Immune tolerance and transplantation

Transplanted individuals in operational tolerance (OT) maintain long term stable graft function after completely stopping immunosuppression. Understanding the mechanisms involved in OT can provide valuable information about pathways to human transplantation tolerance. Here we report that operationally tolerant individuals display quantitative and functional preservation of the B cell compartment in renal transplantation. OT exhibited normal numbers of circulating total B cells, naive, memory and regulatory B cells (Bregs), as well as a preserved B cell receptor repertoire, similar to healthy individuals. In addition, OT also displayed conserved capacity to activate the CD40/STAT3 signaling pathway in Bregs, in contrast, with chronic rejection. Rather than expansion or higher activation, we show that the preservation of the B cell compartment favors operational tolerance.

Significance Apoptotic cell-mediated suppression is critical to prevent inflammatory pathology and fatal autoimmunity. Integral to this process is early recognition by innate phagocytes driving downstream suppressive mechanisms. Though significant progress has been made identifying adaptive immune components involved in apoptotic cell-driven tolerance, early innate mechanisms involved in this process are relatively unknown. Here we report that apoptotic cell capture by CD169 + macrophages promotes rapid expression of the chemokine CCL22, inducing migration and activation of FoxP3 + Tregs and dendritic cells. Moreover, we found CCL22 function is required for generation of stable allograft tolerance and prevention of apoptotic cell-driven autoimmunity. Thus, our findings highlight a previously unknown mechanism whereby stromal macrophages coordinate early cellular interactions required for stable apoptotic cell-driven immune tolerance.

Achieving immunological tolerance in transplantation has been a long sought-after goal since the 1960s. It is, therefore, interesting that the dendritic cells (DC), which are classically known as the most potent stimulators of T cell activation, are now also considered putative tools for tolerance induction. In line with this, much work has been performed using DC for vaccination and immune stimulation. Recently, great interest has been generated regarding the ability of DC to act as immune regulatory cells. Specific subsets of DC or immature DC (iDC) appear to be responsible for maintaining self-tolerance. In this review we will highlight our efforts at elucidating the contribution of DC in transplant tolerant in mice. Specifically, four strategies will be outlined that are currently being used for the generation of DC that have tolerogenic properties in the prevention of allograft rejection. The present study demonstrates that modulated iDC with blunted T cell stimulatory or antigen presentation abilities can afford transplant tolerance by minimizing T cell activation and proinflammatory cytokine production. Moreover, in an alternate strategy, normally matured DC have also been modulated such that alloreactive T cells are specifically targeted for deletion.

An active role of T regulatory cells (Treg) and tolerogenic dendritic cells (Tol-DC) is believed important for the induction and maintenance of transplantation tolerance. However, interactions between these cells remain unclear. We induced donor-specific tolerance in a fully MHC-mismatched murine model of cardiac transplantation by simultaneously targeting T cell and DC function using anti-CD45RB mAb and LF 15-0195, a novel analog of the antirejection drug 15-deoxyspergualin, respectively. Increases in splenic Treg and Tol-DC were observed in tolerant recipients as assessed by an increase in CD4+CD25+ T cells and DC with immature phenotype. Both these cell types exerted suppressive effects in MLR. Tol-DC purified from tolerant recipients incubated with naive T cells induced the generation/expansion of CD4+CD25+ Treg. Furthermore, incubation of Treg isolated from tolerant recipients with DC progenitors resulted in the generation of DC with Tol-DC phenotype. Treg and Tol-DC generated ...

Tolerogenic dendritic cells (Tol-DCs) and regulatory T cells (Treg) are key factors in the induction and maintenance of transplantation tolerance. We previously demonstrated that ex vivo-isolated Tol-DCs promote Treg generation, and vice versa, in an in vitro co-culture system. Here we demonstrate the occurrence of such an immune regulatory feedback loop in vivo. Tol-DC generated in vitro by treatment with LF 15-0195 exhibited features of immature DC and express low levels of MHC class II, CD86 and CD40. These Tol-DCs were capable of augmenting CD4 + CD25 + CTLA4 + and FoxP3 + Treg cell numbers and activity in cardiac allograft recipients. On the other hand, Tol-DCs possessed an ability to generate Treg cells in vitro. The adoptive transfer of these in vitro-generated Treg cells resulted in an increase of Tol-DC in vivo, suggesting that an immune regulatory feedback loop, between Tol-DC and Treg, exists in vivo. Furthermore, the administration of in vitro-generated Tol-DCs or Treg cells prevented rejection of allografts. Coadministration of Tol-DC and Treg synergized efficacy of promoting allograft survival heart transplantation. The present study highlights the therapeutic potential of preventing allograft rejection using in vitro-generated Tol-DCs and Treg.

It's a retrospective study whose aim was to evaluate the incidence and the mortality in a population including 329 patients who received first kidney transplants from a living donor in 269 cases and cadaveric donor in 60 cases at Internal Medicine A department between June 1986 and December 2003. Aetiologies of mortality in our kidney transplant recipients were determined. There were 157 males and 75 females having an average age of 30.8 years. After a period of follow-up of 5.64 years, 51 patients (21.98%) died. Aetiologies of mortality were multiple and were known in approximately 98% of cases. Infections were observed in 25 cases. Cancer was observed in 7 cases (13.72%). Patient survival was not affected by gender, donor age or cause of donor death. Infections represent the major cause of mortality in our patients even many years after kidney transplantation. The maximum of death occurred in the 8th year after kidney transplantation.

