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Behdad Afzali,
Francis C Edozie,
Henrieta Fazekasova,
Cristiano Scottà,
Peter J Mitchell,
James B Canavan,
Shahram Y Kordasti,
Prabhjoat S Chana,
Richard Ellis,
Graham M Lord,
Susan John,
Rachel Hilton,
Robert I Lechler,
Giovanna Lombardi
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ABSTRACT: BACKGROUND AND OBJECTIVES: Cell-based therapy with natural (CD4(+)CD25(hi)CD127(lo)) regulatory T cells to induce transplant tolerance is now technically feasible. However, regulatory T cells from hemodialysis patients awaiting transplantation may be functionally/numerically defective. Human regulatory T cells are also heterogeneous, and some are able to convert to proinflammatory Th17 cells. This study addresses the suitability of regulatory T cells from hemodialysis patients for cell-based therapy in preparation for the first clinical trials in renal transplant recipients (the ONE Study). DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Healthy controls and age- and sex-matched hemodialysis patients without recent illness/autoimmune disease on established, complication-free hemodialysis for a minimum of 6 months were recruited. Circulating regulatory T cells were studied by flow cytometry to compare the regulatory T cell subpopulations. Regulatory T cells from members of each group were compared for suppressive function and plasticity (IL-17-producing capacity) before and after in vitro expansion with and without Rapamycin, using standard assays. RESULTS: Both groups had similar total regulatory T cells and subpopulations I and III. In each subpopulation, regulatory T cells expressed similar levels of the function-associated markers CD27, CD39, HLA-DR, and FOXP3. Hemodialysis regulatory T cells were less suppressive, expanded poorly compared with healthy control regulatory T cells, and produced IL-17 in the absence of Rapamycin. However, Rapamycin efficiently expanded hemodialysis regulatory T cells to a functional and stable cell product. CONCLUSIONS: Rapamycin-based expansion protocols should enable clinical trials of cell-based immunotherapy for the induction of tolerance to renal allografts using hemodialysis regulatory T cells.
Clinical Journal of the American Society of Nephrology 04/2013; · 5.23 Impact Factor
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Expert Review of Molecular Diagnostics 01/2013; 13(1):5-7. · 4.86 Impact Factor
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Cristiano Scotta,
Marianna Esposito,
Henrieta Fazekasova,
Giorgia Fanelli,
Francis C Edozie,
Niwa Ali,
Fang Xiao,
Mark Peakman, Behdad Afzali,
Pervinder Sagoo,
Robert I Lechler,
Giovanna Lombardi
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ABSTRACT: Introduction. Adoptive transfer of ex vivo expanded CD4(+)CD25(+)FOXP3(+) regulatory T cells is a successful therapy for autoimmune diseases and transplant rejection in experimental models. In man, equivalent manipulations in bone marrow transplant recipients appear safe, but questions regarding the stability of the transferred regulatory T cells during inflammation remain unresolved. In this study protocols for the expansion of clinically useful numbers of functionally suppressive and stable human regulatory T cells were investigated. Designs and Methods. Regulatory T cells were expanded in vitro with rapamycin and/or all-trans-retinoic acid and then characterized under inflammatory conditions in vitro and in vivo in a humanized mouse model of graft-versus-host disease. Results. Addition of rapamycin to regulatory T cells cultures confirms the generation of high numbers of suppressive regulatory T cells. Their stability was demonstrated in vitro and substantiated in vivo. In contrast, all-trans-retinoic acid-treatment generates regulatory T cells which retain the capacity to secrete IL-17. However, combined use of rapamycin and all-trans-retinoic acid abolishes IL-17 production and confers a specific chemokine receptor homing profile upon regulatory T cells. The use of purified regulatory T cell subpopulations provided direct evidence that rapamycin can confer an early selective advantage to CD45RA(+) regulatory T cells, while all-trans-retinoic acid favors CD45RA(-) regulatory T cell subset. Conclusions. Expansion of regulatory T cells using rapamycin and all-trans-retinoic acid drug combinations provides a new and refined approach for large-scale generation of functionally potent and phenotypically stable human regulatory T cells, rendering them safe for clinical use in settings associated with inflammation.
