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By encountering the attack of foreign, potentially pathogenic, microorganisms, the immune system responsible for protecting the host by recognizing antigens expressed by those microorganisms, mounting an immune response and eliminate or kill the pathogen. But if an over exuberant response to a pathogen or high reactivity to self-antigens, may result in damage to the host. Therefore, in our body, various mechanisms play a role of control and regulate the immune system to prevent or minimize reactivity to these imbalance coordination.
To avoidance of extensive damage to the body, Regulatory T (Treg) cell populations achieve the active suppression mediation. Fully understanding Tregs will lead us to harnessing the capacity of these cells in order to develop strategies to limit and prevent autoimmune disorders, tolerance to transplantation, and potentially boosting immune activity against cancer cells. We discuss here the varied mechanisms used by Treg cells to suppress the immune system.
The immune system has subsets of T cells, known as regulatory T (TReg) cells, It is a white blood cell that helps end an immune response, to mediating immune suppression. It is essential in the maintenance of immune homeostasis and self-tolerance. Two major Treg population include naturally occurring Treg and interleukin(IL)-10 secreting Treg+.Naturally occuring Tregs (Tregs) are characterized by the expression of CD4, CD25 and FOXP3, So naturally occurring Treg+ also called CD4+CD25+ T cells . A FOXP3 is a transcription factor important in the development of Tregs. as occurs in autoimmune disorders. Such as help control the response by secreting cytokines that inhibit immune responses. a common feature of Treg cells is producing IL-10 (IL-10 Treg) and secreting transforming growth factor (TGF)-β .The activity of these two cytokines is control some of the immune responses in vivo. These cells prevent the immune response from continuing indefinitely.Defects in TReg cell function may be an important factor in the development of autoimmunity or in the failure to control immunopathology, whereas overactive TReg cell function may contribute to the suppression of tumour immunity
TReg cell secretes the immunosuppressive cytokine interleukin-10 (IL-10) and may develop from conventional CD4+ T cells by activation in the presence of IL-10 or may develop from T helper 1 (TH1) or TH2 cell subsets.
Treg cells play a pivotal role in preventing autoimmune diseases, such as type 1 diabetes, and limiting chronic inflammatory diseases, such as asthma and inflammatory bowel disease (IBD), they also block beneficial responses by preventing sterilizing immunity to certain pathogens and limiting anti-tumour immunity.
a key transcription factor, forkhead box P3 (FOXP3), that is required for Treg cells development, it is important in maintaining peripheral tolerance maintenance and function .If patients that lack FOXP3 ,may develop a profound autoimmune-like lymphoproliferative disease
various potential suppression mechanisms of Tregcells can be grouped into four basic ‘modes of action’:
1.suppression by inhibitory cytokines
2.suppression by cytolysis http://www.microbio.uab.edu/mic740/papers/12072009/Shevach.pdf page 638 3. suppression by metabolic disruption 4.suppression by modulation of dendritic-cell (DC) maturation or function 639?
Suppression by inhibitory cytokines
Treg cells may secrete suppressor cytokines that can directly inhibit the function of responder
T cells and myeloid cells. Treg cells express high CD25, the IL-2 receptor a chain, and have the
capacity to compete with effector T cells for IL-2.resulting in cytokine-mediated deprivation of the
effector cells and Bim-mediated apoptosis. Activated Foxp3+ Treg cells may function as cytotoxic
cells and directly kill effector cells in a manner similar to CD8+ cytotoxic cells. Activated Treg cells may express known (e.g., galectin-1) or unknown molecules on their cell surface that can interact with receptors on effector T cells resulting in cell cycle arrest.
Suppression by cytolysis
Cytolysis mediated through secretion of granzymes.CD4+ cells express cytotoxic activity.When the naturally occuring Treg cells are activated,the granzyme A express.Also the granzyme B dependent suppression perforin also suppress B cell function.
Suppression by metabolic disruption
The expression of ectoenzymes CD39 and CD73 generate pericellular adenosine,which bind to the adenoisine receptor 2A.Thus can suppress the T cell effector function and generated induced Treg cell by inhibiting IL-6 expression while promoting TGF β secretion.
Suppression by modulation of dendritic-cell (DC) maturation or function
Major Mechanisms by which TregCells Can Suppress the Function of APC and Indirectly Block the Activation of Foxp3 T Cells
CTLA-4 is expressed by Treg cells downregulates or prevents the upregulation of CD80 and CD86,the major costimulatory molecules on antigen presenting cells(APC). Similarly, LAG-3 on Treg cells can interact with MHC class II on antigen-presenting cells, and binding of LAG-3 to MHC class II molecules expressed by immature DCs results in an inhibitory signal that suppresses DC maturation and immunostimulatory capacity. ExtracellularATP functions as an indicator of tissue destruction and exerts in¬‚ammatory effects on DCs. Catalytic inactivation of extracellular ATP by CD39 represents an anti-in¬‚ammatory mechanism that may be used by Treg cells to prevent the deleterious effects of ATP on antigen-presenting cell function.In contrast, Nrp-1 promotes long interactions between Treg cells and immature Dcs and restricts access of the effector cells to antigen presenting cells. Some of these mechanisms may also be used by Treg cells to suppress responder T cells.
