The influence of chronic stress on T cell immunity
Dissertation
zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.)
des Fachbereiches für Biologie an der Universität Konstanz
vorgelegt von Annette Sommershof
Tag der mündlichen Prüfung: 14.09.2010 1. Referent: Prof. Dr. Alexander Bürkle 2. Referent: Prof. Dr. Marcus Groettrup 3. Referent: Prof. Dr. Volker Stefanski
Konstanzer Online-Publikations-System (KOPS) URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-124695
URL: http://kops.ub.uni-konstanz.de/volltexte/2010/12469/
I
SUMMARY... 1
CHAPTER 1: INTRODUCTION... 4
THE IMMUNE SYSTEM IN RESPONSE TO STRESS... 5
TCD8+ CELLS IN VIRAL INFECTIONS...5
DENDRITIC CELLS...5
SKIN DENDRITIC CELL TYPES...6
LANGERHANS CELL MIGRATION...6
LANGERHANS CELL MIGRATION: ROLE OF CYTOKINES...7
LANGERHANS CELL MIGRATION: ROLE OF CHEMOKINES AND CHEMOKINE RECEPTORS...8
ANTIGEN PROCESSING AND PRESENTATION BY DENDRITIC CELLS...9
DC SIGNALS FOR CLONAL EXPANSION AND DIFFERENTIATION OF NAÏVE T CELLS...11
ROLE OF TCR MEDIATED SIGNALING – SIGNAL 1...11
COSTIMULATION BY THE B7 FAMILY – SIGNAL 2...12
ROLE OF OTHER CORRECEPTORS...13
ROLE OF CYTOKINES - SIGNAL 3...13
EFFECTOR FUNCTION OF TCD8+ CELLS...14
MIGRATION OF T LYMPHOCYTES...14
MOVING INTO SECONDARY LYMPHOID ORGANS...15
MOVING OUT – ROLE OF CHEMOKINE RECEPTOR CCR7 AND ADHESION MOLECULES...15
DIVERSITY OF THE MEMORY T CELL POOL...16
LINEAGE RELATIONSHIP BETWEEN NAIVE, EFFECTOR, AND MEMORY T CELLS...17
LYMPHOCYTIC CHORIOMENINGITIS VIRUS (LCMV)...17
STRESS AND IMMUNITY... 19
INTERACTION OF THE IMMUNE SYSTEM AND THE BRAIN - THE HPA AND SNS AXIS...19
THE SYMPATHETIC NERVOUS SYSTEM (SNS)...19
THE HYPOTHALAMUS-PITUITARY-ADRENAL (HPA) AXIS...20
BIDIRECTIONAL COMMUNICATION BETWEEN THE CNS AND THE IMMUNE SYSTEM...21
GLUCOCORTICOID HORMONES...22
MOLECULAR MECHANISM OF GC-INDUCED IMMUNOSUPPRESSION...22
CELLULAR MECHANISM OF GC-INDUCED IMMUNOSUPPRESSION...23
REGULATION OF THE HPA AXIS AND GC ACTION UNDER CHRONIC STRESS EXPOSURE...25
ANIMAL MODELS OF CHRONIC STRESS AND IMMUNITY...26
SOCIAL STRESS...26
THE SOCIAL DISRUPTION STRESS (SDR) MODEL...26
Table of contents
II CHAPTER 2: ATTENUATION OF THE CYTOTOXIC T LYMPHOCYTE RESPONSE TO
LYMPHOCYTIC CHORIOMENINGITIS VIRUS IN MICE SUBJECTED TO CHRONIC SOCIAL
STRESS... 28
ABSTRACT... 29
INTRODUCTION... 29
RESULTS... 30
DISCUSSION... 40
MATERIALS AND METHODS... 43
CHAPTER 3: IMPAIRED MIGRATION OF SKIN DENDRITIC CELLS IN RESPONSE TO CONTACT SENSITISATION IN MICE SUBJECTED TO CHRONIC SOCIAL STRESS... 48
ABSTRACT... 49
INTRODUCTION... 49
RESULTS... 50
DISCUSSION... 54
MATERIAL AND METHODS:... 57
CHAPTER 4: SUBSTANTIAL REDUCTION OF NAÏVE AND REGULATORY T CELLS FOLLOWING TRAUMATIC STRESS... 59
ABSTRACT... 60
INTRODUCTION... 60
RESULTS... 62
DISCUSSION... 67
MATERIAL AND METHODS... 69
CHAPTER 5: DISCUSSION... 72
REFERENCES... 78
APPENDIX... 97
ABBREVIATIONS... 98
RECORD OF ACHIEVEMENT / EIGENABGRENZUNG... 100
ACKNOWLEDGMENT... 101
1
Summary
Chronic environmental and psychological stress has long been suspected to increase the susceptibility and outcome of numerous infectious and inflammatory diseases. The release of neurotransmitters (catecholamines) and adrenal hormones (glucocorticoids) has been well documented as the basis for a connection between the central nervous system and peripheral components of the immune system. Glucocorticoids, the end products of stress-induced neuroendocrine pathways and the hypothalamic-pituitary-adrenal (HPA) axis, belong to the most potent anti-inflammatory hormones in the body and serve to control immune responses for example during an infection. However, prolonged or excessive elevation of glucocorticoids as occurring during recurrent or chronic stress can negatively impact various aspects of immune cell functions and may therefore contribute to disease development and progression. The precise mechanisms and course of events leading to the suppression of immune functions during chronic stress and how these effects result in certain diseases remains poorly understood.
The aim of the present thesis was to further analyze underlying mechanisms of chronic stress- related immunosuppression focusing on T cell-mediated immunity. In order to mimic recurrent stress experiences, a well-established mouse model of chronic social stress termed
“social disruption stress” (SDR) was chosen for the experiments conducted in chapters 2 and 3. Pharmacological intervention (i.e. the blockage of the respective receptors) in these experiments allowed us to differentiate between the impact of glucocorticoids and catecholamines, the main mediators of stress responses during chronic exposure.
Chapter 2 focuses on the impact of chronic social stress on the outcome of virus-specific cytotoxic TCD8+ cell (CTL) responses in mice after infection with lymphocytic choriomeningitis virus (LCMV). Taking into account that the duration of stress exposure and also the timing of stress relative to an immune challenge can greatly impact the outcome of immune alterations, we directly compare different stress procedures. We show that social stress impacts the generation of IFN-γ-producing, virus-specific TCD8+ splenocytes only when applied prior to virus infection. We further demonstrate that during a prolonged stress exposure glucocorticoid hormones strongly impact the proliferation capacity of TCD8+ cells in the spleen of infected mice. This impairment results most probably from reduced TCD8+
activation as well as an impaired cytokine secretion profile. The reduced expansion of TCD8+
cells appears to be organ specific, as we found no such alterations in the inguinal lymph node or in the blood or peripheral tissues such as the liver and lung. A possible explanation for the organ-specific decline in TCD8+ cell expansion could be an altered migration capacity of splenic TCD8+ cells as demonstrated by adoptive T cell transfer experiments.
Chapter 3 describes the impact of chronic social stress on the migratory capability of skin dendritic cells (DCs). Skin DC and in particular Langerhans cells are known as critical inducers of cutaneous immune responses. Glucocorticoid-mediated impairment of DC function has been in the focus of recent investigations. To directly investigate the consequences of chronic stress on skin DC function, we performed contact allergen-induced skin sensitization assays using the fluorescent dye fluorescein isothiocyanate (FITC), which
Summary
2 allowed us to trace the migration of skin CD11c+ DCs in vivo. Our data reveal that chronic social stress applied prior to skin sensitization suppresses the migratory capability of epidermal CD11c+ DCs to regional lymph nodes. Using an ex vivo ear skin explant model of skin DC migration we further show that the altered migration is presumably a result of an impaired mobilisation of CD11c+ DCs from the skin.
