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Active regulation or suppression of donor reactive cells is emerging as a key mechanism for inducing and maintaining unresponsiveness to donor alloantigens. Accumulating evidence suggests that a balance between immunoregulation and deletion of donor alloantigen reactive T cells can provide effective control of immune responsiveness after organ or cell transplantation. In many settings, immunoregulatory activity is enriched in CD4+ T cells that express high levels of CD25, and common mechanisms appear to be responsible for the activity of regulatory T cells in both transplantation and the control of reactivity to self-antigens.

Regulatory T cells (Treg) are capable of suppressing the proliferation and function of various effector cell populations that contribute to innate and adaptive immune responses (1, 2). The application of Treg as a cellular therapy to treat autoimmune diseases, transplant rejection, and graft-versus-host disease (GVHD) has great potential (3, 4). Adoptive cellular therapy would permit infusion of Treg at a known dose and an appropriate time point, after assessment for purity and functional activity (5). Such a cellular therapy may integrate with existing biological control mechanisms in vivo and therefore be associated with fewer side effects than current immunosuppressive therapies (6). The most appropriate source of Treg for different clinical applications is debated, and both adult peripheral blood (APB) and umbilical cord blood (UCB) have been suggested as sources for Treg for clinical application. There are significant differences in the phenotype of T cells derived from APB compared with UCB (7–10). In UCB, there is a clear delineation of CD25hi Treg from predominantly nonactivated CD25− effector T cells (Teffs), allowing the isolation of Treg with a high purity purely on the basis of CD25hi expression (11, 12). In contrast, APB contains a large proportion of activated CD25+ Teffs, therefore requiring depletion of CD127+ cells (13). More importantly, Treg derived from UCB exhibit a predominantly naive phenotype, with only a small memory population (10, 14). This naive phenotype is associated with a significantly enhanced proliferation potential (15). Proliferating naive Treg developing under the influence of a specific immunological environment may develop into memory Treg with the appropriate tissue-specific homing and functional molecules (16). The activation status of Treg may have a significant impact on their suppressive capacity. Freshly isolated UCB CD4+CD25+ cells have been reported to be incapable of suppressing the proliferation or activation of autologous responder cells in vitro in some systems (8, 17–19), while in vitro-expanded UCB Treg demonstrate suppressive functions in vitro (19–21) and in GVHD models in vivo (21, 22). Nevertheless, in other systems where polyclonally stimulated adult responders are used, or where responders are stimulated with adult-derived antigen-presenting cells (APC), freshly isolated UCB Treg have demonstrated effective in vitro suppression (11, 23–25). The performance of UCB Treg in clinical studies reported to date is promising. Early data from a phase I dose-escalation trial for the infusion of UCB-derived Treg in patients receiving double UCB transplants for the treatment of hematological malignancies demonstrated a reduced incidence of grade II to IV acute GVHD in patients treated with Treg (26). While the frequency of CD25+ cells among the CD4+ fraction is significantly higher in UCB than in APB (7, 10, 27), the absolute number of Treg that can be obtained from a single unit of UCB is substantially lower than the average yield from a standard APB unit. UCB Treg are therefore expanded ex vivo in order to achieve the large numbers required for in vivo use (26). Importantly, however, expanded UCB Treg do not maintain their largely naive phenotype and therefore may behave less efficiently in vivo where differentiation into a specific Treg subtype is beneficial. We therefore investigated an alternative approach whereby multiple allogeneic units of UCB are pooled in order to achieve therapeutic Treg doses. We show that although the in vitro suppressive ability of multiple-unit pooled human UCB-derived Treg was at least as effective as APB-derived Treg, the in vivo efficacy was significantly better on a per-cell basis. Differential immunogenicity, and subsequent survival of Treg after adoptive transfer, contributes to this difference in functional potency of UCB versus APB-derived Treg in vivo.

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.

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.

BackgroundThe 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.MethodsPhenotype, 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.ResultsThe 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.ConclusionOur 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.

