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* Neuroimmunology Research and Tykeson Multiple Sclerosis Research Laboratory, Veterans Affairs Medical Center and Oregon Health & Science University, Portland, OR 97239; Departments of
Neurology and
Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, OR 97239;
Department of Clinical Immunology, Aarhus University Hospital, Skejby Sygehus, Aarhus, Denmark; and
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Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, U.K.
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Abstract |
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1 and 
1 domains of DR2 (DRB1*1501) covalently linked to the encephalitogenic MOG-35-55 peptide (VG312). We found that the MOG/DR2 VG312 RTL could induce long-term tolerance to MOG-35-55 peptide and reverse clinical and histological signs of EAE in a dose- and peptide-dependent manner. Some mice treated with lower doses of VG312 relapsed after cessation of daily treatment, but the mice could be successfully re-treated with a higher dose of VG312. Treatment with VG312 strongly reduced secretion of Th1 cytokines (TNF- and IFN-
) produced in response to MOG-35-55 peptide, and to a lesser degree purified protein derivative and Con A, but had no inhibitory effect on serum Ab levels to MOG-35-55 peptide. Abs specific for both the peptide and MHC moieties of the RTLs were also present after treatment with EAE, but these Abs had only a minor enhancing effect on T cell activation in vitro. These data demonstrate the powerful tolerance-inducing therapeutic effects of VG312 on MOG peptide-induced EAE in transgenic DR2 mice and support the potential of this approach to inhibit myelin Ag-specific responses in multiple sclerosis patients. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One approach to regulating Ag-specific T cell responses to encephalitogenic peptides is to induce nonresponsiveness using TCR ligands containing extracellular domains of class II MHC molecules linked to specific peptide targets. Several such constructs have been developed that involve natural or recombinant 12 and 12 MHC class II domains in association with various encephalitogenic or other pathogenic peptides that were either linked covalently or bound noncovalently (20, 21, 22, 23, 24). These molecular complexes bind not only to the TCR but also to the CD4 molecule on the T cell surface through the 2 MHC domain (25) and were found to inhibit T cell activation and prevent EAE in rodents (22, 26, 27). Our design includes only the minimal TCR interface, which involves only the 1 and 1 MHC domains without CD4 binding covalently linked to peptide (28). These constructs can prevent and treat MBP-induced EAE in Lewis rats (29, 30) and inhibit activation and induce IL-10 secretion in human DR2-restricted T cell clones specific for MBP-85-99 or BCR-ABL b3a2 peptide (CABL) peptides (31, 32). To further evaluate the inhibitory activity and mechanism of the effects of recombinant TCR ligand (RTL) on encephalitogenic T cells in vivo, we designed a MOG-35-55/DR2 construct (VG312) for testing in DR2 mice undergoing MOG peptide-induced EAE. Our results demonstrate potent inhibitory activity resulting in immunological tolerance to the encephalitogenic MOG-35-55 peptide and reversal of clinical and histological signs of EAE.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HLA-DR2 Tg mice bearing chimeric MHC class II molecules were developed as previously described (33). The peptide-binding domain of MHC class II is encoded by human sequences, whereas the membrane proximal portion including the CD4-binding domain is encoded by mouse sequences (DR 1*0101: I-E, and DR1*1501: I-E). The DR1*0101: I-E construct was kindly provided by Dr. D. M. Zaller (Merck Research Laboratories, Rahway, NJ). The DR1*1501: I-E construct was made essentially as described by Woods et al. (33) with the following changes: the < bx;1>pACYC184 vector containing the DRB1*0401 exons 1 and 2 and the Ed exons 3–6 were partially digested with BamHI and treated with Klenow polymerase to remove a BamHI site in the vector. Subsequently, DRB1*1501 exon 2 was cloned into pACYC184, which had been predigested with BamHI and EcoRI to remove DRB1*0401 exon 2. Tg mice were generated by microinjecting the chimeric - and -chain constructs into fertilized eggs from (DBA/2 x C57BL/6)F1 matings. Viable embryos were transferred into pseudo-pregnant females for development to term. Tg offspring were backcrossed twice to the MHC class II knockout mouse, MHCII
(34).
