*Purpose: Constitutive expression of comparatively high levels of DNAX-activating protein of 12KDa (DAP12) by liver myeloid dendritic cells (mDCs) has been implicated in their immune regulatory function. DAP12 knockout (KO) liver mDCs exhibit augmented migratory responses to secondary lymphoid tissue and enhanced ability to elicit alloreactive T cell responses in mice that may contribute to failure of DAP12 KO livers to induce spontaneous transplant tolerance. However, underlying mechanisms are unclear. There is recent evidence that in human and mouse DCs, DAP12 negatively regulates type I interferon (IFN-I) production, that is a high-risk factor for graft loss. IFN-I production can be initiated by retinoic acid-inducible gene I(RIG-I), Toll-like receptors (TLRs), and especially the cyclic GMP-AMP synthase-stimulator of IFN genes (cGAS-STING) pathway, which has been shown to promote liver injury. We postulated that DAP12 may promote liver mDC tolerogenicity by negatively regulating the cGAS-STING-IFN I signaling pathway.

*Methods: Wild-type (WT) and DAP12 KO B6 mouse liver and spleen mDCs (CD11b⁺NK1.1^–CD11c⁺) were freshly-isolated and harvested after 4 hours culture in the absence or presence of 2,3-cGAMP or H-151(STING-specific inhibitor). Phenotypic analysis was performed by flow cytometry to characterize the expression of MHC II, CD80, CD86 and PD-L1. IFN-α and IFN-β mRNA transcripts and cytokine levels in supernatants were quantified using q-PCR and ELISA respectively. The activation of p-STING/STING and its downstream targets p-TBK1/TBK1, p-IRF3/IRF3 were probed by western blot. T cell allostimulatory function of the mDC was assessed by CFSE-MLR.

*Results: DAP12 KO increased p-STING, p-TBK1 and p-IRF3 expression, enhanced MHC II and co-stimulatory molecule expression, but decreased co-inhibitory PD-L1 in the presence or absence of 2,3-cGAMP by both liver and spleen mDC. In addition, IFN-α and IFN-β mRNA levels and secretion in supernatants were augmented. DAP12 KO liver and spleen mDC showed superior ability to stimulate allogeneic T cell proliferation, while H-151-treated DAP12 KO mDCs exhibited reduced p-STING, p-TBK1 and p-IRF3 expression, lower MHC II and co-stimulatory molecule expression, but increased co-inhibitory PD-L1, impaired IFN-α and IFN-β expression and secretion with 2,3-cGAMP exposure in both liver and spleen mDC. The enhanced allogeneic T cell stimulatory ability of DAP12 KO liver and spleen mDC was reversed by STING inhibition via H-151. Notably, the elevation in STING-IFN-I activation in DAP12 KO compared with WT cells was much higher in liver mDC than in spleen mDC.

*Conclusions: DAP12 may promote liver DC tolerogenicity by negatively regulating the STING-IFN I pathway.

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