The Study Of The Effect Of IL-36 In Combination With Inhibition Of PD-1 Signaling On Promoting Tumor Immune Responses

The immune response in tumor microenvironment is generally inhibited and restoring and activating antitumor activeity of tumor infiltrating immune cells by effective means contribute to inhibiting tumor progression.The previous work in our laboratory demonstrated that IL-36(interleukin-36)could improve tumor immune microenvironment and significantly inhibited tumor progression.However,further studies revealed that the T-cells stimulated by IL-36 increased the expression of the immune checkpoint molecular PD-1(Programmed death-1),which mediated the negative feedback inhibitory effect on tumor immunity.Therefore,it may be helpful to promote the tumor immune response by the combination of IL-36? stimulation and blocking the PD-1 signal.The purpose of this study is to establish the gene transfection cell lines co-expressing of IL-36? and sPD-1(Solubel PD-1,an extracellular fragment of PD-1 molecule which can be used to block PD-1 signal by combining PD-L1),and discuss the collaborative role and mechanism of IL-36? combining block PD-1 signal on the tumor immune response through building tumor-bearing mouse models,and then lay a foundation for adopting such a clinical regimens in tumor therapy.Part ? Preparation and identification of gene transfection cell lines co-expressing IL-36? and sPD-1Objective: This research aims to establish tumor cell lines which can stably secrete IL-36? and sPD-1 in the tumor microenvironment and lay a material foundation for further study of their collaborative role in tumor immune responses.Methods: The gene sequence of sPD-1 was designed,synthesized and inserted into plasmid pCDEF3 to construct a recombinant plasmis pCDEF3-sPD-1.Then PCR method and sequencing tehchnique were adopted to confirm the target fragment.In order to get the transfectants B16-sPD-1 and B16-IL-36?-sPD-1,pCDEF3-sPD-1 was transfected into B16 cell line by liposomes and the mixture of pCDEF3-sPD-1 and p CMV4.Pur with a resistant gene to puromycin was transfected into B16-IL-36? cell line,which can secret IL-36? stably.Then the transfected cell lines were selected by G418 or puromycin respectively and the ability of B16-sPD-1 and B16-IL-36?-sPD-1 cell lines secreted sPD-1 was identified by ELISA.At the same time,we make the comparative analysis of secretion expression between these 2 kinds of gene transfection cell lines.The control group B16-vec was obtained by transfecting with empty plasmid.Results: Through designing the gene sequence encoding sPD-1 gene and carrying out gene recombination,we constructed pCDEF3-sPD-1 successfully.Then we successfully obtained a series of stable expression and secretion of sPD-1 B16-sPD-1 and B16-IL-36?-sPD-1 clone cells by transfection,screening and ELISA determination,which lay a material foundation for further research on the synergistic antitumor immune response effect.Conclusion: The transfected cell lines of B16-sPD-1,which can secrete sPD-1 stably,and B16-IL-36?-sPD-1,which can stably co-express IL-36? and sPD-1,were successfully constructed.This transfected cell lines laid a material foundation for further study on the synergistic effect and mechanism of IL-36 ? stimulation and blocking PD-1 signal on tumor immune response.Part ?: The study of the effect of IL-36? in combination with blocking PD-1 on promoting tumor immune responsesObjective: This research aims to study the effect and mechanism of IL-36? and sPD-1 secreted in tumor microenvironment on promoting tumor immune responses and lay a foundation for adopting such a clinical regimens in tumor therapy.Methods: The expression of PD-L1 on the surface of B16 and 4T1 cell lines was detected by flow cytometry,and the expression of PD-1 gene in IL-36? treated T cells or tumor tissues was analyzed by QRT-PCR.B16-vec,B16-sPD-1,B16-IL-36? and B16-IL-36?-sPD-1 gene transfected cells were inoculated into the abdominal subcutaneous tissue of C57BL/6 WT mice respectively.The tumor growth and survival time were measured every 2 days,and the tumor tissues were digested in the early and late stages of tumor growth.The distribution and function of immune cells in mouse tumor microenvironment were detected by flow cytometry and compared with each other.Results: The results showed that PD-L1 could be expressed on B16 cell lines.IL-36? stimulation significantly up-regulated the expression of PD-1 gene in T cells,and the overexpression of IL-36? in tumor cells resulted in an up-regulation of PD-1 gene level in tumor tissues.Compared with the over-expression group of IL-36? alone,IL-36? combined with sPD-1 could significantly inhibit the growth of tumor and prolong the survival time of tumor-bearing mice.The results of immunocyte detection in the early tumor microenvironment of different tumor-bearing mice showed that IL-36 ? combined with sPD-1 could significantly promote the immune response of tumor,compared with IL-36? stimulation alone.The co-expression of IL-36? and sPD-1 in tumor microenvironment could significantly up-regulate the ratio of CD4+ TILs,promote the Ki67 expression of CD4+ TILs and CD8+ TILs,and down-regulate the ratio of CD11b+ Gr-1+ MDSCs.The results of detection in B16-IL-36? and B16-IL-36?-sPD-1 late tumor microenvironment showed that the co-expression of IL-36? and sPD-1 in tumor microenvironment could significantly increase the proportion of CD45+ TILs compared with the group of IL-36? overexpression alone.Co-expression of IL-36? and sPD-1 can also promote nuclear proliferating antigen Ki67 expressed by CD4+ TILs and CD8+ TILs,and enhance the IFN-? expression of CD4+ TILs and CD8+ TILs.Conclusion: The tumor immune response can be promoted by IL-36? combined with blocking PD-1 signal,which can significantly inhibit the growth of tumor and prolong the survival time of mice.