The naturally occurring population of dedicated regulatory T cells that coexpress CD4 and CD25 is known to play a key role in the maintenance of peripheral T-cell tolerance; however, their mechanism of action has remained obscure. Here we report that a member of the family of ␤-galactoside-binding proteins, galectin-1, is overexpressed in regulatory T cells, and that expression is increased after activation. Most importantly, blockade of galectin-1 binding significantly reduced the inhibitory effects of human and mouse CD4 ؉ CD25 ؉ T cells. Reduced regulatory activity was observed in CD4 ؉ CD25 ؉ T cells obtained from galectin-1-homozygous null mutant mice. These results suggest that galectin-1 is a key effector of the regulation mediated by these cells. IntroductionMore than 10 years ago, T lymphocytes, which constitutively coexpress CD4 and CD25 and represent approximately 5% of murine peripheral T cells, were shown to play a key role in the prevention of autoimmunity. 1,2 Similar cells have since been characterized in human peripheral blood. 3,4 These cells differ from recently activated CD4 ϩ T cells expressing CD25 because of their low-level expression of surface markers of activation such as CD69. In contrast to CD4 ϩ CD25 Ϫ T cells, CD4 ϩ CD25 ϩ T cells are hyporesponsive in vitro to polyclonal T-cell-receptor (TCR) stimuli. After activation they do not produce interleukin-2 (IL-2), IL-4, interferon (IFN)-␥, or IL-10, and their anergic state can be partially restored by IL-2. The mechanism of regulation appears to involve the inhibition of transcription of cytokine genes, most notably IL-2 and IFN-␥, leading to reduced proliferation. 5,6 In mouse models, Papiernik et al have demonstrated that CD4 ϩ CD25 ϩ T cells originate in the thymus, where they are induced to express CD25 at the CD4 single-positive stage, before migration to the periphery. 7 Although the generation of CD4 ϩ CD25 ϩ T cells during T-cell differentiation has not yet been fully characterized, it appears that the relatively high affinity of these cells for self-antigens falls between that required for positive and negative selection. 8 The importance of naturally occurring CD4 ϩ CD25 ϩ regulatory T (Treg) cells for maintenance of peripheral T-cell tolerance is well established, but their mechanisms of action remain elusive. Treg cells express high levels of cytotoxic T-lymphocyte-associated protein-4 (CTLA-4), which, it has been suggested, may act by sequestering B7 family molecules and inhibiting the costimulation of neighboring T cells. 9 However, since CD4 ϩ CD25 ϩ Treg cells effect suppression in the absence of B7-expressing antigenpresenting cells, this cannot be a universal explanation. 3 Cellsurface-transforming growth factor ␤-1 (TGF-␤1) has also been implicated, although this has not been reproduced. 10,11 It has recently been reported that Lag-3 contributes to the function of naturally occurring regulatory cells, but the reversal of suppression achieved by Lag-3 blockade was only modest. 5,6,12 Although cytokines such as IL-10 a...

IntroductionTransplantation of cells and organs is regarded as the only therapeutic choice for end-stage failure of several organs and has been made possible by the development of powerful immunosuppressive treatments that prevent transplant rejection. However, most of these drugs nonspecifically target the immune response, leading to unwanted side effects, and have limited ability to prevent chronic rejection. 1,2 The ultimate goal in transplantation is, therefore, the induction of a sustained state of specific tolerance to donor alloantigens with minimization or complete withdrawal of immunosuppression. Many studies in rodents have shown that it is possible to exploit the mechanisms that normally maintain immune homeostasis and tolerance to self-antigens to induce tolerance to alloantigens. 3 Immunologic tolerance involves central and peripheral mechanisms. Central tolerance results from intrathymic deletion of T cells with high avidity for thymically expressed antigens. Peripheral tolerance can be achieved by various mechanisms including deletion of activated/effector T cells, anergy induction, and active regulation of effector T cells. 4,5 CD4 ϩ T cells with regulatory function have been shown to play a critical role in the maintenance of transplantation tolerance. 6 The CD25 ϩ fraction of CD4 ϩ T cells mediates tolerance on adoptive transfer into a naive host. 7-9 CD4 ϩ CD25 ϩ regulatory T (Treg) cells are a naturally occurring population of CD4 ϩ T cells that constitutively express the IL-2 receptor ␣-chain (CD25). 10 These cells were shown to be potent suppressors of activated T cells in vitro 11,12 and to be crucial for the control of autoreactive T cells 13 and of the effector function of alloreactive CD4 ϩ and CD8 ϩ T cells in transplantation models in vivo. 8,14 The precise mechanisms by which these Treg cells exert their suppressive function remain to be defined. Surface molecules such as cytotoxic T lymphocyteassociated antigen 4 (CTLA-4), 15,16 the glucocorticoid-induced tumor necrosis factor receptor (GITR), 17 and cytokines such as TGF-␤ 18-20 and IL-10 8,21 are thought to play roles in different animal models.Several studies have implied that the regulation mediated by Treg cells is dependent on a continuous supply of alloantigens 8,[22][23][24] or tissue-specific target autoantigens, 25,26 suggesting that these cells have specificity for alloantigens or autoantigens. In transplantation, alloreactive CD4 ϩ T cells with indirect allospecificity are thought to play a key role in chronic rejection, and the control of these pathogenic effector cells by donor-specific Treg cells could, therefore, result in transplantation tolerance. [27][28][29][30][31] However, the possibility of using Treg cells as immunotherapy for the induction of antigen-specific tolerance is limited by cell number. Indeed, the entire pool of Treg cells accounts for only 5% to 10% of CD4 ϩ T cells in the peripheral blood of healthy persons. 10,32,33 In this study, we have explored whether in vivo alloresponses could be regulated...

