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Proc Natl Acad Sci U S A. Mar 23, 2004; 101(12): 4228–4233.
Published online Mar 15, 2004. doi:  10.1073/pnas.0400061101
PMCID: PMC384723
Medical Sciences

Epitope-specific immunotherapy induces immune deviation of proinflammatory T cells in rheumatoid arthritis

Abstract

Modulation of epitope-specific immune responses would represent a major addition to available therapeutic options for many autoimmune diseases. The objective of this work was to induce immune deviation by mucosal peptide-specific immunotherapy in rheumatoid arthritis (RA) patients, and to dissect the related immunological mechanisms by using a technology for the detection of low-affinity class II-restricted peptide-specific T cells. A group of patients with early RA was treated for 6 months orally with dnaJP1, a peptide that induces proinflammatory T cell responses in naive RA patients. Immunological analysis at initial, intermediate and end treatment points showed an intriguing change from proinflammatory to regulatory T cell function. In fact, dnaJP1-induced T cell production of IL-4 and IL-10 increased significantly when initial and end treatment points were compared, whereas dnaJP1-induced T cell proliferation and production of IL-2, IFN-γ, and tumor necrosis factor-α decreased significantly. The total number of dnaJP1-specific cells did not change over time, whereas expression of foxP3 by CD4+CD25bright cells increased, suggesting that the treatment affected regulatory T cell function. Thus, rather than clonal deletion, the observed change in immune reactivity to dnaJP1 was the outcome of treatment-induced emergence of T cells with a different functional phenotype. This study contributes to our knowledge of mechanisms and tools needed for antigen-specific immune modulation in humans, thus laying the foundation for exploitation of this approach for therapeutic purposes.

Several attempts at immunotherapy of human autoimmune diseases such as rheumatoid arthritis (RA), type I diabetes, and multiple sclerosis, based either on modulation of individual immune pathways involved in inflammation or on tolerization to various antigens, have shown that this approach may be viable, despite the initial difficulties encountered (16).

Such difficulties may stem from: (i) the multiplicity of the pathogenic mechanisms; (ii) the inherent diversity of the patient population; and (iii) a lack of pretreatment screening to determine whether the antigen was immunologically relevant to perspective patients to be treated. Detailed analysis of the effects of the treatment on the immune system has also been lacking in most of the trials performed to date.

Hence, the following knowledge gaps persist: (i) insufficient understanding of events that lead to autoimmune inflammation; (ii) reliance on clinical outcome as the only measure of efficacy; (iii) lack of surrogate immune markers to be associated with disease activity and efficacy of intervention; and (iv) technical shortcomings in molecular analysis of the immune response, particularly due to difficulties in identifying low-affinity, rare, MHC class II-restricted antigen-specific T cells, despite considerable recent progress in the field (711).

We describe here immune intervention based on a model antigen involved in the pathogenesis of RA. The peptide used (dnaJP1), is derived from the bacterial heat shock protein dnaJ and shares sequence homology with the shared epitope, a 5-aa stretch in common among RA-associated HLA alleles (1214). We proposed that in RA an interplay between HLA and dnaJ-derived peptides maintains and stimulates T cells, which participate in autoimmune inflammation (1518). Modulation of this pathway may affect the autoimmune process and thus, be biologically as well as clinically relevant. We designed a pilot clinical trial to obtain information regarding both safety and immunological efficacy of the treatment. Enrollment criteria for this study included active RA as defined by the American College of Rheumatology (19), a disease duration <5 years and in vitro responsiveness to dnaJP1, defined as T cell proliferation and/or production of proinflammatory cytokines. dnaJP1 was given orally for 6 months. A total of 66.7% of the patients screened met both clinical and immunological entry criteria; 15 patients divided in three different dose groups (0.25, 2.5, and 25 mg daily) were included in the trial (Tables (Tables11 and and2).2). Medical evaluation, including routine laboratory tests and immunological analyses, took place monthly. The objectives of the study were to evaluate tolerability and biological efficacy of the treatment on the immune system of the patients, with particular attention to dnaJP1-specific T cells.

