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Chronic hepatitis C (CHC) infection is associated with increased TIM-3, PD-1 immune checkpoint receptors expression that inhibits adaptive T cells and increases NK cell cytotoxicity against T helper cells, both resulting T cell exhaustion. Elimination of the virus with direct-acting antivirals (DAAs) may modify host immune response via altering these immune checkpoint receptors’ expression. We conducted a prospective study to analyze changes in TIM-3, PD-1 and their ligands galectin-9, PD-L1 expression by peripheral blood T cell subpopulations, NK cell subpopulations, and monocytes by multicolor flow cytometry in 14 CHC patients successfully treated with 12 weeks of dasabuvir, ombitasvir, and paritaprevir/ritonavir plus ribavirin. Blood samples were collected before, at the end of treatment, and 12 and 24 weeks later. Sustained virological response (SVR) was associated with increased percentage of peripheral blood CD3+ T and CD8+ cytotoxic T lymphocytes and decreased percentage of NKbright cells. After DAA treatment, decreased TIM-3 expression by CD4+ T cells, by NKbright, and by NKT cells was found. Expression of immune checkpoint molecules’ ligand PD-L1 by NK cells and by regulatory T cells and galectin-9 by NK cells and monocytes also decreased significantly at SVR. Our data suggest that DAA treatment not only inhibits viral replication but may alter host adaptive and innate immune responses. A decrease in immune checkpoint molecules and their ligands expression both on adaptive and on innate immune cells may contribute to the recovery of exhausted adaptive immune responses and to sustained virological response. Electronic supplementary material The online version of this article (10.1007/s10238-020-00618-3) contains supplementary material, which is available to authorized users.

NKT cell population is a relatively well-characterized immune cell subset. Numerous publications have characterized the phenotypical features of its subpopulations even in human pregnancy. Nevertheless, there have not been studies investigating the distribution of the NKT cells based on the surface presence of the CD8 receptor. Thirty-four pregnant women from the first trimester, 30 from the second, and 36 from the third trimester of pregnancy in addition to 35 healthy non-pregnant women have been involved in the study. PBMCs were isolated from peripheral blood, CD8+ and CD8- NKT cells were then studied by flow cytometry using monoclonal antibodies. Immune checkpoint molecules and intracellular markers were also measured. Substantial differences were revealed in the proportions of the NKT cell subpopulations in the healthy control cohort and the pregnant groups. By comparing the investigated groups, significant changes were detected in the expression levels of PD-L1, TIGIT, CD155, and NKG2D. Further associations were observed through examination of the relative expressions of TIGIT and CD226 in the CD8+ NKT subset. Data suggest that the CD8+ NKT cells are under fine regulation during healthy human pregnancy.

Early-onset preeclampsia is a common obstetrical disease with a potential genetic background and is characterized by the predominance of Th1 immune response. However, although many studies investigated the immunological environment in preeclamptic patients, no information is available about the potential role of the TIGIT/CD226/CD112/CD155 immune checkpoint pathway. A total of 37 pregnant women diagnosed with early-onset preeclampsia and 36 control women with appropriately matched gestational age were enrolled in this study. From venous blood, mononuclear cells were isolated and stored in the freezer. Using multicolor flow cytometry T-, NK cell and monocyte subpopulations were determined. After characterization of the immune cell subsets, TIGIT, CD226, CD112, and CD155 surface expression and intracellular granzyme B content were determined by flow cytometer. Significantly decreased CD226 expression and increased CD112 and CD155 surface expression were detected in almost all investigated T-cell, NK cell, and monocyte subpopulations in women diagnosed with preeclampsia compared to the healthy group. Furthermore, reduced TIGIT and granzyme B expression were measured only in preeclamptic CD8+ T cells compared to healthy pregnant women. A decreased level of the activatory receptor CD226 in effector lymphocytes accompanied with an elevated surface presence of the CD112 and CD155 ligands in monocytes could promote the TIGIT/CD112 and/or TIGIT/CD155 ligation, which mediates inhibitory signals. We assume that the inhibition of the immune response via this immune checkpoint pathway might contribute to compensate for the Th1 predominance during early-onset preeclampsia.

