Research Interests

Type 1 Diabetes

The main theme of my research program is to understand the regulation of autoimmune and anti-viral responses. Our work has shown that the amount of immunopathology or tissue injury is determined not only by the magnitude of a localized or systemic immune process, but also to a large extent by its components or the class(es) of responses it encompasses. Thus, each immune or auto-immune reaction has at least a more aggressive and a more regulatory component that balance each other and in this way have a strong effect on the duration or magnitude of the response and resulting tissue injury. In autoimmune diseases, it is possible to take therapeutic advantage of this paradigm and generate autoreactive regulatory cells (Tregs) by targeted immunization with self-antigens. We have shown that such cells can be induced by mucosal immunization and DNA vaccination. Antigen-induced autoreactive Tregs are able to site-specifically suppress ongoing autoimmune reactions, because they are preferentially retained in the draining lymph node closest to the target organ where they exert their regulatory function. Intriguingly, their induction can be successful in recent-onset diabetes when insulin immunization is combined with a short-term course of non-FC-binding anti-CD3.

Ongoing and future studies are focused on:

Honeymoon project: The lab is continuing a small human study evaluating T-cell responses during the partial remission phase of type 1 diabetes. Preliminary results indicate that this "honeymoon" phase is not associated with classical Treg markers (IL-10 and FoxP3) and that these two markers predict future glucose control in very different ways.

NOD-SCID IL-2R knockout mouse project: The lab is beginning studies to evaluate reconstituting this mouse with human peripheral white blood cells to better allow us to study the human immune system.

The role of key cytokines (TGF-beta, IL-4/IL-5 and IL-10), transcription factors (T-bet) and chemokines (IP-10) in regulating autoimmunity and virally-induced immunopathology. Pertinent issues are modulation of antigen presenting cells, in vivo trafficking of Tregs and their in vitro propagation and antigen specificities (Juedes J. Exp.Med. 2004, Homann Immunity 1999, 2002 and Bot JI 2001).

Dissecting how autoantigen-induced Tregs act in immune-competent hosts in vivo and how they can be used in the clinic to prevent diabetes. Combination of antigen-induced Tregs with systemic therapies such as anti-CD3 that favor a ‘Treg-friendly’ environment will be further developed (see von Herrath JI 2002 and Nature reviews from 2003 and 2004).

The Role of viral immunopathology in autoimmunity. Viral infections can enhance and abrogate autoimmune processes and we will continue to evaluate mechanistically how this occurs. Furthermore, persistent infection systems are being investigated to define approaches that ameliorate immunopathology and facilitate vaccine development (Christen JCI 2-2004 and 11-2004, Christen, Diabetes 2004, Bot J. Virol 2003).

Combination therapy of recent-onset diabetes with insulin immunization and anti-CD3. This new strategy that we pioneered is right now under revision at JCI. Since the murine experiments look very promising in showing strong synergy and life-long reversion after recent-onset diabetes in two animal models (NOD and RIP-LCMV), this strategy will likely be used in clinical trials in the US and Australia starting next year.

Paradigms developed from these studies will not only be useful in selectively suppressing autoimmune diseases, but can also be employed to lower immunopathology that accompanies viral infections.

Generation and characterization of insulin peptide-specific regulatory T cells

M. M. Martinic, J. Oldham, L. M. Togher, C. M. Filippi, D. Bresson, G. Fousteri, J. M. Jasinski, G. S. Eisenbarth and M. G. von Herrath.

Our goal is to generate insulin peptide-specific regulatory T cells (Tregs) in vitro, which suppress overt diabetes in prediabetic and reverse already ongoing disease in diabetic mice. Further we aim to analyze the suppressive properties and mechanisms of these Tregs in vitro and in vivo.