Table 1.
General characteristics of patients enrolled and inclusion criteria for the study
Table 2.
DnaJP1-specific T cell reactivity

Methods

Patients. Eligible patients fulfilled the 1987 revised American College of Rheumatology criteria for RA, had a disease duration of <5 years, had active disease as manifested by at least six joints that were swollen and tender, and had in vitro responsiveness to dnaJP1, defined as any of the following criteria: T cell proliferation expressed as stimulation index >2 and/or production of proinflammatory cytokines expressed as >2% above background of CD3+ T cells producing IFNγ, tumor necrosis factor (TNF)-α, or IL-2 measured by intracellular fluorescence-activated cell sorter (FACS) analysis. Institutional Review Board approval and informed consent was obtained in accordance with regulations. Fifteen patients were included in the study (Tables (Tables11 and and2)2) and were treated for 6 months with dnaJP1 at three different dosages: 0.25, 2.5, and 25 mg. Patients were evaluated every month for a standardized clinical examination (joint scores for swelling and tenderness, Rapid Assessment of Disease Activity in Rheumatology scores), standardized laboratory tests (erythrocyte sedimentation rate, rheumatoid factor, C-reactive protein, and serum chemical values), and an immunological checkup.

T Cell Proliferation Assays. Peripheral blood mononuclear cells (PBMCs) were cultured in triplicate in 200 ml u-shaped bottom wells (Costar, Cambridge, MA) at 5 × 105 cells per well for 120 h with or without antigen. Phytohemagglutinin (2 mg/ml) was used as a positive control. For the final 16 h, cells were pulsed with [3H]thymidine (Amersham Pharmacia International, Bucks, United Kingdom). Thymidine uptake was measured by using a liquid scintillation counter. Results are expressed as a mean cpm of triplicate cultures. The magnitude of the response is expressed as stimulation index: the mean cpm of stimulated/nonstimulated cultures.

Antigens. Peptides were synthesized as C-terminal amides and were purified by reversed-phase HPLC. Peptides were N-biotinylated during synthesis (only one biotin molecule per peptide, 100% biotinylation; Synthetic Biomolecules, San Diego). The following peptides were used: dnaJP1 (QKRAAYDQYGHAAFE), dnaJPv (DERAAYDQYGHAAFE), PADRE (+) (KJVAAWTLKAA-a), and PADRE (-) peptide (UAJAAAATLKAA) (10 mg/ml). dnaJPv is identical to dnaJP1 except for the two N-terminal amino acids. In contrast to dnaJP1, dnaJPv does not induce a proliferative T cell response in patients with RA. It was therefore used as negative control in the proliferation assays. PADRE (+) has good pan-DR binding and antigenic properties, and it has therefore been used as positive control in measuring cytokine production by intracellular FACS analysis. PADRE (-) has a comparable pan-DR-binding capacity but is a poor antigen (2021). It has been therefore used as negative control to identify background in the T cell capture (TCC) assay.

Preparation of Artificial Antigen-Presenting Cells (aAPCs). This preparation is a modification from our published method (22). Briefly, phosphatidylcholine and cholesterol (Sigma) are combined in a glass tube at a molar ratio of 7:2. The solvent is evaporated under an Argon stream for 30 min and is dispersed at a final concentration of 10 mg/ml in 140 mM NaCl and 10 mM Tris·HCl, pH 8 (buffer A) containing 0.5% sodium deoxycholate. Monosialoganglioside-GM1 (Sigma G-7641) is added at a final concentration of 0.28 or 0.55 mM. The solution is sonicated until clear and is stored at -20°C. Liposomes are formed through dialysis at 4°C against PBS in a 10-kDa Slide-A-Lyzer (Pierce) for 48 h. Biotinylated recombinant MHC is incorporated in rafts, engineered on the aAPC surface. The rafts are constructed by mixing biotinylated HLA-DR4 molecules, biotinylated antibodies to CD28 and anti-LFA-1, and biotinylated Cholera toxin subunit B-FITC conjugated (CTB-FITC; Sigma) in the appropriate (equal) molar ratio. Next, neutravidin (NA; Pierce) is added in a molar ratio of four biotinylated moieties per molecule of NA. CTB-FITC is used to visualize T cells bound by the aAPCs. After incubation (1 h at room temperature), the Raft-NA mixture is added to the liposomes for 2 h, again at room temperature washed twice in PBS. Finally, once the aAPCs are generated, they are incubated with the stained cells as described for the tetramers.

