

Cytokine ELISA

Microtiter plates were coated with 50 µl of purified anti-IL-2 (5 µg/ml, clone JES6-1A12; BD PharMingen) or anti-IFN-{gamma} (2 µg/ml, clone R4-6A2) overnight at 4°C, as described (21). Recombinant IL-2 or IFN-{gamma} (BD PharMingen) was used as a standard. Captured cytokines were detected using biotinylated anti-IL-2 (JES6-5H4; BD PharMingen; 100 µg/ml, 100 µl per well) or anti-IFN-{gamma} (clone XMG1.2; BD PharMingen; 100 µg/ml, 100 µl/well) and detected using alkaline phosphatase-conjugated avidin (Sigma-Aldrich) and p-nitrophenylphosphate substrate (Bio-Rad). Colorometric change was measured at 405 nm on a Microplate Autoreader (Bio-Tek Instruments, Winooski, VT).

EAE induction

EAE was induced by immunization on days 0 and 7 with 200 µg of MOG 35-55 emulsified in CFA containing 5 mg/ml heat-inactivated M. tuberculosis (H37 RA; Difco, Detroit, MI) injected s.c. in the hind flank, as described (14). Mice also received 250–500 ng of pertussis toxin i.p. on days 0 and 2. Disease severity was monitored according to the following scale: 0, no disease; 1, flaccid tail; 2, hind limb weakness; 3, hind limb paralysis; 4, forelimb weakness; 5, moribund.

For EAE induction by adoptive transfer, B6 mice were primed with MOG 35-55 peptide, as described above. Single-cell suspensions of lymph nodes and/or spleens were stimulated twice in vitro with either wild-type MOG 35-55 or 45D, and supplemented with IL-2. Forty-eight hours after the last stimulation, cultures were Ficolled, and 3–5 x 106 live cells were injected i.p. into naive mice, as described (22). At the time of adoptive transfer, mice received 200 µg MOG 35-55 emulsified in IFA s.c. in the hind flank. Mice were scored for disease severity according to the scale described above.

Statistical analyses

Statistical analyses were conducted using GraphPad Prism (Software for Science, San Diego, CA). Mean clinical scores were analyzed by Student’s t test, while disease incidence percentages were compared by Fisher’s exact test. Mean high scores and mean day of onset were compared by Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of MHC anchor-substituted peptides and APLs

To assess the potential of MHC variant peptides to regulate polyclonal T cell responses in EAE, variants of MOG 35-55 were generated based on the core epitope defined by Ben-Nun and colleagues as residues 40–48 (23). Based on this binding register, amino acid residues contacting the MHC molecule (residues 1, 4, 6, and 9) are predicted to be at positions 40, 43, 45, and 48, respectively. Previous analyses of optimal I-Ab-binding motifs for the OVA model Ag (24) (unpublished data) were used as a basis to create poor-binding peptide analogs of MOG 35-55 containing amino acid substitutions at MHC anchor residues. These peptides were screened for their MHC affinity in a competition-based ELISA and for their ability to induce proliferation in MOG 35-55-specific T cell lines. Affinities and proliferation-inducing abilities of the MHC anchor-substituted peptides ranged from near wild type to undetectable (Table I).


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  		Table I. Characteristics of MHC anchor-substituted peptides

 
MHC anchor-substituted peptides were then assayed for their ability to induce profound hypoproliferation in MOG 35-55-specific T cells upon restimulation with wild-type Ag (Table I). MOG-specific T cells were cultured with the indicated peptide for 7 days, and live cells were then restimulated with wild-type MOG 35-55. Results indicate that peptides with low, but detectable affinities and abilities to induce proliferation rendered MOG 35-55-specific T cells hypoproliferative upon restimulation. Variants that induce either high levels of proliferation or no proliferation did not significantly inhibit the activation of MOG-specific T cells upon restimulation (Table I). An MHC variant peptide containing a serine to aspartic acid substitution at P6 (45D) and possessing a ~200-fold lower affinity for I-Ab was chosen for further characterization based upon its ability to induce a hypoproliferative state in MOG-specific T cell clones.

In addition, APLs containing either a conservative substitution or an alanine residue at each TCR contact position (P2, 3, 5, 7, and 8) were generated. APLs were also chosen based on their ability to induce weak proliferation in MOG-specific T cells and screened for the ability to induce hypoproliferation in MOG-specific T cell clones upon restimulation with wild-type peptide. In this study, we focus on an APL containing a conservative substitution (valine to alanine) at P8. Results from the competition affinity assay indicate that the affinity of 47A for I-Ab was virtually identical with that of wild-type MOG 35-55 (data not shown), consistent with the classification of 47A as a classical APL.

