Encephalitogenic activity of truncated myelin oligodendrocyte glycoprotein (MOG) peptides and their recognition by CD8+ MOG-specific T cells on oligomeric MHC class I molecules

Deming Sun1,2, Yiping Zhang3, Bingyuan Wei4, Stephen C. Peiper2, Hui Shao1 and Henry J. Kaplan1

1 Kentucky Lions Eye Center, Department of Ophthalmology, and Vision Sciences, and 2 James Graham Brown Cancer Center, University of Louisville, Louisville, KY 40202, USA 3 Department of Neurology, University of California Irvine, Irvine, CA 92697-4275, USA 4 BD Biosciences PharMingen, San Diego, CA 92121, USA

Correspondence to: D. Sun, Kentucky Lions Eye Center, Department of Ophthalmology and Vision Sciences, University of Louisville, 301 E. Muhammad Ali Boulevard, Louisville, KY 40202, USA. E-mail: d0sun001{at}louisville.edu
Transmitting editor: L. Steinman


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have previously demonstrated that the 21-residue peptide pMOG35–55 from myelin oligodendrocyte glycoprotein (MOG) contains an antigenic epitope that activates CD8+ encephalitogenic T cells in C57BL/6 (B6) mice. To identify the core encephalitogenic epitope of CD8+ MOG-specific T cells, we have prepared a panel of highly purified peptides of varying lengths, which span the entire length of pMOG35–55, and tested their binding to recombinant H-2Db dimers and their ability to induce EAE. Two of the truncated peptides, pMOG40–54 and pMOG44–54, strongly bound recombinant H-2Db protein and this complex bound MOG-specific CD8+ T cells. Interestingly, pMOG40–54 retained the full capability of inducing paralytic disease, whereas only a part of the B6 mice immunized with pMOG44–54 developed clinical paralysis and central nervous system (CNS) inflammation. Further deletion of 1 amino acid from either the N- or C-terminus of the peptide pMOG44–54 dramatically reduced binding to recombinant H-2Db, and abolished the induction of paralysis and CNS inflammation. Our results demonstrate that the ability of truncated pMOG35–55 peptides to bind recombinant H-2Db protein does not always correlate with their ability of inducing encephalomyelitis. This approach enables the further identification of the core pathogenic epitope within the pMOG35–55 that activates MOG-specific encephalitogenic CD8+ T cells.

Keywords: CD8+ encephalitogenic T cell, epitope mapping, experimental autoimmune encephalomyelitis, myelin oligodendrocyte glycoprotein, tetramer


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Experimental autoimmune encephalomyelitis (EAE) has been induced in rodents by sensitization to a number of myelin proteins, including myelin basic protein (MBP) (13), proteolipid protein (PLP) (45), myelin oligodendrocyte glycoprotein (MOG) (68), myelin-associated glycoprotein (9) and myelin oligodendrocyte basic protein (10). Previous studies of the functional characteristics of encephalitogenic effector cells during EAE have focused on T lymphocytes that react with MBP and PLP. These studies have led to the general consensus that, unlike autoimmune diabetes in which both CD4+ and CD8+ T cells participate in the causation of disease (1113), effector T cells in EAE are exclusively CD4+ and {alpha}ßTCR+ (14).

In a previous study, we have shown that immunization of B6 mice with a synthetic peptide corresponding to residues 35–55 of MOG (pMOG35–55) persistently induced a MOG-specific CD8+ T cell subset that has an increased ability to induce paralytic demyelinating disease in B6 mice. Such T cells could be readily identified by FACS analysis based on their ability to bind complexes of H-2Db and pMOG35–55 (15). Since typical antigenic epitopes for CD8+ T cells are composed of 9–10 amino acids (16,17), we have analyzed the ability of peptides scanning the length of pMOG35–55 to bind recombinant H-2Db dimers and to induce paralysis in order to identify the core epitope structure of pMOG35–55-specific encephalitogenic CD8+ T cells. In the present study, we show that both of the truncated peptides pMOG40–54 and pMOG44–54 in complexes with H-2Db strongly bound MOG-specific CD8+ T cells in vitro; however, only pMOG40–54 retained a full capability of inducing clinical and pathological changes in B6 mice in vivo, indicating that pMOG44–54 may have lost some stimulatory function necessary for development and/or expansion for CD8+ encephalitogenic T cells, even though it retains the ability to bind these T cells.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals
C57BL/6J (B6) mice were obtained from Jackson Laboratories (Bar Harbor, ME) and housed in an NIH-approved and AAALAC-accredited facility.

