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-gamma, and tumor necrosis factor-alpha decreased significantly. The total number of dnaJP1-specific cells did not change over time, whereas expression of foxP3 by CD4+CD25(bright) 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.

Figures

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 ligand (b) in a standard proliferation assay. Phytohemagglutinin (a, ▴) was used as a positive control for T cell proliferation. The y axis shows the stimulation index: the mean cpm in antigenstimulated cultures divided by the mean cpm in nonstimulated cultures. *, P < 0.001. Intracellular production of IL-2, IFN-γ, and TNF-α in response to dnaJP1 (c, e, and g) or PADRE peptide (d–f and h, respectively) at day 0 assessed at monthly intervals during treatment. Cytokine production is expressed as the percentage of CD3/cytokine double-positive cells measured by FACS in antigen-stimulated cultures, nonstimulated cultures (y axis). Error bars represent SD of the mean. Intracellular production of IL-4 (i) and IL-10 (k) after in vitro culture with dnaJP1 or with PADRE (j and l) at days 0, 56, and 168 of the treatment period.

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 during 48 h with 10 μg/ml dnaJP1 peptide. After in vitro stimulation, PBMCs stained with anti CD4 (phycoerythrin) antibody were incubated with tetramers (FITC) or aAPCs (FITC) loaded with a dnaJP1 or negative control PADRE (-).

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 patients included in this study were expanded in vitro with dnaJP1. Viable cells were harvested, permeabilized, stained for surface markers and intracellular cytokines (IL-4 and IFN-γ), incubated with aAPCs loaded with MHC class II and dnaJP1, and analyzed by FACS. The number of T cells producing either IFN-γ or IL-4 within the dnaJP1-specific population was calculated as percentage of the total number of dnaJP1-specific CD3-positive cells (y axis). Columns represent means (black bars, before treatment; gray bars, after treatment), and error bars represent SD. *, statistical significance.

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 sorted for CD4+/CD25+ cells before and after 48 h in vitro stimulation with dnaJP1. Total RNA was extracted, and the gene expression profile was analyzed by TaqMan. Results are expressed as Ct values, which were normalized according the expression of GAPDH. Induction index is the result of normalization process (arbitrary units) and refers to how many time the gene expression changed compared with not stimulated. Data were evaluated by ANOVA after removing the interaction term and running a main-effects-only model. Columns respresent means, and bars represent SD.