Donor-specific Tolerance Research Articles

A Clinically Feasible Approach to Induce Delayed Tolerance in Recipients of Prior Kidney or Vascularized Composite Allotransplants

Mixed chimerism induces donor-specific tolerance to kidney and vascularized composite allotransplants (VCA). However, simultaneous kidney or VCA and bone marrow transplantation (BMT) is problematic because of the combined risk and time required for conditioning. Here, we developed a delayed tolerance induction strategy with mixed chimerism through BMT in prior kidney or VCA recipients. Wistar Furth rats that received kidney transplantation (KTx) or VCA from allogeneic August-Copenhagen Irish donors were maintained on immunosuppression (IS) for 8 weeks. These recipients were then conditioned with anti-αβ-T-cell receptor and anti-CD8 monoclonal antibodies, total body irradiation, cyclosporine A and mycophenolate mofetil (12 doses), and antilymphocyte serum (one dose); and transplanted with T-cell-depleted donor marrow. All IS was discontinued on day 11 after BMT. Cyclosporine A monotherapy prevented acute rejection of KTx or VCA. However, all allografts were rejected after IS withdrawal in KTx or VCA recipients who were conditioned but did not receive BMT. After delayed BMT, mixed chimerism was initially achieved in all KTx or VCA recipients with 200-, 300-, and 400-cGy total body irradiation. Long-term tolerance to KTx or VCA was achieved in most of these recipients with total IS withdrawal. The tolerance achieved with delayed BMT was donor specific as confirmed by acceptance of donor skin and rejection of third-party skin graft. IS-free donor-specific tolerance can be successfully induced with delayed BMT to previous recipients of kidney transplantation or VCA. These findings have significant clinical implications for transplant recipients who receive an organ from either a living donor or a deceased donor with frozen bone marrow cells available.

Evolving approaches of hematopoietic stem cell-based therapies to induce tolerance to organ transplants: the long road to tolerance.

The immunoregulatory properties of hematopoietic stem cells (HSCs) have been recognized for more than 60 years, beginning in 1945, when Owen reported that genetically disparate freemartin cattle sharing a common placenta were red blood cell chimeras. In 1953, Billingham, Brent, and Medawar demonstrated that murine neonatal chimeras prepared by infusion of donor-derived hematopoietic cells exhibited donor-specific tolerance to skin allografts. Various approaches using HSCs in organ transplantation have gradually brought closer to reality the dream of inducing donor-specific tolerance in organ transplant recipients. Several hurdles needed to be overcome, especially the risk of graft-versus-host disease (GVHD), the toxicity of ablative conditioning, and the need for close donor-recipient matching. For wide acceptance, HSC therapy must be safe and reproducible in mismatched donor-recipient combinations. Discoveries in other disciplines have often unexpectedly and synergistically contributed to progress in this area. This review presents a historic perspective of the quest for tolerance in organ transplantation, highlighting current clinical approaches.

Cell-specific Expression of the Anti-apoptotic Gene XIAP Protects Islet Allografts and Induces Donor-Specific Tolerance

Cell-specific Expression of the Anti-apoptotic Gene XIAP Protects Islet Allografts and Induces Donor-Specific Tolerance

CD4 + CD25 + regulatory T cells-induced tolerance of islet transplantation by targeting OX40/ OX40L costimulation pathway

