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CD4+CD25+ natural regulatory T (nTR) lymphocytes represent a separate, thymus derived T cell lineage that is essential to the maintenance of immunological tolerance in the host. The discovery of Foxp3 as a transcription factor essential to the differentiation of both nTR and induced regulatory T cells (iTR) ushered in detailed studies of the molecular mechanisms of TR cell development, peripheral homeostasis and effector functions. In humans, loss of function mutations in genes that regulate T cell development and function, including FOXP3, IL2RA and STAT5b, have been associated with TR cell deficiency or dysfunction and syndromes of autoimmunity and immune dysregulation, including neonatal type 1 diabetes and polyendocrinopathies. Augmentation of TR cells by immunotherapy and pharmacologic agents is a promising strategy for the treatment of allergic and autoimmune diseases.
Regulatory T (TR) cells represent a distinct T cell lineage that plays a key role in tolerance to self-antigens and prevention of autoimmune diseases, as well as in the inappropriate immune responses involved in allergic diseases 1-3. TR cells are characterized by a set of phenotypic and functional attributes that distinguish them from conventional T cells: they are predominantly CD4+CD25+, T cell receptor (TCR) αβ+. TR cells are anergic and do not produce IL-2 [reviewed in 4]. When activated, they suppress the proliferation and cytokine production of conventional CD4+CD25- T cells as well as that of CD8+ T cells and established Th1 and Th2 cells 5-8. CD4+CD25+ TR cells produce TGF-β and IL-10, two cytokines endowed with immunosuppressive functions and which play critical functions in TR cell biology.
A majority of peripheral TR cells are programmed in the thymus and are know as natural TR (nTR) cells 3. Other TR cells known as induced or adaptive (iTR) cells are derived de novo from a naïve CD4+ precursor pool in peripheral lymphoid tissues following antigenic stimulation in the presence of TGF-β and IL-2. Intense effort has gone into defining the molecular events that guide TR cells through development and lineage commitment, and those that enable the acquisition and maintenance of TR cell phenotypic and functional attributes. These recent advances made in elucidating those pathways are reviewed below.
While TR cells are characterized by the expression of a distinctive combination of surface antigens including CD25, CTLA-4 and GITR, the cardinal hallmark of a TR cell is the forkhead-family transcription factor forkhead box p3, (Foxp3) which is indispensable to their suppressive activity, phenotype stability and survival in the periphery 3.
Foxp3 was initially thought to function as a transcriptional repressor 9. However, it has become clear that Foxp3 may function as either a transcriptional activator or repressor depending on the context 10,11. The transcriptional functions of Foxp3 are enabled by the capacity of its different domains to interact with distinct sets of regulatory proteins to form large macromolecular transcriptional complexes. An N-terminal domain mediates transcriptional activation and repression; zinc finger and leucine zipper domains mediate homo- and hetero-oligomerization of Foxp3 with related members of the Foxp family; and a carboxyl-terminal forkhead domain that mediates binding to specific DNA response elements. It also includes residues that contribute to the physical association of Foxp3 with other transcription factors including the nuclear factor of activated T cells (NFAT) and Runx1/AMl1, both of which contribute to the transcriptional program and suppressive functions of regulatory T cells 12,13. An isoform of Foxp3 is expressed that lacks an N-terminal domain 33 amino acid peptide that is encoded by exon 2. This isoform is ineffective in conferring regulatory function upon expression in conventional T (Tconv) cells 14.
