| BackgroundHepatocellular carcinoma?HCC?is one kind of chronic liver diseases that seriously threatens human health and is commonly treated by surgical resection,transcatheter arterial chemoembolization,radiofrequency ablation and liver transplantation,etc.However,due to the hidden onset and the influence of multiple factors,about 80%of patients have been diagnosed at the middle and late stage of tumors,and often lose the opportunity of treatment.Therefore,the in-depth exploration of the occurrence and development mechanism of HCC and the design of novel treatment strategies will have profound significance for prolonging the survival period of patients,improving the quality of life of patients,reducing the burden of disease.In recent years,immunotherapy has gradually become a hot topic,which can cause more lasting effects than conventional radiotherapy and chemotherapy in patients with advanced cancer.A positive immunotherapy response usually depends on the interaction between tumor cells and the tumor immune microenvironment.Tumor microenvironment?TME?,as the internal environment in which tumor occurs and develops,contains a variety of immune cell subgroups.And,the HCC microenvironment has been widely concerned due to its unique immune tolerant state.The immune cells,such as tumor associated macrophage?TAMs?,myeloid-derived suppressor cells?MDSCs?,dendritic cells?DCs?,cytotoxic T lymphocytes?CTLs?,natural killer cells?NKs?and natural killer T cells?NKTs?,and multiple immune molecules are thought to participate in the formation of tumor immunomicroenvironment and determine the response of HCC to clinical treatment.Co-stimulatory and co-inhibitory molecules expressed on immune cells can precisely regulate the activation status of these immune cells and thus determine the strength and quality of the immune response.These molecules are called immune checkpoints.Targeted inhibitors against inhibitory immune checkpoint molecules?PD-1,CTLA-4,etc.?have become new strategies for tumor immunotherapy and have been approved for clinical application.However,monoclonal antibodies against a single immune checkpoint only have 20%response rate in patients with HCC.Therefore,the in-depth study of the expression of other immune checkpoint molecules in immune cells and their functional status in the HCC microenvironment will bring a new breakthrough for the clinical treatment of HCC.Tim-3,founded in the study of asthma susceptibility genes in 2002,is one of the members of the family of Tim located at chromosome 11B 1.1 in mice and at 5q33.2 in human beings.Similar to PD-1 and CTLA-4,Tim-3 is one of the famous immune checkpoints.As a negative immunomodulatory molecule,Tim-3 is expressed at a variety of immune cell subsets and is involved in the occurrence and development of chronic viral infections,cancer and other diseases.Tim-3 was initially identified as a surface marker for CD4+Thl cells and negatively regulated Thl response by binding to the ligand galectin-9?Gal-9?.In the previous studies,we found that Tim-3 was highly expressed in TAMs and NK cells,and was ectopically expressed in parenchymal cells of HCC,promoting the occurrence and development of HCC.These studies suggested that the expression of Tim-3 in different cells was involved in HCC development.However,the expression of Tim-3 in different subtypes of immune cells and the distribution characteristics of Tim-3 positive cells and their correlation with the clinical characteristics of the tumor have not been systematically analyzed.In the current study,we introduced the tumor microenvironment landscape analysis program to study in situ the immune cell subgroup infiltrating in the HCC microenvironment and the expression and spatial distribution of Tim-3 on these immune cells.The correlation with the prognosis of patients were analyzed.On the other hand,peripheral blood of HCC patients was collected to further verify the important roles of CD4+ T cells in Tim-3-mediated immune regulation.ObjectivesIn this project,we explored the expression and roles of Tim-3 in HCC microenvironment,which might provide new clues for identifying new targets for the diagnosis,treatment and prognosis evaluation of HCC.The specific research purposes to be solved are as follows:1.To detect the infiltration and of major immune cell subsets in the microenvironment of HCC and to analyze their correlation with the prognosis of patients;2.To detect the expression and correlation of Tim-3 in major immune subgroups in HCC microenvironment and to analyze the relationships with the prognosis of patients;3.To analyze the spatial distribution characteristics of the main immune cell subgroups and Tim-3+ cell subgroups in the HCC microenvironment;4.To verify the key role of CD4+ cells in Tim-3-mediated immune regulation.Methods and results1.The expression of Tim-3 in HCC microenvironment and its correlation with prognosis of patientsWe first used multiplexed immunofluorescence staining technology with specific antibodies against CD4/CD8/CD56/CD68/Tim-3 