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Background: Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature cells that suppress immune responses during organ transplantation and participate in mediating long-term graft survival and immune tolerance in animal transplant models. However, their role in regulating transplant tolerance in human subjects is not well understood. In the present study, we investigated the role of MDSCs in mediating long-term graft survival in almost-tolerant kidney transplant recipients (ATKTRs) and the mechanism(s) responsible for increasing MDSC numbers in these recipients., Methods: Peripheral blood mononuclear cells (PBMCs) from whole blood samples were collected from 30 ATKTRs (graft survival, > 10 years after kidney transplant [KTx]) treated with low doses of immunosuppressive drugs and with stable kidney function, 10 short-term graft survival kidney transplant recipients (STKTRs; graft survival, ∼1-3 years post-KTx) with stable kidney function, and 10 healthy donors (HDs). MDSC and regulatory T cell (Tregs) levels were analyzed using multicolor flow cytometry in PBMCs., Results: ATKTRs had significantly higher levels of monocytic MDSCs (P < .001) and CD4 + CD25 + FoxP3 + Tregs than STKTRs and HDs. Furthermore, the M-MDSC levels correlated positively with the survival rates, estimated glomerular filtration rates (eGFRs) of grafts, and the levels of CD4 + CD25 + FoxP3 + Tregs in ATKTRs., Conclusions: Accumulation of high levels of MDSCs was observed in ATKTRs. Changes in MDSC levels may play important roles in mediating transplant tolerance and regulating Tregs. Therefore, we propose that MDSCs may be potentially used for recognizing tolerant transplant recipients and guiding dosage reduction for immunosuppressive drugs for KTx., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Recognition of evolutionarily conserved ligands by Toll-like receptors (TLRs) triggers signaling cascades in innate immune cells to amplify adaptive immune responses. Nearly all TLRs require MyD88 to transduce downstream signaling. MyD88 deficiency has been shown to promote the allograft acceptance in mice. However, direct evidence for therapeutic potential of MyD88 inhibitors remains lacking. Herein, we used a MyD88 inhibitor, namely ST2825, to explore its therapeutic potential and mechanisms in fully allogeneic skin and heart transplant models. Phenotypic maturation of dendritic cells stimulated by TLR ligands was alleviated by ST2825 in parallel with reduced T-cell proliferation in vitro. A short-course treatment with ST2825 significantly prolonged cardiac graft survival (mean survival time = 18.5 ± 0.92 days vs. 7.25 ± 0.46 days). ST2825-treated group had significantly reduced proinflammatory cytokines in allografts compared with control group. ST2825 combined with anti-CD154 induced long-term skin allograft acceptance in about one-third of recipients (>100 days). 'Skin-tolerant' recipients showed attenuated donor-specific IFN-γ responses, intact IL-4 responses, and compromised alloantibody responses. We conclude that MyD88 inhibitor ST2825 attenuates acute cardiac rejection and promotes donor-specific hyporesponsiveness in stringent skin transplant models. The direct evidence suggests that pharmacological inhibition of MyD88 hold promising potential for transplant rejection., (© 2016 Steunstichting ESOT.)

Recent data suggest that activation of aryl hydrocarbon receptor (AhR) by its high-affinity ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) results in expansion of regulatory T (Treg) cells and suppresses the development of autoimmune and allergic diseases in several models. Treg cells have been increasingly documented to suppress allograft rejection and even to establish stable long-term graft acceptance. However, the involvement of TCDD in the regulation of solid organ transplantation rejection is largely unknown. Here, we examined whether activation of AhR with TCDD altered cardiac allograft rejection in an allogeneic heart transplant model. Recipient C57BL/6 (H-2b) mice were administrated with a single intraperitoneal injection of TCDD, and the murine cardiac transplant models from BALB/c (H-2d) to C57BL/6 (H-2b) were built 24 h later. The complete cessation of cardiac contractility was defined as the observation endpoint. The effect of TCDD on T-cell proliferation was assessed by mixed lymphocyte reaction (MLR). Histological and immunohistochemical analyses were performed to estimate the severity of rejection. The phenotype and cytokine profile of lymphocytes were analyzed by flow cytometry and enzyme-linked immunosorbent assay (ELISA). Activation of AhR remarkably prolonged the survival of cardiac allografts to more than 20 days. In vitro, TCDD ugregulated the frequency of CD4+CD25+Foxp3+ Treg cells and suppressed the proliferation of T lymphocytes. In vivo, the prolonged survival time was associated with increased number of Treg cells in allografts and spleens. Furthermore, the secretion of interferon-γ (IFN-γ) and interleukin-17 (IL-17) was reduced to less than 50% of that of the PBS treatment control group by TCDD treatment, whereas IL-10 was elevated to 10-fold of that of the PBS treatment control group. Collectively, our data indicate that activation of AhR with a single dose of TCDD significantly prolonged the survival of fully allogeneic cardiac grafts, and the mechanism underlying this effect might be involved in the induction of Treg cells.

