Study On The Mechanism Of Donor Specific Immune Tolerance That Induced By The Alloreactive NK Cells

Objects: To study the mechanism of donor specific immune tolerance that induced by the alloreactive NK (allo-NK) cell in non-myeloablative bone marrow transplantation.Methods: We used C57BL/6J(H-2D^b) female mice as recipients and C57BL/6JxBALB/c(H-2D^b/d) F1 female mice as donor mice. We used fludarabine and cyclophosphamide as the nonmyeloablative conditioning regimen. The NK cell subset of donor was isolated by magnetic beads separation columns. The purity of NK cells is detected by flowcytometry. Cytotoxicity of purified NK cells subset against Yac-1 lymphoma cell line and autologous lymphocytes and allogeneic lymphocytes were measured using LDH-release assay. The recipient mice were divided into two groups: The mice in group A received the chemotherapy as conditioning regimen and were given bone marrow transplantation. The mice in group B received chemotherapy and donor allogeneic NK cells as conditioning regimen and then were given bone marrow transplantation. We detected the chimerism by detecting the proportion of H-2D cells using flow cytometry within 90 days after bone marrow transplantation. In one-way mixed lymphocyte reaction(MLR), lymphocytes from Fl mice were collected and treated by mitomycin as the stimulators and the lymphocytes from the C57 mice on 7d, 14d, 21d, 28d after transplantion as responsers. The lymphocytes from normal C57 mice instead of the former were used as the responsers and different concentrations of CD4⁺, CD8⁺ and CD4⁺CD25⁺ and CD4⁺ CD25⁺ cells from spleen in either group A or group B on 23 d after transplantation were added in the coculture system to discriminate the regulatory effects of different subsets. Detection of CD4⁺ CD25⁺ CD127^- regulatory T cells of spleen in either group A or group B by flow cytometry. Using real time quantitative RT-PCR amplification, we detected the mRNA expression of IL-2, IL-4, IL-10, TGF-β, IFN-γ, FoxP3 in CD4⁺CD25⁺ and CD4⁺CD25^- cells subsets from spleen and thymus of either group A or group B. Donor specific IFN-γsecretion were measured by ELISA after adding CD4⁺ and CD4⁺CD25⁺ cells from the spleen of either group A of group B into mixed lymphocyte culture. CD11c⁺ cells separated from thymus of either group A or group B on 14d after transplantion and CD3⁺ cells separated from spleen of C57 mice were coincubated in vitro for 5 days, and were compared the propotion and inhibitory activity of the regulatory T cells between two groups using flow cytometry and ELISA.Results: The purity of NK cells in spleen was (74.63±5.5)% after isolation compared to (19.85±4.27)% before isolation. The cytotoxic activity of NK cells from donor F1 mice was (65.52±8.76)% against Yac-1 cells and (60.18±1.03)% against lymphocytes from the recipient C57BL/6J mice, suggested that Allo-NK cells had cytotoxic effect,while NK cells from the recipients had no cytotoxic effect against autologous lymphocytes. The proportion of H-2D^d cells of the mice in group B was significantly higher than that in group A. The proportion of donor cells in spleen was (7.50±0.70)% in group A versus (46.20±5.00)% in group B. The proportion of donor cells in bone marrow was (10.2±2.40)% in group A versus (28.7±5.9) % in group B. Comparison between the two groups are statistically significant, P <0.01; there were still had difference in the two groups on 60 days after the transplation, the proportion of H-2D^d cells in spleen of group B was still higher than group A, however there were no significant difference in the bone marrow. The result of mixed lymphocyte reaction showed that the mouse lymphocytes in group A and B were both less proliferated compared to the normal C57 mice. The proliferation in group B were inhibited more than that in group A, the stimulation index of C57 was (1.48±0.23), (1.14±0.13) in group A and (0.91±0.05) in group B, P<0.01; When different cell subsets were added into the coculture system composed of C57 and Fl, CD4⁺ cells inhibited potently the proliferation rather than CD8⁺ cells, which majorly derived from CD4⁺CD25⁺ cells rather than CD4⁺ CD25^-cells. CD4⁺CD25⁺ CD127^- Treg cells were detected in group B after transplanted 23 days, while did not appear in group A. We used the real time quantitative RT-PCR method to detect mRNA expression of different cytokines and concluded that IFN-γin group A was higer than in group B, while FoxP3 in group B higher than in group A, which also had statistical significance: P<0.05. IL-2 in spleen of group A was higher than group B, while IL-4 in thymus of group A was lower than group B, which also had statistical significance: P<0.05. After coculturing for 5 days, CD4⁺CD25⁺ CD127^- Treg cells could be induced by CD11c⁺ cells from group B rather than group A. And when we added the the Treg cells in MLR reduced IFN-γsecretion was observed.Conclusion: Alloreactive NK cell can promote engraftment in the haploidentical nonmyeloablative bone marrow transplantation, and induce donor specific immune tolerance in host in which Treg cells induced by the CD11c⁺ cells in thymus played a important role.