Foxp3+ TR cells are unique among the effector T cell subsets in being comprised of two developmentally distinct populations: nTR cells, which develop in the thymus and adaptive or induced iTR cells that are induced de novo in the periphery from Tconv cells. The two populations display a close affinity in their regulatory function and phenotype, but are not identical. The phenotypic and genetic attributes of nTR cells are “hard-wired”, with most them persisting even in the absence of Foxp3. This is a reflection of an irreversible commitment to the TR cell lineage that occurs in the course of thymic selection and maturation of nTR cells. In contrast, iTR cells are “plastic”, developing upon antigenic stimulation of Tconv cells in the presence of TGF-β and IL-2 (Figure 1) 15-18. Foxp3, whose expression in iTR cells is induced by the action of TGF-β and T cell receptor signaling, is required for the suppressive functions of iTR cells, similar to the situation of their nTR cells counterparts (D. Haribhai, T.A. Chatila and C.B. Williams; unpublished observations). iTR cells have been demonstrated to develop during induction of oral tolerance to an allergen and may play an important role in tolerance induction in immunotherapy 19,20. Their phenotype, however, is less stable than that of nTR cells. Whereas the Foxp3 locus is stably hypomethylated in nTR cells, it is weakly so in these adaptive iTR cells 21. The suppressive function and Foxp3 expression levels of the latter may accordingly decline over time.
In addition to the Foxp3+ iTR cells, another class of TR cells is the Foxp3−, IL-10+ T regulatory type 1 (Tr-1) cells, derived by the ex-vivo activation of naïve CD4+ T cells in the presence of IL-10 or by IL-10-conditioned dendritic cells 22. Previous studies attempting to track these cells in vivo have been impeded by the lack of clear markers that could distinguish Tr-1 cells from Foxp3+ nTR and iTR cells, which also express IL-10, especially after activation. Recent studies have overcome this limitation by using IL-10 locus-tagged mice, revealing Foxp3− Tr-1-like cells to be particularly abundant in the small and large intestine, where they play an essential role in down-regulating the inflammatory response triggered by the commensal flora 23-25. Foxp3− Tr-1-like cells share with Foxp3+ iTR cells a requirement for TGF-β for their in vivo differentiation, but it remains unclear whether Tr-1 cells branch off from a common differentiation pathway with Foxp3+ iTR cells or arise by a separate pathway. Whereas earlier studies have implicated Tr-1 in mucosal tolerance, the precise contributions of Foxp3+ TR cells and Foxp3− Tr-1 cells to induced tolerance remains to be carefully analyzed with the newer analytical tools now available.
Loss of function mutations in the Foxp3 gene underlie the lymphoproliferative disease of the Scurfy mouse and the homologous autoimmune lymphoproliferative disorder in man, termed Immune dysregulation Polyendocrinopathy Enteropathy −X linked syndrome (IPEX). In humans, loss of function FOXP3 mutations result in the IPEX syndrome 1,26. The hallmark of IPEX is immune dysregulation due to the lack of functional TR cells. It typically presents in a male infant with enteropathy, autoimmune endocrinopathy, immune-mediated cytopenias, and dermatitis. There is also allergic dysregulation that manifests in particular as food allergy, eosinophilia and elevated IgE levels 27,28. The autoimmune endocrinopathy is characterized by early onset type 1 diabetes, frequently beginning in the first year of life. Thyroiditis resulting in either hyper or hypothyroidism is also common. Some of the allergic dysregulation in IPEX results from the presence in circulation of TR cell precursors that secrete large amounts of Th2 cytokines especially IL-4, while it is tempting to speculate that the food allergy is a manifestation of iTR cell deficiency 29-31. Foxp3 deficiency in mice, whether due to natural or induced mutations, gives rise to a fatal autoimmune and inflammatory disorder called Scurfy that has many of the features of IPEX 32. In both mice and humans, female carriers are asymptomatic, consistent with X-linked recessive inheritance.
The immunopathology of Foxp3 deficiency results from unchecked T cell activation due to the lack of regulatory restraint by CD4+CD25+ TR cells 33,34. That Foxp3 is essential for TR cell function is supported by the observation that forced expression of Foxp3 in effector T cells endows them with regulatory properties and some, but not all, of the phenotypic markers of regulatory T cells 35,36. Also, Adoptive transfer of CD4+CD25+ TR cells rescues Scurfy mice from disease, and Foxp3-transduced CD4+CD25- T cells suppress wasting and colitis induced by the transfer of CD4+CD25- T cells into RAG-deficient mice 35,37.
