Amelioration of Established Experimental Autoimmune Encephalomyelitis by an MHC Anchor-Substituted Variant of Proteolipid Protein 139–
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免疫学杂志 2005年第6期
Abstract
Murine experimental autoimmune encephalomyelitis (EAE) is a CD4+ T cell-mediated autoimmune disorder directed against myelin proteins within the CNS. We propose that variant peptides containing amino acid substitutions at MHC anchor residues will provide a unique means to controlling the polyclonal autoimmune T cell response. In this study, we have identified an MHC variant of proteolipid protein (PLP) 139–151 (145D) that renders PLP139–151-specific T cell lines anergic in vitro, as defined by a significant reduction in proliferation and IL-2 production following challenge with wild-type peptide. In vivo administration of 145D before challenge with PLP139–151 results in a significant reduction in disease severity and incidence. Importantly, we demonstrate the ability of an MHC variant peptide to ameliorate established EAE. An advantage to this treatment is that the MHC variant peptide does not induce an acute hypersensitivity reaction. This is in contrast to previous work in the PLP139–151 model demonstrating that anaphylactic shock resulting in death occurs upon rechallenge with the encephalitogenic peptide. Taken together, these data demonstrate the effectiveness of MHC anchor-substituted peptides in the treatment of EAE and suggest their utility in the treatment of other autoimmune disorders.
Introduction
Experimental autoimmune encephalomyelitis (EAE),3 a mouse model of multiple sclerosis (MS), is an organ-specific autoimmune disease directed against myelin-producing cells within the CNS (1, 2, 3). Onset can be induced by immunizing susceptible strains of mice with a peptide derived from myelin proteins such as proteolipid protein (PLP) 139–151 emulsified in CFA (4, 5). Upon immunization, autoreactive CD4+ T cells are activated in the periphery and then cross the blood-brain barrier where they mediate disease, resulting in the demyelination of neuronal axons and subsequent paralysis (1, 6). Because disease is initiated by CD4+ T cells, many studies have focused on developing therapies to regulate the polyclonal Ag-specific CD4+ T cell response.
One strategy employed to ameliorate disease was the use of altered peptide ligands (APLs) (7, 8), which are analogs of the immunogenic peptide that have an amino acid substitution at a TCR contact residue (9, 10). In this regard, the extent of T cell activation can be manipulated in an Ag-specific manner. Although based upon strong rationale and in vivo data of successful treatment of EAE, there are several factors that complicate the use of APLs as a form of immunotherapy. Hypersensitivity reactions have been reported in patients receiving multiple injections of an APL (11). In addition, exacerbation of disease occurred in some patients receiving APL therapy (12). It was suggested that the APL itself became immunogenic, leading to the expansion of APL-specific T cells. Further reports have demonstrated that due to differences in the fine specificity of the TCR, the response to an APL may vary among individual clones within the polyclonal population (13, 14, 15). Instead of delivering an anergic signal, the
APL instead may activate the responding T cell. Nevertheless, these results demonstrate the therapeutic potential of designing peptide analogs to specifically target autoimmune populations of T cells, although APLs themselves may have some limitations.
Previously, altered peptide ligands were exclusively designed to disrupt the interaction between the TCR and peptide-MHC (pMHC) (7, 8, 16, 17). However, recent reports from our laboratory described a model whereby variant peptides were designed to affect the stability of the pMHC complex without altering residues in contact with the TCR (13, 18). This method was shown to be effective at inducing anergy in a polyclonal population of Ag-specific T cells in vitro, in direct contrast to a classically defined APL (13). Given that the PLP139–151-specific T cell response uses a diverse T cell repertoire (19), a more successful approach to treating PLP-induced EAE may be through the generation of an MHC anchor-substituted peptide variant.
In this report, we tested the ability of a new class of variant peptides containing amino acid substitutions at MHC anchor residues and found that we were able to induce anergy in a polyclonal population of PLP139–151-specific T cells in vitro. Immunization with the MHC variant peptide failed to induce symptoms of EAE, confirming our previously published data showing that peptides with a reduced affinity for MHC are unable to fully stimulate autoimmune T cells and thus incapable of mediating disease (13). Additionally, mice preimmunized with the MHC variant peptide were protected against EAE induction as demonstrated by a reduction in disease severity as well as a delay in onset of disease following challenge with the encephalitogenic peptide. Importantly, established EAE was ameliorated following administration of this variant peptide in vivo.
Materials and Methods
Mice
Female SJL/J mice (H-2s) were purchased from the National Cancer Institute (Frederick, MD) and were housed in the Emory University Department of Animal Resources facility according to Institutional Animal Care and Use Committee protocols. Mice were used at 8–12 wk of age.
Peptides
PLP139–151 (HSLGKWLGHPDKF) and 145D (HSLGKWDGHPDKF) were purchased from Biosynth International. Peptides were analyzed by mass spectrometry and HPLC.
Cells and reagents
PLP139–151-specific T cell lines were generated by priming 8-wk-old SJL/J mice in the hind footpad and base of tail with 200 μg of PLP139–151 emulsified in CFA containing 1 mg/ml heat-inactivated Mycobacterium tuberculosis (H37 RA; Difco). Popliteal and inguinal lymph nodes were harvested 10 days later. Lymph node cells (5 x 106) were incubated with irradiated syngeneic splenocytes (2000 rad), 1 μM PLP139–151, and 10 pg/ml IL-2 in a 24-well plate for 7 days. Culture media consisted of RPMI 1640 medium supplemented with 10% FBS (Mediatech), 2 mM L-glutamine, 0.01 M HEPES buffer, 100 μg/ml gentamicin (Mediatech), and 2 x 10–5 M 2-ME (Sigma-Aldrich).
Proliferation assay
PLP139–151-specific T cell lines (5 x 104 per well) were cultured in a 96-well plate with irradiated syngeneic splenocytes (5 x 105 per well) with either no peptide or the indicated concentrations of peptides at 37°C for 48 h. Each well was then pulsed with 0.4 μCi of [3H]thymidine. The plates were harvested 18 h later on a FilterMate harvester (PerkinElmer Life and Analytical Sciences), and the incorporated radioactivity was measured by a Matrix 96 Direct Beta Counter (PerkinElmer Life and Analytical Sciences). All assays were performed in duplicate.
Cytokine ELISA
PLP139–151-specific T cells (3 x 105) were cultured with irradiated syngeneic splenocytes (5 x 106) in the presence of peptides for 24 h (IL-2) or 48 h (IFN- and IL-4). Supernatants were removed and placed on microtiter plates that had been coated with 50 μl of purified anti-IL-2 (5 μg/ml, clone JES6-1A12; BD Pharmingen), anti-IFN- (5 μg/ml, clone R4-6A2; BD Pharmingen), or anti-IL-4 (5 μg/ml, clone 11B11; BD Pharmingen) overnight at 4°C. Recombinant IL-2, IFN-, or IL-4 (BD Pharmingen) was used as a standard. Captured cytokine was detected using biotinylated anti-IL-2 (JES6-5H4, 100 μg/ml, 100 μl/well; BD Pharmingen), biotinylated anti-IFN- (clone XMG1.2, 100 μg/ml, 100 μl/well; BD Pharmingen), or biotinylated anti-IL-4 (clone BVD6-24G2, 100 μg/ml, 100 μl/well; BD Pharmingen) followed by 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). All assays were performed in triplicate.
