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BackgroundMalignancy is a major cause of morbidity and mortality in transplant recipients. Identification of those at highest risk could facilitate pre-emptive intervention such as reduction of immunosuppression. Reduced circulating monocytic HLA-DR density is a marker of immune depression in the general population and associates with poorer outcome in critical illness. It has recently been used as a safety marker in adoptive cell therapy trials in renal transplantation. Despite its potential as a marker of dampened immune responses, factors that impact upon monocytic HLA-DR density and the long-term clinical sequelae of this have not been assessed in transplant recipients.MethodsA cohort study of stable long-term renal transplant recipients was undertaken. Serial circulating monocytic HLA-DR density and other leucocyte populations were quantified by flow cytometry. Gene expression of monocytes was performed using the Nanostring nCounter platform, and 13-plex cytokine bead array used to quantify serum concentrations. The primary outcome was malignancy development during one-year follow-up. Risk of malignancy was calculated by univariate and multivariate proportionate hazards modelling with and without adjustment for competing risks.ResultsMonocytic HLA-DR density was stable in long-term renal transplant recipients (n=135) and similar to non-immunosuppressed controls (n=29), though was suppressed in recipients receiving prednisolone. Decreased mHLA-DRd was associated with accumulation of CD14+CD11b+CD33+HLA-DRlo monocytic myeloid-derived suppressor-like cells. Pathway analysis revealed downregulation of pathways relating to cytokine and chemokine signalling in monocytes with low HLA-DR density; however serum concentrations of major cytokines did not differ between these groups. There was an independent increase in malignancy risk during follow-up with decreased HLA-DR density.ConclusionsDampened chemokine and cytokine signalling drives a stable reduction in monocytic HLA-DR density in long-term transplant recipients and associates with subsequent malignancy risk. This may function as a novel marker of excess immunosuppression. Further study is needed to understand the mechanism behind this association.

Our understanding of the conformational and electrostatic determinants that underlie targeting of human leukocyte antigens (HLA) by anti-HLA alloantibodies is principally based upon in silico modelling. Here we provide a biochemical/biophysical and functional characterization of a human monoclonal alloantibody specific for a common HLA type, HLA-A*11:01. We present a 2.4 Å resolution map of the binding interface of this antibody on HLA-A*11:01 and compare the structural determinants with those utilized by T-cell receptor (TCR), killer-cell immunoglobulin-like receptor (KIR) and CD8 on the same molecule. These data provide a mechanistic insight into the paratope−epitope relationship between an alloantibody and its target HLA molecule in a biological context where other immune receptors are concomitantly engaged. This has important implications for our interpretation of serologic binding patterns of anti-HLA antibodies in sensitized individuals and thus, for the biology of human alloresponses., Anti-human leukocyte antigen (HLA) antibodies are important mediators of alloresponses, but structural insights on antibody:HLA interaction are still lacking. Here the authors provide a 2.4 Å structure of antibody:HLA complex, and also analyse HLA features important for other HLA-interacting molecules, to enhance our understanding of alloimmunity.

The current state-of-the-art technology employed to assess anti-human leukocyte antigen antibodies (Anti-HLA Ab) for donor-recipient matching and patient risk stratification in renal transplantation is the single antigen bead (SAB) assay. However, there are limitations to the SAB assay as it is not quantitative and due to variations in techniques and reagents, there is no standardization across laboratories. In this study, a structurally-defined human monoclonal alloantibody was employed to provide a mechanistic explanation for how fundamental alloantibody biology influences the readout from the SAB assay. Performance of the clinical SAB assay was evaluated by altering Anti-HLA Ab concentration, subclass, and detection reagents. Tests were conducted in parallel by two internationally accredited laboratories using standardized protocols and reagents. We show that alloantibody concentration, subclass, laboratory-specific detection devices, subclass-specific detection reagents all contribute to a significant degree of variation in the readout. We report a significant prozone effect affecting HLA alleles that are bound strongly by the test alloantibody as opposed to those bound weakly and this phenomenon is independent of complement. These data highlight the importance for establishing international standards for SAB assay calibration and have significant implications for our understanding of discordance in previous studies that have analyzed its clinical relevance.

Recent progress in deciphering the mechanisms underlying the concepts of tumor immunosurveillance and immunoevasion has opened new opportunities for the development of effective antitumor therapies. Transplant physicians and immunologists have much to learn from those direct clinical translations of basic science. The 2016 Beaune Seminar in Transplant research brought together researchers from both fields to explore and discuss significant advances in cancer biology, immunotherapies and their potential impacts for the management of cancer in transplant recipients.

The immune system presents a significant challenge to the success of clinical transplantation. For the majority of patients, treatment with a combination of immunosuppressive drugs are essential to prevent rejection. Tissue injury caused by organ retrieval and implantation triggers an innate immune response, which in turn, activates the adaptive immune system capable of responding to both major and minor histocompatibility antigens mismatched between the organ donor and the recipient. Advances in immunosuppressive strategies using small molecules and biological agents have revolutionized the management and outcomes of solid organ transplantation.

In this chapter, we describe the history of transplantation, the multiple cell types, and mechanisms that are involved in rejection and tolerance of a transplanted organ, as well as summarize the common and promising new therapeutics used in transplant patients.

