Tumor cell density dependent IL-8 secretion induces the fluctuation of tregs/CD8 + T cells infiltration in hepatocellular carcinoma: one prompt for the existence of density checkpoint.

BACKGROUND: Tumor cell density is a basic pathological feature of solid tumors. Chemotherapy, radiotherapy, and targeted therapy reduce tumor cell density, whereas unrestricted tumor cell proliferation promotes this feature. The impact of tumor cells on the microenvironment following changes in tumor cell density is still unclear. In this study, we focused on the response of key immune cell subsets to tumor cell density in hepatocellular carcinoma (HCC). METHODS: We determined the density of tumor and immune cells in the same area by section staining. We then identified potential mediators using polymerase chain reaction (PCR), enzyme-linked immunofluorescence assay (ELISA), 3D and co-culture, flow cytometry, and lentivirus intervention. The mechanism of lactate promotion was verified using lactate tests, bioinformatics, western blotting, and the above methods. The IL-8/DAPK1/lactate/regulatory T cell (Treg) axis was verified using a mouse liver cancer model. Tumor mutation burden was calculated using maftools in R. RESULTS: We found that the Treg/CD8 + T cell ratio is not consistent with tumor cell density in HCC, and a decreased Treg/CD8 + T cell ratio in the range of 5000-6000 cells/mm2 may elicit the possibility for immunotherapy in an immunosuppressive microenvironment. We showed that IL-8 mediates this immune fluctuation and promotes the infiltration of Tregs through the DAPK1/pyruvate kinase activity/lactate axis in HCC. Based on tumor ploidy and mutation burden data, we discussed the potential significance of immune fluctuation in the homeostasis of HCC mutation burden and proposed a "density checkpoint" and "entropy model" to describe this phenomenon. CONCLUSIONS: In summary, we report the mode of infiltration of Tregs/CD8 + T cells in response to tumor cell density and provide a new theoretical basis for IL-8 as a therapeutic target and the selection of an immunotherapy window in HCC.

High Systemic Immune-Inflammation Index, Predicting Early Allograft Dysfunction, Indicates High 90-Day Mortality for Acute-On-Chronic Liver Failure after Liver Transplantation.

INTRODUCTION: The aim of the study was to investigate the relationship between systemic immune-inflammation index (SII) and early allograft dysfunction (EAD) and 90-day mortality after liver transplantation (LT) in acute-on-chronic liver failure (ACLF). METHODS: Retrospective record analysis was done on 114 patients who had LT for ACLF. To identify the ideal SII, the receiver operating characteristic curve was used. The incidence of EAD and 90-day mortality following LT were calculated. The prognostic value of SII was assessed using the Kaplan-Meier technique and the Cox proportional hazards model. RESULTS: The cut-off for SII was 201.5 (AUC = 0.728, p < 0.001). EAD occurred in 40 (35.1%) patients of the high SII group and 5 (4.4%) patients of the normal SII group, p < 0.001. 18 (15.8%) deaths occurred in the high SII group and 2 (1.8%) deaths occurred in the normal SII group, p = 0.008. The multivariate analysis demonstrated that SII ≥201.5, MELD ≥27 were independent prognostic factors for 90-day mortality after LT. CONCLUSION: SII predicts the occurrence of EAD and is an independent risk factor for 90-day mortality after LT.

Small-For-Size Syndrome and Graft Inflow Modulation Techniques in Liver Transplantation.

BACKGROUND: Partial liver transplantation has recently been proposed to alleviate organ shortages. However, transplantation of a small-for-size graft is associated with an increased risk of posttransplant hepatic dysfunction, commonly referred to as small-for-size syndrome (SFSS). This review describes the etiology, pathological features, clinical manifestations, and diagnostic criteria of SFSS. Moreover, we summarize strategies to improve graft function, focusing on graft inflow modulation techniques. Finally, unmet needs and future perspectives are discussed. SUMMARY: In fact, posttransplant SFSS can be attributed to various factors such as preoperative status of the recipients, surgical techniques, donor age, and graft quality, except for graft size. With targeted improvement measures, satisfactory clinical outcomes can be achieved in recipients at increased risk of SFSS. Given the critical role of relative portal hyperperfusion in the pathogenesis of SFSS, various pharmacological and surgical treatments have been established to reduce or partially divert excessive portal inflow, and recipients will benefit from individualized therapeutic regimens after careful evaluation of benefits against potential risks. However, there remain unmet needs for further research into different aspects of SFSS to better understand the correlation between portal hemodynamics and patient outcomes. KEY MESSAGES: Contemporary transplant surgeons should consider various donor and recipient factors and develop case-specific prevention and treatment strategies to improve graft and recipient survival rates.

Gly-tRF enhances LCSC-like properties and promotes HCC cells migration by targeting NDFIP2.

