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OBJECTIVE The aim of our study was to investigate the strategy of immune tolerance induced by the demethylating drug decitabine (DAC) combined with multiple allogeneic bone marrow mononuclear cell (BMMNC) infusions. METHODS An animal model with C57BL/6 mice as recipients and a BALB/c mouse as the donor was established. C57BL/6 mice were randomly divided into the following groups: DAC, BMMNC, DAC+BMMNC, and the control groups. Mice in the DAC and DAC+BMMNC groups were given DAC (5 μg/kg) on days 1, 2, 7, 8, 13, and 14. On experimental days 3, 9, and 15, BMMNCs (5 × 106/mouse/time) from BALB/c mice were infused into mice of the BMMNC and DAC+BMMNC groups. One-way mixed lymphocyte reaction (MLR), interleukin-2 reverse test, and in vitro adoptive transfer experiments were performed. The ratio of regulatory T cells in splenocytes, chimera detection, and median survival time of skin grafts were recorded. RESULTS MLR showed that the stimulation index of the DAC+BMMNC group was significantly lower compared with the other 3 groups (P < .01), but still showed a strong proliferative response to the third-party unrelated donor KM mice, with no significant difference compared with the other 3 groups. The MLR low reactivity of the DAC+BMMNC group could be reversed by exogenous interleukin-2. After adding an equal amount of splenocytes from a C57BL/6 mouse in the DAC+BMMNC group, MLR of control group significantly decreased (P < .05). Flow cytometry showed the ratio of regulatory T cells in splenocytes from DAC and DAC+BMMNC groups was significantly higher than that from the BMMNC and control groups (P < .05). Chimeric rate of the DAC+BMMNC (60%) group was significantly higher than that of the BMMNC group (30%). Moreover, survival time of skin grafts in the DAC+BMMNC group was significantly longer than that in the other 3 groups (P < .001). No death or graft-vs-host disease was observed. CONCLUSION DAC combined with multiple allogeneic BMMNC infusion could successfully induce specific immune tolerance in mice, which may provide some new strategies to improve immune tolerance after organ transplant.