| Background and Aims: Hepatocellular carcinoma(HCC)is a common malignant tumor,which seriously threaten human life and health.The early diagnosis rate of HCC is increased with the development of medical levels.Moreover,therapeutic approaches are also improved in response to comprehensive treatments,including surgery,transcatheter arterial chemoembolization,thermal ablation,and molecular targeted drugs.Unfortunately,the overall prognosis of HCC is still poor all over the world.In China,most HCC patients suffer with chronic liver diseases.More than 80% of HCC patients are already in moderate or late stages even in initial consultation,leading to loss of optimal opportunity for treatments.More importantly,the pathogenesis of HCC is still not fully elucidated,which impedes the research of new treatment methods.Immunological factors play important roles in the pathogenesis of HCC.Changes in the numbers of immune cells,cytokines production,and receptors/ligands expression could result in HCC progression.These factors are all the potential targets for restoring anti-cancer immune response.Recently,some scholars have shown that the microwave ablation of liver cancer may lead to some changes in immune function,which may help reduce the suppression of the immune system by tumor tissues,but there is still no research on the relevant mechanism.Interleukin(IL)-7 is an immunoactive cytokine,and belongs to common ? chain cytokine family.IL-7 promotes the development,proliferation,and survival of lymphocytes,and maintains immune homeostasis.IL-7 stimulation elevated the numbers of CD4+ T cells and CD8+ T cells,and increased cytotoxicity of peripheral CD8+ T cells in patients with chronic viral hepatitis,indicating that IL-7 could promote functions of immune system,restore immune defense in cancer patients,and exert anti-cancer activity.However,the role of IL-7 in HCC and the regulatory function/mechanism of IL-7 to tumor specific CD8+ T cells is still not completely understood.Thus,the aim of current study was to observe the changes in the expression levels of interleukin-7 and CD8+T cells in patients with HCC after MWA,to vestigate the expression of IL-7 and IL-7 receptor ? chain CD127 in HCC patients,and to assess the role/regulatory mechanism of recombinant IL-7 stimulation to CD8+ T cells function in HCC patients.Sujects and Methods: 1.Subjects A total of thirty-seven HLA-A2 restricted patients with HCC and sixteen sex and age-matched HLA-A2 restricted healthy individuals were enrolled in China-Japan Union of Jilin University between July 2016 and February 2017.Blood samples were collected from all enrolled subjects,and serum as well as peripheral blood mononuclear cells were isolated.Tissue samples were collected from HCC patients underwent operation.Infiltrating lymphocytes were isolated from normal liver tissues and HCC tissues.CD8+ T cells were then purified.Meanwhile,seventy-four HCC patients with microwave ablation between Feburary 2017 and July 2018 in China-Japan Union of Jilin University were also enrolled.Blood samples were collected,and serum as well as peripheral blood mononuclear cells were isolated.2.Methods(1)Serum IL-7 level was measured in healthy individuals and HCC patients by ELISA,and was compared among different disease stages as well as prior to and post microwave ablation therapy.(2)CD127 expression on peripheral and liver-resident CD8+ T cells was measured by flow cytometry.Percentage of CD8+ T cells was also compared prior to and post microwave ablation therapy.(3)The relative levels of perforin,granzyme B,and Fas L m RNA in CD8+ T cells were measured by real-time PCR,and were compared prior to and post microwave ablation therapy.(4)Hepatocellular carcinoma cell line Hep G2 cells were stimulated with recombinant human IL-7.The bioactivity(proliferation and migration)of Hep G2 cells was assessed in response to IL-7 stimulation.(5)Purified CD8+ T cells were stimulated with recombinant human IL-7.The co-inhibitory molecules PD-1 and CTLA-4 on CD8+ T cells were measured by flow cytometry in response to IL-7 stimulation.(6)The direct contact and indirect contact co-culture systems were set up of CD8+ T cells and Hep G2 cells.CD8+ T cells function was investigated in response to IL-7 stimulation.The cytolytic activity of CD8+ T cells was assessed using the death rate of Hep G2 cells by measurement of lactate dehydrogenase in the cultured supernatants.The non-cytolytic acitivity of CD8+ T cells was assessed by measurement of IFN-? and TNF-? production in the cultured supernatants.Results: 1.IL-7 expression was elevated in HCC patients who underwent microwave ablation therapy in comparison with baseline level.However,there were no remarkable differences in percentage of CD8+ T cells,as well as perforin,granzyme B,and Fas L m RNA relative levels prior to and post microwave ablation therapy.2.Serum IL-7 was significantly reduced in HCC patients in comparison with healthy individuals.There was no remarkable difference of serum IL-7 level among patients with different stages.3.There was no statistical difference of peripheral CD3+CD8+ T cells frequency between healthy individuals and HCC patients.Percentage of CD45RA+CD45ROna?ve CD8+ T cells was decreased in HCC patients,while percentage of CD45RA-CD45RO+ memory CD8+ T cells was increased in HCC patients in comparison with healthy individuals.There was no remarkable difference of CD127 expression on na?ve and memory CD8+ T cells between healthy individuals and HCC patients.4.There was no statistical difference of liver-inflitrating CD3+CD8+ T cells frequency between normal liver tissues and HCC tissues.Percentage of na?ve CD8+ T cells was decreased in HCC tissues,while percentage of memory CD8+ T cells was increased in HCC tissues in comparison with normal liver tissues.There was no remarkable difference of CD127 expression on na?ve and memory CD8+ T cells between normal liver tissues and HCC tissues.5.Recombinant human IL-7 stimulation induced phosphorylation of STAT5 in Hep G2 cells,however,did not affect proliferation and migration of Hep G2 cells.6.PD-1 expression on peripheral CD8+ T cells was notably higher in HCC patients when compared with healthy individuals.PD-1 expression on liver-resident CD8+ T cells was also elevated in HCC tissues when compared with normal liver tissues.Recombinant human IL-7 stimulation reduced PD-1 expression on peripheral and liver-resident CD8+ T cells from HCC patients,however,did not affect CTLA-4 expression on CD8+ T cells.7.Direct contact and indirect contact co-culture systems were successfully set up of CD8+ T cells and Hep G2 cells.In direct contact co-culture system,IL-7 stimulation enhanced the cytolytic and non-cytolytic activity of peripheral and liver-resident tumor-specific CD8+ T cells,which mainly presented as increased target death rate of Hep G2 cells and elevated IFN-?/TNF-? production.In indirect contact co-culture system,Hep G2 cell death rate did not change significantly in response to IL-7 stimulation,although TNF-? production was increased.Conclusion: 1.Microwave ablation led to the elevation of serum IL-7 in HCC patients.2.Tumor-infiltrating CD8+ T cells revealed dysfunctional or exhaustive activity for tumor rejection in HCC patients,which could be partly due to the down-regulation of IL-7 expression.3.IL-7 stimulation promoted cytolytic activity of peripheral and liver-inflitrating CD8+ T cells probably via suppression of PD-1 in HCC patients.IL-7 could be considered as one of the therapeutic candidates for HCC treatment. |
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