1. Top of page
  2. Abstract
  6. Acknowledgements


To identify peptides capable of altering the immune response to type II collagen (CII) in the context of HLA–DR.


Immunizing mice transgenic for the human HLA–DRB1*0101 immune response gene with CII elicits an arthritis (collagen-induced arthritis [CIA]) that resembles rheumatoid arthritis. We have previously identified an immunodominant determinant of CII, CII (263–270), recognized by T cells in the context of DR1. To produce synthetic peptides with the potential of disrupting the DR1-restricted immune response, synthetic analog peptides were developed that contain site-directed substitutions in critical positions. These peptides were used to treat CIA in DR1 transgenic mice.


An analog peptide, CII (256–276, N263, D266), that inhibited T cell responses in vitro, was identified. When DR1 mice were coimmunized with CII and CII (256–276, N263, D266), the incidence and severity of arthritis were greatly reduced, as was the antibody response to CII. Moreover, CII (256–276, N263, D266) was effective in down-regulating the immune responses to CII and arthritis, even when administered 2 weeks following immunization with CII. Spleen and lymph node cells from CII-immunized mice cultured with CII (256–276, N263, D266) in vitro produced increased amounts of interleukin-4 (IL-4) compared with cells cultured with the wild-type peptide, CII (256–276). Furthermore, CII (256–276, N263, D266) was incapable of preventing arthritis in DR1 IL-4−/− mice (genetically deficient in IL-4).


These data establish that CII (256–276, N263, D266) is a potent suppressor of the DR-mediated immune response to CII. Its effect is mediated, at least in part, by IL-4. These experiments represent the first description of an analog peptide of CII recognized by T cells in the context of a human major histocompatibility complex molecule that can suppress autoimmune arthritis.

Collagen-induced arthritis (CIA) is an experimental model of autoimmune-mediated polyarthritis that can be induced in susceptible strains of mice by immunization with type II collagen (CII), the major constituent protein of articular cartilage (1). CIA shares important clinical, histologic, and immunologic features with rheumatoid arthritis (RA) (2). Both CIA and RA are strongly associated with class II immune response genes. Susceptibility to CIA is associated with H-2q and H-2r, whereas susceptibility to RA is associated with the presence of HLA–DRB*0101 (DR1) and HLA–DRB*0401 (DR4). CIA is caused by autoimmunity to CII. Although autoimmune reactions to CII are also present in RA, their significance is not clear. To determine the role of autoimmunity to CII in RA, it may be necessary to suppress the specific immune response in patients and determine if disease activity is affected.

The development of transgenic mice expressing HLA class II molecules has made it easier to address the question of how to suppress DR-mediated specific immune reactions experimentally. We have previously shown that CIA in H-2q mice can be prevented by injection of a peptide analog of the immunodominant epitope of CII recognized by I-Aq. This peptide apparently acts by causing immune deviation. It is important because it can both prevent the development of CIA when administered prior to immunization and alter the course of disease when given after immunization. Development of a similar peptide that can alter DR-mediated immune responses might provide a means of preventing CII autoimmunity in RA patients.

Our studies with HLA–DR transgenic mice show that the mice are susceptible to CIA, and DR1 and DR4 can bind and present peptides derived from human CII (3, 4). Using proliferation and cytokine assays, we identified CII (263–270) (FKGEQGPK) as the core of the immunodominant T cell determinant presented by HLA–DR1 and DR4. Based on these data, we developed synthetic analog peptides containing substitutions at selected residues designed to down-regulate the immune response to CII and CIA in the context of DR1. These peptides were tested for their ability to prevent arthritis in DR1 transgenic mice.


  1. Top of page
  2. Abstract
  6. Acknowledgements


DR1 transgenic mice were produced as previously described (3) and raised in our animal facility. 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. All animals were kept until the age of 7–10 weeks before being used for experiments.