to detect the number and ratio of main immune cells and their Tim-3 expression in HCC tissues.We statistically analyzed their correlation and correlation with patient survival.1.1 Decreased percentages and number of immune cell subsets correlated withpatient survival.We analyzed the proportion and number of immune cell subsets in 93 cases of cancerous tissues and 87 cases of paracancerous tissues,and uncovered the relationship between infiltrating immune cells in cancerous tissues and patient survival.The results showed that the proportion and number of CD4+,CD8+,CD56+and CD68+ cells in the cancerous tissues of HCC patients?n=93?were statistically lower than those in the paracancerous tissues?n=87,p<0.01?.Survival curve analysis showed that patients with CD4+ high proportion had a longer survival time?p=0.0395?.It suggests that CD4+ cell subsets may play an important role in the development of HCC.1.2 The enrichment degree of each immune cell subset in HCC tissues has acertain correlationWe performed spearman correlation analysis for the proportion of immune cell subsets in cancerous tissues.Statistical analysis showed that the proportion of CD4+cells was positively correlated with the that of CD8+?r=0.4265,p<0.0001?,CD56+?r=0.4952,p<0.0001?and CD68+?r=0.5539,p<0.0001?cells in cancerous tissues,while there was no significant correlation between the other immune subgroups.These results suggest that CD4+ cell may have potential regulatory effects on other immune cell subsets in the HCC microenvironment.1.3 Correlattion of combined tumor infiltration immune cell subsets with theprognosis of HCC patientsWe further analyzed the correlation of the combination of tumor infiltration immune cell subsets with the prognosis of HCC patients,and divided the patients into the low proportion of dual immune cell subgroups and the high proportion of dual immune cell subgroups.We found that the proportion of CD4+/CD68+?p=0.0332?was positively correlated with patient survival,while there was no difference among the other groups.It further suggested that CD4+ cell subsets may play an important role in the microenvironment of HCC.1.4 Upregulated Tim-3 in cancerous tissues negatively correlated with patientsurvivalTim-3 as the newly discovered an immune checkpoint molecules,play a negative immunomodulatory role in tumor microenvironment.In order to study the expression of Tim-3 in HCC microenvironment,we compared the proportion of Tim-3+ cells and mean fluorescent intensity?MFI?of Tim-3 in cancerous and paracancerous tissues,and analyzed the relationship of Tim-3 expression with patient survival in cancerous tissue.The results showed that the proportion?p=0.0004?and MFI?p=0.0004?of Tim-3+ cells in cancerous tissues were significantly higher than that in paracancerous tissues,and the patients with low Tim-3 expression had a shorter survival period than those with high Tim-3 expression?p=0.0371?.The results indicated that the expression of Tim-3 in cancerous tissues was significantly correlated with the prognosis of patients.1.5 Upregulated Tim-3 in tumor infiltrating immune cells negatively correlatedwith patient survivalThe previous results showed that Tim-3 was expressed in both tumor cells and a variety of immune cells in HCC tissues.In this study,we explored the expression of Tim-3 on the surface of each immune cell subsets in cancerous tissues and corresponding paracancerous tissues,and analyzed its relationship with the patients survival.The results showed that the expression of Tim-3 in CD4+?p<0.001?,CD8+?p<0.0001?,CD56+?p<0.01?and CD68+?p<0.05?cells in cancerous tissues was significantly higher than that in paracancerous tissues.Survival curve analysis showed that patients with high Tim-3 expression on the surface of CD4+,CD8+ and CD56+cells in cancerous tissues had a shorter survival time?p<0.05?.Combined with the above results,the expression of Tim-3 on the surface of CD4+,CD8+ and CD56+ cells in cancerous tissues may be involved in the regulation of HCC progression.1.6 Positive correlation between the expression of Tim-3 in different tumorinfiltrating immune cellsWe performed spearman correlation analysis on the expression of Tim-3 on the surface of immune cell subsets in cancerous tissues.Statistical analysis showed that the expression of Tim-3 among different immune cell subsets had a significant positive correlation?p<0.0001?.These results suggest that there may be some common factors that can regulate the expression of Tim-3 in each immune cell subset in the HCC microenvironment.1.7 Correlation of combined Tim-3+ tumor infiltration immune cell subsets withthe prognosis of HCC patientsIn order to further verify whether the high expression of Tim-3 on combined tumor infiltration double-immune cell subsets is more negatively correlated with patient survival,we compared the difference of survival time between the double-immune cell subsets with low expression of Tim-3 and the double-immune cell subsets with high expression of Tim-3.Survival curve analysis