In chapter 4 we characterize phenotypic changes in T lymphocyte subsets in the peripheral blood of severely traumatized human patients. Posttraumatic stress disorder (PTSD) is associated with an enhanced susceptibility to various somatic diseases although the exact mechanisms linking traumatic stress to subsequent physical health problems have remained elusive. Our results demonstrate that PTSD patients exhibit a profoundly altered composition of the peripheral T cell compartment characterized by a reduction in naive T lymphocytes, and increased proportion of central (TCM) and effector memory (TEM) cells. Furthermore, we show that subjects with PTSD display a substantial reduction of peripheral regulatory T cells (Treg). To a smaller extent, these findings are also observed in trauma-exposed non-PTSD individuals, indicating a cumulative effect of traumatic stress on T cell distribution.
Zusammenfassung
Chronischer Stress kann durch Umwelteinflüsse oder psychologische Faktoren hervorgerufen werden und steht seit langem im Verdacht, die Anfälligkeit und den Verlauf von infektiösen und entzündlichen Erkrankungen zu verstärken. Die Vermittlung des Stress-Reizes zwischen dem zentralen Nervensystem (ZNS) und den periphären Komponenten des Immunsystems wird durch die Freisetzung von Neurotransmittern wie beispielsweise den Catecholaminen und adrenergen Hormonen wie den Glucocorticoiden erreicht. Glucocorticoide als Endprodukte der stress-induzierten neuroendocrinen Antwort und der Hypothalamus- Hypophysen-Nebennierenrinden-Achse (HPA) gehören zu den potentesten anti- inflammatorischen Hormonen des Körpers und dienen zur Kontrolle von Immunantworten beisspielsweise während einer Infektion. Durch chronischen oder wiederholten Stress kann eine systemische Erhöhung der Glucocorticoid-Konzentrationen eintreten und zu einer Unterdrückung verschiedener Immunfunktionen führen und damit die Entwicklung und den Verlauf von Krankheiten negativ beeinflussen. Allerdings sind die genauen Mechanismen, die zu einer Unterdrückung der Immunfunktionen führen bisher nur wenig verstanden. Das Ziel der vorliegenden Arbeit bestand in der Untersuchung der Mechanismen, die zu einer Unterdrückung des Immunsystems und insbesondere der T-Zell-vermittelten Immunantworten durch chronischen Stress führen. In Kapitel 2 und 3 wurde der Einfluss von wiederholtem, chronischen Stress in einem etablierten Mausmodell (dem so genannten
„social disruption stress“ Modell, SDR) untersucht. Die spezifische Hemmung der jeweiligen Rezeptoren erlaubte es, den Einfluß von Glucocorticoiden und Catecholaminen, den bedeutensten Vermittlern der Stressantwort, zu unterscheiden.
Kapitel 2 beschreibt den Einfluss von chronischem, sozialen Stress auf die Virus-spezifische, TCD8+-Zell-vermittelte Antwort nach Infektion mit dem lymphozytären Choriomeningitis- Virus (LCMV). Da bereits bekannt ist, dass sowohl die Dauer wie auch der Zeitpunkt der
3 Stress-Prozedur eine erhebliche Rolle bei der Auswirkung auf die Immunfunktionen spielt, wurde in unserem Modell der Einfluss verschiedener Stress-Protokolle direkt miteinander verglichen. Wir konnten zeigen, dass andauernder sozialer Stress die Bildung von Virus- spezifischen, IFN-γ-sekretierendenen TCD8+-Zellen beeinträchtigt, allerding nur wenn die Stress-Applikation vor der Virusinfektion erfolgt. Ausserdem konnte gezeigt werden, dass im Falle einer verlängerten Stress-Prozedur Glucocorticoide die Virus-induzierte Proliferation von TCD8+ Zellen in der Milz hemmen. Dabei ist die verringerte Proliferation wahrscheinlich auf eine reduzierte Aktivierung der TCD8+-Zellen sowie eine Hemmung ihrer Zytokin- Sekretion zurückzuführen. Die gehemmte Proliferation der TCD8+-Zellen scheint dabei Organ- spezifisch zu sein, da keine Veränderungen in den inguinalen Lymphknoten, im Blut oder periphären Geweben wie der Leber oder Lunge beobachten werden konnte. Ein möglicher Grund hierfür kann in einer veränderten Fähigkeit zur Migration der TCD8+ Zellen liegen, wie in adaptiven TCD8+-Zell-Transfer-Experimenten demonstriert werden konnte.
Kapitel 3 beschreibt den Einfluss von chronischem, sozialem Stress auf das Migrationsverhalten von dendritischen Zellen in der Haut. Epidermale und Dermale dendritische Zellen, insbesondere epidermale Langerhans-Zellen, sind bekannt für ihre Beteiligung an der Initiierung von T-Zell-vermittelten Immunantworten in der Haut. In neueren Studien wurde vermehrt eine Glucocorticoid-vermittelte Beeinflussung der Funktion von dentritischen Zellen untersucht. Um den direkten Einfluss von chronischem Stress auf die Funktionen der dendritischen Zellen der Haut zu untersuchen, haben wir eine durch ein Kontakt-Allergen (den Fluoreszenz-Farbstoff FITC) induzierte Haut-Sensibilisierung durchgeführt, was eine Untersuchung der Migration von dendritischen CD11c+-Zellen in vivo erlaubte. Unsere Ergebnisse zeigen, dass sozialer Stress, wenn vor der Haut-Sensibilisierung appliziert, die Migrationsfähigkeit von epidermalen, dendritischen CD11c+-Zellen zu den regionalen Lymphknoten hemmt. Durch die Verwendung eines ex vivo-Ohrhaut Explantations-Modells zur Untersuchung der Migration dendritischer Zellen der Haut konnten wir weiterhin zeigen, dass die veränderte Migration vorwiegend das Ergebniss einer geminderten Mobilisierung von dendritischen CD11c+-Zellen in der Haut ist.
In Kapitel 4 beschreiben wir Veränderungen in T-Lymphozyten-Populationen im Blut von schwer traumatisierten, humanen Patienten. Die posttraumatische Belastungsstörung (PTBS bzw. PTSD, aus engl. „posttraumatic stress disorder“) ist mit einer erhöhten Anfälligkeit für diverse somatische Erkrankungen assoziert. Allerdings sind Mechanismen, welche die traumatischen Stressbelastungen mit den physischen Gesundheitsproblemen verbinden bisher nur schwer fassbar. Unsere Ergebnisse zeigen, dass PTSD-Patienten eine erhebliche Veränderung der Zusammensetzung der peripheren T-Zell-Population aufweisen, charakterisiert durch eine Reduzierung von naiven T-Lymphozyten und einen erhöhten Anteil von zentralen und Effektor-Gedächtniszellen (TCM und TEM). Desweiteren konnten wir zeigen, dass PTSD-Patienten eine erhebliche Reduzierung von regulatorischen T-Zellen (Treg) aufweisen. Etwas weniger ausgeprägt konnten diese Befunde auch in traumatisierten, nicht- PTSD-Patienten nachgewiesen werden, was auf einen kumulativen Effekt von traumatischen Erlebnissen auf die Veränderungen der T-Zell-Populationen schließen lässt.
Chapter 1
Introduction
5
The immune system in response to stress
Innate and adaptive components of our immune system are evolved to protect the body from invading pathogens like disease-causing bacteria, fungi, viruses and parasites. The immune system is not operating autonomously but influences and is influenced by the central nervous system (CNS) through a complex, interacting network of nerves, hormones, and neuropeptides. This interaction is thought to serve as a mechanism to fine-tune many immune responses, for instance to prevent the body from harmful excessive immune responses. CNS- immune interactions also play an important role during stress responses. The stress response is essential for the organism since it facilitates all necessary changes (physiological and behavioral) that allow the body to cope with threatful situations (“fight or flight-response”).
