foxp3 treg-specific demethylated region compared to tgf-β-induced tregs. 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. treg 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, treg cells can be infected by hiv, increasing the size of the hiv reservoir directly. thus, treg cells are being investigated as targets for hiv cure research. some treg 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 treg cells may be dysfunctional and driving neuroinflammation in amyotrophic lateral sclerosis due to lower expression of foxp3. ex vivo expansion of treg 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 the recruitment and maintenance of treg cells in the tumor microenvironment 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 treg cells seem to be preferentially trafficked to the tumor microenvironment. while treg 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 treg 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 treg cells in the tumor microenvironment are associated with poor prognosis in many cancers, such as ovarian, breast, renal, and pancreatic cancer. this indicates that treg 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 treg 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 treg cells' ability to suppress general inflammation which is known to trigger cell proliferation and metastasis . these opposite effects indicate that treg 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 treg cells are preferentially trafficked to the tumor microenvironment, the chemotaxis is probably driven by the production of chemokines by the tumor. treg infiltration into the tumor microenvironment is facilitated by the binding of the chemokine receptor ccr4, which is expressed on treg cells, to its ligand ccl22, which is secreted by many types of tumor cells. treg cell expansion at the site of the tumor could also explain the increased levels of treg cells. the cytokine, tgf-β, which is commonly produced by tumor cells, is known to induce the differentiation and expansion of treg cells. forkhead box protein 3 (foxp3) as a transcription factor is an essential molecular marker of treg cells. foxp3 polymorphism (rs3761548) might be involved in the gastric cancer progression through influencing treg 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 treg cells in animal models has shown an increased efficacy of immunotherapy treatments, and therefore, many immunotherapy treatments are now incorporating treg 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 treg 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 treg can be measured in whole blood. the additional measurement of cellular expression of foxp3 protein allowed a more specific analysis of treg cells (cd4+cd25+foxp3+ cells). however, foxp3 is also transiently expressed in activated human effector t cells, thus complicating a correct treg analysis using cd4, cd25 and foxp3 as markers in humans. therefore, the gold standard surface marker combination to defined tregs 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 tregs 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 tregs 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 treg cells more accurately has been described in the literature. this method is based on dna methylation analysis. only in treg cells, but not in any other cell type, including activated effector t cells, a certain region within the foxp3 gene (tsdr, treg-specific-demethylated region) is found demethylated, which allows to monitor treg 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 treg-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".