The Unique Phenotype And Functional Regulation Of CXCR4~+ Decidual NK Cells In Human Early Pregnancy

The establishment of a successful pregnancy has been proven to be very complicated. From immunological point of view, normal pregnancy can be seen as allograft. The embryo expresses paternal antigens foreign to the mother; therefore, the induction of maternal immune tolerance toward the fetus becomes an important event in the maintenance of early pregnancy. The excessive activation of maternal immune cells against embryo which expresses foreign antigen can result in rejection to the fetus and pregnancy failure. The mechanisms of maternal-fetal immune regulation during pregnancy and improvement of the pregnant outcome are of great interest for researchers of reproductive medicine, transplantation immunology and tumor immunology.The maternal-fetal interface is the core part of maternal-fetal immune network. The maternal-fetal interface is composed of trophoblast cells and maternal decidual cells including decidual stromal cells and decidual immune cells. The composition of decidual immune cells is very complicated and special. Decidual NK cells population accounts for 50-70% in all the decidual immune cells in the early pregnancy. For many years, researchers have been working on the biological events which occur at maternal-fetal interface. It has been proven that the maternal-fetal interface exhibits a Th2 bias, and regulatory T cells (Treg) play an important role in preventing from immune rejection to the transplantation. Recently, it has been found that NK cells play more and more important roles in the early pregnancy, but the specific roles underlining the decidual NK cells has yet to be elucidated.The chemokine superfamily includes at least 48 ligands which bind to 18 chemokine receptors. Most chemokines are proteins of 67 to 127 amino acids and play a variety of biological roles through binding to their correspondent receptors. A chemokine may have at least one chemokine receptor, and one chemokine receptor may also bind to a plurality of chemokines as ligands. The interaction between chemokines and chemokine receptors forms a complicated chemokine-receptor network, and present various biological functions in humans. The functions of chemokines and their receptor were studied originally in inflammation. It has been found that chemokines are not only involved in the migration of leukocytes to the inflammatory tissue, but also participate in the body development and tumor progression. Our previous studies have demonstrated the role of CXCL12/CXCR4 axis at maternal-fetal interface. Therefore our study here focuses on the role of different subgroups of decidual NK cells based on CXCR4 expression and their special function at the maternal-fetal interface.Part 1 The unique subgroups of decidual NK cells based on CXCR4 expressionObjective To determine the phenotype, cytokine expression and cytotoxicity of different subgroups of deciduas NK cells based on CXCR4 expression.Methods We collected peripheral blood from normal early pregnancy women, endometrial tissue from normal non-pregnant women and decidual tissue from normal early pregnancies and miscarriage patients; and then we used FCM to analyze the phenotype, cytokine expression and cytotoxicity of peripheral and decidual NK cells based on CXCR4 expression.Results It has been found that the decidual CXCR4+NK cells express lower level of activated receptor NKp30, NKp44 and NKp46, and lower level of inhibitory receptor KIR2DL1, KIR3DL1 and CD94; express lower level of Thl cytokine TNF-a and IFN-γ; higher levels of Th2 cytokines IL-4, IL-10, and higher levels of TGF-β, IL-8; express lower level of perforin and granzyme B compared to decidual CXCR4-NK cells. Compared to endometrial tissue from normal non-pregnant women and decidual tissue from miscarriage patients, CXCR4+decidual NK cells preferentially accumulated in decidual tissue from normal early pregnancy.Conclusions Decidual CXCR4+NK cells display unique phenotype and function and can be observed preferentially accumulated in decidual tissue of the normal early pregnancy.Part 2 The functional modulation of human first-trimester trophoblast cells on peripheral CXCR4+and CXCR4- NK cells.Objective To investigate the functional modulation of human first-trimester trophoblast cells on peripheral CXCR4+and CXCR4- NK cells.Methods MACS was used to purify the peripheral NK and peripheral CXCR4+and CXCR4-NK cells. Co-culture systems of human trophoblast cells and peripheral NK or peripheral CXCR4+and CXCR4- NK cells were established. Flow cytometry was used to analyze the phenotype, cytokine expression and cytotoxicity of peripheral NK cells. Chemotaxis assay was used to analyze the chemotactic effects of trophoblast cells on peripheral NK cells.Results It has been found that human first trimester trophoblast cells up-regulate the expression of NKp30, NKp44, NKp46, KIR2DL1, KIR3DL1 on CXCR4- peripheral NK cells, up-regulate the expression of IFN-Y from CXCR4- peripheral NK