Objective To observe the role of OX40/OX40L costimulation pathway in the CD4+CD25 + regulatory T (Treg) cells-induced tolerance of islet transplantation.Methods Diabetes mellitus was induced in C57BL/6 mice as recipients,and islets from DBA/2 mice were transplanted into the C57BL/6 mice.The recipients were divided into three groups (n =8):treated with IgG as controls,antiOX40L mAb (RM134L) + anti-CD154 mAb (MR1),and RM134L + MR1 + anti-CD25 mAb.The mean survival time (MST) of allograft was observed.The expression of OX40 in T effector cells,Treg cells of wide type and CD154 knock-out mice was detected.The expression of Foxp3 gene in allograft in the absence and absence of anti-OX40L mAb and anti-CD154 mAb was detected.T effector cells and Treg cells were co-cultured at different ratio,and the cell proliferation was assayed.Results The MST in RM134L +MR1 group was ＞ 150 days (n =8),significantly longer than in IgG group ( MST + 19 days,n =5 ) and RM134L + MR1 + anti-CD25 mAb (23 days,n =4) (P ＜0.05).Anti-OX40L mAb and anti-CD154 mAb treatment could successfully induce tolerance,and also induce donor specific tolerance,but the tolerance was blocked by anti-CD25 mAb treatment (P ＜ 0.05 ).OX40 was expressed on the Treg cells surface,whereas the T effector cells had no OX40 expression.The expression level of Foxp3 gene in the allograft treated with anti-OX40L mAb and anti-CD154 mAb was 123 AU,significantly higher than without treatment (21 AU) and syngenic control ( 8 AU) ( P ＜ 0.05 ).After Treg cells were co-cultured with T effector cells at different ratio ( 1∶2 and 1∶ 4),the mean count per minute (CPM) of T effector cells was 63 x 103and 41 × 103 respectively (P ＜0.05),and after addition of OX40,the CPM was 128 × 103 and 135 × 103respectively ( P ＞ 0.05 ).Conclusion CD4 + CD25 + Treg cells were critical to tolerance induction in mice islet transplant recipients through targeting OX40/OX40L costimulation pathway. Key words: Islet transplantation; Regulatory T cells; Tolerance; OX40

Hematopoietic stem cell transplantation induces immunologic tolerance in renal transplant patients via modulation of inflammatory and repair processes

BackgroundInducing donor-specific tolerance in renal transplant patients could potentially prevent allograft rejection and calcineurin inhibitor nephrotoxicity. Combined kidney and hematopoietic stem cell transplant from an HLA-matched donor is an exploratory and promising therapy to induce immune tolerance. Investigtion of molecular mechanisms involved in the disease is needed to understand the potential process of cell therapy and develop strategies to prevent this immunologic rejection.MethodsWe enrolled nine patients in a clinical study in which cryopreserved donor hematopoietic stem cells were infused on days 2, 4, and 6 after kidney transplantation. One month post-transplant, 4 plasma samples were collected from combined transplants (C + Tx), and 8 plasma samples from patients with kidney transplantation alone (Tx). High abundance proteins in plasma were depleted and the two-dimensional liquid chromatography-tandem mass spectrometry coupled with iTRAQ labeling was utilized to identify the protein profiling between the two groups. Clusters of up- and down-regulated protein profiles were submitted to MetaCore for the construction of transcriptional factors and regulation networks.Results and DiscussionAmong the 179 identified proteins, 65 proteins were found in C + Tx with at least a 2-fold change as compared with Tx. A subset of proteins related to the complement and coagulation cascade, including complement C3a,complement C5a, precrusors to fibrinogen alpha and beta chains,was significantly downregulated in C + Tx. Meanwhile, Apolipoprotein-A1(ApoA1), ApoC1, ApoA2, ApoE, and ApoB were significantly lower in Tx compared to C + Tx. Gene ontology analysis showed that the dominant processes of differentially expressed proteins were associated with the inflammatory response and positive regulation of plasma lipoprotein particle remodeling.ConclusionsThus, our study provides new insight into the molecular events in the hematopoietic stem cell-induced immunologic tolerance.

Tolerance Induction by Exosomes from Immature Dendritic Cells and Rapamycin in a Mouse Cardiac Allograft Model

BackgroundDendritic cells (DCs) release bioactive exosomes that play an important role in immune regulation. Because they express low levels of class I major histocompatibility complex (MHC) and co-stimulatory molecules, exosomes derived from donor immature DCs (imDex) prolong allograft survival by inhibiting T-cell activation. However, this effect is limited and does not induce immunological tolerance when imDex are administered alone. Thus, we tested the effect of combined treatment with donor imDex and low-dose rapamycin on inducing tolerance in a mouse cardiac transplantation model.MethodsImDex were obtained from the culture supernatant of immature DCs derived from donor mouse (C57BL/6) bone marrow and were injected with suboptimal doses of rapamycin into recipient mouse (BALB/c) before and after transplantation. The capacity of this treatment to induce immune tolerance was analyzed in vitro and in vivo using the mouse cardiac transplantation model.ResultsDonor imDex expressed moderate levels of MHC class II and low levels of MHC class I and co-stimulatory molecules, but neither imDex nor subtherapeutic rapamycin dose alone induced cardiac allograft tolerance. Combined treatment with imDex and rapamycin, however, led to donor specific cardiac allograft tolerance. This effect was accompanied by decreased anti-donor antigen cellular response and an increased percentage of spleen CD4+CD25+ T cells in recipients. Furthermore, this donor specific tolerance could be further transferred to naïve allograft recipients through injection of splenocytes, but not serum, from tolerant recipients.ConclusionCombined with immunosuppressive treatment, donor imDex can prolong cardiac allograft survival and induce donor specific allograft tolerance.

