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Background: Draining lymph nodes (DLNs) are implicated in alloimmunity and tolerance regulation. The role of DLNs in human organ transplant is unknown due to the lack of relevant clinical tissues., Methods: During a combined kidney and pancreas transplant, the distal vena cava and both right and left common and external iliac vessels were mobilized and exposed. Draining lymph nodes along these vessels were removed sequentially and archived before the pancreas transplant. Similarly, the DLNs along the left iliac vessels were removed and archived before implantation of the kidney allograft., Results: Among 13 patients undergoing kidney and pancreas transplants, 6 had pre- and postreperfusion DLNs clinically archived. The remaining 7 either had only prereperfusion DLNs archived or none. Clinical archiving of DLNs did not alter operative times or parameters., Conclusion: This study describes an approach for clinical archiving of DLNs obtained within the operative field during combined kidney-pancreas transplant in humans. The availability of relevant DLNs is essential for investigating fundamental initial primary immune responses potentially important in allograft alloimmunity and tolerance., Competing Interests: Declaration of Competing Interest All the authors declare no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Renal transplantation is the most effective treatment method for end-stage renal disease (ESRD). However, new treatment modalities are being investigated, such as immunotoleration, to avoid the acute and chronic side effects of immunosuppressant drugs. We report a case in which a man had undergone allogenic stem cell transplantation from his brother 16 years ago due to chronic myeloid leukemia, and who then developed ESRD due to arterial hypertension and underwent renal transplantation (Rtx) from the same brother. The patient was followed up without immunosuppression due to full chimerism., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: To verify whether mesenchymal stem cells protected the islet allograft via modulating follicular B helper T cells (Tfh) cells., Methods: The recipient mice were divided into 5 groups. Group A: the diabetic group (n = 12); Group B: islets alone group (n = 12); Group C: MSCs and islets co-transplanted group (n = 12, MSCs = 0.5 × 10(6)); Group D: MSCs and islets co-transplanted group (n = 12, MSCs = 1 × 10(6)); Group E: MSCs and islets co-transplanted group (n = 12, MSCs = 2 × 10(6)); One control group of normal NOD mice was set as well. ELISA was used to examine the autoantibody level of GAD65 Ab, insulin autoantibodies, and insulin. The Tfh count was determined by fluorescence-activated cell sorting. The insulin expression of islet grafts, the infiltration of lymphocytes, and the Tfh cells were observed via hematoxylin and eosin staining and immunohistochemical staining., Results: There was significant prolonged graft survival and more insulin expression of islet grafts observed in the co-transplant group. A lower level of the Tfh cells and autoantibody GAD65 Ab, insulin autoantibodies were also presented in the co-transplant group (P < .01). The infiltration of lymphocytes in the co-transplant group was notably less than in the islet-alone group (P < .01)., Conclusions: Mesenchymal stem cells were able to protect the islet allograft by regulating the follicular helper T cells., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Galectin-9 (Gal-9), a member of the galectin family, has a variety of biologic activities. However, its role in allografts is not fully clarified yet. The relationship between interleukin-17 (IL-17) and Gal-9 and the role of Gal-9 in T(H)17-cell differentiation also remain unclear. We built a murine cardiac transplantation model, which we treated with Gal-9 and/or EX-527, a specific Sirtuin-1 inhibitor. Afterwards, flow-cytometric analysis and reverse-transcription polymerase chain reaction were used to detect the number of T(H)17 cells and the expression of key factors involved in the differentiation of T(H)17 cells; in addition, the survival times of cardiac transplanted mice in different groups were recorded. The levels of circulating T(H)17 cells were found to increase in the peripheral blood of mice that exhibited acute rejection (AR) after heart transplantation, which was determined to be correlated with the rejection grade. Gal-9 or EX-527 can inhibit the activation and differentiation of T(H)17 cells and effectively suppress T(H)17-cell-mediated AR. These data provide new evidence for the potential regulatory effects of Gal-9 in alloimmune responses in a murine model of heart transplantation, and suggest the combined use of galectin-9 and EX-527 may be a powerful method to induce tolerance of fully mismatched murine cardiac allografts., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Introduction: The achievement of a state of tolerance and minimization of the immunosuppressive load form part of the "Holy Grail" in solid organ transplantation. Galectin-1 recently has been described to be involved in the maintenance of a tolerant environment, but there is no evidence of its role in human liver transplantation. The aim of our study was to measure the serum levels of galectin-1 in stable liver transplant recipients., Methods: Serum levels of galectin-1 were determined in 30 stable liver transplant recipients who had been free of rejection episodes for at least 8 years. Fifteen patients with an acute rejection episode and 34 healthy subjects were used as the control group., Results: The concentrations of galectin-1 were significantly higher in stable liver transplant recipients compared with healthy subjects and with the acute rejection group., Conclusions: These preliminary results indicate that galectin-1 is upregulated in stable liver transplant recipients. Thus, our results extend the recent findings that galectin-1 may play an immune-suppressive role in liver transplantation. It remains to be established whether it might help to induce tolerance in liver transplantation., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Long-term function of living-related kidney allograft depends on multiple variables. The aim of the present study was to assess the influence of donor and recipient gender mismatch on the short and long-term outcomes in human leukocyte antigen (HLA)-identical sibling renal transplants (SRTs) receiving induction therapy and different immunosuppressive regimens., Material and Methods: Twenty-nine recipients who were grafted from their HLA-identical siblings between 1994 and 2008 were divided into 2 groups (same and mismatched) according to gender of donor and recipient. The analyzed variables were age, gender, cholesterol, triglyceride, proteinuria, estimated glomerular filtration rate, weight, body mass index, and serum creatinine at 6, 12, 24, 36, 48, and 60 months, and median follow-up. Univariate and multivariate logistic regression models (when appropriate) were used to evaluate the effects of variables on allograft survival., Results: The number of male donors (P = .001) and recipient age (P = .019) was significantly higher in the same gender group than in mismatched gender group; there were no relationships between remainder analyzed parameters. Multivariate regression analysis revealed that after median follow-up period of 84 months (range, 60-232) the most important potential factors to significantly influence long-term outcomes were male donor (P = .002), recipient age (P < .001), and donor age (P = .021)., Conclusion: Our study demonstrated that male donor, and donor and recipient age affected long-term survival of HLA-identical SRTs, supported with antibody induction therapy and lifetime immunosuppression., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: HLA class I molecules are divided into classic (Ia) and nonclassic (Ib). Nonclassic HLA molecules (E, F, and G) have acquired relevance owing to their immunomodulatory properties and possible repercussions for induction of tolerance in organ transplantation. The objective of this study was to identify the impact of these molecules on transplant success or failure., Methods: A systematic review of literature was performed with the use of MeSH terms in Pubmed. Clinical trials, randomized clinical trials, case-control studies, and reviews from the past 15 years were included., Results: HLA-E*0103/E*0103 genotype is associated with lower risk of graft-versus-host disease, decreased mortality, and greater disease-free survival after bone marrow transplantation. There were no significant associations between HLA-F and clinical outcomes in any of the studies. Elevated serum levels of HLA-G were associated with a lower incidence of rejection in hepatic and renal transplantation during the 1st year and lower T-cell response after bone marrow, liver, and kidney transplantation. Detection of mRNA of HLA-G1 was also associated with less graft rejection., Conclusions: Current literature suggests that nonclassic HLA Ib molecules play an important role in immunotolerance in organ transplantation; however, more studies are required to predict outcomes related to specific genotypes.