Haematologica 12/2012; · 6.42 Impact Factor
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ABSTRACT: Regulatory T cells (CD4(+)CD25(hi)CD127(lo)FOXP3(+) T cells [Tregs]) are a population of lymphocytes involved in the maintenance of self-tolerance. Abnormalities in function or number of Tregs are a feature of autoimmune diseases in humans. The ability to expand functional Tregs ex vivo makes them ideal candidates for autologous cell therapy to treat human autoimmune diseases and to induce tolerance to transplants. Current tests of Treg function typically take up to 120 hours, a kinetic disadvantage as clinical trials of Tregs will be critically dependent on the availability of rapid diagnostic tests before infusion into humans. Here we evaluate a 7-hour flow cytometric assay for assessing Treg function, using suppression of the activation markers CD69 and CD154 on responder T cells (CD4(+)CD25(-) [Tresp]), compared with traditional assays involving inhibition of CFSE dilution and cytokine production. In both freshly isolated and ex vivo expanded Tregs, we describe excellent correlation with gold standard suppressor cell assays. We propose that the kinetic advantage of the new assay may place it as the preferred rapid diagnostic test for the evaluation of Treg function in forthcoming clinical trials of cell therapy, enabling the translation of the large body of preclinical data into potentially useful treatments for human diseases.
Blood 01/2012; 119(8):e57-66. · 9.90 Impact Factor
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Aradhana Rani, Behdad Afzali,
Audrey Kelly,
Lemlem Tewolde-Berhan,
Mark Hackett,
Aditi S Kanhere,
Isabela Pedroza-Pacheco,
Holly Bowen,
Stipo Jurcevic,
Richard G Jenner,
David J Cousins,
Jack A Ragheb,
Paul Lavender,
Susan John
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ABSTRACT: Blockade of IL-2R with humanized anti-CD25 Abs, such as daclizumab, inhibits Th2 responses in human T cells. Recent murine studies have shown that IL-2 also plays a significant role in regulating Th2 cell differentiation by activated STAT5. To explore the role of activated STAT5 in the Th2 differentiation of primary human T cells, we studied the mechanisms underlying IL-2 regulation of C-MAF expression. Chromatin immunoprecipitation studies revealed that IL-2 induced STAT5 binding to specific sites in the C-MAF promoter. These sites corresponded to regions enriched for markers of chromatin architectural features in both resting CD4 and differentiated Th2 cells. Unlike IL-6, IL-2 induced C-MAF expression in CD4 T cells with or without prior TCR stimulation. TCR-induced C-MAF expression was significantly inhibited by treatment with daclizumab or a JAK3 inhibitor, R333. Furthermore, IL-2 and IL-6 synergistically induced C-MAF expression in TCR-activated T cells, suggesting functional cooperation between these cytokines. Finally, both TCR-induced early IL4 mRNA expression and IL-4 cytokine expression in differentiated Th2 cells were significantly inhibited by IL-2R blockade. Thus, our findings demonstrate the importance of IL-2 in Th2 differentiation in human T cells and support the notion that IL-2R-directed therapies may have utility in the treatment of allergic disorders.
The Journal of Immunology 08/2011; 187(7):3721-9. · 5.79 Impact Factor
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ABSTRACT: Organ transplantation is currently the only effective treatment for end-stage organ failure. However, success is limited by the immune response of the recipient to allogeneic tissues (recognized by the direct and indirect alloresponses) and by the morbidity and mortality associated with the immunosuppressive drugs that are used to control alloimmunity. One solution to these problems is the induction of immunological tolerance. In our laboratory, we have selected two strategies to achieve this goal. The first is to expand and/or generate Tregs directly in vivo using infusions of 'tolerogenic' DCs into patients; the second is to purify Tregs from the blood of patients on the waiting list for a transplant, enrich and expand these cells in vitro and then inject back in vivo after transplantation. Here, we have summarized our results both in the murine and human systems on the use of Treg-based strategies to induce tolerance to the transplanted organs.
