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1.  Multiple Unit Pooled Umbilical Cord Blood is a Viable Source of Therapeutic Regulatory T Cells 
Transplantation  2013;95(1):85-93.
Regulatory T cells (Treg) are potentially a useful therapeutic option for the treatment of immunopathological conditions including graft-versus-host disease. Umbilical cord blood (UCB) offers certain advantages over adult peripheral blood (APB) as a source of Treg for cellular therapy but yields far fewer Treg per unit. Pooling of Treg from multiple donors may overcome this challenge.
In this study, we assessed the in vitro and in vivo efficacy of multiple donor pooled UCB or APB-derived Treg.
In vitro, pooled freshly isolated UCB-derived Treg were as suppressive as APB-derived Treg. However, in a mouse model of human skin allodestruction, pooled UCB-derived Treg were more potent at suppressing alloresponses and prolonging skin survival compared with pooled APB-derived Treg. Improved survival of UCB Treg in an in vivo cell survival assay and their lower expression of human leukocyte antigen-ABC suggested that lower immunogenicity may account for their superior efficacy in vivo.
Multiple-unit UCB is therefore a viable source of human Treg for cellular therapy, and pooling of Treg from multiple donors offers a useful strategy for achieving required therapeutic doses.
PMCID: PMC4516307  PMID: 23263503
Regulatory T cells; Treg; Graft-versus-host disease; Skin; Transplantation; Umbilical cord blood; Multiple unit cord blood transplantation
2.  Th17 cells in alemtuzumab-treated patients. The effect of long-term maintenance immunosuppressive therapy. 1,2,3 
Transplantation  2011;91(7):744-750.
Leukocyte depletion at the time of transplantation with alemtuzumab (Campath-1H) has been demonstrated to be a potential strategy for reducing long term exposure to immunosuppressive drugs. While the impact of alemtuzumab treatment on the immune system has been explored, the effects of long-term immunosuppressive therapy in alemtuzumab treated patients still need to be elucidated.
T regulatory cells and Th1/Th17 responses were assessed by flow cytometry and real-time PCR > 3 years after transplantation in 10 kidney recipients treated with alemtuzumab induction. 7 patients were converted to sirolimus monotherapy at 12 months post-transplant while the remaining 3 patients with history of graft rejection were treated with sirolimus and mycophenolate mofetil (MMF). Additionally, we sorted and expanded IL17A-producing CCR6+CD4+ T cells and assessed their susceptibility to suppression by regulatory T (Treg) cells in vitro suppression tests.
3 years of mTOR inhibitor monotherapy correlates with an increase in the number of IL-17A producing cells, compared to patients treated with sirolimus and MMF. In these patients, IL-17A expression was compensated for by an increase in Treg cell frequency and number. Additionally, we demonstrated that both proliferation and cytokine production by Th17 cells can be effectively regulated by Treg cells.
Our results demonstrate that history of rejection and long-term maintenance immunosuppression has an impact on the number of circulating Treg and Th17 cells. But more importantly, we have shown that Treg can effectively regulate Th17cells both in vitro and in vivo.
PMCID: PMC3759143  PMID: 21412187
CAMPATH-1H; IL-17; T regulatory cells; kidney transplant; Sirolimus
In organ transplantation, the composition of the B cell compartment is increasingly identified as an important determinant for graft outcome. Whereas naïve and transitional B cells have been associated with long-term allograft survival and operational tolerance, memory B cells have been linked to decreased allograft survival. Alemtuzumab induction therapy effectively depletes B cells, but is followed by rapid repopulation up to levels exceeding base line. The characteristics of the repopulating B cells are currently unknown. We studied the phenotypic and functional characteristics of B cells longitudinally in 19 kidney transplant recipients, before and at 6, 9 and 12 months after alemtuzumab induction therapy. A transient increase in transitional B cells and cells with phenotypic characteristics of regulatory B cells, as well as a long-term dominance in naïve B cells was found in alemtuzumab treated kidney transplant recipients, which was not influenced by conversion from tacrolimus to sirolimus. At all time-points after treatment, B cells showed unaltered proliferative and IgM-producing capacity as compared to pre-transplant samples, whereas the ability to produce IgG was inhibited long-term. In conclusion, induction therapy with alemtuzumab results in a long-term shift towards naïve B cells with altered phenotypic and functional characteristics.