Cells and reagents
All T cells were cultured in RPMI 1640 medium (Gibco, Grand Island, NY) supplemented with 10% selected FCS (Gibco), 5 x 10–5 M 2-mercaptoethanol and penicillin/streptomycin (100 µg/ml). Synthetic peptides listed in Table 1 were produced by the Shriners Hospital for Children, Montreal, Canada. These peptides were synthesized using standard Fmoc chemistry on an Applied Biosystems model 433A solid-phase peptide synthesizer. All the peptides were HPLC purified. The authenticity of these peptides was confirmed by the amino acid composition analyses and by mini-sequencing of the N-terminal up to 15 amino acids of the HPLC-purified protein. Recombinant dimers of the MHC class I (H-2Db) molecule recombinant human ß2-microglobulin were obtained from BD Biosciences PharMingen (San Diego, CA).



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Fig. 1. The binding activity of MOG-specific T cells to complexes of MHC class I molecules (H-2Db) and truncated peptides derived from pMOG35–55. Staining on pMOG35–55-stimulated immune T cells. Fifteen days after immunization with pMOG35–55/CFA, draining lymph node cells from the immunized B6 mice were stimulated with pMOG35–55 for 72 h. The Ficoll-separated activated T cells were cultured in IL-2-containing medium for 5 days. Such T cells (5 x 105) were dually stained with a complex of PE-labeled recombinant MHC class I (H-2Db) molecules and truncated MOG peptides (y-axis) and FITC-labeled antibodies specific for mouse {alpha}ßTCR (R73), as indicated. The results were analyzed by a FACScan.

 
MOG-specific T cell hybridomas were prepared as we previously reported (15). Briefly, MOG-specific T cell lines were prepared and residual CD4+ T cells were completely removed by magnetic beads conjugated with anti-mouse CD4 antibodies. A mini-fusion between a CD8+ MOG-specific T cell line and a variant line of BW5147 (18) has yielded >40 growing hybridomas, of which 20 were randomly selected for analysis of binding activity against pMOG35–55-bound H-2Db molecules. One of the hybrids (MOG-TH10) was chosen for this study.

Active induction of EAE
Mice were immunized with MOG peptides (200 µg per mouse) emulsified in complete Freund’s adjuvant (CFA) containing 0.6 mg Mycobacterium tuberculosis (H37Ra; Difco, Detroit, MI). One day post-immunization, MOG-sensitized animals received a single dose of 400 ng of Pertussis toxin i.p. (Sigma, St Louis, MO).

Isolation of T cells from B6 mice with EAE actively immunized with MOG peptides
To determine and characterize encephalitogenic T cells from animals immunized with MOG peptides, T cells isolated from the draining lymph nodes and spleens of immunized animals were enriched by passage through nylon wool and re-stimulated in vitro with immunizing peptides (20 µg/ml) presented by irradiated syngeneic spleen cells. After 3 days, activated T cell blasts were separated on a Ficoll gradient and injected (2 x 106, i.p.) into naive B6 mice.

T cell proliferation/inhibition assays
Antigen-presenting cells (APC, irradiated syngeneic spleen cells, 2 x 105/well) were pre-incubated in 96-well flat-bottom microtiter plates with an optimal dose (20 µg/ml) of pMOG35–55. After 1 h, enriched T cells (4 x 105/well), prepared from lymph nodes or spleen by nylon wool adhesion, were seeded and further incubated for 48–72 h. The cultures were then pulsed with 1.0 µCi [3H]thymidine/well for 6 h, harvested and the incorporated isotope quantitated by liquid scintillation.

Depletion of T cell subsets by antibody-coated magnetic beads
Nylon wool-enriched splenic T cells were incubated with 10 µg/ml of either rat anti-mouse CD4 mAb (GK1.5) or normal rat Ig (control) for 30 min. Unbound antibody was removed by twice washing before the addition of magnetic biospheres coupled to goat anti-rat Ig (BioSource, Camarillo, CA), incubation at 4°C and agitation for 30 min. The tube of cells was then placed on a magnetic stand for 5 min and cells free of magnetic particles were collected. The procedure was repeated 2–3 times, as necessary.

Evaluation of EAE
Animals were examined daily for weight loss and clinical signs. Animals were clinically graded as follows: 0 = no signs, 1 = partial loss of tail tonicity, 2 = loss of tail tonicity, difficulty in righting, 3 = unsteady gait and mild paralysis, 4 = hind-limb paralysis and incontinence, and 5 = moribund or death.

