Liver Transplant Tolerance Research Articles

Regulatory T Cells in Autoimmune and Viral Chronic Hepatitis.

In both autoimmune liver disease and chronic viral hepatitis, the injury results from an immune-mediated cytotoxic T cell response to liver cells. As such, it is not surprising that CD4+ regulatory T cells, a key regulatory population of T cells able to curb immune responses, could be involved in both autoimmune hepatitis and chronic viral hepatitis. The liver can induce the conversion of naïve CD4+ T cells to CD4+ regulatory T cells and induce tolerance to locally expressed antigens. This tolerance mechanism is carefully regulated in physiological conditions but any imbalance could be pathological. An overly tolerant immune response can lead to chronic infections while an overreactive and unbridled immune response can lead to autoimmune hepatitis. With the recent advent of monoclonal antibodies able to target regulatory T cells (daclizumab) and improve immune responses and several ongoing clinical trials analysing the impact of regulatory T cell infusion on autoimmune liver disease or liver transplant tolerance, modulation of immunological tolerance through CD4+ regulatory T cells could be a key element of future immunotherapies for several liver diseases allowing restoring the balance between proper immune responses and tolerance.

MiR-152 may silence translation of CaMK II and induce spontaneous immune tolerance in mouse liver transplantation.

Spontaneous immune tolerance in mouse liver transplantation has always been a hotspot in transplantation-immune research. Recent studies revealed that regulatory T cells (Tregs), hepatic satellite cells and Kupffer cells play a potential role in spontaneous immune tolerance, however the precise mechanism of spontaneous immune tolerance is still undefined. By using Microarray Chips, we investigated different immune regulatory factors to decipher critical mechanisms of spontaneous tolerance after mouse liver transplantation. Allogeneic (C57BL/6-C3H) and syngeneic (C3H-C3H) liver transplantation were performed by 6-8 weeks old male C57BL/6 and C3H mice. Graft samples (N = 4 each group) were collected from 8 weeks post-operation mice. 11 differentially expressed miRNAs in allogeneic grafts (Allografts) vs. syngeneic grafts (Syngrafts) were identified using Agilent Mouse miRNA Chips. It was revealed that 185 genes were modified by the 11 miRNAs, furthermore, within the 185 target genes, 11 of them were tightly correlated with immune regulation after Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and Genbank data cross-comparison. Verified by real-time PCR and western blot, our results indicated that mRNA expression levels of IL-6 and TAB2 were respectively down regulated following miR-142-3p and miR-155 augment. In addition, increased miR-152 just silenced mRNA of CaMK II and down-regulated translation of CaMK II in tolerated liver grafts, which may play a critical role in immune regulation and spontaneous tolerance induction of mouse liver transplantation.

Liver transplantation. Immune responses to HCV infection linked to liver transplant tolerance.

Liver transplantation: Immune responses to HCV infection linked to liver transplant tolerance

Operationally Tolerant Pediatric Liver Transplant Recipients Exhibit Features of Immune Activation and Exhaustion.

Experimental models describe immune exhaustion in liver transplant tolerance. It is not known if immune exhaustion is also occurs in clinical operational tolerance. We tested this in a crossectional cohort of pediatric liver transplant recipients with stable allograft function in the absence of immunosuppression (Operationally Tolerant, OT) in comparison to those Weaned off Immunosuppression (WI), continued on Maintenance Immunosuppression (MI) and Healthy Controls (HC). Methods: PBMC from 14 OT, WI, MI patients > 5 years post transplantation and, HC were examined by 13-color FACS for B cell, T cell, monocyte and DC immunophenotyping. Results: (1) In OT recipients, B cell frequencies (p <0.05) were increased and were enriched in both unswitched (CD19+ IgD+ CD27+) (p<0.005) and switched memory B cells (CD19+ IgM+ CD27+ and CD19+ IgG+ CD27+) (p<0.05) with a paucity in transitional B cells (CD24+ CD38+ CD10+) (p<0.05) when compared to MI. Despite these increases in memory B cells, DSA were observed least frequently in OT. (2) CD8 T cells in OT showed an increase in effector memory (CD45RA- CCR7-) compared to MI (p<0.05). CD4 Treg (CD25+ CD127- FoxP3+) frequencies were increased in OT compared to MI (p<0.0005) as previously reported. Both CD4 and CD8 T cells in OT showed an increase in cells expressing CD57 indicative of replicative senescence and these were enriched within CD4 central memory (CD45RA-, CCR7+) and CD8 effector memory when compared to MI (p<0.05). CD57+ T cells upregulated PD1 and 2B4, markers of exhaustion in OT compared to MI (p<0.05). Upon stimulation, IFNg expression in CD4 T cells and IL-2 expression in CD8 T cells in OT were decreased than in MI consistent with exhaustion. Moreover, OT showed decreased CD8 frequencies (p<0.05) and high PD-L1 expression on monocytes (p<0.0005) compare to MI. (3) OT subjects also showed decreased CD8 T cells, reduced expression of T cell cytokines (CD4 IFNg and CD8 IL-2), and increased expression of PDL1 on monocytes when compared to either WI or HC (p<0.0005) suggesting that these observations are unique to operationally tolerant subjects. Conclusions: These data suggest that immune activation occurring off immunosuppression is followed by immune exhaustion in tolerant patients.

