Rapamycin, not cyclosporine, permits thymic generation and peripheral preservation of CD4+ CD25+ FoxP3+ T cells

Graft-versus-host-disease (GVHD) is the most common cause of poor outcome after allogeneic stem cell transplantation (SCT). Of late, exploitation of FOXP3(+) regulatory T-cell (T(REG)) function is emerging as a promising strategy in suppression of GVHD, while preserving graft-versus-leukemia (GVL). Cyclosporine and rapamycin reduce the expansion of effector T cells by blocking interleukin (IL)-2, but signaling by IL-2 is pivotal for T(REG) homeostasis. The resolution of GVHD is critically dependent on thymus-dependent reconstitution of the immunoregulatory system. Thus, there has been concern about the impact of blocking IL-2 signaling by immunosuppressive agents on T(REG) homeostasis. Here we demonstrate in a mouse model that in contrast to rapamycin, cyclosporine compromises not only the thymic generation of CD4(+)CD25(+)FoxP3(+) T cells but also their homeostatic behavior in peripheral immune compartments. Treatment with cyclosporine resulted in a sharp reduction of peripheral CD25(+)FoxP3(+) T cells in all immune compartments studied. Prolonged rapamycin treatment allowed for thymic generation of CD4(+)FoxP3(+) T cells, whereas treatment with cyclosporine led to a reduced generation of these cells. In conclusion, cyclosporine and rapamycin differentially affect homeostasis of CD4(+)FoxP3(+) T(REG) in vivo. As peripheral tolerance induction is a prerequisite for successful treatment outcome after allogeneic SCT, these findings are of potential clinical relevance.     

Identification and expansion of highly suppressive CD8(+)FoxP3(+) regulatory T cells after experimental allogeneic bone marrow transplantation

FoxP3(+) confers suppressive properties and is confined to regulatory T cells (T(reg)) that potently inhibit autoreactive immune responses. In the transplant setting, natural CD4(+) T(reg) are critical in controlling alloreactivity and the establishment of tolerance. We now identify an important CD8(+) population of FoxP3(+) T(reg) that convert from CD8(+) conventional donor T cells after allogeneic but not syngeneic bone marrow transplantation. These CD8(+) T(reg) undergo conversion in the mesenteric lymph nodes under the influence of recipient dendritic cells and TGF-β. Importantly, this population is as important for protection from GVHD as the well-studied natural CD4(+)FoxP3(+) population and is more potent in exerting class I-restricted and antigen-specific suppression in vitro and in vivo. Critically, CD8(+)FoxP3(+) T(reg) are exquisitely sensitive to inhibition by cyclosporine but can be massively and specifically expanded in vivo to prevent GVHD by coadministering rapamycin and IL-2 antibody complexes. CD8(+)FoxP3(+) T(reg) thus represent a new regulatory population with considerable potential to preferentially subvert MHC class I-restricted T-cell responses after bone marrow transplantation.     

Induction of antigen-specific tolerance to bone marrow allografts with CD4+CD25+ T lymphocytes

Thymus-derived regulatory T lymphocytes of CD4(+)CD25(+) phenotype regulate a large variety of beneficial and deleterious immune responses and can inhibit lethal graft-versus-host disease in rodents. In vitro, CD4(+)CD25(+) T cells require specific major histocompatibility complex (MHC)/peptide ligands for their activation, but once activated they act in an antigen-nonspecific manner. In vivo, regulatory T cells are also activated in an antigen-specific fashion, but nothing is known about antigen specificity of their suppressor-effector function. Here we show that CD4(+)CD25(+) regulatory T lymphocytes isolated from naive mice and activated in vitro with allogeneic antigen-presenting cells (APCs) induced specific long-term tolerance to bone marrow grafts disparate for major and minor histocompatibility antigens; whereas "target" bone marrow was protected, third-party bone marrow was rejected. Importantly, in mice injected with a mix of target and third-party bone marrows, protection and rejection processes took place simultaneously. These results indicate that CD4(+)CD25(+) regulatory T cells can act in an antigen-specific manner in vivo. Our results suggest that CD4(+)CD25(+) regulatory T cells could in the future be used in clinical settings to induce specific immunosuppression.     