EAE induction
EAE was induced by immunization of 8-wk-old female SJL/J mice (National Cancer Institute) with 200 μg of PLP139–151 emulsified in CFA containing 5 mg/ml heat-inactivated M. tuberculosis (H37 RA; Difco) on days 0 and 7 s.c. in the hind flank. Mice also received 250 ng of Bordetella pertussis toxin i.p. on days 0 and 2. Mice were scored as follows: 0, no disease; 1, flaccid tail; 2, hind limb weakness; 3, hind limb paralysis; 4, forelimb weakness; and 5, moribund. For treatment protocol, mice were given a s.c. injection of PLP139–151 (200 μg), 145D (200 μg), or PBS at day 21 followed by subsequent injections at day 28 and 35. For induction of anaphylaxis, mice were given an i.p. injection of 100 μg PLP139–151, 145D, 144Q, or OVA 323–339 in PBS at day 35.
Preimmunization protocol
Mice were preimmunized with 200 μg of PLP139–151 or 145D emulsified in CFA containing 5 mg/ml heat-inactivated M. tuberculosis (H37 RA; Difco) s.c. over both hind flanks or were not immunized. Mice were challenged 21 days later with 100 μg of PLP139–151 emulsified in CFA containing 5 mg/ml heat-inactivated M. tuberculosis (Difco). Mice also received 250 ng of B. pertussis toxin i.p. on this day and on day 2 following challenge. Disease severity was monitored as previously described.
Statistical analyses
Statistical analyses were conducted using GraphPad Prism (Software for Science). Mean clinical scores were analyzed by Student’s t test, Mann-Whitney U test, or one-way ANOVA, whereas disease incidence percentages were compared by Fisher’s exact test.
Results
Identification of an MHC anchor-substituted peptide that induces anergy in PLP139–151-specific T cells
Because our laboratory has previously shown that MHC anchor-substituted peptides are effective at regulating polyclonal T cell responses (13, 18), we wanted to identify an MHC variant peptide that could induce anergy in PLP139–151-specific T cell lines. Based upon the identified core epitope of PLP139–151 (20), residues 140, 143, 145, and 148 correspond to the amino acids contacting the MHC molecule (residues 1, 4, 6, and 9, respectively). Peptide analogs of PLP139–151 were rationally designed based on a report that identified positions 145 and 148 as critical MHC binding residues (19). In all, 27 variant peptides were generated and screened for their ability to stimulate proliferation of PLP139–151-specific T cells. One MHC variant peptide (145D) containing a leucine to aspartic acid substitution at P6, induced minimal proliferation in our polyclonal population of PLP139–151-specific lymph node cells, and was chosen for further analysis (Fig. 1A). To determine the effectiveness of treatment with 145D on the PLP-specific T cell response, PLP139–151-specific lines were cultured with the variant peptide and then restimulated with the indicated concentrations of wild-type peptide. We observed a marked reduction in the proliferative capacity of the 145D-treated cells compared with the cells cultured with wild-type Ag (Fig. 1A). There was no reduction in proliferation in PLP139–151-specific T cells following treatment with 100-fold lower concentration of wild-type peptide (data not shown), confirming our previous observations that this effect was not due to low dose tolerance (13, 18). It is also important to note that there was no increase in proliferation in the 145D-treated cells following restimulation with 145D, indicating a unique population of T cells specific for the variant peptide was not generated (Fig. 1A).
FIGURE 1. Induction of anergy in polyclonal PLP139–151-specific T cell lines. PLP139–151-specific T cell lines were cultured with either 1 μM PLP139–151 (filled symbols) or 10 μM 145D (open symbols) for 2 wk. Live cells were then restimulated with the indicated concentrations of PLP139–151 (squares) or 145D (triangles) and irradiated APCs for 72 h. Proliferation was measured by [3H]thymidine incorporation (A), and IL-2 production (24 h) was measured by ELISA (B). IFN- and IL-4 secretion was measured by ELISA from supernatants of PLP139–151 or 145D-cultured cells 48 h after restimulation with PLP139–151 (C). Data are representative of at least four independent experiments.
Previous reports define T cell anergy as a reduction in proliferation accompanied by a decrease in IL-2 production (16, 17, 21). Following challenge with wild-type Ag, PLP139–151-specific T cells that had been cultured with 145D secreted reduced amounts of IL-2 (Fig. 1B). The 145D-treated PLP139–151-specific T cells did retain other effector functions such as the ability to secrete IFN- and IL-4 at similar levels to PLP139–151-cultured T cells (Fig. 1C), indicating that treatment with 145D did not result in deviation of the cytokine profile toward a potentially harmful Th2 response (11, 22). Thus, PLP139–151-specific T cells have a dramatically reduced capacity to proliferate or secrete IL-2 following treatment with 145D, and there is an absence of deviation in cytokine production. These data suggest that autoreactive T cells may be rendered anergic in vivo upon encountering less stable MHC variant peptides and thereby be less capable of mediating EAE.
Immunization with 145D does not induce EAE
To evaluate the stimulatory capacity of 145D, T cell lines were generated from mice primed with 145D and restimulated with either the variant peptide or PLP139–151. Following restimulation with the indicated concentrations of 145D, there was a minimal proliferative response to the priming peptide (Fig. 2A). In addition, these T cells were hyporesponsive upon restimulation with PLP139–151, compared with T cell lines generated upon priming with PLP139–151 (Fig. 1A). These results indicate that 145D does not generate a population of T cells capable of responding to itself or the encephalitogenic peptide in vitro.
FIGURE 2. Immunization with MHC variant peptide 145D does not induce EAE. SJL/J mice were immunized with 145D, and the draining lymph nodes were harvested 10 days later. Lymph node cells were restimulated with the indicated concentrations of PLP139–151 (filled squares) or 145D (open squares) for 72 h. Proliferation was measured by [3H]thymidine incorporation (A). Mice were immunized with PLP139–151 (filled squares) or 145D (open squares) and then scored for the induction of EAE as described under Materials and Methods. All mice immunized with PLP139–151 developed severe symptoms of disease by day 16 postimmunization, whereas 145D-immunized mice failed to exhibit clinical signs of EAE (B and C). Data shown represent results obtained from immunization of 14 mice per group in three independent experiments (B and C).
To assess the ability of 145D to stimulate PLP139–151 T cells in vivo, SJL mice were immunized with either PLP139–151 or 145D and monitored for clinical signs of disease. Mice immunized with 145D did not exhibit symptoms of EAE even up to 100 days postimmunization, whereas all of the mice immunized with wild-type PLP139–151 developed paralysis by day 16 postimmunization (p < 0.0001) (Fig. 2, B and C). An absence of clinical symptoms was corroborated with a lack of histological signs of EAE in 145D-immunized mice, because no cellular infiltration or demyelination was observed upon histological examination of brain and spinal cord tissue (unpublished data). Thus, immunization with 145D fails to activate the endogenous population of PLP139–151-specific T cells in a manner capable of mediating inflammation and demyelination in the CNS.