In this review, we identify important challenges facing physicians responsible for renal and cardiac transplantation in children based on a review of the contemporary medical literature. Regarding pediatric renal transplantation, we discuss the challenge of antibody-mediated rejection, focusing on both acute and chronic antibody-mediated rejection. We review new diagnostic approaches to antibody-mediated rejection, such as panel-reactive antibodies, donor-specific cross-matching, antibody assays, risk assessment and diagnosis of antibody-mediated rejection, the pathology of antibody-mediated rejection, the issue of ABO incompatibility in renal transplantation, new therapies for antibody-mediated rejection, inhibiting of residual antibodies, the suppression or depletion of B-cells, genetic approaches to treating acute antibody-mediated rejection, and identifying future translational research directions in kidney transplantation in children. Regarding pediatric cardiac transplantation, we discuss the mechanisms of cardiac transplant rejection, including the role of endomyocardial biopsy in detecting graft rejection and the role of biomarkers in detecting cardiac graft rejection, including biomarkers of inflammation, cardiomyocyte injury, or stress. We review cardiac allograft vasculopathy. We also address the role of genetic analyses, including genome-wide association studies, gene expression profiling using entities such as AlloMapH, and adenosine triphosphate release as a measure of immune function using the CylexH ImmuKnow TM cell function assay. Finally, we identify future translational research directions in heart transplantation in children.

Background-Blocking CD40-CD40L costimulatory signals induces transplantation tolerance. While B cell depletion prevents alloantibody formation, non-humoral functions of B cells in tolerance have not been well characterized. We investigated whether specific subsets of B cell or B cell derived IL-10 contribute to tolerance. Methods-Wild type C57BL/6, or B cell specific IL-10 -/-(CD19-Cre +/-::IL-10 fl/fl ) mice, received vascularized BALB/c cardiac allografts. BALB/c donor-specific splenocyte transfusion (DST) and anti-CD40L mAb were used as tolerogen. B cells were depleted with anti-mouse CD20 mAb. Various B cell subsets were purified and characterized by flow cytometry, RT-PCR, and adoptive transfer. Results-B cell depletion prevented co-stimulatory blockade induced allogeneic tolerance. Costimulatory blockade increased IL-10 in marginal zone precursor (MZP) B cells, but not other subsets. In particular, costimulatory blockade did not change other previously defined regulatory B cell subsets (Breg), including CD5 + CD1d hi Breg or expression of TIM1 or TIM4 on these Breg or other Breg cell subsets. Costimulatory blockade also induced IL-21R expression in MZP B cells, and IL-21R + MZP B cells expressed even more IL-10. B cell depletion or IL-10 deficiency in B cells prevented tolerance in a cardiac allograft model, resulting in rapid acute cardiac allograft rejection. Adoptive transfer of wild type MZP B cells but not other subsets to B cell specific IL-10 deficient mice prevented graft rejection. costimulatory blockade induces MZP B cell IL-10 which is necessary for tolerance. These observations have implications for understanding tolerance induction and how B cell depletion may prevent tolerance.

B cells are known to control CD4T cell differentiation in secondary lymphoid tissues. We hypothesized that IL-10 expression by marginal zone precursor (MZP) regulatory B cells controls the differentiation and positioning of effector and regulatory T cells during tolerization. Costimulatory blockade with donor-specific transfusion (DST) and anti-CD40L mAb in C57BL/6 mice induced tolerance to allogeneic cardiac allograft. B cell depletion or IL-10 deficiency in B cells prevented tolerance, resulting in decreased follicular regulatory CD4(+) T cells (Tfr) and increased IL-21 expression by T follicular helper (Tfh) cells in the B cell and T cell zones. IL-21 acted with IL-6 to induce CCR6(+) Th17 that caused rejection. Deficiency or blockade of IL-6, IL-21, IL-21R, or CCR6 prevented B cell depletion-induced acute cellular rejection; while agonistic mCCL20-Ig induced rejection. Adoptive transfer of IL-10(+/+) MZP in tolerogen treated CD19-Cre(+/-):IL-10(fl/fl) mice rescued the localizati...

Blocking CD40-CD40L costimulatory signals induces transplantation tolerance. Although B-cell depletion prevents alloantibody formation, nonhumoral functions of B cells in tolerance have not been well characterized. We investigated whether specific subsets of B cell or B cell-derived interleukin (IL)-10 contribute to tolerance. Wild type C57BL/6, or B cell-specific interleukin (IL)-10 (CD19-Cre::IL-10) mice, received vascularized BALB/c cardiac allografts. BALB/c donor-specific splenocyte transfusion and anti-CD40L monoclonal antibody were used as tolerogen. B cells were depleted with antimouse CD20 monoclonal antibody. Various B-cell subsets were purified and characterized by flow cytometry, reverse transcription polymerase chain reaction, and adoptive transfer. B-cell depletion prevented costimulatory blockade-induced allogeneic tolerance. Costimulatory blockade increased IL-10 in marginal zone precursor (MZP) B cells, but not other subsets. In particular, costimulatory blockade di...

Transplantation is a cure for end-stage organ failure but, in the absence of pharmacological immunosuppression, allogeneic organs are acutely rejected. Such rejection invariably results in allosensitization and accelerated rejection of secondary donor-matched grafts. Transplantation tolerance can be induced in animals and a subset of humans, and enables long-term acceptance of allografts without maintenance immunosuppression. However, graft rejection can occur long after a state of transplantation tolerance has been acquired. When such an allograft is rejected, it has been assumed that the same rules of allosensitization apply as to non-tolerant hosts and that immunological tolerance is permanently lost. Using a mouse model of cardiac transplantation, we show that when Listeria monocytogenes infection precipitates acute rejection, thus abrogating transplantation tolerance, the donor-specific tolerant state re-emerges, allowing spontaneous acceptance of a donor-matched second transpl...

