Study Of Maternal-fetal Immunotolerance From Mice With Regnancy Loss Induced By Adoptively Transferring Exosomes Rom T Lymphocytes

Objective: Recurrent spontaneous abortion refers to the pregnancy lossthat happens consecutively for more than three times before20weeks ofpregnancy. It is an obstetric disease with serious hazard to pregnant womenthat threatens their physical and mental health. Normal pregnancy isequivalent to the semi-allogeneic transplantation. Fetus can be viewed as akind of allogeneic antigen which can induce a protective maternal-fetalimmunotolerance, instead of being rejected by its mother. Therefore, thecontrol of immune response produced by embryonic antigen is an importantmethod to mediate maternal-fetal immunotolerance.Till now, adoptive therapy through immunocytes transfering had beeningused for unexplained recurrent spontaneous abortion (URSA) in clinic, whichinduces immunotolerance between pregnant women and semi-allograftembryo antigen. However, the shortcoming of this method limit its clinicalapplication, such as the lower standardization of preparation, the instability oftherapeutic effect and etc. In this study, based on experimental animal models,effects of T lymphocytes and their non-cellular exosomes on embryodevelopment, peripheral and local immunity microenvironment of periph andplacent from mice with pregnancy loss were analyzed comparatively. It isexpected to explore the clinical feasibility of exosomes from T lymphocyte asa non-cellular components that can replace the traditional cell therapy, todevelop a novel idear in induction of immunotolerance and immunotherapy ofrecurrent pregnancy loss.Methods:1Preparation and elementary identification of splenic mononuclear cells andexosomes from unrelated individuals: BALB/c male mice were sacrificed, and the suspension of splenocytes was collected. After coinnoculated with ConAfor72h, the rate of lymphocyte transformation was observed and calculatedunder light microscope. The values of A492were measured through MTTmethod. Exosomes were isolated by sucrose gradient ultracentrifngation andultrafiltration, and them were observed through TEM and SEM. Theconcentrations of proteins in exosomes were measured by ultravioletspectrophotometry for A280.2Establishment of animal model and experimental groups: CBA/J (♀) werebreed independently without mating as non-pregnancy group. The female andmale mice were randomly divided into groups with mating2:1, CBA/J (♀)×BALB/c (♂) as control group of normal pregnancy (Control) and CBA/J (♀)×DBA/2(♂) as URSA experimental animal model. The CBA/J mice fromURSA group were randomly divided into URSA group (On the fourth dayafter pregnancy, were injected intravenously with sodium chloride), CellularTherapy group (On the fourth day after pregnancy, were injectedintravenously with splenocytes from unrelated BALB/c male mice),Non-cellular Therapy group (On the fourth day after pregnancy, were injectedintravenously with exosomes derived from splenocytes).3Observation of embryo development: The CBA/J pregnant mice in eachgroups were sacrificed on14th days, the volumes of placenta were measured,the number of absorbed placenta and the survival placenta were counted, ratesof fetal absorption and pregnancy loss were calculated.4Detection of immune function of peripheral T lymphocytes form pregnantmice: The CBA/J pregnant mice in each groups were sacrificed on14th daysof pregnancy, and the single cell suspension of spleen was prepared. The A492of T lymphocyte transformation induced by ConA were measured throughMTT. The expressions of Th1-type cytokines (IFN-γ, IL-2) and Th2-typecytokines (IL-4, IL-10) were detcted by flow cytometry (FCM) of directimmunofluorescence.5Analysis of immunosuppressions on maternal-fetal interface: The CBA/Jpregnant mice in each groups were sacrificed in14th days of pregnancy, and the placenta tissue were prepared for paraffin imbedding slice. After HEstaining, immunocytes were observed under light microscope with rectangulartrack, and the percentages of large granular lymphocyte cells (LGL), smalllymphocytes and macrophages (Mφ) were calculated. Afterimmunohistochemical staining, the gray scale (GS) were measured underimagine analysis system to show the expressions of immunosuppressors(TGF-β₁, IL-10, COX-2). There is negative relationship between value of GSand the intensity of expression, and the percentages of positive cells