Sonja Reissig

Dependence on nuclear factor of activated T-cells (NFAT) levels discriminates conventional T cells from Foxp3+ regulatory T cells.

Several lines of evidence suggest nuclear factor of activated T-cells (NFAT) to control regulatory T cells: thymus-derived naturally occurring regulatory T cells (nTreg) depend on calcium signals, the Foxp3 gene harbors several NFAT binding sites, and the Foxp3 (Fork head box P3) protein interacts with NFAT. Therefore, we investigated the impact of NFAT on Foxp3 expression. Indeed, the generation of peripherally induced Treg (iTreg) by TGF-β was highly dependent on NFAT expression because the ability of CD4+ T cells to differentiate into iTreg diminished markedly with the number of NFAT family members missing. It can be concluded that the expression of Foxp3 in TGF-β–induced iTreg depends on the threshold value of NFAT rather than on an individual member present. This is specific for iTreg development, because frequency of nTreg remained unaltered in mice lacking NFAT1, NFAT2, or NFAT4 alone or in combination. Different from expectation, however, the function of both nTreg and iTreg was independent on robust NFAT levels, reflected by less nuclear NFAT in nTreg and iTreg. Accordingly, absence of one or two NFAT members did not alter suppressor activity in vitro or during colitis and transplantation in vivo. This scenario emphasizes an inhibition of high NFAT activity as treatment for autoimmune diseases and in transplantation, selectively targeting the proinflammatory conventional T cells, while keeping Treg functional.

A20 deficiency in B cells enhances B-cell proliferation and results in the development of autoantibodies

A20/TNFAIP3 is an ubiquitin‐editing enzyme, important for the regulation of the NF‐κB pathway. Mutations in the TNFAIP3 gene have been linked to different human autoimmune disorders. In human B‐cell lymphomas, the inactivation of A20 results in constitutive NF‐κB activation. Recent studies demonstrate that in mice the germline inactivation of A20 leads to early lethality, due to inflammation in multiple organs of the body. In this report, we describe a new mouse strain allowing for the tissue‐specific deletion of A20. We show that B‐cell‐specific deletion of A20 results in a dramatic reduction in marginal zone B cells. Furthermore, A20‐deficient B cells display a hyperactive phenotype represented by enhanced proliferation upon activation. Finally, these mice develop higher levels of serum immunoglobulins, resulting in an excessive production of self‐reactive autoantibodies.

IL-6 Regulates Neutrophil Microabscess Formation in IL-17A-Driven Psoriasiform Lesions

The lack of a generally accepted animal model for human psoriasis has hindered progress with respect to understanding the pathogenesis of the disease. Here we present a model in which transgenic IL-17A expression is targeted to the skin in mice, achievable after crossing our IL-17A(ind) allele to the K14-Cre strain. K14-IL-17A(ind/+) mice invariably develop an overt skin inflammation bearing many hallmark characteristics of human psoriasis including dermal infiltration of effector T cells, formation of neutrophil microabscesses, and hyperkeratosis. IL-17A expression in the skin results in upregulated granulopoiesis and migration of IL-6R-expressing neutrophils into the skin. Neutralization of IL-6 signaling efficiently reduces the observed pathogenesis in skin of IL-17A-overexpressing mice, with marked reductions in epidermal neutrophil abscess formation and epidermal thickening. Thus, IL-6 functions downstream of IL-17A to exacerbate neutrophil microabscess development in psoriasiform lesions.