Bladder cancer is a current clinical and social problem. At diagnosis, most patients present with nonmuscle-invasive tumors, characterized by a high recurrence rate, which could progress to muscle-invasive disease and metastasis. Bone morphogenetic protein (BMP)-dependent signaling arising from stromal bladder tissue mediates urothelial homeostasis by promoting urothelial cell differentiation. However, the possible role of BMP ligands in bladder cancer is still unclear. Tumor and normal tissue from 68 patients with urothelial cancer were prospectively collected and analyzed for expression of BMP and macrophage markers. The mechanism of action was assessed by experiments with bladder cancer cell lines and peripheral blood monocyte-derived macrophages. We observed expression is associated and favored type II macrophage differentiation. experiments showed that both recombinant BMP4 and BMP4-containing conditioned media from bladder cancer cell lines favored monocyte/macrophage polarization toward M2 phenotype macrophages, as shown by the expression and secretion of IL10. Using a series of human bladder cancer patient samples, we also observed increased expression of in advanced and undifferentiated tumors in close correlation with epithelial-mesenchymal transition (EMT). However, the p-Smad 1,5,8 staining in tumors showing EMT signs was reduced, due to the increased miR-21 expression leading to reduced expression. These findings suggest that BMP4 secretion by bladder cancer cells provides the M2 signal necessary for a protumoral immune environment. In addition, the repression of by miR-21 makes the tumor cells refractory to the prodifferentiating actions mediated by BMP ligands, favoring tumor growth..

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CD4+ CD25+ regulatory T cells (Tregs) have far-reaching immunotherapeutic applications, the realization of which will require a greater understanding of the factors influencing their function and phenotype during ex vivo manipulation. In murine models, IL-2 plays an important role in both the maintenance of a functional Treg population in vivo and the activation of suppression in vitro. We have found that IL-2 maintains optimal function of human CD4+ CD25+ Tregs in vitro and increases expression of both forkhead box protein 3, human nomenclature (FOXP3) and the distinctive markers CD25, cytotoxic T lymphocyte antigen-4 (CTLA-4) and glucocorticoid-induced tumor necrosis factor receptor superfamily member number 18 (GITR). Although IL-2 reduced spontaneous apoptosis of Tregs, this property alone could not account for the optimal maintenance of the regulatory phenotype. The inhibition of phosphatidylinositol 3-kinase (PI3K) signaling by LY294002, a chemical inhibitor of PI3K, abolished the maintenance of maximal suppressive potency by IL-2, yet had no effect on the up-regulation of FOXP3, CD25, CTLA-4 and GITR. Other common gamma chain (gammac) cytokines-IL-4, IL-7 and IL-15-had similar properties, although IL-4 showed a unique lack of effect on the expression of FOXP3 or Treg markers despite maintaining maximal regulatory function. Taken together, our data suggest a model in which the gammac cytokines IL-2, IL-4, IL-7 and IL-15 maintain the optimal regulatory function of human CD4+ CD25+ T cells in a PI3K-dependent manner, offering new insight into the effective manipulation of Tregs ex vivo.

