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Autoimmune diseases such as multiple sclerosis (MS) may result from the failure of tolerance mechanisms to prevent expansion of pathogenic T cells. Our study is the first to establish that MS patients have abnormalities in FOXP3 message and protein expression levels in peripheral CD4+CD25+ T cells (Tregs) that are quantitatively related to a reduction in functional suppression induced during suboptimal T-cell receptor (TCR) ligation. Of importance, this observation links a defect in functional peripheral immunoregulation to an established genetic marker that has been unequivocally shown to be involved in maintaining immune tolerance and preventing autoimmune diseases. Diminished FOXP3 levels thus indicate impaired immunoregulation by Tregs that may contribute to MS. Future studies will evaluate the effects of therapies known to influence Treg cell function and FOXP3 expression, including TCR peptide vaccination and supplemental estrogen. © 2005 Wiley-Liss, Inc.

CD4 + CD25 + regulatory T cells (Treg cells) prevent T cell-mediated autoimmune diseases in rodents. To develop a functional Treg assay for human blood cells, we used FACS- or bead-sorted CD4 + CD25 + T cells from healthy donors to inhibit anti-CD3/CD28 activation of CD4 + CD25 - indicator T cells. The data clearly demonstrated classical Treg suppression of CD4 + CD25 - indicator cells by both CD4 + CD25 + h i g h and CD4 + CD25 + l o w T cells obtained by FACS or magnetic bead sorting. Suppressive activity was found in either CD45RO - (naive) or CD45RO + (memory) subpopulations, was independent of the TCR signal strength, required cell-cell contact, and was reversible by interleukin-2 (IL-2). Of general interest is that a wider sampling of 27 healthy donors revealed an age- but not gender-dependent loss of suppressive activity in the CD4 + CD25 + population. The presence or absence of suppressive activity in CD4 + CD25 + T cells from a given donor could be demonstrated consistently over time, and lack of suppression was not due to method of sorting, strength of signal, or sensitivity of indicator cells. Phenotypic markers did not differ on CD4 + CD25 + T cells tested ex vivo from suppressive vs. nonsuppressive donors, although, upon activation in vitro, suppressive CD4 + CD25 + T cells had significantly higher expression of both CTLA-4 and GITR than CD4 + CD25 - T cells from the same donors. Moreover, antibody neutralization of CTLA-4, GITR, IL-10, or IL-17 completely reversed Treg-induced suppression. Our results are highly consistent with those reported for murine Treg cells and are the first to demonstrate that suppressive activity of human CD4 + CD25 + T cells declines with age.

Although the phenotypic and regulatory properties of the CD4(+)CD25(+) T cell lineage (Treg cells) have been well described, the specificities remain largely unknown. We demonstrate here that the CD4(+)CD25(+) Treg population includes the recognition of a broad spectrum of human TCR CDR2 determinants found in the germline V gene repertoire as well as that of a clonotypic nongermline-encoded CDR3beta sequence present in a recombinant soluble T cell receptor (TCR) protein. Regulatory activity was demonstrated in T cell lines responsive to TCR but not in T cell lines responsive to control antigens. Inhibitory activity of TCR-reactive T cells required cell-cell contact and involved CTLA-4, GITR, IL-10, and IL-17. Thus, the T-T regulatory network includes Treg cells with specificity directed toward self-TCR determinants.