Prof. Dr. med. Univ. Gerold Schuler Myelo

Aus der Hautklinik
der
Friedrich- Alexander- Universität Erlangen-Nürnberg
Direktor: Prof. Dr. med. Univ. Gerold Schuler
Myeloid-derived suppressor cell activation by combined
lipopolysaccharide plus interferon-γγ treatment impairs
dendritic cell development
Inaugural-Dissertation
zur Erlangung der Doktorwürde
der Medizinischen Fakultät
der
Friedrich-Alexander-Universität
Erlangen-Nürnberg
vorgelegt von
Verena Greifenberg
aus
Bamberg
Gedruckt mit Erlaubnis der
Medizinischen Fakultät der Friedrich-Alexander-Universität
Erlangen-Nürnberg
Dekan:
Prof. Dr. J. Schüttler
Referent:
Prof. Dr. M. Lutz
Korreferenten:
Prof. Dr. Dr. A. Gessner
Prof. Dr. N. Romani
Prof. Dr. A. Steinkasserer
Tag der mündlichen Prüfung:
26. November 2009
Für einen lieben Menschen
Für eine Diva
Für eine liebe
Diva
1 ............................................................................................................... SUMMARY 1
1.1..........................................................................................BACKGROUND AND AIMS 1
1.2...............................................................................................................METHODS 1
1.3................................................................................................................RESULTS 1
1.4....................................................................................... PRACTICAL CONCLUSIONS 2
2 ............................................................................................ ZUSAMMENFASSUNG 3
2.1........................................................................................HINTERGRUND UND ZIELE 3
2.2.............................................................................................................METHODEN 3
2.3........................................................................ERGEBNISSE UND BEOBACHTUNGEN 3
2.4......................................................................PRAKTISCHE SCHLUSSFOLGERUNGEN 4
3 .......................................................................................................INTRODUCTION 5
3.1.............................................. STRUCTURE AND FUNCTIONS OF THE IMMUNE SYSTEM 5
3.1.1.........................................Common introduction into the immune system 5
3.1.2...............................................................Regulation of immune response 6
3.2................................................................... MYELOID-DERIVED SUPPRESSOR CELLS 8
3.2.1.................................................................................................. Overview 8
3.2.2................................................................................... Plasticity of MDSC 9
3.2.3.................................................... MDSC in homeostasis and in diesease 9
3.2.3.1....................................................................................... Murine MDSC 10
3.2.3.2.......................................................................................Human MDSC 11
3.2.4..........................................................................In vitro generated MDSC 12
3.2.5............................................................. Induction of generation of MDSC 12
3.2.5.1......................................Factors for expansion and activation of MDSC 12
3.2.5.2................................................................ Signal transduction in MDSC 14
3.2.6...............................................Strategies of MDSC for T-cell suppression 14
3.2.6.1..................................................Mechanisms of suppression by MDSC 15
3.2.6.1.1 ....................................................................... Nitrogen monoxide 15
3.2.6.1.2 ................................................................L-arginine and arginase 15
3.2.6.1.3 ..............................................................Reactive oxygen species 16
3.2.6.1.4 ........................................................Transforming growth factor β 16
3.2.6.2............................. Mechanisms of suppression of both subpopulations 16
3.2.6.3..................................... Role of antigen-specific suppression by MDSC 17
3.2.7..................................................Integration of MDSC in cellular networks 17
3.2.8.................................................................................................... Outlook 18
2
3.2.9............................................................................................... Hypothesis 19
4 ............................................................................................................ EINLEITUNG 20
4.1....................................................... AUFBAU UND FUNKTIONEN DES IMMUNSYSTEMS 20
4.1.1............................................Allgemeine Einführung in das Immunsystem 20
4.1.2.................................................................. Regulation der Immunantwort 22
4.2....................................................... SUPPRESSORZELLEN DER MYELOISCHEN REIHE 23
4.2.1............................................................................. Allgemeine Einführung 24
4.2.2................................................................................ Plastizität der MDSC 25
4.2.3.................................... MDSC in der Homöostase und bei Erkrankungen 26
4.2.3.1....................................................................................... Murine MDSC 26
4.2.3.2......................................................................... MDSC beim Menschen 27
4.2.4..........................................................................In vitro generierte MDSC 28
4.2.5............................................................. Induktion der Bildung von MDSC 29
4.2.5.1............................. Faktoren zur Expansion und Aktivierung von MDSC 29
4.2.5.2................................................................. Signaltransduktion in MDSC 31
4.2.6.......................................... Strategien der MDSC zur T-Zell-Suppression 31
4.2.6.1........................................Mechanismen der Suppression durch MDSC 32
4.2.6.1.1 ......................................................................... Stickstoffmonoxid 32
4.2.6.1.2 .................................................................L-Arginin und Arginase 32
4.2.6.1.3 ..........................................................Reaktive Sauerstoffspezies 33
4.2.6.1.4 ........................................................Transforming growth factor β 33
4.2.6.2..................... Suppressionsmechanismen der beiden Subpopulationen 34
4.2.6.3............Bedeutung der Antigenspezifität der Suppression durch MDSC 34
4.2.7...............................Integration der MDSC in weitere zelluläre Netzwerke 35
4.2.8................................................................................................... Ausblick 35
4.2.9................................................................................................Hypothese 36
5 .................................................................................... VORVERÖFFENTLICHUNG 37
6 ...................................................................................... LITERATURVERZEICHNIS 38
7 .................................................................................ABKÜRZUNGSVERZEICHNIS 52
8 ........................................................................................................ DANKSAGUNG 55
9 .......................................................................................................... LEBENSLAUF 57
1
1
1.1
Summary
Background and aims
Dendritic cells (DC) and myeloid-derived suppressor cells (MDSC) are involved in
the control of the immune response within tumor diease and infections. Here, MDSC
are mainly found in the spleen and bone marrow and barely in lymph nodes.
Morphologically, they are divided into monocytic and polymorphonuclear cells. It is
not known yet which role MDSC play in healthy, untreated mice. MDSC can be
generated
from
murine
bone
marrow
cells
by
application
of
granulocyte/macrophage-colony-stimulating-factor (GM-CSF) and suppress T-cell
response in vitro. The cytokine interferon-γ (IFN-γ) and nitrogen monoxide (NO) are
required for this process. However, further stimulation of bone marrow cells with
GM-CSF generates immature DC. Both DC and MDSC stem from immature myeloid
precursor cells. So far, it is not resolved which factors regulate the differentiation of
these cells to either DC or MDSC.
This dissertation aimed for the identification of different subpopulations of MDSC in
splenocytes of healthy mice. In addition, the modes of stimulation, activation and the
plasticity of MDSC should be analyzed.
1.2
Methods
Therefore, ex vivo isolated splenocytes were characterized with flow cytometry;
based on this, different subpopulations were isolated via cell sorting. Their
suppressive potential was examined with mixed lymphocyte reaction and measuring
of the amount of released NO, cytospins helped to identify their morphology. In vitro
generated and ex vivo isolated immature myeloid cells were also characterized with
flow cytometry after stimulation of mice and/or cells with different cytokines.
1.3
Results
In this dissertation it was shown that the combination of IFN-γ and LPS (bacterial
lipopolysaccharide) inhibited further differentiation of in vitro generated MDSC in the
most efficient way; in addition, their suppressive activity was induced and enhanced
by this treatment. In ex vivo isolated splenocytes of healthy, untreated mice six
different subpopulations could be defined regarding surface markers and
size/granularity/morphology of the cell; two of them were able to suppress T-cell
response: Gr-1high CD11bintermediate cells with ring-shaped nuclei and Gr-1low
CD11bintermediate cells with heterogenous morphology.
2
IFN-γ und LPS did not enhance suppressive activity in splenocytes isolated from
healthy mice. However, the additional induction of a specific immune response with
endogenous or exogenous DC caused an accumulation of Gr-1+CD11b+ cells in the
spleen; hereby the proliferation of T-cells in the spleen was reduced. Furthermore, it
was shown that injection of IFN-γ and LPS inhibited the in vitro differentiation of ex
vivo isolated Gr-1+CD11b+ splenocytes into mature DC. In addition, ex vivo isolated
CD11b+ splenocytes lost their ability to suppress T-cell response after application of
IFN-γ, LPS and GM-CSF to cell culture.
1.4
Practical conclusions
These results suggest that in situations such as chronic inflammation/infection LPS
and IFN-γ both activate the immune response and induce the generation of MDSC
in the spleen to avoid exaggerating systemic reactions. Obviously, the specific T-cell
response in the regional lymph nodes is not influenced by these processes.
These data contribute to a better understanding of the activation of MDSC in vivo. A
possible clinical application could be the selective induction or the injection of MDSC
to avoid chronical inflammation.
3
2
Zusammenfassung
2.1
Hintergrund und Ziele
Dendritische Zellen (DZ) und myeloide Suppressorzellen (MDSC) sind an der
Kontrolle der Immunantwort im Rahmen von Tumoren oder Infektionen beteiligt.
Letztere findet man hierbei vor allem in Milz und Knochenmark und kaum in den
Lymphknoten und teilt sie morphologisch grob in monozytäre und polymorphkernige
Zellen ein. Inwieweit MDSC in gesunden, unbehandelten Mäusen eine Rolle spielen
ist bis dato nicht geklärt. Durch Zugabe von GM-CSF (granulocyte/macrophagecolony-stimulating-factor) kann man aus murinen Knochenmarkszellen MDSC
kultivieren. Diese supprimieren in vitro die T-Zell-Antwort; hierbei sind u.a. das
Zytokin Interferon-γ (IFN-γ) und Stickstoffmonoxid (NO) involviert. Eine längere
Stimulation der Knochenmarkszellen mit GM-CSF führt hingegen zur Bildung reifer
DZ. DZ und MDSC stammen von unreifen myeloiden Vorläuferzellen ab. Bisher ist
nicht geklärt, welche Faktoren die Differenzierung dieser Zellen zu entweder DZ
oder MDSC regulieren.
Diese Arbeit hatte zum Ziel, in Milzzellen gesunder Mäuse verschiedene
Subpopulationen der MDSC zu identifizieren und deren Stimulierbarkeit, Aktivität
und Plastizität näher zu beleuchten.
2.2
Methoden
Hierzu
wurden
ex
vivo
isolierte
Milzzellen
mittels
Durchflusszytometrie
charakterisiert und daraufhin über verschiedene Zellsortiermethoden diverse
Subpopulationen
isoliert.
Diese
wurden
mit
Hilfe
der
gemischten
Lymphozytenreaktion und Messung des NO-Gehalts auf ihr suppressives Potential
und mit Cytospins bezüglich ihrer Morphologie untersucht. Es fanden nach
Zytokinbehandlung in Zellkultur und im Mausmodell auch durchflusszytometrische
Messungen an in vitro generierten und ex vivo isolierten unreifen myeloiden Zellen
statt.
2.3
Ergebnisse und Beobachtungen
In dieser Arbeit wurde gezeigt, dass die Kombination aus IFN-γ und LPS
(bakterielles Lipopolysaccharid) die weitere Differenzierung in vitro generierter
MDSC am effektivsten verhindert und zudem deren suppressorische Aktivität
induziert und verstärkt. In ex vivo isolierten Milzzellen aus gesunden, unbehandelten
Mäusen konnten im Hinblick auf Oberflächenmarker und Zellgröße/-granularität/morphologie sechs verschiedene Subpopulationen charakterisiert werden; davon
4
waren zwei suppressorisch aktiv: Gr-1highCD11bintermediate Zellen mit ringförmigen
Kernen und Gr-1lowCD11bintermediate Zellen mit heterogener Morphologie.
IFN-γ
und
LPS
führten
in
gesunden
Mäusen
zu
keinem
Anstieg
der
suppressorischen Aktivität bei isolierten Milzzellen. Die zusätzliche Induktion einer
spezifischen Immunantwort mittels endogener oder exogener DZ aber bewirkte eine
Akkumulation Gr-1+CD11b+ Zellen in der Milz. Dabei wurde die Proliferation Milzständiger T-Zellen reduziert. Weiterhin wurde gezeigt, dass die Injektion von IFN-γ
und LPS die in vitro-Differenzierung ex vivo isolierter Gr-1+CD11b+ Milzzellen zu
reifen DZ verhindert. Ex vivo isolierte CD11b+
Milzzellen verlieren zudem ihre
Fähigkeit zur Suppression, wenn sie in Kultur IFN-γ, LPS und GM-CSF erhalten.
2.4
Praktische Schlussfolgerungen
Diese Ergebnisse lassen vermuten, dass zum Beispiel im Rahmen chronischer
Entzündungen/bakterieller Infektionen LPS und IFN-γ mit der Aktivierung des
Immunsystems gleichzeitig die Bildung von MDSC in der Milz induzieren, um
überschießende systemische Reaktionen zu vermeiden. Die spezifische T-ZellAntwort in den regionalen Lymphknoten wird hierbei wohl nicht beeinflusst. Diese
Daten tragen zum besseren Verständnis der Aktivierung von MDSC in vivo bei.
Durch gezielte Induktion oder Applikation von MDSC könnte so zum Beispiel bei
Patienten die Entstehung chronischer Entzündungen verhindert werden.
5
3
Introduction
3.1
Structure and functions of the immune system
The vertebral immune system represents a complex arrangement made of cellular
and molecular components as well as organs. It protects the organism from
environmental influences and regulates processes within the body, thereby
sustaining the creature´s integrity.
3.1.1
Common introduction into the immune system
The immune system comprehends two cooperating and partially overlapping
compartments: the unspecific mechanisms of defense of the innate (natural) and the
specific strategies of defense of the acquired immune system.
The first one comprises mechanical (e.g. dermal and mucosal epithelium), microbial
(e.g. bacterial colonization of epithelial layers) and chemical (e.g. pH, enzymes)
barriers as well as molecular (complement system) and cellular (phagocytes, natural
killer cells) components. The innate immune system detects foreign microorganims
and molecules via certain surface receptors and then initiates the elimination of the
pathogens. Mainly macrophages and neutrophil granulocytes ingest foreign material
and trigger an inflammation by secretion of cytokines and chemokines. Immature
dendritic cells (DC) also belong to the family of phagocytes and are able to process
pathogens. They are activated by microbial ligands or proinflammatory cytokines
68,
125
. Mature DC show processed fragments on surface major histocompatibility
complex II (MHC II) molecules and are then called antigen-presenting cells (APC).
Additionally to antigen binding via T-cell-receptor and binding of CD4 (CD=cluster of
differentiation) to MHC II- molecules, costimulatory molecules on DC are required
for an effective activation of T-cells; CD11c can also be found on mature DC 11. After
peripheral activation they migrate to regional lymph nodes. There, the acquired
immune system consisting of B-lymphocytes (humoral defense) and T-lymphocytes
(cellular defense) comes into operation. It is able to specifically recognize presented
antigens and also to establish an immunological memory.
T-lymphocytes detect processed antigen in a specific way via their T-cell receptor
(TCR)
53
. TCR as well as the coreceptors CD4 respective CD8 and costimulatory
molecules like CD28-CD80/CD86 or CD40/CD40-ligand are needed for activation of
T-cells and thereby for a sufficient and effective immune response. Without this
costimulation T-cells can not react on antigen inspite of antigen binding. This
phenomenon is called anergy 137.
6
CD8+ cytotoxic T-cells bind complexes of MHC I-molecules and peptides and
recognize infected or abnormal cells in this way. By secretion of granzyme, perforin
or different cytokines (e.g. interferon γ =IFN-γ) the elimination of pathogens is then
induced.
APC present ingested and processed exogenous antigen on MHC II-molecules,
which are bound by CD4 of T-lymphocytes. Depending on the antigen´s character
the secretion of different cytokine patterns is induced. These cytokines regulate the
differentiation of naive CD4+ T-lymphocytes and control their involvement in either
cell-mediated TH1- or humoral TH2-immune response
106
this process called T-cell-polarization or T-cell-priming
19, 30, 67
. Activated DC take part in
. IFN-γ and interleukin-
12 (IL-12) pave the way for TH1-response with activation of macrophages and
simultaneously inhibit TH2-response 103.
The immunological processes described so far ensure a sufficient elimination of
noxes and pathogens. However, a disproportionate and potentially harmful immune
response (i.e. allergic reaction, autoimmunity, chronic inflammation) has to be
avoided. Another problem is the influence of malignant tumor diseases on the
immune system: tumor cells can influence immune cells to that effect that they are
not able to recognize degenerated cells anymore and cancer can spread out in the
body.
Summing up, immunologic processes in the body have to be strictly controlled and
regulated and abandoned in time to circumvent structural damage. Diverse
mechanisms contribute to the regulation of the immune response.
3.1.2
Regulation of immune response
A central function of the specific immune system is to distinguish between foreign
antigens and antigens produced naturally in the body. In homeostasis self-antigen is
presented in a tolerogenic way to T- and B-lymphocytes, hereby inducing and
sustaining immunologic tolerance.
Formation of so called central tolerance takes place in the thymus. Here,
lymphocytes recognizing self-antigen with their specific TCR are deleted. This is
performed either by “clonal deletion” or inactivation of the affected cell. A small
percentage of autoreactive T-cells can not be removed in this way and reaches the
periphery. In a process called “peripheral tolerance” these remaining, potentially
harmful lymphocytes get eradicated. This can also cause suppression of T-cell
7
response against foreign antigens. Peripheral tolerance is induced by several
intrinsic and extrinsic mechanisms.
Intrinsic processes include for example anergy as described above. The absence of
costimulation inspite of antigen-binding abolishes several signal transduction
cascades and hence inhibits T-cell activation
136
. Normally, exogenous antigen is
presented via MHC II-molecules. Immature DC can induce anergy by ingesting and
presenting surrounding self-antigens (e.g. particles of dropping cells) via MHC Imolecules to T-cells without costimulation
3, 72,87, 92, 152
. This process is called “cross-
+
presentation”. Thereby, CD8 T-cells recognizing self-antigen via MHC I get anergic
or even commit apoptosis 73, 124.
Additionally, coinhibitory molecules on the surface of T-cells (e.g. cytotoxic
T-lymphocyte antigen 4=CTLA-4; programmed death 1=PD-1) can react with their
counterparts on the surface of APC (CD80/ CD86; programmed death ligand 1/2=
PD-L1/ PD-L2). This interaction can inhibit sufficient contact between TCR and
presented self-antigen and hence disturb T-cell activation
165
. Furthermore, kind of
application and amount of featured self or foreign antigen and affinity/avidity of TCR
influence peripheral tolerance 51.
