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Although IL-12 and IL-23 share the common p40 subunit, IL-23, rather than IL-12, seems to drive the pathogenesis of experimental autoimmune encephalomyelitis and arthritis, because IL-23/p19 knockout mice are protected from disease. In contrast, we describe in this study that newly created LacZ knockin mice deficient for IL-23 p19 were highly susceptible for the development of experimental T cell-mediated TNBS colitis and showed even more severe colitis than wild-type mice by endoscopic and histologic criteria. Subsequent studies revealed that dendritic cells from p19-deficient mice produce elevated levels of IL-12, and that IL-23 down-regulates IL-12 expression upon TLR ligation. Finally, in vivo blockade of IL-12 p40 in IL-23-deficient mice rescued mice from lethal colitis. Taken together, our data identify cross-regulation of IL-12 expression by IL-23 as novel key regulatory pathway during initiation of T cell dependent colitis.

Data regarding the role of TGF-β for the in vivo function of regulatory CD4+CD25+ T cells (Treg) are controversial. A transgenic mouse model with impaired TGF-β signaling specifically in T cells was used to assess the role of endogenous TGF-β for the in vivo function of CD4+CD25+ Treg in a murine model of colitis induced by dextran sulfate. Transfer of wild-type, but not transgenic CD4+CD25+ Treg was found to suppress colitis in wild-type mice. In addition, by transferring CFSE-labeled CD4+CD25+ Treg we could demonstrate that endogenous TGF-β promotes the expansion of CD4+CD25+ Treg in vivo. Transgenic mice themselves developed reduced numbers of peripheral CD4+CD25+ Treg and were more susceptible to the induction of colitis, which could be prevented by the transfer of wild-type Treg. These data indicate that TGF-β signaling in CD4+CD25+ Treg is required for their in vivo expansion and suppressive capacity.

In this study, we investigated the role of c-Maf, a transcription factor known to induce IL-4 production, in inflammatory bowel diseases and experimental colitis. Although Crohn′s disease (CD) is associated with low IL-4 production by T-bet-expressing Th1 cells in the lamina propria, surprisingly a higher expression of c-Maf in these cells was found as compared with control patients. The relevance of this finding was further evaluated in an animal model of CD induced by adoptive transfer of CD4+CD62L+ T cells in RAG-deficient mice. In this Th1-mediated model, an increase of c-Maf-expressing T lymphocytes in the lamina propria over time was observed. Interestingly, adoptive transfer of c-Maf transgenic CD4+CD62L+ T cells in RAG-1-deficient mice resulted in an IL-4-dependent inability to induce colitis and suppressed colitis activity induced by wild-type CD4+CD62L+ T cells. In contrast, transfer of CD4+CD62L− T cells from c-Maf transgenic, but not wild-type mice induced colitis and augmented colitis induced by CD4+CD62L+ T cells from wild-type mice in an IL-4-independent pathway, as determined by macroscopic, histologic, and endoscopic criteria. This was associated with an accumulation of CD4+ T-bet+ CD25+ effector Th1 cells in the lamina propria of colitic mice. Our results reveal a novel regulatory role of c-Maf in colitis. Although overexpression of c-Maf in naive T cells prevents Th1-mediated colitis, overexpression of c-Maf in memory T-bet+ Th1 cells regulates CD25 expression and augments such colitis. Targeting of c-Maf in memory T cells in CD appears to be an attractive target for therapeutic interventions.

We generated transgenic mice for STAT-4, a regulatory protein specifically associated with IL-12 signaling, under the control of a CMV promoter. These mice expressed strikingly increased nuclear STAT-4 levels in lamina propria CD4+ T lymphocytes upon systemic administration of dinitrophenyl-keyhole limpet hemocyanin and developed chronic transmural colitis characterized by infiltrates of mainly CD4+ T lymphocytes. The latter cells produced predominantly TNF and IFN-γ but not IL-4 upon activation with αCD3/CD28 or autologous bacterial Ags, consistent with a Th1-type cell response. Furthermore, chronic colitis in STAT-4 transgenic mice could be adoptively transferred to SCID mice by colonic and splenic CD4+ T cells that were activated with Ags from autologous bacterial flora. These data establish a critical molecular signaling pathway involving STAT-4 for the pathogenesis of chronic intestinal inflammation, and targeting of this pathway may be relevant for the treatment of colitis in humans.

