Vaccination with an immunodominant peptide of bovine type II collagen induces an anti-TCR response, and modulates the onset and severity of collagen-induced arthritis

Aki Honda1,2,3,5, Akio Ametani1,5, Takashi Matsumoto1,4, Amane Iwaya1, Hiroshi Kano1, Satoshi Hachimura1, Kensuke Ohkawa1, Shucihi Kaminogawa1, Koji Suzuki2,3, Eli E. Sercarz5 and Vipin Kumar5

1 Applied Biological Chemistry, University of Tokyo, Bunkyo, Tokyo 113-8650, Japan 2 Applied Chemistry, Keio University, Yokohama, Kanagawa 223-8582, Japan 3 Japan Science and Technology Agency, CREST, Kawaguchi, Saitama 332-0012, Japan 4 Research and Development Center, Nippon Meat Packers, Tsukuba, Ibaraki 300-2646, Japan 5 Immune Regulation, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121, USA

Correspondence to: A. Honda; E-mail: aki{at}educ.cc.keio.ac.jp
Transmitting editor: K. Okumura


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
T cell responses directed toward TCR-derived peptides have been shown to be an important regulatory mechanism of protection against autoimmunity. Here, we show that a naturally induced TCR-directed immune response can delay the onset of collagen-induced arthritis (CIA), an animal model of autoimmune rheumatoid arthritis in humans. DBA/1 mice were pretreated with an immunodominant peptide, p245–270, from bovine type II collagen (bCII) and were subsequently immunized with whole bCII for the induction of arthritis. The results showed that preactivation of p245–270-reactive cells delayed the onset and reduced the severity of CIA, compared with animals in the control group. Interestingly, the serum antibody response to bCII and the bCII-specific cytokine were not affected under these conditions. This result indicates that the observed protection was neither directly due to a lower antibody response nor due to the immune deviation of the anti-bCII T cell response. Furthermore, immunization with p245–270, but not bCII, induced a strong response to the B5 peptide, an immunodominant region of the TCR Vß8.2 (amino acids 76–101) that binds very strongly to I-Aq. These data suggest that at a critical phase in the loss of self-tolerance, an effective anti-TCR response, induced naturally, can regulate the pathogenic autoimmune response and thus may provide protection against autoimmunity.

Keywords: anti-TCR response, collagen-induced arthritis, immune regulation


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Collagen-induced arthritis (CIA) is a disease model of human autoimmune rheumatoid arthritis and can be induced by injection of susceptible animals with heterologous type II collagen (CII) in adjuvant. Both MHC and non-MHC genes are involved in the susceptibility to CIA, and H-2q and H-2r mice are the most susceptible haplotypes (1). As in most autoimmune disease models, autoreactive T cells are essential in CIA. CII-reactive CD4+ T cells, derived from DBA/1 mice, were shown to develop attenuated CIA by passive transfer (2,3). Also, by immunohistological techniques, CD4+ and IL-2 receptor-expressing T lymphocytes were regularly detected in the affected joints (4). Antibody to collagen molecules can also transfer attenuated disease (5), indicating that a humoral response to CII is also involved in this disease. Early studies of Osman et al. showed that CII-reactive T cell hybridomas in DBA/1LacJ mice preferentially use the TCR Vß8.2 gene segment (6). Thus, anti-Vß8.2 antibody treatment also resulted in a significant reduction in the incidence of arthritis in DBA/1LacJ mice.

Self-reactive T cells bearing the Vß8.2 gene segment are often utilized in experimental autoimmune disease models, such as experimental autoimmune encephalomyelitis (EAE) (7) or diabetes in non-obese diabetic (NOD) mice (8). In earlier studies it was shown that natural remission of EAE was accompanied by an immune response against the TCR peptide from the Vß8.2 chain (9). Furthermore, we investigated the T cell proliferative response against various TCR Vß8.2-derived peptides and found that the immune response against the B5 peptide corresponding to residues 76–101 of the TCR Vß8.2 chain was spontaneously induced in EAE-susceptible mice (10). We found that CD4+ regulatory T cells that recognize B5 and CD8+ regulatory T cells that were reactive to a different Vß8.2 determinant from the CDR1/2 region (corresponding to 41–50) controlled the disease. Furthermore, we found that injection of B5 peptide has a suppressive effect against CIA (11) as well as EAE. In this study we show that CIA was significantly delayed and inhibited by the injection of p245–270, an immunodominant determinant of bovine CII (bCII) in DBA/1 mice, through activation of the B5-specific regulatory T cells. Notably, the B5 peptide binds to the I-Aq molecule with a high binding affinity. These data suggest that during an autoimmune response to self-CII, feedback TCR–peptide-reactive regulatory T cell responses are also induced which are involved in the maintenance of peripheral self-tolerance.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Female DBA/1LacJ and DBA/1JNCrj (referred to as DBA/1J) mice (6–8 weeks old) were obtained from the Jackson Laboratory (Bar Harbor, ME) and Charles River Japan (Yokohama, Japan) respectively.