Highlights • Tregs are a major focus of investigation in transplantation for immunosuppression minimisation. • Development of alloantigen-specific Tregs has the potential to improve efficacy and safety. • A number of methodologies for production exist including culture with donor alloantigen. • Clinical trials of polyclonal Treg therapy are now moving into Phase II and beyond., Transplantation is limited by the need for life-long pharmacological immunosuppression, which carries significant morbidity and mortality. Regulatory T cell (Treg) therapy holds significant promise as a strategy to facilitate immunosuppression minimization. Polyclonal Treg therapy has been assessed in a number of Phase I/II clinical trials in both solid organ and hematopoietic transplantation. Attention is now shifting towards the production of alloantigen-reactive Tregs (arTregs) through co-culture with donor antigen. These allospecific cells harbour potent suppressive function and yet their specificity implies a theoretical reduction in off-target effects. This review will cover the progress in the development of arTregs including their potential application for clinical use in transplantation, the knowledge gained so far from clinical trials of Tregs in transplant patients, and future directions for Treg therapy.

Background: T follicular helper (Tfh) cells are key players in the production of antibody-producing B cells via the germinal center reaction. Therapeutic strategies targeting Tfh cells are important where antibody formation is implicated in disease, such as transplant rejection and autoimmune diseases. We investigated the impact of the immunosuppressive agent tacrolimus on human Tfh cell differentiation and function in transplant recipients. Methods: Paired blood and lymph node (LN) samples were obtained from 61 transplant recipients immediately prior to organ implantation. Living-donor recipients received a week of tacrolimus prior to kidney transplantation. Deceased-donor recipients served as controls, as tacrolimus was not administered until after the transplant operation. Flow cytometry was used to compare LN and circulating cell subsets. Results: The calcineurin inhibitor (CNIs) tacrolimus specifically suppresses both LN Tfh cells and circulating Tfh cells, but not their regulatory counterparts or other CD4 T cell subsets. Conclusion: Our findings suggest that CNIs may have a more important role in the prevention of antibody formation than previously understood and, therefore, have potential for antibody-associated conditions in which aberrant Tfh function has been implicated in disease.

Cellular therapies with CD4+ T regulatory cells (Tregs) hold promise of efficacious treatment for the variety of autoimmune and allergic diseases as well as posttransplant complications. Nevertheless, current manufacturing of Tregs as a cellular medicinal product varies between different laboratories, which in turn hampers precise comparisons of the results between the studies performed. While the number of clinical trials testing Tregs is already substantial, it seems to be crucial to provide some standardized characteristics of Treg products in order to minimize the problem. We have previously developed reporting guidelines called minimum information about tolerogenic antigen-presenting cells, which allows the comparison between different preparations of tolerance-inducing antigen-presenting cells. Having this experience, here we describe another minimum information about Tregs (MITREG). It is important to note that MITREG does not dictate how investigators should generate or characterize Tregs, but it does require investigators to report their Treg data in a consistent and transparent manner. We hope this will, therefore, be a useful tool facilitating standardized reporting on the manufacturing of Tregs, either for research purposes or for clinical application. This way MITREG might also be an important step toward more standardized and reproducible testing of the Tregs preparations in clinical applications. This work was supported by a grant from the European Cooperation in Science and Technology (COST) for the AFACTT project (Action to Focus and Accelerate Cell-based Tolerance-inducing Therapies; BM1305). COST is part of the EU Framework Programme Horizon 2020. peer-reviewed

Most morbidity associated with malignancy in long-term renal transplant recipients is due to cutaneous squamous cell carcinoma (SCC). Previously identified measures to stratify SCC risk have limited use, however. We hypothesized that an increased proportion of senescent, terminally differentiated CD8(+) T cells would identify renal transplant recipients at elevated SCC risk. Peripheral blood lymphocytes were isolated from 117 stable transplant recipients at high risk of SCC and analyzed phenotypically by flow cytometry. Participants were followed up prospectively for SCC development. The predictive value of variables was assessed using Cox regression. Age at transplant and enrollment, dialysis duration, and previous disease were predictive of SCC development during follow-up. Previously published clinical phenotype-based risk scores lost predictive value with the removal of age as a covariate. The percentage of CD57-expressing CD8(+) T cells was the strongest immunologic predictor of future SCC and correlated with increasing CD8(+) T cell differentiation. We dichotomized participants into those with a majority (CD57hi) and a minority (CD57lo) of CD8(+) T cells expressing CD57; CD57hi participants were more likely to develop SCC during follow-up (hazard ratio, 2.9; 95% confidence interval, 1.0 to 8.0), independent of potential confounders, and tended to develop earlier recurrence. The CD57hi phenotype was stable with time and associated with increasing age and cytomegalovirus seropositivity. Our results show that the CD57hi phenotype is a strong predictor of SCC development and recurrence in this cohort of long-term, high-risk renal transplant recipients. This information may allow identification of recipients who may benefit from intensive dermatologic screening and immunosuppression reduction.

International audience; The effector functions of T lymphocytes are responsible for most autoimmune disorders and act by directly damaging tissues or by indirectly promoting inflammation and antibody responses. Co-stimulatory and co-inhibitory T cell receptor molecules are the primary pharmacological targets that enable interference with immune-mediated diseases. Among these, selective CD28 antagonists have drawn special interest, since they tip the co-stimulation/co-inhibition balance towards efficiently inhibiting effector T cells while promoting suppression by pre-existing regulatory T-cells. After having demonstrated outstanding therapeutic efficacy in multiple models of autoimmunity, inflammation and transplantation, and safety in phase-I studies in humans, selective CD28 antagonists are currently in early clinical development for the treatment of systemic lupus erythematous and rheumatoid arthritis. Here, we review the available proof of concept studies for CD28 antagonists in autoimmunity, with a special focus on the mechanisms of action.

