CD4+25+ regulatory T (Treg) cells maintain immunological self-tolerance through mechanisms that are only in part understood. Previous studies suggest that the glucocorticoid-induced TNFR-related protein (GITR), which is preferentially expressed on the surface of Treg cells, potentially provides a signal that abrogates Treg suppression. In this study, we show that a soluble form of mouse GITR ligand (sGITR-L) induces GITR-dependent NF-κB activation and blocks in vitro suppression mediated by both resting and preactivated polyclonal and Ag-specific Treg cells. Since sGITR-L along with rIL-2 induces proliferation of CD4+25+ cells, it appears that sGITR-L can break the anergic state of Treg cells. Because sGITR-L also up-regulates IL-2 secretion by activated CD4+25 −T cells, these two sGITR-L induced signals synergize to interfere with suppressor activity by CD4+25+ Treg cells.

CD4+CD25+ regulatory T cell selection is initiated by high-specificity interactions with self-peptides in the thymus, although how these cells respond to cytokine-derived signals and to re-exposure to self-peptide:MHC complexes in the periphery is not well understood. We have used a transgenic mouse system, in which the peptide that induces thymic selection of a clonal population of CD4+CD25+ regulatory T cells is known, to show that CD4+CD25+ T cells proliferate in response to their selecting self-peptide in vivo. Moreover, they do not proliferate in response to lymphopenia in the absence of the selecting self-peptide, reflecting a low level of expression of the high affinity receptor for IL-7 (CD127) relative to conventional CD4+ T cells. That their selecting self-peptide is both required for and promotes the peripheral expansion of CD4+CD25+ regulatory T cells may direct their accumulation in sites where the self-peptide is expressed.

We have examined the development of self-peptide-specific CD4+CD25+ regulatory T cells in lineages of transgenic mice that express the influenza virus PR8 hemagglutinin (HA) under the control of several different promoters (HA transgenic mice). By mating these lineages with TS1-transgenic mice expressing a TCR that recognizes the major I-Ed-restricted determinant from HA (site 1 (S1)), we show that S1-specific T cells undergo selection to become CD4+CD25+ regulatory T cells in each of the lineages, although in varying numbers. In some lineages, S1-specific CD4+CD25+ regulatory T cells are highly abundant; indeed, TS1xHA-transgenic mice can contain as many S1-specific CD4+ T cells as are present in TS1 mice, which do not express the neo-self HA. In another lineage, however, S1-specific thymocytes are subjected to more extensive deletion and far fewer S1-specific CD4+CD25+ regulatory T cells accumulate in the periphery. We show that radioresistant stromal cells can direct both deletion and CD4+CD25+ regulatory T cell selection of S1-specific thymocytes. Interestingly, even though their numbers can vary, the S1-specific CD4+CD25+ regulatory T cells in all cases coexist with clonally related CD4+CD25− T cells that lack regulatory function. These findings show that the formation of the CD4+CD25+ regulatory T cell repertoire is sensitive to variations in the expression of self-peptides.

Accumulating evidence indicates that regulatory T cells play a crucial role in preventing autoimmunity. To examine the processes by which regulatory CD4(+) T cells are produced during immune repertoire formation, we have developed transgenic mice that express the influenza virus hemagglutinin (HA) and coexpress major histocompatibility complex class II-restricted T cell receptors (TCRs) with varying affinities for the HA-derived CD4(+) T cell determinant S1. We show that interactions with a single self-peptide can induce thymocytes bearing an autoreactive TCR to undergo selection to become CD4(+) CD25(+) regulatory T cells, and that thymocytes bearing TCRs with low affinity for S1 do not undergo selection into this pathway. We show that CD4(+) thymocytes with identical specificity for the S1 self-peptide can undergo overt deletion versus abundant selection to become CD4(+) CD25(+) regulatory T cells in response to variations in expression of the S1 self-peptide in different lineages of HA transgenic mice. We also show that CD4(+) CD25(+) T cells proliferate in response to their selecting self-peptide in the periphery. Moreover, they do not proliferate in response to lymphopenia in the absence of the selecting self-peptide, reflecting a low level of expression of the high-affinity receptor for IL-7 (CD127) relative to conventional CD4(+) T cells. These studies are determining how specificity for self-peptides directs the thymic selection and peripheral expansion of CD4(+) CD25(+) regulatory T cells. Moreover, the differing responsiveness of CD4(+) CD25(+) regulatory T cells to cytokine- versus self-peptide-mediated signals may direct their accumulation to sites where the self-peptide is expressed.

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