Diverse cell populations complete the intrinsic suppressive mechanisms described
above in an extrinsic way. Since the early 70ies tolerogenic suppressor T-cells are
known. Nowadays, these cells are called regulatory T-cells (Tregs)
48
. They affect
proliferation and differentiation of T- and B-lymphocytes, of natural killer cells (NKcells), but also of monocytes and DC, thereby avoiding excessive immune reactions.
This process requires cell-cell-contact and/or soluble factors
150, 168
. At the moment,
two subpopulations of Tregs can be distinguished: on the one hand natural
CD4+CD25+ Tregs and on the other hand induced respective adaptive Tregs
58, 168
.
Natural Tregs recognize self-antigen via their TCR already in the thymus and get
activated in this way. In the periphery, naive T-cells can be activated by antigen
contact and certain cytokines and differentiate into adaptive Tregs
34, 65
. Within B-
cells certain subpopulations are also known to regulate and suppress immune
response 20, 104.
Cells of the myeloid line, so called myeloid-derived suppressor cells (MDSC), also
contribute to the suppression of immune reactions and will be discussed in the
following chapters.
8
3.2
Myeloid-derived suppressor cells
In the late 70ies a former unknown cell population with suppressive features
awakened the scientific opinion. In bone marrow and spleen of tumor-bearing mice
and in murine lymphatic tissue myeloid cells were detected being able to suppress
T-cell response in vivo and in vitro
126, 148, 154, 155
. These cells were called “natural
suppressor cells” and further characterized in the following decades. Later, they
were nominated as “immature DC” (IDC). Another term used for a long time was
“myeloid suppressor cells” (MSC). Since about 2007, these cells are consistently
described as myeloid-derived suppressor cells (MDSC) to avoid confusions 45.
3.2.1
Overview
During myelopoiesis in murine bone marrow haematopoietic stem cells are
stimulated by different soluble molecules and cell-bound receptors to create
common myeloid precursor cells. Amongst others, these cells generate “immature
myeloid cells” (IMC) without suppressive features.
In healthy individuals, IMC migrate in peripheral lymphatic organs where their
differentiation into mature macrophages, DC or granulocytes takes place. Diverse
pathologic processes like for example inflammation, malignant tumor diseases or
infections inhibit the differentiation of IMC. Thereby, these cells acquire
immunosuppressive qualities and are then called MDSC.
MDSC represent a very heterogenous cell population consisting of various myeloid
precursor cells. Many research groups are interested in their characterization and
classification. All MDSC characterized so far express the surface markers CD11b
(also called CD11b/CD18-complex or Mac-1) and Gr-1
142
. The CD11b receptor is
an αMβ 2-integrin and binds the complement component C3bi. It can be found on
monocytes, macrophages, DC and granulocytes as well as on activated B- and Tcells and natural killer cells 83, 99. Binding ICAM 1 (intercellular adhesion molecule 1),
CD11b mainly regulates adhesion and extravasation of leucocytes
69
. Gr-1, a
glycosyl-phosphatidyl-inositol (GPI)-anchored protein, is a marker of differentiation
in the myeloid lineage and can be found on myeloid precursor cells, granulocytes
and transiently on monocytes
39, 57
. In the meantime, Gr-1 specific antibodies are
known to bind two different epitopes of Gr-1: Ly6G and Ly6C. The application of
epitope-specific antibodies revealed two different subpopulations of MDSC in
splenocytes of tumor-bearing mice: CD11b+Ly6G+Ly6Clow MDSC morphologically
resembling neutrophilic granulocytes (PMN-MDSC) and CD11b+Ly6G-Ly6Chi(gh)
MDSC with monocytic character (MO-MDSC)
107, 174
. However, a separation of
9
splenocytes into two different subpopulations was also possible with the convenient
Gr-1-antibody in a murine tumor model: Gr-1hi and Gr-1int(ermediate). These two
subpopulations differ in their potential to suppress T-cell response and their further
differentiation
170
. Depending on the general conditions, further surface markers like
CD80 (B-7.1), CD115 (macrophage-colony-stimulating factor=M-CSF) or CD124
(IL-4 receptor α chain) can be characterstic for certain subpopulations of MDSC
47,
59, 173
. However, no consistent pattern of surface markers could be defined so far
and it could not be proven that these molecules are directly involved in suppression.
MDSC mainly suppress T-cell response using various surface molecules and
soluble elements, described subsequently. Beside naive and effector T-cells, they
can also influence the function of other cells. They are able to induce the generation
of suppressive Tregs
140
. MDSC have also an effect on the function of NK-cells,
macrophages and even B-cells 22, 142, 147.
3.2.2
Plasticity of MDSC
Many research groups examine the differentiation of MDSC, generating mature
myeloid DC. In addition, so called plasmacytoid DC exist
from bone marrow
63, 182
, spleen
13, 56
or fetal liver
181
110
. Murine cells isolated
can differentiate into myeloid
DC after stimulation with certain cytokines like granulocyte/macrophage-colonystimulating-factor (GM-CSF), tumor necrosis factor α (TNFα), IL-4 or Fms-like
tyrosine kinase 3 ligand (FLT3-ligand). All-trans-retinoid acid (ATRA) can reduce the
amount of MDSC in vivo and in vitro an induce their differentiation into DC,
macrophages or granulocytes 44, 79. The application of 1α, 25-dihydroxyvitamin D3, a
metabolite of vitamin D3, also activates the differentiation of immature myeloid cells
82, 167
. In tumorous tissue, MDSC can differentiate into F4/80+ tumor-associated
macrophages (TAM), which are able to suppress T-cell response secreting certain
cytokines.
There is evidence, that immature myeloid cells are also involved in angiogenesis in
tumor disease, being able to differentiate into endothelial cells
171, 175
. This theory is
supported by the fact that Gr-1+/CD11b+ cells of diseased mice express the enzyme
matrixmetalloproteinase-9 (MMP-9).
3.2.3
MDSC in homeostasis and in diesease
About 20-30% of bone marrow cells in healthy mice are Gr-1+/CD11b+, whereas
only 2-4% of nucleated splenocytes and blood cells belong to this population.
Without stimulation, no such cells can be found in lymph nodes
76
. Particular
10
conditions effect an augmentation respectively a redistribution of Gr-1+/CD11b+ cells
in murine and human organism.
3.2.3.1
Murine MDSC
In various murine tumor models, MDSC can be found to a higher degree in spleen
and tumor tissue
24, 100, 155, 173, 176
. Interestingly, the amount of mature DC is reduced
in tumor diseases, whereas the number of immature precursor cells of DC rises
5, 85
.
DC in patients and mice suffering from tumor disease or from microbial infections
also show functional defects
14, 28, 43, 60
. Consequently, the immune response against
tumor cells is suppressed and they can more or less unhamperedly extend. The
mechanisms of suppression will be further described in following chapters. The
removal of the tumor reduces the number of Gr-1+/CD11b+ cells and restores tumorrelated immune reaction
134
. This confirms the hypothesis that the increase of
suppressive cells is induced by the tumor itself via different mechanisms. The
secretion of cytokines, chemokines and other soluble factors is especially
remarkable in this process (see in detail in further chapters). The depletion of
suppressor cells
144, 158
or their elimination with the chemotherapeutic agent
gemcitabine rebuilds an efficient immune response against tumor cells 156.
In addition, infection with different microorganisms (e.g. Salmonella typhimurium 2,
50
, Candida albicans
Trypanosoma cruzi
101
or Toxoplasma gondii
164
) enhances the
number of myeloid suppressive cells in murine lymphatic organs. Thus, this favours
the expansion of pathogens in the organism. Within a polymicrobial sepsis, MDSC
can induce the suppression and TH2-polarization of the T-cell response. Amongst
others, this activation of MDSC is based on MyD88, an adaptor protein on different
toll-like receptors (TLR) 33.
The inoculation of certain vaccines or antigens provokes an augmentation of MDSC
in the spleen
74, 159
. Using recombinant vaccinia virus, MDSC induce apoptosis in
CD8+ T-cells and thereby soften immune response against the vaccine
23
. The
application of superantigen also augments the amount of MDSC 25.
Within chronic inflammation or autoimmune diseases, the number of MDSC also
rises. In the murine model of multiple sclerosis, called experimental autoimmune
encephalitis (EAE), MDSC accumulate and migrate to the central nervous system
183
. In experimental induced chronic inflammation in skin
94
, gut
55
and eye
70
an
increase of MDSC can be found. This induction of MDSC within chronic
11
inflammation is suspected to facilitate the development of malignant disease. The
activation of TLR and the secretion of different interleukines like IL-1β, IL-10 and
IL-12 are involved in this process 116.
It is assumed that MDSC also take part in the regulation of immune response after
radiation or allogenic bone marrow transplantation. MDSC seem to avoid the
dangerous
“graft-versus-host-disease”
after
infusion
of
donor
supporting the development of a “graft-versus-leukemia-reaction”
lymphocytes
15, 17
.
Furthermore, certain medicaments lead to an extension of MDSC and support their
suppressive activity. As known, the treatment with the chemotherapeutic agent
cyclophosphamide causes severe damage in hematopoietic and lymphatic tissue.
This is due to a direct impairment of T-cells as well as the induction of MDSC
indirectly suppressing proliferation and reagibility of T-cells 8, 113, 138.
3.2.3.2
Human MDSC
Over the years a lot of results argue for the existence of a human counterpart of
murine MDSC. Already in 1995 CD34+ cells were found in tumorous and lymphatic
tissue of patients with head and neck cancer. These cells were able to inhibit the
function of local T-lymphocytes
118
. Later, it was found that patients with tumor
diseases in head and neck region, lung and breast showing reduced amount of DC
in blood had elevated levels of immature myeloid cells which harmed T-cell function
5, 6
. These cells were mainly CD13+, CD33+, CD34+ and CD15-. Regarding their type
of human leukocyte antigen (HLA)-DR and expression of CD11c, these cells can be
divided into two subpopulations: immature monocytes/DC and cells belonging to
earlier stages of myeloid differentiation. Blood of patients with renal cancer
contained myeloid CD11b+/CD15+/CD14- cells also suppressing T-cell response
114,
. Recently, CD11b+/CD14+ myeloid suppressor cells were detected in patients
180
suffering from malignant melanoma
38
. After surgery
61
or a severe trauma
122
,
patients exhibit diverse T-cell dysfunctions. Traumatic stress results in migration of
Gr-1+/CD11b+ cells to the spleen where they affect T-cell function 93.
Hence,
MDSC
are
involved
in
many
different
immunological processes.
Interestingly, there is no definite scheme of suppression, no preassigned “target
cell”, no consistent phenotype and no fixed pattern of released cytokines.
Depending on manifold influences each immunologic process generates a specific
population of MDSC.
12
3.2.4
In vitro generated MDSC
Murine bone marrow cells can be stimulated to differentiate into DC of different
stages of maturation by application of GM-CSF alone or in combination with IL-4.
Depending on concentration of applied cytokines and length of stimulation these
cells possess different immunologic abilites
62, 81, 89, 90, 96
. Lutz et al designed a
method for the in vitro generation of immature myeloid suppressor cells using GMCSF 90. These cells are Gr-1low, CD11b+, CD31+, ER-MP58+, F4/80+ and CD11c- and
exhibit ring-shaped nuclei. Depending on the moment and duration of its application
to culture of bone marrow cells, bacterial lipopolysaccharide (LPS) can either
stimulate or inhibit their differentiation into mature DC
88
. In vitro generated MDSC
are able to suppress immune response of CD4+ and CD8+ T-cells in vitro requiring
cell-cell contact and nitrogen monoxide (NO). IFN-γ also seems to be involved in
suppression
132
. Pretreatment with in vitro generated MDSC reduces the number of
rejections after transplantation of an allogen heart in mice
90
. The application of
immature myeloid Gr-1+ precursor cells suppresses development of autoimmune
diabetes in so called non-obese-diabetic (NOD)-mice
153
. Thus, these in vitro
generated MDSC can be used as model for investigation of in vivo occuring MDSC.
3.2.5
Induction of generation of MDSC
Most knowledge of MDSC is recruited from experimental approaches in murine
tumor model. As described before, tumor disease leads to an accumulation of
MDSC in secondary lymphatic organs (mainly spleen), blood, bone marrow and
tumorous tissue of mice. There, they suppress the function of their target cells and
thus facilitate existence and dissemination of the tumor by weakening the immune
response. The expansion and activation of MDSC is caused by different factors
divided into two main groups: on the one hand substances secreted directly by the
tumor, which stimulate myelopoiesis and inhibit differentiation of myeloid cells. On
the other hand factors secreted from tumor stroma cells or activated T-cells or other
components
of
the
immune
system.
Together
with
particles
of
diverse
42
microorganisms these substances are able to activate MDSC in a direct way .
3.2.5.1
Factors for expansion and activation of MDSC
As a well-known factor, the cytokine GM-CSF induces differentiation of myeloid
precursor cells into granulocytes, macrophages and DC in bone marrow in
combination with other substances
63
. GM-CSF acts via the GM-CSF-receptor
consisting of two subunits α and β 95. Many murine and human tumors secrete a
huge and unphysiological quantity of GM-CSF, thereby generating MDSC. These
13
accumulate in secondary lymphatic organs and in tumor and there suppress T-cell
response
24, 40, 102, 141, 149
recombinant GM-CSF
. This phenomenon was also observed in mice treated with
24
. Supernatant of cell culture with a variant of Lewis lung cell
carcinoma contains GM-CSF. Injection of this supernatant into tumor-bearing mice
enhanced tumor growth, whereas application of neutralizing antibodies against GMCSF and also IL-3 abolished this effect 177. IL-3 and also IL-5 bind to β-subunit of the
GM-CSF-receptor. However, also activated T-cells 1, NK-cells
105
, NK-T-cells
8
and
DC 31 are able to provoke activation of MDSC by secretion of GM-CSF.
Hence, GM-CSF can stimulate immune response by induction of generation and
differentiation of macrophages, granulocytes and mainly DC in mice. This was also
possible in a human model, where GM-CSF was used as vaccine adjuvans
121
.
Exceeding physiological limits concerning quantity and length of stimulation GMCSF can effect the exact opposite and induce a suppression of immune response.
This facilitates expansion of malignant cells and invading pathogens.
Vascular endothelial growth factor (VEGF) is produced by many tumors and
stimulates angiogenesis
160
. This assures full supply of tumor cells with nutritive
substances. Within tumor disease, VEGF reduces the amount of DC and interferes
with their function in vivo and in vitro, whereas the number of immature precursors
of DC increases 41, 46.
Prostaglandin E2 (PGE2) is involved in inflammatory processes and also in tumor
disease. It is assumed that PGE2 secreted by tumors generates a local
inflammatory environment, thereby supporting tumorigenesis 26, 146, 151. Generation of
MDSC is induced via PGE2-receptors in this process. Cyclooxygenase-2-inhibitors
reducing the production of PGE2 diminish accumulation of MDSC and inhibit tumor
growth 146.
IFN-γ, secreted by many components of the immune system, influences the activity
of T-cells as well as MDSC 47, 97, 132. LPS is also involved in induction of MDSC 33.
In addition to the cytokines already mentioned, the immunosuppressive cytokine
transforming growth factor β (TGF-β) 4, 91, 172, 178 as well as stem cell factor (SCF) 119,
IL-1β, IL-4, IL-6, IL-10, IL-12, IL-13, MMP-9, M-CSF (macrophage-colonystimulating-factor) and G-CSF (granulocyte-colony-stimulating-factor=CSF-1) seem
to have influence on generation and function of MDSC 42, 120.
14
3.2.5.2
Signal transduction in MDSC
The substances mentioned above activate certain signal transduction cascades via
cytokine receptors respectively TLR on MDSC. Transmembrane cytokine receptors
on MDSC transmit their signal mostly via so called Janus Kinases (JAK), which
again bind to transcription factors of the STAT (signal transducer and activator of
transcription)-familiy
71
. Regarding MDSC, JAK2 and STAT-3 are known to play an
important role. Activation of STAT-3 causes activation and proliferation of MDSC
while inhibiting their differentiation into mature DC 111, 112. Recently it was shown that
STAT-3 also induces the production of the protein S100A9 (myeloid-related protein
14 respectively Calgranulin B) in myeloid precursor cells; this protein belongs to the
huge family of S100-Ca2+-binding proteins
29
. S100A8 (myeloid-related protein 8
respectively Calgranulin A) also is increased and prevents together with its partner
S100A9 the differentiation of immature myeloid cells and stimulates their
transformation into MDSC. S100A8 and/or S100A9 also seem to be involved in the
recruitment of MDSC to regions with dysplasia and inflammatory changes
162
. Both
proteins bind to the membrane-bound NAPDH (reduced form of nicotinamideadenine-dinucleotide-phosphate)-oxidase-oxygen-complex
and
induce
the
production of reactive oxygen species (ROS) contributing to suppression by MDSC
42
. IFN-γ uses STAT-1 in MDSC; IL-4 and/or IL-13 can activate the transcription
factor STAT-6 in MDSC by binding IL-4-receptor α
42
. STAT-1/-6 induce the
production of arginase-1 and inducible NO-synthase (iNOS), which both participate
in suppression (see 3.2.6). There is evidence that LPS activates the signal
transductor NF-κB (nuclear factor “κ-light-chain-enhancer” of activated B-cells) via
TLR on MDSC stimulating the production of arginase-1 and iNOS 42.
3.2.6
Strategies of MDSC for T-cell suppression
As mentioned above, MDSC suppress the immune response mainly of T-cells which
then can not react in an adequate way to immunological stimuli anymore.
Depending on species, particular organ system and further individual attributes of
the organism, MDSC can use different strategies of suppression. In worst case,
apoptosis of T-cells is caused
10, 23, 101
. MDSC cause changes in T-cell receptor
(mainly in TCR-ζ-chain) and other surface molecules. This can trigger further
reactions, for example the inhibition of activation respectively proliferation of T-cells
80, 93, 101
, the induction of peripheral tolerance
cytokines secreted by T-cells
25, 77
and changes in the pattern of
44
. In most cases, an effective suppression requires
close cell-cell-contact. The suppressive effect of MDSC can be relieved by
separation of cultured MDSC from T-cells using a semi-permeable membrane
44, 64,
15
78, 132
. This suggests that the involved cells interact via certain membran-bound
molecules and/or that rapidly degradable soluble factors play an important role. In
the following chapter the most important mechanisms of suppression of MDSC
detected so far will be described.
