Study Of Maternal-fetal Immunotolerance From Mice With Pregnancy Loss Induced By Peripheral T Lymphocytes

Objective:Repeated pregnancy loss is that spontaneous abortion occurredthree times or more than three times continuously, the incidence ofspontaneous abortion cases is about3-5%, but still50%-60%for unknownreasons, called unexplained recurrent pregnancy loss(URSA), its seriousimpact on the physical and psychological health of the maternal.With thedevelopment of Reproductive Immunology, the research shows that, URSA ismainly related to the immune factors which induces immunotolerancebetween pregnant women and embryo. Pregnancy is an allogeneictransplantation, maternal immune mechanisms identify the fetal, in order toaccept it, the pregnancy is maintained. Therefore, the induction of maternalimmunotolerance to allogeneic fetal antigens is the key to treatment.Domestic and international studies have shown that the current clinicaluse of immune cell adoptive therapy treatment of URSA, which inducematernal immunotolerance between pregnant women and embryo antigenHowever, this method has many limitations. In this study, based onexperimental animal models of abortion to analyze the non-cellularcomponents of the T-cell-derived Exosomes rejusts peripheral T lymphocytesof mice with pregnancy loss induced maternal-fetal immunotolerance,immunological mechanisms, to determine the non-cell adoptive the immunebiologics quality standards, and to lay the foundation to carry out clinical trialsfor the next step.Methods:1Preparation and elementary identification of splenic mononuclear cellsand exosomes from unrelated individuals: BALB/c male mice were sacrificed,and the suspension of splenocytes was collected. After co-innoculated withConA for72h, the rate of lymphocyte transformation was calculated under light microscope. The values of A492were stained through MTT method.Exosomes were obtained by sucrose gradient ultracentrifngation andultrafiltration, and they were observed through TEM and SEM. Theconcentrations of proteins in exosomes were measured by ultravioletspectrophotometry for A280.2Establishment of animal model and experimental groups: The femaleand male mice were randomly divided into4groups with mating2:1, CBA/J(♀)×BALB/c (♂) for normal pregnancy control group (Control) and CBA/J(♀)×DBA/2(♂) for URSA. The CBA/J mice from URSA group on the fourthday after pregnancy were not treatment, were injected intravenously withsplenocytes from unrelated BALB/c male mice, and were injectedintravenously with exosomes derived from splenocytes randomly as URSAgroup, Cellular Therapy group, Non-cellular Therapy group.3Observation of embryonic development: On the14th days the CBA/Jpregnant mice in each groups were sacrificed, the volumes of placenta weremeasured, the number of absorbed placenta and the survival placenta werecounted, rates of fetal absorption and pregnancy loss were calculated.4Analysis of maternal-fetal immunotolerance: The CBA/J pregnant micein each groups were sacrificed on the14thdays of pregnancy, and the singlecell suspension of spleen was prepared. The A492of lymphocyte reaction inone-way and in two-way were measured through MTT.5Detection of immune function of peripheral T lymphocytes formpregnant mice: The CBA/J pregnant mice in each groups were sacrificed onthe14thdays of pregnancy, and the single cell suspension of spleen wasprepared. The expressions of T lymphocyte (CD3CD4CD8CD247CD25IL-17CD28CTLA-4) were detcted by flow cytometry (FCM) ofdirect immunofluorescence, we figured up the expressions percentages ofCD3+CD4+CD8-CD4-CD8+CD3+CD247+CD4+CD25+IL-17+CD4+CD28+CD4+CTLA-4+.6Statistical analysis: SPSS13.0sofeware was used. After analysis ofnormality and variance, the chi-square test was employed to analyze the embryo absorption, pregnancy loss. One-way ANOVA was used to analyzeimmunofunction of T lymphocytes, the expressions of T lymphocyte.Results:1The elementary identification of Exosomes from T lymphocyte1.1The observation of appearance and ultra structure and quantitation ofproteinsExosomes were observed through TEM and SEM, the exosomes from Tlymphocyte were spheres surrounded by the lipid bilayer with round or ovalcup-shape and complete membranes. The diameter of exosomes was about30nm~100nm. Low electron density substances were shown in the middle ofexosomes. The concentrations of protein in exosomes measured by ultravioletspectrophotometry for A280was0.343±0.029.2The effect of adoptive transfer on embryo development from mice withpreganancy lossThe rate of embryo absorption in group of normal pregnancy and URSAwere5.25%and20.78%respectively, and that in group of cellular therapy andnon-cellular therapy were7.81%and3.18%respectively. Compared withuntreated URSA group, the rate of embryo adsorption after both of cellularand non-cellular adoptive transfer decreased significantly (Both P<0.01) to thelevel of normal pregnancy (Both P>0.05). Compared with cellular therapygroup, the rate of embryo adsorption in non-cellular therapy group decreasedsignificantly (P<0.05).The rate of pregnancy loss in Control group and URSA were20%and62%respectively, and that in group of cellular therapy and non-cellulartherapy were22%and22%respectively. Compared with untreated URSAgroup, the rate of pregnancy loss after both of cellular and non-cellularadoptive transfer decreased significantly (Both P<0.01) to the level of normalpregnancy (Both P>0.05), and there was no significant difference between twogroups of treatment (P>0.05).3Analysis of maternal-fetal immunotoleranceOne-way lymphatic reaction, A492of the group of normal pregnancy, untreated URSA,cellular therapy and non-cellular therapy were (0.584±0.060,0.697±0.035,0.411±0.024,0.313±0.191). There was significant different-ce