In order to generate insulin peptide-specific Tregs, we took advantage of insTCR transgenic mice, which express T cell receptor (TCR) transgenic CD4+ T cells specific for the insulinB9-23 peptide (insB) presented on MHC class II H-2IAg7/d molecules. The in vitro generation of insB-specific Tregs involved purification of either CD4+CD25+ or CD4+CD25- insTCR T cells, which were cultured for 1-2 weeks with the insB peptide, syngeneic antigen presenting cells and high doses of IL-2 yielding 25+ and 25- cultures, respectively. Using the classical in vitro suppression assay, only cells derived from the 25+ cultures were able to suppress while cells from the 25- cultures enhanced proliferation and cytokine secretion of CD8+ effector T cells. In vivo, however, cells from both cultures were unable to suppress lymphocytic choriomeningitis virus (LCMV)-induced diabetes. Interestingly, freshly isolated as well as IL-10-cultured CD4+CD25- but not freshly isolated CD4+CD25+ insTCR T cells suppressed spontaneous diabetes in NOD females. We are currently investigating the mechanisms underlying the in vivo suppressive potential of these CD4+CD25- T cells.

Real-time imaging of the pancreas during development of diabetes

K. Coppieters, M. M. Martinic, J. Oldham, A. Althage, Zacarias Garcia, D. Bresson, L. M. Togher, C. Huber, and M. G. von Herrath.

Our aim is to visualize islet antigen-specific T cell trafficking through the pancreas to understand kinetics of diabetes development. We adoptively transferred GFP+ P14 T cells (specific for the lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP)) into RIP-GP mice (express LCMV-GP in the beta-cells of pancreatic islets) followed by real-time imaging of the RIP-GP pancreas using an inverted confocal microscope. Injection of Dithiocarbazone (DTZ) allowed visualization of pancreatic islets in vivo.

One day after transfer, GFP+ cells were distributed evenly throughout the pancreas. Three days later, GFP+ cells started to accumulate specifically around DTZ+ islets. Seven days after transfer, GFP+ cells were not only surrounding but also infiltrating the islets. GFP+ cells located close to a blood vessel moved fast whereas cells within the pancreatic tissue moved slowly and were predominantly found as cell clusters. Thirteen days after transfer, only few GFP+ cells remained in the pancreas. In summary, our approach allowed us to visualize GFP+ cells and their movement in DTZ+ islets.

Currently, we are generating experimental setups inducing lymphocytic infiltration into pancreatic islets with or without complete beta-cell destruction. To ameliorate visualization of beta-cells, we will be using RIP-GPxMIP-GFP and P14xDsRed transgenic mice allowing us to visualize infiltration of DsRed+ P14 cells into GFP+GP+ pancreatic islets. Finally, to improve tissue penetration, we will perform future experiments with a multi-photon microscope, which has a much greater penetration depth than a confocal microscope.

Virally Induced Regulatory Mechanisms in the Prevention of Type 1 Diabetes

Christophe M Filippi, Janine Oldham, Lisa Togher, Tom Wolfe, Marianne Martinic, Evelyn Rodrigo, Urs Christen & Matthias von Herrath

Type 1 Diabetes (T1D) is an autoimmune disease that results from the selective destruction of insulin-producing beta cells in the pancreas. While viral infections may be capable of triggering autoimmunity in genetically susceptible individuals, accumulating evidence indicates that viruses can also prevent T1D. Furthermore, viruses have been suggested to be potent inducers of CD4+CD25+ regulatory T cells (Tregs), which are known to play a crucial role in the prevention of autoimmunity. However, how viral infections may protect from autoimmune diabetes is still under investigation. We found that activation of CD4+CD25+ Tregs by acute lymphocytic choriomeningitis virus (LCMV) infection provides these cells with the capacity to prevent both spontaneous and virally induced T1D in the mouse.

Our goal is to understand how regulatory mechanisms associated with anti-viral immunity can prevent T1D. We believe that polyclonal activation of regulatory responders during virally induced inflammation could serve on one hand to modulate anti-viral immunity and on the other hand to prevent or abort potentially associated autoimmunity. Activation of Tregs during viral infections may be a general feature accounting for the ability of viruses to prevent T1D as well as other autoimmune disorders.