3.2.6.1
3.2.6.1.1
Mechanisms of suppression by MDSC
Nitrogen monoxide
Nitrogen monoxide (NO) is produced by murine DC, macrophages, granulocytes
18
. The inducible NO-synthase iNOS can generate NO from the
and also NK-cells
amino acid L-arginine; it splits L-arginine into citrulline and NO
54
. Many
experimental settings show that T-cell suppression by MDSC depends on NO 8, 37, 97,
132
. The production of iNOS can be induced by the TH1-cytokines IFN-γ, TNFα, IL-1
and endotoxins
27
. An isoform of iNOS, iNOS2, could be detected in myeloid
precursor cells. The inhibition of iNOS2 abolished suppression. The generation of
NO by MDSC also needs IFN-γ (e.g. from activated T-cells) and cell-cell-contact
7, 9,
50, 97
. NO is known to blockade the IL-2 signal transduction cascade of T-cells: this
inhibits the production of IL-2 and proliferation of T-cells
16, 35
. Apoptosis of T-cells
induced by NO was also described 52, 133.
3.2.6.1.2
L-arginine and arginase
As already described, iNOS requires the amino acid L-arginine to generate NO.
Reduction of the concentration of L-arginine in culture medium inhibits the
expression of TCR-ζ-chain, disturbs the proliferation of T-cells and influences the
production of cytokines by T-cells
127-129, 157
. In addition to iNOS, the enzyme
arginase is involved in the metabolism of L-arginine. Arginase catalyzes the
conversion of L-arginine to urea and ornithine
169
. In myeloid cells the isoform
arginase-1 can be induced by the TH2-cytokines IL-4, IL-10, TGFβ and IL-13
well as by prostaglandines
130
and catecholamines
108
as
12
. Immature myeloid precursor
cells also expressing arginase-1 can be found within different diseases
115, 139, 161
.
These cells are able to remove L-arginine from the environment using a cationic
amino acid transporter (CAT2B), thereby causing the T-cell dysfunctions described
above. The application of an inhibitor af arginase-1, nor-NOHA (N -hydroxy-nor-Larginine), abolishes the suppressive effects of MDSC
phosphodiesterase-5-inhibitors
143
and also nitroaspirin
131
. The treatment with
32
represents another
interesting approach to reduce T-cell suppression. These substances decrease the
enzymatic activity of iNOS2 as well as arginase-1 in MDSC and thereby contribute
to an augmented immune response against tumor cells.
16
3.2.6.1.3
Reactive oxygen species
MDSC isolated from tumor-bearing mice and humans are able to produce huge
amounts of ROS 74, 78, 166, first of all hydrogen peroxide (H2O2) and superoxide anion.
The secretion of ROS can be induced by TGFβ, IL-3, IL-6, IL-10, plateled-derivedgrowth-factor (PDGF) and GM-CSF
135
. The enzyme katalase degrades H2O2; this
leads to an inhibition of proliferation of the immature myeloid cells and stimulates
their differentiation into macrophages
74
. Furthermore, ROS secreted by immature
myeloid cells can disturb function and proliferation of T-cells
78, 117
. MDSC isolated
from tumor-bearing mice lose their suppressive activity after inhibition of the
production of ROS in vitro
78
. Together with NO, superoxide can generate
peroxynitrites, which nitrify and nitrosylate certain amino acids
163
. This happens
mainly in inflammatory or tumorous tissue and causes apoptosis and/or
dysfuntion/tolerance of T-cells; changes induced in TCR and in CD8 are responsible
for these processes
21, 109
. Apparently, induced tolerance can be abolished again
and does not affect the memory function of suppressed T-cells 36.
3.2.6.1.4 Transforming growth factor β
Tumor-associated MDSC can cause T-cell dysfuntion by the secretion of TGFβ 158,
179
. Immunization with an oligosaccharide of schistosoma induces a TH2-response in
the organism, favoring the survival of the parasite. In this context, Gr-1+, CD11b+,
F4/80+ cells secrete the antiinflammatory cytokines TGFβ und IL-10, thereby
stimulating TH2-response of T-cells 159.
3.2.6.2
Mechanisms of suppression of both subpopulations
Recently, a separation of murine MDSC into two main subpopulations was
postulated as described in chapter 3.2.1: PNM-MDSC resembling neutrophilic
granulocytes and MO-MDSC with monocytic character
107, 174
. The generation of
granulocytic MDSC is mainly induced in tumor disease. These PMN-MDSC produce
a high amount of ROS and insignificant levels of NO. The opposite effect can be
observed in case of MO-MDSC occuring rather in inflammation. Inspite of different
mechanisms both subpopulations are able to suppress T-cell response in a
sufficient and antigen-specific way. These data support the hypothesis that the
character of pathological processes recruits different subpopulations of MDSC with
differing mechanisms of suppression.
17
3.2.6.3
Role of antigen-specific suppression by MDSC
Undoubtedly, MDSC can suppress T-cell response in vitro in an antigen-unspecific
way
80
. However, results of many in vivo experiments suggest the existence of
antigen-specific mechanisms of suppression. In some approaches, only activated
T-cells previously having been in contact with antigen could be suppressed by
44, 78
. It was also shown that MDSC are able to pick up and process soluble
MDSC
antigens and to present them to T-cells
109
. The role of MHC I-molecules in this
context is still controversially discussed. MHC I-molecules are found on nearly all
44
MDSC
whereas MHC II-molecules can be detected only under certain
circumstances 145. This could also explain the fact that T-cell dysfuntion within tumor
or infectious diseases can be observed mainly in CD8+ cytotoxic T-cells. The
suppressive activity of MDSC can be abolished by blockade of MHC I
44
. In
combination with other surface molecules MHC I-molecules may facilitate the close
contact between T-cell and MDSC necessitated for a sufficient suppression.
Eventually, the specific antigen does not have to be bound on MHC-molecules
47
.
+
On the other hand, an inhibitory effect of MHC II-negative MDSC CD4 T-cells could
be shown 132. This argues against a MHC-dependent, antigen-specific suppression.
Depending on the underlying conditions, MDSC can suppress CD4+ and/or CD8+
T-cells. MHC-molecules can, but do not have to be involved. Suppression can take
place in an antigen-dependent and an antigen-independent way.
3.2.7
Integration of MDSC in cellular networks
In addition to their influence on T-cells MDSC can also interact with other cell types.
For instance TGFβ secreted by MDSC can stimulate proliferation of CD4+CD25+
Tregs via TGFβ−receptor and hence further reduce immune response against tumor
cells
49
. In vitro, CD115+ MDSC induce expression of Foxp3 in CD4+CD25+ cells. In
this context, IFN-γ secreted from activated T-cells seems to stimulate production of
IL-10 and TGFβ which leads to an induction of Tregs
59
. Another interesting aspect
is the interaction between MDSC and macrophages in murine tumor model. This
causes an augmented production of IL-10 by MDSC; IL-12 secretion of
macrophages is reduced and M2-macrophages are preferentially generated. The
induced type 2-response facilitates tumor progression
147
. MDSC seem also to
interfere with NK representing an important link between innate and adaptive
immunity. In vivo and in vitro, MDSC can inhibit cytotoxic effects of NK via direct
cell-cell-contact
86
. Also NK-T-cells obviously have an influence on generation and
function of MDSC
84, 142, 158
. Furthermore, it was shown that Gr-1+ splenocytes
18
isolated from immunized mice are able to stimulate differentiation and proliferation of
antigen-specific B-cells via secretion of IL-4 66, 98.
3.2.8
Outlook
The heterogeneity of the population of myeloid immature cells called MDSC
complicates a classification of these cells. Depending on the disease pattern and
initial position of the examined organism manifold subpopulations can be generated.
They vary in pattern of surface markers as well as in suppressive mechanisms. It
has to be noticed as well that their ability to differentiate and their interaction with
other cells can be influenced in many different ways. Therefore an outlook on their
effect in organisms is very difficult.
Many questions remain to be answered before a therapeutical application of MDSC
is possible.
For example, MDSC could be switched off in a specific way within tumor diseases or
chronic
inflammation,
thereby
intensifying
the
immune
response
against
degenerated cells respectively pathogens. Reduction, depletion or stimulation of
differentiation to mature myeloid stages could be achieved by the application of
certain medicaments.
MDSC could also be used within autoimmune diseases or chronic inflammation
regulating exaggerated immune response. For instance, in vitro generated MDSC
could be applicated, eventually loaded with antigen. Another approach is the
induction of their generation in organism using different medicaments or growth
factors.
So far, MDSC were mainly examined and characterized using murine tumor models.
However, MDSC are also involved in many other pathological processes. The
establishment respectively the further development of animal models for e.g. chronic
inflammation, infections and autoimmune diseases is necessary. Only in this way
myeloid suppressor cells will be characterized in detail. Getting to know MDSC
paves the way for suitable therapeutic strategies.
19
3.2.9
Hypothesis
This work is based on the hypothesis that two mechanisms are initiated within
infection/inflammation: beside the immune response against pathogens regulatory
processes are induced to avoid exaggerating immune reactions. Simultaneously it is
assumed that immature precursors of MDSC already exist in the healthy organism
being activated under pathologic conditions.
This dissertation deals with the identification of different cell populations in the
spleen
of
untreated
mice
regarding
certain
surface
markers
and
cell
size/granularity/morphology. In addition, the effect of LPS and IFN-γ on MDSC was
examined in vitro and in vivo focussing on their potential to differentiate, their
suppressive activity and accumulation.
20
4
Einleitung
4.1
Aufbau und Funktionen des Immunsystems
Das Immunsystem der Vertebraten stellt ein höchst komplexes Gefüge sowohl aus
zellulären und molekularen Bestandteilen als auch aus Organen dar. Es hat zur
Aufgabe, den Organismus vor Einflüssen der belebten Umwelt zu schützen und
körpereigene Prozesse so zu regulieren, dass die Integrität des Lebewesens
erhalten bleibt.
4.1.1
Allgemeine Einführung in das Immunsystem
Das Immunsystem umfasst zwei kooperierende und sich teilweise überschneidende
Kompartimente:
(natürlichen)
die
und
unspezifischen
die
Abwehrmechanismen
spezifischen
Abwehrstrategien
des
angeborenen
des
erworbenen
Immunsystems.
Ersteres beinhaltet sowohl mechanische (z.B. Epithelien der Eintrittspforten),
mikrobiologische (z.B. epitheliale Bakterienflora) und chemische (z.B. pH, Enzyme)
Barrieren als auch molekulare (Komplementsystem) sowie zelluläre (diverse
Phagozyten, natürliche Killerzellen) Bestandteile. Das angeborene Immunsystem
erkennt Mikroorganismen und Moleküle mit Hilfe bestimmter Oberflächenrezeptoren
als fremd und leitet die ersten Schritte zur Elimination des Pathogens in die Wege.
Eine große Rolle spielen hierbei Makrophagen und neutrophile Granulozyten. Diese
phagozytieren körperfremdes Material und schütten daraufhin Zytokine und
Chemokine aus. Sie tragen so zur Entstehung einer entzündlichen Reaktion bei. Ein
weiteres wichtiges Mitglied der Phagozyten stellen die unreifen dendritischen Zellen
(DZ) dar. Diese prozessieren Fremdmaterial an der Eintrittspforte. Die Interaktion
mit mikrobiellen Liganden oder auch der Einfluss proinflammatorischer Zytokine
bewirken eine Aktivierung unreifer DZ
68, 125
. Reife DZ können als sogenannte
Antigen-präsentierende Zellen (APZ) über Major Histocompatibility Complex (MHC)
II Moleküle die prozessierten Fragmente auf ihrer Oberfläche präsentieren und
erhöhen die Anzahl kostimulatorischer Moleküle. Diese sind zusätzlich zur
Antigenbindung über den T-Zell-Rezeptor und zur Bindung von CD4 an
MHCII-Moleküle für eine effektive T-Zell-Aktivierung nötig (siehe weiter unten); auch
CD11c ist auf reifen DZ zu finden
11
. Sie wandern nach der Aktivierung aus der
Peripherie in den nächstgelegenen Lymphknoten.
An dieser Stelle tritt das erworbene Immunsystem in Kraft, das die präsentierten
Antigene spezifisch erkennen und auch ein immunologisches Gedächtnis ausbilden
kann. Diese komplexe Form der immunologischen Überwachung kann nur durch ein
ausgeklügeltes Zusammenspiel verschiedener Zelltypen stattfinden. Diese Aufgabe
21
übernehmen die so genannten Lymphozyten, die man grob in B-Lymphozyten
(humorale Abwehr) und T-Lymphozyten (zelluläre Abwehr) unterteilt.
T-Lymphozyten tragen auf ihrer Oberfläche den T-Zell-Rezeptor (TZR)
erkennt
prozessierte Antigene spezifisch und führt
unter
53
. Der TZR
Mitwirkung
der
Korezeptoren CD4 bzw. CD8 (CD= cluster of differentiation) und weiterer
kostimulatorischer Moleküle (z.B. CD28-CD80/CD86 oder CD40/CD40-Ligand) zur
Aktivierung der T-Zelle. Derart stimulierte T-Zellen stellen potente und zur
Gedächtnisbildung fähige Abwehrzellen dar.
Bleibt die Kostimulation aus, so kann dies trotz Antigen-Bindung dazu führen, dass
T-Zellen nicht auf das Antigen reagieren. Man nennt dieses Phänomen Anergie 137.
CD8+ zytotoxische T-Zellen erkennen über MHC I-Peptid-Komplexe infizierte oder
entartete Körperzellen. Diese zerstören sie mittels bestimmter Mechanismen
(Granzym, Perforin) oder sezernieren Zytokine, die indirekt zur Elimination des
Pathogens beitragen können (z.B. Interferon-γ bzw. IFN-γ). CD4+ T-Zellen binden an
MHC II-Moleküle auf APZ, die einverleibtes und prozessiertes exogenes Antigen auf
diesem Wege zur Schau stellen. Art des Pathogens und das dadurch induzierte
Zytokinmilieu steuern die Differenzierung der naiven CD4+ Zellen, die dann
entweder den Weg einer zellvermittelten TH1- oder einer humoralen TH2Immunreaktion einschlagen
106
. Aktivierte DZ steuern diesen Prozess, der auch T-
Zell-Polarisierung oder T-Zell-Priming genannt wird
Sekretion
von
IFN-γ
und
Interleukin-12
19, 30, 67
(IL-12)
. Hierbei begünstigt die
die
TH1-Antwort
mit
Makrophagenaktivierung, während die humorale TH2-Immunreaktion dadurch
gehemmt wird 103.
Die bisher beschriebenen immunologischen Vorgänge sorgen dafür, dass Noxen
und Pathogene suffizient eliminiert werden können. Gleichzeitig muss aber
gewährleistet werden, dass es hierbei nicht zu einer unverhältnismäßig starken,
überschießenden und potentiell gewebeschädigenden Immunantwort kommt (z.B.
allergische Reaktionen, Autoimmunerkrankungen, chronische Entzündungen). Zum
anderen kann die immunologische Überwachung beispielsweise im Zuge einer
malignen Tumorerkrankung partiell außer Kraft gesetzt werden. Entartete Zellen
werden dann von verschiedenen Bestandteilen des Immunsystems nicht mehr als
fremd erkannt und toleriert, so dass sich der Krebs ungehindert ausbreiten kann.
In Zusammenschau dieser Fakten ist es somit sehr wichtig, dass das
immunologische Geschehen im Organismus streng kontrolliert und reguliert wird, so
22
dass Immunantworten rechtzeitig und adäquat beendet und körpereigene Strukturen
nicht über das normale Maß hinaus in Mitleidenschaft gezogen werden. Hierzu
tragen diverse Regulationsmechanismen bei.
4.1.2
Regulation der Immunantwort
Eine zentrale Eigenschaft des spezifischen Immunsystems stellt die Fähigkeit der
Lymphozyten dar, zwischen körpereigenen und -fremden Antigenen unterscheiden
zu können und auf körpereigene Antigene nicht mit einer Immunantwort zu
reagieren. In der Homöostase werden Selbstantigene auf tolerogene Weise an
T- und B-Zellen präsentiert, so dass hierbei aktiv immunologische Toleranz induziert
und aufrechterhalten wird. Hierbei unterscheidet man die zentrale von der
peripheren Toleranz.
Im Verlauf der zentralen Toleranzbildung im Thymus sollen Lymphozyten
unschädlich gemacht werden, die mit ihrem spezifischen Rezeptor Autoantigene
erkennen. Dies geschieht entweder durch die so genannte „klonale Deletion“ oder
durch Inaktivierung der betroffenen Zelle. Hierbei können jedoch nicht alle
autoreaktiven T-Zellen entfernt werden, so dass immer auch derartige Zellen in die
Peripherie ausgeschwemmt werden. Dort existieren zusätzliche intrinsische und
extrinsische Mechanismen, die zur so genannten „peripheren Toleranz“ führen.
Diese kann sich auch auf körperfremde Antigene beziehen und führt dazu, dass
T-Zellen in ihrer Funktionsweise eingeschränkt, d.h. supprimiert, werden.
Zu den intrinsischen Mechanismen gehört die bereits weiter oben beschriebene
Anergie. Durch das Fehlen der Kostimulation können trotz Antigenbindung
Signaltransduktionskaskaden in der T-Zelle nicht ablaufen, so dass die T-Zellen kein
IL-2 mehr produzieren können und eine Aktivierung ausbleibt
136
Mechanismus sind unreife dendritische Zellen maßgeblich beteiligt
. An diesem
87, 92, 152
. Man
geht davon aus, dass diese immer wieder körpereigene Antigene aus der
Umgebung (z.B. von absterbenden Zellen) aufnehmen und den T-Zellen ohne
Kostimulation über MHC I-Moleküle präsentieren
3, 72
. Diesen Mechanismus nennt
man „Kreuzpräsentation“, weil exogene Antigene normalerweise über MHC IIMoleküle präsentiert werden. Dadurch wird in den CD8+ T-Zellen, die Selbstantigen
erkennen, entweder Apoptose oder Anergie ausgelöst 73, 124.
Des weiteren kann die Interaktion diverser koinhibitorischer Moleküle auf der T-Zelle
(z.B. cytotoxic T-lymphocyte antigen 4=CTLA-4; programmed death 1=PD-1) mit
den entsprechenden Pendants auf der APZ (CD80/CD86; programmed death ligand
1/2= PD-L1/ PD-L2) dazu führen, dass der T-Zell-Rezeptor eventuell dargebotenes
23
Autoantigen nicht mehr suffizient binden kann und eine Aktivierung verhindert wird
165
.
Einfluss auf die periphere Toleranz haben ebenso die Applikationsart und die
Menge des dargebotenen Fremd- oder Eigenantigens und die Stärke der Bindung
des T-Zell-Rezeptors 51.