The IL-4 (C57BL/6-IL-4tm1cgn IL-4 knockout) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and backcrossed onto the arthritis-susceptible DR1 C57BL/10 background for 7 generations before they were intercrossed. Genomic DNA was obtained from blood samples, and polymerase chain reaction (PCR) was used to identify mice homozygous for the IL-4−/− phenotype (5).


For induction of arthritis, mice were immunized with CII at 8–12 weeks of age, as described previously (6). Briefly, CII was dissolved in 0.01N acetic acid and emulsified with an equal volume of Freund's complete adjuvant (CFA). The resulting emulsion was injected subcutaneously at the base of the tail. Each mouse received a total volume of 50 μl containing 50 μg of Mycobacterium tuberculosis and 50 μg of antigen.

Preparation of CII.

Native CII was solubilized from bovine articular cartilage by limited pepsin digestion and purified as described earlier (6).

Synthesis of analog peptides of CII (256–276).

The peptide representing CII (256–276) and its analog peptides containing specific amino acid substitutions were chemically synthesized by a solid-phase procedure described previously, using an Applied Biosystems peptide synthesizer (model 430; Applied Biosystems, Foster City, CA) and purified by high-performance liquid chromatography (7).

Class II peptide binding experiments using purified DR1 molecules.

The ability of CII peptides to bind to the DR1 molecules was determined in competitive binding inhibition assays in which various concentrations of CII peptides were used to compete for DR binding of biotinylated hemagglutinin peptide HA (307–319). Soluble DR1 was purified from culture supernatants of transfected S2 Drosophila cells, as described previously (8). Briefly, the cytoplasmic and transmembrane portions of these molecules were deleted from the complementary DNA (cDNA) using PCR, a new stop codon was inserted immediately prior to the transmembrane domain, and the resulting cDNA was cloned into the Drosophila expression vector pRmHA-3. S2 cells were transfected with a 10:1 ratio of DRB1 and DRA1 to pUChsneo using calcium phosphate precipitation. Soluble DR production was induced by 1 mM CuSO4, and 5 days later the culture supernatant was collected and adjusted to 0.05% octyl glucoside (OcG). The soluble DR was purified by passage of the supernatant over an affinity column coupled with the anti-DR antibody LB 3.1. The column was washed with 0.05% OcG and 0.15M NaCl in phosphate buffer, pH 7.5, followed by 0.05% OcG and 0.5M NaCl in phosphate buffer, pH 7.5. The DR was eluted with 100 mM Tris, 0.5M NaCl, pH 11.2, and the fraction was immediately neutralized with acetic acid. The DR recovered was concentrated using a stirred cell (Amicon, Beverly, MA) and quantitated by absorption at optical density 280 nm and sodium dodecyl sulfate–polyacrylamide gel electrophoresis prior to use.

Class II binding assays were performed as described by Hill et al (8). Briefly, varying concentrations of the competitor peptides were incubated for 4 hours at 37°C, with constant concentrations of biotinylated HA (307–319) peptide (0.5 nM) and 10 nM DR1 in phosphate buffered saline (PBS) containing OcG. After incubation, DR–peptide complexes were transferred and captured by incubating overnight at 4°C on a 96-well microtiter plate initially coated with LB3.1 (anti-DR) and blocked with bovine serum albumin. Excess peptide was removed by washing with PBS containing 0.05% Tween 20. The plates were treated with europium-labeled streptavidin and incubated for 2 hours at room temperature. After washing, the plates were treated with a chelating enhancement solution, which releases europium from streptavidin and forms a highly fluorescent micellar solution. Fluorescence was quantitated using a microplate fluorometer (Delfia model 1234; PerkinElmer Life Science, Wellesley, MA). The concentration of CII peptide inhibiting 50% of the HA peptide binding (IC50) was calculated from the linear portion of the curves. IC50 values represent the average of 2 determinations per peptide.

Measurement of T cell cytokines by enzyme-linked immunosorbent assay (ELISA).

