Decidual γδT cells of early human pregnancy produce angiogenic and immunomodulatory proteins while also possessing cytotoxic potential.

During pregnancy, the maternal immune system must allow and support the growth of the developing placenta while maintaining the integrity of the mother's body. The trophoblast's unique HLA signature is a key factor in this physiological process. This study focuses on decidual γδT cell populations and examines their expression of receptors that bind to non-classical HLA molecules, HLA-E and HLA-G. We demonstrate that decidual γδT cell subsets, including Vδ1, Vδ2, and double-negative (DN) Vδ1-/Vδ2- cells express HLA-specific regulatory receptors, such as NKG2C, NKG2A, ILT2, and KIR2DL4, each with varying dominance. Furthermore, decidual γδT cells produce cytokines (G-CSF, FGF2) and cytotoxic mediators (Granulysin, IFN-γ), suggesting functions in placental growth and pathogen defense. However, these processes seem to be controlled by factors other than trophoblast-derived non-classical HLA molecules. These findings indicate that decidual γδT cells have the potential to actively contribute to the maintenance of healthy human pregnancy.

The Role of Hydrogen-Peroxide (H2O2) Produced by Vaginal Microbiota in Female Reproductive Health.

Female reproductive health is strongly associated with healthy vaginal microbiota, which is thought to be ensured by the dominance of certain Lactobacillus species. Lactobacilli control the vaginal microenvironment through several factors and mechanisms. One of them is their ability to produce hydrogen peroxide (H2O2). The role of Lactobacillus-derived H2O2 in the vaginal microbial community has been intensively investigated in several studies with many designs. However, results and data are controversial and challenging to interpret in vivo. Defining the underlying mechanisms responsible for a physiological vaginal ecosystem is crucial since it could directly affect probiotic treatment attempts. This review aims to summarize current knowledge on the topic, focusing on probiotic treatment possibilities.

Corrigendum: Gamma/Delta T Cells in the Course of Healthy Human Pregnancy: Cytotoxic Potential and the Tendency of CD8 Expression Make CD56+ γδT Cells a Unique Lymphocyte Subset.

[This corrects the article DOI: 10.3389/fimmu.2020.596489.].

The Role of Type I and Type II NKT Cells in Materno-Fetal Immunity.

NKT cells represent a small but significant immune cell population as being a part of and bridging innate and adaptive immunity. Their ability to exert strong immune responses via cytotoxicity and cytokine secretion makes them significant immune effectors. Since pregnancy requires unconventional maternal immunity with a tolerogenic phenotype, investigation of the possible role of NKT cells in materno-fetal immune tolerance mechanisms is of particular importance. This review aims to summarize and organize the findings of previous studies in this field. Data and information about NKT cells from mice and humans will be presented, focusing on NKT cells characteristics during normal pregnancy in the periphery and at the materno-fetal interface and their possible involvement in female reproductive failure and pregnancy complications with an immunological background.

Possible role of natural killer and natural killer T-like cells in implantation failure after IVF.

During implantation, maternal immunoactivation and tolerance are not only limited to the decidua but are also observed in the periphery, predominantly affecting the innate immune system. Since unexplained female infertility, as well as recurrent spontaneous abortion and implantation failure, are thought to be associated with pathological maternal immunotolerance mechanisms, this study focused on immune profile analysis of IVF candidates. Previous studies on peripheral natural killer (NK) cell characteristics of IVF patients have been limited to the comparison of blood samples taken prior to the IVF procedure. This study performed a follow-up study and compared patient's data obtained on the day of oocyte collection with the data 1 week after embryo transfer. The aim was to investigate phenotypic (subpopulations, CD69, T-cell immunoglobulin mucin 3 and NK-activating receptor expression) and functional (perforin and CD107a expression) changes in the peripheral NK and NK T (NKT)-like cell populations. During this short period of time around the IVF procedure, women with failed IVF reflected unfavourable Th1-oriented changes of NK and NKT-like cells. In comparison the follow-up data for women with successful conception remained principally constant. The observed peripheral changes during early pregnancy in the same individual may also have importance in successful embryo implantation.