PMCID: PMC3387484  PMID: 22420490
Campath-1H; depletion; renal transplant; regulatory B cells; conversion
Long-term immunosuppressive therapy represents a huge burden on transplant recipients, but currently cannot be omitted. Improving long-term transplant outcome by immunosuppressive drug withdrawal may be achieved in patients who have developed (partial) immunological unresponsiveness towards their graft, either spontaneously or through tolerance induction. Reliable biomarkers are essential to define such immunological unresponsiveness and will facilitate controlled immunosuppressive drug weaning as well as provide surrogate end-points for tolerance induction trials.
Areas covered
Tolerance biomarkers have been defined for both liver and kidney transplantation and can accurately identify operationally tolerant transplant recipients retrospectively. These two tolerance fingerprints are remarkably different, indicating the involvement of distinct mechanisms. Limited data suggest that tolerance biomarkers can be detected in immunosuppressed transplant recipients. Whether these patients can safely have their immunosuppressive drugs withdrawn needs to be established.
Expert opinion
Mechanistic interpretation of the kidney transplant tolerance biomarker profile dominated by B cell markers remains a challenge in light of experimental evidence suggesting the pivotal involvement of regulatory T cells. Therefore, defining animal models that resemble human transplant tolerance is crucial in understanding the underlying mechanisms. Additionally, to ensure patient safety while monitoring for tolerance, it is essential to develop biomarkers to non-invasively detect early signs of rejection as well.
PMCID: PMC3442251  PMID: 22988481
Tolerance fingerprint; kidney transplantation; liver transplantation; immunosuppressive drug withdrawal; acute rejection
5.  Mesoangioblasts Suppress T Cell Proliferation Through IDO and PGE-2-Dependent Pathways 
Stem Cells and Development  2012;22(3):512-523.
Human mesoangioblasts are vessel-associated stem cells that are currently in phase I/II clinical trials for the treatment of patients with Duchenne muscular dystrophy. To date, little is known about the effect of mesoangioblasts on human immune cells and vice versa. We hypothesized that mesoangioblasts could modulate the function of immune cells in a similar manner to mesenchymal stromal cells. Human mesoangioblasts did not evoke, but rather potently suppressed human T-cell proliferation and effector function in vitro in a dose- and time-dependent manner. Furthermore, mesoangioblasts exert these inhibitory effects uniformly on human CD4+ and CD8+ T cells in a reversible manner without inducing a state of anergy. Interferon (IFN)-γ and tumor necrosis factor (TNF)-α play crucial roles in the initial activation of mesoangioblasts. Indoleamine 2,3-dioxygenase (IDO) and prostaglandin E-2 (PGE) were identified as key mechanisms of action involved in the mesoangioblast suppression of T-cell proliferation. Together, these data demonstrate a previously unrecognized capacity of mesoangioblasts to modulate immune responses.
PMCID: PMC3549627  PMID: 22913669
6.  Tolerance - One Transplant for Life 
Transplantation  2014;98(2):117-121.
A recent TTS workshop was convened to address the question: “What do we need to have in place to make tolerance induction protocols a “standard of care” for organ transplant recipients over the next decade?” In a productive two day meeting there was wide-ranging discussion on a broad series of topics resulting in five consensus recommendations: (1) Establish a registry of results for patients enrolled in tolerance trials; (2) Establish standardized protocols for sample collection and storage; (3) Establish standardized biomarkers and assays; (4) Include children aged 12 and older in protocols that have been validated in adults; (5) a task force to engage third party payers in discussions of how to fund tolerance trials. Future planned workshops will focus on progress in implementing these recommendations and identifying other steps that the community needs to take.
PMCID: PMC4101034  PMID: 24926829
7.  CD4+ regulatory T cells in solid organ transplantation 
Purpose of review:
Solid organ transplantation is the most effective treatment for end-stage organ failure, but the long-term outcomes remain suboptimal. CD4+ regulatory T cells (Treg) are emerging as a potential therapy to facilitate long-term allograft survival. This review provides a general overview of the biology of CD4+ Treg and then goes on to discuss the most relevant and recent experimental and clinical evidence for their therapeutic use in solid organ transplantation.
