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,
,,,
Departments of
*
Pediatrics and
Medicine, University of Tennessee, Memphis, TN 38163; and
Research Service, Veterans Affairs Medical Center, Memphis, TN 38104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) cytokines. We conclude that the T cell cytokine response
most critical for the induction of CIA is that induced against the
corresponding homologous murine T cell determinant and, further, that
the structural differences between the T cell determinants in CB8 and
-10 are important in breaking self tolerance and inducing autoimmune
response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Structural characteristics of CII required for induction of arthritis have been studied in some detail (5). It is known that maintaining CII in its native form during immunization is the most efficient means of inducing arthritis. In rats, native conformation is required (6). However, in mice, a reasonable incidence of arthritis develops after immunization with denatured CII or with large fragments generated by cyanogen bromide digestion (7, 8). Identification of specific arthritogenic epitopes has been hampered by the failure of short peptides to induce arthritis. However, there is a substantial body of evidence supporting the concept that T cell recognition of specific epitopes is critical.
In DBA/1 mice, such an epitope has been identified in the CB11 fragment (CII124–402) of CII (9, 10). This epitope (CII260–267) has been studied in detail and is both immunodominant and in the context of CB11 is arthritogenic for I-Aq mice. B10.RIII mice (I-Ar) respond to different epitopes. The immunodominant epitope for B10.RIII mice is in CB10 (CII552–897). When B10.RIII mice were immunized with intact CII and when T cell proliferation to CB peptides 8–12, which comprise the bulk of CII, was analyzed, the response to CB10 was greater than those elicited against other peptides (11). Moreover, CB10 induced a more profound suppression when administered i.v. as a tolerogen (11). The immunodominance of CB10 in B10.RIII mice has been confirmed by Nabozny and coworkers (12).
However, our previous work has shown that CIA in B10.RIII mice can be induced with CB8 (CII403–551) but not with other CNBr fragments of CII, including CB10 (8). Thus, the largest CNBr peptide of CII and the one that induced the strongest T cell response in vitro was unable to induce arthritis. These data established that immunogenicity and arthritogenicity were at least partially independent properties. Analysis of the of the immune response to CII by B10.RIII mice using overlapping synthetic peptides identified the core of the major T cell determinant in CB10 to be CII610–618 (GPAGTAGAR) and that in CB8 to be CII445–453 (GPAGPAGER) (8, 11). These epitopes are very similar and differ from each other by only two amino acids located at positions 5 and 8. This similarity provided an opportunity to study the structural characteristics that were responsible for arthritogenicity.
In the experiments reported here, we used recombinant DNA technology to generate rCB8, rCB10, and recombinant peptides with alterations in the amino acid residues within the respective T cell determinant cores that differed at the positions where the natural epitopes differed. Using rCB10, we introduced mutations so that the threonine at residue 614 was converted to proline and the alanine at residue 617 to glutamic acid (rCB10T614P,A617E). The resulting mutant rCB10T614P,A617E contained the CB8 T cell determinant but otherwise retained the structure of CB10. Conversely, we introduced mutations into rCB8 so that the proline residue at 449 was converted to threonine and the glutamic acid at position 452 to alanine (rCB8P449T,E452A). The resulting rCB8P449T,E452A contained the CB10 T cell determinant. We then immunized B10.RIII mice with each of the recombinant peptides to answer the question of whether the CB8 epitope would be arthritogenic in another context, i.e., in CB10.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Details of the preparation of rCB10 and rCB8 are being published separately (13). Briefly, chondrocytes were isolated from fetal bovine articular cartilage, and total RNA was isolated from these chondrocytes and used as a template for first strand cDNA synthesis. RT reaction was performed using an RT-PCR kit (Stratagene, La Jolla, CA) according to the manufacturer’s directions. After the first strand cDNA synthesis, 5 µl of RT reaction mixtures was used to amplify the cDNA encoding the CB8 (CII403–551) and CB10 (CII552–897) regions of CII using primers with the following sequence: CB8, 5'-GGGGCTCGAGGGGTCGTGGGCAGCCT-3' (forward),5'-CCCCCTCGAGTTATCCCCTCTCGCCAGGCAT-3' (reverse); and CB10, 5'-GGGATCCGGAATGCCTGGCGAGAGGGGAGCA-3 (forward) and 5'-GCTCTAGACCATGGGGCCTTGTGCACCAGC-3 (reverse). BamHI and XbaI restriction sites were included in the CB10 primer sequences. A TAG stop codon was also introduced in the reverse primers of both CB8 and CB10. The PCR products for CB8 were directly ligated into the PCR II cloning vector (Invitrogen, Carlsbad, CA). The PCR fragments for CB10 were gel purified, digested with BamHI and XbaI restriction enzymes, and subcloned into the BamHI/XbaI sites of the pUC18 vector. Several clones for both CB8 and CB10 were selected and the inserts were bidirectionally sequenced using a DNA sequencing kit (United States Biochemical, Cleveland, OH).
