Investigating The CTLA-4Ig Inducing Th17/Treg Imbalance In Peripheral Blood With Unexplained Recurrent Spontaneous Abortion Patients

ObjectiveTo investigate possible role of CTLA-4Ig inducing Th17/Treg imbalance in peripheral blood with unexplained recurrent spontaneous abortion patients.Methodsâ‘ To collect peripheral blood from unexplained recurrent spontaneous abortion (URSA) patients, then extract peripheral blood mononuclear cells (peripheral blood mononuclear cell, PBMC), the ecimen is divided into three groups which with RPMI1640 medium add for 10ug/ml end PHA, will PBMC match into cell concentration for 2*10⁶cells/ml then join into 96 orifice plate (200ul/hole). Two groups of the CTLA-4Ig0. 4ul(final concentration for 1ug/ml) and CTLA -4Ig4ul (final concentration for 10ug/ml), the third group is only PBMC meanwhile put the samples in 5% CO₂ incubator of 37℃hatch training after 72 hours, collect 100mlPBMC。Flow cytometric analysis the change of CD4+IL-17A+ and CD4+CD25+ FoxP3+cell, to investigate possible role of CTLA-4Ig inducing Th17/Treg imbalance in URSA patients.â‘¡To collect peripheral blood from unexplained recurrent spontaneous abortion (URSA) patients, then extract PBMC,cultured with CTLA-4Ig0.4ul(final concentration for 1ug/ml) and set control group with only PBMCstimulated three days and collected supernatant, By RT-PCR methods for the expression of FoxP3 mRNA and RORγt mRNA of samples.Explore whether CTLA - 4Ig adjust the Treg cells specific transcription factors FoxP3 and Th17 cells specific transcription factor RORγt in URSA patients.Resultsâ‘ Circulating Th17 frequencies (CD4+IL17+/CD4+T cells) were markedly higher in patients with URSA (2.72士0.07%) than those with the group of CTLA-4Ig 1ug/ml (1.16士0.39%) (P<0.05),.But CD4+CD25+FoxP3+ frequencies of URSA were obvious(2.30士0.36%) low than the CTLA-4Ig groups (3.96士0.52%) (P<0.05). while there was no obvious difference between the CTLA-4Igl0ug/ml groups and CTLA-4Ig1ug/ml groups(P>0.05).â‘¡CTLA - 4Ig1ug/ml stimulation training group RORγt mRNA 2 -ΔΔCt value decline 0.306±0.079 to pure PBMC training of control group and compared with significant difference (P < 0.05). Also CTLA - 4Ig group mRNA2 -ΔΔCt FoxP3 value increased 5.162±3.595 to control group compared with significant difference (P < 0.05). For the molecular level CTLA - 4Ig can affect the expression of Th17 and Treg specific transcription factor.ConclusionThe human pregnance is a semi-allograft and so antigenically foreign to the mother. Because the process of implantation may contains the defense mechanisms to prevent allograft rejection and once the immunological tolerance becomes imbalanced will cause pathological pregnancy, such as occur Unexplained recurrent spontaneous abortion (URSA)。Human Th17 cells may play a major role in rejecting conceptus antigens and therefore may harmful to the maintenance of pregnancy. Meanwhile ,Treg may benefit the maintenance of pregnancy.Normal pregnancies Treg cells to maintain populations, and pregnancy URSA patients peripheral blood Treg cells in the lower, while the number Th17 cell increases with peripheral blood for the pregnancy is unfavourable, thereby maintaining miscarriages happen. Above all ,that the balance of Th17/Treg may play a major role in the mechanism of URSA. CTLA-4 plays an important role in T cell activation, differentiation and immune tolerance.For our results CTLA-4Ig can raise Treg cell proliferation, restrain the development of Th17 cell differentiation, formed inside body favors maintain the environment of pregnancy..Our observations idicate that the physiological functionof the costimulatory pathway may be imbalanced in certain pathological pregnancies, such as allogeneic fetal wastage, , which are crucial to the maintenance of normal pregnancy. Thus, treatment with CTLA-4Ig may provide a new more rational immunotherapy for the immune-associated recurrent spontaneous abortion.