Rapamycin, and not cyclosporin A, preserves the highly suppressive CD27+ subset of human CD4+CD25+ regulatory T cells

The immunosuppressive drugs rapamycin and cyclosporin A (CsA) are widely used to prevent allograft rejection. Moreover, they were shown to be instrumental in experimental models of tolerance induction. However, it remains to be elucidated whether these drugs have an effect on the CD4+ CD25+ regulatory T-cell (T(REG)) population, which plays an important role in allograft tolerance. Recently, we reported that alloantigen-driven expansion of human CD4+ CD25+ T(REG)s gives rise to a distinct highly suppressive CD27+ T(REG) subset next to a moderately suppressive CD27- T(REG) subset. In the current study we found that rapamycin and CsA do not interfere with the suppressive activity of human naturally occurring CD4+ CD25+ T cells. However, in contrast to CsA, rapamycin preserved the dominance of the potent CD27+ T(REG) subset over the CD27- T(REG) subset after alloantigen-driven expansion of CD4+ CD25+ T(REG)s in vitro. Accordingly, CD4+ CD25+ T(REG)s cultured in the presence of rapamycin displayed much stronger suppressive capacity than CD4+ CD25+ T(REG)s cultured in the presence of CsA. In addition, CD4+ CD25+ T(REG) cells cultured in the presence of rapamycin, but not CsA, were able to suppress ongoing alloimmune responses. This differential effect of rapamycin and CsA on the CD27+ T(REG) subset dominance may favor the use of rapamycin in tolerance-inducing strategies.     

CTLA4 blockade expands FoxP3+ regulatory and activated effector CD4+ T cells in a dose-dependent fashion

Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) delivers inhibitory signals to activated T cells. CTLA4 is constitutively expressed on regulatory CD4(+) T cells (Tregs), but its role in these cells remains unclear. CTLA4 blockade has been shown to induce antitumor immunity. In this study, we examined the effects of anti-CTLA4 antibody on the endogenous CD4(+) T cells in cancer patients. We show that CTLA4 blockade induces an increase not only in the number of activated effector CD4(+) T cells, but also in the number of CD4(+) FoxP3(+) Tregs. Although the effects were dose-dependent, CD4(+) FoxP3(+) regulatory T cells could be expanded at lower antibody doses. In contrast, expansion of effector T cells was seen only at the highest dose level studied. Moreover, these expanded CD4(+) FoxP3(+) regulatory T cells are induced to proliferate with treatment and possess suppressor function. Our results demonstrate that treatment with anti-CTLA4 antibody does not deplete human CD4(+) FoxP3(+) Tregs in vivo, but rather may mediate its effects through the activation of effector T cells. Our results also suggest that CTLA4 may inhibit Treg proliferation similar to its role on effector T cells. This study is registered at http://www.clinicaltrials.gov/ct2/show/NCT00064129, registry number NCT00064129.     

Inhibition of CD4+CD25+ regulatory T-cell function by calcineurin-dependent interleukin-2 production

CD4+CD25+ regulatory T (Treg) cells control immunologic tolerance and antitumor immune responses. Therefore, in vivo modification of Treg function by immunosuppressant drugs has broad implications for transplantation biology, autoimmunity, and vaccination strategies. In vivo bioluminescence imaging demonstrated reduced early proliferation of donor-derived luciferase-labeled conventional T cells in animals treated with Treg cells after major histocompatibility complex mismatch bone marrow transplantation. Combining Treg cells with cyclosporine A (CSA), but not rapamycin (RAPA) or mycophenolate mofetil (MMF), suppressed Treg function assessed by increased T-cell proliferation, graft-versus-host disease (GVHD) severity, and reduced survival. Expansion of Treg and FoxP3 expression within this population was lowest in conjunction with CSA, suggesting that calcineurin-dependent interleukin 2 (IL-2) production is critically required for Treg cells in vivo. The functional defect of Treg cells after CSA exposure could be reversed by exogenous IL-2. Further, the Treg plus RAPA combination preserved graft-versus-tumor (GVT) effector function against leukemia cells. Our data indicate that RAPA and MMF rather than CSA preserve function of Treg cells in pathologic immune responses such as GVHD without weakening the GVT effect.     

CD25+ regulatory T cells isolated from HIV-infected individuals suppress the cytolytic and nonlytic antiviral activity of HIV-specific CD8+ T cells in vitro

