The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Miyakoshi, A.
Right arrow Articles by Matsumoto, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Miyakoshi, A.
Right arrow Articles by Matsumoto, Y.
The Journal of Immunology, 2003, 170: 6371-6378.
Copyright © 2003 by The American Association of Immunologists

Characterization of the Antigen Specificity and TCR Repertoire, and TCR-Based DNA Vaccine Therapy in Myelin Basic Protein-Induced Autoimmune Encephalomyelitis in DA Rats1

Akira Miyakoshi, Won Kee Yoon, Youngheun Jee and Yoh Matsumoto2

Department of Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Like Lewis rats, DA rats are an experimental autoimmune encephalomyelitis (EAE)-susceptible strain and develop severe EAE upon immunization with myelin basic protein (MBP). However, there are several differences between the two strains. In the present study we induced acute EAE in DA rats by immunization with MBP and MBP peptides and examined the Ag specificity and TCR repertoire of encephalitogenic T cells. It was found that although immunization with MBP and a peptide corresponding to its 62–75 sequence (MBP62–75) induced clinical EAE, the responses of lymph node T cells isolated from MBP-immunized rats to MBP62–75 was marginal, indicating that this peptide contains major encephalitogenic, but not immunodominant, epitopes. The TCR analysis by CDR3 spectratyping of spinal cord T cells revealed that V{beta}10 and V{beta}15 spectratype expansion was always found in MBP62–75-immunized symptomatic rats. On the basis of these findings, we examined the encephalitogenicity of V{beta}10- and V{beta}15-positive T cells. First, the adoptive transfer experiments revealed that V{beta}10-positive T line cells derived from MBP62–75-immunized rats induced clinical EAE in recipients. Second, administration of DNA vaccines encoding V{beta}10 and V{beta}15, alone or in combination, ameliorated MBP62–75-induced EAE. Collectively, it was strongly suggested that V{beta}10- and V{beta}15-positive T cells are encephalitogenic. Analyses of the Ag specificity and T cell repertoire of pathogenic T cells performed in this study provide useful information for designing specific immunotherapies against autoimmune diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Susceptibility to organ-specific autoimmune diseases varies greatly among strains of animals. Lewis rats are highly susceptible to various organ-specific autoimmune diseases including experimental autoimmune encephalomyelitis (EAE)3 and have long been examined to elucidate the pathomechanisms of the disease. Recent analysis using T cell lines and clones established from myelin basic protein (MBP)-immunized rats has demonstrated that encephalitogenic T cells bear CD4 molecules and use a limited number of {beta}-chain families of the TCR such as V{beta}8.2 (1). Furthermore, the complementarity-determining region 3 (CDR3) of TCR of T cell clones is rather short, and some amino acid residues are conservatively preserved (2, 3). More recently, we demonstrated, by CDR3 spectratyping, that the V{beta}8.2 spectratype alone shows oligoclonal expansion in the spinal cord of Lewis rats throughout the course of EAE, whereas irrelevant TCRs become more diverse at later stages of the disease (4, 5). Importantly, the CDR3 sequence of the majority of TCR clones derived from the EAE-specific spectratype is the same as that of encephalitogenic T cell clones. These findings imply that although the phenotype of T cells in the target organ diversifies as the autoimmune disease progresses, the disease-associated TCR spectratype(s) is preserved throughout the course of the disease.

DA rats are another autoimmune disease-susceptible strain. After immunization with MBP or MBP peptides in CFA, rats develop acute EAE (6). However, encephalitogenic epitopes in the MBP molecules for the DA and Lewis strains are different from each other. For example, MBP63–81 caused severe EAE in DA rats, but not in Lewis rats. In contrast, MBP87–99 was inactive in DA rats, but was encephalitogenic for Lewis rats (6). There are also differences between these two strains. DA rats develop clinical EAE after immunization with encephalitogenic Ags, not only with CFA, but also with IFA, whereas Lewis rats develop EAE only after immunization with Ag/CFA (7). In addition, induction of unresponsiveness with encephalitogenic peptide-coupled splenocytes was unsuccessful in DA rats, but not in Lewis rats (7). These findings raised the possibility that TCR usage by encephalitogenic T cells in DA rats is different from that in Lewis rats.

In the present study we attempted to elucidate the nature of the Ag specificity and T cell repertoire in DA rats during the course of EAE and found several interesting results. MBP62–75, only one major encephalitogenic peptide in DA rats, was not immunodominant as defined by Sercarz et al. (8). This is in contrast to Lewis rats, in which the 68–88 sequence of the MBP molecule is an immunodominant and major encephalitogenic epitope. CDR3 spectratyping and sequencing analysis revealed that clonal expansion of encephalitogenic T cells was more diverse in MBP-immunized DA rats, whereas V{beta}10 and V{beta}15 spectratype expansion was always found in MBP62–75-immunized rats. Administration of DNA vaccines encoding V{beta}10 and V{beta}15 significantly ameliorated EAE induced by the MBP peptide, indicating that V{beta}10-positive and V{beta}15-positive T cells are involved in the development of EAE. Characterization of the T cell repertoire in DA rats during EAE will provide useful information for designing specific immunotherapy for human autoimmune diseases, as the Ag reactivity and TCR usage by disease-associated T cells may differ considerably among patients with autoimmune diseases such as multiple sclerosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Rats and reagents

