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Exosomes and the NKG2D receptor-ligand system in pregnancy and cancer: using stress for survival
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology. (Lucia Mincheva-Nilsson)
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Although not obvious at first sight, several parallels can be drawn between pregnancy andcancer. Many proliferative, invasive and immune tolerance mechanisms that supportnormal pregnancy are also exploited by malignancies to establish a nutrient supply andevade or edit the immune response of the host. The human placenta, of crucial importancefor pregnancy success, and its main cells, the trophoblast, share several features withmalignant cells such as high cell proliferation rate, lack of cell-contact inhibition andinvasiveness. Both in cancer and in pregnancy, the immune defense mechanisms,potentially threatening the survival of the tumor or the fetus, are progressively blunted oreven turned into tumor- or pregnancy-promoting players.

Amongst immune mechanisms that are meant to protect the host from cancer and can be apotential threat to the fetus, the NKG2D receptor-ligand system stands out as the mostpowerful, stress-inducible “danger detector” system that comprises the activating NK cellreceptor NKG2D and its ligands, the MIC (MHC class I Chain-related proteins A and B)and ULBP (UL-16 Binding Proteins) families. It is the major cytotoxic mechanism in thebody promoting surveillance and homeostasis. In the present thesis we investigate theNKG2D receptor-ligand system in human early normal pregnancy and in theleukemia/lymphoma cell lines Jurkat and Raji and ask the questions “How is the NKG2Dreceptor-ligand system functioning in pregnancy and tumor? How is the danger of cytotoxicattack of the fetus avoided? Why is the immunosurveillance function compromised incancer patients?”

We developed a method to isolate and culture villous trophoblast from early human normalplacenta and used it to study the NKG2D receptor-ligand system. We discovered that theNKG2D ligand families of molecules MICA/B and ULBP1-5 are constitutively expressedby the syncytiotrophoblast of the chorionic villi. Using immnunoelectron microscopy, westudied the expression of these molecules at the subcellular level and could show for thefirst time that they are preferably expressed on microvesicles in multivesicular bodies(MVB) of the late endosomal compartment and are secreted as exosomes. Exosomes arenanometer sized microvesicles of endosomal origin, produced and secreted by a great7variety of normal and tumor cells. The exosomes are packages of proteins and ribonucleicacids that function as “mail” or “messengers” between cells conveying different biologicalinformation. We isolated and studied exosomes from placental explant cultures. We foundthat they carry NKG2D ligands on their surface and are able to bind and down-regulate thecognate receptor on NK-, CD8+ and T cells. The down-regulation selectively causedimpairment of the cytotoxic response of the cells but did not affect their lytic ability asmeasured by perforin content and gene transcription. Thus, the NKG2D ligand-bearingexosomes suppress the cytotoxic activity of the cells in the vicinity of the placenta, leavingtheir cytolytic machinery intact, ready to function when the cognate receptor isrestored/recycled. These findings highlight the role of placental exosomes in the fetalmaternalimmune escape and support the view of placenta as an unique immunomodulatoryorgan.

Next, we studied the expression and exosomal release of NKG2D ligands by tumor cellsusing the leukemia cell lines Jurkat and Raji as a tumor model. We found that NKG2Dligand-bearing exosomes with similar immunosuppressive properties as placental exosomesare constitutively secreted by the tumor cells, as a mechanism to blunt the cytotoxicresponse of the immune cells and thus protect themselves from cytotoxic attack by the host.Interestingly, we found that thermal- and oxidative stress up-regulates the exosomesecretion and the amount of exosome-secreted NKG2D ligands. Our results imply thattumor therapies that cause stress-induced damage, such as thermotherapy and stripping ofoxygen supply to the tumor, might have a previously unrecognized side effect causingenhanced exosome production and secretion, which in turn suppresses the natural antitumorimmune response and thus should be taken into account when designing an optimaltherapy of cancer patients.

In conclusion, we describe a novel stress-inducible mechanism shared by placenta andtumors as an immune escape strategy. We found that placenta- and tumor-derived NKG2Dligand-bearing exosomes can suppress immune responses to promote the survival and wellbeing of the fetus or the tumor. Our work comprises an important contribution to theelucidation of the NKG2D ligand-receptor system and its mode of operation in the humanbody and opens new perspectives for designing novel therapies for infertility and cancer.

Place, publisher, year, edition, pages
Umeå: Umeå university , 2010. , 64 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1375
Keyword [en]
Exsosomes, NKG2D, MICA, MICB, ULBP, human placenta, trophoblast, syncytiotrophoblast, thermal stress, oxidative stress, leukemia, lymphoma
Research subject
Immunology
Identifiers
URN: urn:nbn:se:umu:diva-37122ISBN: 978-91-7459-072-2 (print)OAI: oai:DiVA.org:umu-37122DiVA: diva2:358014
Public defence
2010-11-12, Betula, Norrlands universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2010-10-23 Created: 2010-10-20 Last updated: 2010-10-23Bibliographically approved
List of papers
1. An efficient optimized method for isolation of villous trophoblast cells from human early pregnancy placenta suitable for functional and molecular studies
Open this publication in new window or tab >>An efficient optimized method for isolation of villous trophoblast cells from human early pregnancy placenta suitable for functional and molecular studies
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2008 (English)In: American Journal of Reproductive Immunology and Microbiology, ISSN 8755-8920, Vol. 60, no 1, 33-42 p.Article in journal (Refereed) Published
Abstract [en]

PROBLEM: The uniqueness of the human placenta cannot be replaced by animal models. In vitro studies are compulsory to elucidate the biology of human placenta and require isolation and purification of villous trophoblasts, which can be used in molecular and functional studies. Constant improvement in the isolation technique is required to obtain a high yield of pure trophoblast cells with high viability and well preserved morphology.

