activated TCR 318 splenocytes of serial dilutions of wild-type galectin-1 (Gal wt)
and ...... Zinkernagel RM, Ohashi PS: Peptide-induced T cell receptor down-.
Research Collection
Doctoral Thesis
Study of selected molecular and cellular aspects of autoimmunity Author(s): Bättig, Patrick Publication Date: 2004 Permanent Link: https://doi.org/10.3929/ethz-a-004779074
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ETH Library
DISS. ETH Nr. 15574
Study
of selected molecular and cellular of
aspects
autoimmunity
A dissertation submitted to the
SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZURICH For the
degree
of
Doctor of Sciences
presented by PATRICK BÄTTIG
Dipl.
Natw. ETH
Born 15th Citizien of
Accepted
May 1974
Hergiswil
on
b. Willisau, LU
the recommendation of
Prof. Dr. S. Werner, examiner Prof. Dr. H.M.
Eppenberger,
co-examiner
Dr. M.F. Bachmann, co-examiner
2004
I
Table of Content
TABLE OF CONTENT
SUMMARY
1
ZUSAMMENFASSUNG
3
INTRODUCTION
5
1.
1.1.
T cell
1.1.1.
2.
3.
5
T CELLS
7
receptor
Genes
the T cell
encoding
1.2.
T cell
1.3.
T cell activation
1.4.
Cytotoxic
1.5.
THi
1.6.
Regulatory
9
receptor
12
development
14
17
lymphocytes (CTL)
T
20
TH2
versus
23
T cells
25
DENDRITIC CELLS 2.1.
Dendritic cell
2.2.
DC function
26
development
27
2.2.1.
Capturing
2.2.2.
Processing
2.2.3.
Migration
2.2.4.
Interaction with T cells
of the
to the
29
antigen
of the
antigen
lymphatic
and
loading
on
to the MHC class molecules
33
organs
36 40
AUTOIMMUNITY
3.1.
Central Tolerance
3.2.
Peripheral
3.3.
Mechanism
41
43
Tolerance
leading
to
45
autoimmunity
45
auto-antigens
3.3.1.
Release of isolated
3.3.2.
Breakdown of T cell anergy
3.3.3.
Chemical Alteration of
3.3.4.
Defective Activation-Induced cell death
3.3.5.
Defective T cell-mediated
3.3.6.
Molecular
Mimicry
47
3.3.7.
Polyclonal
Activators
48
3.3.8.
Exposure
3.3.9.
Genetic Factors
of
Cryptic
45 46
Self-Peptides (AICD)
suppression
Self and
epitope spreading
RESULTS 4.
46 47
48 49
50
EXPRESSION CLONING IDENTIFIES THE CC-CHEMOKINE CCL19 AS
POTENT ACTIVATOR OF DENDRITIC CELLS 4.1.
Abstract
50 51
II
Table of Content
4.2.
Introduction
52
4.3.
Material and Methods
54
4.3.1.
Mice
54
4.3.2.
Construction of normalized cDNA libraries
54
4.3.3.
Generation of
protein library
55
4.3.4.
Preparation
4.3.5.
Functional
4.3.6.
RT-PCR
4.3.7.
Expression
4.3.8.
CFSE in vitro
4.3.9.
Staining
4.3.10.
Induction of
4.4. 4.4.1.
of dendritic cells and T-cells
55
expression screening of DC maturation factors of
rescue
and
55
56
positive supematants
purification
of the recombinant
56
proteins
proliferation assay
57 57
for FCM and ELISA
experimental
autoimmune
57
myocarditis
59
Results Functional
cloning
of CCL19
as
inducer of T cell
proliferation
in DC-T
59
cell mixed culture assays 4.4.2.
CCL19 induces maturation of dendritic cells
4.4.3.
Purified CCL19 enhances
proliferation
61
of T cells in the DC-T cell
co-
culture system 4.4.4.
CCL19 induces
production 4.4.5. 4.5. 5.
62
of
cytokines
Induction of
up-regulation of co-stimulatory
molecules and the
in DCs
64
autoimmunity by CCL19-stimulated
66
DCs
DISCUSSION
68
INDUCTION OF AUTOIMMUNITY AS A FUNCTION OF EPITOPE STRENGTH
AND IMMUNE REGULATION
70
5.1.
Abstract
71
5.2.
Introduction
72
5.3.
Materials and Methods
74
5.3.1.
Mice viruses and cell lines
5.3.2.
Peptides, oligonucleotides, peptide-loaded tetramers,
74 rat anti-mouse
CD40, anti-CD25 Abs and VLPs 5.3.3.
In vitro and in vivo activation of
5.3.4.
In vivo CTL assay
5.3.5.
Assessement of
5.4.
p33-specific
CD8+ T cells
75 75
autoimmunity
Results
76 77
5.4.1.
Generation of VLPs
77
5.4.2.
Weak in vivo
79
5.4.3.
Anti-viral
5.4.4.
Induction of
5.5. 6.
74
containing p33 variant epitopes cross-reactivity of the peptides
protection by
cross-reactive T cells
autoimmunity
Discussion
81 83
86
ORIGINAL ANTIGENIC SIN FOR T CELLS: ANALYZED IN A VIRAL
INDEPENDET MODEL SYSTEM
88
6.1.
Abstract
89
6.2.
Introduction
90
III
Table of Content
92
Materials and Methods
6.3.
92
6.3.1.
Mice viruses and cell lines
6.3.2.
Peptides, oligonucleotides, peptide-loaded
92
tetramers and VLPs
93
Results and discussion
6.4.
ENHANCED APOPTOTIC ACTIVITY OF A STRUCTURALLY OPTIMIZED
7.
98
FORM OF GALECTIN-1
99
7.1.
Abstract
7.2.
Introduction
100
7.3.
Experimental procedures
102 the
sequence of the dimeric and
102
7.3.2.
eukaryotic expression plasmids containing wild-type galectin-1 and purification of the recombinant proteins Expression
102
7.3.3.
Nondenaturing gel electrophoresis
7.3.4.
Reduction
7.3.5.
Hemagglutination assay Measurement of apoptosis by Annexin-Cy5 staining
Construction of
7.3.1.
7.3.6. 7.4.
by acetylation
103
and oxidation of the recombinant
103 104 105
Results
7.4.1.
Dimerized
galectin-1
is still
efficiently
secreted via the unknown
non-
105
and does not form tetramers
classical
pathway
7.4.2.
Covalent dimerization of
activity
galectin-1 strongly
increases the
agglutination 107
protein.
of the
103
proteins
galectin-1 dimers are active and kill thymocytes ten times 109 more efficiently wild-type form. 7.4.4. Covalent galectin-1 dimers kill activated splenocytes more efficiently 112 than the wild-type protein 7.4.3.
Covalent
than the
115
DISCUSSION
7.5.
120
GENERAL DISCUSSION
120
DENDRITIC CELLS AND AUTOIMMUNITY
8.
immunity:
DCs
provide
8.1.
Tolerance
8.2.
Role of DCs in the induction of
8.3.
DCs in various autoimmune disorders
8.4.
Therapeutic
versus
the
120
key
121
autoimmunity
122 123
outlooks
125
MOLECULAR MIMICRY AND AUTOIMMUNITY
9.
APLs
9.2.
Regulatory T
9.3.
Molecular
strength
125
autoimmunity prevention
127
mimicry in autoimmune immune regulation sin:
right
T CELLS AND AUTOIMMUNITY
or
disease
as
function of
wrong, who decides?
signal 128 128 130
the killers
130
GENERAL OUTLOOK
130
10.1. 11.
cells in
Original antigenic
9.4. 10.
and
as
mimicry
model for molecular
9.1.
Killing
Table of Content
IV
REFERENCES
134
ABBREVIATIONS
171
PUBLICATIONS
173
ACKNOWLEDGEMENTS
174
CURRICULUM VITAE
176
1
Summary
SUMMARY system is
The immune
a
tool to
powerful
efficiently recognize non-self antigens, of
resulting
also in auto-reactive
mechanisms to avoid
important checkpoint of T cells
immunity. Given
one was
specificity.
suppress this
performed [1], seeking
to the
leading
maturation factor,
applied
to
investigations
identified. Further
orchestrating
The T cell
receptor
allowing differentiation
thymus. This leads a
is
one
trigger
to
a
of
p33 antigen
analyzed.
Whereas in TCR
as
revealed almost
no
model
from the
of
mouse
islet cells of the pancreas,
Strikingly
after
were
potent DC
a
reveal
adaptive
repertoire,
novel role
a
importance
immune
of
during
maturation in the
such
p33
versus
virus fused to
repertoire of wild-type and A4Y-APLs
APLs. However, the
virus-dependent
viral
immunization induced
were
system.
was
efficiently
cross-reactivity
protection. Analyzing
p33
is
expressed by
autoimmunity
depletion of regulatory T cells, APLs
removed from the
mice
An in vivo killer assay
prevalence.
no
are
Using altered peptide ligands
lymphocytic choriomeningitis TCR
system
where self-reactive T cells
induce diabetes. Thus, cross-reactive T cells fail to mediate T cells
a
prevent autoimmune diseases
model for diabetes in which
p33-VLP
Using
molecules and the
findings
selection
transgenic mice, only p33-
sufficient to lead to dose- and
APL-VLPs failed.
these
molecules in the
antigens, the
cross-reactivity
transgenic
alphaviral Delphi
an
co-stimulatory
central tolerance.
mice showed
recognized, wild-type
key
undergo negative
as
T cell
autoimmunity.
restricted TCR
particles
immunity
for novel maturation factors.
between self and non-self. To
virus-like
regulatory
checkpoints. One
at various
revealed that CCL19 is indeed
upregulation
of the
process described
derived from the
a
system therefore developed
immune responses and demonstrate the
the T cells
self-reactivity,
APLs in
generated randomly,
DC-T-cell-co-culture, the chemokine CCL19
a
DCs in the balance of self-tolerance and
was
for
pro-inflammatory cytokines. Altogether
for CCL19 in
eliminated,
auto-reactivity
importance of DC maturation
the
format assay
gene/well
due to
The immune
are
is the interaction of T cells with dendritic cells, since activation
screen was
secretion of
antigen receptors
tremendous
a
immature DCs results in tolerance while mature DCs may
by
technology
or
system generates
the immune
the
specificities. However,
diversity
In order to
fight intruding pathogens.
the
the ß-
whereas the were
autoimmunity
able to
unless the
2
Summary
Using
the
antigenic sin" during
inducing
model
studied.
was
system of p33
and APLs, the
Original antigenic sin
phenomenon
describes the
strong response against the original virus, but
a
"original
called
concept
that
infection, arising escape mutants evade the immune system by
viral
a
against
same
infection with the
virus and later
original
only
a
weak response
immunization with APL-VLPs simulated initial
experiment,
themselves. In the
no or
immunization mimicked escape
p33-VLP
mutant infection. To characterize the effector function of induced T cell responses,
mice
vaccinated
Interestingly,
challenged
were
initial
APL-VLP
generated by subsequent p33 the
with
prevented
immunization
vaccinia-p33
recombinant
a
protective
a
vaccination. Hence the results
are
virus.
response
in accordance with
concept of the original antigenic sin. Auto-reactive
Therefore the
T
cells
regulation
are
or even
interfere with immune responses, lectin
galectin-1
making acts
as
it
a
in
key players
the
elimination of these T cells would be
especially self-destructing
is known to induce
apoptosis
thymocytes
in
a
possibility
homodimeric molecule,
galectin-1
need for dimerization, two covalent dimer and the
galectin-1
are
proliferating
and
required
of this
to
an
attractive
candidate
for
were
activity.
galectin-1
a
for
To circumvent the
genetically
molecule
new
structurally optimized form of galectin-1 exhibited it
T cells,
is monomeric at concentrations below
monomers
apoptotic activity
to
immune responses. The
candidate for the treatment of autoimmune diseases. But since
1uM, high concentrations of the protein
making
autoimmunity.
of
development
ten-fold
linked to form
was
evaluated. The
increased
therapeutic
a
activity,
intervention.
3
Zusammenfassung
ZUSAMMENFASSUNG Das
eine
riesige
Pathogène.
Es
effizient
erkennen. Da die
zu
mächtiges Werkzeug
ist ein
Immunsystem erzeugt
Diversität
Spezifitäten
von
zu
um
fremde
verhindern oder
das
entwickelte
unterdrücken,
zu
Spezifitäten,
Antigene
zufällig generiert werden, ergeben
aber
sich auch solche mit Selbst-Reaktivität. Um das
Reaktivität
Kampf gegen eindringende
im
um
die Selbst-
verschiedene
Immunsystem
Mechanismen. Ein solcher Mechanismus wirkt bei der Interaktion zwischen T-Zellen
und
Interaktion der
Zellen, denn abhängig
dendritischen zu
Toleranz
Wichtigkeit
DC-Reifung
der
Technologie-Screen identifizieren. Ein
führte
zur
zeigen
um
In weiteren
für DCs ist, der
DCs
zu
auf DC-T-Zell-Kulturen,
potenter T-Zell-Proliferation-
gezeigt werden,
Hochregulierung
dass CCL19
aktivierenden
von
entzündungsfördernden Zytokinen führt.
Rolle
von
CCL19 bei der Kontrolle
Wichtigkeit
von
DCs im
neue
auf und betonen die
zur
für
von
Absonderung
eine
konnte
führt die
In Anbetracht
Reifungsfaktoren
neue
Untersuchungen
DC
alphaviraler Delphi-
Immunität wurde ein
des Chemokins CCL19 als
Reifungsfaktor
Molekülen und
für die
der
(reife DCs).
„Ein-Gen/Well-Format-Assay", angewendet
Induzierungsfaktor. wirklich ein
oder T-Zell Immunität
durchgeführt,
[1]
Identifizierung
zur
Befunde
(unreife DCs)
Reifungsgrad
vom
Gleichgewicht
von
Diese
Immunantworten
zwischen Toleranz und
Autoimmunität.
Rezeptor
Der T-Zell
ermöglicht
zu
verhindern, die
einem
zu
Autoimmunität führen kann, unterziehen sich die T-Zellen der
Selektion während der
Dieser Zustand
Peptid Liganden (APLs)
Choriomeningitis TCR
nur
von
p33-
solche
als
aus
im
als zentrale Toleranz bezeichnet. die
gebunden
vom
an
p33 Antigen
um zu
p33
einem
und APLs
Während
Dosis- und
Virus-abhängigem
des
Lymphocytic
Trotzdem
in
TCR-transgenen
zeigen Wildtyp-Mäuse
/n-wVo-Killer-Assay gefunden.
Mit veränderten
virus-ähnliche Partikel, wurde das
und A4Y-APLs effizent erkannt wurden, In
zu
dem die selbst-reaktiven T-Zellen eliminiert
Modell-Antigene,
Prävalenz.
Selektion führt
Thymus. Negative
Wildtyp-Mäusen analysiert.
Kreuzreaktivität zwischen
ausreichend,
wird
Virus abstammen,
Repertoire
Mäusen keine
Reifung
begrenzten TCR Repertoire,
wurden.
Adaptiven Immunsystem.
das Unterscheiden zwischen Selbst und Fremd. Um Selbst-Reaktivität
Er
negativen
ist eines der Schlüsselmoleküle im
wurde
war
Viren-Schutz
nur
wenig
die Kreuzreaktivität zu
führen. In einem
4
Zusammenfassung
führte
p33-VLP Immunisierung
Immunisierung.
Induktion
regulatorischen
T-Zellen
Zellen
nicht
zur
Eliminierung
der
Mit
von
Diabetes mit APLs
möglich.
Induktion
Autoimmunität
zu
Deshalb kann
man
T Zellen
regulatorischen
aus
dem
antigenic sin"
Immunisierung
schwache Antwort gegen
eine
nur
simulierte die Infektion mit dem
Immunisierung imitierte eine Infektion
System.
Konzept der "original
das
verhinderte
Mäusen
eine
vorangegangene
APL-VLP
den
p33 Immunantwort,
schützenden
„original antigenic sin" Selbst-reaktive Autoimmunität. Die
Möglichkeit,
T-Zellen
Regulation
gezeigt,
Zellen induziert.
Eine
von
aktiv ist und
es
um
ein
zu
stabiles
was
in
Immunisierung
zehnmal
der induzierten T-
Übereinstimmung
Errichtung
die
mit dem
Schlüsselspieler
sind
oder gar die
dass
es
Fähigkeit,
Eliminierung
in
Apoptose
die
es
zu
Autoimmunerkrankungen
einer
Konzept
zu
Dimer
zu
höhere
therapeutische Eingriffe
der
von
dieser T Zellen wäre eine
verhindern. Für das Lektin und
potentiellen
proliferierenden
Kandidaten für die
macht. Aber weil Galectin-1
hauptsächlich
T-
nur
als Dimer
als Monomer
vorliegt,
nötig.
Um das Problem der
wurden zwei Galectin-1 Monomere
genetisch verbunden,
bilden
Aktivität bei
Entwicklung
der
Thymozyten
einem
bei Konzentration unter 1uM
umgehen,
in
und
die
Apoptose-Aktivität
Molekül wurde untersucht. Diese strukturell verbesserte Version eine
p33-
Interessanterweise,
charakterisiert.
sind hohe Konzentrationen des Proteins für die Aktivität
Dimerisierung
Die APL-
Virus und die
Schutzwirkung
selbstzerstörische Immunantworten
um
selbst auslösen.
steht.
