Foxp3+CD4+Regulatory T cells
Foxp3+CD4+ T cells are the best characterized population of regulatory cells and, as already mentioned, there are several types of these cells based on their development. They are indispensable for the maintenance of self-tolerance and immune homeostasis. And indeed their defects cause autoimmune diseases, immunopathologies and allergies .
Their discovery was one of the major findings in immunology and helped to acquire more knowledge regarding the immune system and autoimmunity; it also opened the door to cell therapy based on Treg expansion ex vivo and their transfer into patients to treat autoimmune diseases and inhibit graft-versus-host disease after bone marrow transplantation .
The first evidences for the existence of Foxp3+CD4+ T cells came in 1969 when a neonatal thymeoctomy at day 3 after birth resulted in autoimmune damage of various organs; and this condition was reverted with the transplantation of thymus 7 days later . One year later, it was shown that cells derived from the thymus can suppress immune responses .
Then, in the following years, it was shown that adult thymeoctomy causes an autoimmune disease called thyroiditis  which can be reverted with the injection of CD4+ T cells from syngeneic mice . These results showed that not only there are pathogenic autoreactive T cells but also T cells that suppress autoimmunity. The key problem was to identify a marker to enrich these regulatory cells.
The growing interest for the possibility to identify markers to define Treg pushed researchers to conduct several experiments. One of the most important was to deplete a CD4+ T cell suspension of a population of CD5high cells and to transfer the rest to T-celldeficient athymic nude mice; the result was the development of autoimmunity in several organs . Reconstitution of depleted CD4+ T cell subpopulation inhibited autoimmunity. Indeed, the first identified marker of Treg was CD5 or Lyt-1.
More efforts to search for something more specific led to the identification of CD25 which is the chain of the IL-2 receptor. Transfer of T cells depleted of CD25+ T cell causes autoimmunity in athymic nude mice, while a cotransfer of CD25+CD4+ T cell inhibits the development of autoimmunity .
Indeed IL-2 is a key cytokine for Treg development, survival and function and mice lacking IL-2 develop T cell-mediated fatal lymphoproliferative/inflammatory disease with autoimmune features .
Mice deficient in CD25 or CD122 (the chain of the IL-2 receptor) succumb to a similar disease. In humans, CD25 deficiency is indistinguishable from IPEX (immune dyregulation, polyendocrinopathy, enteropathy, X-linked syndrome), an autoimmune disease characterized by defective Treg . Evidences suggest that the syndrome is due to deficiency or dysfunction of Foxp3+Treg for 3 reasons: the number of these cells is reduced in mice lacking either CD25 or IL-2 , T cell-specific deficiency of Signal Transducer and Activator of Transcription (STAT) 5a and b (which mediate signaling from IL-2 receptor) abrogates the development of Foxp3+Treg causing autoimmunity  and lastly, high dose of neutralizing anti-IL-2 antibody to normal neonatal mice reduces the number of Foxp3+CD25+CD4+ T cells for a limited period .
It is possible to conclude that IL-2 has a pivotal role in immune homeostasis since it is produced by activated non-regulatory T cells (Treg are unable to produce it), and it contributes to differentiation, maintenance, expansion and activation of Treg which in turn limits the expansion of non-regulatory T cells (Figure 2). Disruption of this feedback loop promotes the development of autoimmunity.
Features of Treg stability and activation
Recently, researchers have put a lot of efforts to couple NGS techniques to Treg to unravel their core signature meaning the major genes characterizing their phenotype.
To this regard, Benoist et al.  compared the transcriptome of Treg and conventional T cells (Tconv) from mice and humans at the steady state using single cell RNA-sequencing. They saw two separated clusters for Treg and Tconv, as expected. However, surprisingly, they saw some so-called “furtive Treg” with a transcriptome clustering with the Tconv (26% in mice and 55% in humans). Foxp3 was detected in those cells at similar levels to those of other Treg and canonical Treg signature genes were also overexpressed in these cells compared to surrounding Tconv. However, some of these key genes were present at lower levels compared to other Treg, suggesting that they could be “weaker” Treg with a poorly suppressive phenotype.