Background: The involvement of T-cell immunoglobulin mucin-3 (Tim-3) in the regulation of solid organ transplantation rejection is largely unknown. We used galectin-9 (Tim-3 ligand) to evaluate the effect and mechanisms of Tim-3/galectin-9 pathway in an allogeneic heart transplant model., Methods: The murine cardiac transplant model from BALB/c (H-2d) to C57BL/6 (H-2b) was built. The recipients were administered with galectin-9 for 7 days from day 1 or day 3 posttransplant. The complete cessation of cardiac contractility was defined as the observation endpoint. The effect of galectin-9 on cell proliferation was assessed by mixed lymphocyte reaction. Histology and immunohistochemistry were performed to estimate the severity of rejection. The phenotype and cytokine profile of lymphocytes were analyzed by flow cytometry., Results: In vitro, galectin-9 suppressed the proliferation of lymphocytes mixed lymphocyte reactions in a dose- and beta-galactoside-dependent manner. In vivo, galectin-9 treatment from day 1 to day 3 posttransplant achieved similar survival time of grafts (median survival time, 22.7+/-1.2 vs. 23.0+/-1.0 days). The prolonged survival time was associated with reduced infiltration of CD4 and CD8 lymphocytes in allografts. Furthermore, the intragraft transcriptional profiling in galectin-9-treated group showed reduction of IFN-gamma and IL-17 mRNA but elevation of Ebi-3 and galectin-9 mRNA. Flow cytometry analysis indicated that galectin-9 treatment reduced the ratio of T helper (Th) 1 and Th17 cells in the draining lymph nodes and peripheral blood., Conclusions: A short-course administration of galectin-9 significantly prolonged the survival of fully allogeneic cardiac allografts, which was associated with the suppression of Th1 and Th17 immune responses.

Background: We sought to investigate the effects of donor-specific transfusion (DST) combined with rapamycin (RPM) on engraftment and demonstrate the mechanisms., Methods: B6 was treated with DST (on day -8) combined with RPM (from day -7 to -1, 1.5 microL/g per day). On day 0, B6 splenocytes were harvested to coculture with Balb/c splenocytes pretreated with mitomycin C in 1-way mixed lymphocyte cultures (MLC). After 48 hours, the proliferation, we measured apoptosis and the CD4(+)CD25(+) T-cell ratio of cultured cells. The heterotopic cardiac transplantation model was performed from Balb/c to B6 using the recipients pretreated with this protocol. On day 0, RPM was withdrawn and the transplantations performed. The mean survival time (MST) of the grafts evaluated and in vitro assays measured., Results: Both in vitro and in vivo, the protocol suppressed the proliferation of splenocytes as well as enhanced apoptosis and the CD4(+)CD25(+) T-cell ratio. The MST of the grafts was 35 +/- 14.4 days., Conclusions: Our study showed that DST combined with RPM prolonged cardiac allograft survival even if we withdrew immunosuppressants after transplantation. This result may be associated with inhibition of T-cell proliferation. We also demonstrated that RPM expanded CD4(+)CD25(+) T cells and increased splenocyte apoptosis.

T cell immunoglobulin and mucin domain (Tim)-3 is a molecule expressed on terminally differentiated murine Th1 cells but not on Th2 cells. Identification of Galectin-9 as a ligand for Tim-3 has now firmly established the Tim-3-Galectin-9 pathway as an important regulator of Th1 immunity, which results in apoptosis of Th1 cells. Here, we demonstrate that engagement of Tim-3 by mouse recombinant Galectin-9 remarkably suppresses allograft rejection and improves survival of allogeneic skin grafts. Furthermore, administration of recombinant Galectin-9 decreases Tim-3 positive cells in draining lymph node and selectively inhibits production of IFN-gamma after skin transplantation. At last, even low dose of Galectin-9 (1 microg/ml) can obviously inhibit TCR crosslinking-induced primary T cell proliferation in vitro. These observations suggest that Tim-3-Galectin-9 pathway plays an important role in the termination of productive Th1-immune response and could lead to developing novel therapies in transplant medicine.

CD8+ alloreactive T cells are the key mediators of accelerated rejection. Vigorous CD8+ alloreactive T cells responses against alloantigens, which is the main effector mechanism in acute allograft rejection, has been well described. But the molecular mechanisms to dampen activated CD8+ T cells are largely unknown. On the other hand, Tim-3 is a molecule expressed on terminally differentiated CD4+ Th1 cells. Engaging Tim-3 with its ligand galectin-9 causes an inhibitory signal, resulting in apoptosis of Th1 cells and negatively regulates Th1 type immunity. However, the question whether CD8+ T cells express surface molecular Tim-3 has not been fully elucidated. In this study, we have investigated which CD8+ subset express molecular Tim-3 by flow cytometric assay. In addition, cytotoxic assay was applied to analyze whether CD8+ alloreactive T cells were sensitive to galectin-9 induced apoptosis. Here, our results demonstrated that Tim-3 was expressed on activated CD8+ alloreactive T cells (CD8+CD44highCD62Llow), but not expressed on naïve CD8+ T cells. Furthermore, alloreactive CD8+ cytotoxic T cells were sensitive to galectin-9 induced apoptosis both in vitro and vivo, resulting in attenuation of CD8+ alloreactive T cells mediated cytotoxicity and prolonged survival of skin graft.