Highlights d DC-SIGN + macrophages inhibit CD8 + T cell proliferation and expand CD4 + Foxp3 + Treg d In vivo development of DC-SIGN + macrophages is regulated by IFN-g and CSF1 d IL-10 is essential for DC-SIGN + macrophage-mediated suppression d Simultaneous Fucose-DC-SIGN and HMGB1-TLR4 signaling is required for IL-10 production Authors

Embryonic stem cells (ESCs) hold promise for the treatment of many medical conditions; however, their utility is limited by immune rejection. The objective of our study is to establish tolerance or promote engraftment of transplanted ESCs as well as mature cell populations derived from ESCs. Luciferase (luc(+))-expressing ESCs were utilized to monitor the survival of the ESCs and differentiated progeny in living recipients. Allogeneic recipients conditioned with fractioned total lymphoid irradiation (TLI) and anti-thymocyte serum (ATS) or TLI plus regulatory T cells (Treg) promoted engraftment of ESC allografts after transplantation. Following these treatments, the engraftment of transplanted terminally differentiated endothelial cells derived from ESCs was also significantly enhanced. Our findings provide clinically translatable strategies of inducing tolerance to adoptively transferred ESCs for cell replacement therapy of medical disorders.

Background. Although graft-resident passenger leukocytes are known to mediate acute rejection by triggering direct allorecognition, they may also act in an immunomodulatory fashion and play an important role in tolerance induction. Our purpose in the current study was to utilize rat bone marrow chimeras to evaluate the role of the genotype of passenger leukocytes in both acute rejection and tolerance of liver allografts. Methods. The fate of livers bearing donor-type, recipient-type, and third-party passenger leukocytes was evaluated in the MHC class I and II mismatched rejector combination ACI3 LEW and the acceptor combination PVG3 DA. Results. We report that although treatment of ACI liver donors with lethal irradiation does not lead to prolongation of graft survival in the ACI3 LEW strain combination, ACI livers bearing recipient-type (LEW) or third-party passenger leukocytes (BN) are rejected at a significantly slower rate. We confirm that lethal irradiation of PVG donor animals leads to abrogation of tolerance induction with acute rejection of their livers by DA recipients. However, the majority of PVG livers carrying donor-type (PVG), recipient-type (DA), or third-party (LEW) passenger leukocytes are accepted for >100 days. These DA recipients develop immune tolerance to the donor parenchyma (PVG). Conclusions. Our findings demonstrate that longterm acceptance of liver allografts and tolerance induction is not dependent on the presence of donortype passenger leukocytes and can be achieved with organs carrying donor-type, recipient-type, or thirdparty passenger leukocytes. The importance of the MHC framework on the surface of passenger leukocytes as a critical regulator of the immune response after transplantation of chimeric organs is substantiated by the delayed tempo of rejection of ACI livers bearing recipient-type or third-party passenger leukocytes in the ACI3 LEW strain combination.

Five renal transplant recipients were preoperatively treated with transplant acceptance-inducing cells (TAICs) in a Phase-I safety study of TAICs as an adjunct immune-conditioning therapy in living-donor kidney transplantation. Initially, patients received anti-thymocyte globulin induction therapy in combination with tacrolimus and steroid immunosuppression. Over the course of 12 weeks, steroids were withdrawn and tacrolimus therapy was minimized. Three of the five patients were able to tolerate low-dose tacrolimus monotherapy and one patient was withdrawn from all immunosuppression for over 8 months. No acute or delayed adverse events were associated with the infusion of TAICs. Monitoring of the recipient anti-donor reactivity of TAIC-treated patients in mixed lymphocyte cultures demonstrated that, during periods of clinically stable graft function, recipient T-cell proliferation and cytokine secretion in response to stimulation with donor alloantigen was relatively suppressed. Therefore, although the TAIC-II trial did not provide conclusive evidence of a beneficial effect of preoperative TAIC treatment, the results were encouraging because they suggest that TAICs promote a state of alloantigenspecific unresponsiveness, which might allow safe minimization of pharmacological immunosuppression.

Pre-transplant screening of a woman with end-stage renal disease (ESRD) showed no anti-human leukocyte antigen (HLA) alloantibodies by anti-human globulin-complement-dependent cytotoxicity (AHG-CDC; class I) or enzyme-linked immunosorbent assay (class II). Following a negative AHG-CDC crossmatch, an HLA*01: 01ϩ deceased donor (DD) kidney was transplanted in September 2005. Subsequent screening of pretransplant serum by LABScreen Single Antigen (SA) array showed strong reactivity versus A*01:01. Despite that reactivity, at 5 years post-transplant, the patient has a serum creatinine of 1.6 mg/dl and has never experienced humoral or cellular rejection. Retrospective flow-cytometric crossmatch of pre-and posttransplant sera versus DD cells was negative. Rescreening of multiple pre-and post-transplant sera revealed anti-A1 reactivity persisting from the first through the last samples tested. The patient's anti-A1 was almost two fold more reactive with denatured A*01:01 FlowPRA SA beads after denaturation with acid treatment (pH 2.7) than with untreated beads. Parallel results were observed with pH 2.7 treated versus untreated A1ϩ T cells in FXM. These data highlight the difficulty in interpreting screening results obtained using bead arrays, because of antibodies that appear to recognize denatured but not native class I HLA antigens. We suggest that such bead-positive, flow cytometric crossmatch negative antibodies are not associated with humoral rejection, may not necessarily be detrimental to a graft, and deserve further evaluation before becoming a barrier to transplantation.