Galectin-1 wurde
Behandlung
sich
aber
mit einer Viren-Mutante. Durch die Infektion mit
geimpften
in
Antwort
Immunsystem
originalen Virus,
ursprünglichen
einem rekombinanten Vaccinia-Stamm wurde die Zell
die
braucht zusätzlich
es
der
dass kreuzreaktive T-
folgern,
dadurch enfliehen, dass sie eine starke Antwort gegen den oder
Entfernung
Infektion auftreten, dem
Viren-Mutanten die während einer viralen
keine
APL-VLP
sin" beschreibt das Phänomen, dass die
„Original antigenic
untersucht.
zur
erst nach der
war
gleichen p33/APL-Modellsystem wurde
dem
Gegensatz
im
Autoimmunität reichen,
von
exprimieren p33),
Zellen des Pankreas
transgenen Diabetes-Mausmodell (ß-lnsel
auf
und
ist
deshalb
Autoimmunerkrankungen.
ein
von von
diesem
neuen
Galectin-1 weist
attraktives
Molekül
für
5
Introduction
INTRODUCTION 1.T CELLS T cells the
cells
B
subset of cells
are one
they allow the
immune
pathogens. Additionally, they allows the with the
organism
same
to mount
pathogen. The
constituting the adaptive immunity. Together with
are a
system to specifically respond to various
able to form
stronger
T cell
an
and faster response to
repertoire
can
memory, which
immunological a
second infection
be subdivided in different
subtypes
(Figure 1).
Figure 1. are
T cell
subtypes:
In the
thymus
T cells
develop either
further subdivided into CD4+ and CD8+ T cells. After
in the
periphery
into T
helper cells type
1
periphery into cytotoxic T cells (CTLs). Tregs of the
Tregs develop
in the
can
can
be made
which is the definitive T-cell
or
the
aß
yö
or
T cells. The
aß
the CD4+ T cells
thymus
T cells
develop
type 2 (TH2). The CD8+ T cells develop in the
be of either of the different
thymus and others arise
A first differentiation
(TCR),
(THi)
leaving
into
in the
by
subtypes
of T cells. Some
periphery.
the
expression of the
lineage marker.
There
are
T cell
two defined
receptor
types of
Introduction
TCR:
6
is
one
a
[2,3].
subunit
complex,
heterodimer of
Both
receptors
and form the TCR
blood T cells and their T cells
comprise the
expression of CD4 immune
subset
positive,
is
lymphocytes (CTL). molecules
and
associated with
are
(90-95%).
largest part of
T
the
yö
cytokine production.
according protein
4
to the
(CTLA-4).
Whereas
cells
are
(IFN-y) secretion,
responsible
the
cells
populations
expression of surface
or
a
are
cytotoxic
named
MHC class II
major
by
expressing
can
markers
T cells
are
be further or on
the
subdivided
cytotoxic T-lymphocyte associated on
CD4+ T cells delivers same
ligands
on
a co-
antigen
negative regulatory signal [6]. or
Th2 cells according to their pattern of
characterized
by interleukin-2 (IL-2)
and interferon-
whilst TH2 cells secret IL-4, IL-5, IL-6 and IL-10. THi cells
for cell-mediated
T
A small subset of the aß and
All these
aß TCR
their
second, CD8
The
presented
antigens [5].
expressing
mainly helping inducing
(TH).
cells
of CTLA-4, which binds the
subdivided into THi
cytokine expression. Tm Y
negative".
either CD28
(APCs), provides
The TH cells
by
are
of the
distinguished by
therefore
and
engagement of CD28
stimulatory signal, engagement presenting
helper
recognize
example,
For
expression of
T cells
class I molecules
"double
are
further
are
the CD3
portion (5-10%)
recognize antigen presented by
cells
subdivided into functional subsets basis of
expressing
The CD4+ T cells
histocompability complex (MHC) the
The aß T cells
therefore called T
CD8+
minor
are a
y and 5
a
polypeptides,
function is still controversial, aß TCR
predominantly cytotoxic
the
set of five
a
T cells
complex [4]. yö
CD8. The CD4
and
responses
and ß subunit and the other consists of
physiological
rest
or
a a
immunity,
whereas Th2
are
responsible
are
for extracellular
immunity [7]. An additional subset of T cells
much
debate, reintroduced
regulate
as
were
initially
regulatory
immune responses and may be
T
predominant TregS
co-receptor (CD25). They
mediate their
[8,9].
like
transforming growth
cells
important
be of either subset, but the
cytokines
named suppressor T cells and, after
are
to
(Treg).
These
CD4+ T cells
(TGF-ß)
negatively
prevent autoimmunity. Tregs
regulatory function
factor-ß
cells
or
by
expressing
via the
can
the IL-2
secretion of
direct cell-cell interaction
7
Introduction
1.1.
T cell
important feature
antigen presented
of the
recognition
The
receptor
antibodies the TCR does not to be
processed
similarities. each
to shorter
both
They
generated by
subunit
[12].
a
a
to the
disulphide-linked
the
aß [2] a
The structure of the TCR
comprises
with
short
solved
cytoplasmic
TCR
tail
[13].
variability of the
TCR resides in the N-terminal domains of the
region of
regions,
the
greatest variability corresponds
also known
antigen binding
site
on
an
transmembrane
assembly the
yö
antibodies
CD3
do
[4].
not
are
associated
The CD3
Interestingly,
a
a
and ß
polypeptides.
which is
These
are
to the
analogues
comprise positively charged residues these
residues
are
important
in
for the
transport of the TCR complex [16]. Both the aß and
physically with
components show
no
a
series of
amino acid
called y, 5,
e
and
the
Z, [17-20].
polypeptides, collectively
variability
Functionally
following antigen recognition by
polypeptides,
by
[15].
generate additional diversity.
transduction invariant
region.
and the intracellular
TCR
membrane
immunoglobulin hypervariable
MHC-antigen binding site
An unusual feature is that both subunits
the
(35-47 kDa)
complementary-determining regions (CDRs) [14].
to form
together
clustered
as
to
ß
The aß TCR
The amino acid sequence
peptide
The
[3].
immunoglobulin-like
plasma
transmembrane
a
are
by X-ray crystallography
extracellular into the
receptors
germline genes [10].
(40-50 kDa) and
was
two
clonally distributed;
And the
öy
and the
a
110 amino acids, anchored
domains of about
both
limited number of
a
heterodimer of
polypeptide chain
Each
are
specificity.
own
has
to the TCR.
Ig superfamily. They
receptor with its
antigen
antibodies and TCRs share many
Antigen recognition,
types of TCRs exist,
(Figure 2) [11].
and furthermore the
recognize free antigen
somatic recombination from
Two different
comprises
belong
has
lymphocyte
immune response. In contrast to
specific
a
peptides prior presentation
this difference in
Despite
guarantee
of T cells to
MHC class molecules is the most
on
CD3 TCR.
on
is
different T cells and
important
CD3
called
for
comprises
signal of four
8
Introduction
antigen binding
site
T T
Figure
chain of the T cell receptor and the each
variable and
a
antigen.
Furthermore
a
constant
they
contain
proteins
chains. The CD3 y, 5 and
e
negatively charged
proteins
region.
for the interaction with the
is the
chains
a
from the CD3
The variant
of the CD3 are
transmembrane domain and
each. The ITAM motifs contain
leading
to
a
signal
tyrosine
£
complex is composed of
and two
products of
sequences
only
can
be
a
5, two
£
important and two
TCR, and
small extracellular
an
ITAM
part, also
phosphorylated by specific
Ç
feature
part containing three ITAM
three
[23]. They
domain followed
by
a
closely
are
one
aß heterodimer,
Ç polypeptide chains. The CD3
heterodimers of Y£ and 5e and are
residues which
which is
one
interacts with the
intracellular
long
y,
site for the
binding
primary sequence. An important
chains contain a
and the ß chain contains
segment, one
5)
y and
(or
a
motifs
kinases
transduction to the nucleus.
The CD3-TCR chain and two
Ç
which
a
build up the
consists of
related in their
segment,
The
transmembrane
complex. CD3
closely
transmembrane
complex.
regions together
positively charged
sequence in the intracellular domain. The CD3
negatively charged
the a and the ß
receptor: The T cell receptor complex consist of
2. The T cell
a
homodimer linked
ÇÇ [21,22].
genes
members of the
which
chains
one are
y and
organized
The CD3 y, 5 and
are
related
in
their
Ig superfamily, each containing
transmembrane domain and
a
an
£
5
one
as
chains
primary external
highly conserved cytoplasmic
9
Introduction
tail. The transmembrane domains contain
important for
vitro mutation of these
complex [16]. tyrosine
The
The CD3
Ç
cytoplasmic
a
a
containing
tail
major difference
recognition
are
yö
than
generally
MHC molecules at all
1.1.1.
The
aß
impaired
Genes
not
Mutation
amino
are
experiments
essential for T-cell
are
antigen-MHC,
a
these motifs become
encoding
resistance to
to
such
yö
TCR. The
as
epidermis,
intestinal
a
are
different
genetically
deficient
infections
to be
antigen
recognize
T cells
some
require antigens
the genes for the a,
mouse
chain locus. The
segments leading
epithelia,
T
aß
bearing
are
peripheral
[32,33]. Some
presented by
classical
the T cell receptor
immunoglobulin heavy
segments [37]. In the
aß counterpart [30].
[34,35].
The diversication of the TCR gene
a
only
there may
Furthermore differences in function and
a
ß
ß,
y and Ö chains of the TCR is very
(Figure 3) [36].
chain
sets of V genes and J genes while the
of the
tyrosines
T cells showed that animals which
have
arrangement of
similar to the
the
Research to clear whether
suggest that yö cells do
studies
[24]. Altogether
TCR is similar to that of its
tongue [31].
proposed.
antigen
cells
negatively charged
three ITAMs
especially
abundant in various
are
uterus and
yö
with
is their distinct anatomical locations. 95% of the
epithelium,
in
Ç
majority of the TCR-expressing thymocytes
T cells
classes of
chains consist of
The
within minutes.
The overall structure of the
yö TCR
[26].
segment,
After the TCR is bound to the
[28,29].
cells and the
immuno-receptor
present in the CD3 complex. The tyrosine residues in these motifs
phosporylated
The
one
assembly of the
different chromosome than the other CD3
transmembrane
have shown that the ITAMs and activation
a
phosphorylation by specific protein kinases [27].
for
target
part,
on
unrelated to them
large cytoplasmic
be 10 ITAMS a
the
prevents
(ITAMs) [24,25].
structurally
small extracellular
are
amino acids from the TCR. In
tail of each of the chains carries
gene is encoded
subunits and it is
amino acids
negatively charged
activation motif
acids and
positively charged
the interaction with the
amino acids which
negatively charged
The
a
and y chain have
and 5 loci have sets of V, D and J genes.
occurs
by
recombination between V, D and J
the 5 chain locus is embedded between the V and J loci chain locus carries about 100 different V and also J
great diversity.
The
ß
chain locus and ö chain locus contain
Introduction
10
joins.
segments, which allow
D
additionally
The fact that the D
diversity.
some more
segments
a
are
V-D-J
but also V-J
arrangement
used in all three different
The y chain locus is
arranged similar
locus, with four C genes, each associated with
V-D-D-J
frames adds
antibody light
to the
J gene, and
one
reading
or
one
chain
to four V genes
[38]. mouse
TCR a-chain and 5-cham DNA
LVa1
LVa2
LV„n
{chromosome 14)
LV„1
L
Vgrt //
mouse
v
=
=
Figure
TCR
ß-cham
Il
H
C6
3. Genes
ß
encoding the
T cell
and 5 chain DNA. The
a
chain
(also
recombination to build the variable
V-D-D-J-C
V(D)J
3.
on
join.
region
the surface. The
region.
ß
has sets of V and J genes, which
y)
the
chain
The
(or 5)
product
The locus for the 5 chain is embedded in the
recombination
is
tightly
in
regulated
the recombination at TCR and of
rearrangement
example,
limited
is
are
limited
regulated temporally
TCRß
rearrangement
rearrangement, which one
loci is mediated
TCRs
rearrangements of Ig genes recombination is
Ig
of two alleles
allelic exclusion
occurs
in DP
to
and in
occurs
B a
in
T
a
the cell
can
same
cells,
functional
V-D-J-C,
a
V-J-C
recombinase
whereas
[39].
lymphocyte
specificity. Although
the
complex, complete
Within the cell
stage-dependent DN
a
of
sets of D genes,
of
context
and allele
the
be
are
chain locus.
the
cells
additionally
manner
in
a
lineage,
[40].
before
thymocytes
thymocytes. Moreover,
usually undergoes
(Figure 4) [39].
to
by
on
has
are
product
of the a chain. T cells express the
development, exhibiting lineage, developmental stage,
as
t
receptor: (adapted from Immunology, Janis Kuby) Shown
which allows further variation of the variable
only
C„
Jan
Jn1J„2
DM H-a
6)
functional V-J-C recombination
the
V55
L
enhancer
rearranged by
or a
Jb1 J52
pseudogene
the TCR a,
a
DNA (chromosome
Da1D„2
For
TCRa
given lymphocyte,
rearrangement,
a
process known
Introduction
11
LV„1 German» »chain DNA
LVan
LV51
LVj»
Dj-ID^
Js1 J«2
HJM~4JBHflH{HHHI
5'
Ca
Vs5
L
Cn
J„n
Ja1Ja2
H"HHMHH—1
WÊt~
3'
^ L
Rearranged
Protein
L
V„1
V„2
L
V„
J„
J„r>
C,
a-chain DNA
product aß heterodimer
Cfi2
L
Germ-Hneß-chamDNA
Janis
4. Gene
Kuby)
L VRn
VR1
rearrangement yielding a
-JH1-
Dpi
Cp1
chain
undergoes
Added to the V-J is the C
region joinings: First
a
a
D to J
and
possible.
The
functional
variable V-J
segment building
a
the V to
recombinations
are
tightly regulated
in the context of T cell
aß
TCR
rearrangement
Or is
a
as
diversity
chain. The
ß
mentioned
dependent
development, lineage
is achieved
to the variant
on
cells. However, the
are
different
rejected owing
2.9x1020 possible in
a
life-time of
L V„14
to
are
coding
[41].
two variable-
also VJ
VDDJ
or
products
and is
specificity.
and allele
provides additional immunoglobulin
only by rearrangement
of TCR genes. This may
recognition of MHC by T was
estimated that about
possible. And assuming that 99 % of the sequences
for
auto-antigens
or
other defects, this would still
murine TCRs, and this with less than
a mouse
Immunology,
of the a chain.
undergoes
the RAG gene
generated diversity is tremendous. It
aß TCRs
chain
from
region
alternatively
be linked with the need to maintain tolerance to self and
2.9x1022
C„2
receptor:(adapted
joining, leading
complete DJ.
T cell
In contrast to B cells where somatic mutation
diversity,
-J«2-
Dp2
^M'~-\W^^MmM-m^Hm¥m-^t^Ê-
,
The
VB14
-ö»-iJHIr-E3HHr
Rearranged ß-chain DNA
Figure
L
109
T cells
leaving
the
give
thymus
12
Introduction
1.2.
T cell
development
Lymphoid progenitors (BM)
via the blood to the
migrate
and
for B cell
[42-44]
negative (DN;
no
arise from
further subdivided
CD8),
according
thymus they
In the
thymus.
and natural-killer-cell
CD4 and
stem cells in the bone
hematopoietic
loose the
development [45]. The result is
committed T cell precursor. DN
to their surface
expression of
marrow
potential a
thymocytes
double can
be
CD44 and CD25: DN1,
CD44+CD25-; DN2, CD44+CD25+; DN3, CD44-CD25+; and DN4, CD44-CD25-
(Figure 5) [46]. T cells
can
either express the
starts from DN3 cells
encoded of
a
by
a
and
a
or
aß TCR. The aß is the
DNA-rearrangements,
RAG2) dependent [50,51].
associated with
proximal signal
a
collection of
transduction
development [53].
chain
pre-TCR-a
non-rearranging locus, pairs
set of somatic
(RAG1
expressing
yö
[47-49].
more
The Pre-TCR-a chain,
with the TCR ß chain, which is the which
are
Recombination
Active
At the T cell surface the
signaling
is
required
product
Activating genes
proteins (the CD3/Ç complex) that is
[52].
dominant and
pre-TCR-aß
is
involved in the
for further
thymocyte
Introduction
Figure
5. T cell
progenitors
development
from the bone
marrow
TCR, CD4 and CD8, and
thymocytes
can
in the
are
thymus: (adapted
arrive in the thymus.
be further subdivided into four and
Early
no
CD4 and
of differentiation
stages
[54])
Committed
committed T cells lack
negative (DN;
termed double
CD44+CD25+, DN3, CD44-CD25+;
from Gemain et al
lymphoid
expression of
CD8) thymocytes.
the DN
(DN1, CD44+CD25-; DN2,
DN4, CD44-CD25-). During the progression from DN2 to
DN4, the cells express the pre-TCR, which is composed of the non-rearranging pre-Ta chain and
rearranged
TCR
positive state, yields a
a
ß-chain. After successful pre-TCR
with
replacement
complete aß TCR.
high density
pre-TCR a-chain
The double
positives
with
then interact with cortical
peptides ligands. selection
the
signaling
induced
Insufficient
signaling
results in death
by
on
acute
apoptosis.
