The Role Of PD-L1/PD-1Co-inhibitory Pathway On Liver Tolerance Induction In Mice

Liver transplantation is a marvelous and effective treatment for end-stage liverdiseases；however，the irony is that many patients die of the side effects of globalimmunosuppression. Induction of donor specific transplant tolerance is still a majorgoal for many transplant immunologists and surgeons.The liver is an immune-privileged organ，the transplantation of which favors theinduction of peripheral tolerance. Liver allografts in mice are accepted spontaneouslywithout the requirement of immunosuppression. Furthemore，liver allograft canprotect the subsequent organs of the same donor from acute rejection. Themechanisms underlying liver inherent tolerogenic properties have not been clearlyelucidated. Our recent preliminary studies have demonstrated that the CD4+CD25+Foxp3+T regulatory cells (Treg) play an important role in the induction ofspontaneous transplant tolerance. How Treg cells are induced and how they interactamong Treg and other immune cells remain undefined.Hypothesis:Liver DC induces CD4+CD25+Foxp3+Treg through PD-L1dependentmechanism，and by this mechanism promotes liver transplant tolerance.Aims：The purpose of this study was to elucidate the role of liver DC and theirco-inhibitory molecule PD-L1on Treg induction and liver transplant toleranceinduction in vivoand in vitro by employing orthotopic liver tansplantation andheterotopic heart transplantation models，and PD-L1-/-and Flt3-/-transgeneic mice. Materials and methods:Male C57BL/6(B6；H2b)，C3H/HeJ (C3H；H2k)，B6FL-/-and B6PD-L1-/-micewere employed. Mouse orthotopic liver transplantation was completeted by suture andcuff combination techniques described by Dr. Wei Li. The hepatic artery was notreconstructed. Heterotopic heart transplantation was performed through the end toside anoastomosis of heart aorta and pulmonary artery to abdomal aorta and inferiorvena cava. The microenvironment of liver transplantation in vivo was mimicked for invitro experiments. The donor DC and recipient CD4+T cells were co-cultured in thepresent of IL-2and TGF-β. The purification of CD4T cells and DCs were performedusing negative and positive selection with immunomagnetic beads，respectively.FlowCytometry was used for cell analysis. TUNEL staining was used to detect theapoptosis. The Treg function which was generated in vitro was examined by adoptivetransfer to the heart allograft recipients. In vivo，the liver transplantation wasperformed from PD-L1-/-，or Flt3L-/-mice to C3H recipients，to critically examine therole of liver DC and the PD-L1signal in Treg induction，T cell apoptotic activities，cytokine production and liver graft survivals. Graft survival and immunologicalmonitoring of recipients were evaluated statistically by the Kruskal-Wallis H test andStudent t test for cell numbers，respectively.Results:Our in vitro experiments demonstrated that liver DC was more potent inducer ofCD4+CD25+Foxp3+Treg. In the present of IL-2and TGF-β，50%of CD4+T cellsbecome CD25+Foxp3+in the liver DC co-cultured group compared to25%in thespleen DC co-cultured group（P <0.05）. The DC which expressed high PD-L1molecules，induced more CD4+CD25+Foxp3+Treg in vitro upon coculture withallogeneic CD4T cells compared with spleen DC. The DC from PD-L1-deficientmice failed to expand CD4+CD25+Foxp3+Treg in vitro. Adoptive transfer of CD4+CD25+Foxp3+Treg expanded from liver DC prolonged heart allograft survivalsignificantly greater than spleen DC controls. The heart allograft survivial was25±10 days in the liver DC group vs13.8±5.9days in the spleen DC group (P <0.05).Moreover，liver DC was also more potent to induce activated CD4+T cell apoptosis invitro. The apoptosis ratio of activated CD4+T cells92%in the liver DC group vs49%in the spleen DC group (P <0.01).In vivo experiments revealed that the liver grafts from Flt3L-/-and PD-L1-/-micewere rejected acutely in C3H recipients. The mean graft survival times were6.3±2.3days and6.25±1.0days，respectively，vs55±33days in WT control，P <0.01.Immunohistochemistry revealed significantly reduced Foxp3+cells and increasedIL-2，IL-10，and IFN-producing cells in the liver grafts and recipient spleens ofFlt3L-/-and PD-L1-/-donors.Discussion and Conclusion:CD4+CD25+Foxp3+Treg play an important role in the regulation ofimmunologically related diseases and also the induction of organ transplant tolerance.Induction and expansion of tissue-specific Treg are currently receiving more attentionby immunologists and clinicians. Our study demonstrated that liver DC play a criticalrole in CD4+CD25+Foxp3+Treg induction in vivo and in vitro. In comparision withspleen DC，liver DC was able to expand more CD4+CD25+Foxp3+Treg in vitro andsignificantly prolonged heart allograft survival upon adoptive transfer to the heartallograft recipients. This observation suggests an inherent tolerogeneic property ofliver DC. Our findings have also demonstrated that Flt3-/-and PD-L1-/-liver donorsinduced acute allograft rejection that were associated with reduced Foxp3+Treg aswell as increased IL-10，IL-2，and IFN-production in liver grafts and host spleens，suggesting that liver DC was potent inducers of CD4+CD25+Foxp3+Treg in vivo.The mechanism of liver DC-mediated Treg induction and immune suppression maydepend，at least in part，on the PD-L1signal. In contrast，in the absence of liver DC，other antigen-presenting cells in the liver，such as Kupffer cells，liver sinusoidalendothelial cells，and stellate cells，may compensate to display the role of antigenpresentation，vigorously activating alloreactive T cells，which produce IL-2，IFN-， and IL-10，which mediate acute liver graft rejection.In conclusion，liver DC play a critical role in the induction of CD4+CD25+Foxp3+Treg and spontaneous liver graft acceptance of MHC mismatched liverallografts in mice. The effects of DC on CD4+CD25+Foxp3+Treg induction andexpansion appear to depend on the PD-1/PD-L1signal.