Prolonged Heart Allograft Survival Research Articles

Influence of the Novel ATP-Competitive Dual mTORC1/2 Inhibitor AZD2014 on Immune Cell Populations and Heart Allograft Rejection.

Little is known about how new-generation adenosine triphosphate-competitive mechanistic target of rapamycin (mTOR) kinase inhibitors affect immunity and allograft rejection. mTOR complex (C) 1 and 2 signaling in dendritic cells and T cells was analyzed by Western blotting, whereas immune cell populations in normal and heart allograft recipient mice were analyzed by flow cytometry. Alloreactive T cell proliferation was quantified in mixed leukocyte reaction; intracellular cytokine production and serum antidonor IgG levels were determined by flow analysis and immunofluorescence staining used to detect IgG in allografts. The novel target of rapamycin kinase inhibitor AZD2014 impaired dendritic cell differentiation and T cell proliferation in vitro and depressed immune cells and allospecific T cell responses in vivo. A 9-day course of AZD2014 (10 mg/kg, intraperitoneally, twice daily) or rapamycin (RAPA) (1 mg/kg, intraperitoneally, daily) prolonged median heart allograft survival time significantly (25 days for AZD2014, 100 days for RAPA, 9.5 days for control). Like RAPA, AZD2014 suppressed graft mononuclear cell infiltration, increased regulatory T cell to effector memory T cell ratios and reduced T follicular helper and B cells 7 days posttransplant. By 21 days (10 days after drug withdrawal), however, T follicular helper and B cells and donor-specific IgG1 and IgG2c antibody titers were significantly lower in RAPA-treated compared with AZD2014-treated mice. Elevated regulatory T cell to effector memory T cell ratios were maintained after RAPA, but not AZD2014 withdrawal. Immunomodulatory effects of AZD2014, unlike those of RAPA, were not sustained after drug withdrawal, possibly reflecting distinct pharmacokinetics or/and inhibitory effects of AZD2014 on mTORC2.

Regulation of T cell alloimmunity by PI3K\u03b3 and PI3K\u03b4

Phosphatidylinositol-3-kinases (PI3K) γ and δ are preferentially enriched in leukocytes, and defects in these signaling pathways have been shown to impair T cell activation. The effects of PI3Kγ and PI3Kδ on alloimmunity remain underexplored. Here, we show that both PI3Kγ−/− and PI3KδD910A/D910A mice receiving heart allografts have suppression of alloreactive T effector cells and delayed acute rejection. However, PI3Kδ mutation also dampens regulatory T cells (Treg). After treatment with low dose CTLA4-Ig, PI3Kγ−/−, but not PI3ΚδD910A/D910A, recipients exhibit indefinite prolongation of heart allograft survival. PI3KδD910A/D910A Tregs have increased apoptosis and impaired survival. Selective inhibition of PI3Kγ and PI3Kδ (using PI3Kδ and dual PI3Kγδ chemical inhibitors) shows that PI3Kγ inhibition compensates for the negative effect of PI3Kδ inhibition on long-term allograft survival. These data serve as a basis for future PI3K-based immune therapies for transplantation.

Depletion-Resistant CD4 T Cells Enhance Thymopoiesis During Lymphopenia.

Lymphoablation is routinely used in transplantation, and its success is defined by the balance of pathogenic versus protective T cells within reconstituted repertoire. While homeostatic proliferation and thymopoiesis may both cause T cell recovery during lymphopenia, the relative contributions of these mechanisms remain unclear. The goal of this study was to investigate the role of the thymus during T cell reconstitution in adult allograft recipients subjected to lymphoablative induction therapy. Compared with euthymic mice, thymectomized heart allograft recipients demonstrated severely impaired CD4 and CD8 T cell recovery and prolonged heart allograft survival after lymphoablation with murine anti-thymocyte globulin (mATG). The injection with agonistic anti-CD40 mAb or thymus transplantation only partially restored T cell reconstitution in mATG-treated thymectomized mice. After mATG depletion, residual CD4 T cells migrated into the thymus and enhanced thymopoiesis. Conversely, depletion of CD4 T cells before lymphoablation inhibited thymopoiesis at the stage of CD4- CD8- CD44hi CD25+ immature thymocytes. This is the first demonstration that the thymus and peripheral CD4 T cells cooperate to ensure optimal T cell reconstitution after lymphoablation. Targeting thymopoiesis through manipulating functions of depletion-resistant helper T cells may thus improve therapeutic benefits and minimize the risks of lymphoablation in clinical settings.

