T-regulatory cell

The regulatory T cells (Tregs or T_reg cells), formerly known as suppressor T cells, are a subpopulation of T cells that modulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease. T_reg cells are immunosuppressive and generally suppress or downregulate induction and proliferation of effector T cells. T_reg cells express the biomarkers CD4, FOXP3, and CD25 and are thought to be derived from the same lineage as naïve CD4⁺ cells. Because effector T cells also express CD4 and CD25, T_reg cells are very difficult to effectively discern from effector CD4⁺, making them difficult to study. Research has found that the cytokine transforming growth factor beta (TGF-β) is essential for T_reg cells to differentiate from naïve CD4⁺ cells and is important in maintaining T_reg cell homeostasis.

Mouse models have suggested that modulation of T_reg cells can treat autoimmune disease and cancer and can facilitate organ transplantation and wound healing. Their implications for cancer are complicated. T_reg cells tend to be upregulated in individuals with cancer, and they seem to be recruited to the site of many tumors. Studies in both humans and animal models have implicated that high numbers of T_reg cells in the tumor microenvironment is indicative of a poor prognosis, and T_reg cells are thought to suppress tumor immunity, thus hindering the body's innate ability to control the growth of cancerous cells. Immunotherapy research is studying how regulation of T cells could possibly be utilized in the treatment of cancer.

Populations

T regulatory cells are a component of the immune system that suppress immune responses of other cells. This is an important "self-check" built into the immune system to prevent excessive reactions. Regulatory T cells come in many forms with the most well-understood being those that express CD4, CD25, and FOXP3 (CD4⁺CD25⁺ regulatory T cells). These T_reg cells are different from helper T cells. Another regulatory T cell subset is T_reg17 cells. Regulatory T cells are involved in shutting down immune responses after they have successfully eliminated invading organisms, and also in preventing autoimmunity.

CD4⁺ FOXP3⁺ CD25(high) regulatory T cells have been called "naturally occurring" regulatory T cells to distinguish them from "suppressor" T cell populations that are generated ''in vitro''. Additional regulatory T cell populations include Tr1, T_h3, CD8⁺CD28⁻, and Qa-1 restricted T cells. The contribution of these populations to self-tolerance and immune homeostasis is less well defined. FOXP3 can be used as a good marker for mouse CD4⁺CD25⁺ T cells, although recent studies have also shown evidence for FOXP3 expression in CD4⁺CD25⁻ T cells. In humans, FOXP3 is also expressed by recently activated conventional T cells and thus does not specifically identify human T_regs.

Development

All T cells derive from progenitor cells in the bone marrow, which become committed to their lineage in the thymus. All T cells begin as CD4-CD8- TCR- cells at the DN (double-negative) stage, where an individual cell will rearrange its T cell receptor genes to form a unique, functional molecule, which they, in turn, test against cells in the thymic cortex for a minimal level of interaction with self- MHC. If they receive these signals, they proliferate and express both CD4 and CD8, becoming double-positive cells. The selection of T_regs occurs on radio-resistant hematopoietically derived MHC class II-expressing cells in the medulla or Hassall's corpuscles in the thymus. At the DP (double-positive) stage, they are selected by their interaction with the cells within the thymus, begin the transcription of Foxp3, and become T_reg cells, although they may not begin to express Foxp3 until the single-positive stage, at which point they are functional T_regs. T_regs do not have the limited TCR expression of NKT or γδ T cells; T_regs have a larger TCR diversity than effector T cells, biased towards self-peptides.

The process of T_reg selection is determined by the affinity of interaction with the self-peptide MHC complex. Selection to become a T_reg is a "Goldilocks" process - i.e. not too high, not too low, but just right; a T cell that receives very strong signals will undergo apoptotic death; a cell that receives a weak signal will survive and be selected to become an effector cell. If a T cell receives an intermediate signal, then it will become a regulatory cell. Due to the stochastic nature of the process of T cell activation, all T cell populations with a given TCR will end up with a mixture of T_eff and T_reg – the relative proportions determined by the affinities of the T cell for the self-peptide-MHC. Even in mouse models with TCR-transgenic cells selected on specific-antigen-secreting stroma, deletion or conversion is not complete.

