Immunity In Hepatocellular Carcinoma Research Articles

Yangyin Fuzheng Jiedu Prescription exerts anti-tumor immunity in hepatocellular carcinoma by alleviating exhausted T cells

BackgroundYangyin Fuzheng Jiedu Prescription (YFJP), a formulated Chinese herbal medicine, has been used for several decades in the treatment of hepatocellular carcinoma (HCC). Previous studies have demonstrated its anti-tumor efficacy, but the mechanism of action remains uncharacterized. This study aims to evaluate the therapeutic effect of YFJP on H22 tumor-bearing mice. PurposeThis study aimed to evaluate the therapeutic effect of YFJP on H22 tumor-bearing mice. MethodsA total of 50 male H22 tumor-bearing mice were randomly divided into 6 groups and continuous administered either different doses of YFJP or cyclophosphamide (CTX) or normal saline. for 2 weeks. The tumor appearance was observed by taking photos, and the tumor volume, weight, spleen and thymus index were calculated. Morphology of tumor infiltrating lymphocytes and the CD8+ T lymphocytes were detected through HE staining immunohistochemistry respectively. The frequency of CD3+, CD8+ T cell subsets and co-inhibitory receptors PD-1, TIGIT, Tim-3 on CD8+ T cell in spleen, peripheral blood and tumor tissue was performed by flow cytometry. Meanwhile, the killing and apoptotic functions of CD8+ T cells in tumor tissues were also detected by the same method. The levels of cytokines in peripheral blood were detected by Milliplex map mouse highs sensitivity T Cell kit. The expression of T cell transcription factor T-bet and Eomes in tumor tissues were observed by Western blot. ResultsWe found that YFJP could effectively inhibit the solid tumor growth and spleen indexes, but showed little effect on the body weight in the established mouse model of HCC. Furthermore, we investigated the effect of YFJP on the phenotypic and functional changes of T cells. The results showed that YFJP could maintain the high ratio of CD3+ and CD8+ T cells in the peripheral blood, spleen, and tumor tissues while decreasing the expression of programmed cell death-1 (PD-1), T cell immunoglobulin and ITIM domain (TIGIT), T cell immunoglobulin domain and mucin domain-3 (Tim-3) in CD8+ T cells, respectively. Surprisingly, PD-1/Tim-3 double-positive T cells in the peripheral blood and tumor tissues were significantly decreased. Additionally, YFJP restored the cytotoxicity of tumor-infiltrating T cells and delayed their apoptosis in H22 tumor-bearing mice. In addition, treatment with YFJP significantly decreased the expression of inflammatory and immunosuppressive cytokines (including IL-1β, IL-6, and IL-10) in the serum and tumor tissues whereas enhancing that of effector cytokines TNF-α, and IFN-γ. Moreover, T cell transcription factors T-bet increased and Eomes degraded in the tumor tissues upon YFJP treatment. ConclusionIn conclusion, these results demonstrated that YFJP could simultaneously exert anti-tumor immune response in H22 tumor-bearing mice by alleviating T cell exhaustion and immunosuppression.

CGAS-STING Signaling Pathway and Liver Disease: From Basic Research to Clinical Practice.

The cGAS-STING signaling pathway is an autoimmune inflammatory pathway that can trigger the expression of a series of inflammatory factors represented by type 1 interferon. Recent studies have found that the cGAS-STING signaling pathway played a significant role in liver physiology and was closely related to the progress of liver diseases. For example, activating the cGAS-STING signaling pathway could significantly inhibit hepatitis B virus (HBV) replication in vivo. Moreover, the cGAS-STING signaling pathway was also closely associated with tumor immunity in hepatocellular carcinoma (HCC). This review summarized the role of the cGAS-STING signaling pathway in several common liver diseases, especially the current application of the cGAS-STING signaling pathway in liver disease treatment, and prospected its future research, which provided a new idea for understanding and treating liver diseases.