IRF4 controls a core regulatory circuit of T cell dysfunction in transplantation

Abstract T cell dysfunction has emerged as a key event leading to failure in the control of persistent infections and tumors. However, the transcriptional determinants for T cell dysfunction remain unknown. Here we show that IRF4 is a key transcriptional determinant of T cell dysfunction by restraining the dysfunctional differentiation process. We use a heart transplantation model to decipher the molecular basis of T cell dysfunction. Heart allografts survived indefinitely in Irf4-deficient mice. This stable engraftment is due to the progressive establishment of effector T cell dysfunction. Mechanistically, IRF4 represses PD-1, Helios, and other molecules associated with T cell dysfunction. In particular, IRF4 has a profound impact on epigenetic accessibility of PD-1 cis-regulatory elements. In the absence of IRF4, chromatin accessibility as well as binding of Helios at PD-1 cis-regulatory elements are markedly increased in effector T cells, resulting in enhanced expression of PD-1 and T cell dysfunction. Intriguingly, although the dysfunctional state of Irf4-deficient T cells is initially reversible by blockade of PD-1/PD-L1 pathway, it progressively evolves into a “terminal” irreversible state within 30 days post-transplant. Lastly, we revealed that the MEK1/2 inhibitor, trametinib, dramatically decreases IRF4 expression in T cells, abrogates EAE development, and prolongs heart allograft survival. Our results reveal a previously unappreciated aspect of T cell dysfunction, namely the repression of the dysfunctional differentiation of T cells by IRF4. Central control of T cell dysfunction by IRF4 has a significant potential impact on the course of future work in modulating T cell dysfunction

Erythropoietin Receptor-Mediated Molecular Crosstalk Promotes T Cell Immunoregulation and Transplant Survival.

Although spontaneous kidney transplant acceptance/tolerance occurs in mice and occasionally in humans, mechanisms remain unclear. Herein we test the hypothesis that EPO, a hormone predominantly produced by the adult kidney, has immunomodulating properties that are required for spontaneous kidney graft acceptance. In vitro, in a manner dependent on the EPO receptor and CD131 on antigen-presenting cells, EPO induced the secretion of active TGFβ by antigen-presenting cells, which in turn converted naïve CD4+ T cells into functional Foxp3+ regulatory T cells (Treg). In murine transplant models, pharmacologic downregulation of kidney-derived EPO prevented spontaneous Treg generation. In a controlled, prospective cohort clinical study, EPO administration at doses used to correct anemia augmented the frequency of peripheral CD4+CD25+CD127lo T cells in humans with CKD. Furthermore, EPO directly inhibited conventional T cell proliferation in vitro via tyrosine phosphatase SHP-1-dependent uncoupling of IL-2Rβ signaling. Conversely, EPO-initiated signals facilitated Treg proliferation by augmenting IL-2Rγ signaling and maintaining constitutively quenched IL-2Rβ signaling. In additional murine transplant models, recombinant EPO administration prolonged heart allograft survival, whereas pharmacologic downregulation of kidney-derived EPO reduced the expression of TGFβ mRNA and abrogated kidney allograft acceptance. Together, our findings delineate the protolerogenic properties of EPO in inhibiting conventional T cells while simultaneously promoting Treg induction, and suggest that manipulating the EPO/EPO receptor signaling axis could be exploited to prevent and/or treat T cell-mediated pathologies, including transplant rejection.

Differential depletion of total T cells and regulatory T cells and prolonged allotransplant survival in CD3Ɛ humanized mice treated with polyclonal anti human thymocyte globulin.