The selective induction of immune modulation is of high interest for influencing the course of autoimmune disease. Our studies should facilitate utilization of Treg induction to prevent autoimmunity. Furthermore, our findings support a model explaining the differential ability of viral infections to modulate diabetes, and our work should help evaluate the potential risk of viral infections in genetically susceptible children, thereby addressing current discordance concerning the role of viruses in T1D induction.

IL-10 Receptor Blockade in the Resolution of Chronic Viral Infection

Christophe M Filippi, Mette Ejrnaes, Marianne Martinic, Janine Oldham, Lisa Togher & Matthias von Herrath

Persistent infections can pose severe health risks. In many cases, for example after hepatitis virus infection, only a fraction of infected individuals clear the virus. The resulting chronic infection can lead to liver damage or liver cancer, particularly after hepatitis C virus (HCV) infection. Other viruses such as Epstein Barr virus (EBV) can also cause cancer if systemic reactivation occurs. Numerous chronic infections, in particular HCV, have been associated with systemic increase in IL-10 production. We found that blockade of IL-10 signaling using anti-IL-10 receptor antibodies can completely resolve persistent lymphocytic choriomeningitis virus (LCMV) infection while reducing immunopathology in the mouse. The finding is unique because, to date, immunization with dendritic cells or attempts to directly augment anti-viral effectors with vaccines has mostly failed to show beneficial effects and in some cases was found to augment immunopathology.

Our goal is to understand the immunological mechanisms underlying the beneficial effects of anti-IL-10R treatment in chronic infection, and aid to translate our findings to clinical applications in other chronic viral infections. Mechanistic understanding of IL-10R blockade in persistent LCMV infection will indicate whether IL-10 affects the outcome of infection, the amount of immunopathology, the occurrence of further complications, or indeed whether it could be the actual cause of persistence. Furthermore, preclinical evaluation of combination therapies using anti-IL-10R with other antibodies and viral vaccines will facilitate the therapeutic use of IL-10 blockade as a therapy for the treatment of persistent infections in humans.

Conventional immunotherapy of persistent viral infections has been unsuccessful so far, and we propose that tackling the problem from a different angle may be a crucial step toward successful treatment of chronic infections in humans.

Preclinical models of immunomodulation with peptide epitopes – dose and route, mechanism and enhancement of efficacy.

Georgia Fousteri, Damien Bresson, Mark Peakman, Bart Roep and Matthias von Herrath.

One of the main goals in treatment of autoimmune diseases is the intervention with the immune system without causing immunosuppression. Therapeutic approaches for the treatment of type 1 diabetes which combine decreased doses of immune modulators such the non Fc binding anti-CD3 in conjunction with insulin epitopes (proinsulin) have become of great interest because of the efficacy of the treatment and the reduction of the anti-CD3 immunosuppression side effects. The usage of peptides alone and/or in combination (multi-peptide therapy) has now become of a great interest because of their immunomodulator effect. So, it is proposed that immunosuppression therapies will be conceded by peptide-based therapies due to their specificity, efficacy and risk for lesser side effects.

In our lab, the main goal is the identification of particular peptides and/or combinations of them which have therapeutic potential for the treatment of autoimmune diabetes. Insulin-derived peptides will be administered to prediabetic and recent onset diabetic mice and the disease incidence will be followed. In addition, peptides will be delivered to diabetic prone mice (NOD and Rip-LCMV), by using modulated APCs as carriers. The mechanistic understanding of the peptide based therapy is an important issue, which will be addressed mainly by evaluating the T cell responses ex vivo and the T regulatory cells activity in vitro and in vivo.

In silico modeling of immunological systems.