Adoptive cellular therapies are gaining popularity as a means to treat clinical conditions, with potentially fewer risks and greater efficacy than traditional pharmacological strategies. Regulatory T cells (Tregs) are currently undergoing clinical trials in various immune-mediated pathologies, including transplant rejection and autoimmune conditions. In general, cell therapy relies upon ex vivo expansion of the cell product, in order to administer more cells than can be isolated from one person. In vitro manipulation of cell therapy products, prior to administration to patients, offers the opportunity to enhance the efficacy of the final cell therapy product in other ways. For example, cells can be exposed to reagents that enhance their longevity or functional potency after transfer into the patient. Genetic modification strategies can even permit the design of cells with bespoke functionality. Crucially, in vitro manipulation of therapeutic cells in isolation can exert these influences upon the biology of the therapeutic cells, without systemic exposure of the patient to the reagents being used. Quality control assessments can be integrated into the procedure prior to administration, to protect the patient from the risk of adverse events, should the procedure produce undesirable results. With a particular focus on Tregs, this review surveys the diverse strategies that are being employed to enhance the efficacy of cell therapy via in vitro manipulation of cells, and highlights some emerging technologies that may propel this endeavour in the future.

Regulatory T cells (Tregs) are critical mediators of immune homeostasis and hold significant promise in the quest for transplantation tolerance. Progress has now reached a critical threshold as techniques for production of clinical therapies are optimised and Phase I/II clinical trials are in full swing. Initial safety and efficacy data are being reported, with trials assessing a number of different strategies for the introduction of Treg therapy. It is now more crucial than ever to elucidate further the function and behaviour of Tregs in vivo and ensure safe delivery. This review will discuss the current state of the art and future directions in Treg therapy.

BACKGROUND Donor-specific antibodies create an immunologic barrier to transplantation. Current therapies to modify donor-specific antibodies are limited and ineffective in the most highly HLA-sensitized patients. The IgG-degrading enzyme derived from Streptococcus pyogenes (IdeS), an endopeptidase, cleaves human IgG into F(ab′)2 and Fc fragments inhibiting complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity, which suggests that IdeS might be useful for desensitization. We report on the combined experience of two independently performed open-label, phase 1–2 trials (conducted in Sweden and the United States) that assessed the efficacy of IdeS with regard to desensitization and transplantation of a kidney from an HLA-incompatible donor. METHODS We administered IdeS to 25 highly HLA-sensitized patients (11 patients in Uppsala or Stockholm, Sweden, and 14 in Los Angeles) before the transplantation of a kidney from an HLA-incompatible donor. Frequent monitoring for adverse events, outcomes, donor-specific antibodies, and renal function was performed, as were renal biopsies. Immunosuppression after transplantation consisted of tacrolimus, mycophenolate mofetil, and glucocorticoids. Patients in the U.S. study also received intravenous immune globulin and rituximab after transplantation to prevent antibody rebound. RESULTS Recipients in the U.S. study had a significantly longer cold ischemia time (the time elapsed between procurement of the organ and transplantation), a significantly higher rate of delayed graft function, and significantly higher levels of class I donor-specific antibodies than those in the Swedish study. A total of 38 serious adverse events occurred in 15 patients (5 events were adjudicated as being possibly related to IdeS). At transplantation, total IgG and HLA antibodies were eliminated. A total of 24 of 25 patients had perfusion of allografts after transplantation. Antibody-mediated rejection occurred in 10 patients (7 patients in the U.S. study and 3 in the Swedish study) at 2 weeks to 5 months after transplantation; all these patients had a response to treatment. One graft loss, mediated by non-HLA IgM and IgA antibodies, occurred. CONCLUSIONS IdeS reduced or eliminated donor-specific antibodies and permitted HLA-incompatible transplantation in 24 of 25 patients.

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

In transplantation, the contribution of B cells to the rejection or acceptance of the allograft is a topic of major interest. The presence of donor-specific antibodies in transplant recipients is often associated with decreased graft function and rejection, clearly indicating a pathogenetic role of B cells in transplantation. However, data from studies in humans and rodents suggest that under certain conditions, B cells have the capacity to control or regulate the immune response to a transplanted organ. Although a great deal of attention has been focused on B cells in human and murine models of autoimmunity, our understanding of the role of these cells in transplantation is limited at present. Indeed, results in this setting are controversial and seem to depend on the model system used or the clinical situation studied. Here, we review the current understanding of the various phenotypes and roles that have been associated with immune-regulating B cells. We also discuss the mechanisms employed by subsets of these regulatory B cells to control the immune response in transplant recipients and in animal models of transplantation. © 2014 Macmillan Publishers Limited. All rights reserved.

Organ transplantation results in the activation of both innate and adaptive immune responses to the foreign antigens. While these responses can be limited with the use of systemic immunosuppressants, the induction of regulatory cell populations may be a novel strategy for the maintenance of specific immunological unresponsiveness that can reduce the severity of the detrimental side effects of current therapies. Our group has extensively researched different regulatory T-cell induction protocols for use as cellular therapy in transplantation. In this review, we address the cellular and molecular mechanisms behind regulatory T-cell suppression and their stability following induction protocols. We further discuss the use of different hematopoietically derived regulatory cell populations, including regulatory B cells, regulatory macrophages, tolerogenic dendritic cells, and myeloid-derived suppressor cells, for the induction of transplantation tolerance in light of new clinical trials developing therapies with some of these populations.