Quantitative measurement of murine interferon-γ (IFNγ), IL-4, and IL-10 was performed using a solid-phase ELISA based on the “sandwich” principle. Commercially available kits were used (IFNγ; Gibco BRL, Gaithersburg, MD; IL-4 and IL-10; Endogen, Cambridge, MA). Briefly, spleens and lymph nodes from DR1 transgenic mice immunized with CII or A12 emulsified with CFA 10–14 days previously were individually minced into single-cell suspensions in Hanks' balanced salt solution (HBSS) and washed 3 times with HBSS. Pooled splenocytes and lymph node cells were then adjusted to a concentration of 5 × 106/ml and cultured with 100 μg/ml of antigen (synthetic peptides, collagen, or purified protein derivative [PPD]) in Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY) supplemented with 5% fetal bovine serum (Hyclone, Logan, UT). Supernatants were collected 72–120 hours later and used either fresh or frozen at −70°C. Supernatant samples were incubated in microtiter wells coated with a monoclonal antibody recognizing murine IFNγ, IL-4, or IL-10. Samples were washed and incubated with a preformed detector complex consisting of a biotinylated second monoclonal antibody to the appropriate cytokine and an antibiotin–alkaline phosphatase conjugate. Each sample was tested in duplicate. The absorbance was measured at 405 nm with a spectrophotometer. A standard curve was obtained by plotting the absorbance versus the corresponding concentration of the standards. Values are expressed in pg/ml.

Treatment with analog peptides.

DR1 transgenic mice were treated with analog peptides by dissolving the peptides in PBS and adding them to the CII emulsion so that the peptide was administered at the time of CII immunization, or by administering them intravenously at 333 μg/dose for 3 doses (1 mg total), 166 μg/dose for 3 doses (0.5 mg total), or 33 μg/dose for 3 doses (0.1 mg total) on days 11, 12, and 14 following immunization with CII.

Measuring the incidence and severity of arthritis.

The incidence and severity of arthritis were determined by visually examining each fore paw and hind paw and scoring them on a scale of 1–4, as described previously (6). Scoring was conducted by 2 examiners, 1 of whom was unaware of the identity of the treatment groups. Each mouse was scored 3 times weekly beginning 3 weeks postimmunization and continuing for 8 weeks. The incidence of arthritis (number of animals with 1 or more arthritic limbs) was recorded at each time point. The severity of arthritis in each limb was also recorded.

Measurement of serum antibody titers.

Mice were bled 6 weeks after immunization, and sera were assayed for antibodies reactive with native CII using a modification of an ELISA previously described (9).

Statistical analysis.

The incidence of arthritis in various groups of mice was compared using Fisher's exact test. Antibody levels and mean severity scores were compared using Student's t-test. P values less than 0.05 were considered significant.


  1. Top of page
  2. Abstract
  6. Acknowledgements

Identification of candidate peptides.

We have previously identified a peptide that inhibits CIA in DBA/1 mice (H-2q) (6). This peptide was an analog of the immunodominant epitope but had amino acid substitutions that gave it 2 characteristics: 1) it bound poorly to the I-Aq molecule and, 2) when cultured with CII-sensitized T cells, it failed to induce IFNγ, but was able to induce the secretion of Th2 cytokines. To identify peptides with these characteristics for use in DR1 mice, we synthesized analog peptides representing the immunodominant epitope for DR1. The effect of flanking regions on induction of a response is unknown, so we arbitrarily included several amino acids on both sides of the core epitope. The test peptides were 21 amino acids long. To determine the ability of each of these peptides to bind the DR molecule, we used a soluble DR1–peptide binding assay. Various concentrations of analog peptides were tested for the ability to compete with biotinylated HA (307–319) as the indicator peptide for binding to soluble DR1. HA (307–319) was chosen because it has a relatively high binding affinity for DR1 and provides a highly specific and reasonably sensitive indicator. There was wide variability in the capacity of the synthetic peptides to compete with HA (307–319) (Figure 1). The substitution of asparagine for phenylalanine at position 263 uniformly reduced binding by at least 2 orders of magnitude. This finding is consistent with our previous identification of residue 263 as being the primary anchor residue for the core epitope (3). Phe263 of the core epitope interacts with binding pocket 1 of both DR1 and DR4.

