Naturally arising CD25+ CD4+ regulatory T (Treg) cells, most of which are produced by the normal thymus as a functionally mature T-cell subpopulation, play key roles in the maintenance of immunologic self-tolerance and negative control of a variety of physiological and pathological immune responses. Natural Tregs specifically express Foxp3, a transcription factor that plays a critical role in their development and function. Complete depletion of Foxp3-expressing natural Tregs, whether they are CD25+ or CD25-, activates even weak or rare self-reactive T-cell clones, inducing severe and widespread autoimmune/inflammatory diseases. Natural Tregs are highly dependent on exogenously provided interleukin (IL)-2 for their survival in the periphery. In addition to Foxp3 and IL-2/IL-2 receptor, deficiency or functional alteration of other molecules, expressed by T cells or non-T cells, may affect the development/function of Tregs or self-reactive T cells, or both, and consequently tip the peripheral balance between the two populations toward autoimmunity. Elucidation of the molecular and cellular basis of this Treg-mediated active maintenance of self-tolerance will facilitate both our understanding of the pathogenetic mechanism of autoimmune disease and the development of novel methods of autoimmune disease prevention and treatment via enhancing and re-establishing Treg-mediated dominant control over self-reactive T cells.

Interleukin (IL)-2 plays a crucial role in the maintenance of natural immunologic self-tolerance. Neutralization of circulating IL-2 by anti–IL-2 monoclonal antibody for a limited period elicits autoimmune gastritis in BALB/c mice. Similar treatment of diabetes-prone nonobese diabetic mice triggers early onset of diabetes and produces a wide spectrum of T cell–mediated autoimmune diseases, including gastritis, thyroiditis, sialadenitis, and notably, severe neuropathy. Such treatment selectively reduces the number of Foxp3-expressing CD25+ CD4+ T cells, but not CD25− CD4+ T cells, in the thymus and periphery of normal and thymectomized mice. IL-2 neutralization inhibits physiological proliferation of peripheral CD25+ CD4+ T cells that are presumably responding to normal self-antigens, whereas it is unable to inhibit their lymphopenia-induced homeostatic expansion in a T cell–deficient environment. In normal naive mice, CD25low CD4+ nonregulatory T cells actively transcribe the IL-2 gene and secrete IL-2 protein in the physiological state. IL-2 is thus indispensable for the peripheral maintenance of natural CD25+ CD4+ regulatory T cells (T reg cells). The principal physiological source of IL-2 for the maintenance of T reg cells appears to be other T cells, especially CD25low CD4+ activated T cells, which include self-reactive T cells. Furthermore, impairment of this negative feedback loop via IL-2 can be a cause and a predisposing factor for autoimmune disease.

The emerging notion of environment-induced reprogramming of Foxp3(+) regulatory T (Treg) cells into helper T (Th) cells remains controversial. By genetic fate mapping or adoptive transfers, we have identified a minor population of nonregulatory Foxp3(+) T cells exhibiting promiscuous and transient Foxp3 expression, which gave rise to Foxp3(-) ("exFoxp3") Th cells and selectively accumulated in inflammatory cytokine milieus or in lymphopenic environments including those in early ontogeny. In contrast, Treg cells did not undergo reprogramming under those conditions irrespective of their thymic or peripheral origins. Moreover, although a few Treg cells transiently lose Foxp3 expression, such "latent" Treg cells retained their memory and robustly re-expressed Foxp3 and suppressive function upon activation. This study establishes that Treg cells constitute a stable cell lineage, whose committed state in a changing environment is ensured by DNA demethylation of the Foxp3 locus irrespectively of ongoing Foxp3 expression.

Mouse CD4+CD8+ double-positive (DP) thymocytes differentiate into CD4+ helper-lineage cells upon expression of the transcription factor Th-POK but commit to the CD8+ cytotoxic lineage in its absence. We report the redirected differentiation of class I-restricted thymocytes into CD4+CD8- helper-like T cells upon loss of Runx transcription factor complexes. A Runx-binding sequence within the Th-POK locus acts as a transcriptional silencer that is essential for Th-POK repression and for development of CD8+ T cells. Thus, Th-POK expression and genetic programming for T helper cell development are actively inhibited by Runx-dependent silencer activity, allowing for cytotoxic T cell differentiation. Identification of the transcription factors network in CD4 and CD8 lineage choice provides insight into how distinct T cell subsets are developed for regulating the adaptive immune system.

