Aim: The precise mechanisms of liver transplant tolerance are yet to be elucidated. Non-conventional plasmacytoid dendritic cells (pDCs), important mediators of innate immunity, are also thought to promote immune regulation, including oral tolerance, and tolerance to heart and renal allografts in animal models. pDCs are comparatively numerous in mouse liver compared with secondary lymphoid tissue. However, their role in liver transplantation remains unclear. Our Aim was to assess the influence of donor liver pDCs on the induction of liver transplant tolerance and underlying mechanisms in a fully MHC-mismatched mouse model.
Methods: We determined the phenotype and function of pDCs and T cells by qRT-PCR, flow cytometric analysis and CFSE-MLR, and performed orthotopic liver transplantation from B6 (H2b) donors to C3H (H2k) recipients (1) without immunosuppressive therapy.
Results: Liver pDCs expressed higher levels of the transmembrane adaptor DNAX-activating protein of 12kDa, its co-receptor triggering receptor expressed on myeloid cells 2, IL-10, and a higher programed death ligand 1 (PD-L1)/CD86 ratio, both in the steady-state and following TLR9 stimulation compared with spleen pDCs. Moreover, liver pDCs were inferior stimulators of allogeneic T cell proliferation compared with spleen pDCs. Additionally, liver pDCs markedly suppressed alloantigen- and to a lesser extent, αCD3/CD28-induced T cell proliferation. Mice given pDC-depleted (with anti-PDCA1) allogeneic livers showed significantly poorer graft survival (median survival time ; MST: 25 days) compared to those given untreated allogeneic or pDC-depleted syngeneic livers (MST  >100 days (p<0.05). Following transplantation, pDC-depleted allografts expressed significantly higher granzyme B and perforin levels, and lower co-inhibitory PD-L1 levels compared with untreated allografts. In addition, the number of CD4+CD25+FoxP3+ Treg in grafts and mesenteric lymph nodes of mice given pDC-depleted allogeneic livers was reduced significantly compared with controls. Furthermore, CD8+T cells infiltrating the grafts and in spleen and lymph nodes of the untreated donor group exhibited an exhausted phenotype (PD-1+, TIM-3+), compared with the pDC-depleted donor group. Finally, to investigate the role of PD-L1 in regulation of graft-infiltrating T cell exhaustion and numbers of Treg, we neutralized PD-L1 in recipients of WT B6 liver allografts. Four days after injection, significantly lower levels of exhausted CD8+T cells were observed in the graft and lymphoid tissues of anti-PD-L1-treated recipients. Blocking the PD1-PD-L1 pathway also significantly reduced the incidence of CD4+CD25+FoxP3+ Treg in the graft.
Conclusions: These novel observations link for the first time, the immunoregulatory function of donor liver interstitial pDCs, PD-L1 expression, host alloreactive T cell exhaustion and the induction of mouse liver.