IL17A is a cytokine mainly produced by Th17 cells. The function of the Th17 cell lineage in lung cancer remains to be elucidated. In our study we found increased expression of the Th17 transcription factors RORα4 and RORγt and the cytokine IL-17A in the lungs of patients with lung adenocarcinoma compared to healthy control tissue. In lung cancer tissues IL-17A inversely correlated with the Th1 specific factor T-bet. Targeting of IL-17A in a murine model of lung adenocarcinoma by intranasal application of anti IL17A antibodies resulted in reduction of lung tumor load and prolonged survival of wild-type mice. Studies in T-bet (-/-) mice revealed up-regulation of lung IL-23/IL-23-R and IL-17A/IL-17A-R in T cells infiltrating the tumor. Local anti-IL-17A antibodies treatment improved (50%) survival in treated T-bet (-/-) mice, reduced the number of IL-17R expressing lung CD4+ T cells, induced the number of natural killer cells and CD8+ T cells producing IFNγ compared to wild type littermates-bearing tumor and treated in the same way. These data might indicate that local anti IL-17A antibody therapy could be successful for the treatment of lung tumor also in the absence of T-bet.

IL-6 is a pleiotropic cytokine which induces Th2 type cells. IL-6 binds to IL-6R alpha chain and then induces gp130 dimerization resulting in the intracellular activation of Janus kinase and signal transducers and activators of transcription (STAT) pathways. We previously demonstrated that patients affected by allergic asthma have an increased level of soluble IL-6R which correlates with Th2 CD4+ T-cells in their airways. In a murine model of this disease, we also demonstrated that IL-6 inhibits the development of T-regulatory cells (Tregs) and local blockade of IL-6R resulted in the expansion of Tregs as well as Th1 cells in the lung. T-bet is a Th1-specific transcriptional factor. Mice lacking T-bet show increased asthmatic symptoms. We thus want to study the influence of T-bet on IL-6 mediated inhibition of Tregs. To this aim an antibody against IL-6R was applied to T-bet deficient mice (T-bet (-/-)) during allergen challenge. In preliminary studies we found that local blockade of IL-6R in T-bet (-/-) mice resulted in a reduced AHR and decreased lung eosinophilia. Furthermore we observed selective expansion of the CD4+ CD25+ Foxp3+ Tregs in the draining lymph-nodes taken from anti IL-6R-antibody treated T-bet (-/-) mice, compared to T-bet (-/-) untreated mice. These results suggest that local blockade of IL-6R in the absence of T-bet in allergic asthma could result in amelioration of the disease hallmarks because of inducing Tregs in the local draining lymph nodes.

Human studies have demonstrated that allergen specific immunotherapy (IT) is validated for allergic diseases. IT involves repeated subcutaneous administration of the allergen following allergen sensitization. We now established a mouse model in which subcutaneous applications with allergen suppressed the asthma-like manifestations. In this model we found an antigen dependent decrease of airway hyperresponsiveness, IgE and lung eosinophils mediated inflammation as compared to untreated mice. Consistently, Th2 as well as Th17 type cytokines were found to be reduced in the lung of antigen treated mice. Moreover, we found that this established subcutaneous immunotherapy decreased the number of lung gamma-delta (γδ)+IL17+ T cells. IL-17 production by αβ T helper cells (Th17) and the conditions of their development received much attention over the last few years, however the role of IL-17-producing γδ T cells was not fully investigated. In fact, it has been recently reported that γδ T cells represent a vast majority of IL-17-producing cells in murine lung. Revealing the role of IL-17 and lung γδ+ IL17+ T cells in asthma and in an immunotherapy model will probably lead to therapeutical improvement for this disease.