Gamma/Delta T Cells in the Course of Healthy Human Pregnancy: Cytotoxic Potential and the Tendency of CD8 Expression Make CD56+ γδT Cells a Unique Lymphocyte Subset.

To date, pregnancy is an immunological paradox. The semi-allogenic fetus must be accepted by the maternal immune system, while defense against pathogens and immune surveillance cannot be compromised. Gamma/delta T cells are believed to play an important role in this immunological puzzle. In this study, we analyzed peripheral blood CD56+ γδT cells from pregnant women (1st, 2nd, and 3rd trimester) and non-pregnant women by multicolor flow cytometry. Interestingly, γδT cells represent almost half of CD3+/CD56+ cells. Among γδT cells, the CD56+ population expands in the 2nd and 3rd trimester. CD56+ γδT cells maintained a predominantly CD4-/CD8- or CD8+ phenotype, while CD56- γδT cells were in similar rates CD4-/CD8- or CD4+ during pregnancy. Investigation of the lysosomal degranulation marker CD107a revealed a preserved elevated rate of potentially cytotoxic CD56+ γδT cells in pregnancy, while their cytotoxic strength was reduced. Furthermore, CD56+ γδT cells continuously showed a higher prevalence of PD-1 expression. CD56+ γδT cells' rate of PD-1 increased in the 1st trimester and decreased hereafter back to normal level. We correlated the cytotoxic potential and the expression of the inhibitory immune checkpoint PD-1 and were able to demonstrate that highly cytotoxic cells within this CD56+ γδT population tend to express PD-1, which might allow the inhibition of these cells after binding its ligand in the placenta. These findings should support the understanding of the complex processes, which ensure the maintenance of pregnancy.

TIM-3 and TIM-1 Could Regulate Decidual γδTCR Bright T Cells during Murine Pregnancy.

Pregnancy is an immunological enigma where paternal antigens are present at the fetomaternal interface. What regulates the local immunotolerance, which is necessary to prevent rejection of the conceptus, is still under strong investigation. Gamma/delta T cells are believed to play a role in the local regulation of this immunotolerance towards the semiallogenic fetus. Gamma/delta T cells from the uterus and spleen of pregnant and nonpregnant mice were analyzed by flow cytometry. We confirmed that the rate of γδT cells in the decidua increases during murine pregnancy and half of decidual γδT cells are CD4+. Furthermore, we found a unique association of CD4 or CD8 coreceptor expression with their γδTCR intensity, where in all investigated groups CD4- or CD8-positive γδT cells seemed principally to be γδTCRdim. In addition, compared to peripheral γδT lymphocytes, a greater proportion of decidual γδT cells expressed the cytotoxic marker CD107a and markers of Th1 or Th2 polarization (TIM-3, TIM-1), where decidual γδTCRbright cells were characterized by high TIM-3 and TIM-1 receptor expression. On the other hand, no difference in the expression of CD160, a receptor with dual function affecting cytotoxicity and T cell inhibition, was detected. Within lymphocytes expressing CD107a, TIM-1, or CD160, the rate of γδT cells was significantly higher in the decidua. According to our results, cytotoxic potential of decidual γδTCRbright cells could be regulated by TIM-3 ligation, while the TIM-1 receptor seems to be able to influence the Th1-Th2 balance at the fetomaternal interface. These mechanisms could play a part in the active maternal immunotolerance towards the fetus, allowing an efficient protection against pathogens during healthy murine pregnancy.

Immune Checkpoint Molecules in Reproductive Immunology.

Immune checkpoint molecules, like CTLA-4, TIM-3, PD-1, are negative regulators of immune responses to avoid immune injury. Checkpoint regulators are thought to actively participate in the immune defense of infections, prevention of autoimmunity, transplantation, and tumor immune evasion. Maternal-fetal immunotolerance represents a real immunological challenge for the immune system of the mother: beside acceptance of the semiallogeneic fetus, the maternal immune system has to be prepared for immune defense mostly against infections. In this particular situation, the role of immune checkpoint molecules could be of special interest. In this review, we describe current knowledge on the role of immune checkpoint molecules in reproductive immunology.

Expansion of CD4 phenotype among CD160 receptor-expressing lymphocytes in murine pregnancy.

