Exploring the function of IL-10, BTLA and DCs as regulators of immune responses [Elektronische Ressource] / Nir Yogev

johannes_gutenberg-universitat_mainz - Sonja Reissig

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141 pages

English

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Exploring the function of IL -10, BTLA and
DCs as regulators of immune responses

Dissertation
Zur Erlangung des Grades
Doktor der Naturwissenschaf t

Am Fachbereich Biolog ie
der Johannes Gutenberg -‐Universität Mainz

Nir Yogev

geb. am 31. Dezember 1973 in Kibutz Kineret, Isr ael

Mainz, 2010 ABBREVIATIONS................................ ................................ ................................ ................................ ..IV
1 INTRODUCTION ............................... 1
1.1 INTERLEUKIN -‐10 ........... 4
1.1. 1 IL-10 protein, gene and expression4
1.1. 2 IL-10 receptor and signaling ...........................................................................5
1.1. 3 IL-10R signal transduction...............6
1.1. 4 IL-10 Function........6
1.1.4.1 Antigen presenting cells ............ 6
1.1.4.2 T cell................................s ................. 7
1.1. 5 IL-10 in infectious disease8
1.1. 6 IL-10 in autoimmunity.......................9
1.1. 7 IL-10 and experimental autoimmune encephalomyelitis.................10
1.2 B AND T L YMPHOCYTE ATTENUATOR ................................ ................................ ................................ ....11
1.2. 1 Co -receptors of the CD28 and TNFR superfamilies.............................12
1.2. 2 BTLA structure, expression and signaling...............13
1.3 DENDRITIC CELLS ................................ ........15
2 MATERIALS AND METHOD S................................ ................................ ................................ .....17
2.1 CHEMICALS AND BIOLOGI CAL MATERIAL 17
2.2 M OLECULAR BIOLOGY 19
2.2. 1 Competent cels and isolation of plasmid DNA......19
2.2. 2 Isolation of genomic DNA from ES cells and mouse organs.............19
2.2. 3 RT -PCR and quantitative real -time PCR ...................................................20
2.2. 4 Agarose gel electrophoresis and DNA gel extraction.........................20
2.2. 5 DNA sequencing ..................................................................20
2.2. 6 Quantification of DNA......................21
2.2. 7 Polymerase Chain Reaction (PCR)..............................21
2.2. 8 Southern blot analysis22
2.3 CELL BIOLOGY .............. 23
2.3. 1 Embryonic stem cel culture ..........................................23
2.3. 2 Tat -Cre protein (HTNC) treatment.............................................................24
2.3. 3 EL-4 cell line culture and electroporation...............24
2.3. 4 Preparation of Mouse Embryonic Fibroblasts (MEFs).......................25
2.3. 5 Preparation of c ells from lymphoid organs............25
2.3. 6 Culture of ex vivo lymphocytes.....26
2.3. 7 Cel counting .........................................26
2.3. 8 Adoptive T cel transfer and CFSE labeling.............26
2.3. 9 Flow Cytometry...................................27
2.3.10 Flow Cytometry and Intracelular Cytokine Staining (ICS)...........28
2.3.11 Magnetic cel sorting and FACS sorting.................29
2.3.12 Isolation of CNS infiltrates29
2.3.13 Isolation of splenic DCs..................30
2.3.14 Induction of CD11c -CreER activity in vivo..........................................30 T
2.3.15 In vitro 2D2 -iTreg differentiation and analysis..30
2.3.16 T and iTreg in vitro Differentiation ......................................................30 eff
2.4 IN VIV DOEPLETION OF CD25+ CELLS .....31
2.5 IN VIV DOEPLETION OF DENDRITIC CELLS .............................. 31
2.6 TISSUE PREPARATION FOR IMMUNOH ISTOCHEMISTRY ........32
2.6. 1 Flow -Cytomix ........................................32
2.6. 2 ELISA........................32
II 2.7 M OUSE E XPERIMENTS ................................ ................................ ................................ ............................... 32
2.7. 1 Induction and assessment of EAE................................32
2.7. 2 Induction and assessment of Colitis............................33
2.7. 3 MCMV and MHV Infection ...............................................33
2.8 STATISTICS ................... 34
2.9 M ICE ................................ .............................. 34
3 RESULTS .......... 35
3.1 INTERLEUKIN -‐10 ................................ ........35
3.2 BTLA ............................ 57
3.2. 1 Generation of BTLA over expressing mouse............................................57
3.2. 2 BTLA over expression by dendritic cels...................62
3.2. 3 BTLA over expression by T cels ...................................70
3.3 DENDRITIC CELLS ........82
4 DISCUSSIO................................N ...99
4.1 INTERLEUKIN -‐10 ........99
4.2 B AND T L YMPHOCYTE ATTENUATOR ................................ .101
4.3 DENDRITIC CELLS ................................ .....105
5 REFERENCES ................................ ................................ 109
6 SUMMARY ................................ .....130
7 ZUSAMMENFASSUNG ................ 131
8 ACKNOWLEDGEMENTS ................................ ................................ ............ 132
9 LEBENSLAUF 133
10 PUBLICATIONS ................................ ......................... 134
11 ERKL ÄRUNG ................................ ................................ .............................. 135

