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Human semen promotes CD4+ regulatory T cell activity
Abstracts / Journal of Reproductive Immunology 94 (2012) 5–130
P 016 Preservation of human placenta is feasible and facilitates studies on the local immune regulation in normal and aberrant pregnancies A. van Lochem 1,∗ , E. van Beelen 1 , C. van der Keur 1 , G. Swings 1 , L. van Zijl 1 , F. Claas 1 , S. Scherjon 2 1
Leiden University Medical Center, Immunohematology and Blood transfusion, Leiden, Netherlands 2 St. Lucas Andreas Hospital, Obstetrics, Amsterdam, Netherlands PROBLEM: Previously, we have shown that maternal lymphocytes present in the placenta play an important role in the immunoregulation towards the semi-allogeneic fetus. Currently our focus is on the comparison of immunoregulation in pregnancies complicated by e.g. preeclampsia compared to uncomplicated pregnancies. The standard procedure is to isolate lymphocytes from the decidua (d.) basalis and d. parietalis within five hours after the delivery. However, this results in logistic problems when the delivery takes place during the night, weekends or in a different medical center. Even more difficult to collect are the placentas from women with complicated pregnancies due to the low prevalence and the often unexpected termination of the pregnancy. Therefore, the aim of this study was to investigate the possibility of preservation of the human placenta before isolating the lymphocytes for phenotypic characterization and functional analysis of their immunoregulatory properties. METHODS: Placentas were obtained from women with uncomplicated pregnancy after delivery by elective caesarean section. The tissue was divided into two equal parts: decidual lymphocytes from one part were isolated within five hours according to our standard procedure, whereas the other part was preserved in either Celsior®, a storage solution for solid organ preservation, or phosphate buffered saline (PBS) for 24 hrs after which the decidual lymphocytes were isolated. The phenotype and functional features of the isolated decidual lymphocytes were determined. The focus was on the cell subsets CD4 + CD25bright, CD4 + CD25dim and CD8 + CD28- and the activation markers HLA-DR and CD69 determined using flow cytometry. The functional features of the isolated decidual lymphocytes were assessed in mixed lymphocyte cultures (MLC) in which decidual lymphocytes were stimulated with irradiated (3000 Rad) umbilical cord blood (UCB) from the own child and 3rd party UCB. Furthermore, supernatant was collected for cytokine analysis. RESULTS: Paired analysis of the cell subsets and the activation markers showed no significant difference within the decidual lymphocytes (both d. basalis and d. parietalis) isolated within five hours and isolated after preservation for 24 hrs in Celsior® or PBS. In addition, the phenotype of the decidual lymphocytes showed no difference when isolated after 24 hrs preservation in PBS compared to 24 hrs preservation in Celsior®. The MLC showed no significant difference in proliferation or Th1:Th2 cytokines in the supernatantfor the
45
decidual lymphocytesisolated within five hours and isolated after preservation for 24 hrs in Celsior® or PBS. In addition, the proliferation and cytokine production of the decidual lymphocytes showed no difference when isolated after 24 hrs preservation in PBS compared to 24 hrs preservation in Celsior®. CONCLUSIONS: The phenotype of the cell subsets of interest did not differ when comparing lymphocytes isolated within 5 hours with preservation in either Celsior® or PBS. In addition, the functional features and cytokine production were not affected by preservation. The results obtained after preservation in Celsior® were comparable with PBS, indicating that PBS is already sufficient to make a 24 hour preservation of the placenta for immunoregulation studies possible. The ability to preserve the placenta will simplify