PROBLEM: CD160, a cell surface co-receptor, is capable of up- or downregulating cell proliferation, cytotoxicity or cytokine production on lymphocytes. Our aim was to investigate CD160+ lymphocytes in the periphery and at the maternal-foetal interface during murine pregnancy. METHOD OF STUDY: CD4+ , CD8+ and gamma/delta T-cell phenotype, TIM3 co-expression and cytotoxic activity of CD160+ lymphocytes of pregnant BALB/c mice were analysed by flow cytometry. RESULTS: The percentage of CD160+ lymphocytes in the decidua was unchanged compared to non-pregnant endometrium; however, the ratio of CD4+ cells within the CD160 population was significantly increased. The co-expression of TIM3 co-inhibitory molecule and cytotoxicity of CD160+ cells were increased in the decidua. CONCLUSION: The expansion of CD4-expressing CD160+ decidual lymphocytes is a new observation suggesting a potential regulatory role of T-cell function during mouse pregnancy. The altered immunological character of CD160+ lymphocytes could play a role in the maintenance of murine pregnancy.

Characteristics of peripheral blood NK and NKT-like cells in euthyroid and subclinical hypothyroid women with thyroid autoimmunity experiencing reproductive failure.

Thyroid autoimmunity (TAI) appears to play a crucial role in female infertility, recurrent pregnancy loss and IVF failure. Thyroid autoantibodies against thyroid peroxidase and thyroglobulin have been shown to represent an independent risk factor for infertility and miscarriage. Moreover, thyroxin hormone administration reduces the risk of obstetrical complications in TAI positive women. The aim of our present study was to investigate the immunological background of female infertility and recurrent pregnancy loss in euthyroid and subclinical hypothyroid women with TAI focusing on innate immunity. Phenotypic and functional analysis was carried out on peripheral blood mononuclear cells from healthy donors and TAI patients by flow cytometry. Our findings show Th1 oriented changes of innate immunity in the peripheral blood of women suffering from thyroid autoimmunity. Elevated NK and NKT-like cells ratios and enhanced natural cytotoxicity of TAI positive women reveal an altered immune status with possible negative impact on pregnancy outcome. It is important to notice that immune alterations are already established in the euthyroid phase of autoimmune thyroiditis before endocrine dysfunction develops and only the presence of thyroid autoantibodies indicate TAI condition. For this reason, screening of healthy women of reproductive age for the presence of thyroid autoantibodies would be beneficial not only from the endocrinological aspect but from the reproductive point of view since, although yet unexplained, thyroid hormone administration may improve pregnancy outcome.

HLA-G, pre-eclampsia, immunity and vascular events.

Pre-eclampsia, one of the main complications in pregnancy, is characterised by shallow cytotrophoblast invasion of decidua as well as by vascular endothelial cell dysfunction, leading to a poor perfusion of placenta. A striking feature of pre-eclamptic pregnancies is that expression of HLA-G protein is reduced in term placentas compared with normal pregnancy. How such HLA-G deficient expression may be related to the pre-eclamptic pathology is unknown. Here, we review the major structural characteristics of HLA-G and some of its functions that have been recently characterised. Soluble HLA-G1 isoform down-regulates both CD8(+) and CD4(+) T cell reactivity. HLA-G also modulates innate immunity by binding to several NK and/or decidual receptors, inducing particular cytokine secretion. HLA-G was shown to be less susceptible to human cytomegalovirus-derived US protein down-modulation. Finally, soluble HLA-G1 down-regulates endothelial cell proliferation and migration. In view of these different HLA-G properties, we will briefly discuss how defective HLA-G function may contribute to the low trophoblast invasion and vascular abnormalities observed in pre-eclamptic placentas.

Cell death mechanisms and potentially cytotoxic natural immune cells in human pregnancies complicated by preeclampsia.

