Liver Transplant Tolerance Research Articles

Liver Stromal Cells Contribute to Liver Transplant Tolerance: Involved Cellular and Molecular Mechanisms

Organ transplantation is currently dependent on life-time administration of immunosuppressive drugs to non-specifically suppress immune response, resulting in severe side effects, including infection and cancer. Induction of specific tolerance is an ideal approach, but not generally successful, which however, actually naturally occurs. Thus, ˜1/4 liver allografts in farm pigs are spontaneously accepted, and induce donor specific tolerance. In humans, immunosuppression can be weaned off until completely stopped in ˜1/4 liver transplant patients. The underlying mechanisms are not completely understood. We and others surprisingly found that liver transplant tolerance is dependent on IFN-g stimulation. Mouse liver allograft, otherwise spontaneously accepted, was acutely rejected in IFN-g-/- recipients. In this study, the liver from IFN-gR1-/- mice (B6, H-2b) was transplanted into C3H (H-2k) recipients (n=6). None of them survived >15 days (WT grafts survived >100 days), suggesting that liver transplant tolerance requires intact IFN-g signaling pathway in liver graft. Both flow and histochemical analyses demonstrated markedly increased CD8+ infiltrates in IFN-gR1-/- grafts associated with lower expression of B7-H1 and less infiltrating T cell apoptosis, suggesting a role of B7-H1, an end product of IFN-g signaling in regulating immune response. This was supported by the result that the liver grafts from B7-H1-/- mice were acutely rejected in WT allogeneic recipients, associated with markedly less apoptosis (annexin V) in the graft infiltrating T cells. Liver grafts deficient in IFN-gR1 did not affect much T cell activation (CD44hiCD62Llo), as well as generation of myeloid-derived suppressor cells (MDSC) and Tregs in early phase. However, Tregs were markedly enhanced in long-term survived WT grafts, suggesting that elimination of effectors in early phase is crucial, while the suppressor cells may largely work to maintain tolerance. To identify the cell components in the liver graft responsible for these changes, the graft non-parenchymal cells were isolated at various time points for flow analysis. Almost all graft CD45+ cells were quickly (in one-two days) become recipient origin (H-2k, IFN-gR1+), while CD45- cells remain donor type (H-2b, IFN-gR1-), even in long-term survived grafts, suggesting that liver non-hematopoietic cells may be responsible for these changes. We have studied isolated hepatic stellate cells (HSC) (liver stromal cells, well known for their storing vitamin A and participating in fibrosis) that were exceptionally immunosuppressive. Cotransplantation with HSC effectively protected islet allografts without requirement of immunosuppression. Interestingly, cotransplantation with HSC isolated from IFN-gR1- mice failed to protect islet grafts, associated with markedly more infiltrating CD8+ T cells and less MDSC and Treg cells, indicating that the immune regulatory activity of HSC depends on intact IFN-g signaling pathway. Expression of B7-H1 was upregulated following exposure to IFN-g in a dose dependent manner, suggesting B7-H1 is an end product of IFN-g signaling. HSC isolated from B7-H1-/- mice largely lost their ability to protect islet allgrafts. This was associated with more CD8 T cells graft infiltration. In conclusion, IFN-g/B7-H1 signaling pathway in liver stromal cells play a critical role in establishment of liver transplant tolerance.

Association of Anti-HLA Antibody with Operational Tolerance in Pediatric Living-Donor Liver Transplantation: A Possible Evidence of Humoral Sensitization

Association of Anti-HLA Antibody with Operational Tolerance in Pediatric Living-Donor Liver Transplantation: A Possible Evidence of Humoral Sensitization

