Objective: To study the effects of adoptive transfer of ex vivo expanded homologous CD4⁺;CD25⁺; regulatory T cells (Tregs) on antitumor immunity in mice and explore the potential risk of this therapeutic method.
Methods: Marine CD4⁺;CD25⁺; Tregs were separated from spleens by magnetic activated cell sorting (MACS) and expanded by anti-CD3/CD28-coated microbeads and 1 000 U/mL interleukin 2 (IL-2) in vitro. The purity of fresh and expanded Tregs was determined by FACS. The cells were collected after two rounds of expansion (7 days each), and suppressive activity of expanded Tregs was tested by the mixed lymphocyte reaction (MLR) in vitro. Thereafter, the BALB/c mice were intravenously injected with 1 × 10⁷ expanded Tregs and 24 hours later, they were inoculated intravenously with 1 × 10⁶; B16;F10; tumor cells. The mice injected with tumor cells only were set as the control group. Fourteen days later, the percentage of CD4⁺;CD25⁺;Foxp3⁺; Tregs in peripheral blood was analyzed by flow cytometry and the numbers of pulmonary metastases were counted. Results The purity of expanded Tregs decreased from (96.3 ± 2.88)% to (87.73 ± 2.35)% compared with fresh Tregs, but there was no significant difference in the suppressive activity between fresh Tregs and expanded Tregs (P>0.05). The number of tumor nodules in lung of BALB/c mice was significantly elevated from 14 ± 5 to 73 ± 9 after injected 1 × 10⁷; expanded Tregs compared with the control group (P=0.007), and the same with the mice that were inoculated with 2 × 10⁶; B16;F10; cells alone (P=0.230). What's more, the number of pulmonary metastases was significantly raised from 70 ± 15 to over 300 (P<0.01) in 5 × 10⁵; B16;F10; inoculated C57BL/6 mice if they were injected previously with 8 × 10⁶; expanded Tregs. The percentage of Foxp3⁺; Tregs in peripheral blood significantly increased in the experimental group as compared with normal mice and control group (P<0.05). Conclusion Adoptive transfer of ex vivo expanded Tregs can induce the immune tolerance and inhibit the antitumor immunity at the same time. There was a potential risk in the clinical application of Tregs.