Thymoglobulin (ATG) is a polyclonal rabbit antibody against human thymocytes used as a T cell-depleting agent to prevent or treat allotransplant rejection. The aim of the present study was to investigate the effect of low dose ATG treatment exclusively on T cells using a humanized BALB/c human CD3Ɛ transgenic mouse model expressing both human and murine T cell receptors (TCR). Mice received a single intravenous (i.v.) injection of ATG. Blood and peripheral lymphoid organs were obtained after different time points. We found a significant T cell depletion in this mouse model. In addition, regulatory T cells (Tregs) proved to be less sensitive to depletion than the rest of T cells and the Treg:non-Treg ratio was therefore increased. Finally, we also investigated the effect of ATG in a heterotopic allogenic murine model of heart transplantation. Survival and transplant function were significantly prolonged in ATG-treated mice. In conclusion, we showed (a) an immunosuppressive effect of ATG in this humanized mouse model which is exclusively mediated by reactivity against human CD3Ɛ; (b) provided evidence for a relative resistance of Tregs against this regimen; and (c) demonstrated the immunomodulatory effect of ATG under these experimental circumstances by prolongation of heart allograft survival.

Targeted Delivery of Immunomodulators to Lymph Nodes.

SUMMARYActive-targeted delivery to lymph nodes represents a major advance toward more effective treatment of immune-mediated disease. The MECA79 antibody recognizes peripheral node address in molecules expressed by high endothelial venules of lymph nodes. By mimicking lymphocyte trafficking to the lymph nodes, we have engineered MECA79-coated microparticles containing an immunosuppressive medication, tacrolimus. Following intravenous administration, MECA79-bearing particles showed marked accumulation in the draining lymph nodes of transplanted animals. Using an allograft heart transplant model, we show that targeted lymph node delivery of microparticles containing tacrolimus can prolong heart allograft survival with negligible changes in tacrolimus serum level. Using MECA79 conjugation, we have demonstrated targeted delivery of tacrolimus to the lymph nodes following systemic administration, with the capacity for immune modulation in vivo.

Arsenic Trioxide Induces T Cell Apoptosis and Prolongs Islet Allograft Survival in Mice.

T cell-mediated immune rejection is a key barrier to islet transplantation. Preliminary studies have shown that arsenic trioxide (As2O3) can inhibit T cell responses and prolong heart allograft survival. Here, we sought to investigate the possibility of using As2O3 to prolong islet allograft survival in an acute rejection model of Balb/c to C57B/6 mice. Recipient mice were treated with As2O3 and/or rapamycin after islet allograft transplantation. At day 10 after transplantation, the graft, spleen, lymph nodes, and blood of the recipient mice were recovered for analysis. In vitro, to further examine the mechanism underlying As2O3 protection of islet allografts against T cell-mediated rejection, mixed lymphocyte reaction and apoptosis analyses of T cells were performed. The phosphorylation levels of IκBα and p38 were also evaluated to confirm the proliferation and apoptosis of As2O3-treated T cells. We found that As2O3 prolonged islet allograft survival by reducing inflammatory reactions, influencing cytokine synthesis and secretion and T-cell subset proportions, and inhibiting T-cell responses. Furthermore, As2O3 and rapamycin showed a synergistic effect in suppressing islet allotransplant rejection. Arsenic trioxide may prevent allograft rejection by inhibiting T-cell proliferation and inducing T-cell apoptosis.

Aquaporin 4 blockade is a novel strategy targeting ischemia/reperfusion injury in heart transplantation (TRAN1P.942)

