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«Inaugural-Dissertation zur Erlangung des Doktorgrades Dr. rer. nat. der Fakultät für Biologie an der Universität Duisburg-Essen vorgelegt von ...»

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Molecular Mechanisms of Differential Activation of

Naive T cells by Weak and Strong

Antigen-presenting Cells

Inaugural-Dissertation

zur

Erlangung des Doktorgrades

Dr. rer. nat.

der Fakultät für

Biologie

an der

Universität Duisburg-Essen

vorgelegt von

Eloho Etemire

aus Oteri-Igbide, Delta State, Nigeria.

August 2013

Die Arbeit zugrunde liegenden vorliegenden Experimente wurden durchgeführt beginnend am

Institut für Molekulare und Klinische Immunologie der Otto von Guericke Universität, Magdeburg und schloss am Institut für Expetimentelle Immunologie und Bildung der Universität Duisburg-Essen, Essen.

.

1. Gutachter: Prof. Dr. Matthias Gunzer.

2. Gutachter: Prof. Dr. Wiebke Hansen Vorsitzender des Prüfungsausschusses: Prof. Dr. Shirley Knauer.

Tag der mündlichen Prüfung: Oct 30th 2013.

Publications during thesis Professional articles Eloho Etemire*, Marco Krull*, Mike Hasenberg, Peter Reichardt# and Matthias Gunzer# Transiently reduced PI3K/Akt activity drives the development of regulatory function in antigen-stimulated naïve T cells. PLoS ONE 8(7): e68378. doi:10.1371/journal.pone.0068378 Peter Reichardt*, Irene Patzak*, Kristian Jones, Eloho Etemire, Matthias Gunzer# and Nancy Hogg#. A role for LFA-1 in delaying T lymphocyte egress from lymph nodes. The EMBO Journal (2013) 32, 829–843.

*Co-first authors #Co-Senior authors Oral talks

• Eloho Etemire, Marco Krull, Peter Reichardt and Matthias Gunzer. Early Signaling dynamics driving the generation of induced Tregs from naïve T cell pools. 43rd Annual Meeting of German Society for Immunology (DGfI). Mainz. Sept 2013

• Eloho Etemire, Marco Krull, Peter Reichardt and Matthias Gunzer. Functional Roles for Defective PI3K Signaling and differing Co-stimulatory activity of APCs in T-Cell Plasticity.

MACSQuant users’ day. Bergisch Gladbach. Nov 2012

• Eloho Etemire, Marco Krull, Peter Reichardt and Matthias Gunzer. Functional Roles for Defective PI3K Signaling and differing Co-stimulatory activity of APCs in T-Cell Plasticity.

BIOME Graduate School of Biomedical Science, Hamminkeln. Oct 2012

• Eloho Etemire, Marco Krull, Peter Reichardt and Matthias Gunzer. Molecular Mechanisms for the Differential Activation of Naive T cells by Strong and Weak Antigen Presenting Cells. SFB 854 Annual Retreat, Gommern. Dec 2011 Poster presentations

• Eloho Etemire, Marco Krull, Peter Reichardt and Matthias Gunzer. Early Signaling dynamics driving the generation of induced Tregs from naïve T cell pools. 43rd Annual Meeting of German Society for Immunology (DGfI). Mainz. Sept 2013 3

• Eloho Etemire, Marco Krull, Peter Reichardt and Matthias Gunzer. Functional Roles for Defective PI3K Signaling and differing Co-stimulatory activity of APCs in T-Cell Plasticity.

Universitaet Klinikum Essen Forschung tag, Nov 2012.

• Eloho Etemire, Marco Krull, Peter Reichardt and Matthias Gunzer. Functional Roles for Defective PI3K Signaling and differing Co-stimulatory activity of APCs in T-Cell Plasticity.

European Congress of Immunology, Glasgow. Sept 2012.

• Marco Krull, Eloho Etemire, Peter Reichardt and Matthias Gunzer. Molecular Mechanisms for the Differential Activation of Naive T cells by Strong and Weak Antigen Presenting Cells. SFB 854 Annual Retreat, Gommern. Dec 2010.