(positive selection). Thymocytes
a
TCR
CD4+ T cells. These cells then are
exported
T cells that emerge from
to the
chain, which
cells that express fate of the DP
the TCR with the
MHC-self-
signaling
antigen
in
a
initiates effective maturation
in MHC class I context become MHC class II context
develop
to
peripheral lymphoid sites.
ß selection
recombination at the TCR-a locus
epithelial
a
by neglect. Too much signaling promotes
binding antigen
CD8+ T cells, whereas those that express TCRs that bind
progress to the double
self-peptides. The
by the interaction of
Intermediate level of
that express
the cells
newly rearranged TCR
a
of MHC class I and II molecules associated with
thymocytes depends
negative
of the
expression,
a
undergo
they express the
some
cell divisions and after
mature aß TCR.
They
also start
14
Introduction
to express the
CD4+CD8+
co-receptor molecules
(DP; double-positive)
constitutes 90% of the T cells
positive
T
lymphocytes
are
death
(death by neglect,
molecules survive CD8 cells
migrate
cells. In the
[55].
to
epithelial
cells),
on
whether
into the medulla of the
In the medulla,
they bind
reactive T
lymphocytes
responsiveness
or
during
normal
cells
are
T cell other cell
or
are
to
these double-
programmed
single-positive
class I,
cell
CD4
respectively),
or
and
self-antigens
are
thymus (due
usually anergized
or
thymus
organs and wait for their
with
not
high affinity
cell death
programmed
not deleted in the
in the presence
auto-antigens
cryptic antigens, i.e., self-antigens
secondary lymphatic
1.3.
into either
exposed
via TCR-mediated
development)
thymus,
deleted via
are
MHC class II
tissues. The mature T cells leave the
lymphoid the
to
that
of the
population
thymus [56].
single-positive
(negative selection)
population of
whereas T cells that bind to self-MHC
of self-MHC molecules. Those T cells that bind deleted
a
cells that express MHC class I and II
(positive selection), differentiate
(depending
into
this immature T cell
region
recognize self-MHC
~90 % of the
developing
thymus
In the cortical
exposed
molecules. T cells that do not
CD4 and CD8
to low
present
suppressed
[57].
are
Auto¬
affinity binding in the
in the
thymus
peripheral
via the bloodstream and enter
appointment
with the DCs.
T cell activation
signaling
uses
the
types, including
signal
transduction
B cells. A
pathways
key principle
is the
that
are common
clustering
of
to many
receptors upon
ligand binding. This leads to activation of the associated tyrosine kinases which
phosphorylate tyrosine followed a
by
tails of the clustered
cytoplasmic
the recruitment of additional kinases and
cascade of activated kinases. These
transcription factors in
residues in the
signaling
which lead to the induction of
[58], [59] and [60]).
receptors,
signaling molecules, resulting
in
cascades result in activation of
specific
gene
expression (reviewed
Introduction
Figure
15
6. TCR-mediated
signaling: After ligation
of the TCR with
antigen-MHC complex,
kinase LCK gets activated and starts to phosphorylate the ITAM domains in the CD3
phosphorylation
of the ITAM allows other kinases like ZAP-70 the
phosphorylation
events these kinases
the linker for activated T cells
proteins, which leads
bisphosphate (PIP2)
into
factor
NF-AT.
transcription.
Simultaneously
C
(PKC),
transcription. converts
The
to AP-1
which
DAG
inositol
allows
initiates
activates
the
By
complex. trans-
or
The
auto-
molecules like
of SH2 motif
cleaves
tyrosine
containing
phosphatidylinositol
trisphosphate (IP3). Additionally
LAT
IP3 releases Ca2+ from intracellular stores leading to
NF-AT the
regulation inducing
co-stimulatory
after activation
events.
binding
(PLC-y). PLC-y
enzymes like calcineurin, which
phosphorylation
upregulation, leading kinase
reorganization
and
C
to them.
phosphorylate adapter
LAT allows the
phospholipase
diacylglycerol (DAG)
Ca2+- dependent
The
activated and start to
(LAT). Phosporylated
to the activation of
activation leads to actin the activation of
are
binding
the
to
enter
the
phosphorylates nucleus
Ras/Raf/MAPK
IL-2
pathway
to
transcription
turn
leading
to
on
gene
Jun/Fos
production. Additionally DAG activates protein
transcription factor NF-kB.
molecule CD28 is associated with the
phosphadtidylinositol (PI)
and
the
into
NF-kB
also
turns
phospinositide-3
on
gene
kinase which
phosphatidylinositol 3-phosphate (PIP),
a
16
Introduction
of PIP2. All the
preliminary stage with IL-2
of the most
as one
events end up in the nucleus
signaling
important gene products leading
The TCR is associated with the CD3 an
antigen-MHC complex by
involving tyrosine
kinases of the Src
and CD4
ligation
shown in
Figure
6.
kinases
including
phosphorylation [62,63]. activated T cells of Src
binding
which
then
and
Ç
chains of the CD3
steps schematically
series of
a
gets
by
activated
The
trans-
auto-
or
the linker for
phosphorylation of LAT allows
and a'so leads to actin
the
which leads to the
(SH2) containing proteins,
2
(PLC-y)
C
£
phosphorylates adapter proteins like
homology region
molecules
of the ITAMS allows the association with other
(LAT) (reviewed in) [64].
phospholipase
activation of
the ITAMS initiates
ZAP-70
of
with CD4 and
family, particularly Lck associated
Phosphorylation ZAP-70,
co-stimulatory
of
CD8, and fyn which phosphorylate the ITAMs in the y, Ö,
complex [61]. Phoshorylation of
Recognition
CD8.
or
comprise tyrosine phosphorylation,
events. The earliest events
signaling
initiates the
to auto-activation.
complex
the TCR and
inducing gene transcription,
reorganization
events
[65].
PLC-y signaling
initiates the
by
intracellular
(ER)
Calcineurin
phoshorylates
(NF-AT)
which
stores to activate
causes
the
transcription
jun,
which
immediate
acts with
early
complex,
which also
stimulatory
on
be initiated
can
get
[69,70].
such
transcription
factor NF-kB to the
it to
as
from
calcineurin.
[66]. Additionally
transcription
the
IL-2
by growth
activated
by
gene factor
[67]. Alternatively
the
receptor-bound protein
kinase
(PI-3K),
signals, specifically by
which
on
of fos
promoter AP-1 Ras/Raf/MAPK 2
association with the LAT molecule
molecule CD28, when cross-linked with B7 molecules
Phosphoinositide-3
Ca2+
releases
factor nuclear factor of activated T cells
pathway leading
the APC, also adds additional activation
converting
IP3
into
(PIP2)
bisphosphate
genes for cell division, and activation of the
NF-AT
pathway
downstream
its translocation to the nucleus. Meanwhile DAG activates
DAG also activates the Ras/Raf/MAPK and
further
to
calcium-dependent enzymes
kinase C, which then activates the
protein
leading
trisphosphate (IP3).
inositol
and
both
phosphatidylinositol
of
cleavage
diacylglycerol (DAG)
pathways
distinct
two
(Grb2)-SOS [68].
The
co-
the surface of
the activation of
a
phosphorylates phosphatidylinositol (PI)
phosphatidylinositol 3-phosphate (PIP),
a
preliminary stage of PIP2
17
Introduction
most
activated is IL-2. This allows the T cells to remain activated
important gene
autocrine stimulation since IL-2 receptor activation
is also induced upon T cell
get activation upon antigen recognition
in order to
requirements
still under debate.
it is known that
Although
single TCR-MHC/antigen
a
are
interaction is
sufficient, the exact amount of TCRs necessary to be triggered in order to
activate the T cell is unknown. The
were
classifiable in
virus
(LCMV),
properties
on
against pathogens, modern medicine
they
example after
myeloid
cells
are
important
also
on a
agonistic
an
essential defense
antagonistic
against intracellular pathogens, played
also very
responsible
important for
allogenic
for the destruction of Natural
in the defense
the clearance of tumor cells. In
killer
tissue
cells and also
(NK)
Most CTLs
are
recognize
CD8+ and
recognize antigen presented
peptides after
infection.
and
molecules
on
some
the
them
recognition by
by
cell
CTLs. For
of MHC class I molecules in infected cells,
T cells
lysis.
as
antigen
cytotoxic
T
class II molecules. The most
the surface and
on
Antigen specific CD8+
eliminate
mechanisms to avoid
I
grafts,
cytotoxicity. CTLs recognize specific antigens
role of CTLs is the elimination of virus infected cells.
cells
or
MHC class I molecule, but about 10% of MHC-restricted
CD4+ and
to activate
ability
[73,74].
mediate
MHC class
[75].
their
on
antigens
T cells,
lymphocytic choriomeningitis for
analyzed
were
transplantation.
cells express
these
is
MHC molecules.
presented by presented
organ
can
are
are
from
study of
bacteria. Besides the central role
some
CTLs
the
lymphocytes (CTL)
T
cytotoxicity
viruses and
including
T cells
Cytotoxic
p33 peptide
the
thereof
variants
and
Cell-mediated
cells
Especially
p33-specific
1.4.
requirements. By
antagonists, depending
and
agonists
T cell clones.
specific
story is further complicated by the fact that other
may influence the
stimulatory signals
for
expression
by
[71,72].
The exact
not
of genes in the nucleus. The
transcription
events induce the
signaling
All the
are
Several
Virtually
all nucleated
start to
present viral
then able to viruses
recognize
have
evolved
example, they reduce the expression
shown for the human
cytomegalovirus
18
Introduction
Figure 7.Cytotoxic antigen
on
T cell mediated
the surface of
perforin/granzyme pathway: the content of the vesicles
Ca2+-dependent
manner
a
killing: After
target cell,
it
can
cytotoxic
initiate
leading
to pores in the
target cell
apoptosis program
damage and impairs the repair mechanisms which lead caspase 8 induces the caspase cascade on
killing by
(CTL) two
has
recognized
and
the CTL and binds to Fas
leading
on
to
to
apoptosis
apoptosis.
the surface of the
non-self 1.
The
After activation,
perforin.
polymerizes
in
a
Through
these pores the
Granzyme
A leads to DNA
membrane.
in the cell.
a
pathways.
different
released into the cleft between the two cells. Perforin
granzymes enter the cell and start the
upregulated
T cell
containing granzymes
CTL carry vesicles
are
a
and the granzyme B, similar to
2. The Fas
-FasL-pathway:
FasL is
target cell. This initiates trimerization of
which Fas, allowing the association of transducing molecules like FADD. FADD recruits caspase 8,
gets activated by cleavage
and initiates the caspase cascade
The CTLs have evolved several different
target cells (reviewed in) [76]. Cells
Apoptosis
systematically
is
a
can
leading
pathways
die in two ways
-
to
by necrosis
highly ordered, physiological process,
cytoplasm [77].
of the
or
apoptosis.
in which the cells
are
integrity and cytochrome
Then the nuclear DNA is cleaved into
endonucleases. The cells detach from their
target cell.
to induce cell death in the
disassembled. Mitochondria lose membrane
C leaks into the
apoptosis
fragments by
neighbors and the cytoplasm
and the
19
Introduction
nucleus condense.
fragments
into condensed
phagocytes
and
phagolysomes.
the cell
Finally,
Most
apoptotic
rapidly
are
are a
side of
aspartate residue. They
an
by cleavage
into two
forming
caspase cascade
[78]. Therefore apoptosis
by cleavage
activated In
present in
are
caspases
comparison
or
leading
apoptosis
produced
are
broken
and
in cells
in
the caspases.
on
the C-terminal
proenzyme form and become
a
to the destruction of the cell in
extremely fast
synergistically
necrosis is
a
on
to
apoptotic
an
are
way
then the
need to be
they just
the destruction of the cell.
dying
less ordered event, in which
thereby leading
together
target cell,
in the
inactive proenzyme form, and
an
in
down
are
three subunits. Several caspases
be induced
can
to act
their contents,
releasing
by phagocytosis
up
proteases, which cleave their substrates
activated a
cells attract mononuclear
Apoptotic
for mediation of
Caspases
group of
bodies.
taken
important
blebs and the cell
membrane starts to form
cells fall
apart
macrophage
activation and inflammation
CTLs is the
perforin/granzyme pathway
[79]. pathway
One
(Figure 7,
upper
structurally
and
Granzymes
are a
that
cytotoxicity
of
panel). Perforin
functionally
as
is
related
a
to
pore-forming protein
monomeric the
and
DNA
on
intracellular
degradation [83-85]. Granzyme
the caspases. In order to activate the
granzymes need to enter the cell. This is the
apoptotic pathway
point
where the
released into the cleft between the two cells. Perforin
incorporated channels
into the
target
[86], through
cell membrane and
which granzymes enter the
Granzyme
B
initiates
caspase-independent
a
triggers apoptosis by
[88,89]. Interestingly, despite
or
caspase
cell
death
target
by cathepsin
B which
was
cells the
monomers are
to form transmembrane
cleavage [87],
whereas
apoptosis.
Granzyme
A
the
DNA
target cell, perforin
does
pathway, causing
explained by proteoglycan
same
target
cell and induce
the close contact of CTL and
not affect the CTL. This may be
through binding
polymerize
a
pathways
CTL, perforin and
granzymes
recognition by
in
is
[80-82].
perforin and
synergistically. Upon target
cell
that
B has the
granzymes act are
C9
complement component
collection of serine esterases, which act
trigger apoptosis
specificity
by
used
nicks
in
which inactivates
found to inactivate
perforin
perforin by cleavage
[90,91].
Additionally factor
CTLs may induce
apoptosis through
(TNF) receptor family (Figure 7,
lower
panel).
members of the tumor necrosis
The
ligand
of Fas
(FasL),
one
20
Introduction
member of this group, activation
[92]. Ligation
is
expressed
on
CD4+
mature
and
CD8+ T cells after
of Fas induces trimerization of the Fas molecules
the cell
on
surface, followed by association with adaptor proteins, like FADD which recruits and activates caspases 8 and 10
TNF and on
lymphotoxin,
Fas but
on
which
TNFR-1
[93-96].
can
be released
(CD120a). They
adaptor protein (TRADD, FADD, caspases activation. These which
are
important for the
1.5.
THi
versus
Naive TH cells an
can
immune response
Most CD8+ T cells have vesicles
RIP
on
target cells and act similar
cause
and
immunity,
addition to
RAIDD) recruitment, which
adaptor proteins carry
recruitment of caspases
important opsonizing
in the
so
and
[5]. THi cells,
called death domains
(DD)
(reviewed in) [97].
first characterized
are
class
complement
and TH2
responsible
for cell-
are
responsible for extracellular immunity. In
TNF-ß
[7].
switching. Tm promote
the
fixing
during
by IFN-y secretion,
and IL-2
cells also secrete
antibodies.
macrophage activation, antibody-dependent type hypersensitivity [100].
to
initiates
differentiate into at least two functional classes of cells
antibody
FasL
TH2
whereas Th2 cells
IFN-y, thi
as
receptor trimerization leading
cells, which secrete IL-4 (reviewed in [98,99]). THi cells mediated
containing
The TH cells
production
Furthermore,
cell-mediated
of
they
cytotoxicity
are
and
also
lgG2a,
promote
delayed-
21
Introduction
macrophages
NKT
DCs
mast cells
basophils
IgE lgG1
extracellular immunity
lgG2a
intracellular pathogens
organ-specific autoimmunity
Figure
8. Schematic
[101])
from Liew et al
representation of A precursor
TH
can
induction and
differentiate into THi
cytokine environment, lnterleukin-12 (IL-12) ligation of
the
transcription TH2 program initiates
4
IL-12
and the molecule T-box
with IL-4
as
an
autocrine Tm
autocrine TH2
expressed
promotion
promotion
IL-4, IL-5, IL-6, IL-9, IL-10
IL-13. THi cells induce
defence
against
or
intracellular
pathogens.
TH2 cells induce IgE and lgG1 Associated with TH2 response is
are
allergy.
over
signal
on
the
and
transducer and activator of
(T-bet),
in T cells
is initiated. IL-4 induces
down-regulation of
IL-4 and
IL-4
production
and
IFN-y block THi respectively TH2
lgG2a isotype
Associated with
isotype switch
TH2 cells depending primarily
IL-2 and TNF-ß. The
IFN-y,
THi responses
(adapted
factor. On the other hand GATA3 induces the
factor.
responses. Main mediators of
or
and TH2 cells:
Tm cells, whereas IL-4 promotes TH2 cells. By
STAT6 and GATA3. T-bet induces the
IFN-y production,
opposite,
drives
receptors the Tm program, leading
(STAT4)
over
regulation of THi
typical TH2 cytokines
switch and
are
are
important for the
THi response is organ-specific autoimmunity.
are
important
in
the
extracellular
immunity.
22
Introduction
Th2 cells
characterized
are
by
the
commonly produced [102].