Commentary on “Combined treatment with regulatory T cells and vascularized bone marrow transplantation creates mixed chimerism and donor-specific tolerance to vascularized composite allografts without cytoreductive conditioning”

Transplantation of vascularized composite allografts (VCAs) could significantly affect the liver of the approximately 7 million Americans every year. These Americans would benefit from transplantation because they had undergone or need to undergo reconstructive surgery as the result of oncologic procedures, congenital anomalies, and trauma [ [1] Gander B. Brown C.S. Vasilic D. et al. Composite tissue allotransplantation of the hand and face: a new frontier in transplant and reconstructive surgery. Transplant Int. 2006; 19: 868 Crossref PubMed Scopus (77) Google Scholar ]. Just as important, it may also help bring us a step closer to the “Holy Grail” of transplantation, which is the induction of donor-specific immunologic tolerance. The reason is because, to achieve a state of tolerance, the most promising approach appears to be the creation of mixed hematopoietic chimerism [ [2] Bradley J.A. Induction of transplant tolerance through mixed hematopoietic chimerism. Am J Transplant. 2012; 12: 1073 Crossref PubMed Scopus (3) Google Scholar ]. The significance of VCA transplantation lies in the fact that by definition, VCA already contain hematopoietic tissue, which can lead to chimerism with reduced conditioning. Combined treatment with regulatory T cells and vascularized bone marrow transplantation creates mixed chimerism and induces donor-specific tolerance to vascularized composite allografts without cytoreductive conditioningJournal of Surgical ResearchVol. 178Issue 2PreviewCotreatment with regulatory T cells (Treg) and conventional allogeneic bone marrow transplantation (BMT) successfully induced durable chimerism and tolerance to nonvascularized skin allografts without cytoreductive conditioning in mice. We sought to determine whether Treg treatment combined with vascularized BMT (VBMT) could create mixed chimerism and induce tolerance to vascularized composite allografts (VCAs) without cytoreductive conditioning in rats. Full-Text PDF

Engraftment Potential of Adipose Tissue-Derived Human Mesenchymal Stem Cells After Transplantation in the Fetal Rabbit

Due to their favorable intrinsic features, including engraftment, differentiation, and immunomodulatory potential, adult mesenchymal stem cells (MSCs) have been proposed for therapeutic in utero intervention. Further improvement of such attributes for particular diseases might merely be achieved by ex vivo MSC genetic engineering previous to transplantation. Here, we evaluated for the first time the feasibility, biodistribution, long-term engraftment, and transgenic enhanced green fluorescent protein (EGFP) expression of genetically engineered human adipose tissue-derived MSCs (EGFP(+)-ASCs) after intra-amniotic xenotransplantation at E17 of gestation into our validated pregnant rabbit model. Overall, the procedure was safe (86.4% survival rate; absence of anatomical defects). Stable, low-level engraftment of EGFP(+)-ASCs was confirmed by assessing the presence of the pWT-EGFP lentiviral provirus in the young transplanted rabbit tissues. Accordingly, similar frequencies of provirus-positive animals were found at both 8 weeks (60%) and 16 weeks (66.7%) after in utero intervention. The presence of EGFP(+)-ASCs was more frequent in respiratory epithelia (lung and trachea), according to the route of administration. However, we were unable to detect EGFP expression, neither by real-time polymerase chain reaction nor by immunohistochemistry, in the provirus-positive tissues, suggesting EGFP transgene silencing mediated by epigenetic events. Moreover, we noticed lack of both host cellular immune responses against xenogeneic ASCs and humoral immune responses against transgenic EGFP. Therefore, the fetal microchimerism achieved by the EGFP(+)-ASCs in the young rabbit hosts indicates induction of donor-specific tolerance after fetal rabbit xenotransplantation, which should boost postnatal transplantation for the early treatment/prevention of many devastating congenital disorders.

