Spontaneous Acceptance Of Liver Allografts Research Articles

Mechanisms of liver-induced tolerance.

To highlight the results of the ongoing research on the mechanisms of liver-induced tolerance focusing on results from the last year. The liver is exposed to a massive antigenic burden of dietary and commensal products from the gastrointestinal tract via portal vein, most of which are necessary for survival. To prevent the immune system from destroying these foreign yet beneficial elements, the liver has developed unique mechanisms to suppress immune responses. It is thought that these mechanisms of acquired tolerance may also underlie the spontaneous acceptance of liver allografts observed after transplantation in many species. The fact that isolated hepatocyte transplants are acutely rejected, suggests that nonparenchymal liver cells play a critical role in spontaneous liver allograft acceptance. IFN-γ, a key inflammatory cytokine produced by T effector (Tef) cells, is paradoxically compulsory for spontaneous liver allograft acceptance. Analysis of IFN-γ signaling points to liver mesenchymal nonparenchymal liver cell that eliminate infiltrating Tef cells via expression of B7-H1, IL-10, and tumor growth factor-β, as well as the enhancement of Tregs and MDSCs. Thus, liver mesenchymal cells are thought to promote tolerance by eliminating alloreactive Tef cells and enhancing suppressor cells (T and B). The research during last year offered some key insights into the mechanisms of liver-induced tolerance. Through interactions with activated T cells and B cells via IFN-γ/B7-H1 pathways, liver mesenchymal cells have been shown to be critical components of liver-specific tolerance induction.

Rejection triggers liver transplant tolerance: Involvement of mesenchyme-mediated immune control mechanisms in mice.

Liver tolerance was initially recognized by the spontaneous acceptance of liver allografts in many species. The underlying mechanisms are not completely understood. However, liver transplant (LT) tolerance absolutely requires interferon (IFN)-γ, a rejection-associated inflammatory cytokine. In this study, we investigated the rejection of liver allografts deficient in the IFN-γ receptor and reveal that the liver graft is equipped with machineries capable of counterattacking the host immune response through a mesenchyme-mediated immune control (MMIC) mechanism. MMIC is triggered by T effector (Tef) cell-derived IFN-γ that drives expression of B7-H1 on graft mesenchymal cells leading to Tef cell apoptosis. We describe the negative feedback loop between graft mesenchymal and Tef cells that ultimately results in LT tolerance. Comparable elevations of T-regulatory cells and myeloid-derived suppressor cells were observed in both rejection and tolerance groups and were not dependent on IFN-γ stimulation, suggesting a critical role of Tef cell elimination in tolerance induction. We identify potent MMIC activity in hepatic stellate cells and liver sinusoidal endothelial cells. MMIC is unlikely exclusive to the liver, given that spontaneous acceptance of kidney allografts has been reported, although less commonly, probably reflecting variance in MMIC activity. MMIC may represent an important homeostatic mechanism that supports peripheral tolerance and could be a target for the prevention and treatment of transplant rejection. This study highlights that the graft is an active participant in the equipoise between tolerance and rejection and warrants more attention in the search for tolerance biomarkers.

The Search for Immunosuppressive Therapies to Induce Tolerance in Organ Transplantation

Organ transplantation has become a major therapeutic option for patients with irreversible organ diseases. Immunosuppressive agents are usually required to prevent allograft rejection in patients who undergo an organ transplantation. Such drugs suppress both specific and nonspecific immunity, and render the recipient more susceptible to both infection and malignancy. Therefore, the development of more effective and less toxic immunosuppressive agents could improve the clinical outcomes of organ transplant recipients. Since the early days of clinical and experimental liver transplantation, it has been known that the liver is less likely to be rejected in comparison to other organs and may be tolerogenic even across a fully allogeneic MHC barrier in some specific cases. Spontaneous acceptance of liver allografts has been observed in several species. Orthotopic liver transplantation (OLT) in certain rat strains is accepted without immunosuppressive agents and serum from post-OLT recipients displays immunosuppressive activity. Attempts have been made to identify the immunosuppressive factors that are present in post-OLT serum to elucidate the mechanism of immunological tolerance and to discover novel immunosuppressive agents for potential use in organ transplantation. In this review we will focus on established and recent findings in the identification of immunosuppressive factors in a rat tolerogenic OLT model. The most recent therapeutic methods in organ transplantation and future prospects will be discussed.

