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CD4+CD25high regulatory T cells in human pregnancy

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Abstract

In both rodent and human systems, there is an emerging consensus that immunoregulatory activity specific for donor alloantigens is enriched in the CD4+CD25+ T cell population. The absence of CD4+CD25+ regulatory T (Treg) cells induces severe immunodeficiency with autoimmune disease, dermatitis and fatal infections in humans and mice. CD4+CD25+ Treg cells play a critical role in peripheral tolerance, transplantation tolerance and maternal tolerance to the fetus.

Although both human and mouse CD4+CD25+ Treg have potent regulatory properties, surface phenotypes of human CD4+CD25+ Treg cells are not exactly the same as those of mouse CD4+CD25+ Treg cells. Murine CD4+CD25+ T cells are homogenous and exhibit regulatory function. On the other hand, CD4+CD25high T cells are the only cells which exhibit regulatory function in humans. Humans CD4+CD25low cells have no ability for immunosuppression. CD4+CD25high T cells inhibit the immunostimulation of conventional T cells through cell-to-cell contact or immunosuppressive cytokines such as interleukin 10 and transforming growth factor-β. As another mechanism of immunosuppression, CTLA-4 on CD4+CD25+ regulatory T cells up-regulate indoleamine 2,3-dioxygenase (IDO) expression in dendritic cells which play important roles for immunosuppression. Here, we review the differences between humans and mouse Treg cells and the role of CD4+CD25+Treg during pregnancy.

Introduction

The immune system discriminates between self and nonself, establishing and maintaining unresponsiveness to self. The primary mechanism that leads to tolerance to self-antigens is thymic clonal deletion of self-reactive T cells. However, some self-reactive T cells escape this process and recognize peripheral tissue antigens. Autoreactive T cells are normally present in all individuals, but the incidence of autoimmune diseases is rare. This indicates that a mechanism for peripheral self-tolerance must exist to control pathogenic T cells. Accumulating evidence suggests that populations of regulatory T (Treg) cells function in a critical role to modulate autoimmune responses. Kojima and Prehn (1981) discovered that thymectomy on neonatal day 3 led to the development of multiorgan autoimmune disease in mice. Sakaguchi et al., 1985, Sakaguchi et al., 1995 reported that neonatal thymectomized mice lack CD4+CD25+ T cells and adoptive transfer of this population into day 3 thymectomized animals prevented autoimmune disease. These studies indicated that naturally arising CD4+CD25+ Treg cells play indispensable roles in the maintenance of natural self-tolerance and negative control of pathological, as well as physiological, immune responses (Sakaguchi, 2004, Piccirillo and Shevach, 2004, Bluestone and Abbas, 2003). Importantly, the experiments showed that removal of CD4+CD25+ Treg cells not only elicited autoimmune diseases, but also enhanced immune responses to nonself antigens including xenogeneic proteins and allografts (Wood and Sakaguchi, 2003). Lagging a few years behind the discovery of CD4+CD25+ treg cells in the mouse, CD4+CD25+ Treg cells have also been isolated from human peripheral blood, thymus, lymph nodes and cord blood (Beacher-Allan et al., 2001, Beacher-Allan et al., 2004). The characteristics of CD4+CD25+ Treg are almost the same between mice and humans, but there are also some differences between mouse and human.

Fifty years ago, Medawar (1953) proposed that immunological tolerance should be present during pregnancy to protect against an aggressive maternal allogeneic response directed at the fetus. Recently, Aluvihare et al. (2004) reported that the absence of CD4+CD25+ Treg led to a failure of gestation due to immunological rejection of the fetus, suggesting that CD4+CD25+ Treg cells mediate maternal tolerance to the fetus. In this review, we focus on human CD4+CD25+ Treg in reproduction.

The surface phenotypes of human CD4+CD25+ Treg cells are not exactly the same as those of mouse CD4+CD25+ Treg cells (Table 1). The biggest difference is the expression pattern of CD25 between humans and mice. A subset within the CD4+CD25hlgh T cells in the circulation of normal humans exhibits a strong regulatory function. On the other hand, CD4+CD25low T cells in humans have no ability for immunoregulation. The CD4+CD25high subset is homogeneous as over 95% of the cells express CD45RO, CD62L, glucocorticoid-induced TNF receptor (GITR) and CD122 (IL-2Rβ), but the CD4+CD25low subset contains a more heterogenous mixture of cells (Beacher-Allan et al., 2001, Beacher-Allan et al., 2004). IL-2 is a potent T cell growth factor, and activation signals induce CD25 (IL-2Rα) expression on T cells. Activated CD4+ T cells express CD25, but this is generally transient and of lower magnitude compared with CD4+CD25+ Treg (Beacher-Allan et al., 2001, Sasaki et al., 2004). Other surface markers that are expressed on CD4+CD25+ Treg cells, such as CTLA-4, G1TR, PD-L1, CD45RO and HLA-DR are also found on activated T cells, but their expression is transient in conventional CD4+ T cells. On the other hand, CD4+CD25+ Treg cells constitutively express these surface markers.