Ethylenecarbodiimide-Fixed Donor Splenocyte Infusions Differentially Target Direct and Indirect Pathways of Allorecognition for Induction of Transplant Tolerance

Strategic exposure to donor Ags prior to transplantation can be an effective way for inducting donor-specific tolerance in allogeneic recipients. We have recently shown that pretransplant infusion of donor splenocytes treated with the chemical cross-linker ethylenecarbodiimide (ECDI-SPs) induces indefinite islet allograft survival in a full MHC-mismatched model without the need for any immunosuppression. Mechanisms of allograft protection by this strategy remain elusive. In this study, we show that the infused donor ECDI-SPs differentially target T cells with indirect versus direct allospecificities. To target indirect allospecific T cells, ECDI-SPs induce upregulation of negative, but not positive, costimulatory molecules on recipient splenic CD11c(+) dendritic cells phagocytosing the injected ECDI-SPs. Indirect allospecific T cells activated by such CD11c(+) dendritic cells undergo robust initial proliferation followed by rapid clonal depletion. The remaining T cells are sequestered in the spleen without homing to the graft site or the graft draining lymph node. In contrast, direct allospecific T cells interacting with intact donor ECDI-SPs not yet phagocytosed undergo limited proliferation and are subsequently anergized. Furthermore, CD4(+)CD25(+)Foxp3(+) T cells are induced in lymphoid organs and at the graft site by ECDI-SPs. We conclude that donor ECDI-SP infusions target host allogeneic responses via a multitude of mechanisms, including clonal depletion, anergy, and immunoregulation, which act in a synergistic fashion to induce robust transplant tolerance. This simple form of negative vaccination has significant potential for clinical translation in human transplantation.

Combined treatment with regulatory T cells and vascularized bone marrow transplantation creates mixed chimerism and induces donor-specific tolerance to vascularized composite allografts without cytoreductive conditioning

BackgroundCotreatment with regulatory T cells (Treg) and conventional allogeneic bone marrow transplantation (BMT) successfully induced durable chimerism and tolerance to nonvascularized skin allografts without cytoreductive conditioning in mice. We sought to determine whether Treg treatment combined with vascularized BMT (VBMT) could create mixed chimerism and induce tolerance to vascularized composite allografts (VCAs) without cytoreductive conditioning in rats. MethodsRecipient Lewis rats treated (day 0) with or without naturally sorted Treg (3 × 106) from Lewis rat spleen and lymph nodes received costimulation blockade (anti-CD154 monoclonal antibody, days 0 and 1 and CTLA-4 immunoglobin, days 2, 4, and 6), rapamycin (days −1, 0, and 2), and concurrent transplantation of fully mismatched allogeneic donor VCAs (day 0) from the Brown Norway rat hindlimb containing VBMT. The mixed chimerism level was assessed monthly using flow cytometry. Survival of VCAs and occurrence of graft-versus-host disease were assessed clinically and histologically. ResultsThe combination of Treg and VBMT treatment led to long-term multilineage hematopoietic mixed chimerism (12–18%) and long-term donor-specific tolerance to VCAs (89% acceptance rate). Neither stable mixed chimerism nor VCA acceptance was observed in recipients without Treg treatment. Graft-versus-host disease did not occur in the VBMT recipients. ConclusionsCotreatment with Treg and VBMT created stable mixed chimerism and induced long-term donor-specific tolerance to VCAs without requiring cytoreductive conditioning. This noncytoreductive Treg-VBMT protocol has potential for clinical application in VCAs.

Application of Regulatory T Cells in Transplantation Field

The development of immunosuppressant treatments has enabled remarkable progress in the tissue and organ transplantation field by helping to prevent acute graft rejection. However, complications related to transplantation, such as infection by bacteria and viruses, and the occurrence of cancers resulting from prolonged immune suppression are major obstacles to overcome. Therefore, transplantation immunology research efforts should focus on the induction of donor-specific immune tolerance which preserves patient immune competence which promotes infection and cancer surveillance. Additionally, lifelong administration of immunosuppressants should be forgone in preference to short term therapies. In the 1990s, Dr. Shimon Sakaguchi identified the CD4CD25 regulatory T cells which develop in the thymus, and demonstrated that these cells play crucial roles in the maintenance of immune self tolerance. Studies which followed proved that these regulatory T cells are important to the control of autoimmune disease and prevention of graft rejection. Regulatory T cells have also been found to induce immune tolerance in rodent models. In this review, we discuss several considerations for the use of regulatory T cell therapy in the clinical transplantation field.