HIV infection is characterized by CD4(+) T cell depletion and progressive immune dysfunction; particularly impacted are HIV-specific T cell responses. An important component of immune-mediated control of HIV replication, killing of infected cells, appears to be impaired, in part due to poor cytolytic activity of HIV-specific cytotoxic T cells (CTL). In vitro, several functions of HIV-specific T cells, such as cytokine production, can be enhanced by the depletion of the immunosuppressive CD25(+) FoxP3(+) CD4(+) regulatory (Treg) cell subset. However, the effect of CD25(+) Treg cells on virus-specific cytolytic activity in the context of HIV or any human viral infection has not been investigated. The present study demonstrates that CD25(+) Treg cells isolated from the peripheral blood of HIV-infected subjects significantly suppress HIV Gag-specific cytolytic activity in vitro. In addition, CD25(+) Treg cells suppress effector function (coexpression of TNF-alpha and IFN-gamma) of HIV-specific CD8(+) T cells that proliferate in response to HIV antigen. Finally, the secretion of HIV-inhibitory CC-chemokines by HIV-specific and nonspecific CD8(+) T cells is significantly reduced in the presence of CD25(+) Treg cells. These data suggest that CD25(+) Treg-mediated suppression of the antiviral activity of HIV-specific CD8(+) T cells could impact the ability of HIV-infected individuals to control HIV replication in vivo.     

CD147 (Basigin/Emmprin) identifies FoxP3+CD45RO+CTLA4+-activated human regulatory T cells

Human CD4(+)FoxP3(+) T cells are functionally and phenotypically heterogeneous providing plasticity to immune activation and regulation. To better understand the functional dynamics within this subset, we first used a combined strategy of subcellular fractionation and proteomics to describe differences at the protein level between highly purified human CD4(+)CD25(+) and CD4(+)CD25(-) T-cell populations. This identified a set of membrane proteins highly expressed on the cell surface of human regulatory T cells (Tregs), including CD71, CD95, CD147, and CD148. CD147 (Basigin or Emmprin) divided CD4(+)CD25(+) cells into distinct subsets. Furthermore, CD147, CD25, FoxP3, and in particular CTLA-4 expression correlated. Phenotypical and functional analyses suggested that CD147 marks the switch between resting (CD45RA(+)) and activated (CD45RO(+)) subsets within the FoxP3(+) T-cell population. Sorting of regulatory T cells into CD147(-) and CD147(+) populations demonstrated that CD147 identifies an activated and highly suppressive CD45RO(+) Treg subset. When analyzing CD4(+) T cells for their cytokine producing potential, CD147 levels grouped the FoxP3(+) subset into 3 categories with different ability to produce IL-2, TNF-α, IFN-γ, and IL-17. Together, this suggests that CD147 is a direct marker for activated Tregs within the CD4(+)FoxP3(+) subset and may provide means to manipulate cells important for immune homeostasis.     

Donor-specific transplantation tolerance: the paradoxical behavior of CD4+CD25+ T cells

To investigate the antigen specificity of regulatory T cells capable of preventing transplant rejection, we have developed two different strategies to achieve tolerance to fully mismatched skin grafts in euthymic mice. A combination of nondepleting Abs targeting CD4, CD8, and CD154 (CD40 ligand) induces dominant transplantation tolerance to fully mismatched skin allografts. Such tolerance is antigen-specific, mediated by regulatory T cells, and can be extended through linked suppression to na?ve lymphocytes. The same protocol, when combined with allogeneic bone marrow, enables the development of mixed hematopoietic chimerism and deletional tolerance. Although we cannot exclude that some regulatory T cells may persist in chimeric mice, these cells are insufficient to mediate linked suppression. CD4(+)CD25(+) T cells, whether taken from na?ve mice or from mice tolerized through either treatment protocol, were always able to prevent rejection of skin grafts by na?ve CD4(+) T cells, and did so with no demonstrable specificity for the tolerizing donor antigens. Such data question whether CD4(+)CD25(+) regulatory T cells alone can account for the antigen specificity of dominant transplantation tolerance.     

Activation of the aryl hydrocarbon receptor promotes allograft-specific tolerance through direct and dendritic cell-mediated effects on regulatory T cells

VAF347 is a low-molecular-weight compound, which activates the aryl hydrocarbon receptor (AhR). Herein, we report that oral administration of a water-soluble derivative of VAF347 (VAG539) promotes long-term graft acceptance and active tolerance in Balb/c mice that receive a transplant of MHC-mismatched pancreatic islet allografts. In vivo VAG539 treatment results in increased frequency of splenic CD4(+) T cells expressing CD25 and Foxp3, markers associated with regulatory T (Tr) cells, and in vitro VAF347 treatment of splenic CD4(+) T cells improved CD4(+)CD25(+)Foxp3(+) T-cell survival. Interestingly, transfer of CD11c(+) dendritic cells (DCs), but not of CD4(+) T or CD19(+) B cells, from VAG539-treated long-term tolerant hosts into mice that recently underwent transplantation resulted in donor (C57Bl/6)-specific graft acceptance and in a significantly higher frequency of splenic CD4(+)CD25(+)Foxp3(+) Tr cells. Furthermore, the transfer of CD4(+)CD25(+) T cells from these mice into mice that recently underwent transplantation promoted graft acceptance. Similarly, cell therapy with in vitro VAF347-treated bone marrow-derived mature DCs prevented islet graft rejection, and reduced OVA-specific T-cell responses in OVA-immunized mice. Collectively, our data indicate that AhR activation induces islet allograft-specific tolerance through direct as well as DC-mediated effects on Tr-cell survival and function.     