Rejection is the major barrier to successful transplantation and usually results from the integration of multiple mechanisms. Activation of elements of the innate immune system, triggered as a consequence of tissue injury sustained during cell isolation or organ retrieval as well as ischemia-reperfusion, will initiate and amplify the adaptive response. For cell mediated rejection, T cells require multiple signals for activation, the minimum being two signals; antigen recognition and costimulation. The majority of B cells require help from T cells to initiate alloantibody production. Antibodies reactive to donor HLA molecules, minor histocompatibility antigens, endothelial cells, red blood cells, or autoantigens can trigger or contribute to rejection early as well as late after transplantation. © Springer Science+Business Media, LLC 2013.

There is considerable current interest in the possibility that long-term graft acceptance in clinical solid-organ transplantation might be dependent upon the development of a microchimeric state between the donor and recipient. This possibility has been prompted by the observation that in some transplant patients cells of donor origin can be detected in peripheral sites such as the skin, and it has been proposed that these cells play an essential role in maintaining graft survival. The hypothesis that peripheral microchimerism is an absolute requirement for the long-term survival of solid-organ allografts was tested in a well-characterized model of transplantation tolerance in which adult recipient mice are pretreated 28 days before transplant with a single donor-specific transfusion under the cover of a depleting anti-CD4 antibody. Mice pretreated with this protocol accept donor-specific cardiac allografts (MST > 100 days) but reject those of a third party (MST 16.5 days). The protocol leads to operational tolerance in the long term in that donor-specific skin grafts show prolonged survival while those from a third party strain are rejected acutely. Since peripheral blood contains haematopoietic stem cells we speculated that the success of the anti-CD4/DST protocol might be dependent on the development of microchimerism. To address this possibility the DST was irradiated before administration under anti-CD4 antibody cover in order to prevent donor stem cells in the transfusion from establishing a microchimeric state in the recipient animals. Mice in this group rejected their grafts acutely (MST 12 days), suggesting indeed that stem cells might be very important in the success of this model. However, when the protocol was modified by giving three additional doses of irradiated whole blood to increase the possibility that recipient T cells would be engaged during antibody-induced immunocompromise, graft prolongation was restored (MST > 100 days). These results demonstrate that persistence of donor antigen at the time of anti-CD4 antibody treatment is critical for the induction of unresponsiveness in this model and show that microchimerism is not an absolute requirement for long-term graft survival.

New biomarkers are required to detect acute rejection (AR) in kidney transplant recipients (KTRs) to avoid invasive kidney biopsies. We assess whether a 5-gene panel (DUSP1; NKTR; PBEF1; MAPK9; and PSEN1) in whole blood samples that has previously been shown to identify AR in a paedriatric KTR population is able to distinguish AR in a UK population of adult Caucasian KTR patients.

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.

Transplantation is the only available curative treatment for end-stage organ failure. In recent years, development of efficient immunosuppressive regimens has greatly improved short-term acceptance...

Background: Deciphering the mechanisms of tolerance represents a crucial aim of research in transplantation. We previously identified by DNA chip interleukin (IL)-27 p28 and transforming growth factor (TGF)-β1 as overexpressed 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 T helper 17/Treg axis, suggesting their involvement in tolerance. Methods: We analyzed regulation of IL-27 and TGF-β1 expression in allograft response and their role in tolerance by using blocking anti-TGF-β antibody and by generating an adeno-associated virus encoding IL-27. Results: Here, we confirmed the overexpression of IL-27 and TGF-β1 in tolerated cardiac allografts in two different rodent models. We observed that their expression correlates with inhibition of T helper 17 differentiation and with expansion of regulatory CD4 +CD25 + T cells. We showed in a rat model that anti-TGF-β treatment abrogates infectious tolerance mediated by the transfer of regulatory CD4 +CD25 + T cells. Moreover, overexpression of IL-27 by adeno-associated virus administration in combination with a short-term immunosuppression allows prolongation of cardiac allograft survival and one tolerant recipient. We found that IL-27 overexpression did not induce Foxp3CD4 +CD25 + T-cell expansion but rather IL-10-expressing CD4 + T cells in the tolerant recipient. Conclusions: Taken together, these data suggest that both TGF-β1 and IL-27 play a role in the mechanisms of tolerance. However, in contrast to TGF-β1, IL-27 seems not to be involved in regulatory CD4CD25 T-cell expansion but rather in their mode of action. © 2012 Lippincott Williams and Wilkins.