Preimmunization with 145D results in protection from EAE upon challenge with PLP139–151
Because immunization with the MHC variant peptide did not result in the induction of EAE, the ability of a preimmunization with 145D to modulate disease outcome following challenge with PLP139–151 was analyzed. A high precursor frequency of PLP139–151-specific T cells exists in the periphery of naive SJL/J mice because these T cells are not negatively selected in the thymus (23, 24). Because of this, immunization with 145D might render this population tolerant, thus reducing the ability of these autoreactive T cells to mediate EAE following challenge with PLP139–151. To investigate this possibility, mice were preimmunized with 145D, PLP139–151, or received no preimmunization 3 wk before challenge with the wild-type Ag. Following challenge, the group of mice preimmunized with PLP139–151 exhibited symptoms of EAE that progressively worsened throughout the course of the experiment, exhibiting a mean high score of 3.7 ± 0.2 (Fig. 3A and Table I). Mice receiving no preimmunization also progressed to disease upon challenge with PLP139–151 and had a mean high score of 2.9 ± 0.1. In contrast, mice preimmunized with 145D showed a significant reduction in disease severity, reaching a mean high score of only 1.2 ± 0.3 (Fig. 3A and Table I). Although 145D-preimmunized mice or those receiving no preimmunization had comparative days of disease onset (day 16.4 and 18.7, respectively), there was a significant reduction in the mean clinical score of mice preimmunized with 145D relative to unimmunized controls (p < 0.001) (Fig. 3A). At day 50, mice preimmunized with PLP139–151 had a mean clinical score of 3.2 ± 0.2, and mice receiving no preimmunization had a mean clinical score of 2.4 ± 0.2. However, mice preimmunized with 145D were still protected from EAE at day 50 post-PLP139–151 challenge with a mean clinical score of 0.7 ± 0.3 (Fig. 3A and Table I), and this protection was maintained out to day 125 (unpublished data). The decrease in disease severity in the 145D-preimmunized mice was accompanied by a significant decrease in
disease incidence. All of the mice preimmunized with PLP139–151 or those not preimmunized (15 of 15) displayed symptoms of EAE by day 35 (Fig. 3B), whereas only 7 of 15 of the mice preimmunized with 145D showed signs of disease at any time during the experiment (Fig. 3B and Table I). Even the mice that exhibited symptoms of EAE had a reduced mean clinical score (1.5 ± 0.1) compared with mice preimmunized with PLP139–151 or mice receiving no preimmunization. It is important to note that the effects mediated by 145D are not explained by this peptide having a null effect, because all of the mice receiving no preimmunization developed severe symptoms of disease. Therefore, these data indicate active protection from EAE following preimmunization with 145D.
FIGURE 3. Reduced disease severity and incidence in mice preimmunized with MHC variant peptide 145D. SJL mice were preimmunized with PLP139–151 (filled squares), 145D (open squares), or were not preimmunized (triangles). Mice were challenged 21 days later with PLP139–151 and observed for clinical manifestations of EAE. 145D-preimmunized mice exhibited a significant reduction in disease severity compared with PLP139–151-preimmunized mice (p < 0.0001) or mice not preimmunized (p < 0.001) (A), as well as a significant reduction in disease incidence (p < 0.0001) (B). Data shown represent results obtained from immunization of 15 mice per condition in three independent experiments.
Table I. Preimmunization with the MHC variant peptide 145D protects mice from EAE induced upon challenge with PLP139–151a
Avoidance of acute hypersensitivity reactions following challenge with 145D
A potential concern of using peptide-based therapies to treat EAE or MS is the possibility of hypersensitivity reactions during the course of treatment. Allergic reactions were observed in patients receiving multiple injections of an altered peptide ligand, resulting, in part, in the cessation of the clinical trial (11). In addition, SJL/J mice were shown to be especially susceptible to severe allergic reactions in response to myelin Ags (25). Because of these published observations, the ability of 145D to induce an allergic reaction in SJL/J mice was tested. EAE was induced with PLP139–151, and clinical symptoms of disease were observed in all of the mice immunized. Mice were challenged 5 wk later with PLP139–151, 145D, or a control peptide, OVA323–339, and monitored for symptoms of immediate hypersensitivity reactions such as prostration and shallow breathing, as previously described (25). All of the mice challenged with PLP139–151 developed symptoms indicative of anaphylaxis within 5 min and died within 10 min. Not only was death avoided in mice challenged with the MHC variant peptide, none of these mice exhibited any signs of anaphylaxis (Table II), even following repeated injections with 145D (unpublished data). Additionally, mice immunized with 145D did not develop characteristics associated with immediate hypersensitivity reactions when challenged with 145D or PLP139–151 (Table II). In contrast to the response observed using the 145D MHC variant peptide, all mice (five of five) challenged with 144Q (7), an altered peptide ligand containing a tryptophan to glutamine substitution at a TCR contact residue, succumbed to anaphylactic shock within 10 min of peptide challenge (Table II). These results highlight the importance of screening possible therapeutic peptides for their inability to induce an allergic response and confirm that MHC variant peptides are a viable option for peptide-based therapy.
Table II. Incidence of anaphylactic shock in mice challenged with various peptides
Amelioration of EAE after multiple injections of 145D
To investigate the efficacy of MHC variant peptides as a treatment regimen for disease, 145D was given at day 21 post-EAE induction. At the time of injection, all mice exhibited moderately severe symptoms of disease (clinical score >2.0). As a control, mice were treated with PLP139–151. However, these mice exhibited symptoms associated with anaphylaxis, so this group was not studied further. Symptoms of anaphylaxis were not observed in mice injected with PBS or an irrelevant Ag (unpublished data), thus confirming that the hypersensitivity response occurred in an Ag-specific manner. Mice that received only one injection of soluble 145D or PBS on day 21 following the primary immunization showed no immediate reversal of disease progression (Fig. 4). However, amelioration of EAE was observed in mice receiving subsequent injections of soluble 145D on days 28 and 35 (Fig. 4). There was little variation observed in disease severity following the first and second injection, yet a significant bifurcation in the mean clinical score was observed after the third injection (p < 0.0001). Whereas the mean clinical score of mice injected with PBS progressed from a 2.3 at the onset of treatment to a 3.6 by day 52, mice injected with 145D demonstrated a reversal of disease symptoms. At the time of the initial injection, the mean clinical score was 2.6, and by day 25 there was a 30% reduction in disease severity resulting in a mean clinical score of 1.8 (Fig. 4). Thus, mice receiving injections of 145D exhibited mild hind limb weakness, whereas mice receiving PBS injections displayed symptoms of more severe disease including complete hind limb paralysis and in some cases forelimb paralysis. Importantly, this protection was maintained even after cessation of the injections.
FIGURE 4. Multiple injections of MHC variant peptide 145D lessen EAE severity. EAE was induced in SJL/J mice following the standard immunization protocol. Mice were challenged s.c. 21 days later with 200 μg of soluble PLP139–151, soluble 145D (filled squares), or PBS alone (open squares). Upon soluble PLP139–151 injection, symptoms of allergic horror autotoxicus were observed, and thus, this group was excluded. The remaining groups received two more soluble injections of either 200 μg of 145D (n = 8) or PBS (n = 5) at days 7 and 14 following their initial injection.