Transplant Tolerance and Myeloid Supressor Cells

The successful organ transplantation is currently dependent on chronic administration of immunosuppressive drugs to non-specifically suppress immune response, causing severe side effects, including infection and cancer. Induction of specific tolerance is ideal solution, which, indeed, naturally occurs. Thus, ˜1/4 liver transplants in farm pigs were spontaneously accepted, inducing systemic specific tolerance. In humans, weaning off, leading to complete stop of immunosuppressive has achieved in ˜1/4 liver transplant patients. The underlying mechanisms remain unclear. We have investigated mouse liver transplant tolerance, and noted that, different from liver transplants that are spontaneously accepted, hepatocyte transplants are acutely rejected, suggesting immune regulatory activity of liver non-parenchymal cells. Hepatic stellate cells (HSC) turn out to be potent immunosuppressors. Cotransplantation with HSC effectively protects islet allografts from rejection. We demonstrated in this study that, in contrast to myeloid (CD11b+) cells in islet alone grafts that were CD11c+, CD11b+ cells in HSC/islet grafts expressed low CD11c and costimulatory molecules, but high arginase 1 and iNOS, and markedly inhibited T cell response - sharing many properties with myeloid derived suppressor cells (MDSC). MDSCconsisting of heterogeneous cell populations were initially identified in cancer patients contributing to immune evasion. The impact of MDSC on transplant tolerance has not been extensively studied. MDSC. Induction of MDSC by cotransplantation with HSC was dependent on IFN-g stimulation since HSC deficient in IFN-gR1 largely lost the ability to induce MDSC, and failed to protect islet allografts. The induction of MDSC by HSC was also confirmed by addition of HSC into BM-derived DC culture. The harvested floating cells were predominantly CD11b+CD11c− (not CD11c+), expressed high arginase 1 and iNOS, but low CD40, CD80 and CD86. They expressed Gr-1, F4/80, Ly6C, suggesting heterogeneous nature. Transwell culture data suggested that induction of MDSC by HSC was mediated by soluble factors. They demonstrated potent inhibitory activity against T cell response. Cotransplantation with in vitro-generated MDSC achieved long-term survival in 64% islets allografts as effective as cotransplantation with HSC, associated with reduction of graft CD8 T cells via apoptosis (annexin V), and expansion of antigen-specific Treg cells, which was mediated by B7-H1 expressed on MDSC since the MDSC generated from mice deficient in B7-H1 expression markedly reduced their ability to protect the cotransplanted islet allografts due to failure to induce sufficient Treg cells. In conclusion, MDSC play a crucial role in induction of transplant tolerance. IFN-γ/B7-H1 pathway appears to be crucial in establishing peripheral tolerance. Large amounts of MDSC can be generated in vitro, and can potentially be used for induction of transplant tolerance.

Long Term Follow Up of 30 Operationally Tolerant Liver Transplant Recipients

Introduction: The advances of immunosuppression and post-operative follow up have contributed to an improvement of the long term survival of liver transplant recipients. However, complications related to long term immunosuppression affect both long term survival and quality of life of these patients. Successful withdrawal of immunosuppression has been achieved in many liver transplant recipients. In this study we present a long term follow up of a cohort of patients that had their immunosuppression successfully withdrawn. Methods: We studied the outcome of liver transplant patients who had their immunosuppression successfully withdrawn. A total of thirty operationally tolerant patients were studied (27 men and 3 women). Twenty three of them were weaned off immunosuppression during a withdrawal study that took place about 10 years ago. The remaining seven patients were part of a cohort of patients that were transplanted more recently under Campath-1H induction immunosuppression and were weaned during the last 5 years. Results:Patient Survival:Patient, graft survival: With a follow up until end of January 2012, the post-transplant follow up of the patients was from 7-20 years (average:14.15) and the off immunosuppression follow up was from 1.4-11 years, (average:7.25). There were seven mortalities. Deaths were due to cardiovascular causes (n=5): [myocardial infarct (n=3), cerebrovascular accident (n=1), pulmonary embolism (n=1)]. Other mortalities included biliary obstruction-sepsis (n=1), chronic rejection-sepsis (n=1). Average age of these patients at the time of death was 69.01 years. There were no re-transplants during this period of time. Rejection episodes: A patient presented a moderate rejection 5.3 years after immunosuppression withdrawal and was put back on maintenance immunosuppression. Renal function: Until the end of the follow up period, a patient was on dialysis and another received a kidney transplant 13.9 and 19.26 years post-transplantation respectively. Neoplasms: Six patients presented with squamous cell skin cancer (n=2), throat, kidney, lung cancer and melanoma (one each). Conclusions: Operational tolerance after liver transplantation can be achieved for a long period of time, however is not a permanent state and continuous vigilance and follow up is required to detect rejection episodes.

