IL-9 exerts biological function on antigen-experienced murine T cells and exacerbates colitis induced by adoptive transfer.

IL-9 is involved in various T cell-dependent inflammatory models including colitis, encepahlitis, and asthma. However, the regulation and specificity of IL-9 responsiveness by T cells during immune responses remains poorly understood. Here, we addressed this question using two different models: experimental colitis induced by transfer of naive CD4+ CD45RBhigh T cells into immunodeficient mice, and OVA-specific T cell activation. In the colitis model, constitutive IL-9 expression exacerbated inflammation upon transfer of CD4+ CD45RBhigh T cells from WT but not from Il9r-/- mice, indicating that IL-9 acts directly on T cells. Suprisingly, such naïve CD4+ CD45RBhigh T cells failed to express the Il9r or respond to IL-9 in vitro, in contrast with CD4+ CD45RBlow T cells. By using OVA-specific T cells, we observed that T cells acquired the capacity to respond to IL-9 along with CD44 upregulation, after long-lasting (5 to 12 days) in vivo antigenic stimulation. Il9r expression was associated with Th2 and Th17 phenotypes. Interestingly, in contrast to the IL-2 response, antigen restimulation downregulated IL-9 responsiveness. Taken together, our results demonstrate that IL-9 does not act on naïve T cells but that IL-9 responsiveness is acquired by CD4+ T cells after in vivo activation and acquisition of memory markers such as CD44.

IL-24 contributes to skin inflammation in Para-Phenylenediamine-induced contact hypersensitivity.

Para-Phenylenediamine (PPD) is an aromatic amine used in hair dyes and in temporary black henna tattoos, which is a frequent cause of allergic contact dermatitis (ACD). ACD is a skin inflammatory reaction characterized by modifications such as spongiosis, exocytosis and acanthosis. The aim of this study is to characterize the expression and the role of IL-20-related cytokines, including IL-19, IL-20, IL-22 and IL-24, in ACD. The expression of IL19, IL20, IL22 and IL24 is increased in affected skin from PPD allergic patients compared with uninvolved skin. In addition, the expression of these cytokines positively correlates with clinical symptoms. To assess their role in ACD, we set up a mouse model of PPD-induced allergic contact dermatitis and we showed that, in contrast to Il22-deficient mice, Il22ra1-, Il20rb- and Il24-deficient mice are partially protected against development of PPD-induced contact hypersensitivity. These mice have decreased ear thickening and less acanthosis compared with WT mice after PPD treatment. In addition, the absence of IL-22R, IL-20R2 or IL-24 affects the recruitment of neutrophils into the skin but not the total IgE production. Taken together, these results demonstrate the implication of IL-24 via the IL-20R type II receptor in the inflammatory process of ACD.

Ccr6 Is Dispensable for the Development of Skin Lesions Induced by Imiquimod despite its Effect on Epidermal Homing of IL-22-Producing Cells.

Expression of the chemokine receptor Ccr6 is shared by most IL-22-producing cells, and Ccr6-deficient mice showed decreased IL-22 production and skin inflammation upon IL-23 intradermal injections. To determine whether this observation might be extended to another psoriasis model, we applied imiquimod on Ccr6-deficient mice. Although epidermal IL-22 production was decreased because of a deficient recruitment of γδ T cells in these mice, they were not protected against psoriatic lesions. When primary epidermis or dermis tissue culture cells from nontreated mice were stimulated ex vivo with IL-1α/IL-2/IL-23, we observed that Ccr6 is crucial for Il22 expression from epidermal but not dermal cultures. Taking advantage of Ccr6-LacZ-knock-in mice, we showed that Ccr6 is necessary for the homing of Ccr6-positive cells, probably a γδ T-cell subset, which represents the main potential IL-22 source in the epidermis. Similar results were observed in Rag1-/- epidermis and dermis primary cultures, in which a subset of innate lymphoid cells expressing Ccr6 represents the main potential source of IL-22. Taken together, our data show that Ccr6 is not required for the development of skin lesions induced by imiquimod despite its effect on epidermal homing of IL-22-producing cells.