Interferon γ (IFNγ) is the hallmark cytokine produced by T helper type 1 (Th1) cells, whereas interleukin (IL)-4 is the hallmark cytokine produced by Th2 cells. Although previous studies have revealed the roles of cytokine signaling and of transcription factors during differentiation of Th1 or Th2 cells, it is unclear how the exclusive expression pattern of each hallmark cytokine is established. The DNaseI hypersensitivity site IV within the mouse Il4 locus plays an important role in the repression of Il4 expression in Th1 cells, and it has been named the Il4 silencer. Using Cbfβ- or Runx3-deficient T cells, we show that loss of Runx complex function results in derepression of IL-4 in Th1 cells. Binding of Runx complexes to the Il4 silencer was detected in naive CD4+ T cells and Th1 cells, but not in Th2 cells. Furthermore, enforced expression of GATA-3 in Th1 cells inhibited binding of Runx complexes to the Il4 silencer. Interestingly, T cell–specific inactivation of the Cbfβ gene in mice led to elevated serum immunoglobulin E and airway infiltration. These results demonstrate critical roles of Runx complexes in regulating immune responses, at least in part, through the repression of the Il4 gene.

Naturally arising CD25(+)CD4(+) regulatory T (T(R)) cells can be exploited to establish immunologic tolerance to non-self antigens. In vivo exposure of CD25(+)CD4(+) T cells from normal naive mice to alloantigen in a T cell-deficient environment elicited spontaneous expansion of alloantigen-specific CD25(+)CD4(+) T(R) cells, which suppressed allograft rejection mediated by subsequently transferred naive T cells, leading to long-term graft tolerance. The expanded T(R) cells, which became CD25(low) in the absence of other T cells, stably sustained suppressive activity, maintained expression levels of other T(R) cell-associated molecules, including Foxp3, CTLA-4 and GITR, and could adoptively transfer tolerance to normal mice. Furthermore, specific removal of the T(R) cells derived from originally transferred CD25(+)CD4(+) T(R) cells evoked graft rejection in the long-term tolerant mice, indicating that any T(R) cells deriving from CD25(-)CD4(+) naive T cells minimally contribute to graft tolerance and that natural T(R) cells are unable to infectiously confer significant suppressive activity to other T cells. Similar antigen-specific expansion of T(R) cells can also be achieved in vitro by stimulating naturally present CD25(+)CD4(+) T cells with alloantigen in the presence of IL-2. The expanded CD25(+)CD4(+) T cells potently suppressed even secondary MLR in vitro and, by in vivo transfer, established antigen-specific long-term graft tolerance. Thus, in vivo or in vitro, direct or indirect ways of antigen-specific expansion of naturally arising Foxp3(+)CD25(+)CD4(+) T(R) cells can establish antigen-specific dominant tolerance to non-self antigens, and would also be instrumental in re-establishing self-tolerance in autoimmune disease and antigen-specific negative control of pathological immune responses.

Foxp3 controls the development and function of regulatory T (Treg) cells, but it remains elusive how Foxp3 functions in vivo. Here, we established mouse models harboring three unique missense Foxp3 mutations that were identified in patients with the autoimmune disease IPEX. The I363V and R397W mutations were loss-of-function mutations, causing multi-organ inflammation by globally compromising Treg cell physiology. By contrast, the A384T mutation induced a distinctive tissue-restricted inflammation by specifically impairing the ability of Treg cells to compete with pathogenic T cells in certain non-lymphoid tissues. Mechanistically, repressed BATF expression contributed to these A384T effects. At the molecular level, the A384T mutation altered Foxp3 interactions with its specific target genes including Batf by broadening its DNA-binding specificity. Our findings identify BATF as a critical regulator of tissue Treg cells and suggest that sequence-specific perturbations of Foxp3-DNA interactions can influence specific facets of Treg cell physiology and the immunopathologies they regulate.

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