Final Report Summary - TOLERAGE (Normalisation of immune reactivity in old age - from basic mechanisms to clinical application)
Executive Summary:
Reconstitution of normal immune function in old age is generally based on boosting the response against foreign antigens, e.g. infectious microorganisms. The focus of the TOLERAGE project, however, was to normalise immune reactivity by correcting the immune dysregulation developing with age which forms the basis of important age related diseases. Immune dysregulation is known to start early and to increase with age, causing a high cost to European societies in terms of morbidity and mortality.
Tolerage has paved the way for vaccinations against AT and RA focusing on HSP60 as a main culprit (auto-) antigen. Moreover, rather than inducing tolerance against the whole HSP60 molecule - an approach that may compromise defence against infections - atherogenic and arthritogenic HSP60 epitopes have been defined and will be used as tolerising compounds. The leading role of HSP70 in protecting animal models from more severe RA necessitated a combined focus on both of these heat shock proteins.
Highlights of the TOLERAG project were the following findings
Atherogenic and atheroprotective epitopes of HSP60 were identified, and feeding of anti-CD3 antibody to mice with HSP60 induced atherosclerosis resulted in a marked reduction in lesion size.
Peptide fragments of HSP70 (such as B29) can be developed as novel immunotherapeutic vaccines in rheumatoid arthritis, and some (food derived) compounds have a capacity to co-induce HSP expression and improve vaccination results.
Age-related changes in part controlled by miRNAs in the thymic microenvironment and in gene expression patterns are conducive to subvert the central tolerance process.
Inhibition of local GC synthesis delays/reverses involution although the biological implication of this on autoimmune responses is weak
Induction of apoB100-specific Treg responses reduces the development of atherosclerosis in young mice and blocks disease progression in old animals.
SOCS3 expression in atherosclerosis increases with age. We found that SOCS3-controlled Th17 cells play a major role in the protection against atherosclerosis.
MHV vector-mediated delivery of IL-10 to DCs is feasible both in vitro and in vivo, and MHV vector-encoded IL-10 stimulates CD4+ T cell responses in vivo.
Newly generated iTreg cells are retained in cutaneous lymph nodes via a mechanism involving CCR4/CCL22.
Regional steady state migratory DC-induced iTreg cells protect from autoimmunity
PTX3 levels increase with aging in mice and humans, and are a biomarker for atherosclerosis.
The clinically relevant outcome of this project is a significant advancement in the knowledge of how and when to induce tolerance to auto-antigens responsible for age-relevant disease.
Project Context and Objectives:
Reconstitution of normal immune function in old age is generally based on boosting the response against foreign antigens, e.g. infectious microorganisms. The focus of the TOLERAGE project, however, was to normalise immune reactivity by correcting the immune dysregulation which forms the basis of important age related diseases. Dysregulation is known to start early and to increase with age, causing a high cost to European societies in terms of morbidity and mortality.
TOLERAGE used novel approaches to normalise immune function at old age on three complementary levels. The paradigmatic age related diseases that form the focus of this proposal are atherosclerosis (AT) and rheumatoid arthritis (RA).
1)The first of these was to look into the basic mechanisms of age-related changes in immune function, concentrating on thymic self-antigen display, T cell selection of autoreactive T cells, effects of aging on regulatory T cells (Treg), hormonal influences (particularly glucocorticoids - GC), and the action of cytokines in aging organisms. The knowledge gained of how the immune system ages will eventually be able to be used to focus and fine tune the regulation of the immune system, including inducing tolerance towards HSP60.
2)The second part of the project focused on two paradigmatic age related diseases, atherosclerosis (AT) and rheumatoid arthritis (RA), investigating in detail the effect of aging on the autoimmune reaction to heat shock protein 60 (HSP60), which has been shown to be an important auto-antigen in both AT and RA. In particular, knowledge from research groups engaged in the first part was able to be directly used by other partners to induce tolerance to HSP60 in mouse models of both AT and RA at various ages and disease stages.
3)The third element, the identification of the mechanisms responsible for a normalisation of the immune response after appropriate intervention (e.g. nasal/oral tolerance) on the cellular level, was an important practical component of the project. Investigations elaborating the mechanisms responsible for tolerance addressed the role of central and peripheral deletion of T cells, the action of Treg cells, the balance of immune regulatory cytokines, and hormonal (GC) influences during the aging process.
The developing immune system is increasingly prone to develop into a dysbalanced state, especially in the Western world due to environmental influences. This dysregulation is contributing to raised frequencies of various immune system-mediated diseases such allergies, chronic autoimmune conditions, and inflammatory bowel diseases. One heavily debated issue here is the possibility that a relative lack of exposure to infection causes deficient regulation of the immune system, known as the 'hygiene hypothesis'. Whatever the underlying factors may be, the repeated exposure of the adaptive immune system to microbial antigens is now widely accepted as a factor that contributes to immune fitness and production of immune balance. Since HSP60 is one of the immuno-dominant components of commensal gut microbiota, we have used HSP60 as a prototype microbial-human cross-reactive antigen to promote T cell mediated immune regulation. An immunological approach to prevent and/or treat these autoimmune diseases would require a down-regulation of pathologic immune reactivity to HSP60 or an up-regulation of anti-inflammatory regulation at the level of HSP60 or HSP70. Here the requirement is not only a down regulation of the immune response, but a targeted up-regulation of the response to protective HSP60 and HSP70 epitopes. This can be achieved by special vaccination strategies leading to anti-inflammatory tolerance targeted towards HSP60 or HSP70. It was the aim of TOLERAGE to provide the theoretical basis in vitro and in mouse models for specific tolerance induction to heat shock proteins 60 and 70 to reduce the severity of RA and AT, or even prevent the diseases altogether. In addition, the restoration of tolerance in old mice by means of hormonal, cytokine, soluble antigen, or antigen presenting cell (DC) targeted interventions was also investigated, to help build the foundations of possible treatment regimes.
Progress beyond the state-of-the-art
Vaccination against age-associated, primarily non-infectious, diseases has been a dream of gerontologists and geriatricians for a long time. Partial success in this area, including studies in experimental animals and approaching clinical applications in humans has already been achieved with a vaccine against Alzheimer's disease. Tolerage was to lay the ground for vaccinations against AT and RA focusing on HSP60 as a main culprit (auto-) antigen. Moreover, rather than inducing tolerance against the whole HSP60 molecule - an approach that may compromise defence against infections - atherogenic and arthritogenic HSP60 epitopes have been defined and will be used as tolerising compounds. The leading role of HSP70 in protecting animal models from more severe RA necessitated a combined focus on both of these heat shock proteins. Tolerage has opened a completely new and original line of research that is not only important from a scientific perspective, but also has tremendous social potential taking into account the medical and socioeconomic impact of AT and RA.
In the initial period of TOLERAGE, the expertise available in the consortium was combined to study mechanisms of central and peripheral tolerance in general, and HSP60 in particular. Emphasis was put on DC, Teffector and Tregulatory cells, their interaction with each other, and how this is affected by aging. The age-related alterations in these cells and how they interact, including the mediator involved such as cytokines, chemokines, and glucocorticoids in the thymus and periphery were of particular interest. The knowledge gained from these studies has produced important insights into the mechanisms influencing the aging immune system.
Project Results:
Workpackage 1: Basic mechanisms of central and peripheral tolerance
Introduction and objectives
The two major objectives of WP1 were:
explore how central tolerance towards tissue-specific self-antigens - notably known target auto-antigens of major autoimmune diseases - might change qualitatively or quantitatively during the aging process and thus predispose to age-related autoimmunity.
assess natural tolerance/immunity to the self-antigen Hsp65/70 involved in the auto-immune inflammatory aspect of arthritis and atherosclerosis and induce peripheral tolerance towards Hsp65/70 as a measure to interfere with both diseases.
Results
Three age groups of C57BL/6 mice - 6 weeks, 12 and 18 months - were compared. There was an over-proportional loss of medullary thymic epithelial cells (mTECs) over cortical thymic epithelial cells (cTECs). Within the mTEC lineage we observed a selective loss of the mature subset vs. the immature subset. Since the mature mTECs show the highest level and diversity of pGE, this loss amounts to a proportional reduction in the availability of self-antigens in the thymus. Notably, expression of a selected set of tissue-restricted antigens (TRAs) on a per cell basis including Hsp60 and Hsp70 did not decline with age. We currently, however, cannot exclude more subtle qualitative or quantitative changes of pGE at the whole genome level during aging. Respective gene array expression studies are under way.
Apart from the number of thymic APCs, the efficacy of negative selection is also dependent on the 3D array of mTECs in situ. Our immuno-histological analysis of the aged thymus clearly revealed a highly disarrayed distribution of mTECs along with a loss of a clear cortex/medulla demarcation. It is to be expected that this altered microenvironment affects the scanning process of thymic APCs by thymocytes and thus the efficacy of tolerance induction.
Overall, our detailed analysis of mTECs in the aging thymus revealed clear age-related changes in the composition and developmental dynamics of this compartment, in particular the reduced proportion of mature mTECs and their disarrayed distribution in situ are conducive to compromise central tolerance. Importantly, as far as the observed age-related alterations pertain to ex vivo analysis, the data obtained in mice closely paralleled those in humans.
Taken together these data from WP1 showed that:
Age-related changes in part controlled by miRNAs in the thymic microenvironment and in gene expression patterns are conducive to subvert the central tolerance process.
Peripheral tolerance induction towards intact Hsp65 and peptides of Hsp70 can prevent atherosclerosis and suppress experimental arthritis, respectively.
These new insights open options to interfere with the process of thymic involution, on the one hand, and on the other hand the development of age-related autoimmune diseases.
Workpackage 2.Thymic involution: The role of locally produced glucocorticoids (GC) - Selective inhibition of intra-thymic GC synthesis.
Introduction and objectives
The thymus gland regresses (involutes) during aging, starting early in life and progressing in particular at the time of puberty. T cells are positively and negatively selected in the thymus in order to allow the formation of naive T cells that will not react against self antigens. Central tolerance mechanisms involve the removal of self-reactive T cells and the formation regulatory T cells (Treg) which can induce tolerance in the periphery. Thymic involution leads to a decline in negative selection and in Treg formation as well as in the output of naive T cells. For that reason thymic involution is linked to the development of autoimmune diseases. The reasons why the thymic gland involutes is not clear although there is a consensus that it is caused by a tissue degeneration dependent on changes in hormones, growth factors and cytokines. We have earlier found that the epithelial component in the gland (TEC) synthesize glucocorticoids (GC) in very young mice and that such a synthesis can be detected in the thymocyte population at around the time of puberty. As GC very efficiently kill thymocytes by an apoptosis reaction, it is possible that thymocyte-derived GC play an important role for the thymic involution process.
WP2 aims to show
how the synthesis of the thymocyte and locally produced GCsis regulated
if there is a role for locally produced GC in the thymic involution process
if so, to use this knowledge to delay/prevent involution and then test the effect of this in models for autoimmunity (WP12).
Results
Initial studies established a co-culture method to asses GC synthesis and secretion by co-culturing thymic cells with a reporter cell in which luciferase activity could be activated by co-expressed GR and a GR-controlled reporter gene. The ability of thymic cells to synthesize and secrete corticosterone was confirmed by an ELISA. The ability of thymic cells to synthesize GCs was also demonstrated by the ability of metyrapone, an inhibitor of CYP11B1, the last enzyme involved in the formation of corticosterone, to block GC synthesis. Furthermore, all the necessary enzymes required for the formation of GCs from the mother substance cholesterol was found in thymocytes. Interestingly, the expression of several, including rate limiting, enzymes as well as GC secretion by thymocytes started at the age 4 weeks in the mice, coinciding with the start of the age-dependent thymic involution. GC production increased further with increasing age.
We also studied factors that regulated thymocyte GC production. In contrast to adrenals where ACTH stimulates GC synthesis, ACTH down-regulated GC production from thymocytes as assayed both in culture and in vivo. As high systemic GC levels promote apoptosis of peripheral T cells, the decreased GC production in the thymus and thus a decreased thymocyte apoptosis may represent a way to guarantee feeding of sufficient T cells formed in the thymus into the periphery. Additional studies also demonstrated that testosterone induced local GC production in the thymocytes, and that this testosterone-induced thymic atrophy was at least in part due to testosterone-induced GC synthesis in the thymocytes. This was most likely mediated by TNFa secretion induced by testosterone. The testosterone-stimulated synthesis of GCs in the thymus may contribute to the faster thymic involution seen in males. In contrast, high doses of estrogen administration, which also induces thymic involution, did not mediate this effect by affecting GC synthesis of the thymocytes. We also explored transcription factor expression in thymocytes known to be involved in steroidogenesis in classical steroidogenic tissues. Steroid factor 1 (SF-1), liver receptor homolog 1 (LRH-1) and transcriptional regulating protein of 132 kDa (Trep 132), which are all known to be involved in the regulation of the expression of genes involved in steroidogenesis including GC synthesis, was found to be expressed in thymocytes. However, SF-1 was expressed as a spice variant with a truncated form.
The outcome of this WP clearly demonstrated that de novo local GC synthesis occurs in the thymus, how it is regulated and that it impacts thymocyte homeostasis by enhancing/promoting thymic involution. Inhibition of local GC synthesis delays/reverses involution although the biological implication of this on autoimmune responses requires further studies.