C5aR2 (C5L2/gp77) is a seven-transmembrane spanning receptor that binds to C5a but lacks motifs essential for G protein coupling and associated signal transduction. C5aR2 is expressed on immune cells, modulates various inflammatory diseases in mice, and has been shown to facilitate murine and human regulatory T cell (T) generation in vitro. Whether and how C5aR2 impacts in vivo Tgeneration and pathogenic T cell-dependent disease models have not been established. In this article, we show that murine T cells express and upregulate C5aR2 during induced T(iT) generation and that the absence of T cell-expressed C5aR2 limits in vivo iTgeneration following adoptive transfer of naive CD4T cells intorecipients. Using newly generated C5aR2-transgenic mice, we show that overexpression of C5aR2 in naive CD4T cells augments in vivo iTgeneration. In a model of T-dependent cardiac allograft survival, recipient C5aR2 deficiency accelerates graft rejection associated with lower T/effector T cell rat...

Although certain chemokines and their receptors guide homeostatic recirculation of T cells and others promote recruitment of activated T cells to inflammatory sites, little is known of the mechanisms underlying a third function, migration of Foxp3+ regulatory T (T reg) cells to sites where they maintain unresponsiveness. We studied how T reg cells are recruited to cardiac allografts in recipients tolerized with CD154 monoclonal antibody (mAb) plus donor-specific transfusion (DST). Real-time polymerase chain reaction showed that intragraft Foxp3 levels in tolerized recipients were ∼100-fold higher than rejecting allografts or allografts associated with other therapies inducing prolonged survival but not tolerance. Foxp3+ cells were essential for tolerance because pretransplant thymectomy or peritransplant depletion of CD25+ cells prevented long-term survival, as did CD25 mAb therapy in well-functioning allografts after CD154/DST therapy. Analysis of multiple chemokine pathways showed...

To prevent graft rejection and avoid immunosuppression-related side-effects, we attempted to induce recipient chimerism and graft tolerance in islet transplantation by donor CD34+hematopoietic stem cell (HSC) infusion. Six patients with brittle type 1 Diabetes Mellitus received a single-donor allogeneic islet transplant (8611 ± 2113 IEQ/kg) followed by high doses of donor HSC (4.3 ± 1.9 × 10 6 HSC/kg), at days 5 and 11 posttransplant, without ablative conditioning. An 'Edmonton-like' immunosuppression was administered, with a single dose of anti-TNFa antibody (Infliximab) added to induction. Immunosuppression was weaned per protocol starting 12 months posttransplant. After transplantation, glucose control significantly improved, with 3 recipients achieving insulin-independence for a short time (24 ± 23 days). No severe hypoglycemia or protocol-related adverse events occurred. Graft function was maximal at 3 months then declined. Two recipients rejected within 6 months due to low immunosuppressive trough levels, whereas 4 completed 1-year follow-up with functioning grafts. Graft failure occurred within 4 months from weaning (478 ± 25 days posttransplant). Peripheral chimerism, as donor leukocytes, was maximal at 1month (5.92 ± 0.48%), highly reduced at 1-year (0.20 ± 0.08%), and was undetectable at graft failure. CD25+Tlymphocytes significantly decreased at 3 months, but partially recovered thereafter. Combined islet and HSC allotransplantation using an 'Edmonton-like' immunosuppression, without ablative conditioning, did not lead to stable chimerism and graft tolerance.

Composite tissue allotransplantation holds a great potential for providing increased knowledge of anatomy and microsurgical experience for life-enhancing reconstructions. Many transplant cases around the world have made this a clinical reality at the present time. Composite tissue allotransplants contain multiple tissue types, including bone, muscle, vessels, nerves, skin, and immune cells and bear a huge antigenic load. Although immunosuppressive drugs are applied successfully to prevent allograft rejection, their side effects pose a barrier to worldwide use. Bone marrow therapy in many tolerance induction protocols, therefore, provides a guide to reaching the target of permanent immunotolerance. Multiple studies suggest that bone marrow is immunomodulatory and may facilitate allograft acceptance. In this review, bone marrow based therapy protocols of clinical and experimental models are presented in two major categories: solid organ and composite tissue transplantation.

Solid organ transplantation has become a clinical practice after the development of different immunosuppressive drugs that allowed controlling rejection. The price to be paid for that is the permanent risk of infections and malignancies and a significant drug-associated toxicity. The establishment of transplant tolerance has been the "holy grail" for transplantation medicine since its beginnings. Different experimental approaches and clinical trials resulted in the accumulation of knowledge on mechanisms and strategies that favor the establishment of tolerance without achieving the objective of autonomous allograft tolerance in the clinical field. Development of tolerance in intestinal transplantation constitutes a challenging situation due to several particular features that contribute to the generation of a strong allogeneic response. In the present review, we summarize the different immune mechanisms that may contribute to allograft tolerance. The different barriers that should be bypassed in intestinal transplantation to tolerate the graft are discussed. Finally, we revise the strategies that were applied with different degrees of success in the clinical field including the most promising recent approaches and the forthcoming candidates in the field that might be translated into clinical trials in the near future.

The transplant acceptance-inducing cell (TAIC) is a type of immunoregulatory macrophage with the capacity to specifically dampen allogeneic rejection responses to a degree allowing safe minimization of conventional immunosuppressive therapy. In the first part of this report, the production and phenotype of the human TAIC is described. In the second part, an analysis is given of the TAIC-I clinical trial, in which 12 recipients of renal transplants from deceased donors were treated with donor-derived TAICs as an adjunct immune-conditioning therapy. Conventional immunosuppression was gradually withdrawn from 10 of these 12 patients over a period of 8 weeks, starting in the fourth week after transplantation. All but two patients tolerated cessation of steroid therapy, while the remaining eight patients were first weaned from sirolimus and then, in six cases, were also weaned to low-dose tacrolimus monotherapy. It is concluded that TAIC therapy is both safe and clinically practicable; however, the TAIC-I trial was unable to provide evidence that postoperative TAIC administration has a beneficial effect.

