W. Li

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‐β expression and IL‐4 production. Depletion of recipient CD25+CD4+ T cells using anti‐CD25 mAb (250 μg/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‐β 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.

CTLA4 Engagement is Required for Induction of Murine Liver Transplant Spontaneous Tolerance

Liver transplantation in mice is accepted spontaneously in all strain combinations. The mechanisms remain largely undefined. We hypothesize that signaling via the B7‐CTLA4 receptor pathway is required for induction of liver transplant tolerance. Liver transplantation was performed from B10 (H2b) to C3H (H2k) mice. The recipients received anti‐mouse CTLA4 mAb 0.25 mg i.p. every other day post‐operatively. Liver grafts in anti‐CTLA4 mAb treated recipients were acutely rejected. The allo‐specific proliferative responses, anti‐donor CTL and NK cell activities of GIC and SC and the serum levels of IFN‐γ and IL‐2 from anti‐CTLA4 mAb treated recipients were elevated significantly in comparison to the control mice. The frequency of IFN‐γ and IL‐2 producing cells were markedly increased also in the anti‐CTLA4 treated recipients. The immunohistology of liver grafts from anti‐CTLA4 mAb treated mice showed extensively increased lymphocyte infiltration in the portal and general parenchymal areas, and expanded T‐cell area in the spleen, with a reduction in the frequency of apoptotic cells observed by TUNEL staining compared with control mice. Thus CTLA4 signaling is critical for murine liver transplant tolerance induction. CTLA4 blockade promotes donor specific T‐cell activation, cytotoxicity and Th1 polarization; protects alloreactive T cells from apoptotic death and induces liver allograft acute rejection.

Role of the Liver in Peripheral Tolerance: Induction Through Oral Antigen Feeding

Using a murine liver transplant model, we studied the livers role in peripheral tolerance. Livers from BALB/c mice fed with ovalbumin (OVA) at either a low or high dose were transplanted into syngeneic recipients. Non‐fed recipients were controls. Orthotopic liver transplantation (OLTx) was followed by OVA immunization and delayed‐type hypersensitivity (DTH) challenge. The ex vivo adoptive transfer effect of liver nonparenchymal cells (NPCs) or spleen cells (SCs) from OVA‐fed mice was examined. In vitro proliferative assays and cytokine profiles were conducted on NPCs and SCs from transplant recipients.

γδT Cells Are Involved in Liver Transplant Tolerance

The role of nonconventional T cells in innate and adaptive immunity is just emerging; gammadeltaT cells play important roles in anti-tumor and anti-infectious diseases. The involvement of gammadeltaT cells in immunologic responses to hematopoietic cell transplantation remains controversial; divergent results have been reported depending on the murine strains and model systems. Whether gammadeltaT cells are involved in solid organ transplantation is understudied. We have characterized the gammadeltaT cells in mouse livers and spleens to evaluate their contributions to liver transplant tolerance posttransplantation using a murine allogeneic liver transplant model which induces spontaneous T regulatory cell (Treg)-dependent tolerance. Our studies revealed that gammadeltaT cells comprised about 20% of the population of liver nonparenchymal cells (NPCs). In naïve C3H mice they were CD4, CD8, and NK1.1 negative. The percentage of gammadeltaT cells decreased in spontaneously tolerated liver grafts posttransplantation from 20% in naïve C3H livers to <10% in allografts throughout the time course. In contrast, they increased in liver grafts with rejection induced by anti-CTLA4 plus anti-CD25 mAb administration. CD4 and CD8 expression on gammadeltaT cells dramatically increased in the tolerated but not rejected livers posttransplantation to >20% of CD4(+) and 30% of CD8(+). Our results suggested that gammadeltaT cells are involved in allogeneic immune responses. Whether gammadeltaT cells function as the causal or the effector cells in allograft tolerance rejection warrants further investigation.