werecalculated under light microscope with rectangular track.6Statistical analysis: SPSS13.0sofeware was used. After analysis ofnormality and variance, the chi-square test was employed to analyze theembryo absorption, pregnancy loss and the percentage of positive cells inplacental tissue. One-way ANOVA was used to analyze the transformational Tlymphocytes, the expressions of Th1/Th2cytokines and immunosuppressivemolecules in placenta tissue.Results:1The elementary identification of Exosomes from T lymphocyte1.1The lymphocyte transformation of unrelated individualsThe rate of lymphocyte transformation were78.25±3.50%(n=10) underlight microscope. The A492of control group without ConA and experimentalgroup measured through MTT were0.249±0.073and1.032±0.064respectively (n=10, P<0.01).1.2The observation of appearance and ultra structureExosomes 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 about30nm¹00nm. Low electron density substances were shown in the middle ofexosomes.1.3Quantitation of proteinsThe 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 URSAwere5.26%and28.57%respectively, and that in group of cellular therapy andnon-cellular therapy were8.47%and3.59%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%and52%respectively, and that in group of cellular therapy and non-cellulartherapy were20%and16%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).3The effect of adoptive transfer on immune function of peripheral Tlymphocytes from mice with pregnancy loss3.1Transformational of T lymphocytes induced by ConACompared with A492of cultured splenocytes from non-pregnancy CBA/Jmice (0.195±0.136), the group of normal pregnancy, untreated URSA,cellular therapy and non-cellular therapy were (0.794±0.283,0.812±0.268,0.507±0.114and0.341±0.125) respectively, all of them increased significantly(All P<0.05). There was no significant difference between control and URSAgroup (P>0.05), compared with them, the group of cellular therapy,non-cellular therapy decreased significantly (Both P<0.05), and comparedwith cellular therapy group, non-cellular therapy group decreased significantly(P<0.05).After induced by ConA, Compared with A492of splenocytes fromnon-pregnancy CBA/J mice (1.352±0.315), the group of normal pregnancy, untreated URSA, cellular therapy and non-cellular therapy were (1.009±0.266,1.016±0.205,0.739±0.214and0.509±0.173) respectively, all of themdecreased significantly (All P<0.05). There was no significant differencebetween control and URSA group (P>0.05), compared with them, the group ofcellular therapy, non-cellular therapy decreased significantly (Both P<0.05),and compared with cellular therapy group, non-cellular therapy groupdecreased significantly (P<0.05).3.2Function of Th1/Th2from peripheral T lymphocytes3.2.1The expressions of Th1-type cytokinesCompared with expressions percentages of IFN-γ in splenocytes fromnormal pregnancy (7.48%±0.87%), the group of untreated URSA increasedsignificantly (12.22%±0.91%, P<0.01); the group of cellular therapy andnon-cellular therapy were6.94%±0.97%and6.40%±1.27%respectively, bothof 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).Compared with expressions percentages of IL-2in splenocytes fromnormal pregnancy (6.40%±0.77%), the group of untreated URSA increasedsignificantly (18.90%±1.68%, P<0.01); the group of cellular therapy andnon-cellular therapy were7.20%±1.04%and6.36±0.62%respectively, both ofthem decreased significantly (Both P<0.01) to the level of normal pregnancy(Both P>0.05), and there was no significant difference between two groups oftreatment (P>0.05).3.2.2The expressions of Th2-type cytokinesCompared with expressions percentages of IL-4in splenocytes fromnormal pregnancy (9.26%±0.88%), the group of untreated URSA decreasedsignificantly (5.82%±0.67%, P<0.01); the group of cellular therapy andnon-cellular therapy were8.22%±1.11%and9.98%±1.48%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). Compared with expressions percentages of IL-10in splenocytes fromnormal pregnancy (7.10%±1.07%), the group of untreated URSA decreasedsignificantly (3.98%±0.60%, P<0.01); the group of cellular therapy andnon-cellular therapy were5.80±0.85%and8.22±1.74%respectively, both ofthem increased significantly (Both P<0.01) to the level of normal pregnancy(Both P>0.05), and compared with cellular therapy group, non-cellulartherapy group increased significantly (P<0.05).4The effect of adoptive