Tumor-infiltrating T-Lymphocyte immunity-related immune tolerance and anti–programmed cell death protein 1/ligand of programmed cell death protein 1 therapy for advanced hepatocellular carcinoma

Systemic therapy has become the standard treatment for patients with advanced hepatocellular carcinoma(HCC)whose treatment options are limited.However,the long-term patient response to drugs and the survival outcomes remain a concern.With increasing exploration of the HCC microenvironment,particularly in terms of T lymphocyte immunity,a new era of immunomolecular targeted therapy,based on molecular signaling,has arrived for advanced HCC.In the study of immune tolerance of the intrinsic HCC microenvironment,we found that multiple immunosuppressive mechanisms and immune checkpoint inhibitors,such as anti–programmed cell death protein 1/ligand of programmed cell death protein 1 therapy,have improved clinical outcomes in some patients with advanced HCC.Furthermore,various combination therapies have been investigated,and HCC types have been categorized into different types based on anti–programmed cell death protein 1(PD-1)/ligand of programmed cell death protein 1(PD-L1)treatment.In this paper,we first discuss the tumor-infiltrating T lymphocyte immunity and immune tolerance of HCC.We then clarify the basic mechanism of anti–PD-1/PD-L1 therapy and discuss the types of HCC based on anti–PD-1/PD-L1 therapy.Thereafter,we explain the relevant studies and mechanisms of combination therapy of anti–PD-1/PD-L1 with antiangiogenesis drugs or multikinase kinase inhibitors,anti–T lymphocyte–related signaling pathways in HCC,and other anti-CD8+T cell immune checkpoints.In this way,this review offers a deeper understanding of anti–PD-1/PD-L1 immunotherapy for advanced HCC,in order to provide better individualized treatments for patients with advanced HCC.

Immunotherapy for advanced or recurrent hepatocellular carcinoma

Hepatocellular carcinoma(HCC)is associated with high morbidity and mortality,and is prone to intra-and extrahepatic metastasis due to the anatomical and functional characteristics of the liver.Due to the complexity and high relapse rate associated with radical surgery or radiofrequency ablation,immune checkpoint inhibitors(ICIs)are increasingly being used to treat HCC.Several immunotherapeutic agents,along with their combinations,have been clinically approved to treat advanced or recurrent HCC.This review discusses the leading ICIs in practice and those currently undergoing randomized phase 1-3 trials as monotherapy or combination therapy.Furthermore,we summarize the rapidly developing alternative strategies such as chimeric antigen receptor-engineered T cell therapy and tumor vaccines.Combination therapy is a promising potential treatment option.These immunotherapies are also summarized in this review,which provides insights into the advantages,limitations,and novel angles for future research in establishing viable and alternative therapies against HCC.

Combined TIM-3 and PD-1 blockade restrains hepatocellular carcinoma development by facilitating CD4+ and CD8+T cellmediated antitumor immune responses