Discussion
MS is a chronic, demyelinating disease of the CNS for which there is no cure (1). Thus, designing strategies for the amelioration of EAE and MS is the goal of many therapeutics. Previous work employing Ag-based modalities has focused on disrupting the trimolecular interaction between the TCR-pMHC complex through the use of APLs to treat this autoimmune disease (7, 8, 19, 26). We have generated a variant peptide of PLP139–151 in the current study that contains a single amino acid substitution at the MHC anchor P6. This peptide not only induces anergy in a polyclonal population of PLP139–151-specific T cells (Fig. 1), but also lacks several of the potential problems observed with APLs (Table II).
One inherent problem with the use of APLs is that EAE is mediated by a polyclonal population of T cells that differ in their fine specificity for Ag (13, 14, 15). It may be difficult to identify a TCR contact substitution that would similarly impact all of the clones within the responding polyclonal T cell population. In fact, it has been demonstrated that an APL of myelin basic protein (MBP) Ac1–9 that was inhibitory for one T cell clone resulted in the activation of another clone, leading to the induction of EAE when administered in vivo (14, 15). This demonstrates the advantage of designing MHC variant peptides that have been shown to uniformly affect the polyclonal population. In addition, results from a recent clinical trial showed a correlation between exacerbation of disease and administration of an APL (12). It was suggested that this was due to the expansion of a subset of T cells that were specific for the APL itself. Because of these complications, it may be more beneficial to destabilize the interaction between peptide and MHC. With the exception of MBP Ac1–11 (27), many autoimmune T cells recognize stable pMHC complexes such as PLP139–151·I-As; myelin oligodendrocytic glycoprotein 35–55 (MOG35–55) ·I-Ab; MOG97–108·HLA-DR4; MBP84–102·HLA-DR2 (13, 28, 29, 30). By disrupting this stable interaction, we propose that only a partial signal would be delivered to the T cell resulting in incomplete activation and T cell anergy. This anergic phenotype would be achieved without altering TCR contact residues, thereby avoiding the possibility of generating a unique population of T cells specific for the variant peptide.
Although using an analog of the immunogenic peptide to treat EAE is advantageous in that it presents a way to modulate the immune response in an Ag-specific manner, recent reports have highlighted the need to exert caution when doing this (11, 12, 25). Because EAE is mediated by CD4+ Th1 cells, one strategy to ameliorate disease is to skew the cytokine profile of the autoreactive T cells from a pro-inflammatory (Th1) to an anti-inflammatory (Th2) response, which supports the potential therapeutic benefits of Th2 responses in autoimmunity (7, 31). Although immune deviation was an effective treatment in mice (7, 8, 19), results from a clinical trial using an APL as a therapy demonstrated that patients developed a hypersensitivity reaction that correlated with a strong Th2-like immune response (11). In addition, another report has demonstrated in a PLP139–151-induced model of EAE, an exaggerated form of allergic response occurs whereby mice undergo anaphylactic shock following challenge with the self-peptide (25). Because of this, it was important to determine whether challenge with 145D would induce this state of allergic horror autotoxicus. Our results confirmed that mice challenged with PLP139–151 following disease induction experience fatal anaphylaxis; however, mice challenged with the MHC variant peptide were able to avoid such complications even upon repeated injections (Table II). Although we are currently investigating the mechanism of this phenomenon, these results suggest that MHC variant peptides may be able to evade immediate hypersensitivity reactions by virtue of their decreased potency, thus making them a viable option for the treatment of EAE and MS.
Initially, our experiments were designed to test the ability of 145D to modulate EAE before disease induction. Our results demonstrate that disease severity and time of onset is significantly affected in mice preimmunized with the variant peptide before disease induction with PLP139–151 (Fig. 3). Given the in vitro results, we propose that this is due to the ability of the MHC variant peptide to induce tolerance in PLP139–151-specific T cells, such that these T cells are less capable of becoming activated and mediating disease following challenge with the encephalitogenic peptide. This has important implications for the possible prevention of MS. It is known that certain factors, such as HLA haplotype and other genetic markers, predispose an individual to develop MS (32, 33, 34, 35, 36). As our ability to identify these individuals increases, it may become possible to administer an MHC variant peptide in vivo in an attempt to tolerize the autoreactive T cells before disease onset. This would pose little risk to the individual because decreasing the stability of the pMHC complex also decreases the immunogenicity of the Ag, thereby avoiding induction of disease (Fig. 2, B and C) (13). In addition, because we are altering amino acids in contact with the MHC, we eliminate the possibility of activating a subset of T cells that might be cross-reactive with the peptide analog (Fig. 1A) (13).
Our data also demonstrate that repeated challenges with an MHC anchor-substituted peptide result in the amelioration of EAE. Although a single peptide injection was ineffective at modulating EAE (unpublished data), a significant improvement in disease severity was seen following multiple injections (Fig. 4). Repeated injections may be necessary due to the decreased stability of the pMHC complex, as these complexes may not persist for a sufficient period of time in vivo. This strategy of multiple injections is currently being employed in MS patients receiving injections of glatiramer acetate (copolymer 1) (37), which is a synthetic random copolymer comprised of specified ratios of the amino acids alanine, lysine, glutamate, and tyrosine (38, 39). Although the mechanism is not fully understood, glatiramer acetate is thought to be effective by a combination of pathways. These include tolerance induction, immune deviation, TCR antagonism, and/or MHC blockade (38, 39, 40, 41, 42, 43). In addition, this strategy of multiple injections was shown to be feasible in MS patients receiving APL therapy over the course of several months (11, 12). Therefore, the approach of administering multiple injections of an MHC variant peptide may be translatable into the clinic.
In conclusion, our results demonstrate that an MHC anchor-substituted peptide is an effective therapy for ongoing EAE. This novel approach to altering amino acids associated with the MHC is advantageous in our model for several reasons. First, we are able to effectively tolerize a polyclonal population of autoreactive T cells upon treatment with the peptide analog. Second, we do not observe the outgrowth of a population of T cells specific for our variant peptide as was seen with APLs. Finally, there was no observation of symptoms associated with allergic horror autotoxicus following challenge with the variant peptide. Collectively, these data highlight the potential of MHC anchor-substituted peptides to be used as an effective therapy in any T cell-mediated disease where the target Ag is known.
Disclosures
The authors have no financial conflict of interest.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the National Multiple Sclerosis Society Grant RG 3210A1. C.D.M. was supported by a grant from the Coulter Foundation.
2 Address correspondence and reprint requests to Dr. Brian D. Evavold, Department of Microbiology and Immunology, Emory University, 1510 Clifton Road, Atlanta, GA 30322. E-mail address: evavold{at}microbio.emory.edu
3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; MS, multiple sclerosis; PLP, proteolipid protein; MBP, myelin basic protein; APL, altered peptide ligand; MOG, myelin oligodendrocytic glycoprotein; pMHC, peptide-MHC.
