[Show abstract][Hide abstract] ABSTRACT: Organ transplantation results in the activation of both innate and adaptive immune responses to the foreign antigens. While these responses can be limited with the use of systemic immunosuppressants, the induction of regulatory cell populations may be a novel strategy for the maintenance of specific immunological unresponsiveness that can reduce the severity of the detrimental side effects of current therapies. Our group has extensively researched different regulatory T-cell induction protocols for use as cellular therapy in transplantation. In this review, we address the cellular and molecular mechanisms behind regulatory T-cell suppression and their stability following induction protocols. We further discuss the use of different hematopoietically derived regulatory cell populations, including regulatory B cells, regulatory macrophages, tolerogenic dendritic cells, and myeloid-derived suppressor cells, for the induction of transplantation tolerance in light of new clinical trials developing therapies with some of these populations.
[Show abstract][Hide abstract] ABSTRACT: Owing to the adverse effects of immunosuppression and an inability to prevent chronic rejection, there is a pressing need for alternative strategies to control alloimmunity. In three decades, regulatory T cells (Tregs) have evolved from a hypothetical mediator of adoptively transferred tolerance to a well-defined population that can be expanded ex vivo and returned safely to patients in clinical trials. Herein, we review the historical developments that have permitted these advances and the current status of clinical trials examining Tregs as a cellular therapy in transplantation. We conclude by discussing the critical unanswered questions that face this field in the coming years.
American Journal of Transplantation 02/2014; · 6.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cellular therapy is emerging as a promising alternative to conventional immunosuppression in the fields of haematopoietic stem cell (HSC) transplantation, autoimmune disease and solid organ transplantation. Determining the persistence of cell-based therapies in vivo is crucial to understanding their regulatory function and requires the combination of an extremely sensitive detection technique and a stable, long-lifetime cell labelling agent. This paper reports the first application of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) to perform single cell detection of T cell populations relevant to cellular immunotherapy. Purified human CD4+ T cells were labelled with commercially available Gd-based MRI contrast agents, Omniscan® and Dotarem®, which enabled passive loading of up to 10E8 Gd atoms per cell. In mixed preparations of labelled and unlabelled cells, LA-ICP-MS was capable of enumerating labelled cells at close to the predicted ratio. More importantly, LA-ICP-MS single cell analysis demonstrated that the cells retained sufficient label to remain detectable for up to 10 days post-labelling both in vitro and in vivo in an immunodeficient mouse model.
[Show abstract][Hide abstract] ABSTRACT: Induction of mixed hematopoietic chimerism results in donor-specific immunological tolerance by apoptosis-mediated deletion of donor-reactive lymphocytes. A broad clinical application of this approach is currently hampered by limited predictability and toxicity of the available conditioning protocols. We developed a new therapeutic approach to induce mixed chimerism and tolerance by a direct pharmacological modulation of the intrinsic apoptosis pathway in peripheral T cells. The pro-apoptotic small molecule Bcl-2 inhibitor ABT-737 promoted mixed chimerism induction and reversed the anti-tolerogenic effect of calcineurin inhibitors by boosting the critical role of the pro-apoptotic Bcl-2 factor Bim. A short conditioning protocol with ABT-737 in combination with costimulation-blockade and low-dose cyclosporine A resulted in a complete deletion of peripheral donor-reactive lymphocytes and was sufficient to induce mixed chimerism and robust systemic tolerance across full MHC barriers, without myelosuppression and by using moderate doses of bone marrow cells. Thus, immunological tolerance can be achieved by direct modulation of the intrinsic apoptosis pathway in peripheral lymphocytes - a new approach to translate immunological tolerance into clinically applicable protocols.
[Show abstract][Hide abstract] ABSTRACT: Immune regulation is fundamental to any immune response to ensure that it is appropriate for the perceived threat to the host. Following cell and organ transplantation, it is essential to control both the innate immune response triggered by the injured tissue and the adaptive immune response stimulated by mismatched donor and recipient histocompatibility antigens to enable the transplant to survive and function. Here, we discuss the leukocyte populations that can promote immune tolerance after cell or solid-organ transplantation. Such populations include regulatory T cells, B cells and macrophages, as well as myeloid-derived suppressor cells, dendritic cells and mesenchymal stromal cells. We consider the potential of these regulatory immune cells to develop and function in transplant recipients and their potential use as cellular therapies to promote long-term graft function.
