The Role And Mechanism Of Regulatory T Cell In Maternal-Fetal Tolerance And H-Y Skin Transplantation

PartⅠEffects and Mechanisms of Pregnancy Estrogen and Progesterone on the Immune Reaction in MiceObjective To investigate whether the estrogen (E2), progesterone(P4) within the physiological concentration range of pregnancy could affect the expansion of CD4+CD25+ Foxp3+regulatory T cell. Methods In vitro, we first isolated the CD4+CD25+ regulatory T cells by magenetic columns, and then labeled the regulatory T cells with carboxyfluorescein diacetate succinimidyl ester (CFSE). We treated the labeled regulatory T cells with estrogen (E2) and anti-CD3 antibody and anti-CD28 antibody. Then we used the flow cytometry detect the changes of fluorescence, and analyse the proliferation index of the labeled cells. Furthermore, we detected the function of expanded regulatory T cells. Groups were divided as followed, Group A, labeled E2-untreated Treg with PBS; Group B, E2-treated Tregs with PBS; Group C, labeled E2-untreated Treg with CD3/CD28; Group D, labeled E2-treated Treg with CD3/.CD28. While in the experiments of function of Treg, we use CD4+CD25-Naive T cells as the effective cells (Tef). Group A, coculatured the Tef with PBS; Group B, coculatured the Tef with CD3/CD28; Group C, added the E2-untreated Treg to the culture with the proportion of Tef and Treg 1:1; Group D, insteaded of E2-untreated Treg with E2-treated Treg in the Group C. In vivo, we administrated pregnancy E2/P4 or saline in the ovariectomized and orchiectomized mice. After reconstituting the sex hormone to the physiological concentration range of pregnancy for two weeks, the mice were sacrificed, and lymphocytes were isolated from thymus, spleen, iliac lymph nodes and peripheral blood. The CD4+CD25+ Foxp3+ regulate T cells were detected by flow cytometry and Immunohistochemistry. Results our results revealed that E2 could significantly promote the proliferation index of labeled Treg with the costimulate signals in vitro. E2 alone could not expand the proportion of Tregs, which was not matched with the result in vivo. This phenomenon may involve in broken down of anergy of Treg by E2, and then the costimulate signals promoted the proliferation of the Tregs. In the function experiments E2 could also enhance the inhibitory effect of the effective cells. In vivo, the groups received E2 alone showed a notable increase in the proportion of the Tregs that marked CD4+CD25+ Foxp3+ in spleen, iliac lymph nodes and blood. CD4+CD25+ Foxp3+ cells constituted 10.5% of all CD4+ cells in spleen, 7.63% in iliac lymph nodes and 6.13% in blood, While the proportion in the thymus can't be enhanced compared to the vehicle groups. The P4 administration could improve the proportion of Tregs lightly, but without statistically significant. During the group combine with E2 and P4, each tissue samples showed a same statistically significant trend in the increase of Tregs with E2 alone. Furthermore, no difference was found in the change of Tregs between the ovariectomized and orchiectomized mice. The similar results could be found in immunohistochemistry. Conclusions E2 could promote the proliferation of regulatory T cells with the TCR signals, not only expanded the amout, but also enhancede the function. In vivo, E2 can drive the expansion of the Tregs in the secondary lymphoid organs and peripheral blood, but not P4. Neither of them can improve the proportion of Tregs in thymus. No gender difference could be found in the effect, which may involve in the same distribution of hormone corelative receptors. PartⅡPaternal Antigen-Specific regulatory T cells were involved in the protection in maternal-fetal toleranceObjective We determined whether the function of T regulatorycells (Tregs) on the effector T cells was specific for paternal antigens. Methods The CBA/J×DBA/2J murine models were used as an immunological spontaneous abortion and provoked spontaneously high abortion rates.The MLC was established as follow. Effector cells from spleen of pregnant CBA/J females in abortion group were incubated with MMC treated DBA/2J males'spleen cells. Cultures were added with Tregs from normal pregnant or nonpregnant or further control (Balb/c female mated C57BL/6) mice (ratios of T effector:Treg cells were 10:1,1:1, and 1:5, respectively). Effector cells were stained with CFDA-SE before coculture,72 hours later; cells were processed and analyzed for their proliferation and cytokines. In vivo, Tregs from the normal pregnant, nonpregnant CBA/J females and further control were injected intravenously on day 0 to 2 of pregnancy to CBA/J females mated with DBA/2J abortion group. The abortion rates of each group were documented. Results A statistically significant increased percentage of Treg in peripheral blood from abortion mice when compared to non-pregnant mice (6.59% vs 3.55%, P<0.01), and also significant increased percentage of Treg in peripheral blood from normal pregnant mice when compared to abortion mice (8.34% vs 6.59%, P<0.01). The similar results could be observed in lymphoid nodes (6.86% vs 4.85; 9.62% vs 4.85%, P<0.05; 9.62% vs 6.86%, P<0.05). Cultureed with Tregs from all groups showed comparable ex vivo Treg number depended cytokine secretion such as up-regulated IL-10 and down-regulated IFN-γ.The defective function and insufficient number of Treg cells were involved in the spontaneous abortion combination of DBA/2J-mated CBA/J mice. We found that functional Treg adoptive transfer in vivo did not prevent the rejection of fetal on the abortion prone mate which was undergoing immunological abortion. Full normal pregnancy could only be achieved after adoptive transfer of Treg from normal pregnant mice, which was specific for paternal antigen. Conclusions All of the Treg cells from normal pregnant and nonpregnant CBA/J mice could inhibit the proliferation from abortion mice in vitro whereas in vivo prevention of fetal rejection could only be achieved after adoptive transfer of Treg cells from normal pregnant mice. Our data suggest that pregnancy-induced Treg cells protect the fetal from being rejected in a paternal antigen specific way. Part III Pregnancy Levels of Estrogen and Progesterone enhance regulatory responses to H-Y mismatched skin graft in murine modelObjective To investigate the effects of estrogen (E2) and progesterone (P4) alone or combined with each other to H-Y skin graft and the potential mechanism. Methods The female C57BL/6 mice were ovariectomized and divided into four groups (n=15 in each). The mice were treated consecutively for 14d with subcutaneous injection of saline, E2 and P4 alone and in combination respectively. Before and after H-Y skin grafting, half of each group was sacrificed, and the CD4+CD25+Foxp3+regulatory T cells of peripheral blood and the serum cytokines were detected by flow cytometry and ELISA, respectively. The skin grafts survivals of the other half were observed. Results The administration of sex hormone regardless of E2 and P4 alone or in combination, significantly decreased production of pro-inflammatory cytokines (IFN-y and TNF-a), but increased production of anti-inflammatory cytokines (IL-10 and TGF-β). (P<0.05) E2 alone could significantly augment the proportion of regulatory T cells. In the presence of H-Y antigen, this effect was further enhanced. (P<0.05) By contrast, P4 had no effect on the expression of Foxp3, regardless of the presence of H-Y antigen or not. (P>0.05) The skin grafts survivals were significantly prolonged in the different experimental groups compared to vehicle control group.(P<0.05) Conclusions Pregnancy Levels of E2 and P4 suppresses the inflammatory response and enhances the regulatory response to exogenous antigen, through influencing the levels of cytokines and/or the proportion of regulatory T cells. It may contribute to induce the transplant tolerance.