Experimental Study On The Influence Of The Combined Therapy Of Anti-Tim-3 MAb And Chemotherapeutics Upon Relapse Of Lymphoma After Allogenic Bone Marrow Transplantation And Melanoma In Mice

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Experimental study on the influence of the combined therapy of anti-Tim-3 mAb and cyclophosphamide upon relapse of lymphoma after allogenic bone marrow transplantation in mice modelObjectives:To establish the mouse model of relapse of lymphoma after allogenic bone marrow transplantation.To investigate the antitumor effect of the combined therapy and explore the possible mechanisms underlying this combination medication,thereby providing new avenues for clinical treatment.Methods:1.Female C57BL/6 mice were donor mice,and male BALB/c mice were recipient mice conditioned by a single dose of total body irradiation(8Gy).6 hour later.transplantation was conducted by injection of 0.2mL of PBS including 1 × 107 bone marrow cells,5×106 splenocytes and 1 × 106 A20 cells through the tail vein of the recipient mice.2.The mouse model of relapse of lymphoma after allogenic bone marrow transplantation was concluded to be successful or not,according to the clinical presentation of the mice,peripheral blood cell counting and peripheral blood smears.3.The mice were randomly divided into four groups(isotype control group,anti-Tim-3 mAb group,cyclophosphamide group and combination of cyclophosphamide and anti-Tim-3 mAb group).The survival time of the mice was recorded.Flow cytometry was employed to analyze the percentage of CD4+ T cells,CD8+ T cells,CD3+CD4+CD25+FoxP3+ Treg cells and CD3+CD8+Granzyme B+ T cells in peripheral blood and spleen lymphocytes.Results:The mouse model of relapse of lymphoma after allogenic bone marrow transplantation was set up successfully.Combined therapy of anti-Tim-3 mAb and cyclophosphamide remarkably improved the survival time of the mice(elevated 7.2 days in average,P<0.05).Compared to the contrast,anti-Tim-3 mAb significantly enhanced the percentage of CD4+ T cells in peripheral blood and spleen lymphocytes(10.1%in peripheral blood lymphocytes and 2.2%in spleen lymphocytes,P<0.05);while cyclophosphamide decrease the percentage of CD4+ T cells in spleen lymphocytes(19.1%,P<0.01).Compared to the isotype control group,combined therapy of anti-Tim-3 mAb and cyclophosphamide notably reduced the percentage of CD3+CD4+CD25+FoxP3+ Treg cells(1.55%in peripheral blood lymphocytes and 1.86%in spleen lymphocytes,P<0.01),brought up the percentage of CD3+CD8+Granzyme B+ T cells(6.84%in peripheral blood lymphocytes and 5.47%in spleen lymphocytes,P<0.01)and recover the ratio of CD4/CD8 T cells.Conclusions:CD4+ T cells,CD8+ T cells,Treg cells and CTL cells play considerable roles in the relapse of lymphoma after allogenic bone marrow transplantation.Combined therapy of anti-Tim-3 mAb and cyclophosphamide was proved effective in the treatment of relapse of lymphoma after allogenic bone marrow transplantation.Part ? Combined therapy of anti-Tim-3 mAb and trametinib enhances the efficacy against melanomaObjectives:To investigate the antitumor effect of the combined therapy and explore the possible mechanisms underlying this combination medication,thereby providing new avenues for clinical treatment.Methods:First,we established melanoma animal model in C57BL/6 mouse by injecting B16F10 melanoma cells subcutaneously.The mice were randomly divided into four groups(control group,anti-Tim-3 mAb group,trametinib group and combination of trametinib and anti-Tim-3 mAb group).Then compared the tumor sizes of animals received different treatments and portrayed the growth curve.Flow cytometry was employed to analyze the percentage of CD4+,CD8+ and Tim-3+ T cells in tumor infiltrating lymphocytes.Immunohistochemistry was used to test the positive rate of Ki67 and Caspase-9 cells.Cell viability was evaluated by CCK8 assays.Results:Compared to the contrast.trametinib significantly inhibited the growth of B16-F10 cells,with the highest inhibition rate at 4 h.The inhibition rate of trametinib treated group and the combined drug group was 22.3 ±0.83%and 23.0±1.11%separately(P<0.01).The positive rate of caspase-3 cells in the trametinib treated group and the combined drug group were significantly higher than the control group(P<0.01).However the positive rate of Ki67 cells in the trametinib treated group and the combined drug group were noticeably lower than the control group(P<0.01).It was identified that tumor growth was significantly retarded in the combined drug group compared with the control group,exhibiting 40.8%reduction in tumor size at day 23.Moreover,the frequency of tumor-infiltrating CD8+ T cells was drastically higher in mice treated with anti-Tim-3 mAb compared to control group;while the frequency of Tim-3+ CD8+ T cells was two folds higher in trametinib treated group.Conclusions:Trametinib performances a striking therapeutic efficacy against established B16F10 melanoma by promoting apoptosis and inhibiting cell proliferation.Anti-Tim-3 mAb augmented the antitumor immunity by stimulating CD8+ T cells.Drug combination of trametinib and anti-Tim-3 mAb can generate potent synergistic anti-tumor effect against melanoma.