transfer on immunosuppressions on maternal-fetalinterface from mice with preganancy loss4.1Analysis of immunocytes in placenta tissueLarge granular lymphocyte (LGL), small lymphocytes and othermacrophage immunocytes were scattered in the placental villi tissue of everygroups. Percentage of various types of cells in the control group were17.52%±3.17%,66.00%±2.97%and15.06%±3.27%respectively, URSAgroup were20.16%±4.40%,64.06%±2.86%and12.04%±3.18%respectively,cellular therapy group were19.42%±3.90%,65.56%±2.77%and14.00%±2.80%respectively, non-cellular therapy group were18.14%±3.04%,66.02%±2.82%and14.60%±3.33%respectively. No statistically significant inevery group (All P>0.05). Percentage of total lymphocytes in each group was81.06%±6.30%,76.10%±5.34%,79.56%±4.61%and80.61%±2.35%. Thepercentage expression was no significant difference between groups (AllP>0.05).4.2Expressions of immunosuppressors on maternal-fetal interfaceThe TGF-β₁, IL-10, COX-2were expressed in the cytoplasm oftrophoblast cells, vascular endothelial cell, and immunocytes in placentatissue.4.2.1The expression of TGF-β₁Compared with the percentage of positive cells of TGF-β₁in placentatissue from normal pregnancy (72.46%±11.40%), the group of untreatedURSA decreased significantly (46.31%±11.52%, P<0.01); the group ofcellular therapy and non-cellular therapy were70.02%±7.34%and 72.04%±6.08%respectively, both of them increased significantly (BothP<0.01) to the level of normal pregnancy (Both P>0.05), and there was nosignificant difference between two groups of treatment (P>0.05).Compared with the GS of TGF-β₁in placenta tissue from normalpregnancy (74.36±2.51), the group of untreated URSA decreased significantly(94.00±3.32, P<0.01); the group of cellular therapy and non-cellular therapywere76.20±3.79and74.52±2.38respectively, both of them increasedsignificantly (Both P<0.01) to the level of normal pregnancy (Both P>0.05),and there was no significant difference between two groups of treatment (P>0.05).4.2.2The expression of IL-10Compared with the percentage of positive cells of IL-10in placenta tissuefrom normal pregnancy (69.30%±6.21%), the group of untreated URSAdecreased significantly (43.12%±7.40%, P<0.01); the group of cellulartherapy and non-cellular therapy were65.32%±8.22%and68.98%±7.15respectively, 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 the GS of IL-10in placenta tissue from normalpregnancy (90.60±4.16), the group of untreated URSA decreased significantly(120.04±4.58, P<0.01); the group of cellular therapy and non-cellular therapywere92.96±3.38and90.32±4.22respectively, both of them increasedsignificantly (Both P<0.01) to the level of normal pregnancy (Both P>0.05),and there was no significant difference between two groups of treatment(P>0.05).4.2.3The expression of COX-2Compared with the percentage of positive cells of COX-2in placentatissue from normal pregnancy (82.34%±3.90%), the group of untreated URSAand non-cellular therapy both decreased significantly (44.84%±5.78%,48.24±7.55%, both P<0.01); and there was no significant difference betweenthem (P>0.05). Compared with them, the group of cellular therapy were increased significantly (77.78%±4.78%, P<0.01) to the level of normalpregnancy (P>0.05).Compared with the GS of COX-2in placenta tissue from normalpregnancy (82.12±3.70%), the group of untreated URSA decreasedsignificantly (97.80±4.94, P<0.01); the group of cellular therapy andnon-cellular therapy were83.28±4.44and82.40±6.64respectively, both ofthem increased significantly (Both P<0.01) to the level of normal pregnancy(Both P>0.05), and there was no significant difference between two groups oftreatment (P>0.05).Conclusions:1Adoptive transfer of periphery lymphocytes or their non-cellularcomponents can induce maternal-fetal immunotolerance, which should behelpful for normal pregnancy.2Adoptive transfer of non-cellular components secreted by lymphocytes frompaternal individuals can induce stronger maternal-fetal immunotolerance thanT lymphocytes.3Peripheral lymphocytes from paternal individuals or non-cellularcomponents should initiate Th2shift and local immunosuppressions inplacenta, in order to induce efficient maternal-fetal immunotolerance.4Non-cellular components secreted by T lymphocytes from paternalindividuals could become a biological product to induce maternal-fetalimmunotolerance, which should replace traditional adoptive immunotherapyof lymphocytes.