Inducing and Breaking Tolerogenic Antigen-Presenting Cell Function

London, London
Thursday, 05 July 2012

An event from European Scientific Conferences - Euroscicon "Specialising in communicating cutting edge technology & methodology in the Life Sciences"  Linked In

Inducing and Breaking Tolerogenic Antigen-Presenting Cell Function
Thursday, 05 July 2012 09:00 - 17:00

Watson & Crick Room
The Royal College of Pathologists
2 Carlton House Terrace
London
London
SW1Y 5AF
United Kingdom

Map and Directions

Professional antigen-presenting cells (APC) such as dendritic cells (DC) and macrophages play a critical role in the initiation and maintenance of a T cell-mediated immune response. APC are essential for T cell priming, differentiation and activation in lymphoid tissue and at sites of inflammation. Understanding the molecular mechanisms via which APC function can be controlled may give novel insights into how T cell mediated immunity may be induced or blocked. This meeting will highlight current research aimed at inducing APC with tolerogenic function in order to treat inflammatory disease (e.g. rheumatoid arthritis) as well as work aimed at boosting the immunostimulatory function of APC in the context of cancer. The meeting will also highlight recent progress on the suppressive effects of CD4+ regulatory T cells on APC function, and how these can be overcome.

 

This event  has CPD accreditation and will have a  discussion panel session.  

9:00 – 9:45        Registration

 

9:45 – 10:00      Introduction by the Chairs:  Dr Leonie Taams,  Senior Lecturer in Immunology, King's College London Centre for Molecular and Cellular Biology of Inflammation, Dr Catharien Hilkens, Reader in Immunotherapy, Newcastle University, UK

 

10:00 – 10:30   Developing a dendritic cell-based immunotherapy for rheumatoid arthritis

Dr Catharien Hilkens, Newcastle  Dendritic cells (DC) are antigen-presenting cells that play a critical role in maintaining immune tolerance to self-antigens by controlling the pathogenicity of auto-reactive T cells. DC can be modified ex vivo to induce stable tolerogenic function and have become a promising immunotherapeutic tool for autoimmune diseases such as rheumatoid arthritis (RA). Our goal is to develop and test DC-based therapy for RA. We have generated human DC with potent immunoregulatory activity (termed tolerogenic DC or tolDC) by treating monocyte-derived DC with dexamethasone, vitamin D3 and lipopolysaccharides. These tolDC express high levels of MHC class II and intermediate levels of the co-stimulatory molecules CD80/CD86. Importantly, tolDC display a stable anti-inflammatory cytokine profile with high levels of interleukin (IL)-10 and membrane-bound transforming growth factor (TGF)-ß, and low levels of tumour necrosis factor (TNF)-a, IL-12p70 and IL-23. Despite low expression of the chemokine receptor CCR7, tolDC migrate in response to the chemokine CCL19, required for tolDC migration towards T cell areas in lymphoid tissues (3). tolDC have excellent ability to process and present the RA auto-antigen type II collagen on MHC II, but at the same time modulate T cells in a pro-tolerogenic manner: tolDC polarise naïve T cells towards high IL-10, low interferon (IFN)-g and low IL-17 production, whereas memory T cells are rendered hyporesponsive. The immunosuppressive action of tolDC is partly mediated through TGF-ß. Furthermore, tolDC with similar phenotype and function can be generated from the peripheral blood of RA patients. Equivalent murine tolDC successfully suppress established arthritis in the collagen-induced arthritis model. This therapeutic effect of tolDC is associated with reduced T helper type 17 (Th17) responses in vivo. We have also set up protocols to generate tolDC under current Good Manufacturing Practice (cGMP) conditions and have obtained regulatory approval for a clinical trial with tolDC in RA patients. In conclusion, tolDC with stable and potent tolerogenic properties can be generated from peripheral blood of RA patients. These tolDC are a promising therapeutic tool for the induction of antigen-specific T cell tolerance in RA.