IO and IL-13 also
stimulation of mast cell and
of IL-4 and IL-5 with IL-6, IL-9, IL¬
These cells
and
including lgG1
humoral immune response,
immunity,
production
provide optimal help
and mucosal
IgE isotype switching
eosinophil growth
for
and differentiation and
IgA
synthesis [100]. implicated
Both subsets of Th cells have been cells the
organ-specific autoimmunity
mediate
can
and Th2 cells have been
allergy [103]. Considering
of asthma and
pathogenesis
pathological responses. THi
in
that Tm
implicated
cytokines
versa, the immune response tends to settle either into a
TH2 cells and vice
Tm
in
inhibit or
TH2
type of response. and
Thi
Th2
further
can
be
distinguished by surface marker expression.
Chemokine receptors CXCR3 and CCR5
[104,105] are
[105,106].
Several factors influence the decision whether
Th2- Dependent
on
provided [107,108], affinity,
a
THi
or
the site of
by
the
co-stimulatory
the
the
develops
peptide-density
TH2 response is induced. Furthermore it may depend
For
antigen.
on
cytokine profile
the
With defined in vitro culture either Tm
or
IL-12
APCs
molecules
and on
balance of was
or
binding
the host
cytokines shown to
systems and T cells from TCR transgenic mice, the
TH2 cells
was
on
into THi
[109].
was
extensively
found to be essential for the induction of Th2 cells from
right panel) [110,111],
and
example, high peptide density
favor Thi and low densities favor Th2 responses
development of
CD4+ T cell
antigen presentation,
genetic background and finally evoked
a
properties of the immunogen,
the
Tm cells
on
receptors CXCR4, CCR3, CCR4 CCR7 and CCR8
and whilst chemokine
associated with TH2 cells
preferentially expressed
are
shown to
play
the
studied. Whereas IL-4 naive
same
precursors
role in the
was
(Figure 8,
development of
Tm cells (Figure 8, left panel) [112,113]. IL-12 and IL-4 originate both from the innate immune response. NKT cells, mast cells
macrophages IL-12
and DCs
produce
IL-12
or
basophils
[117-119].
secrete IL-4
[114-116]
In THi differentiation,
and
signaling by
receptor leads to activation of STAT4 (signal transducer and activator of
transcription 4) [120] and induction of
(T-box expressed and IL-5
in T
expression.
cells) [121],
a
which
unique THi transcription factor called T-bet
upregulates IFN-y
In contrast IL-4 induces TH2-cell
and
downregulates
IL-4
development through STAT6
23
Introduction
which activates GATA3
and down
regulation of IFN-y expression.
However, although to be
mixture of both, Tm and Th2-
a
T cells
Regulatory
1.6.
undergo
As mentioned earlier, T cells
high avidity for self-antigens
cells with
complete.
For
that the
postulated
was
guarantee
a
complete
price, namely,
important
responses. One very What
are
regulatory
Although they and
usage and
to
are
not
mechanisms
an
uniform
which
T cells
exported
TNF-receptor family
prevent
of
a
to
high
immune
by Tregs-
to the
related
can
grouped
be
natural
The
periphery,
[128].
where
The
they
thymus
adaptive
into
Treg
cells
Once
generated
have been
postulated
Tregs
induced
express CTLA-4 and GITR
are
Tregs
maintain
CD4+
(glucocorticoid-
protein) [128,129]. Unlike conventional
CD25 upon activation, the
are
show divers TCR
recognizing self-antigens [127].
expression
T cells,
of CD25
peripheral murine CD4+
[130].
T cells,
potent regulatory activity in vitro [9,131] and in vivo [8,132]. They show
partially anergic phenotype, they proliferate poorly growth
includes
auto-reactive
thymus [126]. They
CD4+CD25+ T cells, which constitute -10% of possess
important
To avoid this, the immune
(Figure 9).
in the
development
activation of self-reactive T cells
upregulate
to
is
tolerance
incompleteness
population they
typically CD25+, additionally they
induced
The
mechanism is mediated
potentially capable are
of central
thymus,
cannot be eliminated.
self-antigens
for these
in the
present
not
are
T cells?
the T cell
thymic Treg cells
prevent
and
are
exist which
develop autoimmunity.
naturally occurring regulatory
generated during
the
to
this process
autoimmunity. However,
repertoire [125].
additional
developed
system
of
incompleteness
immune
potential
the
specific
selection process where T
intrathymic
eliminated. This process called central
are
example, self-antigens
hence self-reactive T cell clones It
an
development
tolerance is crucial to avoid the is not
of IL-4 and IL-5
is found in vitro, in vivo responses tend
strong polarization
a
upregulation
to the
[124], leading
[122,123],
is
dependent
on
exogenous IL-2
inhibit autoimmune diabetes in mice
[128]
[133].
upon TCR stimulation and their
CD4+CD25+ T cells
and rats
a
[134].
are
The stimuli that
shown to
are
required
24
Introduction
for
the
of
generation
natural
the
thymus
in
TregS
incompletely defined.
are
Nevertheless, CD28 ligation [135] and strong antigenic signals [136]
shown to
were
be necessary.
natural
adaptive Tregs
Tregs
i
IL-10
IL-4
IL-10
TGF-ß
IL-4
IFN-y
IL-10
T cell subsets:
Figure 9. Regulatory
regulatory
adaptive Tregs. The natural Tr6gs arise during
Negative regulation by GITR.
Adaptive TregS
natural
can
Tregs
T cell
is transduced
be of different
development
in the
by cell-contact
origin. Described
and
thymus,
over
are
and
yö
immune reactions
regulate T cells
are
found to mediate immune
Adaptive regulatory from classical T cell
variable, depending
exert
their
T cells
molecules like CTLA-4 and
in the literature are
THi
or
TH2 cell, which
disease
Tregs
or
are
additional
subtypes. But
or
also CD8+ T cells
from the
natural
setting
Tregs-
and
thymus and
can
be either derived
The level of CD25
the site of
expression
mainly
in
a
cytokine
is
regulatory activity [137].
mediate their function via cell-cell contact, the
function
Tr1 cells
regulation.
originate
populations on
Whereas the natural
Tregs
by IL-10 secretion
and
CD4+CD25+.
regulate immune responses. Furthermore TH3 cells which mediate regulation by TGF-ß, which
TregS
T cells can be subdivided into natural
dependent
manner
adaptive
[138-140].
Furthermore, in contrast to the natural Tregs, they need further antigen exposureinduced differentiation in the
regulatory
T
cells
are
THi
encephalomyelitis (EAE) by
periphery
to be active
which
cells,
secretion of
prevent
[141]. Examples experimental
IFN-y and avoiding
a
of
adaptive
autoimmune
TH2 response [142]. Or
TH2 cells, which suppress THi mediated induction of diabetes by the secretion of IL-4
[143].
The CD4+ TH3 cells suppress induction of
TGF-ß [144].
Other
adaptive Tregs
are
autoimmunity by
the secretion of
CD4+TM (IL-10 mediated) [145,146], CD8+ T
25
Introduction
(by
cells
IL-10
is to inhibit
important feature
regulatory cytokines
or
by
yö
CD8+
or
gets quite obvious that there is
From this list it most
secretion) [147,148]
IL-4
or
T cells
uniform
no
(IL-10 mediated) [149].
population
ongoing immune responses by
of
Tregs-
Their
the secretion of
direct cell-cell interaction.
2. DENDRITIC CELLS
DCs
are
They were
named based
mature DCs. Like
immune
system
their dendrite-like cellular branches, which
on
macrophages
adaptive
response.
Whilst
developed
in
invertebrate
evolutionary relative
is
system
developed during
link between the two branches of the immune
immune
system,
as
disposition already
found in invertebrate
several
defense
evolutionary
system
immunoreactions and
as
to
pathogens,
like as
infections with the innate immune
infection.
possibility
same or a
system
Together
are
with the
to clear its
capturing
of
worms
adaptive
there
immune
developed
adaptive
immune
and
are
strong immune and
old
already
system
[153 649]:
development of
was
is
self-
able to
the
adaptive
allowing
specific
memory. For the
respond specifically
protection against repetitive
The defense mechanisms of the
against pathogens early
system
it
gives
with the DCs
the
after
the vertebrates the
as
an
important key
so, several different
mechanisms and
to the cells of the
system
a
and inducible antimicrobial
receptors
memory allowed
pathogen.
are
parts of the immune system. To do
of DCs
of the innate
and insects has at its
immunological
crucial for the defense
pathogen, presenting
activation of the
system
body from pathogens,
mediator between the two
specialized subtypes
similar
the
antigen
consequence thereof the
immunological
and the
during
of
first time in evolution the vertebrate immune
for
system in vertebrates. The innate
mechanism,
invention
the
were
typical
vertebrate evolution. DCs evolved
recognition, phagocytic cells, encapsulation mechanisms
immune
are
immune
adaptive
the
organisms
factors. The main innovations in vertebrates
part
system [150].
capture antigens, process and present
organisms [151,152], and
new
are
system thereby inducing
immune
immune
innate
the
DCs
phagocytes,
and other
and their main function is to
them to the cells of the
as
APCs found in the immune
potent professional
the most
pathways
of
adaptive immune system and
very diverse.
26
Introduction
DCs
occur
origin,
origin
but evidences
leading
[157,158]. After
produce
the final surface
origin.
criteria
subpopulations. cell
splenic
all the
phenotype of
DCs in the
[167,168].
In the
lymph
can
tissue
DCs, which isoform
are
of CD45,
lymphoid organs
the
potential
[171].
Besides
described in
these
specific
main
by
concept
was
produced thymic
myeloid
cytokines
murine
an
DCs
or
and each
lymphoid
and express
a
into
main
six
CD8- and CD8+
additional DC
population
plasmacytoid DCs, to
produce large
may be derived from cells
which
In addition these cells have been
populations
of
or
are
amounts of
by inducing
that
population
[170]. Finally expression
from dermal of B220,
an
in
all
found
type I interferon
proposed
to be
the differentiation of
Tregs
DCs, additional subsets have been
organs of the mouse; however, their
remains to be determined.
its
epidermal langerhans
involved in the maintenance of T-cell tolerance
[172].
myeloid-restricted
subtypes [159-161]. Therefore
be subdivided into
This DC
in the skin
characterizes the
in response to viral infection
can
[169].
populations,
and have the
with
to derive from
produce DCs,
different
of
nodes there exists
exclusively found
development
be further divided in CD4-CD8- and CD4+CD8-
expresses intermediate levels of CD8 of two other main DC
DC
require
classification
lymphoid
CD8- DCs
myeloid
support this concept [162-165]. Phenotypic and
allowed
have
population [166].
subsets
one
thymic
and
DCs is not determined
factors may
transcription
functional
thought
DCs. But this
"myeloid"
shown that both could
was
The fact that CD8- and CD8+ DCs
different
were
a
myeloid-restricted and lymphoid-restricted
the isolation of
precursors from the BM it precursor could
DCs
Mutant mice, which cannot form T cells, still
recently challenged. CD8+ DCs
and
from
thymus shared early precursors
mouse
"lymphoid"
of
early steps
and all CD8- DCs to derive from
to the terms
develop
all
believed to be of
are
DCs shared
the CD8+ DCs of the
lymphoid-restricted precursors precursors,
Most DCs
some
findings all CD8+
this
lymphoid cells, they
and
(Figure 10).
found that
were
[154,155] and
[156]. After
T cells
myeloid
with the
stem cell in the BM
with B cells
origins.
in different subsets, with different functions and also different
DCs share their
pluripotent
development
Dendritic cell
2.1.
physiological significance
27
Introduction
erythrocyte/ megacaryocyte precursor
granulocyte precursor
myeloid
granulocyte/monocyte
monocyte precursor
precursor
precursor
common
myeloid
monocyte
Wrv-
denved DC precursor
»DC
'DC
Pre-DC
myeloid/lymphoid precursor
rDC
lymphoid derived DC precursorr
'DC
Î B cell precursor common
lymphoid
lymphoid denved thymic DC precursor
precursor
NK cell precursor
Thymic/lympoid precursor
Figure
[173])
10. Theoretical model of the
The differentiation of DCs has been
also
pre-stages leading
the
a
to DCs. On the
thymic lymphoid precursor,
DCs
are
common
myeloid
to DCs. The
monocyte precursor leading
2.2.
proposed
through circulating
derived precursors, and other
development of
which
can
to
(Adapted
from Ardavin et al
proceed directly through myeloid
DC precursors
side there
lymphoid
develop
dendritic cells.
t cell precursor
are a
(pre-DCs). Additionally,
myeloid derived
side offers
and
a
lymphoid
lymphoid there
are
DC precursor and
derived precursor and
into DCs.
DC function involved
in
many
differentiation states, immature main function is to
or
different mature
processes.
(Figure 11).
capture antigens, process
They exist
in
two
major
In the immature state their
them into small
peptides and
to load
I
28
Introduction
them
on
MHC
I
undergo maturation DCs start to molecules
II
or
if induced
migrate
are
molecules. After
to the
up-regulated
has reached the
lymphoid
antigen presenter for
antigen capturing
by appropriate
lymphoid
T and B cells
(imDC) ongoing
During the migration, co-stimulatory
and the secretion of
fully
DCs
factors. Whilst maturation is
tissues.
organs, it has
immature
cytokines
is induced. Once the DC
matured and has become
an
efficient
[174-176].
pathogens (TLR ligands)
cytokines (IL-1p, TNF-a) T cells
maturation
mature DC
immature DC
stimulation of T cell
capture of antigens -
-
-
adsorptive uptake
-
macropinocytosis
-CCR7
phagocytosis: microbes, dying cells
-IL-12, IL-1,TNF-a -
Figure
11. Immature and Mature DC: DCs
are
or
phagocytosis.
capturing
After activation
of
antigen.
and the
upregulation
of
pro-inflammatory cytokines.
co-stimulatory
This is done
by danger signals
T cells, the immature DC mature. Mature DCs stimulate T cell
antigen of
or
high MHC-peptide
found in two different functional states: immature and
mature. The main function of immature DCs is the
uptake, macropinocytosis
immunity
CD40, CD80, CD86, CD54
like
pathogens, cytokines
immunity by
the
molecules like CD40, CD80 and CD86
Furthermore CCR7,
an
by adsorptive
presentation or
of
the secretion
important receptor for migration is upregulated.
29
Introduction
ImDCs have been for
body
long regarded Recent
intruders.
sentinels of the immune
as
studies
tolerance to
self-antigens [177].
imDCs
efficiently capture apoptotic
can
[177]. Second, body, they
as
imDCs
imDCs
patrol
the
actively maintain
present self-antigens to T cells
cells and
continuously produced
are
also
which
Three features of imDCs led to this conclusion. First,
different
constantly sample
can
that
suggest
system,
and
self-antigens
distributed in the
widely
present them
and
to T cells.
Third, imDCs do not undergo maturation when they capture self-antigen under
physiological capacity
conditions.
T
regulatory/suppressor
After infection
uptake
or
recognition receptors, allow them to
such
recognize
findings suggest
as
expression of MHC
class I
imDCs
antigen,
have
the
naïve T cells into IL-10
undergo rapid
maturation. Pattern
the Toll like
receptors (TLR), expressed by imDCs
antigens,
which induces the maturation program
rapidly
DCs
the surface
of
imDCs
that
[178,179].
cells
microbial
[180,181]. Maturing
regulate
recent
peripheral self-tolerance by promoting
to induce
producing
Indeed
and
endocytotic activity and
lose
increase
surface
ll-peptide complexes [182]. Additionally they up
expression of
co-stimulatory
adhesion and
molecules
(CD40,
CD54, CD80 and CD86) [183-185], and secrete pro-inflammatory cytokines like IL-1, IL-12
or
[186],
which drives their
TNF-a
the CCR7 screen
for
responses
[118]. Maturing
migration
ligands CCL21
into the
and CCL19
antigen-specific
lymphatics
[187].
In the
and to the
lymphoid
naïve T cells and induce
primary
lymph
nodes
through
tissue the mature DCs
T cell mediated immune
[188].
2.2.1.
Capturing
of the
antigen
ImDCs residue somewhere in the
antigen coming from bacteria, above, they
consume
virus
apoptotic self
or
body
as
possible pathways
to take up
phagocytosis [189]
similar to
to take up small vesicle
For
waiting
to
capture foreign
alternatively,
as
mentioned
cells to establish self-tolerance. The first
pathogens
or
are
therefore
equipped
droplets coming from apoptotic
example antibody
or
step
to
with different
apoptotic cells. They capture antigen by
macrophages, by macropinocytosis [190],
endocytosis [191]. Especially endocytosis, antigen.
sentinels
other invaders. Or,
fulfill their function is antigen-uptake. ImDCs
defined
receptor CCR7
DCs start to express the chemokine
allows
cells
or
which allows
by receptor-mediated
imDCs, to specifically take up
complement opsonized
bacteria
can
be
30
Introduction
actively
taken up
[192].
of imDC
type lectins,
[193,194]. imDCs
by endocytosis
Other
via the Fc
or
complement receptors
the surface
on
proteins important for receptor-mediated endocytosis specific binding of
which allow sugar
Furthermore microbial
lipopeptides
can
bacteria and
be taken up
by
are
the C-
subsequent uptake TLRs
expressed
on
(TLR1, TLR2) [195,196].