Received for publication October 12, 2004. Accepted for publication December 22, 2004.
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Murine experimental autoimmune encephalomyelitis (EAE) is a CD4+ T cell-mediated autoimmune disorder directed against myelin proteins within the CNS. We propose that variant peptides containing amino acid substitutions at MHC anchor residues will provide a unique means to controlling the polyclonal autoimmune T cell response. In this study, we have identified an MHC variant of proteolipid protein (PLP) 139–151 (145D) that renders PLP139–151-specific T cell lines anergic in vitro, as defined by a significant reduction in proliferation and IL-2 production following challenge with wild-type peptide. In vivo administration of 145D before challenge with PLP139–151 results in a significant reduction in disease severity and incidence. Importantly, we demonstrate the ability of an MHC variant peptide to ameliorate established EAE. An advantage to this treatment is that the MHC variant peptide does not induce an acute hypersensitivity reaction. This is in contrast to previous work in the PLP139–151 model demonstrating that anaphylactic shock resulting in death occurs upon rechallenge with the encephalitogenic peptide. Taken together, these data demonstrate the effectiveness of MHC anchor-substituted peptides in the treatment of EAE and suggest their utility in the treatment of other autoimmune disorders.
Introduction
Experimental autoimmune encephalomyelitis (EAE),3 a mouse model of multiple sclerosis (MS), is an organ-specific autoimmune disease directed against myelin-producing cells within the CNS (1, 2, 3). Onset can be induced by immunizing susceptible strains of mice with a peptide derived from myelin proteins such as proteolipid protein (PLP) 139–151 emulsified in CFA (4, 5). Upon immunization, autoreactive CD4+ T cells are activated in the periphery and then cross the blood-brain barrier where they mediate disease, resulting in the demyelination of neuronal axons and subsequent paralysis (1, 6). Because disease is initiated by CD4+ T cells, many studies have focused on developing therapies to regulate the polyclonal Ag-specific CD4+ T cell response.
One strategy employed to ameliorate disease was the use of altered peptide ligands (APLs) (7, 8), which are analogs of the immunogenic peptide that have an amino acid substitution at a TCR contact residue (9, 10). In this regard, the extent of T cell activation can be manipulated in an Ag-specific manner. Although based upon strong rationale and in vivo data of successful treatment of EAE, there are several factors that complicate the use of APLs as a form of immunotherapy. Hypersensitivity reactions have been reported in patients receiving multiple injections of an APL (11). In addition, exacerbation of disease occurred in some patients receiving APL therapy (12). It was suggested that the APL itself became immunogenic, leading to the expansion of APL-specific T cells. Further reports have demonstrated that due to differences in the fine specificity of the TCR, the response to an APL may vary among individual clones within the polyclonal population (13, 14, 15). Instead of delivering an anergic signal, the
APL instead may activate the responding T cell. Nevertheless, these results demonstrate the therapeutic potential of designing peptide analogs to specifically target autoimmune populations of T cells, although APLs themselves may have some limitations.
Previously, altered peptide ligands were exclusively designed to disrupt the interaction between the TCR and peptide-MHC (pMHC) (7, 8, 16, 17). However, recent reports from our laboratory described a model whereby variant peptides were designed to affect the stability of the pMHC complex without altering residues in contact with the TCR (13, 18). This method was shown to be effective at inducing anergy in a polyclonal population of Ag-specific T cells in vitro, in direct contrast to a classically defined APL (13). Given that the PLP139–151-specific T cell response uses a diverse T cell repertoire (19), a more successful approach to treating PLP-induced EAE may be through the generation of an MHC anchor-substituted peptide variant.
In this report, we tested the ability of a new class of variant peptides containing amino acid substitutions at MHC anchor residues and found that we were able to induce anergy in a polyclonal population of PLP139–151-specific T cells in vitro. Immunization with the MHC variant peptide failed to induce symptoms of EAE, confirming our previously published data showing that peptides with a reduced affinity for MHC are unable to fully stimulate autoimmune T cells and thus incapable of mediating disease (13). Additionally, mice preimmunized with the MHC variant peptide were protected against EAE induction as demonstrated by a reduction in disease severity as well as a delay in onset of disease following challenge with the encephalitogenic peptide. Importantly, established EAE was ameliorated following administration of this variant peptide in vivo.
Materials and Methods
Mice
Female SJL/J mice (H-2s) were purchased from the National Cancer Institute (Frederick, MD) and were housed in the Emory University Department of Animal Resources facility according to Institutional Animal Care and Use Committee protocols. Mice were used at 8–12 wk of age.
Peptides
PLP139–151 (HSLGKWLGHPDKF) and 145D (HSLGKWDGHPDKF) were purchased from Biosynth International. Peptides were analyzed by mass spectrometry and HPLC.
Cells and reagents
PLP139–151-specific T cell lines were generated by priming 8-wk-old SJL/J mice in the hind footpad and base of tail with 200 μg of PLP139–151 emulsified in CFA containing 1 mg/ml heat-inactivated Mycobacterium tuberculosis (H37 RA; Difco). Popliteal and inguinal lymph nodes were harvested 10 days later. Lymph node cells (5 x 106) were incubated with irradiated syngeneic splenocytes (2000 rad), 1 μM PLP139–151, and 10 pg/ml IL-2 in a 24-well plate for 7 days. Culture media consisted of RPMI 1640 medium supplemented with 10% FBS (Mediatech), 2 mM L-glutamine, 0.01 M HEPES buffer, 100 μg/ml gentamicin (Mediatech), and 2 x 10–5 M 2-ME (Sigma-Aldrich).
Proliferation assay
PLP139–151-specific T cell lines (5 x 104 per well) were cultured in a 96-well plate with irradiated syngeneic splenocytes (5 x 105 per well) with either no peptide or the indicated concentrations of peptides at 37°C for 48 h. Each well was then pulsed with 0.4 μCi of [3H]thymidine. The plates were harvested 18 h later on a FilterMate harvester (PerkinElmer Life and Analytical Sciences), and the incorporated radioactivity was measured by a Matrix 96 Direct Beta Counter (PerkinElmer Life and Analytical Sciences). All assays were performed in duplicate.
Cytokine ELISA
PLP139–151-specific T cells (3 x 105) were cultured with irradiated syngeneic splenocytes (5 x 106) in the presence of peptides for 24 h (IL-2) or 48 h (IFN- and IL-4). Supernatants were removed and placed on microtiter plates that had been coated with 50 μl of purified anti-IL-2 (5 μg/ml, clone JES6-1A12; BD Pharmingen), anti-IFN- (5 μg/ml, clone R4-6A2; BD Pharmingen), or anti-IL-4 (5 μg/ml, clone 11B11; BD Pharmingen) overnight at 4°C. Recombinant IL-2, IFN-, or IL-4 (BD Pharmingen) was used as a standard. Captured cytokine was detected using biotinylated anti-IL-2 (JES6-5H4, 100 μg/ml, 100 μl/well; BD Pharmingen), biotinylated anti-IFN- (clone XMG1.2, 100 μg/ml, 100 μl/well; BD Pharmingen), or biotinylated anti-IL-4 (clone BVD6-24G2, 100 μg/ml, 100 μl/well; BD Pharmingen) followed by 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). All assays were performed in triplicate.