Immune tolerance induced by RelB short-hairpin RNA interference dendritic cells in liver transplantation

BackgroundThe induction of specific immune tolerance for alloantigen is the best method for solving transplant rejection. We have previously reported T-cell tolerance induced by RNA interference (RNAi) RelB dendritic cells (DCs), supporting the possibility of immunologic tolerance in liver transplantation. MethodsA stable model of acute rejection was established in Lewis (RT11) rats that had received a liver graft from dark agouti-RT1a rats. To evaluate the immune tolerance of DCs of different maturity, the rats were randomly assigned to four groups (12 donor/recipient pairs): (1) control-DC group, recipient rats without preinjection; (2) RelB short hairpin (sh)RNAi-DC group, recipient rats with preinjection of tolerogenic DCs by way of RelB silencing; (3) imDC group, recipient rats with preinjection of immature DCs; and (4) lipopolysaccharide-DC group, recipient rats with preinjection of mature DCs. The immune tolerance of the grafts was evaluated by liver function tests (aspartate transaminase, total bilirubin), cytokines (interleukin [IL]-2, IL-4, IL-10 and interferon-γ), and histopathologic examination during the 2 wk after transplantation. The survival time of the rats was also observed. ResultsCompared with the other three groups, the graft survival time was significantly prolonged in the RelB shRNAi-DC group. In addition, RelB shRNAi-DCs resulted in the reduced secretion of IL-2 and interferon-γ and increased levels of IL-10 and IL-4. The symptoms of rejection were obviously alleviated in the RelB shRNAi-DC group, and the rejection activity index was still reduced after 2 wk. ConclusionsInjection of RelB-silenced DCs contributed to the reduced incidence of graft rejection and prolonged the graft survival time. The potential mechanisms involved the regulation and induction of immune-incompetent T cell by DCs.

Iron Predicts Tolerance in Liver Transplantation

Selected summary of Bohne F, Martinez-Llordella M, Lozano JJ, et al. Intragraft expression of genes involved in iron homeostasis predicts the development of operational tolerance in human liver transplantation. J Clin Invest 2012;122: 368–382. Lifetime immunosuppressive therapy, which is usually needed to prevent rejection of the graft in solid organ transplantation, may have serious side effects resulting in increased morbidity and mortality. The liver is unique in organ transplantation; up to 20% of transplant recipients can be weaned off immunosuppressive drugs and display “operational tolerance” of the graft in the absence of immunosuppression (Transplantation 2011;91:1065–1974)...

Interleukin-9 in Stable Liver Transplant Recipients

IntroductionInterleukin-9 (IL-9) has recently been described to be involved in the maintenance of a tolerant environment, but there is no evidence of its role in human liver transplantation. The aim of our study was to measure the serum levels of IL-9 in stable liver transplant recipients and examine their influence on immunosuppressant load. MethodsSerum IL-9 levels were determined in 34 healthy subjects and 30 stable liver transplant recipients who were free of rejection episodes for at least 8 years. The results were analyzed according to the blood levels of calcineurin inhibitors (CNIs) at the time of the study: 13 patients showed high concentrations of either cyclosporine or tacrolimus (high CNI: cyclosporine > 80 ng/mL or tacrolimus > 5 ng/mL) and another 17 patients showed low CNI levels. ResultsThe concentrations of IL-9 were significantly higher among liver transplant recipients compared with healthy subjects. In addition, patients with low CNI blood levels showed higher serum levels of IL-9, an effect that was greater with tacrolimus, albeit not significantly. ConclusionsThese preliminary results indicated that increased serum IL-9 concentrations accompanied a lower immunosuppressive load. It remains to be established whether this relates to induction of tolerance in liver transplantation.

