Regulatory T cells and regulatory natural killer (NK) cells play important roles in feto-maternal tolerance
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- Saito, S., Shiozaki, A., Sasaki, Y. et al. Semin Immunopathol (2007) 29: 115. doi:10.1007/s00281-007-0067-2
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In the early pregnancy decidua, lymphocytes express some activation markers on their surface, suggesting that maternal lymphocytes are activated and recognize the semiallograftic fetus. Therefore, the immunoregulation system must work to prevent fetus rejection. Recent data showed that parts of the immunoregulation system such as CD4+CD25+ regulatory T (Treg) cells, Th3 cells, Tr1 cells, regulatory NK cells, and a tryptophan-catabolizing enzyme, indolamine 2,3 deoxygenase, play very important roles in the maintenance of pregnancy. Not only Treg cells but also regulatory NK cells may inhibit maternal T cell or NK cell fetal attack.
KeywordsPregnancyRegulatory T cellRegulatory NK cellTh3 cellTr1 cell
A fetus is a semiallograft; therefore, the process of pregnancy must include mechanisms to prevent allograft rejection. Some mechanisms for the maintenance of pregnancy have been proposed. For example, villous trophoblasts do not express classical major histocompatibility complex (MHC) class I and class II molecules in humans, although some MHC-class I molecules but not MHC-class II molecules are expressed on murine trophoblasts. On the other hand, extravillous trophoblasts in humans express MHC-class I molecules such as human histocompatibility leukocyte antigen (HLA)-C, HLA-E, and HLA-G, which are specific ligands for uterine natural killer (NK) cells [1, 2]. Effector functions of NK cells are finely regulated by a series of inhibitory and activating receptors. Decidual NK cells produce a number of cytokines that promote trophoblast proliferation and differentiation, as well as angiogenetic factors, when NK cells are activated by an activating signal [1–3]. On the other hand, NK cell cytotoxic activity against fetal cells or trophoblasts is regulated by inhibitory signals such as immunoglobulin-like transcripts 2 and killer-immunoglobulin-like receptors (KIR)2DL4, which recognize HLA-G or KIR2DL2, 3, and KIR2DL1, which recognizes HLA-C. Therefore, the balance between the activation system and regulation system in NK cells is important to maintain successful pregnancy.
T cells and NK cells at the feto-maternal interface
The decidual immunological environment is quite different from that in peripheral blood. The population of T cells in early pregnancy decidua is about 10–15%, showing that T cells represent a minor population in the decidua. The major population in early pregnancy decidua is CD16-CD56bright NK cells, known as uterine NK (uNK) cells. Interestingly, both the numbers of T cell and NK cells increase at the decidua basalis (implantation site), while the population of T cells increases at the implantation site , suggesting that T cells accumulate at the feto-maternal interface and play an important role in the maintenance of pregnancy. Both decidual T cells and NK cells express activation markers such as CD69, CD25, CD122, and HLA-DR . After activation, these cells produce a variety of cytokines. T cell immunity is classified into Th1 type immunity and Th2 immunity by the cytokine profile. Therefore, Th1-type cytokines or Th2-type cytokines are produced at the materno-fetal interface by these activated T cells and NK cells.
The Th1/Th2 balance in the peripheral blood of early pregnant women is controversial. Some reports showed a predominant Th2 status during pregnancy [10, 11], while other papers showed that the Th1/Th2 ratio did not change in the peripheral blood during early pregnancy [12, 13]. Systemic Th1/Th2 immunity in early pregnant women might not be very different from nonpregnant subjects. However, it is certain that predominant Th2-type immunity exists in early pregnancy decidua, especially at the implantation site [12, 14]. Regardless, immunoregulation should work at the materno-fetal interface to regulate immunostimulation so that the fetus is protected from activated maternal lymphocyte attack.
CD4+CD25+ Treg cells in alloantigen tolerance
Control of self-reactive T lymphocytes by Treg cells is essential for induction and maintenance of tolerance. These CD4+ T cells include thymus-derived CD4+CD25+ Treg cells, as well as distinct subjects of secondary suppressor T cells, including Th3 cells and Tr1 cells, in the periphery [4, 15]. Recent reports suggest that these CD4+CD25+ Treg cells play essential roles in alloantigen tolerance . CD4+CD25+ Treg cells are involved in tolerance induction to allogeneic skin, pancreatic islets or heart transplants. The majority of such CD4+CD25+ Treg cells are produced by the normal thymus as a functionally distinct and mature subpopulation of T cells. Their repertoire of antigen specificities is as broad as that of naïve T cells, and they are capable of recognizing both self- and nonself-antigens, thus enabling them to control various immune responses .