These data suggest that we could detect CD4+CD25hlgh cells by flow cytometry to evaluate human Treg. As shown in Fig. 1, CD4+ T cells can be classified into CD4+CD25high, CD4+CD25low and CD4+CD25 cells. In the mouse, approximately 10% of CD4+ T cells are CD4+CD25+ Treg cells. However, only 2–6% of CD4+ T cells express high levels of CD25 in humans. It is important that the number of CD4+CD25low cells is higher compared to that of CD4+CD25high cells (Fig. 1). We should evaluate the populations of CD4+CD25high cells in normal subjects and pregnant subjects, as well as pathological conditions such as implantation failure, miscarriage and pre-eclampsia. In mouse studies, 80% suppression of T cell proliferation induced by soluble anti-CD3 is seen at a suppressor:effector ratio of 1:4. In studies of human CD4+CD25+ T cells, the suppression effect by these cells was different between studies. These differences could be explained by the isolation and purification of CD4+CD25+ Treg cells. Many preparations of human CD4+CD25+ Treg cells are contaminated with a significant number of CD4+CD25low activated T cells. For a precise evaluation of human Treg, it is important to check the CD4+CD25high population in human samples.

The most specific and reliable marker for CD4+ Treg cells is the transcription factor Foxp3, which is expressed by the majority of CD4+CD25+ T cells (Hori et al., 2003, Fontenot et al., 2003). GITRhigh T cells, and CTLA-4+ T cells in the CD4+ T cell population, include Foxp3-expressing T cells. Foxp3 transduction indices expression of CD25, CTLA-4, CD103 and GITR which are closely associated with the function of natural CD4+CD25+ Treg cells. When Foxp3 is expressed in CD4+CD25 T cells, these cells differentiate into CD4+CD25+ T cells which have immunoregulation ability (Hori et al., 2003, Fontenot et al., 2003).

Immune dysregulation, polyendocrinopathy, enteropathy and X-linked syndrome (IPEX) was described as an X-linked immunodeficiency syndrome associated with autoimmune disease of multiple endocrine organs, atopic dermatitis, and fatal infections. These symptoms are quite similar to those in scurfy mice. Interestingly, both scurfy mice and IPEX patients have mutations of the gene Foxp3. Both animals (scurfy mice) and humans (males with IPEX) with mutations in the transcription factor Foxp3 do not develop CD4+CD25+ Treg cells. These findings suggest that Foxp3 is an essential transcription factor for induction and development of CD4+CD25+ Treg cells. Recently, Viguier et al. (2004) reported that Foxp3 mRNA is expressed in CD4+CD25high Treg. These cells are over-represented in human metastatic melanoma lymph nodes and inhibit the function of infiltrating T cells. Our recent flow cytometrical data also revealed that the CD4+CD25high population expresses Foxp3 and CTLA-4, but the number of Foxp3 and CTLA-4 expressing cells was low in CD4+CD25low T cells (Fig. 1).