Workpackage 3: T effector/Treg imbalance with increasing age
Introduction and objectives
This part of the project is based on the hypothesis that an imbalance between Teff and Treg cells leads to a defective control of local inflammatory responses with increasing age. In the long-term, impaired immune surveillance may result in increased tissue damage and predispose to opportunistic infections, cancer, as well as immune-mediated diseases such as AT and RA. Teff and Treg compartments will be studied in vivo and in vitro using young and aged mice.
Results
We found reduced suppressive properties of Treg cells isolated from aged vs young mice, only when mice were housed under conventional conditions. No defect of Treg function was observed in aged mice housed under specific pathogen-free conditions.
We showed that induction of apoB100-specific Treg responses reduces the development of atherosclerosis in young mice and blocks disease progression in old animals.
WP4: The role of dendritic cells (DCs) in tolerance induction
Introduction and objectives
Dendritic cells are central players in the decision making process of immune activation vs tolerisation. The major aim of WP4 was to generate safe and efficient coronavirus-based vaccine vectors which are able to deliver antigens in combination with immune-modulating cytokines to transduce dendritic cells and closely related antigen presenting cells, such as macrophages, in vivo.
The major objectives of WP4 were:
To generate and characterize recombinant murine coronavirus-based vectors expressing HSP60 and/or another autoantigen together with an immune-suppresive cytokine such as IL-10 or TGF-beta
To assess the in vitro and in vivo gene transfer of murine coronavirus-based vectors into dendritic cells.
Results
To assess immunogenicity and to ensure maximal safety of coronavirus-based multigene vectors, we have rationally designed a series of vectors based on the mouse hepatitis virus, strain A59 (MHV-A59). With regard to safety, we have introduced three modifications to obtain replication-competent, but attenuated and propagation-deficient MHV-based vectors: (i) deletion of all MHV-encoded accessory genes (NS2, HE, gene4, gene5a), (ii) removal of 99 nucleotides within the replicase-encoded sequence of the non-structural protein 1 (nsp1), and (iii) deletion of the structural gene E to restrict proper particle formation. Since the activation status and the immunstimulatory properties of DCs can be modified by cytokines, we have inserted the gene encoding for the immune-activating murine granulocyte-macrophage colony-stimulating factor (GM-CSF) or the immune-modulatory interleukin 10 (IL-10) between the MHV vector-encoded replicase and spike genes. To demonstrate the feasibility of the approach, we have first generated a model construct containing a well-characterized viral epitope derived from the glycoprotein (GP) of the lymphocytic choriomeningitis virus (LCMV), fused with the enhanced green-fluorescenct protein. The resulting vectors have been termed
MHV-GP
MHV-GP/GM
MHV-GP/10
Furthermore, cloning of MHV-hsp60, MHV-hsp60/GM, MHV-hsp60/10, MHV-hsp70, MHV-hsp70/GM, MHV-hsp70/10 in vaccinia virus shuttle vectors had been completed, but rescue of MHV vector particles had failed for all vectors encoding hsp60 and hsp70. Several rescue strategies were initiated, but none of these strategies resulted in the propagation of sufficient amounts of hsp- or hsp epitope-endoding MHV-based vectors. From these experiments we concluded that the presence of hsp60 or hsp70 in the genome of the coronavirus-based vectors is not compatible with coronavirus replication. We therefore resorted to an alternative strategy and inserted the bacterial antigen b-galactosidase (bgal) into the vectors which could be successfully rescued. We succeeded to amplify to high titers in cells complemented with the viral E-protein the following bgal-encoding vectors:
MHV-bgal
MHV-bgal /10
Next, we assessed the in vivo targeting efficacy of MHV vector and the impact of the cytokine GM-CSF and IL-10 on DCs. We found that MHV-based vectors almost exclusively delivered their antigenic cargo to DCs promoting the induction of transgene-specific T cell responses. GM-CSF substantially improved the immunogenicity of the vaccine with approximately 3-fold higher expansion of antigen-specific CD8+ T cells. Moreover, the MHV-based vaccine could be applied via different routes and still retained its high immunogenicity which is mainly due to an enhanced maturation of DCs and prolonged antigen presentation within these cells. The results of these proof-of-principle studies were published in 2010 in the open access journal mBIO.
Concerning the effects of IL-10, we found only a mildly attenuating effect of the IL-10-encoding MHV vector on the in vivo DC transduction efficacy indicating that the system is well-suited to target both an antigen and the immune-modulatory cytokine IL-10 to DCs. Furthermore, although in vitro experiments showed that specific IL-10 delivery can to some extent inhibit DC maturation, these effects could not be demonstrated in vivo, i.e. unlike in the in vitro experiments, in vivo DC transduction with IL-10-encoding vectors did not significantly impact on DC maturation, i.e. the maturation markers CD86 and CD40 were not down-regulated compared to DCs transduced with the vector not encoding a cytokine. To assess the impact of vector-encoded IL-10 on the induction of CD8+ T cells, we immunized C57BL/6 mice with 106 pfu of the different GFP-GP34-encoding vectors. We confirmed that GM-CSF efficiently enhanced the immunogenicity of the vector, whereas IL-10 led to a mild attenuation. However, the immune-attenuating effect of IL-10 delivered to DCs on CD8+ T cells was not detectable at later time points post immunization. Likewise, immunization with bgal-expressing MHV vectors failed to reveal an immune attenuating effect on CD8+ T cells. Furthermore, IFN-g production by bgal-specific CD8+ T cell was not affected by the absence or presence of IL-10 in the MHV vector indicating that, despite a clearly detectable effect on DCs in vitro, MHV vector-mediated in vivo transfer of IL-10 to DCs only mildly affected the generation of vector-specific CD8+ T cells.
Taken together, the results obtained in WP4 showed that
MHV vector-mediated delivery of IL-10 to DCs is feasible both in vitro and in vivo,
MHV vector-encoded IL-10 negatively affects DC immune functions in vitro, and
MHV vector-encoded IL-10 stimulates CD4+ T cell responses in vivo.
WP5: The role of DC in antigen presentation
Introduction and objectives
A common vision of how dendritic cells (DCs) contribute to the induction and maintenance of peripheral CD4+ T cell tolerance is that, in resting conditions, immature DCs, expressing low levels of signal 1 (specificity) and low or no levels of signal 2 (costimulation), are able to induce T cell unresponsiveness.
The major objectives of WP5 were:
Elucidation whether chronic antigen exposure and/or state of DC activation are parameters relevant for the induction and maintenance of peripheral T cell tolerance
Based on the identified genes differentially expressed in lymph node and splenic DCs, elucidation of the molecular mechanisms through which lymph node DC and/or spleen DCs are able to tolerise auto-reactive T cells.
Results
In order to know whether DCs in general are able to induce T cell tolerance at the steady state or if this is a prerogative of specialized subsets, we took advantage of an experimental system where antigen presentation, in contrast to previous studies, is not a priori confined to a specific DC subpopulation but is extended to all conventional DC subtypes. Specifically, we adopted the 2a T transgenic animal model in which T cell receptor transgenic T cells (2a T cells) recognize a portion of the CH3 region (435-451) of the IgG2ab, the Bpep, in association with the MHC molecule, I-Ad. We also generated a mouse model in which the Bpep was presented by CD11c+ cells that include all conventional DC subtypes.
By performing a systematic study of the behaviour of naive autoantigen-specific T cells after interaction with all conventional CD11c+ DCs in homeostatic conditions we found that DCs are able to induce CD4+ T cell tolerance by promoting the conversion of autoantigen-specific naive T cells into iTreg cells. Among the different DC subtypes, steady state migratory DCs (ssmDCs) possessed unique ability to induce antigen-specific iTreg cells. Diversely, lymphoid tissue resident DCs did not show this capacity. Therefore, iTreg cells developed solely in lymph nodes and not in the spleen, which does not host the migratory DC subtype. We also found that ssmDCs were able to induce iTreg cell conversion in a RA-dependent manner and that newly generated iTreg cells were not able to re-circulate but resided in the lymph nodes where they were generated.
Finally, it was evaluated whether iTreg cells could develop in inflammatory conditions. Using GpG as an inflammatory stimulus we observed that the generation of iTreg cells occurred in lymph nodes irrespective of the fact that the animals were treated with inflammatory substances. Nevertheless, in steady state conditions iTreg cell differentiation was already apparent 5-10 days after antigen encounter. Diversely, in inflammatory conditions, iTreg cells were only visible starting from two weeks after cognate antigen recognition. Interestingly, activated DCs were observable in the lymph nodes until day 6-8 after T cell transfer, therefore the appearance of iTreg cells correlated with the disappearance of activated DCs. Given the transient presence of the inflammatory stimulus, these data strongly suggest that iTreg cells differentiate only when non-activated migratory DCs reach the draining lymph nodes.
Taken together, the results obtained in WP5 showed that
In vivo conventional DCs induce auto-antigen CD4+ T cell tolerance exclusively via the induction of auto-antigen-specific iTreg cells
When in vivo auto-antigen presentation is extended to all CD11c+ conventional DCs, immature lymphoid tissue resident DCs are unable to tolerize CD4+T cells while this is a prerogative of steady state migratory DCs that reach the lymph nodes. These cells induce iTreg cell generation in a RA dependent manner. Consequently, the conversion occurs only in lymph nodes and not in the spleen
Newly generated iTreg cells are retained in cutaneous lymph nodes via a mechanism involving CCR4/CCL22.
Regional steady state migratory DC-induced iTreg cells protect from autoimmunity
WP6 Generation of regulatory dendritic cells in vitro using glucocorticoids (GC).
Introduction and objectives
GC can contribute to the formation of regulatory dendritic cells (DCreg) by inhibiting the maturation of DC, leading instead to the formation of DCreg. We propose an exploration of the use of GC for the generation of mouse DCreg in vitro, and the further applicaton of these cells in the treatment of autoimmune diseases RA and AT.
The formation of regulatory dendritic cells (DCreg) will be induced using GC, which can contribute by inhibiting the maturation of DC, leading instead to the formation of DCreg.
Results
We have devised a method to generate tolerogenic DC in vitro by treatment of cells with the synthetic glucocorticoid dexamethasone (DC-dex) and characterized these by functional assays and phenotypic markers. Bone marrow-derived DC was generated in vitro in the presence of recombinant GM-CSF and IL4, with DCreg produced and by the later addition of dexamethasone to the cultures. DC-dex had an enlarged size and fewer dendritic extensions and supernatants from DC-dex cultures showed a reduction of IL12p40 and an increase in IL-10 synthesis. In contrast, whereas immunogenic DC generated by CpG activation (DC-CpG) proliferated and showed signs of activation and cluster formation, DC-dex expressed downregulated levels of MHC-II and the co-stimulatory molecule CD86, whereas DC-CpG expressed increased levels of both these membrane structures, as expected. The addition of CpG to dexamethasone treated DC for the last 24 h did not reverse the GC induced phenotype to any extent. We conclude that the different DC expressed the expected phenotypes for tolerogenic or immunogenic DC, respectively, and that the DC-dex phenotype was mostly irreversible.
We investigated the effect of DC-dex on T, Treg and NK cell responses in OT-1/Rag-/- mice, which express a transgenic TCR in CD8+ T cells, comparing these responses with the effect of immunogenic, DC-CpG. The OT-1/RAG-/- T cell responses to the different DC were tested in vitro and in vivo. In vitro co-culture of OT-1/RAG-/- cells with peptide treated DC showed that DC-CpG stimulated IFN-gamma synthesis in these as well as IL12p40 secretion. In contrast, DC-dex induced IL-10 synthesis and the expression of Foxp3 as well as a modest IFN-gamma response in these cells. DC-CpG, but not DC-dex, also generated cytotoxic OT-1/RAG-/- T cells which killed EG7 tumor cells, expressing the OVA derived peptide epitope recognized by the transgenic OT-1 cells, but not the parental EL4 cells. DC-dex also generated a Treg response which suppressed the OT-1/RAG-/- T cell proliferation in response to peptide treated antigen-presenting cells. Mice injected with DC-CpG expanded and activated splenic NK cells in B6 mice that had an increased expression of the NKG2D receptor but an essentially unaltered expression of differentiation markers Mac-1 and CD27. In vitro, as well as in vivo, these activated NK cells killed the prototype NK cell target YAC-1. In contrast, DC-dex injection did not lead to any of these responses in splenic NK cells. In conclusion, we have found that GC induced tolerogenic DC do not activate T or NK effector cells but that they induce a Treg response in OT-1 mice. These CD8 positive Tregs most likely express CD122 but not CD25. CD122, being the beta chain of the IL2 receptor, is a marker for natural CD8 positive Tregs that play an important role for the maintenance of T cell homeostasis and which, by producing IL-10, suppress the IFN-gamma production and proliferation of CD8+ T cells.
WP7 Age dependent changes of the cytokine balance.
Introduction and objectives
The main objective of WP7 was to define changes in cytokine balance during aging. In particular, focus has been on the long pentraxin PTX3, a fluid phase pattern recognition receptor which has complex functions in innate immunity, extracellular matrix construction, and regulation of inflammation. Data collected so far suggest that PTX3 may represent a novel marker of innate immune-inflammatory reactions that better represent the events in involved tissues, and in particular the vascular wall. In the context of these studies, the role of PTX3 in pathologies associated with aging has been investigated at a basic research level in humans and in mice. The inflammatory status is the result of a balance between inducers and inhibitors of the inflammatory response. Decoy receptors recognize inflammatory cytokines with high affinity and specificity, but they are incapable of signaling, thus representing a strategy to regulate inflammatory cytokines and chemokines. Work done by this group has identified an isoform of IL-1 receptor antagonist (IL-1ra) and the soluble form of type II IL-1R (sIL-1-RII) as decoy molecules.
