Studies On The Effect Of IFITM3 On Humoral Immune Response And Its Molecular Mechanism After Influenza Vaccine Immunization

ObjectiveInfluenza viruses cause seasonal epidemics or pandemics that pose a threat to human public health.Interferon-induced transmembrane protein 3(IFITM3)is an important antiviral factor,as an interferon stimulating factor,mainly inhibiting viral replication by preventing the viral envelope from fusing with the late lysosome or endosomal membrane.Previous studies indicated that IFITM3 was continuously expressed in dendritic cells(DCs)and CD8+T cells in mouse lungs after influenza virus infection,in order to fight against viral infection.Single-nucleotide polymorphism(SNP)rs12252-C of IFITM3 results in a truncated IFITM3 protein lacking its first 21 amino acids at the N-terminus,which associated with severe illness in adults infected with pandemic influenza H1N1/09 virus.In addition to inhibiting virus replication,IFITM3 may affect the immune response of T cells,but there is no relevant report on whether it will affect the antibody immune response.Our studies had shown that after trivalent inactivated vaccine(TIV)immunization,the antibody level of SNP rs12252-C/C carriers was higher than that of T/T carriers.Therefore,this study focused whether IFITM3 affected the level of antibody response after influenza vaccine immunization and its possible molecular mechanism based on IFITM3 knockout mouse model.Methods1,Transcription activator-like effector nucleases technology was used to construct IFITM3 knockout(Ifitm3-/-)mouse model in this study.Ifitm3-/-and Wild type(WT)mice were immunized with TIV by intraperitoneal injection,and flow cytometry was used to detect immune cells,including T cells,B cells,natural killer cells(NK),dendritic cells(DC),macrophage(M?)and neutrophils at 7d.2,After TIV booster immunization,the orbit blood of Ifitm3-/-and WT mice was collected at Od,7d,14d,21d and 28d to detect HI antibody titers by haemagglutination inhibition(HI),microneutralization(MN)antibody titers by MN test and IgG antibody by enzyme-linked immuno sorbent assay(ELISA).Meanwhile,the amount of germinal center(GC)B cells,plasma cells,plasmablasts cells,memory B cells and T follicular helper cells(TFH)in the spleen of Ifitm3-/-and WT mice were detected by flow cytometry.TIV-specific IgG+antibody secreting cells(ASCs)were detected by enzyme-linked immunospot assay(ELISPOT).3,The expression of transcription factors in B cells of spleen was detected by proteomics,and proteins and gene levels of transcription factors such as BCL6,AID and Blimp-1 were detected by western blot(WB)and fluorescence quantitative PCR to explore its possible mechanism of action.Result1,It was found that on day 7 after TIV booster immunitization,the number of CD4+T cells,DC,M?),and neutrophil cells in Ifitm3-/-mice had no significant change compared with WT mice.The number of CD8+T cells and NK cells in spleen increased,while the number of GC B cells decreased by 3 times in Ifitm3-/-mice compared with WT mice.2,The levels of HI,MN and IgG antibodies against A/H1N1,A/H3N2 and B influenza virases in Ifitm3-/-mice were lower than in WT mice after TIV booster immunization.Meanwhile,compared with WT mice,the number of GC B cells,plasma cells,TIV-specific IgG+ASCs and TFFH cells decreased in Ifitm3-/-mice,while the number of memory B cells increased.After the third TIV immunization,the levels of HI and rgG antibodies in Ifitm3-/-mice increased rapidly,but the levels were still lower than those in WT mice.3,Ifitm3-/-mice had abnormal transcriptioial networks that regulate the differentiation of GC B cells and plasma cells.Plasma cells and plasmablasts pathway was abnormal,and protein and mRNA levels of Blimp-1 decreased in Ifitm3-/-mice.ConclusionIFITM3 affected the number and activation level of spleenic immune cells after TIV booster immunization,and IFITM3 knochout reduced the number of GC B cells and TFH cells,thus decreased the level of antibody response.This study firstly demonstrated that IFITM3 could affect the antibody immune response after influenza vaccine immunization.