2.2.2.
Processing
of the
antigen
and
loading
to the MHC class
on
molecules
After uptake, the
adaptive
immune
antigen
present I
on
virtually
molecules
pathological antigens killing
are
also
of the
peptides
infected
an
presented
to the cells of the
MHC class I
on
ancient
proteins
from the
presentation CTLs.
of
are
cytosol
are
passed
found
in
developmental history [201,202]. incorporated types
of
into the
able to process
of
a
hollow
some
MHC
and the
as
IFN-y. Recently
immunoproteasome
[205,206].
are
important
was
of the
in
are
to
proteasome
the
which
proteolytic
the
subunits of the
indicating
a
close
region
MHC
are
IFN-y [203,204]. Thus, are
expressed
expressed
shown
for the
The
pieces by
cellular stimulation with
immunoproteasomes, which
such
tumor
proteins targeted
cylinder through
genes,
The subunits encoded
proteasome after
cytokines
subunits in the response
in
and cut in smaller
to
or
In the
route starts with the
I
proteasomes exist, the constitutive proteasomes, which
all somatic cells, influence
resulting
vicinity
close
are
foreign antigen, usually leads
The class
protein machinery
units each,
II
body. Peptides presented by MHC class
proteasome [200]. Interestingly
inside the
proteasome
seven
or
MHC class I molecules
[197,198],
endogenously produced proteins.
by specific
cells
presented
degradation by ubiquitinylation (see Figure 12, left side) [199].
activity
two
CD1
are
to be
derived from viruses, intracellular bacteria
The
presented.
consist of four stacks of the
on
derived from
normally
situation
are
presented
peptides
all nucleated cells in the
proteasome, which is for
are
get processed
Whereas
system.
molecules, glycolipids
has to
that the
specificity of
in
under the additional
the immune
31
Introduction
exogenous
antigen
Exogenous pathway (class II MHC)
Endogenous pathway (class I MHC)
Figure
12. Model of
antigens: (adapted
separate antigen presenting pathways for endogenous and exogenous
from
Immunology,
proteasome, the peptide transported molecules.
By vesicle transport
they present its load
phagocytosis. II molecule
transported
And the
gets
to the ER
by
the TAP
peptide-loaded
CD8+ T cells.
antigen gets
Kuby) Endogenous antigen
In
the
cleaved in the
is
transporter, and loaded
MHC class I molecules
exogenous
get
processed by on
endocytic compartments,
exchange
with
peptide,
molecule reaches the cell surface and
a
to the surface, where
pathway, the antigen is taken up by
endocytic compartments. Meanwhile
where the invariant chain
reaction
catalyzed by
presents the antigen
the
the MHC class I
the MHC class
build in the ER and associated with the invariable chain. Then the MHC class II
to the
associated until
to
the
Janis
gets cleaved
to
gets
CLIP, staying
HLA-DM. The loaded MHC class II
to CD4+ T cells.
32
Introduction
majority of antigenic peptides originate
The
The
209].
proteasome is the enzyme complex that generates the
terminus of the MHC class I as
molecules
positively
one
C
termini
[210],
cytosolic
a
leucine
peptidases
TAP1 and TAP2
cytosol
into
transporting manner
(for
multistep followed
a
see) [220].
[221].
In
a
The
a
also
large
a
across
reorganization triggers
proteins belong
Both
number of membrane
membranes in
the
proteins,
ATP-dependent
an
step, the peptide binds
complex [223]. hydrolysis
ATP
to
the TAP heterodimer is
peptide transport by
fast association
peptides from
the
to TAP
It has been
and
a
[222],
suggested
peptide translocation
the membrane.
In the ER the
peptides
I molecule is first
are
complexed
loaded
with
by chaperones, tapasin guides proteasome
MHC
class
I
are
directly
molecule
on
the MHC class I molecules. The MHC class
chaperones,
beta2-microglobulin [224]. After the
the
which is able to cleave
transport of
for the
[218,219].
slow isomerization of the TAP
that this structural
the
ER
transporters, comprising
review
a
of the
lumen
example
For
peptide [211]. Additionally,
precursor
by
trimming
[211-214].
in the ER
or
responsible
are
amino-terminal
or
[215-217].
diverse set of molecules
process
by
described
proteins
the
of ABC
superfamily
were
generated
generally possess hydrophobic
aminopeptidase, a
are
that bind to MHC
optimal peptides
and
cytosol
carboxyl
correct
antigenic peptides
they require
residue from the amino terminus of
ER luminal
across
long
located either in the
report describes
single
by
However many
8-10 residues
are
charged
aminopeptidases
the
ligands.
amino-terminal-extended precursors. Since
class I
newly synthesized proteins [207-
from
which allows
MHC class I molecule is
it to the TAP
transporter,
a
were
the
ER
in
micro vesicles
apparatus and reach the surface of the cell [228], where they TCR of CD8+ T cells, which may be activated to kill the The most
important antigen presenting pathway
Normally, peptides molecules molecules
derived from exogenous
[229,230]. are
and load them
In
contrast
exclusively present on
in the endosome
MHC class II or
to on
MHC
proteins class
APCs. APCs
I
peptides processed
phagosome.
Then
they
are
[225-227].
through
can
the
Then
golgi
interact with the
target cell. in DCs is the class II route.
are
presented by
molecules
actively
(Figure 12, right side).
to the
and stabilized
completed
bound to the MHC class I molecule
leaves the
binding
proper
The
the
take up
MHC class II
MHC
antigens,
class
II
process
antigens get encapsulated
processed
and
degraded
in the late
33
Introduction
endosome
liposome [231].
or
enzymes like
endopeptidases
This
and
performed by pH dependent proteolytic
is
exopeptidases,
MHC class II molecules arrive in the vesicles the
containing
Later the
cathepsins [232].
like the
the
processed peptides
from
pathogens [233]. The MHC class II molecules
transmembrane chains
(alpha
beta)
and
the
binding cleft of
the
proteasome and actively transported
establishment of
processed
to
MHC.
the
as a
mentioned
molecule
of
The
on
is
with beta2 in
are
built
microglobulin
third
a
and
by
an
enzymes into
alpha
and
CLIP,
a
peptide
that
exchanged against
This
a
step is catalyzed and
homologues
to MHC class II
presentation. Namely
of on
chain
the
the class l-like MHC called
homologues
to MHC class I,
class I
complete antigen presenting
to the
lymphatic organs
In order to exert their function, DCs have to
tissue
peptides
to CD4+ T cells.
pathway
a
gets
loaded MHC class II molecule then
glycolipids
to form
for the
important
[243]).
Migration
present their antigen
be
such
The invariant chain
[237,238].
peptide
the MHC
the MHC class II molecule. The
removed
presents the peptide
hydrophobic peptides
(reviewed
2.2.3.
CLIP is then
above there
CD1. CD1 MHC molecules
pairs
then loaded
chaperone [239-242].
on
to the late endosome where the
in the late endosome
reaches the cell surface and
which
are
covers
pathway. However,
to
[235,236].
another molecule called HLA-DM, which is
supported by
presentation
postulated
CTL response
transport from the ER which
being loaded
to the ER from
and
which
[234]
prevents peptides processed by
mixture of the class I and II
reported
been
pathogen-derived peptide
As
invariant chain
an
gets first processed by proteolytic
bound
and acts
a
protective
a
generated,
invariant chain remains
has
later in the
have been
and
the MHC class II molecule and
class II molecule. This avoids
cross-presentation
in the ER out of three chains. Two
synthesized
are
migrate
to the
lymphoid
tissue to
to T and B cells. DCs find their way from the BM to the
by the guidance of chemokines. ImDCs express
a
variety
specific
of chemokine
receptors, including CCR1 [244], CCR5 [245], CCR6 [246,247] and CXCR1 [248], which
may
Differential
participate
in
expression may
the different organs
the
recruitment of DCs
to
the
inflamed
tissue
[249].
also contribute to the selective recruitment of imDCs to
(Figure 13).
34
Introduction
Mlf>-3a secretion CCR6 dowft
^
regulation
c^^—r—~c~ -—-—*—~~
CCR7 up regulation
Figure
13.
Trafficking
blood to tissue
migration, for
of DCs:
(adapted
(here lung) by cytokines
the DCs
capture antigen, get
from
Bleijs
[250])
et al
and chemokines
DC precursors
are
attracted from
(here MIP-3a). Following transendothelial
activated and start to mature.
During maturation
the
receptor
MIP-3a, CCR6 gets down-regulated, and the receptor for CCL19 and CCL21, CCR7 gets
upregulated.
To attract further
DCs, the mature DC starts to secrete MIP-3a. The secretion of the
chemokine renders the mature DC follow the CCL19, CCL21
presents antigen
to T cells
unresponsive
gradient guiding
for the
them to the
inducing proliferation
of
lung homing chemokine
lymph
node. Arrived
antigen specific
T cells.
in
the
and allows the DC to
Lymph
node the DC
35
Introduction
example, CCR6,
For
imDCs in the the
[253].
chemokines is turned
on
yet
-
lymphoid organs. Therefore, unresponsive
chemokines
up-regulation
migration
to the
[252],
areas
and
areas
of
migration [260,264]
[251,252,265,266]. found
[256,257],
maturing DCs migrate
procedures
affects the
back-draft
(HEV)
in
on
for this
tissue-driving
start to secrete these
they
potentating
lymph
mature
the recruitment of
lymphoid and
they
tissue are
pit mice,
node
[264].
found to
CCL21 is
(LN)
as
Both
and
expressed by
were
levels
high
at
stromal cells in T cell
found to be
DCs to the
migration driving a
to the
responsive
CCL19 is secreted
guide
which carry
of CCL21
expression
DCs is crucial for their
which renders them
Peyer's patches [261-263].
with
the
for
receptors
Second
[259,260].
For CCL21 the function
by experiments
mutation
as
receptor
and CCL21
[258]
spleen
of DCs in T cell cell
to make
[255].
tissue
endothelial venules
of LNs,
which acts
of the CCR7
lymphoid
chemokines CCL19
chemokine
the
unresponsive [254].
them
making
imDCs to the inflamed tissue
by high
pathways
there have to be
draining
from the tissue into
emigrate
to these recruitment chemokines, in order to enable them to
chemokines themselves
The
of MIP-3a in
of additional
expression
inflammation
the mature DCs
they down-regulate
First
task:
During
the stream. Indeed, the DCs have evolved two different
against
on
Several organs express different chemokines for the
recruitment of imDCs.
steady-state
expression
In line with this is constitutive
lung [251,252].
and in the liver
lung
receptor of MIP-3a, is expressed at high levels
the
by
a
subset
important
in T
lymphatic organs
force for DCs
was
defect in the CCL21 gene. This
and also
negatively regulates
CCL19
expression [267,268]. After the DCs left the inflamed tissue and enter the follow the CCL21 and CCL19 arrived there,
they
enter the organs
tether to the endothelium
leukocyte
adhesion
[271]
(Figure 14). P-,
E-
or
system, they
lymphatic organs.
Once
L-selectins allow the DCs to
by
antigen-1 (LFA-1)
and very late activation
antigen
4
which bind to the vascular cell adhesion molecules intracellular
molecule-1
(ICAM-1)
and final extravasation
activation of
them to the
vessel
which is essential for the later firm adhesion
[269],
function-associated
(VLA-4) [182,270],
gradient guiding
lymph
specific
and vascular cell adhesion
[272].
T cells starts
In the
[273].
lymphatic organs
molecule-1 the
(VCAM-1)
presentation
and
36
Introduction
3. extravasation
Figure roll
14. Extravasation of DCs into tissue:
along
the endothelia of the tissue. The
CD34 and L-selectin. Later
DC extravasates
2.2.4. The DCs
lymphoid
through
a
J
v.
2. adhesion
(adapted
rolling
firm adhesion takes
from
is initiated
[250])
First the DCs start to
by weak binding
interactions between
Bleijs
et al
place by LFA-1-ICAM-1
interactions. And
finally the
to T cells in
draining
the endothelia into the tissue.
Interaction with T cells
display
the
organs. The TCR
specific recognition Additional molecules
of
captured a
and
processed antigen
and ß chain dimer
peptide presented by
are
enhancing
this
complex of
MHC class I
specific
interaction
T cells is or
responsible
II molecules
(Figure 15).
on
for
DCs.
37
Introduction
DC-SIGN
-^OOcgg^
ICAM-1
TCR
MHC class I
or
II
CD4
or
CD8
CD2
CD28
'«ggai
15.
naive
cell
Tcell
(adapted
from
T cells is activated and allows
cells
further
is
mediates
a
signal
interaction induces activation of the
or
inhibitory
ß2-integrin
specific
adhesion
LFA-1
on
Especially
The transient
complexes
on
cells.
T
on
T cells and ICAM-1
binding
high affinity
Similarly
the CD40-CD40L
by adhesion molecules
[274]. Recently,
[275]
to ICAM-2
allows the T cells to encounter
different APCs. In the absence of
were
MHC molecule is
leading
DCs
on
ICAM-3 is important in the initiation of
DC to the next
cells and DCs
The Interaction of the two
a
novel DC-
receptor (DC-SIGN: DC-specific ICAM-3 grabbing non-integrin)
short-lived and the T cells one
thereby allowing
DC, leading to cytokine secretion.
been identified, which binds with cells.
the DCs
CD80/CD86-CD28/CTLA4 interaction
The
The initial contact between T cell and DCs is mediated like the
lymph nodes
with the MHC molecules, LFA-1
high affinity ICAM-1-LFA-1 interaction.
stimulatory
either
(CTLA-4)
DC-SIGN-ICAM-3 interactions,
mediated
by LFA-3-CD2 interactions.
stabilized
second
a
i IL-2
In the
[250])
et al
Bleijs
subsequent ICAM-1-LFA-1 interactions. After full ligation of the TCR on
(CD 28)
LFA-1
First adhesion is
interact with T cells.
}
T IL-2
active
dendritic
DC-T cell interaction:
CTLA-4
CO40L
ICAM-1
Figure
or
was
migrate
recognized by
to the formation of
an
a
in
lymph
a
recent
immune response
specific interaction
on
the contact is
wandering of the
[278].
the TCR of the T cell,
synapse
T
[277].
T cells from
study, where the movements of
node in situ
immunological
[276]
large number of MHC/peptide
a
to the next DC. This
nicely shown
measured in
a
an
and ICAM-3
has
a
But if the
specific
[279].
peptide
on
T
the
contact is formed
The TCR-MHC
complex,
38
Introduction
together
with CD2-LFA-3 interactions is situated in the center of the synapse and
LFA-1-ICAM-1 interactions LFA-1
are
forming
enhancing
T cells is activated
on
CD2-LFA-3 interactions
bind
superfamily
and
delivering
activation
an
ligand expressed
CD28
to or
tolerance
showed that CD28 Other
ligand
[289].
triggering
B7
molecules CD86
on
are
members
are
or
molecules, is
efficiently
Ig
the
of
lymphocytes [283], thereby
T
CD28 is the main
co-stimulatory
cytokines [287,288].
of IL-2 and other
obligatory,
as
they
may substitute the
OX40/OX40L an
a
the members of the
an
an
This
effective immune response
But studies with CD28 knock-out
is not
like ICOS/ICOSL
for the
CTLA-4
Additionally
DCs is needed to
on
important for induction of
co-stimulatory signals
by CD28/B7
and
CD28 stimulation has been shown to lead to
[286].
interaction is believed to be
preventing
molecules
inhibitory signal [284,285].
T cells
on
and
prolonged expression
increased and
[290].
not sufficient to activate T cells.
[282]. CD80 and
CD86
and
ICAM-1. This
high avidity LFA-1-ICAM-1
the
potent co-stimulatory
activate the T cell. The most
and
are
signal transmitted by co-stimulatory
B7-family, CD80
of the TCR,
[280,281].
However, these interactions second
to interact with
ability
its
immunological junction through
stabilizes the
ring. Following ligation
the outer
are
(KO)
mice
responding
still
to
clearly
antigen
co-stimulatory signal delivered
[291,292]. CTLA-4,
inhibitory receptor, leading
the alternative IL-2
to reduced
production [293]. surface
Other molecules
molecules
by the TCR
CTLs express CD4
complex.
of the MHC in
(reviewed
or
[294]).
are
important
in
major
CD4 and CD8. The two
CD8, respectively. They act
as
that
Molecules
lymphocytes [295].
signal
on
or
co-receptors in the recognition
CD8 interacts with MHC class I and CD4 with MHC class II activate
receptor superfamily. Its ligand, CD40L,
activation
MHC-peptide
the
subsets of T cells, TH
DCs
upon
important. CD40, which is the best described example, is
activated T
of
recognition
the
engagement a
is
expressed
The CD40-CD40L interaction initiates
DCs which leads to the
Furthermore the secretion of
up-regulation
cytokines
a
or
IL-12
on
retrograde
of CD80 and CD86
like IL-1, TNF-a
also
member of the TNF
TNF-a like molecule,
a
are
[296].
by the DCs is
induced, supporting the activation of T cells and guiding their development [297-300]. IL-1 is
a
pluripotent immunomodulatory cytokine
and humoral
immunity
in the
periphery [301].
that has
an
initiating
role in cellular
IL-1 consists of two molecular
species,
39
Introduction
IL-1a
IL-1ß,
and
through
biological
functions
chains,
p40 protein
a
Mouse DCs immune
are
of which
both
and
a
the
exert
similar, but not completely overlapping,
IL-1-type
p35 protein,
I
receptor [302]. IL-12 consists of
which
known to secrete functional IL-12.
response
towards
Tm
a
type [119,298].
receptors play key roles in the development of
regulation, inflammation induce the
production
macrophages starts the
it is
adaptive
and
immune
The
cytokine
to an efficient Th
TNF-a
or
and
ability
[305].
its
immune
ability of TNF-a
and the
the process of inflammation
system, leading
activity [303].
system and in
Due to the
inflammatory cytokines
important during
for
IL-12 is believed to bias the
the immune
autoimmunity [304].
of other
required
both
are
two
to
to activate
This
altogether
CTL response.
