Study Of The Expression Of Perforin And CD28on Maternal-fetal Interface From Mice With Pregnancy Loss Induced By Adoptively Transferring Exosomes From Adult Male T Lymphocytes

Objective: Recurrent pregnancy loss is the spontaneous abortionoccurred three times or more than three times continuously, about60%ofwhich are unexplained recurrent spontaneous abortion(URSA),mainly relatedto the immune factors. Reproductive immunology suggests that URSA iscaused by the deficiency or the lack of tolerance between the pregnant womenand the paternal antigens in the embryo. Therefore, regulating the immuneresponse to the paternal antigens in the embryo and inducing thematernal-fetal immunotolerance are key to patinets with URSA.Presently, the T lymphocytes from husband or unrelated individuals wereused in the immunotherapy on URSA, which had shown the obviousefficiency. However, non-standard preparation, significent individualdifferences and unstable curative effect limited the clinical application. Inrecent years,studies have shown that a kind of membranous non-cellularstructure called Exosomes can be secreted by T lymphocytes along with thegrowth and proliferation which carry the core functional molecules of Tlymphocytes. Its unique advantage of strong stability, well-standardization andhigh effective security produce great research value and broad applicationprospects.URSA experimental animal model was used in this study,expressions of perforin and CD28on maternal-fetal interface from mice withpregnancy loss induced by adoptively transferring Exosomes from adult maleT lymphocytes were analyzed, to explore the immunological mechanism offeto-maternal immunotolerance induced by Exo.Methods:1Preparation and primary identification of Exo derived from adult men Tlymphocyte: The lymphocytes were purified from venous blood of adult men. The proliferation of lymphocytes was induced by PHA, and then Exos wereisolated by sucrose density gradient centrifugation and ultrafiltrationtechnology. Their structure was observed through TEM and SEM. Theconcentrations of proteins in Exo were measured by ultravioletspectrophotometry.2Preparation of mice splenocytes and Exo from unrelated individuals:BALB/c male mice were sacrificed.After grinding,filtration and removal ofred blood cells,the splenocytes were collected and then were induced byConA. Exo were isolated by sucrose density gradient centrifugation andultracentrifngation technology.3Establishment of animal model and experimental groups: The femaleand male mice were randomly divided into groups mating with2:1, 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), ExoTherapy Ⅰ group(On the fourth day after pregnancy, were injectedintravenously with Exo derived from mice splenocytes), Exo TherapyⅡgroup(On the fourth day after pregnancy, were injected intravenously with Exoderived from adult male T lymphocyte).4Observation of embryo fetation: 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.5Analysis of expressions of perforin and CD28on maternal-fetalinterface: The CBA/J pregnant mice in each groups were sacrificed in14thdays of pregnancy, and the placenta were prepared for paraffin imbeddingslice. After immunohistochemical staining, the gray scale (GS) were measuredunder image analysis system to show expressions of perforin and CD28, and there is negative relationship between value of GS and the expression intensity,and the percentages of positive cells were calculated under light microscopewith rectangular track.6Statistical analysis: SPSS13.0sofeware was used. After analysis ofnormality and variance, the chi-square test was employed to analyze theembryo absorption and pregnancy loss. One-way ANOVA was used toanalyze expressions of perforin and CD28in placenta tissue.Results:1The primary identification of ExoExos can be observed clearly through TEM and SEM, which were roundor oval cup-shape with the integrity of double lipid membrane. The diameterof Exo was about30nm~100nm. Low electron density substances were shownin the middle of Exos. The concentrations of protein in Exo from mice andadult men measured by ultraviolet spectrophotometry were0.413±0.067and0.407±0.082respectively(P﹥0.05).2The effect of adoptive transfer on embryo fetation from mice withpreganancy lossThe rate of embryo absorption in group of Control and URSA were5.65%and21.15%respectively, and that in group of