Wnt/β-catenin inhibitor ICG-001 enhances the antitumor efficacy of radiotherapy by increasing radiation-induced DNA damage and improving tumor immune microenvironment in hepatocellular carcinoma

Background and purposeRadiotherapy (RT) has a promising anti-tumor effect depending on its effects on both cancer cells and tumor immune microenvironment (TIME). As one of the most common alterations in hepatocellular carcinoma (HCC), wnt/β-catenin pathway activation, has been reported to induce radioresistance and suppressive TIME. In this study, we aim to explore the effect of wnt/β-catenin inhibitor ICG-001 on radiosensitivity and RT-related TIME of HCC and the underlying mechanism. Materials and methodsC57BL/6 and nude mouse tumor models were used to evaluate the efficacy of different treatments on tumor growth, recurrence and mice survival. Flow cytometry was performed to assess tumor infiltrating lymphocytes (TILs). DNA damage response (DDR) and radioresistance was investigated by colony formation assays, γ-H2AX and micronuclei measurements. ResultsThe addition of ICG-001 to RT exhibited better anti-tumor and survival-prolong efficacy in C57BL/6 than nude mice. TILs analysis revealed that ICG-001 plus RT boosted the infiltration and IFN-γ production of TIL CD8+ T cells, meanwhile reduced the number of Tregs. Moreover, mechanistic study demonstrated that ICG-001 increased the radiation-induced DDR of HCC cells by suppressing p53, thus leading to stronger activation of cGAS/STING pathway. Utilization of cGAS/STING pathway inhibitors impaired the therapeutic effect of combination therapy. Furthermore, combination therapy led to stronger immunologic memory and tumor relapse prevention. ConclusionsOur findings showed that ICG-001 displayed both local and systematic effects by increasing radiosensitivity and improving immunity in HCC, which indicated that ICG-001 might be a potential synergetic treatment for radiotherapy and radioimmunotherapy in HCC patients.

Enhanced radiotherapy efficacy and induced anti-tumor immunity in HCC by improving hypoxia microenvironment using oxygen microcapsules

Hypoxia represents a tremendous barrier for effective radiotherapy in hepatocellular carcinoma (HCC). Treatment strategies for improving hypoxia are of great significance to enhance radiotherapy efficacy. Here, we design polydopamine-nanoparticle-stabilized oxygen microcapsules by interfacial polymerization, which encapsulate oxygen in the core of polydopamine nanoparticle shell, exhibiting excellent biocompatibility and dispersity in aqueous solution. The oxygen microcapsules rapidly increase the oxygen concentration in hypoxia environment and maintain the oxygen concentration for a long time, indicating as an excellent and stable oxygen delivery carrier. Using a mouse model of HCC, local injection of oxygen microcapsules can significantly enhance radiotherapy efficacy. Combined treatments using oxygen microcapsules and radiotherapy reduce the number of tumor associated macrophages (TAMs) while reshaping the pro-tumor M2-type TAMs into anti-tumor M1-type polarization, thus activating T-cell-mediated anti-tumor immune responses (the accumulation of help T lymphocyte 1 cells and cytotoxic T cells) by improving hypoxia in tumors. The results suggest that combined therapy using oxygen microcapsules and radiotherapy, which improves hypoxia in tumors, is a promising strategy against HCC.

Interferon regulatory factor 1(IRF-1) activates anti-tumor immunity via CXCL10/CXCR3 axis in hepatocellular carcinoma (HCC)

Interferon regulatory factor 1 (IRF-1) is a tumor suppressor gene in cancer biology with anti-proliferative and pro-apoptotic effect on cancer cells, however mechanisms of IRF-1 regulating tumor microenvironment (TME) in hepatocellular carcinoma (HCC) remain only partially characterized. Here, we investigated that IRF-1 regulates C-X-C motif chemokine 10 (CXCL10) and chemokine receptor 3 (CXCR3) to activate anti-tumor immunity in HCC. We found that IRF-1 mRNA expression was positively correlated with CXCL10 and CXCR3 through qRT-PCR assay in HCC tumors and in analysis of the TCGA database. IRF-1 response elements were identified in the CXCL10 promoter region, and ChIP-qPCR confirmed IRF-1 binding to promote CXCL10 transcription. IRF-2 is a competitive antagonist for IRF-1 mediated transcriptional effects, and overexpression of IRF-2 decreased basal and IFN-γ induced CXCL10 expression. Although IRF-1 upregulated CXCR3 expression in HCC cells, it inhibited proliferation and exerted pro-apoptotic effects, which overcome proliferation partly mediated by activating the CXCL10/CXCR3 autocrine axis. In vitro and in vivo studies showed that IRF-1 increased CD8+ T cells, NK and NKT cells migration, and activated IFN-γ secretion in NK and NKT cells to induce tumor apoptosis through the CXCL10/CXCR3 paracrine axis. Conversely, this effect was markedly abrogated in HCC tumor bearing mice deficient in CXCR3. Therefore, the IRF-1/CXCL10/CXCR3 axis contributes to the anti-tumor microenvironment in HCC.

