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Background & Aims Autoimmune hepatitis (AIH) is an immune-mediated disease with no curative treatment. Regulatory T cell (Treg) therapy is potentially curative in AIH given the critical role of Tregs in preventing autoimmunity. To work effectively, adoptively transferred Tregs must migrate to and survive within the inflamed liver. We conducted a proof-of-concept study aiming to assess the safety and liver-homing properties of good manufacturing practice (GMP)-grade autologous Tregs in patients with AIH. Methods Autologous polyclonal GMP-grade Tregs were isolated using leukapheresis and CliniMACS, labelled with indium tropolonate and re-infused intravenously to 4 patients with AIH. GMP-Treg homing to the liver was investigated with longitudinal gamma camera and SPECT-CT scanning. GMP-Treg immunophenotype, function and immunometabolic state were assessed during the study. Results We observed that the isolated Tregs were suppressive and expressed CXCR3, a chemokine receptor involved in recruitment into the inflamed liver, as well as Treg functional markers CD39, CTLA-4 and the transcription factor Foxp3. Serial gamma camera and SPECT-CT imaging demonstrated that 22–44% of infused Tregs homed to and were retained in the livers of patients with autoimmune hepatitis for up to 72 h. The infused cells did not localise to any off-target organs other than the spleen and bone marrow. GMP-Tregs were metabolically competent and there were no infusion reactions or high-grade adverse effects after Treg infusion. Conclusion Our novel findings suggest that the liver is a good target organ for Treg cellular therapy, supporting the development of clinical trials to test efficacy in autoimmune hepatitis and other autoimmune liver diseases. Lay summary Autoimmune liver diseases occur when the body’s immune cells target their own liver cells. Regulatory T cells (Tregs) prevent autoimmunity, thus they are a potential therapy for autoimmune liver diseases. In patients with autoimmune hepatitis, Treg infusion is safe, with nearly a quarter of infused Tregs homing to the liver and suppressing tissue-damaging effector T cells. Thus, Tregs are a potentially curative immune cell therapy for early autoimmune liver diseases., Graphical abstract Unlabelled Image, Highlights • Tregs from patients with autoimmune hepatitis are suppressive, possess functional markers CD39 and CTLA-4, and express CXCR3. • Treg infusion in autoimmune liver disease is safe without any side effects. • 22-44% of infused Tregs home to and were retained in the livers of patients with autoimmune hepatitis for up to 72 hours. • GMP-Tregs from patients with autoimmune hepatitis were metabolically competent.