Abstract Aquaporin 4 (AQP4) is the major water channel in the brain and its absence reduces brain edema in murine models of stroke.Recently, human and mouse cardiomyocytes were shown to express AQP4. Our goal was to test whether therapeutic inhibition of AQP4 reduces IRI and improves cardiac allografts survival. B6 mice were transplanted with BALB/c heart allografts subjected to 8 h of cold ischemia. Donors received AQP4 inhibitor 0.5h before harvest; hearts were perfused with and stored for 8 h on ice with the inhibitor. Recipients were given AQP4 inhibitor every 6h for 5 days. In the absence of additional immunosuppression, treatment with AQP4 inhibitor significantly prolonged heart allograft survival (MST 2d, n=24 vs. 6 d, n=5), decreased early T cell infiltration and the intragraft expression of IFNγ, granzyme B, perforin, FASL, CXCR3, ICOS, and CXCL10 in comparison to untreated controls. The frequencies of donor-reactive spleen cells secreting IFNγ in recipients treated with AQP4 inhibitor were also four fold lower than those in untreated controls. Blocking AQP4 during allograft storage and early after transplantation prolongs the survival of fully MHC-mismatched heart allografts subjected to 8 h CIS. The graft prolongation is associated with the reduced early memory T cell infiltration into the graft and decreased anti-donor responses. Our data identify AQP4 as a promising therapeutic target to diminish the detrimental effects of prolonged CIS on transplant outcome.

Transient blockade of delta-like Notch ligands prevents allograft rejection mediated by cellular and humoral mechanisms in a mouse model of heart transplantation.

Rejection remains a major clinical challenge limiting allograft survival after solid organ transplantation. Both cellular and humoral immunity contribute to this complication, with increased recognition of Ab-mediated damage during acute and chronic rejection. Using a mouse model of MHC-mismatched heart transplantation, we report markedly protective effects of Notch inhibition, dampening both T cell and Ab-driven rejection. T cell-specific pan-Notch blockade prolonged heart allograft survival and decreased IFN-γ and IL-4 production by alloreactive T cells, especially when combined with depletion of recipient CD8(+) T cells. These effects were associated with decreased infiltration by conventional T cells and an increased proportion of regulatory T cells in the graft. Transient administration of neutralizing Abs specific for delta-like (Dll)1/4 Notch ligands in the peritransplant period led to prolonged acceptance of allogeneic hearts, with superior outcome over Notch inhibition only in T cells. Systemic Dll1/4 inhibition decreased T cell cytokines and graft infiltration, germinal center B cell and plasmablast numbers, as well as production of donor-specific alloantibodies and complement deposition in the transplanted hearts. Dll1 or Dll4 inhibition alone provided partial protection. Thus, pathogenic signals delivered by Dll1/4 Notch ligands early after transplantation promote organ rejection through several complementary mechanisms. Transient interruption of these signals represents an attractive new therapeutic strategy to enhance long-term allograft survival.

B Cell Activating Factor (BAFF) and a Proliferation Inducing Ligand (APRIL) Mediate CD40-Independent Help by Memory CD4 T Cells

Donor-reactive memory T cells undermine organ transplant survival and are poorly controlled by immunosuppression or costimulatory blockade. Memory CD4 T cells provide CD40-independent help for the generation of donor-reactive effector CD8 T cells and alloantibodies (alloAbs) that rapidly mediate allograft rejection. The goal of this study was to investigate the role of B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) in alloresponses driven by memory CD4 T cells. The short-term neutralization of BAFF alone or BAFF plus APRIL synergized with anti-CD154 mAb to prolong heart allograft survival in recipients containing donor-reactive memory CD4 T cells. The prolongation was associated with reduction in antidonor alloAb responses and with inhibited reactivation and helper functions of memory CD4 T cells. Additional depletion of CD8 T cells did not enhance the prolonged allograft survival suggesting that donor-reactive alloAbs mediate late graft rejection in these recipients. This is the first report that targeting the BAFF cytokine network inhibits both humoral and cellular immune responses induced by memory CD4 T cells. Our results suggest that reagents neutralizing BAFF and APRIL may be used to enhance the efficacy of CD40/CD154 costimulatory blockade and improve allograft survival in T cell-sensitized recipients.

Teach the T cells: How learning can shape immunity

Immune responses can be modulated via associative learning paradigms. We have established a model of behaviorally conditioned suppression of immune functions in rodents and healthy humans, employing the immunosuppressant cyclosporine A (CsA) as an unconditioned stimulus, by demonstrating a behaviorally conditioned suppression of T cell proliferation and cytokine production. This learned immune response is mediated centrally via the insular cortex and the amygdala as well as on the efferent arm via the splenic nerve, through noradrenaline and adrenoceptor-dependent mechanisms and is sufficient to prolong heart allograft survival in rats.