Background - T follicular helper (Tfh) and regulatory (Tfr) cells are key players in the formation of long-lived antibody responses. Their circulating counterparts, cTfh and cTfr, are often used as biomarkers because longitudinal sampling of secondary lymphoid tissues is unfeasible in clinical studies. This is the first study to track cTfh and cTfr cells following therapeutic lymphocyte depletion with alemtuzumab, an anti-CD52 monoclonal antibody, to infer the influence of this treatment on the germinal centre response. Methods - Samples from 61 transplant recipients were taken at the time of transplant, and regular intervals post-transplant for both flow-cytometric immunophenotyping, and Luminex-based donor-specific antibody (DSA) assessment. Results - Patients treated with alemtuzumab (42/61, 69%) had a significantly lower ratio of cTfr to cTfh at all time points post-transplant. We found that, despite a high proportion of Tregs in the recovering cell population, cTfr cells did not repopulate in alemtuzumab treated patients, while cTfh cells reconstituted to higher than pre-transplant levels over the 2-year follow-up. Conclusions - Alemtuzumab has been used as first-line induction immunosuppression and treatment for steroid-resistant rejection in transplantation; our data suggests alemtuzumab-treated patients have a lower cTfr to cTfh ratio even 2 years post-transplant, and may therefore be at higher risk of de novo DSA formation.

Background Human regulatory T cells (Treg) offer an attractive adjunctive therapy to reduce current reliance on lifelong, nonspecific immunosuppression after transplantation. Here, we evaluated the ability of ex vivo expanded human Treg to prevent the rejection of islets of Langerhans in a humanized mouse model and examined the mechanisms involved. Methods We engrafted human pancreatic islets of Langerhans into the renal subcapsular space of immunodeficient BALB/c.rag2−/−.cγ−/− mice, previously rendered diabetic via injection of the β-cell toxin streptozocin. After the establishment of stable euglycemia, mice were reconstituted with allogeneic human peripheral blood mononuclear cells (PBMC) and the resultant alloreactive response studied. Ex vivo expanded CD25highCD4+ human Treg, which expressed FoxP3, CTLA-4, and CD62L and remained CD127low, were then cotransferred together with human PBMC and islet allografts and monitored for evidence of rejection. Results Human islets transplanted into diabetic immunodeficient mice reversed diabetes but were rejected rapidly after the mice were reconstituted with allogeneic human PBMC. Cotransfer of purified, ex vivo expanded human Treg prolonged islet allograft survival resulting in the accumulation of Treg in the peripheral lymphoid tissue and suppression of proliferation and interferon-γ production by T cells. In vitro, Treg suppressed activation of signal transducers and activators of transcription and inhibited the effector differentiation of responder T cells. Conclusions Ex vivo expanded Treg retain regulatory activity in vivo, can protect a human islet allograft from rejection by suppressing signal transducers and activators of transcription activation and inhibiting T-cell differentiation, and have clinical potential as an adjunctive cellular therapy., Supplemental digital content is available in the article.

The transfer of alloreactive regulatory T (aTreg) cells into transplant recipients represents an attractive treatment option to improve long-term graft acceptance. We recently described a protocol for the generation of aTreg cells in mice using a nondepleting anti-CD4 antibody (aCD4). Here, we investigated whether adding TGF-β and retinoic acid (RA) or rapamycin (Rapa) can further improve aTreg-cell generation and function. Murine CD4(+) T cells were cultured with allogeneic B cells in the presence of aCD4 alone, aCD4+TGF-β+RA or aCD4+Rapa. Addition of TGF-β+RA or Rapa resulted in an increase of CD25(+)Foxp3(+)-expressing T cells. Expression of CD40L and production of IFN-γ and IL-17 was abolished in aCD4+TGF-β+RA aTreg cells. Additionally, aCD4+TGF-β+RA aTreg cells showed the highest level of Helios and Neuropilin-1 co-expression. Although CD25(+)Foxp3(+) cells from all culture conditions displayed complete demethylation of the Treg-specific demethylated region, aCD4+TGF-β+RA Treg cells showed the most stable Foxp3 expression upon restimulation. Consequently, aCD4+TGF-β+RA aTreg cells suppressed effector T-cell differentiation more effectively in comparison to aTreg cells harvested from all other cultures, and furthermore inhibited acute graft versus host disease and especially skin transplant rejection. Thus, addition of TGF-β+RA seems to be superior over Rapa in stabilising the phenotype and functional capacity of aTreg cells.

Multiple cell types contribute to the peripheral regulation of T cell alloresponses in haematopoieitc cell transplantation (HCT) and solid organ transplantation (SOT). Of these, regulatory T cells (Tregs) are the principal players and have shown the greatest success in the therapeutic control of detrimental immune responses. Investigations into the induction, location, and mechanism of suppression utilised by Tregs to control alloreactive responses are ongoing. The activation and homing characteristics of Tregs are important to their regulatory capabilities, with activation and homing occurring in the same time and space as conventional T cells. This review discusses these characteristics and recent advances in the field as we move closer to the ultimate goal of utilising Tregs as treatment for allograft rejection and graft-versus-host disease (GvHD). © 2012 Elsevier Ltd.

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.

Since the first solid organ transplant between the Herrick twins in 1954, transplantation immunology has sought to move away from harmful immunosuppressive regimens towards tolerogenic strategies that promote long-term graft survival. This has required a concerted multinational effort with scientists and clinicians working towards a common goal. Reports of immunosuppression-free kidney and liver allograft recipients have provided the proof-of-principle, but intentional generation of tolerance in clinical transplantation is still only achieved infrequently. Recently, there have been an increasing number of encouraging developments in the field in both experimental and clinical studies. In this article, we review the latest advances in tolerance research and consider possible future barriers and solutions in achieving reliable graft acceptance in the long term. © 2013 The Authors Transplant International © 2013 European Society for Organ Transplantation. Published by Blackwell Publishing Ltd.