Classical APL 47A differentially affects proliferation and anergy of MOG 35-55-specific T cell clones

Recent reports using human clones indicate that APLs may be inefficient at controlling polyclonal populations of CNS Ag-specific T cells (17, 18). We therefore sought to determine whether 47A functioned as an APL to affect responses by individual MOG 35-55-specific T cell clones. As shown in Fig. 1, A and B, 47A is a full agonist for clone E, which uses V{beta}8.1/8.2, but induces only minimal proliferation in clone J (V{beta}13). These results indicate that the variable fine specificity of MOG 35-55-specific T cells impacts the ability of classical APL to affect the proliferative response of these cells. Because many T cell-mediated autoimmune diseases are induced by polyclonal populations, the extent to which 47A could stimulate a population containing both responder and nonresponder clones was determined. Fig. 1C demonstrates that the responder T cells dominated over the nonresponders in a polyclonal MOG 35-55-specific T cell population (Fig. 1, C and D).



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  		FIGURE 1. Differential activation of MOG 35-55-specific clones by 47A. Proliferation of MOG 35-55-specific clones and polyclonal lines in response to 47A was determined in T cell proliferation assays in which T cells were incubated with various concentrations of peptide and irradiated APCs for 48 h. Proliferation was determined by [3H]thymidine incorporation. Clone E (V{beta}8) proliferated to near maximal levels in response to stimulation with 47A (A), but clone J (V{beta}13) responded only weakly (B). In a polyclonal population, however, 47A was able to induce near maximal proliferation (C), indicating that responders dominate over nonresponders. The V{beta} usage of the various cells comprising the polyclonal MOG 35-55-specific line is depicted in D.

 
Furthermore, the ability of 47A to induce anergy in clones E and J and in polyclonal cell lines was tested. Treatment with 47A reduced proliferation of clone J cells upon challenge with MOG 35-55 (Fig. 2B). However, treatment with 47A had no effect on either clone E cells or polyclonal cell lines (Fig. 2, A and C). These data suggest that 47A differentially affects individual MOG 35-55-specific T cell clones.



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  		FIGURE 2. Induction of anergy in clone J, but not clone E or polyclonal lines by treatment with 47A APL. MOG 35-55-specific T cell clones E and J were cultured with either wild-type MOG or 47A for 14 days. Live cells were then restimulated with various concentrations of MOG 35-55 and irradiated APCs for 48 h. Proliferation was measured by [3H]thymidine incorporation at 48 h. Culture with 47A resulted in anergy induction in clone J (B), but not in clone E (A) or in polyclonal MOG 35-55-specific T cell lines (C).

 
MHC variant peptide 45D uniformly affects proliferation of multiple MOG 35-55-specific T cell clones

The above results demonstrate that the polyclonal nature of the MOG 35–55-specific T cell population presents an inherent problem in the use of APL to modulate T cell responses. Therefore, the ability of the MHC variant peptide 45D to affect the proliferative response of the same MOG 35-55-specific T cell clones was examined. The 45D supported only minimal proliferation of clones E and J at high doses of Ag (Fig. 3, A and B). These data are representative of four independently derived clones that were tested, and demonstrate that the MHC anchor substitution at P6 similarly affected the proliferative response of multiple MOG-specific clones. The ability of 45D to induce proliferation in polyclonal MOG 35-55-specific lymph node cells was then assessed. As shown in Fig. 3C, 45D induced only minimal proliferation in this polyclonal population at high concentrations of Ag. These data indicate that MHC anchor-substituted peptides may more uniformly affect polyclonal populations of MOG 35-55-specific T cells than classical APLs.



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  		FIGURE 3. Uniform weak activation of MOG 35-55-specific clones by 45D. Proliferation of MOG 35-55-specific T cell clones and polyclonal lines in response to 45D was determined by incubating T cells with various concentrations of peptide and irradiated APCs for 48 h. Proliferation was measured by [3H]thymidine incorporation. Both clones E and J exhibited dramatically reduced proliferation in response to 45D as compared with wild-type peptide (A and B). The 45D also induced minimal proliferation in polyclonal T cell lines (C), indicating that it more uniformly affects MOG 35-55-specific lines and clones.