Antigens
Mouse MOG-35-55 peptide (MEVGWYRSPFSRVVHLYRNGK) was synthesized using solid-phase techniques and purified by HPLC at the Beckman Institute, Stanford University (Palo Alto, CA). Purified protein derivative (PPD) was purchased from the Staten Serum Institute (Copenhagen, Denmark). Con A was purchased from Sigma-Aldrich (St. Louis, MO).
Construction of RTLs
Single chain human RTLs of 
200 aa residues derived from HLA-DR2b were designed using the same principles as for rat RTLs (28) and have been produced in Escherichia coli with and without amino-terminal extensions containing antigenic peptides (32). Similar to the rat constructs, human RTLs exhibited a cooperative two-state thermal unfolding transition, and DR2-derived RTLs with a covalently linked MBP-85-99 peptide or mouse MOG-35-55 peptide showed increased stability to thermal unfolding relative to the empty DR2-derived RTLs.
Induction of active EAE and treatment with RTLs
Tg HLA-DR2 male and female mice between 8 and 12 wk of age were immunized s.c. as described (17) at four sites on the flanks with 0.2 ml of an emulsion comprised of 200 µg of mouse MOG-35-55 peptide in CFA containing 400 µg of Mycobacterium tuberculosis H37RA (Difco, Detroit, MI). In addition, mice were given pertussis toxin (Ptx) from List Biological Laboratories (Campbell, CA) on days 0 and 2 postimmunization (25 and 67 ng per mouse, respectively). Mice were treated i.v. daily for 8 days, beginning 2–4 days after onset of clinical signs, with 100 µl of VG312 (MOG-35-55 peptide/DR2), VG303 (MBP-85-99 peptide/DR2), VG311 (CABL peptide/DR2), or vehicle (Tris, pH 8.5) containing 0–100 µg of various RTL proteins. Actively immunized mice were assessed daily for clinical signs of EAE according to the following scale: 0 = normal; 1 = limp tail or mild hind limb weakness; 2 = limp tail and moderate hind limb weakness or mild ataxia; 3 = limp tail and moderately severe hind limb weakness; 4 = limp tail and severe hind limb weakness or mild forelimb weakness or moderate ataxia; 5 = limp tail and paraplegia with no more than moderate forelimb weakness; and 6 = limp tail and paraplegia with severe forelimb weakness or severe ataxia or moribund condition. The average daily score was determined for each group of mice by summing the individual scores and dividing by the number of mice in the group. Mice that occasionally die from severe EAE are given a score of 6 and are included in the daily score. The cumulative disease index (CDI) was determined by summing the daily clinical scores for each mouse. The mean peak scores, the average daily scores, and the CDI scores ± SD were calculated for the control and experimental groups and were evaluated for statistical differences using the Kruskal-Wallis test for multivariant analyses of nonparametric comparisons.
Proliferation assay
Draining lymph node (LN) cells from HLA-DR2 Tg mice were recovered at the indicated time points following immunization and processed into single-cell suspensions. T cell proliferation responses were assessed by plating 4 x 105 cells per well in a 96-well flat-bottom tissue culture plate in stimulation medium alone (control) or in the presence of Ags. Cultures were incubated for 72 h at 37°C in 7% CO2. Wells were pulsed for the final 18 h with 0.5 µCi per well [3H]thymidine (Amersham, Arlington Heights, IL). The cells were harvested onto glass fiber filters, and [3H]thymidine uptake was measured using a liquid scintillation counter (1205 Betaplate; Wallac, Turku, Finland). Mean cpm ± SD were calculated for triplicate wells.
