Hepatic immunobiology is paradoxical: although the liver possesses unusual tolerogenic properties, it is also the site of effective immune responses against multiple pathogens and subject to immune-mediated pathology. The mechanisms underlying this dichotomy remain unclear. Following previous work demonstrating that the liver may act as a site of primary T cell activation, we demonstrate here that the balance between immunity and tolerance in this organ is established by competition for primary activation of CD8 + T cells between the liver and secondary lymphoid tissues, with the immune outcome determined by the initial site of activation. Using a transgenic mouse model in which antigen is expressed within both liver and lymph nodes, we show that while naive CD8 + T cells activated within the lymph nodes were capable of mediating hepatitis, cells undergoing primary activation within the liver exhibited defective cytotoxic function and shortened half-life and did not mediate hepatocellular injury. The implications of these novel findings may pertain not only to the normal maintenance of peripheral tolerance, but also to hepatic allograft tolerance and the immunopathogenesis of chronic viral hepatitis.

While direct allorecognition underpins both solid organ allograft rejection and tolerance induction, the specific molecular targets of most directly-alloreactive CD8 + T cells have not been defined. In this study, we used a combination of genetically-engineered MHC class I (MHC I) constructs, mice with a hepatocyte-specific mutation in the class I antigen-presentation pathway and immunopeptidomic analysis to provide definitive evidence for the contribution of the peptide cargo of allogeneic MHC I molecules to transplant tolerance induction. We established a systematic approach for the discovery of directly-recognised pMHC epitopes, and identified 17 strongly immunogenic H-2K b -associated peptides recognised by CD8 + T cells from B10.BR (H-2 k ) mice, 13 of which were also recognised by BALB/c (H-2 d ) mice. As few as five different tetramers used together were able to identify a high proportion of alloreactive T cells within a polyclonal population, suggesting that there are immunodominant allogeneic MHC-peptide complexes that can account for a large component of the alloresponse. 5 Results Single chain trimer constructs exclude presentation of endogenous peptides.In preceding studies(1, 3), we have used AAV vectors encoding the donor MHC I HC. Within transduced hepatocytes, allogeneic HC associates with native b2m and the resulting heterodimers are loaded with a repertoire of endogenous peptides (Figure 1A). To express allogeneic MHC I at high levels on recipient hepatocytes while excluding the presentation of naturally processed peptides, we engineered SCT constructs, each encoding the HC of H-2K b , b2m and a single, defined H-2K b -restricted peptide [SIINFEKL (SIIN) or KIITYRNL (KIIT), Figure 1A], and packaged them in hepatocyte-specific AAV2/8 vectors.Sequences are shown in Supplementary Figure 1. Transgene expression in hepatocytes was close to maximal by d7 following intravenous (iv) inoculation, and persisted through to at least d100, no significant increases in serum aspartate aminotransferase (AST) or alanine aminotransferase (ALT) levels were observed, and minimal cellular infiltration was detected by histology (Supplementary Figure 2). SCT molecules were expressed on transduced hepatocytes at equivalent levels to the heterotrimer formed by transgenic H-2K b HC with native b2m and peptide (Figure 1B). To demonstrate exclusion of naturally processed peptides, we co-transduced B10.BR (H-2 k ) hepatocytes with AAV vectors encoding full-length chicken ovalbumin (OVA) and either HC-K b or SCT-K b -KIIT, and stained them with a monoclonal antibody, 25D-1.16, which is specific for the OVA peptide SIINFEKL complexed with K b . K b -SIINFEKL was only detected at the surface of cells co-transduced with HC-K b and not those expressing SCT-K b -KIIT (Figure 1C-D). We extended this analysis to the broader endogenous peptide repertoire of K b -transduced hepatocytes using immunoaffinity purification with the H-2K b -specific antibody K9-178, followed by reverse phase high performance liquid chromatography (RP-HPLC...

Portal tracts are key intrahepatic structures where leukocytes accumulate during immune responses. They contain the blood inflow, which includes portal blood from the gut, and lymphatic and biliary outflow of the liver, and as such represent a key interface for potential pathogen entry to the liver. Myeloid cells residing in the interstitium of the portal tract might play an important role in the surveillance or prevention of pathogen dissemination; however, the exact composition and localization of this population has not been explored fully. Our in-depth characterization of portal tract myeloid cells revealed that in addition to T lymphocytes, portal tracts contain a heterogeneous population of MHCII high myeloid cells with potential antigen presenting cell (APC) function. These include a previously unreported subset of CSF1R-dependent CX3CR1 + macrophages that phenotypically and morphologically resemble liver capsular macrophages, as well as the two main dendritic cell subsets (cDC1 and cDC2). These cells are not randomly distributed, but each subset forms interconnected networks intertwined with specific components of the portal tract. The CX3CR1 + cells were preferentially detected along the outer border of the portal tracts, and also in the portal interstitium adjacent to the portal vein, bile duct, lymphatic vessels and hepatic artery. cDC1s abounded along the lymphatic vessels, while cDC2s mostly surrounded the biliary tree. The specific distributions of these discrete subsets predict that they may serve distinct functions in this compartment. Overall, our findings suggest that portal tracts and their embedded cellular networks of myeloid cells form a distinctive lymphoid compartment in the liver that has the potential to orchestrate immune responses in this organ.

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