DA rats were purchased from Japan SLC, Inc. (Shizuoka, Japan), and were used at 8–12 wk of age. Synthetic peptides corresponding to 43–67, 68–88, and 87–100 sequences of guinea pig MBP (GPBP) were prepared by Multiple Peptide System (San Diego, CA). Synthetic peptides corresponding to the 1–11 (GPBP1–11, ASQKRPSQRHG), 5–25 (GPBP5–25, RPSQRHGSKYLATASTMDHAR), 20–41 (GP20–41, TMDHARHGFLPRHRDTGILDSA), 36–58 (GPBP36–58, LDSIGRFFGSDRAAPKRGSGKDS), 62–75 (GPBP62–75, ARTTHYGSLPQKSQ), 62–84 (GPBP62–84, ARTTHYGSLPQKSQRSQDEN), and 100–120 (GPBP100–120, PSQGKGRGLSLSRFSWGAEGQ) sequences of the GPBP molecule were synthesized using a peptide synthesizer (Shimadzu, Kyoto, Japan). All the peptides used in this study were >90% pure as determined, and purified if necessary, by HPLC. GPBP was prepared as described previously (9).

EAE induction

Active EAE was induced in DA rats by immunization with an emulsion containing 100 µg of GPBP or their fragment peptides in CFA (Mycobacterium tuberculosis; 250 µg/rat) in the hind footpads on both sides. Some rats received i.p. injection of 2 µg of pertussis toxin (PT; Seikagaku, Tokyo, Japan) at the time of immunization. EAE was divided into five clinical stages (grade 1, floppy tail; grade 2, mild paraparesis; grade 3, severe paraparesis; grade 4, tetraparesis; grade 5 death). At different time points rats were killed under ether anesthesia, and several segments of the lumbar spinal cord were snap-frozen in OCT compound (Miles, Elkhart, IN). Frozen sections, 10 µm thick, were cut in a cryostat and used for histological examinations.

Establishment of T cell lines and adoptive transfer of EAE

Adoptive transfer of lymphoid cells to naive rats was performed as described previously (10). Spleens and lymph nodes were removed from rats that had been immunized 8–10 days earlier with GPBP62–75/CFA, and the single-cell suspension was prepared by pressing cells through a steel mesh. Cells were cultured for 3 days in the presence of 5 µg/ml GPBP62–75. Then viable cells at a dose of 2 x 107 were transferred into naive recipients.

GPBP62–75-specific T cell lines (TCL) were established from lymph node cells taken from GPBP62–75-immunized rats by cycle stimulation with GPBP62–75 and mitomycin C-treated thymocytes as APC. Between Ag stimulations, T cells were propagated in culture medium containing 5% Con A supernatant. Adoptive transfer experiments were also performed using V{beta}10-positive T line cells. For this purpose, anti-V{beta}10 mAb (G101)-conjugated Dynabeads (Dynal, Tokyo, Japan) were added to the T cell culture during the propagation period and incubated on ice. Rosetted cells were collected with a magnet and then cultured overnight, after which dissociated Dynabeads were removed with the magnet. This procedure was repeated in the next round. FACS analysis revealed that ~70% of the line cells were positive for G101. Then, TCLs were stimulated with GPBP62–75, and 5 x 106 cells were injected i.v. into naive rats.

Proliferative responses of T cells against MBP and MBP peptides

Proliferative responses of lymph node cells were assayed in microtiter wells by the uptake of [3H]thymidine. After being washed with PBS, lymph node cells (2 x 105 cells/well) were cultured with the indicated concentrations of GPBP or GPBP peptides for 3 days, with the last 18 h in the presence of 0.5 µCi [3H]thymidine (Amersham Pharmacia Biotech, Tokyo, Japan). In some experiments the proliferative responses of GPBP62–75-specific TCLs (3 x 104 cells/well) were assayed in the presence of the Ags and APC (5 x 105 cells/well). The cells were harvested on glass-fiber filters, and label uptake was determined using standard liquid scintillation techniques.

Complementary DNA synthesis and PCR amplification

RNA was extracted from PBL and spinal cord tissue using RNAzol B (Biotecx, Houston, TX). cDNA was then synthesized by RT using ReverTra Ace (Toyobo, Osaka, Japan) and was amplified in a thermal cycler (PerkinElmer, Norwalk, CT) using primer pairs for TCR. Primers for V{beta}1–20 and C{beta} inner were the same as those used in the previous study (4, 11). They were labeled with Cy5 or rhodamine, or remained unlabeled.

CDR3 spectratyping

CDR3 spectratyping was performed as described previously (4, 12). cDNA was amplified with V{beta}-specific and rhodamine-labeled C{beta} inner primers, and undiluted or diluted PCR products were added to an equal volume of formamide/dye loading buffer and heated at 94°C for 2 min. Two microliters of the samples were applied to 6% acrylamide sequencing gels. Gels were run at 30 W for 3.5 h at 50°C. Then, the fluorescence-labeled DNA profile on the gel was directly recorded using an FMBIO fluorescence image analyzer (Hitachi, Yokohama, Japan). Spectratypes revealed by this analysis usually consisted of five to seven bands.

Sequencing of PCR products

cDNA in PCR products or isolated from bands of interest on the acrylamide gel was reamplified, and the PCR products were ligated into pT-Adv vector and cloned using the AdvanTAge PCR Cloning Kit (Clontech, Palo Alto, CA) according to the manufacturer’s instructions. The plasmid DNA was then sequenced using Cy5-labeled C{beta} inner primer and Thermo Sequenase Fluorescent Labeled Primer Cycle Sequencing Kit on an ALFexpress DNA sequencer (Pharmacia Biotech, Tokyo, Japan). The CDR3 length was defined as the region starting from an amino acid residue after the CASS sequence of most V{beta} segments and ending before the GXG box in the J{beta} region as described previously (13).