We investigated peripartum maternal red blood cell (RBC) properties in early-onset preeclampsia (PE). Repeated blood samples were taken prospectively for hemorheological measurements at PE diagnosis (n = 13) or during 26–34 weeks of gestation in healthy pregnancies (n = 24), then at delivery, and 72 h postpartum. RBC aggregation was characterized by M index (infrared light transmission between the aggregated RBCs in stasis) and aggregation index (AI—laser backscattering from the RBC aggregates). We observed significantly elevated RBC aggregation (M index = 9.8 vs. 8.5, AI = 72.9% vs. 67.5%, p &lt, 0.001) and reduced RBC deformability in PE (p &lt, 0.05). A positive linear relationship was observed between AI and gestational age at birth in PE by regression analysis (R2 = 0.554, p = 0.006). ROC analysis of AI showed an AUC of 0.84 (0.68–0.99) (p = 0.001) for PE and indicated a cutoff of 69.4% (sensitivity = 83.3%, specificity = 62.5%), while M values showed an AUC of 0.75 (0.58–0.92) (p = 0.019) and indicated a cutoff of 8.39 (sensitivity = 90.9% and specificity = 50%). The predicted probabilities from the combination of AI and M variables showed increased AUC = 0.90 (0.79–1.00) (p &lt, 0.001). Our results established impaired microcirculation in early-onset PE manifesting as deteriorated maternal RBC properties. The longer the pathologic pregnancy persists, the more pronounced the maternal erythrocyte aggregation. AI and M index could help in the prognostication of early-onset PE, but further investigations are warranted to confirm the prognostic role before the onset of symptoms.

Galectins are one of the critical players in the tumor microenvironment–tumor crosstalk and the regulation of local immunity. Galectin-9 has been in the limelight in tumor immunology. Galectin-9 possesses its multiplex biological functions both extracellularly and intracellularly, plays a pivotal role in the modulation of adaptive and innate immunity, and induces immune tolerance. NK-92MI cell lines against different malignancies were extensively studied, and recently published trials used genetically chimeric antigen receptor-transfected NK-92MI cells in tumor immunotherapy. Besides the intensive research in tumor immunotherapy, limited information is available on their immune-checkpoint expression and the impact of checkpoint ligands on their effector functions. To uncover the therapeutic potential of modulating Galectin-9-related immunological pathways in NK-cell-based therapy, we investigated the dose-dependent effect of soluble Galectin-9 on the TIM-3 checkpoint receptor and NKG2D, CD69, FasL, and perforin expression of NK-92MI cells. We also examined how their cytotoxicity and cytokine production was altered after Gal-9 treatment and in the presence of different serum supplements using flow cytometric analysis. Our study provides evidence that the Galectin-9/TIM-3 pathway plays an important role in the regulation of NK cell function, and about the modulatory role of Galectin-9 on the cytotoxicity and cytokine production of NK-92MI cells in the presence of different serum supplements. We hope that our results will aid the development of novel NK-cell-based strategies that target Galectin-9/TIM-3 checkpoint in tumors resistant to T-cell-based immunotherapy.

Background Our goal with this study was to investigate the contribution of PD-1/PD-L1 immune-checkpoint pathway to maternal immunotolerance mechanisms. Methods Thirteen healthy pregnant women and 10 non-pregnant controls were involved in this project. PBMCs and DICs were isolated from peripheral blood and from decidual tissues. After the characterization of different immune cell subsets, we used fluorochrome-conjugated monoclonal antibodies to measure the expression level of PD-1, PD-L1, NKG2D, and CD107a molecules by flow cytometry. Results We measured significant alternations in the proportion of decidual immune cell subsets compared to the periphery. Elevated PD-1 expression by decidual CD8+ T, CD4+ T, and NKT-like cells were also detected accompanied by the increased PD-L1 expression by decidual CD4+ T, Treg, NKT-like and CD56 + NK cell subsets compared to peripheral blood. The cytotoxic potential was significantly higher in PD-1- decidual immune cells compared to the periphery, however we measured a significantly lower cytotoxicity in the decidual PD-1+ CD8+ T cells compared with the peripheral subsets. An activation receptor NKG2D expression was decreased by the PD-1+ CD8+ T subsets in the first trimester compared to non-pregnant condition but the expression level of the decidual counterparts was significantly elevated compared to the periphery. The cytotoxic potential of decidual PD1/NKG2D double positive CD8+ T cells was significantly decreased compared to the peripheral subsets. Conclusions Based on our results we assume that PD-1/PD-L1 pathway might have a novel role in the maintaining of the local immunological environment. Accompanied by NKG2D activating receptor this checkpoint interaction could regulate decidual CD8 Tc cell subsets and may contribute maternal immunotolerance.