Spontaneous acceptance of mouse kidney allografts is associated with increased Foxp3 expression and differences in the B and T cell compartments

Spontaneous acceptance of organ allografts can identify novel mechanisms of drug-free transplantation tolerance. Spontaneous acceptance occurs in both mouse kidney transplants and rat liver transplants however the early immune processes of mouse kidney acceptance have not been studied. Acceptance of C57BL/6 strain kidney allografts in fully MHC-incompatible B10.BR recipients was compared with rejection (REJ) of heart allografts in the same strain combination. Graft infiltrate and antibody deposition were examined by immunohistochemical staining. Expression of mRNA was measured by quantitative real-time PCR. Apoptosis was examined by TUNEL staining. The majority of kidney allografts were accepted long-term and induced tolerance (TOL) of donor-strain skin grafts, showing that acceptance was not due to immune ignorance. There was an extensive infiltrate of T cells in the TOL kidney that exceeded the level in REJ hearts but subsequently declined. The main differences were deposition of IgG2a antibody in REJ that was absent in TOL, more B cells infiltrating TOL kidneys and a progressive increase in the ratio of CD8 : CD4 cells during rejection. There was also significantly greater Foxp3 mRNA expression in TOL. Kidneys from RAG −/− donors were accepted, showing that donor lymphocytes were not necessary for acceptance. Neutralising antibodies to TGF-β administered from day 0 to day 6 did not prevent TOL. On the basis of cytokine expression and apoptosis there was no evidence for immune deviation or deletion as mechanisms of acceptance. In accord with the findings of spontaneous acceptance of liver allografts in rats, the main difference between mouse kidney TOL and heart REJ was in the B cell compartment. The major difference to rat liver allograft acceptance was that apoptosis of infiltrate did not appear to play a role. Instead, increased Foxp3 expression in TOL kidneys implies that regulatory T cells might be important.

The PD-L1 Signal is Important to Liver Dendritic Cells in Induction of Foxp3+CD4+CD25+ Treg and Liver Transplant Tolerance (141.33)

Abstract Our previous studies have demonstrated that Foxp3+CD25+CD4+ regulatory T cells (Treg) play an important role in the induction of murine liver spontaneous transplant tolerance. How Treg are induced and what their mechanisms are in the regulation of liver transplant tolerance remain undefined. Here, we investigated the role of liver dendritic cells (DCs) on the induction of Treg and liver graft tolerance in vivo and in vitro. Fms-like tyrosine kinase 3 ligand (Flt3L) mutation mice, which have severe reduction in all types of DCs, and PD-L1 deficient mice were used to examine the role of liver DCs and the PD-L1 signal in Treg induction and liver transplant tolerance. Our results showed that liver DCs, which expressed a high number of PD-L1 molecules, induced more Foxp3+CD25+CD4+ Treg in vitro compared to spleen DCs. However, 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 more than in the CD4 treated controls. The liver grafts from Flt3L-/- and PD-L1-/- mice were rejected acutely in the C3H recipients. Foxp3+ cells were reduced, but IL-2, IL-10, and IFN-γ producing cells were significantly increased in the liver graft and recipient spleen sections of Flt3L-/- and PD-L1-/- donors. Thus, liver DCs play a critical role in the induction of Treg, which underpin spontaneous acceptance of liver allografts. The function of DCs on Treg induction appears to depend on the PD-L1 signal.