The engineered Fc-nonbinding (crystallizable fragment-nonbinding) CD3 antibody has lower mitogenicity and a precise therapeutic window for disease remission in patients with type 1 diabetes. Before anti-CD3 can be considered for use in transplantation, the most effective timing of treatment relative to transplantation needs to be elucidated. In this study anti-CD3F(ab′) 2 fragments or saline were administered intravenously for 5 consecutive days (early: d1-3 or delayed: d3-7) to mice transplanted with a cardiac allograft (H2b-to-H2k; d0). Survival of allografts was prolonged in mice treated with the early protocol (MST = 48 days), but most were rejected by d100. In contrast, in mice treated with the delayed protocol allografts continued to survive long term. The delayed protocol significantly inhibited donor alloreactivity at d30 as compared to the early protocol. A marked increase in Foxp3+ T cells (50.3 ± 1.6%) infiltrating the allografts in mice treated with the delayed protocol was observed (p < 0.0001 vs. early (24.9 ± 2.1%)) at d10; a finding that was maintained in the accepted cardiac allografts at d100. We conclude that the timing of treatment with anti-CD3 therapy is critical for inducing long-term graft survival. Delaying administration effectively inhibits the alloreactivity and promotes the dominance of intragraft Foxp3+ T cells allowing long-term graft acceptance. © 2013 The Authors. American Journal of Transplantation Published by Wiley Periodicals Inc.

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, Supplemental digital content is available in the article.

Interferon (IFN)gamma can have paradoxical functions, eliciting inflammatory T helper 1 (Th1)-driven immune responses in some circumstances, and enabling induced regulatory T (Treg) cells to control immune responses in others. Here, we propose a model in which IFNgamma produced rapidly and only transiently by induced Treg cells is crucial to their function in vivo. This early production of IFNgamma by induced Treg cells during an immune response can directly inhibit the activation and proliferation of IFNgammaR1- and IFNgammaR2-bearing T cells. Furthermore, it can indirectly prevent further T-cell activation by creating a microenvironment that influences the function of antigen-presenting cells (APCs) as a result of IFNgamma-induced inducible nitric oxide synthase (iNOS), indoleamine-2,3-dioxygenase (IDO) and heme oxygenase (HO)-1 expression.

Linked unresponsiveness operates to induce specific unresponsiveness to fully mismatched vascularized allografts in recipients pretreated with anti-CD4 antibody and syngeneic bone marrow cells expressing a single donor MHC class I alloantigen. The aim of the study was to evaluate early post transplant cytokine expression in allografts where linked unresponsiveness was required for long term graft survival. CBA (H2(k)) mice were pretreated with CBK (H2(k)+K(b)) bone marrow cells under the cover of anti-CD4 antibody 28 days before transplantation of a CBK or a C57BL/10 (H2(b)) cardiac allograft. In both cases graft survival was prolonged (MST=100 days). Intragraft expression for interferon (IFN)-gamma, interleukin (IL)-2, IL-4, IL-10, IL-12(p40), IL-18, iNOS, transforming growth factor (TGF)-beta(1) and C-beta was determined on day 1.5, 3, 7 and 14 after transplantation. Whereas rejecting allografts displayed a sharp peak in IFN-gamma, IL-2, IL-4 and IL-10 expression, non-rejecting allografts were characterized by an initial TGF-beta(1) and IFN-gamma production. An increasing IL-4 expression towards day 14 was a unique feature of linked unresponsiveness. All non-rejecting allografts were characterized by an increasing IL-12(p40) production towards day 14. In summary, the early cytokine expression pattern in allografts after bone marrow induced operational tolerance is influenced by the quantity of donor alloantigens expressed on the graft as well as on the bone marrow inoculum.

Background. CD25+CD4+ regulatory T cells have been shown to suppress alloimmunity in various experimental settings. Here, we hypothesized that alloantigen-reactive regulatory T cells would reduce the severity of transplant arteriosclerosis. Methods. CD25+CD4+ T cells from CBA mice that were pretreated with C57BL/6 (B.6) blood (donor-specific transfusion, DST) and nondepleting anti-CD4 Ab (YTS 177) were cotransferred with naive CBA CD25-CD4+"effector" T cells into CBA-rag -/- mice. These animals received aorta transplants from B.6 CD31 -/- donors. CBA wild-type recipients of B.6 aorta grafts were pretreated with 177/DST directly. Some animals received 6X 10 5 CD25 + CD4+ T cells from pretreated mice to augment regulation on day -1. Grafts were harvested on day 30. Results. Luminal occlusion of the graft caused by neointima formation was 29.3 ± 19.4% (n=5) after transfer of effector T cells only. Co-transfer of CD25+CD4+ regulators reduced occlusion significantly (2.4±3.3%, n=3; P=0.009). This effect was partially abrogated in the presence of a CTLA4 blocking Ab (11.1±4.7%, n=4; P=0.008). Pretreating immunocompetent CBA recipients of B.6 aortic allografts with 177/DST did not reduce transplant arteriosclerosis significantly (43.0±15.7%, n=5 vs. 56.6±16.8%, n=5; 177/DST vs. controls; P=0.22). However, when pretreated primary CBA recipients received an additional transfer of 6 X 10 5 CD25 + CD4 + T cells procured from other mice pretreated with 177/DST before transplantation, luminal occlusion of the graft was markedly reduced (33.0±7.6%, n=5;P=0.002). Conclusion. Regulatory T cells generated in vivo to alloantigen can prevent CD25 - CD4 + T-cell-mediated transplant arteriosclerosis. In immunocompetent recipients, these cells have potential to be used as cellular immunotherapy to control transplant arteriosclerosis.

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.