 

10:30 – 11:00   Selective ERK activation in dendritic cells for the treatment of arthritis
Dr David Escors, UCL, London
Rheumatoid arthritis is an autoimmune disease characterized by chronic joint inflammation and destruction. However, arthritogenic antigens are unknown and most therapeutic treatments rely on immunosuppressive drugs. We demonstrate that co-delivery of a specific ERK activator with a model antigen induces antigen-specific immune suppression by differentiation of regulatory dendritic cells (DCs) and antigen-specific regulatory T cells (Tregs). Differentiated Tregs strongly proliferate after antigen re-encounter in inflammatory conditions and exhibit antigen-dependent suppressive activities. The suppressive activity of ERK activation depended on secretion of high levels of TGF-beta from mouse and human DCs. In vivo administration of the ERK activator inhibited inflammatory arthritis. 

11:00 – 11:15   Optimization of an iptl-based stable isotope labelling strategy for quantitative proteomics of tolerogenic dendritic cells.

Matthew Buck, Pinnacle Lab, nstitute of Cellular Medicine, Newcastle University, Framlington Place, Newcastle upon Tyne, Tyne & Wear, NE2 4HH

11:15– 11:35    Mid-morning break

 

11:35 – 12:05   Boosting the immunostimulatory function of dendritic cells in an immunosuppressive tumour environment
Dr Sandra Diebold, King's College London, London, UK

 

12:05 – 12:20   Role of pueraria tuberosa dc, on macrophages functions: expression of iNOS and PKC-δ”

Professor Yamini Bhusan Tripathi, Department of Medicinal Chemistry, Institute of Medical Sciences, Banaras Hindu

 

12:20 – 12:50   Targeting regulatory T cell and dendritic cell interaction in vaccination:CCR4 antagonists as molecular adjuvants
Dr Jagadeesh Bayry, France 

CD4+CD25+ regulatory T cells (Tregs) play an indispensable role in maintaining immunological unresponsiveness to self-antigens and in suppressing excessive immune responses deleterious to the host. Since activation state of dendritic cells (DCs) at the time of encounter with antigens determines the outcome of the immune response, limiting the influence of Tregs on DCs at this juncture might lead to an enhanced immune response to poor immunogenic vaccine candidates. Although depletion of Tregs provides a proof of concept for this approach, it is however unlikely that the approach of Treg-depletion per se would be of practical use, since Treg-depletion has been associated with adverse consequences such as localized autoimmune disease. Conversely, we proposed that transient inhibition of Treg function/migration at the time of immunization might be ideal for enhancing immune response to vaccines. We have targeted the interaction between chemokines and their receptors to inhibit transiently the recruitment of Tregs at the site of immunization. Human Tregs express CCR4, a chemokine receptor absent on naïve T cells. CCR4 is the receptor for CCL22 and CCL17, the chemotactic agents for Tregs in vitro and in vivo. Both the chemokines are produced by DC and are crucial in promoting contact between DC and CCR4+ T cells. By in silico technique, we identified small molecular weight antagonists to CCR4 that block the migration of CCR4+ Tregs and enhanced DC-mediated human CD4+ T cell proliferation in an in vitro immune response model and amplified cellular and humoral immune responses in vivo in experimental models when injected in combination with either Modified Vaccinia Ankara expressing Ag85A from Mycobacterium tuberculosis (MVA85A) or recombinant hepatitis B virus surface antigen (rHBsAg) vaccines. In addition, when combined with vaccines, CCR4 antagonists indiced antigen-specific CD8+ T cells and tumor immunity against self-antigens. The significant adjuvant activity observed provides good evidence supporting our hypothesis that CCR4 is a viable target for rational adjuvant design.

 

12:50  - 14:00   Lunch

This is also a good time to fill out your feedback forms

 

14:00  -  14:30  Question and Answer Session and Speakers photo

                            Delegates will be asked to submit questions to a panel of experts.  Questions can be submitted before the event or on the day

 

14:30  -  14:45      The regulation of dendritic cell survival and function by tnf receptors 1 and 2

NJ Maney, Musculoskeletal Research Group, 4th floor Cookson, The Medical School, Newcastle University, Framlington Place, Newcastle Upon Tyne, NE2 4HH

 

14:45 – 15:00   Protection of islet grafts through TGF beta induced suppressive DC