the procedure for the isolation of decidual lymphocytes and make it easier to collect tissue from women that deliver during the night, weekends or in other hospitals and women with complicated pregnancies. doi:10.1016/j.jri.2012.03.318 P 017 Human semen promotes CD4+ regulatory T cell activity E. Balandya ∗ , W. Wieland-Alter, K. Sanders, T. Lahey Dartmouth Medical School, Program in Experimental and Molecular Medicine, Lebanon, United States PROBLEM: Semen contains immunomodulatory factors that may affect maternal immunologic tolerance of conception. Further, semen mediates the expansion of CD4+ regulatory T cells (Treg) in the murine female reproductive tract. The impact of semen on Treg in humans, however, remains unclear. We investigated the impact of human semen on Treg expression of the transcriptional factor FoxP3, proliferation, apoptosis as well as secretion of immunosuppressive cytokines TGF-1 and IL-10. METHODS: We obtained human semen from 20 healthy donors after informed consent and separated seminal plasma (SP) from spermatozoa via centrifugation. We then incubated peripheral blood mononuclear cells (PBMC) with or without 0.5% SP from individual donors for 24, 48 and 72 hours, followed by assessment of CD4+ T cell surface expression of the Treg markers CD49d and CD127 as well as CD4 + CD49d-CD127low Treg intracellular expression of the transcriptional factor FoxP3, proliferation via Ki67 expression and apoptosis via activated caspase-3 expression. We also assessed the impact of SP on Treg expression of signature immunosuppressive cytokines TGF-1 and IL-10 via intracellular cytokine staining (ICS). We acquired data via multiparameter flow cytometry and performed statistical comparisons using non-parametric Mann-Whitney U tests. RESULTS: Compared to medium controls, 24 hour incubation with SP decreased CD4+ T cell expression of CD49d (mean fluorescence intensity [MFI] of 2,282 vs. 2,724, P = 0.0043) and CD127 (MFI of 795 vs. 1,970, P = 0.0043). SP induced an increase in the number of FoxP3- CD49dCD127low Treg (3.90% vs. 1.84% P = 0.0007) but not FoxP3+
46
Abstracts / Journal of Reproductive Immunology 94 (2012) 5–130
CD49d-CD127low Treg (1.40 vs. 1.37%, P = 0.7150). This increase was most likely mediated via conversion of helper T cells into FoxP3- CD49d-CD127low Treg since among FoxP3- CD49d-CD127low Treg there was no difference between SP-treated and media controls in the percentage proliferating cells by Ki67 staining (8.56% vs. 5.53%, P = 0.3095) or the percentage of apoptotic cells by intracellular activated caspase-3 staining (1.33% vs. 1.23%, P = 0.8639). Compared to medium controls, FoxP3CD49d-CD127low Treg incubated with SP exhibited a time dependent increase in TGF-1 expression, peaking at 72 hours (44.78% vs. 3.90%, P = 0.0079). This SP-mediated increase in TGF-1 expression was not evident among FoxP3+ CD49d-CD127low Treg (0.25% vs. 0.53%, P = 0.3095), and SP did not impact expression of IL-10 in either cell population: FoxP3- CD49d-CD127low Treg (0.47% vs. 0.42%, P = 0.8413) and FoxP3+ CD49d-CD127low Treg (0.58% vs. 0.76%, P = 0.5476). Within the FoxP3+ CD49d-CD127low Treg, 24 hour incubation with SP induced an increase in the expression of the immunoregulatory transcriptional factor FoxP3 (MFI of 5,683 vs. 5,137, P = 0.0003). CONCLUSIONS: Human semen promotes the nonproliferative expansion of Treg, enhanced Treg expression of the immunosuppressive cytokine TGF-1 and induction of Treg expression of the immunoregulatory transcriptional factor FoxP3. Understanding the impact of semen on the immunoregulatory activity of Treg in the female reproductive tract could inform the design of new strategies to improve fertility success and immune protection against the sexually transmitted infections, including HIV. doi:10.1016/j.jri.2012.03.319 P 018 Villitis of unknown etiology (VUE) K. Benirschke 1,2 1