Foxp3⁺ T_reg generation in the thymus is delayed by several days compared to T_eff cells and does not reach adult levels in either the thymus or periphery until around three weeks post-partum. T_reg cells require CD28 co-stimulation and B7.2 expression is largely restricted to the medulla, the development of which seems to parallel the development of Foxp3⁺ cells. It has been suggested that the two are linked, but no definitive link between the processes has yet been shown. TGF-β is not required for T_reg functionality, in the thymus, as thymic T_regs from TGF-β insensitive TGFβRII-DN mice are functional.

Thymic recirculation

There was observed, that some FOXP3⁺ T_reg cells are recirculating back to thymus, where they have developed. This T_reg were mainly present in thymic medula, which is the main site of T_reg cells differentiation.
The presence of this cells in thymus or addition into fetal thymic tissue culture suppress development of new T_reg cells by 34–60%, but Tconv cells are not affected. That means, that recirculating T_reg to thymus inhibited just ''de novo'' development of T_reg cells.
Molecular mechanism of this process works due to the ability of T_reg to adsorb IL-2 from the microenvironments, thus being able to induce apoptosis of other T cells which need IL-2 as main growth factor. Recirculating T reg cells in thymus express high amount of high affinity IL-2 receptor α chain (CD25) encoded by ''Il2ra'' gene which gather IL-2 from thymic medulla, and decrease its concentration. New generated FOXP3⁺ T_reg cells in thymus have not so high amount of ''Il2ra'' expression. IL-2 is a cytokine necessary for the development of T_reg cells in the thymus. It is important for T cells proliferation and survival, but in the case of its deficiency, IL-15 may be replaced. However, T_reg cells' development is dependent on IL-2. In humans, there was found population of CD31 negative T_reg cells in thymus. CD31 could be used as a marker of new generated T_reg cells as same as other T lymphocytes. Mature and peripheral T_reg cells have decreased its expression. So it is possible that this regulatory mechanism of thymic T_reg cells development is also functional in humans.

There is probably also positive regulation of thymic T_reg cells development caused by recirculating T_reg cells into thymus. There was found population of CD24 low FOXP3⁺ in thymus with increased expression of IL-1R2 (''Il1r2'') compared with peripheral T_reg cells. High concentration of IL-1β caused by inflammation decrease ''de novo'' development of T_reg cells in thymus. The presence of recirculating T_reg cells in the thymus with high IL1R2 expression during inflammatory conditions helps to uptake IL-1β and reduce its concentration in the medulla microenvironment, thus they are helping to the development of ''de novo'' T_reg cells. High concentration of IL-1β caused by inflammation decrease ''de novo'' development of T_reg cells in thymus. Binding of IL-1β to IL1R2 on the surface of T_reg cells does not cause any signal transduction because there is no present Intracelluar ( TIR) Toll interleukin-1 receptor domain, which is normally present in innate immune cells.

Function

The immune system must be able to discriminate between self and non-self. When self/non-self discrimination fails, the immune system destroys cells and tissues of the body and as a result causes autoimmune diseases. Regulatory T cells actively suppress activation of the immune system and prevent pathological self-reactivity, i.e. autoimmune disease. The critical role regulatory T cells play within the immune system is evidenced by the severe autoimmune syndrome that results from a genetic deficiency in regulatory T cells ( IPEX syndrome – see also below).

The molecular mechanism by which regulatory T cells exert their suppressor/regulatory activity has not been definitively characterized and is the subject of intense research. '' In vitro'' experiments have given mixed results regarding the requirement of cell-to-cell contact with the cell being suppressed. The following represent some of the proposed mechanisms of immune suppression:

* Regulatory T cells produce a number of inhibitory cytokines. These include TGF-β, Interleukin 35, and Interleukin 10. It also appears that regulatory T cells can induce other cell types to express interleukin-10.
* Regulatory T cells can produce Granzyme B, which in turn can induce apoptosis of effector cells. Regulatory T cells from Granzyme B deficient mice are reported to be less effective suppressors of the activation of effector T cells.
* Reverse signalling through direct interaction with dendritic cells and the induction of immunosuppressive indoleamine 2,3-dioxygenase.
* Signalling through the ectoenzymes CD39 and CD73 with the production of immunosuppressive adenosine.
* Through direct interactions with dendritic cells by LAG3 and by TIGIT. This review of T_reg interactions with dendritic cells provides distinction between mechanisms described for human cells versus mouse cells.
* Another control mechanism is through the IL-2 feedback loop. Antigen-activated T cells produce IL-2 which then acts on IL-2 receptors on regulatory T cells alerting them to the fact that high T cell activity is occurring in the region, and they mount a suppressory response against them. This is a negative feedback loop to ensure that overreaction is not occurring. If an actual infection is present other inflammatory factors downregulate the suppression. Disruption of the loop leads to hyperreactivity, regulation can modify the strength of the immune response. A related suggestion with regard to interleukin 2 is that activated regulatory T cells take up interleukin 2 so avidly that they deprive effector T cells of sufficient to avoid apoptosis.
* A major mechanism of suppression by regulatory T cells is through the prevention of co-stimulation through CD28 on effector T cells by the action of the molecule CTLA-4.

