The Study Of CTLA-4Ig And Anti-CD40LmAb (4F1) Blocking Costimulatory Signals In Vitro And Karyotype Analysis Of Aplastic Anemia Patients

PartⅠThe Effect of CTLA-4Ig Fusion Protein and anti-CD40LmAb to the Activation of T cell and to the Levels of Cytokines in Stimulated BMMNCs from Aplastic Anemia Patients in vitroAplastic anemia(AA) is a bone marrow failure syndrome characterized by the immune overreaction of T cell.The clinical finding that most patients respond to combined immunosuppressive therapy implicates the immune pathophysiology of AA. T cells require both primary and costimulatory signals for optimal activation.If there's only Ag-specific signal,while in the absence of costimulatory signal,the T cell will be induced a state of non-responsiveness termed "anergy".This study was designed to observe the effect of CTLA-4Ig and anti-CD40LmAb on cytokine production and on the T cell activation of AA patients in vitro in order to explore the potent therapeutic rule ofblocking-costimulatory signals.Objective:To investigate the cytokine production of BMMNC and the inhibition of the abnormal bone marrow T lymphocyte activitation by the CTLA-4Ig fusion protein/anti-CD40 ligand monoclonal antibody(4F1) in vitro in patients with aplastic anemia(AA). Material and methods:Bone marrow samples were collected from 48 AA patients(SAA,20 patients.NSAA,28 patients) and 16 controls with non-hematopoietic diseases.The bone marrow mononuclear cells(BMMNCs) were prepared by ficoll gradient centrifugation technique.BMMNCs were resuspended in the medium containing RPMI-1640,2 mmol/L L-glutamine,100u/mL penicillin/streptomycin and 15%fetal bovine serum(FBS) that had been inactivated at 56℃.BMMNCs of AA patients were cultured with or without the presence of CTLA-4Ig(10μg/ml) or anti-CD40LmAb(4F 1,20μg/ml) in the 24-well plates.After a period of 5 days incubation,BMMNCs were washed and PHA(20μg/ml) was added to the culture system,then the cells were incubated for additional 48 hours.After that, Cell-free supernatants were aliquoted and stored at -20℃for the determination of IL-2,IFN-γ,IL-10 using commercially available enzyme-linked immunoabsorbent assay(ELISA) kits according to the manufacture's instructions.BMMNCs were harvested and analyzed for immunophenotyping by two-color flow cytometry.The results were expressed in mean+standard deviation((?)±s),Student test and analysis of variance were performed.P value＜0.05 is considerated as significant.Result:1.The stimulated concentrations of IL-2,IFN-γin the SAA and NSAA group were higher than those found in the normal controls(P＜0.05),while the stimulated concentration of IL-10 was lower than that in the normal controls(P＜0.05).2.The stimulated concentrations of IL-2,IFN-γin the SAA group were higher than those found in the NSAA group(P＜0.05),while there's no statistical difference of the stimulated IL-10 concentration between the SAA and NSAA group(P＜0.05). 3.Both CTLA-4Ig and anti-CD40LmAb(4F1) could decrease the stimulated concentrations of IL-2,IFN-γin the SAA and NSAA group,but there were still statistical differences between the AA group(including SAA and NSAA group)and normal control group(P＜0.05).4.Both CTLA-4Ig and anti-CD40LmAb(4F1) could increase the stimulated concentration of IL-10 in the SAA and NSAA group,but there was still statistical difference between the AA group(including SAA and NSAA group)and normal control group(P＜0.05).5.In AA patients(including SAA and NSAA groups),the ratios of the CD3+,CD4+,CD8+,CD25+,CD4+CD25+,CD8+CD25+ cells were statistically decreased in the presence of CTLA-4Ig or anti-CD40LmAb(4F1)(P＜0.05).Conclusion:1.CTLA-4Ig and anti-CD40LmAb(4F1) could decrease the stimulated concentrations of IL-2,IFN-γin the SAA and NSAA group,and could increase the stimulated concentration of IL-10 in the two groups.2.CTLA-4Ig and anti-CD40LmAb(4F1) could decrease the ratio of T cells in AA bone marrow and could inhibite its abnormal activation.PartⅡKaryotype analysis of aplastic anemia patientsAlthough some studies have shown that AA is a polyclonal disorder consistent with the theory that AA is an autoimmune response directed against hematopoietic stem cells.A number of studies have shown that clonal progression of AA occurs during or after immunosuppressive therapy.Long-surviving patients with aplastic anemia(AA) who have been treated with immunosuppressive therapy(IST) have an increased risk of developing myelodysplastic syndrome(MDS) or acute myeloid leukemia(AML).At present,it is uncertain whether the increased survival time displays the natural history of AA as a premalignant disease or whether the secondary disease is related to the therapy itself.This study was designed to investigate whether there was clonal disorder in de novo AA patients.Objective:To investigate whether there is cytogenitic abnormality in the de novo aplastic anemia patientsMaterial and methods:Bone marrow samples were collected from 25 AA patients(SAA,10 patients.NSAA,15 patients) and 20 controls with non-hematopoietic diseases and non-hereditary diseases.Cells from the bone marrow samples were cultured for 24 hours according to routine cytogenetic protocols.The cultured cells were treated with colcemid to disrupt the mitotic spindle apparatus to prevent the completion of mitosis and to arrest the cells in metaphase. Then the harvested cells were treated briefly with a hypotonic solution to cause the nuclei to swell making it easier for technicians to identify each chromesome.After that,the cells were fixed,dropped on a microscope slide,dried,and stained by Giemsa stain.Chromosome analysis was performed on metaphases R-banded.At least twenty metaphase cells were analyzed for each patient.A karyotypically abnormal clone was defined as 2 or more metaphase cells with the same extra chromosome(s) or structural rearrangement(s)or 3 or more metaphase cells with the same missing chromosome. Result:There is no karyotype abnormality founded in de novo AA patients.Conclusion:There is no karyotype abnormality in de novo AA patients we observed.But patient populations should be enlarged and longe time follow-up,FISH and FCM methods are necessary to elevate the accuracy.