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Figure 1. Binding of analog peptides to DRB1*0101. The ability of type II collagen (CII) analog peptides to bind to the DR molecules was determined in competitive binding assays in which various concentrations of CII peptides were used to compete for DR binding of biotinylated hemagglutinin peptide HA (307–319). The 50% inhibition concentration (IC50) values represent the average of 2 determinations per peptide. Peptides with an IC50 of >10,000 are those that inhibited from 0 to 50% at 10,000 nM, the highest concentration tested. Solid bars indicate peptides binding with an affinity of >100 nM. The x axis represents the IC50 value of each peptide. Five of the peptides reached values >10,000 nM. Wt = wild-type.

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It has been suggested that peptides interact with DR molecules primarily through a P1, P4, P6, P9 motif (10). This means that Glu266 (P4) may also be a key amino acid in determining binding affinity. As shown in Figure 1, substitutions at this position affect binding, depending on the particular substitution. Binding affinity may either be increased or decreased. An alanine substitution results in increased binding, whereas an aspartic acid substitution results in decreased binding relative to the wild-type peptide.

To determine the ability of each of the analog peptides to affect the response of CII-reactive T cells, DR1 mice were immunized with CII and the cells were harvested 14 days later for culture with each peptide. Culture supernatants were collected after 72 hours and analyzed for the production of IFNγ, IL-10, and IL-4. As expected, the peptide representing the immunodominant epitope induced a strong response, with high levels of IFNγ and substantial levels of both IL-10 and IL-4. All of the peptides with asparagine substitutions at residue 263 were unable to induce comparable levels of IFNγ. Single residue substitutions at position 266 (Ala266 or Asp266) had less effect on IFNγ production (Table 1). These data confirm the dominant effect of Phe263 on peptide binding to DR1. Peptides with substitutions at this position were uniformly less efficient at inducing IFNγ. Double substitutions at positions 263 and 266 showed a marked divergence of effect. The peptide with Asn263 and Asp266 was very inefficient at inducing IFNγ but was able to induce high levels of IL-10 and exceptionally high levels of IL-4.

Table 1. T cell response to analog peptides*
PeptideIFNγ, pg/mlIL-10, pg/mlIL-4, pg/ml
  • *

    DR1 mice were immunized with human type II collagen. After 14 days, spleen and lymph node cells were harvested and cultured (5 × 106 cells/ml) with 100 μg/ml of collagen analog peptides. Supernatants were collected after 72 hours and assayed for interferon-γ (IFNγ), interleukin-4 (IL-4), and IL-10 by enzyme-linked immunosorbent assay. The cytokine response to wild-type peptide was predominantly a Th1 response. In contrast, the analog peptide A12 induced production of only Th2 cytokines. ND = not done.


Effect of peptides on CIA.

Two analog peptides, CII (256–276, N263, D266) and CII (256–276, N263, A266), were selected for in vivo testing of their ability to prevent CIA. The former peptide was the most promising, based on our finding that it was unable to induce measurable levels of IFNγ in CII-reactive T cells, but retained the ability to induce the Th2 cytokines IL-10 and IL-4. The latter peptide was interesting since it contained substitutions at the same positions but retained the ability to induce measurable levels of IFNγ by CII-reactive T cells. DR1 transgenic mice were immunized with CII, CII + CII (256–276, N263, D266), or CII + CII (256–276, N263, A266) and were observed for the development of arthritis (Table 2 and Figure 2). DR1 mice given CII (256–276, N263, D266) demonstrated a dose-related decrease in the incidence of arthritis and the number of arthritic limbs (Figure 2). Coincident with a decrease in the incidence and severity of arthritis, antibody production to CII was also significantly decreased (Table 2). Moreover, mice receiving CII (256–276, N263, A266) had an incidence of disease comparable with that in control animals given vehicle alone.

