Immune regulation through homotypic T cell, T cell interaction [Elektronische Ressource] = Immunregulation durch homotypische T-Zell-T-Zell-Wechselwirkungen / vorgelegt von Katja Thümmler

Immune regulation through homotypic T cell/T cell interaction Immunregulation durch homotypische T-Zell/T-Zell-Wechselwirkungen Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades Dr. rer. nat. vorgelegt von Katja Thümmler aus Oelsnitz/Vogtland Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Friedrich-Alexander Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 22.04.2010 Vorsitzender der Promotionskommision: Prof. Dr. Eberhard Bänsch Erstberichterstatter: Prof. Dr. Thomas Winkler Zweitberichterstatter: Prof. Dr. Dres. h.c. Joachim R. Kalden Meinen Eltern und Schwestern „Kein Fortschritt ist möglich ohne Erweiterung des Herzens.“ Hans KruppaTable of contents TABLE OF CONTENTS 1 SUMMARY ........................................................................................................... 1 2 ZUSAMMENFASSUNG ........................................................................................ 3 3 INTRODUCTION.................................................................................................. 5 3.1 T cell subsets ........................................................................................................................................................5 3.1.1 Th1/Th2 paradigm ............................................
Publié le : vendredi 1 janvier 2010
Lecture(s) : 15
Source : D-NB.INFO/100248281X/34
Nombre de pages : 93
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Immune regulation through
homotypic T cell/T cell interaction

Immunregulation durch
homotypische T-Zell/T-Zell-Wechselwirkungen








Der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander Universität
Erlangen-Nürnberg
zur
Erlangung des Doktorgrades Dr. rer. nat.



vorgelegt von
Katja Thümmler
aus
Oelsnitz/Vogtland


Als Dissertation genehmigt
von der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander Universität Erlangen-Nürnberg










Tag der mündlichen Prüfung: 22.04.2010

Vorsitzender der
Promotionskommision: Prof. Dr. Eberhard Bänsch

Erstberichterstatter: Prof. Dr. Thomas Winkler
Zweitberichterstatter: Prof. Dr. Dres. h.c. Joachim R. Kalden










