Dokument: Characterization of human innate lymphoid cells (ILCs) in umbilical cord blood

Titel:	Characterization of human innate lymphoid cells (ILCs) in umbilical cord blood
URL für Lesezeichen:	https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=56403
URN (NBN):	urn:nbn:de:hbz:061-20210602-112300-1
Kollektion:	Dissertationen
Sprache:	Englisch
Dokumententyp:	Wissenschaftliche Abschlussarbeiten » Dissertation
Medientyp:	Text
Autor:	Bennstein, Sabrina Bianca [Autor]
Dateien:	[Dateien anzeigen] Adobe PDF [Details] 4,94 MB in einer Datei [ZIP-Datei erzeugen] Dateien vom 27.05.2021 / geändert 27.05.2021
Beitragende:	Prof. Dr. Uhrberg, Markus [Gutachter] Prof. Dr. Lang, Philipp A. [Gutachter]
Stichwörter:	innate lymphoid cells, ILCs, umbilical cord blood
Dewey Dezimal-Klassifikation:	500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie
Beschreibung:	Innate lymphoid cells (ILCs) have been described to be important players within the innate immune system able to protect mucosal barriers and maintaining tissue homeostasis. However, most insights on ILC biology and function were mostly gained by analysing tissue ILCs, while knowledge on circulating ILCs in particular neonatal ILCs is still scarce. In this thesis neonatal ILCs were first characterised by development of an 8-colour flow cytometry panel with a lineage cocktail designed to exclude unwanted cell populations within umbilical cord blood (CB). Once this panel was established, neonatal ILCs were tested for functionality and it became obvious that two ILC subsets - ILC1 and ILC3 - are unresponsive to the described specific cytokine stimulation indicating different functional properties compared to tissue ILCs. As ILC1 were the least well described ILC subset and were thought to be closely related to NK cells, research first focussed on the analysis of ILC1 in comparison to NK cells. Our data show that CB ILC1, here referred to as ILC1-like cells, express several T cell-associated genes such as CD5, CD6, CD28 as well as variable regions of the TCR alpha and beta chains (TRAV/ TRBV). Since the majority of ILC1-like cells did not exert typical ILC1-related effector functions, we hypothesized that they might be an immature cell type. Based on the observation that ILC1-like cells express certain chemokine receptors and that they show a significant reduction in frequency and total cell count with increasing gestational age, we next hypothesized that they potentially migrate into secondary lymphoid tissues for further maturation. Indeed, when subjecting single cell or bulk ILC1-like cells to conditions favouring NK cell development (seeded on the murine stromal OP9-DL1 and in the presence of cytokines), ILC1-like cells differentiated into NKG2A-KIR+ NK cells, representing an advanced stage of NK cell development. Of note, in conditions favouring T cell development no up-regulation of CD3 was observed but increased cell death was noticeable, indicating lack of T cell potential. A complex KIR repertoire with down-regulation of NKG2A was only observed within in vitro cultured ILC1-like cells, but not CD56bright NK cells cultured in parallel. ILC1-like derived NK cells were phenotypically as well as functionality similar to ex vivo CD56dim NK cells due to their intracellular expression of perforin and granzyme B, ability to mobilize cytotoxic granules, and their potential to kill HLA-I-deficient cell lines as well as kill target cells via antibody dependent cell cytotoxicity (ADCC). Hence, our data demonstrated that CB ILC1-like cells constitute a novel NK cell precursor (NKP). The next step was to analyse the transcriptomic signature of CB ILCs. A clear distinction between CB ILCs and NK or T cells were detectable, although CB ILCs -in particular ILC1-like cells- clustered more with T cells, while CB ILC3-like cells clustered more with NK cells. Interestingly, CB ILCs shared a distinct set of differential up-regulated genes compared to CD56bright NK cells: the chemokine receptor CCR4, CD28, SLAMF1, and the transcription factor (TF) ID3. Notably, we observed an unusual ID3/ID2 ratio > 1, which was only seen within CB ILCs and not seen within CB NK cells, tonsillar ILC3 or peripheral blood (PB) ILCs, taken from a published data set. Interestingly, this unique ratio was also seen within CB CD4+ T cells, but not in CB CD8+ T cells or PB CD4+/ CD8+ T cells possibly indicating a shared developmental pathway of CB ILCs and CD4+ cells. We further observed novel differentially expressed genes within each ILC subset, such as the TF ZBTB46 for ILC3-like cells. We next focused on ILC3-like cells. Although ILC3-like cells exhibited no functional capacity (except for LIF production) when stimulated with specific cytokines, we searched for alternative stimulatory pathways. We identified exclusive expression of TLR2 and TLR1 chains on ILC3-like cells, but not on other ILCs, and indeed observed a significant higher proliferation rate in ILC3-like cells stimulated with the TLR2:1 ligand Pam3CSK4 including high secretion of various cytokines indicating a vital role of ILC3-like cells in host innate immune defence pre-birth. We also focussed on the in vitro generation of ILC3, since tissue-resident ILC3 were previously described to support gastrointestinal tissue homeostasis via secretion of IL-22. Their clinical use is however currently hampered by the lack of suitable cell sources: on the one hand access to human tissue ILC3 is ethically difficult as well as logistically challenging, on the other hand circulating ILC3-like cells do not correspond functionally to their respective tissue counterparts. We thus explored the possibility to generate human ILC3 with clinically important features such as IL-22 in vitro using a novel cell culture system developed for the generation of NK cells. The system, which is based on supportive human mesenchymal stem cells (MSC) indeed led to the generation of ILC3 expressing specific ‘markers’: NKp44, CD56, and the TF Ror𝛾t as well as a very high secretion of IL-22 after specific stimulation opening possible novel avenues for the clinical use of ILC3 in suitable clinical settings, such as intestinal bowel disease. Finally, a review was written summarizing the current knowledge of human ILCs and their therapeutic potential. The insights generated within this thesis greatly contributes to a deeper understanding of human neonatal ILCs as well as NK cell development. We generated a staining panel where everyone is able to faithfully identify circulating ILCs. We further generated more insight into neonatal ILC biology, as neonatal ILCs seem to be transcriptionally unique and might share a common developmental pathway with CD4+ T cells with similar ID3/ID2 ratio > 1. Adding to this, CB ILC1-like exhibited T cell-associated molecules as well as expression of various TRAV/ TRBV genes further indicating a close relationship to T cells and possibly also thymic development. We were also the first to show transcriptional analyses on neonatal ILCs. Furthermore, we found a novel CD117- NKP, which phenotypically resembles ILC1 - now called ILC1-like cells - able to differentiate into cytotoxic NK cells with a complex KIR repertoire and able to down-regulate NKG2A. This finding indicates that an assumed linear relationship between CD56bright NK cells and CD56dim NK cells might not be likely, as CD56bright NK cells were only capable of limited up-regulation of KIR receptors and kept their NKG2A expression. Based on our data, we assume that ILC1-like cells and CD56bright NK cells both contribute to the generation of CD56dim NK cells with ILC1-like cells contributing to mature CD56dim NK cells with a broad KIR repertoire and CD56bright NK cells generating less mature NKG2A+ CD56dim NK cells with limited KIR expression.
Lizenz:	Urheberrechtsschutz
Fachbereich / Einrichtung:	Medizinische Fakultät » Institute » Institut für Transplantationsdiagnostik und Zelltherapeutika (ITZ)
Dokument erstellt am:	02.06.2021
Dateien geändert am:	02.06.2021
Promotionsantrag am:	21.01.2021
Datum der Promotion:	25.05.2021