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1.  Th17 cells in alemtuzumab-treated patients. The effect of long-term maintenance immunosuppressive therapy. 1,2,3 
Transplantation  2011;91(7):744-750.
Background
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1097/TP.0b013e31820c85dc
PMCID: PMC3759143  PMID: 21412187
CAMPATH-1H; IL-17; T regulatory cells; kidney transplant; Sirolimus
2.  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.
doi:10.4049/jimmunol.1300553
PMCID: PMC3721124  PMID: 23817421
mice; costimulation; CD4 T cells; Treg; OX40; transplantation
3.  BIOMARKERS OF OPERATIONAL TOLERANCE IN SOLID ORGAN TRANSPLANTATION 
Introduction
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.
doi:10.1517/17530059.2012.680019
PMCID: PMC3442251  PMID: 22988481
Tolerance fingerprint; kidney transplantation; liver transplantation; immunosuppressive drug withdrawal; acute rejection
4.  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.
doi:10.1016/j.smim.2011.04.001
PMCID: PMC3836227  PMID: 21620722
transplantation; T regulatory cells; cellular therapy; drug minimization; translational medicine
5.  Regenerative medicine as applied to solid organ transplantation: current status and future challenges 
Summary
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.
doi:10.1111/j.1432-2277.2010.01182.x
PMCID: PMC3817209  PMID: 21062367
decellularization; extracellular matrix; regenerative medicine; scaffold; solid organ transplantation; stem cells; tissue engineering
6.  CD4+ regulatory T cells in solid organ transplantation 
Current opinion in organ transplantation  2010;10.1097/MOT.0b013e32834017ae.
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.
Summary:
Data in favour of Treg cellular therapy in transplantation are mounting, and we predict that their use in clinical trials is on the horizon.
doi:10.1097/MOT.0b013e32834017ae
PMCID: PMC3198838  PMID: 20881492
Treg; tolerance; rejection; transplantation
7.  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.
doi:10.3389/fimmu.2012.00254
PMCID: PMC3422982  PMID: 22934094
tolerance; immune regulation; cellular therapy; chimerism; regulatory T cell; clinical trials; transplantation
8.  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.
doi:10.1097/TP.0b013e31826d4672
PMCID: PMC3580065  PMID: 23129000
transplantation; biological therapeutics; immunosuppression; T cell activation; rejection; immunoregulation; CTLA-4
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.
Summary
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.
doi:10.1097/MOT.0b013e32833b9bfb
PMCID: PMC3095085  PMID: 20616728
IL-17; Tregs; rejection; plasticity
10.  REGENERATIVE MEDICINE AS APPLIED TO GENERAL SURGERY 
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.
doi:10.1097/SLA.0b013e318243a4db
PMCID: PMC3327776  PMID: 22330032
regenerative medicine; tissue engineering; surgery; extracellular matrix; scaffold; stem cells
11.  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.
doi:10.1097/TP.0b013e3181a19421
PMCID: PMC2796795  PMID: 19424028
ES cells; immunogenicity; pancreatic
12.  Immuno-regulatory function of IL-27 and TGFβ1 in cardiac allograft transplantation. 
Transplantation  2012;94(3):226-233.
Background
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1097/TP.0b013e31825b0c38
PMCID: PMC3442234  PMID: 22790384
Tolerance; regulatory T cells; rodent
13.  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.
doi:10.12688/f1000research.2-24.v1
PMCID: PMC3968899
14.  B CELL REPOPULATION AFTER ALEMTUZUMAB INDUCTION – TRANSIENT INCREASE IN TRANSITIONAL B CELLS AND LONG TERM DOMINANCE OF NAÏVE B CELLS 
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.
doi:10.1111/j.1600-6143.2012.04012.x
PMCID: PMC3387484  PMID: 22420490
Campath-1H; depletion; renal transplant; regulatory B cells; conversion
15.  Conversion to sirolimus in kidney transplant recipients with squamous cell cancer and changes in immune phenotype 
Background
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1093/ndt/gfs474
PMCID: PMC3572772  PMID: 23223314
immune phenotype; mTOR inhibitors; skin cancer; Treg
16.  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.