At least three mechanisms of suppression by CD4+CD25+ Treg cells have been reported. It appears that human CD4+CD25+ Treg cells must be activated through their T cell receptor (TCR) and CD28-mediated costimulation for immunoregulation (Dieckmann et al., 2002). Firstly, some antigens are presented by dendritic cells to CD4+CD25high Treg cells. This stimulation mobilizes the cytosomal CTLA-4 to surface CTLA-4. CTLA-4 is a homologue of the T cell costimulatory molecule CD28. For the full stimulation of T cell proliferation, both the TCR signal and CD28-mediated costimulation are required. If only the TCR signal activates, T cells become anergic. Cell surface CTLA-4 binds B7 complexes, such as CD80 and CD86, because the affinity of CTLA-4 to the B7 complex is more than one hundred times higher than that of CD28 to the B7 complex. As a result, regulatory T cells become anergic. Secondly, CTLA-4 ligation by the B7 complex could trigger a negative signalling pathway or interface with an activating pathway, thus inhibiting activation downstream of the T cell receptor. As a result, regulatory T cells could not proliferate by activation, but inhibited the proliferation of conventional T cells through cell-to-cell contact or humoral factors such as TGF-β and IL-10 (Fig. 2). Recent studies demonstrated that direct contact between CD4+CD25+ T cells and conventional CD4+CD25 T cells is important for immunoregulation (Cao et al., 2003, Sasaki et al., 2004). Another mechanism of immunoregulation of CD4+CD25+ Treg involves CTLA-4 on Treg up-regulating indoleamine 2,3-dioxygenase (IDO) expression in antigen presenting sells (Grohmann et al., 2001). IDO is a tryptophan-catabolizing enzyme expressed by macrophages, dendritic cells and extravillous trophoblasts, and it plays important roles in induction of tolerance and maintenance of allogeneic pregnancy (Munn et al., 1998, Mellor et al., 2001). These data support this view by which CTLA-4 expressing CD4+CD25+ Treg may affect the activity of tolerogenic dendritic cells.

Our group firstly reported that decidual and peripheral blood CD4+CD25high T cells increased during early pregnancy (Sasaki et al., 2003). Aluvihare et al. (2004) firstly reported that CD4+CD25+ Treg were required for the maternal immune system to tolerate a fetal allograft in mice. They showed the proportion of CD4+CD25+ T cells increased in the iliac lymph node, inguinal lymph node, the spleen and blood, and that these cells suppressed alloreactive proliferation in vitro. They showed also that uterine CD4+CD25+ T cells express Foxp3 mRNA. They injected 2 × 107 lymphocytes, or an equal number of cells from a CD25 depleted cell preparation (CD25) into BALB/C nu/nu mice that lacked T cells. All recipient BALB/C nu/nu female were mated with C57BL/6 males on the day after adoptive transfer. As a result, all fetuses were aborted in allogeneic pregnancy when CD25 cells were injected, while this treatment did not induce fetal resorption in syngeneic pregnancy, suggesting that CD25+ cells, and perhaps CD4+CD25+ T cells, mediate maternal tolerance to the fetus.

In human pregnancy, the population of CD4+CD25+ T cells in early pregnancy decidua is elevated (Saito et al., 1992), but regulatory T cells had not been classified at that time. Recently, Somerset et al. (2004) reported an increase in circulating CD4+CD25+ T cells during early pregnancy, peaking the second trimester and then declining postpartum. They showed also that isolated CD4+CD25+ T cells express Foxp3 mRNA. Unfortunately, they only showed the population rate of CD4+CD25+ T cells, but not the population rate of CD4+CD25high T cells which indicates regulatory T cells. They showed that CD4+CD25+ T cells isolated from pregnant women were capable of suppressing [3H]-thymidine uptake in allogeneically stimulated lymphocytes at a CD25 cell to CD25+ cell ratio of 1:1 by approximately 50%. This suppressive effect is rather low compared to Baecher-Allan's report in which over 90% suppression of T cell proliferation was seen at a CD4+CD25high T cell to CD4+CD25 T cell ratio of 1:1 (Beacher-Allan et al., 2001). This difference is most consistent with the possibility that many conventional T cells are contaminated with a significant number of CD25+ effector cells. Sasaki et al. (2004) reported that the population of CD4+CD25high T cells in the peripheral blood of non-pregnant subjects was about 6%. This ratio elevated to 8% in normal early pregnancy subjects, but this elevated CD4+CD25high T cell ratio decreased to a non-pregnancy level in miscarriage cases. They reported also that the population of CD4+CD25high T cells to CD4+ T cells increased to over 20% in early pregnancy decidua, and this population rate decreased to 6% in spontaneous abortion cases. Heikkinen et al. (2004) also observed that about 14% of decidual CD4+ T cells had the CD4+CD25+ phenotype. In contrast, Chao et al. (2002) reported that the expression of CD25 was decreased on decidual CD4+ T cells in normal pregnancy. However, the mean fluorescence intensity of CD25 on decidual CD4+ T cells was almost twice that on peripheral blood, suggesting that CD4+CD25high cells are increased even though the total populations of CD4+CD25low T cells and CD4+CD25high cells are decreased. Vassiliadou et al. (1999) reported that a increased number of CD25+ T cells was detected in the decidua of spontaneous abortion cases based on immunohistochemical methods. Darmochwal-Kolarz et al., 2002 also reported high expression of CD25 on CD4+ T cells in patients with recurrent pregnancy loss. To evaluate the population of CD4+CD25+ Treg cells, we should revaluate the population of CD4+CD25high cells by flow cytometry.