Protein and peptide
bCII used for experiments with DBA/1LacJ mice was purchased from the Institute Jacques (Paris, France). bCII used for experiments with DBA/1J was purified by the salting-out technique after the digestion of bovine joint cartilage with pepsin. Its purity was confirmed by SDS–PAGE analysis. B1 (amino acid 1–30 with an additional C-terminal leucine) and B5 (amino acids 76–101) peptides from mouse TCR Vß8.2, chain, hen egg lysozyme (HEL) 74–90 and ß-Lg119–133 were synthesized by Dr S. Horvath at the California Institute of Technology (Pasadena, CA) and purified by reversed-phase HPLC, as described earlier (12). Peptides of residues 245–270 (p245–270) and 316–333 (p316–333) from bCII were synthesized with a 430A machine (Applied Biosystems, Foster City, CA), and purified with reversed-phase HPLC.

The sequences of peptides used in this study were: p245–270 (bCII), ATGPLGPKGQTGEPGIAGFKGEQGPK; p316–333 (bCII), GFBGADGIAGPKGPBGER; B1 (TCRVß8.2 1–30L), EAAVTQSPRNKVAVTGGKVTLSCNQTNNHNL; B5 (TCRVß8.2 76–101), LILELATPSQTSVYFCASGDAGGGYE; HEL74–90, NLCNIPCSALLSSDITA; HEL93–113, NCAKKIVSDGNGM NAWVAWRN; ß-Lg119–133, CQCLVRTPEVDDEAL.

CIA induction
Ten DBA/1J mice in each group were injected intradermally at the base of the tail with 100 µl of an emulsion containing (i) bCII p245–270 (15 µg/mouse) plus complete Freund’s adjuvant (CFA; Difco, Detroit, MI), (ii) bCII p316–333 (10 µg/mouse) plus CFA, (iii) PBS/CFA or (iv) nothing, and 14 days later they were further injected with bCII (100 µg/mouse) plus incomplete Freund’s adjuvant (IFA; Difco). The peptides and bCII were dissolved in PBS and 0.06% acetic acid respectively, and emulsified with the same volume of adjuvant. The clinical severity of the arthritis was assessed according to an arthritis scale for each limb, which was subjectively graded on a scale of 0–3: 0 = absence of arthritis, 1 = one finger swelling or mild swelling, 2 = two fingers swelling or swelling of tarsus and ankle, and 3 = hard swelling or bony deformity. A sum of the scale for four paws of a single mouse was calculated and a total of this sum in a group of mice was obtained. The arthritic index was defined by dividing this total by the number of mice in the group. Blood was taken every 10 days during the observation period to measure the antibody in the serum. Ten mice in each group were injected with bCII p245–270/CFA, bCII p316–333/CFA, PBS/CFA or nothing and then all the mice were s.c. immunized with bCII for the induction of the disease. We determined the severity of the arthritis by calculating the arthritic index for these four groups of mice (Fig. 1). We also compared the incidence of arthritis by calculating the percentages of arthritic mice and arthritic legs in each group. A mouse was regarded as arthritic when an individual had swelling in at least one leg. The percentage of arthritic mice was obtained by dividing the number of arthritic mice by the total number of mice in each group. The percentage of arthritic legs was obtained by dividing the number of arthritic legs by the number of all mice for each group; arthritic legs were those having swelling or deformity.



