The objective of this project is to define the plasticity and stability of regulatory T cells (Tregs) and the possibility of converting effector cells to functional Tregs, as a prelude to cellular therapy. Our hypothesis is that activated T cells go through a phase of transient Foxp3 expression that is a critical decision point, and stable FoxpS expression is a key determinant of Treg stability. Our approach is to exploit mouse models to address these questions in vivo and in vitro and to develop predictive phenotypic and molecular markers for Treg stability, and use this information to define the stability of human Tregs and to genetically manipulate human T cells in order to program their differentiation pathway. The following Specific Aims are proposed. 1. Functional and phenotypic plasticity of regulatory T cells (Tregs) and effector T cells (TefO. In this Aim, we will generate antigen-specific natural (thymic) and adaptive (peripheral) Tregs using TCR transgenic mice expressing fluorescent reporters for FoxpS and key cytokines (IFN-v, IL-17 and IL-10) and different forms of antigen exposure. Purified Tregs will be transferred into normal recipients and exposed to antigen with and without inflammatory stimuli, or transferred into recipients with active autoimmune reactions. The cells will be followed for FoxpS and cytokine expression and surface phenotype by staining, and unctional responses after sorting. Tregs will be isolated from healthy volunteers and patients with autoimmune disease (initially T1D) and followed in vitro for conversion to effectors in the presence of activating and inflammatory stimuli. A novel transgenic mouse strain that expresses a lineage marker of past roxp3 expression will be used to examine the fates of these cells. The conversion of effector cells into Tregs be examined using the same approaches. 2.'Biochemical basis of Treg stability and conversion. T cells will be examined under conditions of stability and conversion for changes in surface phenotype, expression of Treg-specific genes, and methylation of the :oxp3 locus. The functional consequence of transient FoxpS expression will be analyzed in mouse and human cells using shRNA knockdowns and knockout mice. Attempts will be made to maintain the stability of regs by expressing selected genes in human Tregs.
The importance of Tregs in preventing autoimmune reactions is well established. Defining the differentiation of these cells is central to understanding autoimmunity. Cellular therapy with Tregs also has great potential n these disorders. This project will elucidate the plasticity of Tregs and the key decision points during their differentiation, and develop methods for stabilizing Tregs even during inflammatory reactions. The project trongly complements Project 1 and the broad objectives of the ACEs to develop Treg-based therapies.
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|Sanchez Rodriguez, Robert; Pauli, Mariela L; Neuhaus, Isaac M et al. (2014) Memory regulatory T cells reside in human skin. J Clin Invest 124:1027-36|
|Stumpf, Melanie; Zhou, Xuyu; Chikuma, Shunsuke et al. (2014) Tyrosine 201 of the cytoplasmic tail of CTLA-4 critically affects T regulatory cell suppressive function. Eur J Immunol 44:1737-46|
|Gratz, Iris K; Truong, Hong-An; Yang, Sara Hsin-Yi et al. (2013) Cutting Edge: memory regulatory t cells require IL-7 and not IL-2 for their maintenance in peripheral tissues. J Immunol 190:4483-7|
|Herold, Kevan C; Vignali, Dario A A; Cooke, Anne et al. (2013) Type 1 diabetes: translating mechanistic observations into effective clinical outcomes. Nat Rev Immunol 13:243-56|
|Jeker, Lukas T; Bluestone, Jeffrey A (2013) MicroRNA regulation of T-cell differentiation and function. Immunol Rev 253:65-81|
|de Kouchkovsky, Dimitri; Esensten, Jonathan H; Rosenthal, Wendy L et al. (2013) microRNA-17-92 regulates IL-10 production by regulatory T cells and control of experimental autoimmune encephalomyelitis. J Immunol 191:1594-605|
|Stumpf, Melanie; Zhou, Xuyu; Bluestone, Jeffrey A (2013) The B7-independent isoform of CTLA-4 functions to regulate autoimmune diabetes. J Immunol 190:961-9|
|Jeker, Lukas T; Zhou, Xuyu; Blelloch, Robert et al. (2013) DGCR8-mediated production of canonical microRNAs is critical for regulatory T cell function and stability. PLoS One 8:e66282|
|Rosenblum, Michael D; Gratz, Iris K; Paw, Jonathan S et al. (2011) Response to self antigen imprints regulatory memory in tissues. Nature 480:538-42|
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