Nuclear factor of activated T cells (NFAT) proteins represent a Ca2+/calcineurin- regulated family of transcription factors that control T cell differentiation processes. Specifically NFATc1 and NFATc2 are highly expressed in peripheral T cells and activate the IL-2 and IL-4 promoters, suggesting that the Ca2+-regulated calcineurin/NFAT cascade controls alternative pathways of T cell activation and peripheral tolerance. NFATc1 is strongly activated upon antigen dependent T cell receptor engagement and induces genes that are involved in T cell effector function. In this study we describe that patients affected by lung Adenocarcinoma showed increased NFATc1 mRNA and protein levels in their lungs suggesting an involvement of NFATc1 on lung tumor development. To define the role of NFATc1 on lung cancer we start to analyze NFATc1 deficient mice in a murine model of lung Melanoma. Since NFATc1-deficient mice die at the embryonic stage, we bred conditional NFATc1 mice with CD4-Cre mice to gain insight into the role of NFATc1 in tumor T cell responses. Our preliminary results in this model showed a reduced tumor area in NFATc1-deficient mice compared to wild type mice. We also demonstrated here, that naïve T cells, isolated from NFATc1-deficient mice, produced less IL-17 under Th17- skewing conditions compared to those isolated from wild-type mice indicating a pathogenetic effect mediated by NFATc1 via IL-17 in lung tumor that must be further investigated.

Mutations of the Janus Kinase Tyrosine Kinase 2 (Tyk2) in humans are associated with hypereosinophilia, Hyper-IgE and susceptibility to viral and mycobacterial infections. This syndrome was found to be the result of defective signaling of both IL 12 and IFNalpha as well as abnormal responses to IL 6, IL-10 and IL-23. Here we describe asthmatic Tyk2 deficient mice suffered from extreme airway eosinophilic inflammation and hyper IgE production compared to wild type mice. As these symptoms are associated with an increase in Th2 cytokines IL-4 and IL-5 in the airways, the major goal of the project is to investigate the role of Tyk2 in CD4+ T cell subset differentiation in the lung in a murine model of acute asthma. Th2 and early Th17 effector T cells are important in establishing the inflammation seen in the airways in this disease. In contrast, regulatory T cells and late developed Th17 T cells have a protective function on it. Under Th17 skewing conditions naïve CD4+ T cells from Tyk2 deficient mice showed an IL-6-dependent defect on Th17 cell development. Consistently, we found hyper-proliferation of Th2 cells associated with a constitutive inhibition of the Th1 and Th17 pathway in the lung of asthmatic Tyk2 deficient mice compared to wild type mice. In summary these data suggest that overexpression of Tyk2 could lead to amelioration of the asthmatic symptoms because this would suppress the pathogenic disease-specific CD4+ T effector cells in the lung.

The range of identified effector CD4+ T cell lineages has been expanded to enclose the Th17 cells whose function in lung tumor has not been investigated yet. In this study, we report increased expression of the two newly identified transcription factors of the Th17 lineage, RORA and RORC2, in the lung of patients affected by lung adenocarcinoma. Targeting IL-17A in a murine model of lung adenocarcinoma, resulted in local expansion of CD4+ effector T cells producing IFNγ and reduction of CD4+CD25+FOXP3+ regulatory T cells. In support of a protective role of IFNγ in this model, T-bet (-/-) mice exhibited increased tumor load, decreased survival and increased residual CD4+CD25+FOXP3+ T regulatory cells after anti-IL-17A antibodies treatment as compared to wild type littermates. Thus, blockade of lung Th17 responses requires T-bet to reduce T-regulatory cells for limiting lung cancer.

The transforming growth factor beta TGFβ is known to be a potent tumour suppressor at early stages of cancer. However, cancer cells can lose responsiveness to TGFβ through mutations in the signaling pathway downstream of the TGFβ receptor. TGFβ influences the development of regulatory T cells known to enhance cancer development. We analyzed TGFβ expression in the lung of subjects affected by lung adenocarcinoma. TGFβ mRNA level was increased in the lung of these patients and this value correlated with the progression of the disease. We thus asked whether targeting TGFβ signalling in T cells would inhibit regulatory T cells in a murine model of lung adenocarcinoma. Mice that overexpress a dominant negative form of TGFβ type II receptor lacking the cytoplasmic kinase domain in T cells were used. Histologically, we found a tumour regression in the abscence of TGFβ signalling in T cells. A hyperproliferation of the CD4+ T cells overproducing interferon gamma (IFNγ) was observed. Analysis of the regulatory T cells showed an intact supression function of these cells. In summary, a defective TGFβ-signalling in lung T cells could lead to amelioration of lung adenocarcinoma in mice because inducing hyperproliferation of IFN-gamma producing CD4+ T cells thus limiting the T regulatory component in this disease. This work is supported by the GK1043 in Mainz.