Meinen Eltern und Schwestern


„Kein Fortschritt ist möglich
ohne Erweiterung des Herzens.“
Hans KruppaTable of contents
TABLE OF CONTENTS
1 SUMMARY ........................................................................................................... 1
2 ZUSAMMENFASSUNG ........................................................................................ 3
3 INTRODUCTION.................................................................................................. 5
3.1 T cell subsets ........................................................................................................................................................5
3.1.1 Th1/Th2 paradigm ........................................................................................................................................7
3.1.2 Expanding T cell effector subsets – Th17, Th22, Th9 cells.......................................................................7
3.1.3 Regulatory CD4 T cells ................................................................................................................................9
3.2 Homotypic T cell/T cell interaction................................................................................................................11
4 AIMS OF THE THESIS....................................................................................... 15
5 MATERIALS AND METHODS........................................................................... 16
5.1 Materials.............................................................................................................................................................16
5.1.1 Reagents, enzymes, cytokines....................................................................................................................16
5.1.2 Antibodies ...................................................................................................................................................17
5.1.3 TaqMan Gene Expression Assays for human genome.............................................................................20
5.1.4 Kits...............................................................................................................................................................20
5.1.5 Human cells.................................................................................................................................................21
5.1.6 Mice .............................................................................................................................................................21
5.2 Methods ..............................................................................................................................................................22
5.2.1 Purification of human cells.........................................................................................................................22
5.2.2 Mice and purification of murine cells........................................................................................................23
5.2.3 Flow cytometry ...........................................................................................................................................25
5.2.4 Cell cultures.................................................................................................................................................26
5.2.5 Generation of activated stimulator T cells ................................................................................................26
5.2.5.1 Human T cells .....................................................................................................................................26
5.2.5.2 Murine T cells .....................................................................................................................................26
5.2.6 Co-culture experiments of activated T cells with responder T cells .......................................................27
5.2.7 In vivo expansion of OVA-specific T cells ...............................................................................................27
5.2.8 CFSE labeling for the analysis of cell division.........................................................................................28
5.2.9 Fluorescence microscopy ...........................................................................................................................28
5.2.10 Preparation of total RNA, and amplification of cDNA by real-time PCR............................................29
5.2.11 Cytokine Enzyme-linked Immunosorbent Assay (ELISA)....................................................................30
5.2.12 Proliferation assay.....................................................................................................................................31
5.2.13 Statistical analysis.....................................................................................................................................31
6 RESULTS............................................................................................................ 32
6.1 Prerequisite for homotypic T cell/T cell interaction....................................................................................32
6.1.1 Generation of stimulator CD4 T cells........................................................................................................32
6.1.2 Activated CD4 T cells express surface receptors characteristic for APCs..............................................33
6.1.3 Cytokine secretion after T cell activation under different polarizing conditions ...................................34
6.2 Consequences of homotypic T cell/T cell interaction ..................................................................................35
6.2.1 Proliferation.................................................................................................................................................35
6.2.2 Cytokine production ...................................................................................................................................36
6.3 Phenotypical characterization of T cells resulting from T cell/T cell interaction...................................39 Table of contents
6.3.1 T cells acquire a mild activated phenotype after T cell/T cell interaction ..............................................39
6.4 Mechanisms underlying homotypic T cell/T cell interaction .....................................................................41
6.4.1 T cell/T cell interaction is dependent on cell/cell contact ........................................................................41
6.4.2 Blocking APC-characteristic receptor/ligand interactions.......................................................................43
6.4.3 Allogeneic T cell/T cell interaction ...........................................................................................................44
6.4.4 The role of adhesion receptors in T cell/T cell interaction.......................................................................45
6.4.4.1 The adhesion receptor LFA-1 and its ligand.....................................................................................45
6.4.4.2 Expression of LFA-1 and its ligands on T cells upon homotypic T cell/T cell interaction ...........47
6.4.4.3 The adhesion receptor CEACAM-1 ..................................................................................................47
6.5 Functional characterization of T cells resulting from T cell/T cell interaction ......................................48
6.5.1 Cytokines secreted after T cell contacts and their effect on proliferation of CD25 negative T cells....48
6.5.2 Suppressor phenotype of T cells upon homotypic T cell/T cell interaction............................................49
6.6 Mechanisms underlying the inhibitory effect of Tregs generated by T cell interaction........................51
6.6.1 Inhibition through IL-2 deprivation, cell contact and immuno-modulatory cytokines ..........................51
6.7 Murine T cell/T cell interaction ......................................................................................................................52
6.7.1 Proliferation and cytokine production .......................................................................................................52
6.7.2 Suppressor phenotype.................................................................................................................................53
6.7.3 Homotypic T cell/T cell interaction is antigen-independent in the mouse..............................................54
7 DISCUSSION ...................................................................................................... 58
7.1 Characterization of T cells resulting from homotypic T cell/T cell interactions ....................................58
7.2 Mechanisms mediating T cell/T cell interactions.........................................................................................60
7.3 Functional consequences of T cell/T cell interaction ...................................................................................67
8 CONCLUDING REMARKS ................................................................................ 70
9 ABBREVIATIONS .............................................................................................. 71
10 BIBLIOGRAPHY .............................................................................................. 74
11 ACKNOWLEDGEMENTS ................................................................................ 86
12 CURRICULUM VITAE..................................................................................... 88