Die T-Zell-Antwort kann zusätzlich zu den bereits beschriebenen Mechanismen
auch extrinsisch durch bestimmte Zellen supprimiert werden.
Seit den 70er Jahren ist die Existenz sogenannter Suppressor T-Zellen oder
neuerlich als regulatorische T-Zellen bezeichnete (Tregs) tolerogener T-Zellen
bekannt
48
. Diese beeinflussen die Proliferation und Differenzierung von T- und
B-Lymphozyten, von Natürlichen Killer (NK)-Zellen, aber auch von Monozyten und
DZ. Dies geschieht sowohl über Zell-Zell-Kontakt als auch über lösliche Faktoren 150,
168
. Hierdurch tragen sie dazu bei, überschießende Immunreaktionen zu verhindern.
Zum jetzigen Zeitpunkt unterscheidet man verschiedene Subpopulationen: hierzu
gehören die natürlichen CD4+CD25+ Tregs, zum anderen die induzierten bzw.
adaptiven Tregs
58, 168
. Natürliche Tregs erkennen bereits im Thymus über ihren
T-Zell-Rezeptor Selbstantigen und werden so aktiviert. Adaptive Tregs entstehen
erst in der Peripherie aus naiven T-Zellen. Für diese Differenzierung wird
Antigenkontakt im Rahmen eines bestimmten Zytokinmilieus benötigt 34, 65.
Ebenso wurden unter den B-Zellen Populationen entdeckt, die zur Regulation und
Suppression der Immunantwort fähig sind 20, 104.
Auch der myeloischen Linie entstammen Suppressorzellen, sogenannte myeloidderived suppressor cells, auf die im Folgenden genauer eingegangen wird.
4.2
Suppressorzellen der myeloischen Reihe
Ende der 70er Jahre begannen sich Hinweise auf eine bisher unbekannte
suppressiv aktive Zellpopulation zu häufen. In Knochenmark und Milz von Mäusen
mit Tumorerkrankungen, im lymphatischen Gewebe von neugeborenen Mäusen und
im experimentell manipulierten Lymphgewebe adulter Mäuse wurden myeloische
Zellen gefunden, die die T-Zell-Antwort in vivo und in vitro unterdrücken konnten
126,
148, 154, 155
. Man nannte diese Zellen natürliche Suppressor-Zellen. Im Rahmen
jahrzehntelanger Forschung auf diesem Gebiet wurden die natürlichen SuppressorZellen näher charakterisiert. Man bezeichnete diese im Folgenden dann
fälschlicherweise auch als immature DC oder unreife DZ. Später verwendete man
für diese Zellpopulation hauptsächlich die Bezeichnung myeloid suppressor cells
24
(MSC). Um eine eindeutige wissenschaftliche Nomenklatur zu schaffen und
Verwechslungen auszuschließen, wird seit etwa 2007 der Terminus myeloid-derived
suppressor cells (MDSC) verwendet 45.
4.2.1
Aus
Allgemeine Einführung
murinen
hämatopoietischen
Stammzellen
entstehen
im
Rahmen
der
Myelopoese im Knochenmark durch eine komplexe Interaktion bestimmter löslicher
Moleküle und zellständiger Rezeptoren gemeinsame myeloische Vorläuferzellen.
Aus diesen gehen unter anderem so genannte unreife myeloische Zellen (immature
myeloid cells bzw. IMC) hervor, die keine suppressiven Eigenschaften besitzen.
Letztere wandern im gesunden Individuum in periphere lymphatische Organe und
differenzieren dort zu reifen Makrophagen, DZ oder Granulozyten.
Diverse pathologische Prozesse wie z.B. Entzündung, maligne Tumorleiden oder
Infektionen verhindern über verschiedene Mechanismen die Ausreifung der IMC.
Stattdessen erlangen diese Zellen immunsuppressive Eigenschaften und werden
nun als MDSC bezeichnet.
MDSC stellen eine sehr heterogene Zellpopulation aus diversen myeloischen
Vorläuferzellen dar, deren Charakterisierung und Klassifikation seit Jahren
Gegenstand der Forschung ist.
Gemeinsam ist den MDSC jedoch die Koexpression der Oberflächenmarker CD11b
(auch CD11b/CD18-Komplex oder Mac-1 genannt) und Gr-1
142
. Der CD11b-
Rezeptor gehört zu den αMβ 2-Integrinen und bindet das Komplementfragment C3bi.
Er ist sowohl auf Monozyten, Makrophagen, DZ und Granulozyten als auch auf
aktivierten B- und T-Zellen und natürlichen Killerzellen zu finden
83, 99
. CD11b
reguliert über die Bindung an ICAM 1 (intercellular adhesion molecule 1) unter
anderem Leukozytenadhäsion und Extravasation 69.
Gr-1, ein Glycosyl-Phosphatidyl-Inositol (GPI)-verankertes Protein, repräsentiert
einen Differenzierungsmarker der myeloischen Linie und kann auf myeloischen
Vorläuferzellen, Granulozyten und transient auch auf Monozyten nachgewiesen
werden
39, 57
. Es ist mittlerweile bekannt, dass Gr-1-spezifische Antikörper zwei
verschiedene Epitope von Gr-1 binden: Ly6G und Ly6C. Die Verwendung Epitopspezifischer Antikörper führte zur Identifizierung zweier MDSC-Subpopulationen in
Milzzellen aus Tumor-befallenen Mäusen: CD11b+Ly6G+Ly6Clow
MDSC, die
morphologisch neutrophilen Granulozyten ähneln (PMN-MDSC) und CD11b+Ly6GLy6Chi(gh) MDSC, die monozytären Charakter besitzen (MO-MDSC)
107 174
. Jedoch
gelang in einem murinen Tumormodell auch mittels des herkömmlichen Gr-1-
25
Antikörpers eine Differenzierung der Milzzellen in zwei verschiedene Populationen:
Gr-1hi und Gr-1int(ermediate). Diese beiden Subpopulationen unterscheiden sich in ihrem
Potential zur T-Zell-Suppression und zur weiteren Differenzierung 170.
Weitere Oberflächenmarker wie zum Beispiel CD80 (B7-1), CD115 (macrophagecolony-stimulating-factor=M-CSF) oder CD124 (IL-4R α-Kette) können, abhängig
von
den
jeweiligen
Rahmenbedingungen,
Subpopulationen der MDSC sein
charakteristisch
für
bestimmte
47, 59, 173
. Jedoch konnte diesbezüglich bisher kein
einheitliches Muster definiert und auch nicht nachgewiesen werden, dass diese
Marker direkt an der Suppression beteiligt sind.
MDSC sind hauptsächlich bekannt für ihre Eigenschaft, die T-Zell-Antwort zu
supprimieren. Dies geschieht über eine Vielzahl von Oberflächenmolekülen und
löslichen Elementen, auf die weiter unten eingegangen wird. MDSC können jedoch
neben naiven und Effektor-T-Zellen auch andere Zellen beeinflussen. So kann die
Bildung ebenfalls suppressorisch aktiver Tregs durch MDSC induziert werden
140
.
Auch die Funktion von NK-Zellen, Makrophagen oder auch B-Zellen können MDSC
beeinflussen 22, 142, 147.
4.2.2
Plastizität der MDSC
Von großem Interesse ist die Fähigkeit der MDSC, zu reifen myeloischen DZ zu
differenzieren. Neben diesen existieren noch so genannte plasmazytoide DZ
Murine Zellen aus Knochenmark
63, 182
13, 56
, Milz
oder fetaler Leber
181
110
.
können in
vitro mit Hilfe bestimmter Zytokine wie granulocyte/macrophage-colony-stimulatingfactor (GM-CSF), Tumornekrosefaktor α (TNFα), IL-4 oder Fms-Like TyrosineKinase 3 Ligand (FLT3-Ligand) zu reifen DZ der myeloischen Reihe differenzieren.
All-trans-Retinoidsäure (ATRA) kann in vivo und vitro die Zahl der MDSC reduzieren
und deren Differenzierung zu DZ, Makrophagen oder Granulozyten bewirken
44, 79
.
Die Applikation von 1α, 25-Dihydroxyvitamin D3, einem Metaboliten von Vitamin D3,
induziert ebenfalls die Differenzierung unreifer myeloischer Zellen82,
167
. Weiterhin
+
können MDSC in Tumorgewebe zu F4/80 Tumor-assoziierten Makrophagen (TAM)
differenzieren, die über die Sekretion diverser Zytokine ebenfalls die T-Zell-Antwort
supprimieren 75, 123, 133.
Es gibt Hinweise darauf, dass unreife myeloische Zellen auch an der Angiogenese
im Rahmen von Tumorerkrankungen beteiligt sind bzw. sogar selbst zu
Endothelzellen differenzieren können
171, 175
. Hierfür spricht zum Beispiel, dass
Gr-1+/CD11b+ Zellen aus erkrankten Mäusen das Enzym Matrixmetalloproteinase-9
(MMP-9) exprimieren.
26
4.2.3
MDSC in der Homöostase und bei Erkrankungen
Etwa 20-30% der Zellen im gesunden murinen Knochenmark sind Gr-1+/CD11b+ , in
der Milz und im Blut gehören dazu nur 2-4% der kernhaltigen Zellen, in den
Lymphknoten sind ohne Stimulation keine derartigen Zellen zu finden
76
. Bestimmte
Bedingungen führen zu einem Anstieg bzw. einer Umverteilung der CD11b+Gr-1+
Zellen im murinen und auch humanen Organismus.
4.2.3.1
Murine MDSC
MDSC lassen sich vermehrt in Milz, Knochenmark und Tumorgewebe von Mäusen
mit verschiedensten Tumorleiden nachweisen
24, 100, 155, 173, 176
. Interessanterweise
nimmt die Zahl reifer DZ im Rahmen der Tumorerkrankungen ab und verschiebt sich
zu Gunsten unreifer Vorstufen dendritischer Zellen 5, 85. DZ in Patienten und Mäusen
mit Tumorerkrankungen, aber auch mikrobiellen Infektionen weisen zudem
funktionelle Defekte auf 14, 28, 43, 60. Die Immunantwort gegen die Tumorzellen wird so
mit
Hilfe
unterschiedlicher,
im
Folgenden
näher
beschriebener,
Suppressionsmechanismen durch die MDSC unterdrückt; der Tumor kann sich
somit mehr oder weniger ungehindert ausbreiten. Eine Entfernung des Tumors ist
vergesellschaftet mit einer Abnahme der CD11b+/Gr-1+ Zellen und einer
Wiederherstellung der tumorbezogenen Immunantwort
134
. Dies gibt Hinweis darauf,
dass der Tumor selbst durch bestimmte Mechanismen die Bildung der
Suppressorzellen induziert. Hierbei ist vor allem an von den Tumorzellen sezernierte
Zytokine, Chemokine und andere lösliche Faktoren zu denken (siehe weiter unten).
Die Depletion der Suppressorzellen
Chemotherapeutikum Gemcitabin
156
144,
158
oder deren Elimination mit dem
lässt die Immunantwort gegen den Tumor
wieder aufleben.
Auch Infektionen mit Mikroorganismen wie zum Beispiel Salmonella typhimurium 2,
Trypanosoma cruzi
50
, Candida albicans
101
oder Toxoplasma gondii
164
bewirken
einen Anstieg myeloischer suppressiv aktiver Zellen in murinen lymphatischen
Organen.
Dies
begünstigt
über
die
darauf
folgende
Unterdrückung
der
Immunantwort die Ausbreitung der Erreger im Organismus. Im Rahmen einer
polymikrobiellen Sepsis können MDSC eine Suppression und TH2-Polarisierung der
T-Zell-Antwort induzieren. Diese Aktivierung der MDSC basiert unter anderem auf
MyD88, einem Adaptorprotein an verschiedenen Toll-like Rezeptoren (TLR) 33.
Die Inokulation bestimmter Vakzine bzw. Antigene löst eine Vermehrung der MDSC
in der Milz aus 74, 159. Im Falle vom rekombinanten Vakzinia-Virus induzieren diese in
27
CD8+ T-Zellen Apoptose und mildern somit die Immunantwort gegen die Vakzine ab
23
.
Auch die Applikation von Superantigen führt zu einem Anstieg von MDSC 25.
Der Anteil an MDSC nimmt auch im Rahmen von chronischen Entzündungen bzw.
Autoimmunerkrankungen zu. Im murinen Modell der Multiplen Sklerose, der
experimentellen Autoimmunenzephalitis (EAE), findet sich ein signifikanter Anstieg
der MDSC gepaart mit einer Migration ins zentrale Nervensystem
experimentell induzierte chronisch-entzündliche Prozesse an Haut
Auge
70
183
94
. Auch
, Darm
55
und
führen zu einer Zunahme der MDSC in Mäusen. Es mehren sich zudem
Hinweise, dass chronische Entzündungen über die Induktion von MDSC der
Entstehung maligner Prozesse den Weg ebnen. Dies hängt unter anderem von der
Aktivierung von TLR und verschiedenen Interleukinen wie IL-1β, IL-10 und IL-12
ab116.
Man geht davon aus, dass MDSC ebenso an der Regulation der Immunantwort
nach Bestrahlung und allogener Knochenmarktransplantation beteiligt sind. Sie
scheinen die Entwicklung einer für den Organismus schädlichen „graft-versus-hostdisease“ nach einer Infusion von Donor-Lymphozyten zu Gunsten einer „graftversus -leukemia“-Reaktion zu verschieben 15, 17.
Weiterhin können bestimmte Medikamente die Bildung von MDSC induzieren und
deren suppressive Aktivität fördern. Bekanntermaßen führt eine Behandlung mit
dem
Chemotherapeutikum
Cyclophosphamid
zu
schweren
Schäden
in
hämatopoietischen und lymphatischen Geweben. Man weiß mittlerweile, dass diese
Therapie nicht nur eine direkte T-Zell-Schädigung bewirken, sondern ebenso die
Expansion von CD11b+/Gr-1+ Zellen induzieren und somit indirekt Proliferation und
Reagibilität der T-Zellen unterdrücken kann 8, 113, 138.
4.2.3.2
MDSC beim Menschen
Auch beim Menschen häufen sich Hinweise auf ein Pendant zu den murinen MDSC.
Bereits 1995 wurde beschrieben, dass in Tumor- und Lymphgewebe von Patienten
mit Karzinomen im Kopf- und Halsbereich vermehrt CD34+ Zellen auftreten, die
inhibierend auf die Funktion der ansässigen T-Lymphozyten wirken
118
. Im
Folgenden fand man heraus, dass die verminderte Zahl von DZ im Blut von
Patienten mit Tumorerkrankungen im Kopf-Hals-Bereich und in Brust und Lunge mit
einer Akkumulation unreifer myeloischer Zellen einhergeht, welche zusätzlich die
T-Zell-Funktion beeinträchtigen
5, 6
. Diese Zellen sind größtenteils CD13+, CD33+,
28
CD34+
CD15-
und
und
lassen
sich
anhand
ihres
HLA
(humanes
Leukozytenantigen)-DR-Typs und der CD11c-Expression in 2 Subgruppen einteilen:
unreife Monozyten/DZ und Zellen früherer myeloischer Differenzierungsstadien. Im
Blut
von
Patienten
+
+
mit
Nierenzellkarzinom
wurden
ebenfalls
myeloische
-
CD11b /CD15 /CD14 Zellen nachgewiesen, die die T-Zellantwort unterdrücken
114,
. Bei Patienten mit malignem Melanom fand man vor kurzem CD11b+/CD14+
180
myeloische Suppressorzellen, die im Stande waren, T- Zellen zu supprimieren 38.
Weiterhin leiden Menschen auch nach einem chirurgischen Eingriff
schweren Trauma
+
122
61
oder einem
unter diversen T-Zell-Dysfunktionen. Hierbei wandern
+
CD11b /Gr-1 Zellen nach traumatischem Stress in die Milz und beeinträchtigen dort
T-Zellen in ihrer Funktion 93.
MDSC spielen also in vielen verschiedenen immunologischen Prozessen eine
entscheidende Rolle. Interessanterweise kann man MDSC kein eindeutiges Schema
der Suppression, keine festgelegte „Zielzelle“, keinen einheitlichen Phänotyp und
kein starres Muster an freigesetzten Zytokinen zuteilen. Abhängig von vielfältigen
Einflüssen generiert jedes immunologische Geschehen spezifische MDSC.
4.2.4
In vitro generierte MDSC
Man weiß, dass GM-CSF allein oder in Kombination mit IL-4 in vitro abhängig von
der eingesetzten Menge und der Dauer der Stimulation murine Knochenmarkszellen
zu DZ in verschiedenen Reifestadien und mit unterschiedlichen immunologischen
Fähigkeiten differenzieren lässt
62, 81, 89, 90, 96
. Lutz et al entwickelten eine Methode,
mit der in vitro GM-CSF-abhängig unreife myeloische Suppressorzellen generiert
werden können
90
. Diese CD11c- Zellen weisen ringförmige Kerne auf und sind
unter anderem Gr-1low, CD11b+, CD31+, ER-MP58+ und F4/80+. Je nach Zeitpunkt
und Dauer der Zugabe zu den kultivierten Knochenmarkszellen kann bakterielles
Lipopolysaccharid (LPS) deren Differenzierung zu reifen DZ stimulieren oder diese
88
. In vitro generierte MDSC können die Immunantwort sowohl CD4+ als
verhindern
auch
CD8+
T-Zellen
unterdrücken.
Hierfür
sind
Zell-Zell-Kontakt
und
Stickstoffmonoxid (NO) nötig. Auch IFN-γ scheint eine Rolle bei der Suppression zu
spielen
132
. Eine Vorbehandlung mit in vitro generierten MDSC verringert bei
Mäusen den Prozentsatz der Abstoßungen nach Transplantation eines allogenen
Herzens
90
. In so genannten non-obese-diabetic (NOD)-Mäusen kann die
Behandlung mit unreifen myeloischen Gr-1+ Vorläuferzellen die Entwicklung von
Autoimmundiabetes unterdrücken
153
. Die in vitro generierten MDSC können somit
29
als Modell zur Erforschung der in vivo vorkommenden MDSC herangezogen
werden.
4.2.5
Induktion der Bildung von MDSC
Die meisten experimentellen Ansätze haben sich bisher mit MDSC im murinen
Tumormodell beschäftigt, so dass sich viele Erkenntnisse über die Wirkung der
MDSC aus diesem Ressort rekrutieren.