DNA vaccination

DNA vaccine therapy was performed as reported previously with a few modifications (14). Total RNA was extracted from normal rat PBL and reverse transcribed into cDNA. This cDNA was then amplified using AmpliTaq Gold (PerkinElmer) with primer pairs specific for V{beta}10 (sense, 5'-GGTGGTGGATCCACCATGAGCTATAGGCTCCTAAGC-3'; antisense, 5'-GGTGGTGCGGCCGCCTAGCTGCTGGCACAGAGATACAC-3') or V{beta}15 (sense, 5'-GACTCGAGACCATGTTGCTGCTTCTGCTAC-3'; antisense, 5'-GAGGTACCTCATGCAGCACCACAGAAATAT-3'). These primer pairs cover the full length of the V region of the {beta}-chain and do not include the CDR3 region. All forward primers were designed to include an ATG in-frame. PCR products were cloned into pTargeT plasmid (Promega, Madison, WI) according to the manufacturer’s instructions. Colonies grown in competent cells were picked, and recombinant plasmid DNA was isolated using Mini Prep (Promega). By restriction enzyme digestion with PstI, colonies with an insert with the right direction and length were screened, and the nucleotide sequence of each clone was determined to confirm that inserts had the right sequence with ATG in-frame.

Large-scale preparation of plasmid DNA was performed using the EndoFree Plasmid Mega Kit (Qiagen, Tokyo, Japan). DNA vaccines at a total dose of 100 µg in 100 µl of 0.25% bupivacaine (Sigma-Aldrich, St. Louis, MO) were injected into bilateral tibialis anterior muscles twice on a weekly base. Two weeks after the last vaccination, rats were challenged with GPBP62–75/CFA and PT. In initial studies we determined that transcripts corresponding to the plasmid sequence plus insert existed at the injections sites of the muscle. We also verified the presence of the V{beta}10 protein by Western blot analysis (data not shown). mAbs against rat V{beta}15 are not available at present.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Clinical features of EAE induced in DA rats by immunization with GPBP and GPBP peptides

We first examined the susceptibility of DA rats to EAE when immunized with GPBP or synthetic peptides covering the entire GPBP molecule. Although it was reported previously that DA rats are highly susceptible to immunization with spinal cord homogenate and GPBP (6, 15, 16), substrain differences could exist between rats in Japan and other countries (information from the breeder in Japan). One marked difference between results reported previously and those in this study was that immunization with GPBP or GPBP62–75 in CFA using conventional protocols resulted in the development of very mild EAE (data not shown). We repeated the experiments and obtained essentially the same results. Simultaneous i.p. injection of PT significantly enhanced the development of EAE (Table I). In addition to GPBP and GPBP62–75, immunization with GPBP62–84 induced severe EAE in DA rats. Immunization with GPBP68–88/CFA plus PT induced relatively mild EAE in two of eight rats (Table I). Although GPBP68–88 is an encephalitogenic peptide for DA rats as reported previously (17), its encephalitogenic activity is weak compared with that of GPBP62–75. This relatively weak encephalitogenicity of GPBP68–88 to DA rats was also observed by Bouwer and Hinrichs (18). Similarly, the 100–120 sequence contained an encephalitogenic epitope (19), but showed very mild encephalitogenic activity, as evidenced by the finding that this peptide induced only weight loss. The results obtained in this study confirmed the previous findings (6) and further revealed that only GPBP62–75 contains the major encephalitogenic epitope for DA rats. We also confirmed this finding by the adoptive transfer experiments. Intravenous injection of lymphoid cells that had been taken from GPBP62–75-immunized rats and subsequently stimulated in vitro with the same Ag induced clinically mild, but histologically moderate, EAE in naive recipients (data not shown).


View this table:
[in this window]
[in a new window]
  		Table I. Clinical features of EAE induced in DA rats by immunization with GPBP and GPBP peptidesa

 
Proliferative responses of lymph node cells from DA rats with GPBP- and GPBP62–75 peptide-induced EAE

We next determined whether encephalitogenic peptides, GPBP62–75 and GPBP62–84, are immunodominant or cryptic. Each assay was repeated three times, and representative results are shown. First, DA rats were immunized with GPBP, and the proliferative responses of lymph node T cells from immunized animals to GPBP peptides including GPBP62–75 and GPBP62–84 were assayed on day 12 postimmunization. As shown in Fig. 1A, T cells from GPBP-immunized animals responded only to GPBP and showed marginal responses to synthetic peptides corresponding to the 43–67, 62–75, 68–88, and 87–100 sequences of the GPBP molecules. When lymph node T cells were stimulated with peptides at a higher concentration (100 µg/ml), similar results were obtained (data not shown). In contrast, lymph node T cells taken from DA rats that had been immunized with GPBP62–75 responded strongly to GPBP62–75 and GPBP62–84, but weakly to the entire GPBP molecule (Fig. 1B). Taken together, the epitopes within the 62–75 and 62–84 sequences of the GPBP molecule are judged to be cryptic, as defined by Sercarz et al. (8).



View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 1. Proliferative responses of lymph node cells taken from GPBP-immunized (A) and GPBP62–75-immunized (B) rats on day 12 postimmunization. Lymph node cells (2 x 105 cells/well) taken from GPBP-immunized (A) or GPBP62–75-immunized (B) rats were cultured with the indicated Ags for 72 h, the last 18 h in the presence of [3H]thymidine. The cells were harvested on glass-fiber filters, and the label uptake was determined using standard liquid scintillation techniques. Each symbol represents the mean values of triplicate assays, and SEs were within 10% of the mean values.