Mutagenesis was performed by PCR-based site-directed mutation using a
PCR in vitro mutagenesis kit (Takara Shuzo/Oncor, Gaithersburg, MD).
Three oligonucleotide primers were used to introduce point mutations
into the T cell epitope in CB10 cDNA. These changes resulted in the
substitution of amino acid residue 614 from threonine to proline
(T614
P) and residue 617 from alanine to glutamic acid (E617A).
The sequences of the primers were:
CB10T614P, 5'-CCTGCTGGACCTGCTGGTG-3';
CB10A617E, 5'-ACTGCTGGTGAGCGAGGTGC-3'; and
CB10T614P,A617E,
5'-CCTGCTGGACCTGCTGGTGAGCGAGGTGC-3'. Mutant PCR fragments were
amplified from pUC18-CB10 using the combination of primer M13-M4
(supplied in the kit) and one of the CB10 mutant primers, followed by
amplification by M13-M4 and M13-RV. The resultant individual PCR
products were gel purified, mixed in the same molar ration, and
annealed. Heterogeneous double-stranded DNA was completed using
Taq. The second round of PCR was conducted using M13-M4 and
M13-RV primers to amplify the mutated CB10. The cDNAs were digested
with BamHI and HindIII and ligated into the pUC18
vector to obtain pUC18-CB10T614P,
pUC18-CB10A617E and
pUC18-CB10T614P,A617E. Mutagenesis of CB8 was
performed by PCR-based site-directed mutation in a manner identical
with that performed for CB10, using the following primer:
CB8P449T,E452A,
5'-CTCGCCTCGTGCACCAGCAGTTCCAGCAGG-3'.
Expression of rCB10 and rCB8
To express the recombinant proteins, the relevant CB10 cDNA was cut from pUC18 by digestion with BamHI and HindIII. The resulting fragments were subcloned into the pTricHisA vector (Invitrogen) using directional cloning into the multiple cloning site of the vector. Similarly, mutated CB8 PCR fragments were digested with EcoRI, ligated into the same sites of the pTricHisA expression vector (Invitrogen) and sequenced. Top10 hosts (Invitrogen) were transformed with the constructs, and transformants were isolated on selective agar plates. Selected colonies were grown in Luria-Bertani (LB) medium containing ampicillin. The bacteria were expanded, and expression of the relevant proteins was induced with isopropyl ß-D-thiogalactoside (IPTG). The bacterial cells were harvested, supernatants were clarified, and recombinant proteins were purified by Ni-NTA chromatography following the manufacturer’s protocol (Qiagen, Valencia, CA). Fractions were collected and analyzed by 10% SDS-PAGE, and the fractions of interest were pooled, dialyzed against water, lyophilized, and stored at -20°C. Proteins prepared in this manner were tested for endotoxin using a Limulus assay kit (Sigma. St. Louis, Mo.). If endotoxin was present, the proteins were further purified so that the final endotoxin levels did not exceed 3 U/mg protein.
The proteins were separated by 10% SDS-PAGE and visualized by Coomassie brilliant blue staining. For Western blot analysis, the proteins were separated and electrotransferred onto nitrocellulose membranes. The blots were blocked in 5% nonfat milk/TBS-T buffer (20 mM Tris-HCl (pH 7.6)/150 mM NaCl/0.1% Tween 20) for 2 h and incubated with a specific murine anti-CII antiserum (1:1000 dilution). The blots were washed with TBS-T, incubated with a sheep anti-mouse peroxidase-conjugated Ab (Amersham, Piscataway, NJ), and washed as before. Finally, membranes were subjected to enhanced chemiluminescence detection (ECL kit, Amersham) according to the manufacturer’s protocol.