BACKGROUND: Accumulating evidence demonstrates that tRFs (tRNA-derived small RNA fragments) and tiRNAs (tRNA-derived stress-induced RNA), an emerging category of regulatory RNA molecules derived from transfer RNAs (tRNAs), are dysregulated in in various human cancer types and play crucial roles. However, their roles and mechanisms in hepatocellular carcinoma (HCC) and liver cancer stem cells (LCSCs) are still unknown. METHODS: The expression of glycine tRNA-derived fragment (Gly-tRF) was measured by qRT-PCR. Flow cytometric analysis and sphere formation assays were used to determine the properties of LCSCs. Transwell assays and scratch wound assays were performed to detect HCC cell migration. Western blotting was conducted to evaluate the abundance change of Epithelial-mesenchymal transition (EMT)-related proteins. Dual luciferase reporter assays and signalling pathway analysis were performed to explore the underlying mechanism of Gly-tRF functions. RESULTS: Gly-tRF was highly expressed in HCC cell lines and tumour tissues. Gly-tRF mimic increased the LCSC subpopulation proportion and LCSC-like cell properties. Gly-tRF mimic promoted HCC cell migration and EMT. Loss of Gly-tRF inhibited HCC cell migration and EMT. Mechanistically, Gly-tRF decreased the level of NDFIP2 mRNA by binding to the NDFIP2 mRNA 3' UTR. Importantly, overexpression of NDFIP2 weakened the promotive effects of Gly-tRF on LCSC-like cell sphere formation and HCC cell migration. Signalling pathway analysis showed that Gly-tRF increased the abundance of phosphorylated AKT. CONCLUSIONS: Gly-tRF enhances LCSC-like cell properties and promotes EMT by targeting NDFIP2 and activating the AKT signalling pathway. Gly-tRF plays tumor-promoting role in HCC and may lead to a potential therapeutic target for HCC.

Retinoic acid-inducible gene-1 knockdown induces immature properties in dendritic cells and prolongs the survival time of allograft mice.

BACKGROUND: The mature state of dendritic cells (DCs) determines their ability to regulate immune responses. Retinoic acid-inducible gene-1 (RIG-1) plays a critical role in DC activation and maturation. RIG-1 activation triggers mitogen-activated protein kinase and nuclear factor-kappa B signal transduction. In this study, we aimed to investigate the effects of inhibiting RIG-1 expression in DCs and its potential in inducing immune tolerance. METHODS: DCs were transduced with the recombinant lentiviral vector (Lv) to inhibit RIG-1 expression. A murine islet and skin transplantation model were constructed to find out whether DC-DDX58-RNAi could prolong allograft survival. The phenotypes of DCs and T-cells were analyzed using flow cytometry. Cytokines in serum were detected by the enzyme-linked immunosorbent assay. Protein levels were determined by Western blot. RESULTS: RIG-1-deficient DCs had low expression of costimulatory molecules and major histocompatibility complex and a strong phagocytic ability. DC-DDX58-RNAi induced regulatory T cell differentiation in the transplant recipient spleens. The DC-DDX58-RNAi-treated recipients showed satisfactory islet allograft function and longer survival time. CONCLUSION: Inhibition of RIG-1 with DDX58-RNAi prevented the activation and maturation of the DCs, affected T cell differentiation, protected the biological function of the allograft, and prolonged graft survival. These findings may have important therapeutic implications for new immunomodulatory regimens.

Optimal conditions for adenoviral transduction of immature dendritic cells without affecting the tolerogenic activity of DC-based immunotherapy.

Dendritic cells (DCs) play a pivotal role in maintaining immune tolerance. Using recombinant adenovirus (rAd) to deliver vectors to immature dendritic cells (imDCs) is an important method for studying the tolerogenic function of DCs. We found that using RPMI medium and a higher MOI during transduction increased the expression of CD80, CD86, and MHC-II on the surface of imDCs. Our data reveal a significant increase in the secretion of the pro-inflammatory cytokine IL-6 in the group showing the most pronounced phenotypic changes. In the mouse heart transplant model, imDCs with unstable phenotype and function due to adenoviral transduction resulted in an increased proportion of Th1 and Th17 cells in recipients. However, these effects can be managed, and our proposed optimized transduction strategy significantly minimizes these adverse effects. Our study holds significant implications for the development and optimization of immunotherapy utilizing tolerogenic dendritic cells.

Inhibition of DAI refrains dendritic cells from maturation and prolongs murine islet and skin allograft survival.