–  –  –

Publications during thesis

Table of Contents

Abbreviations

List of Figures

Abstract

1.0 Introduction

1.1 The Mammalian Immune System

1.1.1 The Innate Immune System

1.1.2 The Adaptive Immune System

B Cells

1.1.2.1 1.1.2.2 T Cells

1.1.3 Antigen Presenting Cells (APCs)

1.1.4 Dynamics of CD4+ T cell Activation

1.1.5 The Role of the PI3K/mTOR Pathway in T cell Activation and Function

1.1.6 Function of CD62-L in T cells

1.1.7 The Role of Epigenetics in T cell Differentiation and Function

1.1.8 Aim of Study

2.0 Materials and Methods

2.1 Materials

2.1.1 Chemicals

Buffers and Media

2.1.2 2.1.3 Antibodies and Cell staining

2.1.4 Kits

2.1.5 Primers

2.1.6 Cell Culture Materials and Laboratory ware

–  –  –

2.2 Methods

2.2.1 Mice

2.2.2 Lymphocyte Isolation and purification

2.2.3 Creation of Dendritic cells from mice bone marrow

2.2.4 In vitro T cell activation

2.2.5 Proliferation and Inhibitory Assays

2.2.6 Fluorescence Activated Cell Sorting (FACS)

2.2.7 Protein Detection via Immunoblot

RNA isolation and gene expression analysis

2.2.8 2.2.9 Methylation specific PCR

2.2.10 Immunocytochemistry- Fluorescence Microscopy

2.2.11 Role of adhesion molecules on the stroma of SLOs in T cell activation in vivo

2.2.12 Statistics

3.0 Results

3.1 Critical Previous Work

3.2 Rapamycin and Ly294002 regulate CD62-L in a dose dependent manner

3.3 CD62-L dynamics is independent of lipopolysaccharide (LPS) induced Toll Like receptors (TLR) activation in B cells





3.4 Readout of mTOR induced S6 phosphorylation status as a surrogate marker for PI3K activity in TofB and TofDC

3.5 Induction of Tregs using immature DC (iDC) as weak APC

3.5.1 Generation and characterization of immature dendritic cells (iDCs)

3.5.2 Immature DC triggered T cells show the same Phenotypic Characteristics as TofB............... 74 3.5.3 Functional characterisation of TofiDC and delineation of the involvement of the PI3K and mTOR pathways in acquisition of regulatory function

3.6 Sub-optimal co-stimulation drives acquisition of regulatory function but exerts negligible influence on CD62-L dynamics

3.7 Phospho-site specific defective Akt signaling observed following T-cell triggering by weak APCs

–  –  –

3.9 Clarifying Foxo1 activity during Treg induction

3.9.1 Foxo1 is temporarily excluded at early timepoints from the T cell nucleus following activation but returns at later timepoints

3.9.2 TCR triggering leads to global changes in DNMT expression

3.9.3 Global DNA hypomethylation selectively upregulates CD62-L

3.9.4 CD62-L is independent of HDAC inhibitor activity

3.9.5 Methylation specific PCR of KLF-2 CpG islands in naïve and differentially activated T cells

3.9.6 Role of miRNA let-7b in CD62-L regulation

3.10. Modulation of T cell activation dynamics by the microenvironment

4. Discussion

5.0 Conclusion and outlook

6.0 Zusammenfassung

7.0 References

Supplementary Figures; Gene mapping of the mice KLF-2 locus

Acknowledgement

Curriculum Vitae

Erklärung

–  –  –

LAT linker for Activation of T cells Lck lymphocyte-specific protein tyrosine kinase LPS Lipopolysaccharide MHC Major Histocompatibility Complex miRNA microRNA mRNA messenger RNA mTOR Mammalian Target of Rapamycin NaCl Sodium chloride NEAA Non-Essential Amino Acids NFkB Nuclear factor kappa B

–  –  –

OvGU Otto-von-Guericke University p value probability value PAMP Pathogen Associated Molecular Pattern PBS Phosphate Buffered Saline PCR Polymerase Chain Reaction

–  –  –

ROS Reactive oxygen species RPMI Roswell Park Memorial Institute SDS Sodium Dodecyl Sulphate SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SLOs Secondary lymphoid organs SLP-76 SH2 domain containing leukocyte protein of 76 kDa

–  –  –

Fig 1.1 Mechanism of action for CTL cytotoxicity Fig 1.2 Differentiation of Th1 cells from naïve T cells Fig 1.3 Differentiation of Th2 cells from naïve T cells Fig 1.4 Differentiation of Th17 cells from naïve T cells Fig 1.5 Role of TH17 and TH22 cells in inflammation and immunity Fig 1.6 Numerous mechanisms of action of Tregs Fig 1.7 Schematic of signaling circuit in T cells Fig 1.8 Role of CD62-L in T cell migration to lymph nodes Fig 2.1 Purity of MACS isolated cells Fig 2.2 A one parameter FACS histogram Fig 2.3 A two parameter FACS dot-plot Fig 2.4 Chemical depiction of bisulfite treatment of DNA Fig 3.1 Effect of mTOR and PI3K inhibitor titration on CD62-L expression levels Fig 3.2 Role of TLRs in the regulation of CD62-L in TofB Fig 3.3 pS6 as surrogate readout for PI3K/mTOR signaling Fig 3.4 Characterization of iDCs against mature DCs Fig 3.5 Phenotypic characterization of T cells triggered by iDC Fig 3.6 Functional characterization of TofiDC and delineation of the involvement of the PI3K and mTOR pathways in the acquisition of regulatory function Fig 3.7 Role of co-stimulatory input in acquisition of regulatory behaviour and migratory potential of iTregs Fig 3.8 Analysis of Akt signaling activity as readout of PI3K/mTOR signaling Fig 3.9 PHLPP1upregulation underlies defective PI3K/mTOR signaling Fig 3.10 PHLPP1 is upregulated by PI3K but not Rapamycin inhibition Fig 3.11 Foxo1 is re-expressed in the nucleus of activated T cells Fig 3.12 Confirmation of presence of Foxo1 in the nucleus of activated T cells Fig 3.13 Global mRNA expression levels of DNMTs following differential T cell