Animals
B10.RIII mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and were maintained in groups of six in polycarbonate cages. They were fed standard rodent chow (Ralston Purina, St. Louis, Mo.) and water ad libitum. The environment was specific pathogen free, and sentinel mice were tested routinely for mouse hepatitis and Sendai viruses.
Immunization
Mice were immunized at 8–12 wk of age, as described previously (14). For routine immunization, all Ags were dissolved in 0.01 N acetic acid at a concentration of 4 mg/ml and emulsified with an equal volume of CFA (12). The resulting emulsion was injected intradermally into the base of the tail. Each mouse received a total volume of 50 µl containing 100 µg of Mycobacterium tuberculosis and 100 µg of Ag.
Beginning 3 wk after immunization, the mice were evaluated for the development of arthritis three times weekly by an observer who was unaware of the treatment groups. Each mouse limb was graded on a scale of 0–4 for the development and severity of arthritis. The grading scale has been described previously in detail (14).
Preparation of control Ags
Native CII was solubilized from fetal bovine articular cartilage by limited proteolysis with pepsin and purified, as described in detail earlier (15). The peptides representing various CII sequences were chemically synthesized by a solid phase procedure using a peptide synthesizer (model 432; Applied Biosystems, Foster City, CA) as described previously (16).
Measurement of Ab levels
Mice were bled at 6 wk after immunization and tested for Abs reactive with native CII using a modification of an ELISA previously described (14). In addition, the profile of epitope specificity of autoreactive anti-mouse CII Abs was analyzed using Mimotope-pin bound peptides as described earlier (17, 18). Murine CII peptides were synthesized as 112 individual peptides spanning murine CB10 using the Mimotope noncleavable pin technology (Chiron Mimotopes PTY, Clayton, Victoria, Australia). This synthesis scheme produced individual 15-mers, each overlapping by 3 residues. ELISA on native mCII was used to standardize Ab titers before analysis on Mimotope pins. ELISA plates were coated by overnight incubation at 4°C with murine CII at 5 µg/ml in phosphate buffer. Both Mimotope pins and standard ELISA plates (Ag) were blocked for 30 min with 2% BSA (Fraction V; Sigma) in ELISA Buffer (1.9 mM NaH2PO4, 8.4 mM Na2HPO4, 500 mM NaCl, 0.1% (v/v) Tween 20 (pH 7.4)). Ags were washed (all washes in ELISA buffer) and incubated at 4°C overnight with affinity-purified Abs. After washing, Ags were incubated for 4 h at 4°C with HRP-labeled goat anti-mouse IgG (Sigma) at a dilution of 1:500. ELISAs were developed with OPD substrate (Sigma) for 30 min, and the reactions were stopped with 2.5 N H2SO4. Abs were removed from the Mimotope blocks according to the manufacturer’s instructions. Results are typical of replicate experiments.
T cell hybridomas and IL-2 production assay
Establishment of T cell hybridomas reactive to bovine CB10 and CB8 was previously described (19). The reaction of the hybridomas was measured by culturing them in RPMI 1640 medium in 96-well microtiter plates using a total volume of 0.3 ml containing 4 x 105 syngeneic spleen cells, 105 of T hybridoma cells, and 100 µg of Ag previously dialyzed against RPMI 1640. The cell cultures were maintained at 37°C in 5% humidified CO2 for 20 to 24 h, after which the supernatants were harvested and seven 80-µl 2-fold serial dilutions were made for determination of IL-2 titers. Four thousand IL-2-dependent cells (HT-2) were added to each supernatant dilution, and, after 16 to 24 h, HT-2 cell viability was evaluated by MTT assay. MTT assay was performed by addition of 10 µl/well of MTT solution (5 mg/ml of MTT in PBS) and incubation in 37°C for an additional 3 h. Color was developed by discarding the cell culture supernatant and adding 200 µl of developing solution (75% isopropanol in 0.02 N HCl) and incubation at 4°C for 3 to 12 h. OD690 was measured in an ELISA reader. IL-2 titers were displayed as the last sample dilution that gave half-maximum activity of the HT-2 cells.