AhR modulates the IL-22-producing cell proliferation/recruitment in imiquimod-induced psoriasis mouse model.

IL-22 has a detrimental role in skin inflammatory processes, for example in psoriasis. As transcription factor, AhR controls the IL-22 production by several cell types (i.e. Th17 cells). Here, we analyzed the role of Ahr in IL-22 production by immune cells in the inflamed skin, using an imiquimod-induced psoriasis mouse model. Our results indicate that IL-22 is expressed in the ear of imiquimod-treated Ahr(-/-) mice but less than in wild-type mice. We then studied the role of AhR on three cell populations known to produce IL-22 in the skin: γδ T cells, Th17 cells, and ILC3, and a novel IL-22-producing cell type identified in this setting: CD4(-) CD8(-) TCRß(+) T cells. We showed that AhR is required for IL-22 production by Th17, but not by the three other cell types, in the imiquimod-treated ears. Moreover, AhR has a role in the recruitment of γδ T cells, ILC3, and CD4(-) CD8(-) TCRß(+) T cells into the inflamed skin or in their local proliferation. Taken together, AhR has a direct role in IL-22 production by Th17 cells in the mouse ear skin, but not by γδ T cells, CD4(-) CD8(-) TCRß(+) T cells and ILCs.

IL-22 is required for imiquimod-induced psoriasiform skin inflammation in mice.

Psoriasis is a common chronic autoimmune skin disease of unknown cause that involves dysregulated interplay between immune cells and keratinocytes. IL-22 is a cytokine produced by the TH1, TH17, and TH22 subsets that are functionally implicated in the psoriatic pathology. We assessed the role of IL-22 in a mouse model where psoriasiform skin inflammation is triggered by topical application of the TLR7/8 agonist imiquimod. At the macroscopic level, scaly skin lesions induced by daily applications of imiquimod in wild-type mice were almost totally absent in IL-22-deficient mice or in mice treated with a blocking anti-IL-22 Ab. At the microscopic level, IL-22-deficient mice showed a dramatic decrease in the development of pustules and a partial decrease in acanthosis. At the molecular level, the absence or inhibition of IL-22 strongly decreased the expression of chemotactic factors such as CCL3 and CXCL3 and of biomarkers such as S100A8, S100A7, and keratin 14, which reflect the antimicrobial and hyperproliferative responses of keratinocytes. IL-22 also played a major role in neutrophil infiltration after imiquimod treatment. IL-23 was required for IL-22 production, and γδ TCR lymphocytes represented the major source of IL-22 in lymph nodes from imiquimod-treated mice. However, T cells were not absolutely required for IL-22 production because imiquimod-induced IL-22 expression in the skin is still preserved in Rag2(-/-) mice. Taken together, our data show that IL-22 is required for psoriasis-like lesions in the mouse imiquimod model and is produced by both T cells and innate immune cells.

Dual TCR expression biases lung inflammation in DO11.10 transgenic mice and promotes neutrophilia via microbiota-induced Th17 differentiation.

A commonly used mouse model of asthma is based on i.p. sensitization to OVA together with aluminum hydroxide (alum). In wild-type BALB/c mice, subsequent aerosol challenge using this protein generates an eosinophilic inflammation associated with Th2 cytokine expression. By constrast, in DO11.10 mice, which are transgenic for an OVA-specific TCR, the same treatment fails to induce eosinophilia, but instead promotes lung neutrophilia. In this study, we show that this neutrophilic infiltration results from increased IL-17A and IL-17F production, whereas the eosinophilic response could be restored upon blockade of IFN-γ, independently of the Th17 response. In addition, we identified a CD4(+) cell population specifically present in DO11.10 mice that mediates the same inflammatory response upon transfer into RAG2(-/-) mice. This population contained a significant proportion of cells expressing an additional endogenous TCR α-chain and was not present in RAG2(-/-) DO11.10 mice, suggesting dual antigenic specificities. This particular cell population expressed markers of memory cells, secreted high levels of IL-17A, and other cytokines after short-term restimulation in vitro, and triggered a neutrophilic response in vivo upon OVA aerosol challenge. The relative numbers of these dual TCR lymphocytes increased with the age of the animals, and IL-17 production was abolished if mice were treated with large-spectrum antibiotics, suggesting that their differentiation depends on foreign Ags provided by gut microflora. Taken together, our data indicate that dual TCR expression biases the OVA-specific response in DO11.10 mice by inhibiting eosinophilic responses via IFN-γ and promoting a neutrophilic inflammation via microbiota-induced Th17 differentiation.