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 cellswas 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 pretransplant samples, whereas the ability to produce IgG was inhibited long-term. In conclusion, induction therapy with alemtuzumab results in a long-term shift toward naïve B cells with altered phenotypic and functional characteristics. © Copyright 2012 The American Society of Transplantation and the American Society of Transplant Surgeons.

Establishing immune tolerance in transplant recipients is essential for promoting the long-term survival of an allograft and for preventing the development of harmful graft-versus-host responses. This Review considers the clinical potential of manipulating different immunosuppressive cell populations, including regulatory T cells, B cells and macrophages, in the setting of transplantation. Immune regulation is fundamental to any immune response to ensure that it is appropriate for the perceived threat to the host. Following cell and organ transplantation, it is essential to control both the innate immune response triggered by the injured tissue and the adaptive immune response stimulated by mismatched donor and recipient histocompatibility antigens to enable the transplant to survive and function. Here, we discuss the leukocyte populations that can promote immune tolerance after cell or solid-organ transplantation. Such populations include regulatory T cells, B cells and macrophages, as well as myeloid-derived suppressor cells, dendritic cells and mesenchymal stromal cells. We consider the potential of these regulatory immune cells to develop and function in transplant recipients and their potential use as cellular therapies to promote long-term graft function.

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 centres on the facilitation of donor-cell mixed chimerism in the transplant recipient with the use of bone marrow or haematopoietic stem cell transplantation. The second strategy, utilising 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.

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 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. © 2012 by Lippincott Williams and Wilkins.

Previous work from this laboratory has shown that donor-specific tolerance can be achieved in a mouse heart model if recipients are pretreated with a donor-specific blood transfusion (DST) in combination with a depleting anti-CD4 antibody. The advantage of this approach instead of simply using the antibody alone at the time of transplantation is that the nonspecific immunosuppressive effects of the antibody have largely decayed by the time of transplant such that donor-specific, rather than total, unresponsiveness results. However, this approach would not be applicable to clinical cadaveric transplantation since donor-specific transfusion at a specified time before transplant would not be possible. In an attempt to address these problems we have sought to determine (A) whether the state of unresponsiveness established by the anti-CD4/DST protocol could be maintained by repeated exposure only to the tolerizing antigen; (B) whether unrelated or random transfusion (RT) could substitute for DST in the anti-CD4/antigen pretreatment protocol, and (C) whether these two approaches could be successfully combined to provide an "umbrella unresponsiveness" that could be maintained until the time of transplant. Our data show, first, that antigen rechallenge without further antibody treatment can maintain a state of unresponsiveness to alloantigen; second, that random blood transfusion given under the cover of anti-CD4 monoclonal antibody leads to indefinite allograft survival and true tolerance in the long-term; and third, that once established by random transfusion under anti-CD4 cover, unresponsiveness can be maintained for an extended period by random transfusion alone. These results suggest that random blood transfusion combined with anti-CD4 monoclonal antibody therapy might be considered as a possible approach to the induction of specific unresponsiveness in clinical transplantation.

