Regulatory T Cells and Preventing Graft Versus Host Disease - Research Paper Example

Evaluate the evidence that regulatory T cells may be successfully used to prevent graft versus host disease. Graft versus Host Disease (GvHD) is an immunological response to what the human body recognises as foreign proteins delivered into the body by transplantation. It has been researched and experiments have been carried out to determine whether a certain subsets of regulatory T cells have a positive effect on preventing or reversing GvHD. Experiments carried out were on human and murine models, both are equally reviewed in this project. Experiments evaluated involve evidence that patients with GvHD had a decrease in this subpopulation of Tregs and also evidence that increasing this subpopulation of Tregs reduces the effect of GvHD. A main immunological technique that is used to measure regulatory T cell frequency is flow cytometry; other methods used are immunohistochemistry staining involving studying skin biopsies and ELISAs. All evidence proved strong in correlation with the hypothesis and suggests that CD4 CD25 FOXP3 Tregs could provide a future for the treatment of GvHD. Introduction: Transplantation is the process of obtaining tissues or organs or cells and placing them in the same or different individual. The organs, tissues and cells that are transferred from one individual to another are called grafts. The person who donates the graft is called donor and the one who recieves the graft is called recipient. Heart, kidney, cornea, liver, pancreas, lungs and bone marrow are transplanted from one person to another. There are two types of transplantation: autograft (transplant from one region to another of the same indivudual and Syngraft or Isograft (Transfer of graft between individuals of same species). ( Khan, 2009). Graft versus host disease is the series of events that occurs after the transplantation of the donor T –cells with the stem cell graft. This is a donor T-cell mediated syndrome where the T cells in the graft shows their immune response. This response creates tissue damage. ( Beres and Drobyski, 2013). This is the major difficulty after the stem cell transplantation. The T-cells can recognise the minor and major histocompatibility antigens that are expressed at the host antigen presenting cells. The T-cells gets activated and expands and finally infiltrates and destroys the Graft versus host disease target tissues. The major tissues targeted are liver, gut and skin. This graft versus host hematopoiesis effect is the target for the allogenic stem cell transplantation. (Edinger, 2009). The rejection of the host may occur in first 100 days, where the donor immune cells recognize and attack the host tissues. As the rejection of the foreign tissues increases, the inflammatory lessions are formed at single or multiple locations such as skin, liver and gut. The onset of acute GvHD leads to fatal disease conditions. After 100 days, the acute GvHD becoms chronic GvHD. Chronic GvHD resutls in lupus or scleroderma. Graft rejection occurs when the mature immunocompetent lymphocytes are present in the graft. These mature immunocompetent lymphocytes idenitfies the host tissues as non-self antigens and the host are not able to reject the graft. Many studies conducted in the animals have given us major evidences for GvHD. The mismatches at the major histocompatibility complex (MHC) are more. If the mismatches are many, then the chances for the activation of the immune system are 1000 times stronger than the bacterial infections. In an experiment with mice bone marrow, C57BL/6 (H-2b) strain was used as a donor and B10.BR (H- 2k) strain as recipient, resulted in the onset of GvHD. The experiments have found that CD 4+ cells can independently trigger GvHD. CD8+ are less invovled inthe GvHD. Class I mismatches with the CD8+ cells produced very little effect of graft rejection and if there are more muner of mismatched CD8+ cells , then the chances for lethal graft rejection is more. Major histocompatibility cell receptor interaction with the natural killer cells can act as a seed for the GvHD. The animal studies have concluded that graft rejection will be present in the host, when the organism’s immunity is not destroyed. If the innate immunity of the host is destroyed before the graft replacement, then the chance for GvHD is more. (Trsonkowski et al, 2013). Regulatory T –cells (tregs) are the subset of CD4+ T cells and they constitute less than 1% of the peripheral leukocytes. Suppressed CD4+ cells expresses high levels of Interleukin – 2 receptor alpha chain (CD 25) called as Treg cells. Treg is controlled by the transcription factor called as Foxp3.The Tregs protects our bodies from aberrant autoaggressive immune responses of the effector cells. T- Cell regulators are protective shields against GvHD for the transplantation of peripheral blood cells.Tregs is divided into two types. They are naturally occuring tregs and adaptive tregs. Naturally occuring tregs are very capable and are dedicated to the regulatory function. They are present in the thymus and are also called as autoreactive CD4+ T cells. Treg cells show an excellent pattern of activity when they identify self- antigens. Tregs, on identification of self- antigens, reduce the immune response of other immune cells and protects the system from graft rejection. This is done by the upregulation of Foxp3 transcription factor. The native Tregs produce