As part of a collaborative team we aim to investigate aspects of immunological systems using computational methods that allow for the manipulation of such systems beyond what is possible in vitro/vivo. The approach exploited uses a combination of a stochastic cellular automaton which uses rule sets to replicate immune cell behavior and a differential equation based system which models cell populations. The methods are used in address two different aspect of immunological study; the prediction of in vitro cellular interactions for the refinement of physical experimentation and the modeling of biological phenomena to reveal the mechanisms behind them.The role of viral infections in the development of type 1 diabetes

Virally mediated prevention of type 1 diabetes.

Although type 1 diabetes is a genetic autoimmune disease, it is under the critical influence of epigenetic or environmental parameters, such as infections, that can either promote or diminish autoimmunity. Viruses are prime candidates for constituting an infectious risk factor because they can induce strong cellular immune responses and in some cases infect and damage β cells while causing local inflammation in the pancreas. This has been proposed to account for presentation of β cell antigens to autoreactive T cells, promoting their activation and attack on β cells. On the other hand, epidemiological and experimental evidence indicates that infections, and most notably those mediated by viruses, can efficiently prevent type 1 diabetes (supporting the "hygiene hypothesis"). Whether a particular virus could cause of prevent type 1 diabetes thus still remains unknown and is subject to much debate. In addition, the opposing roles of viral infections in type 1 diabetes are not understood mechanistically. Assessing how viruses protect from this disease could help provide a better understanding of its etiology, but more importantly identifying the mechanisms by which protection occurs could teach us valuable lessons for therapy. Active investigation on this topic is currently undertaken in our group using animal models for type 1 diabetes, such as non-obese diabetic (NOD) mice, which develop spontaneous autoimmune diabetes that mimics many aspects of human disease. We recently reported that viruses that do not inflict damage to β cells protect from type 1 diabetes by triggering immunoregulatory mechanisms. Infection of prediabetic NOD mice with Coxsackievirus B3 or LCMV delayed diabetes onset and reduced disease incidence. Delayed type 1 diabetes onset was due to transient up-regulation of PD-L1 on lymphoid cells, which prevented the expansion of diabetogenic CD8+ T cells expressing PD-1. Reduced type 1 diabetes incidence was caused by increased numbers of invigorated CD4+CD25+ regulatory T cells (Tregs) which produced TGF-β and maintained long-term tolerance. Full protection from type 1 diabetes resulted from synergy between PD-L1 and CD4+CD25+ Tregs. Interestingly, such synergistic phenomena induced by viral infections compare to successful therapies of type 1 diabetes that trigger the direct demise of autoreactive effectors along with their indirect control via Tregs. Our results provided novel mechanistic insight into the role of viruses in type 1 diabetes and should be valuable for prospective studies in humans.

How antiviral immunity and Treg function influence one another?

Tregs play a beneficial role in autoimmunity, and their induction in vivo or infusion constitutes a promising approach for therapy of autoimmune diseases like type 1 diabetes. However, the primary risk of such an approach, as for any immune-based strategy targeting CD8+ T cells in autoimmunity, is interference with non-autoreactive, beneficial T cell responses. While T cells are responsible for the development of type 1 diabetes, they play a decisive role in immune-mediated control over infectious pathogens. Notably, CD8+ T cells are rapidly activated following viral infection and acquire cytotoxic activity which causes the elimination of virally infected cells, thereby clearing the invading virus. It is thus vital that tolerogenic regimens to control autoreactive CD8+ T cells in type 1 diabetes do not affect the ability of other CD8+ T cells to be activated in the face of viral infection. Reciprocally, in strategies enhancing or infusing Tregs in vivo to treat autoimmunity, it is crucial to determine whether viral infections might affect the function of these cells and thus treatment efficacy. We are performing studies in the NOD mouse model for type 1 diabetes to determine whether antiviral immune processes, which transiently hinder immunoregulation, may impair the function of Tregs. Reciprocally, we are assessing whether Treg-based therapy of type 1 diabetes may hinder antiviral immune responses and viral clearance. Our findings suggest that the capacity of viral infections to transiently hinder Treg function enables antiviral immune processes to occur normally while maintaining the long-term efficacy of Tregs with respect to autoimmunity.