Activated hepatic stellate cells (HSCs) possess strong immune inhibitory activity. The present study highlighted the protective role of HSCs in islet transplantation. Recipients were randomly divided into 4 groups: a diabetic group, an HSC-alone group, an islet-alone transplant group, and a cotransplant group. Graft survival was compared among the 4 groups. Serum transforming growth factor β (TGFβ), tumor necrosis factor α, interleukin-1β, and interferon gamma expression levels were measured. The infiltration of lymphocytes was observed via hematoxylin and eosin staining, and immunohistochemical examinations were performed. Results showed that allogeneic HSCs protect islet allografts better than syngeneic HSCs. There was significant prolonged graft survival and a higher level of TGFβ in the cotransplant group (P < .01). The infiltration of lymphocytes in the cotransplant group was notably less than in the islet-alone group (P < .01). The formation of desmin-positive HSC packages was detected in the cotransplant group. In conclusion, allogeneic HSCs can better prolong the survival of islet allografts by stimulating TGFβ expression and forming a biological capsule around the graft., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Many reports have shown that bone marrow-derived mesenchymal stem cells exhibit immunosuppressive effects in allogeneic transplantation. However, few reports have evaluated the immunosuppressive properties of adipose tissue-derived mesenchymal stem cells (ASCs) in vitro and in vivo. In this study, we investigated the immunosuppressive characteristics of ASCs, and investigated whether ASCs originating from donor rats prolong allotransplant survival in a rat hind limb allotransplantation model. T-cell proliferation stimulated by allogeneic stimuli or mitogen with or without ASCs originating from the donor was assessed in vitro. The effects of cellular contact or soluble factors on the inhibition of T-cell proliferation were also evaluated. In the in vivo study, cultured ASCs (1 × 10(5)) that originated from the donor were injected into recipient animals intravenously immediately after operation, followed by 1 dose per day for 3 consecutive days post-transplantation. When immune rejection occurred, the survival time of allotransplants was determined and rejected tissue was histologically and immunochemically assessed for determining regulatory T-cell infiltration. ASCs inhibited the T-cell proliferation stimulated by alloantigen or mitogen in a dose-dependent manner, and recipient T cells proliferated less in animals treated with ASCs than in controls. Although ASCs were separated from T cells, ASCs persisted to elicit a suppressive effect. ASC culture supernatants did not inhibit T-cell proliferation; however, supernatants obtained from the mixed lymphocyte reaction in the presence of ASCs suppressed T-cell proliferation. ASCs prolonged allotransplant survival time, reduced inflammatory cell infiltration, and induced regulatory T cells. In conclusion, ASCs can exhibit in vitro immunosuppressive properties and prolong allotransplant survival time in a rat hind limb composite tissue allotransplantation model, possibly through the induction of regulatory T cells., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Regulatory B cells (Breg) are a subpopulation of B cells that play a suppressive role in the immune system. The mechanism of how these immune cells perform their effects has been explored by experiments in mice and in humans. Intracellular staining for interleukin 10 continues to be a consistent and reproducible method of identifying Breg in mouse and human studies. The lack of Breg is associated with a worsening of several autoimmune diseases such as collagen-induced arthritis, systemic lupus erythematosus, and experimental autoimmune encephalomyelitis in murine studies. The purpose of this review is to provide a concise summary of the role of Breg in the immune system, including the most recently studied cell surface markers associated with Breg, and to describe the role of Breg in the etiology of several autoimmune diseases, the current understanding of Breg development, their role in the development of autoimmune diseases, and their role in inducing tolerance after transplantation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objective: This study aimed to use amniotic fluid stem cells of donors to induce immune tolerance of heterogenous rat kidney graft for investigating the formation mechanism of immune tolerance., Methods: With Wistar rats as donors and Sprague-Dawley (SD) rats as receptors, the heterogenous kidney graft animal model was established, and amniotic fluid stem cells of Wistar rats were isolated and cultured. Moreover, 40 SD rats were randomly divided into 4 groups. Creatinine (Cr), blood urea nitrogen (BUN), interleukin (IL) 2, interferon (IFN) γ, and oxidative stress levels in serum were detected, flow cytometry was used to detect changes of CD4 and CD8 cells, and quantitative changes of urinary protein and pathologic changes of transplanted kidney were observed., Results: BUN, Cr, IL-2, IFN-γ, and oxidative stress levels and urinary protein quantity in rat serum of the test group were significantly lower than those of the control group, creatinine clearance rate was significantly higher than that of the control group, and renal pathologic injury extent was significantly milder than that of the control group., Conclusions: Amniotic fluid stem cells can induce immune tolerance of rat kidney graft and inhibit oxidative stress level, improve kidney function, and alleviate kidney injury., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Allogeneic liver transplantation induces spontaneous tolerance in mice without a requirement for immunosuppression. The underling mechanisms remain unclear. Our recent studies indicated that Foxp3(+)CD25(+)CD4(+) regulatory T (Treg) cells play an important role in the induction of spontaneous transplant tolerance. How Treg cells are induced and their functional mechanisms to regulate the response remain undefined. In this study, we employed a mouse liver transplant model using PD-L1-/-, and Flt3L-/- mice to critically examine the role of liver dendritic cells (DCs) and the PD-L1 signal in Treg induction. Our results showed that liver DCs, which expressed a great number of PD-L1 molecules, induced more Foxp3(+)CD25(+)CD4(+) Treg in vitro upon coculture with allogeneic CD4 T cells compared with spleen DCs. The DCs from PD-L1-deficient mice failed to expand Foxp3(+)CD25(+)CD4(+) Treg in vitro. Adoptive transfer of Foxp3(+)CD25(+)CD4(+)Treg expanded from liver DCs prolonged heart allograft survival significantly greater than spleen cell controls. Moreover, liver grafts from Flt3L-/- and PD-L1-/- mice were rejected acutely in C3H recipients. Immunohistochemistry revealed reduced Foxp3(+) cells and significantly increased IL-2, IL-10, and IFN-γ producing elements in the liver grafts and recipient spleens of Flt3L-/- and PD-L1-/- donors. In conclusion, liver DCs play a critical role in the induction of Foxp3(+)CD25(+)CD4(+) Treg, which may mediate spontaneous acceptance of MHC-mismatched liver allografts in mice. The effects of DCs on Foxp3(+)CD25(+)CD4(+) Treg induction and expansion appear to depend on the PD-L1 signal., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objective: The objective of this study was to investigate the roles of B- and T-lymphocyte attenuator (BTLA) and herpes virus entry mediator (HVEM) in acute and chronic transplant rejection and immune tolerance., Methods: The expression patterns of BTLA/HVEM, interleukin (IL)-2, IL-4, IL-10, and interferon (IFN)-γ were analyzed among patients presenting with acute rejection episodes versus those maintaining stable renal function during therapy with mycophenolate mofetil (MMF), cyclosporine, or tacrolimus (FK506) plus prednisolone., Results: The expressions of BTLA/HVEM in the rejection group were obviously increased compared with the stable group (P < .05), followed by the elevation of serum levels of IL-2 and IFN-γ., Conclusion: The expression levels of BTLA/HVEM can be considered to be early indicators of an acute rejection episode following kidney transplantation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: The liver is considered to be an immune-privileged organ. Several types of liver cells have been implicated in the induction of immunologic tolerance. Hepatic stellate cells (HSCs) seem to participate in hepatic fibrosis and to display immunological properties., Materials and Results: In this study, HSCs isolated from C3H mice were highly positive for GFAP (98.4%) and α-SMA (95.4%). After stimulation by interferon-γ (IFN-γ), HSCs were more active in morphology with enhanced expression of H2-K(K), I-A(K), CD80, and CD54, similar to mature myelogenic dendritic cells (MDCs). Through allogeneic stimulation, C3H HSCs induced proliferation of both CD8(+) and CD4(+) T cells in B6 mice. However, the T cells activated by allogeneic HSCs produced less INF-γ, interleukin (IL)-4, IL-10, and IL-17, but large amount of transforming growth factor-β. These T cells expressed immunoregulatory rather than effector functions. Naïve T cells stimulated by allogeneic HSCs expressed Foxp3 compared with MDCs (8.67% vs 2.14%, P < .05). CD8(+) T cells activated by HSCs lost their allogeneic cytotoxicity, and CD4(+) T cells activated in this fashion suppressed the allogeneic cytotoxicity of CD8(+) T cells activated by MDCs., Conclusion: HSCs seem to act as liver-resident antigen-presenting cells instructing the generation of Foxp3(+) regulatory T cells, a property suggestion of induction of immunologic tolerance., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Effective immunomodulation to induce tolerance to tissue/organ allografts is attained by infusion of donor lymphocytes endowed with killing capacity through ectopic expression of a short-lived Fas-ligand (FasL) protein. The same approach has proven effective in improving hematopoietic stem and progenitor cell engraftment. This study evaluates the possibility of substitution of immune cells for bone marrow cells (BMC) to induce FasL-mediated tolerance to solid organ grafts. Expression of FasL protein on BMC increased the survival of simultaneously grafted vascularized heterotopic cardiac grafts to 90%, as compared to 30% in recipients of naïve BMC. Similar results were obtained for skin allografts implanted into radiation chimeras at 1 week after bone marrow transplantation. Further reduction of preparative conditioning to busulfan resulted in acceptance of donor skin implanted at 2 weeks after transplantation of naïve and FasL-coated BMC, whereas third-party grafts were acutely rejected. The levels of donor chimerism were in the range of 0.7% to 12% at the time of skin grafting, with higher levels in recipients of FasL-coated BMC. It is concluded that FasL-mediated abrogation of alloimmune responses can be effectively attained with BMC. There is no threshold of donor chimerism, but tolerance to solid organs evolves during the process of donor-host mutual acceptance., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The past 5 decades have documented remarkable advances in basic knowledge and clinical expertise in transplantation. The first 12 years of this half century of my participation in the enterprise were consumed with the isolation, chemical characterization, and application of histocompatibility antigens purified from mouse, guinea pig, and human tissues, demonstrating that their specificity was based on unique amino acid sequences in protein structures. Initial unsuccessful attempts to use native molecules to induce tolerance in rat renal or heart transplantation models were followed by limited success when they were administered with a brief perioperative course of cyclosporine (CsA). Production of allochimeric constructs of class I major histocompatibility complex molecules bearing donor-type amino acid substitutions into the host-type C-terminal portion of the α1 helix yielded tolerogens whose activity was not dependent on conditioning with CsA or total lymphoid irradiation (TLI). The allochimeric molecules serve as altered peptide ligands that induce an aberrant T-cell signal 1 response producing transplantation tolerance. The potent activity of CsA in this experimental model was extended to clinical settings. Pharmacologic tools were employed to explore intra- and interindividual variations in drug exposure leading to the development of a better drug formulation. However, the intrinsic nephrotoxicity of CsA necessitated marked 80% reductions in de novo drug exposure as were achieved by exploiting the synergistic pharmacodynamic and pharmacokinetic interactions of CsA with sirolimus. The final decade in this 50-year experience includes editorship of this journal with marked changes in its direction. These experiences have afforded insights into future avenues for preclinical exploration and therapeutic drug development., (Copyright © 2011. Published by Elsevier Inc.)