induced tregs (i- Tregs) during the activation of TGF- beta and IL-2. The role of i-Tregs in immune response is not clearly understood but they are found to play an important in regulating peripheral T cells. Mature T cells are similar to T effector cells and they allow the selection of autoantigens and to some extent alloantigens. The antigens presented by the APCs interact with the T cell receptors and produces the suppressor activity. Tregs compete for autoanitgens on the periphery of the APC and to complete the action, they require a lot of energy. This mechanism is very important for the host cells and graft to identify the host antigens as self antigens and match with the MHC alleles. The Treg- APC interaction prevents the reactions triggered by MHC and non-MHC mismatches at the inflammation site. (Trsonkowski et al, 2013). Tregs are used for the cellular therapy of GvHD. The advantages of Treg were initially studied in animals and it was found that decrease in the Treg of the transplanted material was associated with the lethal effect on graft acceptance. Addition of the donor Tregs during the time of grafting reduced the chance of GvHD. T-cell mediated immune response is the key factor for maintaining immunological tolerance in vivo or unresponsiveness in controlling the T cell homeostasis. CD25+CD4+ foxp3+Treg are naturally occurring and thymus derived molecules, which is very important for therapeutic stratergies. Alloantigen exposure to T-cell Receptors leads to CD4+ cell differentiation and expression of foxp3 depends on age, epigenetic modifcation, genetic background and sex. (Jiang, 2008). 5 to 10 percentage of circulating CD4+ cells comprise of CD25+CD4+ foxp3+ regulatory T lymphocytes. These cells migrate to the inflammatory site and control the immune responses (innate and adaptive).This migration is due to the effector lymphocytes of T helper subsets: Th1, Th2, Th17 and follicular Th cells. The concentration of the CD25+CD4+ foxp3+ cells is inversely proportional to the chronic GvHD. When a study was performed to analyse the expression level of Treg gene and its core, it was observed that there was a marked decrease in the naive Treg, although other treg subsets were preserved. (Koreth and Ritz, 2013). The Treg cells are able to recognise the major and minor Histocompatibility antigens that are expressed by the residual host antigen presenting cells. These Treg cells are activated to destroy the GvHD target tissues. CD4+ CD25+ cells are activated by the Treg cells and they are found to help in the GvHD treatment. The CD4+ CD25+ cells were found to suppress GvHD and they are called as expanded nTreg cells. Native Treg cells are difficult to isolate in large numbers from spleen and sceondary lymphoid tissues hence expanded nTreg cells are used by the system. The suppressive ability of the nTreg cells was due to IL-10 secretion. The adoptive transfer of these nTreg cells are transplorted to the expanded nTreg cells either before or at the time of stem cell transplantation. In the same study, Cd62L n Tregs were found to suppress the alloreactive T cell proliferation in vitro and they are non- functional in vivo. ( Beres and Drobyski, 2013). Haematopoietic stem cell transplantation involves the replacement of genetically defective stem cells with healthy donor cells from bone marrow, peripheral blood or foetal cord blood; all are sources that contain immature stem cells. Transplantation can be autologous, where a patient receives their own stem cells, syngeneicly where stem cells from an identical twin are received or they can be received allogenically, this is the most common form of transplant where patients receive stem cells from a relative or an unrelated donor that they have been closely matched to. Haematopoietic stem cell (HSCs) transplants are a proven successful therapy for cancer patients receiving chemotherapy and radiation treatment and diseases caused by defective blood cells including immunodeficiencies such as severe combined immunodeficiency (SCID). With this being said “Graft versus host disease (GvHD) remains one of the major challenges to the successful outcome of allogenic stem cell transplantation” (GVH3). GvHD is caused by an attack of the immune system and is dependent on how closely human leukocyte associated antigens are matched between donor and patient. Human leukocyte associated antigens are sets of proteins found on the surface of cells, if the immune system recognises the antigens as foreign an attack will occur, in the case of GvHD the graft will identify the host as foreign and cause severe inflammatory disease most commonly characterised by a rash, diarrhoea and liver damage. GvHD is graded into 4 group’s dependant on the severity of the disease, 1 being mild to 4 being severe. Allogenic bone marrow transplantaion has GvHD as the major lefe threatening complication. During bone marrow transplantation, mature donor T cells are transferred and they recognize the differences in the antigens on the antigen presenting cells of the host. The activated donor antihost –specfic T cells mediates tissue destruction. The absence of the autoreactive T cells in the thymus may lead to autoimmune syndrome. This negative selection of the thymus can be suppressed by the use of immunosuppressive drugs. ( Hess, 2006). The next important concept behind the GvHD is the absence of competent peripheral regulatory system. Animal model studies clearly indicate that Treg cells