Staining of Cells for FACS Analysis. Cells are washed twice, stained with phycoerythrin, FITC, or cychrome-labeled monoclonal antibodies for human CD3, CD4, and CD25 (PharMingen) and isotype controls for 20 min at 4°C, are again washed twice and are resuspended in staining buffer.

T Cell Capture and Tetramers. For preparation of tetramers, 25 pmol HLA-DR4/peptide complex is mixed with 16.7 pmol of NA-FITC (Pierce) for 1 h at room temperature. Next, tetramers are incubated for 2 h at 37°C with prestained cells. After two washes, specific T cells are analyzed by FACS. T cell capture: stained cells are incubated with aAPCs for 30 min at room temperature. Before acquisition on the FACScalibur or, when cells are sorted, on the FACSvantage (Becton Dickinson), cells and aAPCs are washed twice and are resuspended in staining buffer.

Intracellular Cytokine Staining. PBMCs are cultured for 96 h with medium or antigen. During the last 4 h of culture, monensin (PharMingen) is added. Cells are stained with monoclonal antibodies for human CD3, are washed and incubated in 100 ml of fixation buffer (PharMingen) for 20 min at 40°C. Fixed cells are washed twice, resuspended in 100 ml of permeabilization buffer; and stained with the following monoclonal antibodies: phycoerythrin or FITC-conjugated anti-human IL-4, anti-human IL-10, anti-human IL-2, anti-human TNF-α, anti-human IFN-γ, and the appropriate isotype controls (PharMingen). Finally, cells are washed twice, resuspended in staining buffer, and are analyzed on a FACScalibur.

mRNA Level Quantification. Cytokine (IFN-γ and IL-10) and transcription factor (foxp3) gene expression levels of TCC-sorted dnaJP1-specific T cells are analyzed by multiplex realtime quantitative PCR (TaqMan). The PCR system we used is an abi prism 7700 thermal cycler (Perkin–Elmer) that is capable of distinguishing and quantitating multiple fluorophores in a single tube so that more than one PCR target can be detected for each cDNA sample. TaqMan probes and primers were designed by using the computer software primer express (PE Biosystems, Foster City, CA). A combination of primers and probes labeled with different dyes have been used; IFN-γ (5′-CCAACGCAAAGCAATACATGA-3′ forward, 5′-TTTTCGCTTCCCTGTTTTAGCT-3′ reverse, 5′-TCCAAGTGATGGCTGAACTGTCGCC-3′ JOE-probe); IL-10 (5′-TGAGAACAGCTGCACCCACTT-3′ forward, 5′-GCTGAAGGCATCTCGGAGAT-3′ reverse, 5′-CAGGCAACCTGCCTAACATGCTTCGA-3′ FAM-probe); foxp3.(5′-TCACCTACGCCACGCTCAT-3′ forward, 5′-TCATTGAGTGTCCGCTGCTT-3′ reverse, 5′-TGGGCCATCCTGGAGGCTCCA/3BHQ1–3′ JOE probe); and GAPDH (5′-CCACCCATGGCAAATTCC-3′ forward, 5′-TGGGATTTCCATTGATGACAAG-3′ reverse, 5′-TGGCACCGTCAAGGCTGAGAACG-3′ Tet-Probe). The parameter provided by the 7700 system software is the threshold cycle (Ct) defined as fractional cycle number at which the fluorescence passes a fixed threshold; the higher the initial amount of mRNA, the sooner accumulated product is detected in the PCR process, and the lower the Ct value (23). The relative mRNA amount for each target gene has been expressed as 1/Ct proband·100/Ct GAPDH. Calculations were performed by the relative standard curve method and results were expressed as induction index (arbitrary units), using as a reference the no-stimulated condition at each time point

Statistical Analysis. A Mann–Whitney U test was used to compare the different data. Kolmogorov–Smirnov statistics were used to analyze FACS cytokine staining. Foxp3 data were evaluated by ANOVA after removing the interaction term and running a main-effects-only model.