CD25+CD4+ regulatory T cells generated by exposure to a model protein antigen prevent allograft rejection: antigen-specific reactivation in vivo is critical for bystander regulation

The importance of CD25(+)CD4(+) regulatory T (Treg) cells in the control of immune responses is established, but their antigen specificity in vivo remains unclear. Understanding Treg-cell specificity requirements will be important if their potential is to be developed for immunotherapy. Pretreatment of recipient mice with donor alloantigen plus anti-CD4 antibody generates CD25(+)CD4(+) Treg cells with the capacity to prevent skin allograft rejection in adoptive transfer recipients. Here we demonstrate that, although this regulation can be antigen-specific, reactivation with the original tolerizing alloantigen allows the Treg cells to suppress rejection of third-party allografts. Aware of the limitations of alloantigen pretreatment, we asked whether graft-protective Treg cells could be generated against unrelated, nongraft antigens. We demonstrate that bystander regulation also extends to CD25(+)CD4(+) Treg cells generated in vivo by exposure to nominal antigens under anti-CD4 antibody cover. Providing these Treg cells are reexposed to the tolerizing antigens before adoptive transfer, they prevent the rejection of fully allogeneic skin grafts. That this might form the basis of a clinically relevant tolerance induction strategy is demonstrated by the fact that, when combined with subtherapeutic anti-CD8 antibody, Treg cells generated in response to nongraft antigens facilitate the acceptance of cardiac allografts in primary recipients.     

Induction and role of regulatory CD4+CD25+ T cells in tolerance to the transgene product following hepatic in vivo gene transfer

Gene replacement therapy is complicated by the risk of an immune response against the therapeutic transgene product, which in part is determined by the route of vector administration. Our previous studies demonstrated induction of immune tolerance to coagulation factor IX (FIX) by hepatic adeno-associated viral (AAV) gene transfer. Using a regulatory T-cell (T(reg))-deficient model (Rag-2(-/-) mice transgenic for ovalbumin-specific T-cell receptor DO11.10), we provide first definitive evidence for induction of transgene product-specific CD4(+)CD25(+) T(regs) by in vivo gene transfer. Hepatic gene transfer-induced T(regs) express FoxP3, GITR, and CTLA4, and suppress CD4(+)CD25(-) T cells. T(regs) are detected as early as 2 weeks after gene transfer, and increase in frequency in thymus and secondary lymphoid organs during the following 2 months. Similarly, adoptive lymphocyte transfers from mice tolerized to human FIX by hepatic AAV gene transfer indicate induction of CD4(+)CD25(+)GITR(+) that suppresses antibody formation to FIX. Moreover, in vivo depletion of CD4(+)CD25(+) T(regs) leads to antibody formation to the FIX transgene product after hepatic gene transfer, which strongly suggests that these regulatory cells are required for tolerance induction. Our study reveals a crucial role of CD4(+)CD25(+) T(regs) in preventing immune responses to the transgene product in gene transfer.     

Foxp3+CD4+CD25+ T cells control virus-specific memory T cells in chimpanzees that recovered from hepatitis C

Hepatitis C virus (HCV) poses a global health problem because it readily establishes persistent infection and a vaccine is not available. CD4(+)CD25(+) T cells have been implicated in HCV persistence because their frequency is increased in the blood of HCV-infected patients and their in vitro depletion results in increased IFN-gamma production by HCV-specific T cells. Studying a well-characterized cohort of 16 chimpanzees, the sole animal model for HCV infection, we here demonstrate that the frequency of Foxp3(+)CD4(+)CD25(+) regulatory T cells (T(Regs)) and the extent of suppression was as high in spontaneously HCV-recovered chimpanzees as in persistently HCV-infected chimpanzees. Foxp3(+)CD4(+)CD25(+) T(Regs) suppressed IFN-gamma production, expansion, and activation-induced cell death of HCV-specific T cells after recovery from HCV infection and in persistent HCV infection. Thus, T(Reg) cells control HCV-specific T cells not only in persistent infection but also after recovery, where they may regulate memory T-cell responses by controlling their activation and preventing apoptosis. However, Foxp3(+)CD4(+)CD25(+) T(Reg) cells of both HCV-recovered and HCV-infected chimpanzees differed from Foxp3(+)CD4(+)CD25(+)T(Reg) cells of HCV-naive chimpanzees in increased IL-2 responsiveness and lower T-cell receptor excision circle content, implying a history of in vivo proliferation. This result suggests that HCV infection alters the population of Foxp3(+)CD4(+)CD25(+) T(Reg) cells.     