In addition, they also saw Tconv and Treg clustering together in three separated minor clusters. One of these contains cells expressing genes associated with residence in B cell follicles (T follicular helper and regulatory cells) and the others contain cells that upregulate a set of genes associated with early response to TCR engagement. These observations mean that even if Treg are generally distinguishable from Tconv, the two populations were imbricated at different levels with an important degree of overlap and that TCR signaling may drive similar programs in both Treg and Tconv. To go deeper with the analysis, they systematically compared Treg with closest Tconv and identified a small set of genes (IL-2ra, IL-2rb, Ikzf2, Ctla4, Capg, Tnfrsf4, Tnfrsf18, Izumo1r, Chchd10, Gpr83 and Foxp3) overexpressed by Treg in all clusters.
Moreover, they also pointed out that in the big cluster of Treg it is possible to identify six clusters, half of which contain resting and the other half activated Treg according to Sell (CD62L) and Ccr7 expression. Among the activated ones, there were Treg expressing genes of early cell activation thus evoked TCR-mediated activation, while others characterized by a diverse set of genes. These data describe well Treg identity which is not so separated from Tconv but also their heterogeneity suggesting that in inflammatory conditions or in different tissues we might find Treg expressing different sets of genes.
Treg stability and plasticity
The molecular and cellular bases of Treg stability remain a key issue of Treg research. To use Treg in clinical for immunosuppression, it is important to know exactly how they develop in terms of Foxp3 expression and epigenetic changes.
What does Treg stability mean, first of all? It could be defined by the maintenance of all the following characteristics: Foxp3 expression, suppressive activity and lack of effector activity. Stable Foxp3 expression is due mainly to specific epigenetic modifications. As already mentioned, the CNS2 at the 5’UTR of Foxp3 must be hypomethilated in Treg allowing transcription ; and this pattern is maintained mainly by IL-2.
Many studies, in the beginning, have suggested that Treg development leads to a terminally differentiated population , suggesting Treg are a highly stable lineage. However, others have suggested that Treg can lose Foxp3 expression and develop a proinflammatory, memory-like phenotype in certain disease states .
Indeed tTreg have been shown to convert to different helper T cells such as Th1, Th17 or Tfh cells . Under lymphopenic conditions, many Treg were found to lose Foxp3 and start to produce IL-2 and IFN-.
The topic is still controversial in part because of different strains of mouse used, then because of different methods used to sort Treg and lastly, it is also possible that T cells transiently upregulate Foxp3 during development leading to a “false labeling” of cells .
Impact of inflammatory cytokines on Treg
A lot of efforts have been put to characterize Treg, meaning their signature, phenotype and functions. However, it was seen that it might be difficult to define a unique subset of Treg. They are different in all the aforementioned aspects based on their localization, activation state and exposure to environmental factors. Of course inflammation is a crucial factor that impact on Treg differentiation and homeostasis. Some cytokines have been further analyzed in relation to their impact on Treg biology. IL-7 is a cytokine produced by lymph node stromal cells important for naïve T cell survival and activation during adaptive immune responses. The binding to its receptor causes STAT5 phosphorylation and expression of antiapoptotic proteins important for T cell development and proliferation. The role of this cytokine on Treg is less clear. It was shown that injections of IL-7 complexes optimize their reactivity to IL-2. Mice deficient for IL-7R have reduced Treg and they exhibit impaired proliferation. In a model of skin allograft tolerance it was seen that IL-7 augments CD25 expression, increasing Treg sensibility to IL-2, depriving Tconv from IL-2 and therefore inhibiting strong allogenic T cell response. Indeed, IL-7 stabilizes Treg suppressive phenotype increasing the expression of CD25, GITR and ICOS. Normally, cTreg express highly CD25 relying deeply on IL-2, while eTreg express less CD25 but higher amount of IL-7R. Therefore, in the presence of inflammation and therefore of high amounts of IL-7, they rapidly upregulate the CD25 consuming more IL-2. This shift in IL-2 consumption could then impact on the cTreg/eTreg balance during inflammation.