It has been reported that the balance of anti- and pro-inflammatory factors is altered in aged people, with IL-1Ra increasing and IL-10 decreasing during aging. The present project thus has also investigated whether other IL-1-related molecules, such as IL-1R type II that acts as a decoy for soluble IL-1, are modulated in aging in humans.
The major objectives of WP8 were:
To standardize the protocol for measurement of PTX3 (both in humans and mice) based on original antibodies and recombinant proteins developed by P6
To define the changes in PTX3 levels during aging both in humans and mice
To define the changes in Il-1ra and sIL-1RII levels during aging both in humans and mice
Results
To start with the measurements of PTX3 in aged and young humans and in age-related pathologies, protocols for PTX3 determination were standardized. We did some modifications of the assay described in already published papers (see for examples Latini R, et al. (2008) Eur Heart J 29 (Suppl 1): 393 (abs). and Peri G, et al. (2000) Circulation 102: 636-641). Main improvements are: a) very good reproducible results were obtained when samples were tested immediately after thawing or 24 hours later, variations between the two measures ranging from 0% to 25% maximum, with a median value of 7%; b) more stability after repeated thawing, the same mean values being obtained after 1, 2 or 3 thawing before measurement; c) the detection limit is 0.1ng/ml.
Having set up the measurement systems for human and murine PTX3, levels were investigated in humans and mice of different ages. While there are no significant differences between children and adults, the levels of PTX3 in the greater than 90 years group are significantly higher (mean; 2.2x0.2 ng/ml) than in the other two groups. Similarly in the mice we had a significant increase in PTX3 levels with aging.
The measurements of sIL-1RII and IL-1ra where then performed in the same blood samples from mice and humans. While no significant changes were observed in IL-1ra levels, sIL-1RII levels significantly decreases with aging both in humans and mice.
Taken together, the main results of WP7 showed:
PTX3 levels increase with aging in mice and humans
sIL-1RII levels decreases with aging in mice and humans
no significant changes were observed for IL-1ra levels in relation to age.
WP8 Rheumatoid arthritis
Introduction and objectives
Rheumatoid arthritis and associated inflammatory conditions are major autoimmune disorders of the ageing population. It was the purpose of WP8 to analyze the role of HSP associated immune-regulation in the incremental susceptibility of the aged individual with respect to these diseases. Moreover, in experimental models a search was made for possible interventions that could lead to improved HSP mediated down-modulation of these inflammatory diseases. The major objectives of WP8 were:
To analyze the role of HSP in the inflammatory process of joint diseases
To define biomarkers of the inflammatory process associated with arthritis
To discover and analyze novel routes of intervention to compensate for the loss of HSP up-regulation of the aged individual
To devise novel therapeutic means to stimulate immune reactivity towards HSP
To analyze the immune-modulatory mechanisms of arthritis in relation to HSP
Results
In the analysis of the role of HSPs in arthritis the primary focus on HSP60 was changed into HSP70 already in the beginning of the Tolerage project. This was done for scientific reasons, mainly based on the fact that immune reactivity in the proteoglycan induced arthritis model seemed dominated by responses to HSP70. In addition, stress dependent up-regulation of HSP70 during inflammation was more significant for HSP70 than for HSP60. In synovium of the inflamed joints and in several lymphoid tissues the up-regulated HSP70 family members were documented and T cell reactivities, which included responses of cells with markers of Tregs, were detected in the spleen and draining lymph-nodes. In addition, antibody profiles were seen to be compatible with immune reactivity for HSPs expressed in the inflammatory tissues.
Besides HSP reactivity being found as a biomarker of the arthritic inflammatory process, PTX3 was also analysed as a possible biomarker. Taking advantage of Ptx3-/- mice, the role of PTX3 has been investigated in a murine model of collagen-induced rheumatoid arthritis. The results evidenced a higher susceptibility to the disease in mice lacking the protein. Moreover, PTX3 levels were measured in different mice models of arthritis. Levels of the protein correlated with the severity of disease in mice, with collagen induced arthritis (CIA). In addition, P6 have measured PTX3 levels in 345 samples collected from mice with PG-induced arthritis (PGIA) treated with different protocols of CD4+CD25+Tcells. When similar experiments were pooled together, a significant correlation was observed between PTX3 levels and clinical score, further supporting the role of PTX3 as biomarker of innate-immune inflammatory reactions.
Especially food- or herb-derived phytonutrients may be attractive compounds to restore optimal Hsp expression in response to stress. In TOLERAGE, we explored three readout systems to monitor Hsp70 expression in a manner relevant for the immune system to evaluate novel Hsp co-inducers. First, intracellular staining and analysis by flow cytometry was used to efficiently detect stress and/or dietary compound induced Hsp70 expression in multiple rodent cell types. This system was used to screen a panel of food-derived extracts with potent anti-oxidant capacity. This strategy yielded the identity of several new enhancers of stress-induced Hsp70 expression, among them carvacrol, found in thyme and oregano. Second, CD4(+) T-cell hybridomas were generated that specifically recognized an immunodominant Hsp70 peptide. These hybridomas were used to show that carvacrol enhanced Hsp70 levels and increased T-cell activation. Third, we generated a DNAJB1-luc-O23 reporter cell line to show that carvacrol increased the transcriptional activation of a heat shock promoter in the presence of arsenite. These assay systems are generally applicable to identify compounds that affect the Hsp level in cells of the immune system.
Carvacrol, a major compound in the oil of many Origanum species, had a notable capacity to co-induce cellular Hsp70 expression in vitro and, upon intragastric administration, in Peyer's patches of mice in vivo. As a consequence, carvacrol specifically promoted T cell recognition of endogenous Hsp70, as demonstrated in vitro by the activation of an Hsp70-specific T cell hybridoma and in vivo by amplified T cell responses to Hsp70. Carvacrol administration also increased the number of CD4+CD25+FoxP3+ T cells systemically in the spleen and locally in the joint, and almost completely suppressed proteoglycan-induced experimental arthritis. Furthermore, protection against arthritis could be transferred with T cells isolated from carvacrol-fed mice, showing the T cell nature of the arthritis suppressing effect of carvacrol. Interestingly enough, these findings illustrate that a food component can boost protective T cell responses to a self stress protein and down-regulate inflammatory disease, i.e. that the immune system can respond to diet.
Taken together, the main results of WP8 showed:
HSP70 is a dominant antigen in rheumatoid arthritis
Carvacrol and similar HSP70 co-inducers can compensate for the loss of HSP reactivity of ageing
Peptide fragments of HSP70 (such as B29) can be developed as novel immunotherapeutic vaccines in rheumatoid arthritis
Blood levels of PTX3 can represent a new useful marker to monitor rheumatoid arthritis
WP9 Atherosclerosis
Introduction and objectives
WP9 dealt with one of the main goals of TOLERAGE, i.e. the development of an HSP60-based vaccine for the prevention of atherosclerosis in a mouse model. Additionally, the role of GC in atherosclerosis and the potential of PTX3 as a new biomarker for atherosclerosis were investigated. The objectives of WP9 were:
To define the pro-atherogenic or atheroprotective HSP60 T cell epitopes in the murine system
evaluate the role of endogenous GC on T cells in atherogenesis
the value of PTX3 as a novel of prognostic marker of AT in humans
Results
In order to investigate the impact of GCs on T cells for the development of atherosclerosis, an mouse model was develop in which GC sensitivity of T cells was altered in the ApoE KO mice. In female Lck-GR/Apo E KO mice no difference in the development of atherosclerosis could be observed, this despite an increased GC sensitivity of the T cells and a reduced T cell number. No clear effect on Treg number or cytokine profile could be detected. In male mice, increased atherosclerosis was observed, contrary to the hypothesis. This was most likely due to an increased general low grade inflammatory activity measured as increased expression of acute phase reactants in the liver. The reason for this observation is not clear but is most likely an unfavorable combination of the particular transgene used to increase GC sensitivity and male specific factors. Based on the female mice where no systemic inflammation was seen, we conclude that a selective increase in GC sensitivity of T cells in itself does not affect atherogenesis. Based on the results from the female mice, we conclude that the increased incidence of AT observed following systemically high and long-term GC exposure is not mediated by GC-effects on T cells but rather through other (e.g. metabolic) mechanisms.
The possible role of PTX3 in the pathogenesis of atherosclerosis has been investigated by generating double-knockout mice lacking PTX3 and Apolipoprotein E. When these mice were fed an atherogenic diet, they developed more aortic lesion compared to single KO animals. In addition, a more pronounced inflammatory profile, as evidenced by microarray analysis of vascular wall specimens, was obtained from double-knockout mice compared to single KO animals. Lack of PTX3 is also associated with higher tissue damage in a model of acute myocardial infarction in mice. Taken together, these results suggest that PTX3 may have a protective role through modulation of the immunoinflammatory balance in the cardiovascular system.
Inflammation plays a major role also in the progression of chronic heart failure (HF), thus a second large cohort was analyzed, derived from two independent studies: the Controlled Rosuvastatin Multinational Trial in HF (CORONA 1233 patients) and the GISSI-Heart failure trial (GISSI-HF). The results confirmed the association between PTX3 levels and age. In addition, baseline elevated PTX3 were associated with higher risk of all-cause mortality or cardiovascular mortality and with fatal events, confirming the usefulness of PTX3 as biomarker in human pathologies.
WP10: Induction of immunologic tolerisation to autoantigens
Introduction and objectives
Within WP10, a number of activities have been integrated to assess whether tolerisation against HSP60 or alternative antigens ameliorates atherosclerosis or rheumatoid arthritis. It was planned to assess the therapeutic usage of arthritogenic and atherogenic T-cell epitopes in atherosclerosis and rheumatoid arthritis model systems. Due to the longer than anticipated time required to produce these epitopes, alternative approaches were established and evaluated. The major, partially revised objectives of WP10 were:
To tolerise mice using MHV-based vectors containing a bacterial antigen and cytokines.
Characterization of the immunotherapeutic capacity of in vitro generated DCreg in vivo.
To determine effects of HSP-inducing compounds in combination with other delivery systems.
To assess whether PTX3 can serve as a correlate for therapy-induced tolerance
To document the efficiency of HSP60-specific Tr1 cells in the treatment of RA and AT in a mouse model.
Results
Since MHV-based vectors expressing either hsp60 or hsp70 could not be generated,the full-length b-galactosidase (bgal) gene, as a substitute bacterial antigen, has been utilized for the in vivo assessment of antigen-specific tolerisation in hypercholesterolemic mice. To assess whether the presence of bgal in the vasculature together with hypercholesterolemia affects the activation pattern of bgal-specific Th cells, transgenic mice expressing the bgal antigen specifically in arterial smooth muscle cells were crossed onto the ApoE-/- background. The results from these experiments revealed that IL-10 encoded by the MHV vector fostered the activation of the bgal-specific T-helper cells, even under conditions of hypercholesterolemia and expression of the antigen in the vasculature. Furthermore, delivery of IL-10 to DCs by the MHV vectors neither favored the differentiation of bgal-specific Th cells towards the Treg phenotype, nor did the global FoxP3 expression in CD4+ T cells change under these conditions. Taken together, these data suggest that in vivo delivery of IL-10 to DCs in atherosclerotic mice did not favour tolerogenic conditions, even when the MHV vectors primed Th cells specific for a vascular antigen.
Since the start of the Tolerage project, published work has shown that tolerogenic DC produced by dexamethasone treatment alone had little effect on collagen induced arthritis (CIA) and that alternatively induced tolerogenic DC (by IL-10) had a very potent effect on plaque formation in hypercholesterolemic mice. For these reasons we have tested DC-dex in another model for autoimmunity (chronic multiple sclerosis, MS) i.e. experimental autoimmune encephalomyelitis (EAE). We found no beneficial effect after treatment with DC-dex or with the tolerogenic macrophages in this EAE model. From this we speculate that DC-dex may lack the capacity to enter the CNS and to exert a tolerogenic function locally. Due to these negative results, we decided to explore alternative ways of producing tolerogenic DC in vitro, by exposing them to combinations of dex, vitamin D3, retinoic acid and TGF as described in the WP 6 report.
To evaluate PTX3 as a correlate for therapy-induced tolerance, we measured PTX3 levels in serum samples from Treg-treated mice suffering from PGIA-induced arthritis. The results obtained did not indicate the existence of a clear relationship between PTX3 plasma levels and control of the disease, at least when the analysis was conducted on samples from a long-established disease as in this setting.
Taken together, the results obtained in WP10 showed that
Vector-mediated in vivo transfer of IL-10 to DCs does not tolerise Th cells reactive against vascular antigens, even under hypercholesterolemic conditions. MHV vector-mediated delivery of IL-10 to DCs is feasible both in vitro and in vivo,
GC-induced DC dex can generate CD8- and CD4-positive Tregs with specificity for model antigens. However, DC-dex did not affect the course of experimental allergic encephalomyelitis, but rendered mice more susceptible to tumour challenge.
DCs treated with carvacrol and exposed to thermal stress induced CD4 Treg cells and attenuated severity of experimental arthritis.