Induced regulatory T cells

Induced regulatory T (iT_reg) cells (CD4⁺ CD25⁺ FOXP3⁺) are suppressive cells involved in tolerance. iT_reg cells have been shown to suppress T cell proliferation and experimental autoimmune diseases. These cells include T_reg17 cells. iT_reg cells develop from mature CD4⁺ conventional T cells outside of the thymus: a defining distinction between natural regulatory T (nT_reg) cells and iT_reg cells. Though iT_reg and nT_reg cells share a similar function iT_reg cells have recently been shown to be "an essential non-redundant regulatory subset that supplements nT_reg cells, in part by expanding TCR diversity within regulatory responses". Acute depletion of the iT_reg cell pool in mouse models has resulted in inflammation and weight loss. The contribution of nT_reg cells versus iT_reg cells in maintaining tolerance is unknown, but both are important. Epigenetic differences have been observed between nT_reg and iT_reg cells, with the former having more stable FOXP3 expression and wider demethylation.

The small intestinal environment is high in vitamin A and is a location where retinoic acid is produced. The retinoic acid and TGF-beta produced by dendritic cells within this area signal for production of regulatory T cells. Vitamin A and TGF-beta promote T cell differentiation into regulatory T cells opposed to T_h17 cells, even in the presence of IL-6. The intestinal environment can lead to induced regulatory T cells with TGF-beta and retinoic acid, some of which express the lectin-like receptor CD161 and are specialized to maintain barrier integrity by accelerating wound healing. The T_regs within the gut are differentiated from naïve T cells after antigen is introduced. It has recently been shown that human regulatory T cells can be induced from both naive and pre-committed Th1 cells and Th17 cells using a parasite-derived TGF-β mimic, secreted by '' Heligmosomoides polygyrus'' and termed ''Hp''-TGM (''H. polygyrus'' TGF-β mimic). ''Hp''-TGM can induce murine FOXP3 expressing regulatory T cells that were stabile in presence of inflammation ''in vivo''. ''Hp''-TGM-induced human FOXP3+ regulatory T cells were stable in the presence of inflammation and had increased levels of CD25, CTLA4 and decreased methylation in the '' FOXP3'' T_reg-Specific demethylated region compared to TGF-β-induced T_regs.

Disease

An important question in the field of immunology is how the immunosuppressive activity of regulatory T cells is modulated during the course of an ongoing immune response. While the immunosuppressive function of regulatory T cells prevents the development of autoimmune disease, it is not desirable during immune responses to infectious microorganisms. Current hypotheses suggest that, upon encounter with infectious microorganisms, the activity of regulatory T cells may be downregulated, either directly or indirectly, by other cells to facilitate elimination of the infection. Experimental evidence from mouse models suggests that some pathogens may have evolved to manipulate regulatory T cells to immunosuppress the host and so potentiate their own survival. For example, regulatory T cell activity has been reported to increase in several infectious contexts, such as retroviral infections (the most well-known of which is HIV), mycobacterial infections (e.g., tuberculosis), and various parasitic infections including '' Leishmania'' and malaria.

T_reg cells play major roles during HIV infection. They suppress the immune system, thus limiting target cells and reducing inflammation, but this simultaneously disrupts the clearance of virus by the cell-mediated immune response and enhances the reservoir by pushing CD4⁺ T cells to a resting state, including infected cells. Additionally, T_reg cells can be infected by HIV, increasing the size of the HIV reservoir directly. Thus, T_reg cells are being investigated as targets for HIV cure research. Some T_reg cell depletion strategies have been tested in SIV infected nonhuman primates, and shown to cause viral reactivation and enhanced SIV specific CD8⁺ T cell responses.

Regulatory T cells have a large role in the pathology of visceral leishmaniasis and in preventing excess inflammation in patients cured of visceral leishmaniasis.