Table 2. Effect of treatment of DR1 transgenic mice with analog peptides*
PeptideAntibodies to bovine CII
  • *

    Groups of DR1 transgenic mice were coimmunized with type II collagen (CII) alone or CII plus 1.2 mg of an analog peptide, and observed for arthritis. Mice were bled 6 weeks after immunization, and sera were individually analyzed by enzyme-linked immunosorbent assay to determine levels of antibody against bovine CII. Values are the mean ± SD units of activity for each group of animals.

  • P ≤ 0.0005 versus no treatment.

None66 ± 11
CII (256–276, N263, A266)49 ± 15
CII (256–276, N263, D266)2 ± 2
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Figure 2. Coimmunization of DR1 transgenic mice with type II collagen (CII) and analog peptides. Groups of DR1 transgenic mice were coimmunized with 50 μg CII alone (▪) (18 mice), 50 μg CII plus 1.2 mg of the analog peptide CII (256–276, N263, D266) (▴) (20 mice), or 50 μg CII plus 1.2 mg of the analog peptide CII (256–276, N263, A266) (•) (10 mice) and observed for arthritis.

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These data established the ability of CII (256–276, N263, D266) to prevent CIA when coadministered with CII. However, the initiating event in human arthritis is often unknown, and administration of a peptide to prevent an immune reaction may not be feasible. We were therefore interested in determining if administering the peptide after immunity was established would be effective. In experiments designed to test that possibility, DR1 transgenic mice were immunized with CII, and then treated intravenously with peptide CII (256–276, N263, D266) on days 11, 12, and 14 at varying doses (Table 3). The incidence of arthritis was reduced in a dose-responsive manner so that administration of 1 mg of CII (256–276, N263, D266) per mouse significantly suppressed arthritis. These data indicate that peptide CII (256–276, N263, D266) significantly down-regulated the immune responses to CII in vivo as well as in vitro, even when administered 2 weeks after the immunization with CII, well after the establishment of immunity to CII.

Table 3. Effect of administration of CII (256–276, N263, D266) after immunization with CII
Treatment*Arthritis incidenceAntibodies to CII
  • *

    Groups of 10 DR1 transgenic mice were immunized with type II collagen (CII) in Freund's complete adjuvant. CII (256–276, N263, D266) was administered intravenously on days 11, 12, and 14 at either 33 μg/dose for a total of 0.1 mg, 166 μg/dose for a total of 0.5 mg, or 333 μg/dose for a total of 1 mg. Control animals were given ovalbumin (OVA) at 333 μg/dose for a total of 1 mg.

  • Six weeks following immunization.

  • Antibody levels were measured by enzyme-linked immunosorbent assay and are reported as the mean ± SD arbitrary units based on comparison with a standard antiserum run simultaneously.

  • §

    P ≤ 0.025 versus OVA treatment, by Student's t-test.

  • P = 0.01 versus OVA treatment, by Fisher's exact test.

OVA, 1 mg7/1042 ± 10
CII (256–276, N263, D266), 0.1 mg5/1033 ± 11
CII (256–276, N263, D266), 0.5 mg3/1024 ± 7§
CII (256–276, N263, D266), 1 mg1/1022 ± 9§

Identification of the method of suppression.

