The Effect And Mechanisms Of T Cell Exhaustion Induced By HCC-derived Exosomes

Object:The liver is the largest solid organ in the body,it carries out a large number of essential tasks,including detoxification and protein synthesis.Due to the crucial role,liver is always exposed to some systemic pathogens.In physiological conditions,the liver can selectively ignore the external substances to form immune tolerance.However,when the liver is exposed to high load of pathogens,toxins or self-antigens for a long time,the immune homeostasis will be disrupted,forming a long-term inflammatory microenvironment and causing liver damage.On the one hand,long-term liver injury may further develop into cirrhosis and liver necrosis;on the other hand,this change of the microenviroment will enhance immune tolerance,resulting in the immune system disorder,eventually developing into liver cancer.As the important member of the immune system,T cells play a critical role in response against pathogens.When T cells are activated,CTL can kill infected cells or clear foreign bodies directly or by secreting cytokines effectively.However,T cells are inefficient if they are overloaded with antigens after prolonged exposure to chronic infection or inflammation.T cell exhaustion can be caused by a variety of factors,including the negative immune factors,immune-negative regulatory cells,as well as the imrnunosuppressive microenvironment.Since T cells play an important role in the anti-tumor immune response,the study on the mechanisms of T cell exhaustion have attracted more and more attention.However,the mechanisms of T cell exhaustion in liver cancer remains unclear.Tumor-associated macrophages,as an important component of tumor-associated stromal cells,play an important role in cancer-related inflammation.In the early stage of inflammation,macrophages can secrete IL-12,IL-1β and other related inflammatory molecules to promote the function of NK cells and T cells,enhance anti-tumor immune response and resist tumor development.In the late stage of tumor development,M2-polarated macrophages can promote angiogenesis by secreting TGF-β,IL-6 and other immune suppression factors,and eventually induce tumor cell proliferation and metastasis.However,in liver cancer,delineating the processes that underlie the phenotypic transition of macrophages will provide novel and insightful understanding of tumor progression.As extracellular nano-level vesicular material,exosomes can be secreted by normal cells,cancerous cells,parenchymal cells and immune cells.Studies have shown that tumor-derived exosomes influence tumor progression by remodeling the tumor microenvironment.However,it is not clear whether exosomes derived from hepatocellular carcinoma cells(HCC)have any effect on macrophages,the major members of the tumor microenvironment.In this study,we speculated that exosomes derived from HCC could reshape macrophages in the microenvironment,thereby inhibit the function of T cells and promote the development of tumors.Our study aim to clarify how exosomes derived from HCC cells regulate the function of macrophages and further reveal which component of exosomes act on macrophages,providing new molecular immune mechanisms for HCC development.Methods:1.A mouse model of primary liver cancer was established by intraperitoneal injection of DEN/CCl4 to determine the cancer status of the mice.The body weight,liver weight and absolute number of immune cells were measured.2.Immune cells from mouse liver were isolated.Flow cytometry(FACS)was used to analyze the proportion of immune cell subsets,including T cells,NK cells,NKT cells,macrophages and MDSC in the liver of healthy mice versus tumor-bearing mice.Meanwhile,FACS was used to detect the expression of inhibitory receptors(TIM-3,TIGIT,CTLA4,PD-1,LAG-3,BTLA4)and functional molecules(IL-2,TNF-a,IFN-y,NKG2D,FasL)on T cells,NK cells and NKT cells.3.Liver parenchymal cells of mice were isolated.FACS was used to detect the expression of immune-related molecules in liver parenchymal cells.4.T cells from mouse spleen were separated and cultured in vitro.After DEN/CCl4 treatment,the expression of inhibitory receptors on the cell surface was analyzed by flow cytometry.5.Exosomes extracted from supernatant of cultured HCC cell lines,mouse serum and human peripheral blood serum by gradient centrifugation.Morphology,markers and particle size distribution of exosomes were detected by transmission electron microscope,Western blot and dynamic light scattering.6.Mouse peritoneal macrophages were extracted and the exosome uptaking by macrophages was confirmed using confocal microscopy7.Western blot and quantitative PCR were used to detect the expression of inflammatory factors in macrophages after exosome "education".8.MTT viability assay and Transwell assay were used to detect the effect of supernatant of macrophages "educated" by HCC-exosomes on the proliferation and migration ability of Hepal-6 cells.9.The effect of macrophage supernatant on the migration ability of splenocyte cells after exosome acclimation was detected by Transwell and FACS assay.10.The polarization markers of macrophages CD206,CCL17,CCL22,Arg-1 and ROS were detected by flow cytometry,fluorescence