Irreversible electroporation induces CD8+ T cell immune response against post-ablation hepatocellular carcinoma growth.

Ablative treatment evokes antitumor immunity, but knowledge on the emerging irreversible electroporation (IRE)-induced immunity in hepatocellular carcinoma (HCC) is limited. To investigate the immune effects induced by IRE and its role in preventing post-ablation HCC progression, a C57BL/6J mouse model bearing subcutaneous H22 hepatoma was employed. IRE treatment significantly suppresses HCC growth, and treated mice are tumor-free after secondary tumor injection and show increased splenic interferon-gamma (IFN-γ)+CD8+ T cells. Additionally, more CD8+ T and dendritic cells, but not CD4+ T, B or NK cells, infiltrate into peri-ablation zones after IRE at day 7. Depletion of CD8+ T cells induces local tumor regrowth and distant metastasis after IRE. Vaccination using IRE-processed H22 lysates prevents tumorigenesis in mice, suggesting a protective immune response. IRE also alleviates immunosuppression by reducing local and splenic Treg and PD-1+ T cells. Regarding mechanism, IRE induces cell necrosis and significant release of danger-associated molecular patterns including ATP, high mobility group box 1 and calreticulin that are pivotal to CD8+ T cell immunity. Together, IRE is a promising approach to evoke CD8+ T cell immunity, which help prevent post-ablation HCC progression.

A Deep Look into the Program of Rapid Tumor Growth of Hepatocellular Carcinoma.

Background and AimsGreat efforts have been made towards increasing our understanding of the pathogenesis involved in hepatocellular carcinoma (HCC), but the rapid growth inherent to such tumor development remains to be explored.MethodsWe identified distinct gene coexpression modes upon liver tumor growth using weighted gene coexpression network analysis. Modeling of tumor growth as signaling activity was employed to understand the main cascades responsible for the growth. Hub genes in the modules were determined, examined in vitro, and further assembled into the growth signature.ResultsWe revealed modules related to the different growth states in HCC, especially the fastest growth module, which is preserved among different HCC cohorts. Moreover, signaling flux in the cell cycle pathway was found to act as a driving force for rapid growth. Twenty hub genes in the module were identified and assembled into the growth signature, and two genes (NCAPH, and RAD54L) were tested for their growth potential in vitro. Genetic alteration of the growth signature affected the global gene expression. The activity of the signature was associated with tumor metabolism and immunity in HCC. Finally, the prognosis effect of the growth signature was reproduced in nine cancers.ConclusionsThese results collectively demonstrate the molecule organization of rapid tumor growth in HCC, which is a highly synergistic process, with implications for the future management of patients.

The EGFR-P38 MAPK axis up-regulates PD-L1 through miR-675-5p and down-regulates HLA-ABC via hexokinase-2 in hepatocellular carcinoma cells.