METHOD OF STUDY: Optimized isolation procedure for human villous trophoblasts based on mild enzymatic treatment, Percoll gradient centrifugation and additional purification step involving positive or negative immunoselection on magnetic beads is described.

RESULTS: A simple and effective isolation protocol gave a reasonably high yield of villous trophoblast cells with high purity and viability, and excellent morphology as assessed by flow cytometry and electron microscopy.

CONCLUSION: This protocol provides an efficient, optimized method for isolation and enrichment of villous trophoblast cells, suitable for phenotypic, ultrastructural, molecular and functional analyses and for establishment of primary cultures.

Keyword
Human placenta;isolation method;Percoll gradient;villous trophoblast
National Category
Immunology in the medical area Obstetrics, Gynecology and Reproductive Medicine
Identifiers
urn:nbn:se:umu:diva-21496 (URN)10.1111/j.1600-0897.2008.00588.x (DOI)18593436 (PubMedID)
Available from: 2009-04-14 Created: 2009-04-14 Last updated: 2014-04-04Bibliographically approved
2. Human placenta expresses and secretes NKG2D ligands via exosomes that down-modulate the cognate receptor expression: evidence for immunosuppressive function
Open this publication in new window or tab >>Human placenta expresses and secretes NKG2D ligands via exosomes that down-modulate the cognate receptor expression: evidence for immunosuppressive function
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2009 (English)In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 183, no 1, 340-351 p.Article in journal (Refereed) Published
Abstract [en]

During mammalian pregnancy maternal-fetal tolerance involves a number of immunosuppressive factors produced by placenta. Recently, placenta-derived exosomes have emerged as new immune regulators in the maternal immune tolerance. Exosomes are membrane nanovesicles with defined morphology, which are secreted from endosomal multivesicular bodies (MVB) upon fusion with the plasma membrane. Previously, we reported that the MHC class I chain-related (MIC) proteins A and B, human ligands of the activating NK cell receptor NKG2D, are expressed by placenta, sorted to MVB of syncytiotrophoblast and probably released via MIC-bearing exosomes. In this report, we show that the second family of human NKG2D ligands, the UL-16 binding proteins (ULBP), is also expressed by placenta. Importantly, this expression was not due to placental CMV infection. Immunoelectron microscopy disclosed that ULBP1-5 are produced and retained in MVB of the syncytiotrophoblast on microvesicles/exosomes. Using human placenta explant cultures and different assays, we demonstrate that exosomes bearing NKG2D ligands are released by human placenta. Isolated placental exosomes carried ULBP1-5 and MIC on their surface and induced down-regulation of the NKG2D receptor on NK, CD8(+), and gammadelta T cells, leading to reduction of their in vitro cytotoxicity without affecting the perforin-mediated lytic pathway. Release of placental NKG2D ligands via exosomes is an alternative mechanism for generation of bioactive soluble form of these ligands. These findings highlight a role for NKG2D ligand-bearing placental exosomes in the fetal immune escape and support the view of placenta as a unique immunosuppressive organ.

National Category
Immunology in the medical area Obstetrics, Gynecology and Reproductive Medicine
Identifiers
urn:nbn:se:umu:diva-36070 (URN)10.4049/jimmunol.0803477 (DOI)19542445 (PubMedID)
Available from: 2010-09-15 Created: 2010-09-15 Last updated: 2014-04-04Bibliographically approved
3. Thermal- and oxidative stress cause enhanced release of NKG2D ligand-bearing immunosuppressive exosomes in leukemia-lymphoma T and B cells
Open this publication in new window or tab >>Thermal- and oxidative stress cause enhanced release of NKG2D ligand-bearing immunosuppressive exosomes in leukemia-lymphoma T and B cells
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(English)Article in journal (Other academic) Submitted
Abstract [en]

Immune evasion from NK surveillance related to inadequate NK-cell function has been suggested as an explanation to the high incidence of relapse and fatal outcome of many blood malignancies. Using Jurkat and Raji cell lines as a model we show that leukemia/lymphoma T and B cells constitutively secrete exosomes carrying MICA/B, ULBP1 and 2, ligands for the activating NK-cell receptor NKG2D. Acting as a decoy, the NKG2D ligand-bearing exosomes downregulate NKG2D receptor-mediated cytotoxicity and impair NK-cell function. Furthermore, we show that thermal and oxidative stress enhances the exosome secretion generating more soluble NKG2D ligands that aggravate the impairment of the cytotoxic response. Taken together, our results might partly explain the clinically observed NK-cell dysfunction in patients suffering from leukemia/lymphoma. The adverse effect of thermal and oxidative stress enhancing the release of immunosuppressive exosomes should be considered when cytostatic and hyperthermal anti-cancer therapies are designed.

Keyword
exosomes, NKG2D, MIC/ULBP, leukemia/lymphoma, oxidative stress, thermal stress, hyperthermia
Research subject
Immunology
Identifiers
urn:nbn:se:umu:diva-37120 (URN)
Available from: 2010-10-20 Created: 2010-10-20 Last updated: 2010-10-23Bibliographically approved

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