40
Introduction
3. AUTOIMMUNITY The immune
system evolved in order to protect higher organisms from infection
tremendous
with
a
and
fungi.
cancer.
of
variety
the innate immune
Additionally,
system also disposes apoptotic cells
waste from the normal cellular household. to
protect the organism,
massive
damage
system,
instead of
Although
the immune
In these
organism by self-destruction.
fighting against pathogens, develops
Paul Ehrlich at the
Autoimmune disorders diseases
Organ-specific leading
massive
to
cytokines such
as
of the last
beginning can are
organ
IL-2 and
organ-specific
characterized
by cell-mediated
destruction.
disorders
are
important
and IL-10 and
by
complex deposition.
antibody
followed
example
for
patients
have
an
organ-specific
to
highly
erythematosus (SLE), body,
nucleus components But
why
or
is there
a
systemic
diseases.
immune
reactions
In contrast
Tissue
cell and
by opsonization
lysis [306].
Some
autoimmune disease is Hashimoto's
systemic disease,
the
from
A classical
repertoire
of
our
These
patients
systemic lupus
react with many
against
[308,309]. in the
with
which react with the
In contrast in the
sera
and
autoimmune
thyroiditis.
of the dominant antibodies is directed
self-reactivity
via cell-
IL-4, IL-5,
as
systemic components.
[307].
Tm
autoimmune
systemic
cytokines such
initiated
is
damage
organ-specific
one
occur
are
widespread circulation of auto-antibodies
localized lesions
since
called
through IgG and IgM
developed auto-antibodies against thyroglobulin
thyroid leading
tissues in the
are
by complement-mediated
demonstrate both
CTLs
killing by
elevated levels of Th2
features
immune
syndromes
as
was
of disease
Important mediators
receptor-mediated killing.
characterized
and
the effector responses tend to
IFN-y and
it
as
by the
century, is real.
be classified in
mediated immune responses such antibodies directed Fc
organism,
to
the immune
cases
to the worst foe itself
induction of autoimmunity. The "horror autotoxicus" of the
by
designed
is
system
other
or
gets beyond control leading
under certain circumstances it
to the
worms
prevent the development of
mechanisms evolved which
Furthermore
protists,
like bacteria, viruses,
microorganisms
immune
system?
cell
41
Introduction
recognize
system requires
immune
Because the
immune
adaptive
the
pathogens,
the different
diversity for
wide
a
This
receptors, leading
to
and T cells constitute
the immune
self-reactivity,
self-tolerance.
immunologic
part of
system
Therefore
tolerance.
pathways
prevention
small number of auto-reactive B
in normal
pool,
since the
production
of
induction
is
self-tolerance
of autoimmune disease and nature
important
an
developed several
to establish and maintain self-tolerance. Tolerance can be
peripheral
broadly
tolerance.
Central Tolerance
Central tolerance refers to the clonal deletion of self-reactive B and T cells their maturation in the BM upper
(B cells) [310,311]
or
during
thymus (T cells) (Figure 15,
the
panel) [55,312-315].
Auto-reactive B cells
already
express
are
eliminated at the stage of immature B cells, when they
antigen specific
an
extracellular molecules and initiate the BCR does not bind
released in the
antigen,
BCR
[316-318]. Functional BCRs bind
the
signaling stays
on
and the B cell will be blocked from further
responded
to
an
ligands
that mimic
development [323,324]. However,
Clonal deletion of
forced to
potentially
auto-antigens [326].
selection for B cells is much less
lines of evidence
can
antigen
auto-antigen
autoreactive B cell clones escape deletion and enter the circulation in cells
be activated with
an
some
anergic
pathogen-derived
It has to be admitted that the
stringent than
If
basal level and the B cell is
If the immature B cell encounters
its BCR, this indicates that it
[325]. Such anergic
to
antigen specific cytoplasmic signaling [319-322].
peripheral circulation.
capable of crosslinking
state
as
direct result from the breakdown of
a
are
of
healthy individuals. To avoid
evolved several mechanisms summarized
classified in two groups: central tolerance and
3.1.
a
the immune cell
Autoimmune disorders
mechanism for the different
normal
somatic
even
danger of auto-reactive antigen
the
actually
frequently observed
auto-antibodies is also this
And
self-reactivity. a
raises
diversity
tremendous
or
to
B cell
system generates
receptors (BCRs) and TCRs randomly by gene rearrangement mutation.
receptors
its
negative
for T cells.
self-reacting T cells has been extensively investigated. Several
suggest
that T
lymphocytes
undergo apoptosis during
that bear
receptors
the maturation in the
for
thymus,
self-antigens a
are
process called
42
Introduction
negative thymus
can
development of the T cells
of the
recapitulation
selection. Detailed
in the
be found in the T cell section.
However the mechanisms
compromise
the immune
leading
to central tolerance
system takes
to assure
a
are
leaky. This is due
complete repertoire of
cells. To further reduce the risk of self destruction the immune mechanism of
peripheral
to
a
B and T
system evolved the
tolerance.
Central bone marrow
thymus
^^^3l0P Antigen JKÊ^k B ce" receptor
£Jr
^^^B
cell
aHHK
B cell
receptor -> mature B cell
^8^8 cell *• mature T cell
Periphery regulatory
clonal deletion
clonal anergy
T cells
FasL« Tcell
receptor Antigen
T cell
immature DC
no
Figure
16. Central and
and B cells the
thymus
cells that
Apoptosis
co-stimulation
peripheral
undergo positive
and
Tolerance. Mechanisms
negative selection
T cells where tested for their
recognize
negative regulation
the MHC molecule
ability are
to
in the
leading
to tolerance: Central tolerance: T
thymus respectively in the bone
recognize antigen presented
rescued
(positive selection);
on
marrow.
In
MHC molecules. T
T cells that
recognize
self-
43
Introduction
antigen presented by MHC molecules are
tested for their self
binding
In the bone
(negative selection). antigen
to
marrow
deleted.
are
specific
T cells become
anergic. Clonal deletion: Selfsurface, which binds to
the Fas molecule on the
constantly activated, upregulate
FasL, leading to induction of apoptosis. Regulatory T cells: regulatory T cells activation of self-reactive T cells by the secretion of contact
with
(for example
lower
extended period of time in
submitogenic provided by
exposure to
and
an
state.
present auto-antigens
to T cells
class I and II molecules and
induction. PD-1 is
mention CTLA-4 and PD-1
Ligation of
CTLA-4
highly expressed
antigens
on
tolerance to self encounters
Hence,
APCs.
an
down-regulated
Fas.
It is
antigen
antigen
non-professional
can no
longer
DCs which
[328,329], display
co-stimulatory
B7
low
proteins
mediators of anergy
for anergy in vivo
Moreover
although
APCs
are
[332]
all cells
be activated
present
is restricted to of
important mediators
is also observed for B cells. If T
and
help,
the
a
B cell
antigen-receptor
is
[334].
by activation-induced cell death (AICD): Pathway of T cell
that
as
on
APCs do not
immature
involving upregulation of Fas ligand that binds
serve
co-stimulatory signal
a
important
as
specific
by
be induced
[179,182,330,331].
required
[333].
can
expression of co-stimulatory molecules
in the absence of
and the cell
postulated
may
T cells
antigens. Clonal anergy
2. Clonal deletion
suicide often
shown to be
anergic
on
their surface the
professional
was
but remains alive
[289,327].
Especially
to
and therefore induce tolerance to self-reactive T cells
publications
cells
antigen-presenting
is
lymphocyte
co-stimulatory receptors
between
activation.
expression of MHC
Other
Anergy
in the absence of
by interactions
on
antigen encounter,
hyporesponsive
a
prior
molecules
continuously sample surface
or
counter-receptors these
upregulate
direct cell-cell
mechanism in which the
a
peptide antigen
cytokines
soluble
T cells and
by
or
panel).
intrinsically functionally inactivated following an
TGF-ß
by several different pathways
tolerance is induced
1. Clonal anergy: T cell anergy is
for
or
influence the
Tolerance
peripheral
As outlined below,
(Figure 15,
like IL-10
cytokines
can
CTLA-4).
Peripheral
3.2.
the B cells
further mechanisms maintain tolerance: Clonal anergy: If self-
immature DCs, the
antigen is presented by reactive T cells,
periphery
In the
Peripheral tolerance:
B cells
specificity,
deleted
are
repetitive
trigger
TCR
for AICD
engagement
[335-342].
All
to the death
receptor
specific
for self-
in T cells
Lymphocytes express
Fas
44
Introduction
(CD95),
homologues to TNF, is expressed
receptor-ligand pair,
the
importance of
by
on
a
[344]
and both
Interestingly
SLE.
also IL-2
cells from mice that lack IL-2
induction of the
IL-2
or
receptor fail
ligand [346]. Consistent
Fas
Peripheral suppression by
postulated to be
to be transmitted
implicated
well. For
as
or
mice
Lpr
to
crucial role in AICD,
as
example
some
T
undergo Fas-mediated AICD
was
with
a
linked to STAT5-mediated
role
a
peripheral
in
of IL-2
show autoimmunity
[347,348].
T cells: A subset of T cells called suppressor
mainly by cytokines,
[127,132].
or
This inhibition has been
however surface
Tregs constitutively
receptors appear
express CTLA-4
signal transmitted by CD28 [6].
which counteracts the activation
Whereas
autoimmune disease
a severe
play
signaling
to inhibit T cell responses
ability
have the
Tregs
highlighted
mice carry mutations in the FasL gene
shown to
was
tolerance, mice deficient in IL-2 production 3.
naturally deleted.
promote apoptosis
of IL-2 to
ability
The
is
spontaneously develop
strains
mouse
pathway
[343], gld
mutation in the Fas gene
have
[338,345].
in activated T cells. The
apoptosis
this mechanism for the elimination of self-reactive T cells is
two mouse strains were Fas-FasL
resembling
lymphocytes. This co-expression of
activated T
leads to Fas mediated
ligand (FasL), structurally
Fas
TNF-receptor-family.
of the
member
a
[349],
Others secrete the
the role of inhibitory cytokines TGF-ß and IL-10 [350,351]. Further evidence for
inhibitory cytokines KO
mice
which
peripheral
in
tolerance arise from observations made in TGF-ß
display inflammatory lesions
similar
to
those
seen
in
some
[352].
autoimmune diseases
Also Th cells have been shown to be
implicated
in the
of
regulation
peripheral
tolerance. For
example, Th2 cells have been implicated in mediating self-tolerance by
the
pathogenic function of self-specific Tm cells. Certainly, cytokines
regulating
produced by Th2 Considering
cells
can
down-regulate Tm responses
how vital the
prevention
of
and vice
autoimmunity
mechanisms evolved to prevent it. Like T helper cells
are
versa
[353-356].
is for survival, several
important regulators
of
humoral and cellular immunity, tolerance of self-reactive T cells is crucial for the
prevention can
T
of autoimmune diseases. However, under certain conditions, tolerance
be broken and
lymphocytes
spontaneous
are
an
autoimmune pathology may result. It has been
the
primary players
in the initiation and
and chemical-induced autoimmune diseases
shown in animal models in which
autoimmunity
suggested
perpetuation
[357,358].
that
of both
This has been
could be transferred
by adoptive
45
Introduction
autoimmunity
transfer. Moreover the transferred
depletion
specific antibodies
of Th cells with
leading
Mechanism
3.3.
Several different mechanisms autoimmune disease is not other factors such
complement [359-361].
autoimmunity.
lead to
only dependent
viral, bacterial,
as
or
Although
events.
leading
mechanisms
to
peripheral
early stages
generally specific
have
tissues
development
of T-cell
for
following availability Induction of
and
isolated
antigen
or
an
or
absent in the
thymic
deletion. These
humans who
thyroglobulin)
stimulatory
in
where vascular and/or
as
in
the
following
access
of
damage
such
and these infections also
immune
response.
develop diabetes after
that results in the
as
availability
viruses may induce similar
provide
the additional stimulus
co-stimulatory molecules important
This
is
best
of
following vasectomy
of orchiditis
case
tissue-tropic pathogens
tissue trauma have been
in the
exemplified in rodents and
[364].
infection with Coxsackie B viruses
Breakdown of T cell anergy
Self-reactive T cells which escape clonal deletion in the if
antigens
anatomically sequestered
are
effective barrier that prevents
via tissue
of soluble mediators and
of the
perpetuation
anergic
thymus during
may lead to the induction of autoimmune diseases.
occur
antigens,
phenomenon,
production
3.3.2.
levels
autoimmune diseases
likely
Infection with
autoimmune of the
of
organ-specific
frequently reported
[362,363].
the
to auto-reactive T cells. Destruction of these basement membranes and
antigen
previously
combination of several
autoimmunity bypass
to
self-antigens
(e.g., myelin basic protein
cellular basement membranes constitute the
self-reactivity.
circumvent the central tolerance.
some
may escape
circulating
low
relatively
a
but
auto-antigens
specificity
Auto-reactive T cells with the
tolerance
Release of isolated
3.3.1.
development of
chemical insult may lead to
pathways leading
most
The
permissive genetic background
on a
Often development of autoimmune disease results from different
the selective
by
autoimmunity
to
can
or
could be reversed
they
meet their
molecules
or
specific antigen
cytokines.
without
thymus
additional
are
rendered
activation
by
co-
broken
by
the
But T cell tolerance may be
46
Introduction
delivery
of
a
co-stimulatory signal.
This may
occur
of B7.1 molecules could be detected in the central
rheumatoid
transgenic a
viral
as
arthritis mouse
peptide
autoimmunity, broken and
be
but if B7.1 is
co-expressed
these
the ß-islet cells,
on
do
mice
a
In this model
the control of the insulin
[367]. Normally,
cells
not
peripheral
promoter develop
tolerance is
deleted.
Self-Peptides
Chemical Alteration of
lymphocytes
periphery.
ß
in
made
were
autoimmunity develops [368].
3.3.3. T
islet
expressed under
with
synovium of patients
insulin-dependent (typel) diabetes mellitus.
model of
on
system of patients [365].
observations
similar
Finally,
(RA) [366].
from the LCMV is
transgene
a
nervous
has been observed in the
induction of B7.1
in tissue
multiple sclerosis (MS) up-regulation
necrosis and local inflammation. For example in
Similarly
resulting
after infections
that bind with low
affinity
However, these T cells
But responses to and
are
to
self-antigens in
thymus
the
may not in
normally functionally anergized
presentation
of these
self-peptides
can
the
be enhanced
under certain conditions. Some metals induce autoimmune disease via the creation
of
new
high-affinity binding
sites for
MHC
molecules
on
self-
chemical-bound
peptides, allowing activation of previously anergized T cells [369]. Expression of appears to be
important step
altered nucleolar
proteins
mercuric chloride
(HgCI2)-induced autoimmunity
an
in
a
in the
development
of
[370].
In
rodent model of SLE
addition, many drugs induce autoimmunity via formation of hapten-induced auto¬ antibodies. For example halothane induce reactions in which
provide help
to
antibody-producing
the native form of the
3.3.4.
B cells that
Defective Activation-Induced cell death
undergo apoptosis
by persistent activation. Therefore,
reactive T cells which then induce
hapten
but not
autoimmunity
mutations in the Fas
or
(AICD)
a
as
pathway
may
pathway
rescue
auto¬
described for the spontaneous
similar
FasL gene
Fas-FasL
via the
defects in this
KO mice for these two molecules, which have
patients rarely
the modified
T cells
self-protein [371-373].
In AICD auto-reactive T cells
induced
recognize
hapten-specific
phenotype are
to the SLE. In SLE
observed, however other
defects in AICD may contribute to human autoimmune diseases. For
report shows that T cells from Lupus patients become resistant
to
example
one
apoptosis through
47
Introduction
region
on
autoimmune T-cell
pathogenic
of
production
In animal models the loss of the The idea that
regulatory
was
model
suppress the
[375].
can
to treat autoimmune diseases
patients.
