Ex Vivo Expanded Regulatory T Cells Inhibit Antitumor Immunity To MHC Matched Or Mismatch Tumor

ObjectiveTo research the effects of ex vivo expanded homologous CD4+CD25+regulatory T cells on antitumor immunity to MHC matched or mismatchedtumor and explore the potential risks of this therapeutic method.Methods1. To establish implanted tumor models of CT26(H2d) and B16F10(H2b) tumor cells in BALB/c (H2d) and C57BL/6(H2b) mice either bysubcutaneous or tail vein injection.2. The CD4+CD25+Tregs were isolated from mice spleens by MACSand were expanded by anti-CD3/CD28monoclonal antibodies andrecombinant IL2in vitro.3. Mixed lymphocytes culture was used to evaluate the suppressiveactivity of Tregs in vitro.4. B16F10tumor cells were inoculated intravenously. In some animals,expanded Tregs were injected into mice through tail vein one day beforetumor inoculation. 5. The purity of Tregs and the percentage of CD4+CD25+Foxp3+Tregsin peripheral blood were analyzed by flow cytometry.6.14days tumor inoculation, the numbers of pulmonary metastaseswere counted under the10×microscope.Results1. In tumor models of CT26(H2d) cells injection, the tumor grew inBALB/c (H2d) mice after injected only5×104cells by subcutaneously orintravenously. However, in C57BL/6mice (H2b), there was no tumorgrowth even intravenously injected tumor cells were added up to5×106,and mice died of acute pulmonary embolisation if intravenously injectedtumor cells over this number. In subcutaneous inoculated model, the rate oftumor growth didn’t reach100%until the cells were added up to1×107.2. In tumor models of B16F10(H2b) cells, there were3±1tumornodules in lungs of BALB/c (H2d) mice after inoculated with5×105cells,and70±15nodule in the lung of C57BL/6(H2b) mice（P＜0.05）.3. The purity of expanded Treg was decreased from96.3±2.88%to87.73±2.35%compared with fresh Treg, but there was no significantlydifference of suppressive activity between fresh Treg and expanded Treg（P＞0.05）.4. The number of tumor nodule in lungs of BALB/c (H2d) micereceived1×106B16F10cells iv was significantly increased from (14±5) to(73±9) after injection of1×107of expanded Treg one day before tumor cell injection(P＜0.05).5. The number of pulmonary metastases was significantly increasedfrom (70±15) to over300in C57BL/6(H2b) mice which were injected with8×106of expanded Treg advanced(P＜0.05).6. The percentage of Foxp3+Treg in peripheral blood was significantlyincreased in tumor burden mice compared with normal control group andincreased drastically in mice which were injected expanded Tregs advanced（P＜0.05）.Conclusions1. The organisms have intrinsic ability to protect body from tumorcells invasion and this was called antitumor immunity was relate to MHCmolecular. The organism showed stronger antitumor ability to tumor cellswith mismatched MHC than with matched MHC.2. The antitumor immunity can be overcome not only by MHCmathced tumor cells but also by MHC mismatched tumor cells in case ofincreasing tumor cells burden.3. MACS were an effective method to get high purity ofCD4+CD25+Treg and those cells can maintain inhibition ability afterexpanded in vitro.4. Adoptive transfer of ex vivo expand Treg inhibit the antitumorimmunity and increase susceptibility to tumor cells.5. The risk of increased susceptibility of MHC matched or mismatched tumor should be addressed when using ex vivo expanded Tregas a cellular therapy.