Mechanisms of human FoxP3+ Treg cell development and function in health and disease

Abstract

Regulatory T (T_reg ) cells represent an essential component of peripheral tolerance. Given their potently immunosuppressive functions that is orchestrated by the lineage-defining transcription factor forkhead box protein 3 (FoxP3), clinical modulation of these cells in autoimmunity and cancer is a promising therapeutic target. However, recent evidence in mice and humans indicates that T_reg cells represent a phenotypically and functionally heterogeneic population. Indeed, both suppressive and non-suppressive T_reg cells exist in human blood that are otherwise indistinguishable from one another using classical T_reg cell markers such as CD25 and FoxP3. Moreover, murine T_reg cells display a degree of plasticity through which they acquire the trafficking pathways needed to home to tissues containing target effector T (T_eff ) cells. However, this plasticity can also result in T_reg cell lineage instability and acquisition of proinflammatory T_eff cell functions. Consequently, these dysfunctional CD4⁺ FoxP3⁺ T cells in human and mouse may fail to maintain peripheral tolerance and instead support immunopathology. The mechanisms driving human T_reg cell dysfunction are largely undefined, and obscured by the scarcity of reliable immunophenotypical markers and the disregard paid to T_reg cell antigen-specificity in functional assays. Here, we review the mechanisms controlling the stability of the FoxP3⁺ T_reg cell lineage phenotype. Particular attention will be paid to the developmental and functional heterogeneity of human T_reg cells, and how abrogating these mechanisms can lead to lineage instability and T_reg cell dysfunction in diseases like immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome, type 1 diabetes, rheumatoid arthritis and cancer.

Keywords: antigen specificity; cell therapy; human immunology; regulatory T cell dysfunction; regulatory T cells.

Figure 1

Mechanisms preserving the stability of the regulatory T cell (T_reg) phenotype. T_reg cell lineage stability is reliant on the strength of forkhead box protein 3 (FoxP3) expression. There are several mechanisms in place to ensure robust FoxP3 expression in T_reg cells. A, T cell receptor (TCR) signaling leads to nuclear factor of activated T cells (NFAT) binding to the CNS2 region of the foxp3 locus for transactivation of gene expression. B, Constitutive High level of CD25 expression, the interleukin (IL)‐2 receptor α, on the T_reg cell surface confers a high sensitivity to IL‐2 in the environment. IL‐2 signaling through Janus kinase (Jak)1 and Jak3 result in signal transducer and activator of transcription (STAT‐5) phosphorylation and dimerization and subsequent translocation into the nucleus. Phosphorylated (p)STAT‐5 binding to the conserved non‐coding DNA sequence (CNS)2 drives FoxP3 expression. C, Transforming growth factor (TGF)‐β signaling through TGF‐βRI and TGF‐βRII result in Smad2/3 phosphorylation, association with the transcription Smad4 and the translocation of the complex into the nucleus. Smad2/3/4 bind to the foxp3 promoter and drive FoxP3 expression. In the presence of TCR signaling, TGF‐β‐driven FoxP3 expression in naïve CD4⁺ conventional T (T_conv) results in induced (i)T_reg/peripheral (p)T_reg induction. D, To enable transcription factor binding to the foxp3 locus enhancer region, certain sites are specifically demethylated in T_reg cells. In the CNS2 enhancer region, this is referred to as the T_reg‐specific demethylated region (TSDR). Demethylation of the TSDR is mediated by the 10–11 translocation (Tet) family demethylases Tet1 and Tet2. DNA methyl transferases (Dnmt) such as Dnmt1 methylate the TSDR and destabilize Foxp3 expression. E, Once FoxP3 is expressed, it heterodimerizes and can associate with many different binding partners (~700), including transcription factors, histone deacetylases and histone acetyl transferases. Binding to these proteins are necessary for transcriptional repression of various genes (il7ra, ifng, il2) and activation of others (il2ra, ctla4, foxp3). F, Significant focus has been devoted to studying the environmental signals controlling FoxP3 expression. Phosphoinositide‐3‐kinase–protein kinase B (PI3K‐Akt) signaling downstream TCR and CD28 signaling is needed for transient mammalian target of rapamycin complex 1 (mTORC1) activation and consolidation of the T_reg cell phenotype. However, chronic activation of mTORC1 (e.g. through environmental signals such as glutamine) result in sustained mTORC1 activation and therefore deregulation of T_reg cells. Thus, mTORC1 inhibitors (e.g. rapamycin) are used in the in‐vitro expansion of T_reg cells.

Figure 2

Mechanisms driving T_reg cell dysfunction in type‐1 diabetes and rheumatoid arthritis. Forkhead box protein 3 (FoxP3) expression is destabilized by extrinsic factors in type‐1 diabetes and rheumatoid arthritis. A, Local deprivation in interleukin (IL)‐2 and diminished sensitivity to IL‐2 increases susceptibility to apoptosis through diminished B‐cell lymphoma 2 (Bcl‐2) production. Furthermore, lack of this positive signal reduces phosphorylated signal transducer and activator of transcription (pSTAT)‐5 activation and occupancy of the foxp3 promoter, leading to diminished FoxP3 expression. As a result, regulatory T cells (T_reg) have a lower suppressive capacity in vitro and can start secreting proinflammatory cytokines. B, High levels of IL‐6 in the inflammatory pannus of rheumatoid arthritis patients trigger STAT‐3 signaling through the IL‐6 receptor (IL‐6R). STAT‐3 occupies the STAT‐5‐binding sites on the foxp3 locus, which attenuates FoxP3 expression. Furthermore, STAT‐3 binding to the rorc promoter enhances retinoic acid orphan receptor (ROR)γt expression, the T helper type 17 (Th17) master transcription factor. As a result, Th17 cells develop preferentially over T_reg cells during disease flares.

Figure 3

Mechanisms promoting regulatory T cell (T_reg) development and immunosuppression in the tumor microenvironment. A, T_reg cells are recruited to the tumor through chemokine attraction. B, Interaction of T_reg cells with antigen‐presenting cells (APCs) through cytotoxic T lymphocyte antigen (CTLA)‐4 deprive T effector (T_eff) cells of co‐stimulatory signals, polarizes dendritic cells (DCs) towards a tolerogenic phenotype and induces the expression of indoleamine 2,3‐dioxygenase (IDO), which catabolizes metabolites, thereby inducing T_eff cell apoptosis. Furthermore, it inhibits the reprogramming of T_reg cells into Th17 cells by suppressing interleukin (IL)‐6 secretion and promotes T_reg cell lineage stability by inhibiting the transient mammalian target of rapamycin complex 1/protein kinase B (mTORC2/Akt) pathway. C, Tumor cells express programmed cell death ligand 1 (PD‐L1), which binds programmed cell death 1 (PD‐1) at the surface of T_reg cells. The PD‐1 pathway stabilizes forkhead box protein 3 (FoxP3) expression by inhibiting the phosphoinositide‐3‐kinase (PI3K)/Akt pathway and synergizes with transforming growth factor (TGF)‐β by diminishing the level of Smad3 necessary to promote the conversion of naïve CD4⁺ T cells into peripheral (p)T_reg cells, while inducing T_eff cell exhaustion.