Flow cytometric detection of T cells binding to pMOG35–55 complexed with recombinant dimers of MHC class I (H-2Db) molecule
Dimeric MHC class I (H-2Db) used in this study is a fusion protein between mouse H-2Db and mouse IgG1. The recombinant protein was added to an equal amount of human ß2-microglobulin at a final concentration of 0.15 mg/ml, in the presence of an excess amount of testing peptide (1 mg/ml). The mixtures were stored at 4°C for 24–48 h before use. Dual-color staining was performed by incubating 5 x 105 cells with peptide dimer complexes of 1.0 µg per staining at 4°C for 30 min. The cells were washed twice in PBS containing 1% BSA and 0.1% sodium azide, and re-stained with a phycoerythrin (PE)-labeled anti-mouse IgG1 antibody, followed by a subsequent staining with FITC-conjugated antibody. The results are presented as FITC (x-axis) versus PE (y-axis) staining.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
MOG-specific CD8+ T cells recognize complexes of pMOG40–54 and pMOG44–54 peptides and dimeric MHC class I (H-2Db) molecules
We have previously reported that a major population of encephalitogenic MOG-specific T cells in the B6 mouse expresses CD8 and {alpha}ßTCR. This CD8+ T cell subset can be readily identified by their ability to bind complexes of pMOG35–55 polypeptide and recombinant H-2Db protein as assessed by flow cytometry (15). The frequency of pMOG35–55-reactive T cells is estimated to be <1% of the total T cell population in the lymphoid organs of immunized mice (not shown). Following antigenic stimulation in vitro and expansion in medium supplemented with IL-2, the percentage of MOG-specific T cells increases rapidly (Fig. 1) (15).

The length of pMOG35–55 polypeptide (21mer) is about twice the size of a peptide that has been shown to fit into the MHC class I groove using molecular and structural approaches (16,17). The binding of MOG-specific CD8+ T cells to recombinant H-2Db dimers in the presence of truncated pMOG35–55 peptide was investigated to identify the core epitope(s) critical to the recruitment of this population in the pathogenesis of encephalomyelitis by MOG peptides. Therefore, we designed a panel of truncated peptides that linearly scanned pMOG35–55 (Table 1). Since MOG-specific CD8+ T cell subsets may vary in their ability to bind peptide–MHC I complexes, MOG-specific T cell hybridomas served as target cells. These cell lines were generated by fusion of highly enriched primary MOG-specific CD8+ T lymphocytes to a variant of the BW5147 thymoma line that lacks both the {alpha} and ß chain of the TCR (19). Several stable T hybridoma cell lines were obtained that specifically bind pMOG35–55 in complex with H-2Db dimers.


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Table 1. Truncated MOG peptides
 
Nylon wool-enriched splenic T cells from mice immunized with pMOG35–55 were stimulated with this peptide and expanded in IL-2-containing medium for 5–7 days. Approximately 40–60% of the {alpha}ßTCR+ lymphoid cells in this population bound PE-labeled pMOG35–55–H-2Db complexes [Fig. 1 and (15)]. The scanning peptides varied greatly in their ability to mediate binding between T cell and dimeric H-2Db protein. The pMOG40–54 peptide interacted with recombinant PE-labeled H-2Db protein and this complex bound primarily MOG-specific CD8+ T cells at high levels, as shown in Fig. 1. This observation was extended in binding experiments using the MOG-TH10 cell, a MOG-specific CD8+ T cell hybridoma, as targets (Fig. 2). Studies of a panel of truncated peptides (Table 1 Panel B) revealed that the peptide pMOG44–54 consistently retained binding activity (Figs 1 and 2), but that further deletion of N- or C-terminal residues dramatically reduced activity. Neither pMOG44–53 nor pMOG45–54 in complex with H-2Db showed consistent binding to CD8+ T cell targets, indicating that Phe44 and Gly54 are required for class I-dependent presentation to most, if not all, MOG-specific CD8+ T cell subsets. The results also showed that the binding activity of the truncated pMOG40–54 and pMOG44–54 was stronger and more frequent compared to the wild-type peptide pMOG35–55, suggesting that Lys55 may exert a negative effect on this interaction. Thus, the 11mer peptide pMOG44–54 (FSRVVHLYRNG) represents the core binding epitope for MOG-specific CD8+ T cells.



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Fig. 2. Binding to complexes of H-2Db and pMOG44–54 by a MOG-specific T cell hybridoma (MOG-TH10) that was dually stained with complexes of PE-labeled recombinant MHC class I (H-2Db) molecules and truncated MOG peptides (y-axis) and FITC-labeled antibodies specific for mouse {alpha}ßTCR.