Cytometric bead array
TNF-, IFN-, IL-2, IL-4, and IL-5 were simultaneously detected in supernatants from the indicated cell source using the mouse Th1/Th2 cytokine cytometric bead array kit from BD Biosciences (San Jose, CA). A total of 50 µl of supernatant, 50 µl of a mixture of capture beads, and 50 µl of Th1/Th2 PE detection reagent were combined and incubated for 2 h at room temperature in the dark. The samples were washed, resuspended in 200 µl of wash buffer, and analyzed by flow cytometry (FACSCalibur; BD Biosciences). Standard curves were generated for each cytokine using a mixed bead standard to interpolate the concentration of cytokine in the cell supernatant in picograms per milliliter.
IL-10 ELISA
Cell culture supernatants collected on day 2 were frozen at -80°C and later evaluated for the presence of IL-10 as described in the IL-10 ELISA kit (Quantikine M; R&D Systems, Minneapolis, MN). Developed plates were read on a kinetic microplate reader (Molecular Devices, Sunnyvale, CA) and quantified using a standard curve.
Assessment of Ab response to peptide Ags by indirect ELISA
Sera were isolated from individual animals, pooled, and frozen at - 80°C. ELISA plates (Nunc Maxisorp; Nalge Nunc International, Roskilde, Denmark) were coated with 10 µg/ml mouse MOG-35-55 peptide in 100 µl of PBS overnight at 4°C. Plates were then washed with 1x PBS/0.05% Tween 20 and blocked with 200 µl of 1x PBS/3%BSA/0.05% Tween 20 for 2 h at 37°C. Plates were washed, and 100 µl of diluted sera (1:25, 1:50, 1:100, 1:200, or 1:500) was added in triplicate and incubated at 37°C for 2 h. Plates were washed, and plate-bound Ab was detected with anti-mouse IgG H and L chain conjugated to streptavidin-HRP (Bethyl Laboratories, Montgomery, TX). Plates were developed for 10 min in the dark using 3,3'5,5'-tetramethylbenzidine as a substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD). The reaction was stopped using tetramethylbenzidine stop solution, and the plates were immediately read at 490 nm on a kinetic microplate reader (Molecular Devices).
Histopathology
The intact spinal cord was removed from mice euthanized 24–38 days after induction of EAE and fixed in 10% phosphate-buffered formalin. The spinal cords were dissected after fixation and embedded in paraffin before sectioning. The sections were stained with either Luxol fast blue/periodic acid-Schiff’s reagent or H&E and analyzed by light microscopy. Semiquantitative analysis of inflammation and demyelination was determined by examining at least 10 sections from each mouse.
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Results |
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As recently described (A. A. Vandenbark, C. Rich, A. Zamora, H. Jacobsen, H. Offner, R. Jones, and L. Fugger, manuscript in preparation), MOG-35-55-induced EAE in Tg DR2 (DRB1*1501) mice is characterized as a moderately severe chronic disease affecting 100% of the mice with ascending paralysis marked by inflammatory, demyelinating CNS lesions. EAE was induced with MOG-35-55 peptide/CFA on day 0 plus Ptx on days 0 and 2. The initial signs of disease could be observed beginning 10 days after induction. To evaluate the clinical potential of human DR2-restricted RTLs, we treated Tg DR2 mice with MOG-induced EAE 2–4 days after onset of clinical signs with VG312 (MOG-35-55/DR2) or control constructs containing different DR2-restricted peptides, VG303 (MBP-85-99/DR2) or VG311 (CABL-peptide/DR2) (31, 32). Each construct was produced and characterized in an identical manner as described. The treatment regimen involved daily i.v. injections of 100, 33, or 10 µg of the indicated construct in 100 µl of Tris buffer, or Tris buffer alone (vehicle) for 8 consecutive days.
