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In recent years, various intervention strategies have reduced malaria morbidity and mortality, but further improvements probably depend upon development of a broadly protective vaccine. To better understand immune requirement for protection, we examined liver-stage immunity after vaccination with irradiated sporozoites, an effective though logistically difficult vaccine. We identified a population of memory CD8+ T cells that expressed the gene signature of tissue-resident memory T (Trm) cells and remained permanently within the liver, where they patrolled the sinusoids. Exploring the requirements for liver Trm cell induction, we showed that by combining dendritic cell-targeted priming with liver inflammation and antigen recognition on hepatocytes, high frequencies of Trm cells could be induced and these cells were essential for protection against malaria sporozoite challenge. Our study highlights the immune potential of liver Trm cells and provides approaches for their selective transfer, expansion, or depletion, which may be harnessed to control liver infections or autoimmunity.

Background & Aims Acute hepatitis is often mediated by cytotoxic T lymphocytes (CTLs); however, the intrinsic parameters that limit CTL-mediated liver injury are not well understood. Methods To investigate whether acute liver damage is limited by molecules that decrease the lifespan or effector function of CTLs, we used a well-characterized transgenic (Tg) mouse model in which acute liver damage develops upon transfer of T cell receptor (TCR) Tg CD8 T cells. Recipient Tg mice received donor TCR Tg T cells deficient for either the pro-apoptotic molecule Bim, which regulates CTL survival, or suppressor of cytokine signaling-1 (SOCS-1), which controls expression of common gamma chain cytokines; the effects of anti-PD-L1 neutralizing antibodies were also assessed. Results Use of Bim-deficient donor T cells and/or PD-L1 blockade increased the number of intrahepatic T cells without affecting the degree and kinetic of acute hepatitis. In contrast, SOCS-1-deficient T cells induced a heightened, prolonged acute hepatitis caused by their enhanced cytotoxic function and increased expansion. Although they inflicted more severe acute liver damage, SOCS-1-deficient T cells never precipitated chronic hepatitis and became exhausted. Conclusions The degree of acute hepatitis is regulated by the function of CD8 T cells, but is not affected by changes in CTL lifespan. Although manipulation of the examined parameters affected acute hepatitis, persistent hepatitis did not ensue, indicating that, in the presence of high intrahepatic antigen load, changes in these factors in isolation were not sufficient to prevent T cell exhaustion and mediate progression to chronic hepatitis.

Liver-resident memory CD8+ T (TRM) cells remain in and constantly patrol the liver to elicit rapid immunity upon antigen encounter and can mediate efficient protection against liver-stage Plasmodium infection. This finding has prompted the development of immunization strategies where T cells are activated in the spleen and then trapped in the liver to form TRM cells. Here, we identify PbRPL6120-127, a H2-Kb-restricted epitope from the putative 60S ribosomal protein L6 (RPL6) of Plasmodium berghei ANKA, as an optimal antigen for endogenous liver TRM cell generation and protection against malaria. A single dose vaccination targeting RPL6 provided effective and prolonged sterilizing immunity against high dose sporozoite challenges. Expressed throughout the parasite life cycle, across Plasmodium species, and highly conserved, RPL6 exhibits strong translation potential as a vaccine candidate. This is further advocated by the identification of a broadly conserved, immunogenic HLA-A∗02:01-restricted epitope in P. falciparum RPL6.

The liver is known for its tolerogenic properties. This unique characteristic is associated with persistent infection of the liver by the hepatitis B and C viruses. Improper activation of cellular adaptive immune responses within the liver and immune exhaustion over time both contribute to ineffective cytotoxic T cell responses to liver-expressed antigens in animal models, and likely play a role in incomplete clearance of chronic hepatitis virus infections in humans. However, under some conditions, functional immune responses can be elicited against hepatic antigens, resulting in control of hepatotropic infections. In order to develop improved therapeutics in immune-mediated chronic liver diseases, including viral hepatitis, it is essential to understand how intrahepatic immunity is regulated. This review focuses on CD8 T cell immunity directed towards foreign antigens expressed in the liver, and explores how the liver environment dictates the outcome of intrahepatic CD8 T cell responses. Potential strategies to rescue unresponsive CD8 T cells in the liver are also discussed.