NKT Cells in the Liver are Important for Peripheral Tolerance Induction

NKT cells are mainly located in the liver in mice. The role of liver inherent NKT cells are still unclear. Method In this study, we employed mouse orthotopic liver transplantation and heterotopic heart transplantation models to critical examine the role of liver NKT cells in liver and peripheral tolerance induction. Results NKT cells were increased in the tolerated liver grafts which expressed higher levels of CD95L and PD-L1, while the CD4+CD25+Foxp3+ regulatory T cells (Treg) were markedly increased in the both liver and spleen at day 7 post transplantation. The heart allograft survival were prolonged significantly in the recipient which accepted donor spleen cells by portal vein (p.v.) injection in contract to the recipient which received the donor spleen cells by tail vein (i.v.) injection. In vitro immunological assay revealed that the number of NKT cells in the liver was increased and Treg were increased in both liver and spleen from the p.v. treated mice significantly. The IL-2 expression was decreased, IL-4 and IL-10 were increased. Further, significantly increased IL-4, IL-10, and IFN-&ggr; production was detected from liver NPCs under GalCer stimulation. Conclusion NKT cells are important to facilitate transplant tolerance induction, appear to play a key role in down regulation of peripheral immune responses and facilitate CD4+CD25+Foxp3+ Treg induction. Nature Science Foundation of China (NSFC) (81170416; 81273264). International cooperation fund of Jilin Science & Technology Committee (2016322).

PD-L1 Signal on Liver Dendritic Cells Is Critical for Foxp3+CD4+CD25+ Treg and Liver Tolerance Induction: 1551

Previous studies have demonstrated that allogeneic liver transplantation induces spontaneous tolerance in mice without requirement for immunosuppression. The underling mechanisms still remain unclear. Our recent studies indicated that Foxp3+CD25+CD4+ regulatory T (Treg) cells play an important role in the induction of liver spontaneous transplant tolerance. How Treg are induced and their functional mechanisms in the regulation of liver transplant tolerance remain undefined. Methods: In this study, we employed mouse spontaneous liver transplantation model, PD-L1-/-, and Flt3L-/mice to critically examine the role of liver dendritic cells (DCs) and the PD-L1 signal in Treg induction and liver transplant tolerance. Results: liver DCs, which expressed a high number of PD-L1 molecules, induced more Foxp3+CD25+CD4+ Treg in vitro in coculture with allogeneic CD4 T cells compared to spleen DCs. After 5-7 days culture at 1:4 ratio of DC:CD4 T cells, approximately 50% of CD4 T cells expressed CD25 and Foxp3 compared to 25% in the spleen DC cocultured group. 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 than in the SCs controls. Moreover, the liver grafts from Flt3L-/and PD-L1-/mice were rejected acutely in the C3H recipients. MST were 6.3±2.3 days and 6.25±1.0 days, respectively, P< 0.001, vs 55±33 in WT controls. Immunohistochemistry staining revealed that Foxp3+ cells were reduced, but IL-2, IL-10, and IFN-γ producing cells were significantly increased in the liver grafts and recipient spleens of Flt3L-/and PD-L1-/donors. Conclusions: liver DCs play a critical role in the induction of Foxp3+CD25+CD4+ Treg, which underpin spontaneous acceptance of MHC-mismatched liver allografts in mice. The function of DCs on Foxp3+CD25+CD4+ Treg induction and expansion appears to depend on the PD-L1 signal. 1552

LIVER DENDRITIC CELLS INDUCES FOXP3+ TREG AND LIVER GRAFT TOLERANCE IN A PD-L1 DEPENDENT MANNER: 1620

W. Li1, R. Bakthavatsalam2, Z. Meng3, J.D. Perkins4, J.D. Reyes4 1Hepato-biliary-pancreatic Surgery, Norman Bethune Medical college of JiLin University, ChangChun/CHINA, 2Transplantation, Dept. Of Surgery, Univ. of Washington, seattle/UNITED STATES OF AMERICA, 3Hepato-biliary-pancreatic Surgery, Norman Bethune Medical School of JiLin University, ChangChun/CHINA, 4Dept. Of Surgery, Div. Of Transplantation, University of Washington, seattle/UNITED STATES OF AMERICA