doi:10.4049/jimmunol.1100519
PMCID: PMC3197898  PMID: 21949025
Cytokines; Dendritic cells; Monocytes/Macrophages; Transplantation; Tolerance/Suppression/Anergy; T cells
17.  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.
doi:10.1089/scd.2012.0386
PMCID: PMC3549627  PMID: 22913669
18.  Inflammatory Ly-6Chi monocytes play an important role in the development of severe transplant arteriosclerosis in hyperlipidemic recipients 
Atherosclerosis  2012;223(2):291-298.
Objective
Transplant arteriosclerosis (TA) restricts long-term survival of heart transplant recipients. Although the role of monocyte/macrophages is well established in native atherosclerosis, it has been studied to a much lesser extent in TA. Plasma cholesterol is the most important non-immunologic risk factor for development of TA but the underlying mechanisms are largely unknown. We hypothesized that monocyte/macrophages might play an important role in the pathogenesis of TA under hyperlipidemic conditions.
Methods
We studied TA in fully mismatched arterial allografts transplanted into hyperlipidemic ApoE−/− recipients compared to wild-type controls. The recruitment of distinct monocyte populations into the grafts was tracked by in vivo labelling with fluorescent microspheres. We used antibody-mediated depletion protocols to dissect the relative contribution of T lymphocytes and monocytes to disease development.
Results
In the hyperlipidemic environment the progression of TA was highly exacerbated and the inflammatory CD11b+CD115+Ly-6Chi monocytes were preferentially recruited into the neointima. The number of macrophage-derived foam cells present in the grafts strongly correlated with plasma cholesterol and disease severity. Depletion of Ly-6Chi monocytes and neutrophils significantly inhibited macrophage accumulation and disease progression. The accelerated monocyte recruitment occurs through a T cell-independent mechanism, as T cell depletion did not influence macrophage accumulation into the grafts.
Conclusions
Our study identifies for the first time the involvement of inflammatory Ly-6Chi monocytes into the pathogenesis of TA, particularly in conditions of hyperlipidemia. Targeted therapies modulating the recruitment and activation of these cells could potentially delay coronary allograft vasculopathy and improve long-term survival of heart transplant recipients.
Highlights
► Inflammatory Ly-6Chi monocytes infiltrate arterial allografts during TA development. ► Hyperlipidemia potently accelerates Ly-6Chi monocyte recruitment into the neointima. ► TA severity correlates well with plasma cholesterol and macrophage accumulation. ► Macrophage-derived foam cell accumulation occurs independently of T lymphocytes. ► Ly-6Chi monocyte and neutrophil depletion inhibits TA development.
doi:10.1016/j.atherosclerosis.2012.05.010
PMCID: PMC3423631  PMID: 22704806
Transplant vasculopathy; Hypercholesterolemia; Inflammation; Monocytes; Macrophages
19.  Ischemia-Reperfusion Injury Accelerates Human Antibody-Mediated Transplant Vasculopathy 
Transplantation  2013;96(2):139-145.
Background
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.
Methods
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.
Results
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).
Conclusions
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.
doi:10.1097/TP.0b013e318295ee32
PMCID: PMC3723087  PMID: 23856999
Donor-specific antibody; Chronic allograft dysfunction; Humanized mouse model; Cold ischemia; Transplant arteriosclerosis
20.  Functional regulatory T cells produced by inhibiting cyclic nucleotide phosphodiesterase type 3 prevent allograft rejection 
Science translational medicine  2011;3(83):83ra40.