Measurement of T cell cytokines: IFN-, IL-4, and IL-10
Quantitative measurement of murine IFN-, IL-4, and IL-10 was
done using a solid-phase ELISA based on the "sandwich" principle.
Commercially available kits were used (IFN-, Life Technologies,
Gaithersburg, MD; and IL-4 and IL-10, Endogen, Boston, MA). Briefly,
spleens and lymph nodes from B10.RIII mice immunized 10 to 14 days
previously with CII or other Ags emulsified with CFA were individually
minced into single cell suspensions in HBSS and washed three times with
HBSS. Pooled splenocytes and lymph node cells were then adjusted to a
concentration of 5 x 106 cells per ml and
cultured with 100 µg/ml of Ag (synthetic peptides, collagen, or
purified protein derivative (PPD)) in DMEM (Life Technologies, Grand
Island, NY) supplemented with 5% FBS (HyClone Laboratories, Logan,
UT). Supernatants were collected from 72 to 120 h later and either
used fresh or after freezing at -70°C. Supernatant samples were
incubated in microtiter wells coated with a mAb recognizing murine
IFN-, IL-4, or IL-10. Samples were washed, incubated with a
preformed detector complex consisting of a biotinylated second mAb to
the appropriate cytokine and an antibiotin-alkaline phosphatase
conjugate. Absorbance was measured at 405 nm with a spectrophotometer.
A standard curve was obtained by plotting the absorbance vs the
corresponding concentration of the standards. Values are expressed as
pg/ml. Each sample was tested in duplicate wells.
Statistical analysis
The incidence of arthritis in various groups of mice was compared using Fisher’s exact test. Ab levels were compared using the Student t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Each of the recombinant peptides was isolated from bacterial
culture, and its purity was determined by SDS-PAGE and analysis for
endotoxin content. The mutated peptides were identical in m.w. to their
respective wild-type (WT) peptides and appeared as single bands by
SDS-PAGE (data not shown). To ascertain that the resulting T cell
determinants were still functional, we tested each peptide using two T
cell hybridomas generated from B10.RIII mice immunized with intact CII.
Splenocytes from unimmunized B10.RIII mice were used as APCs. Hybridoma
A, which responded to CB10, reacted to the WT rCB10 and to a synthetic
peptide representing the immunodominant epitope CII607–621 but failed
to react with rCB10T614A,A617E. Hybridoma B,
which responded to CB8, reacted with the synthetic peptide CII442–456,
and with rCB10T614A,A617E, but did not react with
rCB10 (Fig. 1
). These data confirmed that
we had successfully introduced the CB8 determinant into the CB10
peptide, in effect replacing the original immunodominant
determinant.
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To determine the ability of recombinant peptides to induce
arthritis, groups of 10–12 B10.RIII mice were immunized with each
peptide. WT rCB10 was incapable of inducing arthritis as we have
previously found for CB10 generated from bovine cartilage. In contrast,
the rCB8 was able to induce arthritis in 4 of 12 immunized mice. Mutant
rCB10T614P,A617E was equally as effective as WT
rCB8. The arthritis that developed in
rCB10T614P,A617E-immunized mice was clinically
severe, with involvement of the ankle, dorsum of the foot, and digits
(Fig. 2). Arthritis was confirmed by
histologic examination of selected joints (Fig. 3).
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To confirm the importance of the two substitutions and to further
analyze the importance of the context of the epitope, we immunized mice
with rCB8P449T,E452A. This peptide is identical
to WT CB8 peptide except for mutations to change residue 449 from a
proline to a threonine and residue 452 from glutamic acid to alanine.
Thus, we generated a mutant rCB8 that had an epitope similar to the
immunodominant epitope of CB10. This peptide was no longer capable of
inducing arthritis when used to immunize B10.RIII mice (Table I).