Background Miscarriage, defined as the spontaneous loss of a pregnancy before 24 weeks' gestation, is common with approximately 25% of women experiencing a miscarriage in their lifetime. An estimated 15% of pregnancies end in miscarriage. Miscarriage can lead to serious morbidity, including haemorrhage, infection, and even death, particularly in settings without adequate healthcare provision. Early miscarriages occur during the first 14 weeks of pregnancy, and can be managed expectantly, medically or surgically. However, there is uncertainty about the relative effectiveness and risks of each option. Objectives To estimate the relative effectiveness and safety profiles for the different management methods for early miscarriage, and to provide rankings of the available methods according to their effectiveness, safety, and side-effect profile using a network meta-analysis. Search methods We searched the Cochrane Pregnancy and Childbirth's Trials Register (9 February 2021), ClinicalTrials.gov and the WHO International Clinical Trials Registry Platform (ICTRP) (12 February 2021), and reference lists of retrieved studies. Selection criteria We included all randomised controlled trials assessing the effectiveness or safety of methods for miscarriage management. Early miscarriage was defined as less than or equal to 14 weeks of gestation, and included missed and incomplete miscarriage. Management of late miscarriages after 14 weeks of gestation (often referred to as intrauterine fetal deaths) was not eligible for inclusion in the review. Cluster- and quasi-randomised trials were eligible for inclusion. Randomised trials published only as abstracts were eligible if sufficient information could be retrieved. We excluded non-randomised trials. Data collection and analysis At least three review authors independently assessed the trials for inclusion and risk of bias, extracted data and checked them for accuracy. We estimated the relative effects and rankings for the primary outcomes of complete miscarriage and composite outcome of death or serious complications. The certainty of evidence was assessed using GRADE. Relative effects for the primary outcomes are reported subgrouped by the type of miscarriage (incomplete and missed miscarriage). We also performed pairwise meta-analyses and network meta-analysis to determine the relative effects and rankings of all available methods. Main results Our network meta-analysis included 78 randomised trials involving 17,795 women from 37 countries. Most trials (71/78) were conducted in hospital settings and included women with missed or incomplete miscarriage. Across 158 trial arms, the following methods were used: 51 trial arms (33%) used misoprostol; 50 (32%) used suction aspiration; 26 (16%) used expectant management or placebo; 17 (11%) used dilatation and curettage; 11 (6%) used mifepristone plus misoprostol; and three (2%) used suction aspiration plus cervical preparation. Of these 78 studies, 71 (90%) contributed data in a usable form for meta-analysis. Complete miscarriage Based on the relative effects from the network meta-analysis of 59 trials (12,591 women), we found that five methods may be more effective than expectant management or placebo for achieving a complete miscarriage: · suction aspiration after cervical preparation (risk ratio (RR) 2.12, 95% confidence interval (CI) 1.41 to 3.20, low-certainty evidence), · dilatation and curettage (RR 1.49, 95% CI 1.26 to 1.75, low-certainty evidence), · suction aspiration (RR 1.44, 95% CI 1.29 to 1.62, low-certainty evidence), · mifepristone plus misoprostol (RR 1.42, 95% CI 1.22 to 1.66, moderate-certainty evidence), · misoprostol (RR 1.30, 95% CI 1.16 to 1.46, low-certainty evidence). The highest ranked surgical method was suction aspiration after cervical preparation. The highest ranked non-surgical treatment was mifepristone plus misoprostol. All surgical methods were ranked higher than medical methods, which in turn ranked above expectant management or placebo. Composite outcome of death and serious complications Based on the relative effects from the network meta-analysis of 35 trials (8161 women), we found that four methods with available data were compatible with a wide range of treatment effects compared with expectant management or placebo: · dilatation and curettage (RR 0.43, 95% CI 0.17 to 1.06, low-certainty evidence), · suction aspiration (RR 0.55, 95% CI 0.23 to 1.32, low-certainty evidence), · misoprostol (RR 0.50, 95% CI 0.22 to 1.15, low-certainty evidence), · mifepristone plus misoprostol (RR 0.76, 95% CI 0.31 to 1.84, low-certainty evidence). Importantly, no deaths were reported in these studies, thus this composite outcome was entirely composed of serious complications, including blood transfusions, uterine perforations, hysterectomies, and intensive care unit admissions. Expectant management and placebo ranked the lowest when compared with alternative treatment interventions. Subgroup analyses by type of miscarriage (missed or incomplete) agreed with the overall analysis in that surgical methods were the most effective treatment, followed by medical methods and then expectant management or placebo, but there are possible subgroup differences in the effectiveness of the available methods. AUTHORS' CONCLUSIONS: Based on relative effects from the network meta-analysis, all surgical and medical methods for managing a miscarriage may be more effective than expectant management or placebo. Surgical methods were ranked highest for managing a miscarriage, followed by medical methods, which in turn ranked above expectant management or placebo. Expectant management or placebo had the highest chance of serious complications, including the need for unplanned or emergency surgery. A subgroup analysis showed that surgical and medical methods may be more beneficial in women with missed miscarriage compared to women with incomplete miscarriage. Since type of miscarriage (missed and incomplete) appears to be a source of inconsistency and heterogeneity within these data, we acknowledge that the main network meta-analysis may be unreliable. However, we plan to explore this further in future updates and consider the primary analysis as separate networks for missed and incomplete miscarriage.

Background/aimsIntestinal permeability with systemic distribution of bacterial products are central in the immunopathogenesis of alcoholic liver disease (ALD), yet links with intestinal immunity remain elusive. Mucosa-associated invariant T cells (MAIT) are found in liver, blood and intestinal mucosa and are a key component of antibacterial host defences. Their role in ALD is unknown.Methods/designWe analysed frequency, phenotype, transcriptional regulation and function of blood MAIT cells in severe alcoholic hepatitis (SAH), alcohol-related cirrhosis (ARC) and healthy controls (HC). We also examined direct impact of ethanol, bacterial products from faecal extracts and antigenic hyperstimulation on MAIT cell functionality. Presence of MAIT cells in colon and liver was assessed by quantitative PCR and immunohistochemistry/gene expression respectively.ResultsIn ARC and SAH, blood MAIT cells were dramatically depleted, hyperactivated and displayed defective antibacterial cytokine/cytotoxic responses. These correlated with suppression of lineage-specific transcription factors and hyperexpression of homing receptors in the liver with intrahepatic preservation of MAIT cells in ALD. These alterations were stronger in SAH, where surrogate markers of bacterial infection and microbial translocation were higher than ARC. Ethanol exposure in vitro, in vivo alcohol withdrawal and treatment with Escherichia coli had no effect on MAIT cell frequencies, whereas exposure to faecal bacteria/antigens induced functional impairments comparable with blood MAIT cells from ALD and significant MAIT cell depletion, which was not observed in other T cell compartments.ConclusionsIn ALD, the antibacterial potency of MAIT cells is compromised as a consequence of contact with microbial products and microbiota, suggesting that the ‘leaky’ gut observed in ALD drives MAIT cell dysfunction and susceptibility to infection in these patients.