Wie bereits weiter oben beschrieben, führen Tumorerkankungen in der Maus zur
Akkumulation von MDSC in sekundären lymphatischen Organen (vor allem der
Milz), im Blut, Knochenmark und Tumorgewebe. Dort üben sie suppressive Wirkung
auf die Zielzellen aus und fördern somit über eine Schwächung der Immunantwort
den Fortbestand des Tumors.
Die Expansion und Aktivierung der MDSC wird durch verschiedene Faktoren
bewirkt. Diese kann man in zwei Hauptgruppen einteilen: zum Einen direkt von den
Tumorzellen sezernierte Substanzen, die die Myleopoiese anregen und die
Differenzierung der myeloiden Zellen hemmen. Zum Anderen Faktoren, die
entweder
von
Tumorstromazellen
oder
aktivierten
T-Zellen
bzw.
anderen
Komponenten des Immunsystems ausgeschüttet werden und MDSC direkt
aktivieren 42. Hierzu zählen auch Bestandteile von diversen Mikroorganismen.
4.2.5.1
Faktoren zur Expansion und Aktivierung von MDSC
Einen der bekanntesten Faktoren stellt GM-CSF dar. Dieses Zytokin induziert im
Zusammenspiel mit anderen Faktoren im Knochenmark die Differenzierung von
Granulozyten, Makrophagen und DZ aus myeloischen Vorläuferzellen
63
. GM-CSF
agiert über den GM-CSF-Rezeptor, der aus einer α- und einer β-Untereinheit
besteht 95.
Viele murine und auch humane Tumorarten sezernieren GM-CSF in großer,
unphysiologischer Menge. Sie stimulieren damit die Bildung von MDSC, die in
sekundären lymphatischen Organen und im Tumor selbst akkumulieren und dort
jeweils die T-Zell-Antwort gegen den Tumor unterdrücken
24, 40, 102, 141, 149
. Dieses
Phänomen konnte auch in Mäusen beobachtet werden, die mit rekombinantem
GM-CSF behandelt wurden 24. Wurde der GM-CSF-haltige Zellkulturüberstand einer
Variante des Lewis Lungenzellkarzinoms in Mäuse gespritzt, denen Tumorgewebe
implantiert
wurde,
so
förderte
dies
das
Tumorwachstum.
Die
Injektion
neutralisierender Antikörper gegen GM-CSF und auch IL-3 hob dieses Phänomen
wieder auf
177
. IL-3 bindet ebenso wie IL-5 an die β-Untereinheit des
GM-CSF-Rezeptors.
30
Aber auch aktivierte T-Zellen 1, NK-Zellen 105, NK-T-Zellen 84 und dendritische Zellen
31
können GM-CSF sezernieren und somit im Rahmen ihrer Aktivierung die Bildung
von MDSC auslösen.
GM-CSF kann somit die Immunantwort anregen, indem es die Bildung und
Differenzierung von Makrophagen, Granulozyten und vor allem DZ induziert. Dies ist
in einigen Fällen auch im humanen Modell gelungen, in denen GM-CSF als VakzinAdjuvans verabreicht wurde
121
. Überschreitet die GM-CSF-Menge bzw. die Dauer
der Stimulation im Organismus jedoch die physiologische Grenze, kann sich der
immunologische Abwehrprozess ins Gegenteil kehren. Es kommt zu einer
Unterdrückung der Immunantwort. Dies fördert die Expansion maligne entarteter
Zellen und eindringender Pathogene.
Vascular endothelial growth factor (VEGF) wird von vielen Tumoren produziert und
regt die Angiogenese an, damit der Tumor ausreichend mit Nährstoffen versorgt
werden kann
160
. Man hat in vitro und in vivo beobachtet, dass VEGF im Rahmen
von Tumorerkrankungen die Zahl der DZ reduziert und diese in ihrer Funktion
einschränkt, während die unreife Vorstufen der DZ vermehrt auftreten 41, 46.
Prostaglandin E2 (PGE2) spielt ebenfalls eine große Rolle sowohl bei entzündlichen
Geschehen als auch bei Tumorerkrankungen. Man geht davon aus, dass von
Tumoren sezerniertes PGE2 ein lokales inflammatorisches Umfeld erzeugt, das
wiederum die Tumorgenese unterstützt
26, 146, 151
. Über PGE2-Rezeptoren wird die
Bildung von MDSC induziert. Die Unterdrückung der PGE2-Bildung durch einen
Cyclooxygenase-2-Hemmer reduziert die Akkumulation von MDSC und inhibiert das
Tumorwachstum 146.
Auch das von verschiedenen Komponenten des Immunsystems sezernierte IFN-γ
beeinflusst neben der Aktivität von T-Zellen die von MDSC
47, 97, 132
. LPS spielt
ebenfalls eine Rolle bei der Induktion von MDSC 33.
Neben den genannten Zytokinen scheinen sowohl das immunsuppressive Zytokin
transforming growth factor β (TGF-β)
4, 91, 172, 178
als auch stem cell factor (SCF)
119
,
IL-1β, IL-4, IL-6, IL-10, IL-12, IL-13, MMP-9, M-CSF (macrophage-colonystimulating-factor) und G-CSF (granulocyte-colony-stimulating-factor ; auch CSF-1)
die Bildung und Funktion von MDSC beeinflussen zu können 42, 120.
31
4.2.5.2
Signaltransduktion in MDSC
Die oben genannten Substanzen setzen über Zytokinrezeptoren bzw. TLR auf den
MDSC
bestimmte
Signaltransduktionskaskaden
in
Gang.
Transmembrane
Zytokinrezeptoren auf MDSC vermitteln ihr Signal in der Regel über so genannte
Janus Kinasen (JAK), die wiederum an Transkriptionsfaktoren der STAT (signal
transducer and activator of transcription)-Familie binden
71
. Bei MDSC spielen
bekanntermaßen JAK2 und STAT-3 eine große Rolle. Im murinen Tumormodell
fand man im Vergleich zu den Zellen unbehandelter Mäuse stark erhöhte STAT-3Werte. Die Aktivierung von STAT-3 führt zur Aktivierung und Proliferation von
MDSC und verhindert gleichzeitig deren Differenzierung zu reifen DZ
111, 112
. Jüngst
wurde gezeigt, dass STAT-3 in myeloiden Vorläuferzellen auch die Bildung des
Proteins S100A9 (myeloid- related protein 14 bzw. Calgranulin B) aus der großen
Familie der S100-Ca2+-bindenden Proteine induziert
29
. Zusammen mit seinem
ebenfalls vermehrt gebildeten Partner S100A8 (myeloid-related protein 8 bzw.
Calgranulin A) verhindert S100A9 die Differenzierung der unreifen Zellen und
forciert deren Umwandlung in MDSC. Auch eine Rekrutierung der MDSC in
Regionen mit Dysplasien und inflammatorischen Veränderungen scheint durch
S100A8 und/oder S100A9 bewirkt zu werden
162
. Über einen membranständigen
NADPH (reduzierte Form von Nikotinamid-Adenin-Dinucleotid-Phosphat)-OxidaseKomplex vermitteln S100A8 und S100A9 die Bildung reaktiver Sauerstoffspezies
(ROS), die zur Suppression durch MDSC beitragen 42.
IFN-γ vermittelt seine Wirkung in MDSC über STAT-1; IL-4 und/oder IL-13 können
auf MDSC durch Bindung an den IL-4-Rezeptor α den Transduktionsfaktor STAT-6
aktivieren
42
. Hierbei wird über STAT-1/-6 die Bildung von Arginase-1 und
induzierbarer NO-Synthase (iNOS) induziert, welche bei der Suppression eine
große Rolle spielen (siehe 4.2.6). Es gibt Hinweise, dass LPS über die Bindung an
TLR auf MDSC den Signaltransduktor NF-κB (nuclear factor “κ-light-chainenhancer” of activated B-cells) aktiviert und somit Arginase-1 und iNOS vermehrt
gebildet werden 42.
4.2.6
Strategien der MDSC zur T-Zell-Suppression
Wie bereits erwähnt, supprimieren MDSC hauptsächlich die Immunantwort von
T-Zellen, so dass diese nicht mehr adäquat auf immunologische Stimuli reagieren
können. MDSC können je nach Spezies, Organsystem und weiteren individuellen
Merkmalen des Organismus verschiedene Suppressionsstrategien anwenden. Dies
führt dann im schlimmsten Fall zur Apoptose der T-Zellen
10, 23, 101
. Über durch
MDSC induzierte Veränderungen am T-Zell-Rezeptor (vor allem an der TZR-ζ-
32
Kette) und an anderen Oberflächenmolekülen können weitere Reaktionen ausgelöst
werden: hierzu gehört neben der Inhibierung der T-Zell-Aktivierung
bzw. –proliferation
80, 93, 101
auch die Induktion peripherer Toleranz
Veränderung des Zytokinsekretionsmusters der T-Zellen
25, 77
und die
44
. Eine effiziente
Suppression erfordert, wie bereits erwähnt, in den meisten Fällen engen Zell-ZellKontakt. So kann die suppressive Wirkung der MDSC aufgehoben werden, wenn
man sie in Kultur mit Hilfe semipermeabler Membranen von den T-Zellen trennt 44, 64,
78, 132
. Dies gibt Hinweis darauf, dass die beteiligten Zellen über bestimmte
membrangebundene Moleküle interagieren müssen bzw. schnell degradierbare,
sezernierte Faktoren eine Rolle spielen. Im Folgenden sollen die wichtigsten bisher
charakterisierten Suppressionsmechanismen von MDSC dargestellt werden.
4.2.6.1
4.2.6.1.1
Mechanismen der Suppression durch MDSC
Stickstoffmonoxid
Stickstoffmonoxid (NO) wird von murinen DZ, Makrophagen, Granulozyten und auch
NK-Zellen produziert
18
. NO kann aus der Aminosäure L-Arginin unter anderem
durch die induzierbare NO-Synthase (iNOS) generiert werden; diese spaltet
L-Arginin zu Citrullin und NO
54
. In einer Vielzahl experimenteller Settings wird NO
für die Suppression durch MDSC benötigt
8, 37, 97, 132
. Stimuli, die zu einer Induktion
von iNOS führen, sind unter anderem die TH1-Zytokine IFN-γ, TNFα, IL-1 und
Endotoxine
27
.
Eine
Isoform
der
iNOS,
iNOS2,
konnte
in
myeloischen
Vorläuferzellen nachgewiesen werden. Die Ausschaltung der iNOS2 führt zur
Aufhebung der Suppression. Für die Produktion von NO in MDSC werden zudem
IFN-γ (z.B. aus aktivierten T-Zellen) und Zell-Zell-Kontakt benötigt
7, 9, 50, 97
. Man
weiß mittlerweile, dass NO mit dem IL-2-Signalweg der T-Zelle interferiert. Die
Blockierung der intrazellulären Signaltransduktionskaskaden durch NO verhindert
IL-2-Produktion und somit Proliferation der T-Zelle
Apoptose der T-Zellen wurde beschrieben
4.2.6.1.2
16, 35
. Auch NO-induzierte
52, 133
.
L-Arginin und Arginase
Wie oben erwähnt benötigt iNOS für die Produktion von NO die Aminosäure
L-Arginin. Es ist bekannt, dass die Reduktion des L-Arginin-Gehalts im
Kulturmedium die Expression der TZR-ζ-Kette hemmt, die T-Zell-Proliferation
beeinträchtigt und die Zytokinproduktion der T-Zellen beeinflusst
127-129, 157
. Neben
iNOS ist das Enzym Arginase am Metabolismus von L-Arginin beteiligt. Arginase
katalysiert die Umwandlung von L-Arginin zu Harnstoff und Ornithin
169
. In
myeloischen Zellen kann die Isoform Arginase-1 durch die TH2-Zytokine IL-4, IL-10,
33
TGFβ und IL-13
108
, aber auch durch Prostaglandine
130
und Katecholamine
12
induziert werden. Das Auftreten myeloischer unreifer Vorläuferzellen, die ebenfalls
Arginase-1
beschrieben
exprimieren,
115,
139,
wurde
im
Rahmen
verschiedenster
Erkrankungen
161
. Diese Zellen können mittels eines kationischen
Aminosäuretransporters
(CAT2B)
L-Arginin
aus
der
Umgebung
entfernen,
intrazellulär mittels Arginase-1 abbauen und somit die oben beschriebenen T-ZellDysfunktionen bewirken. Durch die Applikation des Arginase-1-Inhibitors nor-NOHA
(N -hydroxy-nor-L-arginine)
aufgehoben werden
kann
die
supprimierende
Wirkung
der
MDSC
131
. Ein weiterer interessanter Ansatz zur Reduktion der T-Zell-
Suppression durch MDSC ist die Applikation von Phosphodiesterase-5-Inhibitoren
143
und auch Nitroaspirin
32
. Man konnte zeigen, dass diese die enzymatische
Aktivität von sowohl iNOS2 als auch Arginase-1 in MDSC reduzieren und somit zu
einer vermehrten Immunantwort gegen den Tumor beitragen.
4.2.6.1.3
Reaktive Sauerstoffspezies
MDSC aus Mäusen und auch Menschen mit Tumorerkrankungen können große
Mengen an reaktiven Sauerstoffspezies (ROS) produzieren
74, 78, 166
, hierbei vor
allem Wasserstoffperoxid (H2O2) und Superoxidanion. Dies geschieht unter
anderem nach der Applikation von TGFβ, IL-3, IL-6, IL-10, plateled-derived-growthfactor (PDGF) und auch GM-CSF
135
. Man fand heraus, dass der Abbau von H2O2
durch das Enzym Katalase die Proliferation der unreifen myeloischen Zellen hemmt
und deren Differenzierung zu Makrophagen anregt
74
. Des Weiteren wurde gezeigt,
dass ROS aus unreifen myeloischen Zellen Funktion und Proliferation von T-Zellen
beeinträchtigen können
78, 117
. Aus Tumor-befallenen Mäusen isolierte MDSC
verlieren in vitro ihre suppressive Aktivität nach Inhibierung der ROS-Produktion
78
.
Superoxid kann mit NO Peroxynitrite bilden, welche diverse Aminosäuren nitrieren
und nitrosylieren
163
. Dies geschieht vor allem in entzündlichem oder Tumorgewebe
und führt zu Apoptose und/oder Dysfunktion in Form von Toleranzbildung in der
T-Zelle, hauptsächlich aufgrund von Veränderungen am T-Zell-Rezeptor und an
CD8 21, 109.
Anscheinend kann die induzierte Toleranz unter Umständen wieder aufgehoben
werden und affiziert nicht die Gedächtnisbildung der supprimierten T-Zellen 36.
4.2.6.1.4
Transforming growth factorβ
Tumor-assoziierte MDSC bewirken eine T-Zell-Dysfunktion unter anderem über die
Sekretion von TGFβ
158,
179
. Die Immunisierung mit einem Schistosoma-
Oligosaccharid führt im Organismus zu einer TH2-Antwort, die den Fortbestand des
34
Parasiten im Wirt begünstigt. Man fand heraus, dass Gr-1+, CD11b+, F4/80+ Zellen
über die Sekretion der antiinflammatorischen Zytokine TGFβ und IL-10 naive
T-Zellen zu einer TH2-Antwort anregen können 159.
4.2.6.2
Suppressionsmechanismen der beiden Subpopulationen
Wie bereits in Absatz 4.2.1. beschrieben, wurde kürzlich eine Einteilung muriner
MDSC in zwei Hauptgruppen postuliert: PNM-MDSC, die neutrophilen Granulozyten
107, 174
. Die Bildung
ähneln und MO-MDSC, die monozytären Charakter besitzen
granulozytärer MDSC wird vor allem durch Tumore induziert. Diese PMN-MDSC
produzieren in hohem Maße ROS, während NO nur spärlich gebildet wird. Den
umgekehrten Fall kann man bei MO-MDSC beobachten, die man eher in
entzündlichen
Geschehen
finden
kann.
Beide
Populationen
waren
trotz
unterschiedlicher Mechanismen zu einer suffizienten und Antigen-spezifischen
Suppression der T-Zell-Antwort fähig. Diese Daten unterstützen die Hypothese,
dass je nach Art des Krankheitsgeschehens im Körper bestimmte Subpopulationen
der MDSC mit unterschiedlichen Suppressionsmechanismen rekrutiert werden.
4.2.6.3
Bedeutung der Antigenspezifität der Suppression durch MDSC
Unbestritten ist, dass MDSC die T-Zell-Antwort in vitro Antigen-unspezifisch
supprimieren können
80
. Jedoch mehren sich vor allem bei in vivo-Versuchen
Hinweise auf die Existenz Antigen-spezifischer Suppressionsmechanismen. In
einigen experimentellen Ansätzen werden nur aktivierte T-Zellen, die vorher
Antigenkontakt hatten, durch MDSC supprimiert
44, 78
. Es wurde auch gezeigt, dass
MDSC zur Aufnahme und Prozessierung löslicher Antigene fähig sind und sie diese
den T-Zellen präsentieren 109. Die Bedeutung der MHC-Moleküle in diesem Rahmen
wird noch kontrovers diskutiert. So sind MHC I-Moleküle auf nahezu allen MDSC zu
finden 44, während MHC II-Moleküle nur unter bestimmten Bedingungen detektierbar
sind
145
. Dies könnte auch erklären, warum Dysfunktionen im Rahmen von Tumor-
oder Infektionserkrankungen bis dato fast ausschließlich bei CD8+ zytotoxischen
T-Zellen detektiert wurden. Es wurde gezeigt, dass eine Blockade des MHC IMoleküls die suppressive Aktivität von MDSC aufheben kann
44
. Zusammen mit
anderen Oberflächenmolekülen könnten MHC-Moleküle den engen Kontakt
zwischen T-Zelle und MDSC begünstigen, der für eine suffiziente Suppression nötig
ist. Das spezifische Antigen auf MHC-Molekülen muss unter Umständen hierfür aber
nicht gebunden werden
47
. Andererseits konnte eine inhibitorische Wirkung MHC II-
negativer MDSC auf CD4+ T-Zellen nachgewiesen werden
132
. Dies wiederum
spricht gegen eine MHC-abhängige, Antigen-spezifische Suppression.
35
Somit können MDSC abhängig von den Rahmenbedingungen entweder CD4+
und/oder CD8+ T-Zellen supprimieren. MHC-Moleküle können, müssen aber nicht
beteiligt sein. Eine Suppression kann Antigen-abhängig und Antigen-unabhängig
ablaufen.
