Study On The Activation Status Of Follicular Helper T Cells In Type O Pregnant Women With Different ABO Blood Group Antibody Titers

Objective:Hemolytic disease of fetus and newborn(HDFN)is a disease that threatens the health of fetuses and newborns,which is mediated by red blood cell antibodies.The main symptoms of HDFN are fetal and neonatal anemia,edema,and jaundice after birth.In severe cases,HDFN can pose a serious threat to the health and survival of the fetus or newborn.ABO incompatibility accounts for the highest incidence of HDFN in China.When born to mothers with blood type O,newborns with blood types A and B are at risk of developing the disease.In 20%of pregnancies,there is ABO incompatibility between the mother and newborn,and of these cases,2-4%result in HDFN,mainly caused by high-titer ABO antibodies,and previous studies have found that high-titer blood group antibodies are a major independent risk factor for HDFN and are also associated with serious clinical outcomes such as fetal miscarriage and death.Although some progress has been made in the clinical diagnosis and treatment of HDFN,the immune status of pregnant women with different titers and the molecular mechanisms by which blood group O pregnant women produce high-titer antibodies have not yet been fully understood.It has been confirmed that follicular helper T cells(Tfh)is the major lymphocyte subpopulation of that assist B cell differentiation into plasma cells and antibody production,and participate in humoral immune activities such as blood transfusion.There are no reports on whether there is a difference in Tfh expression or activation between blood group O pregnant women with different titers,and changes in immune checkpoints and signaling pathways involved in regulating Tfh cell activation.This study investigates and analyzes the differences in demographic and clinical characteristics of blood group O pregnant women with different titers,and compares the cytokine levels in the peripheral blood of participants,to discover differences in immune status and changes in factors involved in regulating Tfh cell activation between the two groups.Flow cytometry analysis was performed to explore the differences in the distribution of CD4+T cells and Tfh cells in the peripheral blood of pregnant women with different titers,as well as differences in immune checkpoint TIM-3 expression.Furthermore,transcriptome sequencing was performed on Tfh cells in the peripheral blood of blood group O pregnant women with different titers to investigate the difference expression genes and signaling pathways between the two groups,laying a foundation for investigating the mechanism of high-titer blood group antibody production.Methods:1.The microcolumn gel test determined the ABO antibody titers in 100pregnant women with blood type O,dividing them into high and low titer groups based on a cut off value of≥256.We investigated the differences in demographic and clinical characteristics between the two groups of pregnant women.2.Luminex technology was used to detect 48 types of cytokines,including IL-2R alpha,MIG,MIP-1beta,IL-6,IFN-alpha2,IFN-gamma,SDF-1alpha,IL-1ra,MCP-3,IL-16,IL-12p40,LIF,TNF-beta,IL-5,GM-CSF,MIF,TNF-alpha,RANTES,IL-2,IL-1beta,IL-18,Eotaxin,Basic FGF,VEGF,beta-NGF,PDGF-BB,IP-10,IL-13,IL-4,MCP-1,IL-8,MIP-1alpha,IL-10,G-CSF,GRO-alpha,HGF,IL-1alpha,IL-3,SCF,TRAIL,M-CSF,CTACK,IL-15,IL-7,IL-12p70,IL-17,IL-9,and SCGF-beta in the plasma of 5 high-titer and 5 low-titer blood group O healthy pregnant women.Differences in cytokine levels between the two groups of pregnant women,as well as differences in cytokines involved in the activation of Tfh cells,were observed.3.The flow cytometry technique was used to detect the percentage of CD4~+T cells,Tfh(CD4~+CXCR5~+T)cell in peripheral blood of 15 high-titer and 26 low-titer blood type O healthy pregnant women,as well as the expression level of TIM-3 on the surface of CD4~+T cells and Tfh cells.4.Three high-titer and three low-titer healthy blood type O pregnant women were selected,and Tfh cells were sorted from their peripheral blood using flow cytometry for m RNA sequencing.The m RNA expression profile in the Tfh cells of high and low-titer blood type O pregnant women was analyzed.Differential expression gene analysis and