Neointimal hyperplasia after endoluminal injury in mice is dependent on tissue factor- and angiopoietin-2 dependent interferon gamma production by fibrocytes and macrophages.

Background: The intimal hyperplasia (IH) and vascular remodelling that follows endovascular injury, for instance after post-angioplasty re-stenosis, results in downstream ischaemia and progressive end organ damage. Interferon gamma (IFNγ) is known to play a critical role in this process. In mouse models we have previously shown that fibrocytes expressing tissue factor (TF) are recruited early to the site of injury. Through thrombin generation and protease activated receptor-1 (PAR-1) activation, fibrocytes secrete angiopoietin-2, stimulate neointimal cell proliferation, inhibit apoptosis and induce CXCL-12 production, all of which contribute to the progressive IH that then develops. In this study we investigated the relationship between TF, angiopoietin-2 and IFNγ. Methods and results: IH developing in carotid arteries of wild-type mice 4 weeks after endoluminal injury contained a significant proportion of IFNγ+ fibrocytes and macrophages, which we show, using a previously defined adoptive transfer model, were derived from circulating CD34+ cells. IH did not develop after injury in IFNγ-deficient mice, except after transplantation of WT bone marrow or adoptive transfer of WT CD34+ cells. In vitro, CD34+ cells isolated from post-injury mice did not express IFNγ, but this was induced when provided with FVIIa and FX, and enhanced when prothrombin was also provided: In both cases IFNγ secretion was TF-dependent and mediated mainly through protease activated PAR-1. IFNγ was predominantly expressed by fibrocytes. In vivo, all IFNγ+ neointimal cells in WT mice co-expressed angiopoietin-2, as did the small numbers of neointimal cells recruited in IFNγ-/- mice. Adoptively transferred WT CD34+ cells treated with either an anti-TIE-2 antibody, or with siRNA against angiopoetin-2 inhibited the expression of IFNγ and the development of IH. Conclusion: TF-dependent angiopoietin-2 production by newly recruited fibrocytes, and to a lesser extent macrophages, switches on IFNγ expression, and this is necessary for the IH to develop. These novel findings enhance our understanding of the pathophysiology of IH and expose potential targets for therapeutic intervention.

Continuous Risk Score Predicts Waitlist and Post-transplant Outcomes in Hepatocellular Carcinoma Despite Exception Changes.

BACKGROUND & AIMS: Continuous risk-stratification of candidates and urgency-based prioritization have been utilized for liver transplantation (LT) in patients with non-hepatocellular carcinoma (HCC) in the United States. Instead, for patients with HCC, a dichotomous criterion with exception points is still used. This study evaluated the utility of the hazard associated with LT for HCC (HALT-HCC), an oncological continuous risk score, to stratify waitlist dropout and post-LT outcomes. METHODS: A competing risk model was developed and validated using the UNOS database (2012-2021) through multiple policy changes. The primary outcome was to assess the discrimination ability of waitlist dropouts and LT outcomes. The study focused on the HALT-HCC score, compared with other HCC risk scores. RESULTS: Among 23,858 candidates, 14,646 (59.9%) underwent LT and 5196 (21.8%) dropped out of the waitlist. Higher HALT-HCC scores correlated with increased dropout incidence and lower predicted 5-year overall survival after LT. HALT-HCC demonstrated the highest area under the curve (AUC) values for predicting dropout at various intervals post-listing (0.68 at 6 months, 0.66 at 1 year), with excellent calibration (R2 = 0.95 at 6 months, 0.88 at 1 year). Its accuracy remained stable across policy periods and locoregional therapy applications. CONCLUSIONS: This study highlights the predictive capability of the continuous oncological risk score to forecast waitlist dropout and post-LT outcomes in patients with HCC, independent of policy changes. The study advocates integrating continuous scoring systems like HALT-HCC in liver allocation decisions, balancing urgency, organ utility, and survival benefit.

Adenosine A2a receptor inhibition increases the anti-tumor efficacy of anti-PD1 treatment in murine hepatobiliary cancers.