quantitative PCR,ELISA and immunofluorescence technology after HCC-exosome treatment11.Macrophage polarization related signaling pathways STAT3 and STAT1 were detected by Western blot and FACS after HCC-exosome treatment12.Inhibitory molecules(CD 169)and the inhibitory receptor ligands(MHC-Ⅱ,PD-L1,CD80)were analyzed by using flow cytometry13.T cells in the liver and spleen of healthy mice were separated by magnetic beads and incubated with macrophages educated by HCC-exosomes.Flow cytometry was used to detect the expression of inhibitory receptors on the surface of T cells and the secretion of cytokines14.Mouse bone marrow cells were extracted and differentiated into macrophages(BMDM)by adding M-CSF,and the differentiated macrophages were identified by flow cytometry.15.The mice depleted macrophages with chlorine disodium phosphate liposome were transferred with HCC-exosome educated BMDM.5 days later,T cell inhibitory receptors,the secretion of cytokines,cell proliferation and ability to kill tumor cell were analyzed,further determining the effect of HCC-exosome educated macrophages on T cell function16.The expression of miR-146a-5p and SALL4 in liver cancer cells was interfered by the transfection system of LipofectamineTM 2000.Quantitative PCR was used to detect the contents of miRNAs in HCC and HCC-exosomes17.Tail vein high-pressure injection was used to intervene the expression of SALL4 in mouse liver cells,and the gene-silencing efficiency was verified by Western blot,immunohistochemistry,immunofluorescence and other methods.Quantitative PCR assay was used to detect the malignant proliferation related genes in liver cells of healthy mice,HCC mice and HCC mice with SALL4 blocked,so as to determine the role of SALL4 in HCC.Liver tissue damage in mice was detected by H&E staining.18.The binding of SALL4 to the promoter region of mir-146a-5p was verified using ChIP assay.19.Luciferase reporter assay was used to confirm the regulatory effect of SALL4 on miR-146a-5p transcription.Results:1.Establishment of primary HCC mouse modelIn healthy mice,intraperitoneal injection of DEN for 3 times and CCl4 for 12 times induced primary HCC.Through the detection of body and liver weight in mice,we found that the body weight was significantly reduced accompanied by HCC progression.Liver lesions and swelling,at the same time the expression of immune-related molecules on the surface of liver cells was abnormal,and the proportion of immune cells in the liver was aberrant,suggesting that the liver immune microenvironment was disturbed.2.T cells were dysfunction in DEN/CCl4-induced HCC mice.We analyzed the inhibitory receptors and secreted cytokines of T cells,NK cells and NKT cells by FACS.The inhibitory receptors were upregulated on T cells from DEN/CCl4-induced HCC mice,while the production of cytokine was decreased.The expression of inhibitory receptors was up-regulated in NKT cells,but these cells still shown cell function,which was manifested as the increased cytokine secretion.The inhibitory receptors on NK cells were not significantly changed.These results indicated that T cell exhaustion is the main reason for HCC.DEN/CCl4 did not directly influence T-cell exhaustion in vitro.We speculated that the exhaustion of T cells was induced by the microenvironment of HCC.3.Macrophages could uptake exosomes secreted by HCCExosomes were isolated from the supernatant of Hepal-6 cells by ultracentrifugation.Exosomes were verified as small vesicles of approximately 100 nm in size by transmission electron microscopy(TEM),and with the expression of CD63 and CD81.The size distribution of the exosomes was predominantly within the range of 50-150 nm.Exosomes were labeled with DiD dye and incubated with peritoneal macrophages(PMs)from healthy mice.Confocal high content analysis showed that the DiD-labeled exosomes were attached to the cell surface for 15 minutes,and efficiently internalized by PMs after 30 min.4.Exosomes promoted macrophage activation,accelerating tumor proliferation and migrationMacrophages produced more inflammatory factors after exposure to exosomes for 24 hours,including TNF-a and IL-1β.To investigate the effect of macrophages on tumors,macrophages were educated by HCC-exosomes for 24 hours,and the supernatants were collected and used to incubate HCC cells.We found that the"educated" macrophages could promote the proliferation and migration of HCC cells.5.HCC-derived exosomes promoted M2 polarizationAfter HCC-derived exosomes education,the polarization phenotype of macrophages was changed.The proportion of CD11b+F4/80 CD206+macrophages and mRNA levels of M2 phenotype markers including ccl17,ccl22 and arg-1 were significantly increased following exposure to exosomes from Hepal-6 cells.Meanwhile,STAT3 phosphorylation was elevated in PMs incubated with HCC-exosomes,while the phosphorylation of STAT1 decreased accompanied by the reduction of ROS production.It suggested that HCC-derived exosomes promoted polarization of macrophages toward M2 tumor-associated macrophages.6.HCC-exosome“educated" macrophages exhibited immunosuppressive functionThe education of HCC not only reshaped the polarization direction of macrophages,but also affected the immune