Rejection is the major barrier to successful transplantation. The immune response to an allograft is an ongoing dialogue between the innate and adaptive immune system that if left unchecked will lead to the rejection of transplanted cells, tissues, or organs. Activation of elements of the innate immune system, triggered as a consequence of tissue injury sustained during cell isolation or organ retrieval and ischemia reperfusion, will initiate and amplify the adaptive response. T cells require a minimum of two signals for activation, antigen recognition, and costimulation. The activation requirements of naive T cells are more stringent than those of memory T cells. Memory T cells are present in the majority of transplant recipients as a result of heterologous immunity. The majority of B cells require help from T cells to initiate antibody production. Antibodies reactive to donor human leukocyte antigen molecules, minor histocompatibility antigens, endothelial cells, RBCs, or autoantigens can trigger or contribute to rejection early and late after transplantation. Antibody-mediated rejection triggered by alloantibody binding and complement activation is recognized increasingly as a significant contribution to graft loss. Even though one component of the immune system may dominate and lead to rejection being described in short hand as T cell or antibody mediated, it is usually multifactorial resulting from the integration of multiple mechanisms. Identifying the molecular pathways that trigger tissue injury, signal transduction and rejection facilitates the identification of targets for the development of immunosuppressive drugs.

In recent years, substantial advances in T-cell immunosuppressive strategies and their translation to routine clinical practice have revolutionized management and outcomes in autoimmune disease and solid organ transplantation. More than 80 diseases have been considered to have an autoimmune etiology, such that autoimmune-associated morbidity and mortality rank as third highest in developed countries, after cardiovascular diseases and cancer. Solid organ transplantation has become the therapy of choice for many end-stage organ diseases. Short-term outcomes such as patient and allograft survival at 1 year, acute rejection rates, as well as time course of disease progression and symptom control have steadily improved. However, despite the use of newer immunosuppressive drug combinations, improvements in long-term allograft survival and complete resolution of autoimmunity remain elusive. In addition, the chronic use of nonspecifically targeted immunosuppressive drugs is associated with significant adverse effects and increased morbidity and mortality. In this article, we discuss the current clinical tools for immune suppression and attempts to induce long-term T-cell tolerance induction as well as much-needed future approaches to produce more short-acting, antigen-specific agents, which may optimize outcomes in the clinic.

This overview traces the history of regenerative medicine pertinent to organ transplantation, illustrates potential clinical applications reported to date, and highlights progress achieved in the field of complex modular organ engineering. Regenerative medicine can now produce relatively simple tissues such as skin, bladders, vessels, urethras, and upper airways, whereas engineering or generation of complex modular organs remains a major challenge. Ex vivo organ engineering may benefit from complementary investigations in the fields of developmental biology and stem cells and transplantation before its full potential can be realized.

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.

Exposure to alloantigen in vivo or in vitro induces alloantigen reactive regulatory T cells that can control transplant rejection. The mechanisms that underpin the activity of alloantigen reactive regulatory T cells in vivo are common with those of regulatory T cells that prevent autoimmunity. The identification and characterization of regulatory T cells that control rejection and contribute to the induction of immunologic unresponsiveness to alloantigens in vivo has opened up exciting opportunities for new therapies in transplantation. Findings from laboratory studies are informing the design of clinical protocols using regulatory T cells as a cellular therapy.

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. Although 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 polymerase chain reaction more than 4 years after transplantation in 10 kidney recipients treated with alemtuzumab induction. Seven patients were converted to sirolimus monotherapy at 12 months posttransplant, whereas the remaining three patients with history of graft rejection were treated with sirolimus and mycophenolate mofetil. In addition, we sorted and expanded interleukin (IL)-17A-producing CCR6CD4 T cells and assessed their susceptibility to suppression by regulatory T (Treg) cells in in vitro suppression tests. RESULTS: Three years of mammalian target of rapamycin inhibitor monotherapy correlates with an increase in the number of IL-17A producing cells, compared with patients treated with sirolimus and mycophenolate mofetil. In these patients, IL-17A expression was compensated for by an increase in Treg cell frequency and number. In addition, 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. However, more importantly, we have shown that Treg cells can effectively regulate Th17cells both in vitro and in vivo.

