The Relationship Between Treg Cells And Cryoablation Immune Response Of Prostate Cancer

BackgroundPercutaneous cryoablation therapy for prostate cancer can induce cryoablation immune response, while there are big individual differences of the strength and last duration of this immune response, how to improve the strength and last duration of the immune response effectively are the main point and hotpots in this field currently. What and how of Treg cells play the role in cryoablation immune response, and can we change their functional status by the immune adjuvant reagents, vaccines and other drugs to strengthen this immune response and working time, and then increase the survivle rate of petients. This can provide theoretical and experimental evidence for cryoablation combined with immunotherapy which may become the best treatment for prostate cancerObjective1. Making the models of RM-1 prostate cancer to assess the function of Treg and anti-tumor immune response after cryoablation, and study the immune response of combination therapy of cryoablation and Anti-CTLA-4Ab treatment.2. We aim to confirm the immunosuppression fuction of CD4+CD25+Foxp3+Treg to Helper T cell(Th) and Cytotoxic T Lymphocyte(CTL) in vitro.Materials and Methods1. Making the mouse models of RM-1 cell line.2. Tumor-bearing mice were divided into four groups: GroupA-control group, Group B-cryoablation only group, Group C-cryoablation+Anti-CTLA-4Ab group and Group D- Anti-CTLA-4Ab only group. Cryoablation was applied with 2 mm diameter cryosurgery needle from Endocare Argon-Helium cryosurgery system.3. Lung or Tumor draining lymph note(TDLNs) metastasis rate were assessed by HE stain; number changes of Treg, CD4+Th and CD8+CTL cells in tumors, TDLNs and spleens were detected by immunohistochemistry and flow cytometry; Tumor-specific CTL of TDLNs and spleens were measured by LDH assay; serumcytokine(IL-2、TGF-β1) levels were analyzed by enzyme-linked immunosorbent assay(ELISA). Measure the tumor size of all groups and all time points and follow up the overall survival time of all groups.4. Obtain the spleens form 5 Foxp3-GFP(FITC) transgenetic mice, and sort four cell populations of CD4+CD25+Foxp3+Treg、CD4+CD25-T、CD8+T and DC+ cells out respectively. Co-culturing different cell populations for different groups and add Anti-CTLA-4Ab for an intervention factor, detect the apoptosis rate of CTL at different time points of 24 h, 48 h and 72 h, and detect the supernatants cytokine levels of TGF-β1、IFN-γ、IL-4、IL-10 at the same time.Results1. TDLNs and lung metastasis rates of control group, cryoablation group, cryoablation+ Anti-CTLA-4Ab group, only Anti-CTLA-4Ab group on day 21 were 100%、80%、60% 、80% and 100%、80%、60%、80%,respectively.2. Tumor size of cryoablation group and cryoablation+ Anti-CTLA-4Ab group were significantly got to be smallest on day 14(P<0.05), while for cryoablation group, it grown back to the level of day 0 on day 21(P<0.05), for cryoablation+ Anti-CTLA-4Ab group, it did not back to the level of day 0 on day 21.3. The overall survival rate was significantly higher in cryoablation group and cryoablation+ Anti-CTLA-4Ab group than in the control group(P=0.045,P<0.001); it had significant difference in cryoablation+ Anti-CTLA-4Ab group than in cryoablation group and only Anti-CTLA-4Ab group(P=0.025,P=0.002).4. Serum IL-2(pg/ml) levels of cryoablation group and cryoablation+ Anti-CTLA-4Ab group achieved to the highest level on day 14, but returned to the level of day 0 on day 21(P<0.05);Serum IL-2(pg/ml) levels of these two groups decreased to the lowest level on day 14, while increased on day 21 which not increased to the level of day 0(P<0.05).5. Treg、CTLA-4+T、CD4+Th、CD8+CTL cells changes of tumors、TDLNs and spleens detected by immunohistochemistry and flow cytometry, Treg、CTLA-4+T cells decreased to the lowest level on day 14(P<0.05), while CD4+Th andCD8+CTL cells achieved to the highest level on day 14(P<0.05), all of Treg、CTLA-4+T、CD4+Th、CD8+CTL cells started to return to the level of day 0 on day 21.6. Tumor-specific cytolytic activity of cytotoxic T lymphocyte(CTL)(effecter and target cell ratio 40:1) of TDLNs and spleens of cryoablation group and cryoablation+ Anti-CTLA-4Ab group achieved to the highest level on day 14(P<0.05), while decreased on day 21.7. The intratumoral Treg number of tumors、TDLNs and spleens were significantly positively correlated with serum TGF-β1 level and CD4+Th and CD8+CTL cells number or percentage at all sites(primary tumors, spleens, and TDLNs)(P<0.001), negatively correlated with serum IL-2 level(P<0.001).8. Foxp3+Treg/CD8+T ratios in tumors、TDLNs and spleens of cryoablation group did not have significant changes on day 14 and 21(P>0.05); for cryoablation+ Anti-CTLA-4Ab group, Foxp3+Treg/CD8+T ratios decreased to the lowest level on day 14(P<0.05), but increased on day 21(P<0.05); for only Anti-CTLA-4Ab group, Foxp3+Treg/CD8+T ratios achieved to the highest level on day 14, while decreased significantly on day 21(P<0.05).9. After 24 h, 48 h and 72 h co-culture, Treg cells could increase the apoptosis rate of Th and CTL cells(P<0.05), and could also suppress the cytokine production of IFN-γ、IL-4、IL-10(P<0.05). Treg cells had the immunosuppression function for Th and CTL cells.10. At the present of antigen presenting cells, Anti-CTLA-4Ab could suppress the cytokine production of TGF-β1、IL-10 of Treg cells(P<0.05),decreased the function of Treg’s increasing the apoptosis rate of Th and CTL cells(P<0.05). Anti-CTLA-4Ab could suppress the immunosuppression function of Treg cells.Conclusion1. Cryoablation can decreases intratumoral and TLDN Treg effectively and increases Th and CTL cells at the same time.2. Anti-Tumor Immune Response(ATIR) induced by cryoablation is enhanced by combination with immune adjuvant Anti-CTLA-4Ab therapy.3. ATIR induced by cryoablation was time-dependent, achieved to the highest level on day 14, returned to the level of day 0 on day 21; ATIR induced by Anti-CTLA-4Ab antibody still exist on day 21 which is not time-depended.4. ATIR induced by cryoablation was achieved through decreasing Treg number to express ATIR; Anti-CTLA-4Ab antibody was mainly achieved through influencing Treg function to express ATIR.5. Treg cells could increase the apoptosis rate of CD4+Th、CD8+CTL cells and suppress the cytokine production, and then play the antitumor immune fuction by immunosuppressing function for CD4+Th、CD8+CTL;At the present of antigen presenting cells, Anti-CTLA-4Ab could suppress the cytokine production of Treg cells,decreased the function of Treg’s increasing the apoptosis rate of CD4+Th、CD8+CTL cells. Anti-CTLA-4Ab could suppress the immunosuppression function of Treg cells.