The Role of Invariant NKT Cells in Liver Transplant Tolerance in Rats

ObjectiveThe aim of this study was to investigate the role of invariant natural killer T (iNKT) cells in liver transplant tolerance in rats. MethodsAnimals were randomly divided into 3 groups. The α-galactosylceramide (α-GalCer) group underwent injection through the caudal vein; the saline group received the same dose of saline and the control group received no treatment. Ten rats in each group were examined for survival the others were humanely killed on the seventh day posttransplantation. Liver tissues were used to assess histopathologic changes. Real-time polymerase chain reaction (PCR) was performed to determine the relative expressions of messanger RNAs of Th1/Th2-related cytokine (mRNAs) in the liver allograft. The serum levels of related cytokines were determined using enzyme-linked immunosorbent assay (ELISA). ResultsAllograft survival was significantly higher among the α-galactosylceramide α-GalCer group than the saline or control groups. The histopathology showed mild changes in the α-galactosylceramide α-GalCer compared with the other 2 groups. Real-time PCR showed the relative expression of Th1-related cytokine interferen (IFN)-γ mRNA to be significantly lower in the α-galactosylceramide α-GalCer compared with the other 2 groups, while the Th2-related cytokine interleukin (IL)-10 mRNA was much higher. The ELISA results confirmed these differential expressions. ConclusionThe iNKT cells may play a pivotal role in liver transplant tolerance due to their regulatory functions on the Th1/Th2 imbalance. iNKT cells should be considered to be significant targets because of their attractive specificity and induction of liver allograft tolerance.

A Common Peripheral Blood Gene Set for Diagnosis of Operational Tolerance in Pediatric and Adult Liver Transplantation

To identify biomarkers of operational tolerance in pediatric and adult liver transplant recipients, transcriptional profiles were examined from 300 samples by microarrays and Q-PCR measurements of blood specimens from pediatric and adult liver transplant recipients and normal tissues. Tolerance-specific genes were validated in independent samples across two different transplant programs and validated by Q-PCR. A minimal set of 13 unique genes, highly expressed in natural killer cells (p = 0.03), were significantly expressed in both pediatric and adult liver tolerance, irrespective of different clinical and demographic confounders. The performance of this gene set by microarray in independent samples was 100% sensitivity and 83% specificity and the AUC was 0.988 for only three genes by Q-PCR. 26% of adults and 64% of children with excellent liver allograft function, on minimal or dual immunosuppression, showed high prediction scores for tolerance. Novel peripheral transcriptional profiles can be identified in operational tolerance in pediatric and adult recipients of liver allografts, suggesting a high incidence of a pro-tolerogenic phenotype in stable patients on chronic immunosuppression. Given the high incidence of viral infections and malignancies in liver transplant recipients, this gene set provides an important monitoring tool that can move the field toward personalized and predictive medicine in organ transplantation.

Dynamic Changes of Indoleamine 2,3-Dioxygenase of Kupffer Cells in Rat Liver Transplant Rejection and Tolerance

AimsTo study the dynamic changes and the immunologic role of indoleamine 2, 3-dioxygenase (IDO) in Kupffer cells (KCs) after rat liver transplantation. MethodsAnimals were randomly divided into two groups: a rejection group (REJ; LEW to BN) and a tolerance group (TOL; BN to LEW). Liver morphological changes were observed optically with hematoxylin/eosin staining. KCs were isolated from recipients. mRNA and protein expressions of IDO were detected by real-time polymerase chain reaction and Western blotting at 1, 3, 5, and 7 days after transplantation. ResultsThe levels of IDO mRNA and protein in KCs of TOL groups were similar to those in REJ groups at day 1 posttransplantation. However, the expression of IDO mRNA and protein time-dependently increased to much higher levels in the TOL than the in REJ groups at 3, 5, and 7 days posttransplantation (P < .05). The peak was observed at 7 days. ConclusionsThe IDO level of KCs was closely associated with immune tolerance induction. IDO-mediated immune modulation appears to be an attractive means to assess transplant tolerance induction.

Intra-graft expression of genes involved in iron homeostasis predicts the development of operational tolerance in human liver transplantation

Following organ transplantation, lifelong immunosuppressive therapy is required to prevent the host immune system from destroying the allograft. This can cause severe side effects and increased recipient morbidity and mortality. Complete cessation of immunosuppressive drugs has been successfully accomplished in selected transplant recipients, providing proof of principle that operational allograft tolerance is attainable in clinical transplantation. The intra-graft molecular pathways associated with successful drug withdrawal, however, are not well defined. In this study, we analyzed sequential blood and liver tissue samples collected from liver transplant recipients enrolled in a prospective multicenter immunosuppressive drug withdrawal clinical trial. Before initiation of drug withdrawal, operationally tolerant and non-tolerant recipients differed in the intra-graft expression of genes involved in the regulation of iron homeostasis. Furthermore, as compared with non-tolerant recipients, operationally tolerant patients exhibited higher serum levels of hepcidin and ferritin and increased hepatocyte iron deposition. Finally, liver tissue gene expression measurements accurately predicted the outcome of immunosuppressive withdrawal in an independent set of patients. These results point to a critical role for iron metabolism in the regulation of intra-graft alloimmune responses in humans and provide a set of biomarkers to conduct drug-weaning trials in liver transplantation.

