Kumiko Inada

The balance of the immune system between T cells and NK cells in miscarriage.

Immunological dysfunction has been proposed to explain the etiology of recurrent pregnancy loss (RPL). The immunological environment differs between the decidua basalis and decidua parietalis, and also between RPL cases with normal fetal chromosomes and those with abnormal fetal chromosomes. The problem with analyzing decidual tissues from spontaneous abortions is that cause versus effect phenomena are difficult to distinguish. Recent data show that the immune system in a late‐stage miscarriage is completely different from that in an early‐stage miscarriage. If immunocompetent cells can cause RPL, the immunological environment may be a causative factor, especially in an early‐stage miscarriage, at the decidua basalis, and/or in cases of RPL with a normal embryo. Careful examination of the immune system at the decidua basalis in an early‐stage miscarriage in RPL cases with normal fetal chromosomes may reveal useful information. This paper aimed at finding a cause of RPL by analyzing the balance of the immune system between T cells and NK cells in an early‐stage miscarriage.

Characterization of regulatory T cells in decidua of miscarriage cases with abnormal or normal fetal chromosomal content

Decreased regulatory T (Treg) cells have been reported in cases of recurrent pregnancy loss. To understand the role of Treg cells in human pregnancy, we have studied the frequency, localization and characterization of Treg cells in the decidua. The frequency of Foxp3(+) cells among CD3(+)CD8(-) cells at the decidua basalis in cases of miscarriage with a normal embryo karyotype (n=10) was significantly lower than in normally progressing pregnancies (n=10). However, those frequencies in miscarriage with an abnormal embryo karyotype were similar to normally progressing pregnancies. Next, we used flow cytometry to study Treg cell expression of the proliferation marker Ki67 and functional Treg marker CCR5. The frequency of Foxp3(+)CD4(+) T cells in miscarriage with a normal embryo (n=10) was significantly lower than those in normally progressing pregnancies (n=15) and in miscarriage with an abnormal embryo (n=14). In miscarriage with a normal embryo, the population of Ki67(-)Foxp3(+)CD4(+) T cells was significantly smaller than in normal pregnancy. However, the frequencies of Ki67(+)Foxp3(+)CD4(+) cells and CCR5(+)Foxp3(+)CD4(+) cells were not different between the three groups. These data suggest that increased Ki67(-) Treg cells in the decidua basalis may play an important role in the induction of immune tolerance, and that immune-medicated pregnancy loss may be caused by decreased Ki67(-) Treg cells in the implantation site.

Which Types of Regulatory T cells Play Important Roles in Implantation and Pregnancy Maintenance

Regulatory T cells (Treg) play essential roles in implantation and allogeneic pregnancy maintenance in mice and humans. Recent data have shown the heterogeneity of Treg, such as thymic (naturally occurring) Treg, extrathymic (inducible or peripheral) Treg, naïve Treg, effector Treg, resting (non‐proliferating) Treg and activated (proliferating) Treg. Importantly, Foxp3, which was believed to be a specific marker for Treg, is transiently expressed in T cells when conventional T cells are activated and proliferating in humans showing that Foxp3 is not a specific marker for Treg. Therefore, we should evaluate the true Treg level and clarify which types of Treg cells play important roles in implantation and pregnancy maintenance in mice and humans.

Paternal antigen-specific proliferating regulatory T cells are increased in uterine-draining lymph nodes just before implantation and in pregnant uterus just after implantation by seminal plasma-priming in allogeneic mouse pregnancy