Spontaneous acceptance of organ allografts can identify novel mechanisms of drug-free transplantation tolerance. Spontaneous acceptance occurs in both mouse kidney transplants and rat liver transplants however the early immune processes of mouse kidney acceptance have not been studied. Acceptance of C57BL/6 strain kidney allografts in fully MHC-incompatible B10.BR recipients was compared with rejection (REJ) of heart allografts in the same strain combination. Graft infiltrate and antibody deposition were examined by immunohistochemical staining. Expression of mRNA was measured by quantitative real-time PCR. Apoptosis was examined by TUNEL staining. The majority of kidney allografts were accepted long-term and induced tolerance (TOL) of donor-strain skin grafts, showing that acceptance was not due to immune ignorance. There was an extensive infiltrate of T cells in the TOL kidney that exceeded the level in REJ hearts but subsequently declined. The main differences were deposition of IgG2a antibody in REJ that was absent in TOL, more B cells infiltrating TOL kidneys and a progressive increase in the ratio of CD8 : CD4 cells during rejection. There was also significantly greater Foxp3 mRNA expression in TOL. Kidneys from RAG−/− donors were accepted, showing that donor lymphocytes were not necessary for acceptance. Neutralising antibodies to TGF-β administered from day 0 to day 6 did not prevent TOL. On the basis of cytokine expression and apoptosis there was no evidence for immune deviation or deletion as mechanisms of acceptance. In accord with the findings of spontaneous acceptance of liver allografts in rats, the main difference between mouse kidney TOL and heart REJ was in the B cell compartment. The major difference to rat liver allograft acceptance was that apoptosis of infiltrate did not appear to play a role. Instead, increased Foxp3 expression in TOL kidneys implies that regulatory T cells might be important.

Hepatocytes, the predominant cell type in the liver, are the main cell infected by the hepatitis C virus (HCV) and represent important targets for immune therapy. Although early studies suggested that this parenchymal cell expresses low levels of class I MHC molecules, hepatocytes are emerging as important players in intrahepatic immune responses. Not only do they express high levels of molecules important in antigen presentation, but their expression of these molecules in vivo is also polarized towards the lumen of the sinusoids, thus maximising the efficiency of T cell activation. Electron micrographs indicate that interactions between T cells and hepatocytes occur in vivo via fenestrations in the sinusoidal endothelial layer. In this article, we will review the data showing that hepatocytes function as antigen presenting cells in vivo, and explore the fate of T cells activated by this cell type. We propose that primary activation of naïve CD8+ T cells by hepatocytes is a critical event occurring during the very early stages of a HCV infection, that contributes to progression to viral persistence via the removal of virus-specific T cells from the T cell repertoire.

Background & Aims: Chronic infections by hepatotropic viruses such as hepatitis B and C are generally associated with an impaired CD8 T-cell immune response that is unable to clear the virus. The liver is increasingly recognized as an alternative site in which primary activation of CD8 T cells takes place, a property that might explain its role in inducing tolerance. However, the molecular mechanism by which intrahepatically activated T cells become tolerant is unknown. Here, we investigated the phenotype and fate of naive CD8 T cells activated by hepatocytes in vivo. Methods: Transgenic mouse models in which the antigen is expressed in lymph nodes and/or in the liver were adoptively transferred with naive CD8 T cells specific for the hepatic antigen. Results: Liver-activated CD8 T cells displayed poor effector functions and a unique CD25low CD54low phenotype. This phenotype was associated with increased expression of the proapoptotic protein Bim and caspase-3, demonstrating that these cells are programmed to die following intrahepatic activation. Importantly, we show that T cells deficient for Bim survived following intrahepatic activation. Conclusions: This study identifies Bim for the first time as a critical initiator of T-cell death in the liver. Thus, strategies inhibiting the up-regulation of this molecule could potentially be used to rescue CD8 T cells, clear the virus, and reverse the outcome of viral chronic infections affecting the liver.

an increased risk to develop acute myeloid leukemia (AML). While de novo MDS is predominantly found in elderly patients, childhood MDS is rather rare and often associated with genetic disorders and inherited bone marrow failure syndromes. Introduction: Recently, germline mutations in the hematopoietic transcription factor GATA2 were identified in familial cases of MDS. Epigenetic alterations represent a major pathogenetic mechanism in MDS and have been extensively researched in adults, while genomewide studies on deregulated epigenetic processes in childhood MDS are still lacking. Purpose: The goal of the presented study was to analyze aberrant DNAmethylation in familiar childhoodMDSby genome-widemethylation profiling. Materials and Methods: Therefore we obtained DNA methylation profiles across the genome in a cohort of childhood MDS samples as well as monocytes from healthy donors (control) using methylCpG-immunoprecipitation combined with next-generation sequencing. The MDS cohort consisted of 10 patients (5 female, 5 male; median age at diagnosis: 13.7 years; range 6.1-19) carrying different mutations in GATA2 (except for two siblings with an identitical mutation). Results: Our analysis showed that these 10 MDS patients exhibit methylation profiles distinct from healthy controls. Based on the degree of methylation, MDS patients could be clustered into subgroups with distinctive patterns. The subgroup with the highest degree of methylation was exclusively composed of patients with advanced morphological subtype (RAEB or RAEB-t) at diagnosis and aneuploidic karyotypes: monosomy 7 alone or monosomy 7 and trisomy 8. In contrast, most patients with DNA methylation patterns resembling normal control patterns were diagnosed with the less advanced refractory cytopenia subtype. Computational analyses of epigenetic and sequence features of aberrantly methylated regions demonstrated their frequent association with the PcG-mark H3K27me3 in hematopoietic stem cells as well as a distinct composition of transcription factor binding motifs. Conclusions: We present the first genome-wide analyses of aberrant DNA methylation in familiar childhood MDS sharing predisposing mutations in GATA2. Our data suggest that DNA methylation changes might be associated with morphologic subtype and disease progression and warrant further studies in larger cohorts.