Immunotherapy 04/2011; 3(4 Suppl):28-31. · 1.85 Impact Factor
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ABSTRACT: Solid organ transplantation is the standard treatment to improve both the quality of life and survival in patients with various end-stage organ diseases. The primary barrier against successful transplantation is recipient alloimmunity and the need to be maintained on immunosuppressive therapies with associated side effects. Despite such treatments in renal transplantation, after death with a functioning graft, chronic allograft dysfunction (CAD) is the most common cause of late allograft loss. Recipient recognition of donor histocompatibility antigens, via direct, indirect, and semidirect pathways, is critically dependent on the antigen-presenting cell (APC) and elicits effector responses dominated by recipient T cells. In allograft rejection, the engagement of recipient and donor cells results in recruitment of T-helper (Th) cells of the Th1 and Th17 lineage to the graft. In cases in which the alloresponse is dominated by regulatory T cells (Tregs), rejection can be prevented and the allograft tolerated with minimum or no immunosuppression. Here, we review the pathways of allorecognition that underlie CAD and the T-cell effector phenotypes elicited as part of the alloresponse. Future therapies including depletion of donor-reactive lymphocytes, costimulation blockade, negative vaccination using dendritic cell subtypes, and Treg therapy are inferred from an understanding of these mechanisms of allograft rejection.
Kidney international. Supplement 12/2010;
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09/2010; , ISBN: 9780470015902
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The Lancet 06/2010; 375(9733):2278. · 38.28 Impact Factor
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Katie Matthews,
ZiYi Lim, Behdad Afzali,
Laurence Pearce,
Atiyeh Abdallah,
Shahram Kordasti,
Antonio Pagliuca,
Giovanna Lombardi,
J Alejandro Madrigal,
Ghulam J Mufti,
Linda D Barber
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ABSTRACT: A variety of immune pathways can lead to graft-versus-host disease. A better understanding of the type of immune response causing graft-versus-host disease in defined clinical hematopoietic stem cell transplant settings is required to inform development of methods for monitoring patients and providing them tailored care.
Twenty-five patients were recruited presenting with myeloid malignancies and treated with a reduced intensity conditioning transplant regimen with graft-versus-host disease prophylaxis comprising in vivo lymphocyte depletion with alemtuzumab and cyclosporin. A prospective study was performed of lymphocyte subset reconstitution in peripheral blood in relation to the incidence of graft-versus-host disease.
Acute graft-versus-host disease was associated with significantly higher numbers of natural killer cells and donor-derived effector CD4 T cells (CD45RO(+) CD27(-)) early (day 30) after transplantation (p=0.04 and p=0.02, respectively). This association was evident before the emergence of clinical pathology in six out of seven patients. Although numbers of regulatory CD4 T cells (CD25(high) Foxp3(+)) were similar at day 30 in all patients, a significant deficit in those who developed acute graft-versus-host disease was apparent relative to effector CD4 T cells (median of 41 effectors per regulatory cell compared to 12 to 1 for patients without graft-versus-host disease) (p=0.03). By day 180, a functional regulatory CD4 T-cell population had expanded significantly in patients who developed chronic graft-versus-host disease, reversing the imbalance (median of 3 effectors per regulatory cell compared to 9.6 to 1 for patients without graft-versus-host disease) (p=0.018) suggesting no overt absence of immune regulation in the late onset form of the disease.
Imbalance of effector and regulatory CD4 T cells is a signature of graft-versus-host disease in this transplantation protocol.
Haematologica 07/2009; 94(7):956-66. · 6.42 Impact Factor
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ABSTRACT: Regulatory T cells (Tregs) are potential therapeutic tools in averting transplant rejection and promoting lifelong tolerance. However, Tregs can be subverted by inflammatory conditions, resulting in a T helper 17 (Th17) cell response. This review looks at the relationship between Tregs and Th17 cells.
Both naturally occurring and transforming growth factor-beta-induced Tregs can be converted into Th17 cells in the presence of inflammatory cytokines. Transforming growth factor-beta upregulates the Treg transcription factor, forkhead box P3, as well as the Th17 transcription factor, retinoic acid receptor-related orphan receptor gamma. However, forkhead box P3 binds to retinoic acid receptor-related orphan receptor gamma, inhibiting its promotion of IL-17 gene transcription. Inflammatory cytokines can disrupt this interaction through the inhibition of forkhead box P3 expression by signal transducer and activator of transcription 3.