Various formats of forest bathing have been receiving increasing attention owing to their perspectives in health promotion and the treatment of chronic lifestyle diseases. The majority of field studies are still being conducted in the Far Eastern region, and they often make psychological assessments mainly in the green season. In our pretest–posttest field experiment, twelve healthy, working-age volunteers participated in a 2-h leisurely forest walking program, first in the green season (May) and then in the winter season (January), in the Mecsek Hills, next to Pécs, Hungary. Systolic blood pressure decreased after the trips both in late spring and in the winter. Based on changes in the expressions of CD69, an early activation marker, NKG2D, a major recognition receptor, perforin, granzyme B, and TIM-3, an inhibitory immune checkpoint molecule, on CD8+ cytotoxic T, NK, NKdim, NKbright, and NKT cells, we detected the stimulation of NKbright cells and activation of all examined immune cell subsets in the green season. In the winter, a slight activating and an interesting balancing effect regarding TIM-3 could be observed considering our finding that basal (pretest) TIM-3 expression by NK cells was significantly lower in the winter. Our work expands the knowledge on and potentials of forest medicine.

Background: A growing body of evidence supports the importance of PD-1 and PD-L1, especially in the materno-fetal interface, although limited information is available about the peripheral expression of these molecules during the trimesters of pregnancy. Methods: 13 healthy women were enrolled from the 1st, 10 from the 2nd and 12 from the 3rd trimester of pregnancy at the same time, 10 healthy, age-matched nonpregnant women formed the control group. From peripheral blood, mononuclear cells were separated and stored at &ndash, 80 &deg, C. From freshly thawed samples, surface and intracellular staining were performed for flow cytometric analyses. CD107a degranulation assay was used to evaluate the cytotoxicity. Results: significant alternation was detected in PD-1 expression by CD8+T cells and in PD-L1 expression by CD8+T, CD4+T and Treg cells. An interesting relationship was revealed between the PD-1 and PD-L1 expression by the investigated subpopulations in 2nd trimester of pregnancy. Different expression patterns of an activation receptor NKG2D by the PD-1+ CD8+T cells was observed during pregnancy. The notable relationship was further determined in cytotoxicity between PD-1+ and NKG2D+ CD8+T cells throughout pregnancy. Conclusions: the different PD-1 presence and the relationship with NKG2D could contribute to the dynamic changes of the Th1 and Th2 predominance throughout the three trimesters of a healthy pregnancy.

Endometriosis is a gynecological condition that is associated with chronic pelvic inflammation, pain, and infertility. Although substantial evidence supports that immunological alterations contribute to its pathogenesis and we previously posed a pivotal role of Galectin-9 (Gal-9) in this disorder, the involvement of the TIM-3/Gal-9 pathway in the development of endometriosis-associated immunological abnormalities is not yet known. In the present study, multicolor flow cytometry was used to compare the immunophenotype and cell surface expression of TIM-3 and Gal-9 molecules on peripheral blood (PB) and peritoneal fluid (PF) lymphocytes of women with and without endometriosis. We found an altered distribution of different lymphocyte subpopulations, a markedly decreased TIM-3 labeling on all T and NK subsets and a significantly increased Gal-9 positivity on peripheral CD4+ T and Treg cells of the affected cohort. Furthermore, a significantly increased TIM-3 expression on CD4+T-cells and elevated Gal-9 labeling on all T and NK subsets was also revealed in the PF of the examined patients. In conclusion, our results suggest a persistent activation and disturbed TIM-3/Gal-9-dependent regulatory function in endometriosis, which may be involved in the impaired immune surveillance mechanisms, promotes the survival of ectopic lesions, and aids the evolution of reproductive failures in endometriosis.

Problem Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide having several regulatory functions in the nervous system and in peripheral organs including those of the reproductive system. PACAP-deficient male mice have several morphological, biochemical, behavioral defects and show disturbed signaling in spermatogenesis affecting fertility in PACAP KO mice. Reproductive functions such as fertility, mating, and maternal behaviors have been widely investigated, but no immune analyses are available regarding the testicular immune-privileged environment in male PACAP-deficient mice. Method of study We performed detailed immunophenotyping of testicular immune cells and investigated the expression of TIM-3 and PD-1 Immune checkpoint molecules of immune cells together with the detection of galectin-9 and perforin. We investigated the percentage of numerous immune cell populations in the testis of wild-type and PACAP-deficient mice. Results We demonstrated a significant increase in the frequency of testicular CD8+ T cells together with the decrease in Treg cell number obtained from PACAP KO mice compared with wild-type mice. Investigating Immune checkpoint receptors, only PD-1 showed a significantly decreased expression in CD8+ T cells in PACAP KO mice compared with wild-type suggesting an impaired PD-1/PD-L1 pathway. Regarding TIM-3 expression, we did not find any significant difference between the investigated groups. Conclusion We hypothesize that these local changes may result in an immune activation with disturbed testicular immunoregulation in PACAP KO mice; however, determining the exact function requires further investigations. Our data further support the view that besides a systemic immune tolerance, localized active immunosuppression is involved in the regulation of testicular immune privilege.