Recent findings:
There have been major advances in our understanding of Treg, including improvements in methods for their isolation and expansion. Experimental models are providing very important data on the in vitro and in vivo behaviour of Treg in transplantation, while recent clinical trials of Treg cellular therapy in graft-versus-host disease are offering a valuable insight into the efficacy of Treg adoptive cellular therapy.
Data in favour of Treg cellular therapy in transplantation are mounting, and we predict that their use in clinical trials is on the horizon.
PMCID: PMC3198838  PMID: 20881492
Treg; tolerance; rejection; transplantation
8.  Translating tolerogenic therapies to the clinic – where do we stand? 
Manipulation of the immune system to prevent the development of a specific immune response is an ideal strategy to improve outcomes after transplantation. A number of experimental techniques exploiting central and peripheral tolerance mechanisms have demonstrated success, leading to the first early phase clinical trials for tolerance induction. The first major strategy centers on the facilitation of donor-cell mixed chimerism in the transplant recipient with the use of bone marrow or hematopoietic stem cell transplantation. The second strategy, utilizing peripheral regulatory mechanisms, focuses on cellular therapy with regulatory T cells. This review examines the key studies and novel research directions in the field of immunological tolerance.
PMCID: PMC3422982  PMID: 22934094
tolerance; immune regulation; cellular therapy; chimerism; regulatory T cell; clinical trials; transplantation
9.  The impact of TH17 cells on transplant rejection and the induction of tolerance 
Purpose of review
This review aims to provide an overview of the latest evidence for the involvement of Th17 cells in the rejection of solid organ allografts. It will also consider the implications of the relationship between the differentiation pathways of Th17 and regulatory T cells (Tregs), as well as their plasticity in the context of transplantation tolerance.
Recent findings
In the absence of the Th1 lineage in vivo, Th17 cells are capable of rejecting cardiac allografts, showing the capacity of Th17 cells to cause allograft rejection, at least in experimental models. Th17 cells are relatively unsusceptible to suppression by Tregs, although this may be context dependent. Furthermore, addition of inflammatory signals to a Treg inducing environment leads to Th17 development and established Tregs can be converted to Th17 cells under inflammatory conditions.
The capacity of Th17 cells to cause allograft rejection is becoming increasingly clear. However, the role and contribution of Th17 cells in allograft rejection in the presence of the full orchestra of T helper cells remains elusive. The apparent resistance of Th17 to be suppressed by Tregs may pose a hurdle for effective immunosuppression and tolerance inducing protocols. Furthermore, the close developmental pathways of Th17 and Tregs and the ability of Tregs to convert into Th17 cells in the presence of inflammatory signals may impede the establishment of specific unresponsiveness to donor alloantigens in vivo.
PMCID: PMC3095085  PMID: 20616728
IL-17; Tregs; rejection; plasticity
10.  Mesenchymal Stem-Cell Immunosuppressive Capabilities: Therapeutic Implications in Islet Transplantation 
Transplantation  2010;89(3):270-273.
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.
PMCID: PMC4452939  PMID: 20145515
Mesenchymal stem cells; Islet transplantation; Immunosuppression
11.  Mesenchymal Stromal Cells in Transplantation Rejection and Tolerance 
Mesenchymal stromal cells (MSCs) have recently emerged as promising candidates for cell-based immunotherapy in solid organ transplantation (SOT). In addition to immune modulation, MSCs possess proreparative properties and preclinical studies indicate that MSCs have the capacity to prolong graft survival and in some cases induce tolerance. Currently, the application of MSCs in SOT is being evaluated in phase I/II clinical trials. Whereas the mechanisms of action used by MSC immunomodulation have been somewhat elucidated in vitro, the data from preclinical transplant models have been unclear. Furthermore, the optimal timing, dose, and route of administration remain to be elucidated. Importantly, MSCs have the ability to sense their environment, which may influence their function. In this article, we discuss the impact of the local microenvironment on MSCs and the mechanisms of MSC immunomodulation in the setting of SOT.
Early clinical trials suggest that mesenchymal stromal cells (MSCs) may be useful in solid organ transplantation. They can respond to microenvironmental cues, attenuate ischemia reperfusion injury, and prolong graft survival.
