Proportion of peripheral blood and decidual CD4(+) CD25(bright) regulatory T cells in pre-eclampsia

CD4(+) CD25(bright) regulatory T (T(reg)) cells have been identified as a principle regulator of tolerance during pregnancy. In the setting of pre-eclampsia, however, little is known about the dynamics of these cells. In the current study, we determined CD4(+) CD25(bright) T(reg) cells in the peripheral blood using flow cytometry and forkhead box P3 (FoxP3(+)) cells at the placental bed using immunohistochemical staining. Peripheral blood mononuclear cells (PBMC) of 38 pre-eclamptic cases (17 cases Japanese, 21 cases Polish), 40 normal late pregnancy subjects (20 subjects Japanese, 20 subjects Polish), and 21 non-pregnant healthy controls (10 subjects Japanese, 11 subjects Polish) were included. We found the percentage of CD25(bright) cells within the CD4(+) T cell population in PBMC was reduced significantly in both Japanese and Polish pre-eclamptic cases than in normal pregnancy subjects (P < 0.001) and non-pregnant healthy controls (P < 0.001). Also, the percentage of FoxP3(+) cells within CD3(+) T cells in the placental bed biopsy samples of pre-eclamptic cases were decreased compared to those in normal pregnancy subjects. These findings suggest that a decreased number of T(reg) cells was present in pre-eclampsia, and these changes might break the maternal tolerance to the fetus.     

Normal human pregnancy is associated with an elevation in the immune suppressive CD25+ CD4+ regulatory T-cell subset

Summary CD4+ CD25+ T regulatory cells (TReg), suppress antigen-specific immune responses and are important for allograft tolerance. During pregnancy the mother tolerates an allograft expressing paternal antigens (the fetus) requiring substantial changes in immune regulation over a programmed period of time. We analysed whether immune-suppressive TReg cells were altered during pregnancy and therefore might play a part in this tolerant state. The presence of TReg cells was assessed in the blood of 25 non-pregnant, 63 pregnant and seven postnatal healthy women by flow cytometry. We observed an increase in circulating TReg cells during early pregnancy, peaking during the second trimester and then a decline postpartum. Isolated CD25+ CD4+ cells expressed FoxP3 messenger RNA, a marker of TReg cells, and suppressed proliferative responses of autologous CD4+ CD25- T cells to allogeneic dendritic cells. These data support the concept that normal pregnancy is associated with an elevation in the number of TReg cells which may be important in maintaining materno-fetal tolerance.     

Paternal Leukocytes Selectively Increase Secretion of IL-4 in Peripheral Blood During Normal Pregnancies: Demonstrated by a Novel One-Way MLC Measuring Cytokine Secretion

PROBLEM: It has been proposed that immune responses in normal pregnancy are Th2-like, thereby protecting the fetus and placenta from being rejected. Some studies have shown Th2-deviated systemic responses to different antigens and mitogens. The aim of this study was to demonstrate the specific T cell cytokine responses directed toward paternal histocompatibility leukocyte antigen (HLA), because this is the most prominent target for rejection of the feto-placental unit. METHOD OF STUDY: A novel one-way mixed leukocyte culture (MLC) combined with the detection of cytokine secretion with a sensitive ELISPOT assay was developed. Peripheral blood from 11 pregnant women was investigated with respect to allo-reactivity toward paternal leukocytes and pooled leukocytes from unrelated blood donors. This was done at three different occasions during pregnancy and 8 weeks after delivery. Nine age-matched non-pregnant women served as controls. RESULTS: In the second and third trimesters of pregnancy significantly larger numbers of IL-4-secreting cells (Th2) were induced by paternal leukocytes as compared to unrelated leukocytes. CONCLUSIONS: The findings indicate a selective immune deviation toward Th2, which may protect the fetus from rejection and thus may be an important homeostatic mechanism in normal pregnancies.     