Aims: The aims of this study were to study the role of histone deacetylase 11 (HDAC11) in tolerance induction in orthotopic liver transplantation (OLT) in rats and to assess the advantages of gene therapy over the immunosuppressant FK506., Methods: Recipients were assigned to an acute rejection group (AcR; group I), an FK506 intervention group (group II), and a tolerance group (group III). Acute rejection (AcR) was graded by the Banff scheme and we examined postoperative survival. The messenger RNA (mRNA) and protein expressions of histone deacetylase 11 (HDAC11) and interleukin (IL) 10 in liver tissue were detected using real-time polymerase chain reaction (PCR) and Western blots, respectively. Plasma levels of tumor necrosis factor (TNF)-α, IL-2, and IL-10 were measured using enzyme-linked immunosorbent Assays., Results: Group I displayed severe, Group II had less, and Group III had no evidence of AR. The survivals among Group III were longer than those in Group I and Group II. IL-10 expression was promoted by HDAC11-shRNA at 7 days after OLT. Serum IL-2 and TNF-α levels were significantly lower among Group III compared with Groups I and II, whereas IL-10 showed the opposite result., Conclusions: Silence of HDAC11 promotes IL-10 expression and leads to tolerance following OLT in rats. Thus HDAC11 is a promising target for gene therapy to induce tolerance with advantages over immunosuppressive drugs., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Dendritic cells (DCs) are professional antigen-presenting cells able to induce immunity or tolerance. The interactions of immature DCs with naive T lymphocytes induce peripheral tolerance through mechanisms that include anergy or deletion of lymphocytes or the generation of regulatory T cells. Because of the central role of DCs in the immune response, they are potential targets for the induction of experimental tolerance. Thus, the generation of immature (tolerogenic) DCs able to capture and present alloantigens to T cells represents an important aim in our efforts to achieve better transplant acceptance., Methods: In this work, we generated immature DCs by using vitamin D(3) (VD3) during the process of DC differentiation., Results: The VD3-DCs showed an immature phenotype characterized by a low expression of major histocompatibility complex antigens of class II, CD86, and CD80 molecules and the secretion of a tolerogenic cytokine pattern. Furthermore, we showed that VD3-DCs phagocytose apoptotic allogeneic cells efficiently without inducing DC maturation or activation. Most important, our experiments demonstrated that mice treated with VD3 produce immature DCs in vivo, and that DCs from VD3-treated mice immunized with allogeneic apoptotic cells maintained their tolerogenic phenotype., Conclusion: Our results show that allogeneic apoptotic cells in combination with VD3 generate DCs with tolerogenic characteristics that could be used to induce tolerance towards alloantigens., (Copyright © 2011 Elsevier Inc. All rights reserved.)