University of California (UCSD) Medical Center, Pathology, San Diego, United States 2 University, Pathology, San Diego, United States Villitis of unknown etiology (“VUE”) is a relatively common entity in the human placenta, occurring in ∼4.5%. This contrasts with villitis due to CMV infection which is also common. Plasma cells are NOT involved in VUE. We have shown conclusively that in a patient with recurrent VUE the infiltration of cells are MATERNAL T-cells and macrophages. VUE often recurs and causes IUGR. Most commonly the inflammation begins at the basal plate where the villous core is exposed to decidual nK-cells. The possibility arises that there is a mismatch of HLA-antigens that may be expressed by villous core connective tissue. What remains unknown is whether the maternal T-cells reach the fetal circulation and possibly are thus responsible for future events of autoimmune diseases. We suggest that male fetuses need to be examined for maternal T-cells that express XX-karyotypes. Myerson et al. (2006). Ped. Developm. Pathology 9:257265. doi:10.1016/j.jri.2012.03.320
P 019 Immunophenotypic profiles of peripheral blood lymphocytes in women with IVF single and twin pregnancy V. Chernyshov 1,∗ , I. Sudoma 2,3 , B. Dons‘koi 1 1
Institute of Pediatrics, Obstetrics and Gynecology, Lab. of Immunology, Kiev, Ukraine 2 Nadia Clinic for Reproductive Medicine, Kiev, Ukraine 3 National Academy for Postgraduate Education, Kiev, Ukraine INTRODUCTION: Since the 1970s, the twin birth rate has been increased worldwide and one of the main causes is the use of assisted reproductive technologies. IVF twin pregnancies related with higher risk of adverse obstetric outcome. It is important to understand feature of immune phenotype in women with IVF twin pregnancy to differentiate immune deviations in them caused by obstetrical complications or by phenomenon of twin pregnancy. OBJECTIVE: Deviations in immune phenotype of women with IVF single and twin pregnancy. PATIENTS AND METHODS: Blood samples for analysis were taken from 42 women with IVF single pregnancy (SP), 31 women with IVF twin pregnancy (TwP) and 85 women with natural single pregnancy (NSP). Differences in age (25-41 years) and g.w. (6-11 g.w.) of pregnancy between observed groups were not significant. Lymphocyte subsets, T regs (CD4 + CD25 + CD127low/neg), intracellular lymphocytes in CD4+ cells (IFN-g, IL-4, TNF, IL-10), CD56bright cells, NKT cells, expression of activating molecules (CD69, HLA-DR) were studied. Lymphocyte phenotype was detected by flow cytometry using BD Bioscience monoclonal antibodies. RESULTS: We showed that in SP granulocyte absolute count was higher than in NSP and in TwP it was higher in comparison with SP. Percentage of granulocytes was higher in TwP compared to SP. Lymphocyte percentage was lower in TwP than in SP. If to compare normal single pregnancy and IVF single pregnancy it was obtained that percentages of CD3/CD4 and Tregs in SP were higher. Percentages of CD3/HLA-DR, CD3/CD5neg, CD4/HLA-DR and intracellular in CD4: IFN-g, IL-4, TNF were lower and subsets of CD8/HLA-DR, NKT cells, CD56bright and expression of HLADR and 158a on CD56bright cells were also lower in SP. If to compare IVF twin pregnancy with IVF single pregnancy, percentages of CD3/CD5neg, CD4/CD25 and CD4/IL10 were higher in TwP. But percentages of CD3/CD62L, CD4/62L, CD4/158a were lower in TwP. CONCLUSIONS: It is possible to think that successive elevation of granulocyte counts in IVF single and twin pregnancy is a result of more early release of multiple growth factors in IVF pregnancy in general. For IVF single pregnancy in spite of higher percentage levels of T helper cells and T regs, activation of CD4 and CD8 T lymphocytes was suppressed including suppression of surface activating molecules, intracellular cytokines, KIR and NKT cells. As it is known that naïve CD4 T cells use L-selectin (CD62L) expression to facilitate immune surveillance it seems possible that insufficient expression of L selectin in T helper cells in IVF twin pregnancy reflects more complex mechanisms of immune surveillance and its intensive