However, the same mediators that promote these adaptive responses can negatively influence various functions of the innate and adaptive immune system when this responsiveness becomes excessive or inadequate. Such a dysregulation of the immune system that often occurs under chronic or recurrent stress can have significant implications for infectious and inflammatory disease susceptibility and progression.
TCD8+ cells in viral infections
Although in vertebrates the innate immune system controls viruses upon initial encounter, it is the specific adaptive immune response that effectively mediates the clearance of viral pathogens. This is mainly achieved by the activation and expansion of antigen-specific effector TCD8+ lymphocytes that are exquisitely refined in their ability to recognise and lyse infected cells presenting viral antigenic peptides on surface major histocompatibility (MHC) class I molecules (1-2). The generation of cytotoxic TCD8+ cells (CTLs) from naïve TCD8+
precursors in turn depends on the interaction with professional antigen-presenting cells (APCs) that in addition to the MHC I/antigen complex, express high levels of costimulatory molecules required for effective activation of naïve T cells (3). In general, antiviral responses to fast-replicating viruses involve the initial activation of TCD8+ cellsin secondary lymphoid organs, followed by exponential proliferation and differentiation into effector TCD8+ cells with high cytotoxic capacity (day 3-6 post infection). During the subsequent phase of antiviral immune responses (day 6-8) activated cytotoxic TCD8+ cells leave the secondary lymphatic organs and migrate to the site of infection where they specifically kill infected target cells that express their cognate antigen (1). In the late course of a viral infection (day 8-10) when the majority of infected cells are eliminated and viral clearance is achieved, the effector TCD8+
population diminishes rapidly through extensive cell death, while maintaining a pool of long- lived antigen specific memory cells (4).
Dendritic cells
Dendritic cells (DCs) are a family of highly specialized antigen-presenting cells (APCs), most capable of efficiently activating naïve T cells to initiate antigen-specific TCD8+ cell responses (3, 5). Their functional capacity to capture, process and present antigenic peptides on MHC I molecules is a critical property that is modulated during DC maturation process (3, 5). In most
Introduction
6 tissues, DCs are present in a so-called ‘immature’ state, display high endocytic activity but express low levels of MHC and costimulatory molecules and are therefore weak stimulators of naïve T cells (3). Once they have acquired and processed foreign antigens, DCs undergo a number of phenotypic and functional changes resulting in a down-regulation of their phagocytic ability and up-regulation of their antigen-presenting as well as T cell-stimulatory competence (e.g. redistribution of MHC molecules from intracellular endocytic compartments to the DC surface, increase in the surface expression of costimulatory molecules such as CD80 and CD86) (3).
Skin dendritic cell types
Langerhans cells (LC) are a subtype of dendritic cells (DC), residing in an immature state within the epidermis where they comprise between 1-3% of total epidermal cells to form a dense intraepithelial dendritic network (6). Phenotypically, LCs are distinct from conventional DCs in that they express high levels of langerin, a C-type lectin receptor (CD207) that is a potent inducer of the LC-characteristic Birbeck granules (7-8). LCs also constitutively express the epithelial-cell adhesion molecule EpCAM as well as E-cadherin (CDH1), a homotypic adhesion molecule that anchors LCs to neighboring keratinocytes (9). CD205 (also known as DEC205) is another molecule that is constitutively expressed by LCs and is implicated in antigen capture and processing (10).
While LCs represent the most prominent DC population in the epidermis, a number of other DC subsets, including small numbers of plasmacytoid DCs, dermal myeloid DCs (dDC), and the recently described dermal langerin+ DCs (langerin+ dDC) populate the dermal layer of the skin (11-13). Phenotypic characterization of these latter subsets has shown that they are distinct from epidermal LCs, in that they express low levels of the epithelial-cell adhesion molecule EpCAM (14). The two dermal DC populations can be further distinguished phenotypically by their differential expression of CD11b and CD103 (αE-integrin), both expressed at high levels on LC. Taken together, the existence of at least three major subpopulations of DCs in the skin has been established: epidermal langerin+ LCs (EpCAMhigh CD11b+ CD103-), dermal langerin- DCs (EpCAM- CD11bhigh CD103-), and dermal langerin+ DCs (EpCAM- CD11blow CD103+) (11-13).
Langerhans cell migration
Topical exposure to contact allergens and skin irritants initiates the activation and subsequent maturation of LCs in the epidermis. The maturation of LCs is accompanied by phenotypic and functional alterations, including increased expression of costimulatory molecules such as CD83 and CD86, and MHC II antigens enhancing their respective antigen presenting capability (15). On the other hand, altered expression of various adhesion molecules and chemokine receptors enable their mobilisation from the epidermis and homing to skin- draining lymph nodes (LNs) (16). The ability of LCs to migrate from the site of Ag encounter to the area of T cell priming is fundamental for the initiation of cutaneous immune responses
7 (17-19). Consequently, epidermal mobilisation of LCs and their subsequent LN migration are integral processes, orchestrated by a variety of cutaneous cytokines and chemokines.
Langerhans cell migration: role of cytokines
IL-1β and TNF-α are thought to be the most important cytokines for the mobilisation and migration of LC towards the skin-draining LNs (16). Intradermal injection of mice with each cytokine results in a rapid egress of LCs from the epidermis and their subsequent accumulation in the draining LNs (20-22). Moreover, systemic administration of either TNF- α- or IL-1β-neutralizing antibodies prior to skin sensitization causes an almost complete inhibition of allergen-induced LC accumulation in the LN (23-24). Contact sensitization and skin irritation is known to induce an up-regulation of IL-1β and the de novo secretion of TNF- α (25). The current opinion is that topical exposure to allergens induces the secretion of IL-1β by LCs, which in turn stimulates de novo production of TNF-α by keratinocytes (26-27). This view is supported by the fact that the speed of migration induced by IL-1β is slower than that observed with TNF-α (20, 28), as is the observation that IL-1β-induced LC migration can be inhibited by neutralizing anti-TNF-α antibodies (24). Furthermore, IL-1β is believed to provide a second mandatory signal for migration, independent of its role to induce TNF-α secretion. This idea is supported by the observation that TNF-α-induced LC migration is repressed by treatment with neutralizing anti-IL-β antibodies (24).
The mechanism by which IL-1β and TNF-α prompt LC migration include altered expression of adhesion molecules and chemokine receptors on LCs along with a differential responsiveness to the relevant chemotactic ligands in the local microenvironment, which pave the way for LCs to traffic to downstream LNs (16). TNF-α-driven processes include the down-regulation of CCR6 (26-27) and E-cadherin (29) on LCs, allowing their detachment from keratinocytes. Once these mobilisation signals have triggered LC detachment, the cells must migrate through the extracellular matrix and traverse a basement membrane before gaining access to the dermal afferent lymphatics. TNF-α induced up-regulation and activation of adhesion molecules, including α6β1 (30) and CD44 (31) which are important for LC interaction with extracellular matrix proteins such as epidermal laminin are suggested to be involved in this process.
Other cytokines induced by contact allergens in the epidermis, such as IL-10 have been reported to repress the maturation and mobilisation of LCs by inhibiting the production of IL- 1β and TNF-α by cells (32-33) and may therefore facilitate LC retention in the epidermis, providing a counterbalance for controlling cutaneous immune responses and inflammation (32-34).