CD39 Is Critical for Inducing Tolerance in Murine Allogeneic Liver Transplantation.

Background: ATP is abundant in the cell cytosol and important for cell metabolism. Once cells die or are injured, ATP is released into the extracellular space and activates immune cells as a DAMP (damage-associated molecule pattern). CD39 regulates immune activation by hydrolysis of extracellular (e)ATP. We have found that conventional mouse liver myeloid(m) DC express comparatively high levels of surface CD39 and that this contributes to protection of the liver from cold ischemia reperfusion injury after syngeneic liver transplantation (LTx). We hypothesized that CD39 might play an important role in “spontaneous” transplant tolerance after allogeneic mouse LTx. METHODS: 10-12 week-old male B6 WT or CD39 KO (B6 background) livers were transplanted orthotopically into normal 10-12 week-old male C3H mice. Graft rejection was assessed by host survival and histology and also by serum ALT on day 4 after LTx. Incidences of regulatory T cells (Treg) and IFN-γ+ CD8+ T cells in the graft were quantified by flow cytometry at day 4 post LTx. Anti-donor T cell proliferative responses were evaluated by CFSE-MLR. RESULTS: Whereas all the WT B6 liver transplanted C3H mice survived more than 100 days, animals that received CD39KO livers all died within 40 days (MST: WT: >100d vs CD39 KO 8d; p<0.01). Serum ALT at day 4 post LTx was significantly higher in mice given CD39KO livers than in those with WT livers (WT: 480±226 IU/l vs CD39KO 860±484IU/l; p<0.05). The frequency of Treg in liver graft non-parenchymal cells after LTx was significantly lower in CD39KO livers than in WT livers (WT: 3.4±0.5% vs CD39KO 2.6±0.2%; p<0.05). Moreover IFN-γ production by CD8+ liver T cells was significantly higher in CD39KO allografts than in WT allografts (WT: 38.8±1.7% vs CD39KO 46.6±2.0%; p<0.05). Splenic T cells from CD39KO liver transplant recipients exhibited greater proliferation against donor splenocytes than those from WT liver allografted mice (WT: 27.2±5.8% vs CD39KO 36.3±5.7%; p<0.05). CONCLUSION: CD39 deficiency in liver grafts induces stronger anti-donor T cell responses and breaks “spontaneous” tolerance after mouse liver Tx. CD39 may be an important molecule for promoting liver allograft tolerance.

Pediatric liver transplant recipients with operational tolerance exhibit features of immune activation and exhaustion (TRAN3P.869)