 
Stimulation with 45D results in anergy in polyclonal MOG 35-55-specific T cells

Previous work using classical APLs with substitutions at TCR contact residues has shown that stimulation of clonal cell populations by certain APLs can induce anergy (1, 4, 5, 6, 21). However, APL treatment of polyclonal populations has proven less efficacious (25, 26). Because MOG 35-55-induced EAE is mediated by a polyclonal population of CD4+ T cells (23), the effect of 45D treatment on MOG 35-55-primed lymph node cells was analyzed. Upon restimulation with various concentrations of wild-type peptide, the polyclonal population treated with 45D exhibited dramatically reduced proliferation when compared with cells treated with wild-type Ag (Fig. 4A). The hypoproliferation seen in these experiments was not the result of low-dose tolerance, as culture of MOG-specific lines with 100-fold lower concentration of wild-type peptide did not result in a reduction in proliferation upon rechallenge (data not shown). A 100-fold higher concentration of 45D was used to compensate for its lower affinity for I-Ab and allow for a similar number of MHC complexes to be loaded with Ag. Moreover, the reduction in MOG 35-55-specific proliferation after 45D treatment was not due to the selection of noncross-reactive 45D-specific cells during the culture period, because there was no observed increase in proliferation in response to 45D (Fig. 4A).



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  		FIGURE 4. Induction of anergy in polyclonal MOG 35-55-specific T cell lines resulting from treatment with 45D. MOG 35-55-specific T cell lines were cultured with either wild-type MOG (filled symbols) or 45D (open symbols) for 14 days. Live cells were then restimulated with various concentrations of MOG 35-55 (squares) or 45D (diamonds) and irradiated APCs for 72 h. Proliferation was measured by [3H]thymidine incorporation (A), and IL-2 production (18 h) was measured by ELISA (B). IFN-{gamma} and IL-4 secretion was measured by ELISA of supernatants from MOG- or 45D-treated T cells 48 h after restimulation with MOG 35-55 (C). Anergic 45D-treated T cells retained their ability to secrete IFN-{gamma} and did not exhibit an increase in IL-4 production.

 
Consistent with their reduced proliferative capacity, polyclonal MOG 35-55-specific cells treated with 45D exhibited a dramatic reduction in their ability to secrete IL-2 in response to wild-type challenge (Fig. 4B). This anergic phenotype could not be rescued by culture with exogenous IL-2 (data not shown), which is consistent with reports of anergy induced by classical APLs, in which culture with IL-2 was not sufficient to reverse the anergic phenotype (4, 5, 21). Polyclonal, 45D-treated MOG 35-55-specific cells did, however, retain their ability to secrete IFN-{gamma} in response to wild-type Ag, and exhibited no increase in IL-4 production (Fig. 4C). This retention of effector function is consistent with the anergic phenotype induced by APLs or costimulation blockade (4, 5, 21, 27, 28). Thus, after treatment with 45D, polyclonal MOG 35-55-specific T cells lose their ability to both proliferate and secrete IL-2, but retain their cytokine profile. These data suggest that MOG 35-55-specific T cells that have interacted with 45D may be less capable of proliferating in vivo and therefore less able to mediate EAE.

Immunization with 45D does not result in the induction of EAE

Previous studies have assessed the ability of classical APLs to induce EAE (13, 14, 15, 25). One report documented the ability of an APL that antagonized myelin basic protein (MBP)-specific T cell clones in vitro to induce EAE in vivo (25). To test whether 45D was capable of stimulating MOG 35-55-specific T cells in vivo, we assessed whether immunization with 45D resulted in the generation of EAE. As depicted in Fig. 5, 45D-immunized mice manifested no clinical symptoms of EAE, whereas mice immunized with wild-type MOG 35-55 developed severe paralysis by day 15 postimmunization. Histological analysis of brain and spinal cord tissue revealed no evidence of inflammation or demyelination in mice immunized with 45D (data not shown). As expected from its ability to induce maximal proliferation in some MOG 35-55-specific T cell clones, 47A was able to induce EAE disease severity and incidence similar to wild-type MOG 35-55 (Fig. 5).



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  		FIGURE 5. Immunization with 45D does not induce EAE, but immunization with 47A does. B6 mice were immunized with MOG 35-55, 45D, or 47A and monitored for clinical manifestations of disease, as described in Materials and Methods. Although all MOG-immunized mice developed severe disease by day 15 postimmunization, 45D-immunized mice failed to exhibit clinical signs of EAE (A and B). In contrast, immunization with 47A resulted in a disease course similar to that induced by wild-type peptide (A and B).