GSEA pathway enrichment analysis were performed on the sequencing results of Tfh cells,aiming to identify the differences in gene expression and signaling pathway changes between the two groups.Results:1.This study involved 100 pregnant women with O blood type.The participants were divided into two groups based on their anti-A and anti-B antibody titers,with a titer of≥256 used as the cutoff for high and low titers.There were no statistically significant differences between the two groups in terms of age,gestational weeks,parity,gravida,adverse pregnancy outcomes,and whether they were primiparous.Additionally,the distribution of underlying diseases,such as eclampsia,gestational diabetes,obesity,SLE,Hashimoto’s thyroiditis,hypothyroidism,hypoalbuminemia,thrombocytopenia,anemia,cervical incompetence,uterine diseases,adnexal masses,oligohydramnios,placenta previa,and infectious diseases,was not significantly different between the two groups.However,among the four pregnant women with type 2 diabetes,three had higher antibody titers,with titers of 2048,1024,and 256,respectively.This suggests a higher proportion of high titers and higher titers among pregnant women with type 2 diabetes.2.Five high-titer and five low-titer blood type O healthy pregnant women were selected for Luminex liquid bead array detection of 48 cytokines in plasma.Among these cytokines,seven cytokines,including IL-5,IL-2,VEGF,beta-NGF,IL-3,IL-15,and IL-12p70,could not be reliably measured due to being below the lowest detection limit.Among the remaining 41 factors,the expression levels of IL-6,IFN-γ,LIF,Eotaxin,MIG,IL-12(p40),and PDGF-BB were significantly increased in the high-efficiency group.Although there was no statistical difference in the other factors,there was an increasing trend in the high-efficiency group.Among the cytokines related to Tfh cell activation,IL-6 and IFN-γwere significantly increased.3.Flow cytometry was used to examine the expression of CD4~+T cells,CD4~+CXCR5~+T cells(Tfh),and TIM-3 on CD4~+T cells and Tfh cells in the peripheral blood of 16 high-titer and 26 low-titer healthy pregnant women with O blood type.The results showed a significant increase in the percentage of CD4~+T cells in the high-titer group.There was no significant difference in the percentages of Tfh cells,CD4~+T cells,and CD4~+CXCR5~+Tfh cells expressing TIM-3,suggesting no difference in the proportion of Tfh cells between the two groups of pregnant women.4.Three high-titer and three low-titer blood type O pregnant women were sorted by flow cytometry for Tfh cells from peripheral blood and subjected to m RNA sequencing.Differential expression genes were obtained between the high-titer group and the low-titer group,including 561 upregulated genes and 568 downregulated genes.GSEA pathway enrichment of Tfh cell sequencing results showed that the IFN-αsignaling pathway and the IFN-γpathway genes were significantly enriched and activated in the Tfh cells of high-efficiency blood type O healthy pregnant women.Conclusions:1.Using≥256 as the cut off for high and low titers,there were no significant differences in demographic and clinical characteristics between the two groups of blood type O pregnant women.Some data suggested that pregnant women with type 2 diabetes had higher antibody titers and a higher proportion of high-titer individuals.2.In late pregnancy,levels of pro-inflammatory cytokines in high-titer healthy pregnant women tended to increase,with significant increases observed in IL-6,IFN-γ,LIF,Eotaxin,and IL-12.The levels of the chemokine MIG and the neurotrophic factor PDGF-BB also increased.Among them,IL-6 and IFN-γhave been shown to be associated with Tfh activation.3.There were differences in CD4~+T cells in the peripheral blood of high and low-titer healthy pregnant women in late pregnancy,but there were no significant differences in the percentages of Tfh cells or the percentages of TIM-3 expression on CD4~+T cells and Tfh cells.4.Transcriptome sequencing showed that there were gene expression differences in Tfh cells between the two groups of normal pregnant women in late pregnancy with high and low titers.The high-titer group exhibited activation of the IFN-αsignaling pathway and the IFN-γpathway,suggesting Tfh cell activation in high-titer pregnant women.