EAE induction
EAE was induced by immunization of 8-wk-old female SJL/J mice (National Cancer Institute) with 200 μg of PLP139–151 emulsified in CFA containing 5 mg/ml heat-inactivated M. tuberculosis (H37 RA; Difco) on days 0 and 7 s.c. in the hind flank. Mice also received 250 ng of Bordetella pertussis toxin i.p. on days 0 and 2. Mice were scored as follows: 0, no disease; 1, flaccid tail; 2, hind limb weakness; 3, hind limb paralysis; 4, forelimb weakness; and 5, moribund. For treatment protocol, mice were given a s.c. injection of PLP139–151 (200 μg), 145D (200 μg), or PBS at day 21 followed by subsequent injections at day 28 and 35. For induction of anaphylaxis, mice were given an i.p. injection of 100 μg PLP139–151, 145D, 144Q, or OVA 323–339 in PBS at day 35.
Preimmunization protocol
Mice were preimmunized with 200 μg of PLP139–151 or 145D emulsified in CFA containing 5 mg/ml heat-inactivated M. tuberculosis (H37 RA; Difco) s.c. over both hind flanks or were not immunized. Mice were challenged 21 days later with 100 μg of PLP139–151 emulsified in CFA containing 5 mg/ml heat-inactivated M. tuberculosis (Difco). Mice also received 250 ng of B. pertussis toxin i.p. on this day and on day 2 following challenge. Disease severity was monitored as previously described.
Statistical analyses
Statistical analyses were conducted using GraphPad Prism (Software for Science). Mean clinical scores were analyzed by Student’s t test, Mann-Whitney U test, or one-way ANOVA, whereas disease incidence percentages were compared by Fisher’s exact test.
Results
Identification of an MHC anchor-substituted peptide that induces anergy in PLP139–151-specific T cells
Because our laboratory has previously shown that MHC anchor-substituted peptides are effective at regulating polyclonal T cell responses (13, 18), we wanted to identify an MHC variant peptide that could induce anergy in PLP139–151-specific T cell lines. Based upon the identified core epitope of PLP139–151 (20), residues 140, 143, 145, and 148 correspond to the amino acids contacting the MHC molecule (residues 1, 4, 6, and 9, respectively). Peptide analogs of PLP139–151 were rationally designed based on a report that identified positions 145 and 148 as critical MHC binding residues (19). In all, 27 variant peptides were generated and screened for their ability to stimulate proliferation of PLP139–151-specific T cells. One MHC variant peptide (145D) containing a leucine to aspartic acid substitution at P6, induced minimal proliferation in our polyclonal population of PLP139–151-specific lymph node cells, and was chosen for further analysis (Fig. 1A). To determine the effectiveness of treatment with 145D on the PLP-specific T cell response, PLP139–151-specific lines were cultured with the variant peptide and then restimulated with the indicated concentrations of wild-type peptide. We observed a marked reduction in the proliferative capacity of the 145D-treated cells compared with the cells cultured with wild-type Ag (Fig. 1A). There was no reduction in proliferation in PLP139–151-specific T cells following treatment with 100-fold lower concentration of wild-type peptide (data not shown), confirming our previous observations that this effect was not due to low dose tolerance (13, 18). It is also important to note that there was no increase in proliferation in the 145D-treated cells following restimulation with 145D, indicating a unique population of T cells specific for the variant peptide was not generated (Fig. 1A).
FIGURE 1. Induction of anergy in polyclonal PLP139–151-specific T cell lines. PLP139–151-specific T cell lines were cultured with either 1 μM PLP139–151 (filled symbols) or 10 μM 145D (open symbols) for 2 wk. Live cells were then restimulated with the indicated concentrations of PLP139–151 (squares) or 145D (triangles) and irradiated APCs for 72 h. Proliferation was measured by [3H]thymidine incorporation (A), and IL-2 production (24 h) was measured by ELISA (B). IFN- and IL-4 secretion was measured by ELISA from supernatants of PLP139–151 or 145D-cultured cells 48 h after restimulation with PLP139–151 (C). Data are representative of at least four independent experiments.
Previous reports define T cell anergy as a reduction in proliferation accompanied by a decrease in IL-2 production (16, 17, 21). Following challenge with wild-type Ag, PLP139–151-specific T cells that had been cultured with 145D secreted reduced amounts of IL-2 (Fig. 1B). The 145D-treated PLP139–151-specific T cells did retain other effector functions such as the ability to secrete IFN- and IL-4 at similar levels to PLP139–151-cultured T cells (Fig. 1C), indicating that treatment with 145D did not result in deviation of the cytokine profile toward a potentially harmful Th2 response (11, 22). Thus, PLP139–151-specific T cells have a dramatically reduced capacity to proliferate or secrete IL-2 following treatment with 145D, and there is an absence of deviation in cytokine production. These data suggest that autoreactive T cells may be rendered anergic in vivo upon encountering less stable MHC variant peptides and thereby be less capable of mediating EAE.
Immunization with 145D does not induce EAE
To evaluate the stimulatory capacity of 145D, T cell lines were generated from mice primed with 145D and restimulated with either the variant peptide or PLP139–151. Following restimulation with the indicated concentrations of 145D, there was a minimal proliferative response to the priming peptide (Fig. 2A). In addition, these T cells were hyporesponsive upon restimulation with PLP139–151, compared with T cell lines generated upon priming with PLP139–151 (Fig. 1A). These results indicate that 145D does not generate a population of T cells capable of responding to itself or the encephalitogenic peptide in vitro.
FIGURE 2. Immunization with MHC variant peptide 145D does not induce EAE. SJL/J mice were immunized with 145D, and the draining lymph nodes were harvested 10 days later. Lymph node cells were restimulated with the indicated concentrations of PLP139–151 (filled squares) or 145D (open squares) for 72 h. Proliferation was measured by [3H]thymidine incorporation (A). Mice were immunized with PLP139–151 (filled squares) or 145D (open squares) and then scored for the induction of EAE as described under Materials and Methods. All mice immunized with PLP139–151 developed severe symptoms of disease by day 16 postimmunization, whereas 145D-immunized mice failed to exhibit clinical signs of EAE (B and C). Data shown represent results obtained from immunization of 14 mice per group in three independent experiments (B and C).
To assess the ability of 145D to stimulate PLP139–151 T cells in vivo, SJL mice were immunized with either PLP139–151 or 145D and monitored for clinical signs of disease. Mice immunized with 145D did not exhibit symptoms of EAE even up to 100 days postimmunization, whereas all of the mice immunized with wild-type PLP139–151 developed paralysis by day 16 postimmunization (p < 0.0001) (Fig. 2, B and C). An absence of clinical symptoms was corroborated with a lack of histological signs of EAE in 145D-immunized mice, because no cellular infiltration or demyelination was observed upon histological examination of brain and spinal cord tissue (unpublished data). Thus, immunization with 145D fails to activate the endogenous population of PLP139–151-specific T cells in a manner capable of mediating inflammation and demyelination in the CNS.