This report describes the case of patient FR, a 31-year-old recipient of a livingrelated kidney transplant from a donor against whom he was presensitized. Seventeen days prior to transplantation, a central venous infusion of transplant acceptance-inducing cells (TAICs) was administered to the patient. During the 27-month follow up, the patient experienced no acute rejection episodes under an immunosuppressive regime comprising anti-thymocyte globulin (ATG) induction, corticosteroids and tacrolimus. In a similar manner to the kidney transplant recipients treated preoperatively with TAICs in a previous study, patient FR achieved a state of donor-specific hypo-responsiveness. Most remarkably, the deliberate preoperative exposure of a sensitized patient to the sensitizing alloantigen did not heighten his response; on the contrary, after TAIC treatment and transplantation, HLA-specific antibodies were no longer detectable. The case of patient FR provides further evidence of the safety of pre-transplantation treatment with TAICs.

Background: Management of congenital limb aplasia or facial malformations could be improved by composite tissue allotransplantation (CTA), a technique that has never been performed in newborns. For this, however, the induction of donor-specific tolerance would be mandatory, as long-term immunosuppression is not acceptable in this non-lifesaving procedure. Induction of tolerance has been shown to be possible in a newborn CTA rat model but has never been tested in large-animal models. Our goals were to establish a model of CTA in newborn swine to see if tolerance could be obtained without immunosuppression and to assess rejection or tolerance properties via clinical and histologic examinations. We applied a CTA heterotopic knee swine model. We performed two series of surgical procedures: Series 1 was 20 autografts in 6-day-old (1e10) 2,544 kg (1,140e4,060 kg) piglets; Series 2 was 10 allografts without immunosuppression between outbred animals aged 7.8 d (6e10) and weighing 2,770 kg (2,200e3,550 kg). In Series 1, six early deaths and two cases of vascular failure were observed. In Series 2, no spontaneous deaths were observed and all piglets presented clinical and histologic rejection. Conclusions: Our findings strongly suggest that newborn immunologic status is not sufficient for the development of tolerance in large animals without immunologic intervention. Complications and animal death after transplantation correlate with age and weight. Low rates for both vascular failure and postoperative death permit the use of this model in piglets weighing over 2 kg and aged more than 6 d for research on newborn CTA.

Background. The identity of the cells in the human bone marrow that function as effective regulators of in vitro and possibly in vivo cellular immune responses is not well established. Methods. Cell subpopulations were isolated from cadaver donor vertebral-body bone marrow cells (DBMC) by using immuno-magnetic microbeads and were tested as inhibitors (modulators) in cell-mediated lympholysis (CML) and mixed lymphocyte reaction (MLR) responses of normal peripheral blood lymphocytes stimulated with irradiated cadaver donor spleen cells. The inhibition was also mediated by a number of purified subpopulations including pluripotent CD34 ؉ stem cells, and their CD34 negative early progeny of both lymphoid and myeloid lineages. These included DBMC enriched for non-T-cell lymphoid precursors (NT-LP/DBMC; i.e., DBMC depleted of CD3, CD15, and glycophorin-A positive cells) and DBMC positively selected for CD38 ؉ , CD2 ؉ , CD5 ؉ , and CD1 ؉ lymphoid cells (all were depleted of CD3 ؉ cells) as well as CD33 ؉ (but CD15 negative) myeloid precursors. However, positively selected CD19 ؉ B-cells and CD15 ؉ myeloid cells did not inhibit the MLR and CML responses. The NT-LP/DBMC that had been repeatedly stimulated with irradiated allogeneic peripheral blood lymphocytes caused the strongest inhibition of the MLR and CML responses of the same allogeneic cells with 200 times fewer modulator cells needed than uncultured DBMC (P<0.001). Flow cytometric analysis revealed that majority of cells in these cell lines had become CD3 ؉ TcR-␣␤ ؉ CD4 ؉ and CD28 ؉ cells. are immunoregulatory in vitro. Among them, there is at least one subset that can undergo differentiation in vitro into regulatory T cells that can be maintained in long-term cultures.

Background. Even though a number of transplant centers have adopted donor-specific bone marrow cell (DBMC) infusions to enhance donor cell chimerism, to date there has been no direct evidence linking chimerism with tolerance induction in human organ transplant recipients. Methods. Cells of donor phenotype were isolated 1 year postoperatively from the peripheral blood lymphocytes and iliac crest bone marrow of 11 livingrelated-donor (LRD) renal transplant recipients, who had received perioperative donor bone marrow cell infusions. These recipient-derived donor (RdD) cells were characterized phenotypically by flow cytometric analysis and functionally as modulators in mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML) assays. Results. The yield of RdD cells ranged from 0.1 to 0.9% of the starting material with the majority being TcR␣␤, CD3 positive T cells, a substantial percentage of which coexpressed CD28. At 1 year posttransplant almost 50% of the LRD-kidney/DBMC recipients tested so far exhibited donor-specific unresponsiveness in MLR (7/17) and CML (6/13) reactions and this trend was further enhanced at 2-3 years. In the recipients with residual positive antidonor immune responses, the RdD cells inhibited recipient antidonor MLR and CML responses significantly more strongly than freshly isolated and similarly treated iliac crest bone marrow cells from the donor. RdD cells also inhibited the MLR of the recipient to third party allogeneic stimulator cells; however, this nonspecific effect was significantly weaker than specific inhibition. We also established long-term bone marrow cultures stimulated every 2 weeks with irradiated allogeneic feeder cells, that had similar functional properties thus possibly providing us with an in vitro correlate the RdD cells. Conclusions. These results clearly support the notion that the infused donor cells play a positive role in the induction and/or maintenance of transplant tolerance.