Inducement of rat liver transplantation tolerance by hFasL and hTGF-β1 gene modified dendritic cells

Objective To improve immature dendritic cell ( imDC)' s tolerance capability which induces rat allogeneic orthotopic liver transplantation and prolong the survival time of allograft,this research co-modifying imDC with FasL and transforming growth factor-β1 ( TGF-[1 ) gene.Methods One hundred and forty cases of rat allogeneic orthotopic liver transplantation were accomplished in the strain combination of DA to Lewis rats,which were divided equally into 7 groups,including non- injection group ( control group),mDC group,imDC group,empty vector group,FasL group,TGF group and cotransfection group.Then 2 × 106 cells were respectively intraperitoneally injected to Lewis rats with distinct cells,such as mDC,imDC,empty vector-transfected imDC,hFasL-modified imDC,hTGF-β1-transformed imDC and comodified imDC with FasL and TGF-β1 gene.Four Lewis rats from each group were randomly sacrificed on days 3,7 and10 after transplantation.Hepatic function [ alanine aminotransferase (ALT) and total bilirubin (TBIL) ] and cytokines alteration were evaluated in peripheral blood.The pathological changes of the liver graft were observed.Survival analysis were performed for remnant rats,except rats dead for technical factor.Results At day 7,the difference was statistically significant ( P ＜ 0.01 ) between seven groups,and the AR in hepatic graft in cotransfection group were all lighter than those in TGF group and FasL group.At day 10,there was statistical significance between seven groups (P ＜0.01 ),the AR in hepatic graft in cotransfection group were all lighter than those in TGF group and FasL group,further more the AR in hepatic graft in TGF group was lighter than that in FasL group.At day 7,hepatic function of cotransfection group had reverted to normal and the hepatic function of FasL group took a turn for the better,but got worse at day 10;the concentration of ALT and TBIL of FasL group were obviously higher than those of cotransfection group at day 10 (P ＜0.01 and P ＜0.05).At day 10,Liver function of TGF group returned to normal,and ALT and TBIL concentrations of TGF group were significantly lower than those of FasL group ( P ＜ 0.01 and P ＜0.05 ).But compared to those of cotransfection group,the difference was not statistically significant ( P ＞0.05 ).The outcome of enzyme linked immunosorbent assay (ELISA) notified that at day 7,the serum levels of interleukin ( IL)-I,IL-10 and IL-12 in FasL group compared to those in cotransfection group and TGF group,the differences were all statistically significant ( P ＜ 0.05 ),on the contraty,the difference was not statistically significant ( P ＞ 0.05 ) between cotransfection group ane TGF group.The MST of rat recepients in FasL group was 20 days and compared to that in imDC group (23 d) with no statistically significance of difference (P ＞ 0.05),and the ability of TGF Group was limited in extending the rat receptors'survival time (48 d).There were rat recepients with long-term survival ( ＞ 90 d) only in cotransfection group,in which the survival of rat recepients was significantly prolonged than that in FasL group (P ＜0.01 ) and TGF group (P ＜0.01 ).Conclusion lmDC,hFasL or hTGF-β1 single gene-modified imDC all didn't induced long-term allograft survival of receptors in allogeneic rat liver transplantation.The ability of imDC with hFasL and hTGF-β1 co-transfection was significantly enhanced by hFasL and hTGF-β1 in inducing allogeneic rat liver transplantation immunology tolerance,which could significantly extended survival of recipients in allogeneic rat liver transplantation. Key words: Dendritic cells; Immune tolerance; FasL; TGF-β1 ; Liver transplantation

Advantages of Promoting Interleukin-10 by Silence of Histone Deacetylase 11 in Inducing Tolerance in Orthotopic Liver Transplantation in Rats