 
Characterization of T cell lines derived from GPBP62–75-immunized rats

To analyze the nature of GPBP62–75-reactive T cells in more detail, we established GPBP62–75-specific TCLs by repeated stimulation (5–10 cycles) of lymph node T cells taken from GPBP62–75-immunized rats with GPBP62–75. It should be noted that the entire GPBP molecule was not used throughout the procedure for TCL establishment. Using these TCLs, we examined their Ag specificity. A representative result is shown in Fig. 2A. Interestingly, one of the GPBP62–75-specific TCLs (DA24) responded vigorously not only to GPBP62–75, but also to the entire GPBP molecule, clearly indicating that GPBP62–75 is an immunodominant epitope, at least for this TCL. Despite this finding, freshly isolated T cells from GPBP-immunized animals responded marginally to GPBP62–75 (Fig. 1). To explain this discrepancy, we tested the possibility that presentation of GPBP62–75 is suppressed by competitive inhibition by other nonencephalitogenic epitopes in the GPBP molecules. Competition assay using GPBP36–58 and GPBP100–120 demonstrated that addition of these peptides did not suppress the proliferation of GPBP62–75 (Fig. 2B). These findings indicate that although GPBP62–75 was judged to be cryptic on the basis of the findings obtained with lymph node T cells, TCLs that had been selected by repeated stimulation with GPBP62–75 also reacted with the whole GPBP molecule.



View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 2. A, Ag specificity of GPBP62–75-specific T line cells. After four rounds of GPBP62–75 stimulation, the proliferative responses of T line cells to a panel of Ags were assayed in the presence of GPBP, GPBP62–75, or GPBP62–84. GPBP62–75-specific T line cells responded not only to GPBP62–75 and GPBP62–84, but also to the entire GPBP molecule. This finding was confirmed using long term cultured TCL (13 rounds). B, Competition assay. GPBP62–75-specific T line cells were cultured with GPBP, GPBP62–75, GPBP36–58, or GPBP100–120 (10–100 µg/ml) or with GPBP62–75 (33 µg/ml) plus various concentrations of GPBP36–58 or GPBP100–120 (10–100 µg/ml). Each symbol represents the mean values of triplicate assays, and SEs were within 10% of the mean values.

 
CDR3 spectratyping analysis of spinal cord T cells during EAE

To identify clonally or oligoclonally expanded T cells in response to immunization with GPBP and GPBP62–75, both of which contain the major encephalitogenic epitope, we screened T cells that infiltrated the spinal cord by CDR3 spectratyping. For the control, we first examined PBL and lymph node T cells taken from normal DA rats and obtained the normal spectratype profile without any spectratype expansion, as seen in normal Lewis rats (12) (data not shown). A representative spectratype profile of spinal cord T cells of rats with severe EAE is shown in Fig. 3, and the results are summarized in Table II. When GPBP62–75 was used for immunization, spinal cord T cells showed spectratype expansion of V{beta}s including V{beta}7, V{beta}10, and V{beta}15 (indicated by arrows in Fig. 3A). All rats immunized with this Ag showed essentially the same results (Table II). In GPBP62–84-immunized rats, spectratype expansion of a limited number of V{beta}s became less obvious. Although V{beta}15 expansion was observed in two of three rats, other spectratype expansion found in GPBP62–75-immunized rats was not detectable in GPBP62–84-immunized rats (Table II). In rats immunized with the entire GPBP molecule, V{beta}s showing spectratype expansion (arrowheads) became more diversified, and it was difficult to identify Ag-specific spectratype expansion (Fig. 3B and Table II). Of note was that the findings obtained by TCR analysis in DA rats were completely different from those obtained in Lewis rats. In the latter case, V{beta}8.2 spectratype expansion was easily detectable throughout the course of EAE induced by immunization with GPBP and GPBP68–88, which contains the immunodominant epitope for Lewis rats (4, 20).



View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 3. CDR3 spectratyping of spinal cord T cells from DA rats immunized with GPBP62–75 (A) and GPBP (B) emulsified in CFA. Three rats were examined in each group. The spectratype expansion found in all rats examined is indicated by arrows, and that found in some rats is indicated by arrowheads (see also Table II). It was previously demonstrated that T cells in DA rats express not only V{beta}8.2, but also functional V{beta}8.4, which is highly homologous to V{beta}8.2 (35 ).

 

View this table:
[in this window]
[in a new window]
  		Table II. Summary of CDR3 spectratyping analysis in DA rats with clinical EAEa

 
Nucleotide and amino acid sequences of the CDR3 region of activated TCR

Since CDR3 spectratyping analysis revealed that V{beta}7, V{beta}10, and V{beta}15 spectratypes were oligoclonally expanded in all GPBP62–75-immunized animals, we decided to determine the nucleotide and amino acid sequences of the CDR3 region of TCR clones derived from expanded spectratypes to clarify whether expansion of a certain spectratype represents clonal expansion. The results are summarized in Table III. In TCR clones derived from V{beta}7 spectratypes showing expansion (n = 3), there was one predominant CDR3 sequence in each case. However, the length of the CDR3 region was different (3284, 3285, and 3286 had 10, 8, and 9 aa, respectively), indicating that clonal expansion of the V{beta}7 spectratype varies from case to case (Table IIIA). With regard to the V{beta}10 spectratype, all the expanded spectratypes showed the same length of CDR3 region (8 aa), and two of three samples had a common motif in the D region. As shown in Table IIIB, the majority of TCR clones possessed serine at the second position of the CDR3 region, and tyrosine or phenylalanine was frequently used at the first position. In expanded V{beta}15 spectratypes, the identical CDR3 sequence, VNERLFF, was found in all cases examined and was the most predominant in two of three cases (Table IIIC). These findings suggest that V{beta}10 and V{beta}15 spectratype expansion represents clonal expansion of T cells bearing pathogenic TCR.