Allograft rejection is restrained by short-lived TIM-3+PD-1+Foxp3+ Tregs

Tregs play a pivotal role in inducing and maintaining donor-specific transplant tolerance. The T cell immunoglobulin and mucin domain-3 protein (TIM-3) is expressed on many fully activated effector T cells. Along with program death 1 (PD-1), TIM-3 is used as a marker for exhausted effector T cells, and interaction with its ligand, galectin-9, leads to selective death of TIM-3+ cells. We report herein the presence of a galectin-9-sensitive CD4+FoxP3+TIM-3+ population of T cells, which arose from CD4+FoxP3+TIM-3- proliferating T cells in vitro and in vivo and were often PD-1+. These cells became very prominent among graft-infiltrating Tregs during allograft response. The frequency and number of TIM-3+ Tregs peaked at the time of graft rejection and declined thereafter. Moreover, these cells also arise in a tolerance-promoting donor-specific transfusion model, representing a pool of proliferating, donor-specific Tregs. Compared with TIM-3- Tregs, TIM-3+ Tregs, which are often PD-1+ as well, exhibited higher in vitro effector function and more robust expression of CD25, CD39, CD73, CTLA-4, IL-10, and TGF-β but not galectin-9. However, these TIM-3+ Tregs did not flourish when passively transferred to newly transplanted hosts. These data suggest that a heretofore unrecognized graft-infiltrating, short-lived subset of Tregs can restrain rejection.

Dendritic Cell Therapies in Transplantation Revisited: Deletion of Recipient DCs Deters the Effect of Therapeutic DCs

A critical goal in transplantation is the achievement of donor-specific tolerance, minimizing the use of immunosuppressants. Dendritic cells (DCs) are antigen (Ag) presenting cells (APCs) with capability to promote immunity or tolerance. The immune-regulatory properties of DCs have been exploited for generation of tolerogenic/immunosuppressive (IS) DCs that, when transfer systemically, prolong allograft survival in murine models. Surprisingly, the in vivo mechanisms of therapies based on (donor- or recipient-derived) ISDCs in transplantation remain unknown, given that previous studies investigated their effects in vitro, or ex vivo after transplantation. Since once injected, ISDCs are short-lived and transfer Ag to recipient APCs, we assessed the role of recipient DCs by depleting them at the time of ISDC-therapy in a mouse model of cardiac transplantation. The results indicate that, contrary to the accepted paradigm, systemically administered ISDCs reduce the alloresponse and prolong allograft survival, not by themselves, but through conventional DCs (cDCs) of the recipient. These findings raise doubts on the advantages of the currently used ISDC-therapies, since the immune-regulatory properties of the injected ISDC do not seem to be functionally relevant in vivo, and the quiescent/pro-tolerogenic status of cDCs may be compromised in patients with end-stage diseases that require transplantation.

Role of T-Cell-Specific Nuclear Factor κB in Islet Allograft Rejection

Pancreatic islet transplantation has the potential to cure type 1 diabetes, a chronic lifelong disease, but its clinical applicability is limited by allograft rejection. Nuclear factor κB (NF-κB) is a transcription factor important for survival and differentiation of T cells. In this study, we tested whether NF-κB in T cells is required for the rejection of islet allografts. Mice expressing a superrepressor form of NF-κB selectively in T cells (IκBαΔN-Tg mice) with or without the antiapoptotic factor Bcl-xL, or mice with impaired T-cell receptor (TCR)- and B cell receptor-driven NF-κB activity (CARMA1-KO mice) were rendered diabetic and transplanted with islet allografts. Secondary skin transplantation in long-term acceptors of islet allografts was used to test for the development of donor-specific tolerance. Immune infiltration of the transplanted islets was examined by immunofluorescence. TCR-transgenic CD4 T cells were used to follow T-cell priming and differentiation. Islet allograft survival was prolonged in IκBαΔN-Tg mice, although the animals did not develop donor-specific tolerance. Reduced NF-κB activity did not prevent T-cell priming or differentiation but reduced survival of activated T cells, as transgenic expression of Bcl-xL restored islet allograft rejection in IκBαΔN-Tg mice. Abolishing TCR- and B cell receptor-driven activation of NF-κB selectively by CARMA1 deficiency prevented T-cell priming and islet allograft rejection. Our data suggest that T cell-NF-κB plays an important role in the rejection of islet allografts. Targeting NF-κB selectively in lymphocytes seems a promising approach to facilitate acceptance of transplanted islets.