P 016 Preservation of human placenta is feasible and facilitates studies on the local immune regulation in normal and aberrant pregnancies A. van Lochem 1,∗ , E. van Beelen 1 , C. van der Keur 1 , G. Swings 1 , L. van Zijl 1 , F. Claas 1 , S. Scherjon 2 1
Leiden University Medical Center, Immunohematology and Blood transfusion, Leiden, Netherlands 2 St. Lucas Andreas Hospital, Obstetrics, Amsterdam, Netherlands PROBLEM: Previously, we have shown that maternal lymphocytes present in the placenta play an important role in the immunoregulation towards the semi-allogeneic fetus. Currently our focus is on the comparison of immunoregulation in pregnancies complicated by e.g. preeclampsia compared to uncomplicated pregnancies. The standard procedure is to isolate lymphocytes from the decidua (d.) basalis and d. parietalis within five hours after the delivery. However, this results in logistic problems when the delivery takes place during the night, weekends or in a different medical center. Even more difficult to collect are the placentas from women with complicated pregnancies due to the low prevalence and the often unexpected termination of the pregnancy. Therefore, the aim of this study was to investigate the possibility of preservation of the human placenta before isolating the lymphocytes for phenotypic characterization and functional analysis of their immunoregulatory properties. METHODS: Placentas were obtained from women with uncomplicated pregnancy after delivery by elective caesarean section. The tissue was divided into two equal parts: decidual lymphocytes from one part were isolated within five hours according to our standard procedure, whereas the other part was preserved in either Celsior®, a storage solution for solid organ preservation, or phosphate buffered saline (PBS) for 24 hrs after which the decidual lymphocytes were isolated. The phenotype and functional features of the isolated decidual lymphocytes were determined. The focus was on the cell subsets CD4 + CD25bright, CD4 + CD25dim and CD8 + CD28- and the activation markers HLA-DR and CD69 determined using flow cytometry. The functional features of the isolated decidual lymphocytes were assessed in mixed lymphocyte cultures (MLC) in which decidual lymphocytes were stimulated with irradiated (3000 Rad) umbilical cord blood (UCB) from the own child and 3rd party UCB. Furthermore, supernatant was collected for cytokine analysis. RESULTS: Paired analysis of the cell subsets and the activation markers showed no significant difference within the decidual lymphocytes (both d. basalis and d. parietalis) isolated within five hours and isolated after preservation for 24 hrs in Celsior® or PBS. In addition, the phenotype of the decidual lymphocytes showed no difference when isolated after 24 hrs preservation in PBS compared to 24 hrs preservation in Celsior®. The MLC showed no significant difference in proliferation or Th1:Th2 cytokines in the supernatantfor the
45
decidual lymphocytesisolated within five hours and isolated after preservation for 24 hrs in Celsior® or PBS. In addition, the proliferation and cytokine production of the decidual lymphocytes showed no difference when isolated after 24 hrs preservation in PBS compared to 24 hrs preservation in Celsior®. CONCLUSIONS: The phenotype of the cell subsets of interest did not differ when comparing lymphocytes isolated within 5 hours with preservation in either Celsior® or PBS. In addition, the functional features and cytokine production were not affected by preservation. The results obtained after preservation in Celsior® were comparable with PBS, indicating that PBS is already sufficient to make a 24 hour preservation of the placenta for immunoregulation studies possible. The ability to preserve the placenta will simplify the procedure for the isolation of decidual lymphocytes and make it easier to collect