BackgroundImmunotherapy has been shown to be a promising strategy against human cancers. A better understanding of the immune regulation in hepatocellular carcinoma (HCC) could help the development of immunotherapy against HCC. The epidermal growth factor receptor (EGFR) signaling is frequently activated in HCC and plays important roles in tumorigenesis. However, its role in HCC immunity is still largely unknown. This study aimed to investigate the impact of EGFR signaling on programmed death‐ligand 1 (PD‐L1) and human leukocyte antigen class‐I (HLA‐I) expression in HCC cells and its underlying mechanisms.MethodsThe expression of phosphorylated EGFR (p‐EGFR), PD‐L1, and HLA‐I (HLA‐ABC) in HCC specimens was detected by immunohistochemistry, and their correlations were analyzed. PD‐L1 and HLA‐ABC expression in EGFR‐activated HCC cells were detected by quantitative real‐time PCR, Western blotting, and flow cytometry, and T cell‐mediated lysis was performed to test the immunosuppressive effects of PD‐L1 and HLA‐ABC alterations in HCC cells. Furthermore, the underlying mechanisms of EGFR activation‐induced PD‐L1 up‐regulation and HLA‐ABC down‐regulation were explored by animal experiments, luciferase reporter assay, and gene gain‐ and loss‐of‐function studies.Resultsp‐EGFR was positively correlated with PD‐L1 and negatively correlated with HLA‐ABC expression in HCCs. EGFR activation by its ligand EGF up‐regulated PD‐L1 and down‐regulated HLA‐ABC in HCC cells, which was functionally important and could be abolished by the EGFR inhibitor, gefitinib, both in vitro and in vivo. Mechanistically, enhanced P38 mitogen‐activated protein kinase (MAPK) activation down‐regulated microRNA‐675‐5p (miR‐675‐5p) and up‐regulated glycolysis‐related enzyme hexokinase 2 (HK2); miR‐675‐5p down‐regulation enhanced the stability of PD‐L1 mRNA probably via the 3’‐untranslated region (3’‐UTR) of PD‐L1 and thereby caused PD‐L1 accumulation, and HK2 up‐regulation enhanced aerobic glycolysis and mediated a decrease in HLA‐ABC.ConclusionsThe EGFR‐P38 MAPK axis could up‐regulate PD‐L1 through miR‐675‐5p and down‐regulate HLA‐ABC via HK2 in HCC cells. Our study reveals a novel signaling network that may cause immune suppression in HCC and suggests that EGFR signaling can be targeted for HCC immunotherapy.

Immune implication of FAM83D gene in hepatocellular carcinoma

ABSTRACT FAM83D has been demonstrated to contribute to tumorigenesis. However, its immune effects in hepatocellular carcinoma (HCC) have not been reported. This study aimed to identify the immune role of FAM83D in HCC. FAM83D was over-expressed in HCC and contributed to poor prognosis according to the results of data analysis based on The Cancer Genome Atlas (TCGA). Afterward, the levels of immune cells infiltration were found to be correlated with the expression level of FAM83D in HCC. Through TISIDB and cBioPortal network tools, a total of 82 FAM83D-associated genes were screened out, including 12 immunoinhibitors, 20 immunostimulators and 50 tightly co-expressed genes. TCGA cohort was divided into train set and test set on the basis of the proportion of 7:3. According to FAM83D-associated immunomodulators, a four gene predicted model was established using train set via the Cox regression analysis. Survival analysis demonstrated that the overall survival (OS) of high-risk HCC patients was poor compared with the patients in low-risk group. The reliability and predicted power of the risk-score model were identified by a receiver operating characteristic (ROC) curve. A risk-score based nomogram as well as a calibration curve, which were created could be used to anticipate patient’s 1-year, 3-year and 5-year survival probabilities. The test set was used to validate these results. Our findings showed that the FAM83D gene was related with HCC immunity. The immune marker chosen could be a promising biomarker for HCC prognosis.

Correlation Patterns Among B7 Family Ligands and Tryptophan Degrading Enzymes in Hepatocellular Carcinoma.