The data in Table 1 indicate that CII (256–276, N263, D266) induces substantial quantities of IL-4. To determine if IL-4 was essential for its action, we used DR1–IL-4 knockout mice, developed by backcrossing C57BL/6 IL-4−/− mice to DR1 transgenic mice for 7 generations prior to intercross. The resulting mice were immunized with CII or both CII and CII (256–276, N263, D266) and observed for the development of arthritis. The results are shown in Figure 3. DR1 IL-4−/− mice, immunized with CII, had a high incidence of severe arthritis. When these mice were coimmunized with CII and CII (256–276, N263, D266) there was no change in the incidence of arthritis. These data contrast with the significant suppression of arthritis observed using wild-type mice coimmunized with CII and CII (256–276, N263, D266) (Figure 3). Similarly, the number of arthritic limbs in IL-4−/− mice coimmunized with CII and CII (256–276, N263, D266) (43%) was significantly greater than that in wild-type mice (10%; P < 0.0001). Mean severity scores confirmed that CII (256–276, N263, D266) does not suppress arthritis in IL-4−/− mice (Figure 3).

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Figure 3. Analog peptides in IL-4−/− mice. Groups of IL-4−/− mice and their wild-type littermate controls were immunized either with type II collagen (CII) or with CII and the analog peptide CII (256–276, N263, D266) at a ratio of 1:320 (0.6 mg CII [256–276, N263, D266]:50 μg CII). Thirty wild-type mice were immunized with CII (▪), 28 wild-type mice were coimmunized with CII and CII (256–276, N263, D266) (▴), 10 IL-4−/− mice were immunized with CII (⧫), and 10 IL-4−/− mice were coimmunized with CII and CII (256–276, N263, D266) (•). The wild-type mice treated with CII and CII (256–276, N263, D266) differed significantly from the wild-type mice immunized with CII (P = 0.003, P < 0.0001, and P < 0.0005 for arthritis incidence, number of arthritic limbs, and mean severity score, respectively, at the final time point). Neither of the IL-4−/− groups differed significantly from CII-immunized wild-type mice.

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Immunization with CII (256–276, N263, D266) does not induce Th1-mediated autoimmunity.

In another set of experiments, DR1 transgenic mice were immunized with CII (256–276, N263, D266) analog peptide or CII emulsified with CFA and their T cells were cultured in vitro with various antigens. The response of cells from CII (256–276, N263, D266)–immunized mice to CII (256–276) and α1(II) remained predominantly Th2, while their response to PPD was Th1 (Table 4). In contrast, cells from mice immunized with CII generated predominantly a Th1 response to the wild-type peptide. These data suggest that the population of cells reactive with CII (256–276, N263, D266) is predominantly Th2 and that this effect is independent of whether mice are immunized with native CII, the wild-type peptide, or CII (256–276, N263, D266). The ability to induce the secretion of Th2 cytokines may explain the profound suppressive effects CII (256–276, N263, D266) has on the development of CIA.

Table 4. Effect of immunization of DR1 transgenic mice with either CII or CII (256–276, N263, D266)*
Immunogen, antigenIFNγ, pg/mlIL-10, pg/mlIL-4, pg/ml
  • *

    DR1 transgenic mice were immunized with either type II collagen (CII) or CII (256–276, N263, D266). After 14 days, the spleen and draining lymph node cells were harvested and cultured (5 × 106 cells/ml) with 100 μg/ml of the indicated antigen. Supernatants were collected 72 hours later and analyzed for the presence of interferon-γ (IFNγ), interleukin-10 (IL-10), and IL-4. PPD = purified protein derivative.

 CII (256–276)2,97527611
 CII (256–276, N263, D266)<161040
CII (256–276, N263, D266)   
 CII (256–276)17579223
 CII (256–276, N263, D266)<180831
 α1 (II)<168045


  1. Top of page
  2. Abstract
  6. Acknowledgements

The recent development of transgenic mice expressing HLA class II molecules has made it possible to study the DR1-restricted immune responses to collagen in a more systematic manner. Some investigators have been concerned that T cell responses generated by the mouse may not be representative of those generated in humans. Using an HLA–A2.1 transgenic mouse model and T cell responses to a hepatitis C virus, clones from humans (bearing HLA–A2) infected with hepatitis were compared with T cell responses of clones from HLA–A2 transgenic mice infected with the same virus (11). The T cell repertoire was flexible enough to allow a similar, almost identical, response when the same major histocompatibility complex (MHC) molecule was presenting the peptide (11). These data suggest that the HLA molecule plays the primary role in determining which peptides are recognized by T cells and that the transgenic mouse model is a valid model for the study of human HLA-restricted T cell determinants (11).