Asthma is caused by T-helper 2 (Th2)-driven immune-responses in which upregulation of GATA-3 has been shown to play a crucial role in both in humans and in murine models of this disease. T-cell tolerance, induced by respiratory exposure to allergen prior to allergen sensitization, can inhibit the development of airway hyperractivity (AHR), a cardinal feature of asthma. However, the subset of T cells that mediates this protective function in allergic asthma has not been clearly identified yet. We recently set up a tolerogenic model of allergic asthma. In this model, peripheral T cell tolerance is induced by respiratory exposure to allergen and is characterized by decreased Th2 (IL-4,IL-5 and IL-13)-based responses via downregulation of the main Th2 transcription factor GATA-3 in the lung which results in decreased allergen induced AHR and lung eosinophilia. In addition, we found that our tolerogenic model is characterized also by decreased differentiation of Th17 cells. Moreover respiratory exposure to allergen prior to sensitization protected also airway hyperreactivity induced in the absence of T-bet. Thus this model suppressed effector Th2 and Th17 mediated immune responses also in the absence of IFN-gamma and could therefore provide a new avenue also for genetically determined allergic diseases. This work is supported by a SFB 548-B8 project in Mainz, Germany

Tyrosine Kinase 2 is a member of the mammalian Janus kinase (JAK) family of protein tyrosine kinases which consists of three more kinases (JAK1-3). Tyk-2 mutation in humans is associated with a completely defective in signaling in response to IFNα and IL-12, abnormal responses to IL-6, IL-10 and IL-23, susceptibility to viral infection and to mycobacterial infection and Hyper IgE Syndrome. Microscopic analyses showed that asthmatic Tyk2(-/-) mice suffered from extreme airway eosinophilic inflammation as compared to wild type mice. We found that Th1 differentiation is severely impaired in Tyk2(-/-) mice as shown by decreased IFNγ production by lung CD4+ T cells. By contrast, an increase in Th2 cytokines could be measured in the supernatants of lung CD4+ T cells isolated from Tyk2(-/-) mice. Preliminary results on T regulatory cell differentiation revealed an ascent of CD4+CD25+Foxp-3+ T regulatory cells in asthmatic Tyk2(-/-) mice by FACS analysis. Consistent with this finding, quantitative Real-Time PCR also showed an increase of the T regulatory cell-specific transcription factor Foxp3 in Tyk2(-/-) mice. As both the T regulatory inducing IL-10-pathway and the T reg inhibiting IL-6-pathway are blocked in Tyk2 deficient mice only TGFβ might be regulating Foxp-3 expression in T regulatory cells in these mice. We next wish to investigate the suppressive function of T regulatory cells in the absence of Tyk-2 in allergic models of asthma. This work is supported by Graduiertenkolleg 1043 "Antigen-specific Immunotherapy".

Epstein-Barr virus-induced gene 3 codes for a soluble type 1 receptor homologous to the p40 subunit of IL-12 that is expressed by antigen presenting cells following activation. Adoptive intravenous injection of the B16-F10 cell line resulted in a significant reduction of lung tumour metastasis in EBI-3 (−/−) recipient mice compared to wild type. Consistently, we found that EBI-3 (−/−) mice have decreased number of VCAM-1+ endothelial cells. The immunological finding accompanying this therapeutic effect was the orchestrate priming and T cell selection performed by a newly described Dendritic Cell (DC) subset called Interferon-γ producing killer Dendritic Cells (IKDC) which induced the CD8+ T cell response in the lung of EBI-3 deficient mice including Fas and TNF-α programmed tumour cell death. Finally, adoptive transfer of EBI-3 (−/−) DCs primed EBI-3 (−/−) CD8+ T cells into wild type mice decreased the number and size of lung tumour metastasis in recipient mice. Taken together, these data demonstrate that EBI-3 deficiency reduced lung tumour metastasis both because they decrease adhesion of tumour cells to lung vessels and they activate T cell mediated tumour cell apoptosis in the lung.