showed that the patient with high expression of Tim-3 in the CD4+/CD8+ cells?p=0.0082?and CD4+/CD56+ cells?p=0.0257?had a shorter survival time than the patient with the low expression of Tim-3.These results suggest that the expression of Tim-3 on the surface of CD4+/CD8+ and CD4+/CD56+ cells may be more significantly correlated with the progression of HCC.The occurrence and development of tumor is closely related to its microenvironment,involving the interaction between matrix,blood vessels,lymphatic vessels,immune cells and other factors.These cell populations may inhibit or promote tumors,depending on their activation status and relative tumor location.Therefore,it is of great significance to analyze the spatial distribution of each immune cell subgroup and Tim-3+ immune cell subgroup in the microenvironment of HCC.We divided the cancerous tissues into tumoral regions?n=93?and stromal regions?n=60?,and the adjacent non-cancerous tissues were divided into parenchymal regions?n=93?and stromal regions?n=74?.We investigated the distribution of immune subgroups,the expression of Tim-3 on their surfaces in different regions,and analyzed their correlation with patient prognosis.1.8 Spatial distribution of immune cell subsets and their correlation with patient survivalWe calculated the proportion and density of CD4+,CD8+,CD56+ and CD68+cells in each regions,and then analyzed its relationship with patient survival by ANOVA test?multiple data comparison?using GraphPad Prism6.0 software.The results showed that in paracancerous tissues,the proportion and density of CD4+,CD8+ and CD56+ cells in parenchymal regions were significantly lower than that in stroma regions,while the macrophages were significantly higher than that in stroma regions.In cancerous tissues,the proportion and density of CD4+,CD8+ and CD68+cells in tumor regions were significantly lower than that in stroma regions,while there was no significant difference in CD56+ cells.At the same time,we compared respectively the proportion and density of each immune cell subsets in stroma and tumor?parenchyma?regions between cancerous and paracancerous tissues,and it was found that the difference in CD4+ and CD56+ cells between cancerous and paracancerous tissues was mainly in the stroma regions,and there was no difference between the tumor and parenchyma regions.The frequency of CD68+ cells was different between tumor and parenchyma regions,but there was no difference in stroma regions.The proportion and number of CD8+cells were significantly different between both the stromal regions and the tumoral-parenchymal regions.It is speculated the immune cell subsets in different regions of the HCC microenvironment play different roles in the occurrence and development of HCC.Survival curve analysis showed that the high proportion of CD4+ in tumoral regions was positively correlated with the survival prognosis of patients?p=0.0121?,while there was no difference among the other groups.The above results suggest that there is regional regulation in the distribution of immune cell subsets in the stroma and tumor?parenchyma?regions in the microenvironment of HCC,which may play an important role in the progression of HCC.1.9 Correlation between the regional distribution of immune cell subsetsWe analyzed the correlation between the immune cell subsets in different regions of cancerous tissues with patients of HCC.Statistical analysis showed that the proportion of CD4+ cells in the tumor and stroma regions of liver cancerous tissues was positively correlated with the proportion of CD8+,CD56+ and CD68+ cells?p<0.05?,and the correlation was more significant in the tumor regions.In addition,there was a positive correlation between the proportion of CD56+ and CD68+ cells in the stroma regions?p=0.04698?,while there was no significant correlation between the remaining immune subsets.This finding further suggests that CD4+ cells may play an important role in immune microenvironment regulation of HCC.1.10 Correlation between the regional distribution of double immune cell subsetswith patient survivalWe further analyzed the correlation between the infiltration of double immune cell subsets in different regions of cancerous tissues and the prognosis of patients with HCC.Survival curve analysis showed that the proportion of CD4+/CD68+was more positively correlated with the patient survival?p=0.0147?,while there was no difference among the other groups.These results suggest that CD4+ cells combined with CD68+ cells may play an important role in the progression of HCC.1.11 Spatial distribution of Tim-3 expression in cancerous and paracancerous tissues and its correlation with patient survivalWe counted respectively the proportion and MFI of Tim-3+ cells in tumor?parenchyma?and stroma regions between cancerous and paracancerous tissues,and further analyzed the relationship between the expression of Tim-3 in cancerous tissues and patient survival.The results showed that there was no difference in the expression of Tim-3 in