View this table:
[in this window]
[in a new window]
  		Table III. Amino acid sequence of the CDR3 region of V{beta}7 (A), V{beta}10 (B), and V{beta}15 (C) TCR extracted from the spectratypes of spinal cord (SC) T cells from rats with EAEa

 
Adoptive transfer of V{beta}10-positive T cells and TCR-based DNA vaccination suggest that V{beta}10 and V{beta}15 are pathogenic TCRs in GPBP62–75-induced EAE

CDR3 spectratyping and subsequent sequencing of the CDR3 region suggested that V{beta}15-positive and, to a lesser extent, V{beta}10-positive cells are pathogenic T cells in GPBP62–75-induced EAE. To identify pathogenic TCRs in GPBP62–75-induced EAE, we took two different approaches. The first was adoptive transfer experiments using TCLs in which V{beta}10-positive T cells were enriched by positive selection using anti-V{beta}10 mAb-conjugated magnetic beads. FACS analysis revealed that ~70% of cells are V{beta}10 positive (not shown). Intravenous injection of V{beta}10-positive cell-enriched TCL at a dose of 5 x 106 cells induced clinical EAE in all recipients (n = 4), with a mean maximal clinical score of 1.5 ± 0.4, suggesting that V{beta}10-positive T cells are encephalitogenic. We were unable to enrich the V{beta}15-positive T cell population because anti-rat V{beta}15 mAb is not available at present.

The second approach was TCR-based DNA vaccine therapy. We reasoned that if V{beta}15-positive and/or V{beta}10-positive T cells are pathogenic T cells in GPBP62–75-induced EAE, then the regulatory immune responses induced by DNA vaccines encoding V{beta}15 or V{beta}10 genes could prevent or ameliorate the development of EAE, as shown in a previous study (21). The results are summarized in Table IV. Injection of the V{beta}15 DNA vaccine clearly ameliorated the severity of EAE. The clinical severities determined by the maximal clinical score of rats with V{beta}15 DNA vaccine (Table IV, group B) were significantly milder than those of empty vaccine controls (group C). V{beta}10 vaccine also possessed suppressive effects, although its effects were slightly weaker than those of the V{beta}15 DNA vaccine (group A). Furthermore, combination therapy with V{beta}10 and V{beta}15 vaccines completely suppressed the development of EAE in two of six rats, and the mean maximal clinical score of symptomatic rats was significantly milder than that of empty vaccine controls (group C vs group D). The clinical severities of empty vaccine controls were essentially the same as those of unpretreated controls (group D vs group E), indicating that injection of plasmids does not modulate the clinical course of EAE, at least in this experimental model.


View this table:
[in this window]
[in a new window]
  		Table IV. TCR-based DNA vaccine therapy for GPBP62-75-induced EAEa

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we induced EAE in DA rats by immunization with the whole GPBP molecule and its fragment peptides emulsified with CFA and i.p. injection of PT. The use of PT is not usual in DA EAE. However, PT is constantly used in the mouse EAE system, because immunization with Ag/CFA alone induces only mild EAE in a small number of animals. Furthermore, PT was also used in the rat EAE system to identify cryptic, but encephalitogenic, epitopes (22). We used PT in our study for the same reason as in the mouse system, as shown in Table I. We believe that PT does not affect the immunodominancy and encephalitogenicity of peptides and the T cell repertoire because 1) the encephalitogenicity of the peptides shown in this study is essentially the same as that reported previously; and 2) CDR3 spectratyping analysis revealed that the TCR repertoire of lymph node T cells from PT-injected rats is almost the same as that of lymph node T cells from PT-noninjected rats (23) (our unpublished observation in DA rats). Consequently, it was demonstrated that compared with Lewis rats, DA rats exhibit quite different immune responses upon immunization with GPBP. The differences between DA and Lewis rats are summarized in Table V.


View this table:
[in this window]
[in a new window]
  		Table V. Comparison of the nature of encephalitogenic T cells in DA and Lewis rats

 
First, the major encephalitogenic epitope(s) for DA rats resides within the 62–75 sequence of the GPBP molecule, whereas GPBP66–88 is highly encephalitogenic to Lewis rats (Table VA). This difference may be attributable to the difference in the structure of the peptide/MHC complex between DA and Lewis rats. Each peptide/MHC combination in each strain may stimulate pathogenic T cells more efficiently.