Preeclampsia, which occurs in about 2% to 3% of all pregnancies, is a severe multisystem disorder showing symptoms in the second half of human pregnancy. Its prevention, early diagnosis, and treatment are insufficient, since etiology and pathogenesis of the disease are still not totally understood. Recent studies confirm that preeclampsia is the extreme end of a normal inflammatory reaction, which also occurs in healthy pregnancies. This review focuses on maternal immune changes during preeclampsia leading to altered cytotoxic responses. The potential role of perforin/granzyme-, Fas/Fas-ligand-, tumor necrosis factor α (TNF-α)- or TNF-related apoptosis-inducing ligand-mediated apoptotic mechanisms in the pathomechanism is analyzed. The frequency and function of effector cytotoxic cells of natural immunity itself such as natural killer (NK) cells, NKT cells, and γδT cells are also changed both in the periphery and locally in the uterus influencing the outcome of pregnancy. Here, authors conclude that beside exaggerated inflammatory responses, apoptotic and killing mechanisms also seem to be implicated in the pathogenesis of preeclampsia.

Involvement of Galectin-9/TIM-3 pathway in the systemic inflammatory response in early-onset preeclampsia.

BACKGROUND: Preeclampsia is a common obstetrical disease affecting 3-5% of pregnancies and representing one of the leading causes of both maternal and fetal mortality. Maternal symptoms occur as an excessive systemic inflammatory reaction in response to the placental factors released by the oxidatively stressed and functional impaired placenta. The T-cell immunoglobulin domain and mucin domain (TIM) family is a relatively newly described group of molecules with a conserved structure and important immunological functions. Identification of Galectin-9 as a ligand for TIM-3 has established the Galectin-9/TIM-3 pathway as an important regulator of Th1 immunity and tolerance induction. METHODS: The aim of our study was to investigate the expression and function of Galectin-9 and TIM-3 molecules by peripheral blood mononuclear cells and the possible role of Galectin-9/TIM-3 pathway in the immunoregulation of healthy pregnancy and early-onset preeclampsia. We determined TIM-3 and Gal-9 expression and cytotoxicicty of peripheral lymphocytes of early-onset preeclamptic women and healthy pregnant woman using flow cytometry. RESULTS: Investigating peripheral lymphocytes of women with early-onset preeclampsia, our results showed a decreased TIM-3 expression by T cells, cytotoxic T cells, NK cells and CD56(dim) NK cells compared to healthy pregnant women. Interestingly, we found a notably increased frequency of Galectin-9 positive cells in each investigated lymphocyte population in the case of early-onset preeclamptic patients. We further demonstrated increased cytotoxic activity by cytotoxic T and CD56(dim) NK cells in women with early-onset preeclampsia. Our findings showed that the strongest cellular cytotoxic response of lymphocytes occurred in the TIM-3 positive subpopulations of different lymphocytes subsets in early-onset preeclampsia. CONCLUSION: These data suggest that Gal-9/TIM-3 pathway could play an important role in the immune regulation during pregnancy and the altered Galectin-9 and TIM-3 expression could result an enhanced systemic inflammatory response including the activation of Th1 lymphocytes in preeclampsia.

Commitment of decidual haematopoietic progenitor cells in first trimester pregnancy.

PROBLEM The aim of this study was to investigate the phenotype and commitment of decidual haematopoietic progenitor cells (HPCs) in healthy pregnant women and in women with early miscarriage. METHOD OF STUDY Peripheral blood and decidual tissue from healthy and pathological pregnant women were examined for HPCs and lymphoid progenitors using flow cytometric analysis. RESULTS Compared with peripheral blood, we found a significant increase in decidual HPCs in both healthy pregnant women and women with spontaneous abortion. T/NK, natural killer (NK), gamma-delta and NKT cell progenitors were identified in all peripheral blood and decidual samples. In pathologic pregnant women, the ratios of decidual T/NK and NK cell progenitors were significantly increased compared with healthy pregnant controls. CONCLUSION We demonstrated decidual cells with haematopoietic progenitor cell phenotype in human decidua. Increased levels of NK progenitors in the decidua of women with early spontaneous abortion suggest a dysregulation of this pathway that may contribute to pregnancy failure.

Expression profiles of peripheral CD160+ lymphocytes during the course of healthy human pregnancy.