Summary 1
1 SUMMARY
CD4 positive T cells drive specific immune reactions, which are essential for the protection
against invading microorganisms. However, they may also have negative effects like tissue
damage or autoimmune inflammation. Interestingly, activated CD4 T cells express antigen
presenting cell (APC)-specific surface receptors such as CD80, CD86, CD70 and MHC-II,
allowing interaction of activated CD4 T cells with resting CD4 T cells. We tested the
hypothesis, that homotypic T cell/T cell interaction may induce T cell activation and
differentiation from resting T cells. Furthermore, we were interested in the phenotype of the T
cells primed by T cell/T cell contact and in the molecular mechanisms underlying this novel
kind of cellular interactions.
In the first part of the project, we investigated homotypic T cell interaction in vitro. CD4 T
cells were isolated from the peripheral blood of healthy donors and activated under different
polarizing conditions. The resulting effector cells (stimulators) were fixed and co-cultured
together with resting CD4 memory or naive T cells (responders), but in the absence of
specific T cell stimulatory factors such as antibodies to T cell surface molecules, antigens or
mitogens. After several days of culture, the phenotype and function of the resulting cells were
characterized.
Co-culture of activated CD4 T cells with resting memory or naive CD4 T cells resulted in the
proliferation of memory but not of naive responder cells. Fluorescence microscopic
examination of the T cell/T cell interaction revealed the formation of cell clusters of activated
T cells surrounded by the responder cells. The memory responder cells not only proliferated
in response to contact with activated T cells but also secreted distinct cytokines depending on
the polarizing conditions of the activated stimulator T cells. Activated Th1 stimulator cells
induced IL-10 and IFNγ secretion of the memory responder cells in a dose dependent manner,
whereas Th2 stimulator cells induced IL-4 secretion.
The T cell-induced T cell differentiation was strongly dependent on cell contact. Blocking the
adhesion molecule LFA-1 resulted in a profound inhibition of cytokine production and
proliferation of the responder cells. Interestingly, however, neutralization of particular
adhesion receptors, e.g. ICAM-1, -2 or -3, led to different phenotype alterations of the
developing cytokine-producing T cells. Blocking of ICAM-1 diminished production of IFNγ Summary 2
but did not influence IL-4 and IL-10, whereas ICAM-3 was important for IL-4 secretion,
together indicating that ICAM/LFA-1 interaction was not only responsible for the occurrence
of T cell/T cell contact but had also a decisive impact on the phenotype of developing
cytokine-producing cells.
Independent of the polarizing conditions of the stimulator T cells, all T cell populations
resulting from T cell/T cell interaction strongly inhibited the proliferation of CD25 negative T
cells in a dose dependent manner to a similar extent as natural occurring CD25 positive Tregs.
Their inhibitory function could be reverted by exogenous IL-2, and was mediated in part by
cell/cell contact and in part by IL-10.
The second part of this project was focused on the investigation of the function of T cells
generated upon T cell/T cell interaction in vivo. First, the T cell/T cell interaction co-culture
system was adapted to murine cells. Similar to the situation in men, resting memory CD4 T
cells proliferated in response to contact with activated stimulators and secreted IFNγ in the co-
culture of activated Th1 cells, and IL-4 and IL-10 in the co-culture of Th2 cells. The resulting
cell population inhibited the proliferation of CD25 negative T cells in response to anti-CD3