BACKGROUND Immune checkpoint inhibitors(ICIs)targeting programmed cell death protein 1(PD-1)and T cell immunoglobulin and mucin domain-containing protein 3(TIM-3)are beneficial to the resumption of anti-tumor immunity response and hold extreme potential as efficient therapies for certain malignancies.However,ICIs with a single target exhibit poor overall response rate in hepatocellular carcinoma(HCC)patients due to the complex pathological mechanisms of HCC.AIM To investigate the effects of combined TIM-3 and PD-1 blockade on tumor development in an HCC mouse model,aiming to identify more effective immunotherapies and provide more treatment options for HCC patients.METHODS The levels of PD-1 and TIM-3 on CD4+and CD8+T cells from tumor tissues,ascites,and matched adjacent tissues from HCC patients were determined with flow cytometry.An HCC xenograft mouse model was established and treated with anti-TIM-3 monoclonal antibody(mAb)and/or anti-PD-1 mAb.Tumor growth in each group was measured.Hematoxylin and eosin staining and immunohistochemical staining were used to evaluate T cell infiltration in tumors.The percentage of CD4+and CD8+T cells in tissue samples from mice was tested with flow cytometry.The percentages of PD-1+CD8+,TIM-3+CD8+,and PD-1+TIM-3+CD8+T cells was accessed by flow cytometry.The levels of the cytokines including tumor necrosis factor alpha(TNF-α),interferon-γ(IFN-γ),interleukin(IL)-6,and IL-10 in tumor tissues were gauged with enzyme-linked immunosorbent assay kits.RESULTS We confirmed that PD-1 and TIM-3 expression was substantially upregulated in CD4+and CD8+T cells isolated from tumor tissues and ascites of HCC patients.TIM-3 mAb and PD-1 mAb treatment both reduced tumor volume and weight,while combined blockade had more substantial anti-tumor effects than individual treatment.Then we showed that combined therapy increased T cell infiltration into tumor tissues,and downregulated PD-1 and TIM-3 expression on CD8+T cells in tumor tissues.Moreover,combined treatment facilitated the production of T cell effector cytokines TNF-α and IFN-γ,and reduced the production of immunosuppressive cytokines IL-10 and IL-6 in tumor tissues.Thus,we implicated that combined blockade could ameliorate T cell exhaustion in HCC mouse model.CONCLUSION Combined TIM-3 and PD-1 blockade restrains HCC development by facilitating CD4+ and CD8+T cell-mediated antitumor immune responses.

肝硬化结节及小肝癌患者3.0T MRI检查结果分析

目的评价3.0 T MRI检查在肝硬化再生性结节(RN)、异型增生性结节(DN)和小肝癌(SHCC)诊断和鉴别诊断中价值。方法回顾性分析经病理检查确诊的69例RN、DN及SHCC患者的3.0 T MRI平扫及容积采集技术(LAVA)三期动态增强扫描特点。结果 RN主要表现为T2WI低信号,动态增强方式呈"缓慢上升型";DN主要表现为T2WI高、低信号,信号较均匀结节的动态方式以"速升缓降型"为主,可有"结中结"(特征表现);SHCC主要表现为T1WI低信号、T2WI高信号,动态增强方式为"速升速降型"。结论肝硬化结节及小肝癌在3.0 T MRI上各有较为典型的表现,临床可据此进行诊断与鉴别诊断;对其他强化类型的肝脏病灶应结合肿瘤血管的扭曲增粗及包膜等表现排除SHCC。

多排螺旋CT三期增强与3.0T MR多期动态增强扫描诊断肝细胞癌的价值分析

目的探讨3.0T MR多期动态增强扫描与多排螺旋CT三期增强扫描诊断肝细胞癌的价值。方法2015年9月~2017年9月我院收治的98例肝占位患者,分别行MRI和CT增强扫描检查,采用Kappa检验分析三名医师诊断评分之间的一致性,采用受试者工作特征曲线(ROC)并计算曲线下面积(AUC)判断影像学检查指标的诊断效能。结果在98例肝占位患者中,病理学检查诊断HCC 86例,再生结节10例,嗜酸性肉芽肿2例;在86例HCC患者中,存在97个病灶,其中32个病灶<1.0 cm;在32个<1.0 cm的HCC病灶中,MRI动态增强扫描发现31个(96.9%),而多排螺旋CT增强扫描仅发现26个(81.3%,P<0.05),其余65个超过1.0 cm的病灶均被两种扫描方法发现和诊断;ROC曲线分析发现,MRI动态增强扫描和多排螺旋CT增强扫描诊断HCC的AUC分别为0.962和0.824,两者相比具有显著性差异(t=3.106,P<0.05),MRI动态增强扫描诊断HCC的敏感性、特异性和准确性分别为99.0%、91.0%和96.2%,显著优于多排螺旋CT增强扫描检查(分别为93.8%、72.0%和85.3%,x2=19.587,P<0.05)。结论 3.0T MR多期动态增强扫描能够更清晰地反映HCC病灶的细微形态特点和强化特征,对于诊断小HCC的价值优于CT增强扫描。