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.

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.

The abortifacient Mifepristone (RU486) has proven to be a safe, effective, acceptable option for millions of women seeking abortion during the first and second trimester of pregnancy although its precise mechanism of action is not well understood. The main objective of this study was to investigate the impact of low dose Mifepristone administration on placental Galectin-9 (Gal-9) expression, as well as its effect on the cell surface expression of Gal-9, TIM-3 and CD107a molecules by different T and NK cell subsets. A model of Mifepristone-induced immunological changes was established in syngeneic pregnant BALB/c mice. RU486-induced alteration in placental Gal-9 expression was determined by immunohistochemistry. For immunophenotypic analysis, mid-pregnancy decidual lymphocytes and peripheral mononuclear cells were obtained from Mifepristone treated and control mice at the 14.5 day of gestation. TIM-3 and Gal-9 expression by peripheral and decidual immune cells were examined by flow cytometry. Our results revealed a dramatically decreased intracellular Gal-9 expression in the spongiotrophoblast layer of the haemochorial placenta in Mifepristone treated pregnant mice. Although low dose RU486 treatment did not cause considerable change in the phenotypic distribution of decidual and peripheral immune cells, it altered the Gal-9 and TIM-3 expression by different NK and T cell subsets. In addition, the treatment significantly decreased the CD107a expression by decidual TIM-3+ NK cells, but increased its expression by decidual NKT cell compared to the peripheral counterparts. These findings suggest that low dose Mifepristone administration might induce immune alterations in both progesterone dependent and independent way.

Background Lung cancer (LC) is still the most common cause of cancer related deaths worldwide. Non-small cell lung cancer (NSCLC) accounts for 85% of all LC cases but is not a single entity. It is now accepted that, apart from the characteristic driver mutations, the unique molecular signatures of adeno- (AC) and squamous cell carcinomas (SCC), the two most common NSCLC subtypes should be taken into consideration for their management. Therapeutic interventions, however, frequently lead to chemotherapy resistance highlighting the need for in-depth analysis of regulatory mechanisms of multidrug resistance to increase therapeutic efficiency. Methods Non-canonical Wnt5a and canonical Wnt7b and ABC transporter expressions were tested in primary human LC (n = 90) resections of AC and SCC. To investigate drug transporter activity, a three dimensional (3D) human lung aggregate tissue model was set up using differentiated primary human lung cell types. Following modification of the canonical, beta-catenin dependent Wnt pathway or treatment with cisplatin, drug transporter analysis was performed at mRNA, protein and functional level using qRT-PCR, immunohistochemistry, immune-fluorescent staining and transport function analysis. Results Non-canonical Wnt5a is significantly up-regulated in SCC samples making the microenvironment different from AC, where the beta-catenin dependent Wnt7b is more prominent. In primary cancer tissues ABCB1 and ABCG2 expression levels were different in the two NSCLC subtypes. Non-canonical rhWnt5a induced down-regulation of both ABCB1 and ABCG2 transporters in the primary human lung aggregate tissue model recreating the SCC-like transporter pattern. Inhibition of the beta-catenin or canonical Wnt pathway resulted in similar down-regulation of both ABC transporter expression and function. In contrast, cisplatin, the frequently used adjuvant chemotherapeutic agent, activated beta-catenin dependent signaling that lead to up-regulation of both ABCB1 and ABCG2 transporter expression and activity. Conclusions The difference in the Wnt microenvironment in AC and SCC leads to variations in ABC transporter expression. Cisplatin via induction of canonical Wnt signaling up-regulates ABCB1 and ABCG2 drug transporters that are not transporters for cisplatin itself but are transporters for drugs that are frequently used in combination therapy with cisplatin modulating drug response. Electronic supplementary material The online version of this article (doi:10.1186/s12931-017-0537-6) contains supplementary material, which is available to authorized users.