PURPOSE OF REVIEW: This review focuses on the immunogenicity of embryonic stem cell (ESC)-derived progenitors and the impact of the immune response on applications of cell replacement therapy (CRT). Possible strategies to induce immunological tolerance to ESC-derived progenitor cells will also be discussed. RECENT FINDINGS: Evidence for the differential epigenetic control of major histocompatibility (MHC) and antigen processing molecules in ESCs and differentiated ESCs has recently been described. The presence of T cells recognizing the pluripotency-associated transcription factor octamer-binding transcription factor 4 (OCT4) in healthy patient-derived peripheral blood mononuclear cells adds further complexity to the immune response against ESCs and ESC-derived progenitors. SUMMARY: Although ESCs and ESC-derived progenitors appear to exert some level of immune privilege in specific circumstances, these allogeneic cells are indeed recognized by the immune system and can be subject to mechanisms of rejection. Herein, we discuss the importance of the recent reports describing an immunosuppressive capacity of ESCs, and the epigenetic control of MHC in ESCs and how these characteristics may be harnessed in the development of strategies to induce immunological tolerance.

A model was established whereby CS7BL/6 (B6) blood injected i.v. into C3H mice 7 days prior to i.v. injection of syngeneic UV-2237 tumour cells significantly increased the number of pulmonary metastases counted 21 days later as compared with levels observed in mice treated with saline, C3H or NZW blood or SRBC. This regimen of B6 allogeneic blood transfusion of C3H mice also significantly depressed splenic and pulmonary NK activity as assayed by lysis of 51Cr YAC-1 in vitro and by clearance of 111In YAC-1 in vivo respectively. Anti-asialo GMI treatment, which depletes NK activity in vivo, and Poly I:C treatment, which enhances NK activity in vivo, were associated with significantly increased and decreased pulmonary metastasis of UV-2237, respectively, in C3H mice. Depletion of CD4+ and CD8+ T cells had no effect. Cyclophosphamide pretreatment which, among other effects, depletes NK cells, significantly increased pulmonary metastasis of UV-2237 in C3H mice. This was corrected by adoptive transfer of normal C3H spleen cells but not spleen cells from anti-asialo GM 1 -treated C3H mice or B6-blood-transfused C3H mice. Furthermore, a 1:1 mixture of normal C3H spleen cells with spleen cells from B6-blood-transfused C3H mice also failed to reconstitute the cyclophosphamide-pre-treated C3H mice. We conclude that allogeneic blood transfusion augments pulmonary metastasis of the UV-2237 sarcoma in C3H mice and that the mechanism involves suppression of NK activity.

Transplantation tolerance can be induced in the adult mouse by the selective manipulation of the CD4+ T cell subset. C3H/He recipients with prolonged survival (> 120 days) of C57BL/10 cardiac allografts induced by treatment, at the time of transplantation, with the anti-CD4 mAb, YTS 191.1, were skin grafted simultaneously with donor-specific and third-party (BALB/c) skin. The development of donor-specific tolerance was proved by the specific prolongation of C57BL/10 skin graft survival, while third-party grafts were rejected. Further investigation of recipients with long-term surviving primary heart allografts showed that donor-specific tolerance was associated with organ-specific differences. Secondary cardiac allografts were universally accepted, even at 42 days after the primary heart transplant, while prolonged survival of donor-specific skin grafts was not obtained until more than 120 days after primary cardiac transplantation. Analysis of leukocyte reactivity in the mixed leukocyte culture (MLC) showed no correlation between the proliferative response of recipient T cells in vitro to either donor or third-party alloantigen and the survival of either heart of skin allografts. These results illustrate the significant challenge presented when attempting to define and assess accurately the state of transplantation tolerance.