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Fig. 1. The arthritic index of CIA after preimmunization with bCII-derived peptide. Groups of DBA/1J mice (10 in each) were preimmunized with p245–270 (circles) or p316–333 (inverted triangles) or PBS (diamonds) or were not preimmunized (squares) on day 0. CIA was induced on day 14 by injecting mice with bCII plus IFA, as described in the Methods. The data were analyzed by the Mann–Whitney U-test. *Significant difference at P < 0.05.

 
ELISA for detecting anti-bCII antibody in the serum
The titer of anti-bCII antibody in serum from mice treated for the CIA induction previously described was determined by ELISA. bCII was dissolved in PBS at a concentration of 100 µg/ml by heating; it was incubated in the wells of Maxisorp plates (Nunc, Roskilde, Denmark) overnight at 4°C. The solutions were removed and the wells were washed with PBS-Tween (0.05% Tween 20 in PBS). The serum samples were diluted with PBS-Tween and incubated in the wells for 2 h at room temperature. Bound antibody to the solid phase was detected by two sequential sets of incubation with alkaline phosphatase (ALP)-labeled anti-mouse subclass antibody for 2 h and its substrate of p-nitrophenylphosphate disodium salt for ~30 min after washing the wells with PBS-Tween. The conjugates used here were ALP-labeled anti-mouse IgG1, IgG2a and IgG2b (all from Zymed, South San Francisco, CA). Color development was measured at 405 nm.

Cell preparation and culture for proliferation and cytokine release assay
DBA/1J mice were s.c. injected with bCII p245–270 plus CFA H37Ra, PBS plus CFA or nothing. Seven days following the preinjection, the mice were immunized with 100 µg of bCII plus IFA in the base of the tail or treated with nothing. After 10 days, lymphocytes were removed from popliteal and inguinal lymph nodes and cultured at 5 x 105 in each well of a 96-well plate with 200 µl of RPMI 1640 containing 10% FBS with or without B5 (amino acids 76–101, TCRVß8.2) peptide or p245–270. IFN-{gamma} in the supernatant was determined by sandwich ELISA using R4-6A2 (PharMingen, San Diego, CA) as the first antibody and XMG1.2 (PharMingen) as the second antibody. In other experiments, DBA/1LacJ mice were s.c. vaccinated with p245–270 (10 µg/mouse) plus CFA or PBS plus CFA as controls. Lymphocytes were taken and cultured in the protein-free medium X-vivo 20 (Biowhittaker, Walkersville, MD) with either B5 or B1 (amino acids 1–30, 31L, Vß8.2), or without any antigenic peptide. Three days later, 1 µCi [3H]thymidine was added to the culture and incubated overnight. The incorporation of thymidine into cultured cells was measured by liquid scintillation.

Competitive-binding assay of B5 (amino acids 76–101, TCRVß8.2) to I-Aq molecules
I-Aq molecules were purified from spleens of untreated DBA/1J mice as previously described (13). In brief, splenocytes from DBA/1J mice were solubilized and I-Aq molecules were purified with affinity column using anti-I-Aq antibody M5/114.15.2. Binding assay was performed as previously reported (14). The purified I-Aq molecule (14 nM), 2 µM or absence of biotinylated ß-Lg119–133 with various concentrations of B1, B5, ß-Lg119–133 or without competitor peptide were preincubated in 50 mM citrate/phosphate buffer, pH 5.0 containing 0.2% NP-40 and 2 mM EDTA at 37°C for 48 h. Meanwhile, Nunc Maxisorp plates were coated with anti-rat Ig at room temperature for 2 h and with M5/114.15.2 for another 2 h. Non-specific binding was blocked at 4°C for overnight with 50 mM Tris–HCl, pH 7.5 containing 0.3% BSA and 0.1% Tween 20. The incubated mixtures were transferred to the plate and incubated for 1.5 h at room temperature. Biotinylated peptide bound to the I-Aq molecule was detected with AP–streptavidin (Zymed) by development with its substrate, p-nitrophenylphosphate disodium. Color development was measured at 405 nm.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Suppression of CIA by preimmunization with bCII p245–270
CIA can be induced in H-2q DBA/1 mice by injection with bCII plus adjuvant. In our case, a single injection of 6- to 8-week-old female DBA/1J with bCII plus IFA could efficiently induce CIA with almost 100% incidence. Previous reports (15,16) and our results (data not shown) had indicated that p245–270 contained a dominant T cell determinant, judged from the significant proliferative response to p245–270 in lymph node cells from mice immunized with bCII. DBA/1J mice were preimmunized with p245–270 from bCII plus CFA in order to activate T cells specific for the immunodominant determinant in this region. We studied the effect of this preactivation of the dominant T cell population on the incidence of CIA. The data in Fig. 1 indicate that p245–270 preimmunization delayed the onset of arthritis and reduced the severity of the disease, compared with the other three groups of mice. The percentages of arthritic legs and arthritic mice for the four groups showed a similar pattern to that of the arthritic index. We also tested another schedule for studying the effect of p245–270 preimmunization. Before injection with bCII, the mice were treated twice with p245–270 or other peptides. Also, in this experimental set-up (data not shown), the onset of CIA was much delayed and the severity was reduced compared with mice in the other three groups. When we preinjected mice with p245–270, 35 days before injection with bCII (data not shown), the onset of CIA was also suppressed. However, the efficiency of the delay was less than with other protocols having shorter intervals between the p245–270 and bCII injections.

