Project 4

Use of FoxP3 to Induce and Regulate Antigen-specific Tregs

Immunological tolerance has developed to allow organisms to differentiate between self and non-self. Tolerance is achieved primarily through the elimination of auto-reactive clones in the thymus, through a mechanism known negative selection 1. Those clones that escape central tolerance in the thymus are rendered anergic in the periphery upon encounter with antigen under sub-optimal conditions 2. In spite of these two mechanisms for maintaining tolerance, autoreactive T cells can be readily detected in normal individuals. Recently, a regulatory T cell population have been identified and shown to actively suppress immune responses 3. These cells (referred to as Treg cells), characterized by the expression of the cell surface markers CD4 and CD25, inhibit the activation of autoreactive T cells in an antigen-specific, cell-contact-dependent, manner 4-7. In rodents these cells have been shown to develop in the thymus, possibly as a consequence of escape from negative selection 4;8. Their development in the periphery, and possible role in immune responses to foreign antigens, is suggested by their ability to suppress disease in rodent models of inflammatory bowel disease and allergy9-12.

The molecular basis for the development and function of Treg cells remains unclear. Recently the forkhead transcription factor FoxP3 has been implicated in the development and function of Treg cells. Mice carrying the X-linked scurfy mutation develop a fatal lymphoproliferative disease exemplified by a lack of conventional CD4+CD25+ Treg cells 13. In both mice and man, FoxP3 has been shown to expressed predominantly in CD4+CD25+ Treg cells. In addition, in mice, FoxP3 has been shown to be capable of converting naïve CD4+CD25- to Treg cells when introduced via retrovirus or enforced transgene expression 13;14. Thus, in mice, FoxP3 is both necessary and sufficient for the development and function of CD4+CD25+ Treg cells. Similarly, FoxP3 is expressed predominantly in human Treg cells. Additionally, unlike their rodent counterparts, CD4+CD25+ T cells generated as a consequence of in vitro stimulation of CD4+CD25- human T cells express FoxP3 and acquire Treg function.

Thus, expression of FoxP3 correlates with Treg function in man and mouse, and ectopic expression of FoxP3 can lead to the generation of cells with a Treg phenotype. The finding that FoxP3 expression determines Treg cell fate, as well as the ability of ectopic FoxP3 expression to convert naïve T cells into Treg cells, suggests that FoxP3 may be useful in cell-based therapies for autoimmune diseases. However, before this assessment can
be made, additional information on the function of FoxP3 needs to be obtained. As a first step, the experiments in this proposal are designed to test the feasibility of manipulating FoxP3 expression and function as a means of converting both normal and pathogenic T cells to Treg-like cells. In the first Aim we will determine the mechanism that underlies the ability of FoxP3 to act as a transcriptional repressor. Importantly, these experiments will characterize a set of proteins that have been identified as interacting with FoxP3, and thus may be used to regulate FoxP3 function. In aim 2 we will use an in vitro cell culture system to determine whether introduction of FoxP3 into primary antigen-specific human T cells can convert these cells into antigen-specific Treg cells. We will also use animal models of type I diabetes in the mouse to test the ability of ectopic FoxP3 expression to convert diabetogenic T cells into Treg cells. Several FoxP3-interacting proteins have been identified, and the ability of these proteins to enhance FoxP3 function will be assessed. These experiments will provide valuable information on the efficacy of FoxP3 as a tool for therapeutic intervention in autoimmune diabetes.

1. Determine the molecular mechanism of FoxP3-mediated transcriptional repression. Our previous data has shown that FoxP3 acts as a transcriptional repressor. In this aim we will take a series of approaches to determine the molecular mechanism of FoxP3 function, including structure/function studies and the characterization of FoxP3-interacting proteins.
2. Determine whether regulatory T cell function can be modulated through ectopic FoxP3 expression. We have previously shown that expression of FoxP3 correlates with Treg activity. We predict that introduction of FoxP3 into non-regulatory T cells will convert them to a Treg phenotype. We will test this hypothesis in two ways. First, we will use an in vitro system to introduce FoxP3 into human CD4+CD25- T cells. Second, we will use animal models of T1D to ask whether ectopic FoxP3 expression can interdict disease development and/or progression.