Background. CD4 + CD25 ++ CD45RA + cells (naive regulatory T cells [naive-Tregs]) have been identified as a functionally premature form of CD4 + CD25 +++ CD45RA - cells (conventional-Tregs). However, their contribution to transplant tolerance remains to be elucidated. Method. We examined the frequency and the function of conventional and naive-Tregs in the peripheral blood derived from operationally tolerant patients after pediatric living-donor liver transplant (Gr-tol). The data were compared with those of patients who were unable to be weaned off immunosuppression due to rejection (group-intolerance [Gr-intol]), patients in the process of weaning immunosuppression (Gr-weaning) and healthy volunteers (group-healthy volunteers [Gr-vol]). Results. In Gr-tol, the frequency of conventional-Tregs was significantly higher than that in Gr-vol and tended to be higher than that in Gr-intol. The frequency of naive-Tregs was significantly decreased in Gr-intol versus those in Gr-tol, -weaning, and -vol. In mixed lymphocyte reactions, donor-specific hyporesponsiveness of CD4 + cells was observed only in Gr-tol but not in the other groups. Depletion of conventional or naive-Tregs from CD4 + cells demonstrated that the suppressive properties of donor antigen-reactive conventional and naive-Tregs were upregulated compared with those of third-party antigen-reactive conventional and naive-Tregs in Gr-tol only. Conclusions. This is the first report providing detailed evidence that donor-specific naive-Tregs were generated and their suppressive properties were upregulated in the peripheral blood of tolerant patients, whereas their frequency was downregulated in intolerant patients. Therefore, we speculate that not only conventional-Tregs play a role in Tx tolerance but also the role of naive-Tregs is critical.

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.

IFN-γ was originally characterized as a proinflammatory cytokine with T helper type 1 inducing activity, but it is now clear that it also has important immunoregulatory functions. Regulatory T cells play an important role in models of autoimmunity, GVHD, and transplantation, and offer potential as a cellular therapy. In rodent models, in vivo-generated CD25(+)CD4(+) T cells can prevent allograft rejection, but therapeutic exploitation of Treg will more likely depend on protocols that allow the generation or selection of Treg ex vivo. The experiments described in this chapter will show that alloantigen-reactive Treg can be generated/expanded ex vivo using IFN-γ, a cytokine more usually associated with allograft rejection. Although IFN-γ production has hitherto been generally regarded as nonpermissive for allograft survival, we believe this paradoxical "good-bad" role for IFN-γ may reflect an important physiological negative feedback loop.

Identifying transplant recipients in whom immunological tolerance is established or is developing would allow an individually tailored approach to their posttransplantation management. In this study, we aimed to develop reliable and reproducible in vitro assays capable of detecting tolerance in renal transplant recipients. Several biomarkers and bioassays were screened on a training set that included 11 operationally tolerant renal transplant recipients, recipient groups following different immunosuppressive regimes, recipients undergoing chronic rejection, and healthy controls. Highly predictive assays were repeated on an independent test set that included 24 tolerant renal transplant recipients. Tolerant patients displayed an expansion of peripheral blood B and NK lymphocytes, fewer activated CD4+ T cells, a lack of donor-specific antibodies, donor-specific hyporesponsiveness of CD4+ T cells, and a high ratio of forkhead box P3 to alpha-1,2-mannosidase gene expression. Microarray analysis further revealed in tolerant recipients a bias toward differential expression of B cell-related genes and their associated molecular pathways. By combining these indices of tolerance as a cross-platform biomarker signature, we were able to identify tolerant recipients in both the training set and the test set. This study provides an immunological profile of the tolerant state that, with further validation, should inform and shape drug-weaning protocols in renal transplant recipients.

Cutaneous squamous cell cancer (SCC) affects up to 30% of kidney transplant recipients (KTRs) within 10 years of transplantation. There are no reliable clinical tests that predict those who will develop multiple skin cancers. High numbers of regulatory T cells associate with poor prognosis for patients with cancer in the general population, suggesting their potential as a predictive marker of cutaneous SCC in KTRs. We matched KTRs with (n = 65) and without (n = 51) cutaneous SCC for gender, age, and duration of immunosuppression and assessed several risk factors for incident SCC during a median follow-up of 340 days. Greater than 35 peripheral FOXP3(+)CD4(+)CD127(low) regulatory T cells/microl

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.