Abstract Experimental models describe immune exhaustion in liver transplant tolerance. It is not known if immune exhaustion is also occurs in clinical operational tolerance. We tested this in a cross-sectional cohort of pediatric liver transplant recipients with stable allograft function in the absence of immunosuppression (Operationally Tolerant, OT) in comparison to those Weaned off Immunosuppression (WI), continued on Maintenance Immunosuppression (MI) and Healthy Controls (HC). Results: In OT recipients, B cell frequencies (p &amp;lt;0.05) were increased and were enriched in both unswitched (p&amp;lt;0.005) and switched memory B cells (CD19+ IgM+ CD27+ and CD19+ IgG+ CD27+) (p&amp;lt;0.05) compard to MI. CD8 T cells in OT showed an increase in effector memory compared to MI (p&amp;lt;0.05). CD4 Treg frequencies were increased in OT compared to MI (p&amp;lt;0.0005). Both CD4 and CD8 T cells in OT showed an increase in cells expressing CD57, indicative of replicative senescence and these were enriched within CD4 central memory and CD8 effector memory when compared to MI (p&amp;lt;0.05). CD57+ T cells upregulated PD1 and 2B4, markers of exhaustion in OT compared to MI (p&amp;lt;0.05). Upon stimulation, IFNγ expression in CD4 T cells and IL-2 expression in CD8 T cells in OT were decreased compared to MI consistent with exhaustion. Moreover, OT showed decreased CD8 frequencies (p&amp;lt;0.05) and high PDL1 expression on monocytes (p&amp;lt;0.0005) suggesting that this population might contribute to T cell exhaustion in these recipients.

Immune tolerance of rat liver transplantation induced by gene transfer of antisense B7-1

Objective To observe the role of antisense B7-1 in B7-1 passageway and anti-rejection.Methods The ROLT was established by DA→Lewis.All rats were divided into two groups randomly.Group A was perfused with normal saline (1 mL).Group B was perfused with pcDNA3B-B7-1 (1 ml).Both groups were divided to six subgroups randomly (n =10 each).B7-1 expression was detected.RAI was identified.The survival time of recipients was analyzed.Results The expression of B7-1 protein was decreased significantly in group B as compared with group A (0.025 3 ± 0.006 3 vs.0.036 6 ±0.025 2 at first day; 0.058 3 ±0.018 4 vs.0.087 5 ±0.025 1 at 2nd day;0.059 9 ±0.013 5 vs.0.107 5 ± 0.021 1 at 3rd day;0.062 2±0.015 0 vs.0.161 0± 0.016 5 at 4th day; 0.063 6 ± 0.017 9 vs.0.257 1 ±0.025 1 at 5th day,P ＜0.01).The RAI was decreased significantly in group B as compared with group A (1.4 ± 0.5 vs.2.0 ± 0.7 at first day,P ＜ 0.05 ; 1.9 ± 0.6 vs.2.7 ± 0.7 at 2nd day,P＜0.05; 2.0±0.7 vs.4.4±1.0 at 3rd day,P＜0.0l ; 2.3 ±0.7 vs.6.3 ±0.9 at4th day,P＜0.01 ; 2.9 ± 1.2 vs.7.2 ± 1.4 at 5th day,P ＜0.01).The survival time in grop B was significantly longer than in group A [(42.6 ± 13.3) d vs.(8.6±2.3) d,P＜0.01].Conclusion Antisense B7-1 decreased the B7-1 expression and attenuated acute rejection. Key words: Antisense gene B7-1 ; Immune tolerance; Liver transplantation

Protein F-induced immune tolerance in liver transplantation in rats

Liver-specific protein F is commonly used in liver transplantation studies for its allograft immunogenicity. The objective of this study was to investigate immune tolerance induced by protein F in liver transplantation in rats. Healthy inbred male Wistar and Sprague-Dawley (SD) rats were used in this study. The transplant recipient rats were randomly divided into three groups. The SD rats transplanted with liver tissues from SD rats or Wistar rats were defined as intragraft control group (Group A) or acute reaction group (Group B), respectively. The SD rats that received thymic administration of 4mg protein F 1week prior to transplantation with livers from Wistar rats were defined as protein F interference group (Group C). Kamada's two-cuff technique was utilized in the liver transplantation surgeries. The postoperative general condition, transplantation survival time, pathological examination, and serum IFN-γ level (quantified by ELISA) were recorded and compared to evaluate the immune response and outcomes in the recipient rats after liver transplantation. Group A rats exhibited good postoperative condition and prolonged survival (median survival time was 92days). In contrast, Group B rats lost body weight rapidly after liver transplantation, and died starting at day 12 (median survival time was 15days). Compared to Group B, Group C rats showed significantly longer survival (medium survival time was 71days). Our findings indicate that protein F is an important transplantation antigen with allograft immunogenicity, which could successfully induce immune tolerance in liver transplantation.