CD4⁺ regulatory T cells are often associated with solid tumours in both humans and murine models. Increased numbers of regulatory T cells in breast, colorectal and ovarian cancers is associated with a poorer prognosis.

CD70⁺ non-Hodgkin lymphoma B cells induce FOXP3 expression and regulatory function in intratumoral CD4⁺CD25⁻ T cells.

There is some evidence that T_reg cells may be dysfunctional and driving neuroinflammation in amyotrophic lateral sclerosis due to lower expression of FOXP3. ''Ex vivo'' expansion of T_reg cells for subsequent autologous transplant is currently being investigated after promising results were obtained in a phase I clinical trial.

Additionally, while regulatory T cells have been shown to increase via polyclonal expansion both systemically and locally during healthy pregnancies to protect the fetus from the maternal immune response (a process called maternal immune tolerance), there is evidence that this polyclonal expansion is impaired in preeclamptic mothers and their offspring. Research suggests reduced production and development of regulatory T cells during preeclampsia may degrade maternal immune tolerance, leading to the hyperactive immune response characteristic of preeclampsia.

Cancer

Most tumors elicit an immune response in the host that is mediated by tumor antigens, thus distinguishing the tumor from other non-cancerous cells. This causes large numbers of tumor-infiltrating lymphocytes (TILs) to be found in the tumor microenvironment. Although it is not entirely understood, it is thought that these lymphocytes target cancerous cells and therefore slow or terminate the development of the tumor. However, this process is complicated because T_reg cells seem to be preferentially trafficked to the tumor microenvironment. While T_reg cells normally make up only about 4% of CD4⁺ T cells, they can make up as much as 20–30% of the total CD4⁺ population around the tumor microenvironment.

Although high levels of TILs were initially thought to be important in determining an immune response against cancer, it is now widely recognized that the ratio of T_reg to effector T cells in the tumor microenvironment is a determining factor in the success of the immune response against the cancer. High levels of T_reg cells in the tumor microenvironment are associated with poor prognosis in many cancers, such as ovarian, breast, renal, and pancreatic cancer. This indicates that T_reg cells suppress effector T cells and hinder the body's immune response against the cancer. However, in some types of cancer the opposite is true, and high levels of T_reg cells are associated with a positive prognosis. This trend is seen in cancers such as colorectal carcinoma and follicular lymphoma. This could be due to T_reg cells' ability to suppress general inflammation which is known to trigger cell proliferation and metastasis . These opposite effects indicate that T_reg cells' role in the development of cancer is highly dependent on both type and location of the tumor.

Although it is still not entirely understood how T_reg cells are preferentially trafficked to the tumor microenvironment, the chemotaxis is probably driven by the production of chemokines by the tumor. T_reg infiltration into the tumor microenvironment is facilitated by the binding of the chemokine receptor CCR4, which is expressed on T_reg cells, to its ligand CCL22, which is secreted by many types of tumor cells. T_reg cell expansion at the site of the tumor could also explain the increased levels of T_reg cells. The cytokine, TGF-β, which is commonly produced by tumor cells, is known to induce the differentiation and expansion of T_reg cells.

Forkhead box protein 3 ( FOXP3) as a transcription factor is an essential molecular marker of T_reg cells. FOXP3 polymorphism (rs3761548) might be involved in the gastric cancer progression through influencing T_reg function and the secretion of immunomodulatory cytokines such as IL-10, IL-35, and TGF-β.

In general, the immunosuppression of the tumor microenvironment has largely contributed to the unsuccessful outcomes of many cancer immunotherapy treatments. Depletion of T_reg cells in animal models has shown an increased efficacy of immunotherapy treatments, and therefore, many immunotherapy treatments are now incorporating T_reg depletion.

Molecular characterization

Similar to other T cells, regulatory T cells develop in the thymus. The latest research suggests that regulatory T cells are defined by expression of the forkhead family transcription factor FOXP3 (forkhead box p3). Expression of FOXP3 is required for regulatory T cell development and appears to control a genetic program specifying this cell's fate. The large majority of Foxp3-expressing regulatory T cells are found within the major histocompatibility complex (MHC) class II restricted CD4-expressing (CD4⁺) population and express high levels of the interleukin-2 receptor alpha chain (CD25). In addition to the FOXP3-expressing CD4⁺ CD25⁺, there also appears to be a minor population of MHC class I restricted CD8⁺ FOXP3-expressing regulatory T cells. These FOXP3-expressing CD8⁺ T cells do not appear to be functional in healthy individuals but are induced in autoimmune disease states by T cell receptor stimulation to suppress IL-17-mediated immune responses. Unlike conventional T cells, regulatory T cells do not produce IL-2 and are therefore anergic at baseline.