Aim: The aim of this study was to investigate the effects of inhibition of MD-1 expression using nonspecific immunosuppressants and specific antisense oligodeoxynucleotides (AS-ODNs) treatment on skin allograft survival in mice., Methods: C57BL/6 to Balb/c skin allograft model was used in all groups, followed by Cyclosporine (CsA), Tacrolimus (FK506), Mycophenolate Mofetil (MMF), and Sirolimus (SRL) intraperitaneally, as well as AS-ODNs intravenously. Recipients were humanely killed at 11 days after transplantation. MD-1 expression was determined using flow cytometric analysis (FACS). AlamarBlue was used to evaluate proliferation. And serum levels of interleukin (IL)-2 and IL-10 were detected using enzyme-linked immunosorbent assay (ELISA)., Results: Compared with saline controls, the mean survival times (MST) of skin allografts in all of the immunosuppressants and AS-ODNs treated groups were significantly prolonged (P < .05). CsA, MMF, and AS-ODNs inhibited MD-1 expression and lymphocyte proliferation, as well as decreased serum level of IL-2 and increased that of IL-10; FK506, treatment showed all the effects mentioned above but up-regulated the IL-10 level; SRL had no significant influence on either MD-1 expression or IL-2 and IL-10 level, although it equally suppressed the proliferation (P < .05 vs controls). The negative correlation between MD-1 expression and lymphocyte proliferation or IL-2 level was significant, as was the positive correlation between it and IL-10 level (P < .01)., Conclusions: CsA, FK506, MMF, and AS-ODNs can efficiently inhibit MD-1 expression. The effects of the immunosuppressants are seemingly associated with the down-regulation of the IL-2 serum level. MD-1 was theorized to play an important role in rejection promotion, although the precise relationship between it and allograft survival still remains ambiguous.

Introduction: Selective splenectomy when donor antigen-specific activated T cells are sequestered in the recipient spleen may prolong allograft survival because of removal of all of these T cells., Objectives: We investigated the effect on cardiac allograft survival in mice by means of removal of activated specific T cells by splenectomy., Methods: Donor (Balb/c) spleen cells were injected into primed allogeneic recipients (C57BL/6). Selective recipient splenectomy and donor-type cervical heart grafting in Balb/c to C57BL/6 from mice were examined at 0, 24, 48, and 72 hours after donor spleen cell infusion., Results: Control C57BL/6 mice rejected Balb/c heart grafts at 6.86 +/- 0.19 days. Delayed heart grafting plus splenectomy at 24 or 48 hours after donor-type spleen cell infusion significantly prolonged heart allograft survival (24 hours: 15.86 +/- 3.44 days, P < .001; 48 hours: 21.71 +/- 5.22 days, P < .001, respectively). However, 72-hour delayed heart grafting plus splenectomy failed to prevent acute rejection (72 hours: 9.57 +/- 2.51 days, P > .01). Immunohistochemistry showed, at 24 to 48 hours after donor antigen infusion, the recipient spleens characterized by an obvious increase in CD4+ CD8+ T cells in periateriolar lymphoid sheaths, marginal zones, and red pulp compared with the 72-hour group., Conclusions: Transient accumulation of donor-specific activated T cells in the spleen of recipients provide an opportunity to remove all of these T cells by a surgical procedure. As the largest immune organ the spleen is the main place where T cells are activated and regenerated. At 24 or 48 hours when donor-specific T cells were sequestered in the spleen after donor antigen stimulation, selective recipient splenectomy was able to remove the T cells and prolong was allograft survival. Refinement of this protocol may eventually warrant clinical application.

There is a need to derive donor-specific tolerance in clinical organ transplantation, where potential benefits remain overshadowed by chronic rejection and side effects of continual immunosuppressive therapy. It is known that the mature immune system in mice can be reprogrammed to accept a foreign graft as if it were "self." Here we show that, once generated, this state of operational tolerance becomes self-sustaining, imposing itself on new cohorts of lymphocytes as they arise. These new cohorts retain specificity for the tolerizing antigen and can be selectively amplified to tolerate new antigens that have linked expression with the original tolerogen. Regulation is critically dependent upon the continuous presence of tolerizing antigen and is mediated by the CD4+ lymphocyte population. We propose that such natural mechanisms of immune regulation may eventually be exploited for transplantation tolerance, even in fully immune-competent recipients.