HSP70-treated DC suppress peptidoglycan-induced arthritis.
PTX3 levels did not significantly correlate with disease severity and Treg-mediated disease modulation in RA models.
Subcutaneous infusion of adjuvant-free ApoB100-derived peptides to ApoE-/- mice reduced atherosclerosis through the induction of a specific Treg cell response.
In conclusion, the work performed in this work package revealed that Treg cells can exert attenuating function on the course of chronic inflammatory diseases. However, the efficacy of the disease attenuation appears to be context-dependent. Hence, targeted modulation of the effects of Treg and DC-reg cells in diverse disease settings will require further investigations to advance this field towards clinical applicability.
WP11: Mechanisms of tolerance induction
Introduction and Objectives
The main task of this WP was to identify the molecular and cellular mechanisms underlying tolerance induction in age related diseases with a special focus on the role of dendritic cells, macrophages, regulatory T cells (Tregs) and cytokines. To assess the contribution of macrophages and DCs in tolerisation protocols in atherosclerosis particular mouse models were generated that permit long-term deletion of macrophages or DC. The major, partially revised objectives of WP11 were:
To assess the role of DC and macrophages in the tolerisation process
To assses the role of HSPs on Treg induction
To assess the role of cytokines in therapy induced tolerance
Results
Two different genetically modified mouse lines have been generated that permit ablation of macrophages (ApoE-MPI) and DC (ApoE-DCI) under hypercholesterolemic conditions. Following comprehensive time course and depletion dose adjustment experiments, robust protocols for long-term depletion of macrophages or DC were established. The results of the subsequent experiments revealed that DC depletion did not significantly impact on the development of atherosclerotic lesions in ApoE-/- mice. Likewise, depletion of macrophages did not alter the course of the lipid deposition in the aortic wall during the 3 month observation period. Furthermore, cholesterol levels were not significantly changed by the ablation of either DCs or macrophages using the conditions established in this project. Immunization of these mice with IL-10 encoding MHV vectors revealed that DC depletion profoundly diminished the activation of I gal-specific Th cells, whereas macrophage depletion did not significantly alter the priming of I gal-specific Th cells. Importantly, incorporation of IL-10 into the MHV vectors augmented the activation of I gal-specific Th cells, even under conditions of macrophage depletion. Taken together, these data show that DCs are critical for the activation of Th cells in atherosclerosis-prone ApoE-/- mice. However, a long-term effect of DC or macrophage depletion on arterial lipid deposition could not be detected, suggesting that the hypercholesterolemia-dependent atherosclerosis in ApoE-/- mice does not critically depend on the presence of these cells.
Workpackage 12: Role of thymic involution in autoimmunity
Introduction and objectives
Workpackage 12 was designed to test the hypothesis that a delayed, or reversed, thymic involution can result in a better homeostatic T cell control of autoimmune reactions. Thymic involution can be reversed by several different manipulations including inhibition of local thymic GC synthesis using the drug metyrapone (as shown in WP2), castration of male mice and keratinocyte growth factor (KGF) treatment. In addition, further work in WP2 showed that although the CYP11B1 inhibitor metyrapone had a clear positive effect on the thymus, the effect of KGF seemed more profound in terms of feeding naive T cells into the periphery. In order to analyze the effect of delayed or revered thymic involution on autoimmune reactions, the objective was to:
Analyse the effect of KGF in different models for rheumatoid arthritis (RA) and the effect of metyrapone on the development of atherosclerosis (AS) in ApoE-/- mice.
Results
In one model of RA mice were treated with KGF before they were immunized with rat collagen II/CFA. Treatment with KGF was found to delay the onset of CIA in the highly arthritis-susceptible B10.QNcf1 mice. However, the protective effect of KGF was lost after a booster immunization with CII/IFA. At later stages there were no significant differences in the arthritis incidence between the study groups. In this model, we confirmed the positive effect of KGF treatment on the thymus and the spleen. In the periphery we found an outflow of naive T cells and an increased ratio CD4 positive Treg/T cells. In another RA model, mice were treated with a cocktail of monoclonal antibodies (mAbs) specific for major collagen II epitopes followed by an injection of LPS to enhance the incidence and severity of arthritis. KGF treatment was given daily. No protective effect of KGF on collagen antibody induced arthritis (CAIA) was observed.
In order to study the effect of delayed or reversed thymic involution, mediated by inhibition of local GC production, on the development of atherosclerosis, we treated ApoE KO mice that spontaneously develop atherosclerosis with metyrapone. Treatment of C57/Bl6 ApoE mice starting at an age of 10 weeks with a low dose of metyrapone in the drinking water that selectively inhibited intra-thymic GC synthesis and resulted in a reversed/delayed thymic involution did not significantly influence the development of atherosclerosis in this model. Thus, an altered thymic involution mediated by metyrapone did not affect AS development in this model. However, a large heterogeneity among the analyzed mice was seen, indicating that a clearcut conclusion may require larger experimental groups.
We conclude that in the absence of an appropriate arthritis model which more closely reflects the clinical RA development, the question whether the anti-involution effect can delay, or prevent, autoimmune arthritis development remains open. In the ApoE-/- atherosclerosis model, which is a less straight-forward model for autoimmunity, we did not find any evidence for a protective effect of increasing thymic activity with the present approach.
Potential Impact:
To our knowledge, the present project is the first attempting to address the prevention and treatment of two paradigmatic age-related diseases, AT and RA, via down-regulation, rather than boosting, the innate and adaptive immune reaction. The rationale for this approach is based on the already existing knowledge about a Teff/Treg dysbalance that develops during aging and is reflected by a gradual loss of central and peripheral tolerance.
The impact of these investigations is obvious from different points of view. The results of TOLERAGE help to explain auto-tolerance from an age-related perspective for the first time.
- The basic experimental data will be immediately translated into practical application
- The effect of glucocorticoids, the most important clinically applied immunosuppressive drugs, was not analysed on the usual basis of the induction of Teff apoptosis, but rather on the basis of Treg induction.
- The two most important age-dependent diseases, AT and RA, have been chosen as paradigmatic examples to practically apply the gained experimental knowledge.
- Development of a vaccine to these diseases has been seen as the ideal preventative measure for many years.
In summary, the impact of the positive results from TOLERAGE on the understanding of basic immunosenescence and the possible relevance of these data for human medicine is significant.
Partner 1 Medizinische Universität Innsbruck
The results obtained within the framework of TOLERAGE will be disseminated by publications in high impact journals and by presentations at scientific meetings. Special emphasis will be given to fostering cooperative publications and other presentations.
Special attention will be given to the dissemination of results to the European citizens and stakeholders eg. by presentations in printed and audio-visual media. The coordinator and the other partners of the TOLERAGE constorium already have ample experience in the matter. The fact that RA and AT are among the medically and socioeconomically most important age-related diseases - the latter even being the killer number one - these endeavors will meet prepared minds in the lay community.
Partners 1 and 7 have access to patents concerning diagnosis and treatment of AT and RA, respectively, based on the use of HSP60 and peptides thereof, e.g. for induction of oral/nasal tolerance.
Partner 2 Kantonspital St Gallen
The work of Partner 2 has shown that the generation of coronavirus-based vectors is possible and that in vivo targeting of DC by these novel vaccination tools is highly efficient and can be augmented by the immune-stimulatory cytokine GM-CSF. The results of this study have been published in 2010 in the new ASM open-access journal mBIO (2011 impact factor 5.3). Since the intellectual property on coronavirus-based vaccines is already in the public domain, we have decided to further broaden the knowledge base on this approach by publishing in an open access journal with wide distribution in the vaccination field. Further results obtained with the vector system will be published in other high level journals in the vaccination field. Intermediate results of the project have been presented at several international conferences, mainly by the PhD students involved in the project. The work performed in the project has mobilized further resources for research on this topic, for example on the use of cytokine-modified coronavirus-based vectors as vaccine against tumors. Hence, it can be anticipated that the vaccine-platform will be employed for other medically important applications.
Partner 3 Karolinska Institutet
Factors that regulate thymic involution have been characterized. Targeting these processes should provide a tool to counteract thymic involution thereby providing the peripheral immune system with fresh naive T cells for an extended period which would improve cellular immunity and immune regulation. As it is known that the normal age-dependent involution, with a loss of seeding into the peripheral system of naive T cells, contribute to immunosenescence with an increased risk for infection and most likely autoimmunity and cancer, counteracting or reversing this process should reduce the incidence of these diseases in the elderly population.
Partner 4 DKFZ, Heidelberg
Our data document that the age-related alterations of the thymic microenvironment in mice and humans, in particular the selective loss of medullary thymic epithlial cells are conducive to a loss of central tolerance. Since it has been repeatedly shown that ectopic over-expression of self-antigens in thymic APCs restores robust self-tolerance in experimental models, our results point to a potential correction of age-related autoimmunity either by replacing the aged thymus with a new organ via transplantation of thymic epithelial stem cells, which have been identified in this project, or via a more specific restoration of the mTEC compartment, e.g. by cell-specific targeting signalling pathways which induce terminal mTEC differentiation, e.g. the NFkB pathway. These avenues will be pursued within the next years.
Partner 5 Inserm
The work has led to the filing of 3 patents:
We have filed a patent describing a new model of abdominal aortic aneurysm in mice. Discussions are ongoing in order to license the patent to the industry. The model will be useful to test the efficacy of drugs on the prevention of aneurysm rupture, a major health issue in the aged population.
Partner 6 Fondazione Humanitas per la Ricerca
The main efforts of P6 were devoted to the understanding of the possible role of an innate immunity molecule, the long pentraxin PTX3, in pathologies associated with aging. PTX3 is a fluid phase pattern recognition receptor which exerts complex functions in innate immunity, extracellular matrix construction and regulation of inflammation. In the context of TOLERAGE, it has been shown that PTX3 exerts a crucial role in particular in atherosclerosis. Overall the results obtained confirmed that PTX3 may be considered a novel marker of innate-inflammatory reactions that better represent the events in the vascular wall, thus deserving further translational efforts. This is expected to have a significant impact on the diagnosis of atherosclerosis, which will enable targeted treatment of patients with known medications to reduce the risk of heart attack or stroke (eg cholesterol lowering drugs). Whether PTX3 also plays a role in atherogenesis is not yet clear, and this will be followed up in subsequent studies. There may eventually be the opportunity to develop a medication based on any role found for PTX3 in atherosclerosis.
Partner 7 Utrecht University
The Hsp70 peptide B29 that during the project was found to have a protective role in arthritis, has been patented. This patent will allow further exploration of the clinical applicability of this peptide as a therapeutic vaccine in rheumatoid arthritis patients and possibly other chronic inflammatory diseases in the future. In addition these results have been published in PNAS, a high impact journal and presented at several international conferences focused on immunology, vaccination and new therapeutic approaches in autoimmune diseases. The results regarding the induction of HSP specific regulatory T cells via the use of HSP70 co-inducers and the related mechanisms have been published or are in the process of submission.
Partner 8 UNIMIB
From a biological point of view, the results of WP5 provide an extended interpretation of the distinct capacities of the diverse DC subsets in inducing iTreg cells, which mirrors distinct requirements of spleen and lymph nodes to patrol and control T cell auto-reactivity. Specifically, this work implies that immature DCs are not able to tolerize T cells by default (simply because of the absence of signal-2, the co-stimulation) but need to receive specific conditioning. At the steady state the appropriate signals that render DC alternatively activated may be delivered by the organs/tissues where migratory DCs reside. For instance, tissue-specific stimuli could induce the expression of RALDH2.
This study provides important medical implications in terms of improvement of tolerisation or vaccination strategies. For instance, this work may contribute to the identification of optimal conditions for Foxp3+ Treg conversion to contrast autoimmune reactions emphasizing the fundamental aspect of Treg cell homing. Therapeutic vaccination strategies may benefit from the usage of CCR4 blocking agents to contrast the effect of Treg cells, especially when subcutaneous routes are used. This has very wide therapeutic potential across many diseases that have an immune (especially auto-immune) component.
Partner 9 Genopolis
Microarray platforms require analytical pipelines with modules for data pre-processing including: data normalization, statistical analysis for identification of differentially expressed genes, cluster analysis, and functional annotation. Accordingly, we developed the Automated Microarray Data Analysis (AMDA, version 2.3.5) pipeline, to process Affymetrix 3' IVT GeneChips. AMDA has been implemented to integrate the requirements for different platforms. AMDA 2.13 allows the analysis of Affymetrix (cartridges and plates) and whole transcript probe design (Gene 1.0/1.1 ST and Exon 1.0 ST GeneChips), Illumina Bead Arrays, and one-channel Agilent 4-44 arrays. Relative to early versions, it supports various experimental designs and delivers more insightful biological understanding and up-to-date annotations. The developed new platform will provide scientists with a user-friendly tool to analyse the complexity of genomics data coming from different platform. Such instrument is not currently available as a free open source in the scientific community. Genopolis has developed protocols for analysis of ex vivo low number of cells obtained by cell sorting procedure. With this improvement it was possible to study rare cell populations for the understanding of the priming activity characteristic of dendritic cells that are present in lymph nodes as compared with the spleenic environment.