Introduction
8 Langerhans cell migration: role of chemokines and chemokine receptors
In addition to acquiring the capacity to detach from their epidermal environment, maturing LCs must also alter their chemokine receptor repertoire to one endowing their movement towards skin draining LNs. Immature LCs express a numberof chemokine receptors (CCR1, CCR2, CCR5, CCR6 and CXCR1) by which they are attracted to sites of inflammation where they capture and process antigens (26). Upon maturation, LCs lose their responsiveness to most of theinflammatory chemokines through receptor down-regulation butup-regulate other G protein-coupled chemokine receptors, such as CCR4 and CCR7 (35).
By up-regulation of CCR7, LCs acquire responsiveness to the constitutively expressed and selective ligands SLC (secondary lymphoid organ chemokine or CCL21/6C-kine) and ELC (Epstein-Barr virus-inducedmolecule 1 (EBI-1) ligand chemokine or CCL19/MIP-3β) that allow their homing to skin-draining LNs.The important role of CCR7 in the migration of LCs to the draining LNs is well established. Evidence has been drawn from the observation that LCs fail to migrate to LNs in CCR7-deficient (36) and plt (paucity of lymph node T cells) mutant mice, which lack both CCR7 chemotactic ligands SLC/CCL21 and ELC/CCL19 (37).
Although plt mice exhibit a substantial reduction of LC migration to the skin-draining LNs after allergen sensitization, their emigration from the epidermis is not compromised (36-38).
Furthermore, antagonizing the CCR7 ligands SLC/CCL21 and ELC/CCL19 in WT mice does not abrogate migration from the epidermis to the dermis (35). Interestingly, CCR7 ligands are differentially expressed in mice, with both CCL21 isoforms, CCL21-Ser and CCL21-Leu expressed predominantly in the afferent lymphatics, whereas CCL19 and CCL21-Ser localized to the LN paracortex (39). Therefore, it seems reasonable to assume that CCR7 is not only involved in guiding LCs into the dermal lymphatic vessels but might also control LC access deeper into the LN cortex (16). Taken together, these findings have lead to the following conclusions: First, CCR7 regulates the entry of LCs into the dermal lymphatic vessels and their migration to the LN paracortex rather than their mobilisation from the epidermis. Second, CCR7 mobilisation of LCs from the epidermis and the subsequent migration to the dermis occurs in a CCR7-independent manner.
Recent evidence suggests that CXCR4 and its ligand CXCL12 are crucial for LC migration from the epidermis to the dermis (35, 40). Consistent with this idea, migration of LCs from the epidermis to the dermis is abrogated by CXCR4 and CXCL12-blocking antibodies (35).
Moreover, epicutaneous sensitization increases CXCR4 expression on LCs, whereas the CXCR4 ligand, CXCL12, is concomitantly up-regulated in dermal lymphatics (40). Based on these findings, a two-step model for LC migration has been proposed: in a first step LCs exit the epidermis and migrate to the dermis, a process that is CXCR4-CXCL12 dependent. In a second CCR7-regulated step, LCs gain access to the dermal lymphatic vessels and migrate towards the LN (41) (Figure 1).
9 Figure 1: Migration of epidermal Langerhans cells and dermal DCs via afferent lymphatics to draining LNs
The scheme illustrates a proposed model for the migration of epidermal LC via afferent lymphatics en route to the LN. The migratory cascade can be divided into discrete steps, initiated by an inflammatory mobilizing signal (e.g. by epicutaneous contact allergen application) that induces the secretion of IL-1β by LCs, which in turn stimulates TNF-α production by keratinocytes resulting (1) in the detachment of LC from epidermal keratinocytes, (2) interstitial trafficking from the epidermis via the basement membrane to the dermis, (3) transit via intercellular spaces between afferent lymphatic endothelium cells, and (4) migration through the afferent lymph vessels towards draining LNs. CXCR4-CXCL12 interaction controls LC migration from the epidermis to the dermis, while CCR7-CCL21 triggers the recruitment of LCs and both dermal DC populations towards lymphatic vessels. Furthermore, the transit of each DC population via the afferent lymphatics is mediated by a CCL19/21 gradient.
Antigen processing and presentation by dendritic cells
Virus-derived peptides presented by DCs to TCD8+ cells via MHC I molecules result from either cytoplasmatically synthesized proteins (i.e. by viral protein synthesis in the cytoplasm) or endocytosed exogenous proteins that are released from the endosomal compartment into the cytoplasm (3, 42). These peptides are marked in the cytosol for degradation via ubiquitin conjugation and are subsequently digested by the proteasome. The proteasome is a multi- subunit ATP-dependent protease that plays a major role in the degradation of foreign antigens (e.g. viral, bacterial, or fungal), but is also important for normal turnover of cell proteins in the cytosol (43).
Only a small percentage of the peptides released after proteasomal digestion in the cytosol has an optimal binding length for MHC Imolecules and therefore further processing by cytosolic aminopeptidases such as the leucine aminopeptidases (LAPs) (44), puromycin-sensitive aminopeptidases (PSAs) and the bleomycin hydrolase (BH) takes place (45). The generated peptides are then taken up in the cytosol by an endoplasmic reticulum-resident heterodimeric ATP-dependent peptide transporter associated with antigen processing (TAP) and are
Introduction
10 translocated into the lumen of the endoplasmic reticulum (ER). The TAP transporter constitutes only one subunit of the macromolecular peptide-loading complex composed of TAP1/2, tapasin, and the assisting proteins calreticulin and ERp57. Tapasin acts as a bridging molecule linking TAP and MHC I complexes, but also functions by stabilizing the TAP complex, retaining unloaded MHC I molecules in the ER, and controlling the quality of MHC I-bound peptides (46). Some precursor peptides are further trimmed in the ERby the IFN-γ- inducible aminopeptidase ERAP or ERAAP (47-48). The peptides are then loaded onto newly synthesized MHC I/β2-microglobulin dimers and are rapidly transported via the Golgi complex to the cell surface, where they are presented to the T cell receptor (TCR) on TCD8+
cells (Figure 2).
Compared to the numerous peptides encoded by viral pathogens only a small subset is able to generate a specific TCD8+ cell response, with various factors determining whether a specific antigenic epitope is immunodominant or subdominant, including the efficiency of antigen processing, the binding affinity of the peptide for the MHC I molecule, and the magnitude of naïve TCD8+ cells responding to its cognate MHC/peptide complex (49).
In the presence of the inflammatory cytokines interferon gamma(IFN-γ) and tumor necrosis factor alpha (TNF-α) the proteasomal activity and antigenic peptide production is altered, resulting in a gradual replacement of three constitutive catalytic β-subunits β1(δ), β2(MC14), and β5(MB1) of the 20S proteasome by their homologous subunits β1i (LMP2), β2i (MECL1) and β5i (LMP7) to form so-called ‘immunoproteasomes’ (50). As a consequence, cleavage preferences of the proteasome are markedly changed (51-52), resulting in an altered spectrum of epitopes presented to TCD8+ cells. With regard to one of the most studied viral model systems, the murine lymphocytic choriomeningitis virus (LCMV) infection, formation of the immunoproteasome is well established to induce an altered TCD8+ cell response, favoring the generation of TCD8+ cells specific for the LCMV glycoprotein-derived epitope GP33-41, whereas for instance GP276-286 specific TCD8+ cells are less efficiently generated (53-54).