Organ transplantation has become an accepted and successful therapeutic intervention for many patients with end-stage organ disease. Current conventional immunosuppressive regimens achieve very good short-term allograft survival but long-term outcomes are less than adequate. Furthermore, non-speci¢c immunosuppression has its attendant side-e¡ects including increased risks of malignancy and infection as well as drug-speci¢c sequellae. With recent advances in the ¢eld of immunology, promising new therapies have arisen that could potentially eliminate lifelong drug therapy and promote inde¢nite acceptance of the donor tissue. Identi¢cation of co-stimulatory signals essential for T-cell activation has provided exciting new possibilities for controlling the alloimmune response. The compatibility of these new agents with proven conventional therapeutics has yielded mixed results. When used in combination, their immunosuppressive properties appear synergistic. However, if the goal of therapy is sustained, speci¢c T-cell hyporesponsiveness, many conventional agents antagonize the e¡ects of co-stimulatory blockade in several immune tolerance models.

Recent clinical trials have pioneered the successful use of a nonsteroidal immunosuppressive regimen and established a basis for application in a routine clinical setting. In this study, a single islet transplant was not sufficient to regulate blood glucose levels, and a second transplant became necessary. A similar observation was made in our macaque islet transplant study, where animals after the second transplantation have shown trends towards normoglycemia in the presence of mycophenolate mofetil. All five animals that received the second transplant have shown an initial rise in C peptide levels, which rapidly decreased as we tapered the MMF dose from 20 mg/kg BID to 5 mg/kg SID. Two animals of the five that were preconditioned with MMF one week prior to transplantation have shown significantly higher C peptide levels. We believe that it is very important to understand the relationship between the first graft failure and subsequent islet allograft success. Since graft success did not correlate with number of transplanted islets, the correction of blood glucose levels toward normoglycemia after the second transplantation suggests a mechanism by which the allotolerance to second transplant is facilitated by the first islet transplantation. These initial observations suggest approaches to "tolerize" the recipient to accept the second-transplant islets (a) through preconditioning the animal to improve the rate of success for the first transplant or (b) through tolerization to islets in the first transplant to facilitate better engraftment of the second-transplant islets.

An active role of T regulatory cells (Treg) and tolerogenic dendritic cells (Tol-DC) is believed important for the induction and maintenance of transplantation tolerance. However, interactions between these cells remain unclear. We induced donor-specific tolerance in a fully MHC-mismatched murine model of cardiac transplantation by simultaneously targeting T cell and DC function using anti-CD45RB mAb and LF 15-0195, a novel analog of the antirejection drug 15-deoxyspergualin, respectively. Increases in splenic Treg and Tol-DC were observed in tolerant recipients as assessed by an increase in CD4+CD25+ T cells and DC with immature phenotype. Both these cell types exerted suppressive effects in MLR. Tol-DC purified from tolerant recipients incubated with naive T cells induced the generation/expansion of CD4+CD25+ Treg. Furthermore, incubation of Treg isolated from tolerant recipients with DC progenitors resulted in the generation of DC with Tol-DC phenotype. Treg and Tol-DC generated ...

Our previous in vitro data have demonstrated that regulatory mechanisms are involved in tolerance of class I-mismatched renal allografts in miniature swine treated with 12 days of high dose Cyclsporin A. In this study, we attempted to induce tolerance of class I-mismatched kidneys by adoptive transfer of cells and/or kidneys from long-term tolerant animals. Fifteen SLA dd miniature swine received 1.5 Gy whole body irradiation and class I-mismatched (SLA gg) kidneys from naïve pigs with or without cotransplanted kidneys and/or adoptively transferred cells from longterm tolerant (LTT) SLA dd recipients of SLA gg grafts. In addition, three SLA dd miniature swine received class I mismatched kidney with adoptively transferred cells from LTT SLA dd recipients. Naïve kidneys transplanted without a LTT kidney were rejected within 9 days. All recipients of naive kidneys along with cells and kidney grafts from LTT animals showed markedly prolonged survival of the naive renal grafts (day 28, >150 and >150 days). These studies suggest that (1) tolerated kidneys have potent regulatory effects and (2) cells from LTT animals infused in conjunction with kidney grafts augment these regulatory effects. To our knowledge, these studies represent the first demonstration of successful adoptive transfer of tolerance in large animals.

Extrathymically induced Foxp3+ regulatory T (Treg) cells contribute to the pool of Treg cells and are implicated in the maintenance of immune tolerance at environmental interfaces. The impact of T‐cell senescence on their generation and function is, however, poorly characterized. We report here that steady‐state induction of Foxp3 is impaired in aged T cells in vivo. In vitro assays further revealed that this defective generation of Treg cells was independent from the strength of TCR stimulation and arose before T‐cell proliferation. Importantly, they also revealed that this impairment of Foxp3 induction is unrelated to known age‐related T‐cell defects, such as IL‐2 secretion impairment, accumulation of activated T‐cell populations, or narrowing of the T‐cell repertoire. Finally, a loss of extrathymic induction of Foxp3 and tolerance to minor‐mismatched skin graft were observed in aged mice treated by nondepleting anti‐CD4 antibody. The T‐cell intrinsic impairment of Treg‐cell gener...

Allograft rejection is induced by graft tissue infiltration of alloreactive T cells that are activated mainly in secondary lymphoid organs of the host. DOCK2 plays a critical role in lymphocyte homing and immunological synapse formation by regulating the actin cytoskeleton, yet its role in the in vivo immune response remains unknown. We show here that DOCK2 deficiency enables long-term survival of cardiac allografts across a complete mismatch of the major histocompatibility complex molecules. In DOCK2-deficient mice, alloreactivity and allocytotoxicity were suppressed significantly even after in vivo priming with alloantigens, which resulted in reduced intragraft expression of effector molecules, such as interferon-γ, granzyme B, and perforin. This is mediated, at least in part, by preventing potentially alloreactive T cells from recruiting into secondary lymphoid organs. In addition, we found that DOCK2 is critical for CD28-mediated Rac activation and is required for the full activ...