The microenvironment of lymphoid organs can aid healthy immune function through provision of both structural and molecular support. In mice, fibroblastic reticular cells (FRCs) create an essential T-cell support structure within lymph nodes, while human FRCs are largely unstudied. Here, we show that FRCs create a regulatory checkpoint in human peripheral T-cell activation through 4 mechanisms simultaneously utilised. Human tonsil and lymph node-derived FRCs constrained the proliferation of both naïve and pre-activated T cells, skewing their differentiation away from a central memory T-cell phenotype. FRCs acted unilaterally without requiring T-cell feedback, imposing suppression via indoleamine-2,3-dioxygenase, adenosine 2A Receptor, prostaglandin E2, and transforming growth factor beta receptor (TGFβR). Each mechanistic pathway was druggable, and a cocktail of inhibitors, targeting all 4 mechanisms, entirely reversed the suppressive effect of FRCs. T cells were not permanently anergised by FRCs, and studies using chimeric antigen receptor (CAR) T cells showed that immunotherapeutic T cells retained effector functions in the presence of FRCs. Since mice were not suitable as a proof-of-concept model, we instead developed a novel human tissue-based in situ assay. Human T cells stimulated using standard methods within fresh tonsil slices did not proliferate except in the presence of inhibitors described above. Collectively, we define a 4-part molecular mechanism by which FRCs regulate the T-cell response to strongly activating events in secondary lymphoid organs while permitting activated and CAR T cells to utilise effector functions. Our results define 4 feasible strategies, used alone or in combinations, to boost primary T-cell responses to infection or cancer by pharmacologically targeting FRCs.

Autoimmune hepatitis (AIH) is an immune-mediated liver disease currently treated by immunosuppressive medications with significant side effects. Thus, novel mechanistic treatments are greatly needed. We performed prospective deep immunophenotyping of blood immune cells in patients with acute AIH before and after corticosteroid therapy. Blood samples from 26 patients with acute AIH (United Kingdom-AIH Consortium) were phenotyped by flow cytometry at baseline and 4 months after starting corticosteroids. Pretreatment liver tissues were stained for forkhead box P3-positive (FOXP3POS) regulatory T cells (Tregs), clusters of differentiation (CD)56POS natural killer (NK) cells, and chemokine (C-X-C motif) ligand 10. Chemokine secretion by cultured primary hepatocyte and biliary epithelial cells was measured by enzyme-linked immunosorbent assay. Functional coculture assays with stimulated NK cells and Tregs were performed. CD161 ligand, lectin-like transcript-1 expression by intrahepatic immune cells was demonstrated with flow cytometry. Frequencies of NKbright cells declined with therapy (P < 0.001) and correlated with levels of alanine aminotransferase (P = 0.023). The Treg:NKbright ratio was lower pretreatment, and Tregs had an activated memory phenotype with high levels of CD39, cytotoxic T lymphocyte antigen 4, and FOXP3 but also high programmed death ligand 1, indicating exhaustion. Coculture experiments suggested the Tregs could not efficiently suppress interferon-γ secretion by NK cells. Both Tregs and NK cells had high expression of liver infiltration and T helper 17 plasticity-associated marker CD161 (P = 0.04). Pretreatment and CD161pos NK cells expressed high levels of perforin and granzyme B, consistent with an activated effector phenotype (P < 0.05). Lectin-like transcript 1, a ligand for CD161, is expressed on intrahepatic B cells, monocytes, and neutrophils. Conclusion: Activated effector NK cells, which correlate with biochemical measurements of hepatitis, and exhausted memory Tregs are increased in the blood of patients with treatment-naive AIH and decline with corticosteroid therapy. Inadequate regulation of NK cells by exhausted FOXP3pos Tregs may play a role in AIH pathogenesis and contribute to liver injury. (Hepatology Communications 2018;2:421-436).

There is no effective treatment for autoimmune biliary diseases. Therefore, understanding their immunopathology is crucial. The biliary epithelial cells (BEC), expressing TLR-4, are constantly exposed to gut microbes and bacterial wall LPS, and in settings of inflammation, the immune infiltrate is dense within the peribiliary region of human liver. By dual immunohistochemistry, we affirm human intrahepatic T cell infiltrate includes CCR6+CD4+ and AhR+CD4+ T cells with potential for plasticity to Th17 phenotype. Mechanistically, we demonstrate that Th1 and Th17 inflammatory cytokines and LPS enhance human primary BEC release of the CCR6 ligand CCL20 and BEC secretion of Th17-polarizing cytokines IL-6 and IL-1β. Cell culture assays with human BEC secretome showed that secretome polarizes CD4 T cells toward a Th17 phenotype and supports the survival of Th17 cells. BEC secretome did not promote Th1 cell generation. Additionally, we give evidence for a mutually beneficial feedback of the type 17 cell infiltrate on BEC, showing that treatment with type 17 cytokines increases BEC proliferation, as monitored by Ki67 and activation of JAK2-STAT3 signaling. This study identifies human BEC as active players in determining the nature of the intrahepatic immune microenvironment. In settings of inflammation and/or infection, biliary epithelium establishes a prominent peribiliary type 17 infiltrate via recruitment and retention and enhances polarization of intrahepatic CD4 cells toward Th17 cells via type 17 cytokines, and, reciprocally, Th17 cells promote BEC proliferation for biliary regeneration. Altogether, we provide new insight into cross-talk between Th17 lymphocytes and human primary biliary epithelium in biliary regenerative pathologies.