paracancerous tissues.The expression of Tim-3 in tumor regions was higher than that in stroma regions.At the same time,we respectively compared the positive ratios and MFI of Tim-3 + in the stroma and tumor?parenchyma?regions of cancerous and paracancerous tissues.It was found that the positive rate and MFI of Tim-3 in the tumor regions of cancer tissues were significantly higher than that in the adjacent parenchyma,while the MFI of Tim-3 in the stroma regions in cancerous results was slightly higher than that in paracancerous tissues,and there was no significant difference in the proportion of positive cells.In addition,the expression of Tim-3 in the tumor regions was significantly negatively correlated with the prognosis of patients?p=0.0065?,while there was no significant difference in survival between the two groups in the stroma regions?p=0.2541?.These results suggest that the expression of Tim-3 in tumor regions may have a higher correlation with the progression of HCC.1.12 Correlation analysis between the spatial distribution Tim-3+ immune cell subsets and patient survivalsWe counted the expression of Tim-3 on the surface of immune cell subsets in the tumor?parenchyma?and stroma regions between the cancerous and paracancerous tissues,and analyzed the correlation between the expression of Tim-3 on the surface of immune cell subsets in different regions of cancerous tissues with patient survival.The results showed that in paracancerous tissues,the expression of Tim-3 on the surface of CD4+,CD8+ and CD56+ cells in parenchyma regions was higher than that in stroma regions,while the expression of Tim-3 on the surface of CD68+ cells was significantly lower than that in the stroma regions.In cancerous tissues,the expression of Tim-3 in CD4+ cells in tumor region was significantly higher than that in stroma regions,but the MFI of Tim-3 on the surface of CD8+ and CD56+ cells was significantly higher than that in the stroma regions,with no difference in the proportion of positive cells.While the expression of Tim-3+ in CD68+ cells was significantly lower than that in stroma regions,with no difference in MFI.The proportion of Tim-3+ on the surface of CD4+ cells in the stroma regions of cancer tissues was significantly higher than that on the surface of paracancerous tissues,but there was no significant difference in the proportion of positive cells.The proportion of Tim-3+ on the surface of CD8+ and CD68+ cells was significantly higher than that in the stroma regions,but there was no difference in MFI,and there was no significant difference in the expression of Tim-3 on the surface of CD56+ cells.The expression of Tim-3 on the surface of each immune cell subsets was higher than that im the paracancer parenchyma.These results suggest that the expression of Tim-3 in CD4,CD8+,CD56+ and CD68+ cells in tumor areas may be involved in the progression of HCC.Survival analysis showed that the patient with low expression of Tim-3 in CD4,CD8+ and CD56+ cells in tumor and stroma regions had better prognosis,and this difference was more obvious in tumor regions,while the expression of Tim-3 on the surface of CD68+ cells had no significant correlation with patient survival.Furthermore,the expression of Tim-3 on the surface of CD4+,CD8+ and CD56+ cells in the tumor regions of HCC patients may be the main factor affecting the progression of HCC.1.13 Correlation between the expression of Tim-3 in each immune cell subsets in tumor and stroma regionsWe performed spearman correlation analysis on the expression of Tim-3 in immune subsrts in different regions of cancerous tissues.The results showed that the expression of Tim-3 in each immune cell subsets in tumor and stroma regions was positively correlated,and there was more positive correlation in tumor regions?p<0.0001?.This further suggests that there may be a common factor regulating the expression of Tim-3 in each immune cell subgroup in the HCC microenvironment.1.14 Correlation between the expression of Tim-3 in combined immune cell subsets with patient survivalWe further analyzed the correlation between the expression of Tim-3 on the surface of two immune cell subsets in different regions of cancerous tissues with patient prognosis.Survival analysis showed that the group of patients with low expression of Tim-3 in CD4+/CD8+ cells,CD4+/CD56+ cells and CD4+/CD68+ cells had a better prognosis than the high expression group in tumor and stroma regions.The difference of the former two groups is more apparent in the tumor area,after a set of difference is more obvious in interstitial area.There was no significant difference in the expression of Tim-3 with patient prognosis in the other groups.This indicated that the double high expression of Tim-3 on CD4+ and CD8+,CD4+ and CD56+ cell surface in the tumor region of HCC tissue,and the double high expression of Tim-3 on the surface of CD4+ and CD68+ in the interstitial region might be closely related to the progression of HCC.2.Effect of CD4+ cells on Tim-3 immunomodulatory function