11 Figure 2: Scheme of the classical MHC class I-restricted antigen processing and presentation by DCs Cytosolic viral proteins are marked for degradation by ubiquitination and are subsequently degraded into peptide fragments by the proteasome. The peptides, partially further processed by cytosolic aminopeptidases, are then transported via the TAP into the lumen of the endoplasmatic reticulum (ER) where they can bind to nascent MHC class I molecules associated with β2-microglobulin (β2m). Assisting proteins of the peptide-loading complex like tapasin, calreticulin and ERp57 increase the efficiency of peptide loading. The complex of MHC I heavy chain, β2m and peptide fragment then leaves the ER and is transported via the Golgi to the cell surface, where it can be recognized by the TCR of TCD8+ cells. The combinatory signal of cognate MHC/peptide complex and costimulatory molecules provided primarily by B7 molecules induces the stimulation of naive TCD8+ which become activated and initiate a program of clonal expansion and effector differentiation. Fully activated cytotoxic effector TCD8+ (CTL) cells mediate their function by secretion of effector molecules like perforin and granzymes to induce apoptosis in their target cells.
DC signals for clonal expansion and differentiation of naïve T cells Role of TCR mediated signaling – signal 1
The activation of naive T cells is triggered through engagement of the TCR/CD3 complex and its cognate MHC/peptide complexes on DCs. One of the most immediate consequences of T cell receptor (TCR) stimulation is the phosphorylation of the immunoreceptor tyrosine-based activationmotifs (ITAM) within the associated cytosolic TCR-ζ and CD3-γ,-δ, -ε chains by the src family protein kinases p56lck and p59fyn (55). While p59fyn is weakly associated with the cyoplasmatic domain of the ζ and CD3 chains, p56lck is associated with the cytoplasmatic domain of the co-receptor molecules CD4 or CD8. Binding of the MHC/peptide complex to its cognate TCR complex and co-receptors results in the clustering of the latter and helps to stimulate signal transduction by bringing p56lck together with ITAMS in the cytoplasmatic domain of the TCR complex. The lymphocyte common antigen CD45, a receptor-linked protein tyrosine phosphatase plays a crucial role for supporting signal transduction from
Introduction
12 TCRs by catalysing the tyrosine dephosphorylation of the positive regulatory autophosphorylation sites of p56lck and p59fyn, thereby activating the protein kinases (56-57).
TCR-ζ chain phosphorylation by p56lck and p59fyn then recruits the protein kinase ZAP-70 to the receptor complex. ZAP-70 becomes activated and in turn phosphorylates the scaffold proteins LAT (linker of activated T cells) and SLP-76. Phospholipase C-γ (PCL-γ) is one of the key signalling molecules recruited by the phosphorylation of LAT and SLP. Activated PCL-γ initiates different key downstream signalling pathways including 1) protein kinase C (PKC) which subsequently activates the transcription factor NFκB; 2) a Ras/MAP kinase cascade and downstream induction of the transcription factor AP-1, and 3) the phosphatase calcineurin pathway that activates the nuclear factor of activated T cells (NFAT) (58). All three transcription factors (NFκB, AP-1, and NFAT) can bind to distinct cis-regulatory elements in the promotor region of the interleukin-2 (IL-2) gene and are essential to activate its transcription (59-60). IL-2 is one of two major growth hormones for T cells and it sustains proliferation of effector TCD8+ cells in an autocrine manner when produced by TCD8+ cells (61).
IL-2 is also provided by activated TCD4+ cells to support clonal expansion and acquisition of effector function by the TCD8+ cells (27-29).
Activation of PKC through stimulation of the TCR complex induces expression of CD69 (Leu-23) via NFκB activation (62). CD69 is one of the earliest inducible cell surface molecules newly synthesized and expressed during T cell activation. The induction of CD69 results in increased transcription of IL-2, TNF-α, INF-γ, and up-regulation of CD25 expression (62-65), the IL-2Rα chain that enables high-affinity binding of IL-2.
Costimulation by the B7 family – signal 2
Stimulation of the TCR complex alone is not sufficient for induction of T cell activation, proliferation, and survival but is controlled by several factors that are provided by the presence of costimulatory signals (66). In the earliest stages of TCD8+ activation this costimulatory signalling is provided through B7.1 (CD80) and B7.2 (CD86) molecules expressed on mature DCs that bind the transmembrane glycoprotein CD28 which is constitutively expressed on T cells (67). The importance for costimulation is emphasized by findings that stimulation of TCD4+ Th1 or TCD8+ cells in the absence of CD28 induces a state of anergy. Anergic T cells are characterized by reduced cytokine synthesis (including IL-2), a lack of proliferation, and failure to differentiate into effector cells when reencountered with their cognate antigen (68-69).
The biological consequences of B7/CD28 costimulation are numerous and include control of the T cell cycle, survival and differentiation, as well as amplification of the membrane- proximal signalling generated by TCR ligation(70). B7/CD28 costimulation in the presence of a TCR signal results in increased transcription of the IL-2 gene and up-regulation of CD25 expression (71-73). TCD8+ cells from CD28-deficient mice exhibit an impaired proliferative response and IL-2 secretion in response to mitogen stimulation, which is only partially restored by the addition of exogenous IL-2 (74). B7/CD28 signalling further promotes T cell
13 division by mediating the subsequent progression of T cells into the S phase via both IL-2- dependent and IL-2-independent regulatory mechanisms (75). In addition, CD28 can promote T cell survival by enhancing the nuclear translocation of NF-κB, which positively effects the expression of anti-apoptotic genes including Bcl-xl (76).
Role of other correceptors
In addition, inducible costimulatory molecules including CD40, 4-1BB, OX40, inducible costimulator (ICOS), and LFA-1 prolong or sustain activation of T cells (57). CD40/CD40L- dependent signaling for instance enhances B7 expression on DCs (11–13), thereby increasing the level of costimulation available to the T cells. OX40 (CD134) and 4-1BB (CD137) members of the TNFR family provide costimulation upon engagement with their ligands OX40L and 4-1BBL, and the latter preferentially induces TCD8+ proliferation and production of IFN-γ (77).
Other counter-receptors such as cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed death-1 (PD-1) provide inhibitory signals, thereby limiting the expansion and activation of T cells. CTLA-4 is engaged by both B7-1 and B7-2 ligands and its function involves negative signalling (78-79) and competitive antagonism of B7/CD28-mediated costimulation (80), resulting in a decrease in proliferationand IL-2 production (81).
Role of cytokines - signal 3
Several studies support that the strength and duration of TCR and costimulatory signalling determines the extent of subsequent TCD8+ clonal expansion (82-85). This concept is supported by the finding that T cell proliferation can be induced within 6 hours of stimulation if naïve T cells are stimulated with high dose of antigen in the presence of co-stimulation, but requires as long as 40 hours if stimulation occurs in the presence of low doses of antigen and costimulation (86). However, after an initial single period of TCR/CD28 stimulation, it is assumed that TCD8+ cells initiate a program for their autonomous clonal expansion and development into functional effector TCD8+ cells without a requirement for continued signaling through the Ag receptor (87). This view is emphasized by the observation that in vitro- activated TCD8+ cells undergo further short-term expansion and acquire full effector function when these cells are transferred in vivo into an antigen-free environment (88).
Inflammatory cytokines such as IL-12 or type I IFN (IFN-α or IFN-β) produced by macrophages and/or activated DCs respectively synergize with signals from the TCR and costimulatory receptors, thereby critically influencing the continued TCD8+ cell division and even more important their development into fully differentiated effector cells (83, 88-92). In the absence of these additional signals (“signal 3 cytokine”), TCD8+ clonal expansion can be compromised due to poor survival of the expanding cells that also do not acquire cytolytic activity or the ability to produce IFN-γ. For example, type I IFNs act directly on TCD8+ cells to allow clonal expansion in response to LCMV infection. This was demonstrated by examining the responses of adoptively transferred TCR transgenic TCD8+ cells specific to the GP33-41
Introduction
14 epitope of LCMV. In comparison to wildtype TCD8+ cells, clonal expansion of cells that were deficient for the type I IFN receptor was reduced by greater than 99%, and this outcome was shown to be due to poor survival instead of a proliferation defect (93-94). Other investigators have highlighted the role of IL-2 for effector differentiation in the later phase of clonal expansion. For instance, expansion of IL-2Rα-/- TCD8+ cells appears to be normal in secondary lymphoid organs after viral stimulation while being impaired in peripheral, non-lymphoid tissues (61).