Mesenchymal stromal/stem cells (MSCs) possess immunomodulatory and reparative properties. Through specific interactions with immune cells that participate in both innate and adaptive responses, MSCs exposed to an inflammatory microenvironment can downregulate many immune effector functions. Clinical trials focusing on MSCs to treat graft-versus-host disease (GvHD) and autoimmune diseases are underway. Current analyses suggest that MSCs will improve cell and solid organ transplantation by ameliorating rejection and possibly eliminating the requirement for prolonged regimens of conventional immunosuppressive drugs. This review examines the in vitro and in vivo evidence for the clinical use of bone marrow derived MSCs.

PURPOSE OF REVIEW: Donor reactive regulatory T cells (Treg) play an important role in tolerance induction and maintenance in experimental transplant models. In this review we focus on the formation of the donor reactive Treg pool and explore the potential of these cells for therapeutic application in clinical transplantation. RECENT FINDINGS: Donor reactive Treg can arise by both conversion of alloreactive nonregulatory cells and expansion of naturally occurring Treg (nTreg) cross-reactive with donor alloantigen but the quantitative contribution of each of these pathways is at present unclear. However, the fact that donor reactive Treg can be driven both in vivo and ex vivo by alloantigen challenge of nonregulatory precursors is encouraging as it demonstrates that the functional potential of these cells for use in clinical transplantation will not be limited by fortuitous cross-reactivity between nTreg and donor alloantigens. Treg can be generated in vivo by transplantation or alloantigen challenge in combination with Treg-permissive immunosuppression, or ex vivo by phenotypic selection or by polyclonal or antigen-specific stimulation. A number of ex-vivo protocols exist for the enrichment of Treg in the laboratory and in many cases these cells have demonstrable function both in vitro and in relevant graft-versus-host disease (GVHD) or organ transplant models. The challenge now is to understand the clinical opportunities and limitations that these populations present. SUMMARY: Combined with appropriate immunosuppression, Treg generated/expanded in vivo or ex vivo may hold the final key to operational tolerance in clinical setting.

BACKGROUND: 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. METHODS AND RESULTS: 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-gamma (IFN-gamma). MHC Class II was induced on IPCCs, but not ES cells, after exposure to IFN-gamma. Transplantation of syngeneic or allogeneic IPCCs was insufficient to trigger up-regulation of MHC class I within three days after transplantation. DISCUSSION: 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.

Background Operational tolerance is defined as long-term acceptance of a transplanted organ after complete cessation of immunosuppression (IS), but may not always protect against antigen-dependent changes in graft morphology. Method IS free patients after living-donor liver transplantation (LDLT) underwent protocol biopsy (tolerance group [Gr-Tol]) and were evaluated for rejection and fibrosis. The degree of fibrosis was compared with those in the patients on maintenance IS group (Gr-IS) and the base line normal liver group (Gr-BS). When bridging fibrosis or progression of fibrosis was observed, IS was reintroduced or increased in Gr-Tol or in the patients in the weaning process. Results Neither acute nor chronic rejection was observed. The degree of fibrosis, however, was significantly greater in Gr-Tol than those in Gr-IS and Gr-BS. In Gr-Tol, the number of graft infiltrating FOXP3 cells was significantly increased, the interval between LDLT and biopsy plus the donor age was significantly longer, and recipient age at LDLT was significantly younger, compared with those in Gr-IS. However, none of these three parameters correlated with the degree of fibrosis. In 7 of 11 patients in whom IS was reintroduced or increased, the improvement of fibrosis was observed by the subsequent biopsy. Conclusion Grafts of operationally tolerant patients after LDLT did not exhibit acute or chronic rejection, but they exhibited fibrosis. It remains elusive whether fibrosis observed in tolerant grafts is antigen dependent. The finding that after [corrected] the reintroduction or the increase of IS fibrosis was improved supported the possibility that fibrosis in operationally tolerant patients was antigen dependent.