4.2.7
Integration der MDSC in weitere zelluläre Netzwerke
Bisher wurde nur der Einfluss der MDSC auf T-Zellen geschildert. Es ist jedoch
bekannt, dass MDSC auch mit anderen Zellarten interagieren können.
So
kann
TGFβ
aus
MDSC
über
den
TGFβ-Rezeptor
auf
CD4+CD25+
regulatorischen T-Zellen deren Proliferation anregen und somit die Immunantwort
gegen den Tumor weiter reduzieren
49
. CD115+ MDSC induzieren in vitro die
Expression von Foxp3 in CD4+CD25+ Zellen. IFN-γ aus aktivierten T-Zellen scheint
hierbei MDSC zur Produktion von IL-10 und TGFβ anzuregen und auf diesem Wege
die Induktion der Tregs in die Wege zu leiten 59.
Einen weiteren interessanten Aspekt stellt die Interaktion zwischen MDSC und
Makrophagen im murinen Tumormodell dar. Diese führt über vermehrte Produktion
von IL-10 durch MDSC zu einer Reduktion der IL-12-Sekretion aus Makrophagen
und fördert die Bildung von M2-Makrophagen. Die Typ-2-Antwort wiederum
begünstigt die Tumorprogression 147.
Auch mit NK, die einen wichtigen Mediator zwischen angeborener und erworbener
Immunität darstellen, scheinen MDSC zu interferieren. So können MDSC in vivo und
in vitro über direkten Zell-Zell-Kontakt die zytotoxische Wirkung natürlicher
Killerzellen inhibieren 86.
Es gibt des Weiteren Hinweise darauf, dass NK-T-Zellen die Bildung und
Funktionsweise von MDSC beeinflussen können 84, 142, 158.
Zudem wurde gezeigt, dass Gr-1+ Milzzellen aus immunisierten Mäusen über die
Sekretion von IL-4 Differenzierung und Proliferation Antigen-spezifischer B-Zellen
stimulieren können 66, 98.
4.2.8
Die
Ausblick
Heterogenität
der
als
MDSC
beschriebenen
myeloischen,
unreifen
Zellpopulation macht es schwer, diese in ein bestimmtes Schema einzufügen. Je
nach Krankheitsbild und Ausgangslage des untersuchten Organismus können
unterschiedlichste Subpopulationen generiert werden. Diese unterscheiden sich
sowohl im Muster der Oberflächenmarker als auch in den angewandten
Suppressionsstrategien.
Ebenso
muss
beachtet
werden,
dass
deren
Differenzierungsfähigkeit und die Interaktion mit anderen Zellen auf vielfältige Art
36
und Weise beeinflusst werden kann und somit eine Vorhersage über deren Wirkung
im Organismus erschwert.
Viele offene Fragestellungen müssen somit geklärt werden, bevor man MDSC auch
therapeutisch anwenden kann.
So könnte man MDSC zum Beispiel im Rahmen von Tumorerkankungen oder
chronischen Infektionen gezielt ausschalten und so die Immunantwort gegen
entartete Zellen bzw. Pathogene verstärken. Dies könnte durch medikamentöse
Reduktion, Depletion oder Anregung der Differenzierung zu reifen myeloischen
Stadien bewirkt werden.
Ein
anderer
denkbarer
Ansatz
wäre
der
Einsatz
von
MDSC
bei
Autoimmunerkrankungen oder chronischen Entzündungen. Hierbei könnten diese
die überschießende Immunantwort regulieren. Es ist vorstellbar, sowohl in vitro
generierte, eventuell Antigen-beladene, MDSC zu applizieren als auch deren
Bildung im Organismus durch Medikamente oder Wachstumsfaktoren zu induzieren.
Bisher wurden MDSC hauptsächlich anhand muriner Tumormodelle charakterisiert.
Jedoch sind MDSC wie oben beschrieben auch in zahlreiche andere pathologische
Geschehen involviert. Die Etablierung bzw. der weitere Ausbau von Tiermodellen für
z.B. chronische Entzündung, Infektionen und autoimmunologische Prozesse ist
notwendig, um die Natur der Suppressorzellen aus der myeloischen Reihe genauer
erforschen und daraus geeignete Therapiestrategien ableiten zu können.
4.2.9
Hypothese
Die vorliegende Arbeit geht von der Hypothese aus, dass im Rahmen einer
Infektion/Entzündung neben der Immunantwort gegen die Pathogene gleichzeitig
ein regulatorischer Mechanismus in Gang gesetzt wird, der eine überschießende
Immunreaktion verhindert. Gleichzeitig wird angenommen, dass bereits im
gesunden Organismus unreife Vorstufen von MDSC vorhanden sind, die erst unter
pathologischen Konditionen aktiviert werden.
In dieser Dissertation sollen anhand diverser Oberflächenmarker und der Zellgröße/granularität/-morphologie verschiedene Zellpopulationen in der Milz unbehandelter
Mäuse identifiziert und auf deren suppressorisches Potential untersucht werden.
Einen weiteren Schwerpunkt stellt die Untersuchung des Effekts von LPS und IFNγ
auf Differenzierungspotential, suppressorische Aktivität und Akkumulation von
MDSC sowohl in vitro als auch in vivo dar.
37
5
Vorveröffentlichung
Eur. J. Immunol. 2009 Oct;39(10):2865-76.
Myeloid-derived suppressor cell activation by combined lipopolysaccharide
plus interferon-γγ treatment impairs dendritic cell development
Verena Greifenberg1*, Eliana Ribechini2*, Susanne Rößner1, Manfred B. Lutz2,#
1
Department of Dermatology, University Hospital Erlangen, Germany,
2
Institute for
Virology and Immunobiology, University of Würzburg, Germany
Comment in:
Eur. J. Immunol. 2009 Oct;39(10):2670-2.
* these authors contributed equally to the study
#
correspondence: [email protected]
Abbreviations: BM, bone marrow; DC, dendritic cells; LPS, lipopolysaccharide;
MDSC, myeloid-derived suppressor cells;
Running title: Myeloid-derived suppressor cell activation
Keywords: myeloid-derived suppressor cells, interferon, lipopolysaccaride, dendritic
cells
Eur. J. Immunol. 2009. 39: 2865–2876
DOI 10.1002/eji.200939486
Immunomodulation
Myeloid-derived suppressor cell activation by combined
LPS and IFN-c treatment impairs DC development
Verena Greifenberg1, Eliana Ribechini2, Susanne Rößner1
and Manfred B. Lutz2
1
2
Department of Dermatology, University Hospital Erlangen, Erlangen, Germany
Institute for Virology and Immunobiology, University of Würzburg, Würzburg, Germany
Myeloid-derived suppressor cells (MDSC) and DC are major controllers of immune
responses against tumors or infections. However, it remains unclear how DC development
and MDSC suppressor activity both generated from myeloid precursor cells are regulated.
Here, we show that the combined treatment of BM-derived MDSC with LPS plus IFN-c
inhibited the DC development but enhanced MDSC functions, such as NO release and Tcell suppression. This was not observed by the single treatments in vitro. In the spleens of
healthy mice, we identified two Gr-1lowCD11bhighLy-6ChighSSClowMo-MDSC and Gr-1highCD11blowPMN-MDSC populations with suppressive potential, whereas Gr-1highCD11bhigh
neutrophils and Gr-1lowCD11bhighSSClow eosinophils were not suppressive. Injections of
LPS plus IFN-c expanded these populations within the spleen but not LN leading to the
block of the proliferation of CD81 T cells. At the same time, their capacity to develop into
DC was impaired. Together, our data suggest that spleens of healthy mice contain two
subsets of MDSC with suppressive potential. A two-signal-program through combined LPS
and IFN-c treatment expands and fully activates MDSC in vitro and in vivo.
Key words: DC . IFN . LPS . Myeloid-derived suppressor cells
See accompanying commentary by Bronte
Introduction
Under steady state conditions myeloid/monocytic (Mo) precursor
cells expressing the Gr-1 and CD11b markers have the capacity to
differentiate into neutrophils, macrophages or DC in the presence
of GM-CSF or other myeloid growth factors [1]. This can be also
mimicked in vitro [2]. The resulting immature or semi-mature DC
present self-antigens in a tolerogenic fashion, which results in
protection from autoimmunity [3]. After microbial or inflammatory activation, DC can induce T-cell immune responses by
presenting the foreign antigens in a different context, i.e. by
upregulating costimulatory molecules and releasing proinflammatory cytokines [4, 5]. The termination or prevention of these
Correspondence: Dr. Manfred B. Lutz
e-mail: [email protected]
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
immune responses is controlled by various mechanisms including
the modulation of mature DC functions, such as by Foxp31 or
IL-10-producing regulatory T cells [3, 6]. Also myeloid-derived
suppressor cells (MDSC) have been described to control immune
responses and their major role in modulating CD81 T-cell
responses against tumors is well established [7–9]. However,
little is known about the mechanisms that regulate MDSC
activation versus DC development from the same myeloid
precursors.
MDSC function is mostly associated with the splenic expansion of Gr-11 CD11b1 cells and is suggested to result from a
specific activation of such myeloid precursor cells [1]. Data
from tumor-bearing mice and infection models indicate that
MDSC require activation to exert their suppressor function
[8–10]. Proinflammatory cytokines, such as IL-1b, IL-6 and
These authors contributed equally to this work.
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Eur. J. Immunol. 2009. 39: 2865–2876
Verena Greifenberg et al.
Prostaglandin E2 have been described to mediate accumulation of
MDSC at tumor sites, despite the fact that it still occurred in their
absence [11–16]. More recently, the inflammatory mediators
S100A8 and S100A9 proteins have been identified as the major
MDSC attractants at tumor sites [17, 18]. Furthermore, the
combination of S100A8/S100A9 is also effective in blocking
myeloid differentiation into macrophages and DC [18]. The
expansion and activation of MDSC has also been described after
surgical trauma [19] and in polymicrobial sepsis, where LPS
injection alone could expand Gr-11 CD11b1 cells in the spleen
[20]. IFN-g has been demonstrated to promote MDSC activity by
inducing NO production and to interfere with DC development in
vitro [2, 21]. We have shown before that LPS is able to interfere
with DC in vitro where it blocks not only DC maturation, but also
DC development, when added throughout the DC cultures [22].
How bacterial LPS or inflammatory IFN-g either alone or in
combination can influence MDSC expansion versus suppressive
activity has not been investigated.
More recent data indicate that two subsets of MDSC can be
distinguished in tumor-bearing mice [23, 24]. While both subsets
suppress T-cell proliferation, one subset resembled monocytes
and was thus termed Mo-MDSC, while the other had more
similarities with PMN granulocytes and was therefore called
PMN-MDSC [23]. Recently, we identified two similar populations
in the BM of healthy normal mice (ER and MBL, unpublished
observations); however, whether both subsets of MDSC can be
isolated and activated from spleens of healthy mice is not known.
Here, we report that combined LPS/IFN-g treatment further
enhances the suppressive function of in vitro generated MDSC. We
identify MO-MDSC and PMN-MDSC in the spleens of healthy mice
and show that, after isolation, they bear the capacity to suppress
T cells in vitro. Injection of LPS/IFN-g into healthy mice led to the
activation of MO-MDSC and PMN-MDSC suppressor activity in the
spleen blocking CD81 T cell proliferation. We also show that
injection of LPS/IFN-g leads to the expansion of splenic myeloid
precursors, blocking their development into DC. Together, we
provide evidence for a dual activation program for MDSC in vitro
and in vivo, which can be elicited by a microbial LPS signal in
conjunction with IFN-g.
Results
Treatment of day 3 BM-MDSC with LPS/IFN-c induces
NO production and impairs DC development
The development of DC from BM precursor cells in vitro is
impaired in the presence of LPS [22]. Since MDSC are also early
products within BM-DC cultures and their suppressive function
through NO release depends on IFN-g [2], we wondered how LPS
and IFN-g in combination affects MDSC function and DC
development.
Day 3 BM cultures, known to suppress T-cell proliferation
in vitro, were treated with various cytokines or LPS alone or in
combination. After 24 h, neither TNF, IL-6 nor IL-1b induced NO
release. However, NO production was induced by either LPS or
IFN-g, but LPS and IFN-g in combination elicited the highest
release (Fig. 1A). In addition, the same combination dramatically
affected the subsequent DC development. The cultures were
treated on day 3 with IFN-g, LPS or LPS/IFN-g, further cultured
with GM-CSF until day 8, and then analyzed for the surface
expression of CD11c, MHC class II and CD86 molecules. Whereas
IFN-g did not influence the development of immature MHC
class IIlowCD86neg DC or spontaneously matured MHC class
IIhighCD86pos DC in the cultures, LPS showed the expected inhibition and the LPS/IFN-g combination completely blocked MHC
Figure 1. Combined treatment of BM cells with LPS/IFN-g induces high NO production and blocks DC development. Day 3 MSDC generated from
C57BL/6 mice were cultured in 24 well plates using 1 106 cells/well and stimulated over night with LPS, TNF, IL-1b or IL-6 either alone or in
combination with IFN-g. (A) On day 4 the cell supernatants were tested for NO production by Griess reaction. (B) Replicate wells were further
cultured to develop into DC. On day 8 cells were analyzed for MHC class II, CD86 and CD11c by flow cytometry to determine the percentage of DC
using mAb against MHC class II, CD86 and CD11c. Data are representative of three independent experiments with similar results.
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eji-journal.eu
Eur. J. Immunol. 2009. 39: 2865–2876
class II and CD86 expression, despite the surprising fact that the
proportion of CD11c1 cells was the same under all conditions
(Fig. 1B). Taken together, our data show that combined
LPS/IFN-g treatment of day 3 BM-MDSC cultures greatly induces
NO release and blocks DC development.
Combined LPS/IFN-c treatment enhances the
suppressive capacity of in vitro generated MDSC
Next, we wanted to examine the effect of LPS/IFN-g on the
suppressive capacity of day 3 BM-GM-CSF cultures. For this
purpose, we stimulated day 3 BM-GM-CSF cultures with IFN-g,
LPS or the LPS/IFN-g combination overnight or left them
untreated. Then, the cells were titrated into an allogeneic MLR
Immunomodulation
(allo-MLR) to test their suppressive potential. The untreated cells
served as controls and suppressed the T-cell proliferation as
described before [2], while day 4 cells had a stronger suppressive
capacity (Fig. 2A). Cells stimulated with IFN-g alone partially,
and cells treated with LPS alone completely lost their suppressive
function. In contrast, cells stimulated with LPS/IFN-g dramatically increased their suppressive potential as compared with
the control day 3 or day 4 MDSC (Fig. 2A). To test whether
this activation of suppressor function by LPS/IFN-g would
be maintained, we cultured the LPS/IFN-g treated cells for
another 5 days with GM-CSF and then used them as suppressors
in an allo-MLR. The results indicated that cultures treated
with LPS/IFN-g completely lost their suppressive potential
(Fig. 2B). Thus, LPS/IFN-g treatment of MDSC activates their
suppressive potential transiently. Further, culture in GM-CSF led
to impaired DC development and does not maintain the
suppressive function.
Characterization of six spleen cell subsets differentially expressing Gr-1 and CD11b
Figure 2. Only the LPS/IFN-g combined treatment enhances the
suppressive capacity of in vitro generated day 3 MDSC. Day 3 BM cells
of C57BL/6 mice were stimulated overnight with IFN-g, LPS or combined LPS/IFN-g. As positive suppressive controls the MDSC of days 3
or 4 were left untreated. (A) Capacity of stimulated and control cells to
suppress allo-MLR. Proliferation was tested after 3 days by [3H]Thymidine incorporation. (B) The LPS/IFN-g-treated cells were washed
at day 4 and further cultured until day 8. Suppressive capacity was
tested by titrating the cells into an allo-MLR. Proliferation was tested
after 3 days by [3H]-Thymidine incorporation. Data are representative
of four independent experiments with similar results.
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Previous reports indicated that the MDSC activity of Gr-11 CD11b1
cells could only be measured with cells isolated from tumor-bearing
mice or animals with infections. In the following, we wanted to
elucidate whether Gr-11 CD11b1 cells from healthy mice already
have MDSC potential. From our previous experiments, we knew
that BM-derived MDSC represent Gr-1low, CD11b1, CD11cneg and
F4/801 myeloid cells with ring-shaped nuclei [2]. More recent
results from others obtained using the spleens of tumor-bearing
mice [23, 24], and our own results from the BM (ER and MBL,
unpublished observations), indicate that there are at least two
MDSC subsets with differential suppressive capacity present in
mice. To identify the putative suppressive cell populations in the
spleens of healthy mice, we stained fresh splenocytes with mAb
against Gr-1 and CD11b and isolated different fractions by
magnetic bead technology or cell sorting. For analysis forward
scatter (FSC) and side scatter (SSC), and the morphological
analysis of cytospin preparations were also used. Spleens of healthy
mice consisted of at least six different subpopulations (Fig. 3). Gr1high cells with an SSCint profile can be divided into Gr-1highCD11bint cells with ring-shaped nuclei (Fig. 3, population P1), with
similarities to the BM-derived MDSC, and Gr-1highCD11bhigh cells
with PMN shape (P2), indicative for neutrophils. Gr-1lowCD11bint
CD1151 splenocytes comprise two different subpopulations with
respect to their granularity. Within these, we found SSChigh (P3)
and SSClow (P4) cells, which consisted of eosinophils as indicated
by their red granular staining by eosin in the cytoplasm (Fig. 3, P3,
arrows labelled E), cells with myelomonocytic morphology (Fig. 3,
P3, arrows labelled M) and small non-granular cells with a
lymphocyte-like morphology (Fig. 3, P4, arrows labelled L). Gr1neg CD11blowCD1151 SSClow cells represent the fifth splenic
subpopulation that also showed a myelomonocytic morphology
(P5) and Gr-1pos CD11bneg CD115neg SSClow cells the sixth (P6)
with a lymphocyte-like morphology.
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Eur. J. Immunol. 2009. 39: 2865–2876
Figure 3. Splenocytes of C57BL/6 mice can be divided into six different subpopulations with regard to their surface markers CD11b and Gr-1,
granularity and morphology. Freshly isolated spleen cells of C57BL/6 mice were stained with mAb against CD11b and Gr-1 and analyzed by flow
cytometry. The dot plot shows the pattern of distribution of spleen cells regarding Gr-1 versus CD11b. Gated subsets are further shown as FSC
versus SSC profiles or were sorted and cytospin preparations were stained with H&E dye. The resulting six different populations are termed P1-P6.
In the cytospin preparation of P3/P4 cells with various morphologies appear as eosinophils (E), myelomonocytic cells (M) or lymphoid-like cells (L).