List of Websites:
Professor Georg Wick, Medizinische Universitat Innsbruck, Austria
Tel: +43-512-9003-70960
Fax: +43-512-9003-73960
E-mail: georg.wick@i-med.ac.at
Project website address: http://www.tolerage.eu
Reconstitution of normal immune function in old age is generally based on boosting the response against foreign antigens, e.g. infectious microorganisms. The focus of the TOLERAGE project, however, was to normalise immune reactivity by correcting the immune dysregulation developing with age which forms the basis of important age related diseases. Immune dysregulation is known to start early and to increase with age, causing a high cost to European societies in terms of morbidity and mortality.
Tolerage has paved the way for vaccinations against AT and RA focusing on HSP60 as a main culprit (auto-) antigen. Moreover, rather than inducing tolerance against the whole HSP60 molecule - an approach that may compromise defence against infections - atherogenic and arthritogenic HSP60 epitopes have been defined and will be used as tolerising compounds. The leading role of HSP70 in protecting animal models from more severe RA necessitated a combined focus on both of these heat shock proteins.
Highlights of the TOLERAG project were the following findings
Atherogenic and atheroprotective epitopes of HSP60 were identified, and feeding of anti-CD3 antibody to mice with HSP60 induced atherosclerosis resulted in a marked reduction in lesion size.
Peptide fragments of HSP70 (such as B29) can be developed as novel immunotherapeutic vaccines in rheumatoid arthritis, and some (food derived) compounds have a capacity to co-induce HSP expression and improve vaccination results.
Age-related changes in part controlled by miRNAs in the thymic microenvironment and in gene expression patterns are conducive to subvert the central tolerance process.
Inhibition of local GC synthesis delays/reverses involution although the biological implication of this on autoimmune responses is weak
Induction of apoB100-specific Treg responses reduces the development of atherosclerosis in young mice and blocks disease progression in old animals.
SOCS3 expression in atherosclerosis increases with age. We found that SOCS3-controlled Th17 cells play a major role in the protection against atherosclerosis.
MHV vector-mediated delivery of IL-10 to DCs is feasible both in vitro and in vivo, and MHV vector-encoded IL-10 stimulates CD4+ T cell responses in vivo.
Newly generated iTreg cells are retained in cutaneous lymph nodes via a mechanism involving CCR4/CCL22.
Regional steady state migratory DC-induced iTreg cells protect from autoimmunity
PTX3 levels increase with aging in mice and humans, and are a biomarker for atherosclerosis.
The clinically relevant outcome of this project is a significant advancement in the knowledge of how and when to induce tolerance to auto-antigens responsible for age-relevant disease.
Project Context and Objectives:
Reconstitution of normal immune function in old age is generally based on boosting the response against foreign antigens, e.g. infectious microorganisms. The focus of the TOLERAGE project, however, was to normalise immune reactivity by correcting the immune dysregulation which forms the basis of important age related diseases. Dysregulation is known to start early and to increase with age, causing a high cost to European societies in terms of morbidity and mortality.
TOLERAGE used novel approaches to normalise immune function at old age on three complementary levels. The paradigmatic age related diseases that form the focus of this proposal are atherosclerosis (AT) and rheumatoid arthritis (RA).
1)The first of these was to look into the basic mechanisms of age-related changes in immune function, concentrating on thymic self-antigen display, T cell selection of autoreactive T cells, effects of aging on regulatory T cells (Treg), hormonal influences (particularly glucocorticoids - GC), and the action of cytokines in aging organisms. The knowledge gained of how the immune system ages will eventually be able to be used to focus and fine tune the regulation of the immune system, including inducing tolerance towards HSP60.
2)The second part of the project focused on two paradigmatic age related diseases, atherosclerosis (AT) and rheumatoid arthritis (RA), investigating in detail the effect of aging on the autoimmune reaction to heat shock protein 60 (HSP60), which has been shown to be an important auto-antigen in both AT and RA. In particular, knowledge from research groups engaged in the first part was able to be directly used by other partners to induce tolerance to HSP60 in mouse models of both AT and RA at various ages and disease stages.
3)The third element, the identification of the mechanisms responsible for a normalisation of the immune response after appropriate intervention (e.g. nasal/oral tolerance) on the cellular level, was an important practical component of the project. Investigations elaborating the mechanisms responsible for tolerance addressed the role of central and peripheral deletion of T cells, the action of Treg cells, the balance of immune regulatory cytokines, and hormonal (GC) influences during the aging process.
The developing immune system is increasingly prone to develop into a dysbalanced state, especially in the Western world due to environmental influences. This dysregulation is contributing to raised frequencies of various immune system-mediated diseases such allergies, chronic autoimmune conditions, and inflammatory bowel diseases. One heavily debated issue here is the possibility that a relative lack of exposure to infection causes deficient regulation of the immune system, known as the 'hygiene hypothesis'. Whatever the underlying factors may be, the repeated exposure of the adaptive immune system to microbial antigens is now widely accepted as a factor that contributes to immune fitness and production of immune balance. Since HSP60 is one of the immuno-dominant components of commensal gut microbiota, we have used HSP60 as a prototype microbial-human cross-reactive antigen to promote T cell mediated immune regulation. An immunological approach to prevent and/or treat these autoimmune diseases would require a down-regulation of pathologic immune reactivity to HSP60 or an up-regulation of anti-inflammatory regulation at the level of HSP60 or HSP70. Here the requirement is not only a down regulation of the immune response, but a targeted up-regulation of the response to protective HSP60 and HSP70 epitopes. This can be achieved by special vaccination strategies leading to anti-inflammatory tolerance targeted towards HSP60 or HSP70. It was the aim of TOLERAGE to provide the theoretical basis in vitro and in mouse models for specific tolerance induction to heat shock proteins 60 and 70 to reduce the severity of RA and AT, or even prevent the diseases altogether. In addition, the restoration of tolerance in old mice by means of hormonal, cytokine, soluble antigen, or antigen presenting cell (DC) targeted interventions was also investigated, to help build the foundations of possible treatment regimes.
Progress beyond the state-of-the-art
Vaccination against age-associated, primarily non-infectious, diseases has been a dream of gerontologists and geriatricians for a long time. Partial success in this area, including studies in experimental animals and approaching clinical applications in humans has already been achieved with a vaccine against Alzheimer's disease. Tolerage was to lay the ground for vaccinations against AT and RA focusing on HSP60 as a main culprit (auto-) antigen. Moreover, rather than inducing tolerance against the whole HSP60 molecule - an approach that may compromise defence against infections - atherogenic and arthritogenic HSP60 epitopes have been defined and will be used as tolerising compounds. The leading role of HSP70 in protecting animal models from more severe RA necessitated a combined focus on both of these heat shock proteins. Tolerage has opened a completely new and original line of research that is not only important from a scientific perspective, but also has tremendous social potential taking into account the medical and socioeconomic impact of AT and RA.
In the initial period of TOLERAGE, the expertise available in the consortium was combined to study mechanisms of central and peripheral tolerance in general, and HSP60 in particular. Emphasis was put on DC, Teffector and Tregulatory cells, their interaction with each other, and how this is affected by aging. The age-related alterations in these cells and how they interact, including the mediator involved such as cytokines, chemokines, and glucocorticoids in the thymus and periphery were of particular interest. The knowledge gained from these studies has produced important insights into the mechanisms influencing the aging immune system.
Project Results:
Workpackage 1: Basic mechanisms of central and peripheral tolerance
Introduction and objectives
The two major objectives of WP1 were:
explore how central tolerance towards tissue-specific self-antigens - notably known target auto-antigens of major autoimmune diseases - might change qualitatively or quantitatively during the aging process and thus predispose to age-related autoimmunity.
assess natural tolerance/immunity to the self-antigen Hsp65/70 involved in the auto-immune inflammatory aspect of arthritis and atherosclerosis and induce peripheral tolerance towards Hsp65/70 as a measure to interfere with both diseases.
Results
Three age groups of C57BL/6 mice - 6 weeks, 12 and 18 months - were compared. There was an over-proportional loss of medullary thymic epithelial cells (mTECs) over cortical thymic epithelial cells (cTECs). Within the mTEC lineage we observed a selective loss of the mature subset vs. the immature subset. Since the mature mTECs show the highest level and diversity of pGE, this loss amounts to a proportional reduction in the availability of self-antigens in the thymus. Notably, expression of a selected set of tissue-restricted antigens (TRAs) on a per cell basis including Hsp60 and Hsp70 did not decline with age. We currently, however, cannot exclude more subtle qualitative or quantitative changes of pGE at the whole genome level during aging. Respective gene array expression studies are under way.
Apart from the number of thymic APCs, the efficacy of negative selection is also dependent on the 3D array of mTECs in situ. Our immuno-histological analysis of the aged thymus clearly revealed a highly disarrayed distribution of mTECs along with a loss of a clear cortex/medulla demarcation. It is to be expected that this altered microenvironment affects the scanning process of thymic APCs by thymocytes and thus the efficacy of tolerance induction.
Overall, our detailed analysis of mTECs in the aging thymus revealed clear age-related changes in the composition and developmental dynamics of this compartment, in particular the reduced proportion of mature mTECs and their disarrayed distribution in situ are conducive to compromise central tolerance. Importantly, as far as the observed age-related alterations pertain to ex vivo analysis, the data obtained in mice closely paralleled those in humans.
Taken together these data from WP1 showed that:
Age-related changes in part controlled by miRNAs in the thymic microenvironment and in gene expression patterns are conducive to subvert the central tolerance process.
Peripheral tolerance induction towards intact Hsp65 and peptides of Hsp70 can prevent atherosclerosis and suppress experimental arthritis, respectively.
These new insights open options to interfere with the process of thymic involution, on the one hand, and on the other hand the development of age-related autoimmune diseases.
Workpackage 2.Thymic involution: The role of locally produced glucocorticoids (GC) - Selective inhibition of intra-thymic GC synthesis.
Introduction and objectives
The thymus gland regresses (involutes) during aging, starting early in life and progressing in particular at the time of puberty. T cells are positively and negatively selected in the thymus in order to allow the formation of naive T cells that will not react against self antigens. Central tolerance mechanisms involve the removal of self-reactive T cells and the formation regulatory T cells (Treg) which can induce tolerance in the periphery. Thymic involution leads to a decline in negative selection and in Treg formation as well as in the output of naive T cells. For that reason thymic involution is linked to the development of autoimmune diseases. The reasons why the thymic gland involutes is not clear although there is a consensus that it is caused by a tissue degeneration dependent on changes in hormones, growth factors and cytokines. We have earlier found that the epithelial component in the gland (TEC) synthesize glucocorticoids (GC) in very young mice and that such a synthesis can be detected in the thymocyte population at around the time of puberty. As GC very efficiently kill thymocytes by an apoptosis reaction, it is possible that thymocyte-derived GC play an important role for the thymic involution process.
WP2 aims to show
how the synthesis of the thymocyte and locally produced GCsis regulated
if there is a role for locally produced GC in the thymic involution process
if so, to use this knowledge to delay/prevent involution and then test the effect of this in models for autoimmunity (WP12).
Results
Initial studies established a co-culture method to asses GC synthesis and secretion by co-culturing thymic cells with a reporter cell in which luciferase activity could be activated by co-expressed GR and a GR-controlled reporter gene. The ability of thymic cells to synthesize and secrete corticosterone was confirmed by an ELISA. The ability of thymic cells to synthesize GCs was also demonstrated by the ability of metyrapone, an inhibitor of CYP11B1, the last enzyme involved in the formation of corticosterone, to block GC synthesis. Furthermore, all the necessary enzymes required for the formation of GCs from the mother substance cholesterol was found in thymocytes. Interestingly, the expression of several, including rate limiting, enzymes as well as GC secretion by thymocytes started at the age 4 weeks in the mice, coinciding with the start of the age-dependent thymic involution. GC production increased further with increasing age.
We also studied factors that regulated thymocyte GC production. In contrast to adrenals where ACTH stimulates GC synthesis, ACTH down-regulated GC production from thymocytes as assayed both in culture and in vivo. As high systemic GC levels promote apoptosis of peripheral T cells, the decreased GC production in the thymus and thus a decreased thymocyte apoptosis may represent a way to guarantee feeding of sufficient T cells formed in the thymus into the periphery. Additional studies also demonstrated that testosterone induced local GC production in the thymocytes, and that this testosterone-induced thymic atrophy was at least in part due to testosterone-induced GC synthesis in the thymocytes. This was most likely mediated by TNFa secretion induced by testosterone. The testosterone-stimulated synthesis of GCs in the thymus may contribute to the faster thymic involution seen in males. In contrast, high doses of estrogen administration, which also induces thymic involution, did not mediate this effect by affecting GC synthesis of the thymocytes. We also explored transcription factor expression in thymocytes known to be involved in steroidogenesis in classical steroidogenic tissues. Steroid factor 1 (SF-1), liver receptor homolog 1 (LRH-1) and transcriptional regulating protein of 132 kDa (Trep 132), which are all known to be involved in the regulation of the expression of genes involved in steroidogenesis including GC synthesis, was found to be expressed in thymocytes. However, SF-1 was expressed as a spice variant with a truncated form.
The outcome of this WP clearly demonstrated that de novo local GC synthesis occurs in the thymus, how it is regulated and that it impacts thymocyte homeostasis by enhancing/promoting thymic involution. Inhibition of local GC synthesis delays/reverses involution although the biological implication of this on autoimmune responses requires further studies.