Imaging of blood flow in narrow channels and close to vessel walls is important in cardiovascular research for understanding pathogenesis. Our aim was to provide novel nanoprobes with visible emission and long lifetimes as trackers of flow.Gold nanoparticles coated with an iridium complex were prepared. Luminescence imaging was used to monitor their flows in different hematocrit blood and in murine tissues.The velocities are independent of hematocrit level and the nanoparticles entering blood circulation can be clearly detected in vessels in lungs, mesentery and the skeletal muscle.The work introduces for the first time iridium-based yellow-green luminescence with nanoparticle size of 100 nm for visualizing and monitoring flows with much higher resolution than conventional alternatives.

Anemia is a feature of CKD and a complication of renal transplantation, often caused by impaired production of erythropoietin. The kidney is a target organ for human cytomegalovirus (hCMV) in such patients, but it is not known whether hCMV effects erythropoietin production. We found that kidneys from patients with CKD were positive for hCMV protein and that blood levels of hCMV IgG inversely correlated with red blood cell count. In mice, systemic murine cytomegalovirus infection decreased serum erythropoietin levels. In human erythropoietin-producing cells, hCMV inhibited hypoxia-induced expression of erythropoietin mRNA and protein. hCMV early gene expression was responsible, as ultraviolet-inactivated virus had no effect and valganciclovir treatment showed that late gene expression was nonessential. Hypoxia-induced gene transcription is controlled by the transcription factors hypoxia-inducible transcription factor (HIF)-1α and HIF2α, which are constitutively produced but stable only under low oxygen conditions. We found that hCMV inhibited constitutive production of HIF2α mRNA. HIF2α is thought to be the master regulator of erythropoietin transcription. Single-cell analysis revealed that nuclear accumulation of HIF2α was inhibited in hCMV-infected cells, and the extent of inhibition correlated with hCMV protein expression. Our findings suggest that renal hCMV infection could induce or exacerbate anemia in patients.

Contradictory studies report either pro- or anti-inflammatory endothelial cell (EC) responses to human cytomegalovirus (hCMV) infection, hindering the validation of a potential link between this virus and associated inflammatory pathologies. Clarifying this issue, we report that hCMV induces a biphasic response. Early after inoculation, hCMV promoted lymphocyte and, to a lesser extent, neutrophil capture under in vivo relevant shear stresses. In contrast, later stages of infection rendered EC refractory to basal, or cytokine-induced, leukocyte recruitment.

Autoimmune hepatitis (AIH) is an immune mediated liver injury. The precise aetiology of AIH is still unknown but current evidence suggests both genetic and environmental factors are involved. Breakdown in peripheral self-tolerance, and impaired functions of FOXP3(+) regulatory T cell along with effector cell resistance to suppression at the tissue level seem to play an important role in AIH immunopathogenesis. AIH is predominantly a T lymphocytes driven disease but B lymphocytes are also involved in the immunopathology. Innate immune cells are crucial in the initial onset of disease and their response is followed by adaptive T (Th1, Th17, and cytotoxic T cells) and B cell responses evidenced by liver histology and peripheral blood serology. Standard treatment regimens involving steroid and immunosuppressive medications lead to global immune suppression requiring life-long therapy with many side effects. Biologic therapies have been attempted but duration of remission is short-lived. Future direction of diagnosis and treatment for AIH should be guided by "omics" and the immunology profile of the individual patient and clinicians should aim to deliver personalised medicine for their patients. Cell therapy such as infusion of autologous, antigen-specific, and liver-homing regulatory T cells to restore hepatic immune tolerance may soon be a potential future treatment for AIH patients.