The induction of peripheral tolerance to alloantigen is accompanied in many cases by a decrease in the production of cytokines such as IL-2 and IFN gamma, yet a sustained production of cytokines such as IL-10 and IL-4. Whether or not this altered pattern of cytokine production in tolerant animals is causally related to the induction and/or maintenance of the tolerant state has yet to be fully determined, although experiments blocking selectively the action of IL-2 with CD25 antibodies suggest that manipulation of cytokine production may at least be a route to tolerance. Alternative methods for directly influencing the cytokine balance are sought and recent experiments on the CD28/CTLA-4-B7 interaction suggest a possible approach.

Decellularized (acellular) scaffolds, composed of natural extracellular matrix, form the basis of an emerging generation of tissue-engineered organ and tissue replacements capable of transforming healthcare. Prime requirements for allogeneic, or xenogeneic, decellularized scaffolds are biocompatibility and absence of rejection. The humoral immune response to decellularized scaffolds has been well documented, but there is a lack of data on the cell-mediated immune response toward them in vitro and in vivo. Skeletal muscle scaffolds were decellularized, characterized in vitro, and xenotransplanted. The cellular immune response toward scaffolds was evaluated by immunohistochemistry and quantified stereologically. T-cell proliferation and cytokines, as assessed by flow cytometry using carboxy-fluorescein diacetate succinimidyl ester dye and cytometric bead array, formed an in vitro surrogate marker and correlate of the in vivo host immune response toward the scaffold. Decellularized scaffolds were free of major histocompatibility complex class I and II antigens and were found to exert anti-inflammatory and immunosuppressive effects, as evidenced by delayed biodegradation time in vivo; reduced sensitized T-cell proliferative activity in vitro; reduced IL-2, IFN-γ, and raised IL-10 levels in cell-culture supernatants; polarization of the macrophage response in vivo toward an M2 phenotype; and improved survival of donor-derived xenogeneic cells at 2 and 4 wk in vivo. Decellularized scaffolds polarize host responses away from a classical TH1-proinflammatory profile and appear to down-regulate T-cell xeno responses and TH1 effector function by inducing a state of peripheral T-cell hyporesponsiveness. These results have substantial implications for the future clinical application of tissue-engineered therapies.

We present evidence that donor-reactive CD4+ T cells present in mice tolerant to donor alloantigens are phenotypically and functionally heterogeneous. CD4+ T cells contained within the CD45RBhigh fraction remained capable of mediating graft rejection when transferred to donor alloantigen-grafted T cell-depleted mice. In contrast, the CD45RBlow CD4+ and CD25+CD4+ populations failed to induce rejection, but rather, were able to inhibit rejection initiated by naive CD45RBhigh CD4+ T cells. Analysis of the mechanism of immunoregulation transferred by CD45RBlow CD4+ T cells in vivo revealed that it was donor Ag specific and could be inhibited by neutralizing Abs reactive with IL-10, but not IL-4. CD45RBlow CD4+ T cells from tolerant mice were also immune suppressive in vitro, as coculture of these cells with naive CD45RBhigh CD4+ T cells inhibited proliferation and Th1 cytokine production in response to donor alloantigens presented via the indirect pathway. These results demonstrate that alloantigen-specific regulatory T cells contained within the CD45RBlow CD4+ T cell population are responsible for the maintenance of tolerance to donor alloantigens in vivo and require IL-10 for functional activity.

Naturally occurring FOXP3(+) CD4(+) Treg have a crucial role in self-tolerance. The ability to generate similar populations against alloantigens offers the possibility of preventing transplant rejection without indefinite global immunosuppression. Exposure of mice to donor alloantigens combined with anti-CD4 antibody induces operational tolerance to cardiac allografts, and generates Treg that prevent skin and islet allograft rejection in adoptive transfer models. If protocols that generate Treg in vivo are to be developed in the clinical setting it will be important to know the origin of the Treg population and the mechanisms responsible for their generation. In this study, we demonstrate that graft-protective Treg arise in vivo both from naturally occurring FOXP3(+) CD4(+) Treg and from non-regulatory FOXP3(-) CD4(+) cells. Importantly, tolerance induction also inhibits CD4(+) effector cell priming and T cells from tolerant mice have impaired effector function in vitro. Thus, adaptive tolerance induction shapes the immune response to alloantigen by converting potential effector cells into graft-protective Treg and by expanding alloreactive naturally occurring Treg. In relation to clinical tolerance induction, the data indicate that while the generation of alloreactive Treg may be critical for long-term allograft survival without chronic immunosuppression, successful protocols will also require strategies that target potential effector cells.

Regulatory macrophages (M regs) were administered to two living-donor renal transplant recipients. Both patients were minimized to low-dose tacrolimus monotherapy within 24 wk of transplantation and subsequently maintained excellent graft function. After central venous administration, most M regs remained viable and were seen to traffic from the pulmonary vasculature via the blood to liver, spleen, and bone marrow. By 1 y posttransplantation, both patients displayed patterns of peripheral blood gene expression converging upon the IOT-RISET signature. Furthermore, both patients maintained levels of peripheral blood FOXP3 and TOAG-1 mRNA expression within the range consistent with nonrejection. It is concluded that M regs warrant further study as a potential immune-conditioning therapy for use in solid-organ transplantation. The results of this work are being used to inform the design of The ONE Study, a multinational clinical trial of immunomodulatory cell therapy in renal transplantation. Copyright © 2011 by The American Association of Immunologists, Inc.