PMCID: PMC3633184  PMID: 23637312
12.  Variation in MHC expression between undifferentiated mouse ES cells and ES cell derived insulin-producing cell clusters 
Transplantation  2009;87(9):1300-1304.
The progeny of embryonic stem (ES) cells may eventually be used to replace damaged tissues in transplantation, yet their immunogenicity remains ill-defined. The major histocompatibility complex (MHC) is a determinant of immunogenicity in transplantation. Herein, we show differences in MHC expression between mouse ES cells and ES cell derived insulin producing cell clusters (IPCCs), including a relatively higher expression of MHC Class I in IPCCs and a faster, more dramatic induction of MHC Class I in IPCCs following challenge with interferon-γ (IFN-γ). MHC Class II was induced on IPCCs, but not ES cells, after exposure to IFN-γ. Transplantation of syngeneic or allogeneic IPCCs was insufficient to trigger up-regulation of MHC class I within three days after transplantation. These data highlight differences in MHC expression between ES cells and a fully differentiated ES cell derived tissue and suggest how the progeny of ES cells may be susceptible to rejection after transplantation.
PMCID: PMC2796795  PMID: 19424028
ES cells; immunogenicity; pancreatic
13.  Ischemia-Reperfusion Injury Accelerates Human Antibody-Mediated Transplant Vasculopathy 
Transplantation  2013;96(2):139-145.
The pathogenesis of transplant vasculopathy (TV) is a multifactorial process. We hypothesized that ischemia-reperfusion injury and antibody-mediated damage contribute to the development of TV.
Human vessels were procured from nine separate donors undergoing cardiac surgery and stored in saline solution on ice until transplantation. BALB/c Rag2-/-IL-2Rγ-/- mice were transplanted with a human vessel graft on day 0. Purified anti–human leukocyte antigen class I antibody (W6/32), isotype control antibody, or saline was injected into recipient mice weekly until day 42, at which point the degree of intimal expansion (IE) of vessels was assessed by histologic analysis.
We found that a prolonged cold ischemia time (6–12 hr) alone did not induce IE. In mice that received antibody where vessels were transplanted within 6 hr of procurement, no IE was observed. By contrast, in vessels exposed to more than 6 hr cold ischemia, both W6/32 antibody (30.4%±6.9%) and isotype control antibody (39.5%±6.0%) promoted significant IE (P<0.05 vs. saline [12.4%±1.7%]). Importantly, the isotype control antibody did not cross-react with human tissue. Interestingly, the number of mouse Fc-receptor–positive cells was significantly increased in human vessels exposed to more than 6 hr cold ischemia but only in the presence of antibody (P<0.05).
Antibody, regardless of its specificity, may promote IE in human vessels that are injured through cold ischemia via interaction with Fc-receptor–positive cells. This highlights the importance of controlling the degree of cold ischemia in clinical transplantation in an effort to reduce the risk of TV development.
Supplemental digital content is available in the article.
PMCID: PMC3723087  PMID: 23856999
Donor-specific antibody; Chronic allograft dysfunction; Humanized mouse model; Cold ischemia; Transplant arteriosclerosis
14.  Ex Vivo Expanded Human Regulatory T Cells Can Prolong Survival of a Human Islet Allograft in a Humanized Mouse Model 
Transplantation  2013;96(8):707-716.
Human regulatory T cells (Treg) offer an attractive adjunctive therapy to reduce current reliance on lifelong, nonspecific immunosuppression after transplantation. Here, we evaluated the ability of ex vivo expanded human Treg to prevent the rejection of islets of Langerhans in a humanized mouse model and examined the mechanisms involved.
We engrafted human pancreatic islets of Langerhans into the renal subcapsular space of immunodeficient BALB/c.rag2−/−.cγ−/− mice, previously rendered diabetic via injection of the β-cell toxin streptozocin. After the establishment of stable euglycemia, mice were reconstituted with allogeneic human peripheral blood mononuclear cells (PBMC) and the resultant alloreactive response studied. Ex vivo expanded CD25highCD4+ human Treg, which expressed FoxP3, CTLA-4, and CD62L and remained CD127low, were then cotransferred together with human PBMC and islet allografts and monitored for evidence of rejection.