Mechanisms of dysfunctional T cell immunity in Hepatocellular Carcinoma (HCC) need to be well defined. B7 family molecules provide both co-stimulatory and co-inhibitory signals to T cells while tryptophan degrading enzymes like Indoleamine 2,3 dioxygenase (IDO) and Tryptophan 2,3 Dioxygenase (TDO) mediate tumor immune tolerance. It is necessary to identify their in situ correlative expression, which informs targets for combined immunotherapy approaches. We investigated B7 family molecules, IDO, TDO and immune responsive effectors in the tumor tissues of patients with HCC (n = 28) using a pathway-focused quantitative nanoscale chip real-time PCR. Four best correlative expressions, namely (1) B7-1 & PD-L2, (2) B7-H2 & B7-H3, (3) B7-2 & PD-L1, (4) PD-L1 & PD-L2, were identified among B7 family ligands, albeit they express at different levels. Although TDO expression is higher than IDO, PD-L1 correlates only with IDO but not TDO. Immune effector (Granzyme B) and suppressive (PD-1 and TGF-β) genes correlate with IDO and B7-1, B7-H5, PD-L2. Identification of the in situ correlation of PD-L1, PD-L2 and IDO suggest their cumulative immuno suppressive role in HCC. The distinct correlations among B7-1, B7-2, B7-H2, and B7-H3, correlation of PD-1 with non-cognate ligands such as B7-1 and B7-H5, and correlation of tumor lytic enzyme Granzyme B with IDO and PD-L2 suggest that HCC microenvironment is complexly orchestrated with both stimulatory and inhibitory molecules which together neutralize and blunt anti-HCC immunity. Functional assays demonstrate that both PDL-1 and IDO synergistically inhibit T cell responses. Altogether, the present data suggest the usage of combined immune checkpoint blocking strategies targeting co-inhibitory B7 molecules and IDO for HCC management.

BRCA1 Is a Novel Prognostic Indicator and Associates with Immune Cell Infiltration in Hepatocellular Carcinoma

The breast cancer gene 1 (BRCA1) is a tumor suppressor, and mutations or epigenetic inactivation will increase the risk of breast cancer oncogenesis. The current research aimed to explore the relationship between BRCA1 expression, prognosis, and tumor immunity in hepatocellular carcinoma (HCC). In this study, BRCA1 expression was analyzed via multiple online databases and its association with clinical characteristics, prognosis and genetic alterations was identified using the original The Cancer Genome Atlas-liver hepatocellular carcinoma cohorts. DNA methylation sites and their prognostic values were analyzed using MethSurv. The correlations between BRCA1 and immune infiltration were investigated via Tumor Immune Estimation Resource. As results, BRCA1 was significantly upregulated in tumor tissues in multiple HCC cohorts. Besides, high BRCA1 expression was correlated with race, advanced T stage, clinical stage, poor tumor grade, MSI status, and worse prognosis. Notably, BRCA1 expression was positively correlated with infiltration levels of B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and dendritic cells. The current findings imply that BRCA1 is associated with prognosis and immune infiltration, laying foundations for in-depth research on the role of BRCA1 in HCC.

Immune Checkpoint Inhibitors as Monotherapy or Within a Combinatorial Strategy in Advanced Hepatocellular Carcinoma.

In advanced-stage hepatocellular carcinoma (HCC), systemic treatment represents the standard therapy. Target therapy has marked a new era based on a greater knowledge of molecular disease signaling. Nonetheless, survival outcomes and long-term response remain unsatisfactory, mostly because of the onset of primary or acquired resistance. More recently, results from clinical trials with immune targeting agents, such as the immune checkpoint inhibitors (ICIs), have shown a promising role for these drugs in the treatment of advanced HCC. In the context of an intrinsic tolerogenic liver environment, since HCC-induced immune tolerance, it is supported by multiple immunosuppressive mechanisms and several clinical trials are now underway to evaluate ICI-based combinations, including their associations with antiangiogenic agents or multikinase kinase inhibitors and multiple ICIs combinations. In this review, we will first discuss the basic principles of hepatic immunogenic tolerance and the evasive mechanism of antitumor immunity in HCC; furthermore we will elucidate the consistent biological rationale for immunotherapy in HCC even in the presence of an intrinsic tolerogenic environment. Subsequently, we will critically report and discuss current literature on ICIs in the treatment of advanced HCC, including a focus on the currently explored combinatorial strategies and their rationales. Finally, we will consider both challenges and future directions in this field.

CD8+CXCR5+T cells infiltrating hepatocellular carcinomas are activated and predictive of a better prognosis.

