Inhibitory Effects Of Tacrolimus On Effector T Cells From Patients With Severe Aplastic Anemia

ObjectiveTo investigate the inhibitory effects of tacrolimus on effector T cells from patients with severe aplastic anemia (SAA) in vitro.MethodsMononuclear cells were separated and extracted by lymphocyte separation medium from10ml bone marrow of16SAA patients each.CD8+HLA-DR+cells were sorted by immunomagnetic separation from the bone marrow mononuclear cells (BMMNC) of16SAA patients,and were cultured in different concentrations of IL-2alone or with tacrolimus for72hours.Cell proliferation effect was measured with MTT method.T lymphocytes were separated from the SAA patients mentioned above by lymphocyte separation medium and cultured alone or with IL-2, FK506or FK506plus CsA for18hours.The expression of tumor necrosis factor-β (TNF-β) in CD8+HLA-DR+T cells was analyzed by flow cytometry. The relationship between the expression of TNF-β and the clinical data,inclμding percentage of reticulocytes and lymphocytes in peripheral blood and the ratio of CD4+T cell to CD8+T cell,was also analyzed.Result1The purity of CD8+HLA-DR+T cells of the SAA patients in our study reached85%or more.2Cell proliferation When the concentration of IL-2was greater than or eqμal to20u/ml,the OD values(0.538±0.142、0.548±0.148、0.559±0.144) which represent the cell proliferation were significantly higher than that of the blank culture hole(0.505±0.153)(p<0.05). The OD values were significantly decreased after cultured with FK506(0.386±0.124、0.418±0.182、0.445±0.161)(p<0.05)3The expression of TNF-β Compared with control group,the expression of TNF-β was significantly higher in IL-2group(P<0.05),significantly lower in FK506group and FK506plus CsA group(P<0.05),there was no significant difference between the FK506group and FK506plus CsA group (P>0.05)4The expression of TNF-β in SAA was negatively correlated with the percentage of reticulocyte and the ratio of CD4+T cell and CD8+T cell,positively correlated with the percentage of lymphocyte in the peripheral blood count.Conclμsion1IL-2could enhance the proliferation and expression of TNF-β of the CD8+HLADR+T cells of SAA patients,which could be inhibited by FK506.2IL-2could significantly accelerate the secretion of TNF-β of CD8+HLA-DR+T cells of SAA patients and,FK506might inhibit this effect, while FK506and FK506plus CsA displayed similar effects.3The expression of TNF-β in SAA was negatively correlated with the percentage of reticulocyte and the ratio of CD4+T cell and CD8+T cell,positively correlated with the percentage of lymphocyte in the peripheral blood count,which all prompted again that TNF-β played an important role in bone marrow damage induced by the CD8+effective T cells.