cellular therapy, ExoTherapy-Ⅰ, Exo Therapy-Ⅱwere8.01%、3.41%and2.05%,respectively.Compared with untreated URSA group, the rate of embryo adsorption afterboth of cellular and non-cellular adoptive transfer decreased significantly(both P<0.01). The rate of embryo adsorption in cellular therapy group andExo Therapy-Ⅰ group decreased significantly to the level of Controlgroup(P<0.05).While Exo Therapy-Ⅱgroup decreased more obviously belowthe level of normal pregnancy(P<0.05).The rate of pregnancy loss in Control group and URSA were26%and66%respectively, and that in group of cellular therapy, Exo Therapy-Ⅰ, ExoTherapy-Ⅱ were24%、18%、14%respectively. Compared with untreatedURSA group, the rate of embryo adsorption after both of cellular andnon-cellular adoptive transfer decreased significantly (Both P<0.01). In which the rate of embryo adsorption in cellular therapy group and Exo Therapy-Ⅰgroup decreased significantly to the level of Control group(P<0.05). ExoTherapy-Ⅱ group decreased more obviously below the level of normalpregnancy (P<0.05).3The effect of adoptive transfer on expressions of perforin onmaternal-fetal interface from mice with preganancy lossCompared with the percentage of positive cells of perforin in placentatissue from Control group(18.7±2.0%), the group of untreated URSAincreased significantly (29.9±2.6%, P<0.0005); the group of cellular therapy,Exo Therapy-Ⅰ, Exo Therapy-Ⅱwere17.7±1.2%、13.0±1.9%，11.1±2.1%，P<0.0005respectively, all of them decreased significantly to the level ofControl group(both P>0.05),Compared with cellular therapy group andControl group, the percentage of positive cells of perforin in placenta tissue innon-cellular therapy group decreased significantly (both P>0.05), and therewas no significant difference between two non-cellular groups of treatment(P>0.05).Compared with the GS of perforin in placenta tissue from Control group(130.9±6.1), the group of untreated URSA increased significantly(93.9±9.9,P<0.0005), the group of cellular therapy, Exo Therapy-Ⅰ, ExoTherapy-Ⅱwere134.0±5.9,154.2±8.2,154.7±9.3respectively (P<0.0005), allof them decreased significantly to the level of Control group(bothP>0.05),Compared with cellular therapy group and Control group, the GS ofperforin in placenta tissue in non-cellular therapy group decreasedsignificantly (P>0.05),and there was no significant difference between twonon-cellular groups of treatment (P>0.05).4The effect of adoptive transfer on expressions of CD28onmaternal-fetal interface from mice with preganancy lossCompared with the percentage of positive cells of CD28in placentatissue from Control group (52.7±5.3%), the group of untreated URSAincreased significantly (65.8±8.2%, P<0.005); the group of cellular therapy,Exo Therapy-Ⅰ, Exo Therapy-Ⅱwere50.2±4.7%,34.7±5.6%,25.8±4.4%， P<0.05、P<0.001、P<0.001） respectively, all of them decreased significantly(both P<0.01) to the level of normal pregnancy (both P>0.05), Compared withcellular therapy group and Control group, the percentage of positive cells ofCD28in placenta tissue in non-cellular therapy group decreased significantly(P<0.05). Among which Exo Therapy-Ⅱgroup decreased more obviously.(P<0.05).Compared with the GS of CD28in placenta tissue from Controlgroup(117.9±2.7), the group of untreated URSA increased significantly(96.7±6.8，P<0.05); the group of cellular therapy，Exo Therapy-Ⅰ, ExoTherapy-Ⅱ were117.3±2.2,130.9±4.7,143.9±7.1，P<0.05、P<0.001、P<0.001respectively, all of them decreased significantly to the level of Controlgroup(both P>0.05), Compared with cellular therapy group and Control group,the GS of CD28in placenta tissue in non-cellular therapy group decreasedsignificantly P<0.05). Among which Exo Therapy-Ⅱ group decreased moreobviously.(P<0.05).Conclusions:1Periphery lymphocytes from unrelated paternal individuals or their Exocan both induce maternal-fetal immunotolerance and benefits the process ofpregnance. And Exo can induce stronger effect than T lymphocytes.2Both of peripheral lymphocytes from unrelated paternal individuals andtheir Exo can initiate efficient maternal-fetal immunotolerance throughregulating expressions of periforin and CD28in placenta, and reducing thetoxicity of T and NK cells to embryo antigen and the placenta cells viacommon or similar pathway.3Exo derived from human lymphocytes can induce strongermaternal-fetal immunotolerance than that from mice splenocytes.