Dr Maja Wallberg, Cambridge Institute for Medical Research, Cambridge, UK

In Type 1 Diabetes, the insulin producing beta cells are destroyed by the immune system which leaves the patient in need of exogenous insulin for survival. Calculating correct dosage and maintaining good glycaemic control is a challenge, and many Type 1 Diabetes patients experience episodes hyper- or hypoglycaemia which in some cases can be life threatening. One way of restoring glucose control is to transplant new beta cells from a donor, a procedure now routinely done at several centres world wide. While this procedure may restore endogenous insulin production, immunosuppressive treatment is needed to prevent the recipient from rejecting the donor derived islets. We have investigated the possibilities of using a brief pulse of expression of the immunosuppressive cytokine Transforming Growth Factor beta to achieve long term tolerance to the grafted islets. We found that expression of TGF beta could prevent rejection of islets from syngeneic donors through affecting DC activation in the graft, and thus reactivation of T cells in the graft draining lymph node. Interferon gamma production from T cells in graft draining lymph nodes was reduced, and in vivo proliferation of adoptively transferred insulin specific CD8+ T cells was inhibited. TGF beta reduced expression of costimulatory molecules CD80 and CD86 on DC, but did not affect levels of MHC class I and II. Exposure to TGF beta also reduced antigen uptake of bone marrow derived DC in vitro, and reduced their capacity to activate both CD4+ and CD8+ T cells. Once TGF beta expression was switched off, the grafts were eventually all rejected, demonstrating that temporary exposure to TGF beta cannot permanently disarm an established immune memory response.

 

15:00  - 15:30   Afternoon Tea/Coffee

 

15:30  16:00  CD4+ T cell-monocyte cross-talk and immune regulation of rheumatoid arthritis
Leonie TaamsKings College London, London, UK

Rheumatoid arthritis (RA) is a painful and debilitating disease affecting 0.5-1% of the Western population. The disease is characterised by chronic inflammation of the synovial lining, leading to damage and destruction of the underlying joint tissue and bone. Immune cells such as CD14+ monocytes and CD4+ T cells are abundantly present at the site of inflammation and are involved in the chronic inflammatory process. Our lab has been interested for many years in the cross-talk between monocytes and CD4+ T cells and how these interactions influence the ensuing immune response. Our previous work has demonstrated that activated monocytes can potently promote a pro-inflammatory Th17 cell response, which may be particularly important in the context of RA (Evans PNAS 2007; Evans PNAS 2009, Gullick PLoSOne 2010). Recent data from our lab indicates that activated monocytes may also influence CD4+CD25+CD127low regulatory T cell (Treg) function. We therefore wish to obtain a better understanding of how monocyte activation can be controlled. Data will be discussed demonstrating the mechanisms via which both CD4+ effector and CD4+ regulatory T cells can control monocyte activation. In addition, we will discuss recent data on therapeutic manipulation of monocytes/monocyte-derived factors leading to regulation of the pro-inflammatoryTh17 response

 

16:00 – 16:30   Inducing and Breaking Tolerogenic Antigen-Presenting Cell Function

Dr Steve Cobbold, University of Oxford, UK

A short treatment with mAbs that block T cell function is able to induce immunological tolerance in mouse models of transplantation and autoimmune disease.  Data will be presented to show that this tolerance is induced and maintained by the de novo generation of antigen specific, foxp3+ Treg in the periphery which are required to act continuously to maintain a tolerogenic microenvironment for antigen presentation within the tolerated tissue itself. The molecular mechanisms that constitute this microenvironment, including cytokines, amino acid catabolism, adenosine generation, and infectious tolerance, will be discussed.