The plasticity of the Treg population during inflammation presents a challenge to the use of Tregs as a therapeutic tool in solid organ transplantation. Investigations into the Th17-Treg axis have identified a number of potential pharmacological targets to avoid the risk of conversion to Th17 cells, but further work must be done before we can separate the benefits of Treg therapy from the hazards of the Th17 response.
Current opinion in organ transplantation 06/2009; 14(4):326-31. · 1.22 Impact Factor
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03/2009: pages 441 - 451; , ISBN: 9781444303391
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ABSTRACT: Despite advances in immunosuppression, allograft rejection remains a significant challenge to the long-term success of solid-organ transplantation. Whilst allorecognition pathways are clearly central to rejection, the effector mechanisms of this process are less defined. T helper (Th) type 17 cells are a recently described CD4 T-cell subset, and have been implicated in a range of autoimmune and inflammatory conditions that were previously thought to be Th1 mediated. In light of these developments, this review examines the relative roles of these subsets in allograft rejection.
Th1 cells are characterized by production of the cytokine interferon-gamma, which has recently been described as having both pro- and anti-inflammatory effects, including a role in regulatory T-cell function. A number of clinical studies show that serum and intragraft interferon-gamma levels positively correlate with episodes of acute rejection, although increased interleukin-17 expression has also been reported in transplants undergoing rejection. Interestingly, a complex interplay between Treg and Th17 development has recently been demonstrated, with transforming growth factor-beta being necessary for both.
Current data indicate the presence of both subsets during allograft rejection, although their precise role is unclear. An improved understanding of the factors that influence the differentiation and function of these cell types will assist in the development of future immunomodulatory therapies.
Current opinion in organ transplantation 03/2009; 14(1):23-9. · 1.22 Impact Factor
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Shahram Y Kordasti, Behdad Afzali,
Ziyi Lim,
Wendy Ingram,
Janet Hayden,
Linda Barber,
Katie Matthews,
Rajani Chelliah,
Barbara Guinn,
Giovanna Lombardi,
Farzin Farzaneh,
Ghulam J Mufti
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ABSTRACT: Immunological responses are increasingly recognised as being important in the initiation and progression of myelodysplastic syndrome (MDS). Indeed, autoimmune diseases commonly occur in association with MDS, particularly in subtypes with a low risk of leukaemic transformation. This study showed for the first time that the numbers of CD3(+) CD4(+) IL-17 producing T cells (Th17) were markedly increased in low risk MDS compared with high risk MDS (P < 0.01). An inverse relationship between the numbers of Th17 cells and naturally occurring CD4(+)CD25(high) FoxP3(+) regulatory T cells (Tregs) were also described. The Th17:Tregs ratio was significantly higher in low risk disease (P < 0.005) compared with high risk MDS and was correlated with increased bone marrow (BM) apoptosis (P < 0.01). Tregs from MDS patients suppressed interferon-gamma (IFN-gamma) secretion by effector CD4(+) T cells but had no effect on interleukin (IL)-17 production. In addition, the serum levels of IL-7, IL-12, RANTES and IFN-gamma are significantly elevated in low risk MDS, while inhibitory factors, such as IL-10 and soluble IL-2 receptor, are significantly higher in high risk disease. The 'unfavourable' Th17:Tregs ratio in low risk MDS may explain the higher risk of autoimmunity and the improved response to immune suppression in patients with low risk MDS compared to those with high risk disease.
British Journal of Haematology 02/2009; 145(1):64-72. · 4.94 Impact Factor
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ABSTRACT: Here, we review the pathways of allorecognition and their potential relevance to the balance between regulatory and effector responses following transplantation.
Transplantation between nonidentical members of the same species elicits an immune response that manifests as graft rejection or persistence. Presentation of foreign antigen to recipient T cells can occur via three nonmutually exclusive routes, the direct, indirect and semi-direct pathways. Allospecific T cells can have effector or regulatory functions, and the relative proportions of the two populations activated following alloantigen presentation are two of the factors that determine the clinical outcome. Regulatory T cells have been the subject of significant research, and there is now greater understanding of their recruitment and function in the context of allorecognition.
A greater understanding of the mechanisms underlying allorecognition may be fundamental to appreciating how these different populations are recruited and could in turn inform novel strategies for immunomodulation.
