| ObjectMalignant lymphoma is the general name of a large class of lymphatic hematopoietic malignancies.It is divided into two categories:Hodgkin’s and non-Hodgkin’s lymphoma.Malignant lymphoma is the top ten in all malignant tumors.In recent years,with the improvement of life,the incidence of malignant lymphoma is increasing year by year.PD-1 antibody,as an effective immunotherapy,has been applied to a variety of malignant tumors,of which relapsed refractory Hodgkin’s lymphoma can reach a total of 87%total efficiency.But for some patients,the effect of PD-1 antibody treatment is poor,so it is imminent to find a kind of drug to improve the response rate of PD-1 antibody.Through controlling the regulation of epigenetic and acetylation level of histone to control the gene expression,Histone deacetylase inhibitors(HDAC,Chidamide)increase the ability of cell immunity and enhance the antitumor immune response.The aim of this experiment is to explore the mechanism of Chidamide in vitro and the effect of Chidamide combined with PD-1 antibody in regulating tumor immune microenvironment,so that to provide theoretical basis for combination therapy strategy of Chidamide and PD-1 antibody in the treatment of malignant lymphoma.Methods1)Comparison the ability of Chidamide to induce apoptosis of different lymphoma cell lines.2)After 8 and 24 hours that Chidamide added into each cell lines,extracting protein of different cell lines.Analyzing cell line expression of total histone,acetylated histone and acetylated alpha-tubulin by Western Blot,so that we can clarify the mechanism of Chidamide on different lymphoma cell lines.3)Detecting the expression of PD-L1 molecules on the surface of different lymphoma cell lines at different time points with Chidamide.4)We selected EL4 cells to subcutaneously inject at armpit of healthy black mice which had normal immunity(C57BL/6).Lymphoma model mice were divided into 4 groups randomly,including blank control group,single PD-1 antibody group,single Chidamide group and combined group.Injected each group at the corresponding dose and interval time,respectively.Each group was executed two mice per two week randomly to analysis the amount of infiltration T cells and inhibitory T cells in the tumor microenvironment and spleen.5)After the formation of tumor,we injected quantitative dose at correspond time point to each group,and made registration of death,measure the tumor volume of mice daily with vernier caliper by the same researcher from the first day given drug to the end of the experiment,After treatment for 3 weeks,all the mice were executed,stripped tumor samples of each group and measure weight of all masses respectively.After the end of the experience,we analyze the changes of mice tumor load and survival by SPSS software.6)After three weeks of giving drugs,all the mice were executed.The fresh tissues removed from each group was fixed by formalin and embedded by paraffin.The expression level of PD-1 and PD-L1 on tumor cell surface was analyzed by immunohistochemistry.Results1)For T-cell lymphoma cell lines,including EL4,HUT78,Jurkat and Karpas,with the time of adding Chidamide prolonging,the apoptosis ratio of cell lined increased.On the contrary,for B-cell lymphoma,including LY8 and Raji,there was no significant change in the proportion of apoptotic cells with the prolongation of the time of drug action.2)Choosing four cell lines of T-cell lymphoma which Chidamide can induce significant apoptosis,adding corresponding concentration of Chidamide for 8 and 24 hours.According to the results of Western Blot,the acetylation of alpha-tubulin of Karpas cell lines has increased significantly,the acetylation of histone of EL4 cell lines also has increased remarkably.For Jurkat and HUT78 cell lines,the level of acetylation of two groups of proteins increased in varying degrees,which indicted that Chidamide play the key role by improving acetylation level of protein to control the expression of gene.3)Testing the expression of PD-L1 of each T-cell lymphoma cell lines which mixing with Chidamide for 12,24 and 36 hours and blank control group by flow cytometry.The results show that the expression of PD-L1 on EL4 cell line increases with the time of drug action,the level of PD-L1 expression on the cell surface of HUT78 and Jurkat cell lines is always lower,and high expression of PD-L1 on the cell surface of Karpas cell line can find at any time.4)For the number of CD8+ T cells,after using each drug for 7 days,the maximum number of T cells in spleen tissue was the blank control group,followed by Chidamide group,however single PD-1 antibody group had the lowest number of effective lymphocyte.In the tumor tissue,single Chidamide group achieved highest number of CD8+T cells,followed by the blank control group,and the number of effective CD8+T cell in combined group was lowest.After 21 days of drug application,the level of effective T cells both in spleen tissue and tumor tissue in the blank control group decreased to the lowest level,while the level of CD8+T cells in the two single drug groups were not significantly different.The level of effective cells in the spleen and tumor tissues of the combined group reached the highest level.For the number of regulatory T cells,after application of drugs for seven days,the number of T suppressor cell in the spleen tissue,single PD-1 antibody group achieved the highest level,and the combined group reached the highest level in tumor tissue.Twenty-one days later,the number of regulatory T cells of single PD-1 antibody group in the spleen tissue was the lowest,and in the tumor tissues,the highest level of regulatory T cell was blank control group,the lowest was combined group.5)Compared with the tumor weight of each group,the mean weights for blank control group,single PD-1 antibody group,single Chidamide group and combined group were 9.79g,6.93g,6.17g and 5.16g,respectively.The combination group and Chidamide group both had significant difference compared with blank control group(p<0.05).The result of comparative analysis of survival in each group indicated that the median survival time of the blank control group,single drug PD-1 antibody group,single drug Chidamide group and combined group was 17.59 days,20.62 days,21.35 days and 21.86 days,respectively(p<0,05).For the tumor volume of each group,which measured every day,the record suggested that there was significant difference in tumor volume between the single PD-1 antibody group and the single Chidamide group and between single PD-1 antibody group and combination group at fourth days after injection drugs(p<0.05).And this difference has been maintained to the fourteenth days of given drugs.However,there was no significant difference between single PD-1 antibody group and blank control group(p<0.05).6)After 3 weeks of medication,the results of immunohistochemistry showed that the expression level of PD-1 in each group is similar,there was no statistic difference between each group.For the expression of PD-L1,the difference is significant,single Chidamide group has the highest expression level of PD-L1.The image of immunohistochemistry staining showed strong positive,followed by the control group and single PD-1 antibody group,which showed positive,and combined group had weakly positive expression of PD-L1.Conclusion1 Chidamide can effectively induce apoptosis of T cell lymphoma cell lines.It regulated the formation of protein by controlling the acetylation level of histone and alpha-tubulin.2 With the prolongation of the time of drug action,Chidamide can effectively improve the cell surface expression of PD-L1 on EL4 cell line.3 Chidamide can quickly adjust the tumor microenvironment and infiltration of immune cell in vivo,resulting in a large number of tumor infiltrating lymphocyte cells attracted into tumor microenvironment,also can increase the expression level of PD-L1 on tumor cell,enhancing the immunogenicity of tumor and immune response of body.4 According to the macro analysis of in vivo experiments,the combination group showed an absolute advantage in tumor load and survival time compared to other experimental groups and blank control group,there exist significant difference. |
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