Data are representative of three independent experiments with similar results.
The six different subpopulations assigned P1–P6 in Fig. 3
were further analyzed for their expression of a selected panel
markers (Fig. 4). As expected the P1 and P2 expression patterns
were consistent with early myeloid cells (Ly-6C1 F4/801), while
the P3 population expressed CCR3, which is characteristic for
eosinophils. Within the P4 gate fractions of CD11c1 DC and
Ly-6Chigh monocytes could be detected. In the subpopulation P5,
a few cells expressed not only CD11c, but also MHC class II, NK
1.1 and DX5 (data not shown, a remarkable profile that could
represent the NK cell subset with DC features that was originally
named NK-DC or IK-DC [25]. The Gr-11 CD11bneg splenocytes
within gate P6 expressed the B-cell marker CD45-R/B220 (data
not shown) and a small subset the PDCA-1 marker, and therefore
constituted a mixture of B cells and plasmacytoid DC [26].
Having addressed the morphology and surface marker profile of
these subpopulations, we wanted to assess the suppressive
capacities of these subsets.
Two subsets of suppressive spleen cells in healthy
mice
Since suppression of T-cell proliferation is the best functional
evidence for MDSC, we separated the spleen subpopulations by
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
magnetic beads or cell sorting and tested their capacity to
suppress T-cell proliferation in an allo-MLR. In vitro generated
day 3 BM-MSDC served as control suppressor cells. In a first step,
we sorted fresh spleen cells into Gr-1pos or CD11bpos splenocytes
by magnetic bead separation (MACS) and utilized them in
titrated numbers as potential suppressor cells. While the Gr-1pos
fraction could not suppress T-cell proliferation substantially, the
CD11bpos cells were strongly suppressive (Fig. 5A). In the next
step, we isolated different subpopulations with a cell sorter to
examine their suppressive potential. First, we compared
Gr-1highCD11bint (P1, ‘‘ring’’ cells) with Gr-1highCD11bhigh cells
(P2, granulocytes). As expected, the Gr-1highCD11bint subpopulation P1 was able to suppress T-cell proliferation, while splenic
granulocytes (P2) did not influence T-cell proliferation
(Fig. 5B). The latter was expected but has not been appreciated
so far because usually all Gr-11 CD11b1 spleen cells have been
considered to have MDSC function. This, however, does not
exclude that neutrophils may exert suppressive functions in other
assays or experimental settings in vivo.
In the next step, we wanted to know whether there were any
differences regarding suppressive potential between the MDSC
(P1) and the Gr-1lowCD11bint fraction (P31P4) comprising cells
of the myeloid lineage in diverse differentiation stages (Fig. 5C).
Both subpopulations were able to suppress T-cell proliferation as
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Figure 4. Surface marker expression of the individual splenic Gr-11 CD1b1 subsets P1-P6. Spleen cells were triple stained on their cell surface for
Gr-1, CD11b and the indicated markers or the respective isotype controls. Analysis gates were set according to their differential Gr-1 and CD11b
expression as indicated in Fig. 3 and designated P1–P6. The mAb-stained (solid line) or the respective isotype control (filled graph) histograms are
shown for the indicated populations. Data are representative of three independent experiments with similar results.
efficiently as control day 3 BM-MDSC. Additional separation of
the P3 and P4 subsets by their FSC/SSC profiles as shown in
Fig. 3 indicates that the P4 subset is responsible for the
suppressive effect within the Gr-1lowCD11bint fraction (Fig. 5D).
When we used Gr-1negCD11blow splenocytes (P5; presumed
NKDC) as a potential suppressor population (Fig. 5E), no
reduction of T-cell proliferation was obtained in this setting. In
addition, Gr-1pos CD11bneg cells (P6) also did not show
suppressive activity in this type of assay (data not shown).
To summarize, we identified two suppressive subpopulations
in the spleens of healthy mice by their differential expression of
CD11b and Gr-1, FSC, SSC and morphology. The suppressive
populations found here correlate with previous descriptions of
such cells in the spleens of tumor-bearing mice [23, 24] and BM
of healthy mice (ER and MBL, unpublished observations). The
ring-shaped Gr-1highCD11bint MDSC correlate with the described
PMN-MSDC and the morphologically more heterogeneous
Gr-1lowCD11bint myeloid cells resemble the MO-MDSC [23].
Accumulation and activation of MDSC after injection of
LPS/IFN-c
Since LPS/IFN-g enhanced the MDSC potential in vitro, the
question remained how it would affect the suppressive activity
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
of the MDSC subsets in vivo. To investigate this, mice were
left untreated or injected at days 0, 2 and 4 with LPS/IFN-g. On
day 7 splenic Gr-11 CD11b1 cells were sorted from spleens
of these mice and tested for their potential to inhibit an
allo-MLR in vitro. To our surprise, the suppressive capacity of
all isolated subsets in the inhibition of an allo-MLR in vitro
remained comparable and was not enhanced by LPS/IFN-g
treatment (data not shown) as we had observed using the
BM-derived MDSC.
To analyze the suppressive capacity after LPS/IFN-g injection
in vivo, mice were injected with LPS/IFN-g and then immunized
s.c. with OVA-loaded and CpG-matured DC (exogenous DC) or
OVA and CpG in incomplete Freund’s adjuvant (IFA; endogenous
DC). Control mice were left untreated or received only LPS/IFN-g
or the immunizations alone. To monitor specific T-cell suppression,
all mice received CFSE-labeled OVA-specific, TCR-transgenic CD81
OT-I cells. Injection of LPS/IFN-g led to higher frequencies of both
Gr-11 CD11b1 cells in the spleens but not LN (Fig. 6A). Both Gr1highCD11bint (P1) and Gr-1lowCD11bint (P3/4) MDSC splenic
subsets, and also Gr-1highCD11bhigh granulocytes (P2) were
increased by this treatment, while immunization by exogenous or
endogenous DC alone led to only minor increases of the MDSC
subpopulations (Fig. 6A). Further subgating of the Gr1lowCD11bint cells by their FSC/SSC profiles into P3 and P4
populations after LPS/IFN-g injection indicated a drop in P3 cell
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Figure 5. Gr-1highCD11bint and Gr-1lowCD11bint splenocytes are able to suppress T-cell proliferation in vitro. Freshly isolated spleen cells of C57BL/6
mice were stained with mAb against CD11b and Gr-1 and sorted (A) by magnetic beads using Gr-1 or CD11b mAb or (B-D) by FACS for the indicated
antibodies and for their FSC/SSC profile for the populations P3 and P4 to isolate the different splenic subpopulations. As suppressive positive
control cells, day 3 BM-MDSC were used. Cells were then added as potential suppressor cells in triplicates at graded concentrations into an alloMLR. Proliferation was tested after 3 days by [3H]-Thymidine incorporation. Data show mean7SEM (n 5 3) and are representative of 8 independent
experiments.
numbers from 29.276.7% (n 5 2) in untreated animals to
9.073.0% (n 5 3) in LPS/IFN-g treated animals (n 5 3) and
thereby an increase of P4 cells from 56.6715.2% (n 5 2) to
81.371.5% (n 5 3). This may indicate a specific expansion of
myelomonocytic P4 cells but not the eosinophils within P3.
Analysis of the injected OT-I T cells revealed that vigorous proliferation was induced after both types of immunizations, but preinjections with LPS/IFN-g inhibited the proliferation of OT-I cells
in the spleens (Fig. 6B). Correlated with the absence of MDSC in
the LN, the proliferation of OT-I cells in these organs was largely
unaffected (Fig. 6B). These data indicate that MDSC accumulation
in lymphoid organs after LPS/IFN-g injection was associated with
impaired CD81 T-cell proliferation in the same organ. To further
investigate whether the depletion of Gr-11 cells would reconstitute
CD81 T-cell proliferation, we injected Gr-1 antibodies into the
immunized control mice and mice injected with LPS/IFN-g.
Unfortunately, the adoptively transferred OT-I cells were depleted
after Gr-1 injection (data not shown), similar as described for
CD81 memory T cells [27].
investigated the splenic CD11c1 DC frequency 6, 9 or 12 days
after LPS/IFN-g treatment. The total number of spleen cells
increased after this treatment until day 9 (Fig. 7A), similar to the
absolute numbers of splenic DC, which resulted in a stable
percentage of DC during the whole period investigated (Fig. 7B).
This would indicate that the splenic DC populations are not
affected by LPS/IFN-g injections. However, since the turnover of
spleen DC is very high with half-lifes of 1.5–2.9 days [28], and the
splenic DC pool may not be recruited from BM precursors but a
splenic precursor pool [29], potential effects of the LPS/IFN-g
treatment may be rapidly corrected for in the splenic DC pool. In
order to test the capacity of the Gr-11 CD11b1 cells regarding their
potential to differentiate into DC, these cells were sorted from mice
injected with LPS, IFN-g, LPS/IFN-g or untreated animals. After in
vitro culture with GM-CSF for 3 days the animals injected with LPS
or LPS/IFN-g generated far fewer CD11c1 CD86neg immature DC
as compared with the control or IFN-g injection alone (Fig. 7C),
similar to that which we observed in vitro. Thus, activation by LPS/
IFN-g blocks the capacity of splenic MDSC to develop into DC.
Impaired capacity of Gr-11 CD11b1 cells to develop
into DC after LPS/IFN-c injections
GM-CSF culture modulates the suppressive capacity of
LPS/IFN-c treated Gr-11 CD11b1 cells
To evaluate whether LPS/IFN-g activation would affect the
potential of Gr-11 CD11b1 cells to develop into DC, we
The question remained whether LPS/IFN-g activated Gr-11
CD11b1 cells would lose their suppressive capacity after further
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Eur. J. Immunol. 2009. 39: 2865–2876
Figure 6. LPS/IFN-g injections accumulate Gr-11CD11b1 MDSC subsets in the spleen but not LN leading to suppression of CD81 T-cell proliferation
only in the spleen. Mice were injected with a combination of LPS/IFN-g i.p. at days 0, 2 and 4 or remained untreated. At day 10, they were
immunized s.c. into the footpads with IFA/CpG/OVA peptide or mature OVA peptide-loaded DC. CFSE1 OT-I cells were transferred i.v. at the same
day 10. The animals were sacrificed 4 days after the immunization/OT-I-cell transfer. LN and spleen were removed and analyzed for the subsets P1
and P3/P4 of Gr-11 CD11b1 cells (A) and Vb51 OT-I-cell proliferation (B). The data are representative of two independent experiments.
culture in GM-CSF. To test this, the same experimental setting as
shown above (Fig. 7C) was used to obtain Gr-11 CD11b1 cells
from spleens of mice that were injected with LPS, IFN-g, LPS/IFN-g
or PBS as a control. These cells were then cultured for 3 days in
GM-CSF and then tested for their capacity to suppress an allo-MLR
(Fig. 8A). While cells from control mice and IFN-g treated mice
remained suppressive, the Gr-11 CD11b1 cells from the other
two groups seemed to be less suppressive. Given that, for this
experiment, the high turnover of cells in the spleen may influence
the populations, we directly tested the effect of GM-CSF on the
splenic Gr-11 CD11b1 cells ex vivo. CD11b1 cells were sorted with
magnetic beads from spleens of untreated mice and cultured for
24 h with GM-CSF only or in addition with LPS, IFN-g, LPS/IFN-g
before they were titrated into an allo-MLR to test their suppressive potential. Only the control cells retained suppressive capacity
but the LPS, IFN-g, LPS/IFN-g plus GM-CSF treated cells completely lost this function (Fig. 8B). These data indicate that
pretreatment with GM-CSF and more strongly simultaneous
treatment of splenic Gr-11 CD11b1 cells conteracts their suppressive potential.
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Discussion
Here, we showed that two specific subsets of splenic Gr-11 CD11b1
cells of healthy naive mice bear the potential to become MDSC and
suppress T-cell proliferation. The combined LPS/IFN-g signaling
but none of the single components led to the activation of myeloid
precursors into functionally suppressive MDSC, which impaired
their developmental potential into DC. Additional exposure to GMCSF counteracted the LPS/IFN-g effects.
Our earlier findings indicated that LPS signals on myeloid
precursor cells interfered with DC development in vitro. This LPS
blocking generated CD11c1 cells that expressed only little surface
MHC class II and no costimulatory molecules. Consequently,
these immature DC were functionally tolerogenic and induced
T-cell anergy in vitro [22]. Here, we extended our findings
in vitro by showing that combined LPS/IFN-g treatment enforced
this effect, while IFN-g alone showed no changes. After injection
of LPS, Gr-11 CD11b1 cells accumulated in the spleen as reported
before [20] and, similar to our in vitro findings, these cells
showed reduced capacity to develop into CD11c1 DC. These data
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Figure 7. In vivo activation of Gr-1/CD11b cells by LPS or LPS/IFN-g impairs their capacity to develop into DC. Mice were injected i.p. with LPS/IFN-g
or with PBS at days 0, 2 and 4. At days 6, 9 and 12 the spleen cell numbers were counted (A), stained for CD11c and analyzed by flow cytometry
(B). In another experimental setting the mice were injected i.p. with LPS, IFN-g, the combination, or with PBS at days 0, 2, and 4 and at day 7 the
splenic Gr-11CD11b1 cells from individual mice of each group were isolated by cell sorting and cultured in the presence of GM-CSF. At day 10 FACS
analyses were performed with the indicated markers (C) to analyze their precursor/granulocyte by Gr-1/CD11b or immature/mature DC phenotype
by CD86/CD11c. The data are representative dot plots of duplicate mice per experiment. Three experiments with similar results for all individual
mice have been performed.
indicated that LPS could expand Gr-11 CD11b1 cells and block
DC development, but not whether LPS alone is sufficient to
activate their suppressor function.
NO has been shown to be one of the suppressive tools secreted
by MDSC [1]. Here, LPS transiently induced the NO production in
our BM-MDSC cultures. However, when the NO was washed off
from the cells they lost their suppressive potential after continued
culture in GM-CSF. These findings indicate that LPS treatment of
myeloid precursors leads to a partial activation as indicated by
NO release that allows a transient suppressive activity. This also
may indicate that the suppressive mechanism that could be
observed in the allo-MLR by using LPS plus IFN-g for MDSC
stimulation could be mediated through a mechanism other than
NO production or involve a second wave of NO production that
may, however, depend on IFN-g.
IFN-g is the major cytokine released by CD41 Th1 cells and
CD81 CTL and has been also shown to control MDSC activity
[1, 30]. Our results with in vitro generated MDSC indicated that
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their suppressive activity could be partially abrogated by blocking
IFN-g [2], similar to that which others observed in vivo [30].
Treatment of BM-MDSC cultures with IFN-g led to low NO
production, but after washing, the blocking activity in an alloMLR was reduced when compared with untreated cells. Injections
of IFN-g did not induce T-cell suppression in vivo and did not
increase the number of myeloid cells in the spleen. Thus, these
data indicate that neither myeloid cell accumulation in the spleen
nor MDSC activity could be induced by IFN-g alone.
The combined in vitro treatment or in vivo injection of LPS/
IFN-g led to an accumulation of myeloid cells in culture or in the
spleen but with an impaired capacity to develop into DC. When
LPS/IFN-g-treated day 3 BM cell cultures were further propagated with GM-CSF they not only showed an impaired capacity to
develop into DC, but also lost their suppressive potential. This
GM-CSF effect was also observed with splenic MDSC ex vivo. At
this point, it is speculative whether activated MDSC may further
undergo a special myeloid development toward tolerogenic DC,
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Figure 8. GM-CSF counteracts the suppressive potential of LPS/IFN-g activated MDSC. (A) In analogy to the experimental setting shown in Fig. 7C,
mice were injected i.p. with LPS, IFN-g, the combination, or as a control with PBS at days 0, 2 and 4. At day 7 the splenic Gr-11 CD11b1 cells from
individual mice of each group were isolated by cell sorting and cultured in the presence of GM-CSF. At day 10 cells were harvested, counted and
their suppressive function was tested by titrating them into an allo-MLR. (B) Freshly isolated and magnetic bead-sorted CD11b1 cells from spleens
of untreated mice were simultaneously treated with GM-CSF plus either LPS, IFN-g or the combination or remained untreated for 24 h. Capacity to
suppress allo-MLR was tested by adding graded doses of the cells. Data show mean7SD (n 5 3) and are representative of three independent
experiments.
since they acquire the CD11c marker without leading to fully
functional immunogenic DC, similar to that observed with LPS
treatment alone [22].
Only the simultaneous presence of LPS plus IFN-g activated
MDSC suppression in vitro. During the course of a bacterial
infection, LPS will be detectable to immune cells at the earliest
time points, initiating an anti-microbial immune response.
Subsequently, as a result of the adaptive immune response IFN-g
producing T cells will be generated, which produce IFN-g only
locally when they are restimulated by antigens at the infection
site. The simultaneous treatment of mice with LPS/IFN-g may
reflect a chronic infection [31–33] or sepsis [20], where pathogens carrying LPS or other immunostimulatory molecules are
systemically present, together with high levels of IFN-g. In this
situation, MDSC activation may occur as a beneficial mechanism
to control immunopathology in the host. We can only speculate
why the single treatments with either LPS or IFN-g have opposing
effects as compared with the combination of both. However, the
single detection of either LPS or IFN-g by MDSC also results in
useful mechanisms. Recognition of pathogen alone requires an
immune response and should not be associated with increased
suppression. Similarly, detection of IFN-g by MDSC is just part of
a normal ongoing immune response where suppression would
also be counter-productive.
The suppression of T cells in the spleen but not in the LN
would further support this model, as only the systemic T-cell
activation is blocked in the spleen but not in the local tissuedraining LN where T-cell activation may continue. Although
increased frequencies of MDSC in LN have been observed by
others using models for sepsis, or tumors, the percentages
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remained low around 2–4% as compared with about 20% in
the spleens [20, 34]. Under the conditions tested here, there
was clearly no induction of Gr-11 CD11b1 cell in the LN.
The slightly lower rate of CD81 T-cell proliferation detected in
the LN of our mice must therefore reflect the reduced recirculation of T cells from the spleen. Together, this may indicate that
the major site of suppression in mice is the spleen rather than
the LN.
It is a matter of debate whether MDSC exist in the steady state.
Our ex vivo and in vivo data demonstrate that two subsets of Gr-11
CD11b1 cells with suppressive potential exist within the spleen,
indicating preformed MDSC in the steady state. However, our
previous findings indicated that blocking IFN-g within the alloMLR that is undergoing suppression by BM-derived MDSC limited
NO production and partially reconstituted T-cell proliferation [2].