IL-9 promotes IL-13-dependent paneth cell hyperplasia and up-regulation of innate immunity mediators in intestinal mucosa.

IL-9 contributes to lung inflammatory processes such as asthma, by promoting mast cell differentiation, B cell activation, eosinophilia, and mucus production by lung epithelial cells. The observation that IL-9 overexpressing mice show increased mast cell numbers in the intestinal mucosa suggests that this cytokine might also play a role in intestinal inflammation. In colons from IL-9 transgenic mice, the expression of Muc2, a major intestinal mucin gene, was up-regulated, together with that of CLCA3 chloride channel and resistin like alpha, which are goblet cell-associated genes. Additional IL-9 up-regulated genes were identified and included innate immunity genes such as angiogenin 4 and the PLA2g2a phospholipase A(2), which are typical Paneth cell markers. Histochemical staining of Paneth cells by phloxine/tartrazine showed that IL-9 induces Paneth cell hyperplasia in Lieberkühn glands of the small intestine, and in the colonic mucosa, where this cell type is normally absent. Expression of Paneth cell markers, including angiogenin 4, PLA2g2a, and cryptdins, was induced in the colon of wild-type mice after two to four daily administrations of IL-9. By crossing IL-9 transgenic mice with IL-13(-/-) mice, or by injecting IL-9 into IL-4R(-/-) mice, we showed that IL-13 was required for the up-regulation of these Paneth cell-specific genes by IL-9. Taken together, our data indicate that Paneth cell hyperplasia and expression of their various antimicrobial products contribute to the immune response driven by TH2 cytokines, such as IL-9 and IL-13 in the intestinal mucosa.

Comparative prime-boost vaccinations using Semliki Forest virus, adenovirus, and ALVAC vectors demonstrate differences in the generation of a protective central memory CTL response against the P815 tumor.

Tumor-specific Ags are potential target molecules in the therapeutic treatment of cancer. One way to elicit potent immune responses against these Ags is to use recombinant viruses, which activate both the innate and the adaptive arms of the immune system. In this study, we have compared Semliki Forest virus (SFV), adenovirus, and ALVAC (poxvirus) vectors for their capacity to induce CD8(+) T cell responses against the P1A tumor Ag and to elicit protection against subsequent challenge injection of P1A-expressing P815 tumor cells in DBA/2 mice. Both homologous and heterologous prime-boost regimens were studied. In most cases, both higher CD8(+) T cell responses and better tumor protections were observed in mice immunized with heterologous prime-boost regimens, suggesting that the combination of different viral vectors is beneficial for the induction of an effective immune response. However, homologous immunization with SFV provided potent tumor protection despite a rather moderate primary CD8(+) T cell response as compared with mice immunized with recombinant adenovirus. SFV-immunized mice showed a rapid and more extensive expansion of P1A-specific CD8(+) T cells in the tumor-draining lymph node after tumor challenge and had a higher frequency of CD62L(+) P1A-specific T cells in the blood, spleen, and lymph nodes as compared with adenoimmunized mice. Our results indicate that not only the magnitude but in particular the quality of the CD8(+) T cell response correlates with tumor protection.

IL-13 mediates in vivo IL-9 activities on lung epithelial cells but not on hematopoietic cells.