Human islets transplanted into diabetic immunodeficient mice reversed diabetes but were rejected rapidly after the mice were reconstituted with allogeneic human PBMC. Cotransfer of purified, ex vivo expanded human Treg prolonged islet allograft survival resulting in the accumulation of Treg in the peripheral lymphoid tissue and suppression of proliferation and interferon-γ production by T cells. In vitro, Treg suppressed activation of signal transducers and activators of transcription and inhibited the effector differentiation of responder T cells.
Ex vivo expanded Treg retain regulatory activity in vivo, can protect a human islet allograft from rejection by suppressing signal transducers and activators of transcription activation and inhibiting T-cell differentiation, and have clinical potential as an adjunctive cellular therapy.
Supplemental digital content is available in the article.
PMCID: PMC3864182  PMID: 23917725
Regulatory T cells; Islet transplantation; Allograft; Humanized mouse model; Cellular therapy
15.  A diametric role for OX40 in the response of effector/memory CD4+ T cells and regulatory T cells to alloantigen 
OX40 is a member of the TNFR superfamily that has potent costimulatory properties. Although the impact of blockade of the OX40-OX40L pathway has been well documented in models of autoimmune disease, its effect on the rejection of allografts is less well defined.
Here we show that the alloantigen-mediated activation of naïve and memory CD4+ T cells results in the induction of OX40 expression and that blockade of OX40-OX40L interactions prevents skin allograft rejection mediated by either subset of T cells. Moreover, a blocking anti-OX40 was found to have no effect on the activation and proliferation of T cells, but rather effector T cells failed to accumulate in peripheral lymph nodes and subsequently migrate to skin allografts. This was found to be the result of an enhanced degree of cell death amongst proliferating effector cells. In clear contrast, blockade of OX40-OX40L interactions at the time of exposure to alloantigen enhanced the ability of regulatory T cells to suppress T cell responses to alloantigen by supporting rather than diminishing regulatory T cell survival.
These data show that OX40-OX40L signalling contributes to the evolution of the adaptive immune response to an allograft via the differential control of alloreactive effector and regulatory T cell survival. Moreover, these data serve to further highlight OX40 and OX40L as therapeutic targets to assist the induction of tolerance to allografts and self-antigens.
PMCID: PMC3721124  PMID: 23817421
mice; costimulation; CD4 T cells; Treg; OX40; transplantation
16.  Moving to tolerance: Clinical Application of T regulatory cells 
Seminars in immunology  2011;23(4):10.1016/j.smim.2011.04.001.
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.
PMCID: PMC3836227  PMID: 21620722
transplantation; T regulatory cells; cellular therapy; drug minimization; translational medicine
17.  Regenerative medicine as applied to solid organ transplantation: current status and future challenges 
In the last two decades, regenerative medicine has shown the potential for “bench-to-bedside” translational research in specific clinical settings. Progress made in cell and stem cell biology, material sciences and tissue engineering enabled researchers to develop cutting-edge technology which has lead to the creation of nonmodular tissue constructs such as skin, bladders, vessels and upper airways. In all cases, autologous cells were seeded on either artificial or natural supporting scaffolds. However, such constructs were implanted without the reconstruction of the vascular supply, and the nutrients and oxygen were supplied by diffusion from adjacent tissues. Engineering of modular organs (namely, organs organized in functioning units referred to as modules and requiring the reconstruction of the vascular supply) is more complex and challenging. Models of functioning hearts and livers have been engineered using “natural tissue” scaffolds and efforts are underway to produce kidneys, pancreata and small intestine. Creation of custom-made bioengineered organs, where the cellular component is exquisitely autologous and have an internal vascular network, will theoretically overcome the two major hurdles in transplantation, namely the shortage of organs and the toxicity deriving from lifelong immuno-suppression. This review describes recent advances in the engineering of several key tissues and organs.
PMCID: PMC3817209  PMID: 21062367
decellularization; extracellular matrix; regenerative medicine; scaffold; solid organ transplantation; stem cells; tissue engineering
18.  Costimulation blockade: Current perspectives and implications for therapy 
Transplantation  2013;95(4):527-535.