Expression of intracellular Th1 and Th2 cytokines in women with recurrent spontaneous abortion,implantation failures after IVF/ET or normal pregnancy

PROBLEM: We aimed to investigate absolute counts of intracellular T helper 1 (Th1) and Th2 cytokine expressing T-cell subpopulations in women with three or more recurrent spontaneous abortions (RSA), multiple implantation failures after in-vitro fertilization and embryo transfer (IVF/ET) (three or more) or during normal pregnancy. METHOD OF STUDY: Absolute cell counts and percentages of CD3+, CD3+/CD4+, and CD3+/CD8+ T-cell populations expressing intracellular cytokines [interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-4 and IL-10] was studied by four-color flow cytometry in 15 RSA and 13 implantation failure patients. Eighteen fertile non-pregnant and 47 normal pregnant women were also compared with regard to intracellular cytokine expression. RESULTS: Interleukin-10 producing CD3+/CD8+ T-cell counts were significantly lower in women with RSA (P < 0.05) and implantation failures (P < 0.05), and TNF-alpha producing CD3+/CD4+ T-cell counts were higher in women with RSA (P < 0.05) and implantation failures (P < 0.005) than those of non-pregnant fertile controls. During normal pregnancies, first trimester IL-4 expressing CD3+, CD3+/CD4+ T-cell counts (P < 0.05) and IFN-gamma expressing CD3+ T-cell counts (P < 0.05) were significantly higher than those of third trimester (P < 0.05). First trimester TNF-alpha expressing CD3+/CD8+ T-cell counts were significantly higher than those of second and third trimester women (P < 0.05). However, there are no differences in cytokine expression between non-pregnant and first trimester pregnant women. CONCLUSION: Absolute counts of IFN-gamma, IL-4, and TNF-alpha expressing T cells decrease with the progress of gestation (third trimester) during normal pregnancies. In women with implantation failures, absolute cell counts of TNF-alpha expressing CD3+/ 4- cells reflects the presence of dominant Th1 immune response. A significantly increased Th1 cytokine expression may be the underlying immune etiology for reproductive failures.     

Dendritic cells partially abrogate the regulatory activity of CD4+CD25+ T cells present in the human peripheral blood

The factors that influence the functionality of human CD4(+)CD25(+) regulatory T cells are not well understood. We sought to characterize the effects of dendritic cells (DCs) on the in vitro regulatory activity of CD4(+)CD25(+) T cells obtained from peripheral blood of healthy human donors. Flow cytometry showed that a higher proportion of CD4(+)CD25(+(High)) T cells expressed surface glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) and CTL-associated antigen 4 than CD4(+)CD25(-) or CD4(+)CD25(+(Med-low)) T cells. Intracellular Foxp3 was equivalently expressed on CD4(+)CD25(+(All)), CD4(+)CD25(+(High)), CD4(+)CD25(+(Med-low)) and CD4(+)CD25(-) T cell populations, irrespective of GITR and CTL-associated antigen 4 expression. CD4(+)CD25(+) T cells were isolated and then cultured in vitro with CD4(+)CD25(-) responder T cells and stimulated with anti-CD3 antibodies, and immature dendritic cells (iDCs), mature dendritic cells (mDCs), PBMCs or PBMCs plus anti-CD28 antibodies to provide co-stimulation. In addition, secretion of the T(h)1 cytokine IFN-gamma, IL-2 and the immunoregulatory cytokines, IL-10 and transforming growth factor (TGF)-beta, were also assessed in these cultures. We found that iDCs and mDCs were capable of reversing the suppression of proliferation mediated by CD4(+)CD25(+) regulatory T cells. However, the reversal of suppression by DCs was not dependent upon the increase of IFN-gamma and IL-2 production or inhibition of IL-10 and/or TGF-beta production. Therefore, DCs are able to reverse the suppressive effect of regulatory T cells independent of cytokine production. These results suggest for the first time that human DCs possess unique abilities which allow them to influence the functions of regulatory T cells in order to provide fine-tuning in the regulation of T cell responses.     

Antigen-specific,CD4+CD25+ regulatory T cell clones induced in Peyer's patches

Since intestine is exposed to numerous exogenous antigens such as food and commensal bacteria, the organ bears efficient mechanisms for establishment of tolerance and induction of regulatory T cells (T(reg)). Intestinal and inducible T(reg) include T(r)1-like and T(h)3 cells whose major effector molecules are IL-10 and transforming growth factor (TGF)-beta. These antigen-specific T(reg) are expected to become clinical targets to modify the inflammatory immune response associated with allergy, autoimmune diseases and transplantation. In the present study, we characterized the antigen-specific T(reg) induced in the intestine by orally administering high-dose beta-lactoglobulin (BLG) to BALB/c mice. Seven days after feeding, only Peyer's patch (PP) cells among different organs exerted significant suppressive effect on antibody production upon in vitro BLG stimulation. This suppressive effect was also prominent in six BLG-specific CD4(+) T cell clones (OPP1-6) established from PP from mice orally administered with high doses of BLG and was partially reversed by antibodies to TGF-beta. Intravenous transfer of OPP2 efficiently suppressed BLG-specific IgG1 production in serum following immunization, indicating the role of such T(reg) in the systemic tolerance after oral administration of antigen (oral tolerance). OPP clones secrete TGF-beta, IFN-gamma and low levels of IL-10, a cytokine pattern similar to that secreted by anergic T cells. OPP clones bear a CD4(+)CD25(+) phenotype and show significantly lower proliferative response compared to T(h)0 clones. This lower response is recovered by the addition of IL-2. Thus, antigen-specific CD4(+)CD25(+) T(reg), which have characteristics of anergic cells and actively suppress antibody production are induced in PP upon oral administration of protein antigen.     