There has been significant progress in the field of heart transplantation over the last 45 years. The 1-yr survival rates following heart transplantation have improved from 30% in the 1970s to almost 90% in the 2000s. However, there has been little change in long-term outcomes. This is mainly due to chronic rejection, malignancy, and the detrimental side effects of chronic immunosuppression. In addition, over the last decade, new challenges have arisen such as increasingly complicated recipients and antibody-mediated rejection. Most, if not all, of these obstacles to long-term survival could be prevented or ameliorated by the induction of transplant tolerance wherein the recipient&#39;s immune system is persuaded not to mount a damaging immune response against donor antigens, thus eliminating the need for chronic immunosuppression. However, the heart, as opposed to other allografts like kidneys, appears to be a tolerance-resistant organ. Understanding why organs like kidneys and liv...

Operational tolerance after liver transplantation has been described in around 20% of the recipients. These patients are able to maintain a normal graft function in the absence of immunosuppressive drugs, thus being free of adverse effects that are common and frequently severe. Here we present a well-documented case of operational tolerance after liver transplantation and discuss current concepts on this topic with emphasis on recent findings that will potentially allow for identifying graft-tolerant patients.

Administration of donor-derived immature dendritic cells (DC) treated with transforming growth factor-beta (TGF-β) to prevent allograft rejection is not applicable for clinical use. We therefore attempted to explore the use of recipient-derived DC pulsed with donor antigens via the indirect pathway (cross-priming). DC were propagated from C3H (H2 k) bone marrow (BM) with GM-CSF and IL-4. TGF-β (0.2ng/ml) was added at the initiation of culture and the resultant (TGF-β DC) were pulsed with B10 (H2 b) splenocyte lysate. Expression of MHC class I and II were not affected, while CD40, CD80 and CD86 co-stimulatory molecules on DC was significantly inhibited by treatment with TGF-β. C3H DC pulsed with B10 antigens stimulated proliferate responses in C3H T cells was inhibited when DC were treated with TGF-β, and the CTL activity was also inhibited. This correlated with reduced IFN-γ and increased IL-10 production. A single injection of TGF-β DC prolonged allograft survival (MST 18 days vs. 10 days in no-DC treatment control, p<0.05). These data indicate that an approach utilizing recipient DC as a "vaccine" strategy is possible.

The prevalent renal transplant population presents an opportunity to observe the adaptive changes in the alloimmune response over time, but such studies have been limited by uncertainties in the conventional biopsy diagnosis of T cell-mediated rejection (TCMR) and antibody-mediated rejection (ABMR). To circumvent these limitations, we used microarrays and conventional methods to investigate rejection in 703 unselected biopsies taken 3 days to 35 years post-transplant from North American and European centers. Using conventional methods, we diagnosed rejection in 205 biopsy specimens (28%): 67 pure TCMR, 110 pure ABMR, and 28 mixed (89 designated borderline). Using microarrays, we diagnosed rejection in 228 biopsy specimens (32%): 76 pure TCMR, 124 pure ABMR, and 28 mixed (no borderline). Molecular assessment confirmed most conventional diagnoses (agreement was 90% for TCMR and 83% for ABMR) but revealed some errors, particularly in mixed rejection, and improved prediction of failure. ABMR was strongly associated with increased graft loss, but TCMR was not. ABMR became common in biopsy specimens obtained .1 year post-transplant and continued to appear in all subsequent intervals. TCMR was common early but progressively disappeared over time. In 108 biopsy specimens obtained 10.2-35 years post-transplant, TCMR defined by molecular and conventional features was never observed. We conclude that the main cause of kidney transplant failure is ABMR, which can present even decades after transplantation. In contrast, TCMR disappears by 10 years post-transplant, implying that a state of partial adaptive tolerance emerges over time in the kidney transplant population.

We have reported a beneficial effect of donor vertebral body bone marrow cells (DBMC) infusions in cadaver renal allograft recipients in a 6-year follow-up, but with a transient increase in early (6 month) postoperative CMV infections and concomitant suppressed immunoglobulins (Ig) production. We also found that although there was no difference between the DBMC-infused and non-infused (control) groups in the development of donor-specific antibody, we now describe an additional difference seen in the percent reactive antibody (PRA) reactivity against a panel of HLA antigens that developed postoperatively. We hypothesize that (allogeneic) antigen presenting cells in the DBMC, systemically infused, caused the generation of recipient T suppressor (T4-suppressor) cells, thereby "inducing" a negative influence on B cell Ig production. We tested this notion in vitro by incubating PBL from CMV IgG positive laboratory volunteers with either (allogeneic) T-cell depleted DBMC or donor spleen cells (DSPC) from (the same) cadaver donors. After 7 days, the (responding) T cells were collected using magnetic beads and placed in culture with purified B cells freshly obtained from the same (autologous) CMV positive volunteer. To these cultures were added either media or 40 ng of CMV antigen. After 3, 5, 7, and 9 days, the expression of surface anti-CMV Ig was measured by flow cytometry using a panel of fluorescent markers double-labeled for activated B cells (CD20, CD19, and HLA DRw) and CMV-FITC. We also determined the phenotype of the cultured T cells using anti-CD3, CD4, and CD62L specific monoclonal antibodies. B cells that had been in contact with autologous T cells derived from DBMC cultures (T BM) were less likely to express anti-CMV surface Ig than those cultured with DSPC (T SP). The flow cytometry analysis revealed an increase in the number of T4 suppressor cells (CD3 ϩ , CD4 ϩ , CD62L ϩ) in the T BM group, whereas the T4 helper phenotype (CD3 ϩ , CD4 ϩ , CD62L Ϫ) predominated in the T SP group. These in vitro findings support the notion that (allogeneic) DBMC infusions can induce a T4 suppressor (regulatory) influence and thereby indirectly affect B-cell function.