Effector function of TCD8+ cells
Fully differentiated effector TCD8+ cells control virus infections by secreting effector cytokines such as TNF-α (95) and in particular IFN-γ (96) andby using either or both of two cytolytic pathways (97-98).The first, a granule exocytosis pathway, is mediated by perforin and two serine proteases (granzyme A and granzyme B) that act by initiating a caspase cascade in the target cell, leading to apoptotic death (99-100). Perforin mainly exerts its function of causing cell lysis by penetrating the target cell membrane (101). Recent work however has suggested that perforin rather functions by enabling the granzymes to escape from endosomes into the cytosol of the target cell (102-103). The second is a Fas-mediated apoptotic pathway that functions by binding of the Fas ligand (FasL)on TCD8+ cells to the Fas-receptor on target cells.
Perforin-dependent cytotoxicity mediated by effector TCD8+ cells has been shown to be crucial for clearance of non-cytopathic viruses such as LCMV (104-105), while the Fas-dependent pathway seems not essentialfor clearance of this virus (106).
The precise mechanisms howIFN-γ exerts its antiviral effects are not fully understood. It is thought that IFN-γ acts mainly by restricting intracellular viral replication (107), thereby reducing the rate with which a virus establishes productive infection in new host cells. A role for TCD8+ cell-derived IFN-γ as an essential effector mechanism in the control of acuteLCMV infection has been confirmed by the observation that viral clearance is delayed in IFN-γ receptor-deficient mice (62). Similarly, treatment with neutralizing anti-IFN-γ mAb has been demonstrated to result in reduced virus elimination from the spleen and liver, which is accompanied by a decreased generation of effector TCD8+ cells (108-109).
Migration of T lymphocytes
Under steady state non-inflammatory conditions naïve T cells constantly circulate through the bloodstream, the secondary lymphoid organs including spleen, mesenteric and peripheral lymph nodes (LNs), and then subsequently return to the bloodstream. This process is directed by various adhesion molecules and chemokine receptors (110). The circulatory path continues until activation and differentiation into effector TCD8+ cells in secondary lymphoid tissues.
Effector TCD8+ cells then acquire the capacity to traffic to inflammatory peripheral tissues.
15 Moving into secondary lymphoid organs
Role of adhesion molecules
Naïve T cells leave the blood and enter peripheral lymph nodes via specialized postcapillary vessels called high endothelial venules (HEV) in a complex process that involves rolling, adhesion and extravasation through the endothelium. The interactions of the lymphocyte adhesion receptor L-selectin (CD62L) and the β2 integrin LFA-1 (CD11a/CD18) with their respective ligands, the vascular adressins (e.g. GlyCAM-1 and CD34) and intercellular adhesion molecule ICAM-1/-2 play a key role in this process (110). In contrast to LNs, HEV- like vessels are not present in the spleen. T cells access the splenic red pulp directly from the blood while subsequently traversing the boundary marginal sinus and entering the T cell zones (the periarteriolar lymphatic sheath - PALS) of the white pulp in a CD62L-independent manner (111-113).
Role of the chemokine receptor CCR7
The chemokine receptor CCR7, expressed at high levels on naïve T cells and activated mature DCs plays a key role in the recruitment of T cells into lymph nodes and the splenic white pulp (114-115). The secondary lymphoid organ chemokine SLC/CCL21 is constitutively expressed by cells of the HEVs and at lower levels by stromal cells within T cell zones of the spleen, lymph nodes, and Peyer's patches (114, 116-117). Expression of ELC/CCL19, the second ligand for CCR7 is present in cells throughout the T cell zones ofsecondary lymphoid tissues but does not appear to be produced by HEVs (118). The importance of CCR7 and its ligands in vivo has been demonstrated inmice lacking expression of CCR7 and “paucity of lymph node T cell” (plt/plt) mice that lack the SLC/CCL21 and ELC/CCL19 genes expressed in lymphoid organs. Both animal models exhibit greatly reduced migration of T cells into LNs and have further shown to display an abnormal distribution in secondary lymphoid organs (36, 119). For instance, T cells do not accumulate in the white pulp of the spleen where T cells are normally located, instead they accumulate around the sinuses in the red pulp where red blood cells are usually found.
Once present in T cell zones of secondary lymphoid organs, naïve T cells continue to migrate through this area, which enables them to screen DCs for their cognate antigen. The expression of ELC/CCL19 by activated DCs within the T cell zone is thought to enhance T-cell attraction,thereby providing an optimal co-localization of antigen-presenting DCs and naïve T cells resulting in a more efficient T cell priming and effector generation (120).
Moving out – Role of chemokine receptor CCR7 and adhesion molecules
Once fully activated TCD8+ cellslose their attraction for the lymph node and splenic T cell compartments by simultaneous down-regulation of secondary lymphoid organ homing receptors such as CD62L and CCR7 (121). The up-regulation of a new range of surface molecules such as CD44, LFA-1 and/or α4β1 integrin enables them to migrate to sites of inflammation in non-lymphoid tissues like the liver and lung. These traffic signals that direct
Introduction
16 effector TCD8+ to peripheral tissues are highly complex and are implicated to be organ-specific (121).
The distinct migration pattern of naïve and effector TCD8+ cells in correlation to their CCR7 expression profile has been confirmed in adaptive transfer experiments using TCR-transgenic TCD8+ cells from mice specific for the GP33 epitope of LCMV. Only naïve (CCR7+), but neither memory nor effector TCD8+ (CCR7- CD62low CD44high)cells homed to LNs of non- infected recipient mice, while similar numbers of naïve, memory, and effector cells entered the spleen, although with distinct splenic locations: naïve cells predominantly localised in the PALS, while effector cells were completely excluded from these areas but instead accumulated in the red pulp (122).
Diversity of the memory T cell pool
When TCD8+ responses have occurred and the antigen is cleared from the system, most of the Ag-specific cytotoxic TCD8+ cells (90-95%) undergo programmed cell death and only a small fraction (5-10%) survives and differentiates into long-lived, antigen-specific memory TCD8+
cells. These memory cells rapidly re-acquire effector functions without needing a third signal (90, 123).
Memory T cells are heterogeneous and can be divided in terms of their surface phenotype, development, effector function, and trafficking properties (124). The existence of at least three different subpopulations of memory cells has been established: central memory T cells (TCM), and two distinct types of effector memory cells that are distinct from central memory T cells by expressing low levels of the lymph node homing receptor CCR7. These two populations of effector memory cells can further be distinguished by their differential surface expression of CD45RA, the high molecular weight isoform of the receptor-type protein tyrosine phosphatase CD45 (124).
Central memory cells (TCM) are CD45RO+ memory cells that constitutively express CCR7 and CD62L; both receptors are required for their extravasation through HEVs and the migration to T cell areas of secondary lymphoid organs. They recirculate between the blood and the lymphoid compartment and are thought to provide a poolof antigen-experienced cells with a high proliferative capacity but a lower activation threshold than naïve cells, thus allowingfor a more rapid generation of effector cells during recallresponses (125).Incontrast, effector memory T (TEM) cells have lost the constitutive expression of CCR7 but have acquired chemokine receptors and adhesion molecules, allowing their homing to peripheral tissues and to sites of inflammation. TEM cells are thought to enable a recall response in the tissue, as these cells survey non-lymphoid organs in search ofcognate antigens and, while able to produce IFN-γ upon antigen recognition, are only moderately cytotoxic (125). The peripheral TEMRA population (124) is most prominent within TCD8+ cells, while within TCD4+
cells only few TEMRA cells (1-2% of TCD4+ cells) can be detect. Like the TEM population, TEMRA
cells have lost constitutive expression of CCR7, but re-express CD45RA. Functionally they
17 share properties with the TEM population in that they display an immediate effector function and low proliferative capacity. Compared to TEM cells they exhibit an even lower expansion potential and higher expression of perforin and are therefore considered to be “terminally differentiated” effector memory cells (124).