Suppression of CIA is independent of alterations in the antibody response or immune deviation of the anti-bCII response
We next examined the antibody response to bCII in the serum of individual mice from the four groups. The antibody titer versus the days after the first treatment of mice was plotted. After 21 days from the first treatment, the IgG1 and IgG2a titers to bCII increased. Although the onset and the severity of CIA were altered by bCII p245–270 preimmunization, the IgG1 antibody response to bCII was similar to that of the other three groups of mice (Fig. 2A). The response of IgG2a possessing complement-binding activity has been proposed to be more relevant to CIA than the IgG1 response (17). However, the mice preinjected with p245–270 showed an IgG2a titer specific for bCII that was similar to the other groups (Fig. 2B). These observations indicate that the suppression of CIA by p245–270 preimmunization was independent of the antibody responses to bCII.




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Fig. 2. Antibody responses against bCII. Anti-bCII IgG1 (A) and IgG2a (B) antibody amounts in the serum of individual mice were measured. Mice were the same as those shown in Fig. 1. Each symbol indicates absorbance for each individual mouse by ELISA and the average values for each day are linked with a straight line. Each panel indicates the following group: ‘bCII’ = mice immunized with bCII plus IFA without any preimmunization; ‘PBS + bCII’ = mice preimmunized with PBS plus CFA on day 0 and immunized with bCII on day 21; ‘p316–333 + bCII’ = mice preimmunized with p316–333 on day 0 and immunized with bCII on day 21; ‘p245–270 + bCII’ = mice preimmunized with p245–270 on day 0 and immunized with bCII on day 21.

 
Furthermore, we measured the amount of IFN-{gamma} secreted by T cells specific for p245–270 (Fig. 3). Since the balance of the cytokines produced by Th1/Th2 subsets of the Th cells plays an important role in the development of CIA (18), we considered that the regulation caused by preimmunization with p245–270 might have been due to Th1/Th2 deviation. However, the IFN-{gamma} response against p245–270 was clearly shown in mice preimmunized with p245–270 before the immunization with bCII in a dose-dependent manner. The IFN-{gamma} response against p245–270 could not be detected in mice immunized with bCII without preimmunization with p245–270. A slight response was shown in mice immunized with p245–270 without further immunization. We also tested IL-2 and IL-4 included in the same supernatant with a bioassay. No significant difference between the two groups was apparent (data not shown). These results implied that regulation of CIA by preimmunization with p245–270 was not due to Th1/Th2 deviation.



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Fig. 3. The IFN-{gamma} production of lymphocytes specific for p245–270. The triangles indicate the response of DBA/1J mice immunized with p245–270 on day 0 without further treatment; circles, preimmunized with p245–270 on day 0 and immunized with bCII on day 7; squares, immunized with bCII without preimmunization; diamonds, preimmunized with PBS on day 0 and immunized with bCII on day 7. Lymphocytes were taken on day 17 and cultured with different concentrations of p245–270 or without antigen.

 
We then considered the possibility that preimmunization with p245–270 regulated CIA via the activation of a natural immune regulatory system (11). We have shown earlier that a TCR-specific immune regulatory response inhibited CIA. We set out to examine whether immunization of DBA/1J mice with p245–270 could activate a TCR-specific response.