in HCC patientsThe results of the first part of this study suggest that CD4+ cells may play an important regulatory role in the microenvironment of HCC,and this effect may be related to the expression of Tim-3.To further clarify the roles of CD4+ cells in Tim-3-mediated immune regulation,we collected the peripheral blood mononuclear cells?PBMCs?of HCC patients,and/or removed CD4+ cells in PBMCs,and/or blocked Tim-3,and finally detected the changes in functions or phenotype of CD8+T,NK,NKT and mononuclear cells by flow cytometry.2.1 Effect of blocking Tim-3 on immune cell subsets in peripheral bloodWe isolated the PBMCs from 11 patients of HCC,and/or blocked Tim-3,and then stimulated with PMA and ionomycin,and collected the cells labeled with fluorescence antibodys.And we finally detected the changes in functions or phenotype of CD8+T,NK,NKT and mononuclear cells by flow cytometry.The results showed that the proportion of CD4+T T,CD8+ T,NK and NKT cells have no change after blocking Tim-3 in PBMCs,but the ability to secrete TNF-a and IFN-y is significantly enhance ed.Besides,the ability of CD8+ T,NK,and NKT cells to express CD107a is also significantly increases,which explains that blocking Tim-3 can enhance the production of cytokines and killing activity in CD4+ T and CD8+ T,NK and NKT cells of HCC.However,the proportion of monocytes decreased signifacantly,and the expression of CD206 was also down-regulated,but there was no significant change in the proportion of MDSCs,which suggested that Tim-3 may regulate the polarization process of monocytes.2.2 Effect of CD4+ cells removal on immune cell subsets in peripheral bloodWe isolated the PBMCs from 11 patients of HCC,and/or removed the CD4+ cells by means of magnetic bead sorting,and stimulated by PMA and ionomycin.Then,we collected the cells labeled with fluorescence antibodys,and we finally detected the changes in functions or phenotype of CD8+T,NK,NKT and mononuclear cells by flow cytometry.The results showed that the average removal rate of CD4+ cells was up to 82.77%after magnetic bead separation,and the proportion of CD8+T,NK and NKT cells in lymphocytes was significantly increased after removing CD4+ in PBMCs,and the ability to express TNF-?,IFN-y and CD107a was significantly enhanced.These results suggest that CD4+ cells can inhibit the function of CD8+T,NK and NKT cells in PBMCs of patients with HCC.After removing CD4+ cells,the proportion of monocytes in PBMCs was significantly reduced,and the expression of CD206 on the surface was also significantly down-regulated.However,there was no significant change in the proportion of MDSCs,whith suggested that CD4+ cells may be involved in the regulation of to M2-type monocyte polarization.2.3 Effect of CD4+ cells removal on Tim-3-mediated immune regulationWe isolated the PBMCs from 11 patients of HCC,and/or removed the CD4+ cells by means of magnetic bead sorting,and/or then blovked Tim-3,and stimulated by PMA and ionomycin.Finally,we collected the cells labeled with fluorescence antibodys and detected the changes in functions or phenotype of CD8+T,NK,NKT and mononuclear cells by flow cytometry.The results showed that after removing CD4+ cells,the promoting effect of blocking Tim-3 on the secretion of TNF-a,IFN-?and CD107a by CD8+T,NK and NKT cells was significantly reduced,and the effect of Tim-3 blocking on monocyte phenotype was also significantly reduced.These results suggest that CD4+ cells play an important role in the immunoregulatory activity of Tim-3.ConclusionBased on the above experimental results,we preliminarily confirmed the expression and role of Tim-3 in the microenvironment of HCC,and made the following conclusions:1.The infiltration of immune cells in HCC tissue was significantly lower than that in the adjacent tissue,and the infiltration of CD4+ cells in the tumoral region was significantly positively correlated with the infiltration of other immune cell subgroups,and positively correlated with the prognosis of patients;2.Tim-3 expression in cancerous tissues was significantly higher than that in adjacent tissues,and was negatively correlated with prognosis.3.The expression of Tim-3 on immune cells in cancerous tissues was significantly higher than that on immune cell in adjacent tissues,and the expression ofTim-3 in CD4+ cells was positively correlated with that in other immune cells,and was significantly negatively correlated with the prognosis of patients.4.CD4+ cells may be involved in the immune regulation of Tim-3 in HCC.Innovations and significancesIn this study,we firstly introduced the tumor microenvironment landscape analysis,analyzed the expression of Tim-3 on a variety of immune cell subgroups in HCC tissue,and the relationship with the prognosis of patients.And we firstly proveded that the influence of CD4+ cells on immune regulatory function of Tim-3.It might provided a systematic basis for the application of Tim-3 targeted immune intervention strategy in HCC. |
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