T Regulatory cells (Treg) play an important role in the induction and maintenance of immunological tolerance to self and alloantigens. Recent findings in experimental transplant models have demonstrated that Treg can control acute and delayed allograft rejection. Preclinical attempts to use Treg as a cellular therapy have been successfully undertaken demonstrating the safety and feasibility of such treatment, suggesting that they have therapeutic potential., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Introduction: Various methods have been tried to induce operational tolerance in organ transplantation. We present a single-center experience using 6 tolerance induction protocols (TIP) in living-related renal transplantation., Methods: We evaluated 6 TIP protocols: (1) peripheral blood stem cells employed (n = 38); (2) midified the protocol by portal infusion (n = 292); (3) the second protocol plus TIP+DST+BM+intrathymic and intramarrow infusion plus low-dose, nonmyeloablative conditioning employed (n = 174), (4) the third protocol of TIP plus cultured hematopoietic stem cells (HSC) with target-specific irradiation (n = 290); (5) TIP 4 plus thymus, intramarrow infusion, and target-specific irradiation converted to total lymphoid irradiation (TLI) (n = 366); and (6) TIP 5 plus bortzomib-TLI (n = 165). Patient/donor demographics were comparable., Results: We evaluated patient and graft survival, rejection episodes, recurrence, drug toxicity, and chimerism revealed; groups 4 and 5 showed better survival, graft function, chimerism, and decreased rejection episodes compared with previous protocols. Serum creatinine (mg/dL) at 1 year was 1.5, 1.39, 1.5, 1.51, 1.46, and 1.41, and at 5 years, 1.69, 1.72, 1.82 and 1.59, in groups 1-6, respectively. Chronic rejection episodes were 10.5%, 14.1%, 10.4%, 9.3%, 3.5%, 1.7%, and 1.8% respectively. Patient survival of groups 1, 2, and 3 at 1, 5, and 10 years was 86.5%, 56.8%, and 40.1%; 89.4%, 69.1%, and 56.4%; and 89.6%, 67.7%, and 64.6%, respectively; of group 4 for 1 and 5 years was 92.4% and 81.8%; for groups 5 and 6 for 1 year was 94% and 96.3%, respectively. The death-censored graft survival of groups 1, 2, and 3 at 1, 5, and 10 years was 91.9%, 70.3%, and 64.7%; 89%, 66%, and 57.6%; and 86.7%, 67%, and 42.5%, respectively. In group 4 for 1 and 5 years was 87.9% and 74.7%; and for groups 5 and 6 for 1 year was 94% and 96.5%, respectively., Conclusion: TIP results showed improved graft/patient survivals, minimum immunosuppression, and fewer rejection episodes and recurrence., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background and Aim: The drugs routinely administered to prevent rejection often cause lethal side effects. Tolerant patients, therefore, should be identified to minimize these problems. The aim of this analysis was to identify clinical variables that may be associated with tolerance., Methods: We recruited 522 heart transplants (HT), excluding combined procedures, retransplantations, pediatric recipients, and subjects who died in the first year to obtain a cohort of 375 patients. Two groups were distinguished by the presence of echocardiographic, clinical, or pathological evidence of rejection in the first year (15 echocardiograms and 10 protocol biopsies per patient); 99 tolerant patients were compared with 276 nontolerant patients. We analyzed clinical variables related to morbidity and mortality., Results: The univariate analysis showed few differences between the groups. The multivariate analysis showed that only major histocompatibility complex (MHC)-A and MHC-DR matched recipients were significantly associated with tolerance. Thus, the likelihood of tolerance was increased by 1.7- and 2.8-fold if 1 or 2 MHC-I matches were present and by 3.4- and 3.7-fold if 1 or 2 MHC-DR matches were present, respectively survival curves showed significant differences (P=.034). Most deaths in both groups were related to immunosuppressive drugs; among tolerant subjects, deaths were due to infection and neoplasms and among nontolerant patients, deaths were due to chronic rejection, neoplasms, and infection., Conclusions: The only clinical parameter that can determined whether a HT recipient was tolerant was MHC-A and MHC-DR matching. If there is matching, a reduced immunosuppressive load should be prescribed to prevent drug toxicity., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Dendritic cells (DCs) play a crucial role in T cell allorecognition in the context of organ transplantation and allogeneic hematopoietic stem cell transplantation (allo-HSCT). This DC function is believed to be directly involved in allo-HSCT morbidity and mortality. DC functions range from immunoaggressive responses to the promotion of tolerance, reflecting functional plasticity. This unique characteristic offers a rationale to propose generation of tolerogenic DCs as a therapeutic tool for graft-versus-host disease (GVHD). The accumulated preclinical findings support the concept that glucocorticoid-induced leucine zipper (GLIZ) expression redirects mature DC function toward a tolerogenic mode, driving differentiation of antigen-specific regulatory T cells. Taking into account the unique role of DCs within the allo-HSCT context, novel preventive and curative therapeutics for GVHD might be based on the selective induction of GILZ expression in vivo., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Introduction: Many studies suggest that CD4(+)CD25(high) T regulatory cells (Tregs) have a crucial role in downregulating the immune response to alloantigens. In this study, we investigated the possible influence of immunosuppressive therapy, including rapamycin and calcineurin inhibitors (CNIs; tacrolimus), on level of Tregs in liver allograft recipients., Materials and Methods: We assessed 47 liver transplant recipients with stable liver function for ≥2 years, dividing them into 2 groups: Patients receiving rapamycin (n = 15), and those receiving tacrolimus (n=32). Thirty-eight, age-matched healthy subjects were used as normal controls. We examined the expression of CD4, CD25, and Foxp3 in peripheral blood T cells. Flow cytometry was performed with a FACSCalibur instrument with data analysis using Cell Quest software., Results: Rapamycin significantly increased the prevalence of Tregs, including the percentage of CD4(+)CD25(high) T cells in total lymphocytes and among total CD4(+) T cells, compared with the healthy subjects and the CNI group. The prevalence of Tregs in the CNIs group was significantly lower than that of controls. Foxp3 was expressed in >95% of CD4(+)CD25(high)T cells, whereas it was in <20% of CD4(+)CD25(low) T cells and not expressed among CD4(+)CD25(-) T cells., Conclusions: Immunosuppressive therapy (rapamycin or CNIs) may have a different roles in tolerance induction among liver transplant recipients. Namely, rapamycin promoted the induction of a profile consistent with alloantigen tolerance; CNIs hampered this progression., (2010 Elsevier Inc. All rights reserved.)

It has been shown that mast cells (MC) are absolutely required for transplant acceptance. However, only a few of the numerous mediators produced by MC have been proposed as potential mechanisms for the observed immunosuppression. The role of proteases in acquired immune tolerance as such has not yet been addressed. In this study, we have shown the requirement for MC protease 6 (MCP6), an MC-specific tryptase, to establish tolerance toward an allogeneic skin graft. The substrate for MCP6 is interleukin (IL)-6, cytokine generally considered to indicate transplant rejection. Herein we have shown an inverse correlation between MCP6 and IL-6. High expression of MCP6 is accompanied by low levels of IL-6 when the allograft is accepted, whereas low expression of MCP6 in combination with high levels of IL-6 are observed in rejecting grafts. Moreover, tolerance toward an allogeneic graft cannot be induced in MCP6(-/-) mice. Rejection observed in these mice was comparable to that of MC-deficient hosts; it is T-cell mediated. These findings suggest that MCP6 actively depletes the local environment of IL-6 to maintain tolerance., (2010. Published by Elsevier Inc.)

Pharmacological aspecific immunosuppression, despite being widely used in solid organ transplantation recipients, is unable to completely prevent allograft rejection. It promotes the occurrence of sometimes life-threatening infections. Due to their immunosuppressive and anti- inflammatory properties, there is great interest in the therapeutic use of bone marrow (BM)-derived mesenchymal stromal cells (MSC). Large animal models play a crucial role to investigate the biological and functional properties of MSCs as novel cellular therapy. In the current study we sought to isolate expand ex vivo, and phenotypically characterize MSC derived from BM of 4 Large White 6-month-old piglets. Porcine MSC (pMSC) were characterized for their in vitro differentiation capacity. pMSC were successfully isolated from all BM samples. They showed spindle-shaped morphology and a stable doubling time on culture. They were positive for CD90, CD29, CD105, and negative for CD45 and CD11b. Furthermore, they differentiated, upon specific in vitro conditions toward adipogenic and osteogenic lineages. The optimization of methods for the isolation and characterization of pMSC may be useful to elucidate their biological and functional properties. The anatomy and physiology of the pig, which is similar to humans, make this animal model more attractive than small animals to test the safety and efficacy of MSC in the context of solid organ transplantation., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Outcomes after liver transplantation are outstanding; however, the limiting factor is the shortage of organs. Recently, the utilization of donors after cardiac death has been encouraged; however, such transplants are associated with a high complication rate, mainly a high incidence of biliary complications, particularly ischemic cholangiopahty, a serious complication that often leads to retransplantation. The second problem is the morbidity associated with the use of immunosuppressive drugs. In this manuscript, the current status of clinical protocols for induction of tolerance is briefly discussed. Furthermore, the future of research in transplantation will involve basic scientists and clinical scholars working in concert as has been developed at Stanford School of Medicine with the creation of the Institute for Immunity, Transplantation and Infection., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Liver transplantation has been a positive impact on both the survival and the quality of life of patients with advanced liver diseases. Progressive, spectacular improvements in the results of liver transplantation have been observed since the preliminary studies by Thomas Starzl in the United States and Roy Calne in Europe. This improvement is related to better knowledge of the natural history of liver diseases, allowing more adequate recipient selection, improvement of surgical techniques, progress in postoperative management, availability of potent antibacterial, antiviral, and antifungal drugs, as well as introduction of new immunosuppressive agents and protocols. These advances have occurred in the short interval of 45 years, suggesting future improvements in the liver transplantation field. The main investigative efforts in liver transplantation have been directed as follows: First attenuation of disproprortion between the numbers of available liver grafts versus waiting list recipients, by increasing the donor pool applying bioartificial support systems, or rendering grafts compatible by the use of stem cells. Second, improved knowledge about the biology of primary liver tumors establishes indications for and optimal moments of transplantation. Third, application of individualized immunosuppressive protocols, adapted to clinical status of the recipient, as well as the development of more selective, less toxic new immunosuppressive agents., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Objective: To evaluate in a canine model the induction of tolerance to renal transplantation after splenectomy and splenosis., Materials and Methods: This prospective, experimental, comparative, longitudinal study included 4 experimental groups, each comprising 4 dogs. Group 1 (control group) underwent renal transplantation only; group 2 underwent renal transplantation and splenectomy; group 3 underwent renal transplantation and splenosis; and group 4 underwent renal transplantation, splenectomy, and splenosis. Survival and degree of rejection were compared between the 4 groups., Results: Splenosis improved renal function after transplantation, as indicated by increased serum creatinine concentration (group 3, 6.2 mg/dL vs group 1, 12.9 mg/dL). Comparison of weighted survival curves (corrected for degree of rejection) demonstrated a significant difference between group 2 (66.0 days) and group 4 (66.2 days) vs group 1 (52.7 days) and group 3 (41.2 days) (P = .05, Wilcoxon rank sum test)., Conclusion: These results suggest that in this experimental model of renal transplantation, splenosis and splenectomy induce clinical tolerance, as indicated by improved renal function and prolonged recipient survival.