III Abbreviations

Ab antibody
ALT al a ni ne transaminase
APC antigen presenting cell or allophycocyanin
approx. approximately
BAC bacterial artificial chromosome
Bio biotinylated
β-‐ME β-‐mercaptoethanol
bp base pair
BMDCs bone marrow derived dendritic cells
BSA bovine serum albumin
°C temperature in celsius degrees
CD cluster of differentiation
CFA Complete Freund’s Adjuvant
CFSE c arboxyfluorescein diacetate succinimidyl ester
cDNA complementary DNA
CNS central nervous system
cpm counts per minute
Cre site-‐specific recombinase (causes recombination)
d day/s
d.p.i d ay s post immunization
DC dendritic cel l
DMEM Dulbecco’s modified Eagle medium
DN d oub l e nega tive
DNA desoxyribonucleic acid
dNTP desoxynucleotide triphosphate
DP dou b l e positive
DTA Diphtheria toxin A
DTT dithiothritol e
DTR Diphtheria toxin receptor
Dtx Dipht toxin
EAE experimental autoimmune encephalomyelitis
EDTA ethylene-‐diaminetetraacetic acid
ELISA enzyme -‐linked immuno -‐sorbent assay
ES embryonic stem
EtOH ethanol
FACS fluorescence activated cell sorting
FCS fetal calf seru m
Fig. Figure
FITC fluorescein isothiocyanate
Flp site-‐specific recombinase, product of yeast FLP1-‐gene
FoxP3 forkhead box protein 3
FRT Flp recombination target
IV gr gram
hr hour/s
HEPES N-‐2-‐hydroxyethylpiperazine-‐N’-‐2-‐ethansulfonic acid
iDTR inducible Diphtheria toxin receptor
i.p. intraperitoneally
i.v. intravenously
ICS intracellular staining
IFN-‐γ interferon-‐γ
Ig immunoglobulin
IL interleukin
kb kilobase pair
l liter
LN lymph node/s
P recognition sequence for Cre (locus of -‐ing over of phaX ge P1 )
LPS lipopolysaccharide
Ly6C lymphocyte antigen 6 complex, locus C
M molar
MACS magnetic activated cell sorter
MFI mean fluorescence intensity
MgCl2 magnesium chloride
MHC major histocompatibility complex
min minute
ml millilite r
mM millimolar
MOG myelin oligodendrocyte glycoprotein
mRNA messenger RNA
MS multiple sclerosi s
M Φ macrophage/s
NaCl sodium chloride
n nano
NaOH sodium hydroxide
neo neomycin resistance gene
ng nanogram
o/n over night
OD optical density
pDC Plasmacytoid dendritic cell
PBS phosphate buffered saline
PCR polymerase chain reaction
PE phycoerythrine
Ptx Pertussis toxin
RAG recombinase activating gene
RNA ribonucleic acid
rpm revolutions per minute
RT room temperature
sec seconds
SA streptavidine
V
loxs.c. subcutaneously
sc spinal cord
SDS sodium dodecyl sulfate
SN supernatant
SP single positive
SSC sodium chloride/sodium citrate buffer
TAE Tris -‐acetic acid-‐EDTA buffer
TAM Tamoxifen
Taq polymerase from Thermus aquaticus
TCR T cell receptor
TE Tris -‐EDTA buffer
TEC thymic epithelial ce ll
tg transgenic
TGF-‐β transforming growth factor-‐β
Th helper T cel ls
T effector T cel ls eff
Tregs regulatory T cell s
Tris 2-‐amino -‐2-‐(hydroxymethyl-‐)1,3 -‐propandiole
U units
UV ultraviolett
V volts
vs versus
v/v volume per volume
w/v weight per volume
WT wild type
µg microgram
µl microliter
µM micromolar
3’ three prime end of DNA sequences
5’ five prime end of DNA sequences

