IL‐10‐modulated dendritic cells from birch pollen‐ and hazelnut‐allergic patients facilitate Treg‐mediated allergen‐specific and cross‐reactive tolerance

Background Approximately 70% of individuals allergic to birch pollen (Bet v 1.01 [Bet]) develop a secondary food allergy (e.g., hazelnut: Cor a 1.04 [Cor]), due to allergen cross-reactivity. However, standard immunotherapy for type I allergies often does not improve the food allergy sufficiently. We analyzed the allergen-specific and cross-reactive suppressive capacity of primary human regulatory T cells (Treg) induced by autologous IL-10-modulated dendritic cells (IL-10 DC) in vitro and in vivo. Methods CD4+ T cells of patients with birch pollen and associated hazelnut allergies were differentiated into Bet-specific or non-specific induced Treg (iTreg). After Bet- or Cor-specific restimulation the phenotype, proliferation, and suppressive capacity of iTreg subsets were analyzed. iTreg function was further investigated in humanized mouse models of airway and intestinal allergy, generated by engraftment of peripheral blood mononuclear cells from allergic donors into immunodeficient animals. Results After IL-10 DC priming and allergen-specific restimulation (Bet or Cor), non-specific control iTreg remained anergic, whereas Bet-specific iTreg proliferated extensively and exhibited a regulatory phenotype (enhanced expression of CTLA-4, PD-1, TNFR2, IL-10). Accordingly, activated Bet-specific iTreg displayed a high capacity to suppress Bet- and Cor-induced responder Th2 cell responses in vitro, indicating induction of both allergen-specific (birch) and cross-reactive tolerance (hazelnut). In vivo, the beneficial effect of Bet-specific iTreg was verified in humanized mouse models of allergic airway and intestinal inflammation, resulting in reduced allergen-induced clinical symptoms, and immune responses. Conclusion Human IL-10 DC-induced iTreg facilitate allergen-specific and cross-reactive tolerance. Therefore, they are potential candidates for regulatory cell therapy in allergic and autoimmune diseases

Interferon-α Abrogates Tolerance Induction by Human Tolerogenic Dendritic Cells

BACKGROUND: Administration of interferon-α (IFN-α) represents an approved adjuvant therapy as reported for malignancies like melanoma and several viral infections. In malignant diseases, tolerance processes are critically involved in tumor progression. In this study, the effect of IFN-α on tolerance induction by human tolerogenic dendritic cells (DC) was analyzed. We focussed on tolerogenic IL-10-modulated DC (IL-10 DC) that are known to induce anergic regulatory T cells (iTregs). METHODOLOGY/PRINCIPAL FINDINGS: IFN-α promoted an enhanced maturation of IL-10 DC as demonstrated by upregulation of the differentiation marker CD83 as well as costimulatory molecules. IFN-α treatment resulted in an increased capacity of DC to stimulate T cell activation compared to control tolerogenic DC. We observed a strengthened T cell proliferation and increased IFN-γ production of CD4(+) and CD8(+) T cells stimulated by IFN-α-DC, demonstrating a restoration of the immunogenic capacity of tolerogenic DC in the presence of IFN-α. Notably, restimulation experiments revealed that IFN-α treatment of tolerogenic DC abolished the induction of T cell anergy and suppressor function of iTregs. In contrast, IFN-α neither affected the priming of iTregs nor converted iTregs into effector T cells. CONCLUSIONS/SIGNIFICANCE: IFN-α inhibits the induction of T cell tolerance by reversing the tolerogenic function of human DC

IL-10–modulated human dendritic cells for clinical use: identification of a stable and migratory subset with improved tolerogenic activity

Abstract Dendritic cells (DCs) are key regulators of protective immune responses and tolerance to (self-)Ags. Therefore, the scientific rationale for the use of tolerogenic DC therapy in the fields of allergies, autoimmunity, and transplantation medicine is strong. In this study, we analyzed the tolerogenic capacity of IL-10–modulated DC (IL-10DC) subpopulations to identify a DC subset that combines potent immunosuppressive activities with valuable immune properties for clinical implementation. IL-10DCs consist of two phenotypically distinct subpopulations: CD83highCCR7+ IL-10DCs and CD83lowCCR7− IL-10DCs. Suppressor assays with activated effector T cells revealed that CD4+ regulatory T cells generated by CD83high IL-10DCs (iTreg+) exhibited a significantly higher suppressive capacity compared with CD4+ regulatory T cells generated by CD83low IL-10DCs (iTreg−). In this context, iTreg+ displayed a more activated phenotype (proliferation, cytokine production) compared with iTreg−. In contrast to CD83low IL-10DCs, CD83high IL-10DCs exerted a strong migratory capacity toward the secondary lymphoid organ chemokine CCL21 and retained a functionally stable phenotype under inflammatory conditions. In addition, CD83high IL-10DCs expressed significantly higher levels of surface and soluble CD25. Functional analysis demonstrated that IL-10DC–related soluble CD25 efficiently inhibited the proliferation of activated T cells and that blockade of CD25 function abolished the induction of regulatory T cells by IL-10DCs, indicating a critical role for IL-10DC–related CD25 in shifting the immune response toward an iTreg− controlled tolerance reaction. In conclusion, the selective use of the CD83high IL-10DC subset may result in a higher efficacy of tolerance induction in vivo and may support the development of novel DC vaccination strategies for transplantations, as well as for allergic and autoimmune diseases.</jats:p

IFN-α did not interfere with priming or perpetuation of T cell anergy.