CD8+ T cells are thought to be the primary cytotoxic lymphocytes exerting antitumor effects. However, few studies have focused on the antitumor effects of CD8+ T cell-mediated humoral immunity or on interactions between CD8+ T cells and B cells in hepatocellular carcinoma (HCC). We found that the frequency of IL-21-producing CD8+CXCR5+ T cells was higher in HCC tumor tissue than in peritumoral tissue or peripheral blood from the same patients or in blood from healthy donors. Moreover, CD8+CXCR5+ T cells migrated in response to supernatants from primary HCC (HCC-SN) cells, and HCC-SN cells also powerfully induced CXCR5 expression in CD8+ T cells and IL-21 expression in CD8+CXCR5+ T cells. CD8+CXCR5+ T cells from HCC patients, but not those from healthy individuals, stimulated CD19+ B cells to differentiate into IgG-producing plasmablasts. These findings reveal that CD8+CXCR5+ T cells strongly infiltrate HCC tumors, and their infiltration is predictive of a better prognosis. Surprisingly, moreover, CD8+CXCR5+ T cells produced IL-21, which induced B cells to differentiate into IgG-producing plasmablasts and to play a key role in humoral immunity in HCC.

Fludarabine as an Adjuvant Improves Newcastle Disease Virus-Mediated Antitumor Immunity in Hepatocellular Carcinoma

In addition to direct oncolysis, oncolytic viruses (OVs) also induce antitumor immunity, also called viro-immunotherapy. Limited viral replication and immune-negative feedback are the major hurdles to effective viro-immunotherapy. In this study, we found that use of an adjuvant of fludarabine, a chemotherapeutic drug for chronic myeloid leukemia, increased the replication of Newcastle disease virus (NDV) by targeting signal transducer and activator of transcription 1 (STAT1), which led to enhanced oncolysis of hepatocellular carcinoma (HCC) cells. Moreover, fludarabine accelerated ubiquitin-proteasomal degradation by enhancing ubiquitylation rather than proteasomal activity. This resulted in accelerated degradation of phosphorylated STAT3 and indoleamine 2, 3-dioxygenase 1 (IDO1), whose expression was induced by NDV infection. In addition, fludarabine significantly increased the NDV-induced infiltration of NK cells and decreased the number of NDV-induced myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment. The aforementioned effects of fludarabine significantly improved NDV-mediated antitumor immunity and prolonged survival in mouse model of HCC. Our findings indicate the utility of fludarabine as an adjuvant for oncolytic anticancer viro-immunotherapy.

GITR ligation enhances functionality of tumor-infiltrating T cells in hepatocellular carcinoma.

No curative treatment options are available for advanced hepatocellular carcinoma (HCC). Anti‐PD1 antibody therapy can induce tumor regression in 20% of advanced HCC patients, demonstrating that co‐inhibitory immune checkpoint blockade has therapeutic potential for this type of cancer. However, whether agonistic targeting of co‐stimulatory receptors might be able to stimulate anti‐tumor immunity in HCC is as yet unknown. We investigated whether agonistic targeting of the co‐stimulatory receptor GITR could reinvigorate ex vivo functional responses of tumor‐infiltrating lymphocytes (TIL) freshly isolated from resected tumors of HCC patients. In addition, we compared GITR expression between TIL and paired samples of leukocytes isolated from blood and tumor‐free liver tissues, and studied the effects of combined GITR and PD1 targeting on ex vivo TIL responses. In all three tissue compartments, CD4+FoxP3+ regulatory T cells (Treg) showed higher GITR−expression than effector T‐cell subsets. The highest expression of GITR was found on CD4+FoxP3hiCD45RA− activated Treg in tumors. Recombinant GITR‐ligand as well as a humanized agonistic anti‐GITR antibody enhanced ex vivo proliferative responses of CD4+ and CD8+ TIL to tumor antigens presented by mRNA‐transfected autologous B‐cell blasts, and also reinforced proliferation, IFN‐γ secretion and granzyme B production in stimulations of TIL with CD3/CD28 antibodies. Combining GITR ligation with anti‐PD1 antibody nivolumab further enhanced tumor antigen‐specific responses of TIL in some, but not all, HCC patients, compared to either single treatment. In conclusion, agonistic targeting of GITR can enhance functionality of HCC TIL, and may therefore be a promising strategy for single or combinatorial immunotherapy in HCC.