Amputations and unsalvageable injuries with devastating tissue loss are common in the combat wounded. Reconstructive transplantation in the civilian setting using vascular composite allotransplants (VCAs) with multiple tissues (skin, muscle, nerve, bone) combined with long-term multidrug immunosuppression has been encouraging. However, skin rejection remains a critical complication. Adipose-derived stromal/stem cells (ASCs) are easily obtained from normal individuals in high numbers, precluding ex vivo expansion. The reparative function and paracrine immunomodulatory capacity of ASCs has gained considerable attention. The present study investigated whether ASCs facilitate long-term skin allograft survival. ASCs were isolated from fresh human subcutaneous adipose lipoaspirate. Full-thickness skin grafts from BALB/c mice were transplanted onto the dorsal flanks of C57BL/6 mice treated with five doses of anti-CD4/CD8 monoclonal antibodies (10 mg/kg) on days 0, +2, +5, +7, and +14 relat...

OBJECTIVEThe objective of this study was to determine whether tolerance to neonatal porcine islet (NPI) xenografts could be achieved by short-term administrations of anti–LFA-1 and anti-CD154 monoclonal antibodies (mAbs).RESEARCH DESIGN AND METHODSDiabetic B6 mice received NPI transplants and short-term injections of combined anti–LFA-1 and anti-CD154 mAbs. Mice with long-term islet graft function were treated with depleting anti-CD25 mAb or re-transplanted with a second-party NPI. At the end of the study, grafts from mice with long-term islet function were examined. Their spleen cells were characterized and used for in vitro proliferation and adoptive transfer studies.RESULTSAll mAb-treated NPI recipients maintained normoglycemia for &gt;100 days post-transplantation. Only 5 of 50 mice rejected their grafts before 300 days post-transplantation. Intact islets, foxp3+ immune cells, as well as interleukin (IL)-10 and transforming growth factor (TGF)-β regulatory cytokine transcripts w...

We have previously shown that tolerance of kidney allografts across a full major histocompatibility complex (MHC) barrier can be induced in miniature swine by a 12-day course of high-dose tacrolimus. However, that treatment did not prolong survival of heart allografts across the same barrier. We have now tested the effect of cotransplanting an allogeneic heart and kidney from the same MHC-mismatched donor using the same treatment regimen. Heart allografts (n = 3) or heart plus kidney allografts (n = 5) were transplanted into MHC-mismatched recipients treated with high-dose tacrolimus for 12 days. As expected, all isolated heart allografts rejected by postoperative day 40. In contrast, heart and kidney allografts survived for >200 days with no evidence of rejection on serial cardiac biopsies. Heart/kidney recipients lost donor-specific responsiveness in cell-mediated lympholysis and mixed-lymphocyte reaction assays, were free of alloantibody and exhibited prolonged survival of donor, but not third-party skin grafts. Late (>100 days) removal of the kidney allografts did not cause acute rejection of the heart allografts (n = 2) and did not abrogate donor-specific unresponsiveness in vitro. While kidney-induced cardiac allograft tolerance (KI-CAT) has previously been demonstrated across a Class I disparity, these data demonstrate that this phenomenon can also be observed across the more clinically relevant full MHC mismatch. Elucidating the renal element(s) responsible for KICAT could provide mechanistic information relevant to the induction of tolerance in recipients of isolated heart allografts as well as other tolerance-resistant organs.

Current immunosuppressive medications used after transplantation have significant toxicities. Foxp3 + T-regulatory (Treg) cells can prevent allograft rejection without compromising protective host immunity. Interestingly, inhibiting the class III histone/protein deacetylase Sirtuin-1 can augment Foxp3 + Treg suppressive function through increasing Foxp3 acetylation. Here we determined whether Sirtuin-1 targeting can stabilize biological allograft function. BALB/c kidney allografts were transplanted into C57BL/6 recipients with a CD4-conditional deletion of Sirtuin-1 (Sirt1 fl/fl CD4 cre) or mice treated with a Sirtuin-1 specific inhibitor (EX-527), and the native kidneys removed. Blood chemistries and hematocrit were followed weekly. Sirt1 fl/fl CD4 cre recipients showed markedly longer survival and improved kidney function. Sirt1 fl/fl CD4 cre recipients exhibited donor specific tolerance, accepted BALB/c, but rejected third-party C3H cardiac allografts. C57BL/6 recipients of BALB/c renal allografts that were treated with EX-527 showed improved survival and renal function at 1, but not 10 mg/kg/day. Pharmacologic inhibition of Sirtuin-1 also improved renal allograft survival and function with dosing effects having relevance to outcome. Thus, inhibiting Sirtuin-1 can be a useful asset in controlling T-cell mediated rejection. However, effects on non-T cells that could adversely affect allograft survival and function merit consideration.

Toll-like receptors (TLRs) belong to a family of pattern-recognition receptors for microbial products and endogenous molecules released by stressed cells. Experimental studies show that TLRs are involved in the process of acute allograft rejection and that their activation can prevent transplantation tolerance. Herein, we review the expression of TLRs and the impact of TLR signaling in different cell types in grafted organs including antigen-presenting cells, T and B lymphocytes, epithelial and endothelial cells. We then discuss the involvement of TLRs in the different phases of the rejection phenomenon and the impact of TLR-mediated events on regulatory circuits which dampen alloimmune responses.