At least in animal models this
allergic pathogenesis
gives
rise to
of CD4+ T cells in response to
a
in
of cells, which use
autoimmunity.
by generation
approach
was
prevent autoimmune colitis
TregS
with low
low levels of IL-2 and
levels of IL-10,
generated Tregs
the successful
by causing
limit the function of auto-reactive T and B cells is
from
peripheral
in
In
can
be
easily
of these cells
as a
But
a
proliferative IL-4, which
prevent colitis second
study
naïve CD4+ T cells and showed that
murine asthma model
lungs [376].
no
and
antigen
by pathogenic CD4+CD45RB(high) T cells.
transfer of these cells in
population
were
The authors show that chronic activation of both human and
proliferation
induced in SCID mice the authors
auto-antibodies to DNA
shown that these cells where able to
capacity, producing high
prevented
Tregs
defined
are
house dust mite-induced
not
representing
by unique surface
a
uniform
markers. Therefore
tool to treat autoimmune diseases still needs
improvement.
3.3.6.
Molecular
Mimicry
Many peptide fragments perhaps
induce
of infectious
organ-specific
agents
found that
a
membrane
protein
are
homologous
autoimmune responses.
rheumatic heart disease sometimes follows was
lupus-susceptibility
lead to the induction of
can
murine CD4+ T cells in the presence of IL-10
and
a
suppression
Tregs
possibility
of such cells to
application
successful. It
T cells
It offers the
quite attractive.
further
in
apoptosis [374].
Defective T cell-mediated
3.3.5.
a mouse
inhibitor of the Fas-FasL
chromosome 1. Further the authors could show that COX-2 inhibitors
able to suppress the
and
an
is located
encoding COX-2
the gene
pathway. Notably,
a
which is
upregulation of cyclooxygenase-2 (COX-2),
the
on
the
a
streptococcal
with host
proteins
It is well known that
infection. And indeed it
ß-hemolytic streptococcus
bacterium has
high degree of homology with cardiac myosin, and antibodies that target the
bacterium also cross-react with cardiac muscle and induce rheumatic fever Yersinia
enterocolitica,
a
bacterium
normally associated
with
food
[377,378]. poisoning
outbreaks, has also been associated with various autoimmune diseases. Increased
48
Introduction
patients
levels of antibodies to Yersinia have been demonstrated in disease
or
variety of thyroid antigens [381,382]. concept
more
even
with MS. There, it
patients
derived from
strongly
myelin
peptides
with viral
basic
peptides derived from
a
this
T cell clones,
protein-reactive
found that T cell clones
was
than
supporting
data
compelling
most
The
from studies with
coming
are
These antibodies cross-react with
thyroiditis [379,380].
autoimmune
with Graves
seem
the
to react
myelin
basic
protein [383].
Polyclonal Activators
3.3.7.
Superantigens
activate T cells
trough
This distinctive mode of T cell activation, bind to of T
a
wide
with the
together
MHC/peptide specificity [384].
of their
superantigen stimulates cytokine production
expression
in human T cell lines
associated
TCR-ß
[385] In
[386].
chains
unspecific activation of contribute to the
3.3.8.
B cells
antigen-specific
rodent
a
of
type
large numbers
Mycoplasma
The
regulates MHC
lymphocytes
model,
to
class II
with arthritis-
Mycoplasma
arthritidis
polyclonal B-cell activation and/or
in
[387]. Thus, autoimmunity
may result from
superantigens. Such
mechanism may
T and/or B cells via
pathogenesis
and up
and stimulates T
superantigen stimulated Th cells, resulting differentiation of
ability of superantigens
MHC class II molecules, leads to activation of
irrespective
cells
arthritidis
variety of
the variable domain of the TCR ß chain.
I diabetes in
some cases
a
[388].
Exposure of Cryptic Self and epitope spreading
During the past years it got obvious that molecular sequestration of antigens is much
more
antigenic
common
than the anatomic
determinants
are
sequestration.
effectively processed
development the T cells specific for self-antigens the T cells for
antigens
are
cryptic self-antigens
presented
in
an
optimal
induced. But the mechanisms for are
not well understood
rendering
to T cells.
either deleted
immune response
an a
are
presented
or
if the
against self
cryptic antigen optimal
few
During
anergized.
get deleted. Therefore
did not way,
and
protein just
For each
for
But
cryptic can
be
presentation
[389].
Another mechanism
important
epitope spreading. During
the
for the
exaggeration
development of
an
of autoimmune diseases is
autoimmune disease
a
shift
can
be
49
Introduction
observed from the also start to
antigenic epitope originally inducing autoimmunity
trigger autoimmunity.
response shifted from the tissue
As shown for EAE in mice, where the autoimmune
original antigen
to
epitopes released
immunopathogenesis
due to the
damage
to others which
of
relapsing
result of acute
as a
clinical
episodes [390].
Genetic Factors
3.3.9.
Familial studies
suggest
clear association between
a
diseases, particularly those with
an
genetics and
autoimmune
organ-specific pathology [391]. Further, although
concordance rates between identical twins
can
be
relatively
low
depending
on
the
disease, this may be explained by non-identity in immune repertoires because of TCR and
Ig gene recombination,
mutation of B-cell
to
tend
are
no
and
clinical
also
with
complex. Certain haplotypes (HLA-B8, certain
non-MHC
of
arises from studies of
TCR-ß
haplotypes
at
in
[394,395]).
sibling pairs a
(reviewed
in
development of
haplotypes
genes
have examined the
certain autoimmune diseases in the human
(reviewed
DR2-
found
in
contribute
to
are
of disease.
number
a
diseases
is not sufficient for
autoimmunity-associated
signs
autoimmunity. Many studies
for the TCR
autoimmune
specific MHC haplotype
a
disease,
individuals with There
the MHC gene
associated
be
[392,393]). However, autoimmune
receptor assembly, and somatic
in
receptors. The most clearly established genetic association is with
specific alleles within DR5)
variations
frequency
The
with
possibility
population strongest
higher
may
that
segregation [396]. TCR-gene polymorphisms
disease
susceptibility [397].
germline
genes
evidence for TCR involvement MS.
They shared specific
than would be
random
predisposition for
may lie in the
recurrent/relapsing
much
that
expected
based
on
have also been associated with
50
Results
RESULTS 4. EXPRESSION
CHEMOKINE
CLONING CCL19
AS
POTENT
CC-
THE
IDENTIFIES
ACTIVATOR
OF
Lässing1,
Roger
DENDRITIC CELLS
Patrick
Bättig1,
Beerii1,
Klaus
Monika
Bauer1,
Dietmeier1,
Kopf3, Philippe Saudan1
Urs
Lidia
Eriksson2,
Ivanova1,
and Martin F.
1Cytos Biotechnology AG, Wagistr 25, department
Christiane
Thomas
Ruedl1,
Pfister1,
Ute
Lorenz
Vogt1,
Manfred
Bachmann1
8952 Zurich-Schlieren, Switzerland
of Research and Medicine,
University Hospital, Petersgraben 4,
CH-
4031 Basel, Switzerland
3Molecular Biomedicine, Department Institute of
Technology,
of
Environmental
CH-8092 Zurich, Switzerland
Keywords: DCs, Cytokines, CCL19, Autoimmunity
Sciences,
Swiss
Federal
51
Results
Abstract
4.1.
(DCs)
Dendritic cells
are
key regulators
They capture antigen, process where T cell
priming
the identification of
system,
proteins
isolated
we
production of
the
DCs
autoimmune as
an
migrate
chemokine CCL19
a
in
were
up-regulation
potent
only
highly immunogenic if
pulsed
natural
with
adjuvant
orchestrate DC
to
lymphoid
organs
alpha-viral expression cloning technology proliferation as
of
in
in
a
DC-T cell co-culture
proliferation by inducing molecules and the
co-stimulatory
vivo
IL-12 and
and were
myosin-derived peptides.
Our
TNF-a.
able
but also
regulate
to
CCL19cause
findings suggest
for the activation of DCs, and
migration
for
potent inducer of proliferation.
pro-inflammatory cytokines IL-1ß,
myocarditis
chemokines not
potential.
Using
that enhance T cell
resulting
maturation of DCs,
CCL19
the
it and, upon activation,
studies showed that CCL19 enhanced T cell
Subsequent
stimulated
occurs.
for the induction of immune responses.
their
suggest
that
immunogenic
52
Results
Introduction
4.2.
Dendritic cells
They
are
(DCs)
are
key
initiators of innate and
potent antigen processing and presenting cells
their
target tissues, taking up that is
residence at sites of
is
[182,401], antigen
processed
and
I
or
the
change
tissue and to
peripheral
during
of
regions
migrate
II
to
specific CTLs and Th cells
receptor-mediated endocytosis
via
DCs
molecules
via
on
After
[402].
the cell
receiving
inflammatory stimuli,
a
migration [187,404].
them to leave
Arrived in the T
induce both activation and
can
presentation
of
immunogenic peptides
respectively [270,399].
cellular immune response to
cancer
in association
Besides
protection and
regulation, maturation, [405,406].
of
proliferation
In contrast, in the
activation stimulus, DCs induce T cell tolerance rather than activation
[177,178,407,408]. Thus, regulation protective
presented
are
DCs appear to be central to the
against pathogens,
an
of
uptake
draining lymphoid organs [403]. Upregulation
with self-MHC class I and II molecules,
absence of
a
maturation renders DCs sensitive to two chemoattractants, MIP-
lymphoid organs,
maintenance of
After
expression pattern of chemokine receptors, allowing
3b/CCL19 and 6kine/CCL21, which direct the
of
via
inducing signals either directly from pathogens or
maturation
of CCR7
or
in
potential pathogen entry
peptides thereof
surface associated with the MHC class
cell
immune reactions
antigen capture [273].
for
specialized
antigen by phagocytosis, macropinocytosis
the
to
Immature DC precursors exit the BM and circulate via the bloodstream to reach
physiological stage
DCs
unique ability
with the
boosting secondary
stimulate primary immune responses and
[400].
adaptive immunity [398,399].
T cell responses
Pathogen-mediated
as
APC
well
of DC-activation is central for the establishment as
for the maintenance of T cell tolerance is
activation
usually transduced
via
the
[409]. like
Toll
with receptor (TLR) family [410-412]. TLR recognize invariable patterns associated
pathogens
like LPS
(TLR5) [416]
or
(TLR4) [413,414], double-stranded
bacterial DNA
(TLR9) [417,418].
RNA
(TLR3) [415], flagellin
But APC activation is also induced
by pro-inflammatory cytokines like TNF-a [419], IL-1ß [420,421] secreted from the
particular CD40L
surrounding and
inflamed tissue. Members of the TNF
Trance/RANKL
are
maturation of DCs both in vitro and in vivo
additional
and
or
IL-7
[422]
superfamily,
potent stimuli for
in
the
[423-425]. Recently, chemokine receptors
53
Results
been
have
can
shown
Specifically, ligation
to
in
protective
T cell responses is
CD86 level,
leading
measles virus engages the CD46 molecule
on
generation
of
viruses interfere with
by lowering
to reduced T cell co-stimulation
production
led to
for the
highlighted by the fact that several
activation of DCs. Poxviruses inhibit DCs maturation
DC-maturation.
of
induction
in
of DC-maturation
importance
The
of IL-12
directly involved
by toxoplasmosis-derived cyclophilin
of CCR5
[426].
DCs
be
expression of
the
[427]. Along
similar line,
a
DCs, inhibiting the expression of IL-12
[428]. To
DCs,
identify
we
novel
performed
Sindbis virus
as
splenocytes
and
single-stranded
proteins capable an
RNA virus with
of interest
plus
replication competent encoding genes
for DCs and demonstrate that CCL19
autoimmune disease in
induction of DC maturation chemokines may not their
ability
to
prime
is
a
an
the viral
or even
report here the identification of
We used naive
to
alphavirus
replicase,
it is
genus.
proteins
possible
to
is
a
Using
and the
generate
libraries of interest.
mouse
can
activation of
protein library [1]. Sindbis
RNA molecule encodes the structural
protein
an
a
positive polarity belonging
other encodes the
by
splenocytes normalized against
infection of BHK cells
generated by
one
in vivo
T cell responses
expression cloning campaign.
carrier of cDNA from activated
where
We
regulating
based
alphavirus
bipartite system,
a
of
CCL19
as a
potent maturation
be used to activate DCs
myocarditis
model. These
ex
factor
vivo to induce
findings
indicate that
important property of CCL19 and suggest that
only organize
T cell responses.
the
migration
of DCs but also
directly regulate
54
Results
Material and Methods
4.3.
Mice
4.3.1.
Netherlands)
mice
C57BL/6
and
BALB/c
purchased
were
and used at the age of 8-14 wk. DO11.10
TCR-transgenic
obtained from the breeding colony at the Institut für Labortierkunde
Zurich, Zurich, Switzerland). Animals
produced from polyA+
was
[429]. Photobiotinylated polyA+
RNA from
the normalization. Therefore 10 mg RNA
irradiated for 20 minutes using
photobiotin
biotinylated and
was
resuspended
polyA+
hybridization
in
was
The
mediated in RNA
2 mg tester cDNA
oligo-dT (20mer),
an
hamster
used
as
driver for
photobiotin
acetate
Phillips Reflector Floodlamp.
buffer
was
ethanol
was
precipitated, 25 mM
carried out in
an
step, nucleic acid
initial denaturation
for 16 hours at 42 °C.
Free
mixed with 2 mg
(80% formaldehyde,
EDTA). Hybridization
(Roche)
library plasmid with
vitro transcription was
was
mixed with 30 mg
driver RNA. Double-stranded cDNA
pDelSfi.
the
Unhybridized single-
purified by streptavidin-mediated removal of RNA/DNA hybrids
pDHEB [430] linearized
Baby
was
After
proceed
the restriction endonuclease Sfi1
Library
splenocytes
formaldehyde hybridization
allowed to
was
naive
300W
a
250 mM NaCI, 5 mM
stranded cDNA
vector
of
template switch protocol
using
RNA and 0.2 mg
Eppendorf Mastercycler personal.
excess
(University
of C57BL/6 mice.
splenocytes
mouse
RNA
hybridization,
removed. For
driver
Hepes pH 7.5,
and
were
kept under SPF conditions.
were
isolated from LCMV-activated
was
Tester cDNA
and
mice
Construction of normalized cDNA libraries
4.3.2.
RNA
The
(Horst,
Harlan
from
produced by PCR, digested
and cloned into the
linearized with Notl
EcoRI
were
using
co-electroporated
kidney (BHK)
was
with
the an
cells. 18 hours
harvested and the viral titers determined.
or
subjected
alphaviral expression
Pad and the to
helper plasmid
RNA
SP6
mMessage mMachineTM equimolar
amount of
post transfection,
with
helper
cell
polymerasekit
(Ambion).
RNA into
supernatants
107
were
55
Results
Generation of
4.3.3.
BHK cells
protein library
infected with the normalized viral library at
were
infection of 0.1. Two hours later, cells
presence of
infection cells
were
incubated for 3 with 50
plate
stored
at
to avoid
detached and stained for the
expression of Sindbis
pi supernatant containing replication competent °C.
-80
The
remaining supernatants
shown in
schematically
Immature
lymph
mouse
nodes of
with 4nM
using
Biotec). marrow
MACS
Bone
of
CD4+-T cells
were
isolated from
in
assay. The
order to
procedure
spleens and mesenteric
[431]. Purified DCs
pulsed
were
BALB/c mice
or
LPS
ug/ml). Alternatively
to
instructions of the
the
(BM-DCs)
described elsewhere
as
were
pulsed
for 4h
(0.1 ug/ml; Sigma-Aldrich; BD
these
PharMingen)
samples
45 min at 37°C in DPBS
were
of naïve DO11.10
spleens
isolated from
were
derived dendritic cells
(clone3/23, 5ug/ml;
Alrich) for
made and
was
irradiated
proliferation
described
as
according
beads
marrow
naive
systems)
CD40
(DC)
BALB/c mice
For DC maturation, cultures R&D
then
OVA-peptide (Ovalbumin (OVA) 323-339; ISQAVHAAHAEINEAGR).
of
OVA-peptide-specific mice
were
of dendritic cells and T-cells
dendritic cells
naive
proteins
virus
1A.
Figure
Preparation
4.3.4.
viruses
UV
were
inactivate viruses and then used for the DC-T cell is
post¬
°C, until full cytopathic effect had developed. A master
at 37
days
8 hours
superinfection.
plate containing BHK cells. Plates
cell sorted into 96-well
single
and
washed and further incubated in the
were
neutralizing anti-Sindbis antibody
a
multiplicity of
a
or
supplier (Miltenyi
prepared
from bone
18h with TNF-a
(50 ng/ml;
were
[432]. or
E. coli,
with
026:B6)
FLAG-CCL19
treated with 10
without anti-
or
(0.33 ug/ml
ug/ml proteinase
K
or
0.99
(Sigma-
(containing Ca2+ and Mg2+) and afterwards boiled
at95°Cfor10min.
Functional expression screening of DC maturation factors
4.3.5.
Maturation of dendritic cells
proliferation
in the presence of
pulsed dendritic cells/well irradiated viral
spleen library
were
was
specific antigen. Briefly,
added
and
capacity
the
one
protein
plates
were
to induce T cell
30000 naïve
seeded in 96-well round bottom
supernatant containing was
determined via their
OVA-peptide
plates.
