Several subsets of Foxp3+ regulatory T (Treg) cells work in concert to maintain immune homeostasis. However, the molecular bases underlying the phenotypic and functional diversity of Treg cells remain obscure. We show that in response to interferon-γ, Foxp3+ Treg cells upregulated the T helper 1 (TH1)-specifying transcription factor T-bet. T-bet promoted expression of the chemokine receptor CXCR3 on Treg cells, and T-bet+ Treg cells accumulated at sites of TH1-mediated inflammation. Furthermore, T-bet expression was required for the homeostasis and function of Treg cells during type-1 inflammation. Thus, within a subset of CD4+ T cells, the activities of Foxp3 and T-bet are overlaid, resulting in Treg cells with unique homeostatic and migratory properties optimized for suppression of TH1 responses in vivo.

CD4+Foxp3+ regulatory T cells (T reg) are essential for maintaining self-tolerance, but their functional mechanisms and sites of action in vivo are poorly defined. We examined the homing receptor expression and tissue distribution of T reg cells in the steady state and determined whether altering their distribution by removal of a single chemokine receptor impairs their ability to maintain tissue-specific peripheral tolerance. We found that T reg cells are distributed throughout all nonlymphoid tissues tested, and are particularly prevalent in the skin, where they express a unique CCR4+CD103hi phenotype. T reg cell expression of CCR4 and CD103 is induced by antigen-driven activation within subcutaneous lymph nodes, and accumulation of T reg cells in the skin and lung airways is impaired in the absence of CCR4 expression. Mice with a complete loss of CCR4 in the T reg cell compartment develop lymphocytic infiltration and severe inflammatory disease in the skin and lungs, accompanied by peripheral lymphadenopathy and increased differentiation of skin-tropic CD4+Foxp3+ T cells. Thus, selectively altering T reg cell distribution in vivo leads to the development of tissue-specific inflammatory disease.

SUMMARY Foxp3+ regulatory T (Treg) cells limit inflammatory responses and maintain immune homeostasis. Although comprised of several phenotypically and functionally distinct subsets, the differentiation of specialized Treg cell populations within the periphery is poorly characterized. We demonstrate that the development of T-bet+ Treg cells that potently inhibit T helper (Th)1 cell responses was dependent on the transcription factor STAT1, and occurred directly in response to interferon-γ produced by effector T cells. Additionally, delayed induction of the IL-12Rβ2 receptor component following STAT1 activation helped ensure that Treg cells do not readily complete STAT4-dependent Th1 cell development and lose their ability to suppress effector T cell proliferation. Thus, we define a pathway of abortive Th1 cell development that results in the specialization of peripheral Treg cells, and demonstrate that impaired expression of a single cytokine receptor helps maintain Treg cell suppressive function in the context of inflammatory Th1 cell responses.

Tolerance against self-and harmless environmental antigens involves several levels of immune regulation. Whereas deletional mechanisms attempt to purge the B and T cell repertoire of self-reactive specifi cities, antigen-specifi c suppression of autoimmunity by CD4 + Foxp3 + regulatory (T reg) T cells also plays a critical role in maintaining self-tolerance. The importance of T reg cells is highlighted by the severe autoimmunity in humans and mice rendered T reg cell -defi cient because of lesions in the FOXP3 gene, which in mice can be prevented by adoptive transfer of purifi ed T reg cells ( 1 -3 ). In addition, neonatal or adult ablation of T reg cells, or adoptive transfer of T reg cell -depleted CD4 + T cells to immunodefi cient mice rapidly triggers severe autoimmune or infl ammatory disease ( 4 -6 ). Collectively, these data demonstrate that T reg cells are required for both the establishment and maintenance of self-tolerance in vivo.T reg cells can be subdivided based on expression of homing receptors thought to direct their migration to lymphoid versus nonlymphoid tissues ( 7 ). However, the relative contributions of the various T reg cell subsets to the maintenance of self-tolerance are not well defi ned. Several reports have demonstrated that T reg cells function within nonlymphoid sites to restrict T cell responses to foreign antigens during experimentally induced acute infl ammation or infection ( 8 -11 ). In addition, we have shown that mice whose T reg cells lack the chemokine receptor CCR4 develop spontaneous infl ammatory disease in skin and lungs ( 12 ). However, although CCR4 helps direct T cell migration to the skin and lung airways, it may also facilitate interactions between T reg cells and antigenpresenting cells within secondary lymphoid tissues. Thus, the infl ammatory disease in these animals is likely the result of impaired T reg cell function in both lymphoid and nonlymphoid organs, and the importance of nonlymphoid T reg cells in maintaining tolerance in the absence of a strong infl ammatory stimulus has not been adequately addressed.Surface expression of functional E-and Pselectin ligands (E-/P-lig) is required for optimal migration of eff ector T cells to infl amed skin ( 13 ). Interestingly, a high percentage of circulating human and mouse T reg cells express Eand/or P-lig, and Foxp3 + T reg cells make up a large fraction of the CD4 + T cells in normal skin from both humans and mice ( 12,14 ). This Cutaneous immune responses must be tightly controlled to prevent unwanted infl ammation in response to innocuous antigens, while maintaining the ability to combat skin-tropic pathogens. Foxp3 + regulatory T (T reg) cells are potent immune regulators and are found at high frequency in both human and mouse skin. Although T reg cells migrate to the skin and can dampen immune responses during experimentally induced infl ammation or infection, the importance of cutaneous T reg cells for maintaining normal immune homeostasis in the skin has not been addressed. To selectively block T reg cell...

The matricellular glycoprotein, secreted protein acidic and rich in cysteine (SPARC), has complex biological activities and is important for lens epithelial cell function and regulation of cataract formation. To understand how SPARC influences lens epithelial cell activity and homeostasis, we have studied the subcellular distribution of SPARC in murine lens epithelial cells in vitro. We demonstrate that endogenous SPARC is located in the cytoplasm of either quiescent or dividing lens epithelial cells in culture. However, cytoplasmic SPARC was translocated into the nuclei of immortalized lens epithelial cells upon a significant reduction of intracellular SPARC in these cells. Recombinant human (rh) SPARC added to the culture media was quickly and efficiently internalized into the cytosol of SPARC-null lens epithelial cells. Moreover, cytoplasmic rhSPARC was also translocated into the nucleus after exogenous rhSPARC was removed from the culture media. The translocation of SPARC into the nucleus was therefore triggered by the reduction of SPARC protein normally available to the cells. A mouse SPARC-EGFP chimeric fusion protein (70 kDa) was expressed in lens epithelial cells and 293-EBNA cells, and was observed both in the cytoplasm and culture medium, but not in the nucleus. SPARC does not appear to have a strong nuclear localization sequence. Alternatively, SPARC might pass through the nuclear pore complex by passive diffusion. SPARC therefore functions not only as an extracellular protein but also potentially as an intracellular protein to influence cellular activities and homeostasis.