VI 1 INTRODUCTION

Immune homeostasis depends on the existence of equilibrium between
responses that control infection and tumour growth, and reciprocal responses that
prevent inflammation and autoimmune diseases. Protection against infection is
fundamental to the survival of all multicellular organisms and is mediated by the
immune system, which has evolved both innate and adoptive mechanisms to deal
with invading microorganisms. The effector mechanisms used by the host to control
infection include production of proinflammatory cytokines and chemokines,
recruitment of inflammatory cells to the site of infection and activation of cytotoxic
T lymphocytes (CTL) and natural killer (NK) cells, which lyse infected host cells.
Although these responses help to eliminate or slow the spread of the pathogen, if
they are not tightly controlled, they can result in severe infon lammand ticollatera l
tissue damage (Artavanis -‐Tsakonas et al., 2003) .
As the cells and molecules of thimmue ne system that respon d to pathogen-‐
derived antigens might also respond to self-‐antigens, autoimmune disease can result
if this reactivity is not tightly cont(von rolled Herrath and Harrison, 2003).
Therefore, inflammation and immune response to pathogens are regulated by
various host suppressor mechanisms, including the production of anti -‐inflammato ry
cytokines, co -‐inhibitory signals and regulatory cel ls.

+CD4 T cells, also known as T helper (Th) cells, play an important role in
orchestrating adaptive immune responses to various infectious agents. They are
also involved in the induction of autoimmun e and allergic diseases. Upon T cell
receptor (TCR)-‐mediated cell activation, naïve CD4 T cells can differentiate into at
least four major lineages, Th1, Th2, Th17 and iTregwh cielchls par, ticipate in
different types of immune responses. Networks of cytokines and transcription
+factors are critical for determining C TD4 cell fates and effector cytokine
production. The major determinant for Th cell differentiation is the cytokine milieu
1 at the time of antigen encounter, although the nature of cognate antign e and its
affinity to the TCR as well as the available -‐st icomulants, many of which regulate
initial cytokine production, can influence Th cell fa te.
Interleukin 10 -‐(IL10) is a cytokine that modulates both innate and adoptive
immunity, primarily by exertng i anti-‐inflammatory effects. IL-‐10 is produced by a
+variety of cell types of both innate and adoptive origin. During many infections, CD4
T cells produce both IFNγ and IL -‐10, as the -‐10 IL produced by effector Th1 cells
helps limit the collateral damagcea used by exaggerated inflammation. However,
this control may limit the effectiveness of the immune response, resulting in a
failure to fully eliminate pathoge ns.