The increasing demand for liver transplantation and the decline in donor organs has highlighted the need for alternative novel therapies to prevent chronic active hepatitis, which eventually leads to liver cirrhosis and liver cancer. Liver histology of chronic hepatitis is composed of both effector and regulatory lymphocytes. The human liver contains different subsets of effector lymphocytes that are kept in check by a subpopulation of T cells known as Regulatory T cells (Treg). The balance of effector and regulatory lymphocytes generally determines the outcome of hepatic inflammation: resolution, fulminant hepatitis, or chronic active hepatitis. Thus, maintaining and adjusting this balance is crucial in immunological manipulation of liver diseases. One of the options to restore this balance is to enrich Treg in the liver disease patients. Advances in the knowledge of Treg biology and development of clinical grade isolation reagents, cell sorting equipment, and good manufacturing practice facilities have paved the way to apply Treg cells as a potential therapy to restore peripheral self-tolerance in autoimmune liver diseases (AILD), chronic rejection, and posttransplantation. Past and on-going studies have applied Treg in type-1 diabetes mellitus, systemic lupus erythematosus, graft versus host diseases, and solid organ transplantations. There have not been any new therapies for the AILD for more than three decades; thus, the clinical potential for the application of autologous Treg cell therapy to treat autoimmune liver disease is an attractive and novel option. However, it is fundamental to understand the deep immunology, genetic profiles, biology, homing behavior, and microenvironment of Treg before applying the cells to the patients.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS), an endothelial cell (EC) neoplasm characterized by dysregulated angiogenesis and inflammation. KSHV infection of EC causes production of proinflammatory mediators, regarded as possible initiators of the substantial mononuclear leukocyte recruitment seen in KS. Conversely, KSHV immune evasion strategies exist, such as degradation of EC leukocyte adhesion receptors by viral proteins. Here, we report the effects of KSHV infection of primary EC on recruitment of flowing leukocytes. Infection did not initiate adhesion of any leukocyte subset per se . However, on cytokine-stimulated EC, KSHV specifically inhibited neutrophil, but not PBL or monocyte, transmigration, an observation consistent with the inflammatory cell profile found in KS lesions in vivo . This inhibition could be recapitulated on uninfected EC using supernatant from infected cultures. These supernatants contained elevated levels of human interleukin 6 (hIL-6), and both the KSHV- and the supernatant-induced inhibitions of neutrophil transmigration were abrogated in the presence of a hIL-6 neutralizing antibody. Furthermore, preconditioning of EC with hIL-6 mimicked the effect of KSHV. Using RNA interference (RNAi), we show that upregulation of suppressor of cytokine signaling 3 (SOCS3) was necessary for this effect of hIL-6. These studies reveal a novel paracrine mode of KSHV immune evasion, resulting in reduced recruitment of neutrophils, a cell type whose antiviral and antitumor roles are becoming increasingly appreciated. Moreover, the findings have implications for our understanding of the contribution of hIL-6 to the pathogenesis of other inflammatory disorders and tumors in which this cytokine is abundant.

Stromal cells may regulate the recruitment and behaviour of leukocytes during an inflammatory response, potentially through interaction with the endothelial cells (EC) and the leukocytes themselves. Here we describe new in vitro methodologies to characterise the effects of stromal cells on the migration of lymphocytes through endothelium and its underlying matrix. Three-dimensional tissue-like constructs were created in which EC were cultured above a stromal layer incorporating fibroblasts either as a monolayer on a porous filter or dispersed within a matrix of collagen type 1. A major advantage of these constructs is that they enable each step in leukocyte migration to be analysed in sequence (migration through EC and then stroma), as would occur in vivo. Migrated cells can also be retrieved from the constructs to identify which subsets traffic more effectively and how their functional responses evolve during migration. We found that culture of EC with dermal fibroblasts promoted lymphocyte transendothelial migration but not onward transit through matrix. A critical factor influencing the effect of fibroblasts on recruitment proved to be their proximity to the EC, with direct contact tending to disrupt migration. Comparison of the different approaches indicates that choice of an appropriate 3-D model enables the steps in lymphocyte entry into tissue to be studied in sequence, the regulatory mechanism to be dissected, and the effects of changes in stroma to be investigated., Highlights • New models of tissue using endothelial cells, fibroblasts and collagen matrix • Imaging of T-cell migration through tissue-like constructs • Fibroblasts promote T-cell migration through endothelial cells. • Fibroblasts contract collagen gel, restricting T-cell entry. • In vitro tools to test agents with the potential to alter leukocyte recruitment

Endothelial cells (EC) can present antigen to either CD8 + T lymphocytes through constitutively expressed major histocompatibility complex class I (MHC-I) or CD4 + T lymphocytes through gamma interferon (IFN-γ)-induced MHC-II. Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS), an EC neoplasm characterized by dysregulated angiogenesis and a substantial inflammatory infiltrate. KSHV is understood to have evolved strategies to inhibit MHC-I expression on EC and MHC-II expression on primary effusion lymphoma cells, but its effects on EC MHC-II expression are unknown. Here, we report that the KSHV infection of human primary EC inhibits IFN-γ-induced expression of the MHC-II molecule HLA-DR at the transcriptional level. The effect is functionally significant, since recognition by an HLA-DR-restricted CD4 + T-cell clone in response to cognate antigen presented by KSHV-infected EC was attenuated. Inhibition of HLA-DR expression was also achieved by exposing EC to supernatant from KSHV-inoculated EC before IFN-γ treatment, revealing a role for soluble mediators. IFN-γ-induced phosphorylation of STAT-1 and transcription of CIITA were suppressed in KSHV-inoculated EC via a mechanism involving SOCS3 (suppressor of cytokine signaling 3). Thus, KSHV infection resulted in transcriptional upregulation of SOCS3, and treatment with RNA interference against SOCS3 relieved virus-induced inhibition of IFN-γ-induced STAT-1 phosphorylation. Since cell surface MHC-II molecules present peptide antigens to CD4 + T lymphocytes that can function either as direct cytolytic effectors or to initiate and regulate adaptive immune responses, inhibition of this antigen-presenting pathway would provide a survival advantage to the virus.

