Study On The Anti-tumor Effect Of WT1-DNA Vaccine With Multiple Low Dose Cyclophosphamide

ObjectiveObjective:1 To compare the number and function alteration of CD4~+CD25~+ regulatory Tcell treated with single or multiple low dose cyclophosphamide in murine, anddetermine whether low dose cyclophosphamide can be used to down regulate Treg cellrepeatedly.2 To investigate the antitumor effect of DNA vaccine and the combination effectwhen they administrated with multiple low dose cyclophosphamide, by injecting DNAvaccine with multiple low dose cyclophosphamide in murine.Methods:Part 1Thirty-two male C57BL/6J mice were randomly divided into four groups, control,once, twice and thrice treated groups. Each group was injected intraperitoneally with20mg/kg CTX or equal volume PBS every ten days. Three days after the last injection,the ratio of Treg cells to CD4~+ T cells in spleen was detected by flow cytometry; Theproliferation of T and B lymphocytes were detected by CCK-8 test; and thephagocytosis of macrophages was detected by neutral red test.Part 21 Extract WT1-DNA plasmid and control plasmid abundantly.2 The prophylactic trial of tumor Sixty male C57BL/6J mice were randomlydivided into five groups, PBS control, empty plasmid control, CTX control, DNAvaccine and combination treated groups. Each group was injected intraperitoneally with20mg/kg CTX or equal volume PBS every ten days. Three days after each injection,mice were injected intramuscularly with either 100 ug WT1-DNA plasmid, controlplasmid or equal volume PBS, repeat three times. Seven days after the last injection, theCTX and combination groups were injected intraperitoneally with 20mg/kg CTX again,the others were injected equal volume PBS. Three days after these injections, four micewere killed in every group. The ratio of Treg cells to CD4~+ T cells, CD3~+CD4~+ T cells tototal lymphocyte, CD3~+CD8~+ T cells to total lymphocyte, and the secretion of IFN-γamong CD4~+ and CD8~+T cell in spleen was detected by flow cytometry. At the sametime, other spleen cells were cultured for three days with WT1 peptide, then cytotoxicactivity of spleen cells to FBL3 were performed. The other mice were injectedsubcutaneously with FBL3 cells. Observe and record the growth rate and the volume of tumor.3 The therapy trial of tumor Fifty male C57BL/6J mice were randomly dividedinto five groups, the same as prophylactic trial. Each mouse was injectedsubcutaneously with FBL3 cells on day 1. On day 2 were injected intraperitoneally with20mg/kg CTX or equal volume PBS. On day 4 were injected intramuscularly witheither 100 ug WT1-DNA plasmid, empty plasmid or equal volume PBS. The mice wereboosted ten days later. Observe and record the growth rate and the volume of tumor.ResultsResults:Part 11 Compared to control group, the percentage of Treg cell account for CD4~+T cellsof treated groups decreased sharply (P＜0.05), but there was no difference between thetreated groups (P＞0.05);2 The proliferation of T and B lymphocytes and the phagocytosis of macrophageswere all stronger significantly than control group(P＜0.05), but there was no differencebetween the treated groups(P＞0.05);Part 21 The result of prophylactic trial1.1 Compared to PBS control group, the percentage of Treg cell account forCD4~+T cells of CTX treated group and combination group decreased sharply (P＜0.05),but there was no difference between these groups (P＞0.05); There was no differencebetween PBS control, empty plasmid and DNA vaccine groups (P＞0.05).1.2 Compared to PBS control group, the percentage of CD3~+ T cell account fortotal lymphocyte of CTX, DNA vaccine and combination groups raised sharply (P＜0.05), but there was no difference between PBS and empty plasmid groups (P＞0.05);There was significantly difference between CTX, DNA vaccine and combination groups(P＞0.05), meanwhile the ratios combination group＞DNA vaccine group＞CTXgroup.1.3 Compared to PBS control group, the percentage of CD3~+CD4~+ T cell accountfor total lymphocyte of DNA vaccine and combination groups raised sharply (P＜0.05),but there was no difference between these groups (P＞0.05); There was no differencebetween PBS, CTX and empty plasmid groups (P＞0.05).1.4 Compared to PBS control group, the percentage of CD3~+CD8~+ T cell accountfor total lymphocyte of CTX, DNA vaccine and combination groups raised sharply (P＜0.05), but there was no difference between PBS and empty plasmid groups (P＞0.05);There was significantly difference between CTX, DNA vaccine and combination groups (P＞0.05), meanwhile the ratios combination group＞DNA vaccine group＞CTXgroup.1.5 After stimulated by WT1-126 or WT1-235 peptide, the percentage ofCD8~+IFN-γ~+T cell account for CD8~+T cell of DNA vaccine and combination groupsraised sharply compared to PBS control group (P＜0.05), but there was no differencebetween PBS, empty plasmid and CTX groups (P＞0.05); There was no differencebetween DNA vaccine and combination groups too; But the percentage of CD4~+IFN-γ~+Tcell account for CD4~+T cell of each group has no difference (P＞0.05).1.6 After stimulated by WT1-126 or WT1-235 peptide, the killing rates of spleencells of CTX, DNA vaccine and combination groups raised sharply compared to PBScontrol group (P＜0.05), but there was no difference between PBS and empty plasmidgroups (P＞0.05); There was significantly difference between CTX , DNA vaccine andcombination groups (P＞0.05), meanwhile the rates combination group＞DNA vaccinegroup＞CTX group.1.7 The result of forming-time and volume of tumor Compared to PBS controlgroup, the median tumor-free survival time of CTX, DNA vaccine and combinationgroups raised sharply (P＜0.05), but there was no difference between PBS and emptyplasmid groups (P＞0.05); There was significantly difference between CTX, DNAvaccine and combination groups (P＞0.05), meanwhile the median tumor-free survivaltime combination group＞DNA vaccine group＞CTX group. It is coincidence with theoutcome of tumor volume.2 The result of therapy trialMice of CTX group and combination group survival without tumor until the lastobervation day. Compared to PBS control group, the median tumor-free survival time ofDNA vaccine group raised sharply (P＜0.05), but there was no difference between PBSand empty plasmid groups (P＞0.05). It is coincidence with the outcome of tumorvolume.ConclusionConclusion:1 There is no difference in decreasing Treg cell between the single andmulti-administration with low dose cyclophosphamide, also no difference in regulatingthe function of immunocells subsequently. Low dose cyclophosphamide could be usedto down regulate Treg cell repeatedly.2 Multiple administration with low dose cyclophosphamide can down regulatethe number and the function of Treg cell, and up regulate the percentage of CD3~+ andCD3~+CD8~+ T cells subsequently, but it doesn't influent the CD3~+CD4~+ T cells. Multiple administration with low dose cyclophosphamide has anti-tumor effectiveness.3 WT1-DNA vaccine can up regulate the number of T lymphocyte, both CD4~+and CD8~+ T lymphoyte raised. This vaccine can induce WT1 specific CTLs, enhancethe cytotoxity of spleen cells and delay tumor-free survival time. Moreover, comparedto CTX it has stronger anti-tumor effectiveness.4 The combination of multiple low dose cyclophosphamide and DNA vaccinehas the better anti-tumor effectiveness.