Summary The liver displays inherent tolerogenicity and liver allografts promote tolerance more readily than other organs. Compared to circulating or secondary lymphoid tissue dendritic cells (DC), liver conventional myeloid (m) DC produce lower IL-12, secrete more IL-10, induce alloAg-specific T cell hyporesponsiveness and prolong allograft survival. Liver DC are refractory to Toll-like receptor (TLR) agonism and nuclear factor (NF)κβ activation,- a critical determinant of DC maturation. Underlying mechanisms may contribute to their regulatory function and the inherent tolerogenicity of hepatic allografts. Importantly, our new data show that selective deletion of donor mDC prevents ‘spontaneous’ liver transplant tolerance in mice. Mechanisms underlying unresponsiveness to TLR agonism in DC in general, and in liver DC, in particular, are poorly defined. We have identified the signaling adaptor DNAX-activating protein of 12KDa (DAP12), that can mediate inhibition of TLR activation and that is upregulated in liver mDC, as a critical novel regulator of liver DC function and liver transplant tolerance. Consequently, we hypothesize that negative regulators of TLR signaling, in particular IL-1R-associated kinase (IRAK)-M (that we show is upregulated with DAP12 in liver mDC) confer resistance to maturation and tolerogenic capacity. We further postulate that anti- inflammatory IL-10 and TGFβ, produced in the liver, potentiate liver DC tolerogenicity through molecular ‘crosstalk’ between their respective signaling pathways (Smad for TGFβ and STAT3 for IL-10) and the TLR signaling pathway. The tolerogenic function attributed to liver mDC may contribute to induction/maintenance of liver transplant tolerance in the face of continuous TLR agonism and other pro-inflammatory stimuli. Our studies will use innovative approaches and cutting edge technology, including intravital imaging and generation of novel, chimeric donor mouse livers, to provide new mechanistic insight into the molecular regulation of liver DC function and its impact on alloreactive T cell responses and liver transplant tolerance. We will also evaluate the therapeutic potential of negative regulators of TLR signaling/liver DC maturation for restoration/promotion of transplant tolerance. We propose the following aims: AIM 1: To elucidate the role of DAP12 and inducible regulation of TLR signaling in the development of mouse liver mDC tolerogenicity, and the influence of anti- and pro-inflammatory factors; AIM 2: To establish using innovative in vivo approaches, the roles of donor mDC, TLR4, and DAP12-IRAK-M expression by liver mDC in control of T cell responses and tolerance to liver allografts.