Introduction CD56 + Natural killer cells are the principal effector cells of the innate immune system and have a well-established role in tumour surveillance and anti-viral immunity. Expression of NKp46 has been shown to correlate closely with the severity of liver inflammation, viral resistance to IFN treatment and the attenuation of liver fibrosis. CD56 + NKp46 cells expressing IL-17 and IL-22 have also been described as a family of innate lymphoid cells in humans. Although the role of intrahepatic NK cells has been well described, little is known about the function and phenotype of intrahepatic NKp46 subsets. Thus, We aim to investigate the phenotypic characteristics of CD56 + NKp46 cells in the inflamed human liver, with a view to exploring their functional role. Methods Liver infiltrating lymphocytes were freshly isolated from explanted human liver tissue from our transplant program and phenotyped with multicolor flow cytometry. Cellular localization was investigated by immunohistochemistry and confocal microscopy Results Human liver infiltrating NK cells reside predominantly around biliary epithelial cells at the portal tract close to regulatory T cells. We observed two populations of liver-infiltrating CD3 neg CD19 neg CD56 pos cells distinguished by different levels of NKp46, NKp46 mid (15% ±4.8 SD) and NKp46 high (11% ±1.2 SD) neither subset expressed NKp44. The chemokine receptor expression of NKp46 mid and NKp46 high populations was: CCR6 (12% ± 3 vs. 7%± 2.4), CCR9 (20% ± 5.6 vs. 9% ± 0.9), CX3CR1 (18% ± 14 vs. 10% ± 1) CXCR3 (47%±14.4 vs 38%±11.0) and CXCR6 19% ± 4.0 vs. 14% ± 4.6). Both populations expressed IL-18R (42% ± 5.4 vs 7% ± 1.0), IL-23R (19% ± 6.0 vs. 11% ± 2.5), surface receptor CD161 (61% ± 12.1 vs 85% ±4.8) and the integrin receptor CD103 (4% ± 1.35 vs. 16% ± 1.7). The NKp46 high population was highly enriched with the activation marker CD69 (77%±18%). NKp46 cells were also shown to express TNF-α (29% ± 7.5), IFN-γ (70% ± 7.0), Granzyme B (23% ± 11.0) and Perforin (23% ±11.1) along with transcription factor Tbet (19% ± 9.1). Conclusion We hereby report novel subsets of liver infiltrating CD56 + NKp46 cells, which localise around the portal tract biliary epithelium in the inflamed human liver. These populations have distinct cytokine, chemokine and CD103 expression, which may explain their recruitment, positioning and effector functions in the inflamed hepatic microenvironment. Disclosure of Interest None Declared.

Introduction B cells classically provide humoral immunity in the form of antibody production as part of the adaptive immune response. Regulatory and antigen presenting functions of B cells have been reported before and autoantibodies are associated with autoimmune liver diseases. B cell depletion in animal models of PBC has highlighted the regulatory roles of B cells in ameliorating disease. Some evidence of efficacy of anti-B cell therapy using rituximab in human autoimmune liver diseases further supports a role for B cells. Mature B cells (Bm) subpopulations had been described in Sjogren’s syndrome. However, little is known about the localisation, subsets, phenotype and function of B cells in human liver diseases. Methods In this study we characterised the frequencies of B cell subsets in the blood and liver of patients with inflammatory and autoimmune liver diseases. Results Frequencies of naive mature BM1 cells were reduced in the liver compared to blood (7.5% ± 2.3 vs. 20.2% ±2.8 p = 0.0022) and IgD neg CD27 neg subset was increased in diseased livers compared to diseased blood (22.9% ± 6.8 vs. 6.0% ± 1.1 p = 0.0013). B cells localise close to the bile ducts in PBC and reside around hepatocytes in AIH. Frequencies of regulatory B cells (CD19 pos CD24 hi CD38 hi ) were significantly reduced in diseased blood vs. control blood (1.8% ± 0.4 vs. 3.6% ± 0.5 p = 0.01) similar to recent observation in acute rheumatoid arthritis. However this population is increased in the diseased liver compared with blood (6.2% ± 0.07 vs. 1.8% ± 0.4 p = 0.007), suggesting enrichment of regulatory B cells within the inflamed liver. Liver infiltrating B cells were capable of IL-10 production. Conclusion We have characterised for the first time the heterogeneity of B cell subsets and presence of regulatory B cells and IL-10 secreting B cells in human diseased livers. We showed that B cells reside close to bile ducts along with other immune cells; thus B cells may play a role in biliary pathology. Disclosure of Interest None Declared.