PROBLEM: CD160 receptor is expressed by natural killer (NK) and T-cell subsets, and after activation, it could enhance cytotoxicity or pro-inflammatory cytokine production on NK cells. Here, we investigated the phenotype of peripheral CD160+ cells during healthy pregnancy. METHOD OF STUDY: We analyzed the expression of CD69 activation marker, gamma/delta TCR, and NKG2A or NKG2D NK cell receptors on CD160+ lymphocytes of non-pregnant and healthy pregnant women at four different stages of pregnancy by flow cytometry. RESULTS: In our hands, CD160 receptor-positive lymphocytes were present during pregnancy; however, they had different characteristics depending on gestational age. During implantation, CD160+ cells showed low activation rate, decreased NK receptor expression while 40% of Vδ2 + T cells expressed CD160 receptor. In turn, all the above parameters increased as pregnancy proceeds. CONCLUSION: Our results indicate that CD160+ lymphocytes could be able to play a role in the maintenance of healthy pregnancy.

Immunoactivation in preeclampsia: Vdelta2+ and regulatory T cells during the inflammatory stage of disease.

Recent data suggest a dominant role of the innate, rather than the adaptive immune system in pregnancy-related immunoregulation. gamma/delta T cells, that comprise a minor subpopulation of human peripheral blood lymphocytes, represent a link between the innate and the acquired immune systems. However little is known about how they function in preeclampsia, which is suggested to be associated with a Th1 predominant immune response. The aim of our study was to investigate the presence and phenotype of Vdelta2+ cells and of regulatory T cells in the pathogenesis of preeclampsia. Since Vdelta2+ T cell function has been shown to be altered in patients with preeclampsia we investigated the expression of perforin, Fas and TIM-3 by Vdelta2+ T cells and the possible role of activating and inhibitory NK cell receptors as well as of regulatory T cells. Vdelta2+ T cells of preeclamptic patients demonstrated an increased perforin and IFNgamma production, which could be explained by dysregulation of NK cell receptor expression. These Th1 polarized cells were less susceptible to apoptosis than Vdelta2+ T cells from healthy pregnant women. Our data suggest that activated Vdelta2+ T cells of preeclamptic women have an increased cytotoxic potential, which may be due to altered expression of NK cell inhibitory and activating receptors. In this study we report a series of observations, which taken together suggest the role of multiple pathways in generating an exaggerated systemic inflammatory response observed in the clinical stage of preeclampsia.

V-chain preference of gamma/delta T-cell receptors in peripheral blood during term labor.

PROBLEM: An altered function of the maternal immune system creates a favorable environment for the developing fetus during pregnancy. At term, new regulatory mechanisms are activated, to initiate labor. Earlier we showed that in peripheral blood of pregnant women gamma/delta T cells of cytotoxic phenotype are replaced by those of a non-cytotoxic phenotype. Here we studied the Vgamma and Vdelta chain usage of peripheral gamma/delta T cells from women in labor. METHOD OF STUDY: Vgamma and Vdelta chain expression on peripheral blood lymphocytes obtained at the 3rd trimester of pregnancy and during parturition were examined by immuncytochemistry and flow cytometry. RESULTS: Increased % of Vgamma9/Vdelta2 and decreased % of Vgamma4/Vdelta1 T cells were found in peripheral blood during labor, together with unaltered percentages of single Vgamma+ or Vdelta+ cells. The initially high Vgamma4/Vdelta1 to Vgamma9/Vdelta2 ratio decreased during labor. CONCLUSION: The initiation of labor is characterized by an altered V-chain usage of gamma/delta T cells.

The role of invariant NKT cells in pre-eclampsia.

PROBLEM: Recent data suggest a dominant role of the innate, rather than the adaptive immune system in pregnancy-related immunoregulation. Invariant NKT (iNKT) cells represent a link between the innate and the acquired immune systems; however, little is known about how they function in pre-eclampsia. The aim of our study was to investigate the possible role of iNKT cells in the pathogenesis of pre-eclampsia. METHOD OF STUDY: Human peripheral blood samples were obtained from pre-eclamptic, healthy pregnant- and non-pregnant women. Freshly separated peripheral blood mononuclear cells were immediately labeled with anti-perforin-, anti-CD69-, anti-CD95-, anti-NKG2A-, anti-NKG2D-, anti-IFN-gamma and anti iNKT antibodies and analyzed by flow cytometry. RESULTS: Pre-eclamptic patients demonstrated increased CD69, perforin and IFN-gamma expression, which could be explained by dysregulation of NK cell receptor expression. These Th1 polarized cells were less susceptible to apoptosis than iNKT cells from healthy pregnant women. CONCLUSION: Our data suggest that activated iNKT cells of pre-eclamptic women have an increased cytotoxic potential, which may be because of altered expression of NK cell inhibitory and activating receptors.