The second and most interesting finding was obtained in the analysis of the Ag specificity and encephalitogenicity of MBP peptides in DA rats. As shown in Table I, immunization with GPBP62–75 constantly induced severe EAE in DA rats, but the proliferative responses of lymph node T cells from GPBP62–75-immunized animals to GPBP were marginal. Conversely, lymph node T cells taken from GPBP-immunized rats did not respond to GPBP62–75 (Fig. 1). These findings indicate that GPBP62–75 is only one major encephalitogenic peptide, but contains a cryptic epitope. The Ag specificity of DA rats shown in this study is in sharp contrast to that found in Lewis rats in which GPBP68–88 is an encephalitogenic and, at the same time, immunodominant epitope, as evidenced by the finding that immunization of Lewis rats with the whole GPBP molecule induces strong responses of lymph node T cells to the 68–88 sequence (24). Interestingly, GPBP62–75-specific TCLs established from GPBP62–75-immunized animals responded to both GPBP and GPBP62–75. One of the explanations for this finding is that the frequency of GPBP62–75-reactive T cells in DA rats appears to be low compared with that of GPBP68–88-reactive T cells in Lewis rats. In the previous study (6) it was reported that the 31–50 and 41–60 sequences of MBP induced vigorous responses of lymph node T cells. In contrast, we observed the marginal responses using GPBP36–58 and GPBP43–67. Although the precise reasons remain unclear, the differences in the substrain and/or peptides used may be attributable to this difference.

It is well known that cryptic epitopes induce organ-specific autoimmune diseases such as encephalomyelitis (22) and uveoretinitis (25). Since these cryptic epitopes are able to induce T cell-mediated autoimmune diseases, the epitopes must be presented in vivo to epitope-specific pathogenic T cells. Lipham et al. (25) suggested that cryptic epitopes defined by the in vitro assay are processed in vivo by extracellular endoproteases and presented to responding T cells. Moreover, Viner et al. (26) observed that a cryptic epitope defined in a similar way was predominantly presented in vivo on the MHC class II molecules. The present and previous studies imply that the immunodominancy and cripticity of a certain peptide defined by in vitro assays do not always associate with its autoimmune disease-inducing ability. However, the immunodominancy of autoimmune disease-inducing Ags may play an important role in the formation of the TCR repertoire of pathogenic T cells (see below).

Finally, it was shown that the TCR repertoire of GPBP- and GPBP peptide-immunized DA rats determined by CDR3 spectratyping was different from that of Lewis rats. While oligoclonal expansion of the V{beta}8.2 spectratype was found throughout the course of GPBP and GPBP68–88-induced EAE in Lewis rats (4, 20), the spectratype pattern in DA rats immunized with GPBP varied from case to case (Tables II and VC). In contrast, V{beta}10 and V{beta}15 spectratype expansion was always found in GPBP62–75-immunized DA rats. To demonstrate that these T cells are encephalitogenic in GPBP62–75-induced EAE, we took two different approaches. First, we have shown that injection of the V{beta}10-positive T line cells induced clinical EAE in recipients. Second, vaccination with V{beta}15 and V{beta}10 DNA, alone or in combination, ameliorated the clinical severities of GPBP62–75-induced EAE. These findings strongly suggest that V{beta}10 and V{beta}15 are pathogenic TCRs. However, the suppressive effects of DNA vaccination in DA rats were slightly milder than those found in Lewis rats. In the latter case, 50–70% of rats pretreated with DNA vaccine encoding the V{beta}8.2 gene that is mainly used by Lewis encephalitogenic T cells did not develop clinical EAE upon challenge with GPBP (Y. Matsumoto et al., manuscript in preparation). Differences in the immunodominancy of encephalitogenic epitopes in the GPBP molecule and the degree of the diversity of the TCR usage by encephalitogenic T cells may be attributable to the difference in the outcome of the therapy.

Little is known about the relationship between the presence or the absence of the immunodominant epitope and the TCR repertoire formation in organ-specific autoimmune diseases. However, information about this issue is currently accumulating. Kääb et al. (27, 28) reported using rat SCID chimeras that MBP-specific T cells in SCID mice transferred with T precursor cells from the fetal liver of Lewis rats along with Lewis thymuses frequently use V{beta}8.2 and have the short CDR3 region in the TCR {beta}-chain, whereas in SCID mice reconstituted with rat fetal liver cells alone, MBP-specific T cells use a random repertoire of the V{beta} gene with the long CDR3. This finding clearly indicates that the thymus plays a central role in the formation of the MBP-specific TCR repertoire in Lewis rats. Moreover, recent analyses using mutant mice lacking MBP (29, 30) and mice with proteolipid protein-induced EAE (31, 32) have revealed that thymic expression of myelin autoantigens deletes T cells responding to the immunodominant epitope of the autoantigen and inversely correlates with the susceptibility to EAE. Other studies also demonstrated that increased thymic expression of autoantigens correlates with resistance to autoimmune diseases (33, 34). Investigations of MBP expression in the thymus of DA and Lewis rats are currently underway in our laboratory.

In summary, we demonstrate in this study that in contrast to Lewis rats, DA rats have a less immunodominant, but encephalitogenic, epitope in the MBP molecule and display a more diversified TCR repertoire. Interestingly, however, TCR-based immunotherapies were effective to some extent under certain conditions. Analysis of the Ag specificity and TCR repertoire of animal models and patients with organ-specific autoimmune diseases provides useful information for developing specific immunotherapies.


   Acknowledgments
 
We thank Dr. N. Tanuma for critical reading of the manuscript, and Y. Kawazoe and K. Kohyama for technical assistance.