 
Treatment of polyclonal MOG 35-55-specific T cells reduced encephalitogenicity upon adoptive transfer

Because treatment of MOG 35-55-specific T cells with 45D resulted in anergy in vitro, the ability of 45D-treated cells to adoptively transfer EAE was assessed. MOG 35-55-specific cells were generated, as described in Materials and Methods, and were stimulated with Ag (MOG 35-55 or 45D) and IL-2 48 h before adoptive transfer. At the time of transfer, mice were challenged with wild-type MOG 35-55 in IFA. Results indicate that 45D treatment significantly diminishes the encephalitogenicity of adoptively transferred MOG 35-55-specific T cells, even after challenge with wild-type Ag. The 45D-treated recipients exhibited a reduction in disease severity and disease incidence, as well as a delay in the day of onset of disease as compared with wild-type treated recipients (Fig. 6 and Table II). These data strongly suggest that 45D may be a potential candidate for the regulation of MOG 35-55-specific T cell responses in vivo, and implicate the use of MHC anchor-substituted peptides as a possible method of controlling unwanted immune responses.



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  		FIGURE 6. Reduced encephalitogenicity of 45D-treated T cells upon adoptive transfer. Naive B6 mice received MOG- or 45D-treated T cells (5 x 106) and a hind flank challenge of MOG 35-55 in IFA. Mice were then scored for the induction of EAE. Recipients of 45D-treated cells exhibited a delay in disease onset (A), decreased disease severity (A), and decreased disease incidence (B).

 

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  		Table II. Clinical features of EAE induced by adoptive transfer of MOG- or 45D-treated T cellsa

 
To characterize the phenotype of 45D-treated cells after adoptive transfer and peptide challenge, adoptive transfer recipients were sacrificed at day 30 and splenocytes were restimulated and assayed for proliferation and cytokine production. In accordance with results from in vitro experiments, splenocytes from 45D-treated recipients exhibited significantly reduced proliferation in response to Ag as compared with recipients of MOG-treated cells (Fig. 7A). However, these splenocytes retained their ability to secrete IFN-{gamma} (Fig. 7B), as is characteristic of anergic T cells. No increase in IL-4 secretion was observed (Fig. 7C), indicating that a shift in cytokine phenotype was not responsible for the reduction in disease severity and incidence in 45D-treated T cell recipients.



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  		FIGURE 7. Reduced proliferative response, but retention of cytokine production of 45D-treated T cells isolated from adoptive transfer recipients. Splenocytes were isolated from adoptive transfer recipients 30 day postchallenge and assayed for proliferation and cytokine secretion in response to MOG 35-55. Splenocytes from recipients of 45D-treated cells exhibited reduced proliferation relative to splenocytes isolated from recipients of MOG-treated cells (A), as measured by [3H]thymidine incorporation. However, splenocytes from recipients of 45D-treated cells exhibited similar levels of IFN-{gamma} and IL-4 secretion (B and C, respectively), indicating there was no change in the effector phenotype in these mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A major concern in the use of variant peptides as a treatment for polyclonal autoimmune diseases such as EAE is the variation in clonal TCR fine specificity for Ag (25, 26). It may prove more difficult than originally anticipated for an APL to regulate all T cells in a polyclonal response. In addition, recent clinical trials using an APL of MBP 87–99 demonstrated that the APL itself may become an immunogen, resulting in the expansion of APL-specific T cells that correlated with exacerbation of disease (17, 18). To address these concerns, this study examined the ability of an MHC anchor-substituted peptide, specifically, an analog of MOG 35-55, to have an effect on a polyclonal population of MOG-specific T cells. Our results indicate that a single amino acid substitution at the MHC anchor P6 similarly affected several clones (Fig. 3), whereas a change at a TCR contact residue differently affected two independent clones (Figs. 1 and 2). The MHC anchor-substituted peptide was able to induce anergy in a polyclonal population of MOG 35-55-specific T cells.

These results are relevant to other EAE models, MS, and potentially other T cell-mediated autoimmune diseases, because several studies have documented the polyclonal nature of the responding T cell populations. Because individual T cell clones may have different fine specificities, it may be necessary to fully characterize the response of individual T cell clones to a particular APL. One study documented an APL that was inhibitory for one MBP Ac1–9-specific clone, but not another, thus allowing expansion of this clone and the clinical manifestation of EAE (25, 26). These data, along with our study, highlight the potential difficulties in predicting clonal T cell responses to an APL. Previous studies using peptide analogs of proteolipid protein 139-151 to ameliorate disease in the SJL/J strain have documented an APL capable of inhibiting the response of a T cell population expressing multiple V{beta} segments (15). Our data, as well as those of Wraith and colleagues (25, 26), demonstrate that extensive analyses may be required to identify such APLs. In this study, we propose a method to circumvent the complicated situation resulting from the presence of multiple Ag fine specificities in a MOG-specific T cell population. Because amino acid substitutions at MHC anchors may not induce major changes in recognition of the TCR contact residues, the majority of Ag-specific T cell clones should be similarly affected by the reduction in peptide affinity for the MHC molecule.