Clinical Immune Tolerance in Liver Transplantation: Present and Future

Clinical Immune Tolerance in Liver Transplantation: Present and Future

Immunosuppression withdrawal following liver transplantation: The older, the wiser… but maybe too late

Immunosuppression withdrawal following liver transplantation: The older, the wiser… but maybe too late

Characteristics of Recipients Who Achieved Spontaneous Operational Tolerance in Adult Liver Transplantation

BackgroundChronic immunosuppression is associated with unwanted adverse effects and increased morbidities. Long-term acceptance of transplanted organs without the requirement for immunosuppression, or operational tolerance, remains an important goal in clinical transplantation. MethodsWe reviewed the characteristics of recipients who achieved spontaneous operational tolerance after liver transplantation (OLT) among a consecutive series of 1014 adult recipients in a single center. ResultsWe observed 5 cases (0.5%) of operational tolerance. All cases were men who underwent transplantations for hepatitis B virus-related liver cirrhosis. The mean time from OLT to achievement of spontaneous operational tolerance was 83.1 ± 62.9 months (range, 21.3–156.2). Characteristics common to all tolerant recipients were superior graft quality and good pretransplant recipient condition: specifically, high graft–recipient weight ratio (median, 1.18; range, 1.15–2.69), low hepatic macrosteatosis (median, 3; range, 0–15), low score of model for end-stage liver disease (median, 13; range, 7–21), and no history of preoperative intensive care.

Operational tolerance in liver transplantation: Shall we predict or promote?

An increased frequency of suc-cessful immunosuppression withdrawal is associatedwith an increasing length of time between the with-drawal of immunosuppression and transplantation aswell as a lack of inflammation on prewithdrawal biopsysamples. This state is not true tolerance, but it hasbeen labeled operational tolerance.

Biomarkers of tolerance

As the induction and maintenance of donor-specific tolerance is a central aim in solid organ transplantation, it is essential that clinicians are able to identify and monitor tolerance accurately and reliably. This review highlights recent advances in defining sets of biomarkers in noninvasive samples that may guide minimization and withdrawal of immunosuppression in tolerant recipients. Recent studies in liver and kidney transplant recipients have identified distinct biomarker profiles that are associated with operational tolerance. Although there is some heterogeneity in the findings of these studies, these have suggested novel cellular mechanisms for the development of tolerance. Multiple platforms such as microarray gene expression analysis, flow cytometry, and immune cell functional assays have been used to discover and validate composite sets of biomarkers, which identify recipients with operational tolerance both in liver and kidney transplantation. These studies suggest that distinct cellular and molecular mechanisms lead to the development of tolerance in different transplanted organs. These putative biomarker profiles now need to be validated prospectively in trials of immunosuppression withdrawal and in novel approaches to induce transplant tolerance.

Intragraft Vδ1 γδ T Cells With a Unique T-Cell Receptor Are Closely Associated With Pediatric Semiallogeneic Liver Transplant Tolerance

T-cell receptor Vδ2 γδ T cells (Vδ2 cells) participate in host defense, whereas Vδ1 γδ T cells (Vδ1 cells) may regulate immune responses. Vδ1 cells appear to play a role in fetomaternal tolerance and our aim was to examine their role in liver transplant tolerance. To determine whether Vδ1 cells increase within accepted grafts after semiallogeneic pediatric liver transplantation, the Vδ1/Vδ2 ratio was assessed at the transcriptional level and the complementarity-determining region 3 loop of the δ chain of Vδ1 cells was sequenced in biopsies from immunosuppression-free (n=6) or almost free (n=3) liver transplant recipients, referred to as group tolerance (Gr-Tol; n=9). The results were compared with biopsies from grafts of recipients on maintenance immunosuppression due to concern of rejection (Gr-IS; n=11). Chronically rejected grafts (Gr-CR; n=6) and normal livers (Gr-NL; n=8) were also examined. The Vδ1/Vδ2 ratio was the highest in Gr-Tol (0.07±0.06) compared with Gr-IS (0.03±0.02; P=0.04), Gr-CR (0.01±0.02; P=0.008), and Gr-NL (0.02±0.04; P=0.01). There was an identical complementarity-determining region 3 sequence (100% homologous) among all recipients in Gr-Tol, which was dominant in six of nine recipients. This sequence was not seen in Gr-IS or Gr-CR, although it was observed in five of six normal livers. A unique Vδ1-bearing T-cell clone accumulates within accepted human liver grafts. It might be useful as a biomarker of tolerance and the identification of its ligand might aid in the development of a novel strategy for tolerance induction.