   Footnotes
 
1 This work was supported in part by Grants-in-Aid from the Ministry of Education, Japan. Back

2 Address correspondence and reprint requests to Dr. Yoh Matsumoto, Department of Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Musashidai 2-6 Fuchu, Tokyo 183-8526, Japan. E-mail address: matyoh{at}tmin.ac.jp Back

3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; CDR3, complementarity-determining region 3; MBP, myelin basic protein; GPBP, guinea pig MBP; PT, pertussis toxin; TCL, T cell line. Back

Received for publication November 7, 2002. Accepted for publication April 25, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Burns, F. R., X. Li, N. Shen, H. Offner, Y. K. Chou, A. A. Vandenbark, E. Heber-Katz. 1989. Both rat and mouse T cell receptors specific for the encephalitogenic determinant of myelin basic protein use similar V{alpha} and V{beta} chain genes even though the major histocompatibility complex and encephalitogenic determinants being recognized are different. J. Exp. Med. 169:27.[Abstract/Free Full Text]
  2. Gold, D. P., H. Offner, D. Sun, S. Wiley, A. A. Vandenbark, D. B. Wilson. 1991. Analysis of T cell receptor {beta} chains in Lewis rats with experimental allergic encephalomyelitis: conserved complementarity determining region 3. J. Exp. Med. 174:1467.[Abstract/Free Full Text]
  3. Zhang, X., E. Heber-Katz. 1992. T cell receptor sequences from encephalitogenic T cells in adult Lewis rats suggest an early ontogenic origin. J. Immunol. 148:746.[Abstract]
  4. Kim, G., N. Tanuma, T. Kojima, K. Kohyama, Y. Suzuki, Y. Kawazoe, Y. Matsumoto. 1998. CDR3 size spectratyping and sequencing of spectratype-derived T cell receptor of spinal cord T cells in autoimmune encephalomyelitis. J. Immunol. 160:509.[Abstract/Free Full Text]
  5. Kim, G., K. Kohyama, N. Tanuma, H. Arimoto, Y. Matsumoto. 1998. Persistent expression of autoimmune encephalomyelitis (EAE)-specific V{beta}8.2 TCR spectratype in the central nervous system of rats with chronic relapsing EAE. J. Immunol. 161:6993.[Abstract/Free Full Text]
  6. Stepaniak, J. A., N. A. Wolf, D. Sun, R. H. Swanborg. 1997. Interstrain variability of autoimmune encephalomyelitis in rats: multiple encephalitogenic myelin basic protein epitopes for DA rats. J. Neuroimmunol. 78:79.[Medline]
  7. Lenz, D. C., N. A. Wolf, R. H. Swanborg. 1999. Strain variation in autoimmunity: attempted tolerization of DA rats results in the induction of experimental autoimmune encephalomyelitis. J. Immunol. 163:1763.[Abstract/Free Full Text]
  8. Sercarz, E. E., P. L. Lehmann, A. Ametani, G. Benichou, A. Miller, K. Moudgil. 1993. Dominance and crypticity of T cell antigenic determinants. Annu. Rev. Immunol. 11:729.[Medline]
  9. Deibler, G. E., R. E. Martenson, M. W. Kies. 1972. Large scale preparation of myelin basic protein from central nervous tissue of several mammalian species. Prep. Biochem. 2:139.[Medline]
  10. Matsumoto, Y., M. Fujiwara. 1987. The immunopathology of adoptively transferred experimental allergic encephalomyelitis (EAE) in Lewis rats. I. Immunohistochemical examination of developing lesion of EAE. J. Neurol. Sci. 77:35.[Medline]
  11. Tsuchida, M., Y. Matsumoto, H. Hirahara, H. Hanawa, K. Tomiyama, T. Abo. 1993. Preferential distribution of V{beta}8.2-positive cells in the central nervous system of rats with myelin basic protein-induced autoimmune encephalomyelitis. Eur. J. Immunol. 23:2399.[Medline]
  12. Kim, G., K. Kohyama, N. Tanuma, Y. Matsumoto. 1998. Diagnosis and assessment of preclinical and clinical autoimmune encephalomyelitis using peripheral blood TCR. Eur. J. Immunol. 28:2751.[Medline]
  13. Rock, E. P., P. R. Sibbald, M. M. Davis, Y.-H. Chien. 1994. CDR3 length in antigen-specific immune receptors. J. Exp. Med. 179:323.[Abstract/Free Full Text]
  14. Waisman, A., P. Ruiz, D. L. Hirschberg, A. Gelman, J. R. Oksenberg, S. Brocke, F. Mor, I. R. Cohen, L. Steinman. 1996. Suppressive vaccination with DNA encoding a variable region gene of the T-cell receptor prevents autoimmune encephalomyelitis and activates Th2 immunity. Nat. Med. 2:899.[Medline]
  15. Lorentzen, J. C., S. Issazadeh, M. Storch, M. I. Mustafa, H. Lassman, C. Linington, L. Klareskog, T. Olsson. 1995. Protracted, relapsing and demyelinating experimental autoimmune encephalomyelitis in DA rats immunized with syngeneic spinal cord and incomplete Freund’s adjuvant. J. Neuroimmunol. 63:193.[Medline]
  16. Diab, A., J. Zhu, B. G. Xiao, M. Mustafa, H. Link. 1997. High IL-6 and low IL-10 in the central nervous system are associated with protracted relapsing EAE in DA rats. J. Neuropathol. Exp. Neurol. 56:541.[Medline]
  17. Stepaniak, J. A., K. E. Gould, D. Sun, R. H. Swanborg. 1995. A comparative study of experimental autoimmune encephalomyelitis in Lewis and DA rats. J. Immunol. 155:2762.[Abstract]