Backgrounds & Aims: The efficacy of immune checkpoint inhibitor (ICI) therapy for liver cancer remains limited. As the hypoxic liver environment regulates adenosine signaling, we tested the efficacy of adenosine A2a receptor (A2aR) inhibition in combination with ICI treatment in murine models of liver cancer. Methods: RNA expression related to the adenosine pathway was analyzed from public databases. Peripheral blood mononuclear cells of 13 patients with hepatocellular carcinoma (HCC) were examined by flow cytometry. The following murine cell lines were used: SB-1, RIL175, and Hep55.1c (liver cancer), CT26 (colon cancer), and B16-F10 (melanoma). C57BL/6 and BALB/c mice were used for orthotopic tumor models and were treated with SCH58261, an A2aR inhibitor, in combination with anti-PD1 therapy. Results: RNA expression of ADORA2A in tumor tissues derived from patients with HCC was higher than in tissues from other cancer types. A2aR+ T cells in peripheral blood from patients with HCC were highly proliferative after immunotherapy. Likewise, in an orthotopic murine model, A2aR expression on T cells increased following anti-PD1 treatment, and the expression of A2aR on T cells increased more in tumor-bearing mice compared with tumor-free mice. The combination of SCH58261 and anti-PD1 led to activation of T cells and reductions in tumor size in orthotopic liver cancer models. In contrast, SCH58261 monotherapy was ineffective in orthotopic liver cancer models and the combination was ineffective in the subcutaneous tumor models tested. CD4+ T-cell depletion attenuated the efficacy of the combination therapy. Conclusion: A2aR inhibition and anti-PD1 therapy had a synergistic anti-tumor effect in murine liver cancer models. Impact and implications: Adenosine A2a receptor (A2aR)-expressing T cells in the liver increased in tumor-bearing mice and after anti-PD1 treatment. The combination of an A2aR inhibitor and anti-PD1 treatment had potent anti-tumor effects in two murine models of orthotopic liver cancer. Adenosine A2a receptor blockade promotes immunotherapy efficacy in murine models, highlighting putative clinical benefits for advanced stage liver cancer patients.

Human myofibroblasts increase the arrhythmogenic potential of human induced pluripotent stem cell-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have the potential to remuscularize infarcted hearts but their arrhythmogenicity remains an obstacle to safe transplantation. Myofibroblasts are the predominant cell-type in the infarcted myocardium but their impact on transplanted hiPSC-CMs remains poorly defined. Here, we investigate the effect of myofibroblasts on hiPSC-CMs electrophysiology and Ca2+ handling using optical mapping of advanced human cell coculture systems mimicking cell-cell interaction modalities. Human myofibroblasts altered the electrophysiology and Ca2+ handling of hiPSC-CMs and downregulated mRNAs encoding voltage channels (KV4.3, KV11.1 and Kir6.2) and SERCA2a calcium pump. Interleukin-6 was elevated in the presence of myofibroblasts and direct stimulation of hiPSC-CMs with exogenous interleukin-6 recapitulated the paracrine effects of myofibroblasts. Blocking interleukin-6 reduced the effects of myofibroblasts only in the absence of physical contact between cell-types. Myofibroblast-specific connexin43 knockdown reduced functional changes in contact cocultures only when combined with interleukin-6 blockade. This provides the first in-depth investigation into how human myofibroblasts modulate hiPSC-CMs function, identifying interleukin-6 and connexin43 as paracrine- and contact-mediators respectively, and highlighting their potential as targets for reducing arrhythmic risk in cardiac cell therapy.

CSF-1R+ Macrophages Control the Gut Microbiome-Enhanced Liver Invariant NKT Function through IL-18.

The gut microbiome is an important modulator of the host immune system. In this study, we found that altering the gut microbiome by oral vancomycin increases liver invariant NKT (iNKT) cell function. Enhanced iNKT cytokine production and activation marker expression were observed in vancomycin-treated mice following both Ag-specific and Ag-independent in vivo iNKT stimulations, with a more prominent effect in the liver than in the spleen. Fecal transplantation studies demonstrated that the iNKT functional regulation is mediated by altering the gut microbiome but uncoupled from the modulation of iNKT cell population size. Interestingly, when stimulated in vitro, iNKT cells from vancomycin-treated mice did not show increased activation, suggesting an indirect regulation. iNKT cells expressed high levels of IL-18 receptor, and vancomycin increased the expression of IL-18 in the liver. Blocking IL-18 by neutralizing Ab or using genetically deficient mice attenuated the enhanced iNKT activation. Liver macrophages were identified as a major source of IL-18. General macrophage depletion by clodronate abolished this iNKT activation. Using anti-CSF-1R depletion or LyzCrexCSF-1RLsL-DTR mice identified CSF-1R+ macrophages as a critical modulator of iNKT function. Vancomycin treatment had no effect on iNKT cell function in vivo in IL-18 knockout macrophage reconstituted mice. Together, our results demonstrate that the gut microbiome controls liver iNKT function via regulating CSF-1R+ macrophages to produce IL-18.