We have developed a method to generate alloreactive regulatory T cells in vitro in the presence of interferon (IFN)-gamma and donor antigen presenting cells (APCs). We hypothesized that these IFN-gamma-conditioned T cells (Tcon) would reduce transplantation-associated arteriosclerosis. Tcon were generated from mouse (CBA.Ca, H-2(k)) CD4(+) T cells cultured in the presence of IFN-gamma for 14 days. These cultures were pulsed with bone marrow-derived B6 (H-2(b)) APC. 1 x 10(5) CD25(-)CD4(+) effector T cells from naive H-2(k) mice were then cotransferred with 4 x 10(5) Tcon into CBA-rag(-/-) mice. One day later, these mice received a fully allogenic B6 CD31(-/-) abdominal aorta transplant. Transfer of CD25(-)CD4(+) effectors resulted in 29.7 +/- 14.5% luminal occlusion of allogeneic aortic grafts after 30 days. Cotransfer of Tcon reduced this occlusion to 11.7 +/- 13.1%; P < 0.05. In addition, the CD31(-) donor endothelium was fully repopulated by CD31(+) recipient endothelial cells in the absence of Tcon, but not in the presence of Tcon. In some experiments, we cotransplanted B6 skin with aortic grafts to ensure enhanced reactivation of the regulatory cells, which led to an additional reduction in vasculopathy (1.9 +/- 3.0% luminal occlusion). In the presence of Tcon, CD4(+) T cell infiltration into grafts was markedly reduced by a regulatory mechanism that included reduced priming and proliferation of CD25(-)CD4(+) effectors. These data illustrate the potential of ex vivo generated regulatory T cells for the inhibition of transplant-associated vasculopathy.

Background. Liver grafts transplanted across a major histocompatibility barrier are accepted spontaneously and induce donor specific tolerance in some species. Here, we investigated whether liver allograft acceptance is characterized by, and depends upon, the presence of donor reactive CD25 + CD4 + regulatory T cells. Methods. CD25 + and CD25 - CD4 + T cells, isolated from CBA. Ca (H2 k ) recipients ofC57BL/10 (B10; H2 b ) liver and heart allografts 10 days after transplantation, were transferred into CBA. Rag1 -/- mice to investigate their influence on skin allograft rejection mediated by CD45RB high CD4 + effector T Cells. Results. Fully allogeneic B10 liver allografts were spontaneously accepted by naive CBA.Ca recipient mice, whereas B10 cardiac allografts were acutely rejected (mean survival time = 7 days). Strikingly, however, CD25 + CD4 + T cells isolated from both liver and cardiac allograft recipients were able to prevent skin allograft rejection in this adoptive transfer model. Interestingly, CD25 - CD4 + T cells isolated from liver graft recipients also showed suppressive potency upon adoptive transfer. Furthermore, depletion of CD25 + CD4 + T cells in primary liver allograft recipients did not prevent the acceptance of a secondary donor-specific skin graft. Conclusions. Our data provide evidence that the presence of CD25 + CD4 + regulatory T cells is not a unique feature of allograft acceptance and is more likely the result of sustained exposure to donor alloantigens in vivo.

Introduction: Intrathymic inoculation of donor alloantigen and concomitant immunosuppressive treatment can induce immune unresponsiveness to alloantigen. To examine the role of non-deletional mechanisms in the development of unresponsiveness, fractionated splenocytes were injected into only 1 lobe of the thymus. Methods and results Untreated CBA (H2 k ) mice or controls pre-treated with anti-CD4 monoclonal antibody alone (on Day −28 and −27 relative to transplantation) acutely rejected C57BL/10 (H2 b ) cardiac allografts. Intrathymic inoculation of unfractionated splenocytes, resting B (rB) cells, or dendritic cells into both thymic lobes with the antibody resulted in indefinite survival of cardiac allografts. In contrast, when donor rB cells or dendritic cells were delivered into a single lobe of the thymus with the antibody, only rB cells induced indefinite prolongation of graft survival; unfractionated splenocytes or dendritic cells were markedly less effective. Mice that had 1 of the 2 thymic lobes removed were able to reject grafts even when treated with the antibody 27 days before transplantation. Therefore, T-cell export from 1 thymic lobe was sufficient to induce graft rejection. Finally, adoptive transfer of splenocytes from mice with long-term surviving primary grafts resulting from the intrathymic injection of rB cells significantly prolonged a graft from the same donor strain in a naive syngeneic recipient. Conclusion Taken together, these data suggest that regulatory mechanisms generated by intrathymic injection of a non-professional antigen presenting cell, in this study donor rB cells, suppressed the rejection response mediated by T cells exported from the uninjected lobe.

Over the past 20 years, natural killer T (NKT) cells have been shown to play an important role in both innate and adaptive immune responses. In this review, the potential role of NKT cells in transplantation will be discussed, particularly their role in rejection and the induction of a state of tolerance.

Background Specific immunological unresponsiveness to alloantigens can be induced in vivo by treating mice with a donor alloantigen in combination with a non-depleting anti-CD4 antibody. This tolerance induction protocol enriches for alloantigen reactive regulatory T cells (Treg). We previously demonstrated that alpha-1,2-mannosidase, an enzyme involved in the synthesis and processing of N-linked glycoproteins, is highly expressed in tolerant mice, in both graft infiltrating leukocytes and peripheral blood lymphocytes. Principal Findings In this study we have identified that alpha-1,2-mannosidase expression increases in CD25+CD4+ Treg when they encounter alloantigen in vivo. When alpha-1,2-mannosidase enzyme activity was blocked, Treg retained their capacity to suppress T cell proliferation in vitro but were unable to bind to physiologically relevant ligands in vitro. Further in vivo analysis demonstrated that blocking alpha-1,2-mannosidase in Treg resulted in the migration of significantly lower numbers to the peripheral lymph nodes in skin grafted mice following adoptive transfer, where they were less able to inhibit the proliferation of naive T cells responding to donor alloantigen and hence unable prevent allograft rejection in vivo. Significance Taken together, our results suggest that activation of alloantigen reactive Treg results in increased alpha-1,2-mannosidase expression and altered N-glycosylation of cell surface proteins. In our experimental system, altered N-glycosylation is not essential for intrinsic Treg suppressive capacity, but is essential in vivo as it facilitates Treg migration to sites where they can regulate immune priming. Migration of Treg is central to their role in regulating in vivo immune responses and may require specific changes in N-glycosylation upon antigen encounter.