Results and Discussion

Mucosal Immune Therapy with dnaJP1 Is Safe. Treatment with dnaJP1 was well tolerated because no significant side effects were reported. The clinical characteristics of the patients were monitored closely to identify any treatment-related clinical deterioration. Physicians recorded affected joint scores and numbers and overall disease activity. Self-assessment by patients was performed by using the Rapid Assessment of Disease Activity in Rheumatology (24). Both physician- and patient-generated data showed a marked improvement from baseline. Because this study was not aimed at evaluating clinical efficacy, and no placebo treated control was included in this pilot trial, we refrain from showing the clinical data, which we interpret only as indicating that the treatment was safe and that certainly did not worsen the disease. Based on the encouraging preliminary data from this phase I trial, future research is warranted to evaluate the clinical efficacy of the treatment.

Mucosal Tolerization to dnaJP1 Leads to a Peptide-Specific Immune Deviation in PBMCs from Patients with RA. Immunological evaluation showed remarkable treatment-specific changes in responsiveness to dnaJP1. dnaJP1-induced T cell production of IL-10 and IL-4 increased significantly from baseline at the second month and continued throughout the treatment period (P < 0.001 at day 168 compared with day 0, Fig. 1). Conversely, we observed a strong decline in both T cell proliferation and production of proinflammatory cytokines IFN-γ and TNF-α in response to dnaJP1 stimulation in vitro (Fig. 1). These changes were already statistically significant for all parameters considered at the second month and persisted throughout the length of the study. All treatment groups showed similar trends in treatment-induced immune deviation, suggesting that the dosages tested were within the biological range necessary to induce immune deviation. Controls comprised mitogens (phytohemagglutinin) and irrelevant peptides, including dnaJPv, an altered peptide ligand not stimulatory in patients, and PADRE, a designer pan-DR-binding peptide immunogenic in humans irrespective of DR restriction (Fig. 1). The control experiments showed that the immune modulation obtained was antigen-specific, and that the patients' physiologic immune responses remained intact.

Fig. 1.
Treatment-induced modulation of T cell responses to dnaJP1. PBMC immune responses to dnaJP and controls from patients enrolled in the trial (n = 15). T cell-proliferative response after in vitro culture with dnaJP1 (a) or an irrelevant altered peptide ...

The fact that the responses to unrelated antigens did not change over time ruled out the possibility of a treatment-independent, random change in the quality of immune responsiveness (i.e., Th1 vs. Th2/3) in all patients treated. Thus, the changes were the outcome of a true immune deviation from a proinflammatory (IFN-γ, TNF-α, and T cell proliferation) to a more regulatory (IL-4 and IL-10) functional phenotype of the dnaJP1-specific T cell repertoire.

Mucosal Therapy with dnaJP1 Does Not Lead to Clonal Deletion of dnaJP1-Specific Cells. The persistence of T cell recognition of dnaJP1 in treated patients, as shown by production of regulatory cytokines after initiation of treatment, suggests that immune deviation, rather than clonal deletion of peptide-specific T cells took place. To address this question directly, we enumerated dnaJP1-specific T cells by TCC, a recently described method, based on aAPCs. We and others (22, 2527) showed that these aAPCs can effectively bind low-affinity polyclonal MHC class II-restricted CD3+ T cells, thus providing an excellent tool to characterize antigen-specific CD4+ T cells. This task is very difficult to achieve by using state-of-the-art MHC tetramer technology.