Insights into the Mechanism Responsible for Spontaneous Acceptance of Liver Allografts (141.41)

Abstract Transplantation of fully histoincompatable livers between strains of rodents often results in spontaneous acceptance of the graft. Lewis rats reject DA livers in 14 days while DA recipients of Lewis livers have long-term graft survival. We sought to determine the mechanism of this spontaneous graft survival. NK cells and subsets of T cells express NKG2D. On NK cells, NKG2D functions as a stimulatory receptor that induces effector functions. We examined NKG2D receptor and ligand expression post-transplant. Groups of Lewis rats received livers from DA (rejection combination) or Lewis donors (syngeneic) and DA rats received livers from Lewis (non-rejection combination) or DA donors (syngeneic). NKG2D expression was low in syngeneic grafts but increased 7-fold in Lewis→DA grafts and 17-fold in DA→Lewis grafts by day 7 post-transplant. Ligands of NKG2D were not expressed in normal liver but were up regulated in all liver grafts by day 1 post-transplant, suggesting that ischemia/reperfusion injury and trauma induce expression of these ligands. RAE1L and RRLT were significantly up regulated specifically in DA→Lewis liver grafts by day 7 post-transplant. In marked contrast, the expression of RAE1L and RRLT in Lewis→ DA liver grafts was similar to the levels detected in syngeneic liver grafts suggesting that signals that lead to an increase in NKG2D ligand expression are absent in this strain combination. In conclusion, the absence of NKG2D ligand expression in the liver graft may play a role in preventing rejection, and decreasing the expression of these proteins may be a useful strategy to promote graft non-responsiveness. Supported by AI044095

Unique Aspects of Rejection and Tolerance in Liver Transplantation

Spontaneous acceptance of liver allografts occurs in several species. However, tolerance is rare in human transplant patients even though rejection is relatively easily reversed. Histological features of acute rejection in liver transplantation are similar to those in other organs. Nevertheless, mechanisms of rejection of liver transplants may differ in degrees and cellular involvement. Liver-specific cell populations, such as Kupffer cells (KCs), liver sinusoidal epithelial cells (LSECs), and hepatic stellate cells (HSCs), may contribute to liver tolerogenicity. Other mechanisms, such as microchimerism, soluble major histocompatibility complex (MHC molecules), donor human leukocyte antigen (HLA)-C genotype, and regulatory T cells, may participate in inducing tolerance. The low incidence of hyperacute or antibody-mediated rejection in liver might be linked to the infrequency of chronic rejection of liver transplants. Understanding the mechanisms of liver transplant rejection/tolerance and the availability of better immune monitoring could help develop strategies to recognize tolerance and reduce rejection.

New Insights into Mechanisms of Spontaneous Liver Transplant Tolerance: The Role of Foxp3-Expressing CD25+CD4+ Regulatory T Cells

Liver allografts in mice are accepted across MHC barriers without requirement for immunosuppressive therapy. The mechanisms underlying this phenomenon remain largely undefined. In this study, we investigated the role of Foxp3-expressing CD25(+)CD4(+) regulatory T cells (Treg) in the induction of murine liver transplant tolerance. Foxp3(+)CD25(+)CD4(+) T cells were increased in liver grafts and recipient spleens from day 5 to day 100 posttransplantation, associated with enhanced CTLA4 and TGF-beta expression and IL-4 production. Depletion of recipient CD25(+)CD4(+) T cells using anti-CD25 mAb (250 microg/day) induced acute liver allograft rejection. This was associated with a decreased ratio of Foxp3(+) Treg: T effector cells, decreased IL-4 and elevated IL-10 and IL-2 production by graft-infiltrating T cells, and reduced apoptotic activity of graft-infiltrating CD4(+) and CD8(+) T cells in anti-CD25-mAb-treated recipients. Thus, the data suggest that Foxp3(+)CD25(+)CD4(+)Treg are involved in spontaneous acceptance of liver allografts in mice. The ratio of Treg to T effector cells appears to determine liver transplant outcome. CTLA4, IL-4, TGF-beta and apoptosis of graft-infiltrating T cells are also associated with liver transplant tolerance and may contribute, at least in part, to the mechanisms of Treg-mediated immune regulation in this model.