Preimmunization with 145D results in protection from EAE upon challenge with PLP139–151
Because immunization with the MHC variant peptide did not result in the induction of EAE, the ability of a preimmunization with 145D to modulate disease outcome following challenge with PLP139–151 was analyzed. A high precursor frequency of PLP139–151-specific T cells exists in the periphery of naive SJL/J mice because these T cells are not negatively selected in the thymus (23, 24). Because of this, immunization with 145D might render this population tolerant, thus reducing the ability of these autoreactive T cells to mediate EAE following challenge with PLP139–151. To investigate this possibility, mice were preimmunized with 145D, PLP139–151, or received no preimmunization 3 wk before challenge with the wild-type Ag. Following challenge, the group of mice preimmunized with PLP139–151 exhibited symptoms of EAE that progressively worsened throughout the course of the experiment, exhibiting a mean high score of 3.7 ± 0.2 (Fig. 3A and Table I). Mice receiving no preimmunization also progressed to disease upon challenge with PLP139–151 and had a mean high score of 2.9 ± 0.1. In contrast, mice preimmunized with 145D showed a significant reduction in disease severity, reaching a mean high score of only 1.2 ± 0.3 (Fig. 3A and Table I). Although 145D-preimmunized mice or those receiving no preimmunization had comparative days of disease onset (day 16.4 and 18.7, respectively), there was a significant reduction in the mean clinical score of mice preimmunized with 145D relative to unimmunized controls (p < 0.001) (Fig. 3A). At day 50, mice preimmunized with PLP139–151 had a mean clinical score of 3.2 ± 0.2, and mice receiving no preimmunization had a mean clinical score of 2.4 ± 0.2. However, mice preimmunized with 145D were still protected from EAE at day 50 post-PLP139–151 challenge with a mean clinical score of 0.7 ± 0.3 (Fig. 3A and Table I), and this protection was maintained out to day 125 (unpublished data). The decrease in disease severity in the 145D-preimmunized mice was accompanied by a significant decrease in
disease incidence. All of the mice preimmunized with PLP139–151 or those not preimmunized (15 of 15) displayed symptoms of EAE by day 35 (Fig. 3B), whereas only 7 of 15 of the mice preimmunized with 145D showed signs of disease at any time during the experiment (Fig. 3B and Table I). Even the mice that exhibited symptoms of EAE had a reduced mean clinical score (1.5 ± 0.1) compared with mice preimmunized with PLP139–151 or mice receiving no preimmunization. It is important to note that the effects mediated by 145D are not explained by this peptide having a null effect, because all of the mice receiving no preimmunization developed severe symptoms of disease. Therefore, these data indicate active protection from EAE following preimmunization with 145D.
FIGURE 3. Reduced disease severity and incidence in mice preimmunized with MHC variant peptide 145D. SJL mice were preimmunized with PLP139–151 (filled squares), 145D (open squares), or were not preimmunized (triangles). Mice were challenged 21 days later with PLP139–151 and observed for clinical manifestations of EAE. 145D-preimmunized mice exhibited a significant reduction in disease severity compared with PLP139–151-preimmunized mice (p < 0.0001) or mice not preimmunized (p < 0.001) (A), as well as a significant reduction in disease incidence (p < 0.0001) (B). Data shown represent results obtained from immunization of 15 mice per condition in three independent experiments.
Table I. Preimmunization with the MHC variant peptide 145D protects mice from EAE induced upon challenge with PLP139–151a
Avoidance of acute hypersensitivity reactions following challenge with 145D
A potential concern of using peptide-based therapies to treat EAE or MS is the possibility of hypersensitivity reactions during the course of treatment. Allergic reactions were observed in patients receiving multiple injections of an altered peptide ligand, resulting, in part, in the cessation of the clinical trial (11). In addition, SJL/J mice were shown to be especially susceptible to severe allergic reactions in response to myelin Ags (25). Because of these published observations, the ability of 145D to induce an allergic reaction in SJL/J mice was tested. EAE was induced with PLP139–151, and clinical symptoms of disease were observed in all of the mice immunized. Mice were challenged 5 wk later with PLP139–151, 145D, or a control peptide, OVA323–339, and monitored for symptoms of immediate hypersensitivity reactions such as prostration and shallow breathing, as previously described (25). All of the mice challenged with PLP139–151 developed symptoms indicative of anaphylaxis within 5 min and died within 10 min. Not only was death avoided in mice challenged with the MHC variant peptide, none of these mice exhibited any signs of anaphylaxis (Table II), even following repeated injections with 145D (unpublished data). Additionally, mice immunized with 145D did not develop characteristics associated with immediate hypersensitivity reactions when challenged with 145D or PLP139–151 (Table II). In contrast to the response observed using the 145D MHC variant peptide, all mice (five of five) challenged with 144Q (7), an altered peptide ligand containing a tryptophan to glutamine substitution at a TCR contact residue, succumbed to anaphylactic shock within 10 min of peptide challenge (Table II). These results highlight the importance of screening possible therapeutic peptides for their inability to induce an allergic response and confirm that MHC variant peptides are a viable option for peptide-based therapy.
Table II. Incidence of anaphylactic shock in mice challenged with various peptides
Amelioration of EAE after multiple injections of 145D
To investigate the efficacy of MHC variant peptides as a treatment regimen for disease, 145D was given at day 21 post-EAE induction. At the time of injection, all mice exhibited moderately severe symptoms of disease (clinical score >2.0). As a control, mice were treated with PLP139–151. However, these mice exhibited symptoms associated with anaphylaxis, so this group was not studied further. Symptoms of anaphylaxis were not observed in mice injected with PBS or an irrelevant Ag (unpublished data), thus confirming that the hypersensitivity response occurred in an Ag-specific manner. Mice that received only one injection of soluble 145D or PBS on day 21 following the primary immunization showed no immediate reversal of disease progression (Fig. 4). However, amelioration of EAE was observed in mice receiving subsequent injections of soluble 145D on days 28 and 35 (Fig. 4). There was little variation observed in disease severity following the first and second injection, yet a significant bifurcation in the mean clinical score was observed after the third injection (p < 0.0001). Whereas the mean clinical score of mice injected with PBS progressed from a 2.3 at the onset of treatment to a 3.6 by day 52, mice injected with 145D demonstrated a reversal of disease symptoms. At the time of the initial injection, the mean clinical score was 2.6, and by day 25 there was a 30% reduction in disease severity resulting in a mean clinical score of 1.8 (Fig. 4). Thus, mice receiving injections of 145D exhibited mild hind limb weakness, whereas mice receiving PBS injections displayed symptoms of more severe disease including complete hind limb paralysis and in some cases forelimb paralysis. Importantly, this protection was maintained even after cessation of the injections.
FIGURE 4. Multiple injections of MHC variant peptide 145D lessen EAE severity. EAE was induced in SJL/J mice following the standard immunization protocol. Mice were challenged s.c. 21 days later with 200 μg of soluble PLP139–151, soluble 145D (filled squares), or PBS alone (open squares). Upon soluble PLP139–151 injection, symptoms of allergic horror autotoxicus were observed, and thus, this group was excluded. The remaining groups received two more soluble injections of either 200 μg of 145D (n = 8) or PBS (n = 5) at days 7 and 14 following their initial injection.