Ex Vivo Perfusion Culture of Large Blood Vessels in a 3D Printed Bioreactor.

Vascular disease forms the basis of most cardiovascular diseases (CVDs), which remain the primary cause of mortality and morbidity worldwide. Efficacious surgical and pharmacological interventions to prevent and treat vascular disease are urgently needed. In part, the shortage of translational models limits the understanding of the cellular and molecular processes involved in vascular disease. Ex vivo perfusion culture bioreactors provide an ideal platform for the study of large animal vessels (including humans) in a controlled dynamic environment, combining the ease of in vitro culture and the complexity of the live tissue. Most bioreactors are, however, custom manufactured and therefore difficult to adopt, limiting the reproducibility of the results. This paper presents a 3D printed system that can be easily produced and applied in any biological lab, and provides a detailed protocol for its setup, enabling users' operation. This innovative and reproducible ex vivo perfusion culture system enables the culture of blood vessels for up to 7 days in physiological conditions. We expect that adopting a standardized perfusion bioreactor will support a better understanding of physiological and pathological processes in large animal blood vessels and accelerate the discovery of new therapeutics.

Peri-operative thrombophilia in patients undergoing liver resection for colorectal metastases.

BACKGROUND: Routine chemical venous thromboembolism (VTE) prophylaxis for liver surgery remains controversial, and often delayed post-operatively due to perceived bleeding risk. This study asked whether patients undergoing hepatectomy for colorectal metastases (CRM) were at risk from VTE pre-operatively, and the impact of hepatectomy on that risk. METHODS: Single-centre prospective observational cohort study of patients undergoing open hepatectomy for CRM, comparing pre-, peri- and post-operative haemostatic variables. RESULTS: Of 336 hepatectomies performed October 2017-December 2019, 60 resections in 57 patients were recruited. There were 28 (46.7%) major resections, with median (interquartile range [IQR]) blood loss 150.0 (76.3-263.7) mls, no blood transfusions, post-operative VTE events or deaths. Patients were prothrombotic pre-operatively (high median factor VIIIC and increased thrombin generation velocity index), an effect exacerbated post-hepatectomy. Major hepatectomies had a significantly greater median drop in Protein C, rise in Factor VIIIC and von Willebrand Factor, versus minor resections (p = 0.001, 0.005, 0.001 respectively). Patients with parenchymal transection times greater than median (40 min), had significantly increased median (IQR) PMBC-TFmRNA expression [1.65(0.93-2.70)2ddCt], versus quicker transections [0.99(0.69-1.28)2ddCt, p = 0.020]. CONCLUSIONS: Patients with CRM are prothrombotic pre-operatively, an effect exacerbated by hepatectomy, particularly longer, complex resections, suggesting chemical thromboprophylaxis be considered early in the patient pathway.

Age-Dependent Decline in Common Femoral Artery Flow-Mediated Dilation and Wall Shear Stress in Healthy Subjects.

Femoral artery (FA) endothelial function is a promising biomarker of lower extremity vascular health for peripheral artery disease (PAD) prevention and treatment; however, the impact of age on FA endothelial function has not been reported in healthy adults. Therefore, we evaluated the reproducibility and acceptability of flow-mediated dilation (FMD) in the FA and brachial artery (BA) (n = 20) and performed cross-sectional FA- and BA-FMD measurements in healthy non-smokers aged 22−76 years (n = 50). FMD protocols demonstrated similar good reproducibility. Leg occlusion was deemed more uncomfortable than arm occlusion; thigh occlusion was less tolerated than forearm and calf occlusion. FA-FMD with calf occlusion was lower than BA-FMD (6.0 ± 1.1% vs 6.4 ± 1.3%, p = 0.030). Multivariate linear regression analysis indicated that age (−0.4%/decade) was a significant independent predictor of FA-FMD (R2 = 0.35, p = 0.002). The age-dependent decline in FMD did not significantly differ between FA and BA (pinteraction agexlocation = 0.388). In older participants, 40% of baseline FA wall shear stress (WSS) values were <5 dyne/cm2, which is regarded as pro-atherogenic. In conclusion, endothelial function declines similarly with age in the FA and the BA in healthy adults. The age-dependent FA enlargement results in a critical decrease in WSS that may explain part of the age-dependent predisposition for PAD.