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Several studies have demonstrated that complement-fixing
autoantibodies to CII are required for the development of arthritis and
that qualitative differences occur in the Ab responses between
susceptible and nonsusceptible strains of mice (18). We
compared the level of Abs generated in response to each of the
recombinant peptides by ELISA. Ab levels were measured using plates
coated with native bovine CII and serum collected 6 wk after
immunization. There was an overall correlation of Ab levels with
arthritis in that all of the arthritogenic peptides also generated high
Ab levels. In contrast, the nonarthritogenic peptides generated lower
levels (Table I
). To compare the topographical distributions of the
autoantibodies binding the CB fragments, mice were immunized with
either rCB10 or mutant rCB10T614P,A617E, and sera
from individual mice were collected, pooled, and selected using an
affinity column specific for native mouse CII. Murine CII-reactive
autoantibodies were then cultured with a panel of overlapping peptides
immobilized on Mimotope pins spanning the sequence of murine CB10.
Interestingly, when sera from mice immunized with mutant
rCB10T614P,A617E or rCB10 were analyzed for the
presence of Ab binding sites, the same topographical distribution of Ab
binding sites was detected (Fig. 4).
These data suggest that the two mutations created within
rCB10T614P,A617E did not cause Ab specificity to
vary from that observed following immunization with rCB10. Although
sera from mice immunized with rCB10T614P,A617E
had a higher titer of Ab recognizing the determinants within the CB10
region, there were no qualitative differences in the epitope
specificity of autoantibodies.
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These data support the concept that there are arthritogenic
epitopes in CII that can be distinguished from immunogenic epitopes. It
is also evident that the primary sequence of the epitope is critical.
We have shown that GPAGPAGER forms the core sequence of an epitope that
is arthritogenic for B10.RIII mice in the context of CB10 or CB8. In
addition, GPAGTAGAR forms the core of an epitope that is immunogenic
but not arthritogenic in identical contexts. T cell cytokine responses
to each determinant remained to be examined. B10.RIII mice were
immunized with bovine CB8, CB10, mutant
rCB10T614P,A617E, or mutant
rCB8P449T,E452A. Spleen and lymph node cells
obtained from these mice were cultured in vitro with synthetic peptides
CII442–456 and CII607–621, and supernatants were collected and
analyzed for the IL-10, IL-4 and IFN- content by ELISA. As shown in
Table II, T cell responses following
immunization with either CB8 or CB10 were predominantly Th1 to the
immunizing determinant, with the greater response occurring to the
nonarthritogenic CB10. On the other hand, immunization with mutant
rCB10T614P,A617E more closely resembled the
pattern observed following immunization with CB8. The response was
predominantly Th1, and a greater response was directed against the CB8
determinant (CII442–456). Th2 cytokine responses, IL-10 and IL-4, were
also greater to the CB8 rather than CB10 determinants although lesser
quantities were produced compared with IFN-. These results suggest
that the new T cell determinant within mutant
rCB10T614P,A617E induces a cytokine profile more
comparable to that generated by CB8 rather than that generated by CB10.
In contrast, immunization with rCB8P449T,E452A
led to cytokine responses more closely resembling the pattern observed
following immunization with CB10.
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) response when cultured with murine
CII442–456 (CB8 determinant). On the other hand, immunization
with CB10 induced only a Th2 response when cultured with murine
CII607–621 (CB10 determinant). Interestingly, the T cell cytokine
response induced following immunization with
rCB10T614P,A617E more closely resembles that
induced by immunization with CB8 in that the predominant cytokine
response is IFN- when cultured with the murine CB8 determinant and
in that greater IFN- and IL-10 responses are generated against the
CB8 than the CB10 determinants. Furthermore, cells from mice immunized
with rCB8P449T,E452A showed a profile resembling
that of nonarthritogenic CB10 (Table III).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using the recombinantly produced peptides, we identified core T cell determinants that differed by only two amino acids but that had profoundly different consequences when used as immunogens. Both were immunogenic, but only one was capable of inducing arthritis. It should be noted that the arthritogenic epitope was not sufficient by itself to induce disease, since immunization with synthetic peptides comprising only that epitope does not cause CIA (Data not shown). However, when expressed as a part of a collagenous peptide, the sequence GPAGPAGER is both necessary and sufficient for the induction of CIA in B10.RIII mice.
We then analyzed the in vitro response of T cells to both epitopes to identify critical differences. Analysis of responses following immunization with CB8, CB10, mutant rCB8P449T,E452A or rCB10T614P,A617E revealed that, when tested using synthetic peptides representing bovine (immunizing) determinants, there were no detectable differences in the cytokines tested. The critical T cell responses following immunization with these peptides appear to be those generated against the homologous murine determinants, rather than to the immunizing determinant.