 
The mean fluorescence of MOG-TH10 cells by the pMOG44–54–H-2Db complex was consistently higher than that observed with PE staining of pMOG45–54–H-2Db. The mean fluorescence intensity of CD3 or TCR was also higher in MOG-TH10 cells incubated with pMOG44–54, suggesting that exposure to this peptide may up-regulate the TCR complex or induce aggregation of the responder T cells.

Encephalitogenic nature of pMOG40–54 and pMOG44–54
A key question is whether the ability of truncated peptides that scan pMOG35–55 sequences and bind MOG-specific CD8+ T cells in complex with H-2Db–PE reflects their ability to induce paralytic disease in vivo. Table 2 summarizes the outcome of EAE induction in B6 mice by pMOG35–55 and individual truncated peptides. Paralysis developed in mice immunized with pMOG35–55, pMOG40–54 and pMOG44–54, even though partially, but not those receiving pMOG44–53 and pMOG45–54. We have also determined the ability of these peptides to induce activated T lymphocytes that can adoptively transfer EAE. Three groups (n = 10) of B6 mice were immunized with pMOG40–54, pMOG44–54 or pMOG35–55 (200 µg emulsified in 50 ml CFA). T cells specific for the immunizing peptides (2 x 106) were isolated 2–3 weeks after the immunization and transferred to groups of naive B6 mice. As summarized in Table 3, paralysis developed in two out of 10 mice that received pMOG44–54-specific T cells and in 14 of 16 mice that received pMOG40–54-specific T cells. The paralysis following transfer from mice immunized with pMOG40–54 was complete, whereas it was partial in those receiving T cells elicited by pMOG44–54. We also determined the central nervous system (CNS) inflammatory reaction induced by the peptides using a routine pathological examination method as well as Fast Luxol blue staining. As demonstrated in Fig. 3(A–C), pMOG40–54 induced an intense CNS inflammatory infiltration that was similar in composition and magnitude to that observed in mice receiving pMOG35–55 (15); inflammation induced by pMOG44–54 was marginal.


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Table 2. Comparison of the induction of EAE by MOG peptides
 

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Table 3. Encephalitogenic activity of T cells specific for MOG peptides
 


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Fig. 3. CNS inflammation induced by T cells specific for MOG peptides, and the responses of T cells obtained from pMOG35–55- pMOG40–54- and pMOG44–54-immunized B6 mice. (Top panle) Syngeneic B6 mice were administered 2 x 106 (A) pMOG35–55-, (B) pMOG40–54- or (C) pMOG44–54-specific T cells. Eight days later, when the mice developed clinical signs and became paralytic, they were killed and perfused intracardially through the left ventricle with ice-cold PBS followed by 10% buffered formalin. Paraffin sections were stained with Fast Luxol blue to assess inflammation and demyelination of diseased animals and control mouse. Original magnification x40. (Bottom panel) Nylon wool-enriched splenic T cells (4 x 105/well) prepared from (D) pMOG35–55-, (E) pMOG40–54- and (F) pMOG44–54-immunized B6 mice were stimulated in 96-well plates in the presence of indicated peptides (20 µg/ml) and 2 x 105 irradiated syngeneic spleen cells (APC) for 3 days. The cultures were then pulsed with 1.0 µCi [3H]thymidine for 6 h before harvest. Results are reported as the means of triplicate cultures. SD < 15% in all experiments. Results of one representative experiment of three separate experiments.

 
The truncated MOG peptide pMOG40–54 was more potent in eliciting T cell responses than pMOG44–54
We determined whether T cells induced by the truncated peptides share antigenic specificity with those T cells induced by pMOG35–55 and whether the binding ability of the truncated MOG peptides paralleled their T cell stimulating activity. To test this, B6 mice were randomly grouped and immunized with pMOG40–54, pMOG44–54 and pMOG35–55. Two weeks after immunization, T cells obtained from the spleen and draining lymph nodes of immunized mice were enriched and confronted with a panel of selected MOG-derived peptides (see Table 1) in the presence of irradiated APC. As demonstrated in Fig. 3(D–F), T cells obtained from pMOG35–55-immunized mice respond to pMOG35–55 and pMOG40–54, but not to truncated pMOG46–54. Likewise, mice that were immunized with pMOG44–54 and pMOG40–54 responded to pMOG35–55 and pMOG40–54, but not to pMOG46–54. Once again, these results demonstrated that the 11mer pMOG44–54 (FSRVVHLYRNG) is the minimal, functionally constant, length of the MOG35–55 peptide that binds to CD8+ MOG-specific T cells and stimulates T cell proliferation in vitro.