Innate immune mechanisms of Treg enhancement in type 1 diabetes.

In type 1 diabetes, the inflammatory events induced on anti-infectious immunity could enable enhanced presentation of β cell antigens to autoreactive T cells. On the other hand, infections and inflammation may play a protective role in type 1 diabetes, and we recently reported that viruses can trigger the expansion of invigorated CD4+CD25+ Tregs that prevent autoimmune diabetes by synergizing with PD-L1. Dendritic cells (DCs) and innate receptors such as Toll-like receptors (TLRs) promote inflammation and immunity, but can on the other hand also modulate CD4+CD25+ Tregs. DCs are highly efficient in promoting immune responses and can trigger autoimmunity, but on the other hand these cells can also induce or expand Tregs in vitro or in vivo. TLRs are "danger-sensing" molecules that play a central part in triggering inflammation and immunity, but these molecules can also suppress autoimmune diabetes when triggered in the absence of β cell antigen. Furthermore, CD4+CD25+ Tregs not only express different TLRs but are also functionally regulated directly and indirectly through TLR signaling. We are currently investigating how innate immune mechanisms involving DCs and TLRs alter immunoregulation in type 1 diabetes. Notably, we are assessing the mechanisms involved in our recent discovery that viral infections can protect from autoimmune diabetes by expanding and invigorating Tregs. This work should help understand type 1 diabetes etiology and favor the development or amelioration of immune-based therapeutic interventions to treat this disease.

Vaccination and therapy of viral infection

Treatment of persistent viral infection.

Chronic viral infections such as HIV, CMV and HCV pose serious health problems. For example long-term infection by HCV can lead to liver damage or liver cancer. Unfortunately, once patients are afflicted with a hepatitis virus infection, only a fraction of individuals are able to clear the virus. Furthermore, antiviral therapies usually ameliorate but do not overcome the chronic infection. Conventional immunotherapies attempting to lower the viral antigenic load (interferons or antiviral drugs) have demonstrated clear beneficial effects, but for the most part immune-based strategies have failed to show efficacy and in fact may have augmented immunopathology in some cases. Interestingly, in certain persistent viral infections, IL-10 production is increased systemically. Whether IL-10 could be the actual cause of persistence is a subject of strong interest, and we have recently made a significant discovery whereby IL-10 receptor blockade in mice completely resolved a persistent viral infection while reducing immunopathology. We are currently exploring this result in terms of mechanism, effectiveness, and synergism with current conventional therapeutics. By examining the combinatorial effects of IL-10 receptor blockade in conjunction with other conventional treatments, significant advancement toward resolving persistent infections in humans could be achieved. The results of these studies will aid in the amelioration of clinical strategies to treat chronic infections.

The role of TLR9 in DNA vaccination.

One of the requirements for efficient vaccination against infection is to achieve the best combination of an adequate adjuvant with the antigenic information to deliver. While plasmid DNA is a promising tool bearing the unique potential to activate humoral and cellular immunity, an actual challenge is to increase plasmid immunogenicity in human vaccination protocols where efficacy has proven rather limited. Previous work has shown that the bacterial DNA backbone of the plasmid has potent adjuvant properties because it contains CpG motifs, which are particular activating nucleotidic sequences. Among TLRs, which are key sensors of microbial products, TLR9 can detect CpG motifs and confer activation of antigen-presenting cells such as dendritic cells. However, whether the immunogenic properties of plasmid DNA involve TLR9 signaling has not been clearly established. Our current work aims at determining whether TLR9 directs the effectiveness of vaccination against lethal viral infection using plasmid DNA. Our results point to a role for this molecule depending on the vaccine regimen, notably in regimens generally used to vaccinate humans. This work suggests that the TLR9 signaling pathway is involved in efficacy of plasmid DNA vaccination and should remain a focus in the development or amelioration of vaccines to treat infections in humans.