between control and URSA group (P<0.05), compared with them, thegroup of cellular therapy, non-cellular therapy decreased significantly (BothP<0.05), and There was no significant difference between cellular therapygroup and non-cellular therapy group (P>0.05).Two-way lymphatic reaction, A492of the group of normal pregnancy,untreated URSA, cellular therapy and non-cellular therapy were(0.551±0.308,0.478±0.116,0.702±0.126,0.563±0.134). There was significantdifference between control and URSA group (P<0.05), compared with them,the group of cellular therapy decreased significantly (P<0.05), there was nosignificant difference between control and non-cellular therapy group(P>0.05), and There was significant difference between cellular therapy groupand non-cellular therapy group (P<0.05).4Detection of function of peripheral T lymphocytes4.1Changes in the expressions of CD4CD8Compared with expressions percentages of CD3in splenocytes fromnormal pregnancy (33.47%±11.78%), the group of URSA increased signific-antly (43.43%±5.41%P<0.01); the group of cellular therapy andnon-cellular therapy were42.17%±7.43%and41.62%±7.21%respectively,both of them had no differences significantly (Both P>0.05), and there was nosignificant difference between two groups of treatment (P>0.05).Compared with expressions percentages of CD4+CD8-in splenocytesfrom normal pregnancy (23.09%±3.02%), the group of untreated URSAincreased significantly (29.02%±5.74%P<0.01); the group of cellulartherapy and non-cellular therapy were20.25%±7.70%and22.86%±8.98%respectively, both of them decreased significantly to the level of normalpregnancy (Both P>0.05), and there was no significant difference between twogroups of treatment (P>0.05).Compared with expressions percentages of CD4-CD8+in splenocytesfrom normal pregnancy (30.64%±1.55%), the group of untreated URSA decreased significantly (27.24%±1.70%, P<0.01); the group of cellulartherapy and non-cellular therapy were32.45%±2.12%and31.72%±0.98%respectively, both of them increased significantly (Both P<0.01) to the level ofnormal pregnancy (Both P>0.05), and there was no significant differencebetween two groups of treatment (P>0.05).Compared with expressions percentages of CD4/CD8in splenocytesfrom normal pregnancy (1.04%±0.21%), the group of untreated URSAincreased significantly (1.50%±0.63%, P<0.01); the group of cellular therapyand non-cellular therapy were1.06%±0.11%and1.06%±0.29%respectively,both of them decreased significantly (Both P<0.01) to the level of normalpregnancy (Both P>0.05), and there was no significant difference between twogroups of treatment (P>0.05).4.2Changes in the expressions of CD3+CD247+Compared with expressions percentages of CD3+CD247+in splenocytesfrom normal pregnancy (3.77%±1.79%), the group of untreated URSAdecreased significantly (0.13%±0.06%P<0.01); the group of cellular therapyand non-cellular therapy were3.30%±0.99%and3.57%±0.78%respectively,both of them increased significantly (Both P<0.01) to the level of normalpregnancy (Both P>0.05), and there was no significant difference between twogroups of treatment (P>0.05).4.3Changes in expression of the regulatory T cellsCompared with expressions percentages of CD4CD25in splenocytesfrom normal pregnancy (1.68%±0.75%), the group of untreated URSAdecreased significantly (1.26%±0.85%, P<0.01); the group of cellular therapyand non-cellular therapy were2.00%±0.89%and2.10%±0.70%respectively,both of them increased significantly (Both P<0.01) to the level of normalpregnancy (Both P>0.05), and compared with cellular therapy group,non-cellular therapy group increased significantly (P<0.05).Compared with expressions percentages of IL-17+in splenocytes fromnormal pregnancy (2.49%±0.14%), the group of untreated URSA decreasedsignificantly (1.21%±0.11%,P<0.01); the group of cellular therapy and non-cellular therapy were2.24%±0.52%and2.38%±1.02%respectively, bothof them increased significantly (Both P<0.01) to the level of normalpregnancy (Both P>0.05), and compared with cellular therapy group,non-cellular therapy group increased significantly (P<0.05).4.4Changes in expressions of positive and negative co-stimulatory factor cellCompared with expressions percentages of CD4+CD28+in splenocytesfrom normal pregnancy (4.91%±1.21%), the group of untreated URSAdecreased significantly (1.73%±0.84%, P<0.01); the group of cellular therapyand non-cellular therapy were4.05%±1.23%and4.12%±1.28%respectively,both of them increased significantly (Both P<0.01) to the level of normalpregnancy (Both P>0.05), and there was no significant difference between twogroups of treatment (P>0.05).Compared with expressions percentages of CD4+CTLA-4+in splenocytesfrom normal pregnancy (0.82%±0.08%), the group of untreated URSAincreased significantly (3.08%±0.37%, P<0.01); the group of cellular therapyand non-cellular therapy were0.77%±0.11%and1.20%±0.30%respectively,both of them decreased significantly (Both P<0.01) to the level of normalpregnancy (Both P>0.05), and there was no significant difference between twogroups of treatment (P>0.05).Conclusions:1Adoptive transfer of periphery lymphocytes or their non-cellular comp-onents can induce maternal-fetal immunotolerance. Adoptive transfer of Tlymphocytes have a stronger effect of the maternal-fetal immune toleranceinduction.2Peripheral T lymphocytes and their secretion of non-cellular compon-ents Exosomes can be caused by a common or similar pathway the maternalperipheral T lymphocyte function changes, induced of maternal-fetal immune-otolerance effectively.3Further clarify the immunological mechanisms of maternal-fetalimmuno tolerance induced by the males peripheral T lymphocytes and theirsecretion of non-cellular components Exosomes, provide a theoretical basis for future clinical application.