Predicting Operational Tolerance in Pediatric Living-Donor Liver Transplantation by Absence of HLA Antibodies

The role of anti-human leukocyte antigen (HLA) antibodies in operational tolerance (OT) after pediatric living-donor liver transplantation (LDLT) remains inconclusive. We investigated whether the presence of HLA antibodies impeded the development of OT. We retrospectively examined the prevalence of anti-HLA antibodies in pediatric LDLT recipients before transplantation and at 3 weeks after transplantation and analyzed the significance of those antibodies in relation to later OT. Forty pediatric LDLTs were performed between April 1996 and December 2000 and followed up through July 2011, with sera available for measurement of HLA antibodies. Seventeen patients achieved OT (mean follow-up, 4571.9±544.7 days) and 23 patients did not achieve OT (mean follow-up, 4532.0±425.4 days). Protocol liver biopsy was done for 14 OT patients and 16 non-OT patients. Their sera were tested for anti-HLA class I and II antibodies using the LABScreen single antigen beads test, in which a 1000 mean fluorescence value was considered positive. The prevalence of antibodies after transplantation in non-OT patients was higher than in OT patients (95.2% vs. 73.3%; P<0.001). The highest mean fluorescence intensity of antibodies was significantly higher in non-OT patients than in OT patients. The prevalence of HLA-B, HLA-C, HLA-DQ, and HLA-DR antibodies was significantly higher in non-OT patients than in OT patients. The highest mean fluorescence intensity of HLA-A, HLA-B, and HLA-DQ observed in non-OT patients was significantly higher than those in OT patients. In our study, posttransplantation HLA antibodies were associated with the future absence of OT. A prospective study with more patients is necessary to confirm the predictive value of HLA antibodies for OT.

Liver transplant tolerance and its application to the clinic: can we exploit the high dose effect?

The tolerogenic properties of the liver have long been recognised, especially in regard to transplantation. Spontaneous acceptance of liver grafts occurs in a number of experimental models and also in a proportion of clinical transplant recipients. Liver graft acceptance results from donor antigen-specific tolerance, demonstrated by the extension of tolerance to other grafts of donor origin. A number of factors have been proposed to be involved in liver transplant tolerance induction, including the release of soluble major histocompatibility (MHC) molecules from the liver, its complement of immunosuppressive donor leucocytes, and the ability of hepatocytes to directly interact with and destroy antigen-specific T cells. The large tissue mass of the liver has also been suggested to act as a cytokine sink, with the potential to exhaust the immune response. In this review, we outline the growing body of evidence, from experimental models and clinical transplantation, which supports a role for large tissue mass and high antigen dose in the induction of tolerance. We also discuss a novel gene therapy approach to exploit this dose effect and induce antigen-specific tolerance robust enough to overcome a primed T cell memory response.

Nuclear Antigens and Auto/Alloantibody Responses: Friend or Foe in Transplant Immunology

In addition to cellular immune responses, humoral immune responses, mediated by natural antibodies, autoantibodies, and alloantibodies, have increasingly been recognized as causes of organ transplant rejection. In our previous studies, we have demonstrated the induction of antinuclear antibodies against histone H1 and high-mobility group box 1 (HMGB1), in both experimental and clinical liver transplant tolerance. The active induction of antinuclear antibodies is usually an undesirable phenomenon, but it is often observed after liver transplantation. However, the release of nuclear antigens and its suppression by neutralizing antibodies are proposed to be important in the initiation and regulation of immune responses. In this review article, we summarize the current understanding of nuclear antigens and corresponding antinuclear regulatory antibodies (Abregs) on infection, injury, inflammation, transplant rejection, and tolerance induction and discuss the significance of nuclear antigens as diagnostic and therapeutic targets.