A number of different methods are employed in research to identify and monitor T_reg cells. Originally, high expression of CD25 and CD4 surface markers was used (CD4⁺CD25⁺ cells). This is problematic as CD25 is also expressed on non-regulatory T cells in the setting of immune activation such as during an immune response to a pathogen. As defined by CD4 and CD25 expression, regulatory T cells comprise about 5–10% of the mature CD4⁺ T cell subpopulation in mice and humans, while about 1–2% of T_reg can be measured in whole blood. The additional measurement of cellular expression of FOXP3 protein allowed a more specific analysis of T_reg cells (CD4⁺CD25⁺FOXP3⁺ cells). However, FOXP3 is also transiently expressed in activated human effector T cells, thus complicating a correct T_reg analysis using CD4, CD25 and FOXP3 as markers in humans. Therefore, the gold standard surface marker combination to defined T_regs within unactivated CD3⁺CD4⁺ T cells is high CD25 expression combined with the absent or low-level expression of the surface protein CD127 (IL-7RA). If viable cells are not required then the addition of FOXP3 to the CD25 and CD127 combination will provide further stringency. Several additional markers have been described, e.g., high levels of CTLA-4 (cytotoxic T-lymphocyte associated molecule-4) and GITR (glucocorticoid-induced TNF receptor) are also expressed on regulatory T cells, however the functional significance of this expression remains to be defined. There is a great interest in identifying cell surface markers that are uniquely and specifically expressed on all FOXP3-expressing regulatory T cells. However, to date no such molecule has been identified.

The identification of T_regs following cell activation is challenging as conventional T cells will express CD25, transiently express FOXP3 and lose CD127 expression upon activation. It has been shown that T_regs can be detected using an activation-induced marker assay by expression of CD39 in combination with co-expression of CD25 and OX40(CD134) which define antigen-specific cells following 24-48h stimulation with antigen.

In addition to the search for novel protein markers, a different method to analyze and monitor T_reg cells more accurately has been described in the literature. This method is based on DNA methylation analysis. Only in T_reg cells, but not in any other cell type, including activated effector T cells, a certain region within the gene (TSDR, T_reg-specific-demethylated region) is found demethylated, which allows to monitor T_reg cells through a PCR reaction or other DNA-based analysis methods.
Interplay between the Th17 cells and regulatory T cells are important in many diseases like respiratory diseases.

Recent evidence suggests that mast cells may be important mediators of T_reg-dependent peripheral tolerance.

Epitopes

Regulatory T cell epitopes ('Tregitopes') were discovered in 2008 and consist of linear sequences of amino acids contained within monoclonal antibodies and immunoglobulin G (IgG). Since their discovery, evidence has indicated Tregitopes may be crucial to the activation of natural regulatory T cells.

Potential applications of regulatory T cell epitopes have been hypothesised: tolerisation to transplants, protein drugs, blood transfer therapies, and type I diabetes as well as reduction of immune response for the treatment of allergies.

Genetic deficiency

Genetic mutations in the gene encoding FOXP3 have been identified in both humans and mice based on the heritable disease caused by these mutations. This disease provides the most striking evidence that regulatory T cells play a critical role in maintaining normal immune system function. Humans with mutations in FOXP3 develop a severe and rapidly fatal autoimmune disorder known as Immune dysregulation, Polyendocrinopathy, Enteropathy X-linked (IPEX) syndrome.

The IPEX syndrome is characterized by the development of overwhelming systemic autoimmunity in the first year of life, resulting in the commonly observed triad of watery diarrhea, eczematous dermatitis, and endocrinopathy seen most commonly as insulin-dependent diabetes mellitus. Most individuals have other autoimmune phenomena including Coombs-positive hemolytic anemia, autoimmune thrombocytopenia, autoimmune neutropenia, and tubular nephropathy. The majority of affected males die within the first year of life of either metabolic derangements or sepsis. An analogous disease is also observed in a spontaneous FOXP3-mutant mouse known as "scurfy".

References

External links

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