Our data also demonstrate that an MHC anchor-substituted peptide with a 200-fold lower affinity for I-Ab effectively induces anergy in a polyclonal MOG 35-55-specific T cell population. We propose that this is a result of 45D having a decreased t1/2 relative to wild-type peptide, resulting in the disruption of the TCR:peptide:MHC complex before the full transmission of a stimulatory signal through the TCR is achieved. Thus, as with APLs, this partial signal results in anergy. Because culture of MOG 35-55-specific T cells with a low concentration of wild-type peptide did not result in anergy (data not shown), we conclude that the observed phenomenon is not due to low-dose tolerance (21, 29). It is highly unlikely that secondary TCR contact changes are solely responsible for this observation (30), because this amino acid change similarly affected all tested clones. In light of our findings that TCR contact changes can differentially affect individual T cell clones, a single secondary change at a TCR contact residue would not be expected to impact polyclonal populations. Hence, our findings strongly favor the conclusion that an MHC anchor change is responsible for the altered T cell responses.

The 45D was chosen for characterization of the ability of MHC anchor-substituted peptides to affect autoreactive T cell responses in EAE based upon its ability to induce anergy in MOG-specific T cell clones. Analysis of the relationship between peptide:MHC affinity and T cell response revealed that some MHC anchor changes generated peptides with higher affinities functioned as agonists to support normal T cell responses (Table I). Other peptides possessing affinities undetectable in our assay induced neither proliferation nor anergy in MOG-specific T cells (Table I), indicating that they are essentially null in terms of TCR recognition. Further identification and characterization of 45D and other anergy-inducing peptides such as 43R are ongoing and aim to determine the optimal peptide:MHC kinetics for the induction of anergy. This type of analysis would be extremely advantageous in that it would allow the rapid identification of anergy-inducing peptides. In addition, once anchor residues conferring affinity within the optimal range are identified, these residues could then be used in any epitope restricted by a particular MHC allele to create anergy-inducing peptides.

The anergy observed in this system, like that induced by APLs (4, 5, 21), was not reversible with the addition of exogenous IL-2 (data not shown). This is in contrast to the anergy observed using costimulation blockade (27, 31), suggesting that there are fundamentally different mechanisms responsible for these phenotypes (28). Furthermore, 45D-treated anergic cells retained their ability to secrete effector cytokines such as IFN-{gamma} and low levels of IL-4 (Fig. 4c), which is consistent with anergic phenotypes induced by both APLs and costimulation blockade. Current studies are underway to identify the molecular changes and signaling defects associated with the induction of anergy in this system.

Because most T cells respond optimally to peptides possessing high affinity for MHC, destabilization of the peptide:MHC complex by altering anchor residues usually decreases the immunogenicity of the epitope (32, 33, 34). Our results demonstrate that T cell recognition of a destabilized peptide:MHC complex failed to generate a response sufficient to produce EAE. This is in contrast to the MBP Ac1–9 system, in which unstable peptide:MHC complexes are highly immunogenic and induce severe disease (35, 36). In this system, however, stabilization of the peptide:MHC complex inhibits disease induction due to central deletion of Ag-specific T cells (36). The difference in these systems most likely lies in the fact that MOG 35-55-specific T cells optimally recognize a stable ligand, and therefore are not fully activated peptide ligands possessing low affinity for MHC.

In conclusion, our data provide evidence that MHC anchor-substituted peptides may have utility in mitigating the functionality of polyclonal populations of encephalitogenic T cells. By modifying MHC anchor residues in an encephalitogenic epitope, we were able to circumvent the complications arising from the existence of many individual clones with variable fine specificities. Although we describe the application of this principle in an EAE model, the approach may have general applicability in modulating other aberrant immune responses in which the target Ag is known.


   Acknowledgments
 
We acknowledge Aron E. Lukacher and members of the Evavold lab for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by the National Multiple Sclerosis Society Grant RG 3210A1. M.L.F. was supported in part by a fellowship from the National Science Foundation. Back

2 Address correspondence and reprint requests to Dr. Brian Evavold, Department of Microbiology and Immunology, Emory University, 1510 Clifton Road, Atlanta, GA 30322. E-mail address: evavold{at}microbio.emory.edu Back

3 Abbreviations used in this paper: APL, altered peptide ligand; EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein; MOG, myelin oligodendrocyte glycoprotein; MS, multiple sclerosis. Back

Received for publication March 7, 2003. Accepted for publication May 28, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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