Donor Specific Transfusion Combined With Anti-CD134L and Rapamycin Inhibits Accelerated Rejection Mediated By Alloantigen-Primed Memory Cells in Mice.

Background Donor-reactive memory cells threaten the survival of transplanted organs, and hamper maintenance of transplant tolerance. In this study, we investigated the inhibitory effect of donor splenocyte transfusion(DST) combined with anti-CD134L and rapamycin on memory cells-mediated accelerated rejection in an allogeneic heart transplantation model in mice. Methods Male Balb/c and C57BL/6 mice were used as the donor and recipient. After sensitization with donor skin allografts, splenocytes from the alloantigen-primed C57BL/6 mice were prepared and adoptively transferred (5×106) to a naïve recipient followed by heart transplantation (HTx). Thus, we established a memory cells-mediated accelerated rejection model of heart transplantation. Mitomycin C-treated donor splenocytes were transfused to HTx recipients seven days prior to transplantation, and short-term low dose of rapamycin and anti-CD134L were initiated after transplantation. Graft survival and pathology of rejection was examined. Memory cells proliferation, functionand the proportion of regulatory T and B cell populations were also examined. Results The mean survival time (MST) of grafts in thecontrolgroup was 5.3 days,and more than 40 days in the group treated with DST/anti-CD134L/rapamycin. The DST/anti- CD134L/rapamycinregimen prevented lymphocytic infiltration in the grafts, inhibited memory cells proliferation in the spleen, reduced donor specific antibody level, and increased proportion of CD4+Foxp3+regulatory T cells (Tregs) as well as CD1d+CD5+regulatoryB cells (Bregs). Conclusions Our data suggest that DST combined with rapamycin and anti-CD134L can synergistically inhibit accelerated rejection mediated by memory cells and thus prolong heart allograft survival in mice. Both regulatory T cells and regulatory B cells may play important roles in modulation of memory cells.

Dendritic Cells, Propagated From Bone Marrow With Low Dose GM-CSF, Expand FOXP3+ Regulatory T Cells and Prolong Heart Allograft Survival.

Dendritic Cells, Propagated From Bone Marrow With Low Dose GM-CSF, Expand FOXP3+ Regulatory T Cells and Prolong Heart Allograft Survival.

Induction of Immunological Tolerance With In Vitro Generated Suppressive Donor Blood Monocytes.

Our previous studies showed that incubation of dendritic cells (DCs) with the chemotherapeutic drug mitomycin C (MMC) renders them immunosuppressive. Donor-derived MMC-DCs injected into the recipient before transplantation prolonged heart allograft survival in rats. Whereas generation of DCs is labour-intensive, peripheral blood mononuclear cells (PBMCs) can be easily harvested. Here we analyse under which conditions DCs can be replaced by PBMCs and study their mode of action. When injected into rats, MMC-incubated donor PBMCs (MICs) strongly prolonged heart allograft survival. Removal of monocytes from PBMCs abrogated their suppressive effect, showing that monocytes are the active cell population. Suppression of rejection was donor-specific. Recipients treated with MICs showed an increased number of CD4+CD25+FoxP3+ Tregs in their peripheral blood and FoxP3-cell infiltration of heart allograft. Most important, tolerance could be transferred to syngeneic recipients with blood cells, whereas depletion of Tregs annihilated the effect. This argues for mediation of suppression by CD4+CD25+FoxP3+ Tregs. Cell culture studies showed that MMC-treated monocytes give rise to myeloid cells, which do neither resemble monocytes, nor DCs but show morphological and phenotypic similarities with monocytic myeloid-derived suppressor cells (MDSCs) occurring in some forms of cancer. Donor-derived MICs also prolonged kidney allograft survival in pigs. MICs were applied for the first time in a patient with therapy-resistant rejection of a haploidentical stem cell transplant, showing that they can be easily generated and used in humans. In conclusion, we describe here a simple method for in vitro generation of monocytic suppressor cells for potential use in clinical organ transplantation.