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The intensity of antigen-specific T cell responses is usually measured by limiting dilution analysis (LDA), which determines the number of antigen-specific T cells (2022). Because the assay does not detect those T cells that fall into a refractory phase of the T cell cycle, LDA may underestimate the number of T cells recognizing certain peptides. To complement LDA, a new experimental tool has been discovered to directly enumerate antigen-specific T cells by their binding of peptide–MHC complexes, thus obviating the need to rely on effector T cell activities.

Staining antigen-specific T cells with fluorescent-labeled tetrameric MHC–peptide complexes has provided a powerful experimental tool to characterize the intensity of an immune response (2326). The binding assays have been applied to quantify virus-specific cytotoxic T cells (25,27,28), T cells that are specific for tumor-associated antigen (29) and CD4+ autoreactive T cells (3033). In such studies, peptide–MHC complexes were most often engineered as tetramers, with addition of a substrate peptide for BirA-dependent biotinylation to the C-terminus of the heavy chain of the MHC molecule. After folding in vitro the heavy chain fusion protein in the presence of ß2-microglobulin and a specific peptide ligand was biotinylated on the lysine within the BirA substrate peptide. Tetramers were produced by mixing the biotinylated peptide–MHC complex with PE-labeled deglycosylated avidin at a molar ratio of 4:1 (27,34).

Dimeric MHC molecules can also be used. For the dimeric peptide–MHC complex the recombinant light (ß2-microglo bulin) and heavy chains can be folded in vitro with different antigenic peptides, whereas their tetrameric counterparts are pre-conjugated with one chosen antigenic peptide. In the present study, we applied this unique feature of dimeric MHC molecules to define epitopes of antigen-specific CD8+ T cells. In a previous report, we have demonstrated that synthetic peptides encompassing residues 35–55 of the MOG readily activated a CD8+{alpha}ßTCR+ encephalitogenic T cell subset(s) in B6 mice and that this subset of MOG-specific T cells induced a paralytic disorder and caused CNS inflammation (15). Remarkably, as few as 106 CD8+ MOG-specific T cells administered to naive B6 mice could be readily retrieved 6–10 months after adoptive transfer (15), indicating that the CD8+ encephalitogenic T cells survived for long periods in vivo and their appearance was closely associated with progression of disease. Notably, MOG induced a chronic progressive disease and pathologic demyelination, which closely resembled secondary progressive MS, the most common form of disabling MS (7,35).

Our previous observation showed that CD8+ MOG-specific T cells recognize H-2Db dimers when loaded with an encephalitogenic peptide (pMOG35–55). Such binding activity can easily be monitored by a fluorescent-binding assay (15). We first asked whether such binding assays could be exploited for prediction of the functional epitope of myelin-specific CD8+ T cells. We were particularly interested to learn whether all MOG peptides that possessed binding activity to MOG-specific CD8+ T cells were able to induce paralytic pathology in B6 mice. Surprisingly, the truncated MOG peptides bound better in the absence of the lysine at position 55. Peptides pMOG35–53 and pMOG44–53 did not differ appreciably in binding, indicating that the amino acids at positions 35–43 did not play a critical role in binding. In contrast, the lack of glycine at position 54 in these two peptides dramatically decreased their binding activity, indicating that this amino acid was functionally critical. The fact that pMOG35–53 lost most of its binding activity reinforced the conclusion that the glycine at position 54 was functionally essential.

We next determined whether truncation of pMOG40–54 from the N-terminal modified the peptide’s functional activity. The second panel of peptides (Table 1 Panel B) was designed by further truncation of the N-terminus of pMOG40–54 while preserving the C-terminus (residue 54) of the peptide. Our results have shown that the minimal peptide length required for binding to CD8+ MOG-specific T cells was the MOG peptide containing residues 44–54 (FSRVVHLYRNG).