Discussion
MS is a chronic, demyelinating disease of the CNS for which there is no cure (1). Thus, designing strategies for the amelioration of EAE and MS is the goal of many therapeutics. Previous work employing Ag-based modalities has focused on disrupting the trimolecular interaction between the TCR-pMHC complex through the use of APLs to treat this autoimmune disease (7, 8, 19, 26). We have generated a variant peptide of PLP139–151 in the current study that contains a single amino acid substitution at the MHC anchor P6. This peptide not only induces anergy in a polyclonal population of PLP139–151-specific T cells (Fig. 1), but also lacks several of the potential problems observed with APLs (Table II).
One inherent problem with the use of APLs is that EAE is mediated by a polyclonal population of T cells that differ in their fine specificity for Ag (13, 14, 15). It may be difficult to identify a TCR contact substitution that would similarly impact all of the clones within the responding polyclonal T cell population. In fact, it has been demonstrated that an APL of myelin basic protein (MBP) Ac1–9 that was inhibitory for one T cell clone resulted in the activation of another clone, leading to the induction of EAE when administered in vivo (14, 15). This demonstrates the advantage of designing MHC variant peptides that have been shown to uniformly affect the polyclonal population. In addition, results from a recent clinical trial showed a correlation between exacerbation of disease and administration of an APL (12). It was suggested that this was due to the expansion of a subset of T cells that were specific for the APL itself. Because of these complications, it may be more beneficial to destabilize the interaction between peptide and MHC. With the exception of MBP Ac1–11 (27), many autoimmune T cells recognize stable pMHC complexes such as PLP139–151·I-As; myelin oligodendrocytic glycoprotein 35–55 (MOG35–55) ·I-Ab; MOG97–108·HLA-DR4; MBP84–102·HLA-DR2 (13, 28, 29, 30). By disrupting this stable interaction, we propose that only a partial signal would be delivered to the T cell resulting in incomplete activation and T cell anergy. This anergic phenotype would be achieved without altering TCR contact residues, thereby avoiding the possibility of generating a unique population of T cells specific for the variant peptide.
Although using an analog of the immunogenic peptide to treat EAE is advantageous in that it presents a way to modulate the immune response in an Ag-specific manner, recent reports have highlighted the need to exert caution when doing this (11, 12, 25). Because EAE is mediated by CD4+ Th1 cells, one strategy to ameliorate disease is to skew the cytokine profile of the autoreactive T cells from a pro-inflammatory (Th1) to an anti-inflammatory (Th2) response, which supports the potential therapeutic benefits of Th2 responses in autoimmunity (7, 31). Although immune deviation was an effective treatment in mice (7, 8, 19), results from a clinical trial using an APL as a therapy demonstrated that patients developed a hypersensitivity reaction that correlated with a strong Th2-like immune response (11). In addition, another report has demonstrated in a PLP139–151-induced model of EAE, an exaggerated form of allergic response occurs whereby mice undergo anaphylactic shock following challenge with the self-peptide (25). Because of this, it was important to determine whether challenge with 145D would induce this state of allergic horror autotoxicus. Our results confirmed that mice challenged with PLP139–151 following disease induction experience fatal anaphylaxis; however, mice challenged with the MHC variant peptide were able to avoid such complications even upon repeated injections (Table II). Although we are currently investigating the mechanism of this phenomenon, these results suggest that MHC variant peptides may be able to evade immediate hypersensitivity reactions by virtue of their decreased potency, thus making them a viable option for the treatment of EAE and MS.
Initially, our experiments were designed to test the ability of 145D to modulate EAE before disease induction. Our results demonstrate that disease severity and time of onset is significantly affected in mice preimmunized with the variant peptide before disease induction with PLP139–151 (Fig. 3). Given the in vitro results, we propose that this is due to the ability of the MHC variant peptide to induce tolerance in PLP139–151-specific T cells, such that these T cells are less capable of becoming activated and mediating disease following challenge with the encephalitogenic peptide. This has important implications for the possible prevention of MS. It is known that certain factors, such as HLA haplotype and other genetic markers, predispose an individual to develop MS (32, 33, 34, 35, 36). As our ability to identify these individuals increases, it may become possible to administer an MHC variant peptide in vivo in an attempt to tolerize the autoreactive T cells before disease onset. This would pose little risk to the individual because decreasing the stability of the pMHC complex also decreases the immunogenicity of the Ag, thereby avoiding induction of disease (Fig. 2, B and C) (13). In addition, because we are altering amino acids in contact with the MHC, we eliminate the possibility of activating a subset of T cells that might be cross-reactive with the peptide analog (Fig. 1A) (13).
Our data also demonstrate that repeated challenges with an MHC anchor-substituted peptide result in the amelioration of EAE. Although a single peptide injection was ineffective at modulating EAE (unpublished data), a significant improvement in disease severity was seen following multiple injections (Fig. 4). Repeated injections may be necessary due to the decreased stability of the pMHC complex, as these complexes may not persist for a sufficient period of time in vivo. This strategy of multiple injections is currently being employed in MS patients receiving injections of glatiramer acetate (copolymer 1) (37), which is a synthetic random copolymer comprised of specified ratios of the amino acids alanine, lysine, glutamate, and tyrosine (38, 39). Although the mechanism is not fully understood, glatiramer acetate is thought to be effective by a combination of pathways. These include tolerance induction, immune deviation, TCR antagonism, and/or MHC blockade (38, 39, 40, 41, 42, 43). In addition, this strategy of multiple injections was shown to be feasible in MS patients receiving APL therapy over the course of several months (11, 12). Therefore, the approach of administering multiple injections of an MHC variant peptide may be translatable into the clinic.
In conclusion, our results demonstrate that an MHC anchor-substituted peptide is an effective therapy for ongoing EAE. This novel approach to altering amino acids associated with the MHC is advantageous in our model for several reasons. First, we are able to effectively tolerize a polyclonal population of autoreactive T cells upon treatment with the peptide analog. Second, we do not observe the outgrowth of a population of T cells specific for our variant peptide as was seen with APLs. Finally, there was no observation of symptoms associated with allergic horror autotoxicus following challenge with the variant peptide. Collectively, these data highlight the potential of MHC anchor-substituted peptides to be used as an effective therapy in any T cell-mediated disease where the target Ag is known.
Disclosures
The authors have no financial conflict of interest.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the National Multiple Sclerosis Society Grant RG 3210A1. C.D.M. was supported by a grant from the Coulter Foundation.
2 Address correspondence and reprint requests to Dr. Brian D. Evavold, Department of Microbiology and Immunology, Emory University, 1510 Clifton Road, Atlanta, GA 30322. E-mail address: evavold{at}microbio.emory.edu
3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; MS, multiple sclerosis; PLP, proteolipid protein; MBP, myelin basic protein; APL, altered peptide ligand; MOG, myelin oligodendrocytic glycoprotein; pMHC, peptide-MHC.
Received for publication October 12, 2004. Accepted for publication December 22, 2004.
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