Manipulation of tissue factor-mediated basal PAR-2 signalling on macrophages determines sensitivity for IFNγ responsiveness and significantly modifies the phenotype of murine DTH.

Background: Tissue factor (TF) generates proteases that can signal through PAR-1 and PAR-2. We have previously demonstrated PAR-1 signalling primes innate myeloid cells to be exquisitely sensitive to interferon-gamma (IFNγ). In this work we explored how TF mediated PAR-2 signalling modulated responsiveness to IFNγ and investigated the interplay between PAR-1/-2 signalling on macrophages. Methodology: We characterised how TF through PAR-2 influenced IFNγ sensitivity in vitro using PCR and flow cytometry. and how it influenced oxazolone-induced delayed type hypersensitivity (DTH) responses in vivo. We investigated how basal signalling through PAR-2 influenced PAR-1 signalling using a combination of TF-inhibitors and PAR-1 &-2 agonists and antagonists. Finally, we investigated whether this system could be targeted therapeutically using 3-mercaptopropionyl-F-Cha-Cha-RKPNDK (3-MP), which has actions on both PAR-1 and -2. Results: TF delivered a basal signal through PAR-2 that upregulated SOCS3 expression and blunted M1 polarisation after IFNγ stimulation, opposing the priming achieved by signalling through PAR-1. PAR-1 and -2 agonists or antagonists could be used in combination to modify this basal signal in vitro and in vivo. 3-MP, by virtue of its PAR-2 agonist properties was superior to agents with only PAR-1 antagonist properties at reducing M1 polarisation induced by IFNγ and suppressing DTH. Tethering a myristoyl electrostatic switch almost completely abolished the DTH response. Conclusions: TF-mediated signalling through PARs-1 and -2 act in a homeostatic way to determine how myeloid cells respond to IFNγ. 3-MP, an agent that simultaneously inhibits PAR-1 whilst delivering a PAR-2 signal, can almost completely abolish immune responses dependent on M1 polarisation, particularly if potency is enhanced by targeting to cell membranes; this has potential therapeutic potential in multiple diseases.

Cocoa flavanol consumption improves lower extremity endothelial function in healthy individuals and people with type 2 diabetes.

Background: diabetes and age are major risk factors for the development of lower extremity peripheral artery disease (PAD). Cocoa flavanol (CF) consumption is associated with lower risk for PAD and improves brachial artery (BA) endothelial function. Objectives: to assess if femoral artery (FA) endothelial function and dermal microcirculation are impaired in individuals with type 2 diabetes mellitus (T2DM) and evaluate the acute effect of CF consumption on FA endothelial function. Methods: in a randomised, controlled, double-blind, cross-over study, 22 individuals (n = 11 healthy, n = 11 T2DM) without cardiovascular disease were recruited. Participants received either 1350 mg CF or placebo capsules on 2 separate days in random order. Endothelial function was measured as flow-mediated dilation (FMD) using ultrasound of the common FA and the BA before and 2 hours after interventions. The cutaneous microvasculature was assessed using optical coherence tomography angiography. Results: baseline FA-FMD and BA-FMD were significantly lower in T2DM (FA: 3.2 ± 1.1% [SD], BA: 4.8 ± 0.8%) compared to healthy (FA: 5.5 ± 0.7%, BA: 6.0 ± 0.8%); each p < 0.001. Whereas in healthy individuals FA-FMD did not significantly differ from BA-FMD (p = 0.144), FA-FMD was significantly lower than BA-FMD in T2DM (p = 0.003) indicating pronounced and additional endothelial dysfunction of lower limb arteries (FA-FMD/BA-FMD: 94 ± 14% [healthy] vs. 68 ± 22% [T2DM], p = 0.007). The baseline FA blood flow rate (0.42 ± 0.23 vs. 0.73 ± 0.35 l min-1, p = 0.037) and microvascular dilation in response to occlusion in hands and feet were significantly lower in T2DM subjects than in healthy ones. CF increased both FA- and BA-FMD at 2 hours, compared to placebo, in both healthy and T2DM subgroups (FA-FMD effect: 2.9 ± 1.4%, BA-FMD effect 3.0 ± 3.5%, each pintervention< 0.001). In parallel, baseline FA blood flow and microvascular diameter significantly increased in feet (3.5 ± 3.5 µm, pintervention< 0.001) but not hands. Systolic blood pressure and pulse wave velocity significantly decreased after CF in both subgroups (-7.2 ± 9.6 mmHg, pintervention = 0.004; -1.3 ± 1.3 m s-1, pintervention = 0.002). Conclusions: individuals with T2DM exhibit decreased endothelial function that is more pronounced in the femoral than in the brachial artery. CFs increase endothelial function not only in the BA but also the FA both in healthy individuals and in those with T2DM who are at increased risk of developing lower extremity PAD and foot ulcers.