 

16:30 – 17:00    Chairman’s summing up

KeywordsERK, regulatory T cell, Dendritic cells; tolerance; autoimmunity; cancer; vaccine,Treg,arthritis,RA, Treg, dendritic cells, transplantation tolerance, foxp3, molecular mechanisms, Th17, CD4, RA, rheumatoid arthritis, CD127, CD25

 

Information about the chairs

The main focus of research in  Leonie Taams,  research group is to identify key cellular processes and molecular mechanisms involved in the regulation of inflammation in humans, with a specific interest in the interactions between CD4+ T cells and monocytes. The lab hopes to use this knowledge to identify novel pathways and/or approaches to target inflammation in humans.  Research in the Taams laboratory is/has been funded by the Biotechnology and Biological Sciences Research Council (BBSRC), the Medical Research Council (MRC), Arthritis Research UK, the Innovative Medicines Initiative (IMI), the Department of Health (DoH) via the National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre (BRC) award to Guy’s & St Thomas’ NHS Foundation Trust (GSTFT) in partnership with King’s College London (KCL) and King’s College Hospital (KCH) NHS Foundation Trust, and industrial collaborative funding.  In addition to her research activities, Leonie is research project module organiser for the MSc Immunology, Chair of the MSc Immunology Exam Board and Deputy Program Director of the MSc Immunology. She is a member of the Higher Education Academy, and serves on a number of (inter)national meeting committees.

 

Catharien Hilkens  did her PhD research at the University of Amsterdam, where she worked on regulation of T cell immunity by dendritic cells. She then was awarded an EMBO fellowship to work on understanding how cytokines regulate T cell- and dendritic cell- function at the Imperial Cancer Research Fund (now Cancer Research UK) laboratories in London. In October 2003 she joined Newcastle University, where she runs a research group studying mechanisms underlying immune tolerance, and the development of dendritic cells as an immunotherapeutic tool.


About the Speakers

David Escors got his PhD from the Autonomous University of Madrid, Spain in molecular virology in 2002. There he  got interested in the development of viral vectors for gene therapy. During his first post-doc in the National Centre for Biotechnology, Spain, he was involved in the development of coronavirus-derived gene vectors. In 2005 he joined UCL as a Marie Curie Fellow, and got interested in the manipulation of intracellular signalling pathways in dendritic cells to manipulate immune responses. In 2008 he  obtained an Arthritis Research UK Fellowship to manipulate dendritic cells for the treatment of arthritis.

 

Steve Cobbold is currently the Reader in Cellular Immunology at the Sir William Dunn School of Pathology, Oxford working on the mechanisms of immune tolerance with particular focus on regulatory T cells. He studied Biochemistry at Oxford, and developed the first immunosuppressive mAbs during his PhD in Cambridge with Herman Waldmann.  As part of the Therapeutic Immunology Group, he contributed to the development of CAMPATH, the first humanized therapeutic antibody. He was a scientific founder of TolerRx Inc., and co-founded BioAnaLab Ltd., which was successfully sold to Millipore in 2009. He has published more than 250 articles and patents.

 

Sandra Diebold: research interests - employing viral nucleic acid as adjuvants for cancer immunotherapy with a focus on studying the influence of innate immune activation on the induction of adaptive anti-tumour responses.

 

 

Jagadeesh Bayry is a senior research scientist (Equivalent of Associate Professor) at Institut National de la Santé et de la Recherche Médicale (French National institute of Health and Medical Research) (INSERM). He received his Veterinary Medicine degree with a specialization of  Virology and Immunology from the Indian Veterinary Research Institute. He obtained a PhD from the Université Pierre et Marie Curie, Paris in 2003. He later carried out postdoctoral research at the Edward Jenner Institute for Vaccine Research, University of Oxford, UK. In  2006, he was recruited as a scientist by INSERM. His research is aimed at understanding the mechanisms of immune tolerance, the mechanisms of action of intravenous immunoglobulin and the host-pathogen interaction. He has authored more than 130 papers in leading journals. He is an Academic Editor of PLoS ONE and Scientific Reports, editorial board member of several journals, expert member of AERES (French Agency for Evaluation of Research and Higher Education) and expert referee for Panel LS6 "Immunity and Infection" panel of European Research Council.

 

 


POSTERS

 

 

MODULATION OF HUMAN DENDRITIC CELLS BY ADENOSINE RECEPTORS AND THE CAMP SECOND MESSENGER PATHWAY.