Regulatory T cells (Tregs) manipulated ex vivo have potential as cellular therapeutics in autoimmunity and transplantation. Although it is possible to expand naturally occurring Tregs, 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 under conditions that lead to the generation or expansion of donor-reactive, adaptive Tregs. Here we demonstrate that stimulation of mouse CD4+ T cells by immature allogeneic dendritic cells (DCs) combined with pharmacological inhibition of phosphodiesterase 3 (PDEi) results 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+ TCR transgenic T cells and inhibited both effector cell proliferation and T cell priming for IFN-γ production. Notably, PDE inhibition also enhanced the enrichment of human Foxp3+ CD4+ T cells driven by allogeneic APC. These cells inhibited T cell proliferation in a standard in vitro mixed lymphocyte assay and importantly, attenuated the development of vasculopathy mediated by autologous PBMC 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 Tregs that uses a clinically approved agent, making pharmacological PDE inhibition a potential strategy for Treg-based therapies
doi:10.1126/scitranslmed.3002099
PMCID: PMC3321352  PMID: 21593400
21.  In vivo Prevention of Transplant Arteriosclerosis by ex vivo Expanded Human Regulatory T Cells 
Nature medicine  2010;16(7):809-813.
Transplant arteriosclerosis (TA) is the hallmark of chronic allograft dysfunction (CAD) affecting transplanted organs in the long term [1,2]. These fibroproliferative lesions lead to neointimal thickening of arteries in all transplanted allografts [2]. Luminal narrowing then leads to graft ischemia and organ demise. To date, there are no known tolerance induction strategies that prevent TA [3,4]. Therefore, this study was designed to test the hypothesis that human regulatory T cells (Treg cells) expanded ex vivo could prevent TA. Here we show the comparative capacity of Treg cells, sorted via two separate strategies, to prevent TA in a clinically relevant chimeric humanized mouse system. We found that the in vivo development of TA in human arteries was prevented with the treatment of ex vivo expanded human Treg cells. Additionally, we show that Treg cells sorted based on the low expression of CD127 (IL-7Rα) provide a more potent therapy to conventional Treg cells. Our results demonstrate, for the first time, that human Treg cells can inhibit TA by impairing effector function and graft infiltration. We anticipate our findings to serve as a foundation for the clinical development of therapeutics targeting TA in both allograft transplantation and other immune-mediated causes of vasculopathy [5].
doi:10.1038/nm.2154
PMCID: PMC2929438  PMID: 20473306
22.  T regulatory cells and the control of alloimmunity: from characterisation to clinical application 
Current Opinion in Immunology  2010;22(5):662-668.
T regulatory cells (Treg) play an important role in the induction and maintenance of immunological tolerance. Recent findings in experimental transplant models combined with the development of functional reporter mice have opened new avenues to study Treg biology and their therapeutic potential. In particular, recent advances in understanding Treg function and lineage stability revealed unexpected plasticity of this lineage. Nevertheless, pre-clinical and pilot clinical trials using Treg cells as cellular therapies have been initiated suggesting the safety and feasibility of such treatment.
doi:10.1016/j.coi.2010.08.011
PMCID: PMC3025322  PMID: 20869224
23.  In Vivo Quantification of Vcam-1 Expression in Renal Ischemia Reperfusion Injury Using Non-Invasive Magnetic Resonance Molecular Imaging 
PLoS ONE  2010;5(9):e12800.
Rationale and Objective
Vascular cell adhesion molecule-1 (VCAM-1) is upregulated in ischemia reperfusion injury (IRI), persisting after restoration of blood flow. We hypothesized that microparticles of iron oxide targeting VCAM-1 (VCAM-MPIO) would depict “ischemic memory” and enable in vivo assessment of VCAM-1 expression.