Platelets control liver tumor growth through P2Y12-dependent CD40L release in NAFLD.

Platelets, the often-overlooked component of the immune system, have been shown to promote tumor growth. Non-alcoholic fatty liver disease (NAFLD) is a common disease in the Western world and rising risk for hepatocellular carcinoma (HCC). Unexpectedly, we observed that platelets can inhibit the growth of established HCC in NAFLD mice. Through pharmacological inhibition and genetic depletion of P2Y12 as well as in vivo transfusion of wild-type (WT) or CD40L-/- platelets, we demonstrate that the anti-tumor function of platelets is mediated through P2Y12-dependent CD40L release, which leads to CD8+ T cell activation by the CD40 receptor. Unlike P2Y12 inhibition, blocking platelets with aspirin does not prevent platelet CD40L release nor accelerate HCC in NAFLD mice. Similar findings were observed in liver metastasis models. All together, our study reveals a complex role of platelets in tumor regulation. Anti-platelet treatment without inhibiting CD40L release could be considered for liver cancer patients with NAFLD.

3D Printed Bioreactor Enabling the Pulsatile Culture of Native and Angioplastied Large Arteries.

Routine interventions such as balloon angioplasty, result in vascular activation and remodeling, often requiring re-intervention. 2D in vitro models and small animal experiments have enabled the discovery of important mechanisms involved in this process, however the clinical translation is often underwhelming. There is a critical need for an ex vivo model representative of the human vascular physiology and encompassing the complexity of the vascular wall and the physical forces regulating its function. Vascular bioreactors for ex vivo culture of large vessels are viable alternatives, but their custom-made design and insufficient characterization often hinders the reproducibility of the experiments. The objective of the study was to design and validate a novel 3D printed cost-efficient and versatile perfusion system, capable of sustaining the viability and functionality of large porcine arteries for 7 days and enabling early post-injury evaluations. MultiJet Fusion 3D printing was used to engineer the EasyFlow insert, converting a conventional 50 ml centrifuge tube into a mini bioreactor. Porcine carotid arteries either left untreated or injured with an angioplasty balloon, were cultured under pulsatile flow for up to 7 days. Pressure, heart rate, medium viscosity and shear conditions were adjusted to resemble arterial in vivo hemodynamics. Tissue viability, cell activation and matrix remodeling were analyzed by immunohistochemistry, and vascular function was monitored by duplex ultrasound. Culture conditions in the EasyFlow bioreactor preserved endothelial coverage and smooth muscle organization and extracellular matrix structure in the vessel wall, as compared to static culture. Injured arteries presented hallmarks of early remodeling, such as intimal denudation, smooth muscle cell disarray and media/adventitia activation in flow culture. Duplex ultrasound confirmed continuous pulsatile blood flow conditions, dose-dependent vasodilator response to nitroglycerin in untreated vessels and impaired dilator response in angioplastied vessels. The scope of this work is to validate a low-cost, robust and reproducible system to explore the culture of native and injured large arteries under pulsatile flow. While the study of vascular pathology is beyond the scope of the present paper, our system enables future investigations and provides a platform to test novel therapies and devices ex vivo, in a patient relevant system.

Pericytes' Circadian Clock Affects Endothelial Cells' Synchronization and Angiogenesis in a 3D Tissue Engineered Scaffold.

