The Antibody PD-1 And CTLA-4 Combined With Macs IFN-? Positive T Lymphocytes Research Melanoma Treatment

Objective: This experiment set up tumor model by injecting B16 melanoma to mouse back.The primitive cells derived from bong marrow was induced to matured and loaded melanoma antigen DC cells,then co-cultured with haploidentical T lymphocytes(C57BL/6).3 to 5 days later,the activated IFN-? secreting T lymphocytes were collected by magnetic beads sorting.Then the activated IFN-? secreting T lymphocytes adds checkpoint inhibitor(PD-1and CTLA-4 inhibitors)and incubated for half an hour.Then the collected incubated T lymphocytes were subjected to the following cytotoxic assay in vivo respectively.The collected T lymphocytes were injected to tumor-carrying C57BL/6 mice models,signs of the side effects of the checkpoint inhibitor and the lethal effect to the B16 melanoma cells during the next two or three weeks,after treated with different reagents such as the checkpoint inhibitor incubated with effector T lymphocytes,checkpoint inhibitor and control,and the levels changing of IL-10 mRNA and TGF-? mRNA in mouse tumor tissue was also detected with RT-PCR assay.The inhibitor cells(Tregs)in spieen was checked out with FCM.The survival period in different mice groups was observed within 80 days.This study explored the lethal effect to the B16 melanoma by treatment with immune checkpoint inhibitor combined with Macs,and the study provide more fundmental datas for constructing an innovative remedy also known as tumor microenvironment interfering induced adoptive cellular immunotherapy.Methods:1 We recovery and cultivate B16 melanoma for the use of setting up B16-carrying mice model.2 Induction and cultivate DC cells : The primitive cells derived from bong marrow was induced with rmGM-CSF,rmIL-4 and B16 melanoma antigen.Then we used TNF-? to induced matured DC cells.3 Preparation of magnetic beads selected effector T lymphocytes cells: Spleen cells were isolated by density gradient centrifugation,the collected T lymphocytes were adsorption with nylon wool column.2 day's later,the DCs sensitized by B16 antigen was added to the culturing T lymphocytes co-culturing for 3-4 days to obtain activated effector T lymphocytes cells.Then the effector T cells which secretion IFN-? were enriched by magnetic bead sorting.4 MACS purified CTL were incubated with immune checkpoint inhibitor: The magnetic bead sorting of IFN-?-positive cells incubated with PD-1 and CTLA-4 inhibitor at 37 ° for half an hour.5 The experiment groups: The carrying-tumor mouses were randomized divided into 3 groups,each group included 40 carrying-tumor mouses.PBS group(group A): mouse was injected by vein with 0.2ml PBS;Immune checkpoint inhibitor combined with Macs group(group B):mouses injected with MACS purified CTL combined with immune checkpoint inhibitor 1×106 per 0.2ml by vein;Immune checkpoint inhibitor treatment group(group C):PD-1 and CTLA-4 inhibitors were injected by intraperitoneal at 20 ug per inhibitor.Observed the mouse survival time and the range of tumor in different treatment groups.6 Killing mouses to obtain tumor tissue used to performed RT-PCT examined at 1,4,7,10,14 day after treatment.We observed the range of IL-10 mRNA and TGF-? mRNA by RT-PCT.Then comparing the effect in different treatment.7 Collecting mouse spleen to make single cell suspension which used for monitoring content and changes in Tregs by FCM.Comparision of inhibitor cells in spleen with different methods.8 Collecting mouse tumor tissue,liver,lung and small intestine to used for biopsy to observed pathological changes.Whether MACS purified CTL combined with immune checkpoint inhibitor induced side effects of immune checkpoint inhibitor:reactive hepatitis,enteritis and so on.Results: 1 It's about 7 days for successfully setting up B16-carrying mice model;2 The mature dendritic cells appear suspending,having dendritic prominencies on the 6th day;3 The range of tumor volumes: Mouse injected with PBS developed more huge tumor volume.MACS combined with immune checkpoint inhibitor DC-CTLs and immune checkpoint inhibitor treatment can significantly inhibit tumor growth from day 7 to day 20;4 The time of survival periods of A,B and C groups: There is obvious statistical significance different in each group(P<0.05);5 The range of IL-10 mRNA and TGF-? mRNA levels examined by RT-PCR: The treatment of MACS combined with immune checkpoint and treatment with immune checkpoint all can significantly reduced the tumor tissue levels of IL-10 mRNA and TGF-? mRNA but there is no significantly difference in these treatment groups(P>0.05);6 MACS combined immune checkpoint inhibitor therapy and checkpoint inhibitor all can decline the inhibitor cell(Tregs)in mouse spleen;7 Biopsy results:MACS combined with immune checkpoint inhibitor group does not respond to hepatitis,enteritis and other immune checkpoint inhibitor side effects.Reactive hepatitis,enteritis appear in immune checkpoint inhibitor group.Conclusions:1 Compared with PBS group,MACS combined with immune checkpoint inhibitor group and immune checkpoint inhibitor treatment can significantly inhibit tumor developing after treatment 7 to 20 day.The tumor developed speed is no significantly difference between Immune checkpoint inhibitor combined with MACS and immune checkpoint inhibitor group.MACS combined with immune checkpoint inhibitor significantly inhibited tumor growth.2 Treatment with MACS combined with immune checkpoint inhibitor group or immune checkpoint inhibitor treatment all can increase the time of tumor mouse survival period.The time of survival period of the tumor mouse received MACS combined with immune checkpoint inhibitor treatment was longer than that of immune checkpoint inhibitor group chemotherapy.3 The immunotherapy of MACS combined with immune checkpoint inhibitor and immune checkpoint inhibitor all can decreased the levels of tumor tissue IL-10 mRNA and TGF-? mRNA,but there is no significantly difference between these two treatment groups.4 MACS combined immune checkpoint inhibitor therapy and checkpoint inhibitor all can decline the inhibitor cell(Tregs)in mouse spleen5 MACS combined with immune checkpoint inhibitor group does not respond to hepatitis,enteritis and other immune checkpoint inhibitor side effects.