Specific CD8^+ T cell response immunotherapy for hepatocellular carcinoma and viral hepatitis

Hepatocellular carcinoma (HCC), chronic hepatitis B (CHB) and chronic hepatitis C (CHC) are characterized by exhaustion of the specific CD8+ T cell response. This process involves enhancement of negative co-stimulatory molecules, such as programmed cell death protein-1 (PD-1), cytotoxic T-lymphocyte antigen-4 (CTLA-4), 2B4, Tim-3, CD160 and LAG-3, which is linked to intrahepatic overexpression of some of the cognate ligands, such as PD-L1, on antigen presenting cells and thereby favouring a tolerogenic environment. Therapies that disrupt these negative signalling mechanisms represent promising therapeutic tools with the potential to restore reactivity of the specific CD8+ T cell response. In this review we discuss the impressive in vitro and in vivo results that have been recently achieved in HCC, CHB and CHC by blocking these negative receptors with monoclonal antibodies against these immune checkpoint modulators. The article mainly focuses on the role of CTLA-4 and PD-1 blocking monoclonal antibodies, the first ones to have reached clinical practice. The humanized monoclonal antibodies against CTLA-4 (tremelimumab and ipilimumab) and PD-1 (nivolumab and pembrolizumab) have yielded good results in testing of HCC and chronic viral hepatitis patients. Trelimumab, in particular, has shown a significant increase in the time to progression in HCC, while nivolumab has shown a remarkable effect on hepatitis C viral load reduction. The research on the role of ipilimumab, nivolumab and pembrolizumab on HCC is currently underway.

Roles of Tregs in development of hepatocellular carcinoma:A meta-analysis

AIM: To assess systematically the association between regulatory T cells (Tregs) and hepatocellular carcinoma (HCC).

Changes of CD4^+ CD25^+ Regulatory T Cells in Peripheral Blood in Patients with Hepatocellular Carcinoma Before and after TACE

This study investigated the changes of CD4＋ CD25＋ regulatory T cells （Tregs） in periph-eral blood of patients with hepatocellular carcinoma before and after transcatheter arterial chemoem-bolization （TACE）. The proportion of CD4＋ CD25＋ Tregs among CD4＋ T lymphocytes in peripheral blood of 33 patients with hepatocellular carcinoma was determined by flow cytometry before, 1 week and 1 month after TACE. And 25 healthy volunteers served as control. One month after TACE, the patients were divided into two groups： 22 in group A, who were in stable condition or getting better; and 10 in group B, who were deteriorating. One patient died and was excluded. The results showed that the percentage of CD4＋CD25＋ Tregs among CD4＋ T lymphocytes did not significantly change in the 33 patients 1 week after TACE as compared with that before TACE, however, the difference was significant （P〈0.01） between the patients with hepatocellular carcinoma and the healthy subjects. The percentage of CD4＋ CD25＋ Tregs among CD4＋ T lymphocytes in group A 1 month after TACE was decreased significantly in comparison with that before and 1 week after TACE （P〈0.01）, whereas, that in group B was increased significantly 1 month after TACE （P〈0.01）. It was concluded that patients with hepatocellular carcinoma had a higher proportion of CD4＋CD25＋ Tregs in peripheral blood. TACE did not significantly affect the level of CD4＋ CD25＋ Tregs within short time （such as 1 week）. The proportion of CD4＋CD25＋ Tregs in peripheral blood 1 month after TACE was related to the prognosis of hepatocellular carcinoma.