Angiogenesis, the formation of new capillaries from existing ones, is a fundamental process in regenerative medicine and tissue engineering. While it is known to be affected by circadian rhythms in vivo, its peripheral regulation within the vasculature and the role it performs in regulating the interplay between vascular cells have not yet been investigated. Peripheral clocks within the vasculature have been described in the endothelium and in smooth muscle cells. However, to date, scarce evidence has been presented regarding pericytes, a perivascular cell population deeply involved in the regulation of angiogenesis and vessel maturation, as well as endothelial function and homeostasis. More crucially, pericytes are also a promising source of cells for cell therapy and tissue engineering. Here, we established that human primary pericytes express key circadian genes and proteins in a rhythmic fashion upon synchronization. Conversely, we did not detect the same patterns in cultured endothelial cells. In line with these results, pericytes' viability was disproportionately affected by circadian cycle disruption, as compared to endothelial cells. Interestingly, endothelial cells' rhythm could be induced following exposure to synchronized pericytes in a contact co-culture. We propose that this mechanism could be linked to the altered release/uptake pattern of lactate, a known mediator of cell-cell interaction which was specifically altered in pericytes by the knockout of the key circadian regulator Bmal1. In an angiogenesis assay, the maturation of vessel-like structures was affected only when both endothelial cells and pericytes did not express Bmal1, indicating a compensation system. In a 3D tissue engineering scaffold, a synchronized clock supported a more structured organization of cells around the scaffold pores, and a maturation of vascular structures. Our results demonstrate that pericytes play a critical role in regulating the circadian rhythms in endothelial cells, and that silencing this system disproportionately affects their pro-angiogenic function. Particularly, in the context of tissue engineering and regenerative medicine, considering the effect of circadian rhythms may be critical for the development of mature vascular structures and to obtain the maximal reparative effect.

Metformin treatment rescues CD8+ T-cell response to immune checkpoint inhibitor therapy in mice with NAFLD.

BACKGROUND & AIMS: Non-alcoholic steatohepatitis (NASH) represents the fastest growing underlying cause of hepatocellular carcinoma (HCC) and has been shown to impact immune effector cell function. The standard of care for the treatment of advanced HCC is immune checkpoint inhibitor (ICI) therapy, yet NASH may negatively affect the efficacy of ICI therapy in HCC. The immunologic mechanisms underlying the impact of NASH on ICI therapy remain unclear. METHODS: Herein, using multiple murine NASH models, we analysed the influence of NASH on the CD8+ T-cell-dependent anti-PD-1 responses against liver cancer. We characterised CD8+ T cells' transcriptomic, functional, and motility changes in mice receiving a normal diet (ND) or a NASH diet. RESULTS: NASH blunted the effect of anti-PD-1 therapy against liver cancers in multiple murine models. NASH caused a proinflammatory phenotypic change of hepatic CD8+ T cells. Transcriptomic analysis revealed changes related to NASH-dependent impairment of hepatic CD8+ T-cell metabolism. In vivo imaging analysis showed reduced motility of intratumoural CD8+ T cells. Metformin treatment rescued the efficacy of anti-PD-1 therapy against liver tumours in NASH. CONCLUSIONS: We discovered that CD8+ T-cell metabolism is critically altered in the context of NASH-related liver cancer, impacting the effectiveness of ICI therapy - a finding which has therapeutic implications in patients with NASH-related liver cancer. LAY SUMMARY: Non-alcoholic steatohepatitis represents the fastest growing cause of hepatocellular carcinoma. It is also associated with reduced efficacy of immunotherapy, which is the standard of care for advanced hepatocellular carcinoma. Herein, we show that non-alcoholic steatohepatitis is associated with impaired motility, metabolic function, and response to anti-PD-1 treatment in hepatic CD8+ T cells, which can be rescued by metformin treatment.

NAFLD indirectly impairs antigen-specific CD8+ T cell immunity against liver cancer in mice.

Non-alcoholic fatty liver disease (NAFLD) has become an important etiology leading to liver cancer. NAFLD alters adaptive T cell immunity and has a profound influence on liver cancer development. However, it is unclear how NAFLD affects tumor antigen-specific T cell response. In this study, we generated a doxycycline-inducible MHC-I and -II antigen-expressing HCC cell line which allowed us to investigate tumor antigen-specific T cell response in two NAFLD mouse models. The system proved to be an effective and efficient way to study tumor antigen-specific T cells. Using this model, it was found that NAFLD impairs antigen-specific CD8+ T cell immunity against HCC. The effect was not due to reduced generation or intrinsic functional changes of tumor antigen-specific CD8+ T cells but caused by accumulated macrophages in the liver environment. The findings suggest that targeting macrophages in NAFLD-driven HCC may improve therapeutic outcomes.