The disease-inducing activity of the truncated MOG peptides does not completely match the binding activity. Encephalitogenic activity was completely abolished in the absence of residues 44–45, even though variable binding activity remained. In addition, pMOG44–54 retained a strong binding activity, yet only some of the naive B6 mice immunized with this peptide were rendered paralytic. Only three out of 10 mice immunized with pMOG44–54 developed clinical EAE, compared to 80–90% of the mice immunized with pMOG35–55 or pMOG40–54. Proliferation assays agreed with the relative abilities of peptides to induce disease since the partially truncated 15mer pMOG40–54 induced a strong T cell response in vivo and in vitro, while the 11mer pMOG44–54 was weaker. It appears that residues 40–43 play a major role in eliciting a strong T cell response, but are not mandatory for binding. Conceivably, peptides >12 amino acids may activate both CD8+ and CD4+ T cells—with the latter providing ‘help’ for the activation and/or expansion of CD8+ T cells, whereas the binding assay does not require help fro CD4+ T cells. Thus, we conclude that: (i) the binding assays are a useful tool for revealing core antigenic epitopes of CD8+ T cells and (ii) the optimal encephalitogenic peptide of MOG may be longer than the peptide needed for the binding of CD8+ T cells.

Thus far, we were unable to define separate antigenic epitopes specific for CD4 or CD8 T cells, because pMOG40–54 retained both CD4 and CD8 stimulatory activity, whereas pMOG44–54 dramatically lost activity in stimulating either T cells (data not shown). The available data appear to support the notion that only the MOG peptides capable of activating both CD4 and CD8 cells acquired encephalitogenic activity. Indeed, such a hypothesis has also been supported by our recent study using established CD4+ pMOG35–55-specific T cell lines, which responded to both pMOG35–55 and pMOG40–54, but not pMOG44–54. It remains undetermined whether pMOG40–54 contains two separate epitopes for CD4 and CD8 T cells respectively.

In summary, we have determined the binding activity of truncated MOG peptides to CD8+ encephalitogenic T cells and H-2Db molecules, and compared their binding activity with the encephalitogenic potential of the peptides. Our results showed that the pMOG44–54 (FSRVVHLYRNG) is the minimal binding epitope for the CD8+ MOG-specific T cell. Addition of amino acids at positions 40–43 augmented T cell stimulation and disease induction, but not necessarily the binding activity of the peptide. Our studies indicated that the binding assay of using dimeric MHC molecules is a useful tool for prediction of the functional epitope of CD8+ autoreactive T cells. The optimal or maximal induction of CD8+ autoreactive T cells may require the help of CD4+ T cells.