B. Laugel1,2; P. Farmer2; M.L. Curchod2; D. Cole1; M. Guerrier2; C. Salvat2 and A.K. Sewell1
1 Institute of Infection and Immunity, Cardiff University School of Medicine, Heath Park, Cardiff, CF14 4XN Wales, UK; 2 Merck Serono S.A., Geneva Research Center, 9 chemin des Mines, Genève, Switzerland

Corresponding author: laugelbf@cf.ac.uk
Multiple endogenous mechanisms that regulate immune and inflammatory processes contribute to the maintenance of peripheral tolerance and prevent chronic inflammation in mammals. Yet pathogens and tumors are able to exploit these homeostatic pathways to foster immunosuppressive microenvironments and evade immune surveillance. The release of adenosine in the extra-cellular space contributes to these phenomena by exerting a broad range of immunomodulatory effects.               
We used gene expression micro-array to interrogate the effects of adenosine receptor (AR) triggering on monocyte-derived DC (mo-DC) differentiation and functions. Global transcriptional analysis suggested that the effects of adenosine only marginally affected cellular differentiation. Nevertheless, several immunomodulatory factors (ILT-3, ILT-4 and CD25) and myeloid differentiation markers (SCF, CD4 and CD123) were differentially regulated by AR agonists and cAMP elevating agents on the surface of immature DCs. Collectively these changes were reminiscent of the phenotype associated with tolerogenic DC subsets. In addition this skewed differentiation resulted in an altered cytokine profile upon LPS maturation; the most notable feature being the near complete absence of secretion of the IL-12 family cytokines IL-12p70, IL-23 and IL-27. These phenotypic changes translated into a defective capacity to prime antigen-specific CD8 T cells in vitro.

Our study thus offers insights into the developmental and immunomodulatory processes leading to the establishment of tolerogenic functions in DCs by adenosine and by other factors triggering the cAMP second messenger pathway. The presence of such cues in the cellular environment of developing DCs may subvert immunity by suppressing the initiation of CD8 T cell responses

 

OPTIMIZATION OF AN IPTL-BASED STABLE ISOTOPE LABELLING STRATEGY FOR QUANTITATIVE PROTEOMICS OF TOLEROGENIC DENDRITIC CELLS.

Matthew Buck1,2, Joe Gray1, Catharien Hilkens2 & John Isaacs2

1Pinnacle Lab, 2Institute of Cellular Medicine, Newcastle University, Framlington Place, Newcastle upon Tyne, Tyne & Wear, NE2 4HH

Tolerogenic dendritic cells are monocyte-derived dendritic cells (DC) cultured such that they adopt an immunoregulatory phenotype. In vitro, these cells are able to induce and maintain T cell tolerance through deviation of naive T cells to an anti-inflammatory phenotype and induction of anergy in memory T cells. Equivalent cells suppress established arthritis in murine models. Recently, two tolDC trials with tolerogenic DC in RA and type I diabetes patients have been carried out and other trials are in progress or are imminent.

In spite of these promising data, we are yet to rigorously explore the basis of the phenotype of tolerogenic DC and lack markers to unequivocally distinguish them from other types of DC. The aim of this PhD project is to address these questions using a quantitative proteomics approach. Briefly, I will isolate the membrane proteomes of mature/immature and tolerogenic DC and utilize stable isotope labelling to label the peptides present in one proteome relative to those in the other. The two proteomes will then be combined and analysed using high resolution LC/MS/MS. Differentially expressed plasma membrane proteins identified in this body of work will subsequently be further investigated for putative roles in tolerance induction.  SILAC (stable isotope labelling with amino acids in cell culture) and isobaric tags (iTRAQ, TMT) are the most well-known and routinely used labelling methods for quantitative proteomics. However, SILAC can only be used to label cell lines whilst isobaric tagging reagents are not compatible with all mass spectrometers. Neither method is suitable for this particular project. We have thus developed and optimized an isobaric peptide termini labelling (IPTL)-based approach as an effective and economical alternative to SILAC and iTRAQ / TMT and evaluated its performance in quantifying the proteins present in simple and complex samples across a wide dynamic range. We now intend to use this method to address the questions outlined above.