The tumour microenvironment shapes innate lymphoid cells in patients with hepatocellular carcinoma.

OBJECTIVE: Hepatocellular carcinoma (HCC) represents a typical inflammation-associated cancer. Tissue resident innate lymphoid cells (ILCs) have been suggested to control tumour surveillance. Here, we studied how the local cytokine milieu controls ILCs in HCC. DESIGN: We performed bulk RNA sequencing of HCC tissue as well as flow cytometry and single-cell RNA sequencing of enriched ILCs from non-tumour liver, margin and tumour core derived from 48 patients with HCC. Simultaneous measurement of protein and RNA expression at the single-cell level (AbSeq) identified precise signatures of ILC subgroups. In vitro culturing of ILCs was used to validate findings from in silico analysis. Analysis of RNA-sequencing data from large HCC cohorts allowed stratification and survival analysis based on transcriptomic signatures. RESULTS: RNA sequencing of tumour, non-tumour and margin identified tumour-dependent gradients, which were associated with poor survival and control of ILC plasticity. Single-cell RNA sequencing and flow cytometry of ILCs from HCC livers identified natural killer (NK)-like cells in the non-tumour tissue, losing their cytotoxic profile as they transitioned into tumour ILC1 and NK-like-ILC3 cells. Tumour ILC composition was mediated by cytokine gradients that directed ILC plasticity towards activated tumour ILC2s. This was liver-specific and not seen in ILCs from peripheral blood mononuclear cells. Patients with high ILC2/ILC1 ratio expressed interleukin-33 in the tumour that promoted ILC2 generation, which was associated with better survival. CONCLUSION: Our results suggest that the tumour cytokine milieu controls ILC composition and HCC outcome. Specific changes of cytokines modify ILC composition in the tumour by inducing plasticity and alter ILC function.

Dissection of pleiotropic effects of variants in and adjacent to F8 exon 19 and rescue of mRNA splicing and protein function.

The pathogenic significance of nucleotide variants commonly relies on nucleotide position within the gene, with exonic changes generally attributed to quantitative or qualitative alteration of protein biosynthesis, secretion, activity, or clearance. However, these changes may exert pleiotropic effects on both protein biology and mRNA splicing due to the overlapping of the amino acid and splicing codes, thus shaping the disease phenotypes. Here, we focused on hemophilia A, in which the definition of F8 variants' causative role and association to bleeding phenotypes is crucial for proper classification, genetic counseling, and management of affected individuals. We extensively characterized a large panel of hemophilia A-causing variants (n = 30) within F8 exon 19 by combining and comparing in silico and recombinant expression analyses. We identified exonic variants with pleiotropic effects and dissected the altered protein features of all missense changes. Importantly, results from multiple prediction algorithms provided qualitative results, while recombinant assays allowed us to correctly infer the likely phenotype severity for 90% of variants. Molecular characterization of pathogenic variants was also instrumental for the development of tailored correction approaches to rescue splicing affecting variants or missense changes impairing protein folding. A single engineered U1snRNA rescued mRNA splicing of nine different variants and the use of a chaperone-like drug resulted in improved factor VIII protein secretion for four missense variants. Overall, dissection of the molecular mechanisms of a large panel of HA variants allowed precise classification of HA-affected individuals and favored the development of personalized therapeutic approaches.

Anti-PD-1 in Combination With Trametinib Suppresses Tumor Growth and Improves Survival of Intrahepatic Cholangiocarcinoma in Mice.