First, we compared the efficiency of TCC and tetramer technology in measuring dnaJP1-specific T cells in PBMCs from patients with RA. For this purpose, we engineered aAPCs with GM-1 ganglioside as part of the lipid bilayer. GM-1 binds one molecule of biotinylated CTB. In each raft, one molecule of NA anchors the biotinylated molecules to the aAPC surface through one biotinylated CTB, whereas the remaining three free valences of NA are saturated with MHC/peptide complex consisting of HLA DRB1*0401 molecules loaded with dnaJP1 or the appropriate control peptide [PADRE (-)], anti-CD28 antibody, and anti-LFA-1 antibody. Detection of dnaJP1-specific T cells from dnaJP1-stimulated PBMC cultures was possible by using aAPCs, and was more effective then tetramer staining of the same samples (Fig. 2). Thus, as in the other models tested before, TCC provided a very effective tool to enumerate and characterize dnaJP-specific T cells in PBMCs from patients with RA (22, 25, 27).

Fig. 2.
Comparison between tetramer and T cell capture technology for the detection of dnaJP1-specific T cells in PBMCs from patients with RA. PBMCs from a representative HLA-DRB1*0401-positive RA patient under treatment with dnaJP1 peptide were put in culture ...

We then applied TCC to identify dnaJP1-specific T cells in PBMCs from patients included in this study. By using this approach, we compared the number of dnaJP1-specific T cells as the percentage of total T cells before and after mucosal tolerization with dnaJP1. This number did not change significantly after treatment (before treatment: 6.5 ± 2.5% vs. after treatment: 8.0 ± 2.5%, P = 0.3, n = 6).

These experiments demonstrated that although treatment with dnaJP1 led to a significant decrease in antigen-induced proinflammatory cytokine production and T cell-proliferative capacities, this result was not due to a loss in total number of antigen-specific T cells.

Mucosal Therapy with dnaJP1 Leads to a Functional Shift of dnaJP1-Specific Cells. It remained, however, still to be addressed whether dnaJP1-specific T cells may have been rendered functionally incompetent by the treatment. We combined TCC with intracellular cytokine staining to monitor the changes in cytokine production within the population of antigen-specific T cells. After immunotherapy with dnaJP1, we observed a significant increase in the percentage of cells producing IL-4 within the total dnaJP1-specific CD3+ population (before treatment: 8.4 ± 5.4%, after treatment: 23.1 ± 3.8%, P < 0.05, n = 6). A corresponding significant reduction in percentage of CD3+/dnaJP1-specific cells producing IFN-γ was found (before treatment: 15.8 ± 6.7%, after treatment: 7.9 ± 1.4%, P = 0.05, n = 6; Fig. 3).

Fig. 3.
Characterization of peptide-specific dnaJP1 human MHC class II-restricted T cells with TCC. Cytokine production by dnaJP1-specific MHC class II-restricted cells after in vitro activation with dnaJP1 before and after treatment with dnaJP1. PBMCs from six ...

Several mechanisms could explain the discrepancy between the significant decline in the number of polyclonal T cells producing IFN-γ after treatment and the persistence of IFN-γ-producing, dnaJP1-specific T cells (Figs. (Figs.11 Left and and3).3). Most likely, different populations of dnaJP1-specific T cells are affected by the treatment (28). Among these cells, T regulatory-1-like T cells that produce IFN-γ and IL-10 may be of importance (2930).

To explore this hypothesis, dnaJP1-specific T cells were sorted from selected samples (n = 4) at baseline and at the end of treatment. mRNA was extracted and analyzed by real-time-PCR (TaqMan), for simultaneous expression of IFN-γ and IL-10. We found dnaJP1-specific T cells, which produce IFN-γ and IL-10 at the same time, at the end of the treatment period (IFN-γ: 0.081 ± 0.0054, IL-10: 0.075 ± 0.013, 1/Ct proband·100/Ct GAPDH, n = 4), supporting the hypothesis that the immune deviation observed may, at least in part, be depending on regulatory cells.