Pretreatment of Transfused Donor Splenocytes and Allografts With Mitomycin C Attenuates Acute Rejection in Heart Transplantation in Mice

ObjectiveThe aim of this study was to investigate the effect of pretreatment of donor splenocytes and grafts with mitomycin C (MMC) on heart allograft survival, as well as to demonstrate the mechanism of function. MethodsDonor splenocytes from Balb/C mice were incubated with MMC (40 μg/mL) in vitro and then transfused into recipients (C57BL/6 mice). The heart allograft was perfused with MMC before harvest. Graft survival and histopathology were examined. Lymphocyte activation, regulatory T cells, and donor splenocyte apoptosis were examined with the use of flow cytometry. ResultsMMC incubation in vitro induced apoptosis of donor splenocytes (15.5 ± 2.3% vs 23.2 ± 4.2%; P < .01). Either intravenous injection of MMC-treated donor splenocytes or transplantation of allograft pretreated with MMC prolonged heart allograft mean survival time from 7 ± 0.8 days to 20.5 ± 1.9 days or 10 ± 0.9 days, respectively (both P < .01). A combination of MMC-pretreated donor splenocyte transfusion and allografts showed the best effect on prolongation of graft survival (28.5 ± 1.8 days). Activation of CD4+ T cells in spleen and peripheral lymph nodes of recipients was significantly inhibited by either MMC-splenocyte transfusion or the combination treatment. Meanwhile, the percentage of CD4+CD25+Foxp3+ regulatory T cells in the spleen was increased in the MMC-splenocyte transfusion group (15.5 ± 1.1% vs 18.2 ± 0.9%; P < .05). ConclusionsBoth injection of MMC-conditioned donor splenocytes and MMC-conditioned allograft have effects on prolongation of heart allograft survival in mice, and MMC-conditioned donor splenocytes might play an essential role. MMC pretreatment induced regulatory T cells likely through induction of donor splenocyte apoptosis, and thus it inhibited T-cell activation.

Control Of Allograft Rejection With Mitomycin C Incubated Blood Cells

Immunosuppressive treatment in allogeneic transplantation is an effective way to control allograft rejection. But unfortunately it can cause serious side effects, such as infections or malignancies. Cell therapeutic approaches for induction of specific tolerance are on the rise and may have fewer side effects. We observed that monocytes or peripheral blood mononuclear cells (PBMC) of healthy persons after culture with dendritic cell-inducing cytokines and differentiation blockade with the chemotherapeutic drug mitomycin C (MMC) show the ability to suppress allogeneic T cell responses. Interestingly these cells resemble morphologically as well as phenotypically myeloid-derived suppressor cells (MDSC) which occur in cancer patients impairing the anti-tumor immunity. FACS- and gene expression analyses of these MMC incubated blood cells revealed downregulation of the immunostimulatory cell surface molecules CD80, CD83, CD86 and HLA-DR, and upregulated transcription of immunosuppressive and pro-apoptotic genes such as iNOS, arginase-1, IL-10, COX2, TGF-β, placental growth factor, immunoglobulin-like transcript 3, adrenomedullin, SMAD5, LRDD and of the transcription factor C/EBPβ. For use in preclinical transplantation models in vitro culture of MMC treated donor-derived blood cells with dendritic-cell inducing cytokines was not applicable and was therefore omitted. The tolerance inducing properties of donor-derived MMC incubated blood cells were investigated in two different animal models of solid organ transplantation. When injected into rats, donor-derived PBMC incubated with MMC strongly prolonged heart allograft survival. Suppression of rejection was donor-specific. Recipients showed an increased number of CD4+CD25+FoxP3+ regulatory T-cells and tolerance could be transferred to syngeneic recipients with blood or spleen cells. Donor-derived MMC incubated blood cells also prolonged kidney allograft survival in pigs. Furthermore, in a first clinical use a 6-year-old girl with relapse of acute lymphoblastic leukemia and imminent rejection of a third haploidentical stem cell transplant received donor-derived CD3/CD19-depleted peripheral blood stem cells, which have been treated in vitro with MMC, in order to prohibit graft rejection. We describe a simple method for in vitro generation of tolerance inducing donor-derived blood cells for potential use in clinical organ transplantation. Disclosures: No relevant conflicts of interest to declare.