In-vitro activation of cytotoxic T lymphocytes by fusion of mouse hepatocellular carcinoma cells and lymphotactin gene-modified dendritic cells

AIM： To investigate the in-vitro activation of cytotoxic T lymphocytes （CTLs） by fusion of mouse hepatocellular carcinoma （HCC） ceils and lymphotactin gene-modified dendritic cells （DCs）. METHODS： Lymphotactin gene modified DCs （DCLptn） were prepared by lymphotactin recombinant adenovirus transduction of mature DCs which differentiated from mouse bone marrow cells by stimulation with granulocyte/macrophage colony-stimulating factor （GM- CSF）, interleukin-4 （IL-4） and tumor necrosis factor alpha （TNF-α）. DCLptn and H22 fusion was prepared using 50% PEG. Lymphotactin gene and protein expression levels were measured by RT-PCR and ELISA, respectively. Lymphotactin chemotactic responses were examined by in-vitro chemotaxis assay. In-vitro activation of CTl_s by DCLptn/H22 fusion was measured by detecting CD25 expression and cytokine production after autologous T cell stimulation. Cytotoxic function of activated T lymphocytes stimulated with DCLptn/H22 cells was determined by LDH cytotoxicity assay. RESULTS： Lymphotactin gene could be efficiently transduced to DCs by adenovirus vector and showed an effective biological activity. After fusion, the hybrid DCLptn/H22 cells acquired the phenotypes of both DCLptn and H22 cells. In T cell proliferation assay, flow cytometry showed a very high CD25 expression, and cytokine release assay showed a significantly higher concentration of IFN-α, and IL-2 in DCLptn/H22 group than in DCLptn, DCLptn＋H22, DC/H22 or H22 groups. Cytotoxicity assay revealed that T cells derived from DCLptn/H22 group had much higher anti-tumor activity than those derived from DCLptn, H22, DCLptn ＋ H22, DC/H22 groups. CONCLUSION： Lymphotactin gene-modified dendritoma induces T-cell proliferation and strong CTL reaction against allogenic HCC cells. Immunization-engineered fusion hybrid vaccine is an attractive strategy in prevention and treatment of HCC metastases.

Dendritic cells pulsed with hsp70-peptide complexes derived from human hepatocellular carcinoma induce specific anti-tumor immune responses

AIM： To investigate the anti-tumor effect of dendritic cells （DCs） pulsed with hsp70-peptide complexes derived from human hepatocellular carcinoma （HCC） cells on human T cells. METHODS： Hsp70-peptide complexes were purified from human HCC cells with column chromatography using ADP-agarose and DEAE-Sepharose. DCs were derived from peripheral blood mononuclear cells of healthy donors in the presence of human GM-CSF and IL-4. The anti-tumor effect of DCs pulsed with hsp70-peptide complexes on human T-cell was assayed by CTL and enzyme-linked immunospot （ELISPOT） tests. RESULTS： Hsp70-peptide complexes derived from human HCC cells activated phenotypic and functional maturation of DCs. The matured DCs stimulated a high level of autologous T-cell proliferation and type Ⅰ cytokine secretion, and induced HCC-specific cytotoxic T lymphocytes （CTLs）, which specifically killed HCC cells by a MHC class Ⅰ restricted mechanism. CONCLUSION： Hsp70-peptide complexes derived from human HCC cells can serve as a potent tumor antigen source for pulsing DCs, the pulsed DCs are very effective in activating specific T-cell responses against HCC cells. 2005 The WJG Press and Elsevier Inc. All rights reserved

Risk for hepatocellular carcinoma in the course of chronic hepatitis B virus infection and the protective effect of therapy with nucleos(t)ide analogues