[Show abstract][Hide abstract] ABSTRACT: Decreasing the incidence of chronic rejection and reducing the need for life-long immunosuppression remain important goals in clinical transplantation. In this article, we will review how regulatory T cells (Treg) came to be recognized as an attractive way to prevent or treat allograft rejection, the ways in which Treg can be manipulated or expanded in vivo, and the potential of in vitro expanded/generated Treg for cellular therapy. We will describe the first regulatory T cell therapies that have been or are in the process of being conducted in the clinic as well as the safety concerns of such therapies and how outcomes may be measured.
Seminars in Immunology 05/2011; 23(4):304-13. · 5.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Regulatory T cells (T(regs)) manipulated ex vivo have potential as cellular therapeutics in autoimmunity and transplantation. Although it is possible to expand naturally occurring T(regs), an attractive alternative possibility, particularly suited to solid organ and bone marrow transplantation, is the stimulation of total T cell populations with defined allogeneic antigen-presenting cells (APCs) under conditions that lead to the generation or expansion of donor-reactive, adaptive T(regs). Here we demonstrate that stimulation of mouse CD4(+) T cells by immature allogeneic dendritic cells combined with pharmacological inhibition of phosphodiesterase 3 (PDE) resulted in a functional enrichment of Foxp3(+) T cells. Without further manipulation or selection, the resultant population delayed skin allograft rejection mediated by polyclonal CD4(+) effectors or donor-reactive CD8(+) T cell receptor transgenic T cells and inhibited both effector cell proliferation and T cell priming for interferon-γ production. Notably, PDE inhibition also enhanced the enrichment of human Foxp3(+) CD4(+) T cells driven by allogeneic APCs. These cells inhibited T cell proliferation in a standard in vitro mixed lymphocyte assay and, moreover, attenuated the development of vasculopathy mediated by autologous peripheral blood mononuclear cells in a functionally relevant humanized mouse transplant model. These data establish a method for the ex vivo generation of graft-reactive, functional mouse and human T(regs) that uses a clinically approved agent, making pharmacological PDE inhibition a potential strategy for T(reg)-based therapies.
Science translational medicine 05/2011; 3(83):83ra40. · 10.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Exposure to alloantigen in vivo or in vitro induces alloantigen reactive regulatory T cells that can control transplant rejection. The mechanisms that underpin the activity of alloantigen reactive regulatory T cells in vivo are common with those of regulatory T cells that prevent autoimmunity. The identification and characterization of regulatory T cells that control rejection and contribute to the induction of immunologic unresponsiveness to alloantigens in vivo has opened up exciting opportunities for new therapies in transplantation. Findings from laboratory studies are informing the design of clinical protocols using regulatory T cells as a cellular therapy.
[Show abstract][Hide abstract] ABSTRACT: Naturally occurring FOXP3(+) CD4(+) Treg have a crucial role in self-tolerance. The ability to generate similar populations against alloantigens offers the possibility of preventing transplant rejection without indefinite global immunosuppression. Exposure of mice to donor alloantigens combined with anti-CD4 antibody induces operational tolerance to cardiac allografts, and generates Treg that prevent skin and islet allograft rejection in adoptive transfer models. If protocols that generate Treg in vivo are to be developed in the clinical setting it will be important to know the origin of the Treg population and the mechanisms responsible for their generation. In this study, we demonstrate that graft-protective Treg arise in vivo both from naturally occurring FOXP3(+) CD4(+) Treg and from non-regulatory FOXP3(-) CD4(+) cells. Importantly, tolerance induction also inhibits CD4(+) effector cell priming and T cells from tolerant mice have impaired effector function in vitro. Thus, adaptive tolerance induction shapes the immune response to alloantigen by converting potential effector cells into graft-protective Treg and by expanding alloreactive naturally occurring Treg. In relation to clinical tolerance induction, the data indicate that while the generation of alloreactive Treg may be critical for long-term allograft survival without chronic immunosuppression, successful protocols will also require strategies that target potential effector cells.
European Journal of Immunology 03/2011; 41(3):726-38. · 4.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: IFN-γ was originally characterized as a proinflammatory cytokine with T helper type 1 inducing activity, but it is now clear that it also has important immunoregulatory functions. Regulatory T cells play an important role in models of autoimmunity, GVHD, and transplantation, and offer potential as a cellular therapy. In rodent models, in vivo-generated CD25(+)CD4(+) T cells can prevent allograft rejection, but therapeutic exploitation of Treg will more likely depend on protocols that allow the generation or selection of Treg ex vivo. The experiments described in this chapter will show that alloantigen-reactive Treg can be generated/expanded ex vivo using IFN-γ, a cytokine more usually associated with allograft rejection. Although IFN-γ production has hitherto been generally regarded as nonpermissive for allograft survival, we believe this paradoxical "good-bad" role for IFN-γ may reflect an important physiological negative feedback loop.
[Show abstract][Hide abstract] ABSTRACT: We have developed a method to generate alloreactive regulatory T cells in vitro in the presence of interferon (IFN)-gamma and donor antigen presenting cells (APCs). We hypothesized that these IFN-gamma-conditioned T cells (Tcon) would reduce transplantation-associated arteriosclerosis. Tcon were generated from mouse (CBA.Ca, H-2(k)) CD4(+) T cells cultured in the presence of IFN-gamma for 14 days. These cultures were pulsed with bone marrow-derived B6 (H-2(b)) APC. 1 x 10(5) CD25(-)CD4(+) effector T cells from naive H-2(k) mice were then cotransferred with 4 x 10(5) Tcon into CBA-rag(-/-) mice. One day later, these mice received a fully allogenic B6 CD31(-/-) abdominal aorta transplant. Transfer of CD25(-)CD4(+) effectors resulted in 29.7 +/- 14.5% luminal occlusion of allogeneic aortic grafts after 30 days. Cotransfer of Tcon reduced this occlusion to 11.7 +/- 13.1%; P < 0.05. In addition, the CD31(-) donor endothelium was fully repopulated by CD31(+) recipient endothelial cells in the absence of Tcon, but not in the presence of Tcon. In some experiments, we cotransplanted B6 skin with aortic grafts to ensure enhanced reactivation of the regulatory cells, which led to an additional reduction in vasculopathy (1.9 +/- 3.0% luminal occlusion). In the presence of Tcon, CD4(+) T cell infiltration into grafts was markedly reduced by a regulatory mechanism that included reduced priming and proliferation of CD25(-)CD4(+) effectors. These data illustrate the potential of ex vivo generated regulatory T cells for the inhibition of transplant-associated vasculopathy.
American Journal Of Pathology 07/2010; 177(1):464-72. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Intravenous immunoglobulins (IVIg) therapy is effective as a treatment for T-cell-mediated immune diseases, but whether and how IVIg suppress allogeneic T-cell responses is largely unknown.
In vitro, human CD4(+), CD4(+)CD25(-), or CD4(+)CD25(+) T cells were stimulated with allogeneic antigen-presenting cells (APCs), and mouse CBA/Ca (H2(k)) CD4(+) or CD4(+)CD25(-) T cells were stimulated with C57BL/10 (H2(b)) splenocytes, in the presence or absence of IVIg. Proliferation, binding of IVIg, expression of activation markers, and ZAP70-phosphorylation were determined. In vivo, 1x10(5) CD4(+) or CD4(+)CD25(-) T cells of CBA/Ca mice were adoptively transferred into CBA/RAG1(-/-) mice, which were 1 day later transplanted with skin grafts of C57BL/10 mice. IVIg was administered intravenously and skin graft survival was determined.
IVIg bound to the surface of human and mouse CD4(+)Foxp3(+) regulatory T cells (Tregs). IVIg binding resulted in functional activation of Tregs, as detected by increased expression of surface activation markers, enhanced ZAP70-phosphorylation, and increased capacity to suppress allogeneic T-cell proliferation. IVIg inhibited allogeneic T-cell proliferation in the presence of Tregs, but this effect was abrogated on selective depletion of CD25(+) cells from responder T cells. IVIg prevented T-cell-mediated rejection of fully mismatched skin grafts in CBA/RAG1(-/-) mice reconstituted with CD4(+) T cells, but this effect was lost on selective depletion of CD4(+)CD25(+) cells from transferred T cells, indicating that IVIg induced dominant allograft protection mediated by Tregs.
Our data show that IVIg suppress allogeneic T-cell responses by direct activation of Tregs. IVIg treatment, which has been proven safe, may have therapeutic potential in tolerance-inducing strategies in transplant medicine.
[Show abstract][Hide abstract] ABSTRACT: Mesenchymal stem cells (MSCs) are known to be capable of suppressing immune responses and offer therapeutic potential for achieving transplantation tolerance. This review will discuss the impacts of MSCs on transplant immunity and focus on the potential role of MSCs in protecting islet grafts from both rejection and autoimmune attack.
[Show abstract][Hide abstract] ABSTRACT: Mesenchymal stem cells (MSCs) are known to be capable of suppressing immune responses, but the molecular mechanisms involved and the therapeutic potential of MSCs remain to be clarified.
We investigated the molecular mechanisms underlying the immunosuppressive effects of MSCs in vitro and in vivo.
Our results demonstrate that matrix metalloproteinases (MMPs) secreted by MSCs, in particular MMP-2 and MMP-9, play an important role in the suppressive activity of MSCs by reducing surface expression of CD25 on responding T-cells. Blocking the activity of MMP-2 and MMP-9 in vitro completely abolished the suppression of T-cell proliferation by MSCs and restored T-cell expression of CD25 as well as responsiveness to interleukin-2. In vivo, administration of MSCs significantly reduced delayed-type hypersensitivity responses to allogeneic antigen and profoundly prolonged the survival of fully allogeneic islet grafts in transplant recipients. Significantly, these MSC-mediated protective effects were completely reversed by in vivo inhibition of MMP-2 and MMP-9.
We demonstrate that MSCs can prevent islet allograft rejection leading to stable, long-term normoglycemia. In addition, we provide a novel insight into the mechanism underlying the suppressive effects of MSCs on T-cell responses to alloantigen.
[Show abstract][Hide abstract] ABSTRACT: Donor reactive regulatory T cells (Treg) play an important role in tolerance induction and maintenance in experimental transplant models. In this review we focus on the formation of the donor reactive Treg pool and explore the potential of these cells for therapeutic application in clinical transplantation.
Donor reactive Treg can arise by both conversion of alloreactive nonregulatory cells and expansion of naturally occurring Treg (nTreg) cross-reactive with donor alloantigen but the quantitative contribution of each of these pathways is at present unclear. However, the fact that donor reactive Treg can be driven both in vivo and ex vivo by alloantigen challenge of nonregulatory precursors is encouraging as it demonstrates that the functional potential of these cells for use in clinical transplantation will not be limited by fortuitous cross-reactivity between nTreg and donor alloantigens. Treg can be generated in vivo by transplantation or alloantigen challenge in combination with Treg-permissive immunosuppression, or ex vivo by phenotypic selection or by polyclonal or antigen-specific stimulation. A number of ex-vivo protocols exist for the enrichment of Treg in the laboratory and in many cases these cells have demonstrable function both in vitro and in relevant graft-versus-host disease (GVHD) or organ transplant models. The challenge now is to understand the clinical opportunities and limitations that these populations present.
Combined with appropriate immunosuppression, Treg generated/expanded in vivo or ex vivo may hold the final key to operational tolerance in clinical setting.
Current opinion in organ transplantation 06/2009; 14(4):432-8. · 3.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Blockade of the CD40-CD154 costimulatory pathway can prolong allograft survival, but does not prevent the development of transplant arteriosclerosis in several models. In this study, we investigated the mechanisms of CD40-CD154-independent transplant arteriosclerosis in major histocompatibility complex (MHC)-class I-mismatched aortic allografts.
MHC class I-mismatched CBK (H2k+Kb) donor aortas were transplanted into CBA (H2k) recipients who can only recognize the graft through CD8+ T cells and CD4+ T cells responding to the class I MHC mismatch through the indirect pathway of allorecognition. Recipients were treated with anti-CD154 antibody (MR1) alone or in combination with anti-CD8 (YTS169) or anti-interleukin (IL)-4 (11B11) antibodies. Grafts were analyzed by histology on days 30 and 60 and for intragraft mRNA expression on day 14 after transplantation.
Repeated treatment with anti-CD154 alone or in combination with anti-CD8 antibody did not prevent intimal proliferation compared with untreated controls (65%+/-6%, 62%+/-9%, and 71%+/-7% luminal occlusion, respectively, 60 days after transplantation). In both treatment groups, the expression of IL-4, IL-5, and eotaxin was increased compared with control grafts, and an eosinophilic infiltration was observed. Neutralizing IL-4 in combination with CD40-CD154 blockade and CD8+ T-cell depletion abrogated transplant arteriosclerosis (9%+/-4% luminal occlusion 60 days after transplantation).
Prolonged treatment with anti-CD154 was not able to prevent the development of transplant arteriosclerosis in MHC class I-mismatched aortic allografts, in the presence or absence of CD8+ T cells. This CD40-CD154 pathway resistant transplant arteriosclerosis was mediated by IL-4, because neutralizing IL-4 in addition to CD40-CD154 costimulation blockade and CD8+ T-cell depletion prevented its development.
[Show abstract][Hide abstract] ABSTRACT: Interferon (IFN)-gamma was originally characterized as a pro-inflammatory cytokine with T helper type 1-inducing activity, but subsequent work has demonstrated that mice deficient in IFN-gamma or IFN-gamma receptor show exacerbated inflammatory responses and accelerated allograft rejection, suggesting that IFN-gamma also has important immunoregulatory functions. Here, we demonstrate that ex vivo IFN-gamma conditioning of CD4 T cells driven by allogeneic immature dendritic cells (DC) results in the emergence of a Foxp3(+) regulatory T-cell (Treg)- dominant population that can prevent allograft rejection. The development of this population involves conversion of non-Treg precursors, preferential induction of activation-induced cell death within the non-Treg population and suppression of Th2 and Th17 responses. The suppressive activity of IFN-gamma is dependent on the transcription factor signal transducer and activator of transcription 1 and is mediated by induced nitric oxide. These data indicate not only how IFN-gamma could be used to shape beneficial immune responses ex vivo for possible cell therapy but also provide some mechanistic insights that may be relevant to exacerbated inflammatory responses noted in several autoimmune and transplant models with IFN-gamma deficiency.
European Journal of Immunology 10/2008; 38(9):2512-27. · 4.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent successes of therapeutic intervention in chronic inflammatory diseases using epigenetic modifiers such as histone deacetylase inhibitors and inhibitors of DNA methylation suggest that epigenetic reprogramming plays a role in the aetiology of these diseases. The epigenetic signature of a given immune cell is reflected in the history of modifications from different signals the cell has been subjected to during differentiation. Like other cells, differentiating immune cells are dependent on a complex combination of inter- and intracell signalling as well as transcription machineries to modulate their epigenomes in order to mediate differentiation. Despite extensive research into these processes, the link between cellular signalling and epigenetic modulation remains poorly understood. Here, we review recent progress and discuss key factors driving epigenetic modulation in chronic inflammation.
The International Journal of Biochemistry & Cell Biology 09/2008; 41(1):176-84. · 4.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Regulatory T cells (Treg) play important roles in preventing autoimmunity, graft-versus host disease and transplant rejection. In rodent transplant models, tolerance induction strategies can induce graft protective CD25CD4 Treg in vivo but therapeutic exploitation of active regulation will more likely depend on protocols that allow generation or selection of regulatory cells ex vivo for use as a cellular therapy. We have used adoptive transfer skin and islet allograft models to identify, develop and evaluate ex vivo protocols that generate donor-reactive, adaptive Treg.
Naïve CDA CD4 T cells were stimulated with allogeneic antigen-presenting cell under neutral conditions or with cytokine modification, restimulated under identical conditions and subsequently analyzed for cytokine profile, phenotypic markers characteristic of Treg and in vivo regulatory function.
Without modification, CD4 T cells default to a Th2 phenotype characterized by a dominant interleukin-4 response which is profoundly detrimental to allograft survival. However, addition of exogenous interferon-gamma suppresses interleukin-4 production without priming for effector function, induces suppressor of cytokine signaling-1 and results in up-regulation of Foxp3 and CD62L. The generation of these populations is enhanced by, but is independent of, the presence of naturally occurring endogenous Treg. Most importantly, when tested for regulatory function in vivo, these cells prevent rejection of both skin and islet allografts mediated by effector T cells.
These data reveal an unexpected role for interferon-gamma in the generation of Treg ex vivo and suggest a possible route for the generation of regulatory cells for therapeutic use.