Workpackage 3: T effector/Treg imbalance with increasing age
Introduction and objectives
This part of the project is based on the hypothesis that an imbalance between Teff and Treg cells leads to a defective control of local inflammatory responses with increasing age. In the long-term, impaired immune surveillance may result in increased tissue damage and predispose to opportunistic infections, cancer, as well as immune-mediated diseases such as AT and RA. Teff and Treg compartments will be studied in vivo and in vitro using young and aged mice.
Results
We found reduced suppressive properties of Treg cells isolated from aged vs young mice, only when mice were housed under conventional conditions. No defect of Treg function was observed in aged mice housed under specific pathogen-free conditions.
We showed that induction of apoB100-specific Treg responses reduces the development of atherosclerosis in young mice and blocks disease progression in old animals.
WP4: The role of dendritic cells (DCs) in tolerance induction
Introduction and objectives
Dendritic cells are central players in the decision making process of immune activation vs tolerisation. The major aim of WP4 was to generate safe and efficient coronavirus-based vaccine vectors which are able to deliver antigens in combination with immune-modulating cytokines to transduce dendritic cells and closely related antigen presenting cells, such as macrophages, in vivo.
The major objectives of WP4 were:
To generate and characterize recombinant murine coronavirus-based vectors expressing HSP60 and/or another autoantigen together with an immune-suppresive cytokine such as IL-10 or TGF-beta
To assess the in vitro and in vivo gene transfer of murine coronavirus-based vectors into dendritic cells.
Results
To assess immunogenicity and to ensure maximal safety of coronavirus-based multigene vectors, we have rationally designed a series of vectors based on the mouse hepatitis virus, strain A59 (MHV-A59). With regard to safety, we have introduced three modifications to obtain replication-competent, but attenuated and propagation-deficient MHV-based vectors: (i) deletion of all MHV-encoded accessory genes (NS2, HE, gene4, gene5a), (ii) removal of 99 nucleotides within the replicase-encoded sequence of the non-structural protein 1 (nsp1), and (iii) deletion of the structural gene E to restrict proper particle formation. Since the activation status and the immunstimulatory properties of DCs can be modified by cytokines, we have inserted the gene encoding for the immune-activating murine granulocyte-macrophage colony-stimulating factor (GM-CSF) or the immune-modulatory interleukin 10 (IL-10) between the MHV vector-encoded replicase and spike genes. To demonstrate the feasibility of the approach, we have first generated a model construct containing a well-characterized viral epitope derived from the glycoprotein (GP) of the lymphocytic choriomeningitis virus (LCMV), fused with the enhanced green-fluorescenct protein. The resulting vectors have been termed
MHV-GP
MHV-GP/GM
MHV-GP/10
Furthermore, cloning of MHV-hsp60, MHV-hsp60/GM, MHV-hsp60/10, MHV-hsp70, MHV-hsp70/GM, MHV-hsp70/10 in vaccinia virus shuttle vectors had been completed, but rescue of MHV vector particles had failed for all vectors encoding hsp60 and hsp70. Several rescue strategies were initiated, but none of these strategies resulted in the propagation of sufficient amounts of hsp- or hsp epitope-endoding MHV-based vectors. From these experiments we concluded that the presence of hsp60 or hsp70 in the genome of the coronavirus-based vectors is not compatible with coronavirus replication. We therefore resorted to an alternative strategy and inserted the bacterial antigen b-galactosidase (bgal) into the vectors which could be successfully rescued. We succeeded to amplify to high titers in cells complemented with the viral E-protein the following bgal-encoding vectors:
MHV-bgal
MHV-bgal /10
Next, we assessed the in vivo targeting efficacy of MHV vector and the impact of the cytokine GM-CSF and IL-10 on DCs. We found that MHV-based vectors almost exclusively delivered their antigenic cargo to DCs promoting the induction of transgene-specific T cell responses. GM-CSF substantially improved the immunogenicity of the vaccine with approximately 3-fold higher expansion of antigen-specific CD8+ T cells. Moreover, the MHV-based vaccine could be applied via different routes and still retained its high immunogenicity which is mainly due to an enhanced maturation of DCs and prolonged antigen presentation within these cells. The results of these proof-of-principle studies were published in 2010 in the open access journal mBIO.
Concerning the effects of IL-10, we found only a mildly attenuating effect of the IL-10-encoding MHV vector on the in vivo DC transduction efficacy indicating that the system is well-suited to target both an antigen and the immune-modulatory cytokine IL-10 to DCs. Furthermore, although in vitro experiments showed that specific IL-10 delivery can to some extent inhibit DC maturation, these effects could not be demonstrated in vivo, i.e. unlike in the in vitro experiments, in vivo DC transduction with IL-10-encoding vectors did not significantly impact on DC maturation, i.e. the maturation markers CD86 and CD40 were not down-regulated compared to DCs transduced with the vector not encoding a cytokine. To assess the impact of vector-encoded IL-10 on the induction of CD8+ T cells, we immunized C57BL/6 mice with 106 pfu of the different GFP-GP34-encoding vectors. We confirmed that GM-CSF efficiently enhanced the immunogenicity of the vector, whereas IL-10 led to a mild attenuation. However, the immune-attenuating effect of IL-10 delivered to DCs on CD8+ T cells was not detectable at later time points post immunization. Likewise, immunization with bgal-expressing MHV vectors failed to reveal an immune attenuating effect on CD8+ T cells. Furthermore, IFN-g production by bgal-specific CD8+ T cell was not affected by the absence or presence of IL-10 in the MHV vector indicating that, despite a clearly detectable effect on DCs in vitro, MHV vector-mediated in vivo transfer of IL-10 to DCs only mildly affected the generation of vector-specific CD8+ T cells.
Taken together, the results obtained in WP4 showed that
MHV vector-mediated delivery of IL-10 to DCs is feasible both in vitro and in vivo,
MHV vector-encoded IL-10 negatively affects DC immune functions in vitro, and
MHV vector-encoded IL-10 stimulates CD4+ T cell responses in vivo.
WP5: The role of DC in antigen presentation
Introduction and objectives
A common vision of how dendritic cells (DCs) contribute to the induction and maintenance of peripheral CD4+ T cell tolerance is that, in resting conditions, immature DCs, expressing low levels of signal 1 (specificity) and low or no levels of signal 2 (costimulation), are able to induce T cell unresponsiveness.
The major objectives of WP5 were:
Elucidation whether chronic antigen exposure and/or state of DC activation are parameters relevant for the induction and maintenance of peripheral T cell tolerance
Based on the identified genes differentially expressed in lymph node and splenic DCs, elucidation of the molecular mechanisms through which lymph node DC and/or spleen DCs are able to tolerise auto-reactive T cells.
Results
In order to know whether DCs in general are able to induce T cell tolerance at the steady state or if this is a prerogative of specialized subsets, we took advantage of an experimental system where antigen presentation, in contrast to previous studies, is not a priori confined to a specific DC subpopulation but is extended to all conventional DC subtypes. Specifically, we adopted the 2a T transgenic animal model in which T cell receptor transgenic T cells (2a T cells) recognize a portion of the CH3 region (435-451) of the IgG2ab, the Bpep, in association with the MHC molecule, I-Ad. We also generated a mouse model in which the Bpep was presented by CD11c+ cells that include all conventional DC subtypes.
By performing a systematic study of the behaviour of naive autoantigen-specific T cells after interaction with all conventional CD11c+ DCs in homeostatic conditions we found that DCs are able to induce CD4+ T cell tolerance by promoting the conversion of autoantigen-specific naive T cells into iTreg cells. Among the different DC subtypes, steady state migratory DCs (ssmDCs) possessed unique ability to induce antigen-specific iTreg cells. Diversely, lymphoid tissue resident DCs did not show this capacity. Therefore, iTreg cells developed solely in lymph nodes and not in the spleen, which does not host the migratory DC subtype. We also found that ssmDCs were able to induce iTreg cell conversion in a RA-dependent manner and that newly generated iTreg cells were not able to re-circulate but resided in the lymph nodes where they were generated.
Finally, it was evaluated whether iTreg cells could develop in inflammatory conditions. Using GpG as an inflammatory stimulus we observed that the generation of iTreg cells occurred in lymph nodes irrespective of the fact that the animals were treated with inflammatory substances. Nevertheless, in steady state conditions iTreg cell differentiation was already apparent 5-10 days after antigen encounter. Diversely, in inflammatory conditions, iTreg cells were only visible starting from two weeks after cognate antigen recognition. Interestingly, activated DCs were observable in the lymph nodes until day 6-8 after T cell transfer, therefore the appearance of iTreg cells correlated with the disappearance of activated DCs. Given the transient presence of the inflammatory stimulus, these data strongly suggest that iTreg cells differentiate only when non-activated migratory DCs reach the draining lymph nodes.
Taken together, the results obtained in WP5 showed that
In vivo conventional DCs induce auto-antigen CD4+ T cell tolerance exclusively via the induction of auto-antigen-specific iTreg cells
When in vivo auto-antigen presentation is extended to all CD11c+ conventional DCs, immature lymphoid tissue resident DCs are unable to tolerize CD4+T cells while this is a prerogative of steady state migratory DCs that reach the lymph nodes. These cells induce iTreg cell generation in a RA dependent manner. Consequently, the conversion occurs only in lymph nodes and not in the spleen
Newly generated iTreg cells are retained in cutaneous lymph nodes via a mechanism involving CCR4/CCL22.
Regional steady state migratory DC-induced iTreg cells protect from autoimmunity
WP6 Generation of regulatory dendritic cells in vitro using glucocorticoids (GC).
Introduction and objectives
GC can contribute to the formation of regulatory dendritic cells (DCreg) by inhibiting the maturation of DC, leading instead to the formation of DCreg. We propose an exploration of the use of GC for the generation of mouse DCreg in vitro, and the further applicaton of these cells in the treatment of autoimmune diseases RA and AT.
The formation of regulatory dendritic cells (DCreg) will be induced using GC, which can contribute by inhibiting the maturation of DC, leading instead to the formation of DCreg.
Results
We have devised a method to generate tolerogenic DC in vitro by treatment of cells with the synthetic glucocorticoid dexamethasone (DC-dex) and characterized these by functional assays and phenotypic markers. Bone marrow-derived DC was generated in vitro in the presence of recombinant GM-CSF and IL4, with DCreg produced and by the later addition of dexamethasone to the cultures. DC-dex had an enlarged size and fewer dendritic extensions and supernatants from DC-dex cultures showed a reduction of IL12p40 and an increase in IL-10 synthesis. In contrast, whereas immunogenic DC generated by CpG activation (DC-CpG) proliferated and showed signs of activation and cluster formation, DC-dex expressed downregulated levels of MHC-II and the co-stimulatory molecule CD86, whereas DC-CpG expressed increased levels of both these membrane structures, as expected. The addition of CpG to dexamethasone treated DC for the last 24 h did not reverse the GC induced phenotype to any extent. We conclude that the different DC expressed the expected phenotypes for tolerogenic or immunogenic DC, respectively, and that the DC-dex phenotype was mostly irreversible.
We investigated the effect of DC-dex on T, Treg and NK cell responses in OT-1/Rag-/- mice, which express a transgenic TCR in CD8+ T cells, comparing these responses with the effect of immunogenic, DC-CpG. The OT-1/RAG-/- T cell responses to the different DC were tested in vitro and in vivo. In vitro co-culture of OT-1/RAG-/- cells with peptide treated DC showed that DC-CpG stimulated IFN-gamma synthesis in these as well as IL12p40 secretion. In contrast, DC-dex induced IL-10 synthesis and the expression of Foxp3 as well as a modest IFN-gamma response in these cells. DC-CpG, but not DC-dex, also generated cytotoxic OT-1/RAG-/- T cells which killed EG7 tumor cells, expressing the OVA derived peptide epitope recognized by the transgenic OT-1 cells, but not the parental EL4 cells. DC-dex also generated a Treg response which suppressed the OT-1/RAG-/- T cell proliferation in response to peptide treated antigen-presenting cells. Mice injected with DC-CpG expanded and activated splenic NK cells in B6 mice that had an increased expression of the NKG2D receptor but an essentially unaltered expression of differentiation markers Mac-1 and CD27. In vitro, as well as in vivo, these activated NK cells killed the prototype NK cell target YAC-1. In contrast, DC-dex injection did not lead to any of these responses in splenic NK cells. In conclusion, we have found that GC induced tolerogenic DC do not activate T or NK effector cells but that they induce a Treg response in OT-1 mice. These CD8 positive Tregs most likely express CD122 but not CD25. CD122, being the beta chain of the IL2 receptor, is a marker for natural CD8 positive Tregs that play an important role for the maintenance of T cell homeostasis and which, by producing IL-10, suppress the IFN-gamma production and proliferation of CD8+ T cells.
WP7 Age dependent changes of the cytokine balance.
Introduction and objectives
The main objective of WP7 was to define changes in cytokine balance during aging. In particular, focus has been on the long pentraxin PTX3, a fluid phase pattern recognition receptor which has complex functions in innate immunity, extracellular matrix construction, and regulation of inflammation. Data collected so far suggest that PTX3 may represent a novel marker of innate immune-inflammatory reactions that better represent the events in involved tissues, and in particular the vascular wall. In the context of these studies, the role of PTX3 in pathologies associated with aging has been investigated at a basic research level in humans and in mice. The inflammatory status is the result of a balance between inducers and inhibitors of the inflammatory response. Decoy receptors recognize inflammatory cytokines with high affinity and specificity, but they are incapable of signaling, thus representing a strategy to regulate inflammatory cytokines and chemokines. Work done by this group has identified an isoform of IL-1 receptor antagonist (IL-1ra) and the soluble form of type II IL-1R (sIL-1-RII) as decoy molecules.