Genomic and transcriptional Profiling of tumor infiltrated CD8+ T cells revealed functional heterogeneity of antitumor immunity in hepatocellular carcinoma

ABSTRACTAs key players in HCC antitumor response, the functions of tumor infiltrated CD8+ T cells are significantly affected by surrounding microenvironment. A detailed profiling of their genomic and transcriptional changes could provide valuable insights for both future immunotherapy development and prognosis evaluation. We performed whole exome and transcriptome sequencing on tumor infiltrated CD8+ T cells and CD8+ T cells isolated from other tissue origins (peritumor tissues and corresponding PBMCs) in eight treatment-naive HCC patients. The results demonstrated that transcriptional changes, rather than genomic alterations were the main contributors to the functional alterations of CD8+ T cells in the process of tumor progression. The origins of CD8+ T cells defined their transcriptional landscape, while the tumor infiltrated CD8+ T cells shared more similarity with peritumor-derived CD8+ T cells compared with those CD8+ T cells in blood. In addition, tumor infiltrated CD8+ T cells also showed larger transcriptional heterogeneity among individuals, which was modulated by clinical features such as HBV levels, preoperative anti-viral treatment and the degree of T cell infiltration. We also identified multiple inter-connected pathways involved in the activation and exhaustion of tumor infiltrated CD8+ T cells, among which IL-12 mediated pathway could dynamically reflect the functional status of CD8+ TILs and activation of this pathway indicated a better prognosis. Our results presented an overview picture of CD8+ TILs’ genomic and transcriptional landscape and features, as well as how the functional status of CD8+ TILs correlated with patients’ clinical course.

Genomic and Transcriptional Profiling of Tumor Infiltrated CD8 T Cells Revealed Functional Heterogeneity of Antitumor Immunity in Hepatocellular Carcinoma

As a key player in HCC antitumor response, the functions of tumor infiltrated CD8 T cells are significantly affected by surrounding microenvironment. A detailed profiling of their genomic and transcriptional changes could provide valuable insights for both future immunotherapy development and prognosis evaluation. We performed whole exome and transcriptome sequencing on tumor infiltrated CD8 T cells and CD8 T cells isolated from other tissue origins (peritumor and corresponding PBMCs) in eight treatment-naive HCC patients. The results demonstrated that transcriptional changes, rather than genomic alterations were the main contributors to the functional alterations of CD8 T cells in the process of carcinogenesis. The origins of CD8 T cells defined their transcriptional landscape, while tumor infiltrated CD8 T cells shared more similarity with peritumor-derived CD8 T cells compared with those CD8 T cells in blood. In addition, tumor infiltrated CD8 T cells also showed larger transcriptional heterogeneity among individuals, which was modulated by clinical features such as HBV levels, preoperative anti-viral treatment and the degree of T cell infiltration. We also identified multiple inter-connected pathways involved in the activation and exhaustion of tumor infiltrated CD8 T cells, among which IL-12 mediated pathway could dynamically reflect the functional status of CD8 TILs and activation of this pathway indicated a better prognosis. Our results presented an overview picture of CD8 TILs' genomic and transcriptional landscape and features, as well as how the functional status of CD8 TILs correlated with patients' clinical course. Funding: This work was supported by the Science and Technology major project of Fuzhou (Grant No. RongKe 16 in 2016); the Scientific Foundation of Fujian Health and family planning Department (Grant No. 2018-1-92); the Scientific Foundation of Fuzhou City (Grant No. 2017-S-131-3); the Startup Fund for scientific research, Fujian Medical University (Grant No.2016QH085); the Scientific Foundation of Fuzhou Health Department (Grant No. 2017-S-wq38); the Science and Technology development project of central government guiding local government (Grant No.2017L3017). Declaration of Interests: All authors declare no competitive interests related to this study. Ethical Approval Statement: A written informed consent was obtained from each participant, and all of the study protocols were approved by the Institution Review Board of Mengchao Hepatobiliary Hospital of Fujian Medical University.