Immunological Issues in Clinical Composite Tissue Allotransplantation

Composite tissue allografts are made of histogenetically different tissues and although skin seems to be the most antigenic of them, it is unknown whether the dominant immune response is really directed against the skin or we have insufficient information on the involvement of the other components of these allografts. The first clinical signs of acute rejection manifest on the skin and microscopically the earliest lesions consist in a dermal perivascular lymphocytic infiltrate, predominantly made of CD3+/CD4+ T cells. On the basis of the histological changes, a specific score (Banff score 2007) has been established to assess the severity of rejection. Despite the high incidence of acute rejection episodes, they can be completely reversed when promptly diagnosed and treated. C4d deposits in the skin and circulating donor-specific antibodies are rarely detected, suggesting that humoral rejection does not play a significant role in composite tissue allotransplantation. Chronic rejection features are still unknown. The majority of recipients have been maintained on an immunosuppressive regimen consisting of tacrolimus, steroids, and mycophenolate mofetil. The complications in composite tissue allotransplantation are similar to those usually reported after solid organ transplantation and have prompted different strategies to minimize the maintenance immunosuppression or to induce donor-specific tolerance. Furthermore, to what extent the immunosuppression can be tapered is unknown, as well as the influence of donor bone-marrow infusion in tolerance and chronic rejection. The increasing number of patients and the longer follow-up hopefully will allow answering these questions.

Establishment of nonmyeloablative bone marrow chimerism by double negative Treg cells through inducing T cell clonal deletion and suppressing NK cell function (126.1)

Abstract In bone marrow transplantation, T cells and NK cells play important role for graft rejection. In addition, graft-versus-host-disease and establishment of stable chimeric states without complete marrow ablation remain as major obstacles post bone marrow transplantation. In this study, we aimed to establish mixed chimerism in a non-irradiation condition. Our data indicate that adoptive transfer of donor-derived TCRαβ+CD3+CD4-CD8-NK1.1-(double negative, DN) Treg cells prior to C57BL/6 to BALB/c bone marrow transplantation, in combination with cyclophosphamide but not cyclosporine, FK506, or rapamycin, established stable mixed chimerism that led to acceptance of C57BL/6 skin allografts and rejection of 3rd party C3H skin grafts. Adoptive transfer of CD4+ and CD8+ T cells, but not DN-Treg cells, induced graft-versus-host diseases in this regimen. The recipient T cell alloreacitve responsiveness was reduced in the DN-Treg cell-treated group and T cell receptor Vβ2, Vβ7 and Vβ8 clonal deletions were observed in both CD4+ and CD8+ T cells. Furthermore, DN-Treg cell treatment suppressed NK cell-mediated donor bone marrow rejection in perforin-dependent manner. Taken together, our results suggest that adoptive transfer of DN-Treg cells can control both adoptive and innate immunity and promote a stable mixed chimerism and donor-specific tolerance in the non-irradiation regimen.

Inhibition of p300 impairs Foxp3+ T-regulatory (Treg) function and promotes anti-tumor immunity (127.40)

Abstract Foxp3+ Treg cells limit autoimmunity and maintain immune homeostasis but also restrict immune responses to cancer. Thus, Tregs are a promising target for modulation of host anti-tumor immune responses. We have shown that targeting of Foxp3+ Tregs using histone/protein deacetylase inhibitors (HDACi) promotes Foxp3 acetylation and increases Treg production and suppression function. By contrast, there are no studies concerning the role of histone/protein acetyltransferases (HAT) targeting in Tregs. HATs are classified into 3 main families; GNAT, MYST and p300/CBP. Here, we found that conditional deletion of p300 in Tregs caused lymphoproliferation, weight loss, arthritis and dermatitis in mice. Likewise, deletion of p300 in Tregs, or its pharmacologic inhibition, decreased Foxp3+ Treg production and impaired Treg-mediated suppression of homeostatic proliferation, and blocked Treg-dependent donor-specific allograft tolerance. In contrast with these data, p300 targeting decreased TC1 lung tumor volume in immunocompetent C57BL/6 mice, but had no direct effect on TC1 tumor cell proliferation in vitro, or on the growth of TC1 implanted tumors in RAG1-/- immunodeficient mice. We conclude that in real world tumor models using immunocompetent mice, p300 targeting impaired Treg function without overt impairing effector T cell responses or inducing autoimmunity. We thereby identify p300 as a critical target for modulation of Foxp3+ Treg function in the context of anticancer therapy.