Introduction: Central to allograft rejection is the T cell-mediated adaptive immune response initiated by activated dendritic cells (DCs). Previous studies have shown that the DNA-dependent activator of IFN regulatory factors (DAI) is involved in the maturation and activation of DCs. Therefore, we hypothesized that inhibition of DAI could prevent DCs from maturation and prolong murine allograft survival. Methods: Donor mouse bone marrow-derived dendritic cells (BMDCs) were transduced with the recombinant adenovirus vector (AdV-DAI-RNAi-GFP) to inhibit DAI expression (DC-DAI-RNAi), and the immune cell phenotype and function of DC-DAI-RNAi upon lipopolysaccharide (LPS) stimulation were evaluated. Then DC-DAI-RNAi was injected into recipient mice before islet transplantation and skin transplantation. The survival times of islet and skin allograft were recorded and the proportions of T cell subsets in spleen and secretion levels of cytokines in serum were measured. Results: We identified that DC-DAI-RNAi inhibited the expression of main co-stimulatory molecules and MHC-II, exhibited strong phagocytic ability, and secreted high levels of immunosuppressive cytokines and low levels of immunostimulating cytokines. Recipient mice treated with DC-DAI-RNAi had longer islet and skin allograft survival times. In the murine islet transplantation model, we observed an increase in Treg cells proportion, a reduction in Th1 and Th17 cells proportions in spleen, and similar trends in their secreted cytokines in serum in the DC-DAI-RNAi group. Conclusion: Inhibition of DAI by adenovirus transduction inhibits the maturation and activation of DCs, affects the differentiation of T cell subsets as well as their secreted cytokines, and prolongs allograft survival.

Dysregulation of innate cell types in the hepatic immune microenvironment of alcoholic liver cirrhosis.

Introduction: The risk of alcoholic cirrhosis increases in a dose- and time-dependent manner with alcohol consumption and ethanol metabolism in the liver. Currently, no effective antifibrotic therapies are available. We aimed to obtain a better understanding of the cellular and molecular mechanisms involved in the pathogenesis of liver cirrhosis. Methods: We performed single-cell RNA-sequencing to analyze immune cells from the liver tissue and peripheral blood form patients with alcoholic cirrhosis and healthy controls to profile the transcriptomes of more than 100,000 single human cells and yield molecular definitions for non-parenchymal cell types. In addition, we performed single-cell RNA-sequencing analysis to reveal the immune microenvironment related to alcoholic liver cirrhosis. Hematoxylin and eosin, Immunofluorescence staining and Flow cytometric analysis were employed to study the difference between tissues and cells with or without alcoholic cirrhosis. Results: We identified a fibrosis-associated M1 subpopulation of macrophages that expands in liver fibrosis, differentiates from circulating monocytes, and is pro-fibrogenic. We also define mucosal-associated invariant T (MAIT) cells that expand in alcoholic cirrhosis and are topographically restricted to the fibrotic niche. Multilineage modeling of ligand and receptor interactions between the fibrosis-associated macrophages, MAIT, and NK cells revealed the intra-fibrotic activity of several pro-fibrogenic pathways, including responses to cytokines and antigen processing and presentation, natural killer cell-mediated cytotoxicity, cell adhesion molecules, Th1/Th2/Th17 cell differentiation, IL-17 signaling pathway, and Toll-like receptor signaling pathway. Discussion: Our work dissects unanticipated aspects of the cellular and molecular basis of human organ alcoholic fibrosis at the single-cell level and provides a conceptual framework for the discovery of rational therapeutic targets in liver alcoholic cirrhosis.

Parvovirus B19-Associated Severe Anemia in Adult Liver Transplant Recipients: A Case Series and Review of the Literature.

Background: Parvovirus B19 (B19V) infection is a rare cause of severe anemia in liver transplant recipients. However, few studies have systematically reviewed reported cases and summarized experience in managing this disease. Objective: We described a retrospective case series of eight adult liver transplant recipients with B19V-associated severe anemia and performed a literature review of epidemiology, etiology, clinical courses, diagnosis, treatment options available, and outcomes of B19V-associated anemia in adult liver transplant recipients. Patients and Methods: We systematically reviewed articles describing adult liver transplant recipients with B19V-associated anemia from PubMed and ScienceDirect databases from database inception to May 2022. Results: Eight articles containing 23 cases were identified in addition to eight cases from our center for a total of 31 patients (mean age, 45.7 ± 9.7 years; 74.2% male). Eighty-seven percent developed transfusion-dependent anemia within two months after liver transplantation (LT). Fever and progressive anemia are among the major manifestations. Intravenous immunoglobulin (IVIG)-based therapy was given to all patients and the treatment protocols varied among different centers. Except for two cases who died of comorbidities, 17 patients obtained long-term recovery from anemia after one course of treatment and six (19%) experienced relapses that were reversed by repeated courses of IVIG therapy. Two recipients presented with IVIG-associated side effects and two developed acute cellular rejection (ACR) after reduction of immunosuppression. Conclusions: B19V infection should be suspected early as a cause of severe anemia of unknown etiology in adult liver transplant recipients. The clearance of B19V typically lags behind recovery of anemia, and inadequate clearance of virus after cessation of IVIG appears to be a potential risk of anemia recurrence. Moreover, more attention should be paid to the side effects of high-dose IVIG infusion and ACR because of reduction of immunosuppression.