It has been reported that the balance of anti- and pro-inflammatory factors is altered in aged people, with IL-1Ra increasing and IL-10 decreasing during aging. The present project thus has also investigated whether other IL-1-related molecules, such as IL-1R type II that acts as a decoy for soluble IL-1, are modulated in aging in humans.
The major objectives of WP8 were:
To standardize the protocol for measurement of PTX3 (both in humans and mice) based on original antibodies and recombinant proteins developed by P6
To define the changes in PTX3 levels during aging both in humans and mice
To define the changes in Il-1ra and sIL-1RII levels during aging both in humans and mice
Results
To start with the measurements of PTX3 in aged and young humans and in age-related pathologies, protocols for PTX3 determination were standardized. We did some modifications of the assay described in already published papers (see for examples Latini R, et al. (2008) Eur Heart J 29 (Suppl 1): 393 (abs). and Peri G, et al. (2000) Circulation 102: 636-641). Main improvements are: a) very good reproducible results were obtained when samples were tested immediately after thawing or 24 hours later, variations between the two measures ranging from 0% to 25% maximum, with a median value of 7%; b) more stability after repeated thawing, the same mean values being obtained after 1, 2 or 3 thawing before measurement; c) the detection limit is 0.1ng/ml.
Having set up the measurement systems for human and murine PTX3, levels were investigated in humans and mice of different ages. While there are no significant differences between children and adults, the levels of PTX3 in the greater than 90 years group are significantly higher (mean; 2.2x0.2 ng/ml) than in the other two groups. Similarly in the mice we had a significant increase in PTX3 levels with aging.
The measurements of sIL-1RII and IL-1ra where then performed in the same blood samples from mice and humans. While no significant changes were observed in IL-1ra levels, sIL-1RII levels significantly decreases with aging both in humans and mice.
Taken together, the main results of WP7 showed:
PTX3 levels increase with aging in mice and humans
sIL-1RII levels decreases with aging in mice and humans
no significant changes were observed for IL-1ra levels in relation to age.
WP8 Rheumatoid arthritis
Introduction and objectives
Rheumatoid arthritis and associated inflammatory conditions are major autoimmune disorders of the ageing population. It was the purpose of WP8 to analyze the role of HSP associated immune-regulation in the incremental susceptibility of the aged individual with respect to these diseases. Moreover, in experimental models a search was made for possible interventions that could lead to improved HSP mediated down-modulation of these inflammatory diseases. The major objectives of WP8 were:
To analyze the role of HSP in the inflammatory process of joint diseases
To define biomarkers of the inflammatory process associated with arthritis
To discover and analyze novel routes of intervention to compensate for the loss of HSP up-regulation of the aged individual
To devise novel therapeutic means to stimulate immune reactivity towards HSP
To analyze the immune-modulatory mechanisms of arthritis in relation to HSP
Results
In the analysis of the role of HSPs in arthritis the primary focus on HSP60 was changed into HSP70 already in the beginning of the Tolerage project. This was done for scientific reasons, mainly based on the fact that immune reactivity in the proteoglycan induced arthritis model seemed dominated by responses to HSP70. In addition, stress dependent up-regulation of HSP70 during inflammation was more significant for HSP70 than for HSP60. In synovium of the inflamed joints and in several lymphoid tissues the up-regulated HSP70 family members were documented and T cell reactivities, which included responses of cells with markers of Tregs, were detected in the spleen and draining lymph-nodes. In addition, antibody profiles were seen to be compatible with immune reactivity for HSPs expressed in the inflammatory tissues.
Besides HSP reactivity being found as a biomarker of the arthritic inflammatory process, PTX3 was also analysed as a possible biomarker. Taking advantage of Ptx3-/- mice, the role of PTX3 has been investigated in a murine model of collagen-induced rheumatoid arthritis. The results evidenced a higher susceptibility to the disease in mice lacking the protein. Moreover, PTX3 levels were measured in different mice models of arthritis. Levels of the protein correlated with the severity of disease in mice, with collagen induced arthritis (CIA). In addition, P6 have measured PTX3 levels in 345 samples collected from mice with PG-induced arthritis (PGIA) treated with different protocols of CD4+CD25+Tcells. When similar experiments were pooled together, a significant correlation was observed between PTX3 levels and clinical score, further supporting the role of PTX3 as biomarker of innate-immune inflammatory reactions.
Especially food- or herb-derived phytonutrients may be attractive compounds to restore optimal Hsp expression in response to stress. In TOLERAGE, we explored three readout systems to monitor Hsp70 expression in a manner relevant for the immune system to evaluate novel Hsp co-inducers. First, intracellular staining and analysis by flow cytometry was used to efficiently detect stress and/or dietary compound induced Hsp70 expression in multiple rodent cell types. This system was used to screen a panel of food-derived extracts with potent anti-oxidant capacity. This strategy yielded the identity of several new enhancers of stress-induced Hsp70 expression, among them carvacrol, found in thyme and oregano. Second, CD4(+) T-cell hybridomas were generated that specifically recognized an immunodominant Hsp70 peptide. These hybridomas were used to show that carvacrol enhanced Hsp70 levels and increased T-cell activation. Third, we generated a DNAJB1-luc-O23 reporter cell line to show that carvacrol increased the transcriptional activation of a heat shock promoter in the presence of arsenite. These assay systems are generally applicable to identify compounds that affect the Hsp level in cells of the immune system.
Carvacrol, a major compound in the oil of many Origanum species, had a notable capacity to co-induce cellular Hsp70 expression in vitro and, upon intragastric administration, in Peyer's patches of mice in vivo. As a consequence, carvacrol specifically promoted T cell recognition of endogenous Hsp70, as demonstrated in vitro by the activation of an Hsp70-specific T cell hybridoma and in vivo by amplified T cell responses to Hsp70. Carvacrol administration also increased the number of CD4+CD25+FoxP3+ T cells systemically in the spleen and locally in the joint, and almost completely suppressed proteoglycan-induced experimental arthritis. Furthermore, protection against arthritis could be transferred with T cells isolated from carvacrol-fed mice, showing the T cell nature of the arthritis suppressing effect of carvacrol. Interestingly enough, these findings illustrate that a food component can boost protective T cell responses to a self stress protein and down-regulate inflammatory disease, i.e. that the immune system can respond to diet.
Taken together, the main results of WP8 showed:
HSP70 is a dominant antigen in rheumatoid arthritis
Carvacrol and similar HSP70 co-inducers can compensate for the loss of HSP reactivity of ageing
Peptide fragments of HSP70 (such as B29) can be developed as novel immunotherapeutic vaccines in rheumatoid arthritis
Blood levels of PTX3 can represent a new useful marker to monitor rheumatoid arthritis
WP9 Atherosclerosis
Introduction and objectives
WP9 dealt with one of the main goals of TOLERAGE, i.e. the development of an HSP60-based vaccine for the prevention of atherosclerosis in a mouse model. Additionally, the role of GC in atherosclerosis and the potential of PTX3 as a new biomarker for atherosclerosis were investigated. The objectives of WP9 were:
To define the pro-atherogenic or atheroprotective HSP60 T cell epitopes in the murine system
evaluate the role of endogenous GC on T cells in atherogenesis
the value of PTX3 as a novel of prognostic marker of AT in humans
Results
In order to investigate the impact of GCs on T cells for the development of atherosclerosis, an mouse model was develop in which GC sensitivity of T cells was altered in the ApoE KO mice. In female Lck-GR/Apo E KO mice no difference in the development of atherosclerosis could be observed, this despite an increased GC sensitivity of the T cells and a reduced T cell number. No clear effect on Treg number or cytokine profile could be detected. In male mice, increased atherosclerosis was observed, contrary to the hypothesis. This was most likely due to an increased general low grade inflammatory activity measured as increased expression of acute phase reactants in the liver. The reason for this observation is not clear but is most likely an unfavorable combination of the particular transgene used to increase GC sensitivity and male specific factors. Based on the female mice where no systemic inflammation was seen, we conclude that a selective increase in GC sensitivity of T cells in itself does not affect atherogenesis. Based on the results from the female mice, we conclude that the increased incidence of AT observed following systemically high and long-term GC exposure is not mediated by GC-effects on T cells but rather through other (e.g. metabolic) mechanisms.
The possible role of PTX3 in the pathogenesis of atherosclerosis has been investigated by generating double-knockout mice lacking PTX3 and Apolipoprotein E. When these mice were fed an atherogenic diet, they developed more aortic lesion compared to single KO animals. In addition, a more pronounced inflammatory profile, as evidenced by microarray analysis of vascular wall specimens, was obtained from double-knockout mice compared to single KO animals. Lack of PTX3 is also associated with higher tissue damage in a model of acute myocardial infarction in mice. Taken together, these results suggest that PTX3 may have a protective role through modulation of the immunoinflammatory balance in the cardiovascular system.
Inflammation plays a major role also in the progression of chronic heart failure (HF), thus a second large cohort was analyzed, derived from two independent studies: the Controlled Rosuvastatin Multinational Trial in HF (CORONA 1233 patients) and the GISSI-Heart failure trial (GISSI-HF). The results confirmed the association between PTX3 levels and age. In addition, baseline elevated PTX3 were associated with higher risk of all-cause mortality or cardiovascular mortality and with fatal events, confirming the usefulness of PTX3 as biomarker in human pathologies.
WP10: Induction of immunologic tolerisation to autoantigens
Introduction and objectives
Within WP10, a number of activities have been integrated to assess whether tolerisation against HSP60 or alternative antigens ameliorates atherosclerosis or rheumatoid arthritis. It was planned to assess the therapeutic usage of arthritogenic and atherogenic T-cell epitopes in atherosclerosis and rheumatoid arthritis model systems. Due to the longer than anticipated time required to produce these epitopes, alternative approaches were established and evaluated. The major, partially revised objectives of WP10 were:
To tolerise mice using MHV-based vectors containing a bacterial antigen and cytokines.
Characterization of the immunotherapeutic capacity of in vitro generated DCreg in vivo.
To determine effects of HSP-inducing compounds in combination with other delivery systems.
To assess whether PTX3 can serve as a correlate for therapy-induced tolerance
To document the efficiency of HSP60-specific Tr1 cells in the treatment of RA and AT in a mouse model.
Results
Since MHV-based vectors expressing either hsp60 or hsp70 could not be generated,the full-length b-galactosidase (bgal) gene, as a substitute bacterial antigen, has been utilized for the in vivo assessment of antigen-specific tolerisation in hypercholesterolemic mice. To assess whether the presence of bgal in the vasculature together with hypercholesterolemia affects the activation pattern of bgal-specific Th cells, transgenic mice expressing the bgal antigen specifically in arterial smooth muscle cells were crossed onto the ApoE-/- background. The results from these experiments revealed that IL-10 encoded by the MHV vector fostered the activation of the bgal-specific T-helper cells, even under conditions of hypercholesterolemia and expression of the antigen in the vasculature. Furthermore, delivery of IL-10 to DCs by the MHV vectors neither favored the differentiation of bgal-specific Th cells towards the Treg phenotype, nor did the global FoxP3 expression in CD4+ T cells change under these conditions. Taken together, these data suggest that in vivo delivery of IL-10 to DCs in atherosclerotic mice did not favour tolerogenic conditions, even when the MHV vectors primed Th cells specific for a vascular antigen.
Since the start of the Tolerage project, published work has shown that tolerogenic DC produced by dexamethasone treatment alone had little effect on collagen induced arthritis (CIA) and that alternatively induced tolerogenic DC (by IL-10) had a very potent effect on plaque formation in hypercholesterolemic mice. For these reasons we have tested DC-dex in another model for autoimmunity (chronic multiple sclerosis, MS) i.e. experimental autoimmune encephalomyelitis (EAE). We found no beneficial effect after treatment with DC-dex or with the tolerogenic macrophages in this EAE model. From this we speculate that DC-dex may lack the capacity to enter the CNS and to exert a tolerogenic function locally. Due to these negative results, we decided to explore alternative ways of producing tolerogenic DC in vitro, by exposing them to combinations of dex, vitamin D3, retinoic acid and TGF as described in the WP 6 report.
To evaluate PTX3 as a correlate for therapy-induced tolerance, we measured PTX3 levels in serum samples from Treg-treated mice suffering from PGIA-induced arthritis. The results obtained did not indicate the existence of a clear relationship between PTX3 plasma levels and control of the disease, at least when the analysis was conducted on samples from a long-established disease as in this setting.
Taken together, the results obtained in WP10 showed that
Vector-mediated in vivo transfer of IL-10 to DCs does not tolerise Th cells reactive against vascular antigens, even under hypercholesterolemic conditions. MHV vector-mediated delivery of IL-10 to DCs is feasible both in vitro and in vivo,
GC-induced DC dex can generate CD8- and CD4-positive Tregs with specificity for model antigens. However, DC-dex did not affect the course of experimental allergic encephalomyelitis, but rendered mice more susceptible to tumour challenge.
DCs treated with carvacrol and exposed to thermal stress induced CD4 Treg cells and attenuated severity of experimental arthritis.