ROLE OF PUERARIA TUBEROSA DC, ON MACROPHAGES FUNCTIONS: EXPRESSION OF iNOS AND PKC-δ”

Professor Yamini Bhusan Tripathi, Department of Medicinal Chemistry, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005, India

The secondary metabolites of various plants, needs deep scientific study, to understand their mechanism of action for their claimed therapeutic effects. Ayurvedic system of Indian Medicine, believes in treating oxidative stress and inflammation as the 1st step in treatment of metabolic disorders and in this process, macrophage play important role. Several plants are already in clinical use. Here, we have explored the effects of semi-purified fraction and isolated compound of tubers of Pueraria tuberosa DC. Pueraria tuberosa DC, (Fabaceae)(PT), on macrophage functions. PT preparations are in clinical use for inflammation, diabetes and also to prevent aging process. The rat peritoneal macrophages were isolated and attached cells were cultured for 17hr in complete media (5% FBS) at 370C in CO2 incubator, with and without LPS and PMA. The test drugs (PTY-1 and other kinase inhibitors e.g. H-7, genistein and quercetin) were added in different experimental conditions and degree of expression of iNOS and PKC-δ were assessed by Western blot analysis. The PTY-1 inhibited iNOS and PKC-δ expression in concentration dependent manner. The post-treatment kinetics also indicated its specific action on PKC expression. Further, in vivo studies with rats, has shown its anti-inflammatory and antioxidant properties. Thus, it could be suggested that the claimed antiaging and anti-inflammatory property of PT preparations is through its inhibitory property for PKC and iNOS.

 

THE REGULATION OF DENDRITIC CELL SURVIVAL AND FUNCTION BY TNF RECEPTORS 1 AND 2

NJ Maney, A Krippner-Heidenreich, CMU Hilkens, Musculoskeletal Research Group, 4th floor Cookson, The Medical School, Newcastle University, Framlington Place, Newcastle Upon Tyne, NE2 4HH

Tumour Necrosis Factor (TNF) is a key mediator of inflammatory diseases such as rheumatoid arthritis, and plays a central role in dendritic cell (DC) biology. To investigate the role of TNF in regulating DC survival and immunostimulatory function, we made use of TNFR-selective ligands to dissect the individual contributions of the two TNF receptors; TNFR1 (usually associated with pro-inflammatory effects) and TNFR2 (associated with both pro- and anti-inflammatory effects).

We demonstrate that human monocyte-derived DC survival could be prolonged by selective stimulation of either TNFR1 or TNFR2 as shown by the detection of Annexin V/ViaProbe and also by reduced intracellular levels of active caspase-3. An antagonistic TNFR1-specific antibody (H398) was used to block TNFR1 which confirmed that protection via TNFR2 was independent of TNFR1-mediated signalling. We have also used a DNA binding assay to demonstrate that TNFR1 activates the classical p65 NFkB pathway whereas TNFR2 activates the alternative p100/p52 NFkB pathway highlighting differences in signalling downstream of the receptors. Moreover, TNFR1-selective, but not TNFR2-selective stimulation resulted in increased expression of the DC maturation markers CD83, CD86 and PD-L1.

Thus, our data demonstrate that signalling through either TNFR1 or TNFR2 protects DC from cell death, whereas TNFR1- but not TNFR2-mediated signalling induces DC maturation. Understanding the immunoregulatory properties of signalling through these two TNF receptors is important for the design of more targeted anti-TNF therapy.

 

IMMUNE MODULATION BY VITAMIN D3 [1,25(OH)2D3]-TREATED DENDRITIC CELLS IN MULTIPLE SCLEROSIS

W.P. Lee1, A.H. Nuyts1, S. Anguille1, K. Lenders1, T. Possemiers1, B. Willekens2, Z.N. Berneman1, N. Cools1

1Antwerp University Hospital, Lab of Experimental Hematology, Antwerp, Belgium.
2Antwerp University Hospital, Division of Neurology, Antwerp, Belgium.