Relationship Between PD-L1 Expression and CD8+ T-cell Immune Responses in Hepatocellular Carcinoma.

As PD-1/PD-L1 immune checkpoint inhibitors exhibited promising clinical outcomes in various types of solid tumors, PD-1/PD-L1 blockades have been explored for the treatment of hepatocellular carcinoma (HCC). However, the association of PD-L1 with antitumor immunoregulation is not clearly defined in HCC. Here, we evaluated the characteristics of PD-L1 expression, CD8 T-cell infiltration and their relationship in HCC. A total of 411 resected tumor specimens from HCC patients were immunostained for PD-L1 and CD8. Only 78 (19%) cases showed ≥5% membranous PD-L1 expression on tumor cells, although a significantly positive correlation was found between PD-L1 expression and CD8 T-cell densities. Moreover, patients with higher tumor PD-L1 expression also showed a higher hepatitis B virus load, which was also related to increased CD8 infiltration. Survival analysis suggested that both tumor and stroma PD-L1 status did not significantly affect overall survival or recurrence-free survival in patients. Although high CD8 T-cell density was overall associated with better overall survival and recurrence-free survival, its favorable prognostic value was eliminated by high tumor PD-L1 expression. Further flow cytometric and enzyme-linked immunosorbent assay (ELISA) results from the coculture of HCC cell lines with specific CD8 cytotoxic T lymphocytes (CTLs) demonstrated that CD8 CTLs remarkably upregulated PD-L1 expression on tumor cell lines by HLA class-I specificity, and the overexpression of tumor PD-L1 impaired interferon-γ secretion by CD8 CTLs in a negative feedback regulation mechanism. In conclusion, our findings reveal an interaction between PD-L1 expression and CD8 T-cell immunity in HCC, although PD-L1 is not a prognostic factor for the patients.

Lambda phage-based vaccine induces antitumor immunity in hepatocellular carcinoma

Background and aimsHepatocellular carcinoma (HCC) is a difficult to treat tumor with a poor prognosis. Aspartate β-hydroxylase (ASPH) is a highly conserved enzyme overexpressed on the cell surface of both murine and human HCC cells.MethodsWe evaluated therapeutic effects of nanoparticle lambda (λ) phage vaccine constructs against ASPH expressing murine liver tumors. Mice were immunized before and after subcutaneous implantation of a syngeneic BNL HCC cell line. Antitumor actively was assessed by generation of antigen specific cellular immune responses and the identification of tumor infiltrating lymphocytes.ResultsProphylactic and therapeutic immunization significantly delayed HCC growth and progression. ASPH-antigen specific CD4+ and CD8+ lymphocytes were identified in the spleen of tumor bearing mice and cytotoxicity was directed against ASPH expressing BNL HCC cells. Furthermore, vaccination generated antigen specific Th1 and Th2 cytokine secretion by immune cells. There was widespread necrosis with infiltration of CD3+ and CD8+ T cells in HCC tumors of λ phage vaccinated mice compared to controls. Moreover, further confirmation of anti-tumor effects on ASPH expressing tumor cell growth were obtained in another murine syngeneic vaccine model with pulmonary metastases.ConclusionsThese observations suggest that ASPH may serve as a highly antigenic target for immunotherapy.

The Promise of Immunotherapy in the Treatment of Hepatocellular Carcinoma.

Advanced hepatocellular carcinoma (HCC) has presented a therapeutic challenge. Despite its heterogeneity, which is partially related to its various etiologies, it frequently arises in a background of chronic inflammation, which makes it a potentially excellent candidate for immunotherapeutic approaches. There is evidence of antitumor immunity in HCC as manifested by the cell infiltrate and its association with prognosis, the presence of tumor-associated antigens, and the reports of immune-mediated spontaneous regressions. However, both the liver itself and the tumor environment possess a diverse armamentarium of mechanisms that suppress antitumor immunity. Here, we describe the rationale for immunotherapy in HCC and discuss the emerging clinical data from various immunotherapeutic approaches including checkpoint inhibition, cell therapy, oncolytic viral therapy, and various combinatorial approaches. We also highlight the potential for various modalities to be adapted across different stages of the disease.