E. Papaemmanuil1, M. Gerstung1, L. Malcovati2, S. Tauro3, G. Gundem1, P. Van-Loo1, A. Pellagati4, M. Groves3, N. Cross5, C. Gambacorti-Passerini6, A. Green7, J. Boultwood4, P. Vyas8, E. Hellstrom-Lindberg9, D. Bowen10, M. Cazzola2, M.R. Stratton1, P.J. Campbell1. 1Cancer Genome Project, Wellcome Trust Sanger Institute, Cambridge, United Kingdom; 2Haematology Oncology, Fondazione IRCCS Policlinico San Matteo & University of Pavia, Pavia, Italy; 3Haematology, Ninewells Hospital, Dundee, United Kingdom; 4LRF Molecular Haematology Unit, Nuffield Department of Clinical Laboratory Sciences, Oxford, United Kingdom; 5Wessex Regional Genetics Laboratory, University of Southampton, Southampton, United Kingdom; 6Health sciences, Milano Biccoca, Milano, Italy; 7Haematology, University of Cambridge, Cambridge, United Kingdom; 8Molecular Heamatology Unit and Dept of Haematology, Weatherall Institute of Molecular Medicine and John Radcliffe Hospita, Oxford, United Kingdom; 9Medicine Hematology, Karolinska Institutet, Stockholm, Sweden; 10Haematology, St. James’s Institute of Oncology, Leeds, United Kingdom

Introduction: Although several staging systems exist for HCC, the most widely accepted system is the Barcelona Clinic Liver Cancer (BCLC) staging system. All patients with vascular invasion and/or distant metastases are categorized as BCLC C with similar with prognosis and treatment. The aim of this study is to compare the clinical presentation and prognosis of HCC patients who present with BCLC C. Methods: We conducted a retrospective analysis of a prospective database of patients diagnosed with HCC between January 2005 and December 2011. All patients who presented with extrahepatic metastasis (EHM) or vascular invasion (VI), consisting of either a portal venous or hepatic venous thrombus, at presentation were included for analysis. Patients with either a Child Pugh score C or ECOG performance . 2 were excluded from analysis. Demographics, tumor characteristics, treatment regimens, and survival were compared between the following groups of patients: patients with both VI and EHM (group C1), patients with VI but without EHM (group C2), and patients with EHM but without VI (group C3). Differences between groups were determined with oneway ANOVA and Chi-squared tests. Survival curves were generated using Kaplan-Meier plots.Results: Of the 448 patients diagnosed with HCC, 130 patients presented at BCLC C stage, 45 patients in group C1, 54 patients in group C2, and 31 patients in group C3. There was no difference in age, gender, race/ethnicity, presence of symptoms, or etiology of cirrhosis between the three groups of patients. Patients with both EHM and VI (Group C1) had a significantly higher AFP than the other groups (mean, 93491+/164819,19488+/-49375, and 13948+/-5248, p,0.001). These patients also had significantly worse survival than those with VI or EHM alone (2.3 mths vs. 5.9 mths, p ,0.001). Patients with VI alone (group C2) and those with EHM alone (group C3) had similar survival (5.7 vs. 6.1 mo, p= 0.62). On multivariate analysis treatment, AFP , 400 ng/ml, absence of both EHM and VI, and lack of symptoms on presentation were associated with prolonged overall survival. Conclusion: Patients with BCLC stage C tumors comprise a heterogeneous group of patients with different prognoses. Patients who present with both VI and EHM have significantly worse prognosis than those who present with VI or EHM alone.

activity predominantly in higher-risk MDS patients, with CR rates approaching 50%, and overall response rates of two-thirds. Adverse event profiles appear similar. However, the only comparative phase 2 study demonstrated similar activity of azacitidine + entinostat to azacitidine alone, with trilineage response rates of 24% and 31%, respectively. Subpopulations of patients defined by disease biology who appear to have better responses to combinations are emerging. Advanced phase studies comparing combination strategies to standard single agents are being developed or are actively accruing patients. Dosing of combination drugs and schedules are being modified in an effort to enhance activity. Conclusion: Combination drug strategies represent the next frontier in MDS therapeutics, though it has yet to be demonstrated prospectively their superiority to established single agent approaches.