Tregcells might modulate the maturation and/or function of dendritic cells (DCs) required for effector T-cell activation ,Tregcells may also modulate the function of monocytes and macrophages
By finding therapeutic potential of Treg cells,the isolation of highly purified functional Tregs is very important processing.When isolation combined with expansion methods for Treg cells that can be used for clinical applications. For example,good use in clinical trials for a variety of conditions, such as graft-versus-host disease, autoimmune diseases, and organ rejection. be important both in tumor therapy (depletion) and autoimmune diseases
A simple three-step isolation procedure leaves cells bead-free. The isolated cells can be directly analyzed by flow cytometry or used in any downstream experiment. The combination of negative isolation of CD4+
cells followed by positive isolation of CD25+cells secures high purity, yield, and viability of the small Treg population. Highly functional Treg cells with up to 97% purity CD4+CD25+expression can be isolated.
Three-step procedure for isolation of Treg cells. Step 1, untouched CD4+T cells are negatively isolated using Depletion Dynabeads®. Step 2, CD25+cells are positively isolated using Dynabeads® CD25. Step 3, beads are released from the CD4+CD25+cells using the provided release reagent (FlowComp™ release buffer for the mouse product, DETACHaBEAD® release reagent for the human product. Note that DETACHaBEAD® reagent also releases the CD25 antibody from the cell surface)
Magnetic Sorting of Treg cells
1) Harvest 5 Spleens from 5 to 12 week old mice. 2) After mashing the spleens, pass the cells through the nylon strainer in the medium. 3)After washing the cells with PBS/BSA, osmotically lyse the erythrocytes and incubate the single cell suspensions with FITC-conjugated anti-CD4 and biotin-conjugated anti-CD25 for 30 minutes on ice. (each 2µg/ml concentration in 3ml volume of PBS/BSA) 4) After washing in 40ml of PBS/BSA, resuspend the cells in 2ml of PBS/BSA and incubate with 120µl of anti-FITC microbeads for 15 minutes in the refrigerator. This is followed by washing with PBS/BSA. 5) PurifyCD4+ T cells by magnetic isolation using the Auto MACS sorter (Miltenyi Biotec) usingPOSSELD2program. Every incubation step is followed by PBS/BSA washing.
6) Release the Anti FITCbeads by incubating with release reagent (available in the multisort kit) followed by the depletion of cell that are bound to beads. This is performed by the DEPLETE program in the sorter. The negative fraction after this sort is used for further sorting. 7) For isolation of CD4+CD25+ Tregs, after releasing the beads, incubate the purified CD4+ T cell suspension (500µl) with 2.5µl of α-biotin microbeads followed by POSSELD2 separation using the Auto MACS. 8) In all the experiments 90 to 95% of these cells were positive for CD4 and CD25. The negative fractions were depleted of CD25+ cells to obtain CD4+CD25– cells(used as Tcon or Tresp cells in the assay).
Indicated below are combinations of markers which can be used for the isolation of various sets of human Tregs
IL-10 was originally described as a cytokine produced by TH2 cells
regulatory T cells were originally implicated in the regulation of autoimmune and/or inflammatory pathologies. it is clear that they may also have a role in regulating the response to infectious pathogens and/or allergens to minimize immune pathology
TH1 and TH2 cells can clearly function as “regulatory” cells by their production of IL-10 and by their ability to reciprocally regulate each other ,they also mediate effector functions
the naturally occurring CD4+CD25+ Treg cells çš„ä»‹ç´¹:
the naturally occurring CD4+CD25+ Treg cells that develop in the thymus and are Foxp3 dependent mainly important for responses to self-antigens and/or antigens encountered in the thymus, or can they also regulate inflammatory pathologies associated with gut flora, alloantigens,tumors and infectious diseases
Disscussion-if multiple suppressor mechanisms exist, how might these be integrated and used productively by Tregcellsin vivo? We would explore this question. Tregcells possess many mechanisms that could be used but only one or two that are really crucial and consistently important in a variety of regulatory settings where different mechanisms become more or less important depending on the background or context in which the Tregcells reside and the type of target cell that they have to repress. For example, some cell types may be inhibited primarily by cytokines, whereas others are most effectively suppressed through lysis by Tregcells. Alternatively, different mechanisms may be more effective in different tissue compartments or in different disease settings.
Saving time and money by eliminating the majority of unwanted cells and allowing for faster sorting of highly purified Tregs is the challenge for researcher.low cytometric sorting is the most reliable method to achieve this level of purity,” says Stronge. “However, the low frequency of Tregs in human peripheral blood means that flow sorting can take many hours, which can also translate to high equipment cost .The method of isolating human CD25hiCD4+foxP3+ Tregs without beads or antibodies attached are good.CD4+ cells are isolated by negative isolation followed by a positive isolation of the CD25 high-expressing cell ??
both types of Treg cells use different mechanisms to regulate proliferation in vitro or in vivo and to
control in vivo immune responses and/or pathologies,Treg cells may use multiple mechanisms to suppress immune responses. important both in tumor therapy (depletion) and autoimmune diseases