Current opinion in organ transplantation 09/2008; 13(4):438-44. · 1.22 Impact Factor
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The Lancet 09/2007; 370(9586):482; author reply 482-3. · 38.28 Impact Factor
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The Lancet 08/2007; 370(9582):192. · 38.28 Impact Factor
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Shahram Y Kordasti,
Wendy Ingram,
Janet Hayden,
David Darling,
Linda Barber, Behdad Afzali,
Giovanna Lombardi,
Marcin W Wlodarski,
Jaroslaw P Maciejewski,
Farzin Farzaneh,
Ghulam J Mufti
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ABSTRACT: Foxp3+ regulatory T cells (Tregs) play a central role in maintaining immune tolerance. A reduction in the function of Tregs is a key feature of autoimmune diseases, whereas their expansion in malignant diseases leads to the suppression of host antitumor responses. We analyzed the absolute number of CD4+ and CD8(+) Tregs in the peripheral blood of 52 patients with myelodysplastic syndrome (MDS) and show a significant correlation between increased number of CD4+ Tregs and MDS subgroups with 5% or more bone marrow blasts (P < .001), high International Prognostic Scoring System (IPSS) score (P < .001), and disease progression (P < .001), whereas no correlation between CD8+ Tregs and prognostic variables was observed. The CD4+ Tregs showed a polyclonal spectratype, and the percentage of the naive subset was significantly higher in the high-risk patients compared with low-risk or healthy age-matched donors (P = .032). Our data suggest that CD4+ Treg expansion is a feature of high-risk MDS and progression to aggressive subtypes of the disease.
Blood 08/2007; 110(3):847-50. · 9.90 Impact Factor
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ABSTRACT: CD4+ CD25+ regulatory T cells (Tregs) have far-reaching immunotherapeutic applications, the realization of which will require a greater understanding of the factors influencing their function and phenotype during ex vivo manipulation. In murine models, IL-2 plays an important role in both the maintenance of a functional Treg population in vivo and the activation of suppression in vitro. We have found that IL-2 maintains optimal function of human CD4+ CD25+ Tregs in vitro and increases expression of both forkhead box protein 3, human nomenclature (FOXP3) and the distinctive markers CD25, cytotoxic T lymphocyte antigen-4 (CTLA-4) and glucocorticoid-induced tumor necrosis factor receptor superfamily member number 18 (GITR). Although IL-2 reduced spontaneous apoptosis of Tregs, this property alone could not account for the optimal maintenance of the regulatory phenotype. The inhibition of phosphatidylinositol 3-kinase (PI3K) signaling by LY294002, a chemical inhibitor of PI3K, abolished the maintenance of maximal suppressive potency by IL-2, yet had no effect on the up-regulation of FOXP3, CD25, CTLA-4 and GITR. Other common gamma chain (gammac) cytokines-IL-4, IL-7 and IL-15-had similar properties, although IL-4 showed a unique lack of effect on the expression of FOXP3 or Treg markers despite maintaining maximal regulatory function. Taken together, our data suggest a model in which the gammac cytokines IL-2, IL-4, IL-7 and IL-15 maintain the optimal regulatory function of human CD4+ CD25+ T cells in a PI3K-dependent manner, offering new insight into the effective manipulation of Tregs ex vivo.
International Immunology 07/2007; 19(6):785-99. · 3.41 Impact Factor
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ABSTRACT: Anemia in the setting of chronic kidney disease is a well-recognized phenomenon that is associated with decreasing renal function and deficiency of or resistance to erythropoietin. However, anemia in the post-renal transplantation setting has received comparatively less attention in the literature. In this review, we aim critically to appraise the available literature regarding posttransplantation anemia, concentrating in particular on the prevalence of posttransplantation anemia, its etiopathogenesis, potential effects on morbidity and mortality, and the rationale for intervention and treatment. Despite deficiencies in the literature, we conclude that posttransplantation anemia is a common phenomenon that can occur either early or late posttransplantation, and its causation is usually multifactorial and includes contributions notably from poor or decreasing renal function, immunosuppression, and iron deficiency. Conversely, there is a shortage of well-conducted prospective studies looking at the morbidity attributable to posttransplantation anemia and a lack of trial evidence to determine whether intervention improves patient morbidity and outcome.
American Journal of Kidney Diseases 11/2006; 48(4):519-36. · 5.43 Impact Factor
