Control of regulatory T cell lineage commitment and maintenance

Abstract

Foxp3-expressing regulatory T (Treg) cells suppress pathology mediated by immune responses against self and foreign antigens and commensal microorganisms. Sustained expression of the transcription factor Foxp3, a key distinguishing feature of Treg cells, is required for their differentiation and suppressor function. In addition, Foxp3 expression prevents deviation of Treg cells into effector T cell lineages and confers dependence of Treg cell survival and expansion on growth factors, foremost interleukin-2, provided by activated effector T cells. In this review we discuss Treg cell differentiation and maintenance with a particular emphasis on molecular regulation of Foxp3 expression, arguably a key to mechanistic understanding of biology of regulatory T cells.

Figure 1. Differentiation of thymic and induced Treg cells

A, Most Foxp3⁺ thymic Treg (tTreg) cells differentiate from Foxp3-negative CD4⁺ SP thymocytes. The process of tTreg cell differentiation as defined by induction of Foxp3 requires: 1) increased strength of T cell receptor (TCR) stimulation by self-peptide-MHC complexes presented by thymic epithelial cells (TEC) or dendritic cells (DC), 2) CD28 signaling induced by CD80 and CD86 ligand expressed on antigen presenting cells, and 3) high affinity IL-2 receptor and other γc cytokine receptor signaling. Treg cell homeostasis is dependent on exocrine IL-2 produced by effector T cells. B, Foxp3⁺ Treg cells can also be induced from peripheral naive CD4⁺ T cells (iTreg). Conditions favoring the peripheral induction of Foxp3 include chronic low dose antigen stimulation under tolerizing conditions. iTreg cells are likely prominent in the gut-associated lymphoid tissue where chronic exposure to food, commensal, or environmental antigens likely facilitates their generation. Suboptimal co-stimulation is critical for differentiation of iTreg cells with a particularly important role for the immunomodulatory cytokine TGF-β. Additionally, IL-2 and the vitamin A metabolite retinoic acid (RA) facilitate induction of Foxp3 in peripheral Foxp3⁻ CD4 T cells. CD103⁺ DC, which produce RA and TGF-β are potent inducers of Foxp3 expression in activated T cells. These dendritic cells are present in high numbers in the gut where they likely limit immune inflammation through the generation of iTreg cells.

Figure 2. Signaling pathways, transcription factors, and regulatory DNA elements that control Foxp3 expression

A, T cell receptor (TCR) and CD28 costimulatory signals are important for induction of Foxp3. Downstream of the TCR-CD28 signal, the NFκB pathway via PDK1, PKC-θ, the BCL-10, MALT1, CARMA1 complex and IKK is critical for Foxp3 expression in the thymus, whereas CREB, NFAT, and AP1 have been demonstrated to bind to regulatory DNA elements at the Foxp3 locus. In contrast to thymic differentiation of Treg cells, weaker TCR-CD28 signals appear to favor peripheral induction of Foxp3, as evidenced by increased induction of Foxp3 among peripheral naïve CD4 T cells with early withdrawal of PI3K-Akt-mTOR signaling, and reduced CD28 signaling. TCR and CD28 signaling together with IL-2 and TGF-β are likely important for the survival and proliferation of Treg cell precursors in addition to their putative direct effects on Foxp3 expression. STAT5, activated downstream of the IL-2 receptor, binds to the promoter and an intronic regulatory DNA element within the Foxp3 locus and may have a role in transcriptional regulation of Foxp3. TGF-β dependent synergistic binding of NFAT and Smad to conserved non-coding sequence 1 (Foxp3-CNS1) is important for the peripheral induction of Foxp3. Transcription of the Foxp3 gene is likely dependent on other regulatory DNA elements in addition to the Foxp3 promoter. Foxp3-CNS2 may function in the stable maintenance of Foxp3 expression. This function of CNS2 is dependent on demethylation of the CpG island located at this region, indicating that methyl-sensitive transcription factor binding likely mediates stable expression of Foxp3. Some in vitro generated iTreg cells and activated human CD4 T cells only transiently express Foxp3 perhaps due to a heavily methylated state at CNS2. Permissive chromatin modifications such as methylation of histone 3 at lysine residue 4 (H3K4me) and acetylation of histone 3 (H3Ac) at the promoter and CNS1 associate with active transcription of Foxp3, while CNS2 chromatin is only found in a permissive state, with demethylated CpG motifs, in mature Treg cells that stably express Foxp3. The maintenance DNA methyltransferase I (DNMT1) likely represses Foxp3 expression through the propagation of methylation marks on CpG dinucleotides at CNS2. Receptors and extracellular signals are represented in yellow boxes and signaling intermediates and transcription factors in green. Regulatory DNA elements are highlighted in yellow. Grey boxes with green dotted outlines contain factors thought to be specifically important for peripheral Foxp3 induction.