Lineage relationship between naive, effector, and memory T cells
Longitudinal studies performed in mice have suggested a linear differentiation pathway for T cell development, whereby memory T cells are direct offspring of effector cells (126).In light of this opinion, a linear differentiation pathway with TNaïve Æ TEffector Æ TEM Æ TCM
differentiation has been demonstrated following acute LCMV infection in mice (127-128).
This model proposes that memory T cell development does not occur until the antigen is removed or greatly decreased in concentration. These results are in contrast to the finding of a persistence and stability of both TEM and TCM subsets in humans (129).
Figure 3: Schematic model of T cell differentiation according to the “progressive differentiation model”
According to the model of progressive T cell differentiation, the strength and duration of antigenic stimulation as well as the type and amount of cytokines offered to naïve T cells during their priming phase will either favour their differentiation into short-lived effector T cells (TEff) or into cells with a memory phenotype that are devoid of immediate effector function - with strong signals (as occurs during the early stage of immune responses) turning differentiation towards (TEff) and weak signals (late stages of immune responses) imprinting central memory T cells (TCM) (dashed arrows indicate potential contributions of cytokines). After elimination of their cognate antigen some of the effector cells (TEff) will persist as effector memory cells (TEM). Thus, both memory cell types (TCM and TEM) are maintained in the memory pool, with TEM mediating immediate protection in non-lymphoid tissues, and TCM cells mediating reactive memory and home to T cell areas of secondary lymphoid organs.
In humans and in some settings of viral infection in mouse models, a “progressive differentiation model” has been proposed in which naïve T cells can directly develop a memory stage without traversing an effector state (85, 124, 130). According to this model the
Introduction
18 differentiation depends on the applied signal strength and duration of the stimulation during the priming period (Figure 3). Short duration of antigenic stimulation is thought to favour the development of an intermediate level of differentiation that will persist as TCM cells, whereas a longer duration of stimulation favours the differentiation into TEffector cells (124). Next, at the end of the immune response, intermediates give rise to TCM cells, whereas TEM arise from fully differentiated TEffector cells. This model postulates that memory progenitor cells are already detectable early during immune responses concomitant with the development of an effector T cell pool and that TCM cells can further differentiateinto TEM cells following Ag stimulation and into TEMRA in responseto homeostatic cytokines (131).
Lymphocytic Choriomeningitis Virus (LCMV)
The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a prominent model to study immunological mechanisms of both acute and persistent viral infection. The natural host of the noncytopathic virus is the mouse. LCMV is an enveloped virus with a bisegmented negative-strand (NS) RNA genome. The viral genome encodes four proteins on two ambisense RNA sequences, the L (long) and S (short) RNA segments which have an approximate size of 7.2 and 3.4 kb, respectively (132-133). The S RNA encodes the 63 kDa nucleoprotein (NP) and the 75 kDa glycoprotein precursor (134). The glycoprotein precursor (GPC) is further processed to a 6 kDa signal peptide and the two glycoproteins GP-1 (40 to 46 kDa) and GP-2 (35 kDa), which build the spikes on the virion envelope as trimer or tetramer- structures (135). Host cell infection involves the interaction with the α-dystroglycan receptor (136) and internalisation of the virions within vesicles, followed by the GP-mediated fusion with cellular membranes and delivery of the nucleocapsids into the cytoplasm. The L RNA segment codes for the 200 kDa virus-specific RNA polymerase (L) and an 11 kDa RING finger protein (Z) (133).
Infection of C57BL/6 (H-2b) mice with LCMV induces a strong and protective cytotoxic T cell (CTL) response. In C57BL/6 mice, this response is strongly dominated by CTLs specific for the GP-derived epitopes GP33-41/Db and GP34-41/Kb, the NP-derived NP396-404/Db epitope (137), and the RNA polymerase (L)-derived epitope L455-463 (138). In addition the generation of CTLs against the subdominant epitopes GP276-286/Db, GP92-101/Db, GP118- 125/Kb (137) and the recently identified GP70-77, NP166-175, NP235-243 (139), GP166- 173, GP221-228, GP365-372 and GP44-52 (138) have been characterized.
Intraperitoneal (i.p.) or intravenous (i.v.) infection of adult immunocompetent mice with low doses of LCMV-WE (200pfu i.v.) results in a well-characterized acute infection during which the virus replicates systemically in tissues including the spleen, leading to a peak virus titer on day 4 after infection. Both type I (α and β) IFNs as well as IFN-γ are induced early after infection and play a role in controlling viral replication. IFN-α and IFN-β secretion occurs in close correlation with the activation of NK-cells. However, several lines of evidence indicate that NK cells are not capable of controlling the virus infection in vivo (140-141). Experiments with CD4-deficient or CD4-depleted mice further demonstrated that T help is not important
19 for the LCMV-specific CTL response in the acute phase (142-143). Virus-specific antibody production occurs early after infection, whereas neutralising antibodies can be detected 20 to 60 days after infection and are important for a long-term elimination of the virus (144).
LCMV-specific CTL response peaks between day 7 to day 9 after infection, leading to effective elimination of virus infected cells (145). The indispensable role of CD11c+ DCs duringTCD8+ cell priming is well established and very limited DC numbers (as few as 1000) are sufficient to generate potent CTL activation (146). Moreover, conventional CD11chigh CD8+ splenic DCs(sDC) are thought to be the most important DC subpopulation for TCD8+
cell priming andhave been shown to efficiently present Ag to and stimulate the proliferation of naive LCMV-specificTCD8+ cells (147).
Stress and immunity
The pioneering experimental report on an interaction between psychological stress and the immune system was published by Selye in 1936, showing that chronic stress results in an atrophy of the thymus (148). Since then, numerous epidemiological studies have revealed that severe psychological stress has strong suppressive effects on the immune system (149). Such suppression of the immune system has significant implications on disease susceptibility and progression. Investigations in humans have revealed that chronic stress contributes to many illnesses including cardiovascular diseases and cancer (150-151). Moreover, substantial evidence has linked chronic or recurrent exposure to stress with exacerbation of inflammatory and autoimmunity diseases such asthma, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease and psoriasis (152-153) and an increased susceptibility to infectious diseases. The relationship between psychological stress and a higher vulnerability to infectious diseases has also been confirmed experimentally by laboratory stressors demonstrating an impaired responsiveness to Hepatitis B (154-157) and influenza virus vaccination (158-159). In addition, it was shown that human volunteers who were inoculated with five different strains of respiratory viruses showed a dose-dependent relationship between stress and clinical symptoms after infection (160).
Interaction of the immune system and the brain - the HPA and SNS axis
The hypothalamus-pituitary-adrenal (HPA) axis and the sympathetic nervous system (SNS) are the two major pathways by which the immune system is modulated during psychological stress (Figure 4).