CD4+CD25+ Treg cells are crucial to the maintenance of tolerance in pregnancy. However, the factors regulating CD4+CD25+ Treg cells are largely unknown. Recently, Polanczyk et al. (2004) reported that estrogen augmented Foxp3 expression in vitro and in vivo, and treatment with estrogen increased the CD4+CD25+ Treg cell number in mice. These data suggest that estrogen promotes maternal tolerance to the fetus by increasing the number of CD4+CD25+ Treg cells.

The factors regulating the accumulation of CD4+CD25+ Treg cells in the decidua are also unknown. CD4+CD25+ Treg cells express chemokine receptors, CCR4 and CCL22, which are produced by tumor cells and microenvironmental macrophages, and mediate trafficking of CD4+CD25+ Treg cells to tumors (Curiel et al., 2004). CCL22, a ligand for CCR4, induces the accumulation of CD4+CD25+ Treg cells, so CCL22 at the feto-maternal interface might induce the accumulation of CD4+CD25high Treg cells at the decidua.

CD4+CD25+ Treg cells express intracellular CTLA-4 and, after activation, express CTLA-4 on their surface. These surface CTLA-4 molecules play an important role in immunoregulation. Decidual CD4+CD25high T cells express a high frequency of intracellular CTLA-4 and 5–7% of these cells express surface CTLA-4 (Heikkinen et al., 2004, Sasaki et al., 2004), suggesting that decidual CD4+CD25high T cells are stimulated by some antigens such as fetal antigens. Because antigen stimulation by dendritic cells induced the surface expression of CTLA-4 (Fig. 1), these activated CD4+CD25high Treg cells should mediate maternal tolerance to the fetus. Recently, Sindram-Trujillo et al. (2003) reported that labor is associated with a decrease of CD4+CD25+ cells in decidua, suggesting that the disappearance of the CD4+CD25+ T cell population may contribute to the induction of labor, although they did not check CD4+CD25high cells.

In normal pregnancy, CD4+CD25high Treg cells play an important role in the maintenance of pregnancy. The recruitment or function of CD4+CD25high Treg might be impaired in pathological conditions such as recurrent spontaneous abortion, preterm labor and pre-eclampsia. Infection or inflammation are known to cause these disease (Sacks et al., 1999). Interestingly, CD4+CD25+ Treg cells express toll like receptors (TLR)-4, 5, 7 and 8 (Caramalho et al., 2003). TLRs play an essential role in innate host defence, as well as control of adaptive immune responses. Recent data showed that microbial induction of the Toll pathway blocks the suppressive effect of CD4+CD25+ Treg, allowing activation of pathogen-specific adaptive immune responses. This block of suppressor activity is dependent in part on IL-6 (Pasare and Medzhitov, 2003). IL-6 also inhibits the tolerogenic function of CD8α+ dendritic cells expressing IDO (Grohmann et al., 2001). IL-6 down-regulates IFN-γR expression on CD8+ dendritic cells, and Munn's group reported that the enzyme IDO is important for materno-fetal tolerance (Munn et al., 1998, Mellor et al., 2001). It is interesting that an inflammatory cytokine, IL-6, inhibits the suppressive effect of both CD4+CD25+ Treg cells and IDO-expressing dendritic cells. Recently, Yang et al. (2004) made an interesting report. Dendritic cell-based tumor vaccines ablate tumor-specific T cell tolerance only after removal of CD4+CD25+ Treg cells. However, persistent TLR signals and dendritic cell-based tumor vaccines could ablate tumor-specific T cell tolerance in the presence of CD4+CD25+ Treg cells. Therefore, persistent TLR signals are required to reverse CD4+CD25+ Treg-mediated CD8+ T cell tolerance. These data suggest that chronic inflammation in recurrent spontaneous abortion, preterm labor land pre-eclampsia cases might ruin the suppressive effects of CD4+CD25+ Treg cells resulting in induction of fetal rejection responses. Further studies are needed to clarify these points.

We have focused on immunostimulation systems, such as the Thl/Th2 balance, during pregnancy. However, a further subtype of T cells with immunosuppressive function, termed Treg cells, has been identified. We should clarify the role of not only immunostimulation systems such as Th1 and Th2 cells, but also immunoregulation systems in human reproduction.

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