  18. Bouwer, H. G. A., D. J. Hinrichs. 1997. Experimental allergic encephalomyelitis and vaccination-induced resistance in DA rats. Cell. Immunol. 175:92.[Medline]
  19. Smeltz, R. B., N. A. Wolf, R. H. Swanborg. 1998. Delineation of two encephalitogenic myelin basic protein epitopes for DA rats. J. Neuroimmunol. 87:43.[Medline]
  20. Matsumoto, Y., Y. Jee, G. Kim, N. Tanuma. 2000. Fine TCR repertoire analysis of spinal cord T cells responding to the major and minor epitopes of myelin basic protein during rat autoimmune encephalomyelitis. J. Neurosci. Res. 59:145.[Medline]
  21. Matsumoto, Y., Y. Jee, M. Sugisaki. 2000. Successful TCR-based immunotherapy for autoimmune myocarditis with DNA vaccines after rapid identification of pathogenic TCR. J. Immunol. 164:2248.[Abstract/Free Full Text]
  22. Mor, E., I. R. Cohen. 1995. Pathogenicity of T cells responsive to diverse cryptic epitopes of myelin basic protein in the Lewis rat. J. Immunol. 155:3693.[Abstract]
  23. Jee, Y., Y. Matsumoto. 2001. Two-step activation of T cells, clonal expansion and subsequent Th1 cytokine production, is essential for the development of clinical autoimmune encephalomyelitis. Eur. J. Immunol. 31:1800.[Medline]
  24. Matsumoto, Y., K. Kawai, M. Fujiwara. 1990. Analysis of the T cell repertoire for myelin basic protein in thymus-grafted and other types of chimera: evidence that major histocompatibility complex molecules on accessory cells rather than T cell specificity mainly regulate susceptibility to autoimmune encephalomyelitis. Eur. J. Immunol. 20:2119.[Medline]
  25. Lipham, W. J., T. M. Redmond, H. Takahashi, J. A. Berzofsky, B. Wiggert, G. J. Chder, I. Gery. 1991. Recognition of peptides that are immunopathogenic but cryptic: mechanisms that allow lymphocytes sensitized against cryptic peptides to initiate pathogenic autoimmune processes. J. Immunol. 146:3757.[Abstract]
  26. Viner, N. J., C. A. Nelson, E. R. Unanue. 1995. Identification of a major I-Ek-restricted determinant of hen egg lysozyme: limitation of lymph node proliferation studies in defining immunodominance and crypticity. Proc. Natl. Acad. Sci. USA 92:2214.[Abstract/Free Full Text]
  27. Kääb, G., G. Brandl, A. Marx, H. Wekerle, M. Brandl. 1996. The myelin basic protein-specific T cell repertoire in (transgenic) Lewis rat/SCID mouse chimeras: preferential V{beta}8.2 T cell receptor usage depends on an intact Lewis thymic microenvironment. Eur. J. Immunol. 26:981.[Medline]
  28. Kääb, G., I. Haarmann, H. Wekerle, M. Bradl. 1998. The myelin basic protein-specific T cell repertoire in Lewis rats: T cell receptor diversity is influenced both by intrathymic milieu and by extrathymic peptide presentation. Eur. J. Immunol. 28:1499.[Medline]
  29. Targoni, O. S., P. V. Lehmann. 1998. Endogenous myelin basic protein inactivates the high avidity T cell repertoire. J. Exp. Med. 187:2055.[Abstract/Free Full Text]
  30. Harrington, C. J., A. Paez, T. Hunkapiller, V. Mannikko, T. Brabb, M. Ahearn, C. Beeson, J. Goverman. 1998. Differential tolerance is induced in T cells recognizing distinct epitopes in myelin basic protein. Immunity 8:571.[Medline]
  31. Klein, L., M. Klugmann, K. Nave, V. K. Tuohy, B. Kyewski. 2000. Shaping of the autoreactive T-cell repertoire by a splice variant of self protein expressed in thymic epithelial cells. Nat. Med. 6:56.[Medline]
  32. Anderson, A. C., L. B. Nicholson, K. L. Legge, V. Turchin, H. Zaghouani, V. K. Kuchroo. 2000. High frequency of autoreactive myelin proteolipid protein-specific T cells in the periphery of naive mice: mechanisms of selection of the self-reactive repertoire. J. Exp. Med. 191:761.[Abstract/Free Full Text]
  33. Egwuagu, C. E., P. Charukamnoetkanok, I. Gery. 1997. Thymic expression of autoantigens correlates with resistance to autoimmune disease. J. Immunol. 159:3109.[Abstract]
  34. Liu, H., A. J. MacKenzie-Graham, S. Kim, R. R. Voskuhl. 2001. Mice resistant to experimental autoimmune encephalomyelitis have increased thymic expression of myelin basic protein and increased MBP specific T cell tolerance. J. Neuroimmunol. 115:118.[Medline]
  35. Asmuss, A., K. Hofmann, T. Hochgrebe, G. Giegerich, T. Huenig, T. Herrmann. 1996. Alleles of highly homologous rat T cell receptor {beta}-chain variable segments 8.2 and 8.4: strain-specific expression, reactivity to superantigens, and binding of the mAb R78. J. Immunol. 157:4436.[Abstract]



This article has been cited by other articles:


Home page
 Infect. Immun.Home page
D. Gorton, B. Govan, C. Olive, and N. Ketheesan
B- and T-Cell Responses in Group A Streptococcus M-Protein- or Peptide-Induced Experimental Carditis
Infect. Immun., May 1, 2009; 77(5): 2177 - 2183.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Miyakoshi, A.
Right arrow Articles by Matsumoto, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Miyakoshi, A.
Right arrow Articles by Matsumoto, Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS