In JoVE (1)

Other Publications (42)

Articles by Andrew Bushell in JoVE

 JoVE Immunology and Infection

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

1Division of Respirology, Departments of Medicine and Immunology, Toronto Lung Transplant Program, Multiorgan Transplant Program, Toronto General Research Institute, University of Toronto and University Health Network, 2Latner Thoracic Surgery Laboratories, Toronto General Research Institute, University Health Network, 3National Institutes of Health Research, Oxford Biomedical Research Centre, Translational Immunology Laboratory, NDORMS, Kennedy Institute of Rheumatology, University of Oxford, 4Transplantation Research Immunology Group, Nuffield Department of Surgical Sciences, John Radcliffe Hospital, University of Oxford


JoVE 55283

Other articles by Andrew Bushell on PubMed

CD25+CD4+ Regulatory T Cells Prevent Graft Rejection: CTLA-4- and IL-10-dependent Immunoregulation of Alloresponses

Journal of Immunology (Baltimore, Md. : 1950). Feb, 2002  |  Pubmed ID: 11801641

Specific and selective immunological unresponsiveness to donor alloantigens can be induced in vivo. We have shown previously that CD25+CD4+ T cells from mice exhibiting long-term operational tolerance to donor alloantigens can regulate rejection of allogeneic skin grafts mediated by CD45RB(high)CD4+ T cells. In this study, we wished to determine whether donor-specific regulatory cells can be generated during the induction phase of unresponsiveness, i.e., before transplantation. We provide evidence that pretreatment with anti-CD4 Ab plus a donor-specific transfusion generates donor-specific regulatory CD25+CD4+ T cells that can suppress rejection of skin grafts mediated by naive CD45RB(high)CD4+ T cells. Regulatory cells were contained only in the CD25+ fraction, as equivalent numbers of CD25-CD4+ T cells were unable to regulate rejection. This pretreatment strategy led to increased expression of CD122 by the CD25+CD4+ T cells. Blockade of both the IL-10 and CTLA-4 pathways abrogated immunoregulation mediated by CD25+ T cells, suggesting that IL-10 and CTLA-4 are required for the functional activity of this population of immunoregulatory T cells. In clinical transplantation, the generation of regulatory T cells that could provide dynamic control of rejection responses is a possible route to permanent graft survival without the need for long-term immunosuppression.

Intercellular Cell Adhesion Molecule-1 and Selectin Ligands in Acute Cardiac Allograft Rejection: a Study on Gene-deficient Mouse Models

Journal of Leukocyte Biology. Feb, 2002  |  Pubmed ID: 11818453

Cell adhesion molecules and their ligands are essential for regulating lymphocyte recirculation and leucocyte emigration into an inflamed or injured tissue. Vascular endothelial selectins as mediators of leucocyte rolling and intercellular cell adhesion molecule-1 (ICAM-1) have been found to be up-regulated on activated endothelium during acute allograft rejection. This study was designed to investigate whether ICAM-1 or selectin-ligand deficiency, or a combination of both, affected graft survival during acute cardiac allograft rejection. To this goal, we performed cardiac transplantation using mice deficient in genes for ICAM-1 or alpha(1,3)fucosyltransferase Fuc-TVII, representing a model for general absence of selectin-ligand expression, and a newly developed strain with a double mutation in Fuc-TVII and ICAM-1 alleles. Transplantation of a heart from ICAM-1 -/- or Fuc-TVII/ICAM-1 double-mutated mice into allogeneic recipients resulted in limited (2-2.5 days) but nevertheless significant prolongation of the graft survival (P<0.01 and P<0.01 in log-rank test) compared with the survival of unmodified hearts. When ICAM-1 -/- hearts were transplanted into Fuc-TVII -/- recipients, the median survival time was prolonged by 8 days (P<0.01). These data indicate that endothelial ICAM-1 is involved in adhesion events during acute cardiac allograft rejection but reveal that the loss of one type, selectin/leucocyte ligand or selectin/endothelial ligand interaction, does not markedly affect graft survival, thereby suggesting a role for other compensatory adhesion molecule/ligand interactions.

The Role of the Graft in Establishing Tolerance

Frontiers in Bioscience : a Journal and Virtual Library. May, 2002  |  Pubmed ID: 11991840

At the present time, clinical solid organ transplantation continues to rely on the use of non-specific immunosuppressive protocols in order to prevent graft rejection. However, these regimens bring with them complications related both to the global immunosuppression that they cause, and to toxicity related to individual drugs. The pursuit of protocols that will allow graft-specific tolerance thus remains a major goal of research both in animal models and in clinical practice. There is evidence that the graft itself may play an active part in establishing and maintaining donor-specific hyporesponsiveness and ultimately tolerance; the aim of this review is to analyze this role in more detail.

TGF-beta and Graft Vasculopathy: Where to from Here?

Transplantation. May, 2002  |  Pubmed ID: 12042635

Regulatory T Cells in Transplantation

Current Opinion in Immunology. Oct, 2002  |  Pubmed ID: 12183157

There has recently been an explosion of renewed interest in regulatory T cells, particularly those within the CD4(+)CD25(+) population. It is becoming increasingly apparent that these cells exist not only as naturally occurring cells that may contribute to the maintenance of self-tolerance, but they also have the potential to prevent rejection of allografts in experimental models. Such cells have now been identified in humans as well as in rodents.

Syngeneic Bone Marrow Transduced with a Recombinant Retroviral Vector to Express Endoplasmic Reticulum Signal-sequence-deleted Major Histocompatibility Complex Class-I Alloantigen Can Induce Specific Immunologic Unresponsiveness in Vivo

Transplantation. Feb, 2003  |  Pubmed ID: 12605123

Long-term survival of fully allogeneic cardiac grafts can be induced in mice through transduction of recipient bone marrow cells (BMCs) with a recombinant retroviral vector encoding a single full-length major histocompatibility complex (MHC) class I alloantigen. This study investigated whether cell surface expression of the transduced MHC antigen was necessary for the induction of specific unresponsiveness. METHOD The signal sequence for translocation into the endoplasmic reticulum was deleted from H-2K (SDELKb). Syngeneic BMCs from CBA.Ca (H2k) recipients were transduced with an MFG retroviral vector encoding either wild-type Kb or the mutant SDELKb and reinfused in conjunction with an anti-CD4 therapy. Four weeks later, the recipients underwent transplantation with a fully allogeneic C57BL/10 cardiac graft. Graft survival and the development of transplant arteriosclerosis were assessed.

Pretransplant Blood Transfusion Without Additional Immunotherapy Generates CD25+CD4+ Regulatory T Cells: a Potential Explanation for the Blood-transfusion Effect

Transplantation. Aug, 2003  |  Pubmed ID: 12923427

Preoperative blood transfusion has had a significant historic impact on graft outcome in clinical kidney transplantation, and the effect has been widely replicated in many experimental transplant models. Although the mechanisms underlying the blood-transfusion effect are poorly understood, one possibility is that preexposure to alloantigen results in the induction of regulatory cells with the capacity to control the effector arm of the immune response.

Regulatory Cells in Transplantation

Novartis Foundation Symposium. 2003  |  Pubmed ID: 14609219

Regulatory T cells can play an important role in both the induction and maintenance of tolerance to donor alloantigens in vivo. Regulatory activity specific for donor alloantingens is enriched amongst CD4+CD25+ T cells in some settings and can be induced by manipulating the immune system before transplantation. Donor alloantigen-specific CD4+CD25+ regulatory T cells can control aggressive CD4+ as well as CD8+ T cells thereby preventing rejection and can mediate linked unresponsiveness. In vivo, donor alloantigen specific CD4+CD25+ cells are dependent on interleukin (IL)10 and CTLA4 for functional activity. These populations of regulatory cells induced by manipulating the adult immune system therefore have properties in common with naturally occurring regulatory T cells. The active regulation/suppression of immune responsiveness to donor alloantigens offers a way to silence aggressive immune responses directed to donor alloantigens thereby preventing damage to the graft from being inflicted. The generation of regulatory T cells with defined alloantigen specificity could provide dynamic control of rejection responses and offers a potential route to permanent graft survival without the need for life-long non-specific immunosuppression.

Alloantigen-induced CD25+CD4+ Regulatory T Cells Can Develop in Vivo from CD25-CD4+ Precursors in a Thymus-independent Process

Journal of Immunology (Baltimore, Md. : 1950). Jan, 2004  |  Pubmed ID: 14707064

The capacity of naturally occurring autoreactive CD25+CD4+ regulatory T cells (Treg) to control immune responses both in vivo and in vitro is now well established. It has been demonstrated that these cells undergo positive selection within the thymus and appear to enter the periphery as committed CD25+CD4+ Treg. We have shown previously that CD25+CD4+ Treg with the capacity to prevent skin allograft rejection can be generated by pretreatment with donor alloantigen under the cover of anti-CD4 therapy. Here we demonstrate that this process does not require an intact thymus. Furthermore, generation of these Treg is not dependent on the expansion of CD25+CD4+ thymic emigrants, because depletion of CD25+ cells before pretreatment does not prevent Treg development, and Treg can be generated from CD25-CD4+ precursors. Taken together, these results clearly demonstrate that CD25+CD4+ Treg can be generated in the periphery from CD25-CD4+ precursors in a pathway distinct to that by which naturally occurring autoreactive CD25+CD4+ Treg develop. These observations may have important implications for the design of protocols, both experimental and clinical, for the induction of tolerance to autoantigens or alloantigens in adults with limited thymic function.

A Simple Method to Cure Established Tumors by Inflammatory Killing of Normal Cells

Nature Biotechnology. Sep, 2004  |  Pubmed ID: 15300260

We describe a simple technology used to cure an established metastatic disease. Intradermal injection of plasmid DNA encoding a transcriptionally targeted cytotoxic gene, along with hsp70, not only promoted tissue-specific, inflammatory killing of normal melanocytes, but also induced a CD8(+) T-cell-dependent, antigen-specific response in mice that eradicated systemically established B16 tumors. This CD8(+) T cell response was subsequently suppressed in vivo within a few days. The data demonstrate that deliberate destruction of normal tissue can be exploited to generate immunity against a malignant disease originating from that tissue. This approach obviates the need to identify tumor antigens and does not require complex isolation of tumor cells or their derivatives. In addition, it provides a model system for studying the mechanisms underlying the etiology and control of autoimmune diseases. Finally, despite targeting normal tissue, therapy could be separated from development of overt autoimmune symptoms, suggesting that the strategy may be valuable against tumors derived from both non-essential and essential tissue types.

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

Blood. Jun, 2005  |  Pubmed ID: 15713793

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.

The Generation of CD25+ CD4+ Regulatory T Cells That Prevent Allograft Rejection Does Not Compromise Immunity to a Viral Pathogen

Journal of Immunology (Baltimore, Md. : 1950). Mar, 2005  |  Pubmed ID: 15749860

In all but a small minority of cases, continued survival of solid organ grafts after transplantation depends on lifelong, nonselective immunosuppression that, although effective, results in increased rates of infection, cancer, and vascular disease. Therapeutic strategies that engage or mimic self-tolerance may allow prolonged allograft survival without the disadvantages of nonspecific immunotherapy. Pretreatment of recipient mice with donor alloantigen combined with transient modulation of the peripheral T cell pool with anti-CD4 Ab leads to the indefinite survival of MHC-incompatible cardiac allografts without further therapy. Tolerance is dependent on CD25+ CD4+ regulatory T cells that arise from naive CD25- precursors and regulate rejection via both IL-10 and CTLA-4. Although these cells are clearly effective at controlling rejection, the proven ability of recently activated CD25+ cells to mediate bystander regulation raises the possibility that tolerized individuals might also have a reduced capacity to respond to environmental pathogens. We have examined anti-influenza responses in tolerized primary heart recipients, secondary recipients following adoptive transfer of regulatory populations, and tolerized mice in which bystander regulation has been deliberately induced. Neither virus-specific CTL activity in vitro nor the clearance of virus in vivo was significantly diminished in any of these treatment groups compared with infected unmanipulated controls. The data suggest that the induction of dominant allograft tolerance dependent on regulatory T cells does not necessarily result in attenuated responses to pathogens providing further support for the development of tolerance induction protocols in clinical transplantation.

CD25+CD4+ Regulatory T Cells Develop in Mice Not Only During Spontaneous Acceptance of Liver Allografts but Also After Acute Allograft Rejection

Transplantation. Nov, 2006  |  Pubmed ID: 17102772

Liver grafts transplanted across a major histocompatibility barrier are accepted spontaneously and induce donor specific tolerance in some species. Here, we investigated whether liver allograft acceptance is characterized by, and depends upon, the presence of donor reactive CD25CD4 regulatory T cells.

Regulation of Transplant Arteriosclerosis by CD25+CD4+ T Cells Generated to Alloantigen in Vivo

Transplantation. Jun, 2007  |  Pubmed ID: 17565319

CD25+CD4+ regulatory T cells have been shown to suppress alloimmunity in various experimental settings. Here, we hypothesized that alloantigen-reactive regulatory T cells would reduce the severity of transplant arteriosclerosis.

Interferon Gamma: Friend or Foe?

Transplantation. Jul, 2007  |  Pubmed ID: 17632413

Interferon gamma (IFN-gamma) can elicit an inflammatory TH1-driven immune response but has also been found to be necessary for long-term allograft survival induced by costimulation blockade. Recently, we have found that regulatory T cells rapidly and transiently produce IFN-gamma creating a microenvironment that can influence the function of antigen presenting cells (APCs), T-cell proliferation and activation as well as T-cell effector mechanisms, thereby controlling immune responses locally. Moreover, addition of IFN-gamma to cocultures of T cells and APCs can drive the generation of T cells with regulatory activity. Thus, the influence of IFN-gamma on the immune response to a transplant is likely to be context dependent.

Dependency of the Trans Vivo Delayed Type Hypersensitivity Response on the Action of Regulatory T Cells: Implications for Monitoring Transplant Tolerance

Transplantation. Aug, 2007  |  Pubmed ID: 17700166

The trans vivo delayed-type hypersensitivity (DTH) assay has been used for monitoring the immune status of clinical transplant recipients. Here we tested the hypothesis that the assay can reveal control of allograft rejection by CD25CD4 regulatory T cells (Treg).

Functional Dichotomy of NK Cells in Organ Transplantation

Expert Review of Clinical Immunology. May, 2007  |  Pubmed ID: 20477669

Evaluation of: Yu G, Xu X, Vu MD, Kilpatrick ED, Li XC. NK cells promote transplant tolerance by killing donor antigen-presenting cells. J. Exp. Med. 203, 1851-1858 (2006). Natural killer (NK) cells have the potential to display different functional activities after transplantation. The traditional view is that NK cells have the capacity to contribute to rejection by facilitating the activation/differentiation of leukocytes that destroy the graft. By contrast, in the article under review, a novel role for NK cells was identified in the setting of costimulation blockade where alloreactive NK cells of recipient origin were found to have the capacity to kill donor-derived antigen-presenting cells, thereby reducing T-cell priming and promoting long-term skin graft acceptance.

Investigation into the Onset and Progression of Transplant Arteriosclerosis in a Mice Aortic Retransplantation Model

Microsurgery. 2008  |  Pubmed ID: 18253942

Long-term function of vascularized human organ grafts is often limited by transplant arteriosclerosis and can lead to graft failure. Here, we have analyzed the impact of an initial rejection episode on the later development of transplant arteriosclerosis. Following transplantation of allogeneic abdominal aortic segments in mice, aortic grafts were retransplanted into either immunodeficient or syngeneic recipients. Retransplantation of grafts from immunocompetent into immunodeficient mice as early as 2 days after the primary transplant resulted in intimal proliferation and obstruction of the graft lumen 30 days after the primary transplant. In contrast, retransplantation of the grafts into donor syngeneic B10 recipients within 7 days did not result in the development of transplant arteriosclerosis. These data suggest that the adaptive immune system can induce intimal proliferation by an initial lethal hit that is sustained by the innate response. However our data demonstrate that development of chronic rejection can be inhibited, in this case by retransplantation into a syngeneic host.

Interferon-gamma Conditioning Ex Vivo Generates CD25+CD62L+Foxp3+ Regulatory T Cells That Prevent Allograft Rejection: Potential Avenues for Cellular Therapy

Transplantation. Aug, 2008  |  Pubmed ID: 18724229

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.

Exogenous IFN-gamma Ex Vivo Shapes the Alloreactive T-cell Repertoire by Inhibition of Th17 Responses and Generation of Functional Foxp3+ Regulatory T Cells

European Journal of Immunology. Sep, 2008  |  Pubmed ID: 18792404

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.

Neutralizing Interleukin-4 Prevents Transplant Arteriosclerosis Mediated by Indirect Pathway T Cells Under CD40-CD154 Costimulation Blockade

Transplantation. Dec, 2008  |  Pubmed ID: 19077898

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.

Inflammatory Signalling As Mediator of Epigenetic Modulation in Tissue-specific Chronic Inflammation

The International Journal of Biochemistry & Cell Biology. Jan, 2009  |  Pubmed ID: 18793748

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.

Donor Reactive Regulatory T Cells

Current Opinion in Organ Transplantation. Aug, 2009  |  Pubmed ID: 19448539

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.

Mesenchymal Stem Cells Prevent the Rejection of Fully Allogenic Islet Grafts by the Immunosuppressive Activity of Matrix Metalloproteinase-2 and -9

Diabetes. Aug, 2009  |  Pubmed ID: 19509016

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.

The Discovery of Immunological Tolerance: Now More Than Just a Laboratory Solution

Journal of Immunology (Baltimore, Md. : 1950). Jan, 2010  |  Pubmed ID: 20028657

Mesenchymal Stem-cell Immunosuppressive Capabilities: Therapeutic Implications in Islet Transplantation

Transplantation. Feb, 2010  |  Pubmed ID: 20145515

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.

Intravenous Immunoglobulins Promote Skin Allograft Acceptance by Triggering Functional Activation of CD4+Foxp3+ T Cells

Transplantation. Jun, 2010  |  Pubmed ID: 20463648

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.

CD4+ Regulatory T Cells Generated in Vitro with IFN-{gamma} and Allogeneic APC Inhibit Transplant Arteriosclerosis

The American Journal of Pathology. Jul, 2010  |  Pubmed ID: 20472892

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.

Regulatory T Cell Enrichment by IFN-γ Conditioning

Methods in Molecular Biology (Clifton, N.J.). 2011  |  Pubmed ID: 20941618

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.

Induction of Transplantation Tolerance Converts Potential Effector T Cells into Graft-protective Regulatory T Cells

European Journal of Immunology. Mar, 2011  |  Pubmed ID: 21243638

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.

Immunologic Unresponsiveness to Alloantigen in Vivo: a Role for Regulatory T Cells

Immunological Reviews. May, 2011  |  Pubmed ID: 21488894

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.

Functional Regulatory T Cells Produced by Inhibiting Cyclic Nucleotide Phosphodiesterase Type 3 Prevent Allograft Rejection

Science Translational Medicine. May, 2011  |  Pubmed ID: 21593400

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.

Moving to Tolerance: Clinical Application of T Regulatory Cells

Seminars in Immunology. Aug, 2011  |  Pubmed ID: 21620722

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.

Regulatory Immune Cells in Transplantation

Nature Reviews. Immunology. May, 2012  |  Pubmed ID: 22627860

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.

Targeting Apoptosis to Induce Stable Mixed Hematopoietic Chimerism and Long-term Allograft Survival Without Myelosuppressive Conditioning in Mice

Blood. Aug, 2013  |  Pubmed ID: 23869083

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 proapoptotic small-molecule Bcl-2 inhibitor ABT-737 promoted mixed chimerism induction and reversed the antitolerogenic effect of calcineurin inhibitors by boosting the critical role of the proapoptotic 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 major histocompatibility complex 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.

Single Cell Tracking of Gadolinium Labeled CD4+ T Cells by Laser Ablation Inductively Coupled Plasma Mass Spectrometry

Analytical Chemistry. Nov, 2013  |  Pubmed ID: 24080112

Cellular therapy is emerging as a promising alternative to conventional immunosuppression in the fields of hematopoietic 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 labeling 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 labeled with commercially available Gd-based magnetic resonance imaging (MRI) contrast agents, Omniscan and Dotarem, which enabled passive loading of up to 10(8) Gd atoms per cell. In mixed preparations of labeled and unlabeled cells, LA-ICP-MS was capable of enumerating labeled cells at close to the predicted ratio. More importantly, LA-ICP-MS single cell analysis demonstrated that the cells retained a sufficient label to remain detectable for up to 10 days post-labeling both in vitro and in vivo in an immunodeficient mouse model.

Induction of Transplantation Tolerance Through Regulatory Cells: from Mice to Men

Immunological Reviews. Mar, 2014  |  Pubmed ID: 24517428

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.

Varying Degrees of Ventricular Unloading in the Heterotopic Rat Heart Transplant Model Demonstrated by Magnetic Resonance Imaging

International Journal of Biomedical Science : IJBS. Dec, 2014  |  Pubmed ID: 25598751

Left ventricular assist device placement is an increasingly common treatment for cardiac failure, resulting in cardiac unloading and potentially reversing the remodelling changes seen in heart failure. A popular animal model for human ventricular unloading is the rodent heterotopic non-working heart transplant; the volume loading status of this preparation is important to interpreting the resulting reverse remodelling yet has not been previously investigated. This study was designed to assess the variability of left ventricular volume loading in the rodent transplant model.

Alloreactive Regulatory T cells Generated with Retinoic Acid Prevent Skin Allograft Rejection

European Journal of Immunology. Feb, 2015  |  Pubmed ID: 25381698

CD4(+) CD25(+) Foxp3(+) regulatory T (Treg) cells mediate immunological self-tolerance and suppress immune responses. Retinoic acid (RA), a natural metabolite of vitamin A, has been reported to enhance the differentiation of Treg cells in the presence of TGF-β. In this study, we show that the co-culture of naive T cells from C57BL/6 mice with allogeneic antigen-presenting cells (APCs) from BALB/c mice in the presence of TGF-β, RA, and IL-2 resulted in a striking enrichment of Foxp3(+) T cells. These RA in vitro-induced regulatory T (RA-iTreg) cells did not secrete Th1-, Th2-, or Th17-related cytokines, showed a nonbiased homing potential, and expressed several cell surface molecules related to Treg-cell suppressive potential. Accordingly, these RA-iTreg cells suppressed T-cell proliferation and inhibited cytokine production by T cells in in vitro assays. Moreover, following adoptive transfer, RA-iTreg cells maintained Foxp3 expression and their suppressive capacity. Finally, RA-iTreg cells showed alloantigen-specific immunosuppressive capacity in a skin allograft model in immunodeficient mice. Altogether, these data indicate that functional and stable allogeneic-specific Treg cells may be generated using TGF-β, RA, and IL-2. Thus, RA-iTreg cells may have a potential use in the development of more effective cellular therapies in clinical transplantation.

Hurdles in Therapy with Regulatory T Cells

Science Translational Medicine. Sep, 2015  |  Pubmed ID: 26355029

Improper activation of the immune system contributes to a variety of clinical conditions, including autoimmune and allergic diseases as well as solid organ and bone marrow transplantation. One approach to counteract this activation is through adoptive therapy with regulatory T cells (Tregs). Efforts to manufacture these cells have led to good maunfacturing practice-compliant protocols, and Treg products are entering early clinical trials. Here, we report the stance of the European Union Cooperation in Science and Technology Action BM1305, "Action to Focus and Accelerate Cell-based Tolerance-inducing Therapies-A FACTT," which identifies hurdles hindering Treg clinical applications in Europe and provides possible solutions.

Regulatory T Cells: First Steps of Clinical Application in Solid Organ Transplantation

Transplant International : Official Journal of the European Society for Organ Transplantation. Jan, 2016  |  Pubmed ID: 25981203

Solid organ transplantation is the treatment of choice for patients with end-stage organ failure. To prevent rejection of the transplanted organ continuous treatment with immunosuppressive medication is needed. Immunosuppression may be harmful to the transplant recipient, increasing the risk of cancer, infections and cardiovascular disease. To improve transplant and patient survival, there is a need for an immune-modulatory regimen that is not only potent in preventing rejection of the transplanted organ, but has less side effects compared to current immunosuppressive regimens. Increasingly, transplantation research focusses on regulatory T cell (Treg) therapy to achieve this aim, in which Treg are used as a strategy to allow reduction of immunosuppression. Currently, the first clinical trials are underway investigating the safety and feasibility of Treg therapy in renal transplantation. This review gives an overview of the rationale of using Treg therapy in transplantation, previous experience with Treg therapy in humans, and the expected safety, potential efficacy and cost-effectiveness of Treg therapy in solid organ transplantation.

Distinctive Expression of Bcl-2 Factors in Regulatory T Cells Determines a Pharmacological Target to Induce Immunological Tolerance

Frontiers in Immunology. 2016  |  Pubmed ID: 26973650

Distinctive molecular characteristics of functionally diverse lymphocyte populations may represent novel pharmacological targets for immunotherapy. The intrinsic apoptosis pathway is differently regulated among conventional and regulatory T cells (Tregs). Targeted pharmacological modulation of this pathway with a small molecule Bcl-2/Bcl-xL inhibitor (ABT-737) caused a selective depletion of effector T cells and a relative enrichment of Tregs in vivo. Treatment with ABT-737 resulted in a tolerogenic milieu, which was exploited to alleviate graft-versus-host disease, to prevent allograft rejection in a stringent fully MHC-mismatched skin transplantation model and to induce immunological tolerance in combination with bone marrow transplantation. This concept has the potential to find various applications for immunotherapy, since it allows pharmacologic exploitation of the immunomodulatory properties of Tregs without the need for cell manipulation ex vivo.

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