The sympathetic nervous system (SNS)
The sympathetic nervous system, a major component of the autonomic nervous system, is a fast-acting response to stress that can be detected within seconds after a stress stimulus. The SNS exerts its function by the release of the sympathetic neurotransmitters, the catecholamines. The SNS originates in the central nervous system (CNS) (in nuclei within the brain stem and spinal cord) and gives rise to preganglionic efferent fibres that leave the CNS through the thoracolumbar region of the spinal cord (161). Most of the sympathetic
Introduction
20 preganglionic fibres travel to ganglia located in the paravertebral chains, where they synapse with postganglionic neurons. From there, the long postganglionic neurons extend across most of the body and their nerve terminals innervate nearly every organ in the body including primary and secondary lymphoid organs (162-163). In general, T cell zones, macrophages and plasma cells are richly innervated, while follicular zones of B cells are poorly innervated (162). These postganglionic sympathetic neurons are noradrenergic fibres, meaning that they act by locally releasing norepinephrine (NE).
Additionally, preganglionic sympathetic fibres that end in the adrenal medulla secrete acetylcholine, which activates the secretion of epinephrine (adrenaline) and norepinephrine (NE, noradrenaline) directly into the blood stream, where they exert their action as circulating hormones (161). The adrenal medulla, unlike the postganglionic sympathetic nerve terminals, releases mainly epinephrine, and to a much lesser extend NE. This response is also known as the sympathetic adreno-medullary response of the SNS. Thus, the principal end products of the SNS are catecholamines (NE and epinephrine) with norepinephrine acting both as a neurotransmitter when released via nerve fibres and as a hormone when it is released by the adrenal medulla into the blood along with adrenalin (161).
The hypothalamus-pituitary-adrenal (HPA) axis
The activation of the hypothalamus-pituitary-adrenal (HPA) axis takes place somewhat slower (usually within 3-5 min of stress onset). The main components of the HPA axis are the paraventricular nucleus (PVN) in the hypothalamus, the anterior pituitary gland located at the base of the brain, and the adrenal glands (164). Upon activation of the HPA axis corticotrophin-releasing hormone (CRH) and arginine-vasopressin (AVP) are secreted from the PVN of the hypothalamus into the hypophyseal portal blood, which in turn stimulate the expression of adrenocorticotropin hormone (ACTH) in the anterior pituitary gland. CRH seems to play a permissive role in ACTH secretion, whereas AVP has synergistic or additive effects, but very little ACTH secretory activity on its own. ACTH then circulates in the bloodstream where it acts on the adrenal cortex to induce the expression and release of adrenal steroid hormones, in particular glucocorticoids (GCs) into the blood (for an overview see (165)). Glucocorticoids (cortisol in humans and most mammals, corticosterone in rats and mice) represent the final effector molecules of the HPA axis that mediate their function primarily by acting directly on immune cells, which they access via the blood.
In addition, GCs play an important role in regulating the activity of the HPA axis under basal and stress conditions, by exerting a negative feedback control directly on the pituitary and also on the synthesis and secretion of CRH and AVP. For example GCs potently inhibit pituitary ACTH secretion and down-regulate the action of CRH through binding to their receptors (166). Moreover the secretion and effects of CRH and AVP are influenced by neurotransmitters in the hypothalamus.
21 Figure 4: Signalling between the central nervous system (CNS) and the immune system through the HPA axis and the SNS
The hypothalamus-pituitary-adrenal (HPA) axis exerts most of its influence systemically through a release of glucocorticoid hormones (GC). The first part of this axis, the hypothalamus, is located in the forebrain and neuroendocrine cells in the hypothalamus release corticotropin- releasing hormone (CRH) that stimulates the release of adrenocorticotropic hormone (ACTH) from the anterior pituitary. The adrenal cortex responds to ACTH by releasing glucocorticoid hormones into the circulation. The sympathetic nervous system (SNS) transmits sympathetic information to peripheral targets by releasing norepinephrine (NE) from noradrenergic nerve terminals that end in all primary and secondary lymphoid organs, and/or systemically by releasing epinephrine (along with some NE) from the adrenal medulla into the circulation. Dotted lines represent negative regulatory GC feedback pathways, blue lines represent bi-directional communication of peripheral immune events to the brain that involve the secretion of inflammatory cytokines like TNF-α, IL-1 and IL-6.
(Figure modified from Esther M. Sternberg, Nature Reviews Immunology, 2006.)
Summing up, the CNS regulates the immune system through two major classes of effector molecules: glucocorticoids, which are regulated in the hormonal stress response by the HPA axis, and the catecholamines norepinephrine and epinephrine, which are released either by the sympathetic adreno-medullary system or via postganglionic nerve fibres. Both branches closely interact with each other and have positive reverberating feedback loops at different levels. For instance, reciprocal neural connections exist between the CRH and noradrenergic neurons, with CRH and NE stimulating each other.
Bidirectional communication between the CNS and the immune system
Bidirectional communication between the CNS and the immune system allows the immune system to signal to the brain through neural and humoral routes. In fact, certain cytokines and in particular the pro-inflammatory cytokines TNF-α, IL-1 and IL-6 are known to activate both the SNS and the HPA-axis (167-168). For example TNF-α, IL-1 and IL-6 stimulate hypothalamic CRH and/or AVP secretion, resulting in the secretion of GCs. How these inflammatory cytokines pass the blood-brain barrier to reach the hypothalamic CRH and AVP neurons is unclear. However, this feedback loop constitutes an important mechanism by
Introduction
22 which GCs serve as a regulatory mechanism to prevent excessive activation of the immune response during infection (169-170). Studies in mice using viruses that elicit both strong early pro-inflammatory and later T cell responses (LCMV clones 13 and WE, influenza, HSV-1) have confirmed the release of endogenous GCs, whereas viruses that induce little or no inflammation do not stimulate significant GC induction (171). In this context it has been demonstrated that endogenous secretion of GCs protect the hostagainst cytokine-mediated pathologies during murine CMV infection (172). More specifically it has been shown that if GCs are removed by adrenalectomy, IL-12, IFN-γ, TNF-α, and IL-6 production increases and the mice die due to septic shock. Moreover, experimental evidence in mice have confirmed that an increase of endogenous GC levels plays a protective role for the host during experimentally induced autoimmune encephalomyelitis (173)and arthritis (174).
Glucocorticoid hormones
GC hormones are long known for their immunosuppressive effects and clinically, GCs and their synthetic analogues are used as potent immunosuppressive agents. Many of the immunomodulatory effects of stress have been attributed to the action of GC stress hormones, therefore the cellular and molecular mechanisms of GC-mediated immunosuppression will be introduced in the following paragraphs.
Molecular mechanism of GC-induced immunosuppression
GCs belong to the family of steroid hormones and their action is mainly mediated through binding to the respective cytoplasmic receptors. There are two main receptors in the cytoplasm for GCs, the glucocorticoid receptors (GR) and the mineralocorticoid receptor (MR). GCs have a higher affinity for MR than for GR (175), thus at low levels, GCs bind preferentially to the MR, only at high levels, e.g. during stress exposure, the GRs are occupied (176). The most accepted mechanism by which GCs enter the cell is through passive diffusion facilitated by their relative small size and lipophilic nature. However, it has also been proposed that GCs can mediate their action by binding to membrane-associated glucocorticoid response receptors (mGCR) and that this interaction might participate in a GC- mediated apoptosis (177-179), but the precise mechanism has not yet been identified.
The glucocorticoid receptors (GRs) belong to the nuclear hormone receptor superfamily that are present in the cytoplasm in an inactive state and form multi-protein complexes with hsp90 and other chaperons. Upon GC binding, GRs dissociate from this complex and translocate as a homodimer to the nucleus where they bind via a zinc finger motif in their DNA-binding domain to the glucocorticoid response element (GRE) (180). The bound GR homodimer then modulates gene expression directly by either up-regulation or down-regulation of target genes, depending on the GRE sequence and promoter context. A direct down-regulation of gene expression occurs mainly via binding to so-called negative glucocorticoid response elements (nGRE) (Figure 5).