The Immunosuppressant Protosappanin A Promotes Dendritic Cell-Mediated Expansion of Alloantigen-Specific Tregs and Prolongs Allograft Survival in Rats

Protosappanin A (PrA), an immunosuppressive ingredient of the medicinal herb Caesalpinia sappan L, prolongs heart allograft survival in rats, possibly by impairing the function of antigen-presenting cells (APCs). We examined the effects of PrA on the maturation and function of dendritic cells (DCs), a potent class of APCs, and the downstream cell–cell and intracellular signaling pathways mediating the immunosuppressive activity of PrA. PrA inhibited LPS-stimulated maturation of Wistar rat DCs in vitro as reflected by reduced expression of costimulatory molecules (CD80 and CD86) and reduced expression of TLR4 and NF-κB, two critical signaling components for antigen recognition. PrA also enhanced the release of IL-10 and decreased the release of IL-12 from DCs, but had no effect on the production of TGF-ß. In mixed cultures, Wistar DCs pretreated with PrA impaired the proliferation of Sprague Dawley (SD) rat T cells while promoting the expansion of SD rat CD4+CD25+ regulatory T cells (Tregs). Both oral PrA treatment and infusion of PrA-pretreated Wistar DCs prolonged cardiac allograft survival (Wistar donor, SD recipient) and expanded recipient CD4+CD25+Foxp3+ Tregs. Donor spleen cells, but not spleen cells from a third rat strain (DA), supported the expansion of recipient CD4+CD25+Foxp3+ Tregs and suppressed recipient T cell proliferation. We conclude that PrA triggers a tolerogenic state in DCs that allows for the induction of alloantigen-specific Tregs and the suppression of allograft rejection in vivo.

PD-L1 Signal on Liver Dendritic Cells Is Critical for Foxp3+CD4+CD25+ Treg and Liver Tolerance Induction in Mice

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.

Regulatory T Cells (Tregs) in Neonatal Tolerance: Allogeneic Tregs Regulate the Neonatal Immune System and Prolong Heart Graft Survival

Purpose Semi-allogeneic (F1; H-2k/b) splenocytes (SC) injected into newborn mice (H-2k) induce immunological tolerance whereas allogeneic SC (H-2b) lead to graft- vs -host disease (GVHD). This study tested injection of purified allogeneic Tregs in our model of neonatal tolerance to cardiac allografts. Methods and Materials CD4 + CD25 + Tregs were isolated using StemCell Technologies EasySep® purification kits. Injected labeled syngeneic, F1 or allogeneic SC, or purified allogeneic Tregs, were tracked in newborn mice for 6 days by OV100 small animal imaging system. Treg interactions and fates were studied by confocal microscopy, and their ability to prolong heart allograft survival in adults. Results By whole body and organ imaging syngeneic and F1 SC tracked to lymphoid organs while allogeneic SC initially tracked there but then proliferated and distributed throughout the mice consistent with GVHD. Injected Tregs also trafficked to lymphoid organs but unlike allogeneic SC did not proliferate. Allogeneic Tregs survived post-injection in spleen suggesting they were not being attacked by the host immune system. In neonatal spleen, allogeneic Tregs interacted with host Tregs either directly or via host dendritic cells indicating a possible role in iTreg formation. Injected allogeneic Tregs prolonged survival of both donor-type and third-party heart grafts to 100 days in 50% of transplanted mice (n=6). A >100 day functioning donor-type heart graft showed mild-moderate cellular infiltrate of macrophages and T cells including CD8+ T cells, with focal regions of myocyte damage. Conclusions Injection of neonates with purified allogeneic Tregs prolongs survival of donor-type and third party heart grafts transplanted as adults. Prolonged third-party graft survival suggests suppression of the host immune system in a localized “non-specific” manner; alternatively given the presumed clonal diversity of allogeneic Tregs, some form of specific/cross-reactive suppression may be occurring.