function of macrophages.While analyzing the phenotype of HCC-exosome "educated" macrophages,we noticed that the proportion of CD 169 macrophages,which was associated with suppressive activity of macrophages and immune tolerance,was significantly upregulated in the PMs treated by HCC-exosomes compared with the control group.However,the antigen presenting MHC-Ⅱ was downregulated by HCC-exosome treatment,while the ligands related to macrophage dysfunction such as PD-L1 and CD80 were upregulated.These results suggested that HCC-exosome "educated" macrophages could induce immunosuppressive function,and affect other cell functions through cell surface molecules.7.Macrophages educated with HCC-derived exosomes inhibited T cell responseWe observed that the inhibitory receptors PD-1,TIGIT and CTLA4 on CD3+T cells were increased,and the production of IL-2,IFN-γ and TNF-α in CD3+T cells was lowered by co-culturing with PMs treated with HCC-exosomes compared with T cells co-cultured with untreated PMs in vitro.The T cells were exhausted when co-cultured with HCC-exosome "educated",macrophages.We transferred with control or HCC-exosome treated BMDMs to the mice depleted macrophages.Compared with the hepatic T cells from mice transferred with control BMDMs,the inhibitory receptors Tim-3 and TIGIT were upregulated on T cells from mice transferred with HCC-exosome treated BMDMs,while the production of IL-2 and TNF-α was decreased.Meanwhile,the cytolysis activity of T cells against HCC cells and the expansion ability of T cells were suppressed by transferring HCC-exosome treated BMDMs.Thus,HCC-exosome "educated" macrophages displayed immunosuppressive activity by inducing T cell exhaustion.8.MiR-146a-5p in HCC-derived exosomes exerted key role on macrophage M2-polarizationWe analyzed several miRNAs involved in immunoregulation in exosomes derived from HCC cell lines.The miR-146a-5p expression was remarkably higher in Hepal-6 and H22 exosomes than healthy mouse hepatocytes.The transfection of miR-146a-5p inhibitors could inhibit M2 polarization.We found the overexpression of miR-146a-5p in Hepal-6 cells significantly increased the level of miR-146a-5p in the secreted exosomes,which promoted the M2 polarization.In contrast,miR-146a-5p inhibiting vector(inh-146a)decreased the expression of miR-146a-5p in the Hepal-6-derived exosomes,which suppressed the M2 polarization.Similar phenomena were observed in human HCC cell lines.It indicated that delivery of miR-146a-5p by HCC-derived exosomes is involved in M2 macrophage polarization.9.Transcription factor SALL4 controlled the expression of miR-146a-5p.We found SALL4 expression was increased during HCC development accompanied by the increasing number of M2-like macrophages,while silencing SALL4 via hydrodynamic injection of sh-SALL4 vector in DEN/CCl4-induced HCC mice significantly decreased the number of M2-like macrophages.Meanwhile the level of miR-146-5p in the exosomes was elevated by DEN/CCl4 treatment,and prevented by sh-SALL4.ChIP analysis clearly demonstrated that SALL4 could bind to miR-146a-5p promoter.In addition,we found the transcription activity of miR-146a-5p promoter was enhanced by overexpressing of SALL4 but repressed by silencing of SALL4.These data indicate that direct binding of SALL4 to the miR-146a-5p promoter regulated M2 polarization.10.Blocking SALL4 could prevent HCC progressWe analyzed the changes of inhibitory receptors in DEN/CCl4-induced HCC mice treated by sh-SALL4.The results showed that the administration of sh-SALL4 significantly lowered the inhibitory receptors on T cells of DEN/CCl4-treated HCC mice,but enhanced the production cytokines.Additionally,silencing SALL4 could obviously reduce malignant proliferation in hepatocytes and the tumor diameter,accompanying with low degree of malignancy in DEN/CCl4-induced HCC.These results confirm that SALL4 activation enhances the expression of inhibitory receptors in T cells and promotes T cell exhaustion accelerating HCC progression.Conclusion:We found that the T cells were exhausted with the proportion of macrophages up-regulated in DEN/CCl4-induced HCC.Hepatocellular carcinoma(HCC)-derived exosomes could remodel macrophages by activating NF-κB signaling and inducing pro-inflammatory factors to promote the proliferation and migration of HCC cells.Meanwhile,HCC derived exosomes activated STAT3 and suppressed STAT1 signaling pathway resulting in M2-polarized tumor-associated macrophages.In addition,expression of IFN-y and TNF-a was inhibited,while the expression of inhibitory receptors such as PD-1 and CTLA-4 was upregulated in T cells by HCC-derived exosome educated macrophages.Data also revealed that HCC exosomes were enriched with miR-146a-5p and promoted M2-polarization.Further investigation demonstrated that the transcription factor Sal-like protein-4(SALL4)was critical for regulating miR-146a-5p in HCC exosomes and M2-polarization.Mechanistically,SALL4 could bind to the promoter of miR-146a-5p,and directly controlled its expression in exosomes.Blocking the SALL4/miR-146a-5p interaction in HCC reduced the expression of inhibitory receptors on T cells,reversed T cell exhaustion,and delayed HCC progression in DEN/CCl4-induced HCC mice.