tissue from women that deliver during the night, weekends or in other hospitals and women with complicated pregnancies. doi:10.1016/j.jri.2012.03.318 P 017 Human semen promotes CD4+ regulatory T cell activity E. Balandya ∗ , W. Wieland-Alter, K. Sanders, T. Lahey Dartmouth Medical School, Program in Experimental and Molecular Medicine, Lebanon, United States PROBLEM: Semen contains immunomodulatory factors that may affect maternal immunologic tolerance of conception. Further, semen mediates the expansion of CD4+ regulatory T cells (Treg) in the murine female reproductive tract. The impact of semen on Treg in humans, however, remains unclear. We investigated the impact of human semen on Treg expression of the transcriptional factor FoxP3, proliferation, apoptosis as well as secretion of immunosuppressive cytokines TGF-1 and IL-10. METHODS: We obtained human semen from 20 healthy donors after informed consent and separated seminal plasma (SP) from spermatozoa via centrifugation. We then incubated peripheral blood mononuclear cells (PBMC) with or without 0.5% SP from individual donors for 24, 48 and 72 hours, followed by assessment of CD4+ T cell surface expression of the Treg markers CD49d and CD127 as well as CD4 + CD49d-CD127low Treg intracellular expression of the transcriptional factor FoxP3, proliferation via Ki67 expression and apoptosis via activated caspase-3 expression. We also assessed the impact of SP on Treg expression of signature immunosuppressive cytokines TGF-1 and IL-10 via intracellular cytokine staining (ICS). We acquired data via multiparameter flow cytometry and performed statistical comparisons using non-parametric Mann-Whitney U tests. RESULTS: Compared to medium controls, 24 hour incubation with SP decreased CD4+ T cell expression of CD49d (mean fluorescence intensity [MFI] of 2,282 vs. 2,724, P = 0.0043) and CD127 (MFI of 795 vs. 1,970, P = 0.0043). SP induced an increase in the number of FoxP3- CD49dCD127low Treg (3.90% vs. 1.84% P = 0.0007) but not FoxP3+
46
Abstracts / Journal of Reproductive Immunology 94 (2012) 5–130
CD49d-CD127low Treg (1.40 vs. 1.37%, P = 0.7150). This increase was most likely mediated via conversion of helper T cells into FoxP3- CD49d-CD127low Treg since among FoxP3- CD49d-CD127low Treg there was no difference between SP-treated and media controls in the percentage proliferating cells by Ki67 staining (8.56% vs. 5.53%, P = 0.3095) or the percentage of apoptotic cells by intracellular activated caspase-3 staining (1.33% vs. 1.23%, P = 0.8639). Compared to medium controls, FoxP3CD49d-CD127low Treg incubated with SP exhibited a time dependent increase in TGF-1 expression, peaking at 72 hours (44.78% vs. 3.90%, P = 0.0079). This SP-mediated increase in TGF-1 expression was not evident among FoxP3+ CD49d-CD127low Treg (0.25% vs. 0.53%, P = 0.3095), and SP did not impact expression of IL-10 in either cell population: FoxP3- CD49d-CD127low Treg (0.47% vs. 0.42%, P = 0.8413) and FoxP3+ CD49d-CD127low Treg (0.58% vs. 0.76%, P = 0.5476). Within the FoxP3+ CD49d-CD127low Treg, 24 hour incubation with SP induced an increase in the expression of the immunoregulatory transcriptional factor FoxP3 (MFI of 5,683 vs. 5,137, P = 0.0003). CONCLUSIONS: Human semen promotes the nonproliferative expansion of Treg, enhanced Treg expression of the immunosuppressive cytokine TGF-1 and induction of Treg expression of the immunoregulatory transcriptional factor FoxP3. Understanding the impact of semen on the immunoregulatory activity of Treg in the female reproductive tract could inform the design of new strategies to improve fertility success and immune protection against the sexually transmitted infections, including HIV. doi:10.1016/j.jri.2012.03.319 P 018 Villitis of unknown etiology (VUE) K. Benirschke 1,2 1