After Mucosal Tolerance Induction, in Vitro Activation with dnaJP1 Leads to Increased Expression of foxP3 by CD4+CD25bright Cells. To corroborate the concept that tolerization to dnaJP1 did not alter total numbers but rather the function of T cells, we focused on cells with a regulatory function (Treg), which are being increasingly proposed as central in immune regulation. A combination of functional and phenotypical markers, centered on CD4/CD25-double positive T cells was used to identify these cells. Recent work did not find differences in number of CD25bright T cells between synovial fluids from human rheumatoid arthritis patients and normals (3132). Because it is well known that Treg are difficult to expand in response to antigens, we focused on function rather than absolute numbers. The hypothesis, consistent with the our data, was that restoration of Treg function could be one of the consequences of our immunotherapy. For this purpose, we studied expression of the forkhead transcription factor foxP3. FoxP3 expression affects positively development and function of Treg (3335). It is therefore been proposed as a functional marker of these cells.

In the experiments shown here, we tested whether tolerization to dnaJP1 would induce foxP3 expression as an indication of restored Treg function.

Samples before and after tolerization from two patients were cultured in vitro with dnaJP1. CD4+CD25bright cells were sorted and assessed for foxP3 expression by TaqMan. Significantly increased (P < 0.001) Foxp3 expression by CD4+CD25bright cells after tolerization was achieved (Fig. 4).

Fig. 4.
Increased expression of foxP3 by CD4+/CD25+ T cells of patients after treatment with dnaJP1. Two representative patients were analyzed for foxP3 transcription factor expression. PBMCs obtained at initial evaluation and at the end of the treatment were ...

Altogether, these data demonstrate that the functional switch from proinflammatory to tolerogenic responses to dnaJP1 was an active phenomenon of immune deviation mediated by functionally competent antigen-specific T cells, probably consisting of various populations with diverse and integrated regulatory functions (3638).

Conclusion

We have characterized a T cell-dependent, proinflammatory pathway that can be specifically and safely modulated in patients with RA. Our findings show that epitope-specific mucosal therapy does not lead to a change in the number of epitope-specific T cells, but rather to a functional readjustment of the responding antigen-specific T cells, based on a functional change from a proinflammatory to a regulatory phenotype. This finding is in agreement with a recent report (39), showing that committed Th1 cells can still undergo a phenotypic change, which previously was considered to be impossible.

We believe that the peptide studied here is part of a pool of antigens that share characteristics such as their availability at the site of inflammation, strong proinflammatory effect on T cell responses in RA and, in certain instances, interspecies homologies (40). Peptides derived from heat shock protein fulfill this profile. dnaJP1, hence, is particularly attractive in this context, not only for its homology with the shared epitope but also for its potential role in autoimmune inflammation as a heat shock protein-derived peptide. This finding may explain the fact that that the small proportion of shared epitope-negative patients in our study had detectable responses to the peptide and was tolerized by the treatment.

Immune intervention of a defined epitope-specific proinflammatory pathway has detectable and desirable biologic effects. Epitope-specific immune modulation may therefore be a therapeutic option whose viability is being tested in a double-blind, placebo-controlled trial of clinical efficacy.

Acknowledgments

We thank Dr. W. Kwok (Virginia Mason, Seattle), who provided recombinant HLA-DR4. This work was supported by National Institutes of Health Grants 5P50 AR44850-04, N01-AR-9-2241, 2R01 AI41721-05, and 1R01AR48084-01, the Royal Netherlands Academy of Arts and Sciences, and the Dutch Organization for Scientific Research.

Notes

Abbreviations: RA, rheumatoid arthritis; PBMC, peripheral blood mononuclear cell; TNF, tumor necrosis factor; FACS, fluorescence-activated cell sorter; aAPC, artificial antigenpresenting cell; CTB, Cholera toxin subunit B; NA, neutravidin; TCC, T cell capture; Ct, threshold cycle; Treg, cells with a regulatory function.

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