CD160-activating NK cell effector functions depend on the phosphatidylinositol 3-kinase recruitment.

CD160 NK cell-activating receptor is a glycosyl-phosphatidylinositol-anchored molecule that, upon specific engagement, triggers both cytotoxicity and a unique cytokine production [IFN-gamma, tumor necrosis factor-alpha (TNF-alpha) and IL-6] through an undefined signaling pathway. In the current study, we have identified several signaling molecules recruited after mAb-specific CD160 engagement in freshly isolated human circulating NK cells. Using confocal microscopy, we found that CD160 engagement induces the recruitment and co-localization of phosphorylated molecules with redistributed, capped CD160 at the cell surface. We then demonstrated that phosphatidylinositol 3-kinase (PI3K) signaling molecule is required for CD160-mediated cytotoxicity and cytokine release. First, we observed by confocal microscopy that engagement of CD160 induces its polarization and co-localization with PI3K. Second, we showed that pharmacological inhibitors of PI3K abrogate both CD160-mediated cytotoxicity and IFN-gamma, TNF-alpha and IL-6 cytokine release. We further found that CD160 engagement induced marked phosphorylation of Akt, as evidenced by western blotting. We identified additional CD160-mediated signaling molecules recruited downstream and upstream of PI3K. Both induction of phosphorylated ERK molecules after CD160-specific engagement and prevention of CD160-induced cytokine release by MEK pharmacological inhibitor indicate that ERK downstream pathway is implicated. Similarly, we identified that Syk molecule upstream of PI3K is involved in the signaling cascade mediated by CD160 engagement. Two different Syk-specific inhibitors blocked CD160-mediated cytokine release, and CD160-specific engagement induced the enhancement of phosphorylated Syk proteins. These data demonstrate that PI3K is a crucial signaling element for both effector functions of the CD160 NK cell-activating receptor.

Recognition of nonclassical HLA class I antigens by gamma delta T cells during pregnancy.

The healthy trophoblast does not express classical HLA-A and HLA-B products; therefore, an MHC-restricted recognition of trophoblast-presented Ags is unlikely. In the decidua and also in peripheral blood of healthy pregnant women, gammadelta T cells significantly increase in number. We investigated the possible role of gammadelta T cells in recognition of trophoblast-presented Ags. PBL and isolated gammadelta T cells from healthy pregnant women as well as from those at risk for premature pregnancy termination were conjugated to choriocarcinoma cells (JAR) transfected with nonclassical HLA Ags (HLA-E, HLA-G). To investigate the involvement of killer-inhibitory/killer-activatory receptors in trophoblast recognition, we tested the effect of CD94 block on cytotoxic activity of Vdelta2(+) enriched gammadelta T cells to HLA-E- and/or HLA-G-transfected targets. Lymphocytes from healthy pregnant women preferentially recognized HLA(-) choriocarcinoma cells, whereas those from pathologically pregnant patients did not discriminate between HLA(+) and HLA(-) cells. Normal pregnancy Vdelta2(+) T cells conjugated at a significantly increased rate to HLA-E transfectants, whereas Vdelta2(+) lymphocytes from pathologically pregnant women did not show a difference between those and HLA(-) cells. Blocking of the CD94 molecule of Vdelta2(+) lymphocytes from healthy pregnant women resulted in an increased cytotoxic activity to HLA-E-transfected target cells. These data indicate that Vdelta2(+) lymphocytes of healthy pregnant women recognize HLA-E on the trophoblast, whereas Vdelta1 cells react with other than HLA Ags. In contrast to Vdelta2(+) lymphocytes from healthy pregnant women, those from women with pathological pregnancies do not recognize HLA-E via their killer-inhibitory receptors and this might account for their high cytotoxic activity.