Immunization of DBA/1J mice with bCII p245–270 induces a T cell response against the dominant TCR peptide (B5: amino acids 76–101, Vß8.2)
Lymphocytes from individual DBA/1J mice immunized with p245–270 or control mice were examined for a TCR-specific proliferative response (Fig. 4A and B). Mice immunized with p245–270 showed proliferative responses to the B5 peptide, but not to the control peptide B1 (amino acids 1–30L, Vß8.2). We found similar data indicating a response to B5 in a repeated experiment using pooled lymphocytes from another set of three mice. Interestingly, lymphocytes from mice injected with PBS plus CFA also proliferated against B5, although the response was much weaker than those from mice immunized with p245–270. This result is consistent with the data in Fig. 1, which showed that PBS plus CFA or control peptide plus CFA delayed the onset of CIA. We also found that splenocytes from some, but not all, individuals showed T cell proliferative responses against B5 even without any treatment (unpublished data). These facts imply that the TCR-specific response represents a physiological immune regulatory system that is very easily triggered. Previous reports suggested that B5-specific CD4+ regulatory T cells are required to produce a Th1 response for effective regulation and prevention of autoimmune disease (19). We measured the B5-specific IFN-{gamma} response of lymphocytes from the mice that were treated with the same schedule as the CIA induction (Fig. 4C). The result indicated that immunization only once with p245–270 induced a significant IFN-{gamma} response to B5 and that preimmunization with p245–270 before immunization with bCII induced the strong IFN-{gamma} response to the B5 peptide, suggesting that immunization with p245–270 before the induction of CIA induced an effective regulatory response. On the other hand, no IFN-{gamma} was detected from other groups, those preinjected with PBS or nothing, before the immunization with bCII.




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Fig. 4. B5-specific proliferative responses of lymphocytes. The assay was started 10 days after immunization of four individual mice with p245–270 (mouse 1: squares; mouse 2: circles) or PBS plus CFA (mouse 3: triangles; mouse 4: diamonds). The cells were incubated with various concentrations of B5 peptide Vß8.2 (amino acids 76–101, TCR Vß8.2) (A) or B1 (amino acids 1–30, L31, TCR Vß8.2) (B). B5-specific IFN-{gamma} production of lymphocytes from DBA/1J mice immunized with p245–270 on day 0 without further treatment (triangles), preimmunized with p245–270 on day 0 and immunized with bCII on day 7 (circles), immunized with bCII without preimmunization (squares) or preimmunized with PBS on day 0 and immunized with bCII on day 7 (diamonds). Lymphocytes were taken on day 17 and cultured with graded concentrations of B5 peptide or without antigen (C).

 
B5 (amino acids 76–101, Vß8.2) peptide binds to MHC class II I-Aq molecules
Next, we investigated whether TCR peptide B5 is able to bind to I-Aq molecules by a competitive-binding assay using the biotinylated ß-Lg119–133 peptide (Fig. 5). In this experiment, increasing amounts of B1 peptide (Vß8.2, amino acids 1–30L), B5 or non-labeled ß-Lg119–133 peptide were added as competitors to the mixture of I-Aq and biotinylated ß-Lg119–133. The result indicated that peptides of B5 and ß-Lg119–133 could bind to I-Aq molecules, while B1 did not (the slight inhibition by B1 peptide is a background effect). It could be shown that B5 and ß-Lg119–133 competed for binding to I-Aq, suggesting that this TCR peptide binds well to I-Aq molecules and, thereby, can be presented to appropriate CD4+ regulatory T cell populations, as has been shown in EAE (12).



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Fig. 5. Competitive-binding assay of I-Aq. The amount of a biotinylated peptide bound to I-Aq in the presence of B1 (circles), B5 (squares), ß-lg119–133 (diamonds), without biotinylated peptide (triangle) or competitor (inverted triangle).