BACKGROUND & AIMS: Intrahepatic cholangiocarcinoma (iCCA) accounts for a fraction of primary liver cancers but has a 5-year survival rate of only 10%. Immune checkpoint inhibitors are effective in treating many solid cancers, but immune checkpoint inhibitor monotherapy has no clear benefit in iCCA. Mitogen-activated kinase (MEK) inhibitors, such as trametinib, have shown promising results in preclinical studies for iCCA by inhibiting cell proliferation and modifying the tumor microenvironment. This study aimed to show the potential benefit of combining trametinib with anti-programmed cell death protein 1 (PD-1) therapy in different iCCA mouse models. METHODS: Here, we assessed the in vitro cytotoxicity of trametinib in mouse (SB1 and LD-1) and human (EGI-1) cholangiocarcinoma cell lines. We examined the efficacy of single-agent trametinib, anti-PD-1, and a combination of both in subcutaneous, orthotopic, and plasmid-induced iCCA mouse models. Flow cytometry analysis was used to elucidate changes in the tumor immune microenvironment upon treatment. Whole-exome sequencing (WES) was performed on the SB1 tumor cell line to correlate this preclinical model with iCCAs in patients. RESULTS: Trametinib reduced tumor cell growth of SB1, LD-1, and EGI-1 tumor cells in vitro. Trametinib treatment led to up-regulation of major histocompatibility complex (MHC-I) and programmed cell death ligand 1 (PD-L-1) (programmed cell death ligand 1) on tumor cells in vitro. The combination of trametinib and anti-PD-1 reduced tumor burden in several iCCA tumor models and improved survival in SB1 tumor-bearing mice compared with either agent alone. Immunoprofiling of tumor-bearing mice showed an increase of hepatic effector memory CD8+ and CD4+ T cells, as well as an increased degranulation of CD8+ T cells, indicating enhanced cytotoxicity. WES and somatic mutational analysis showed no mutations of KRAS, BRAF, and ERK in SB1 tumor cells, and showed a similar genetic signature of SB1 found in a cohort of patients with iCCA. CONCLUSIONS: Altogether, our study shows that trametinib improves the immunogenicity of tumor cells by up-regulating MHC-I surface expression. The combination with anti-PD-1 results in optimal treatment efficacy for iCCA. WES of SB1 cells suggests that KRAS wild-type iCCAs also respond to this combination therapy.

Conditional probability of graft survival in liver transplantation using donation after circulatory death grafts - a retrospective study.

The use of livers from donation after circulatory death (DCD) is historically characterized by increased rates of biliary complications and inferior short-term graft survival (GS) compared to donation after brain death (DBD) allografts. This study aimed to evaluate the dynamic prognostic impact of DCD livers to reveal whether they remain an adverse factor even after patients survive a certain period following liver transplant (LT). This study used 74 961 LT patients including 4065 DCD LT in the scientific registry of transplant recipients from 2002-2017. The actual, 1 and 3-year conditional hazard ratio (HR) of 1-year GS in DCD LT were calculated using a conditional version of Cox regression model. The actual 1-, 3-, and 5-year GS of DCD LT recipients were 83.3%, 73.3%, and 66.3%, which were significantly worse than those of DBD (all P < 0.01). Actual, 1-, and 3-year conditional HR of 1-year GS in DCD compared to DBD livers were 1.87, 1.49, and 1.39, respectively. Graft loss analyses showed that those lost to biliary related complications were significantly higher in the DCD group even 3 years after LT. National registry data demonstrate the protracted higher risks inherent to DCD liver grafts in comparison to their DBD counterparts, despite survival through the early period after LT. These findings underscore the importance of judicious DCD graft selection at individual center level to minimize the risk of long-term biliary complications.

PAR-1 signaling on macrophages is required for effective in vivo delayed-type hypersensitivity responses.

Delayed-type hypersensitivity (DTH) responses underpin chronic inflammation. Using a model of oxazolone-induced dermatitis and a combination of transgenic mice, adoptive cell transfer, and selective agonists/antagonists against protease activated receptors, we show that that PAR-1 signaling on macrophages by thrombin is required for effective granuloma formation. Using BM-derived macrophages (BMMs) in vitro, we show that thrombin signaling induced (a) downregulation of cell membrane reverse cholesterol transporter ABCA1 and (b) increased expression of IFNγ receptor and enhanced co-localization within increased areas of cholesterol-rich membrane microdomains. These two key phenotypic changes combined to make thrombin-primed BMMs sensitive to M1 polarization by 1000-fold less IFNγ, compared to resting BMMs. We confirm that changes in ABCA1 expression were directly responsible for the exquisite sensitivity to IFNγ in vitro and for the impact on granuloma formation in vivo. These data indicate that PAR-1 signaling plays a hitherto unrecognized and critical role in DTH responses.