Changes in the proteasome function after induction of donor-specific tolerance in rats with ovarian allograft

Induction of donor-specific tolerance in a recipient is one of the methods for enhancing acceptance of the grafts of endocrine glands in the absence of immunodepressants, which interfere with hormone production. This paper describes changes in the proteasome pool in the rat liver, spleen, and graft during the development of donor-specific tolerance after intraportally infusing the recipient with donor splenocytes with subsequent allografting of ovarian tissue into the renal capsule. It has been demonstrated that the shift in the balance in the liver and graft proteasome pools towards the variants with the LMP2 subunit determines the development of immunological tolerance and graft retention. On the contrary, an increase in the forms with the LMP7 subunit induces the immune response and graft rejection.

A role for anti-CD45RB monoclonal antibody treatment upon dendritic cells

Selective interference with CD45RB isoform by monoclonal antibody (anti-CD45RBmAb) reliably induces donor-specific tolerance. Dendritic cells (DCs) are the most potent antigen-presenting cells that are capable of activating naïve T cells. The purposes of the present study were to investigate the roles of anti-CD45RBmAb on the phenotypes and functioning of DCs and to further illustrate the mechanism of anti-CD45RBmAb-inducing immunologic tolerance. DCs from C57BL/6 mice were cultured and treated with various doses of anti-CD45RB monoclonal antibody. Cell phenotype, cycle and phagocytic ability were detected by flow cytometry. The production of IL-10 and IL-12 in the supernatants of mature DCs was measured with ELISA. Exosomes (Dex) were recovered from the supernatant of DCs cultured for 6 days in depleted medium, and effects of DCs and Dex on the ability of T-cell proliferation were detected by mixed lymphocyte culture. Anti-CD45RBmAb could inhibit DCs maturation in a dose-dependent manner, and the effects of exosomes (Dex) on DCs enhance or inhibition proliferation of T cells were also in a dose-dependent manner. Anti-CD45RBmAb could profoundly inhibit the maturation and functioning of DCs and generate tolerogenic dendritic cells (tDCs) as well as Dex, suggesting mechanistic contributions to tolerance development from the DCs through interactions with T cells.

Double negative Treg cells promote nonmyeloablative bone marrow chimerism by inducing T‐cell clonal deletion and suppressing NK cell function

The establishment of immune tolerance and prevention of chronic rejection remain major goals in clinical transplantation. In bone marrow (BM) transplantation, T cells and NK cells play important roles for graft rejection. In addition, graft-versus-host-disease (GVHD) remains a major obstacle for BM transplantation. In this study, we aimed to establish mixed chimerism in an irradiation-free condition. Our data indicate that adoptive transfer of donor-derived T-cell receptor (TCR) αβ(+) CD3(+) CD4(-) CD8(-) NK1.1(-) (double negative, DN) Treg cells prior to C57BL/6 to BALB/c BM transplantation, in combination with cyclophosphamide, induced a stable-mixed chimerism and acceptance of C57BL/6 skin allografts but rejection of third-party C3H (H-2k) skin grafts. Adoptive transfer of CD4(+) and CD8(+) T cells, but not DN Treg cells, induced GVHD in this regimen. The recipient T-cell alloreactive responsiveness was reduced in the DN Treg cell-treated group and clonal deletions of TCRVβ2, 7, 8.1/2, and 8.3 were observed in both CD4(+) and CD8(+) T cells. Furthermore, DN Treg-cell treatment suppressed NK cell-mediated BM rejection in a perforin-dependent manner. Taken together, our results suggest that adoptive transfer of DN Treg cells can control both adoptive and innate immunities and promote stable-mixed chimerism and donor-specific tolerance in the irradiation-free regimen.