Hepatocellular carcinoma(HCC) is a major health problem worldwide, representing one of the leading causes of death. Chronic hepatitis B virus(HBV) infection(CHB) is the most important etiologic factor of this tumor, accounting for the development of more than50% of the cases in the world. Primary prevention ofHCC is possible by hepatitis B vaccination conferring protection from HBV infection. However, according to the World Health Organization Hepatitis B Fact sheet N° 204(update of July 2014) globally there exists a large pool of > 240 million people chronically infected with HBV who are at risk for development of HCC. These individuals represent a target population for secondary prevention both of cirrhosis and of HCC. Since ongoing HBV replication in CHB is linked with the progression of the underlying liver disease to cirrhosis as well as with the development of HCC, effective antiviral treatment in CHB has also been evaluated in terms of secondary prevention of HCC. Currently, most patients with active CHB are subjected to long term treatment with the first line nucleos(t)ide analogues entecavir and tenofovir. These compounds are of high antiviral potency and have a high barrier to HBV resistance compared to lamivudine, adefovir dipivoxil and even telbivudine. Many studies have shown that patients under antiviral treatment, especially those in virological remission, develop less frequently HCC compared to the untreated ones. However, the risk for development of HCC cannot be eliminated. Therefore, surveillance for the development of HCC of patients with chronic hepatitis B must be lifelong or until a time in the future when new treatments will be able to completely eradicate HBV from the liver particularly in the early stages of CHB infection. In this context, the aim of this review is to outline the magnitude of the risk for development of HCC among patients with CHB, in the various phases of the infection and in relation to virus, host and environmental factors as evaluated in the world literature. Moreover, the benefits of antiviral treatment of CHB with nucleos/tide analogs, which have changed the natural history of the disease and have reduced but not eliminated the risk of HCC are also reviewed.

Role of the tissue microenvironment as a therapeutic target in hepatocellular carcinoma

Hepatocellular carcinoma is difficult to treat,primarilybecause the underlying molecular mechanisms drivingclinical outcome are still poorly understood.Growingevidence suggests that the tissue microenvironmenthas a role in the biological behavior of the tumor.Themain clinical issue is to identify the best target fortherapeutic approaches.Here,we discuss the hypothesis that the entire tissue microenvironment might beconsidered as a biological target.However,the tissuemicroenvironment consists of several cellular and biochemical components,each of which displays a distinctbiological activity.We discuss the major components ofthis environment and consider how they may interactto promote tumor/host crosstalk.

Implications of biomarkers in human hepatocellular carcinoma pathogenesis and therapy

Hepatocellular carcinoma(HCC)is one of the most frequent tumors worldwide and accounts for approximately one-third of all malignancies.In the past decade,advances have been made to improve the prognosis of HCC,including improvement in the clinical diagnosis of early-stage HCC using molecular biomarkers and molecular-targeted therapy to treat advanced HCC.However,the diagnosis,pathogenesis and targeted therapy of HCC are not completely independent,and should be comprehensively studied.For example,a number of tumor markers provide useful clinical information not only for prognosis,but also in pathogenesis and treatment efficacy.Therefore,this review will focus on the role of several specific biomarkers implicated in the pathogenesis of HCC and several promising molecular-targeted drugs that target the biomarkers of HCC.

Role of transforming growth factor-beta1-smad signal transduction pathway in patients with hepatocellular carcinoma

AIM： To explore the role of transforming growth factorbeta1 （TGF-β1）-smad signal transduction pathway in patients with hepatocellular carcinoma. METHODS： Thirty-six hepatocellular carcinoma specimens were obtained from Qidong Liver Cancer Institute and Department of Pathology of the Second Affiliated Hospital of Nanjing Medical University. All primary antibodies （polyclonal antibodies） to TGF-β1, type H Transforming growth factor-beta receptor （TβR-Ⅱ）, nuclear factor-kappaB （NF-KB）, CD34, smad4 and smad7, secondary antibodies and immunohistochemical kit were purchased from Zhongshan Biotechnology Limited Company （Beijing, China）. The expressions of TGF-β1, TβR-Ⅱ, NF- KB, smad4 and smad7 proteins in 36 specimens of hepatocellular carcinoma （HCC） and its adjacent tissue were separately detected by immunohistochemistry to observe the relationship between TGF-β1 and TβR-Ⅱ, between NF-KB and TGF-β1, between smad4 and smad7 and between TGF-β1 or TβR-Ⅱ and microvessel density （MVD）. MVD was determined by labelling the vessel endothelial cells with CD34. RESULTS： The expression of TGF-β1, smad7 and MVD was higher in HCC tissue than in adjacent HCC tissue （P〈0.01, P〈0.05,P〈0.01 respectively）. The expression of TβR-Ⅱ and smad4 was lower in HCC tissue than in its adjacent tissue （P〈0.01, P〈0.05 respectively）. The expression of TGF-β1 protein and NF-KB protein was consistent in HCC tissue. The expression of TGF-β1 and MVD was also consistent in HCC tissue. The expression of TIER- Ⅱ was negatively correlated with that of MVD in HCC tissue. CONCLUSION： The expressions of TGF-IB1, TβR- Ⅱ, NF-KB, smad4 and smad7 in HCC tissue, which are major up and down stream factors of TGF-β1-smad signal transduction pathway, are abnormal. These factors are closely related with NVD and may play an important role in HCC angiogenesis. The inhibitory action of TGF-β1 is weakened in hepatic carcinoma cells because of abnormality of TGF-β1 receptors （such as TIBR- Ⅱ） and postreceptors （such as smad4 and smad7）. NF-KB may cause activation and production of TGF-β1.

