Immune regulation of autoimmune disease can function at two sites: at the secondary lymphoid organs or in the target organ itself. In this study, we investigated the natural resolution of autoimmune pathology within the CNS using murine experimental autoimmune encephalomyelitis (EAE). Recovery correlates with the accumulation of IL-10-producing CD4+CD25+ T cells within the CNS. These CD4+CD25+ cells represent as many as one in three of CD4+ cells in the CNS during recovery, they are FoxP3+ and express other markers associated with regulatory cells (CTLA-4, GITR, and αEβ7), and they have regulatory function ex vivo. Depletion of CD25+ cells inhibits the natural recovery from EAE. Also, depletion of CD25+ cells after recovery removes the resistance to reinduction of EAE observed in this model. Furthermore, passive transfer of CNS-derived CD4+CD25+ cells in low numbers provides protection from EAE in recipient mice. These are the first data demonstrating the direct involvement of CD4+CD25+ regulatory T cells in the natural resolution of autoimmune disease within the target organ.

Transfer of CD4+ T cells to immune-deficient mice in the absence of the CD25+ subset leads to the development of colitis, indicating that regulatory cells capable of controlling a bacteria-driven inflammatory response are present in normal mice. Cells with this function are present in the thymus as well as in the periphery of germ-free mice, suggesting they may be reactive with self-antigen. These cells resemble CD4+CD25+ cells that inhibit organ-specific autoimmunity, suggesting that a similar subset of regulatory T cells may control responses to self and foreign antigens. Development of colitis is dependent on accumulation of activated CD134L+ dendritic cells (DC) in the mesenteric lymph nodes, which is inhibited by CD4+CD25+ cells, indicating that regulatory T cells may control DC activation in vivo. Whilst inhibition of T-cell activation in vitro by CD4+CD25+ cells does not involve interleukin-10 and transforming growth factor-beta, these cytokines are required for the suppression of colitis. It may be that control of responses that activate the innate immune system requires multiple mechanisms of immune suppression. Recently, we identified CD4+CD25+ cells with immune suppressive activity in the thymus and peripheral blood of humans, raising the possibility that dysfunction in this mechanism of immune regulation may be involved in the development of autoimmune and inflammatory diseases.

CD4+CD25+ T cells in mice and rats are capable of transferring protection against organ‐specific autoimmune disease and colitis and suppressing the proliferation of other T cells after polyclonal stimulation in vitro. Here we describe the existence in humans of CD4+CD25+ T cells with the same in vitro characteristics. CD4+CD8–CD25+ T cells are present in both the thymus and peripheral blood of humans (∼ 10 % of CD4+CD8– T cells), proliferate poorly in response to mitogenic stimulation and suppress the proliferation of CD4+CD25– cells in co‐culture. This suppression requires cell contact and can be overcome by the addition of exogenous IL‐2. CD4+CD25+ cells from thymus and blood were poor producers of IL‐2 and IFN‐γ, and suppressed the levels of these cytokines produced by CD4+CD25– cells. However, CD4+CD25+ PBL produced higher levels of IL‐4 and similar amounts of IL‐10 as CD4+CD25– cells. Regulatory CD4+CD25+ T cells have an activated phenotype in the thymus with expression of CTLA‐4 and CD122 (IL‐2Rβ). The fact that CD4+CD25+ regulatory T cells are present with a similar frequency in the thymus of humans, ratsand mice, suggests that the role of these cells in the maintenance of immunological tolerance is an evolutionarily conserved mechanism.

Previously we have shown that autoimmune diabetes, induced in rats by a protocol of adult thymectomy and split-dose gamma irradiation, can be prevented by the transfer of a subset of CD4+ T cells with a memory phenotype (CD45RC−), as well as by CD4+CD8− thymocytes, from syngeneic donors. Further studies now reveal that in the thymus the regulatory cells are observed in the CD25+ subset of CD4+CD8− cells, whereas transfer of the corresponding CD25− thymocyte subset leads to acceleration of disease onset in prediabetic recipients. However, in the periphery, not all regulatory T cells were found to be CD25+. In thoracic duct lymph, cells that could prevent diabetes were found in both CD25− and CD25+ subsets of CD4+CD45RC− cells. Further, CD25− regulatory T cells were also present within the CD4+CD45RC− cell subset from spleen and lymph nodes, but were effective in preventing diabetes only after the removal of CD25− recent thymic emigrants. Phenotypic analysis of human thymocytes showed the presence of CD25+ cells in the same proportions as in rat thymus. The possible developmental relationship between CD25+ and CD25− regulatory T cells is discussed.