The molecular entities present on a cell or organ transplant that trigger the innate immune response and link to the adaptive immune system are increasingly recognized as a key influence on early graft function and for determining the microenvironment that will guide longer-term graft outcomes. The 2014 Beaune Seminar in Transplant Research discussed the evidence for triggers, sensors, and modulators of innate and adaptive immunity in response to alloantigens, challenged the conventional view, developed novel hypotheses, and highlighted the potential for therapeutic manipulation of these responses.

Assessment of donor-specific alloreactive memory/effector T cell responses using an IFN-γ Elispot assay has been suggested to be a novel immune-monitoring tool for evaluating the cellular immune risk in renal transplantation. Here, we report the cross-validation data of the IFN-γ Elispot assay performed within different European laboratories taking part of the EU RISET consortium. For this purpose, development of a standard operating procedure (SOP), comparisons of lectures of IFN-γ plates assessing intra- and interlaboratory assay variability of allogeneic or peptide stimuli in both healthy and kidney transplant individuals have been the main objectives. We show that the use of a same SOP and count-settings of the Elispot bioreader allow low coefficient variation between laboratories. Frozen and shipped samples display slightly lower detectable IFN-γ frequencies than fresh samples. Importantly, a close correlation between different laboratories is obtained when measuring high frequencies of antigen-specific primed/memory T cell alloresponses. Interestingly, significant high donor-specific alloreactive T cell responses can be similarly detected among different laboratories in kidney transplant patients displaying histological patterns of acute T cell mediated rejection. In conclusion, assessment of circulating alloreactive memory/effector T cells using an INF-γ Elispot assay can be accurately achieved using the same SOP, Elispot bioreader and experienced technicians in kidney transplantation.

The impact of donor-specific HLA alloantibodies (DSA) on short- and long-term liver transplant outcome is not clearly defined. While it is clear that not all levels of allosensitization produce overt clinical injury, and that liver allografts possess some degree of alloantibody resistance, alloantibody-mediated adverse consequences are increasingly being recognized. To better define the current state of this topic, we assembled experts to provide insights, explore controversies and develop recommendations for future research on the consequences of DSA in liver transplantation. This article summarizes the proceedings of this inaugural meeting. Several insights emerged. Acute antibody-mediated rejection (AMR), although rarely diagnosed, is increasingly understood to overlap with T cell-mediated rejection. Isolated liver allograft recipients are at increased risk of early allograft immunologic injury when preformed DSA are high titer and persist posttransplantation. Persons who undergo simultaneous liver-kidney transplantation are at risk of renal AMR when Class II DSA persist posttransplantation. Other under-appreciated DSA associations include ductopenia and fibrosis, plasma cell hepatitis, biliary strictures and accelerated fibrosis associated with recurrent liver disease. Standardized DSA testing and diagnostic criteria for both acute and chronic AMR are needed to distil existing associations into etiological processes in order to develop responsive therapeutic strategies.

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.

The significance of cytokine production by CD4(+) regulatory T (T reg) cells after antigen exposure in vivo and its impact on their regulatory activity remains unclear. Pretreatment with donor alloantigen under the cover of anti-CD4 therapy generates alloantigen reactive T reg cells that can prevent rejection of donor-specific skin grafts that are mediated by naive CD45RB(high)CD4(+) T cells. To examine the kinetics and importance of cytokine gene transcription by such alloantigen-reactive T reg cells, pretreated mice were rechallenged with donor alloantigen in vivo. CD25(+)CD4(+) T cells, but not CD25(-)CD4(+) T cells, showed a fivefold increase in IFN-gamma mRNA expression within 24 h of re-encountering alloantigen in vivo. This expression kinetic was highly antigen-specific and was of functional significance. Neutralizing IFN-gamma at the time of cotransfer of alloantigen reactive T reg cells, together with CD45RB(high)CD4(+) effector T cells into Rag(-/-) skin graft recipients, resulted in skin graft necrosis in all recipients; the generation and function of alloantigen-reactive T reg cells was impaired dramatically in IFN-gamma-deficient mice. These data support a unique role for IFN-gamma in the functional activity of alloantigen-reactive T reg cells during the development of operational tolerance to donor alloantigens in vivo.

Transplantation is the most effective treatment for end-stage organ failure, but organ survival is limited by immune rejection and the side effects of immunosuppressive regimens. T cells are central to the process of transplant rejection through allorecognition of foreign antigens leading to their activation, and the orchestration of an effector response that results in organ damage. Long-term transplant acceptance in the absence of immunosuppressive therapy remains the ultimate goal in the field of transplantation and many studies are exploring potential therapies. One promising cellular therapy is the use of regulatory T cells to induce a state of donor-specific tolerance to the transplant. This article first discusses the role of T cells in transplant rejection, with a focus on the mechanisms of allorecognition and the alloresponse. This is followed by a detailed review of the current progress in the field of regulatory T-cell therapy in transplantation and the translation of this therapy to the clinical setting.