| Naturally occurring CD4+CD25+ regulatory T cells (nTregs), represent 5-10% of total CD4+ T cells in the peripheral circulation. They play an important role in controlling immune responses, silencing self-reactive T cells and mediating immunological self-tolerance and immune homeostasis. Tregs have been shown to be capable of suppressing CD4+CD25-effector T cell-mediated cellular responses through two possible ways:contact dependent and cytokine mediated mechanisms.In addition to high level expression of CD25, Tregs constitutively express a distinct set of cell surface and intracellular molecules. Predominant amongst is the the transcription factor Foxp3 (forkhead box P3) which is required for Treg development and function, and is sufficient to induce a Treg phenotype in murine CD4+CD25- T cells. Mutations in Foxp3 cause severe, multi-organ autoimmunity in both humans and mice. In mice Foxp3 has been shown to be both necessary and sufficient for the generation of suppressive CD4+CD25+ Tregs. It is a definitive marker of regulatory function because forced expression of Foxp3 in Foxp3- T cells leads to the acquisition of regulatory function and non-regulatory cells do not express Foxp3. Similarly in mice, Foxp3 expression was not upregulated in CD4+CD25-Foxp3- effector T cells following TcR stimulation. This combination of findings had led to the proposal that Foxp3 is a lineage specific marker of Tregs in mice. In humans T cells the situation is not so clear cut. Foxp3 expression is clearly important for the maintenance of self-tolerance as mutations in Foxp3 transcription leads to a rare X-linked/immune/polyendcrinopathy/enteropathy syndrome (IPEX) which manifests as a fatal disease with multiple autoimmune syndromes. However in human T cell biology, expression of Foxp3 is not always associated with suppressor function. Induction of Foxp3 in CD4+CD25- T cells with TCR stimulation and TGF-p did not result in anergic or suppressive activity. Unlike in their murine counterparts ectopic expression of Foxp3 in human CD4+ T cells was not an effective method for generating suppressive Treg in vitro. Furthermore activated non suppressive T cell clones express significant levels of Foxp3 suggesting that Foxp3 is not sufficient for Treg function and may also be implicated in T cell activation. These studies question the relevance of ongoing Foxp3 expression once Treg reach the peripheral. Naturally occurring CD4+CD25+ Treg are derived in the thymus and it has been shown that Foxp3 is an important element that identifies natural Treg during thymus selection. Once in the periphery these cells take on a regulatory role. However it is not clear from the published data that Foxp3 is an essential element in the maintenance of their regulatory phenotype. Substantial expansion of naturally occurring CD4+CD25+ Treg in culture is now possible and expanded Treg has been proposed as a therapeutic strategy for the treatment of autoimmune diseases and prevention of allograft rejection. Essential to this strategy is the development of cell markers whose expression and level of expression would be predictive of suppressive function. Hence it is important to determine whether Foxp3 is a transcription factor whose expression is essential for human Treg function and whether the level of Foxp3 suppression correlates with suppressive activity.CD4+CD25+ Tregs mediated suppression through cell contact dependent and /or cytokine mediated mechanisms. Our previous study showed the enhanced suppression shown by expanded Tregs was associated with the enhanced production of the suppressive cytokine IL-10, suggesting that IL-10 may be involved mechanistically in the regulation of xenogeneic response. However, the precise molecular mechanism by which Tregs suppress the activation and proliferation of other T cells is currently controversial. Some studies support the role of cytokines such as IL-10 and others emphasize the contribution of cell-to-cell cognate interactions with effector T cells on APC. Although many studies have reported that the in vitro suppression of alloimmune responses by natural Tregs was IL-10 independent and cell-cell contact dependent, the importance of IL-10 in expanded human Treg-mediated suppression of xenoresponse remains to be investigated.Gene targeting methods represent an important approach to study the function of individual single genes. RNA interference (RNAi) by small interfering RNA (siRNA) has been shown to specifically knock down a gene of interest in numerous cell types. Since siRNA can be introduced into mammalian cells by various methods, siRNA-mediated gene silencing has become a powerful tool to investigate gene function both in vitro and in vivo. In this study, a lipid-based siRNA delivery system was used to target and silence Foxp3 and IL-10 of in vitro expanded human Tregs. The aim was to determine the importance of Foxp3 and IL-10 expression for maintaining human Treg phenotype and their suppressive function in the human xeno and alloimmune response. Aims:Foxp3 is required for CD4+CD25+ T cells (Tregs) development and murine Treg function. The precise role of Foxp3 in regulating natural human Treg function remains to be defined by direct gene targeting approach.Methods:In vitro expanded human Tregs were transfected with Foxp3 siRNA by lipofectamine 2000. Foxp3 knockdown and Treg phenotype were evaluated by FACS, real-time PCR, Western-blotting and immunofluorescence. Treg suppressive function assay was performed by culturing human CD4+CD25- T cells with xeno- and allo PBMC in the presence or absence of Tregs in a coculture or transwell system for 5 days prior to assessment of proliferation and xeno and allo-cytotoxicity of CD4+CD25- T cells. The production of effector cytokines by xeno- and allo-reactive CD4+CD25- T cells as well as suppressive cytokines by Tregs in their cocultures was also examined.Results:Foxp3 knockdown resulted in impaired Treg phenotype and nonresponsiveness, downregulated expression of Treg function molecules, and reduced production of suppressive cytokines. These changes were correlated with diminished Treg-mediated suppression of CD4+CD25- T cells proliferation, xeno-and allo-cytotoxicity and effector cytokine production in xeno- and allogeneic response.Conclusions:Foxp3 expression is absolutely required for maintaining natural human CD4+CD25+ T cells phenotype and suppressive function in xeno- and alloimmune response in vitro. Aims:Cellular rejection of xenografts is predominantly mediated by CD4+ T cells. CD4+CD25+ T regulatory (Tregs) have been shown to be able to suppress CD4+ T cell-mediated anti-pig xenogeneic response in vitro. However, the precise mechanism(s) involved remain to be identified. In this study, we investigated whether IL-10 is required for human Tregs to suppress xenogeneic response in vitro.Methods:In vitro expanded human natural Tregs were transfected with IL-10 or non-specific siRNA by Lipofectamine 2000. Transfected Tregs were then analyzed for IL-10 gene and protein expression as well as their phenotypic characteristics. Human CD4+CD25- T cells were stimulated with allo or pig PBMC in the presence or absence of Tregs in a coculture or transwell system for evaluation of Treg suppressive activity. The production of effector cytokines by xeno-reactive CD4+CD25- T cells as well as suppressive cytokines by Tregs in their cocultures was examined by ELISA.Results. SiRNA mediated IL-10 knockdown resulted in substantially reduced production of IL-10 by human Tregs, leading to impaired Treg-mediated suppression of xeno-but not allo-reactive CD4+CD25- T cell proliferation. IL-10 knockdown had no impact on Treg phenotype and their production of suppressive cytokines.Conclusions:This study demonstrates that IL-10 is required for expanded human Tregs to suppress xeno-but not allo-immune response in vitro.
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