Introduction Mucosal-Associated Invariant T (MAIT) cells are characterised by expression of the semi-invariant TCR α-chain Vα7.2-Jα33 and high expression of CD161 and shown to play a role at mucosal barriers. They display a limited T cell receptor repertoire being restricted by the MHC class 1-related molecule, MR1 but secrete high levels of pro-inflammatory cytokines suggesting they may play an important role in liver inflammation. We have shown before that the majority of MAIT cells in circulation are CD8 + MAIT cells. Recently, presence of MIAT cells have been described within human liver perfusate. However, very little is known about the phenotype and functions of liver infiltrating MAIT cells. In this study we investigated the frequencies and phenotypes of human liver infiltrating MAIT cells in healthy donors and diseased livers. Methods Peripheral blood and explanted liver infiltrating lymphocytes were freshly isolated and phenotyped by multicolour flow cytometry. The MAIT population was defined as CD3 + CD16 Hi Va7.2 + . Results There was no difference in frequencies of circulating CD3 Pos CD161 Hi Va7.2 Pos MAIT cells between patients with inflammatory liver disease and healthy controls (1.4 ± 0.7% vs. 2.3 ± 1.0%) and the majority were CD8 Pos (82.3 ± 3.1%) with a smaller population of CD4 Pos (2.7 ± 0.6%) and double negative CD8 Neg CD4 Neg cells (14.8 ± 2.9%). Total CD3 Pos CD161 Hi Va7.2 Pos MAIT frequencies were not significantly altered in inflamed liver tissue compared to blood (4.4 ± 1.0% vs. 1.4 ± 0.7%). However, in the inflamed liver, the CD8 + subset was reduced (61.2 ± 6.2 vs. 82.3 ± 3.1, P = 0.006) while the CD4 + MAIT subset was increased (15.9 ± 5.6 vs. 2.7 ± 0.6, P = 0.02). CXCR3, liver homing chemokine receptor was highly enriched on circulating and liver infiltrating CD3 Pos CD161 Hi Va7.2 Pos MAIT cells (>75%). Liver infiltrating MAIT cells expressed chemokine receptors CCR5 (78.4 ± 7.2), CX3CR1 (51.3 ± 10), CCR6 (46.3 ± 14.8) and CXCR6 (36 ± 6.2%). Interestingly they expressed high levels of the integrin β7 (39.1 ± 3.6) and CD103 (19.6 ± 5.5%), which are associated with mucosal immune responses. They also expressed the cytokine receptors IL23R (27.1 ± 8.5%) and IL18Rα (76.7 ± 5%). Conclusion We have described for the first time that CD3 Pos CD161 Hi Va7.2 Pos MAIT cells are present in inflamed human liver and express high levels of CXCR3 receptor implicated in lymphocyte recruitment to the liver and three other chemokine receptors CX3CR1 and CCR6 and CXCR6 that are associated with homing to portal tracts and bile ducts. Thus MAIT cells may play a role in biliary pathology. Disclosure of Interest None Declared.

Introduction Acute liver failure (ALF) is a sterile inflammation with a high mortality. The immunological response to acute liver injury is initiated by the infiltration of innate neutrophils and is followed by an adaptive T cells response. T cells in drug induced skin lesions have been shown to express neutrophil chemoattractant and there is also evidence that regulatory T cells (Tregs) in peripheral blood secrete IL-8. However, little is known on the ability of liver infiltrating T cell subsets to retain neutrophils in ALF or the function of Tregs. Our aim is to investigate the phenotypic features and cytokine profiles of circulating and liver infiltrating T cell subsets from ALF patients with a view of assessing their functional ability to retain neutrophils. Methods Distribution and localisation of Liver infiltrating lymphocytes and neutrophils were assessed by immunohistochemistry. Peripheral blood and liver infiltrating lymphocytes were isolated from ALF patients. Intracellular-cytokines expression profiles of the lymphocytes subsets were assessed by flow cytometry. Functional status of T cells including Tregs was assessed by pSTAT5 signalling. Results Immunohistochemistry revealed high numbers of neutrophils in ALF compared to chronic diseased livers (109 ± 12.2 vs. 7.1 ± 2.67; p = 0.01) and normal liver (109 ± 12.2 vs. 0.8 ± 0.33; p = 0.0002). Dual immunohistochemistry showed co-localisation of lymphocytes and neutrophils in areas of hepatic necrosis. Neutrophil chemoattractant IL-8 expression in peripheral blood was higher in lymphocyte subsets of ALF patients compared to normal donor, CD3 (2.3 ± 0.54% vs. 0.93 ± 0.4%; p = 0.23), CD4 (2.8 ± 2.2% vs. 1.1 ± 0.7%; p = 0.41), CD8 (2.2 ± 1% vs. 0.61 ± 0.09%; p = 0.43) and Treg (0.8 ± 0.4% vs. 0.2 ± 0.02%; p = 0.48). There was an up-regulation of IL-8 production in Tregs after 7 days of ex vivo expansion compared to day zero (1.63 ± 1% vs. 0.77 ± 0.42%; p = 0.44). IFN-γ expression in ALF peripheral blood compared with normal blood was (23.1 ± 6.2% vs. 11.1 ± 8.4%; p = 0.38) for CD3 (11.72 ± 2.3% vs. 7.8 ± 6.26; p = 0.5) for CD4 and (14.21 ± 3.04 vs. 9.95 ± 8.44; p = 0.58) for CD8 after day seven of ex vivo expression. Importantly, Tregs from both blood and explanted liver of ALF were functional indicated by STAT5 phosphorylation in response to IL-2. Conclusion We demonstrated for the first time that lymphocyte subsets including Tregs in ALF produce IL-8, which may contribute to the retention of neutrophils in areas of hepatic necrosis in ALF. We also showed that Tregs and other T cells are functionally responsive to IL-2 in both blood and explanted liver tissue of ALF patients. Disclosure of Interest None Declared.

