Graphical Abstract Highlights d TCR stimulation is essential for liver Trm cell formation d Liver Trm cells require IL-15 d Local antigen presentation and inflammation enhance liver Trm cell formation d Newly formed liver Trm cells do not displace existing Trm cell populations SUMMARY Liver tissue-resident memory T (Trm) cells migrate throughout the sinusoids and are capable of protecting against malaria sporozoite challenge. To gain an understanding of liver Trm cell development, we examined various conditions for their formation.Although liver Trm cells were found in naive mice, their presence was dictated by antigen specificity and required IL-15. Liver Trm cells also formed after adoptive transfer of in vitro-activated but not naive CD8 + T cells, indicating that activation was essential but that antigen presentation within the liver was not obligatory. These Trm cells patrolled the liver sinusoids with a half-life of 36 days and occupied a large niche that could be added to sequentially without effect on subsequent Trm cell cohorts. Together, our findings indicate that liver Trm cells form as a normal consequence of CD8 + T cell activation during essentially any infection but that inflammatory and antigenic signals preferentially tailor their development.

Highlights d The Plasmodium ribosomal protein RPL6 is expressed during liver-stage infection d RPL6 can be targeted by specific liver T RM cells for efficient parasite elimination d Prime-and-trap vaccination targeting RPL6 induces effective protection against malaria d RPL6 is highly conserved across global P. falciparum clinical isolates

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...

Hepatitis C virus (HCV) is a significant contributor to the global disease burden, and development of an effective vaccine is required to eliminate HCV infections worldwide. CD4+ and CD8+ T cell immunity correlates with viral clearance in primary HCV infection, and intrahepatic CD8+ tissue-resident memory T (TRM) cells provide lifelong and rapid protection against hepatotropic pathogens. Consequently, we aimed to develop a vaccine to elicit HCV-specific CD4+ and CD8+ T cells, including CD8+ TRM cells, in the liver, given that HCV primarily infects hepatocytes. To achieve this, we vaccinated wild-type BALB/c mice with a highly immunogenic cytolytic DNA vaccine encoding a model HCV (genotype 3a) nonstructural protein (NS5B) and a mutant perforin (pVAX-NS5B-PRF), as well as a recombinant adeno-associated virus (AAV) encoding NS5B (rAAV-NS5B). A novel fluorescent target array (FTA) was used to map immunodominant CD4+ T helper (TH) cell and cytotoxic CD8+ T cell epitopes of NS5B in vivo, which were subsequently used to design a KdNS5B451-459 tetramer and analyze NS5B-specific T cell responses in vaccinated mice in vivo. The data showed that intradermal prime/boost vaccination with pVAX-NS5B-PRF was effective in eliciting TH and cytotoxic CD8+ T cell responses and intrahepatic CD8+ TRM cells, but a single intravenous dose of hepatotropic rAAV-NS5B was significantly more effective. As a T-cell-based vaccine against HCV should ideally result in localized T cell responses in the liver, this study describes primary observations in the context of HCV vaccination that can be used to achieve this goal. IMPORTANCE There are currently at least 71 million individuals with chronic HCV worldwide and almost two million new infections annually. Although the advent of direct-acting antivirals (DAAs) offers highly effective therapy, considerable remaining challenges argue against reliance on DAAs for HCV elimination, including high drug cost, poorly developed health infrastructure, low screening rates, and significant reinfection rates. Accordingly, development of an effective vaccine is crucial to HCV elimination. An HCV vaccine that elicits T cell immunity in the liver will be highly protective for the following reasons: (i) T cell responses against nonstructural proteins of the virus are associated with clearance of primary infection, and (ii) long-lived liver-resident T cells alone can protect against malaria infection of hepatocytes. Thus, in this study we exploit promising vaccination platforms to highlight strategies that can be used to evoke highly functional and long-lived T cell responses in the liver for protection against HCV.

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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.

The env gene products of nine AKR dual-tropic murine leukemia viruses were compared by peptide mapping and were assayed for expression on the cell surface of infected fibroblasts. Seven virus isolates expressed the env gene polyprotein on the cell surface. The env gene products of six of the seven viruses had identical peptide maps. The analysis of structure and expression of env gene products carried out in this study characterizes a subset of dual-tropic murine leukemia viruses shown by others to be thymotropic.

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 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-2Kb-associated peptides recognised by CD8+ T cells from B10.BR (H-2k) mice, 13 of which were also recognised by BALB/c (H-2d) mice. As few as five different tetramers used together were able to identify almost 40% of alloreactive T cells within a polyclonal population. To our knowledge, this represents the first example of such an approach in the context of direct allorecognition.