Hepatocellular carcinoma progression in hepatitis B virus-related cirrhosis patients receiving nucleoside(acid)analogs therapy:A retrospective cross-sectional study

BACKGROUND Antiviral therapy cannot completely block the progression of hepatitis B to hepatocellular carcinoma(HCC).Furthermore,there are few predictors of early HCC progression and limited strategies to prevent progression in patients with HBV-related cirrhosis who receive nucleos(t)ide analog(NA)therapy.AIM The study aim was to clarify risk factors and the diagnostic value of alphafetoprotein(AFP)for HCC progression in NA-treated hepatitis B virus(HBV)-related cirrhosis patients.METHODS In this retrospective cross-sectional study,we analyzed the clinical data of 266 patients with HBV-related cirrhosis who received NA treatment between February 2014 and April 2020 at Zhejiang Provincial People’s Hospital.The patients were divided into two groups,145 who did not progress to HCC(No-HCC group),and 121 who progressed to HCC during NA treatment(HCC group).The logistic regression analysis was used to analyze the risk factors of HCC progression.The diagnostic value of AFP for HCC was evaluated by receiver operating characteristic(ROC)curve analysis.RESULTS Univariate analysis showed that age≥60 years(P=0.001),hepatitis B and alcoholic etiology(P=0.007),smoking history(P<0.001),family history of HBV-related HCC(P=0.002),lamivudine resistance(P=0.011),HBV DNA negative(P=0.023),aspartate aminotransferase>80 U/L(P=0.002),gamma-glutamyl transpeptidase>120 U/L(P=0.001),alkaline phosphatase>250 U/L(P=0.001),fasting blood glucose(FBG)≥6.16(mmol/L)(P=0.001)and Child-Pugh class C(P=0.005)were correlated with HCC progression.In multivariate analysis,age≥60 years[hazard ratio(HR)=3.089,95%confidence interval(CI):1.437-6.631,P=0.004],smoking history(HR=4.001,95%CI:1.836-8.716,P<0.01),family history of HBV-related HCC(HR=6.763,95%CI:1.253-36.499,P<0.05),lamivudine resistance(HR=2.949,95%CI:1.207-7.208,P=0.018),HBV DNA negative(HR=0.026,95%CI:0.007-0.139,P<0.01),FBG≥6.16 mmol/L(HR=7.219,95%CI:3.716-14.024,P<0.01)were independent risk factors of HCC progression.ROC of AFP for diagnosis of HCC was 0.746(95%CI:0.674-0.818).A cutoff value of AFP of 9.00 ug/L had a sensitivity of 0.609,and specificity of 0.818 for diagnosing HCC.CONCLUSION Age≥60 years,smoking history,family history of HCC,lamivudine resistance,HBV DNA negative,FBG≥6.16 mmol/L were risk factors of HCC progression.Serum AFP had limited diagnostic value for HCC.