The programmed cell death protein 1 (PD-1) receptor has been reported to downregulate T cell activation effectively via binding to its ligands PD-L1 or PD-L2 in a negative co-stimulatory manner. Little is known about the involvement of PD-1 mediated immunoregulation in pregnancy and in pregnancy-related disorders. In this work, we investigated the possible role of the PD-1 co-stimulatory pathway in the pathogenesis of the clinical phase of early-onset preeclampsia characterized by a systemic maternal inflammatory response. We performed a cross-sectional study for comparative analysis of phenotypic and functional characteristics of peripheral blood mononuclear cells in women with early-onset preeclampsia and third-trimester healthy pregnant controls. According to our findings, enhanced expression of either PD-1 or its ligand PD-L1, or both, on the cell surface of effector cells (T cells, natural killer (NK) cells, natural killer T (NKT)-like cells) and Tregs could be observed, but PD-1 expression did not correlate with effector cells exhaustion. These results suggest the failure of the axis to downregulate Th1 responses, contributing thereby to the exaggerated immunoactivation observed in early-onset preeclampsia.

Background Angiogenesis is important both in normal tissue function and disease and represents a key target in lung cancer (LC) therapy. Unfortunately, the two main subtypes of non-small-cell lung cancers (NSCLC) namely, adenocarcinoma (AC) and squamous cell carcinoma (SCC) respond differently to anti-angiogenic e.g. anti-vascular endothelial growth factor (VEGF)-A treatment with life-threatening side effects, often pulmonary hemorrhage in SCC. The mechanisms behind such adverse reactions are still largely unknown, although peroxisome proliferator activator receptor (PPAR) gamma as well as Wnt-s have been named as molecular regulators of the process. As the Wnt microenvironments in NSCLC subtypes are drastically different, we hypothesized that the particularly high levels of non-canonical Wnt5a in SCC might be responsible for alterations in blood vessel growth and result in serious adverse reactions. Methods PPARgamma, VEGF-A, Wnt5a, miR-27b and miR-200b levels were determined in resected adenocarcinoma and squamous cell carcinoma samples by qRT-PCR and TaqMan microRNA assay. The role of PPARgamma in VEGF-A expression, and the role of Wnts in overall regulation was investigated using PPARgamma knock-out mice, cancer cell lines and fully human, in vitro 3 dimensional (3D), distal lung tissue aggregates. PPARgamma mRNA and protein levels were tested by qRT-PCR and immunohistochemistry, respectively. PPARgamma activity was measured by a PPRE reporter system. The tissue engineered lung tissues expressing basal level and lentivirally delivered VEGF-A were treated with recombinant Wnts, chemical Wnt pathway modifiers, and were subjected to PPARgamma agonist and antagonist treatment. Results PPARgamma down-regulation and VEGF-A up-regulation are characteristic to both AC and SCC. Increased VEGF-A levels are under direct control of PPARgamma. PPARgamma levels and activity, however, are under Wnt control. Imbalance of both canonical (in AC) and non-canonical (in SCC) Wnts leads to PPARgamma down-regulation. While canonical Wnts down-regulate PPARgamma directly, non-canonical Wnt5a increases miR27b that is known regulator of PPARgamma. Conclusion During carcinogenesis the Wnt microenvironment alters, which can downregulate PPARgamma leading to increased VEGF-A expression. Differences in the Wnt microenvironment in AC and SCC of NSCLC lead to PPARgamma decrease via mechanisms that differentially alter endothelial cell motility and branching which in turn can influence therapeutic response. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2943-4) contains supplementary material, which is available to authorized users.

INTRODUCTION: Immunoregulation implies the activation of negative pathways leading to the modulation of specific immune responses. Co-inhibitory receptors (such as PD-1 and TIM-3) represent possible tools for this purpose. PD-1 and TIM-3 have been demonstrated to be present on immune cells suggesting general involvement in immunosuppression such as fetomaternal tolerance. The aim of our study was to investigate the expression pattern of PD-1, TIM-3, and its ligand Gal-9 on different immune cell subsets in the peripheral blood and at the fetomaternal interface in pregnant mice.METHODS: TIM-3 and PD-1 expression by peripheral and decidual immune cells from pregnant BALB-c mice in 2 weeks of gestational age were measures by flow cytometry. Placental galectin-9 expression was determined by immunohistochemically and RT-PCR.RESULTS: Gal-9 was found to be present in the spongiotrophoblast layer of the haemochorial placenta. Decidual NK, NKT and γ/δ T cells showed increased PD-1 expression and reduced cytotoxic potential when compared to the periphery. TIM-3 expression by NK cells and γ/δ T cells is similar both in the periphery and in the decidua, notably, their relative TIM-3 expression is increased locally which is associated with reduced lytic activity. Decidual NKT cells exhibit a reduced TIM-3 expression with increased relative receptor expression and a slightly increased cytotoxicity when compared to the periphery.DISCUSSION: Our data reveals a particularly complex, tissue and cell type specific immunoregulatory mechanism by the investigated co-inhibitory receptors at the fetomaternal interface.