The pits and pearls in translating operational tolerance biomarkers into clinical practice

This review highlights the importance and the role of key biomarker studies in liver and kidney transplant tolerance, discusses the most recent findings with respect to organ-type and cell-type specificity in blood and tissue, and points out the novel research directions in the field of immunological tolerance that involve both adult and pediatric recipients. Recent studies indicate that biomarkers for solid organ transplant tolerance are distinct with respect to organ type and cell type, suggesting distinct tolerogenic mechanisms for different organs. In both liver and kidney transplant tolerant recipients, novel cellular mechanisms have been proposed for natural killer cells, B cells, and dendritic cells in the maintenance of stable operational tolerance. Major advances have been made with respect to the understanding of mechanisms and the process of discovery and early validation of peripheral blood biomarkers for operational transplant tolerance both in kidney and liver transplantation. These studies have shed light on the findings that these tolerance mechanisms may be organ specific, as the peripheral blood transcriptional profiling attempts by microarrays and PCR reveal distinct differences and suggest roles for specific cell types. Although these studies are mostly in adults and limited in children, the first tolerance gene signature for pediatric liver transplant tolerance suggests that there are common mechanisms, yet distinct peripheral biomarkers across age. Prospective trials and organ integrative studies are now needed to further develop these biomarkers for future clinical application in addition to expanding novel approaches such as the investigation of miRNAs to better understand the tolerance mechanisms.

Effect of Kupffer cells on immune tolerance in liver transplantation

ObjectiveTo observe the effect of Kupffer cells on immune tolerance in liver transplantation. MethodsThe rats were randomly divided into A, B and C groups. A group was sham operation group. The donor rats of group B had intraperitoneal injection of 1 nmol Kuppffer cells every other day for three days before liver transplantation. Rats of group C were injected with equal saline. The rat liver transplantation models were established by modified Kamada's two-cuff technique. The rats were sacrificed after 24 hours. The concentrations of ALT and AST in serum were measured with the biochemical analyzer. The level of IL-2 and TNF-α in serum were measured by ELISA method. The apoptotic indexes were detected by immunohistochemical assay. ResultsThe concentration of ALT, AST, IL-1 and TNF-α in A, B and C groups were increased successively. The levels of group C were significantly higher than that of group B and A (P<0.05), and the levels of group B were significantly higher than that of group A (P<0.05). The apoptotic indexes of three groups were 3.40±0.37, 14.70±2.54 and 26.33±3.65, respectively, with significant difference among three groups (P<0.05). ConclusionsPretreatment with Kupffer cells can reduce liver injury and raise liver transplantation immune tolerance.

Hepatic stellate cells promote immunotolerance following orthotopic liver transplantation in rats via induction of T cell apoptosis and regulation of Th2/Th3-like cell cytokine production

Hepatic stellate cells (HSCs) have been demonstrated to have immunoinhibitory activity. The aim of this study was to investigate the role of HSCs in the development of immunotolerance following liver transplantation. A rat liver transplantation tolerance model [donor Lewis into recipient Dark Agouti (DA)] and rejection (donor DA into recipient Lewis) was established. On the 7th day following transplantation, the HSCs and T cells were isolated from the rats of either the tolerance or rejection group and cultured together. The apoptosis rate of the T cells was determined 24 h later by flow cytometry following staining with anti-CD3 mAb and Annexin V-FITC/PI. Additionally, the FasL expression of HSCs was determined by flow cytometry following staining with anti-FasL mAb. The protein levels of IL-2, TNF-α, TGF-β and IL-10 in the supernatant collected from mixed lymphocyte reaction cultures of HSCs and T cells for 5 days were measured using ELISA assays. HSCs isolated from the tolerance group had a higher T-cell apoptosis induction activity compared with those of the rejection group. The activity of the HSCs was partially reversed by FasL blocking mAb. Accordingly, the FasL expression level of HSCs in the tolerance group was revealed to be higher than that of the rejection group. Moreover, HSCs stimulated IL-10 and TGF-β production in the tolerance group. This study suggests that HSCs are involved in liver transplantation immune tolerance via the induction of T-cell apoptosis partially mediated by the Fas/FasL pathway and the activation of Th2/Th3-like cell cytokine production.