are very essential for the down regualtion of GvHD. The regulatory T cells are important for the production of allo- and self- ajor histocompatibility complex antigens. CD4+ regulatory cell expresses CD25+ cells to enable a combined role in the control of immune response. Recent studies clearly indicates that antigen-specific CD4 (+) CD25 (+) Foxp3 (+) T cells are found to play an important –ole in the control of GvHD reactions mediated by the autoreactive and alloreactive lymphocytes. Thus these regulatory cells appear to increase the occurence and stabilisation of donor antihost and donor anti donor self tolerance. ( Hess, 2006). The immune system undergoes a drastic change in tolerance level to self- antigens adter autologous bone marrow transplantaion. The use of drugs such as cyclosporine inhibited thymic – dependent clonal deletion and immune system function was altered. Hence the autoaggression is cleared in the host only by the reconstitution of CD4+ regulatory T cells. A common peptide antigen frame work is presented by the major histocompatibility complex class II molecules and they are recognised by the pathogenic effector T cells and antigen – specific regulatory T cells. CD4+ regulatory T cells are found to have two subsets of common peptide natigen framework (CLIP) reactive T cells requiring different C- terminal and N- ternima lDomain for activation. The regulatory function is mediated by the C-terminal flanking region. Fox P3 nuclear transcription factor is expressed by the CD4+ cells and they play crucial role in expressing the tolerance ability to major histocompatibility complex class II antigens. Regulatory T cells can lead to apoptosis of the pathogenic auto reactive lymphocytes by suppressing the production of type I cytokines. ( Hess and Thoburn, 2006). Studies have found that donor HSC- derived Thy 1 + T reg cells containing mainly of CD 4 (+) CD 25 (+) cells play an important role in the suppression reaction in GvHD, when donor lymphocyte infusion was done to donor after 28 days from hematopoietic stem cell Transplantation. Thy 1(+) was found to be less important for the suppression of GvHD but the deletion of the Thy 1 (+) cells led to lethal GvHD. The lethal GvHD was observed at day 28, 35 and 42 after HSC and removal of Thy 1 (+) cells. After 100 days, the lethal GvHD was not observed in the host. The reason may be the loss of donor lymphovyte infusion (DLI) in the graft- versus- host (GVH). The infusion of the host- type dendritic cells has restored the DLI induced Graft – versus – leukemia and graft –versus- host effect. The complexity of GvHD after DLI was very little because of the increase in CD 25(+) cells of the regulatory T cells. (Xia, Truitt and Johnson, 2006). Regulatory T cells (Tregs) are a subpopulation of T cells that account for less than 5% of cell; the majority arise spontaneously from the thymus to control responses to tissue specific auto antigens and to antigens by suppressing T cell responses rather than activating them. 2 groups are recognised, natural regulatory T cells and induced regulatory T cells. Recent advances and experiments described in this report have shown that the subset of T cells, Tregs specifically CD4+ CD25+ that express the protein forkhead box protein 3 (Foxp3), have been proven to mediate GvHD and “suggests that enriching or generating Treg cells in preparations of donor HSCs might provide a possible therapy for GvHD in the future” FoxP3 transcription factor interferes with gene transcription preventing IL 2 production. (Janeway, 2001). Human Treg cells are involved in immunoregulation and parenchymal homeostasis during the autoimmune inflammation and infection. Treg cells alter the signalling pathways under the signalling of T-cell Receptors.CD4+ CD25+FoxP3+ cells express the Interleukin – 10 molecules and exert their function using the cyclic adenosine – monophosphate. The recent study has also found that CD4+HLA-G+ cells are the potent tTreg population and can modulate the polyclonal adaptive immune responses in vivo and are the potential candidates for future clinical applications. (Prankratz et al, 2014). Treg cells are found in many inflammatory disorders. The studies have found that interleukin-2 is essential for Treg cell growth, activity and survival. Treg plays an important role in the immune tolerance and prevention of unusual disorders. Most of the stratergies are based on the adoptive transfer of Treg cells. In many animal models for auto immunity, the enhancement of Treg cells has reversed the organ damage. Adoptive transfer of Treg cells was found to prevent the disease, but the state of inflammation is not clear. Since the large scale isolation and expansion of the Treg cells are difficult to achieve in vivo, the use of drugs to enhance the Treg cells were practised. The use of low dose interleukin-2 subcutaneously daily was found to increase the Treg cells concentration and reduce the effects in GvHD. The increased concentration of Treg cells was biologically very active, ex vivo. The drawback of this trial was that all the patients are not benefitted by this technique. (Koreth et al, 2011). Allogenic immune response is still the major problem after allogenic stem cell transplantation. The use of biomarkers for the identification and treatment of GvHD is associated with the immune responses of Treg cells. Treg cells can prevent GvHD by the suppressive action of alloreactive donor T cells. To confirm the suppressive