Clinical operational tolerance (COT) is a clinical condition obtainable with difficulty after solid organ transplantation (SOT). It is characterized by perfectly normal graft function in the total absence of maintenance immunosuppression. Major benefits deriving from the onset of COT are the reduction of risk for immunosuppression-related side effects and the improved quality of life. Currently, COT can be safely achieved in stable liver transplant recipients; it remains a challenge after renal transplantation. Only 1 case of COT has been reported after lung transplantation; no cases have been described after other types of SOT. Overall, mechanisms of COT are unclear and strategies to induce COT cannot be applied on a regular base to a large cohort of SOT recipients. Due to the failure of molecularly based tolerogenic protocols, great hope relies in the adoption of cell-based strategies.

Dendritic cells (DCs) are bone marrow-derived, professional antigen-presenting cells, with inherent tolerogenic function. The ability of immature or maturation-resistant DCs to regulate alloantigen-specific T-cell responses and to promote tolerance induction has been well demonstrated in organ and bone marrow transplantation. Recent data suggest that DCs can also promote long-term survival of composite tissue allografts in the absence of continued immunosuppressive drug therapy.

Background: Despite the widely accepted implication of antidonor antibodies and complement in solid organ transplantation, their role in reconstructive allotransplantation is not clear. The aim of this study was to analyze the humoral immune response using a rat orthotopic limb transplantation model., Methods: We used the Brown Norway to Lewis rat orthotopic hind-limb transplant model: Group 1, isografts; group 2, allografts with daily continuous cyclosporine treatment to prevent acute rejection; and group 3, allografts undergoing multiple episodes of acute rejection. Samples were taken at 30, 60, and 90 days. Serum was analyzed by FACS for antidonor antibodies. Tissue deposition of antibodies and complement was investigated by immunofluorescence., Results: By day 90, animals in group 3 had undergone 19 (+/-3.2) acute rejection episodes. There was no difference in the occurrence of serum antidonor antibodies between the three groups at any time point. However, at 90 days, anti-third-party antibodies were significantly greater among group 3. There was no difference in antibody or complement deposition in muscles between the 3 groups., Conclusion: Despite the increased antibody against a third party after multiple rejection episodes in this animal model, there was no clear evidence of an antibody-mediated alloresponse in limb transplantation.

Facial disfigurement in children with congenital craniofacial defects can lead to decreased self-esteem and poor self-perception. Traditional methods of reconstruction can fail to achieve a normal appearance in patients with severe disfigurements. Composite tissue allotransplantation (CTA) in children could offer a unique reconstructive opportunity. A discussion of the usage of CTA for congenital craniofacial defects is thus warranted. Treatment of severe craniofacial clefts, Treacher-Collins syndrome, hemifacial microsomia, and some vascular anomalies can yield unsatisfactory results, even after multiple surgeries. CTA provides the advantage of intact vascularized bone that would not need to be reshaped to fit the defect, with the correct donor match. CTA also provides reconstruction with similar tissue type in regions of the central midface such as the nose, lips, and eyelids. With advances in transplant immunology to devise mechanisms to decrease immunosuppression and induce donor antigen-specific tolerance, CTA may be a future reality in the pediatric population.

The role of nonconventional T cells in innate and adaptive immunity is just emerging; gammadeltaT cells play important roles in anti-tumor and anti-infectious diseases. The involvement of gammadeltaT cells in immunologic responses to hematopoietic cell transplantation remains controversial; divergent results have been reported depending on the murine strains and model systems. Whether gammadeltaT cells are involved in solid organ transplantation is understudied. We have characterized the gammadeltaT cells in mouse livers and spleens to evaluate their contributions to liver transplant tolerance posttransplantation using a murine allogeneic liver transplant model which induces spontaneous T regulatory cell (Treg)-dependent tolerance. Our studies revealed that gammadeltaT cells comprised about 20% of the population of liver nonparenchymal cells (NPCs). In naïve C3H mice they were CD4, CD8, and NK1.1 negative. The percentage of gammadeltaT cells decreased in spontaneously tolerated liver grafts posttransplantation from 20% in naïve C3H livers to <10% in allografts throughout the time course. In contrast, they increased in liver grafts with rejection induced by anti-CTLA4 plus anti-CD25 mAb administration. CD4 and CD8 expression on gammadeltaT cells dramatically increased in the tolerated but not rejected livers posttransplantation to >20% of CD4(+) and 30% of CD8(+). Our results suggested that gammadeltaT cells are involved in allogeneic immune responses. Whether gammadeltaT cells function as the causal or the effector cells in allograft tolerance rejection warrants further investigation.

Background: We have previously reported the successful induction of renal allograft tolerance in non-human primates using a nonmyeloablative conditioning regimen to produce a mixed-chimeric state in the recipient. In the present study, we applied this same technique to lung allotransplantation in cynomolgus monkeys., Methods: Nine pairs of fully major histocompatibility complex (MHC)-mismatched cynomolgus monkeys were used. The conditioning regimen consisted of total body irradiation, thymic irradiation, and antithymocyte globulin. The recipients underwent lung and bone marrow transplantation, followed by anti-CD154 monoclonal antibody (mAb), and a 1-month course of cyclosporine. The regimen included anti-CD8 mAb in the last 5 recipients and alpha 1-antitripsin in the last 3 recipients. The results were compared with 8 recipients that received kidney allografts using the same regimen., Results: Transient chimerism developed in all lung recipients, as was previously seen in the kidney recipients. Nonetheless, the lung recipients rejected their allografts significant earlier than the kidney recipients (P < .01)., Conclusions: Despite the successful induction of mixed chimerism in recipients of fully MHC-mismatched lung allografts, we have not observed long-term graft survival, as has been seen in an analogous kidney model. Strategies to overcome this problem include organ-specific modifications of the transplant regimen.