T cells must be activated before they can elicit damage to allografts, through interaction of their T cell receptor (TCR) with peptide-MHC complex, and through accessory molecules. Signalling through accessory molecules or costimulatory molecules is a critical way for the immune system to fine tune T cell activation. An emerging therapeutic strategy is to target selective molecules involved in the process of T cell activation using biological agents, which do not impact TCR signalling, thus only manipulating the T cells which recognise alloantigen. Costimulatory receptors and their ligands are attractive targets for this strategy and could be used both to prevent acute graft rejection as well as for maintenance immunosuppression. Therapeutic agents targeting costimulatory molecules, notably belatacept, have made the progression from the bench, through non-human primate studies and into the clinic. This Overview describes some of the most common costimulatory molecules, their role in T cell activation, and the development of reagents which target these pathways and their efficacy in transplantation.
PMCID: PMC3580065  PMID: 23129000
transplantation; biological therapeutics; immunosuppression; T cell activation; rejection; immunoregulation; CTLA-4
19.  A Shift towards Pro-Inflammatory CD16+ Monocyte Subsets with Preserved Cytokine Production Potential after Kidney Transplantation 
PLoS ONE  2013;8(7):e70152.
The presence of monocyte-macrophage lineage cells in rejecting kidney transplants is associated with worse graft outcome. At present, it is still unclear how the monocyte-macrophage related responses develop after transplantation. Here, we studied the dynamics, phenotypic and functional characteristics of circulating monocytes during the first 6 months after transplantation and aimed to establish the differences between kidney transplant recipients and healthy individuals.
Phenotype, activation status and cytokine production capacity of classical (CD14++CD16−), intermediate (CD14++CD16+) and non-classical (CD14+CD16++), monocytes were determined by flow cytometry in a cohort of 33 healthy individuals, 30 renal transplant recipients at transplantation, 19 recipients at 3 months and 16 recipients at 6 months after transplantation using a cross-sectional approach.
The percentage of both CD16+ monocyte subsets was significantly increased in transplant recipients compared to healthy individuals, indicative of triggered innate immunity (p≤0.039). Enhanced production capacity of tumor necrosis factor-α, interferon-γ and interleukin-1β was observed by monocytes at transplantation compared to healthy individuals. Remarkably, three months post-transplant, in presence of potent immunosuppressive drugs and despite improved kidney function, interferon-γ, tumor necrosis factor-α and interleukin-10 production capacity still remained significantly increased.
Our data demonstrate a skewed balance towards pro-inflammatory CD16+ monocytes that is present at the time of transplantation and retained for at least 6 months after transplantation. This shift could be one of the important drivers of early post-transplant cellular immunity.
PMCID: PMC3726371  PMID: 23922945
Annals of Surgery  2012;255(5):867-880.
The present review illustrates the state of the art of regenerative medicine (RM) as applied to surgical diseases and demonstrates that this field has the potential to address some of the unmet needs in surgery. RM is a multidisciplinary field whose purpose is to regenerate in vivo or ex vivo human cells, tissues or organs in order to restore or establish normal function through exploitation of the potential to regenerate, which is intrinsic to human cells, tissues and organs. RM uses cells and/or specially designed biomaterials to reach its goals and RM-based therapies are already in use in several clinical trials in most fields of surgery. The main challenges for investigators are threefold: Creation of an appropriate microenvironment ex vivo that is able to sustain cell physiology and function in order to generate the desired cells or body parts; identification and appropriate manipulation of cells that have the potential to generate parenchymal, stromal and vascular components on demand, both in vivo and ex vivo; and production of smart materials that are able to drive cell fate.
PMCID: PMC3327776  PMID: 22330032
regenerative medicine; tissue engineering; surgery; extracellular matrix; scaffold; stem cells
21.  Immuno-regulatory function of IL-27 and TGFβ1 in cardiac allograft transplantation. 
Transplantation  2012;94(3):226-233.
Deciphering the mechanisms of tolerance represents a crucial aim of research in transplantation. We previously identified by DNA chip, IL-27 p28 and TGFβ1, as over-expressed in a model of rat cardiac allograft tolerance mediated by regulatory CD4+CD25+ T cells. The role of these two molecules on the control of the inflammatory response remains controversial. However, both are involved in the regulation of the Th17/Treg axis suggesting their involvement in tolerance.