Aims The aims of this study were to study the role of histone deacetylase 11 (HDAC11) in tolerance induction in orthotopic liver transplantation (OLT) in rats and to assess the advantages of gene therapy over the immunosuppressant FK506. Methods Recipients were assigned to an acute rejection group (AcR; group I), an FK506 intervention group (group II), and a tolerance group (group III). Acute rejection (AcR) was graded by the Banff scheme and we examined postoperative survival. The messenger RNA (mRNA) and protein expressions of histone deacetylase 11 (HDAC11) and interleukin (IL) 10 in liver tissue were detected using real-time polymerase chain reaction (PCR) and Western blots, respectively. Plasma levels of tumor necrosis factor (TNF)-α, IL-2, and IL-10 were measured using enzyme-linked immunosorbent Assays. Results Group I displayed severe, Group II had less, and Group III had no evidence of AR. The survivals among Group III were longer than those in Group I and Group II. IL-10 expression was promoted by HDAC11-shRNA at 7 days after OLT. Serum IL-2 and TNF-α levels were significantly lower among Group III compared with Groups I and II, whereas IL-10 showed the opposite result. Conclusions Silence of HDAC11 promotes IL-10 expression and leads to tolerance following OLT in rats. Thus HDAC11 is a promising target for gene therapy to induce tolerance with advantages over immunosuppressive drugs.

The impact of CD4+ CD25+ regulatory T cells on the mouse spontaneous liver transplant tolerance

Objective To examine the contribution of CD4^＋CD25^＋ regulatory T cells to liver transplant tolerance. Methods After injection of anti-CD25 monoclonal antibody （mAb, PC61）, mouse orthotopic liver transplantation was performed and survivals were determined. The paraffin-embedded sections of hepatic allografts were cut and stained with hematoxylin and eosin （HE）. Furthermore, the effect ofCD4^＋CD25^＋regulatory T cells on proliferative response of CD4^＋ T cells and cyto toxicity of CD8^＋ T ceils was examined by depleting these regulatory T cells. Results Depletion of these cells in the recipients but not in the donors before liver transplantation caused rejection. Histological analyses of hepatic allografts with PC61 treatment showed extensive leukocyte infiltration and tissue destruction, whereas those in the control group showed minimal changes. Moreover, elimination of CD4^＋CD25^＋ T cells resulted in the enhancement of both proliferative response of CD4^＋ T cells and eytotoxicity of CD8^＋ T cells against donor-type alloantigen. Conclusions These results suggest that CD4^＋CD25^＋regulatory T cells were important for tolerance induction to hepatic allografts. Key words: CD4^＋CD25^＋regulatory T cells; Liver transplantaton; Immunologieal tolerance

HLA-G Level on Monocytoid Dendritic Cells Correlates With Regulatory T-Cell Foxp3 Expression in Liver Transplant Tolerance

Human leukocyte antigen (HLA)-G is a nonclassical HLA class I molecule expressed as membrane-bound and soluble isoforms. Interaction of HLA-G with its receptor, immunoglobulin-like transcript 4 on dendritic cells (DCs) down-regulates their T-cell stimulatory ability. We examined expression of HLA-G, immunoglobulin-like transcript 4, other immune regulatory molecules (inducible costimulator ligand and glucocorticoid-induced tumor necrosis factor-related receptor ligand), and the activation marker CMRF44 on circulating monocytoid dendritic cell (mDC) and plasmacytoid dendritic cell by monoclonal antibody staining and flow cytometry. Three groups of stable liver transplant recipients: operationally tolerant (TOL), prospective immunosuppressive drug weaning, and maintenance immunosuppression (MI) were studied, together with healthy controls (HC). Serum HLA-G levels were measured by enzyme-linked immunosorbent assay. In TOL patients, monocytoid dendritic cell (mDC) but not plasmacytoid dendritic cell expressed higher HLA-G than in MI patients or HC. In TOL patients, the incidence of CD4(+)CD25(hi)CD127(-) regulatory T cells (Treg) and the intensity of Treg forkhead box p3 (Foxp3) expression were significantly higher than in the MI group. HLA-G expression on circulating mDC correlated significantly with that of Foxp3 in the TOL group. There was no correlation between immunosuppressive drug (tacrolimus) dose or trough level and HLA-G expression or Treg frequency or Foxp3 expression. The incidence of patients with circulating HLA-G levels more than 100 ng/mL was highest in the TOL group, although statistical significance was not achieved. Higher HLA-G expression on circulating mDC in TOL recipients compared with MI or HC, suggests a possible role of HLA-G in immune regulation possibly mediated by enhanced host Treg Foxp3 expression.