Nucleos(t)ide analogs in the prevention of hepatitis B virus related hepatocellular carcinoma

Hepatocellular carcinoma(HCC) is among the most common cancer types and causes of cancer related mortality worldwide.Almost 50% of all HCC cases globally are attributable to chronic hepatitis B virus(HBV) infection.The incidence rates of HCC in untreated Asian subjects with HBV infection was estimated to be 0.2% in inactive carriers,0.6% for those with chronic hepatitis without cirrhosis,and 3.7% for those with compensated cirrhosis.In Western populations,HCC incidences are reported to be 0.02% in inactive carriers,0.3% in subjects with chronic hepatitis without cirrhosis,and 2.2% in subjects with compensated cirrhosis.Despite effective antiviral treatment options which are able to transform chronic hepatitis into an inactive carrier state,the risk of HCC cannot be fully ruled out to exclude those patients from surveillance.Newer nucleos(t)ide analogues(NAs) as entecavir and tenofovir are very potent in terms of sustained virological suppression which leads to improved liver histology.However,they do not have any influence on the ccc DNA or integrated DNA of HBV in the liver.Nonetheless,viral replication is the only modifiable component among the established risk factors for HBV-related HCC with the current treatment options.In this review,it was aimed to summarize cumulative evidence behind the concept of prevention of HBV related HCC by NAs,and to discuss remaining obstacles to eliminate the risk of HCC.

Nutrition deprivation affects the cytotoxic effect of CD8 T cells in hepatocellular carcinoma

Hepatocellular carcinoma(HCC) is the most common type of liver cancer and the third leading cause of cancer-related death worldwide. Factors including carcinogens, infection of hepatitis viruses, alcohol abuse, and metabolic disorders such as non-alcoholic fatty liver disease mainly contribute to HCC initiation and progression. Immunotherapy is one of the most powerful tools for unresectable HCC treatment in patients. CD8T cells are a major immune component in the tumor microenvironment with cytotoxic effects against cancer cells. However, these CD8T cells commonly display an exhaustion phenotype with high expression of programmed cell death protein 1, T-cell immunoglobulin and mucindomain containing-3, and/or lymphocyte-activation gene 3, producing low levels of perforin(PRF1) and granzyme B(GZMB), as well as anti-tumor cytokines, such as interferon gamma and tumor necrosis factor alpha. In the referenced study, the authors also showed that deprivation of glutamine decreased the antitumor function of CD8T cells, as well as the production of PRF1 and GZMB. However, the role of each amino acid in T cell function and exhaustion may depend on tumor type and tumor microenvironment, including the source of other nutrients. Overall, amino acids or other nutrient metabolites in the tumor microenvironment play a pivotal role in both tumor growth and immune response.

Glutamine deprivation impairs function of infiltrating CD8^(+)T cells in hepatocellular carcinoma by inducing mitochondrial damage and apoptosis