In the mouse, CD4+CD25+ T cells are homogeneous, and they exhibit a strong immunoregulatory function. However, in humans, CD4+CD25+ T cells are heterogeneous. A subset within CD4+CD25high or CD25bright cells exhibits a regulatory function, but the CD4+CD25low subset exhibits no such immunoregulatory function . Other surface phenotypes such as CD122, CD132, glucocorticoid-induced tumor necrosis factor receptor superfamily-related gene, cytotoxic T lymphocyte antigen (CTLA)-4, programmed death-ligand 1, CD62, CD38, CD45, and CD103 are expressed on both human and murine CD4+CD25+ Treg cells . The development and function of CD4+CD25+ Treg cells is controlled by Foxp3 . A mutation of Foxp3 results in loss of CD4+CD25+ Treg cells and massive multiorgan autoimmunity in scurfy mice and immune dysregulation, polyendocrinopathy, enteropathy, X-linked patients. Furthermore, decreased Foxp3 expression in CD4+CD25+ Treg cells in various immune disorders such as graft-versus-host disease, myasthenia gravis, and multiple sclerosis has been reported. A large number (∼700 genes) of Foxp3-bound genes are up- or down-regulated in Foxp3+ T cells, suggesting that Foxp3 acts as both a transcription activator and repressor . Foxp3 can cooperate in a DNA-binding complex with nuclear factor of activated T cell to regulate the transcription of several known target genes . For example, Foxp3 down-regulates the transcription of Gpr171, Dusp6, Gadd45b, Jak2, IkBNS, ZAP70, Prpn22, Itk, Il2, Pou2af1, Myc, Tglf, Slfn2, and Ucp2 and up-regulates Ly6a, CD2, S100a6, and Irf8. Interestingly, mutation of PTPN22 has been reported in type 1 diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus, and Grave’s disease. These mutations might inhibit the differentiation of CD4+CD25+ Treg cells, resulting in autoimmune disease. Recent data suggest that CD4+CD25+ Treg cells play an essential role in the maintenance of pregnancy.
CD4+CD25+ Treg cells in pregnancy
The second mechanism is when the cytokines such as TGF-β and IL-10 produced by CD4+CD25+ Treg cells ihhibit T cell activation (Fig. 2), while the third mechanism is when CTLA-4 expressed on CD4+CD25+ Treg cells induces indolamine 2, 3-dioxygenase (IDO) expression in dendritic cells (DCs) and macrophages (Mϕ; Fig. 2). This interaction induces interferon (IFN)-γ production by DCs resulting in IDO induction in DCs and Mϕ . Tryptophan-catabolizing enzyme activity during pregnancy protects developing fetuses from the maternal immune response . It has been reported that treatment with an IDO inhibitor in allogeneic pregnant mice induced abortion, although this inhibitor did not induce abortion in syngeneic pregnant mice .
We used a trans-well culture system to clarify which mechanism is important for immunoregulation by decidual CD4+CD25+ Treg cells, cell-to-cell interaction, or immunosuppressive cytokines . Purified decidual CD4+CD25+ Treg cells and irradiated antigen-presenting cells (APCs) were seeded on the upper well, and conventional CD4+CD25− T cells and APCs were seeded on the lower well. Then, these cells were stimulated with an anti-CD3 monoclonal antibody. If immunosuppressive cytokines are key molecules that induce immunoregulation, CD4+CD25+ Treg cells should inhibit the DNA synthesis of conventional T cells in a trans-well culture system because immunoregulatory cytokines produced by CD4+CD25+ Treg cells easily pass through this membrane. Our data revealed that CD4+CD25+ Treg cells had no immunoregulatory ability in the trans-well system, suggesting that cell-to-cell contact between CD4+CD25+ Treg cells and conventional T cells is necessary for immunoregulation . Recent data suggested that B7 (CD80/CD86) on macrophages and DCs bind CTLA-4 on CD4+CD25+ Treg cells and that this interaction induces IDO expression  (Fig. 2). CTLA-4 is usually located on lysosomes in CD4+CD25+ Treg cells. When CD4+CD25+ Treg cells recognize the antigen, lysosomal CTLA-4 quickly moves to the cell surface. The decidual CD4+CD25high Treg cells express surface CTLA-4 in normal pregnancy, although peripheral CD4+CD25high Treg cells do not express surface CTLA-4 in humans . Interestingly, surface CTLA-4 expression on decidual CD4+CD25high Treg cells down-regulates in miscarriage cases . This finding has two important implications. One is that decidual CD4+CD25high Treg cells may recognize fetal antigens and induce fetal antigen-specific tolerance at the materno-fetal interface. The other is that surface CTLA-4 on CD4+CD25high Treg cells may induce IDO expression in decidual DCs or macrophages. We studied whether CTLA-4 or IFN-γ induces IDO expression in DCs and macrophages . Both IFN-γ and CTLA-4 augmented IDO expression in peripheral blood and decidual DC and macrophages, and these expressions in pregnant women were significantly higher than those in nonpregnant women. Interestingly, these IDO expressions decreased in miscarriage cases . Not only the number of CD4+CD25high Treg cells but also the induction of IDO in DCs and macrophages by CTLA-4 decrease in miscarriage cases.