Instruction of naive CD4+ T-cell fate to T-bet expression and T helper 1 development: roles of T-cell receptor-mediated signals

Using T-cell receptor (TCR) transgenic mice, we demonstrate that TCR stimulation of naive CD4(+) T cells induces transient T-bet expression, interleukin (IL)-12 receptor beta2 up-regulation, and GATA-3 down-regulation, which leads to T helper (Th)1 differentiation even when the cells are stimulated with peptide-loaded I-A(b)-transfected Chinese hamster ovary cells in the absence of interferon-gamma (IFN-gamma) and IL-12. Sustained IFN-gamma and IL-12 stimulation augments naive T-cell differentiation into Th1 cells. Intriguingly, a significant Th1 response is observed even when T-bet(-/-) naive CD4(+) T cells are stimulated through TCR in the absence of IFN-gamma or IL-12. Stimulation of naive CD4(+) T cells in the absence of IFN-gamma or IL-12 with altered peptide ligand, whose avidity to the TCR is lower than that of original peptide, fails to up-regulate transient T-bet expression, sustains GATA-3 expression, and induces differentiation into Th2 cells. These results support the notion that direct interaction between TCR and peptide-loaded antigen-presenting cells, even in the absence of T-bet expression and costimulatory signals, primarily determine the fate of naive CD4(+) T cells to Th1 cells.     

Expansion of regulatory CD8+ CD25+ T cells after neonatal alloimmunization

Transplantation tolerance induced by neonatal injection of semi-allogeneic spleen cells is associated with a pathological syndrome caused by T helper type 2 (Th2) differentiation of donor-specific CD4(+) T lymphocytes. We have shown previously that this Th2-biased response is inhibited by host CD8(+) T cells. Herein, we demonstrate that upon neonatal immunization with (A/J × BALB/c)F(1) spleen cells, BALB/c mice expand a population of CD8(+) T cells expressing both CD25 and forkhead box P3 (FoxP3) markers. In this setting, CD8(+) CD25(+) T cells predominantly produce interferon (IFN)-γ and interleukin (IL)-10 and are efficient in controlling IL-4, IL-5 and IL-13 production by donor-specific CD4(+) T cells in vitro. CD8(+) FoxP3(-) T cells are single producers of IFN-γ or IL-10, whereas CD8(+) FoxP3(+) T cells are double producers of IFN-γ and IL-10. We further demonstrate that IFN-γ and IL-10 are two major cytokines produced by CD8(+) T cells involved in the in vivo regulation of Th2-type pathology. In this setting, we conclude that neonatal alloimmunization induces the expansion of several regulatory CD8(+) T cells which may control Th2 activities via IFN-γ and IL-10.     

Activated CD4+ CD25+ T cells suppress antigen-specific CD4+ and CD8+ T cells but induce a suppressive phenotype only in CD4+ T cells

CD4(+) CD25(+) regulatory T cells are increasingly recognized as central players in the regulation of immune responses. In vitro studies have mostly employed allogeneic or polyclonal responses to monitor suppression. Little is known about the ability of CD4(+) CD25(+) regulatory T cells to suppress antigen-specific immune responses in humans. It has been previously shown that CD4(+) CD25(+) regulatory T cells anergize CD4(+) T cells and turn them into suppressor T cells. In the present study we demonstrate for the first time in humans that CD4(+) CD25(+) T cells are able to inhibit the proliferation and cytokine production of antigen specific CD4(+) and CD8(+) T cells. This suppression only occurs when CD4(+) CD25(+) T cells are preactivated. Furthermore, we could demonstrate that CD4(+) T-cell clones stop secreting interferon-gamma (IFN-gamma), start to produce interleukin-10 and transforming growth factor-beta after coculture with preactivated CD4(+) CD25(+) T cells and become suppressive themselves. Surprisingly preactivated CD4(+) CD25(+) T cells affect CD8(+) T cells differently, leading to reduced proliferation and reduced production of IFN-gamma. This effect is sustained and cannot be reverted by exogenous interleukin-2. Yet CD8(+) T cells, unlike CD4(+) T cells do not start to produce immunoregulatory cytokines and do not become suppressive after coculture with CD4(+) CD25(+) T cells.     