Increased IL-9 expression, either systemically or under the control of lung-specific promoter, induces an asthma-like phenotype, including mucus overproduction, mastocytosis, lung eosinophilia, and airway hyperresponsiveness. These activities correlate with increased production of other Th2 cytokines such as IL-4, IL-5, and IL-13 in IL-9 Tg mice. To determine the exact role of IL-13 in this phenotype, mice overexpressing IL-9 were crossed with IL-13-deficient mice. In these animals, IL-9 could still induce mastocytosis and B lymphocyte infiltration of the lungs. Although IL-9-induced eosinophilia in the peritoneal cavity was not diminished in the absence of IL-13, IL-13 was required for IL-9 to increase eotaxin expression and lung eosinophilia. Mucus production and up-regulation of lung epithelial genes upon IL-9 overexpression were completely abolished in the absence of IL-13. Using hemopoietic cell transfer experiments with recipients that overexpressed IL-9 but were deficient in the IL-9 receptor (IL-9R), we could demonstrate that the effect of IL-9 on lung epithelial cells is indirect and could be fully restored by transfer of hemopoietic cells expressing IL-9R. Mucus production by lung epithelial cells was only up-regulated when hemopoietic cells simultaneously expressed functional IL-9R and IL-13 genes, indicating that IL-13 is not a cofactor but a direct mediator of the effect of IL-9 on lung epithelial cells. Taken together, these data indicate that IL-9 can promote asthma through IL-13-independent pathways via expansion of mast cells, eosinophils, and B cells, and through induction of IL-13 production by hemopoietic cells for mucus production and recruitment of eosinophils by lung epithelial cells.

Interleukin-17 inhibits tumor cell growth by means of a T-cell-dependent mechanism.

Interleukin 17 (IL-17) is a proinflammatory cytokine produced by activated CD4(+) memory T cells. We previously showed that IL-17 increased the growth rate of human cervical tumors transplanted into athymic nude mice. To address the possible role of T cells in the biologic activity of IL-17 for tumor control, we grafted 2 murine hematopoietic immunogenic tumors (P815 and J558L) transfected with a complementary DNA encoding murine IL-17 into syngeneic immunocompetent mice. We found that growth of the 2 IL-17-producing tumors was significantly inhibited compared with that of mock-transfected tumors. In contrast to the antitumor activity of IL-17 observed in immunocompetent mice, we observed no difference in the in vivo growth of IL-17-transfected or mock-transfected P815 cells (P815-IL-17 and P815-Neo, respectively) transplanted into nude mice. We then showed that IL-17 increased generation of specific cytolytic T lymphocytes (CTLs) directed against the immunodominant antigens from P815 called A, B, C, D, and E, since all mice injected with P815-IL-17 developed a P815-specific CTL response, whereas only 6 of 16 mice immunized with P815-Neo had a specific CTL response against the antigens. The induction of CTLs was associated with establishment of a tumor-protective immunity. These experiments suggest that T lymphocytes are involved in the antitumor activity of IL-17. Therefore, IL-17, like other cytokines, appears to be a pleiotropic cytokine with possible protumor or antitumor effects on tumor development, which often depends on the immunogenicity of tumor models.

The production of a new MAGE-3 peptide presented to cytolytic T lymphocytes by HLA-B40 requires the immunoproteasome.

By stimulating human CD8(+) T lymphocytes with autologous dendritic cells infected with an adenovirus encoding MAGE-3, we obtained a cytotoxic T lymphocyte (CTL) clone that recognized a new MAGE-3 antigenic peptide, AELVHFLLL, which is presented by HLA-B40. This peptide is also encoded by MAGE-12. The CTL clone recognized MAGE-3--expressing tumor cells only when they were first treated with IFN-gamma. Since this treatment is known to induce the exchange of the three catalytic subunits of the proteasome to form the immunoproteasome, this result suggested that the processing of this MAGE-3 peptide required the immunoproteasome. Transfection experiments showed that the substitution of beta5i (LMP7) for beta5 is necessary and sufficient for producing the peptide, whereas a mutated form of beta5i (LMP7) lacking the catalytically active site was ineffective. Mass spectrometric analyses of in vitro digestions of a long precursor peptide with either proteasome type showed that the immunoproteasome produced the antigenic peptide more efficiently, whereas the standard proteasome more efficiently introduced cleavages destroying the antigenic peptide. This is the first example of a tumor-specific antigen exclusively presented by tumor cells expressing the immunoproteasome.