The specific function of an individual’s immune system in different
physiological and pathological setting is regulated by the action of opposing factors
or systems. Common examples are the polarization of T helper cells into Th1 and
Th2 subsets, and the balance between effector T-‐cell (T) activation and regulatory eff
T-‐cell (T) activation. At the molecular level, -‐sctoimulatory members of the B7 and reg
TNF superfamilies can have both stimulatory and inhibitory effect on -‐Tcell
activation.
Co -‐signalling molecules are ce-‐lsulrface glycoproteins that can direct,
modulate and fine -‐tune T-‐cell receptr o signals. On the basis of their functional
outcome, co -‐signalling molecules can be divided into co-‐stimulators and co -‐
inhibitors, which promote or suppress T cell activation, respectively. By expression
at the appropriate time and location, co -‐signalling molecules control the priming,
growth, differentiation and functional maturation of a T cell response .
Traditionally, co-‐signalling molecules were termed as receptors or ligands to
distinguish the direction of signal transmission. However this nomenclatu ire s
entirely operational and does not reflect the intrinsic nature of these molecules. A
ligand could be either a -‐csotimulator or co-‐inhibitor, depending on the specific
receptor it interacts with: for example, the binding of CD80/CD86 to CD28 transmit
a co -‐stimulatory signal, whereas the binding of CD80/CD86 to CTLA4 transmit a-‐ co
2 inhibitory signal. Moreover, a ligand could become a receptor (reverse signalling) as
it receives a -‐coinhibitory or -‐costimulatory signal, a process that had been
described for CTLA4, P-‐D1 and BTLA (Dong et al., 2003; Fallarino et al., 2003;
Grohmann et al., 2002; Nguyen et al., 200. 2)
Dendritic cells (DCs) considered are as the most potent antigen presenting
cells (APCs) and as suc, hplay a central role in the orchestration of the various forms
of immunity and tolerance. Their immune -‐regulatory role relies mainly on the
ligation of specific receptors that initiate and modulate DC maturation, resulting in
the development of functionall y different effector DC subsets that selectively
promote Th -‐1, Th-‐2, Th-‐17 or Treg cell responses.

This thesis focuses on different aspects of immune regulation, both at the
cellular and molecular levels. More specifically, this work concesn otraten the
importance of Interleukin-‐10, B and T Lymphocyte Attenuator (BTLA), and dendritic
cells in respect to immune regulation, with special emphasis on autoimmunity.

3 1.1 Interleukin -10

Interleukin -‐10 (I-‐L10) is a cytokine that modulates both innate nd a adaptive
immunity, primarily by exerting ant-‐iinflammatory effects. Originally identified as a
Th2 cell derived fact(Fiorentor ino et al., 1989), IL-‐10 was later found to be secreted
by a variety of haemopoietic cell types including activated macrophages (de Waal
Malefyt et al., 1991; Maeda et al., , 19dendr95)itic cells (Anderton et al., 2002;
Iwasaki and Kelsall, 1999; Khanna et al., 2000; McGuirk et al., 2002; Mizoguchi et al.,
2002; O'Garra et al., 1992; Stumbles et al., 1B9 9ce8l)ls and mast cel(Mlsasuda et
al., 2002. )IL-‐10 is also produced by regulatory T cells (Tregs), Th1 (O'Gacelrral s
and V ieira, 2007; Trinchieri, , and2007) Th17 cells( Awasthi et al., 2007; Fitzgerald
et al., 2007; McGeachy et al., 2007; Stumhofer et al., as2 0we07)ll as Th9 ce lls
(Dardalhon et al., 2008; Veldhoen et a. lIn ., 2addition, 008) keratinocytes can be
induced to secrete IL-‐10 by contact allergen or UV irradiati(Pinton o et al., 2006) .
1.1.1 IL-10 protein, gene and expression
IL-‐10 protein is a homodimer composed of two interpenetrating polypeptide
chains, similar to interferon gamma (IFγN) (Syto et al., 1998; Walter and
Nagabhushan, 1995). IL-‐10 open reading frames (ORF) encode a secreted protein of
178 amino acids with rather a we-‐lclonserved sequence of about 73% homology
shared by human and mice.
The murine IL-‐10 (mIL -‐10) gene is encoded in five exons, located on
chromosome 1 (Kim et al., 199. 2Ac)tivation of IL-‐10 gene expression results in a 1.4
kb mRNA, which can be regulated by the transcription factors Sp1 and Sp(Tone 3 et
al., 2000 a)s well ast athe posttranscriptional levels (Powell et al., 2000), indicating
that the IL-‐10 gene is transcribed to some degree constitutively and subject to
control by alteration of posttranscriptional RNA degradation mechanis ms.
It has been suggested that Toll Like Receptor (TL-‐2 R)agonists are specialized
in inducing IL-‐10 expression by antigen presenting cell(s Agrawal et al., 2003; Dillon
et al., 2004; Hu et al., 2006; Netea et a)l. ., IL-‐200410 production is also induced by
TLR4, TLR9 and TLR3 ligands (Boonstra et al., 2006) . Following TLR stimulation,
4