Liver transplant tolerance: a new look at an old puzzle (169.17)

Abstract We explored the tolerogenicity of liver allografts (Ltx) by characterizing the phenotype and persistence of alloreactive T cells after spontaneously tolerant Ltx in mice. Using adoptive transfer of TCR-tg direct and indirect donor-reactive T cells, we found that LTx (BALB→B10) induced a stronger CD4 T cell response than either skin or heart Tx. Moreover, large numbers of donor-reactive T cells persist 21 days after Ltx and do not upregulate Foxp3, even though tolerance to subsequent allospecific skin grafts is established. In vivo MLR confirmed the persistence of endogenous effector CD4 T cells at 21 and 250 days post-Ltx. Overall, these findings are consistent with an ongoing effector alloresponse rather than immune regulation after Ltx. In contrast, the CD8 donor-reactive T cells were significantly reduced, though still detectable, after Ltx. However, post-Ltx animals selectively eliminate allogenic target cells in an in vivo CTL assay. Thus, deletion of effector CD8 T cells may help protect liver allografts, but cannot completely account for Ltx tolerance. Finally, CD4, CD8, and Total T cells isolated from day 90 post-Ltx mice reject donor skin grafts when transferred into Rag1-/- hosts, indicating that Ltx tolerance cannot be transferred via the immune compartment. These data demonstrate the magnitude and persistence of the anti-donor T cell responses after Ltx and opposes either deletional tolerance or regulatory T cell induction as mechanisms for Ltx tolerance.

The dynamic changes of Th17/Treg cytokines in rat liver transplant rejection and tolerance

Acute rejection is still a major cause of early graft loss and a risk factor for long-term recipient post-transplant survival. Recently, CD4 + CD25 + Foxp3 + regulatory T (Treg) cells and Th17 cells have been described as two distinct subsets with opposing effects on autoimmunity and transplant immunity. We investigated the existence of Th17/Treg functional imbalance between tolerance and rejection groups during rat liver transplantation. Then, Th17/Treg functions on different levels were investigated comparatively between those two groups, including related cytokine secretion and key transcription factors. REJ groups revealed significant increase in Th17-related cytokine (IL-17, IL-6 and IL-23) and transcription factor (RORγt) levels and remarkable decrease in Treg-related cytokine (IL-10 and TGF-β1) and transcription factor (Foxp3) levels when compared to day-matched TOL groups from day 3 post-transplantation. Results indicated Th17/Treg functional imbalance between tolerance and rejection groups during rat liver transplantation, suggesting a potential role of Th17/Treg imbalance in pathogenesis of acute transplant rejection.

Desarrollo de inmunotolerancia en el trasplante hepático

The liver is a privileged organ and has a lower incidence of rejection than other organs. However, immunosuppressive regimens are still required to control the alloreactive T-lymphocyte response after transplantation. These treatments may lead to severe complications, such as infectious diseases, cancers, cardiovascular diseases and chronic renal insufficiency. In clinical transplantation there is increasing evidence that some liver transplant recipients who cease taking immunosuppressive (IS) drugs maintain allograft function, suggesting that tolerance is already present. This strategy is feasible in 25–33% of liver transplant recipients. A series of experimental and clinical observations indicates that liver allografts can even provide “tolerogenic” properties for other organ grafts. In the clinical setting, clinical operational tolerance (COT) is defined as the absence of acute and chronic rejection and graft survival with normal function and histology in an IS-free, fully immunocompetent host, usually as an end result of a successful attempt at IS withdrawal. The exact mechanisms involved in achieving transplant tolerance remain unknown, although animal models suggest a possible role for regulatory T cells (Treg). Recent data have demonstrated an increase in the frequency of CD4+ CD25 high T cells and FoxP3 transcripts during IS withdrawal in operationally tolerant liver transplant recipients. The data obtained from transcriptional profiling of the peripheral blood of IS-free liver transplant recipients suggest that there is a molecular signature of tolerance that could be employed to identify tolerant liver transplant recipients and that innate immune cells are likely to play a major role in the maintenance of COT after liver transplantation.