We analyzed regulation of IL-27 and TGFβ1 expression in allograft response and their role in tolerance by using blocking anti-TGFβ antibody and by generating an adeno-associated virus encoding IL-27.
Here, we confirmed the over-expression of IL-27 and TGFβ1 in tolerated cardiac allografts in two different rodent models. We observed that their expression correlates with inhibition of Th17 differentiation and with expansion of regulatory CD4+CD25+ T cells. We showed in rat that anti-TGFβ treatment abrogates infectious tolerance mediated by the transfer of regulatory CD4+CD25+ T cells. Moreover, over-expression of IL-27 by adeno-associated virus administration in combination with a short-term immuno-suppression allows prolongation of cardiac allograft survival and one tolerant recipient. We found that IL-27 over-expression did not induce Foxp3+CD4+CD25+ T cell expansion but rather IL10 expressing CD4+ T cells in the tolerant recipient.
Taken together, these data suggest that both TGFβ1 and IL-27 play a role in the mechanisms of tolerance. However, in contrast to TGFβ1, IL-27 seems not to be involved in regulatory CD4+CD25+ T cell expansion but rather in their mode of action.
PMCID: PMC3442234  PMID: 22790384
Tolerance; regulatory T cells; rodent
22.  Human iPSC-derived mesoangioblasts, like their tissue-derived counterparts, suppress T cell proliferation through IDO- and PGE-2-dependent pathways 
F1000Research  2013;2:24.
Human mesoangioblasts are currently in a phase I/II clinical trial for the treatment of patients with Duchenne muscular dystrophy. However, limitations associated with the finite life span of these cells combined with the significant numbers of mesoangioblasts required to treat all of the skeletal muscles in these patients restricts their therapeutic potential. Induced pluripotent stem cell (iPSC)-derived mesoangioblasts may provide the solution to this problem. Although, the idea of using iPSC-derived cell therapies has been proposed for quite some time, our understanding of how the immune system interacts with these cells is inadequate. Herein, we show that iPSC-derived mesoangioblasts (HIDEMs) from healthy donors and, importantly, limb-girdle muscular dystrophy 2D patients exert immunosuppressive effects on T cell proliferation.  Interferon gamma (IFN-γ) and tumour necrosis factor alpha (TNF-α) play crucial roles in the initial activation of HIDEMs and importantly indoleamine 2,3 dioxygenase (IDO) and prostaglandin E2 (PGE-2) were identified as key mechanisms involved in HIDEM suppression of T cell proliferation. Together with recent studies confirming the myogenic function and regenerative potential of these cells, we suggest that HIDEMs could provide an unlimited alternative source for mesoangioblast-based therapies.
PMCID: PMC3968899  PMID: 24715949
23.  Homing of Regulatory T Cells to Human Skin Is Important for the Prevention of Alloimmune-Mediated Pathology in an In Vivo Cellular Therapy Model 
PLoS ONE  2012;7(12):e53331.
Regulatory T cell (Treg) therapy for immune modulation is a promising therapeutic strategy for the treatment and prevention of autoimmune disease and graft-versus-host disease (GvHD) after bone marrow transplantation. However, Treg are heterogeneous and express a variety of chemokine receptor molecules. The optimal subpopulation of Treg for therapeutic use may vary according to the pathological target. Indeed, clinical trials of Treg for the prevention of GvHD where the skin is a major target of the anti-host response have employed Treg derived from a variety of different sources. We postulated that for the effective treatment of GvHD-related skin pathology, Treg must be able to migrate to skin in order to regulate local alloimmune responses efficiently. To test the hypothesis that different populations of Treg display distinct efficacy in vivo based on their expression of tissue-specific homing molecules, we evaluated the activity of human Treg derived from two disparate sources in a model of human skin transplantation. Treg were derived from adult blood or cord blood and expanded in vitro. While Treg from both sources displayed similar in vitro suppressive efficacy, they exhibited marked differences in the expression of skin homing molecules. Importantly, only adult-derived Treg were able to prevent alloimmune-mediated human skin destruction in vivo, by virtue of their improved migration to skin. The presence of Treg within the skin was sufficient to prevent its alloimmune-mediated destruction. Additionally, Treg expressing the skin homing cutaneous lymphocyte antigen (CLA) were more efficient at preventing skin destruction than their CLA-deficient counterparts. Our findings highlight the importance of the careful selection of an effective subpopulation of Treg for clinical use according to the pathology of interest.