<p>(A) CD4⁺ T cells were cocultured with allogeneic mDC or IL-10 DC and incubated with concentrations of IFN-α as indicated. After 5 days, T cell proliferation was assessed by [³H] thymidine incorporation, results are depicted in absolute cpm +/− SD (left panel) and in percentages +/− SD (100% = T cell proliferation induced by mDC) (right panel). One representative experiment of 3 is shown. (B) CD4⁺ T cells primed with allogeneic mDC or IL-10 DC were restimulated with anti-CD3/anti-CD28 in the presence of increasing concentrations of IFN-α as indicated. T cell proliferation was detected after 72 hours of restimulation as described. Results are shown in absolute cpm +/− SD (left panel) and in percentages +/− SD (normalized to proliferation induced by mDC = 100%) (right panel). One representative experiment of 2 is demonstrated. n.s. not significant.</p

IL-10 DC generated in the presence of IFN-α induced an enhanced T cell activation.

<p>(A, B) CD4⁺ or CD8⁺ T cells were stimulated in primary culture with allogeneic mDC, IL-10 DC or IL-10 DC cultured with 10⁴ U/mL IFN-α. (A) T cell proliferation after restimulation was assessed as described and pooled data of 6 (CD4⁺ T cells) or 3 (CD8⁺ T cells) independent experiments are demonstrated. Proliferation is demonstrated in % +/−SD (normalized to 100% proliferation induced by control mDC). (B) IFN-γ and IL-10 production were detected in the supernatants of CD4⁺ T cells at day 5 of primary culture by ELISA. Mean values of cytokine levels in pg/ml +/− SD of 6 (IFN-γ) or 5 (IL-10) independent experiments are shown. * p<0.05; ** p<0.01; n.s. not significant.</p

IFN-α treatment of IL-10 DC abolished suppressor function of iTregs.

<p>(A, B) CD4⁺ or CD8⁺ T cells were cultured in primary culture with allogeneic mDC, IL-10 DC or IL-10 DC treated with 10⁴ U/mL IFN-α, respectively. Restimulation experiments were performed with anti-CD3/anti-CD28. T cell proliferation is depicted in % (normalized to 100% proliferation induced by mDC). Mean values +/− SD of 4 (CD4⁺) or 3 (CD8⁺ T cells) independent experiments are shown. (B) Supernatants were obtained after 72 h and analyzed for IFN-γ production by ELISA. One representative experiment is demonstrated for CD4⁺ and CD8⁺ T cells, respectively. (C) Suppressor assay: CD4⁺ T cells primed with allogeneic mDC (iTeff), IL-10 DC (iTregs) or IL-10 DC treated with 10⁴ U/mL IFN-α (iTregs + IFN-α), respectively, or freshly isolated CD4⁺CD25^high nTregs were cocultured with CD4⁺CD25^low effector T cells and stimulated by 0.5 µg/mL anti-CD3 in the presence of irradiated PBMC. T cell proliferation was determined by [³H] thymidine incorporation. Suppressor activity is shown in proliferation of Teff + cocultured T cell population/poliferation of Teff ×100 in % +/− SD. One representative experiment of 3 is demonstrated. Results are shown in absolute cpm +/− SD for triplicates. * p<0.05; ** p<0.01; *** p<0.001, n.s. not significant.</p

Immunophenotype of IL-10 DC after IFN-α treatment.

<p>(A, B) Immature DC were stimulated at day 5 of culture with a maturation cocktail (mDC) or a maturation cocktail supplemented with IL-10 either with or without 10⁴ U/mL IFN-α. (A) Dot plots of one representative experiment and (B) expression of surface molecules of DC of 6 independent experiments are depicted. (C) mDC and IL-10 DC and IL-10 DC +/−10⁴ U/mL IFN-α were generated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022763#s4" target="_blank">Materials and Methods</a>. At day 7, supernatants were collected and production of IL-12 p40 was assessed by ELISA. Mean values +/− standard deviation (SD) of 4 independent experiments are shown; nd: not detected. * p<0.05; ** p<0.01; *** p<0.001, n.s. not significant.</p

Interferon-α suppresses cAMP to disarm human regulatory T cells

Abstract IFN-α is an antineoplastic agent in the treatment of several solid and hematologic malignancies that exerts strong immune- and autoimmune-stimulating activity. However, the mechanisms of immune activation by IFN-α remain incompletely understood, particularly with regard to CD4+CD25highFoxp+ regulatory T cells (Treg). Here, we show that IFN-α deactivates the suppressive function of human Treg by downregulating their intracellular cAMP level. IFN-α–mediated Treg inactivation increased CD4+ effector T-cell activation and natural killer cell tumor cytotoxicity. Mechanistically, repression of cAMP in Treg was caused by IFN-α–induced MAP–ERK kinase (MEK)/extracellular signal-regulated kinase (ERK)–mediated phosphodiesterase 4 (PDE4) activation and accompanied by downregulation of IFN receptor (IFNAR)-2 and negative regulation of T-cell receptor signaling. IFN-α did not affect the anergic state, cytokine production, Foxp3 expression, or methylation state of the Treg-specific demethylated region (TSDR) within the FOXP3 locus associated with a stable imprinted phenotype of human Treg. Abrogated protection by IFN-α–treated Treg in a humanized mouse model of xenogeneic graft-versus-host disease confirmed IFN-α–dependent regulation of Treg activity in vivo. Collectively, the present study unravels Treg inactivation as a novel IFN-α activity that provides a conceivable explanation for the immune-promoting effect and induction of autoimmunity by IFN-α treatment in patients with cancer and suggests IFN-α for concomitant Treg blockade in the context of therapeutic vaccination against tumor antigens. Cancer Res; 73(18); 5647–56. ©2013 AACR.</jats:p