The immune system maintains self-tolerance through the interplay of several mechanisms. One of the key mechanisms is the suppression of effector cells through regulatory T cells (Tregs). Recent progress has allowed the identification, analysis, and experimental manipulation of various subpopulations of Tregs. Several major issues regarding Tregs have emerged in relation to organ transplantation, including the questions of whether immunosuppressive drugs influence Tregs and whether Tregs can be used therapeutically to achieve graft acceptance or even tolerance. Although none of the available immunosuppressive drugs have been specifically designed to influence Tregs, they nevertheless do so through various pathways. Notably, current evidence suggests that rapamycin has a favorable effect on Tregs in organ transplant recipients, whereas calcineurin inhibitors (CNI) have markedly negative effects on Tregs. The idea of inducing tolerance through the therapeutic administration of Tregs has received much attention in recent years. To obtain sufficient numbers of recipient-derived Tregs, various strategies are being investigated to expand sorted Treg populations. Rapamycin has been found to significantly promote the in vitro expansion of murine and human Tregs; it may thereby help to generate clinically relevant quantities of Tregs. However, no data have yet been published that would demonstrate that the therapeutic use of Tregs induces transplantation tolerance across full major histocompatibility complex barriers in immunocompetent mice displaying a polyclonal T-cell repertoire.

Background: By consensus, HLA-G has a role in the induction of tolerance via interaction with dendritic cells and manipulation of co-stimulatory molecule expression. The purpose of this study was to demonstrate that HLA-G modified dendritic cells exhibit a decreased ability to stimulate T-cells in vitro due to increased expression of B7-1, which provides regulatory signalling to T-cells as a consequence of binding CD28 and CD152 ligands., Methods: Bone-marrow cells were cultured from Brown Norway (BN) rat femurs and sorted with anti-rat dendritic cell Ab (OX62) microbeads. Isolated dendritic cells (DCs) were treated with HLA-G tetramer for 3 days. Initially, cells were plated with media, alloantigenic splenocytes, and T-cells and then observed in mixed lymphocyte reaction for thymidine uptake. Also, the cells were analyzed by flow cytometry using antibodies for MHC-II (IA), CD80, and CD86., Results: This study displays that HLA-G-modified DCs decrease induction of an alloproliferative response. In addition, this study demonstrated that HLA-G tetramer treatment decreases CD86 and permits expression of CD80 without altering MHC-II expression., Discussion: This study specifically investigated the role of B7-1 (CD80), showing that HLA-G treatment of immature DCs allows expression of B7-1 (CD80) without altering MHC-II expression and simultaneously decreases B7-2 (CD86) expression. Therefore, a low level of B7-2 (CD86) allows attenuation of T-cell response after activation, both of which are beneficial to immune tolerance. The selective expression of B7-1 (CD80) is important and may serve as a guide for future therapies aimed at transplant tolerance.

Myeloid differentiation factor 88 (MyD88) is a key adaptor of Toll-like receptor signaling, which plays a critical role in dendritic cell (DC) function. However, its role in the induction of transplant tolerance by immature DCs is unknown. In this study, we silenced MyD88 expression of bone marrow-derived immature DCs with small interference RNA demonstrating that this maneuver significantly prolonged the survival of intestinal allografts. This study provided evidence that the absence of MyD88 enhances the tolerogenicity of DCs and suggested that inhibiting innate immunity may be a potential strategy to facilitate induction of transplant tolerance by DCs.

Background: We have used anti-CD154 monoclonal antibody (mAb; MR1) and rapamycin (rapa) to induce tolerance to islet xenografts. The aim of this study was to investigate whether classical anergy and/or regulation by interleukin (IL)2-dependent CD25+ T regulatory cells played roles in the induction and maintenance of tolerance in this model., Methods: Streptozotocin-induced diabetic mice were transplanted with rat islets. We performed the following groups: control group, islet transplantation without therapy; rapamycin group, 0.2 mg/kg by oral gavage on days 0, 1, 2, and every other day to day 14; anti-CD154 mAb (MR1) group, 0.5 mg intraperitoneally on days 0, 2, and 4; combination therapy group with rapa and MR1. We then administered in addition to the combination therapy with early (from days 0 to 14 [for IL2] or to 28 [for anti-IL2 mAb and anti-CD25 mAb] post-transplantation) or late (from days 100 to 114 [for IL2] or to 128 [for anti-IL2 mAb and anti-CD25 mAb] posttransplantation) recombinant IL2 (2000 U, intraperitoneally twice a day), a neutralizing anti-IL2 mAb (S4B6-1, 0.3 mg intraperitoneally twice weekly), and a depleting anti-CD25 mAb (PC61, 0.3 mg intraperitoneally twice weekly), respectively. Histology was performed at time of rejection., Results: Rapa and MR1 therapy alone significantly prolonged xenograft survival compared to the control group: median graft survival was 34 days versus 17 days (P<.05) and 98 days versus 17 days (P<.05), respectively, but rejection still occurred. Combination therapy with MR1 and rapa allowed indefinite graft survival (median graft survival [MGS]>200 days, P<.001). When exogenous IL2 was administered early with MR1 and rapa, rapid rejection developed in 18 of 18 mice (MGS 7 days), whereas when IL2 was given late, only 3 of 10 developed rejection. Early administration of anti-IL2 mAb led to rejection in 10 of 10 mice (MGS 42 days), whereas late administration led to rejection in only one of four mice. Early administration of anti-CD25 mAb led to rejection in eight of nine mice (MGS 49 days), whereas late administration led to rejection in only three of seven mice., Conclusions: Rapa and MR1 allowed indefinite graft survival of islet xenografts. Classical anergy and regulation by IL2-dependent CD25+ T regulatory cells were critical in the induction of tolerance in the immediate posttransplantation period and less important for maintenance of tolerance.

In previous studies, CD8+FOXP3+ T cells have been shown to upgrade the inhibitory receptors on dendritic cells (DC) in heart transplantation patients. The inhibitory receptor were immunoglobulin-like transcripts (ILT)-3 and ILT-4, which were highly expressed on tolerogenic dendritic cells. Our study focused on the CD8+FOXP3+ T cells from allogeneic renal transplant recipients, seeking to dissert their function in inducing ILT-3- and ILT-4-expressing dendritic cells. We analyzed 11 quiescent renal transplant recipients and their respective donors. Reverse transcriptase polymerase chain reaction analysis showed that the levels of Foxp3 mRNA in CD8+ T cells after renal transplantation were significantly higher than those in CD8+ T cells before transplantation. We further investigated the capacity of allospecific CD8+FOXP3+ T cells to induce upregulation of the inhibitory receptors ILT-3 and ILT-4 on DC. Data from FACS showed that CD8+FOXP3+ T cells induced greater expression of ILT-3 and ILT-4 on DC in an allospecific fashion. This study confirmed that Foxp3 gene expression was enhanced among quiescent renal transplant patients, suggesting that CD8+FOXP3+ T cells play an important role in unresponsiveness related to upregulated ILT-3 and ILT-4 receptors on DC.

Introduction: Autoimmune disease represents a (AD) breakdown of natural tolerance against autoreactive antigens leading to a high mortality and morbidity. The reaction is usually polyclonal; T- and B-cell components of the hematopoietic system are responsible for disease progression. Allogeneic/autologous hematopoietic stem cell transplantation (HSCT) are the current modalities for treating drug-resistant AD., Patients and Methods: We present a single-center retrospective evaluation of allogeneic HSCT with nonmyeloablative, low-intensity conditioning in nine patients (five males, four females) with pemphigus vulgaris (PV) and 27 patients with systemic lupus erythematosus (SLE; 3 males, 24 females). The mean follow-up period was 4.24 years for PV and 4.9 years for SLE. Cytokine-mobilized HSC from unmatched related donors, with mean dose of 21.3 x 10(8) nucleated cells/kg body weight (BW; mean CD34(+) count, 6 x 10(6)/kg BW) was administered in to the thymus as well as the portal and peripheral circulations of recipients. Cyclosporine (4 +/- 1 mg/kg BW per day) and prednisolone (10 mg/kg BW per day) were administered for 6 months to protect mixed chimerism. A subset of patients with cross-gender donors were analyzed for peripheral blood chimerism at 1 month post-HSCT and every 3 months thereafter., Results: Sustained clinical remission with peripheral lymphohematopoietic chimerism of 0.7 +/- 0.3% was observed in PV, whereas SLE relapsed after mean of 7.35 months of disease-free interval associated with fall in chimerism from 5 +/- 3% to < or =0.08 +/- 0.03%., Conclusion: HSCT was effective to achieve early clinical remission of PV; and in SLE relapsed after a 7.35-month disease-free interval accompanied by a fall in mixed lymphohematopoietic chimerism.

