Transforming growth factor-β (TGF-β) signaling in naive T cells induces expression of the transcription factor Foxp3, a 'master' regulator of regulatory T cells (T reg cells). However, the molecular mechanisms leading to Foxp3 induction remain unclear. Here we show that Itch −/− T cells were resistant to TGF-β treatment and had less Foxp3 expression. The E3 ubiquitin ligase Itch associated with and promoted conjugation of ubiquitin to the transcription factor TIEG1. Itch cooperated with TIEG1 to induce Foxp3 expression, which was reversed by TIEG1 deficiency. Functionally, 'TGF-β-converted' T reg cells generated from TIEG1-deficient mice were unable to suppress airway inflammation in vivo. These results suggest TIEG and Itch contribute to a ubiquitindependent nonproteolytic pathway that regulates inducible Foxp3 expression and the control of allergic responses.Allergic asthma is a chronic inflammatory disease of the airways characterized by higher serum immunoglobulin E (IgE) concentrations, airway hyper-responsiveness, excessive airway mucus production, lung eosinophilia and airway remodeling. Differentiation of CD4 + T cells into T helper type 2 (T H 2) cells renders them able to produce the cytokines interleukin 4 (IL-4), IL-5 and IL-13, which are critical for driving asthmatic pathogenesis 1,2 . The induction of T H 2 differentiation is regulated by many factors, including the properties of the antigens, the duration of stimulation, and genetic factors. In addition, the T H 2-mediated immune responses are counterbalanced by tolerogenic mechanisms that prevent excessive reactions. One of the tolerance mechanisms involves regulatory T cells (T reg cells), which limit allergic responses 3 . In mouse models of allergic asthma, repeated delivery of antigen to the respiratory tract results in inhibition of allergic responses to the same antigen after subsequent challenges 4 . Published studies have shown that repeated treatment with a low dose of antigen induces the generation of Foxp3 + CD4 + CD25 + T reg cell population, which accounts for the attenuation of T H 2-mediated airway inflammation 5,6 . T reg cells represent a unique subpopulation of CD4 + T cells that suppress the effector function of other types of T cells and

The transcription factor Foxp3 is essential for optimal regulatory T (T reg) cell development and function. Here, we show that CD4+ T cells from Cbl-b RING finger mutant knockin or Cbl-b–deficient mice show impaired TGF-β–induced Foxp3 expression. These T cells display augmented Foxo3a phosphorylation, but normal TGF-β signaling. Expression of Foxo3a rescues Foxp3 expression in Cbl-b–deficient T cells, and Foxo3a deficiency results in defective TGF-β–driven Foxp3 induction. A Foxo3a-binding motif is present in a proximal region of the Foxp3 promoter, and is required for Foxo3a association. Foxo1 exerts similar effects as Foxo3a on Foxp3 expression. This study reveals that Foxo factors promote transcription of the Foxp3 gene in induced T reg cells, and thus provides new mechanistic insight into Foxo-mediated T cell regulation.

A main role for interleukin-4 (IL-4) is in humoral immunity, and follicular helper CD4(+) T (Tfh) cells may be an intrinsic IL-4 source. Here we demonstrate that conserved noncoding sequence 2 (CNS2) is an essential enhancer element for IL-4 expression in Tfh cells but not in Th2 cells. Mice with a CNS2 deletion had a reduction in IgG1 and IgE production and in IL-4 expression in Tfh cells. Tracking of CNS2 activity via a GFP reporter mouse demonstrated that CNS2-active cells expressed several markers of Tfh cells: CXCR5, PD-1, and ICOS; the transcriptional master regulator Bcl6; and the cytokines IL-21 and IL-4. These CNS2-active cells were mainly localized in B cell follicles and germinal centers. The GFP(+) Tfh cells were derived from GFP(-) naive T cells after in vivo systemic immunization. These results indicate that CNS2 is an essential enhancer element required for IL-4 expression in Tfh cells controlling humoral immunity.

CORRESPONDENCEYun-Cai Liu: yuncail@liai.org Abbreviations used: iT reg cell, induced T reg cell; mTOR, mammalian target of rapamycin; nT reg cell, naturally occuring T reg cell; PI3K, phosphoinositide 3-kinase; T eff, effector T cell; T reg cell, regulatory T cell. Transcription factors

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