Similar data have been acquired in vivo [27]. This clearly indicates
that during the allo-MLR, factors such as IFN-g plus presumably
others are instantly produced to activate MDSC function. We could
observe this especially at high antigen doses during the in vitro
stimulation of TCR-transgenic T cells [2]. Enhancing the doses of
IFN-g in vivo by injection in combination with the presence of
tumor-derived or microbial factors such as LPS then may further
unfold the full potential of MDSC and increase their numbers in
the spleen. Thus, MDSC suppressor activity requires activation,
which may have more than one activation level.
However, Gr-11 CD11b1 cells isolated from mice that were
pre-injected with LPS/IFN-g and tested ex vivo for suppression did
not show an elevated suppressive potential as compared with
untreated mice (data not shown). The reasons for this discrepancy are unclear but could also be related to the high cellular
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turnover within the spleen. Particularly, the LPS/IFN-g injections
impaired the OT-I-cell proliferation, indicating that MDSC
activity increased. Taken together, we observed the increased
OT-I T-cell suppression in the spleen after LPS/IFN-g injections
without a higher intrinsic suppressive potential of the splenic
MDSC, but increased frequencies of the two splenic MDSC
subsets. These data indicate that MDSC activity in vivo may be
predominantly regulated by their number in the spleen.
The elimination of MDSC would be a tool to investigate their
role in vivo. However, injecting Gr-1 antibody to deplete MDSC in
the steady state was not successful in our hands since the antibody depleted activated CD81 OT-I T cells, similar to that which
has been described before for memory CD81 T cells by others
[27]. Genetic models to specifically ablate MDSC or their
functions will clarify this point in the future.
Finally, it is of note that here we identified the Gr-1high
CD11bhigh expressing neutrophils and the Gr-11 CD11blowSSChigh
eosinophils as non-suppressive populations in the spleen of
healthy mice. This fact is worth mentioning since many reports
generalize all Gr-11 CD11b1 cells in the spleen as candidates for
MDSC activity. However, differentiated neutrophilic granulocytes
and eosinophils do not seem to have suppressive potential in this
type of assay.
In conclusion, our data indicate that healthy mice contain two
splenic subsets of Gr-11 CD11b1 cells with suppressive capacity.
Their full activation requires two simultaneous signals such as
LPS/IFN-g. This activation by the simultaneous presence of these
factors resembles chronic infections or sepsis and is triggered to
inhibit immunopathology.
Materials and methods
Mice
C57BL/6 and BALB/c mice between 4–12 wk of age were used for
generating single cell suspensions of BM, spleen and LN as well as
for the in vivo experiments (C57BL/6 mice). Animals were
purchased from Charles River, Germany, or obtained from our
internal breeding facilities in Erlangen or Würzburg, Germany.
All experiments were performed according to the animal
protection laws and under control and with permission of the
local authorities (Regierung von Mittelfranken AZ: 621.2531.3206/02, TS-99/14; Regierung von Unterfranken AZ: 54-2531.0108/07).
Media and reagents
For cell culture R10 medium was used consisting of RPMI 1640
(Lonza) supplemented with 100 U/mL penicillin (Sigma),
100 mg/mL streptomycin (Sigma), 2 mM L-glutamine (Sigma),
50 mM b-mercaptoethanol (Sigma) and 10% heat-inactivated FBS
(PAA, Cölbe, Germany).
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Isolation and preparation of cells and treatments
in vitro
To generate single cell suspensions of spleens, LN or BM organs
were removed under sterile conditions and the popliteal,
inguinal, axillary, cervical and mesenteric LN were disrupted
with glass slides and resuspended in PBS (Lonza) as described in
detail before [35]. Stimulation of MDSC were performed over
night with 0.1–1 mg/mL LPS (Sigma), 500 U/mL TNF, 200 U/mL
IL-1b or 200 U/mL IL-6 either alone or in combination with
100 U/mL IFN-g (all from Preprotech).
BM-derived MDSC and DC cultures
The culture of BM cells from C57BL/6 mice to generate d3 MSDC
or DC was performed as described previously [2]. Shortly, fresh
BM cells were cultured in 10 mL R10 medium with 10% culture
supernatant of a murine GM-CSF transfected cell line (equivalent
to 4200 U/mL). After 3 or 4 days of culture MSDC could be
harvested as non-adherent cells.
Flow cytometry and cell sorting for MDSC
Cells were stained with PerCP-conjugated mAb against Gr-1 and
either PE- or FITC-CD11b or FITC- or PE-conjugated mAb
against CD11c, F4/80, CD45R/B220, DX5, NK1.1, MHC class II
(I-A; M5/114), or CCR3-AlexaFluor647 (all BD Pharmingen,
Hamburg, Germany) or for Ly-6C (ER-MP20, AbD Serotec), or
CD115-APC, PDCA-1-AlexaFluor647 (eBiosciences) or the appropriate fluorochome-conjugated mAb or supernatants as isotype
controls at 2–5 mg/mL in PBS containing 0.1% sodium azide and
5% FBS for 30 min on ice in the dark. Samples were washed once
in staining buffer, measured and analyzed with a FACScan
(Becton Dickinson, Heidelberg, Germany).
For some experiments fresh spleen cells were sorted either by
MACS technology (Miltenyi, Bergisch-Gladbach, Germany)
or with a Mo-Flo highspeed sorter (Cytomation, Freiburg,
Germany) or a FACS-Vantage (BD). The purity of sorted cells was
generally above 90%. Sorted cells were then cultured as
indicated.
Allo-MLR
The different Gr-11 CD11b1 splenocyte populations from C57BL/
6 mice were sorted and cultured as triplicates in a 96 well flatbottomed plate (Falcon) in R10 medium at titrated numbers. In
vitro generated untreated day 3 or 4 BM-derived MDSC served as
positive control. LN cells from BALB/c mice (4 105 well) were
used as responder population. Mature day 9 DC from C57BL/6
mice were used as stimulator cell population (1 104 well). After
3 days of culture cells were pulsed with 1 mCi [3H]-thymidine
(Amersham) in HL-1 medium for 16 h and harvested onto
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Eur. J. Immunol. 2009. 39: 2865–2876
filtermats with an ICH-110 harvester (Inotech, Dottikon, Switzerland). Filters were counted in a 1450 microplate counter (Wallac,
Turku, Finland).
Measurement of NO as nitrite production
NO was measured as nitrite production using the Griess reaction
[36]. Briefly, 50 mL of cell culture supernatant were put into a
96 well ELISA-plate (Corning) as duplicates with titrated NaNO2
(Sigma) in R10 medium serving as a standard. An aqueous
solution of 0.1% naphtylethylendiamine dihydrochloride (Sigma)
and 1% sulfanilamide (Sigma) in 5% conc. H3PO4 (Merck,
Darmstadt, Germany) in water were mixed 1:1 and 50 mL of this
solution were added to 50 mL of the samples. The evoked color
reaction was measured after 10 min in the ELISA reader
(Molecular Devices) at 492 nm and nitrite concentrations were
calculated from the sodium nitrite standard curve.
Cytospins
Sorted splenocytes (2 105) were resuspended in 200 mL R10
medium and centrifuged onto a microscope slide using a
Cytospin-3 (Shandon, Life Sciences International, Astmoor,
UK). Then, slides were stained with hematoxylin/eosin dye
according to standard protocols.
10 mM OVA peptide for 4 h. For preparation of OT-I cells, a single
cell suspension from spleens and LN was prepared, lysed for
erythrocytes with 0.8% ammoniumchloride, washed and then
labeled with CFSE (5 mM in PBS for 15 min, RT; Molecular
Probes). OT-I cells (1.3 107 cells per mouse) were transferred
i.v. at the same day 10. The animals were sacrificed at day
14 and the single cell suspensions of spleens and LN used
for flow cytometry analysis with biotinylated mAb against Vb5
detected by an streptavidin-PerCP conjugate (both BD Pharmingen).
Acknowledgements: We thank Gerold Schuler for his continuous
support, Thomas Hünig for support and helpful discussions,
Gudrun Schell for expert technical assistance and Christian
Linden for cell sorting. This work was supported by the
Interdisciplinary Centre for Clinical Research (IZKF) Erlangen
for VG, DFG through LU851/4-1 for SR and MBL and the
Collaborative Research Centre SFB479 for ER and MBL.
Conflict of interest: The authors declared no financial or
commercial conflict of interest.
References
Ex vivo analysis of spleen cells after injection of
LPS/IFN-c
1 Gabrilovich, D. I. and Nagaraj, S., Myeloid-derived suppressor cells as
regulators of the immune system. Nat. Rev. Immunol. 2009. 9: 162–174.
2 RöXner, S., Voigtländer, C., Wiethe, C., Hänig, J., Seifarth, C. and
Female C57BL/6 mice received 1 mg IFN-g and 10 mg LPS i.p. per
mouse on day 0. IFN-g- and, where indicated, LPS injections were
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Abbreviations: Allo-MLR: allogenic MLR FSC: forward scatter IFA:
incomplete Freund’s adjuvant MDSC: myeloid-derived suppressor
cell Mo: monocytic SSC: side scatter
Full correspondence: Dr. Manfred B. Lutz, Institute for Virology and
Immunobiology, University of Würzburg, Versbacherstr.7, Würzburg,
Germany
Fax: 149-937-201-49243
e-mail: [email protected]
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Identification of discrete tumor-induced myeloid-derived suppressor cell
Received: 2/4/2009
Revised: 6/7/2009
Accepted: 14/7/2009
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7
Abkürzungsverzeichnis
APZ/APC
Antigen-präsentierende Zellen/antigen-presenting cells
ATRA
all-trans-Retinoidsäure/all-trans-retinoid acid
BM
Knochenmark/bone marrow
bzw.
beziehungsweise
CAT2B
kationischer Aminosäuretransporter 2B/cationic amino acid
transporter 2B
CCR
Chemokinrezeptor/chemokine receptor
CD
cluster of differentiation
CFSE
carboxyfluorescein-diacetate-succinimidyl-ester
CpG
Cytosin-phosphatidyl-Guanosin
CSF-1
colony-stimulating-factor-1
CTLA-4
cytotoxic T-lymphocyte antigen 4
DZ/DC
dendritische Zellen/dendritic cells
e.g.
for example (exempli gratia)
EAE
experminentelle Autoimmunenzephalitis/experimental
autoimmune encephalitis
ELISA
enzyme-linked immunosorbent assay
FACS
Durchflusszytometrie/fluorescence activated cell sorting
FCS
fetales Kälberserum/fetal calf serum
FITC
fluorescein isothiocyanate
FLT3- Ligand
Fms-like tyrosine-kinase 3 ligand
FSC
Vorwärtsstreulicht/forward light scatter
GM-CSF
granulocyte/macrophage-colony-stimulating-factor
GPI
Glycosyl-Phosphatidyl-Inositol
H2O2
Wasserstoffperoxid/ hydrogen peroxide
H3PO4
ortho-Phosphorsäure/orthophosphoric acid
HLA
humanes Leukozytenantigen/human leukocyte antigen
ICAM 1
intercellular adhesion molecule 1
IDC
unreife dendritische Zellen/immature dendritic cells
IFA
inkomplettes Freund-Adjuvans/incomplete Freund´s
adjuvant
IFN
Interferon
IL
Interleukin
IMC
unreife myeloide Zellen/immature myeloid cells/
53
iNOS
induzierbare NO-Synthase/inducible NO-synthase
JAK
Janus Kinase
LN
Lymphknoten/lymph node
LPS
Lipopolysaccharid(e)
mAB
monoklonaler Antikörper/monoclonal antibody
MACS
Magnet-aktivierte Zelltrennung/magnetic bead cell sorting
MDSC
myeloid-derived suppressor cells
MHC
major histocompatibility complex
MLR
gemischte Leukozytenreaktion/mixed leukocyte reaction
MMP
Matrixmetalloproteinase
NADPH
reduzierte Form von Nikotinamid-Adenin-DinucleotidPhosphat/reduced form of nicotinamide-adeninedinucleotide-phosphate
NF-κB
nuclear factor “κ-light-chain-enhancer” of activated B-cells
NK- Zellen/NK-cells
natürliche Killerzellen/natural killer cells
NO
Stickstoffmonoxid/nitrogen monoxide
NOD-Maus
non-obese-diabetic-Maus
nor-NOHA
N -hydroxy-nor-L-arginine
OT
OVA-spezifischer TZR/OVA specific TCR
OVA
Ovalbumin
PBS
Phosphat-gepufferte Salzlösung/phosphate-buffered saline
PD
programmed death
PDCA
plasmacytoid dendritic cell antigen
PDGF
plateled-derived-growth-factor
PD-L
programmed death ligand
PE
phycoerythrin
perCP
peridinin-chlorophyll protein complex
PGE2
Prostaglandin E2
PMN
polymorphkernig/polymorphonuclear
ROS
reaktive Sauerstoffspezies/reactive oxygen species
SCF
stem cell factor
SSC
Seitwärtsstreulicht/sideward light scatter
STAT
signal transducers and activators of transcription
TAM
Tumor-assoziierte Makrophagen/tumor-associated
macrophages
TGF
transforming growth factor
TLR
Toll-like Rezeptor/toll-like receptor
54
TNF
Tumornekrosefaktor/tumor necrosis factor
Treg
regulatorische T-Zellen/regulatory T-cells
TZR/TCR
T-Zell-Rezeptor/T-cell-receptor
v.a.
vor allem
VEGF
vascular endothelial growth factor
z.B.
zum Beispiel
55
8
Danksagung
An erster Stelle möchte ich mich bei Herrn Professor Gerold Schuler bedanken,
dass er mir die Anfertigung meiner Dissertation in den Laboratorien der Hautklinik
Erlangen ermöglichte.
Großer Dank geht an das Interdisziplinäre Zentrum für Klinische Forschung
Erlangen, das meine Promotion mit einem Doktorandenstipendium unterstützte.
Besonderen Dank spreche ich meinem Betreuer Herrn Professor Manfred Lutz für
die langjährige Betreuung meiner Promotion aus, die auch „überregional“
funktionierte und bedanke mich für seine Geduld.
Bedanken möchte ich mich auch bei den Referenten Prof.Dr.Dr. André Gessner und
Prof.Dr. Alexander Steinkasserer aus Erlangen und Prof.Dr. Nikolaus Romani aus
Innsbruck, die Zweitgutachten für diese Arbeit erstellten.
Unvorhersehbare Umstände führten dazu, dass Teile der Arbeit in Würzburg
durchgeführt werden müssen. Vielen, vielen Dank an Dr. Eliana Ribechini, dass sie
neben ihrem eigenen Projekt auch an der Fragestellung dieser Dissertation
mitarbeitete.
Vielen Dank auch an das gesamte Laborteam, das sich im Laufe der Jahre ja
ziemlich gewandelt hat. Besonders möchte ich mich bei Susa Rößner bedanken, die
immer ein offenes Ohr für mich hatte, mir mit Rat und Tat zur Seite stand und auch
ab und zu eine Verstellung des Radiosenders tolerierte. Danken möchte ich auch
vor allem Dr. Jens Hänig und Dr. Lisa Zinser - beide waren ebenfalls jederzeit
hilfsbereit und nie genervt, wenn ich Fragen stellte. Danke auch an all die Menschen
in den anderen Labors, denen ich im Laufe der Zeit immer wieder mal kleinere und
größere Fragen stellen musste, die diese immer bereitwillig beantworteten. Danke
für die wirklich nette Atmosphäre im Laborbereich dieser Klinik.
Bedanken möchte ich mich auch bei den Dres. Pichler: Danke, Dr. Matthias Pichler,
dass Du „trotzdem“ immer da warst und bist und hoffentlich bleibst. Der Weg wird
ebener und gerader werden, jedoch nie eine Autobahn. Danke, Dr. Klemens Pichler,
dass Du meine Technikwüste in eine Oase verwandelt hast.
56
Das Promotionsprojekt konnte in der Form nur stattfinden, weil ich mich neben
meinem Studium nicht um dessen Finanzierung kümmern musste, sondern seitens
meiner Eltern unterstützt wurde. Dafür möchte ich mich herzlich bei Euch bedanken!
Nun müsst Ihr nur noch Eure Visitenkarte ändern…Vielen Dank auch besonders an
meine Schwester, die mir bei vielen kleinen und größeren Problemen immer
geholfen hat und sich durch den Wust gelesen hat. Danke Julia! Danke Oma, dass
Du immer an mich gedacht hast.
Danke an zwei gewisse, starke Damen aus Nürnberg, die das sehen, was andere
nicht sehen und meinen Blick dahingehend schärfen konnten.
Danke an Dr. Weibchen, dass Du stark geblieben bist und Dich nicht unterkriegen
hast lassen. In Kooperation mit Dr. Nasi wird es ein phantastisches, lebenslanges
Projekt werden, glaub mir!
57
9
Lebenslauf
Persönliche Daten:
Name
Verena Greifenberg
Geburtsdatum/-ort
12. Oktober 1978/Bamberg
Eltern
Rita Greifenberg, geb. Gruber (*12.01.1954)
Günter Greifenberg (*15.06.1952)
Geschwister
Julia Greifenberg (*11.12.1982)
Familienstand
ledig
Staatsangehörigkeit
deutsch
Berufliche Tätigkeit:
seit 01.09.2009
Assistenzärztin am Institut für Medizinische
Mikrobiologie, Immunologie und Hygiene
(TU München)
Hochschulstudium:
05/2009
Approbation als Ärztin
04/2009
Ärztliche Prüfung, zweiter Abschnitt
04/2005-02/2009
Humanmedizin, klinischer Abschnitt
an der Friedrich- Alexander- Universität Erlangen
03/2005
Ärztliche Prüfung, erster Abschnitt
10/2004-02/2005
Humanmedizin
Vervollständigung des vorklinischen Abschnitts
an der Friedrich-Alexander-Universität Erlangen
11/2003-06/2004
Diplomarbeit der Molekularen Medizin
an der Klinik für Psychiatrie und Psychotherapie
Erlangen
Thema: Etablierung und Charakterisierung der Zelllinie
NTera-2 zur Entwicklung eines Modellsystems für
antidepressive Therapien
10/1999-06/2004
Molekulare Medizin (Hauptstudium)
an der Friedrich-Alexander-Universität Erlangen
Abschluss: Diplom
05/1999-07/1999
Humanmedizin
an der Friedrich-Alexander-Universität Erlangen
Schulbildung:
09/1989-06/1998
Franz-Ludwig-Gymnasium Bamberg
Abschluss: Allgemeine Hochschulreife