Mature dendritic cells differentiated in the presence of interferon-beta and interleukin-3 prime functional antigen-specific CD8 T cells

Dendritic cell (DC)-based immunization represents a promising approach for the immunotherapy of cancer. The optimal conditions required to prepare DCs remain to be defined. Monocytes incubated in the presence of interferon (IFN)-beta and interleukin (IL)-3 give rise to a distinct type of DCs (IFN-beta/IL-3 DCs) that are particularly efficient at eliciting IFN-gamma and IL-5 production by allogeneic helper T cells. We assessed the capacity of this new type of DCs to prime antigen-specific naive CD8(+) T cells and compared them to the conventional DCs differentiated in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-4 (GM-CSF/IL-4 DCs). We demonstrate that IFN-beta/IL-3 DCs matured by TLR3 or CD40 ligation efficiently prime Melan-A(26-35)-specific CD8(+) T cells in vitro, at a similar level as GM-CSF/IL-4 DCs. Activated antigen-specific CD8(+) T cells produced IFN-gamma and displayed potent cytotoxic activity against peptide-pulsed target cells. Expansion of CD8(+) T cell numbers was generally higher following priming with CD40-L than with polyinosinic-polycytidylic acid (poly I:C) matured DCs. Cytolytic activity was induced by both maturing agents. These data indicate that IFN-beta/IL-3 DCs represent a promising cell population for the immunotherapy of cancer.     

Simple enumerations of peripheral blood natural killer (CD56+ NK) cells, B cells and T cells have no predictive value in IVF treatment outcome

BACKGROUND: To evaluate the association between the absolute counts of the peripheral natural killer (NK) cells (including total CD56(+) NK cells, CD56(dim) NK cells and CD56(bright) NK cells), B cells and T cells on the implantation rate and miscarriage rate after IVF treatment. METHODS: This was a prospective observation study. A total of 138 patients who underwent IVF treatment from December 2002 to July 2003 were recruited to the study. Blood samples were obtained on the day of vaginal oocyte retrieval prior to the procedure. The absolute counts of lymphocytes, NK cells, B cells and T cells were identified by flow cytometry. These absolute counts and their relationships to IVF treatment outcome and miscarriage rate were analysed. RESULTS: There were no significant differences with regard the mean values of absolute lymphocyte count, T cell count, B cell count and NK cell count (including total CD56(+) NK, CD56(dim) NK and CD56(bright) NK cells) between the pregnant and non-pregnant groups and also between the ongoing pregnancy and miscarriage groups. The cause of infertility, duration of infertility, basal FSH levels, number of previous failed IVF treatments, number of previous miscarriages and stimulation characteristics were not significantly different between the pregnant and non-pregnant groups. Previous studies have suggested that women with a history of recurrent miscarriage and those with infertility accompanied by recurrent failed IVF treatments are associated with a peripheral blood NK cell percentage >12%, therefore further analysis of peripheral CD56(+) NK cell levels <12% (group A) and >12% (group B) was performed. There was no significant difference in implantation rate (group A: 17.0%; group B: 23.2%), pregnancy rate (group A: 36.6%; group B: 47.7%) or miscarriage rate (group A: 23.3%; group B: 28.6%). CONCLUSION: There were no significant differences between simple enumerations of peripheral blood NK cells (including total CD56(+) NK, CD56(dim) NK and CD56(bright) NK cells), B cells and T cells with IVF treatment outcome and pregnancy outcome. Women who had a peripheral NK cell level >12% did not have higher number of previous pregnancy losses. Importantly their pregnancy rate was not reduced and their miscarriages were not increased compared to women who had a peripheral NK cells level <12%.     