stimulation in a dose dependent manner in vitro. Transfer of cells generated upon T cell/T cell
contacts into BALB/c mice reconstituted with DO11.10 OVA-TCR transgenic T cells
prevented the expansion of antigen-specific effectors upon antigen challenge, together
indicating, that homotypic T cell interaction results in the generation of T cells with a potent
regulatory capacity. By this means, effector T cells activated under physiologic conditions
may engage a negative feedback mechanism to keep sustained immunity under control and
prevent tissue damage. Zusammenfassung 3
2 ZUSAMMENFASSUNG
CD4-T-Zellen steuern spezifische Immunreaktionen, die essentiell für den Schutz gegen
eindringende Mikroorganismen sind. Dennoch können diese auch negative Effekte wie
Gewebeschädigung oder autoimmune Entzündungen hervorrufen. Interessanterweise
exprimieren aktivierte CD4-T-Zellen Rezeptoren, welche charakteristisch für Antigen-
präsentierende Zellen sind wie z.B. CD80, CD86, CD70 und MHC-II. Diese Rezeptoren
ermöglichen Wechselwirkungen von aktivierten CD4-T-Zellen mit ruhenden T-Zellen. Wir
untersuchten die Hypothese, dass homotypische T-Zell/T-Zell-Wechselwirkungen die
Aktivierung und Differenzierung von ruhenden T-Zellen induziert. Außerdem interessierten
wir uns für den Phänotyp der T-Zellen, die nach Kontakt zu T-Zellen entstehen und die
molekularen Mechanismen, die dieser neuen Art von Zell-Wechselwirkungen zu Grunde
liegen.
Im ersten Teil des Projekts untersuchten wir homotypische T-Zell-Wechselwirkungen in
vitro. CD4-T-Zellen wurden aus dem peripheren Blut gesunder Spender isoliert und unter
verschiedenen polarisierenden Bedingungen aktiviert. Die sich daraus ergebenden Effektor
Zellen (Stimulatoren) wurden fixiert und zusammen mit Gedächtnis- oder Naiven-CD4-
Zellen (Responder) in Abwesenheit von spezifischen T-Zell-Stimulanzien, wie Antikörpern
gegen T-Zell-Oberflächenmoleküle, Antigenen oder Mitogenen, kokultiviert. Nach einigen
Tagen in Kokultur wurden der Phänotyp und die Funktion der entstandenen Zellen analysiert.
Die Kokultur der aktivierten CD4-T-Zellen mit ruhenden Gedächtnis-oder Naiven-CD4-T-
Zellen löste die Proliferation der Gedächtnis- aber nicht der Naiven-Responder-Zellen aus.
Bei der fluoreszenzmikroskopischen Untersuchung der T-Zell/T-Zell-Wechselwirkung
wurden Zellcluster aus Responder-Zellen gefunden, die von aktivierten T-Zellen umgeben
waren. Nach Kontakt mit den aktivierten T-Zellen proliferierten die Gedächtnis-Responder-
Zellen nicht nur, sondern sezernierten außerdem bestimmte Zytokine in Abhängigkeit von den
Bedingungen, unter denen die Stimulator-Zellen aktiviert wurden. Aktivierte Th1-Stimulator-
Zellen induzierten dosisabhängig bei den Gedächtnis-Responder-Zellen die Sekretion von IL-
10 und IFNγ, während Th2-Stimulator-Zellen zur IL-4 Sekretion führten.
Die T-Zell-Differenzierung, die durch Kontakt mit T-Zellen induziert wurde, war stark
kontaktabhängig. Die Hemmung des Adhäsionsmoleküls LFA-1 resultierte in einer starken Zusammenfassung 4
Inhibition der Zytokin-Produktion und Proliferation der Responder-Zellen. Interessanterweise
führte jedoch die Blockade bestimmter Adhäsionsrezeptoren, wie ICAM-1,-2 oder -3 zu
unterschiedlichen Veränderungen im Phänotyp der sich entwickelnden Zytokin-
produzierenden T-Zellen. Die Blockade von ICAM-1 verringerte die IFNγ-Produktion, hatte
allerdings keinen Einfluss auf IL-4 und IL-10, wohingegen ICAM-3 wichtig für die IL-4
Sekretion war. ICAM/LFA-1-Wechselwirkungen scheinen also nicht nur verantwortlich für
das Auftreten der T-Zell/T-Zell-Kontakte zu sein, sondern haben auch einen entscheidenden
Einfluss auf den Phänotyp der sich entwickelnden Zytokin produzierenden Zellen.
Unabhängig von den Bedingungen, unter denen die Stimulator-Zellen aktiviert wurden,
inhibierten alle T-Zell-Populationen, die nach T-Zell/T-Zell-Wechselwirkung entstanden