The murine homologue of the CII610–618 immunodominant determinant clearly binds to the I-Ar molecule and is capable of stimulating T cells primed with bovine CII. The only difference in the sequences of the bovine CII445–453 and the murine CII445–453 is a conservative substitution of a serine (mouse) residue for an alanine (bovine) residue at position 447. This residue has previously been identified as a T cell interaction site in the antigenic peptide, whereas CII610–618 (bovine) and the murine CII610–618 have three differences. These results suggest that the critical response is influenced by the degree of difference between the immunizing Ag and the corresponding murine homologue.
One of the consequences of this altered response is in the level of Ab produced. Each of the arthritogenic peptides induced higher levels of Ab than the nonarthritogenic peptides. The transfer of autoimmune arthritis to naive mice by both polyclonal and mAbs has previously demonstrated the importance of autoantibody production for the initiation of arthritis (16, 17, 18). High levels of circulating autoantibody to murine CII invariably accompany the development of CIA (19). However, the degree of similarity between the autoantibody binding patterns induced by the arthritogenic rCB10T614P,A617E and unmodified rCB10 was unexpected. In recent studies by Brand et al. (18) using DBA/1 mice, a unique subpopulation of autoantibodies that bound to a specific determinant was identified only in CIA-susceptible B10.Q, B10.QßBR, and DBA/1 mice. These data suggested that the number and positions of binding sites available might play an important role in initiating disease. Nevertheless, our present data suggest that immunization with either CB8 or CB10 appears to induce an adequate number of Ab specificities for induction of arthritis. The differences appear to be quantitative rather than qualitative.
T cells can be functionally classified as Th1 or Th2 depending on the
cytokines they produce (24). Th1 cells make predominantly
IL-2, IFN-, and TNF-
and mediate inflammatory reactions
(25). Th2 cells produce IL-4, IL-10, and TGF-ß and
suppress some autoimmune responses (26, 27, 28). In general, a
Th1 response predominates in the development of organ-specific
autoimmune diseases. Our data are consistent with that hypothesis in
that spleen cells from animals given an arthritogenic challenge with
CII consistently produced high levels of IFN-. In addition, when the
nonarthritogenic epitope was analyzed for reactivity with the murine
homologue, a Th2 response predominated. These data show that the change
in only two amino acids caused a shift in the Th1 vs Th2 cytokine
profile. Similar alterations in Th profile due to alterations in
peptide ligand have been shown in the autoimmune disease experimental
autoimmune encephalomyelitis (29).
Our data do not address the question of whether the most important response is a proinflammatory Th1 response to the CB8 epitope or an antiinflammatory Th2 response to the CB10 epitope. The data are consistent with either possibility. In addition, additional experiments using individual substitutions at both the 5 and 8 positions will be required to determine the relative contribution of the individual amino acids.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Linda Kay Myers, Department of Medicine, 956 Court Avenue, Room G326, Memphis, TN 38163.
3 Abbreviations used in this paper: CIA, collagen-induced arthritis; CII, type II collagen; CB8, a peptide of 149 amino acid residues representing CII 403–551 generated by cyanogen bromide cleavage of type II collagen; CB10, a peptide of 346 residues representing CII 552–897 generated by cyanogen bromide cleavage of type II collagen; WT, wild type.
Received for publication May 11, 1999. Accepted for publication October 20, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1(II)-CB11 in H-2q mice. J. Immunol. 152:3088.[Abstract]
2 chain as the proteoglycan subunit of type IX collagen. J. Biol. Chem. 261:6742. repertoire in the T cell response to a tolerogenic determinant of type II collagen. J. Immunol. 158:6013.[Abstract]
receptor. J. Immunol. 161:1542.This article has been cited by other articles:
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  A. Honda, A. Ametani, T. Matsumoto, A. Iwaya, H. Kano, S. Hachimura, K. Ohkawa, S. Kaminogawa, K. Suzuki, E. E. Sercarz, et al. 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 Int. Immunol., May 1, 2004; 16(5): 737 - 745. [Abstract] [Full Text] [PDF]
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