The Tumor Suppressor CYLD Controls the Function of Murine Regulatory T Cells

CYLD was originally identified as a tumor suppressor gene mutated in familial cylindromatosis, an autosomal dominant predisposition to multiple benign neoplasms of the skin known as cylindromas. The CYLD protein is a deubiquitinating enzyme that acts as a negative regulator of NF-κB and JNK signaling through its interaction with NEMO and TNFR-associated factor 2. We have previously described a novel mouse strain that expresses solely and excessively a naturally occurring splice variant of CYLD (CYLDex7/8). In this study, we demonstrate that CYLD plays a critical role in Treg development and function. T cells of CYLDex7/8 mice had a hyperactive phenotype manifested by increased production of inflammatory cytokines and constitutive activation of the NF-κB pathway. Furthermore, the amount of Foxp3+ regulatory T cells in these mice was markedly enhanced in thymus and peripheral organs. Importantly, these regulatory T cells displayed decreased expression levels of CD25 and CTLA-4 associated with impaired suppressive capacity. Hence, our data emphasize an essential role of CYLD in maintaining T cell homeostasis as well as normal T regulatory cell function, thereby controlling abnormal T cell responses.

Overexpression of Bcl-3 inhibits the development of marginal zone B cells.

The transcription factor Bcl‐3 functions as a proto‐oncogene via regulation of cell proliferation and apoptosis. Bcl‐3 is an atypical member of the IκB family and plays a central role in the immune response through interactions with the NF‐κB subunits p50 and p52. To investigate the impact of Bcl‐3 on B‐cell maturation and regulation, we generated mice that overexpress Bcl‐3 specifically in B cells. Interestingly, these mice lack marginal zone B cells and exhibit a significant reduction in the number of B‐1 B cells. Further, B cells from these mice are impaired in their proliferative capacity. Our data demonstrate that the overexpression of the transcription factor Bcl‐3 inhibits germinal center formation, marginal zone B‐cell development, and affects the B‐1 B‐cell compartment.

Activated glycoprotein A repetitions predominant (GARP)–expressing regulatory T cells inhibit allergen-induced intestinal inflammation in humanized mice

BACKGROUND Recently, we developed a humanized mouse model of allergen-induced IgE-dependent gut inflammation in PBMC-engrafted immunodeficient mice. OBJECTIVE In the present study, we wanted to investigate the role of regulatory T (Treg) cells and their activation status in this model. METHODS Nonobese diabetic-severe combined immunodeficiency-γc(-/-) mice were injected intraperitoneally with human PBMCs from allergic donors together with the respective allergen or NaCl as control in the presence or absence of different concentrations of CD4(+)CD25(+) Treg cells of the same donor. After an additional allergen boost 1 week later, mice were challenged with the allergen rectally on day 21 and gut inflammation was monitored by a high-resolution video mini-endoscopic system evaluating translucency, granularity, fibrin production, vascularity, and stool. RESULTS Allergen-specific human IgE in mouse sera, which was detectable only in PBMC plus allergen-treated mice, was strongly inhibited by coinjection of Treg cells at a ratio of at least 1:10. Consequently, the presence of Treg cells significantly decreased IgE-dependent allergen-induced gut inflammation after rectal allergen challenge. In addition, Treg cells reduced allergen-specific proliferation and cytokine production of recovered human CD4(+) T cells in vitro. Activation of Treg cells before injection further increased all inhibitory effects. Prevention of gut inflammation also occurred by the administration of glycoprotein A repetitions predominant, a molecule expressed by activated Treg cells, whereas its blockade completely abrogated inhibition by Treg cells. CONCLUSIONS These results demonstrate that allergen-specific gut inflammation in human PBMC-engrafted mice can be avoided by enhancing the numbers or activity of autologous Treg cells, which is of great interest for therapeutic intervention of allergic diseases of the intestine.

The ubiquitin-specific protease USP8 is critical for the development and homeostasis of T cells

The modification of proteins by ubiquitin has a major role in cells of the immune system and is counteracted by various deubiquitinating enzymes (DUBs) with poorly defined functions. Here we identified the ubiquitin-specific protease USP8 as a regulatory component of the T cell antigen receptor (TCR) signalosome that interacted with the adaptor Gads and the regulatory molecule 14-3-3β. Caspase-dependent processing of USP8 occurred after stimulation of the TCR. T cell–specific deletion of USP8 in mice revealed that USP8 was essential for thymocyte maturation and upregulation of the gene encoding the cytokine receptor IL-7Rα mediated by the transcription factor Foxo1. Mice with T cell–specific USP8 deficiency developed colitis that was promoted by disturbed T cell homeostasis, a predominance of CD8+ γδ T cells in the intestine and impaired regulatory T cell function. Collectively, our data reveal an unexpected role for USP8 as an immunomodulatory DUB in T cells.