Problem: The T-cell immunoglobulin 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 negative regulator of Th1 immunity and tolerance induction. Data about the TIM-3/Gal-9 pathway in the pathogenesis of human diseases is emerging, but their possible role during human pregnancy is not precisely known. The aim of our study was to investigate the number, phenotype and functional activity of TIM-3+ peripheral blood mononuclear cells during healthy human pregnancy. Methods of Study: 57 healthy pregnant women [first trimester (n = 16); second trimester (n = 19); third trimester (n = 22)] and 30 non-pregnant controls were enrolled in the study. We measured the surface expression of TIM-3 by cytotoxic T cells, NK cells and NK cell subsets as well as Galectin-9 expression by regulatory T cells by flow cytometry. We analyzed the cytokine production and cytotoxicity of TIM3+ and TIM3- CD8 T and NK cells obtained from non-pregnant and healthy pregnant women at different stages of pregnancy by flow cytometry. Serum Galectin-9 levels were measured by ELISA. Results: Our results show that the numbers of peripheral NK and cytotoxic T cells and their TIM-3 expression do not change between the first, second and third trimesters of pregnancy. Compared to non-pregnant individuals, regulatory T cells show higher level of Galectin-9 expression as pregnancy proceeds, which is in line with the level of Galectin-9 in the patients sera. Cytotoxic T cells, NK cells and NK cell subsets expressing TIM-3 molecule show altered cytokine production and cytotoxicity during pregnancy compared to non-pregnant individuals. Conclusion: Our results indicate that Galectin-9 expressing regulatory T cells, TIM-3+ cytotoxic T cells and NK cells could play an important role in the maintenance of healthy pregnancy.

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 CD56dim 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 CD56dim 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.

To investigate killer inhibitory and activating receptor expression by natural killer (NK), natural killer T-like (NKT-like) and CD8+ T lymphocytes in patients with chronic hepatitis C virus (HCV) infection with elevated and with persistently normal alanine aminotransferase (PNALT).The percentage of peripheral blood Treg cells, KIR2DL3, ILT-2, KIR3DL1, CD160, NKG2D, NKG2C expressing NK, T and NKT-like cells, cytokine production and NK cytotoxicity were determined by flow cytometry. Twenty-one patients with chronic HCV infection with elevated alanine aminotransferase, 11 HCV carriers with persistently normal alanine aminotransferase and 15 healthy volunteers were enrolled.No significant differences were observed in the percentage of total T, NK or NKT-like cells between study groups. Comparing the activating and inhibitory receptor expression by NK cells obtained from HCV carriers with PNALT and chronic HCV hepatitis patients with elevated alanine aminotransferase, NKG2D activating receptor expression was the only receptor showing a significant difference. NKG2D expression of NK cells was significantly lower in patients with elevated alanine aminotransferase. The expression of CD160, NKG2D and NKG2C activating receptor by CD8+ T cells were significantly lower in patients with chronic HCV hepatitis than in healthy controls and in HCV carriers with PNALT. Plasma TGF-β1 levels inversely correlated with NKG2D expression by NK cells. In vitroTGF-β1 treatment inhibited NK cells cytotoxic activity and downregulated NKG2D expression. CD8+ T cells from HCV carriers with PNALT showed significantly elevated expression of CD160, NKG2D and NKG2C activating receptors compared to chronic HCV patients with elevated alanine aminotransferase. Enhanced expression of inhibitory KIR2DL3 receptor, and decreased ILT-2 expression on NK cells were also found in chronic hepatitis C patients compared to healthy controls.Our study demonstrated a complex dysregulation of activating and inhibitory receptor expression, such as decreased NKG2D and CD160 activating receptor expression and increased KIR2DL3 inhibitory receptor expression by NK and cytotoxic T cells and may provide further mechanism contributing to defective cellular immune functions in chronic hepatitis C. Increased NKG2D receptor expression in HCV patients with persistently normal ALT suggests an important pathway for sustaining NK and CD8 T cell function and a protective role against disease progression.