Liver sinusoidal endothelial cell function in rejected and spontaneously accepted rat liver allografts

Studies have suggested that liver sinusoidal endothelial cells (LSEC) may play an important role in tolerance induction. In this study, we evaluated the functional difference of LSEC in rejection and spontaneous acceptance of liver allografts by using rat liver transplant model. LSEC function was determined by circulating hyaluronic acid (HA) levels and fluorescein isothiocyanate-labeled formaldehyde-treated serum albumin (FITC-FSA) uptake. Additional parameters include the number of circulating lymphocytes and LSEC apoptosis. In spontaneously accepted group, we found (i) significantly lower serum HA levels (P = 0.002), (ii) a more rapid uptake of FITC-FSA, and (iii) a reduced number of circulating CD8a+ cells when compared with the rejection group. Strikingly, HA levels in spontaneously accepted group are even lower than syngeneic control group. Further investigation revealed that interleukin-1beta, a cytokine that promotes LSEC function, was higher in DA than in Lewis rats. In summary, our study demonstrates that LSEC function is better preserved in spontaneously accepted rat liver allografts than in those which are rejected. These findings warrant further studies to verify if LSEC actively contributes to liver transplant outcome or just a target of different immunologic responses.

CD25+CD4+ Regulatory T Cells Develop in Mice Not Only During Spontaneous Acceptance of Liver Allografts but Also After Acute Allograft Rejection

Liver grafts transplanted across a major histocompatibility barrier are accepted spontaneously and induce donor specific tolerance in some species. Here, we investigated whether liver allograft acceptance is characterized by, and depends upon, the presence of donor reactive CD25CD4 regulatory T cells. CD25 and CD25CD4 T cells, isolated from CBA. Ca (H2) recipients of C57BL/10 (B10; H2) liver and heart allografts 10 days after transplantation, were transferred into CBA. Rag1 mice to investigate their influence on skin allograft rejection mediated by CD45RBCD4 effector T Cells. Fully allogeneic B10 liver allografts were spontaneously accepted by naive CBA.Ca recipient mice, whereas B10 cardiac allografts were acutely rejected (mean survival time=7 days). Strikingly, however, CD25CD4 T cells isolated from both liver and cardiac allograft recipients were able to prevent skin allograft rejection in this adoptive transfer model. Interestingly, CD25CD4 T cells isolated from liver graft recipients also showed suppressive potency upon adoptive transfer. Furthermore, depletion of CD25CD4 T cells in primary liver allograft recipients did not prevent the acceptance of a secondary donor-specific skin graft. Our data provide evidence that the presence of CD25CD4 regulatory T cells is not a unique feature of allograft acceptance and is more likely the result of sustained exposure to donor alloantigens in vivo.

In vivo immune modulatory activity of hepatic stellate cells in mice

Accumulating data suggest that hepatic tolerance, initially demonstrated by spontaneous acceptance of liver allografts in many species, results from an immune regulatory activity occurring in the liver. However, the responsible cellular and molecular components have not been completely understood. We have recently described profound T cell inhibitory activity of hepatic stellate cells (HSCs) in vitro. In this study, we demonstrate in vivo evidence of immune modulatory activity of HSCs in mice using an islet transplantation model. Co-transplanted HSCs effectively protected islet allografts from rejection, forming a multi-layered capsule, which reduced allograft immunocyte infiltrates by enhancement of apoptotic death. The immune modulation by HSCs appeared to be a local effect, and regulated by inducible expression of B7-H1, an inhibitory molecule of B7 family. This may reflect an intrinsic mechanism of immune inhibition mediated by liver-derived tissue cells. In conclusion, these results may lead to better understanding of liver immunobiology and development of new strategies for treatment of liver diseases.

Immune regulatory activity of liver‐derived dendritic cells generated in vivo

Hepatic tolerance is demonstrated by spontaneous acceptance of liver allografts in mice. Hepatic dendritic cells (DC) play a crucial role in determining immunity or tolerance. In this study, we adopted an approach to transfect gene(s) into the mouse liver by tail-vein injection of plasmid-carrying genes. Transfection with GM-CSF expanded liver CD11c+ myeloid DC (LMDC), while liver B220+CD11c- lymphoid DC (LLDC) were expanded after transfection of IL-3 and CD40L. Flow analysis revealed that these liver DC subsets were phenotypically mature following overnight culture. However, in contrast to LMDC, LLDC induced hyporesponsiveness in allogeneic T-cells, with suppressed secretion of both IL-2 and IFN-gamma, and prolonged cardiac allograft survival. This immune regulatory DC population in the liver may play a role in modulating T-cell immunity in the liver.