A newly discovered subset of T helper cells, Th17 cells, has been implicated in a number of models of autoimmunity. However, their role in transplantation remains undefined. This is despite studies showing a link between IL-17, a cytokine secreted by Th17 cells, and acute allograft rejection. This chapter summarises current evidence for a role of Th17 cells in transplant rejection, tolerance and ischaemia reperfusion injury.

The effects of using perioperative cyclosporine in conjunction with pretreatment with donor spleen cells or 3M KCl solubilized extracts of donor antigen were investigated in a LEW-to-DA rat renal allograft model. Cyclosporine given orally in a dose of 10 mg/kg/day around the time of transplantation (days -1, 0, +1), did not prolong renal allograft survival (median survival time [MST]--10 days). However when used in combination with pretreatment with either 10(8) donor spleen cells (1 day before transplantation), or 10(5) donor spleen cells (7 days before transplantation), pretreatment regimens that were in themselves ineffective, DA recipients accepted Lewis renal allografts indefinitely (MST greater than 100 days). Soluble antigen was prepared by 3M KCl extraction from donor spleen cells. Absorption assays were used to quantify the amount of class I major histocompatibility complex antigen in the preparation, and amounts of antigen equivalent to that expressed by 10(6)-10(8) donor spleen cells were used for pretreatment. These soluble antigen preparations given either 1 or 7 days before transplantation with or without perioperative cyclosporine did not prolong allograft survival of either homozygous or heterozygous donors (MST 10 days).

The immunosuppressive drugs currently in use in clinical transplantation are undoubtedly very effective at controlling graft rejection. However, their use is associated with a large number of side-effects, both immunological and non-immunological, particularly in the longer term. From an immunological point of view, the major disadvantage of these agents is that their mode of action is immunologically non-specific, resulting in blanket or pan-immunosuppression of the recipient's immune system. Thus, not only is the recipient's immune response against the organ graft suppressed, but responses to all other antigenic stimuli such as viral infections are also prevented. The transplant recipient can therefore become severely immunocompromised as a result of the drug therapy and is susceptible to opportunistic infections and an increased incidence of cancer. One of the aims for the design of new immunosuppressive therapy is to develop protocols that are both effective and immunologically specific, such that only the immune response to the transplanted organ is suppressed. The optimal approach would be to induce tolerance to the donor histocompatibility antigens before transplantation, permanently paralysing the ability of the recipient's immune system to mount a rejection response against the graft. Some of the approaches for the induction of immunological tolerance currently being explored in clinical transplantation will be discussed. These include total lymphoid irradiation (TLI) and donor bone marrow transfusion combined with anti-lymphocyte globulin (ALG) post-transplantation. In addition some new approaches for the induction of tolerance before transplantation currently being investigated in experimental systems will be presented.

Tolerance to alloantigen may be induced in rats by administration of blood followed by transplantation of a renal allograft. The mechanism of this tolerance was investigated by directly analyzing the functional activity of graft-infiltrating cells. We have previously shown cytotoxic T lymphocyte infiltration of, and major histocompatibility complex induction on, grafts of tolerant animals. We now report that cells isolated from the grafts of tolerant rats show a reduced expression of the p55 interleukin 2 receptor (IL-2R) chain on the cell surface compared with that seen on the cells of untreated animals. Scatchard analysis further reveals low expression of high affinity IL-2R. This is due to reduced transcription of both IL-2R alpha and beta chain mRNAs and results in a reduced ability of cells to proliferate in response to IL-2. Cells isolated from tolerant animals are unable to make biologically active IL-2 in culture, whereas cells from untreated animals make high levels. This is not reflected at the mRNA level as the IL-2 gene is induced in both tolerant and untreated animals to similar levels. The induction of tolerance is abrogated by administration of recombinant IL-2 to animals at the time of transplantation. Thus, we conclude that an altered regulation of the IL-2 pathway results in tolerance in these alloantigen-treated and transplanted animals.

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