Methodology and Findings
Mice subject to unilateral, transient (30 minutes) renal ischemia and subsequent reperfusion received intravenous VCAM-MPIO (4.5 mg iron/kg body weight). Contrast agent bound rapidly (<30 minutes) in IRI-kidneys and appeared as intensely low signal areas by MRI in vivo. Automated segmentation and quantification yielded MPIO contrast volumes of 5991±354×106 µm3 in IRI vs. 87±7×106 µm3 in kidneys with no surgical intervention (P<0.001); 90±8×106 µm3 in IRI kidneys exposed to control (IgG-MPIO) and 625±80×106 µm3, in IRI kidneys pre-treated with a blocking dose of VCAM-1 antibody (P<0.001). In keeping with quantitative MRI data, VCAM-1 mRNA expression in IRI was 65-fold higher than in kidneys without surgical intervention (3.06±0.63 vs. 0.05±0.02, P<0.001). Indeed VCAM-1 mRNA expression and VCAM-MPIO contrast volume were highly correlated (R2 = 0.901, P<0.01), indicating that quantification of contrast volume reflected renal VCAM-1 transcription. Serial imaging showed VCAM-MPIO accumulation at target within 30 minutes, persisting for ≥90 minutes, while unbound VCAM-MPIO was cleared rapidly from blood, with sequestration by mac-3 positive Kupffer cells in the liver and monocyte/macrophages in the spleen.
Conclusions
(1) VCAM-MPIO detected VCAM-1 expression and defined its 3-dimensional distribution, revealing “ischemic memory” in renal IRI; (2) automated volumetric quantification of VCAM-MPIO accurately reflected tissue levels of VCAM-1 mRNA; and (3) VCAM-MPIO bound rapidly to target with active sequestration of unbound MPIO in the liver and spleen.
doi:10.1371/journal.pone.0012800
PMCID: PMC2943468  PMID: 20877722
24.  Mesenchymal Stem Cells Prevent the Rejection of Fully Allogenic Islet Grafts by the Immunosuppressive Activity of Matrix Metalloproteinase-2 and -9 
Diabetes  2009;58(8):1797-1806.
OBJECTIVE
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.
RESEARCH DESIGN AND METHODS
We investigated the molecular mechanisms underlying the immunosuppressive effects of MSCs in vitro and in vivo.
RESULTS
Our results demonstrate that matrix metalloproteinases (MMPs) secreted by MSCs, in particular MMP-2 and MMP-9, play an important role in the suppressive activity of MSCs by reducing surface expression of CD25 on responding T-cells. Blocking the activity of MMP-2 and MMP-9 in vitro completely abolished the suppression of T-cell proliferation by MSCs and restored T-cell expression of CD25 as well as responsiveness to interleukin-2. In vivo, administration of MSCs significantly reduced delayed-type hypersensitivity responses to allogeneic antigen and profoundly prolonged the survival of fully allogeneic islet grafts in transplant recipients. Significantly, these MSC-mediated protective effects were completely reversed by in vivo inhibition of MMP-2 and MMP-9.
CONCLUSIONS
We demonstrate that MSCs can prevent islet allograft rejection leading to stable, long-term normoglycemia. In addition, we provide a novel insight into the mechanism underlying the suppressive effects of MSCs on T-cell responses to alloantigen.
doi:10.2337/db09-0317
PMCID: PMC2712800  PMID: 19509016
25.  Characteristics of the Early Immune Response Following Transplantation of Mouse ES Cell Derived Insulin-Producing Cell Clusters 
PLoS ONE  2010;5(6):e10965.
Background
The fully differentiated progeny of ES cells (ESC) may eventually be used for cell replacement therapy (CRT). However, elements of the innate immune system may contribute to damage or destruction of these tissues when transplanted.
Methodology/Principal Findings
Herein, we assessed the hitherto ill-defined contribution of the early innate immune response in CRT after transplantation of either ESC derived insulin producing cell clusters (IPCCs) or adult pancreatic islets. Ingress of neutrophil or macrophage cells was noted immediately at the site of IPCC transplantation, but this infiltration was attenuated by day three. Gene profiling identified specific inflammatory cytokines and chemokines that were either absent or sharply reduced by three days after IPCC transplantation. Thus, IPCC transplantation provoked less of an early immune response than pancreatic islet transplantation.
Conclusions/Significance
Our study offers insights into the characteristics of the immune response of an ESC derived tissue in the incipient stages following transplantation and suggests potential strategies to inhibit cell damage to ensure their long-term perpetuation and functionality in CRT.
doi:10.1371/journal.pone.0010965
PMCID: PMC2881030  PMID: 20532031

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