University of California (UCSD) Medical Center, Pathology, San Diego, United States 2 University, Pathology, San Diego, United States Villitis of unknown etiology (“VUE”) is a relatively common entity in the human placenta, occurring in ∼4.5%. This contrasts with villitis due to CMV infection which is also common. Plasma cells are NOT involved in VUE. We have shown conclusively that in a patient with recurrent VUE the infiltration of cells are MATERNAL T-cells and macrophages. VUE often recurs and causes IUGR. Most commonly the inflammation begins at the basal plate where the villous core is exposed to decidual nK-cells. The possibility arises that there is a mismatch of HLA-antigens that may be expressed by villous core connective tissue. What remains unknown is whether the maternal T-cells reach the fetal circulation and possibly are thus responsible for future events of autoimmune diseases. We suggest that male fetuses need to be examined for maternal T-cells that express XX-karyotypes. Myerson et al. (2006). Ped. Developm. Pathology 9:257265. doi:10.1016/j.jri.2012.03.320
P 019 Immunophenotypic profiles of peripheral blood lymphocytes in women with IVF single and twin pregnancy V. Chernyshov 1,∗ , I. Sudoma 2,3 , B. Dons‘koi 1 1
Institute of Pediatrics, Obstetrics and Gynecology, Lab. of Immunology, Kiev, Ukraine 2 Nadia Clinic for Reproductive Medicine, Kiev, Ukraine 3 National Academy for Postgraduate Education, Kiev, Ukraine INTRODUCTION: Since the 1970s, the twin birth rate has been increased worldwide and one of the main causes is the use of assisted reproductive technologies. IVF twin pregnancies related with higher risk of adverse obstetric outcome. It is important to understand feature of immune phenotype in women with IVF twin pregnancy to differentiate immune deviations in them caused by obstetrical complications or by phenomenon of twin pregnancy. OBJECTIVE: Deviations in immune phenotype of women with IVF single and twin pregnancy. PATIENTS AND METHODS: Blood samples for analysis were taken from 42 women with IVF single pregnancy (SP), 31 women with IVF twin pregnancy (TwP) and 85 women with natural single pregnancy (NSP). Differences in age (25-41 years) and g.w. (6-11 g.w.) of pregnancy between observed groups were not significant. Lymphocyte subsets, T regs (CD4 + CD25 + CD127low/neg), intracellular lymphocytes in CD4+ cells (IFN-g, IL-4, TNF, IL-10), CD56bright cells, NKT cells, expression of activating molecules (CD69, HLA-DR) were studied. Lymphocyte phenotype was detected by flow cytometry using BD Bioscience monoclonal antibodies. RESULTS: We showed that in SP granulocyte absolute count was higher than in NSP and in TwP it was higher in comparison with SP. Percentage of granulocytes was higher in TwP compared to SP. Lymphocyte percentage was lower in TwP than in SP. If to compare normal single pregnancy and IVF single pregnancy it was obtained that percentages of CD3/CD4 and Tregs in SP were higher. Percentages of CD3/HLA-DR, CD3/CD5neg, CD4/HLA-DR and intracellular in CD4: IFN-g, IL-4, TNF were lower and subsets of CD8/HLA-DR, NKT cells, CD56bright and expression of HLADR and 158a on CD56bright cells were also lower in SP. If to compare IVF twin pregnancy with IVF single pregnancy, percentages of CD3/CD5neg, CD4/CD25 and CD4/IL10 were higher in TwP. But percentages of CD3/CD62L, CD4/62L, CD4/158a were lower in TwP. CONCLUSIONS: It is possible to think that successive elevation of granulocyte counts in IVF single and twin pregnancy is a result of more early release of multiple growth factors in IVF pregnancy in general. For IVF single pregnancy in spite of higher percentage levels of T helper cells and T regs, activation of CD4 and CD8 T lymphocytes was suppressed including suppression of surface activating molecules, intracellular cytokines, KIR and NKT cells. As it is known that naïve CD4 T cells use L-selectin (CD62L) expression to facilitate immune surveillance it seems possible that insufficient expression of L selectin in T helper cells in IVF twin pregnancy reflects more complex mechanisms of immune surveillance and its intensive