Background and objectives Fibroblasts actively regulate the recruitment of leucocytes by endothelial cells, acting in a pro- or anti-inflammatory manner depending on their site of origin.1 Effects of fibroblasts on leucocyte migration through tissue and their subsequent fate remain unclear. Here, the authors explored the concept that fibroblasts from different tissues modulate the migration patterns of peripheral blood lymphocyte (PBL) in a site specific manner. Materials and methods Fibroblasts were isolated from the skin, bone marrow and synovium of patients with rheumatoid arthritis (RA) undergoing joint replacement surgery. Two forms of co-cultures were developed, appropriate to answer specific questions: (1) endothelial cells and fibroblasts cultured on separate 3.0 µm porous filters, one above the other; (2) endothelial monolayers formed on a filter above a collagen gel in which fibroblasts were incorporated. In the multi-filter model, PBL adhesion and migration was assessed at 24 h by flow cytometry. In the collagen construct, PBL migration into the gel and their depth of penetration was assessed at intervals over 48 h. Results The authors used the multi-filter model to assess the ability of fibroblasts to influence PBL migration through resting endothelial monolayers. Fibroblasts, synovial and dermal, enhanced the ability of endothelial cells to support T cell adhesion, and significantly promoted their onward transendothelial migration. Although fibroblasts generate CXCL12 and IL-6, blockade of either protein had no effect on T cell migration. Interestingly, fibroblasts appeared to restrain onward motion of recruited T cells. This indicated that the process of migration changed the migratory phenotype of recruited T cells and/or that fibroblasts were providing ‘stop’ signals resulting in T cell retention within the fibroblast layer. Next, the authors employed the multi-cellular collagen gel construct to visually observe the effects of fibroblasts on PBL migration into and retention within tissue. Once again, all fibroblast types promoted PBL transendothelial migration, while reducing their onward penetration of the collagen gel. Notably, migrating PBL tracked closely to fibroblast rich regions of the gel, indicating that fibroblasts are capable of retaining recruited cells. Conclusions Collectively these data suggest a novel regulated step in lymphocyte migration, where stromal-derived ‘go’ and ‘stop’ signals control lymphocyte migration into and within tissues, respectively.

We investigated the migration of human leukocytes through endothelial cells (EC), and particularly their underlying basement membrane (BM). EC were cultured for 20days on 3μm-pore filters or collagen gels to form a distinct BM, and then treated with tumour necrosis factor-α, interleukin-1β or interferon-γ. Neutrophil migration through the cytokine-treated EC and BM was delayed for 20-day compared to 4-day cultures. The BM alone obstructed chemotaxis of neutrophils, and if fresh EC were briefly cultured on stripped BM, there was again a hold-up in migration. In studies with lymphocytes and monocytes, we could detect little hold-up of migration for 20-day versus 4-day cultures, in either the filter- or gel-based models. Direct microscopic observations showed that BM also held-up neutrophil migration under conditions of flow. Treatment of upper and/or lower compartments of filters with antibodies against integrins, showed that neutrophil migration through the endothelial monolayer was dependent on β(2)-integrins, but not β1- or β(3)-integrins. Migration from the subendothelial compartment was supported by β1- and β(2)-integrins for all cultures, but blockade of β(3)-integrin only inhibited migration effectively for 20-day cultures. Flow cytometry indicated that there was no net increase in expression of β1- or β3-integrins during neutrophil migration, and that their specific subendothelial function was likely dependent on turnover of integrins during migration. These studies show that BM is a distinct barrier to migration of human neutrophils, and that β(3)-integrins are particularly important in crossing this barrier. The lesser effect of BM on lymphocytes and monocytes supports the concept that crossing the BM is a separate, leukocyte-specific, regulated step in migration.

Highlights • We examined infection and transmission of rKSHV.219 in endothelial cell cultures. • The number of rKSHV.219 infected/GFP positive endothelial cells increased over time. • This increase was not due to horizontal transmission or cellular proliferation. • LANA analysis revealed an infection threshold was required for GFP expression. • GFP was a reliable indicator of infection only at later time points post infection., Kaposi's sarcoma-associated herpesvirus (KSHV) is the aetiologic agent of Kaposi's sarcoma (KS), a tumour of endothelial cell origin. The study of KS development was aided by the generation of a recombinant GFP (latent)/RFP (lytic)-expressing KSHV (rKSHV.219) by Vieira and O’Hearn (2004). In this study the first data characterising primary endothelial cell infection and transmission with this virus is presented. Infection was predominantly latent and the percentage of GFP-positive cells increased over time. Neither horizontal transmission of infection, nor cellular proliferation, explained this increase. Analysis of latency-associated nuclear antigen (LANA-1) expression revealed that a threshold level of infection was required for GFP expression early post infection. At later time points GFP correlated more closely with LANA-1 expression, likely due to the accumulation of GFP over time. This study provides methodological guidance for the use of rKSHV.21. In addition, it highlights potential problems associated with the use of fluorescent proteins as markers of viral infection.