    Abbreviations
 
APC—antigen-presenting cell

CFA—complete Freund’s adjuvant

CNS—central nervous system

EAE—experimental autoimmune encephalomyelitis

LDA—limiting dilution assay

MBP—myelin basic protein

MOG—myelin/oligodendrocyte glycoprotein

pMOG35-55—synthetic peptide representing residues 35–55 of MOG

MS—multiple sclerosis

PE—phycoerythrin

PLP—proteolipid protein


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Martenson, R. E., Deibler, G. E., Kies, M. W., Levine, S. and Alvord, E. C. 1972. Myelin basic proteins of mammalian and submammalian vertebrates: encephalitogenic activities in guinea pigs and rats. J. Immunol. 109:262.[ISI][Medline]
  2. McFarlin, D. E., Blank, S. E., Kibler, R. F., McKneally, S. and Shapira, R. 1973. Experimental allergic encephalomyelitis in the rat: response to encephalitogenic proteins and peptides. Science 179:478.[ISI][Medline]
  3. McFarlin, D. E., Blank, S. E., Kibler, R. F., McKneally, S. and Shapira, R. 1973. Experimental allergic encephalomyelitis in the rat: response to encephalitogenic proteins and peptides. Science 179:478.[ISI][Medline]
  4. Yoshimura, T., Kunishita, T., Sakai, K., Endoh, M., Namikawa, T. and Tabira, T. 1985. Chronic experimental allergic encephalomyelitis in guinea pigs induced by proteolipid protein. J. Neurol. Sci. 69:47.[CrossRef][ISI][Medline]
  5. Sobel, R. A., Van der Veen, R. C. and Lees, M. B. 1986. The immunopathology of chronic experimental allergic encephalomyelitis induced in rabbits with bovine proteolipid protein. J. Immunol. 136:157.[Abstract/Free Full Text]
  6. Bernard, C. C. A., Johns, T. G., Slavin, A., Ichikawa, M., Ewing, C., Liu, J. and Bettadapura, J. 1997. Myelin oligodendrocyte glycoprotein: a novel candidate autoantigen in multiple sclerosis. J. Mol. Med. 75:77.[CrossRef][ISI][Medline]
  7. Kerlero de Rosbo, N., Mendel, I. and Ben-Nun, A. 1995. Chronic relapsing experimental autoimmune encephalomyelitis with a delayed onset and an atypical clinical course, induced in PL/J mice by myelin oligodendrocyte glycoprotein (MOG)-derived peptide: Preliminary analysis of MOG T cell epitopes. Eur. J. Immunol. 25:985.[ISI][Medline]
  8. Linington, C., Berger, T., Perry, L., Weerth, S., Hinze-Selch, D., Zhang, Y., Lu, H., Lassmann, H. and Wekerle, H. 1993. T cells specific for the myelin oligodendrocyte glycoprotein mediate an unusual autoimmune inflammatory response in the central nervous system. Eur. J. Immunol. 23:1364.[ISI][Medline]
  9. Zhang, Y., Burger, D., Saruhan, G., Jeannet, M. and Steck, A. J. 1993. The T-lymphocyte response against myelin-associated glycoprotein and myelin basic protein in patients with multiple sclerosis. Neurology 43:403.[Abstract]
  10. Holz, A., Schaeren-Wiemers, N., Schaefer, C., Pott, U., Colello, R. J. and Schwab, M. E. 1996. Molecular and developmental characterization of novel cDNAs of the myelin-associated/oligodendrocytic basic protein. J. Neurosci. 16:467.[Abstract]
  11. Bendelac, A., Carnaud, C., Boitard, C. and Bach, J.-F. 1987. Syngeneic transfer of autoimmune diabetes from diabetic NOD mice to healthy neonatal requirement for both L3T4+ and Lyt2+ T cells. J. Exp. Med. 166:823.[Abstract]
  12. Nagata, M. and Yoon, J.-W. 1994. Prevention of autoimmune type I diabetes in BioBreeding (BB) rats by a newly established, autoreactive T cell line from acutely diabetic BB rats. J. Immunol. 153:3775.[Abstract/Free Full Text]
  13. Nagata, M., Santamaria, P., Kawamura, T., Utsugi, T. and Yoon, J. W. 1994. Evidence for the role of CD8+ cytotoxic T cells in the destruction of pancreatic beta-cells in nonobese diabetic mice. J. Immunol. 152:2042.[Abstract/Free Full Text]
  14. Steinman, L. 1991. The development of rational strategies for selective immunotherapy against autoimmune demyelinating disease. Adv. Immunol. 49:357.[ISI][Medline]
  15. Sun, D., Whitaker, J. N., Huang, Z., Liu, D., Coleclough, C., Wekerle, H. and Raine, C. S. 2001. Myelin antigen-specific CD8+ T cells are encephalitogenic and produce severe disease in C57BL/6 mice. J. Immunol. 166:7579.[Abstract/Free Full Text]
  16. Rötzschke, O., Falk, K., Deres, K., Schild, H., Norda, M., Metzger, J., Jung, G. and Rammensee, H. G. 1990. Isolation and analysis of naturally processed viral peptides as recognized by cytotoxic T cells. Nature 348:252.[CrossRef][ISI][Medline]
  17. Rammensee, H. G., Falk, K. and Rötzschke, O. 1993. Peptides naturally presented by MHC class I molecules. Annu. Rev. Immunol. 11:213.[CrossRef][ISI][Medline]
  18. Born, W. K., White, J., O‘Brien, R. and Kubo, R. 1988. Development of T-cell receptor expression: studies using T cell hybridomas. Immunol. Res. 7:279.[ISI][Medline]
