The Pathogenic Role Of CD4~+ Stem Cell-like Memory T Cells In Unexplained Recurrent Spontaneous Abortion

Background:Fetal chromosome karyotype abnormality is the main pathogenic factor of recurrent spontaneous abortion（RSA）.Still,the cause of the remaining 50%of recurrent abortions is not clear.The imbalance of maternal immune status plays a vital role in unexplained recurrent spontaneous abortion（URSA）.Numerous studies have confirmed the close association between autoimmune diseases or autoantibodies with unexplained recurrent spontaneous abortion.Stem cell-like memory T(T_SCM)cells are a group of memory T cells with the ability of self-renewal and the potential of multidirectional differentiation.The pathogenic role of T_SCM cells in autoimmune diseases has been demonstrated.However,the relationship between URSA and T_SCMcells has not been reported.In a previous investigation of us,we found that the percentage of circulation T_SCM has been increased significantly.Therefore,we speculate that T_SCM cells may be amplified in the peripheral blood of patients with URSA and play a role in initiating the occurrence and maintaining development of the disease.Methods:1.After establishing the RSA inclusion criteria,patients’embryonic villus was collected with informed consent.Fetal chromosome karyotype analysis was detected to exclude RSA caused by fetal chromosome abnormalities.Then peripheral blood of URSA patients was collected.2.After the peripheral blood plasma and blood cells of URSA patients were isolated, the levels of T_SCM population and its CD4⁺and CD8⁺subsets in peripheral blood of URSA patients were detected by flow cytometry（FCM）,and the levels of na（?）ve T （T_N）cells,central memory T(T_CM)cells and effector memory T(T_EM)cells,as well as the levels of natural killer（NK）cells and B cells were analyzed.3.The levels of autoantibodies in the peripheral blood plasma of patients with URSA were analyzed by enzyme-linked immunosorbent assay（ELISA）.4.Matings were performed as trios（one DBA/2N male,two CBA/J female,in 6-8- week old）for two cages and used as the unexplained spontaneous recurrent abortion model group.The 6-8week old CBA/J female mice and BALB/c male mice were closed at 2:1 and used as normal pregnancy control.Mice were checked each morning for a vaginal plug and that day was designated day 0.5 of gestational day （gd）.The embryo absorption rate was calculated on gd14.5.The single-cell suspensions of peripheral blood,spleen,distal uterine lymph nodes,uterine draining lymph nodes and uterus were isolated,and the ratios of T_SCM and CD4⁺T_SCM cells in mice were analyzed by FCM.5.The mice of normal pregnancy control were divided into two groups:normal pregnancy treated with physiological saline group and normal pregnancy treated with IL-7 group.The day of plug detection was defined as gestational day（gd）0.5.The pregnant CBA/J mice were injected intraperitoneally on gd4.5,gd6.5,gd8.5, respectively.The IL-7 group was injected with saline diluted IL-7 solvent 200μg per day,and the placebo group was injected with the same amount of saline.The embryo absorption rate was calculated by gd14.5,and the single cell suspensions of peripheral blood,spleen,distal uterine lymph nodes,uterine draining lymph nodes and uterus were isolated,and the ratio of T_SCM and CD4⁺T_SCM cells in mice was analyzed by FCM.6.Real-time fluorescence quantitative PCR（q RT-PCR）was used to analyze the levels of inflammatory cytokines IL-6 and TNF-αin each group of mice.Results:1.Excluding the RSA patients with abnormal karyotype of fetal chromosome,the proportion of T_SCM cells in peripheral blood of residual URSA was significantly higher than that of the control group,and the increased T_SCM cells were mainly enriched in CD4⁺T_SCM group.The proportion of the downstream of T_SCM,T_CM cells, also increased to some extent.But there was no significant change in TN and TEM cells.The ratio of NK cells increased significantly,but B cells had no significant change.The level of autoantibodies in plasma increased in varying degrees.2.The embryo absorption rate of the mouse model of URSA was significantly higher than that of the normal pregnancy group,indicating that the model was successfully constructed.The proportion of T_SCM and CD4⁺T_SCM cells in peripheral blood spleen and uterus of the abortion model mice increased,but the changes in distal uterine lymph nodes and uterine draining lymph nodes were not obvious.3.Compared with placebo group,IL-7 group showed a significant increase in the proportion of T_SCM and CD4⁺T_SCM cells in peripheral blood,spleen,distal uterine lymph nodes,uterine draining lymph nodes and uterus,indicating that IL-7 induced systemic expansion of T_SCM and CD4⁺T_SCM cells in mice.The embryo resorption rate of IL-7 group was significantly higher than that of placebo group,indicating that excessive T_SCM induced by IL-7 led to the increase of embryo resorption rate and the change of pregnancy outcome in mice.4.The results of q RT-PCR showed that the expression of IL-6 and TNF-αin IL-7 group was significantly higher than that in placebo group.Conclusion:The increase of CD4⁺T_SCM cells in URSA may be one of the etiologies.T_SCM cells are expected to become detection indicators and therapeutic targets of URSA.