Elisa Cardilicchia

Identification of a novel subset of human circulating memory CD4+ T cells that produce both IL-17A and IL-4

BACKGROUND IL-17A has been suggested to play a pathogenic role in bronchial asthma and other allergic disorders. OBJECTIVE Study of the relationship between human IL-17A-producing CD4(+) T(H) cells (T(H)17) and IL-4-producing CD4(+) T(H) (T(H)2) cells. METHODS T-cell clones generated from the CCR6(+)CD161(+) fraction of human circulating CD4(+) T cells, which contains virtually all T(H)17 cells, as well as circulating CD4(+) T cells from both healthy subjects and patients with asthma, were assessed by flow cytometry for their cytokine production profile. RESULTS A small proportion of CCR6(+)CD161(+)CD4(+) T-cell clones showed the ability to produce both IL-17A and IL-4 (T(H)17/T(H)2). T(H)17/T(H)2 clones also produced IL-5, IL-8, IL-9, IL-13, IL-21, and IL-22 and displayed the ability to induce the in vitro secretion of IgE. A very few T(H)17/T(H)2 cells were found among circulating CD4(+) T cells from normal subjects, but their proportions were significantly increased in the circulation of patients with chronic asthma. T(H)17/T(H)2 cells could not be derived from naive umbilical cord blood CD4(+) T cells under any experimental condition. However, when circulating memory CCR6(+)CD161(+)CD4(+) T cells were cloned under appropriate polarizing conditions, T(H)17/T(H)2 clones originated in the presence of IL-4, suggesting that an IL-4-rich microenvironment may induce the shifting of memory T(H)17 cells into T(H)17/T(H)2 cells. CONCLUSION Because of its peculiar functional properties and the increased numbers in the circulation of patients with bronchial asthma, this previously unknown population of T(H)17/T(H)2 cells may play some role in the pathogenesis of this disease.

Perspectives in vaccine adjuvants for allergen-specific immunotherapy

The design of more powerful adjuvants is a tool of crucial interest to ameliorate vaccination strategies to reduce injections and/or dose of antigen, induce local immunity and obtain better protection. Effective anti-infectious vaccines should elicit protective TH1 responses, cytotoxic CD8+ cells and antibody-forming cells. However, cytokine microenvironment is a key point also in targeted therapeutic vaccinations, such as allergen-specific immunotherapy, where the interference with an already-existing but inappropriate immunity is required. In this case, safe, appropriately conditioning and potentially orally available adjuvants together with delivery to appropriate subsets of dendritic cells would be highly appreciated to properly boost innate immune cells. In fact, aluminium hydroxide, although safe, has been classically associated with the induction of a TH2 response to co-formulated antigens. Thus, detoxified lipopolysaccaride (MPL-A), CpG oligonucleotides, imidazoquinolines and adenine derivatives acting via innate sensors may represent improvements in therapeutic vaccinations for allergy as able to interfere with pathogenic TH2 cells with eventual induction of TH1 differentiation.

A novel allergen-adjuvant conjugate suitable for specific immunotherapy of respiratory allergy.

BACKGROUND Several approaches to find a better adjuvant, focus immunomodulation, and reduce allergenicity are under investigation to improve the efficacy and safety of specific immunotherapy. OBJECTIVE We performed an investigation of the in vitro and in vivo effects of a purified allergen chemically conjugated to a novel 8-OH modified adenine as an adjuvant. METHODS Purified group 2 major allergen from house dust mite chemically conjugated to 4-(6-amino-9-benzyl-8-hydroxy-9H-purin-2-ylsulfanyl)-butyric acid succinimidyl ester was analyzed by using mass spectrometry. The adduct (nDer p 2-Conj) was assayed for Toll-like receptor activation on transfected HEK293 cells, stimulation of innate cells, and effects on the functional phenotype of specific T-cell lines and clones by means of flow cytometry, real-time PCR, and expression of TH-related transcription factors. Lung cells and sera of nDer p 2-Conj-sensitized C57Bl/6 mice were studied by means of cytology, histology, real-time PCR, and ELISA. RESULTS nDer p 2-Conj stimulated IL-12 and IFN-α production from monocytes and plasmacytoid dendritic cells, respectively, retaining the ability to trigger Toll-like receptor 7 exclusively, and expanded human allergen-specific lymphocytes with reduced ability to produce T(H)2-related cytokines and increased IFN-γ levels, as based on GATA-3/T-bet expression. In vivo adduct-sensitized mice exhibited reduced eosinophil infiltration and IL-13 expression in the airways, IFN-γ upregulation together with IgE downregulation, and an increase in allergen-specific IgG(2a) levels in sera. The conjugate exhibited reduced ability to activate human FcεRI(+) cells without inducing T(H)17 cells or autoantibodies. CONCLUSIONS The codelivery of an allergen with a modified adenine as a conjugate inducing modulatory cytokines from innate cells redirects in vitro and in vivo pathogenic TH2 responses without eliciting harmful effects.