The potential use of CD4

The molecular-mimicry theory proposes that immune crossreactivity between microbial and self-antigen is the initiating event in the activation of autoaggressive immune responses leading to autoimmune disease. In support of this possibility, it is now accepted that T cell recognition of antigen is highly degenerate. However, it is to be expected that the immune system would have evolved mechanisms to counter such a potential danger. We studied the influence of CD4 ؉ CD25 ؉ regulatory T cells (Treg) on the ability of suboptimal T cell receptor ligands to provoke autoimmunity. By using CD4 ؉ T cell-driven experimental autoimmune encephalomyelitis as a model, it was found that depletion of CD4 ؉ CD25 ؉ Foxp3 ؉ Treg allowed pathology to develop in response to suboptimal T cell stimulation. These data demonstrate the importance of Treg in raising the threshold of triggering of autoreactive T cell responses, thus limiting the risk of autoimmune disease due to molecular mimicry.experimental autoimmune encephalomyelitis (EAE) ͉ multiple sclerosis ͉ regulation ͉ tolerance E xtensive flexibility in T cell receptor (TCR) recognition of peptide-MHC (pMHC) complexes has been proposed to be essential to provide effective immune surveillance of all possible pathogen-derived pMHC complexes (1). The logical extension of this cross-reactivity is that the peripheral T cell repertoire should contain a sizeable population of cells that are capable of responding in a cross-reactive manner to both pathogen-derived antigens (Ags) and self-molecules. This concept is at the heart of the molecular-mimicry theory, in which self-reactive T cells, activated initially by infectious pathogens, subsequently provoke a self-destructive response in an organ expressing a crossreactive self-Ag (2). However, there is considerable debate over the validity of this theory as a general mechanism for the induction of autoimmune disease (3), and mechanisms that limit this potential risk are likely to exist. Naturally occurring regulatory T cells (Treg) may have a role by raising the activation threshold of T cell responses, potentially providing one mechanism by which weakly self-reactive T cells can be maintained in the T cell repertoire without inducing overt autoimmunity.Treg activity is enriched in the subset of CD4 ϩ T cells expressing CD25 in mice (4), rats (5), and humans (6), leading to the now widespread use of this marker to define a naturally occurring population of Treg. The influence of Treg on peripheral tolerance is shown most vividly by the widespread autoimmune and inflammatory lesions that are evident in humans and mice that lack these cells because of mutations in the Tregspecific transcription factor Foxp3 (7-10). Although conclusive evidence regarding the specificity of CD4 ϩ CD25 ϩ Foxp3 ϩ Treg remains elusive, they are known to have a broad TCR repertoire (11), and there is evidence to suggest that CD25 ϩ Treg developing in the thymus are selected to have high affinity for self-Ags expressed on thymic epithelium (12)(13)(14). Negative...

Tumor necrosis factor-alpha (TNFalpha) is a potential mediator of beta cell destruction in insulin-dependent diabetes mellitus. We have studied TNF-responsive pathways leading to apoptosis in beta cells. Primary beta cells express low levels of the type I TNF receptor (TNFR1) but do not express the type 2 receptor (TNFR2). Evidence for TNFR1 expression on beta cells came from flow cytometry using monoclonal antibodies specific for TNFR1 and TNFR2 and from RT-PCR of beta cell RNA. NIT-1 insulinoma cells similarly expressed TNFR1 (at higher levels than primary beta cells) as detected by flow cytometry and radio-binding studies. TNF induced NF-kappaB activation in both primary islet cells and NIT-1 cells. Apoptosis in response to TNFalpha was observed in NIT-1 cells whereas apoptosis of primary beta cells required both TNFalpha and interferon-gamma (IFNgamma). Apoptosis could be prevented in NIT-1 cells by expression of dominant negative Fas-associating protein with death domain (dnFADD). Apoptosis in NIT-1 cells was increased by coincubation with IFNgamma, which also increased caspase 1 expression. These data show that TNF-activated pathways capable of inducing apoptotic cell death are present in beta cells. Caspase activation is the dominant pathway of TNF-induced cell death in NIT-1 cells and may be an important mechanism of beta cell damage in insulin-dependent diabetes mellitus.

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