Objectives: The mechanisms and treatment of chronic rejection in pulmonary allotransplantation remain elusive. We have induced robust tolerance to class I-disparate lung allografts in miniature swine using an intensive 12-day course of tacrolimus. Here, we tested whether a tolerant state can be induced in swine receiving fully mismatched lung allografts., Methods: Orthotopic left lung allografts were performed using MHC class I-disparate (group 1: n = 3) or fully disparate (group 2: n = 6) donors. The recipients received a 12-day postoperative course of tacrolimus (continuous intravenous infusion; target level = 35-50 ng/mL) as their only immunosuppression., Results: All swine in group 1 maintained their grafts long term without developing any lesions of chronic rejection (>497, >432, >451 days). These recipients exhibited donor-specific hyporesponsiveness in cell-mediated lymphocytotoxity (CML) and mixed lymphocyte reaction (MLR) assays. In group 2, five of the six recipients maintained their grafts long term (sacrificed on postoperative days 515, 389, 429, 481, and 438 with viable grafts). Isolated lesions of obliterative bronchiolitis were occasionally seen on biopsy, and donor-specific hyporesponsiveness on assays was consistently observed. The remaining recipient rejected its graft on day 103 with histologic findings of obliterative bronchiolitis., Conclusions: We report long-term graft acceptance without chronic immunosuppression in five of six recipients across a full MHC disparity, albeit with some evidence of obliterative bronchiolitis. These data suggest that the class II disparity inherent in a fully mismatched transplant increases the requirement for tolerance induction.

Aims: The potency of immunosuppression is a critical factor in small bowel transplantation (SBTx). FTY720 altered lymphocyte trafficking and prevented the donor T cells from migrating into target organs, resulting in the prolongation of recipient survival in acute graft-versus-host disease (GVHD) of SBTx. However, the effect of FTY720 on donor T cells in the chronic phase of GVHD following SBTx remains unclear., Methods: Heterotopic SBTx was performed in a WF-to-F1 (WF x ACI) rat combination. Recipients were given FTY720 for 14 days after SBTx. The subpopulations of donor-derived T cells and the cytokine production in the target tissues were evaluated on postoperative day 150., Results: FTY720 treatment significantly prolonged recipient survival over 150 days without any clinical signs of GVHD. The numbers of donor-derived CD4+ and CD8+ T cells in the peripheral blood, mesenteric lymph nodes, and Peyer's patches of recipients were maintained at low levels on postoperative 150, which were almost similar to the levels on postoperative day 14. In the host lamina propria, however, a significant higher number of donor T cells (CD4+, 18.4 +/- 4.3 x 10(4); CD8+, 13.9 +/- 3.6 x 10(4)) were still observed on postoperative day 150. Production of interferon-gamma was significantly reduced in target tissues by FTY720 treatment both in the acute and chronic phase. However, interleukin-4 and interleukin-10 production, which was significantly higher on day 14, returned to the level of naive rats in the chronic phase., Conclusions: A 14-day treatment of FTY720 induced tolerance in our SBTx model. Down-regulation of both Th1 and Th2 immune response was observed in the chronic phase.

The liver has long been considered a tolerogenic organ that favors the induction of peripheral tolerance. The mechanisms underlying liver tolerogenicity remain largely undefined. In this study, we characterized Foxp3-expressing CD4+ CD25+ regulatory T cells (Treg) in liver allograft recipients and examined the role of Treg in inherent liver tolerogenicity by employing the mouse spontaneous liver transplant tolerance model. Orthotopic liver transplantation was performed from C57BL/10 (H2b) to C3H/HeJ (H2k) mice. The percentage of CD4+ CD25+ Treg was expanded in the liver grafts and recipient spleens from day 5 up to day 100 posttransplantation, associated with high intracellular Foxp3 and CTLA4 expression. Immunohistochemistry further demonstrated significant numbers of Foxp3+ cells in the liver grafts and recipient spleens and increased transforming growth factor beta expression in the recipient spleens throughout the time courses. Adoptive transfer of spleen cells from the long-term liver allograft survivors significantly prolonged donor heart graft survival. Depletion of recipient CD4+ CD25+ Treg using anti-CD25 monoclonal antibody (250 microg/d) induced acute liver allograft rejection, associated with elevated anti-donor T-cell proliferative responses, CTL and natural killer activities, enhanced interleukin (IL)-2, interferon-gamma, IL-10, and decreased IL-4 production, and decreased T-cell apoptotic activity in anti-CD25-treated recipients. Moreover, CTLA4 blockade by anti-CTLA4 monoclonal antibody administration exacerbated liver graft rejection when combined with anti-CD25 monoclonal antibody. Thus, Foxp3+ CD4+ CD25+ Treg appear to underpin spontaneous acceptance of major histocompatability complex- mismatched liver allografts in mice. CTLA4, IL-4, and apoptosis of alloreactive T cells appear to contribute to the function of Treg and regulation of graft outcome.

Comparison of intragraft gene expression changes in tolerant cardiac allograft models may provide the basis for identifying pathways involved in graft survival. Our laboratory has previously demonstrated that tolerance to the gal alpha1,3 gal epitope, the major target of rejection of wild-type pig hearts in human cardiac transplantation, can be achieved after transplantation with bone marrow transduced with a lentiviral vector expressing alpha1,3 galactosyltransferase. We now present intracardiac gene expression changes associated with long-term tolerance in this model. Biotin-labeled cRNA was hybridized to Affymetrix GeneChip 430 2.0 Mouse Genome Arrays. Data were subjected to functional annotation analysis to identify genes of known function in which expression was increased or decreased by at least 2-fold (t-test, P < .05) in tolerant gal+/+ wild-type hearts as compared to transplanted syngeneic controls. Tolerant hearts demonstrated increased expression of genes associated with the stress response, modulation of immune function and cell survival (HSPa9a, CD56, and Akt1s1), and decreased expression of several immunoregulatory genes (CD209, CD26, and PDE4b). These data suggest that tolerance may be associated with activation of immunomodulatory and survival pathways.

It is well known that interferon-gamma (IFN-gamma) not only plays a critical role in antigen-dependent but also in antigen-independent tissue injury; however, it is not clear how tolerance induction affects the actions of IFN-gamma in the transplant setting. To address this question, we compared the effects of IFN-gamma on porcine recipients of near-syngeneic, rejecting, and tolerant heart transplants. IFN-gamma was infused continuously into the left anterior descending artery of hearts transplanted into 3 groups of major histocompatibility complex (MHC) inbred miniature swine, each treated with a 12-day course of cyclosporine A (CyA). Group 1 recipients received a MHC class I disparate heart, group 2 recipients received a near-syngeneic heart, and group 3 recipients were cotransplanted with a MHC class I disparate heart and kidney, which uniformly induces tolerance to both grafts. An additional group of animals was not transplanted but received intracoronary IFN-gamma infusion into their native hearts. IFN-gamma perfusion not only accelerated the acute rejection of MHC class I disparate hearts (mean survival time = 19 +/- 7.21 vs 38 +/- 8.19 days, P = .025), but caused near-syngeneic heart transplants, which otherwise survive indefinitely, to reject within 35 days (n = 3). In contrast, IFN-gamma perfusion had no demonstrable effects on interstitial rejection, the development of vascular lesions, or graft survival in tolerant heart plus kidney allograft recipients (n = 4) or in autologous hearts (n = 2). These results suggest that tolerance induction mitigates the damaging effects of IFN-gamma itself and that the beneficial effects of tolerance induction on acute and chronic rejection may extend to antigen-independent factors like ischemia/reperfusion injury.