Suppression of Histone Deacetylase 11 Promotes Expression of IL-10 in Kupffer Cells and Induces Tolerance Following Orthotopic Liver Transplantation in Rats

Suppression of histone deacetylase 11 (HDAC11) can promote IL-10 expression in mouse macrophages RAW264.7 and induce immune tolerance. This study is to further investigate the role of HDAC11 in tolerance induction via Kupffer cells (KCs) following orthotopic liver transplantation (OLT) in rats. KCs isolated from BALB/c mice were divided into pHDAC11, adHDAC11, and pCV group (treated with HADC11-shRNA, adenovirus encoding HDAC11, and control vector, respectively). IL-10 expression was determined after lipopolysaccharide treatment. The expression of MHC-II and co-stimulatory molecules on KCs surface was evaluated by flow cytometry. T cell proliferation was measured by [(3)H]-thymidine incorporation after culturing with aforementioned three groups, treated KCs, respectively. OLT was performed in rats after Ad-HDAC11 and pHDAC11 treatment. Blood samples were collected for biochemical studies, and postoperative survival was examined. IL-10 expression was inhibited and promoted by Ad-HDAC11 and HDAC11-shRNA in KCs, respectively. MHC-II and co-stimulatory molecules on KCs surface as well as T cell proliferation were significantly inhibited and induced in pHDAC11 and Ad-HDAC11 compared with pCV, respectively. Serum IL-2, TNF-α, and IFN-γ levels were significantly lower in pHDAC11 and higher in Ad-HDAC11 compared with pCV, respectively, while IL-4 and IL-10 were the reverse. Postoperative survival, liver function, and histology were different among the three groups. Suppression of HDAC11 can promote IL-10 expression in KCs and induce tolerance following OLT in rats. Consequently, HDAC11 may be a key component of this immune regulation system and a promising target for development of novel drugs of gene therapy for inducing tolerance in clinical liver transplantation.

Elevated HLA-G Expression On Myeloid Dendritic Cells Correlates With Regulatory T Cell Foxp3 Expression In Operationally Tolerant Pediatric Liver Transplant Recipients

Introduction: Human leukocyte antigen (HLA)-G is a non-classical HLA class I molecule expressed as membrane-bound and soluble isoforms. Interaction of HLA-G with its receptor, Ig-like transcript (ILT)4 on professional antigen (Ag)-presenting dendritic cells (DC) down-regulates their T cell costimulatory ability. We examined the expression of HLA-G and ILT4, together with other immune regulatory molecules, on circulating DC subsets in three groups of clinically stable, pediatric liver recipients: operationally tolerant (TOL: 26); prospective immunosuppressive drug weaning (PW: 28) and maintenance immunosuppression (MI: 24), and in healthy controls (HC: 28). Methods: PBMC were stained with a lineage monoclonal antibody (mAb) cocktail, andwith mAbs, directed against blood DC Ag (BDCA)-1 (CD1; monocytoid [m]DC),and BDCA-2 (CD303; plasmacytoid [p]DC), as well as with mAbs against HLA-G and ILT4 (CD85d) or CMRF-44, inducible costimulatory ligand (ICOSL) or glucocorticoid-induced tumor necrosis factor-related protein ligand (GITRL), then analyzed by flow cytometry. Serum HLA-G levels were determined by ELISA. Results: Both circulating mDC and pDC in HC expressed surface HLA-G and ILT4. The incidence of mDC expressing HLA-G (but not ILT4) was significantly higher than that of pDC, although fluorescence intensity (MFI) was lower for HLA-G and ILT4 on mDC, compared with pDC. In TOL patients, mDC but not pDC expressed higher HLA-G (both% positive cells and MFI) than in PW or MI patients or HC. By contrast, ILT4, CMRF-44, GITRL or ICOSL expression on either pDC or mDC did not differ significantly between the groups. In TOL patients, the incidence of regulatory T cells (Treg) and the intensity of Foxp3 expression by Treg were significantly higher than in MI or HC groups. In addition, the intensity of HLA-G expression on circulating mDC correlated significantly with that of Foxp3 in the TOL group. The incidence of patients with circulating HLA-G levels>100pg/ml was greatest in the TOL group. Conclusion: Higher HLA-G expression on circulating mDC,- the predominant DC subset, in TOL pediatric liver transplant recipients compared with MI or HC, suggests a possible role of HLA-G in immune regulation. This may be correlated with enhanced Foxp3 expression by host Treg. The findings also suggest the potential of HLA-G expression on mDC as a biomarker of operational pediatric liver transplant tolerance.