As shown in Fig. 1, treatment with VG312 at doses of 100 or 33 µg/injection rapidly reversed established clinical signs of EAE (score 2.5) to an average daily score of <0.5 U by the end of the 8-day treatment period (combined data from four separate experiments). This low degree of disability was maintained without further RTL injections over the remainder of the observation period, which in one experiment lasted for 5 wk after treatment was stopped. In contrast to the reversal of EAE mediated by the higher doses of VG312, control groups receiving vehicle or 100 µg/injection VG303 (MBP-85-99/DR2) or VG311 (CABL-peptide/DR2) developed moderately severe chronic EAE (score of 4). The clinical improvement observed in VG312-treated mice was reflected by a marked reduction of inflammatory lesions and demyelination in spinal cord tissue (Fig. 2).
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To evaluate the effects of VG312 treatment, LN cells were collected and tested for T cell responses to the encephalitogenic MOG-35-55 peptide. As shown in Fig. 6, the proliferation responses of LN cells from VG312-treated mice stimulated ex vivo with three concentrations of MOG-35-55 peptide were profoundly (>90%) inhibited compared with responses of LN cells from vehicle-treated mice. In contrast, treatment with VG312 produced moderate inhibition of responses to PPD (28%) and Con A (34%) (Fig. 6). Surprisingly, T cell proliferation responses appeared to be enhanced at the 10- and 2-µg concentrations of MOG-35-55 peptide in Tg DR2 mice treated with VG311 compared with those treated with vehicle (Fig. 6). Of importance mechanistically, addition of IL-2 to unresponsive T cell cultures did not enhance responses to MOG-35-55 peptide or PPD beyond the effects of IL-2 alone (data not shown), indicating that the lack of response to MOG-35-55 peptide was not caused by functional anergy. The strongly reduced T cell proliferation responses to MOG-35-55 peptide were reflected by a concomitant reduction in secretion of TNF- and particularly IFN- (Fig. 6), as well as IL-2 (data not shown), as measured by cytokine bead analysis. In addition, treatment with VG312 but not VG311 reduced consistently, but to a lesser degree, the secretion of TNF-, IFN- (Fig. 6), and IL-2 (data not shown) by LN cells stimulated with PPD and Con A, indicating bystander suppression. Other cytokines measured in this assay, including IL-4 and IL-5 as well as IL-10 measured by ELISA, were not detected at significant levels in either the vehicle-treated or VG312-treated mice in response to MOG-35-55 peptide, PPD, or Con A (data not shown).
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Although the sequence of VG312 is based on the germline sequences of human HLA-DR2 (DRA:DRB1*1501) and mouse (self) MOG-35-55 peptide, alterations required to generate a single chain RTL construct (e.g., linker regions and amino acid substitutions) might be immunogenic in Tg DR2 mice undergoing treatment for EAE (32). To evaluate immunogenic components of VG312, LN cells and Abs were tested for recognition of VG312, VG311 (with a different bound peptide), VG302 (empty RTL without peptide or linker), and MOG-35-55 peptide. As shown in Fig. 6, LN cells from VG312-treated Tg DR2 mice developed significant proliferation and cytokine responses to VG312, thus indicating that this construct was immunogenic in vivo.
The pattern of serum Ab responses to RTL components revealed a substantial baseline recognition of the empty DR2 moiety (VG302) and VG311 in vehicle-treated mice that was strongly enhanced in VG312 and VG311-treated Tg DR2 mice with EAE (Fig. 7). However, Abs specific for the encephalitogenic MOG-35–55 peptide were also present in VG312- and vehicle-treated mice (Fig. 7). The combination of these components resulted in a vigorous Ab response to the VG312 molecule in VG312- and vehicle-treated mice. Similarly, a vigorous Ab response was observed to VG311 in VG311-treated mice, with Ab recognition of the CABL peptide being present only in antiserum from the VG311-treated mice (data not shown). To further evaluate the possible neutralizing effects of Abs induced in the RTL-treated mice, antisera from various groups of donors were incubated with splenocytes from MOG-35-55-immunized mice in the presence of Con A, PPD, or various dilutions of MOG-35-55 peptide. We found that antiserum from VG312-treated mice did not have any detectable neutralizing effect on T cell responses to MOG-35-55 peptide or control Ags, and may have had a slight enhancing effect compared with antisera from VG303- or vehicle-treated mice (data not shown). No activating effects of these antisera were noted in the absence of Ag or mitogen stimulation.