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Regulation of immune responses using peptides derived from the determinant region of CII has been shown as an effective method to control CIA. Development of CIA likely requires responses of both T cells and antibodies. Although p245–270 contains the dominant determinant of CII in H-2q mice (20), the peptide fragment that contains p245–270 cannot induce CIA (21) since it does not contain the appropriate B cell epitope of CII. However, i.v. or neonatal administration of peptide containing this determinant from chicken CII could inhibit CIA in DBA/1 mice (16). It has also been shown that oral administration of human CII or chicken CII determinant peptide inhibited CIA in DBA/1 mice (22). Previously we screened overlapping peptides derived from bCII by intranasal administration and found that the peptide corresponding to 253–272 of bCII could most effectively inhibit CIA (23). In these cases, the administration of peptides reduced either the T cell response or the antibody response against CII or the immunizing peptide due to tolerization of pathogenic lymphocytes.

Administration of peptide with adjuvant is a general protocol for induction of antigen-specific T cells. In this report we induced activation of bCII-reactive T cells by immunization of DBA/1 mice with peptide plus adjuvant (Fig. 3). Thus, regulation of CIA in our case was caused by the activation of regulatory T cells, but not by the induction of tolerance. Several reports showed that regulation of CIA due to tolerization accompanies reduction of the CII-specific IgG2a response (16,24). However, we have shown that bCII-specific IgG1 and IgG2a did not change by immunization with bCII p245–270 (Fig. 2), which also shows that our protocol induced the activation of T cells. CD4+ T cells mainly differentiate into either Th1- or Th2-type effectors. Th1-type cells produce IL-2, IFN-{gamma} and tumor necrosis factor-ß, and support cell-mediated immunity, whereas Th2-type cells produce IL-4, IL-5, IL-6, IL-10 and IL-13, and enhance humoral immune responses (2527). The Th1 response has been considered to play a major role in the pathogenesis of CIA (28). However, the role of IFN-{gamma} may be different at different stages of the diseases, as the administration of IFN-{gamma} can inhibit CIA (29). It is suggested from our data that regulation of CIA through activation of CII-reactive pathogenic T cells by immunization with the dominant CII peptide may not be due to the immune deviation of the anti-bCII response. Notably, the IFN-{gamma} response raised by p245–270 was shown to be increased in the mice preimmunized with p245–270, and yet the disease was significantly delayed and inhibited.

Our data suggest that TCR peptide-reactive regulatory T cells that are naturally induced following expansion of bCII-reactive Vß8.2+ cells are able to inhibit the disease. Osman et al. established T cell clones that were specific for p245–270 from DBA/1 mice for the characterization of the pathogenic TCR repertoire and found that the Vß8.2 gene segment was preferentially utilized (58.3%) (6). It has also been demonstrated that the TCR Vß8.2 gene segment is preferentially utilized in the EAE of B10.PL mice (H-2u) (7), in the uveoretinitis of B10.A (H-2k) mice (30) as well as the EAE of Lewis rats (31). Other studies also implicate the role of Vß8.2+ T cells in the pathogenesis of NOD mice, a model for insulin-dependent juvenile diabetes (32). Both in EAE and CIA, it has been shown that administration of antibodies specific for Vß8.2 significantly protects animals from disease (7,33,34). Therefore, we have examined the possibility that CIA was inhibited by an immune regulatory mechanism involving TCR Vß8.2 determinants.

We have already shown that immunization of DBA/1LacJ mice with B5 peptide, corresponding to amino acids 76–101 of the TCRVß8.2 framework region, results in significant protection of the animals from CIA (11). Response to the B5 peptide was found to be naturally induced during the course of EAE in B10.PL mice. Both CD4+ and CD8+ regulatory T cells were necessary when remission of EAE was to occur. Furthermore, CD4+ regulatory T cells were shown to be specific for the B5 peptide, whereas CD8+ regulatory T cells were specific for a distinct peptide from the CDR1/2 region of the Vß8.2 chain. EAE was inhibited by priming a Th1-type response against B5, whereas EAE was enhanced by Th2-type priming (19). We have hypothesized that both CD4+ and CD8+ regulatory T cells recognize each of the TCR-derived peptides that were processed in professional antigen-presenting cells (APC) through the turnover of effector T cells. IFN-{gamma} secreted by CD4+ regulatory T cells was considered to be necessary for the recruitment/activation of CD8+ regulatory T cells and the consequent inhibition of effector T cells. CD8+ T cell activation probably occurs through a CD40 signal that enhances antigen processing and up-regulates co-stimulatory molecules on APC.