Secretory phospholipase A2-IID is an effector molecule of CD4+CD25+ regulatory T cells

Suppression by natural CD4⁺CD25⁺ regulatory T cells (Tregs) is one mechanism by which tolerance is maintained. However, the way in which Tregs mediate suppression is not well understood. Here, we show that secreted phospholipase A2 (sPLA2)-IID is selectively produced by Tregs. sPLA2-IID is a potent mediator of Treg function, because it strongly suppressed proliferation of CD4⁺ and CD8⁺ T cells in vitro and in vivo in a manner independent of its catalytic activity. Furthermore, sPLA2-IID promoted the differentiation of Tregs, presumably via attenuating signaling through the PI3K/Akt/mammalian target of rapamycin pathway. Importantly, administration of a sPLA2-IID-Fc fusion protein inhibited disease development in murine models of colitis and multiple sclerosis, suggesting that sPLA2-IID''s immunosuppressive function might be exploited therapeutically.     

Efficient generation of human alloantigen-specific CD4+ regulatory T cells from naive precursors by CD40-activated B cells

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) play an important role in the induction and maintenance of immune tolerance. Although adoptive transfer of bulk populations of Treg can prevent or treat T cell-mediated inflammatory diseases and transplant allograft rejection in animal models, optimal Treg immunotherapy in humans would ideally use antigen-specific rather than polyclonal Treg for greater specificity of regulation and avoidance of general suppression. However, no robust approaches have been reported for the generation of human antigen-specific Treg at a practical scale for clinical use. Here, we report a simple and cost-effective novel method to rapidly induce and expand large numbers of functional human alloantigen-specific Treg from antigenically naive precursors in vitro using allogeneic nontransformed B cells as stimulators. By this approach naive CD4(+)CD25(-) T cells could be expanded 8-fold into alloantigen-specific Treg after 3 weeks of culture without any exogenous cytokines. The induced alloantigen-specific Treg were CD45RO(+)CCR7(-) memory cells, and had a CD4(high), CD25(+), Foxp3(+), and CD62L (L-selectin)(+) phenotype. Although these CD4(high)CD25(+)Foxp3(+) alloantigen-specific Treg had no cytotoxic capacity, their suppressive function was cell-cell contact dependent and partially relied on cytotoxic T lymphocyte antigen-4 expression. This approach may accelerate the clinical application of Treg-based immunotherapy in transplantation and autoimmune diseases.     

Expansion of CD4+CD25+ regulatory T cells from cord blood CD4+ cells using the common ��-chain cytokines (IL-2 and IL-15) and rapamycin

Rapamycin has important roles in the modulation of regulatory T cells. We tried to expand CD4(+)CD25(+) regulatory T cells (Treg cells) from umbilical cord blood (CB) CD4-positive cells using interleukin (IL)-15 or IL-2 with transforming growth factor (TGF)-β and rapamycin. We were able to obtain more than 500-fold expansion of CD4(+)CD25(+) cells from CB CD4(+) cells using IL-15 and TGF-β with rapamycin. These expanded CD4(+)CD25(+) cells expressed forkhead box P3 (FoxP3) mRNA at a level about 100-fold higher and could suppress allogeneic mixed lymphocyte culture (MLC) by more than 50%. Early after rapamycin stimulation, CB CD4(+) cells showed increased expression of FoxP3 and a serine/threonine kinase Pim2 and sustained expression of negative phosphoinositide 3-kinase regulator phosphatase and tensin homolog deleted on chromosome 10 (PTEN). On the other hand, CD4(+)CD25(+) cells expanded with rapamycin for 8 days showed much higher levels of FoxP3 mRNA expression and decreased expression of PTEN. A comparison of IL-15 stimulation and IL-2 stimulation showed slightly higher efficiency of IL-15 for expansion of CD4(+)CD25(+) cells, and for FoxP3 expression, IL-15 also showed significantly higher efficacy for inhibition of MLC. The combination of the common γ-chain cytokine IL-15, TGF-β, and rapamycin may be a useful means for expanding Treg cells. Pim2 expression early after stimulation with rapamycin may be important for conferring rapamycin resistance for growth of Treg cells. IL-15 is not less useful than IL-2 for expansion of Treg cells.