HSP70-treated DC suppress peptidoglycan-induced arthritis.
PTX3 levels did not significantly correlate with disease severity and Treg-mediated disease modulation in RA models.
Subcutaneous infusion of adjuvant-free ApoB100-derived peptides to ApoE-/- mice reduced atherosclerosis through the induction of a specific Treg cell response.
In conclusion, the work performed in this work package revealed that Treg cells can exert attenuating function on the course of chronic inflammatory diseases. However, the efficacy of the disease attenuation appears to be context-dependent. Hence, targeted modulation of the effects of Treg and DC-reg cells in diverse disease settings will require further investigations to advance this field towards clinical applicability.
WP11: Mechanisms of tolerance induction
Introduction and Objectives
The main task of this WP was to identify the molecular and cellular mechanisms underlying tolerance induction in age related diseases with a special focus on the role of dendritic cells, macrophages, regulatory T cells (Tregs) and cytokines. To assess the contribution of macrophages and DCs in tolerisation protocols in atherosclerosis particular mouse models were generated that permit long-term deletion of macrophages or DC. The major, partially revised objectives of WP11 were:
To assess the role of DC and macrophages in the tolerisation process
To assses the role of HSPs on Treg induction
To assess the role of cytokines in therapy induced tolerance
Results
Two different genetically modified mouse lines have been generated that permit ablation of macrophages (ApoE-MPI) and DC (ApoE-DCI) under hypercholesterolemic conditions. Following comprehensive time course and depletion dose adjustment experiments, robust protocols for long-term depletion of macrophages or DC were established. The results of the subsequent experiments revealed that DC depletion did not significantly impact on the development of atherosclerotic lesions in ApoE-/- mice. Likewise, depletion of macrophages did not alter the course of the lipid deposition in the aortic wall during the 3 month observation period. Furthermore, cholesterol levels were not significantly changed by the ablation of either DCs or macrophages using the conditions established in this project. Immunization of these mice with IL-10 encoding MHV vectors revealed that DC depletion profoundly diminished the activation of I gal-specific Th cells, whereas macrophage depletion did not significantly alter the priming of I gal-specific Th cells. Importantly, incorporation of IL-10 into the MHV vectors augmented the activation of I gal-specific Th cells, even under conditions of macrophage depletion. Taken together, these data show that DCs are critical for the activation of Th cells in atherosclerosis-prone ApoE-/- mice. However, a long-term effect of DC or macrophage depletion on arterial lipid deposition could not be detected, suggesting that the hypercholesterolemia-dependent atherosclerosis in ApoE-/- mice does not critically depend on the presence of these cells.
Workpackage 12: Role of thymic involution in autoimmunity
Introduction and objectives
Workpackage 12 was designed to test the hypothesis that a delayed, or reversed, thymic involution can result in a better homeostatic T cell control of autoimmune reactions. Thymic involution can be reversed by several different manipulations including inhibition of local thymic GC synthesis using the drug metyrapone (as shown in WP2), castration of male mice and keratinocyte growth factor (KGF) treatment. In addition, further work in WP2 showed that although the CYP11B1 inhibitor metyrapone had a clear positive effect on the thymus, the effect of KGF seemed more profound in terms of feeding naive T cells into the periphery. In order to analyze the effect of delayed or revered thymic involution on autoimmune reactions, the objective was to:
Analyse the effect of KGF in different models for rheumatoid arthritis (RA) and the effect of metyrapone on the development of atherosclerosis (AS) in ApoE-/- mice.
Results
In one model of RA mice were treated with KGF before they were immunized with rat collagen II/CFA. Treatment with KGF was found to delay the onset of CIA in the highly arthritis-susceptible B10.QNcf1 mice. However, the protective effect of KGF was lost after a booster immunization with CII/IFA. At later stages there were no significant differences in the arthritis incidence between the study groups. In this model, we confirmed the positive effect of KGF treatment on the thymus and the spleen. In the periphery we found an outflow of naive T cells and an increased ratio CD4 positive Treg/T cells. In another RA model, mice were treated with a cocktail of monoclonal antibodies (mAbs) specific for major collagen II epitopes followed by an injection of LPS to enhance the incidence and severity of arthritis. KGF treatment was given daily. No protective effect of KGF on collagen antibody induced arthritis (CAIA) was observed.
In order to study the effect of delayed or reversed thymic involution, mediated by inhibition of local GC production, on the development of atherosclerosis, we treated ApoE KO mice that spontaneously develop atherosclerosis with metyrapone. Treatment of C57/Bl6 ApoE mice starting at an age of 10 weeks with a low dose of metyrapone in the drinking water that selectively inhibited intra-thymic GC synthesis and resulted in a reversed/delayed thymic involution did not significantly influence the development of atherosclerosis in this model. Thus, an altered thymic involution mediated by metyrapone did not affect AS development in this model. However, a large heterogeneity among the analyzed mice was seen, indicating that a clearcut conclusion may require larger experimental groups.
We conclude that in the absence of an appropriate arthritis model which more closely reflects the clinical RA development, the question whether the anti-involution effect can delay, or prevent, autoimmune arthritis development remains open. In the ApoE-/- atherosclerosis model, which is a less straight-forward model for autoimmunity, we did not find any evidence for a protective effect of increasing thymic activity with the present approach.
Potential Impact:
To our knowledge, the present project is the first attempting to address the prevention and treatment of two paradigmatic age-related diseases, AT and RA, via down-regulation, rather than boosting, the innate and adaptive immune reaction. The rationale for this approach is based on the already existing knowledge about a Teff/Treg dysbalance that develops during aging and is reflected by a gradual loss of central and peripheral tolerance.
The impact of these investigations is obvious from different points of view. The results of TOLERAGE help to explain auto-tolerance from an age-related perspective for the first time.
- The basic experimental data will be immediately translated into practical application
- The effect of glucocorticoids, the most important clinically applied immunosuppressive drugs, was not analysed on the usual basis of the induction of Teff apoptosis, but rather on the basis of Treg induction.
- The two most important age-dependent diseases, AT and RA, have been chosen as paradigmatic examples to practically apply the gained experimental knowledge.
- Development of a vaccine to these diseases has been seen as the ideal preventative measure for many years.
In summary, the impact of the positive results from TOLERAGE on the understanding of basic immunosenescence and the possible relevance of these data for human medicine is significant.
Partner 1 Medizinische Universität Innsbruck
The results obtained within the framework of TOLERAGE will be disseminated by publications in high impact journals and by presentations at scientific meetings. Special emphasis will be given to fostering cooperative publications and other presentations.
Special attention will be given to the dissemination of results to the European citizens and stakeholders eg. by presentations in printed and audio-visual media. The coordinator and the other partners of the TOLERAGE constorium already have ample experience in the matter. The fact that RA and AT are among the medically and socioeconomically most important age-related diseases - the latter even being the killer number one - these endeavors will meet prepared minds in the lay community.
Partners 1 and 7 have access to patents concerning diagnosis and treatment of AT and RA, respectively, based on the use of HSP60 and peptides thereof, e.g. for induction of oral/nasal tolerance.
Partner 2 Kantonspital St Gallen
The work of Partner 2 has shown that the generation of coronavirus-based vectors is possible and that in vivo targeting of DC by these novel vaccination tools is highly efficient and can be augmented by the immune-stimulatory cytokine GM-CSF. The results of this study have been published in 2010 in the new ASM open-access journal mBIO (2011 impact factor 5.3). Since the intellectual property on coronavirus-based vaccines is already in the public domain, we have decided to further broaden the knowledge base on this approach by publishing in an open access journal with wide distribution in the vaccination field. Further results obtained with the vector system will be published in other high level journals in the vaccination field. Intermediate results of the project have been presented at several international conferences, mainly by the PhD students involved in the project. The work performed in the project has mobilized further resources for research on this topic, for example on the use of cytokine-modified coronavirus-based vectors as vaccine against tumors. Hence, it can be anticipated that the vaccine-platform will be employed for other medically important applications.
Partner 3 Karolinska Institutet
Factors that regulate thymic involution have been characterized. Targeting these processes should provide a tool to counteract thymic involution thereby providing the peripheral immune system with fresh naive T cells for an extended period which would improve cellular immunity and immune regulation. As it is known that the normal age-dependent involution, with a loss of seeding into the peripheral system of naive T cells, contribute to immunosenescence with an increased risk for infection and most likely autoimmunity and cancer, counteracting or reversing this process should reduce the incidence of these diseases in the elderly population.
Partner 4 DKFZ, Heidelberg
Our data document that the age-related alterations of the thymic microenvironment in mice and humans, in particular the selective loss of medullary thymic epithlial cells are conducive to a loss of central tolerance. Since it has been repeatedly shown that ectopic over-expression of self-antigens in thymic APCs restores robust self-tolerance in experimental models, our results point to a potential correction of age-related autoimmunity either by replacing the aged thymus with a new organ via transplantation of thymic epithelial stem cells, which have been identified in this project, or via a more specific restoration of the mTEC compartment, e.g. by cell-specific targeting signalling pathways which induce terminal mTEC differentiation, e.g. the NFkB pathway. These avenues will be pursued within the next years.
Partner 5 Inserm
The work has led to the filing of 3 patents:
We have filed a patent describing a new model of abdominal aortic aneurysm in mice. Discussions are ongoing in order to license the patent to the industry. The model will be useful to test the efficacy of drugs on the prevention of aneurysm rupture, a major health issue in the aged population.
Partner 6 Fondazione Humanitas per la Ricerca
The main efforts of P6 were devoted to the understanding of the possible role of an innate immunity molecule, the long pentraxin PTX3, in pathologies associated with aging. PTX3 is a fluid phase pattern recognition receptor which exerts complex functions in innate immunity, extracellular matrix construction and regulation of inflammation. In the context of TOLERAGE, it has been shown that PTX3 exerts a crucial role in particular in atherosclerosis. Overall the results obtained confirmed that PTX3 may be considered a novel marker of innate-inflammatory reactions that better represent the events in the vascular wall, thus deserving further translational efforts. This is expected to have a significant impact on the diagnosis of atherosclerosis, which will enable targeted treatment of patients with known medications to reduce the risk of heart attack or stroke (eg cholesterol lowering drugs). Whether PTX3 also plays a role in atherogenesis is not yet clear, and this will be followed up in subsequent studies. There may eventually be the opportunity to develop a medication based on any role found for PTX3 in atherosclerosis.
Partner 7 Utrecht University
The Hsp70 peptide B29 that during the project was found to have a protective role in arthritis, has been patented. This patent will allow further exploration of the clinical applicability of this peptide as a therapeutic vaccine in rheumatoid arthritis patients and possibly other chronic inflammatory diseases in the future. In addition these results have been published in PNAS, a high impact journal and presented at several international conferences focused on immunology, vaccination and new therapeutic approaches in autoimmune diseases. The results regarding the induction of HSP specific regulatory T cells via the use of HSP70 co-inducers and the related mechanisms have been published or are in the process of submission.
Partner 8 UNIMIB
From a biological point of view, the results of WP5 provide an extended interpretation of the distinct capacities of the diverse DC subsets in inducing iTreg cells, which mirrors distinct requirements of spleen and lymph nodes to patrol and control T cell auto-reactivity. Specifically, this work implies that immature DCs are not able to tolerize T cells by default (simply because of the absence of signal-2, the co-stimulation) but need to receive specific conditioning. At the steady state the appropriate signals that render DC alternatively activated may be delivered by the organs/tissues where migratory DCs reside. For instance, tissue-specific stimuli could induce the expression of RALDH2.
This study provides important medical implications in terms of improvement of tolerisation or vaccination strategies. For instance, this work may contribute to the identification of optimal conditions for Foxp3+ Treg conversion to contrast autoimmune reactions emphasizing the fundamental aspect of Treg cell homing. Therapeutic vaccination strategies may benefit from the usage of CCR4 blocking agents to contrast the effect of Treg cells, especially when subcutaneous routes are used. This has very wide therapeutic potential across many diseases that have an immune (especially auto-immune) component.
Partner 9 Genopolis
Microarray platforms require analytical pipelines with modules for data pre-processing including: data normalization, statistical analysis for identification of differentially expressed genes, cluster analysis, and functional annotation. Accordingly, we developed the Automated Microarray Data Analysis (AMDA, version 2.3.5) pipeline, to process Affymetrix 3' IVT GeneChips. AMDA has been implemented to integrate the requirements for different platforms. AMDA 2.13 allows the analysis of Affymetrix (cartridges and plates) and whole transcript probe design (Gene 1.0/1.1 ST and Exon 1.0 ST GeneChips), Illumina Bead Arrays, and one-channel Agilent 4-44 arrays. Relative to early versions, it supports various experimental designs and delivers more insightful biological understanding and up-to-date annotations. The developed new platform will provide scientists with a user-friendly tool to analyse the complexity of genomics data coming from different platform. Such instrument is not currently available as a free open source in the scientific community. Genopolis has developed protocols for analysis of ex vivo low number of cells obtained by cell sorting procedure. With this improvement it was possible to study rare cell populations for the understanding of the priming activity characteristic of dendritic cells that are present in lymph nodes as compared with the spleenic environment.
List of Websites:
Professor Georg Wick, Medizinische Universitat Innsbruck, Austria
Tel: +43-512-9003-70960
Fax: +43-512-9003-73960
E-mail: georg.wick@i-med.ac.at
Project website address: http://www.tolerage.eu