waren, dosisabhängig die Proliferation von CD25 negativen T-Zellen ähnlich stark wie CD25
positive regulatorische T-Zellen. Diese Inhibition konnte durch exogenes IL-2 aufgehoben
werden und war teilweise kontaktabhängig und teilweise durch IL-10 vermittelt.
Der zweite Teil der Arbeit richtete sich auf die Untersuchung der in vivo-Funktion der T-
Zellen, die durch T-Zell/T-Zell-Wechselwirkung entstanden. Zunächst wurde das Kokultur
System auf die Untersuchung von T-Zell/T-Zell-Wechselwirkungen bei murine T-Zellen
angepasst. Ähnlich wie bei humanen T-Zellen, proliferierten die Gedächtnis-CD4-T-Zellen
nach Kontakt mit aktivierten Stimulatoren und sezernierten IFNγ nach Kokultur mit
aktivierten Th1-Zellen, oder IL-4 und IL-10 nach Kokultur mit Th2-Zellen. In vitro inhibierte
die resultierende T-Zell-Population dosisabhängig die Proliferation von anti-CD3-stimulierten
CD25 negativen T-Zellen. Überträgt man T-Zellen, die durch T-Zell/T-Zell-Wechselwirkung
generiert wurden, auf BALB/c Mäuse, die zuvor DO11.10 OVA-TCR transgene T-Zellen
erhielten, so verhinderte dies die Expansion der antigenspezifischen Effektor-Zellen nach
Antigenkontakt. Zusammengefasst weist dies darauf hin, dass homotypische T-Zell/T-Zell-
Wechselwirkungen T-Zellen mit starker regulatorischer Kapazität hervorbringen. Dadurch
erlangen Effektor T-Zellen, die unter physiologischen Bedingungen aktiviert wurden, die
Möglichkeit, in einem negativen Feedback-Mechanismus überschießende Immunreaktionen
zu kontrollieren und Gewebeschädigungen zu verhindern. Introduction 5
3 INTRODUCTION
3.1 T cell subsets
The adaptive immune response is initiated once antigen presenting cells (APCs) internalize
and cleave the pathogen in the peripheral tissue. The internalized pathogenic proteins are
degraded into short peptide fragments and loaded on polymorphic peptide binding
glycoproteins from within the major histocompatibility complex (MHC). T cells can
recognize a large variety of antigens presented by MHC class I or II peptides on the APCs by
a specific and highly variable antigen recognition structure, the T cell receptor (TCR). In the
peripheral circulation, about 95% of the T cells express a TCR consisting of α and β chains
(αβ T cells), and only 5% of the T cells express a γδ TCR. αβ T cells are further divided into
2 subgroups concerning the co-receptors of the TCR, CD4 and CD8, that bind to conserved
parts of two different classes of MHC, MHC-II and MHC-I, respectively. CD4 T cells
recognize extracellular antigen presented by MHC-II molecules, initiate and drive both an
adaptive cellular and humoral immune response. In contrast, CD8 T cells recognize
endogenously synthesized peptides presented by MHC-I molecules, differentiate into
cytotoxic T lymphocytes and promote the effector phase of a cellular immune response.
Sufficient activation of naive T cells requires at least two signals, besides the TCR binding
also a second so called co-stimulatory signal. These co-stimulatory signals are provided by
molecules on the APCs such as CD80, CD86 or CD40 that bind their respective ligands CD28
and CD40L on the T cell (Schwartz, 1992), (Schwartz, 1990). Upon activation, naive T cells
start to proliferate, and differentiate into specialized effector T cells. The differentiation of T
cells into distinct effector subsets is controlled by the nature of an innate immune response to
an antigen. The interplay of a variety of extrinsic factors such as the nature of the antigen, the
strength of MHC-antigen/TCR interaction, co-stimulatory receptors on the surface of the
APC, and the cytokine milieu induces the activation of lineage specific transcription factors in
naive T cells resulting in the secretion of the typical effector cytokines (summarized in Fig.
1).

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