The herpes simplex virus thymidine kinase gene type 1 (HSV-Tk) ganciclovir (GCV) system is a novel therapeutic strategy for the modulation of graft-versus-host disease (GVHD), a major complication of allogeneic stem cell transplantation (allo-SCT). Retroviral-mediated gene transfer of the HSV-Tk gene into donor T lymphocytes before allo-SCT may allow their in vivo selective depletion after treatment with GCV. The expression of the HSV-Tk gene was analyzed in vitro in CEM cells, a human lymphoblastoid cell line, transduced with 2 different vectors, each containing the HSV-Tk gene and a selectable marker gene. GCV-resistant clones were identified within the clones expressing the marker gene. Characterization of the molecular events leading to this resistance revealed a 227-bp deletion in the HSV-Tk gene due to the presence of cryptic splice donor and acceptor sites within the HSV-Tk gene sequence. Furthermore, it was confirmed that this deletion was present in human primary T cells transduced with either vector and in 12 patients who received transduced donor T cells, together with a T-cell-depleted allo-SCT. In vivo circulating transduced T cells containing the truncated HSV-Tk gene were identified in all patients immediately after infusion and up to 800 days after transplantation. In patients who received GCV as treatment for GVHD, a progressive increase in the proportion of transduced donor T cells carrying the deleted HSV-Tk gene was observed. These results suggest that the limitations within the HSV-Tk/GCV system can be improved by developing optimized retroviral vectors to ensure maximal killing of HSV-Tk-transduced cells.

Mesenchymal stem cells (MSCs) are multipotent stromal cells with immunosuppressive properties. They have emerged as a very promising treatment for autoimmunity and inflammatory diseases such as rheumatoid arthritis. Recent data have identified that GM-CSF-expressing CD4 T cells and Th17 cells have critical roles in the pathogenesis of arthritis and other inflammatory diseases. Although many studies have demonstrated that MSCs can either prevent or suppress inflammation, no studies have addressed their modulation on GM-CSF-expressing CD4 T cells and on the plasticity of Th17 cells. To address this, a single dose of human expanded adiposederived mesenchymal stem cells (eASCs) was administered to mice with established collageninduced arthritis. A beneficial effect was observed soon after the infusion of the eASCs as shown by a significant decrease in the severity of arthritis. This was accompanied by reduced number of pathogenic GM-CSF 1 CD4 1 T cells in the spleen and peripheral blood and by an increase in the number of different subsets of regulatory T cells like FOXP3 1 CD4 1 T cells and IL10 1 IL17 2 CD4 1 T cells in the draining lymph nodes (LNs). Interestingly, increased numbers of Th17 cells coexpressing IL10 were also found in draining LNs. These results demonstrate that eASCs ameliorated arthritis after the onset of the disease by reducing the total number of pathogenic GM-CSF 1 CD4 1 T and by increasing the number of different subsets of regulatory T cells in draining LNs, including Th17 cells expressing IL10. All these cellular responses, ultimately, lead to the reestablishment of the regulatory/inflammatory balance in the draining LNs. STEM CELLS 2015;33:3493-3503 SIGNIFICANCE STATEMENTWe identify, for the first time, a novel mechanism by which adipose-derived mesenchymal stem cells modulate ongoing immune responses by promoting an early adaptive T cell signature characterized by decreased levels of pathogenic GM-CSF-secreting CD41, superscript T cells, increased levels of regulatory T cells and plasticity of effector Th17 cells towards an IL10-driven anti-inflammatory response thus shifting the inflammatory/regulatory balance from GM-CSF inflammatory predominance to IL10 regulatory predominance. Altogether, these data will offer much needed further insight into the mechanisms of action of mesenchymal stem cells for their translation to the clinic.

BackgroundApplication of mesenchymal stem/stromal cells (MSCs) in treating different disorders, in particular osteo-articular diseases, is currently under investigation. We have already documented the safety of administrating human adipose tissue-derived stromal MSCs (hASCs) in immunodeficient mice. In the present study, we investigated whether the persistence of MSC is affected by the degree of inflammation and related to the therapeutic effect in two inflammatory models of arthritis.Methodology/Principal FindingsWe used C57BL/6 or DBA/1 mice to develop collagenase-induced osteoarthritis (CIOA) or collagen-induced arthritis (CIA), respectively. Normal and diseased mice were administered 2.5×105 hASCs in the knee joints (IA) or 106 in the tail vein (IV). For CIA, clinical scores were monitored during the time course of the disease while for CIOA, OA scores were assessed by histology at euthanasia. Thirteen tissues were recovered at different time points and processed for real-time PCR and Alu sequence detection. Immunological analyses were performed at euthanasia. After IV infusion, no significant difference in the percentage of hASCs was quantified in the lungs of normal and CIA mice at day 1 while no cell was detected at day 10 taking into account the sensitivity of the assay, indicating that a high level of inflammation did not affect the persistence of cells. In CIOA mice, we reported the therapeutic efficacy of hASCs at reducing OA clinical scores at day 42 when hASCs were not detected in the joints. However, the percentage and distribution of hASCs were similar in osteoarthritic and normal mice at day 1 and 10 after implantation indicating that moderate inflammation does not alter hASC persistence in vivo.Conclusions/SignificanceWhile inflammatory signals are required for the immunosuppressive function of MSCs, they do not enhance their capacity to survive in vivo, as evaluated in two xenogeneic inflammatory pre-clinical models of arthritis.