IL-33 is part of the IL-1 family and its receptor exists in two forms: membrane-bound and soluble. It is usually considered an epithelial cytokine that promotes type 2 immune responses, however recent studies showed its role in basal tissue regulation, organ injury and repair and immunity to microbes.
Suppression by inhibitory cytokines
The two main cytokines involved are IL-10 and TGF- which are not only part of suppression mediated by Treg but also stimulate the development of pTreg, which renders them attractive therapeutic targets.
In allergy and asthma models, evidences suggest both tTreg and pTreg control these diseases which depend, in part, on IL-10 . After antigen challenge, adoptively transferred Treg stimulate Tconv to produce high amount of IL-10 and therefore to control the disease. However, after transfer of IL-10-deficient Treg, the disease was still controlled by IL-10 which means that the production of this cytokine by Treg is not necessary for the suppression observed . In contrast, IL-10 production by Treg seems essential for the prevention of colitis in mouse model of IBD . Moreover, the tumor microenvironment promotes the generation of Treg that mediate IL-10-dependent, cellcontact independent suppression . In addition, papers suggest a role for Tregderived IL-10 in the induction of feto-maternal tolerance and B-cell enhanced recovery from EAE .
The importance of TGF- for tTreg has been also a controversial topic. There are studies suggesting that TGF- produced by Treg may directly participate in the suppression of Tconv. For instance, Tconv resistant to TGF--mediated suppression cannot be controlled by Treg . In addition, TGF- produced by Treg has been found important in the control of immune responses against M. tuberculosis , suppression of allergic responses  and prevention of colitis in an IBD model .
A new inhibitory cytokine was then added in mediated Treg suppression, IL-35. Indeed IL- 35 is sufficient for Treg activity as ectopic expression of IL-35 confers regulatory activity on naïve T cells and recombinant IL-35 suppresses Tconv proliferation in vitro .
Table of contents :
1. The immune tolerance
1.1 Central tolerance
1.2 Peripheral tolerance
2. Foxp3+CD4+Regulatory T cells
2.2 Phenotypic markers
2.3 Features of Treg stability and activation
2.4 Treg development
2.4.1 Development of tTreg
2.4.2 Development of pTreg
2.5 Treg stability and plasticity
2.5.1 Treg stability
2.5.2 Class control
2.6 Role of tTreg and pTreg
2.6.2 Allergies and asthma
2.6.6 tTreg and pTreg
2.7 Impact of inflammatory cytokines on Treg
2.8 Mechanisms of suppression
2.8.1 Suppression by inhibitory cytokines
2.8.2 Suppression by cytolysis
2.8.3 Suppression by metabolic disruption
2.8.4 Suppression by targeting dendritic cells
3. Role of Tumor Necrosis Factor Receptor Super Family (TNFRSF) in T cells
3.1 The discovery of the TNFSF
3.4 General features on the roles of TNFRSF members
3.4.1 TNFRSF members and Tconv
3.4.2 TNFRSF members and Treg
3.4.3 TNFRSF members and disease
3.5.1 Expression and main features
3.5.2 Role in AD
3.5.3 Role in cancer
3.5.4 Role in Tconv
3.5.5 Role in Treg
3.6.1 Expresion and main features
3.6.2 Role in AD
3.6.3 Role in cancer
3.6.4 Role in Tconv
3.6.5 Role in Treg
3.7.1 Expression and main features
3.7.2 Role in AD
3.7.3 Role in cancer
3.7.4 Role in Tconv
3.7.5 Role in Treg
3.8.1 Expression and main features
3.8.2 Role in AD
3.8.3 Role in cancer
3.8.4 Role in Tconv
3.8.5 Role in Treg
3.9.1 Expression and main features
3.9.2 Role in AD
3.9.3 Role in Tconv
3.9.4 Role in Treg
3.10 Other TNFRSF members
AIM OF WORK
1. The importance of Treg and TNFRs
2. Role of TNFRs members in Treg biology
3. Role of TNFRs in iTreg