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Discussion |
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The treatment regime with VG312 used in our study profoundly inhibited T cell responses to the encephalitogenic MOG-35-55 peptide, including proliferation and secretion of the proinflammatory cytokines TNF-, IFN-, and IL-2. The Th2 cytokines IL-4, IL-5, and IL-10 were not detected in mice developing EAE and were not induced after treatment with VG312. In a previous study using VG303 and VG311 (31), in vitro treatment of human DR2-restricted T cell clones specific for the respective cognate peptides MBP-85-99 and CABL produced partial agonist signaling through the TCR, resulting in default induction of IL-10. Our current study of DR2 mice did not detect any induction of IL-10 in vivo using VG312, thus indicating possible differences in signaling pathways in mouse vs human T cells. However, T cell proliferation and inflammatory cytokine responses to PPD and Con A were moderately to strongly reduced in VG312- vs vehicle-treated mice, indicating possible bystander suppression exerted by VG312. However, it remains to be seen whether the inhibitory mechanism affects bystander T cell responses to other encephalitogenic determinants.
Attempts to overcome possible anergy and thus rescue MOG-35-55-specific T cells with IL-2 did not enhance T cell responses to MOG-35-55 peptide. Yet, nonresponsiveness to the MOG-35-55 peptide persisted for up to 5 wk after cessation of treatment, strongly suggesting either potent suppression or a deleting mechanism. These results are not inconsistent with a mechanism of anergy leading to eventual late apoptosis proposed by Appel, Wucherpfennig, and colleagues (35) to explain inhibition of human T cells with four-domain DR2/MBP-85-99 dimers.
The DR2 RTL constructs were found to be immunogenic in the treated DR2 mice, inducing both T cell and Ab responses, primarily to the covalently linked highly antigenic MOG-35-55 peptide. However, some T cell and Ab reactivity was directed against the DR2 moiety itself, presumably to minor sequence changes required for correct folding of the single chain construct or to linker sequences attaching the MOG-35-55 peptide to the DR2 moiety or joining the DR1 and the DR 1 domains (32). These T cell and Ab responses did not appear to affect the therapeutic activity of VG312 on EAE. However, antiserum from VG312-treated mice appeared to moderately enhance responses of MOG-specific T cells in vitro compared with antisera from VG303- or vehicle-treated mice, suggesting some Ab recognition of the MOG peptide/DR2 complex that facilitated activation.
In conclusion, our study demonstrates for the first time the potent therapeutic effects of a rationally designed minimal TCR ligand in a humanized model of EAE. These results strongly support the application of this novel class of peptide/MHC class II constructs, and specifically the VG312 construct directed against the DR2-restricted MOG-35-55 peptide for treatment of T cell-mediated autoimmune diseases such as MS.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Arthur A. Vandenbark, Veterans Affairs Medical Center, R&D-31, 3710 SW US Veterans Hospital Road, Post Office Box 1034, Portland, OR 97239.
3 Abbreviations used in this paper: Tg, transgenic; MOG, myelin oligodendrocyte glycoprotein; EAE, experimental autoimmune encephalomyelitis; RTL, recombinant TCR ligand; PPD, purified protein derivative; MBP, myelin basic protein; MS, multiple sclerosis; CDI, cumulative disease index; LN, lymph node; Ptx, pertussis toxin; CABL, BCR-ABL b3a2 peptide.
Received for publication February 2, 2003. Accepted for publication April 28, 2003.
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References |
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*0101, *0401) transgenic mice. Proc. Natl. Acad. Sci. USA 95:8082.
production and reduces the severity of experimental autoimmune encephalomyelitis in cytokine knockout mice. J. Immunol. 167:542.1 domain of the MHC class II protein HLA-DR1. J. Immunol. 161:5472.This article has been cited by other articles:
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