Paternal antigen-specific regulatory T (PA-specific Treg) cells play an important role in feto-maternal tolerance. To detect the PA-specific Tregs, female BALB/c mice were mated with male DBA/2 mice. Mls Ia antigen on DBA/2 mice is recognized by the T-cell receptor Vβ6; thus, CD4(+)Foxp3(+)Vβ6(+) cells are recognized as PA-specific Treg cells. CD4(+)CD25(+)Vβ6(+) cells effectively suppressed the allo-reactive proliferation of lymphocytes compared with that of CD4(+)CD25(+)Vβ6(-) cells. Vβ6(+) PA-specific Treg cells expressed CCR4 and CCR5 on their surface. The frequency of Ki67(+) PA-specific Treg cells among Treg cells was significantly increased in draining lymph nodes on day 3.5 post-coitus (pc; 6.8±1.1%, p<0.05) and day 5.5 pc (7.2±1.1%, p<0.05) in allogeneic pregnant mice compared with that in nonpregnant mice (2.7±0.2%). The frequency of Ki67(+) PA-specific Treg cells in the uterus increased significantly after day 5.5 pc in allogeneic pregnant mice compared with that in nonpregnant mice (8.8±2.8% vs. 1.2±1.3%, p<0.05). However, Ki67(-)PA-specific Tregs did not change during pregnancy. To analyze the role of seminal fluid or sperm in Treg expansion, female BALB/c mice were mated with vasectomized DBA/2 male mice (VAS) or seminal vesicle-excised DBA/2 male mice (SVX). The frequency of Ki67(+) PA-specific Treg cells did not increase in draining lymph nodes or uterus in BALB/c×DBA/2 (SVX) allogeneic mating mice. These findings suggest that the priming by seminal fluid is important for the induction of proliferating PA-specific Tregs in uterine-draining lymph nodes just before implantation and pregnant uterus after implantation, resulting in successful implantation and the maintenance of allogeneic pregnancy.

Accurate Prediction of the Stage of Histological Chorioamnionitis before Delivery by Amniotic Fluid IL-8 Level.

To estimate the stage of histological chorioamnionitis (h‐CAM) antenatally using clinical data.

Helios-positive functional regulatory T cells are decreased in decidua of miscarriage cases with normal fetal chromosomal content

Regulatory (Treg) T cells play essential roles in the maintenance of allogeneic pregnancy in mice and humans. Recent data show that Foxp3 expression occurs in both immuno-suppressive Treg and -nonsuppressive effector T (Teff) cells upon activation in humans. Samstein et al. (2012) reported that inducible Treg (iTreg) cells enforce maternal-fetal tolerance in placental mammals. Therefore, we should reanalyze which types of Treg cell play an important role in the maintenance of allogeneic pregnancy. In this study, we studied the frequencies of naïve Treg cells, effector Treg cells, Foxp3(+) Teff cells, Helios(+) naturally occurring Treg (nTreg) cells, and Helios(-) iTreg cells using flow cytometry. The frequencies of effector Treg cells and Foxp3(+) Teff cells among CD4(+)Foxp3(+) cells in the decidua of miscarriage cases with a normal embryo karyotype (n=8) were significantly lower (P=0.0105) and significantly higher (P=0.0258) than those in normally progressing pregnancies (n=11), respectively. However, these frequencies in miscarriages with an abnormal embryo karyotype (n=15) were similar to those in normally progressing pregnancies. The frequencies of these cell populations in the three groups were unchanged in peripheral blood; on the other hand, most of the effector Treg cells in the decidua were Helios(+) nTreg cells and these frequencies were significantly higher than those in peripheral blood, while those among effector Treg and naïve Treg cells in the decidua and peripheral blood were similar among the three groups. These data suggest that decreased Helios(+) effector nTreg might play an important role in the maintenance of pregnancy in humans.

Role of Paternal Antigen-Specific Treg Cells in Successful Implantation.

Maternal lymphocytes recognize fetal antigens, so tolerance is necessary to prevent rejection. Seminal plasma is important for induction of paternal antigen‐specific Treg cells in the uterine draining lymph nodes and the pregnant uterus. Elimination of Treg cells during implantation or early pregnancy induces implantation failure or fetal resorption in mice. Immunosuppressive therapy with an anti‐TNF antibody or the immunosuppressive agent tacrolimus improves the pregnancy rate in women with repeated implantation failure and recurrent pregnancy loss of unknown etiology, suggesting that Treg cells play an essential role in successful implantation and pregnancy in humans.