CD4+CD25+ Treg cells increase in regional lymph nodes of the uterus such as para-aortic lymph nodes and pelvic lymph nodes before implantation. CD4+CD25+ Treg cells have already increased on successful mating day 2 in mice [5, 8]. Implantation occurs on day 4.5 in mice, so increased CD4+CD25+ Treg cells in regional lymph nodes occurs before implantation, and these cells could play some roles in the maintenance of allogeneic pregnancy. Indeed, anti-CD25 monoclonal antibody treatment in an allogeneic pregnancy mice model on day 0 decreased the pregnant mice per total mated allogeneic pregnancy mice but not in syngeneic mice [5, 32]. This finding suggests that CD4+CD25+ Treg cells prevent fetal rejection at the implantation phase. In humans, Jasper et al.  reported reduced endometrial Foxp3 mRNA in unexplained infertility cases, but mRNAs encoding T cell transcription factors T-box expressed in T cell (T-bet) and GATA3, associated with differentiation of Th1 and Th2 cells, respectively, were unchanged. Previous studies showed that reduced CD4+CD25+ Treg cells before implantation induces fetal rejection. However, it has not been clarified whether reduced CD4+CD25+ Treg cells in the early pregnancy period induce fetal rejection or not. In our preliminary study, larger amounts of anti-CD25 monoclonal antibodies were needed to induce abortion in pregnant mice at days 4.5 and 7.5 of gestation. Early-stage embryos could be very susceptive to maternal immune cell attack. After successive implantation, the fetus has some defence mechanisms to prevent fetal rejection; therefore, a high amount of anti-CD25 antibodies might be needed to induce abortion. Recent data demonstrated that CD4+CD25+ Treg cell migration requires l-selectin expression . Interestingly, up-regulation of l-selectin on circulating CD56bright NK cells during the periovulatory window (days 10–12) induced the promotion of CD56bright NK cells homing in on the uterus in humans . l-Selectin expression on CD4+CD25+ Treg cells may up-regulate during the periovulatory period, and circulating CD4+CD25+ Treg cells might accumulate to the uterus in the implantation period.
Regulatory NK cells at the feto-maternal interface
MHC class I-specific tolerance and MHC class II-specific tolerance during pregnancy
CD4+CD25+ Treg cells play very important roles for induction of both MHC class I- and MHC class II-specific tolerance . On the other hand, MHC class I-dependent reactivity in allograft tolerance is induced by NK cells but not by T cells NK T cells . Maternal lymphocytes directly contact trophoblasts. MHC class II antigens and polymorphic MHC class I antigens such as HLA-A and HLA-B are not expressed on trophoblasts. Only HLA-C antigens are expressed on extravillous trophoblasts (EVT), while nonpolymorphic HLA-G and HLA-E are expressed on EVT. On this point, some researchers question whether MHC class I- or MHC class II-specific tolerance is present during pregnancy, but we detected anti-MHC class I and MHC class II antibodies in the serum of pregnant women suggesting that maternal lymphocytes recognize fetal MHC class I and class II antigens. It has been reported that MHC class II antigens, HLA-DR, and HLA-DQ are expressed on the endoplasmic reticulum in trophoblastic cell debris [40, 41]. Several grams of trophoblastic cell debris per day are released into the maternal circulation . Fetus-derived lymphocytes are also present in maternal circulation . These findings support the idea that MHC class I- and class II-specific tolerance play very important roles in the maintenance of pregnancy. We recently report regulatory NK cells in leukemic mice , and supportive data were also reported . CD4+CD25+ Treg cells may induce MHC class I- and MHC class II-specific tolerance during pregnancy, while regulatory NK cells may induce MHC class I-specific tolerance during pregnancy.
These balances are disturbed in miscarriage cases. The decidual Th1/Th2 balance and NK1/NK2 balance shift to a type 1 dominant state, and the immunoregulation system decreases to a nonpregnant level. In such an immunological environment, an allogeneic fetus could easily be rejected by the maternal immune system.
However, much remains to be done to fully understand how T cells and NK cells function in pregnancy.
This research was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan [Grant-in Aid for Scientific Research (B)-17390447 and (C)-18591797, and Grant-in-Aid for Exploratory Research 18659482] and the 21st Century COE Program.