  19. White, J., Blackman, M. A., Bill, J., Kappler, J., Marrack, P., Gold, D. P. and Born, W. K. 1989. Two better cell lines for making hybridomas expressing specific T cell receptors. J. Immunol. 143:1822.[Abstract/Free Full Text]
  20. Sun, D., Wilson, D. B., Cao, L. and Whitaker, J. N. 1997. The role of regulatory T cells in Lewis rats resistant to EAE. J. Neuroimmunol. 78:69.[CrossRef][ISI][Medline]
  21. Cohen, J. A., Essayan, D. M., Zweiman, B. and Lisak, R. P. 1987. Limiting dilution analysis of antigen-reactive lymphocytes isolated from the central nervous system of Lewis rats with experimental allergic encephalomyelitis. Cell. Immunol. 108:203.[ISI][Medline]
  22. Matsumoto, Y., Kawai, K., Tomita, Y. and Fujiwara, M. 1990. Limiting-dilution analysis of the frequency of myelin basic protein-reactive T cells in Lewis, PVG/c and BN rats. Implication for susceptibility to autoimmune encephalomyelitis. Immunology 69:215.[ISI][Medline]
  23. Muralikrishna, K., Altman, J. D., Suresh, M., Sourdive, D. J. D., Zajac, A. J., Miller, J. D., Slansky, J. and Ahmed, R. 1998. Counting antigen-specific CD8 T cells—a reevaluation of bystander activation during viral infection. Immunity 8:177.[ISI][Medline]
  24. Ogg, G. S., Jin, X., Bonhoeffer, S., Dunbar, P. R., Nowak, M. A., Monard, S., Segal, J. P., Cao, Y., Rowland-Jones, S. L., Cerundolo, V., Hurley, A., Markowitz, M., Ho, D. D., Nixon, D. F. and McMichael, A. J. 1998. Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA. Science 279:2103.[Abstract/Free Full Text]
  25. Flynn, K. J., Riberdy, J. M., Christensen, J. P., Altman, J. D. and Doherty, P. C. 1999. In vivo proliferation of naive and memory influenza-specific CD8+ T cells. Proc. Natl Acad. Sci. USA 96:8597.[Abstract/Free Full Text]
  26. Spencer, J. V. and Braciale, T. J. 2000. Incomplete CD8+ T lymphocyte differentiation as a mechanism for subdominant cytotoxic T lymphocyte responses to a viral antigen. J. Exp. Med. 191:1687.[Abstract/Free Full Text]
  27. Altman, J. D., Moss, P. A. H., Goulder, P. J. R., Barouch, D. H., McHeyzer-Williams, M. G., Bell, J. I., McMichael, A. J. and Davis, M. M. 1996. Phenotypic analysis of antigen-specific T lymphocytes. Science 274:94.[Abstract/Free Full Text]
  28. Callan, M. F., Tan, L., Annels, N., Ogg, G. S., Wilson, J. D., O‘Callaghan, C. A., Steven, N., McMichael, A. J. and Rickinson, A. B. 1998. Direct visualization of antigen-specific CD8+ T cells during the primary immune response to Epstein-Barr virus in vivo. J. Exp. Med. 187:1395.[Abstract/Free Full Text]
  29. Romero, P., Dunbar, P. R., Valmori, D., Pittet, M., Ogg, G. S., Rimoldi, D., Chen, J. L., Lienard, D., Cerottini, J. C. and Cerundolo, V. 1998. Ex vivo staining of metastatic lymph nodes by class I major histocompatibility complex tetramers reveals high numbers of antigen-experienced tumor-specific cytolytic T lymphocytes. J. Exp. Med. 188:1641.[Abstract/Free Full Text]
  30. Wong, F. S., Karttunen, J., Dumont, C., Wen, L., Visintin, I., Pilip, I. M., Shastri, N., Pamer, E. G. and Janeway, C. A. 1999. Identification of an MHC class I-restricted autoantigen in type 1 diabetes by screening an organ-specific cDNA library. Nat. Med. 5:1026.[CrossRef][ISI][Medline]
  31. Radu, C. G., Anderton, S. M., Firan, M., Wraith, D. C. and Ward, E. S. 2000. Detection of autoreactive T cells in H-2u mice using peptide–MHC multimers. Int. Immunol. 12:1553.[Abstract/Free Full Text]
  32. Lukacher, A. E., Moser, J. M., Hadley, A. and Altman, J. D. 1999. Visualization of polyoma virus-specific CD8+ T cells in vivo during infection and tumor rejection. J. Immunol. 163:3369.[Abstract/Free Full Text]
  33. Lee, P. P., Yee, C., Savage, P. A., Fong, L., Brockstedt, D., Weber, J. S., Johnson, D., Swetter, S., Thompson, J., Greenberg, P. D., Roederer, M., Davis, and MM 1999. Characterization of circulating T cells specific for tumor-associated antigens in melanoma patients. Nat. Med. 5:677.[CrossRef][ISI][Medline]
  34. Belz, G. T., Altman, J. D. and Doherty, P. C. 1998. Characteristics of virus-specific CD8+ T cells in the liver during the control and resolution phases of influenza pneumonia. Proc. Natl Acad. Sci. USA 95:13812.[Abstract/Free Full Text]
  35. Malipiero, U., Frei, K., Spanaus, K. S., Agresti, C., Lassmann, H., Hahne, M. X. T. J., Eugster, H. P. and Fontana, A. 1997. Myelin oligodendrocyte glycoprotein-induced autoimmune encephalo myelitis is chronic/relapsing in perforin knockout mice, but monophasic in Fas- and FasL-deficient lpr and gld mice. Eur. J. Immunol. 27:3151.[ISI][Medline]