50ul of UV-
of the normalized activated
mouse
incubated at 37°C. As controls
56
Results
supernatants containing virally expressed TNF-a BHK cells
plates
added to each well and the
(1mCi/well)
harvester
multiple-well scintillation
resulting second
counter.
high
in
was
rescue
performed
on
SUPERSCRIPTTM II RNase H-
Complementary amplified by
were
a
dendritic cells in
reanalysed
a
supernatants from wells which showed increased RNA
RNA
PCR
supernatant using
using
cDNA
into
transcribed
was
the
transcriptase (Invitrogen Life Technologies).
reverse
removed
was
isolated from
was
RNA
(Qiagen).
Kit
RNA
Viral
of
a
positive supernatants
of
rates. Therefore viral
proliferation
into CD4+ cells
in
determined
was
maturation
to
3H-thymidine
assay for confirmation.
RT-PCR
RT-PCR
supernatants leading
Viral
were
harvested using
were
3H-thymidine incorporation
and
3H-thymidine incorporation
proliferation
4.3.6.
QIAmp
added for the final 14 h of culture. Cells
was
days.
further incubated for 2.5
were
CD4+ cells
OVA-specific
used. After 18-20 hours 50000 naïve
were
from SR5-infected
supernatants
or
by
genes
were
System (Roche) and
PCR
using Expand High Fidelity
The
treatment.
H
RNase
then
sequenced. Further
subcloning of CCL19 bearing
primers
into
a
pCEP-N-FLAG-C
atcgcgGATCCCGGTGCTAATGATGCGGAAGA-3')
and
(5'-ctatactagctagctcaAGACACAGGGCTCCTTCTGG-3') construct
was
then used for
Expression
4.3.7.
293-EBNA cells
were
were
Nhel-site at the C-terminus
and
purification of the recombinant proteins
seeded at
then transfected with
selection. The resistant
supernatants
were
Agarose-sepharose
This
performed.
was
a
density
of
4x106 cells/
lipofectamine
90mm
day prior
1
2000
collected
Affinity
population
and
Gel
an
then further
was
in DMEM
to the
(Invitrogen) according
affinity purification
(SIGMA)
manufactuers manual. For elution the
was
plate
to transfection. The
manufacturer's recommendation. One day after transfection cells
puromycin
(5'-
protein production.
(GIBCO BRL) containing 10% FCS (GIBCO BRL) cells
a
specific
N-terminus
at
BamHI-site
a
vector with gene
were
passed
expanded.
with
performed
under
Serum-free
ANTI-FLAG®
M2-
to
the
according
Flag-tagged protein FLAG peptide (Asp-Tyr-
57
Results
100
Lys-Asp-Asp-Asp-Asp-Lys) the
against PBS,
determined
were
were
CFSE
was
as
PBS
used. After extensive
was
stored in PBS at 4°C. The
protein concentrations
proliferation
assay
(Eugene, OR). BM-DCs
from Molecular Probes
purchased
of at least 90%. The T cells
labelling,
days incubation
pg/ml; FACS
BD
were
the cells
cells
subsequently
a
were
purity
at 37°C for 10
washed with PBS at 4°C. After four
harvested, stained with PE labelled anti-CD4 mab (0.1
were
and the
PharMingen)
by incubation
labelled 0.5 uM CSFE
were
were
transgenic CD4+ T cells
described above for 4h. Then DO11.10
added, obtained by positive MACS MicroBeads isolation (Miltenyi Biotec) with
min. After
dialysis
by Bradford (Biorad).
CFSE in vitro
4.3.8.
activated
proteins
in
ug/ml
CD4+ T cells
were
analysed
staining by
for CSFE
analysis.
Staining
4.3.9.
for FCM and ELISA
following antibodies
The
staining: FITC-conjugated anti-CD86,
used for
were
FITC-conjugated anti-CD80, PE-conjugated anti-CD11c, allophycocyanin-conjugated BD anti-TNF-a, biotin-conjugated anti-IL-1ß, biotin-conjugated anti-IL-12 Abs (all Dead cells
Pharmingen), ALEXA633-conjugated Streptactin (IBA). PI
and
gated
standard
Intracellular
out.
cytokine stainings
performed according
to
protocols. produced
For detection of IL-1 ß and IL-12 18h with indicated stimuli. Then the for IL-1 ß
were
stained with
were
(R&D Systems) (both
1ß and IL-12
R&D
systems)
Induction of
4.3.10.
BM-DCs
were
cultures
experimental
supernatants
as
were
heart muscle
specific peptide derived
subsequently
with different stimuli
cultured for
analyzed by sandwich ELISA As standards recombinant IL-
autoimmune
[432].
myocarditis,
from
a-myosin
including
0.1
myocarditis
In order to minimize
supplemented
autoimmune
were
used.
described
were
were
(BD PharMingen).
experimental
generated
maturation of DCs, Induction of
and IL-12
from BM-DCs, cells
with
IL-10
DCs
were
H chain
For the
(10 ng/ml). a
murine
were
treated
loaded with
[433].
ug/ml LPS plus
spontaneous
5
DCs
pg/ml anti-CD40
ab
58
Results
or
0.33
ug/ml Flag-CCL19
for 4 h and
2 and 4. Non-stimulated DCs
sacrificed and the hearts
were
were
injected i.p.
injected
as
(2x105 cells/mouse)
negative control.
removed and the
grade
of
On
on
day
myocarditis
days 0
10 mice
was
and
were
determined
by histology. A vague
diagnosis. Myocarditis to 4
(0,
myocytes
was
scored
cellularity
on a
was
considered
not
inflammatory
inflammatory
cells
or
cells
involving
involved; and 4, >30% of
a
surrounding >30
individual
cross-section
cells
a
0
between
myocytes; 2, larger foci of
myocytes; 3, >10% of
myocardial
sufficient for
semi-quantitative scale using grades from
inflammatory infiltrates; 1, small foci of inflammatory
no or
in interstitial
increase
>100
myocardial cross-section
involved).
59
Results
Results
4.4.
Functional
4.4.1.
cloning
of CCL19
as
inducer of T cell
in
proliferation
DC-T cell mixed culture assays As DCs
crucial for the induction of immune responses,
are
novel DC maturation factors We an
generated
using
an
alphaviral expression
vector.
spleen
Originally
cDNA
the RNA
library and transferred
a
as
viral
described in Materials and Methods. The cDNA library
library by
in vitro
transcription
and
it into
spleens of
extracted from
was
LCMV-infected BALB/c mice, transcribed in cDNA and normalized RNA
identify
alphaviral screening technology.
normalized activated
a
set out to
we
against splenocyte
was
electroporation together
transformed into
with
a
viral
helper
construct.
Following of 0.1 and This
the
single infected
led to the
cells
were
generation of the
expression
of
to
supernatants
were
harvested and
derived DCs
were
treated with the
The next
sorted
one
remaining
were
within
3
protein.
over
T cells
The
protein containing
night
were
plate
were
shown in
genes in total
Figure
1B.
by UV-light. Spleen pulsed
and
with OVA
added. Proliferation
was
calculated and every well which showed
the virus recovered and the cDNA sequence determined
was
plates.
where each well
virus inactivated
supernatants
MOI
days,
count/min value two standard deviations above the average
assay. CCL19
an
3H-Thymidine incorporation.
The average counts per
library 10000
infected with
subconfluent BHKs in 96 well
on
different
day OVA-specific CD4+
assessed 2.5 days later by
1A BHK cells
protein library
a
corresponds
peptide.
Figure
shown in
protocol
were
plate
were
further evaluated,
was
(Figure 1B). Out
of the
proliferation
in the
tested for induction of T cell
identified two times. One
a
CCL19
was
identified is
60
Results
BHK cells infected with
Sindbis virus genes from
encoding library
AMS
an
UV treatment
Sorting
of
infected
Transfer of
3
days propagation
cells
e
"10°
10'
10!
10'
supernatant
->
ï
10'
Transfer of
supernatant to OVA-pulsed
Addition of
imDcs
e *§
0
specific
Activation
OVA
of DCs
CD4+ T cells
0
Antigen specific °
Proliferation of T cells
B 64000
®
48000
•
.IV*-,*...*.* .***....
......... *
H
.
32000 3
s
*C
%
*....*..
w
^v~*
.
*
•
«
O
.*.
*
Ü
16000
24
12
Outline of the
Figure 1(17). campaign: activated one
A. BHK cells were infected with
mouse
cell/well
on
infected wells DCs. After
screening strategy and CCL19
spleen library.
Infected cells
BHK cells seeded in 96 well
were
over
a
CD4+
(stained plates.
with anti-sindbis
on
were one
(encircled point).
well showed
on
the
RNA from the
the 96 well
plate.
an
sorted
were
supernatant of
the
OVA-peptide pulsed spleen-derived
H3-thymidine
into
T cells
were
proliferating
added
T cells.
substantially increased proliferation of OVA-specific
positive
well
was
step into DNA and sequenced. The virus encoded gene turned axis location
specific antibody)
night treatment OVA-specific MACS-beads purified CD4+ D011.10 incorporation of
screening
encoding genes from
days propagation
After 3
harvested, UV treated and transferred
plate is shown
T cells
of the results of the
MOI of 0.1 with Sindbis virus
to the wells. Read-out for activation of DCs was
B. A 96 well
as one
96
84
72
60
48
36
extracted, transcribed by
out to be CCL19. Y-axis
Pointed line indicates counts/min average of the
plate.
a
RT-PCR
counts/min, X-
61
Results
CCL19 induces maturation of dendritic cells
4.4.2.
To reconfirm that CCL19 is
system, the RNA from the virus cloned into the the
supernatant
factors, the
alphaviral
we
not
was
shown).
was
recovered, transcribed in DNA and the sequence
vector. Virus
a
possible
tested directly
From this
was
(Figure 2A).
retested
wanted to exclude
supernatant
(Data
was
we
in the DC-T cell co-culture
inducing proliferation
on
BHK cells
generated, As
we were
showing
infected and
interested in DC activation
direct effect of CCL19
T cells
were
no
on
effect
T cells. Therefore
on
the
proliferation
concluded that CCL19 influences DCs directly. To
test, whether CCL19 may enhance T cell proliferation by activating DCs,
spleen
derived DCs with the
expression of CD86
are
CD40 and CD86
both
supernatants
treated with
Therefore
a
over
night
by FACS staining
important molecules
DC maturation. We found with
supernatants
with
and
analyzed
treated
them for surface
specific antibodies. CD40 and
for T cell activation and
significantly higher part
we
are
up-regulated
upon
of mature DCs in cultures treated
from SR-CCL19 infected BHK cells
compared
to DC cultures
supernatants from empty virus infected BHK cells (Figure 2B and 2C).
we
conclude that CCL19 indeed induces maturation in DCs.
62
Results
B
SR5
15000
E 10000 c 3
O
Ü 5000
SR5-TNF-Q
SR5-
SR5-TNF
SR5
CCL19
10u
10'
10'
10°
1. o
40
o
&
20
t
Immunization
Peptide
p33
A4Y
V4Y
G4Y
P33
A4Y
V4Y
G4Y
on
target cells
s«
o
2L
Immunization
p33
p33
p33
p33
p33
p33
p33
A4Y
V4Y
G4Y
S4Y
Peptide on target cells
p33
A4Y
V4Y
G4Y
p33
S4Y
p33
p33
p33
p33
p33
Figure 2(23). Induction of peptide-specific cytotoxic variants-VLP: C57BL/6 mice VLP with 20 nmol later
CpGs.
As
were a
control mice
samples
were
CD8+ T cells
used per group,
were
efficiency of
with PBS and 20 nmol
induced CTL responses
stained with APL-tetramers and anti-CD8
percentage peptide specific Four mice
the
injected
were
error
are
bars
shown
on
represent
by immunization
with
the
antibody
and
error
were
S4Y-
determined. A.
analysed by FACS.
of the
to naïve CD8+ T cells
or
CpGs. Eight days
Y-axis, and the peptide
the standard
and
p33-
G4Y-
30ug of p33-, A4Y-, V4Y-,
immunized i.v. with either
frequencies of specific T-cells and
Blood
T cells
(data
on
mean.
not
The
the X-axis.
The different
shown).
B. and
tetramers showed less than 0.2 %
unspecific binding
C. A 1:1 mixture of CFSE labelled,
peptide pulsed splenocytes (CSFEhigh) and unpulsed splenocytes
(CFSElow) and
were
spleen and
injected
LN
response of each determined
(C).
are
as
target cells. After 4 h the mice
were
killed
analyzed for the presence CSFEhigh (pulsed) and CFSElow (unpulsed) cells. peptide against itself (B) and
In vivo
specific lysis is shown target cells
i.v. into the immunized mice
cytotoxicity on
the
was
determined
as
cross
reactivity of
the different
peptides
were
described in material and methods. Percentage
Y-axis, the VLPs used for immunisation and the peptides loaded
shown on the X-axis. Circles
lysis in each group. Three mice
the
The
were
represent
individual mice and lines show average
used per group. One of two
comparable experiments
on
specific
is shown.
81
Results
The
ability
next. Mice
and 9
were
were
primed
with
challenged
challenged naive
for the
with
mice
more
a
peptides
under less
was
able to confer
results
were
stringent conditions,
reduced viral dose,
(Figure 3B).
assessed
CpG oligonuleotides
expressing
p33-VLPs efficiently controlled viral replication (Figure
leading
from
(not shown).
mice
were
immunized and
A4Y and
complete protection
partial protection
was seen
with the other
V4Y
(Figure 3B). Protection
from LCMV infection followed
Whereas
p33,
A4Y and V4Y induced
good protection, G4Y and
do
so
(Figure 3C). Thus,
albeit with limited
variant
efficiency.
peptides
were
In order to
agonistic peptide pattern.
high
to 100-fold lower viral titers in ovaries
Under these circumstances,
strongly cross-reactive peptide
protection
obtained if the VLPs
into mice in the presence of anti-CD40 antibodies
protection
was
with recombinant vaccinia virus
challenge (Figure 3A). Similar
test anti-viral
of
mice
In contrast, none of the variant
injected
protection
immunized with the various VLPs together with
days later,
dose virus
T cells
of cross-reactive T cells to mediate anti-viral
LCMV-GP. Mice
3).
protection by cross-reactive
Anti-viral
5.4.3.
are
a
similar
S4Y failed to
able to mediate anti-viral
protection
82
Results
Vaccinia: high titer
0
©•
Ä
•e-
T7
0
I
o
o
O) o
e-
G4Y
V4Y
A4Y
p33
Immunization: PBS
Vaccinia: low titer
B
o. U)
o
Immunization: PBS
p33
A4Y
V4Y
G4Y
S4Y
2.
£
LCMV
XX O
°
o
a
o
Immunization: PBS
Figure 3(24). Pathogen and
Groups of
three C57BL/6 mice
S4Y-VLP and 20 nmol
priming
mice
were
(B) of Vacc-GP were
dose
or
CpGs.
a
*
£
p33
A4Y
V4Y
G4Y
dependent protection after
were
As
&
.,
j
S4Y
A4Y- and V4Y-VLP immunization:
immunized i.v. with either 30 ug of
p33-, A4Y-, V4Y-,
control mice received PBS and 20 nmol
challenged i.p. either
with
i.v. with 200 PFU LCMV
a
high
(C).
isolated, and viral titers in the organs
lines show average viral titers in each group.
dose 1.5
Five
were
x
106
days later
PFU
ovaries
determined. Circles
(A)
CpGs.
or a
Nine
low dose 3
(Vacc-GP)
or
G4Y-
or
days after x
104
PFU
spleens (LCMV)
represent individual
mice and
83
Results
Induction of autoimmunity
5.4.4.
expressing LCMV-GP under
mice
Transgenic
first set of
experiments,
presented by
activated DCs
diabetes if
given
alone
[460]. Thus,
In addition,
(Figure 4).
peptides
as
A4Y
or
V4Y,
(Figure 4). Thus,
no
rat
insulin
[367].
even
autoimmunity could
responses
were
induced with
days
were
an
In
for
failed to induce
injected together
onset between d-7
(not shown).
later
unless
heavily infiltrated early
fused to VLPs failed to induce diabetes also if
anti-CD40 antibodies such
few
a
cell
T
p33-VLPs
islet cells
pancreatic
after onset of diabetes and virtually absent variant
if
rapidly
was
poor
expected, p33-VLPs
as
(not shown). However,
with anti-CD40 antibodies, diabetes and d-13
induce
p33-VLPs
p33-VLPs.
with
of the
tested whether RIP-GP mice develop diabetes upon
we
immunization
control
diabetes upon infection with LCMV
promoter (RIP-GP) develop autoimmune a
the
In contrast,
applied together
with
strongly cross-reactive peptides,
be induced.
600,
,
500
|
400
.§.
300
I
2M 100
|
„] 5
,
,
,
,
7
9
11
13
—A—P33
15
-1-A4Y -0-V4Y -K-G4Y -»-S4Y
time
(days)
but not by Figure 4(25). Induction of increased blood glucose level and autoimmunity by p33 variant
peptides: RIP-GP mice
were
immunized i.v. with either 100 ug of
S4Y-VLP and 100 ug anti-CD40 antibodies. Blood different intervals
starting
but not the variant
5
glucose
days after immunization.
peptides induced diabetes
and
p33-, A4Y-, V4Y-,
G4Y-
or
levels in these mice were monitored at
Three animals
were
used per group. P33-VLPs
significant elevation of blood glucose levels
(p
H-*
«?,
'/5
'/*
o„
o
*