Emerging evidence indicates that dendritic cells (DC) play a central role in the initiation and progression of multiple sclerosis (MS). Since DC are the most professional antigen-presenting cells (APC) and have the ability to stimulate naïve T cells, they play a crucial role in the induction of tolerance in autoimmunity. The aim of this study is to investigate and exploit the capacity of tolerogenic DC to restore or modulate pathogenic responses in MS. Indeed, DC can be modified ex vivo using immune-suppressive agents to induce stable tolerogenic function. Therefore, we studied the effects of the active form of vitamin D3, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], on the differentiation, maturation and function of monocyte-derived DC. Important immunoregulatory and anti-inflammatory properties have been attributed to this hormone. First, by using a dilution series, we demonstrated that 10-5M 1,25(OH)2D3 was the optimal concentration for differentiation of stable immature DC. Next, the phenotype of vitD3-treated DC was evaluated and compared to the phenotype of conventional (i.e. IL-4-treated) DC and other immune-suppressing (i.e. IL-10-treated) DC. In addition, the in vitro stability of the tolerogenic phenotype was investigated, as tolerance induction by immature DC might be counteracted by concomitant DC activation in a pro-inflammatory micro-environment in vivo. Our results reveal that DC, differentiated in the presence of 10-5M 1,25(OH)2D3, fail to up-regulate expression of CD83, of the costimulatory molecules CD86 and CD80 and of MHC II molecules, upon stimulation with pro-inflammatory signals, as compared to conventional IL-4-treated DC. While the secretion of pro-inflammatory cytokines (IL-1beta, TNF-alpha and IL-6) and importantly IL-12p70 was abolished, we could not detect IL-10 secretion by vitD3-treated DC. However we observed increased secretion of TGF-β by these DC. Of utmost importance, we demonstrated the inability of vitD3-treated DC to stimulate myelin-specific T cells in DC/T cell co-cultures compared to conventional DC using IFN-γ ELISPOT.

In conclusion, our data underline the possible applicability of the use of vitD3-treated DC as interesting cellular mediators in order to correct the immunological imbalance in MS.

 

Dendritic cells in multiple sclerosis: key players in the immune pathogenesis, key players for new cellular immunotherapies

N. Cools1, A.H. Nuyts1, W.P. Lee1, N. Deckx1, P. Cras2, B. Willekens2, G. Nagels3, M.B. D'hooghe3, Z.N. Berneman1.
1Antwerp University Hospital, Lab of Experimental Hematology, Antwerp, Belgium.
2Antwerp University Hospital, Division of Neurology, Antwerp, Belgium.
3National Center for Multiple Sclerosis, Division of Neurology, Antwerp, Belgium.

Purpose/Objective

Much has been done to understand the pathogenesis of multiple sclerosis (MS). Whereas several studies have focused on the role of the adaptive immune system in the disease pathogenesis of MS, recent data suggest that dendritic cells (DC), which are innate immune cells, also contribute to the pathogenesis of MS. Due to their specialized antigen-presenting capacity, DC play an important role in polarizing the T cell response, thereby regulating the balance between immunity and tolerance.

Materials and Methods

DC were generated in vitro from circulating monocytes of MS patients and healthy controls. Subsequently, phenotype, cytokine profile and T cell stimulatory capacity were investigated. Additionally, DC were exposed to pro-inflammatory signals to address their maturation-resistant phenotype.

Results

Our recent observations indicate that both circulating as well as in vitro generated DC of MS patients display an altered phenotype and function as compared to age- and gender-matched healthy controls, thereby influencing the effector function of T cells. Of interest, we also demonstrate that migratory patterns of DC in MS are altered, as evidenced by aberrant expression of migratory molecules, i.e. CD62L, CCR5 and CCR7, by DC of MS patients as compared to healthy controls. Furthermore, current immune-modulating therapies affect DC phenotype and function, hence underscoring their pivotal role in MS pathogenesis. In light of these observations, we subsequently aimed to modulate DC function in order to restore the immunological imbalance in MS. We demonstrated that treatment of DC with immunomodulatory cytokines (such as IL-10) results in a more stable tolerogenic DC phenotype. Indeed, modulation of DC by IL-10 treatment arrests DC in a semi-mature state and prevents the up-regulation of costimulatory molecules as well as the secretion of pro-inflammatory cytokines. Moreover, we deliver proof-of-principle that tolerogenic DC generated from MS patients are capable of suppressing myelin-specific T cell responses.

Conclusions

From the results of this study, it can be envisaged that we have provided the first step in the development of a new form of immunotherapy for the treatment of MS.


Registration Web Site:  www.regonline.co.uk/apc2012



 

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