Foxp3 deficiency is permissive to the development in the thymus of TR cell precursors that share many of the phenotypic and genetic attributes of TR cells. However, unlike Foxp3-sufficient TR cells, Foxp3-deficient TR cell precursors fail to mediate suppression 29,31. Once in the periphery, they acquire the attributes of an activated cytotoxic cell phenotype. They express high levels of mRNA encoding granzymes, some killer cell markers and a mixed Th1 and Th2 cytokine profile. In particular, they secrete large amounts of IL-4 and other Th2 cytokines, which may account for much of the allergic dysregulation associated with Foxp3 deficiency 27,29,31. Circulating Foxp3-deficient TR cell precursors also exhibit a high rate of apoptotic death, possibly due to suboptimal responses to growth factors such as IL-2. The continued requirement for Foxp3 expression to maintain the phenotype of mature TR cell in the periphery was demonstrated in experiments in which the acute inactivation of the Foxp3 locus rapidly lead to the loss of TR cell regulatory function. Acute Foxp3 deficiency also alters the transcriptional program of TR cells in a manner reminiscent of that of Foxp3-deficient TR cell precursors 38.
IPEX like syndromes in humans and in mice also arise from mutations along the IL-2 signaling pathway, including loss of function mutations in IL-2 receptor alpha chain (CD25) and the IL-2-responsive transcription factor STAT5b, the latter with an associated phenotype of resistance to growth hormone 39-41. CD25 deficiency has been associated with early onset of type 1 diabetes in humans, consistent with the phenotype observed in IPEX syndrome due to Foxp3 mutations. More broadly, the CD25 and IL-2 gene loci have been associated with type 1 diabetes in humans (see chapter 3) and mice, respectively, indicating the essential function of this pathway in maintaining tolerance to islet β-cells (Figure 2) 42,43.
Manipulation of TR cells is an attractive strategy for immunotherapy, as suggested by adoptive transfer of TR cells to treat experimental autoimmune diseases 44,45. Similar treatment strategies are now being developed in humans in whom it is envisaged that cellular therapy with antigen-specific TR cells may allow the development of long-term immune modulation without the problem of general immunosuppression and systemic toxicity 46,47. Other approaches include the derivation for therapeutic use of human cell lines that stably express ectopic Foxp3, which converts both naïve and antigen-specific memory CD4+ T cells into cells with TR cell-like properties 48. TR cell therapy may also be rendered more effective by combining it with immunomodulatory drugs that spare TR cells while targeting conventional T cells. One such drug is rapamycin, an inhibitor of the mTor pathway, which plays an important role in T cell proliferation and survival. The mTor pathway is relatively inactive in TR cells, which employ an alternative IL-2 receptor-coupled STAT5 pathway to mediate cell growth and proliferation 49,50. A second approach is to use histone deacetylase inhibitors, which promote the development of Foxp3+ iTR cells 51.
Impressive progress has been made in elucidating molecular mechanisms underlying TR cell development and differentiation. The stage is now set to tackle some important outstanding issues in the field. These include the nature of the molecular circuitry governing the early development of nTR cells in the thymus, the mechanisms underlying suppressive function of TR cells, the respective roles of nTR and iTR cells in dominant tolerance, and the nature of the molecular pathways mediating their non-redundant functions. The striking allergic dysregulation and food allergy associated with the IPEX syndrome indicates a fundamental role for TR cells in tolerance to allergens, especially foods. Pharmacological interventions that aim to bolster TR cell development and function, alone or together with immunotherapy, offer the potential for novel therapeutic strategies in the treatment of allergic and autoimmune diseases.
This work was supported by National Institutes of Health grants R01AI065617 and R21AI80002.
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