–  –  –

Fig 3.15 HDAC activity is dispensable for CD62-L regulation Fig 3.16 Methylation patterns of KLF-2 CpG Islands in naïve and 72h differentially activated T cells Fig 3.17 CD62-L regulation is independent of let-7b Fig 3.18 Modulation of T cell activation in ICAM deficient in vivo microenvironment.

–  –  –

Regulatory T cells (Tregs) are a special branch of T cells which function primarily to keep immune responses at a non harmful level and are principally of two types: naturally occurring Tregs (nTreg) which develop in the thymus and induced Tregs (iTregs) which develop from naïve T cell pools in the periphery. Tregs are widely depicted by their expression of the Foxp3 transcription factor and there is an increasing awareness of the existence and activity of non-Foxp3 Tregs which also play a role in modulating immune responses. iTregs have been extensively studied in recent years and while much is now known about their biology, there however exists a knowledge gap about initial signaling events which underlie the process of naïve T cell conversion into iTregs in the periphery. With the aid of transgenic T cells specific for the pOVA peptide, this study investigated the processes involved in the conversion of naïve T cells into CD4+ CD25+ Foxp3- iTregs following activation of the T cell receptors (TCRs) on these T cells by weak antigen presenting cells (APC).

Results of this study showed that after activation by weak APC, iTregs initially shed the lymph node homing molecule CD62-L and after 24h begin to be re-express this molecule again and by 72h, had returned to values as high as in naïve T cells. This was in contrast to T cells triggered by mature dendritic cells (DCs) which shed CD62-L just as iTregs but maintained consistently low amounts of CD62-L after the initial shedding phase. Based on pharmacological dissection, a differential role for the PI3K/mTOR signaling pathway was implicated in the dynamics of CD62-L regulation between iTreg and effector T cells. After TCR triggering by weak APC, iTregs exhibited an attenuated phopsho-status at the hydrophobic motif of Akt; Ser473 and this signaling profile was found to be associated with upregulated levels of the novel molecule PHLPP1 which has been reported to specifically target the hydrophobic motif of Akt. Lack of co-stimulatory molecules was also identified as playing a critical role in acquisition of regulatory function in T cells triggered by weak APC and augmented CD28 stimulation was able to abrogate regulatory function but interestingly had no effect on CD62-L expression, thus emphasizing the importance of suboptimal CD28 signaling for conferment of regulatory behaviour in iTregs but not in modulating its migratory potential as assessed by its CD62-L expression level. This work also highlighted the differential requirements of PI3K and mTOR signaling for acquisition of regulatory function as pharmacological inhibition of PI3K but not mTOR drove T cells from effector lineage into regulatory cell lineage.

14 In the final part of the thesis, we began investigating the role of adhesion molecules of the microenvironment stroma in T cell activation. Adaptively transferred transgenic OT 2 cells were equally activated by transferred antigen laden wildtype dendritic cells with regards to CD25 expression but the percentage of proliferating cells was significantly lower in Intercellular adhesion molecule (ICAM) deficient mice which lack ICAM expression on the stroma of the lymph node compared to wildtype thus suggesting that in situations of ICAM deficiency, T cell immune response might be delayed or insufficient compared to wild type conditions.

In conclusion, this present work apart from giving insight into early signaling events in iTreg generation also proposes that there exists a temporal window of sensitivity after TCR triggering within which the signaling threshold of the PI3K/Akt axis determines cells fate lineage into effector or regulatory T cell fate lineage and provides hint that in vivo, adhesion molecules on the stroma of lymph nodes play a role in efficiency of T cell responses.

151.0 INTRODUCTION

1.1 The Mammalian Immune System The immune system is a pathogen fighting system found in invertebrates (at a basic rudimentary level) down through to vertebrates which have evolved a more complex system.



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