The role of etanercept on the expression of markers of T helper 17 cells and their precursors in skin lesions of patients with psoriasis vulgaris.

Very recently, it has been demonstrated that CD161, retinoic acid—related orphan receptor γt (RORγt) and CC-chemokin receptor 6 (CCR6) can be considered good surface markers to detect T helper 17 cells and their precursors, T cell populations that are considered to play an important role in the pathogenesis of psoriasis. In the present study, we evaluate the clinical involvement by calculating the PASI score and the number of CD4+, CD161+, RORγt+ and CCR6+ cells before and after a 12-week course with etanercept or acitretin in patients with moderate-to-severe, plaque-type psoriasis vulgaris. Ten patients were given etanercept 50 mg twice weekly and 10 patients acitretin 0.4 mg/kg per day, both for 12 weeks. At the baseline and at the end of the treatment PASI was calculated, and skin biopsies were taken to evaluate the expression of CD4, CD161, RORyt and CCR6 by immunohistochemistry. As controls, 10 patients with atopic dermatitis (AD) were included in the study. After 12 weeks, PASI was significantly lower than at the baseline for both groups. However, etanercept-treated patients showed lower PASI than acitretin-treated ones. While CD4+ cell numbers were similar in both diseases, all the other markers, that are considered more specific for Th17 cells and their precursors, were more expressed in psoriasis than in AD. Furthermore, only etanercept, but not acitretin, was able to significantly reduce CD161+, RORγt+ and CCR6+ cells in skin lesions of patients with psoriasis. Our study provides further evidence of the role of Th17 pathway in the pathogenesis of psoriasis. Furthermore, our findings suggest that etanercept is able to downregulate the expression of the recently recognized markers of Th17 cells and their precursors CD161, RORγt and CCR6, while acitretin is not. This activity on the Th17 lineage may contribute to the efficacy of etanercept in the treatment of psoriasis.

Treatment with 8-OH-modified adenine (TLR7 ligand)-allergen conjugates decreases T helper type 2-oriented murine airway inflammation

A strategy to improve allergen‐specific immunotherapy is to employ new adjuvants stably linked to allergens. The study is addressed to evaluate the in vivo and in vitro effects of allergens [natural Dermatophagoides pteronyssinus 2 (nDer p 2) and ovalbumin (OVA)] chemically bound to an 8‐OH‐modified adenine. Humoral and cellular responses were analysed in allergen‐sensitized and challenged mice by using conjugates (Conj) in a therapeutic setting. The in vitro activity of the conjugates on cytokine production induced by bone marrow dendritic cells and the co‐culture system was also investigated. The nDer p 2‐Conj treatment in nDer p 2‐primed and challenged BALB/c mice reduced the numbers of eosinophils in bronchoalveolar lavage fluid and lung, airway allergen‐driven interleukin‐13 (IL‐13) production in lung mononuclear cells and IgE, in comparison with nDer p 2‐treated mice. The increase of IgG2a paralleled that of interferon‐γ (IFN‐γ) and IL‐10 in allergen‐stimulated spleen cells. Similar effects were elicited by treatment with OVA‐Conj in an OVA‐driven BALB/c model. The nDer p 2‐Conj or OVA‐Conj redirected memory T helper type 2 cells towards the production of IL‐10 and IFN‐γ also in C57BL/6 mice and when subcutaneously administered. Interleukin‐10, IL‐12 and IL‐27 were produced in vitro by Conj‐stimulated bone marrow dendritic cells, whereas IL‐10 and IFN‐γ were up‐regulated in co‐cultures of CD11c+ and CD4+ T cells from Conj‐treated mice stimulated with allergen. Cytofluorometric analysis indicated that the Conj expanded IFN‐γ‐ and IL‐10‐ producing memory T cells. The Conj effects on IL‐10−/− and IL‐12−/− mice confirmed the role of IL‐10 and IFN‐γ in inducing a protective and balanced redirection the T helper type 2‐mediated airway inflammation.