Regulating Factors On Damage Of Hematopoiesis By Effector T Cells Of Severe Aplastic Anemia Patients

ObjectiveTo explore the effects of tumor necrosis factor-β (TNF-β) on damage of hematopoietic cells by CD8+HLA-DR+effector T cells of the patients with severe aplastic anemia(SAA), and then prove the role of TNF-Pin immunopathogenesis of SAA and explore the importance of CD8+HLA-DR+effector T cells on immunopathogenesis of SAA further.MethodsFirst Section CD8+HLA-DR+T cells as effector cells were sorted from bone marrow mononuclear cells(BMMNC) of SAA patients by magnetic activated cell sorting system (MACS). CD3+cells MACS depleted BMMNC of normal controls as target cells were co-cultured with the effector cells(1:1) in different concentrations of TNF-β(0ng/ml,15ng/ml,25ng/ml and50ng/ml) for72hours. The percentage of Annexin V positive cells were measured by flow cytometry. The quantitive assay of IL-10, IFN-y in the culture supernatant was also performed using the enzyme linked immunosorbent assay kit.Second Section CD8+HLA-DR+T cells were sorted from bone marrow mononuclear cells(BMMNC) of SAA patients by magnetic activated cell sorting system (MACS) and were divided into three group:interleukin-2(IL-2) group (in different concentrations of OU/ml,0.1U/ml,1U/ml,100U/ml and1000U/ml); CsA group (400ng/ml of CsA was added in each well containing IL-2); receptor antagonist group (in each well was IL-2receptor antagonist added with8ug/ml based on IL-2).Then cell proliferation rate was evaluated using MTT asseay after cultured for72hours. Bone marrow mononucleu cells of the patients with SAA divided into CsA group, IL-2group and control group were cultured for18hours and another4hours following Phorbol ester(PMA) administration. The expressions of tumor necrosis factor-β(TNF-β) in CD8+HLA-DR+T cells were analyzed by flow cytometry.ResultsFirst Section1. The apoptotic rate(AR) of target cells in control group and experimental groups were (49.85±20.33)%,(51.65±20.34)%,(55.94±20.19) %and (57.72±19.45)%. AR of target cells in25ng/ml and50ng/ml TNF-βgroups were significiantly higher than those in control group and15ng/ml TNF-βgroup (P<0.05). There were no significiant difference between those in control group and15ng/ml TNF-βgroup, also between those in50ng/ml and25ng/ml TNF-βgroups (all P>0.05)2. The levels of IL-10in the culture supernatant in different groups were (217.99±29.47) ng/ml、(216.47±29.00) ng/ml、(212.15±13.19) ng/ml and (218.01±28.61) ng/ml. There were no difference among them (P>0.05)3. The levels of IFN-y in the culture supernatant of50ng/ml TNF-βgroup [(593.50±48.18)ng/ml] was significiantly higher than those of control group [(544.85±69.77)ng/ml],15ng/ml TNF-βgroup [(567.38±74.51)ng/ml] and25ng/ml TNF-pgroup [(544.05±79.69)ng/ml](P<0.05)Second Section1.The cell proliferation of IL-2wells in concentrations of10U/L、100U/L and1000U/L were significantly higher than control wells, CsA group and receptor antagonist group(P<0.05),there was no statistic difference between CsA group and receptor antagonist group(P>0.05).2. The expressions of TNF-βof CD8+HLA-DR+T cells of IL-2group were (60.77±26.37)%, higher than those of control group(41.41±24.63)%, whereas CsA group’s(23.99±20.16)%were lower than control group’s(P<0.05).Conclusion1、TNF-βcould enhance the apoptosis of hematopoietic cells induced by effector T cells of SAA patients in dose-respanse manner. High level of TNF-Pmight also promote CD8+HLA-DR+T cell secrete IFN-ywhichcould collaborative by damage hematopoiesis.2、IL-2could significantly stimulate the proliferateions of CD8+HLA-DR+T cells of SAA patients and accelerate their secretion of TNF-βin vitro, CsA and receptor antagonist might inhibit this proliferation, meanwhile the expressions of TNF-βwere suppressed by CsA significantly.