cells, and up-regulate the expression of perforin and GraB from CXCR4- peripheral NK cells. Human first trimester trophoblast cells down-regulate the expression of NKp30, KIR2DL1, KIR3DL1 on CXCR4+peripheral NK cells; down-regulate the expression of TNF-α on CXCR4+peripheral NK cells, up-regulate the expression of IL-4, IL-10, TGF-β and IL-8 from CXCR4+peripheral NK cells, down-regulate the expression of perforin and GraB from CXCR4+peripheral NK cells. Trophoblast cells could up-regulate the expression of CD56 and down-regulate the expression of CXCR4 on peripheral NK cells, which thus makes the phenotype of peripheral NK cells shift to the decidual NK cells. Furthermore, trophoblast cells recruit peripheral NK cells to maternal-fetal interface via the CXCL12/CXCR4 interaction.Conclusions Trophoblast cells induce the phenotype of peripheral NK cells shift towards the decidual NK cells. Decidual NK cells display Th2 bias and immune-tolerance phenotype. Trophoblast cells recruit peripheral NK cells to preferentially accumulate at maternal-fetal interface via the CXCL12/CXCR4 axis.Part 3 CXCR4+decidual NK cells induce naive CD4+T cells to a Th2 bias and induce maternal-fetal immune-toleranceObjective To demonstrate the role of CXCR4+decidual NK cells in Th2 bias and establishment of maternal-fetal immune-tolerance at maternal-fetal interface.Methods CXCR4+decidual NK cells, CXCR4- decidual NK cells or peripheral NK cells were co-cultured with naive CD4+T cells for 5 days. The expression of the membrane receptors and the intracellular staining of cytokines and transcription factor were analyzed by FCM. CXCR4+decidual NK cells, CXCR4- decidual NK cells and peripheral NK cells were co-cultured with CD4+CD25-T cells for 5 days, intracellular staining of transcription factor were analyzed by FCM.Results CXCR4+decidual NK cells instructed naive CD4+T cells to produce significantly higher level of IL-4, to up-regulate transcription factor GATA-3, meanwhile, to produce lower level of IFN-γ, to down-regulate transcription factor T-bet, thus attributing to a Th2 immune bias. CD4+CD25- T cells induced by CXCR4+decidual NK cells showed higher expression of Foxp3, beneficial to boost Treg expansion and maintain normal pregnancy.Conclusions CXCR4+decidual NK cells could induce naive CD4+T cells to a Th2 bias, and CXCR4+decidual NK cells could induce CD4+CD25- T cells express higher level of Foxp3, which both contributes to immune-tolerance at maternal-fetal interface.Part 4 Decidual NK cells defects at maternal-fetal interface leads to Thl bias at maternal fetal interface and spontaneous abortionObjective To verify the importance of normal decidual NK cells at maternal fetal interface by way of animal model.Methods After normal pregnancy CBAJ♀×BALB/c♂ matings and abortion-prone CBA/J♀×DBA/2♂ matings were established, we cultured the primary decidual immunocompetent cells at day 10.5 of gestation, and analyzed the expression of Thl and Th2 cytokines on CD4+T cells at maternal-fetal interface. After CBA/J♀× BALB/c♂ NK-depleted matings were established, we cultured the primary decidual immunocompetent cells at day 10.5 of gestation, and analyzed the expression of Thl and Th2 cytokines on CD4+T cells of NK depleted matings and normal pregnancies at maternal-fetal interface. Embryo absorption rate of every group was also calculated.Results At day 10.5 of gestation, it was found that the embryo absorption rate of abortion-prone CBA/J♀×DBA/2♂ matings was higher, and levels of CXCR4+NK cells was lower, Th1 bias was observed on abortion-prone CBA/JJ♀×DBA/2♂ matings compared to normal pregnancy CBA/J♀×DALB/2♂ matings. At day 10.5 of gestation, it was found that the embryo absorption rate of CBA/J♀×BALB/2♂ NK depleted matings was higher, and Th1 bias was observed on CBA/J♀×BALB/c ♂ NK depleted matings compared to normal pregnancy CBA/J♀×BALB/c♂ matings.Conclusions CXCR4+ NK cells was lower, and Th1 bias can be observed on abortion-prone CBA/J♀×DBA/2♂ matings; NK cell defects in normal CBA/J♀× BALB/♂ matings can result in Th1 bias and lead to miscarriage, which suggests that the decidual NK cells is critical in establishment of normal pregnancy.In summary, decidual CXCR4+ NK cells display Th2 bias and immune-tolerance phenotype, and preferentially accumulated at maternal-fetal interface during normal early pregnancy. Trophoblast cells mediate the phenotype of peripheral NK cells shift towards the decidual NK cells and recruit peripheral NK cells to preferentially accumulate at maternal-fetal interface via the CXCL12/CXCR4 axis. CXCR4+ decidual NK cells could induce naive CD4+T cells to a Th2 bias, and induce CD4+ CD25- T cells express higher level of Foxp3, which both contributes to immune-tolerance at maternal-fetal interface. CXCR4+ NK cells was down regulated in abortion-prone CBA/J♀×DBA/2♂ matings; NK cell defects in normal CBA/J♀ ×BALB/c♂ matings can result in Thl bias and miscarriage. Therefore, decidual NK cells, especially the CXCR4+decidual NK cells, are critical in a successful early pregnancy.