Our results show that immunization of DBA/1J mice with p245–270 induces an anti-B5 response accompanied by IFN-{gamma} secretion. As shown earlier (11), the immunization of DBA/1J mice with B5 down-regulated the immune response against bCII and p245–270. We have also established B5-specific T cell lines from DBA/1 LacJ. Their responses to B5 were inhibited by anti-CD4 or anti-MHC class II antibodies (Koh, unpublished data). We propose that the expansion of Vß8.2+ effectors and their turnover in vivo lead to processing and presentation of a B5-like peptide by APC. It is evident from our data that B5 is likely to be presented by APC, because it displays a strong binding affinity for the I-Aq molecule (Fig. 5). The expansion of effector T cells bearing Vß8.2 was not strong enough to induce effective regulation through bCII immunization; however, it becomes strong when the dominant determinant is used as the immunogen.

Jolly et al. reported that the expression of TCR Vß8.2 from the unrearranged gene in a murine lymphoid precursor cell line is higher than that of other Vß members of the repertoire at an early phase of T cell differentiation (35). It may be possible that Vß8.2 plays an unknown role in the body and the anti-B5 response may be a general regulatory system for the control of the immune response.

In EAE, a B5-specific T cell response was induced during the natural remission of the disease. We induced such a response through the activation of Vß8.2+ T cells specific for the dominant determinant region of the antigen, bCII. Induction of the regulation by activating the dominant determinant-specific response has not been reported by others. Considering both of these cases with EAE and CIA, we can propose that the TCR peptide B5-specific T cell represents an important focus of the immune regulation system. Thus, B5-specific responses could be frequently induced by immune responses towards self-antigen, especially when the Vß8.2 T cells comprise a significant portion of the response.

The inflammation of limbs in mice suffering from CIA usually settles gradually, although they cannot be completely cured due to irreversible bony deformities. However, a reduction in the inflammation is the important aspect for therapy of rheumatoid arthritis. It is possible that eventual reduction in the inflammation is caused by a naturally induced anti-TCR response following immunization with bCII. Immunization with bCII may only have induced a weaker anti-TCR response compared to immunization with p245–270. As a consequence, immunization with bCII induces strong CIA, whereas immunization with p245–270 leads to the regulation of the disease. These data further suggest that the production of the clinical manifestations of autoimmunity depend upon the balance between the pathogenic effector and regulatory populations. Each population undergoes its own development and then interaction will be decisive in determining the clinical outcome, as proposed earlier (36).

Autoimmune rheumatoid arthritis in humans and CIA have several similarities, e.g. susceptibility depends on the genetic background in both diseases. However, common T cell repertoires have not been identified in rheumatoid arthritis patients (37,38) except for a few reports showing a predominant TCR usage of V{alpha}11, V{alpha}14, V{alpha}28 and Vß7, Vß9, Vß17 in the early phase of rheumatoid arthritis or a common usage of the TCR Vß14 in juvenile rheumatoid arthritis (39,40). Some reports have demonstrated successful vaccination with peptides derived from the TCR in rheumatoid arthritis patients (41,42). However, it is difficult to find an effective vaccine region for each patient, because one has to identify the dominant TCR repertoire for each patient. Our vaccination method, which is based upon vaccination with the dominant autoantigenic determinant, is superior in that we do not need to identify the dominant TCR repertoire for each patient. We assume that it is possible to find a functional TCR peptide region corresponding to the immunoregulatory B5 region commonly shared by different strains of mice.


    Acknowledgements
 
This is manuscript #624 from the La Jolla Institute for Allergy and Immunology. We thank Dr Emanual Maverakis for the critical reading of the paper. V. K. is supported by the Arthritis Foundation, the National Multiple Sclerosis Society and the National Institutes of Health.


    Abbreviations
 
ALP—alkaline phosphatase

APC–antigen presenting cell

bCII—bovine type II collagen

CFA—complete Freund’s adjuvant

CIA—collagen-induced arthritis

CII—type II collagen

EAE—experimental autoimmune encephalomyelitis

HEL—hen egg lysozyme

IFA—incomplete Freund’s adjuvant

NOD—non-obese diabetic


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Holmdahl, R., Jansson, L., Andersson, M. and Larsson, E. 1988. Immunogenetics of type II collagen autoimmunity and susceptibility to collagen arthritis. Immunology 65:305.[ISI][Medline]
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