We have identified a synthetic analog peptide, CII (256–276, N263, D266), that is capable of reducing the incidence and severity of arthritis as well as the antibody response to CII when administered to CIA-susceptible DR1 transgenic mice. This peptide was effective in down-regulating the immune responses to CII and arthritis, even when administered 2 weeks following CII immunization. CII (256–276, N263, D266) represents the first peptide analog of collagen that has been shown to suppress arthritis in the context of a human HLA molecule that is known to be associated with RA. This peptide may have therapeutic potential.

Although the precise mechanism by which CII (256–276, N263, D266) exerts its effect is not clear, it may act as an altered peptide ligand (APL). Investigators using other model antigens have indicated that APLs that differ from their respective wild-type peptides can influence T cell activation and cytokine production by cloned T cells (12, 13). For example, an APL containing a single amino acid substitution at the T cell receptor (TCR) contact residue of a cytochrome c peptide was shown to induce immune deviation to a Th2 response, compared with the wild-type agonist peptide, which induced a Th1 response (14, 15). APLs containing a substitution of TCR contact residues in other peptides resulted in marked variability in the TCR recognition of these peptides (15–17). A hemoglobin peptide containing specific amino acid substitutions caused T cells to secrete IL-4 without proliferating (12). Alterations in the structure of hemoglobin 64–76 induced T cells to become cytolytic without proliferating or secreting cytokines (18). These data suggest that minor variations in the peptide binding affinity or in the physicochemical properties of amino acid residues involved in MHC binding can lead to disparate immunologic responses (12, 14, 16, 18, 19).

These observations have stimulated considerable interest in the use of APLs as immunotherapeutic agents in experimental models of autoimmune diseases (20, 21). An APL of myelin proteolipid protein, generated by substitution at a principal TCR contact residue of the encephalitogenic peptide, has been shown to prevent autoimmune encephalitis (22) in mice. Those investigators demonstrated that T cell clones reactive with this APL secreted Th2 cytokines (IL-4 and IL-10) (22). Coimmunization of susceptible mice with myelin basic protein and an APL containing a single substitution at an anchor residue for I-Au similarly blocked the development of experimental autoimmune encephalomyelitis (23). An APL of an acetylcholine receptor peptide effectively down-regulated experimental autoimmune myasthenia gravis in C57BL/6 mice (24). The suppression was accompanied by up-regulated secretion of transforming growth factor β (TGFβ).

The use of APL in the treatment of human disease is limited. Two trials of APL used to treat multiple sclerosis were terminated early because of concerns about disease activity increasing (20, 25). One of these showed that patients treated with the APL had an increase in immunity both to the APL and to myelin basic protein. For this reason, we directly tested the immune potential of CII (256–276, N263, D266) by using it to immunize DR1 transgenic mice. Even when given with CFA, this peptide failed to induce significant Th1 immunity to itself or to intact CII. These data suggest that CII (256–276, N263, D266) may behave somewhat differently than the APL used to treat multiple sclerosis.