PMCID: PMC3534052  PMID: 23300911
24.  Conversion to sirolimus in kidney transplant recipients with squamous cell cancer and changes in immune phenotype 
Conversion to sirolimus from calcineurin inhibitor- (CNI), azathioprine- (AZA) and mycophenolate-based regimens reduces the risk of development of squamous cell carcinoma of the skin (SCC) in kidney transplant recipients (KTRs). Sirolimus conversion may also be protective by permitting beneficial changes in immune phenotype. It is not known how sirolimus will affect immune phenotype in KTRs with SCC.
Thirty-two KTRs with SCC were enrolled into this single-blinded randomized study and 13 KTRs randomized to sirolimus (4–10 ng/mL) and prednisolone 5 mg/day.
Six-month post conversion to sirolimus FOXP3+ CD127lowCD25highCD69−, the number of T cells (putative Treg) increased significantly (P = 0.008). Natural killer (NK) and CD56bright NK cells also increased significantly (P = 0.039 and 0.02). T-cell number only significantly increased in those KTRs where CNI was ceased as part of the conversion to mammalian target of rapamycin inhibitors (mTORi's) (P = 0.031) implying CNI cessation rather than mTORi initiation induced an increase in T-cell number. Increases in the NK cell number was only significant in those KTRs where AZA was ceased (P = 0.040), implying AZA cessation rather than mTORi initiation caused the NK cell number to increase. At 6 months, sirolimus conversion reduces new SCC/year, rate ratio 0.49 (95%CI: 0.15–1.63), P = 0.276. On therapy analysis and intention-to-treat analysis over 24 months, the rate ratios were 0.84 and 0.87, respectively, and did not reach significance.
Conversion to mTORi from CNI may reveal a pre-existing high Treg phenotype by unmasking CNI inhibition of FOXP3 expression. Cessation of AZA leads to increased NK cell number. High FOXP3+ T-cell number on conversion to mTORi may predict those KTRs who continue to accrue SCC.
PMCID: PMC3572772  PMID: 23223314
immune phenotype; mTOR inhibitors; skin cancer; Treg
25.  IL-33 expands suppressive CD11b+ Gr-1int and regulatory T cells (Treg), including ST2L+ Foxp3+ cells, and mediates Treg-dependent promotion of cardiac allograft survival 
IL-33 administration is associated with facilitation of Th type-2 (Th2) responses and cardioprotective properties in rodent models. However, in heart transplantation, the mechanism by which IL-33, signaling through ST2L, the membrane-bound form of ST2, promotes transplant survival is unclear. We report that IL-33 administration, while facilitating Th2 responses, also increases immunoregulatory myeloid cells and CD4+ Foxp3+ regulatory T cells (Treg) in mice. IL-33 expands functional myeloid-derived suppressor cells (MDSC), -CD11b+ cells that exhibit intermediate (int) levels of Gr-1 and potent T cell suppressive function. Furthermore, IL-33 administration causes a St2-dependent expansion of suppressive CD4+ Foxp3+ Treg, including a ST2L+ population. IL-33 monotherapy following fully allogeneic mouse heart transplantation resulted in significant graft prolongation, associated with increased Th2-type responses and decreased systemic CD8+ IFN-γ+ cells. Also, despite reducing overall CD3+ cell infiltration of the graft, IL-33 administration markedly increased intragraft Foxp3+ cells. Whereas control graft recipients displayed increases in systemic CD11b+ Gr-1hi cells, IL-33-treated recipients exhibited increased CD11b+ Gr-1int cells. Enhanced ST2 expression was observed in the myocardium and endothelium of rejecting allografts, however the therapeutic effect of IL-33 required recipient St2 expression and was dependent on Treg. These findings reveal a new immunoregulatory property of IL-33. Specifically, in addition to supporting Th2 responses, IL-33 facilitates regulatory cells, particularly functional CD4+ Foxp3+ Treg that underlie IL-33-mediated cardiac allograft survival.
PMCID: PMC3197898  PMID: 21949025
Cytokines; Dendritic cells; Monocytes/Macrophages; Transplantation; Tolerance/Suppression/Anergy; T cells

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