We previously demonstrated that cardiac allografts to fully tolerant chimeric mice developed cardiac allograft vasculopathy (CAV). Here we begin to examine which components of the immune system are responsible for the pathogenesis of CAV in such tolerant recipients. B10.A/B6 mixed chimeric mice were created by receiving injections of bone marrow cells from B10.A (H-2k) mice given to C57BL/6 (B6; H-2b) mice with some preparations. B10.A skin grafts were first placed onto B10.A/B6 mixed chimeric recipients. When the donor strain skin grafts had survived perfectly for at least 56 days, B10.A hearts were transplanted heterotopically into B10.A/B6 mixed chimeric recipients. Hearts were examined for the presence of CAV 56 days later. To determine the effector cells that contribute to the development of CAV, they were treated weekly with a combination of anti-CD4/CD8 monoclonal antibodies (mAbs) or anti-NK1.1 mAb continuing until 56 days. 14 B10.A cardiac transplants of 18 otherwise untreated B10.A/B6 chimeric recipients developed CAV; concurrent B6 isografts were unaffected (0/7). In chimeric recipients treated with anti-CD4/8 mAbs, the prevalence of CAV was greatly reduced (0/6, P < .01 compared to the untreated group). Anti-NK1.1 mAb was not effective in the prevention of CAV (4/5). These data suggest that T cells may contribute in some way to the development of CAV that occurs in those fully tolerant recipients. Host T cells that may still be responsive to non-major histocompatability complex antigens, including tissue-specific antigens presented not on skin but on heart, may also be responsible for the development of CAV in tolerant animals.

Objective: We recently reported that autoreactive antibodies (Abs) against nuclear histone H1 was transiently induced at an early phase after orthotopic liver transplantation (OLT) in a tolerogenic rat OLT model and possessed immunosuppressive activity. It was also reported that nuclear antigen, high-mobility group box 1 (HMGB1) protein was one of the initiators of the immune reaction. The present study sought to evaluate the role of antinuclear Abs in experimental and clinical liver transplantation., Materials and Methods: We prepared 3 animal models: natural tolerance model (DA liver into PVG); acute rejection model (DA liver into LEW); and drug-induced tolerance model (acute rejection model + cyclosporine [CsA]). In addition, we examined clinical samples, including 1 drug-free patient, to measure the antihistone H1/HMGB1 titers at various times after OLT., Results: In a natural tolerance model, antihistone H1 and HMGB1 Ab was induced during the rejection and the tolerance induction phases, respectively. Those Ab responses were also confirmed in a drug-induced tolerance model, whereas no such responses were shown in an acute rejection model. In our clinical drug-free patient, antihistone H1/HMGB1 titer was significantly higher after cessation of CsA than that in healthy volunteers., Conclusions: Antinuclear Ab is actively expressed in accordance with overcoming rejection episodes with subsequent tolerance induction in both a natural tolerance model and a drug-induced tolerance model. We also observed a similar tendency in our clinical drug-free patient. These results suggested that antinuclear Abs may be useful markers to determine the timing to withdraw immunosuppressants.

The holy grail in clinical transplantation is the establishment of long-term donor-specific transplantation tolerance with the minimum use of immunosuppressive agents. CD4+ CD25+ regulatory T cells (Tregs) play a crucial role in the prevention of autoimmunity, and appear to mediate transplantation tolerance. Harnessing Tregs for potential adoptive cell therapy to promote donor-specific transplantation tolerance is promising. Here we show that human CD4+ CD25+ Tregs with indirect allospecificity for an HLA-A2 (103-120) peptide can be generated from purified peripheral blood CD4+ CD25+ by priming with HLA-DR0101+ A2- autologous dendritic cells (DCs) pulsed with the A2 peptide in vitro. The antigen specificity for the A2 peptide was demonstrated in functional assays and flow cytometric analysis using a fluorescent tetramer composed of HLA-DR0101 and the A2 peptide. The CD4+ CD25+ Tregs with indirect allospecificity for the A2 peptide showed potent suppression of an indirect alloresponse by effector CD4+ CD25- T-helper cells. Importantly, the selected CD4+ CD25+ Tregs can be expanded substantially to meet a therapeutic end after T-cell receptor (TCR) stimulation by CD3/CD28 antibody-coated beads in the presence of high doses of interleukin-2 (IL-2). The expanded CD4+ CD25+ Tregs highly expressed Foxp3, and retained their suppressive properties and maintained expression of lymphoid homing receptor CD62L. Taken together, these data pave the way for clinical studies using in vitro generated and expanded human CD4+ CD25+ Tregs with indirect allospecificity as therapeutic reagents to promote donor-specific transplantation tolerance.

Unlabelled: We generated an human embryonic stem cell (hESC) line to augment chimerism-associated tolerance. A 40-year-old African with chronic glomerulonephritis-chronic renal failure with 100% G6PD enzyme deficiency presented for renal transplantation with a 27-year-old, 6/6 HLA-matched sister as a willing donor., Method: We generated an hESC line from the donor's oocytes using long ovarian stimulation protocol simultaneously with tolerance induction protocol. A nuclear transfer (NT)-hESC line was derived by transferring a donor cumulus cell into an enucleated oocyte, subjected to electrical fusion, and cultured for 5 days. ESCs hatched from the blastocyst on day 6 were cocultured with her unmodified bone marrow for 2 days and suspended in Ringer's lactate. Five milliliters of suspension were collected for cell counting, viability, pluripotency, flow cytometry, and karyotyping. The remaining suspension was infused into the periphery of the recipient. Transplantation was performed 1 week later following a negative lymphocytotoxicity cross-match test using no immunosuppression. Peripheral blood chimerism (PBC) was studied using fluorescent in situ hybridization technique. Allograft biopsy was performed on day 7., Results: NT-hESC CD34+ count was 7.6%, viability 100%, karyotyping normal, pluripotency markers: SSEA-1, SSEA-4, OCT-3/4, TRA-1/60:positive; 12% PBC was noted at 1 week after transplantation. Serum creatinine was 1.2 mg%, graft biopsy was unremarkable, and G6PD enzyme deficiency was corrected to 0% at 100 days posttransplant. Liver function tests and hematology profile were unremarkable for graft-versus-host disease., Conclusion: This is the first report of tolerance induction using NT-hESC-induced hematopoietic chimerism with synergistic use of adult bone marrow. It was safe and effective.

Transplantation is standard therapy for many patients suffering from kidney, liver, or heart failure. In contrast, transplantation of the intestine remains a high-risk procedure, which is performed in a minority of patients with short bowel syndrome. The difficulty is the strong alloimmune response caused by intestinal grafts and the complications of the profound immunosuppression. We tested a new clinical immunomodulatory protocol using donor-specific blood transfusion, a strategy that was popular before the introduction of cyclosporine and was recently shown to promote development of regulatory cells. Low-dose steroids and low-dose tacrolimus were administered based on previous observations that tolerance requires an intact immune system, that over-immunosuppression is counterproductive, and that high doses of calcineurin inhibitors block development of regulatory cells whereas low doses promote it. Finally, inflammation within the intestinal graft was minimized to reduce the additional stimulants that the innate immunity of the transplanted intestine exert on the adaptive immune response. Under this protocol, freedom from rejection was achieved in four consecutive intestinal transplant recipients using extremely low immunosuppression.