action of treg cells, the patients with and without acute and chronic GvHD were used for the clinical study. CD44, CD69, FAS, PIP5Ky and cell cycle regulators like cyclin B1, A2 and E2 are used as the potential biomarkers. (Ukena et al., 2012). Recently T-helper cells (TH) – 17 cells are foundto play an important role in mediating many auto immune diseases. The increase in number of TH – 17 cells was observed in the patients suffering from GvHD with an inflammatory process. The concentration of TH- 17 cells was very less in the patients without GvHD. TH- 17 cells contain Interleukin (IL) – 17 and interferon (IFN)- gamma and the subsets of IL-17 and IFN -17 and expressed IL- 23 receptor. ELISA method was used to identify the cytotoxic plasma levels. Monitoring of the patients was done with flow cytometry, confocal microscopy followed by ELISA. ( Dander et al, 2009). “Experiments on the transplantation of allogenic HSCs in mice” have shown that “Depletion of either CD4 CD25 Treg cells in the recipient or of the same class in the HSC s before transplantation accelerated the onset of GvHD and subsequent death. In contrast, supplementing the graft with fresh CD4 CD25 Tregs or such cells prevented death from GVHD”. (Janeway, 2001). This project will investigate statistics of Treg frequency in patients with GvHD and also provide statistics that show the use of Tregs, expanded through the use of drugs, can have a positive impact on supressing GvHD. This project will also make comparisons with human and mouse models, stating the advantages and disadvantages. Results: Treg frequency has a direct effect on GvHD patietns who had HSCs transplantation. This was confirmed by Magenau et al in hos research paper, where the patients with HSCs transplantation were studied for the effect of Treg. 215 patients were monitored and the samples were taken from them at four different time intervals ranging from day 20 – day 100. The study was a blind control study. Out of 215 patients, 125 patients had allogenic grafts and 90 had autologous grafts. Flow cyotmetry was used to measure the concentration of CD25+CD4+ foxp3 cells using 4 differnet markers. The first hypothesis suggested is that Treg frequencies are similar in patients who did not develop GvHD in either autologous or allogenic HSC transplantations. With a strong P value of 0.84, this hypothesis can be proven. In some cases of allogenic grafts with no GvHD the P value is 0.04. Regulatory T-cells are very important supressing the GvHD post allogenic stem cell transplantation. Treg cells are also found to impair the antitumor immunity. Since longitudinal Treg studies are not possible, the use of flow cytometry for the complete analysis is practised now. Identification of the Treg cells and their types in the GvHD patietns and replase was carried out in 239 peripheral blood samples of 16 patietns for two years after stem cell transplatation. The analysis was performed using 10 color flow cytometric panel and ten healthy individuals were also included in the study. It was identified that CD127 expression was less in the early post- stem cell transplation studies and the percentage of CD127 increased after the first year. The Treg concentration was high at grade I of GvHD and less at grades II- IV of GvHD. They have also classifed Tregs into three types: 40% naive, 51% central memory and 9% effector memory Tregs. (Bremm et al, 2011). The studies conclude that impairment of Treg is an early event and the reduced numberof Treg cells are even present in the Grade I acute GvHD. Hence the stability of Treg Foxp3+ cells expression and availability of Th1 and Th17 cytokine secretion are based on the proinflammatory milieu in GvHD. By supplying low doses of IL-2 can increase the clinical responses of GvHD patients and restore Treg homeostasis. (Koreth, 2013). The potential of CD4+ CD25+ Treg cells are emerging as the best treatment approach for GvHD. Optimising the Treg cell concentration can suppress the proliferative activity of CD4+ cells and CD8+ T cells against alloantigenic stimulation both invitro and in vivo. They will thus increase the transplantation tolerance and reduce the occurenceof GvHD. The use of Treg as the prognostic cellular biomarker and diagnostic tool for acutre GvHD was investigated in rat and mice models and the trial are being performed in human HSC transplantation too. The mRNA expression of FoxP3 at the peripheral blood mononuclear cells of hte paitents with acute GvHD and Chronic GvHD was very less than the patients without GvHD. In the skin biopsies, the concentration of FoxP3+ Treg cells was high in the patients who responded well to GvHD treatment. (Leventhal et al, 2012). Similarly, Treg Cells are found to show an importnat role in the maintenance of tolerance after allogenic stem cell transplantation. CD8+Foxp3+T cells are found to play an important role in the Treg population during the early Graft versus host disease. CD4+ cells were expressed on the cell surface in the GvHD patients. These cells are induced due to the mispairing in the MHC cells of donor and reciepient. CD4+ Treg cell number was more than the induced-Treg (I- Treg) cells. The mice that lack Cd4+ and CD8+ induced Tregs, died because of GvHD mortality. CD4+ iTreg cells were found in GvHD recipients in vivo and the ability of these cells in showing host reactivity is not clearly understood. (Beres and Drobyski, 2013). 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