BACKGROUND The functions of infiltrating CD8^(+)T cells are often impaired due to tumor cells causing nutrient deprivation in the tumor microenvironment.Thus,the mechanisms of CD8^(+)T cell dysfunction have become a hot research topic,and there is increased interest on how changes in metabolomics correlate with CD8^(+)T cell dysfunction.AIM To investigate whether and how glutamine metabolism affects the function of infiltrating CD8^(+)T cells in hepatocellular carcinoma.METHODS Immunohistochemical staining and immunofluorescence were performed on surgically resected liver tissues from patients.Differentially expressed genes in infiltrating CD8^(+)T cells in hepatocellular carcinoma were detected using RNA sequencing.Activated CD8^(+)T cells were co-cultured with Huh-7 cells for 3 d.The function and mitochondrial status of CD8^(+)T cells were analyzed by flow cytometry,quantitative real-time polymerase chain reaction,and transmission electron microscopy.Next,CD8^(+)T cells were treated with the mitochondrial protective and damaging agents.Functional alterations in CD8^(+)T cells were detected by flow cytometry.Then,complete medium without glutamine was used to culture cells and their functional changes and mitochondrial status were detected.RESULTS There were a large number of infiltrating PD-1+CD8^(+)T cells in liver cancer tissues.Next,we cocultured CD8^(+)T cells and Huh-7 cells to explore the regulatory effect of hepatoma cells on CD8^(+)T cells.Flow cytometry results revealed increased PD-1 expression and decreased secretion of perforin(PRF1)and granzyme B(GZMB)by CD8^(+)T cells in the co-culture group.Meanwhile,JC-1 staining was decreased and the levels of reactive oxygen species and apoptosis were increased in CD8^(+)T cells of the co-culture group;additionally,the mitochondria of these cells were swollen.When CD8^(+)T cells were treated with the mitochondrial protective and damaging agents,their function was restored and inhibited,respectively,through the mitochondrial damage and apoptotic pathways.Subsequently,complete medium without glutamine was used to culture cells.As expected,CD8^(+)T cells showed functional downregulation,mitochondrial damage,and apoptosis.CONCLUSION Glutamine deprivation impairs the function of infiltrating CD8^(+)T cells in hepatocellular carcinoma through the mitochondrial damage and apoptotic pathways.

Effects of Radiofrequency Ablation on Lymphocyte Subsets and Type 1/Type 2 T Cell Subpopulations in Patients with Hepatocellular Carcinoma

Objective： To evaluate whether radiofrequency ablation （RFA） might have an influence on immune status in hepatocellular carcinoma （HCC） patients. Methods： We measured the T lymphocytes, B lymphocyte and NK cells, and determined the population of Thl, Th2, Tcl and Tc2 of peripheral blood samples taken from 26 HCC patients before and after RFA. Results： The proportion of Typel cells （Thl and Tcl） and NK cells were significantly increased after RFA, especially in patients of the following subgroups： male, age〉55 years, pathological grade Ⅰ-Ⅱ tumor, clinical stage Ⅰ-Ⅱ or Child-Pugh A and B. Conclusion： TypeⅠ cells and NK cells in HCC patients were increased in a short period after RFA.

Functions of three ubiquitin-conjugating enzyme 2 genes in hepatocellular carcinoma diagnosis and prognosis

BACKGROUND Liver cancer ranks the third cause of cancer-related death worldwide.The most common type of liver cancer is hepatocellular carcinoma(HCC).The survival time for HCC patients is very limited by years due to the lack of efficient treatment,failure of early diagnosis,and poor prognosis.Ubiquitination plays an essential role in the biochemical processes of a variety of cellular functions.AIM To investigate three ubiquitination-associated genes in HCC.METHODS Herein,the expression levels of ubiquitin-conjugating enzymes 2(UBE2)including UBE2C,UBE2T,and UBE2S in tumor samples of HCC patients and nontumor controls at the Cancer Genome Atlas(TCGA)database,was comprehensively analyzed.The relationship of UBE2 gene expression level with cancer stage,prognostic outcome,and TP53 mutant status was studied.RESULTS Our results showed that UBE2C,UBE2T,and UBE2S genes were overexpressed in HCC samples compared to non-tumor tissues.Dependent on the cancer progression stage,three UBE2 genes showed higher expression in tumor tissues at all four stages compared to non-tumor control samples.Furthermore,a significantly higher expression of these genes was found in stage 2 and stage 3 cancers compared to stage 1 cancer.Additionally,overexpression of those genes was negatively associated with prognostic outcome and overall survival time.Patients with TP53 mutation showed a higher expression level of three UBE2 genes,indicating an association between UBE2 expression with p53 function.CONCLUSION In summary,this study shed light on the potential roles of UBE2C,UBE2T,UBE2S on diagnostic and prognostic biomarkers for HCC.Moreover,based on our findings,it is appealing to further explore the correlation of those genes with TP53 mutation in HCC and the related mechanisms.