The deubiquitinating enzyme CYLD regulates the differentiation and maturation of thymic medullary epithelial cells

The cross talk between thymocytes and the thymic epithelium is critical for T‐cell development and the establishment of central tolerance. Medullary thymic epithelial cells (mTECs) are located in the thymic medulla and mediate the elimination of self‐reactive thymocytes, thereby preventing the onset of autoimmunity. Previous studies identified the deubiquitinating enzyme CYLD as a critical regulator of T‐cell development by activating proximal T‐cell receptor signaling during the transition of double‐positive to single‐positive thymocytes. Here we evaluated the impact of the naturally occurring short‐splice variant of the cyld gene (sCYLD) on the development and maturation of mTECs. We found that thymi of CYLDex7/8 mice, solely expressing sCYLD, displayed a reduced number of mature mTECs caused by a developmental block during the transition of immature to mature mTECs. Further, we could demonstrate an impaired negative selection of thymocytes in these mice. Our data demonstrate that inefficient negative selection in the thymus of CYLDex7/8 mice result from a defect in mTEC maturation.

Mutated cylindromatosis gene affects the functional state of dendritic cells

Cylindromatosis gene (CYLD) is a ubiquitously expressed deubiquitinating enzyme, which interacts with members of the NF‐κB signaling pathway and attenuates NF‐κB and JNK signaling. Here, we report that DC derived from transgenic mice, which solely express a naturally occurring CYLD isoform (CYLDex7/8), display a higher content of nuclear RelB and express elevated levels of NF‐κB family members as well as of known NF‐κB‐target genes comprising costimulatory molecules and pro‐inflammatory cytokines, as compared with WT DC. Accordingly, unstimulated CYLDex7/8 DC exhibited a significantly higher primary allogenic T‐cell stimulatory capacity than WT DC and exerted no tolerogenic activity. Transduction of unstimulated CYLDex7/8 DC with relB‐specific shRNA reduced their T‐cell stimulatory capacity. Treatment with the synthetic glucocorticoid dexamethasone known to inhibit NF‐κB and AP‐1 activity reverted the pro‐immunogenic phenotype and function of CYLDex7/8 DC and re‐established their tolerogenic function. DC derived from CYLD knockout mice showed no functional alterations compared with WT DC. Therefore, although complete loss of CYLD may be compensated for by other endogenous NF‐κB inhibitors, CYLDex7/8 acts in a dominant negative manner. Our findings raise the question of whether genetic defects associated with increased NF‐κB activity may result in disturbed maintenance of peripheral tolerance.

Cylindromatosis (Cyld) gene mutation in T cells promotes the development of an IL-9-dependent allergic phenotype in experimental asthma

Cylindromatosis (CYLD) is a ubiquitously expressed deubiquitinating enzyme which removes activating ubiquitin residues from important signaling molecules of the NF-κB pathway. In CYLDex7/8 transgenic mice, a naturally occurring short isoform (sCYLD) is overexpressed in the absence of full length CYLD, leading to excessive NF-κB activity. Herein, we investigated the impact of the CYLDex7/8 mutation selectively in T cells on the development of experimental allergic airway disease induced by sensitization and challenge with ovalbumin. Compared with their wildtype littermates, mice bearing the T cell-specific mutation (CD4+CYLDex7/8) display stronger eosinophilia and mucus production in the lungs and higher IgE serum levels. The reason for these observations is excessive production of T cell-derived IL-9, a cytokine to whom allergy-promoting properties were ascribed. Consequently, blockade of IL-9 in CD4+CYLDex7/8 mice alleviates the development of disease symptoms. Thus, by polarization of the T cell cytokine response, sCYLD can favor the development of allergic airway disease.