Unlike APL used in studies of other models of autoimmune disease, CII (256–276, N263, D266) contains 2 amino acid substitutions, both MHC-binding residues. Jardetzky and colleagues, using a panel of endogenous peptides, described 4 pockets of the class II molecule that can interact with residues of peptides (10). Our data suggest that the phenylanine residue at position 263 and the aspartic acid residue at position 266 most likely bind to the P1 and the P4 binding pockets predicted by the crystal structure data. We find that an aspartic acid substitution at position 266 exhibits decreased binding to DR1 relative to the native peptide, while an alanine substitution has increased binding. Although the binding differences are small, the difference in the effectiveness of the peptides in preventing arthritis is substantial. It is clear that the differences in effectiveness are related entirely to relative binding efficiencies. Although the predominant effect of the CII (256–276, N263, D266) substitution at residue 266 appears to be mediated by a decrease in binding to DR1, we cannot rule out the possibility that an aspartic acid at 266 might affect the neighboring 267 TCR contact residue. Jardetzky and colleagues also predicted that residues P5 (corresponding to CII 267) and P8 (corresponding to CII 270) are the residues most likely to interact with the TCR, since they are positioned to point away from the DR1 binding sites. Based on a comparison of the effect of CII (256–276, N263, D266) with that of peptides that can suppress CIA in mice with other MHC genotypes, all of the effective peptides have decreased MHC binding.

It is evident that there is not a total absence of binding since CII (256–276, N263, D266) does mediate a T cell response. In fact, it seems to stimulate increased production of IL-4 relative to that induced by other analogs. IL-4 is a pleiotropic T cell–derived cytokine. Although it was originally identified as a B cell growth factor, it is now known to be a potent antiinflammatory cytokine (26). It is produced by activated CD4+ T cells and stimulates proliferation, differentiation, and activation of several cell types (27). Major functions include suppression of metalloproteinase production (28), protection against extracellular matrix degradation, and inhibition of osteoclast activity, which blocks bone resorption in vitro (29). Treatment of autoimmune arthritis with the gene for IL-4 has successfully ameliorated CIA in several different studies (28, 30, 31). One would expect that the increased IL-4 detected by culture of CII-autoimmune T cells with CII (256–276, N263, D266) is indicative of in vivo production of this cytokine and protection against inflammation and arthritis. In addition to the in vitro data that show CII (256–276, N263, D266) stimulates production of IL-4, the in vivo data using IL-4−/− mice confirm that its action is mediated through an IL-4–dependent mechanism. These data are consistent with those from a previous report by Yoshino and Yoshino, who used the anti–IL-4 (11B11) antibody to disrupt suppression in another animal model of arthritis, specifically antigen-induced arthritis, using oral administration of the antigen (32). However, these data do not rule out the possibility that other Th2 cytokines, such as IL-10 or TGFβ, may play a role.

Susceptibility to RA is strongly associated with the expression of specific HLA class II alleles, especially HLA–DR1 and DR4 (33–35). Polyarticular juvenile RA is similarly associated with DR1 (36). Evidence has recently been presented showing a correlation of T cell responses to CII (262–270) and the presence of DRB1*0101 and *0401 in RA patients (37, 38). While this does not ensure that a similar parallelism exists between these DR transgenic mice and RA patients, it certainly provides a reasonable basis for development of reagents that might have therapeutic importance. There are strong theoretical and practical reasons to suggest that immunologically specific therapy for autoimmune diseases is preferable to the use of immunologically nonspecific drugs and antibodies. The analog peptide we have identified represents a promising specific immunotherapy for patients with autoimmunity to CII mediated by DR1.


  1. Top of page
  2. Abstract
  6. Acknowledgements

The authors thank Theresa Tran, Sarah Rowe, and Karen Whittington for excellent technical support.


  1. Top of page
  2. Abstract
  6. Acknowledgements
  • 1
    Courtenay JS, Dallman MJ, Dayan AD, Martin A, Mosedale B. Immunisation against heterologous type II collagen induces arthritis in mice. Nature 1980; 283: 6668.
  • 2
    Trentham DE, Townes AS, Kang AH. Autoimmunity to type II collagen: an experimental model of arthritis. J Exp Med 1977; 146: 85768.
  • 3
    Rosloniec EF, Brand DD, Myers LK, Whittington KB, Gumanovskaya M, Zaller DM, et al. An HLA-DR1 transgene confers susceptibility to collagen-induced arthritis elicited with human type II collagen. J Exp Med 1997; 185: 111322.
  • 4
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