The Role Of Ferritin Light Chain(FTL)in The Pathogenesis Of Immuno-related Pancytopenia

Objective: We screened out the potential antigen of IRP was Ferritin light chain(FTL)by SEREX in the previous study.On basis of preliminary studies,antigenic epitopes of FTL were further screened and identified.And we further analyzed the correlation between FTL and the indicators,and verified the antigenicity of FTL in immuno-related pancytopenia(IRP).Contents:We purified serum antibodies from IRP patients and healthy people separately,and used phage random peptide library to screen the positive clones.The sequencing results of the positive clones were compared with the amino acid sequence of the ferritin light chain.And then we extended the peptides of FTL by T-cell epitope prediction software,and screened the antigen peptides that can be recognized by Th2 cells but not by Th1 cells through Elispot technology.The differences between FTL and FTL-mRNA levels in the IRP case group and the control group were further analyzed.At last,we analyzed the immune status of T and B lymphocytes,and the correlation between the expression level of FTL and clinical indicators.Methods: 1.To screen FTL epitopes and synthesize antigen peptides.We first purified the serum antibodies of IRP patients and healthy people,then we combined phage random peptide library with ELISA to select phage-positive clones that could bind to IRP patient’s serum antibodies,but could not bind to the serum of the healthy people.The sequencing results of the positive clones were compared with the amino acid sequence of the ferritin light chain.We extended the antigen peptides by T cell epitope prediction software,and synthesized the antigen peptide VNLYLQASYTYLSLG,which was used for subsequent Elispot experiments.2.To study the effect of antigen peptide on Th2 cells.PBMCs from newly diagnosed IRP patients were extracted by lymphocyte separation medium and stored in the refrigerator at-80℃.When we started the experiment,we resuscitated the cells and examined cell survival rate.Next,we placed them in IL-4 and IFN-γ plates respectively after counting,and to test them on the machine after 48 hours of incubation.3.To study the effects of FTL in IRP patients,and the correlation between the expression level of FTL and clinical indicators.In this experiment,we used flow cytometry to detect the expression of FTL on BMMNCs from newly treated IRP patients,recovering IRP patients,and case-control groups.ELISA methods were used to detect FTL antigen concentrations in serum of newly treated IRP patients,recovered IRP patients,and normal controls,and WB was used to detect FTL protein.The expression of FTL-mRNA in the BMMNCs was detected by Q-PCR method.At last,we analyzed the immune status of T and B lymphocytes,and the correlation between the expression level of FTL and clinical indicators.Results 1.We successfully used phage random peptide library combined with ELISA to pick phage-positive clones for sequencing.Combined with the ferritin light chain sequence and T cell epitope prediction software,we screened out possible FTL epitopes: VNLYLQASYTYLSLG.2.In this experiment,through ELISPOT,it was found that peptide 1: VNLYLQASYTYLSLG can significantly stimulate the production of IL-4 and cannot stimulate the production of IFN-r,which suggested that peptide 1 can obviously activate Th2 cells.3.1)The expression levels of FTL on the membrane of bone marrow mononuclear cells by flow cytometry in the untreated IRP group(4.62±4.61)higher than that of the case-control group(0.68±0.46),and the difference was statistically significant(P<0.05).The levels of serum FTL in the untreated IRP group(954.85±743.34)ng/ml and recovered IRP group(303.22±300.79)ng/ml detected by ELISA were higher than the normal control group(70.46±82.63)ng/ml,the difference was statistically significant(P<0.05).The expression levels of FTL-mRNA in bone marrow mononuclear cells of the untreated group,the recovered group and the case-control group were(2.00±1.30),(1.27±0.65)and(1.10±0.49),respectively.The difference between the untreated IRP group and the case-control group were statistically significant.Through WB,the level of FTL protein in the untreated IRP group was significantly higher than that in the case-control group.2)Immune status of T and B lymphocytes in IRP patients and its correlation with clinical indicators:The ratio of CD3+CD4+/CD3+ 、 CD3+CD4+/CD3+CD8+ 、 CD19+/PBMCs and the expression levels of IL-6、IL-10 of untreated IRP patients were significantly higher than that of case-controls(P<0.05).The proportion of CD5+CD19+/CD19+ cells and the levels of IL-6、IL-10 in the untreated and recovered IRP group were higher than that in the case-control group,and the difference was statistically significant(P <0.05).There was no significant difference in the expression of IL-2,TNF-α,IFN-γ between the untreated IRP group,recovered IRP group and case-control group.(P> 0.05).There was positive correlation between the ratio of CD5+CD19+/CD19+ and HBe Ab.There was negative correlation between the ratio of CD5+CD19+/CD19+ and C4.The expression of IL-4 was positively correlated with Ig A and ASO,and the expression of IL-6 was positively correlated with CRP,HBe Ag,Ferritin.The FTLmRNA levels were positively correlated with ASO.FTL levels detected by ELISA were negatively correlated with RBC and PLT,and positively correlated with ferritin,CRP and HBe Ag.Conclusions:1.FTL is the "target antigen" for some IRP patients.2.The polypeptide of VNLYLQASYTYLSLG is a "target antigen" epitope.3.The target antigen is abnormally overexpressed on the bone marrow cell membrane of IRP patients and is related to the severity of disease.