The Potent Role of Graft-Derived NKR-P1+TCRαβ+ T (NKT) Cells in the Spontaneous Acceptance of Rat Liver Allografts

The mechanism involved in the spontaneous acceptance of liver allografts in some rat strain combinations remains unclear. Immunoregulatory NKR-P1TCRalphabetaT (NKT) cells primarily produce IL-4 and IFN-gamma, and enhance the polarization of immune responses to Th2 and Th1, respectively. The aim of this study was to clarify the role of graft-derived NKT cells in inducing the spontaneous acceptance of rat orthotopic liver transplantation (OLTx) The experimental groups were divided as follows: Group 1, BN to LEW "low responder (acceptor)" combination; Group 2, DA to LEW "high responder (rejector)" combination; naïve BN (Group 3) or LEW recipients (Group 4) with liver allografts from irradiated BN donors. The recipients had liver allografts from irradiated donors reconstituted from the following cell populations 24 hr before harvesting, spleen cells (SPCs, Group 5), IgSPCs (Group 6), IgNKR-P1SPCs (Group 7), and IgTCRabSPCs (Group 8) In Group 1, the percent of graft-derived NKT cells harvested on day 7 posttransplant were significantly higher than in Group 2. In the case of BN liver allografts that had been irradiated and reconstituted with cell populations including NKT cells (Groups 5 and 6), the mean graft survival (MST) was extended to 39.2+/-5.7 and 38.8+/-8.0 days, respectively. In contrast, when NKT cells were excluded (Groups 7 and 8), the grafts were acutely rejected within MST of 17.8+/-4.0 and 18.8+/-7.7 days, respectively. The concentrations of IL-10 and TGF-beta, but not IL-4 in IgGICs culture supernatants were predominant in the acceptor, whereas those with IFN-gamma predominated in the rejector. Graft-derived NKT cells might be responsible for spontaneous acceptance in the rat OLTx.

Basic concepts in transplant immunology

Subsequently,spontaneousliverallograftacceptancewasalsoobservedintransplants done in several allogeneic rat strain combina-tions and most allogeneic mouse strain combinations.Moreover, other experimental studies in rodents demon-strated that allogeneic liver transplantation providesimmune“protection”againstsubsequentcardiac,kidney,pancreas, islet, and skin grafts from the same donor.

Evidence of regulatory T lymphocytes that constitute peripheral blood microchimerism following rat liver transplantation

IT has been well established that, unlike other vascularized allografted organs, hepatic transplants in various species such as pigs or rats of some strain combinations are spontaneously accepted across a whole MHC class I plus class II barrier without active immunotherapy. In addition, spontaneous acceptance of a liver allograft is accompanied by systemic immunological tolerance of other grafts from donor strains, but not of a third party. Furthermore, it has been shown that the tolerance induced by liver allografting is able to reverse ongoing rejection of heart. The mechanisms underlying this unique phenomenon might be of potential use in a clinical setting. In this respect, spontaneous liver allograft acceptance has been suggested to be due to some characteristics of the liver per se, such as its ability to make large amounts of soluble class I antigen, or its complex with antibodies, which may interfere with rejection. Alternatively, it has been suggested that passenger leukocytes within the transplanted liver result in host microchimerism and thereby induce tolerance. If liver passenger leukocytes induce tolerance, then they may bear different characters than those from other organs, since the original concept of passenger leukocytes indicated that the immunogenicity of transplanted organs was attributed to these cells. Thus, it has been shown that several maneuvers, such as removal of passenger leukocytes from these grafts by irradiation and/or pretreatment of the donor by radiomimetic chemical or by parking the graft in an intermediate host syngeneic with the recipient, prolongs allograft survival. In this study we examined whether and to what extent a population of liver passenger leukocytes are important in liver allograft acceptance and its relationship to microchimerism in the peripheral blood by treating them by donor irradiation.