Increased interferon-γ- and interleukin-4-synthesizing subsets of circulating T lymphocytes in pregnant asthmatics

BACKGROUND: Pregnancy frequently interferes with the course of bronchial asthma, and asthmatic pregnant women experience less successful pregnancies. T lymphocytes synthesizing IL-4 or IFN-gamma are important in allergic mechanisms of the airways as well as in materno-fetal immunity. OBJECTIVE: We hypothesized that pregnancy (a T helper-2 polarized state) of asthmatics will enhance the number of circulating T2 lymphocytes, but decrease the subset-producing IFN-gamma (T1 lymphocytes) and thereby cause a culminating T2 dominance with possible clinical consequences. METHODS: IL-4- or IFN-gamma-producing T lymphocytes were determined by flow cytometry in healthy (n=8) and asthmatic (n=13) non-pregnant women and healthy (n=18) and asthmatic (n=48) pregnant women of similar chronological and gestational (2nd-3rd trimester) age and asthma severity (Global Initiative for Asthma II-III). RESULTS: In the blood of non-pregnant women--healthy or asthmatic--the numbers of IL-4- and IFN-gamma+ T cells were very low (<10/microL blood). In contrast, in asthmatic pregnant women, the cell counts were 182+/-27 and 39+/-6 for IFN-gamma+ and IL-4+ T cells/microL blood, respectively (both P<0.05 vs. respective control values of non-pregnant asthmatics). Within the asthmatic pregnant group, significant negative correlations were revealed between the numbers of IFN-gamma+ or IL-4+ T cells and maternal peak expiratory flow as well as birth weight of newborns (both P<0.05). CONCLUSION: These data show a previously unknown immunological interference between asthma and pregnancy. The culminating proliferation of IFN-gamma+ and IL-4+ T lymphocytes may potentially impair maternal airway symptoms as well as fetal development.     

Reduced levels of both circulating CD4+ CD25+ CD127(low/neg) and CD4+ CD8(neg) invariant natural killer regulatory T cells in stable heart transplant recipients

A cross-regulation between two regulatory T cell (T(reg) ) subsets [CD4(+) CD25(+) and invariant natural killer (NK) T - iNK T] has been described to be important for allograft tolerance induction. However, few studies have evaluated these cellular subsets in stable recipients as correlates of favourable clinical outcome after heart transplantation. T(reg) and iNK T cell levels were assayed by flow cytometry in peripheral blood samples from 44 heart transplant recipients at a 2-year interval in 38 patients, and related to clinical outcome. Multi-parameter flow cytometry used CD4/CD25/CD127 labelling to best identify T(reg) , and a standard CD3/CD4/CD8/Vα24/Vβ11 labelling strategy to appreciate the proportions of iNK T cells. Both subtypes of potentially tolerogenic cells were found to be decreased in stable heart transplant recipients, with similar or further decreased levels after 2 years. Interestingly, the patient who presented with several rejection-suggesting incidents over this period displayed a greater than twofold increase of both cell subsets. These results suggest that CD4(+) CD25(+) CD127(low/neg) T(reg) and iNK T cells could be involved in the local control of organ rejection, by modulating immune responses in situ, in clinically stable patients. The measurement of these cell subsets in peripheral blood could be useful for non-invasive monitoring of heart transplant recipients, especially in the growing context of tolerance-induction trials.     

妊娠期调节性T细胞介导保护性效应需要同种抗原的刺激

越来越多的证据表明,人和小鼠妊娠期CD4+CD25+调节性T细胞(Treg)参与介导母胎免疫耐受。然而,目前对于妊娠期间调控Treg变化的因素还不清楚,对同基因交配(BALB/c×BALB/c)和异基因交配(BALB/c×C57)小鼠以及妊娠妇女CD4+CD25+Treg进行了分析,以探讨雌、孕激素和胎儿同种异基因抗原对Treg的影响。发现异基因孕鼠外周淋巴器官CD4+CD25+T细胞于早孕、中孕期明显高于非孕对照,而在同基因孕鼠只有少数个例升高;这些CD4+CD25+T细胞均表达Foxp3蛋白,在体外具有无反应性和免疫抑制能力;而且异基因孕鼠淋巴细胞对父方同种抗原的应答强度明显弱于同基因孕鼠。对去势的雌鼠给予雌二醇或孕酮,使其在血清中浓度接近中孕水平,未发现外周血CD4+CD25+Treg的明显变化。妊娠妇女CD4+CD25highTreg在早孕、中孕期显著升高,临近分娩期降至非孕水平,而此时CD4+CD25lowT细胞反而增加。以上结果表明,异基因抗原的存在对于妊娠期CD4+CD25+Treg介导对胎儿的免疫保护效应具有重要作用,而CD4+CD25+Treg的消失可能与分娩的发动有一定关系。