EARLY INTRAGRAFT EXPRESSION OF CYTOTOXIC T CELL MOLECULES DURING LIVER ALLOGRAFT ACCEPTANCE

411 Although spontaneous acceptance of liver allografts is observed in many experimental models, the mechanisms involved are not well understood. The purpose of this study was to determine the intragraft expression of cytotoxic T cell molecules (Granzyme B, FasL and perforin) during liver allograft acceptance. BALB/c (H-2d) mice received liver grafts from syngeneic and allogeneic CBA (H-2k) donors. No immunosuppressive therapy was administered. Survival of BALB/c to BALB/c syngenic grafts (n=23) was 87% and 56% at 30 days and long-term (>100 days), respectively. The majority of early graft loss was due to bile duct complications related to technical aspects of the model. In comparison, CBA to BALB/c liver allografts (n=13) had a 30 day survival rate of 69% with several long term survivors. Allograft livers harvested at day 7 post-transplant demonstrated moderate rejection (RAI=7) with a mononuclear cellular infiltrate centered around the portal triads. However, allografts harvested at day 10 post-transplant demonstrated early signs of resolving rejection (RAI=5). Semi-quantitative RT-PCR analysis of mRNA extracted from CBA livers demonstrated strong induction of cytotoxic T cell genes at days 5 and 7 after transplantation with decreased perforin expression at day 10. In addition, BALB/c recipients produced a vigorous anti-donor class I antibody response to CBA liver grafts. Thus, graft acceptance is not due to a host deficiency in cytotoxic or humorally-mediated responses. The spontaneous acceptance of liver allografts may be related to its unique ability to regenerate and thus, recover from early injury produced by cytotoxic T cells and humorally-mediated immune mechanisms. (Table)Table

Implication of gamma-delta T cells in the human immune response to cytomegalovirus

Spontaneous acceptance of organ allografts can identify novel mechanisms of drug-free transplantation tolerance. Spontaneous acceptance occurs in both mouse kidney transplants and rat liver transplants however the early immune processes of mouse kidney acceptance have not been studied. Acceptance of C57BL/6 strain kidney allografts in fully MHC-incompatible B10.BR recipients was compared with rejection (REJ) of heart allografts in the same strain combination. Graft infiltrate and antibody deposition were examined by immunohistochemical staining. Expression of mRNA was measured by quantitative real-time PCR. Apoptosis was examined by TUNEL staining. The majority of kidney allografts were accepted long-term and induced tolerance (TOL) of donor-strain skin grafts, showing that acceptance was not due to immune ignorance. There was an extensive infiltrate of T cells in the TOL kidney that exceeded the level in REJ hearts but subsequently declined. The main differences were deposition of IgG2a antibody in REJ that was absent in TOL, more B cells infiltrating TOL kidneys and a progressive increase in the ratio of CD8 : CD4 cells during rejection. There was also significantly greater Foxp3 mRNA expression in TOL. Kidneys from RAG−/− donors were accepted, showing that donor lymphocytes were not necessary for acceptance. Neutralising antibodies to TGF-β administered from day 0 to day 6 did not prevent TOL. On the basis of cytokine expression and apoptosis there was no evidence for immune deviation or deletion as mechanisms of acceptance. In accord with the findings of spontaneous acceptance of liver allografts in rats, the main difference between mouse kidney TOL and heart REJ was in the B cell compartment. The major difference to rat liver allograft acceptance was that apoptosis of infiltrate did not appear to play a role. Instead, increased Foxp3 expression in TOL kidneys implies that regulatory T cells might be important.

High rate of spontaneous liver allograft acceptance in the mouse despite full MHC incompatibility: Investigatios on the mechanisms of tolerance induction

High rate of spontaneous liver allograft acceptance in the mouse despite full MHC incompatibility: Investigatios on the mechanisms of tolerance induction