In vivo imaging of lymphocytes in the CNS reveals different behaviour of naïve T cells in health and autoimmunity

<p>Abstract</p> <p>Background</p> <p>Two-photon laser scanning microscopy (TPLSM) has become a powerful tool in the visualization of immune cell dynamics and cellular communication within the complex biological networks of the inflamed central nervous system (CNS). Whereas many previous studies mainly focused on the role of effector or effector memory T cells, the role of naïve T cells as possible key players in immune regulation directly in the CNS is still highly debated.</p> <p>Methods</p> <p>We applied <it>ex vivo </it>and intravital TPLSM to investigate migratory pathways of naïve T cells in the inflamed and non-inflamed CNS. MACS-sorted naïve CD4+ T cells were either applied on healthy CNS slices or intravenously injected into RAG1 -/- mice, which were affected by experimental autoimmune encephalomyelitis (EAE). We further checked for the generation of second harmonic generation (SHG) signals produced by extracellular matrix (ECM) structures.</p> <p>Results</p> <p>By applying TPLSM on living brain slices we could show that the migratory capacity of activated CD4+ T cells is not strongly influenced by antigen specificity and is independent of regulatory or effector T cell phenotype. Naïve T cells, however, cannot find sufficient migratory signals in healthy, non-inflamed CNS parenchyma since they only showed stationary behaviour in this context. This is in contrast to the high motility of naïve CD4+ T cells in lymphoid organs. We observed a highly motile migration pattern for naïve T cells as compared to effector CD4+ T cells in inflamed brain tissue of living EAE-affected mice. Interestingly, in the inflamed CNS we could detect reticular structures by their SHG signal which partially co-localises with naïve CD4+ T cell tracks.</p> <p>Conclusions</p> <p>The activation status rather than antigen specificity or regulatory phenotype is the central requirement for CD4+ T cell migration within healthy CNS tissue. However, under inflammatory conditions naïve CD4+ T cells can get access to CNS parenchyma and partially migrate along inflammation-induced extracellular SHG structures, which are similar to those seen in lymphoid organs. These SHG structures apparently provide essential migratory signals for naïve CD4+ T cells within the diseased CNS.</p

Modulation of dendritic cell immunobiology via inhibition of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase

The maturation status of dendritic cells determines whether interacting T cells are activated or if they become tolerant. Previously we could induce T cell tolerance by applying a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor (HMGCRI) atorvastatin, which also modulates MHC class II expression and has therapeutic potential in autoimmune disease. Here, we aimed at elucidating the impact of this therapeutic strategy on T cell differentiation as a consequence of alterations in dendritic cell function. We investigated the effect of HMGCRI during differentiation of peripheral human monocytes and murine bone marrow precursors to immature DC in vitro and assessed their phenotype. To examine the stimulatory and tolerogenic capacity of these modulated immature dendritic cells, we measured proliferation and suppressive function of CD4+ T cells after stimulation with the modulated immature dendritic cells. We found that an HMGCRI, atorvastatin, prevents dendrite formation during the generation of immature dendritic cells. The modulated immature dendritic cells had a diminished capacity to take up and present antigen as well as to induce an immune response. Of note, the consequence was an increased capacity to differentiate naïve T cells towards a suppressor phenotype that is less sensitive to proinflammatory stimuli and can effectively inhibit the proliferation of T effector cells in vitro. Thus, manipulation of antigen-presenting cells by HMGCRI contributes to an attenuated immune response as shown by promotion of T cells with suppressive capacitie

A framework for parameter estimation and model selection from experimental data in systems biology using approximate Bayesian computation.

As modeling becomes a more widespread practice in the life sciences and biomedical sciences, researchers need reliable tools to calibrate models against ever more complex and detailed data. Here we present an approximate Bayesian computation (ABC) framework and software environment, ABC-SysBio, which is a Python package that runs on Linux and Mac OS X systems and that enables parameter estimation and model selection in the Bayesian formalism by using sequential Monte Carlo (SMC) approaches. We outline the underlying rationale, discuss the computational and practical issues and provide detailed guidance as to how the important tasks of parameter inference and model selection can be performed in practice. Unlike other available packages, ABC-SysBio is highly suited for investigating, in particular, the challenging problem of fitting stochastic models to data. In order to demonstrate the use of ABC-SysBio, in this protocol we postulate the existence of an imaginary reaction network composed of seven interrelated biological reactions (involving a specific mRNA, the protein it encodes and a post-translationally modified version of the protein), a network that is defined by two files containing 'observed' data that we provide as supplementary information. In the first part of the PROCEDURE, ABC-SysBio is used to infer the parameters of this system, whereas in the second part we use ABC-SysBio's relevant functionality to discriminate between two different reaction network models, one of them being the 'true' one. Although computationally expensive, the additional insights gained in the Bayesian formalism more than make up for this cost, especially in complex problems

Regulatorische CD4+ und CD8+ T-Zellen als Kandidaten für regenerative Therapien in einem Mausmodell der Multiplen Sklerose

Multiple sclerosis (MS) is the most common chronic inflammatory disease of the central nervous system in Europe and North America with unknown etiology and only limited treatment options available. Studies using the animal model experimental autoimmune encephalomyelitis (EAE) have shown, that CD4 T helper (Th) cells of the Th1 or Th17 subtype are the essential mediators of the disease, whereas the contribution of CD8 T cells is controversially debated. In parallel to the pathogenic role of T cells in MS and EAE, also a regulatory role for T cells is acknowledged. The overall aim of this work was to better understand the phenotype and the role of regulatory T cells of the CD4- and CD8-subset, in order to apply them as a therapeutic approach in EAE. Therefore, the phenotype of these cells was studied and modified in vitro, and their behavior was monitored in vivo using two-photon laser scanning microscopy. First, it was investigated if and how the immunomodulatory drug atorvastatin can shift the T cell response into a regulatory direction. Atorvastatin directly inhibited proliferation of naïve CD4 T cells, but did not directly induce regulatory T cells. In contrast, co-culture of CD4 T cells with immature dendritic cells (DC), generated in the presence of atorvastatin, induced IL-10 Treg cells with increased suppressive potential. The treatment of DC with atorvastatin and the modulation of DC-T cell interactions might therefore be a promising starting-point for future therapies. Furthermore, the role of CD8 T cells in MOG-induced EAE was investigated. Using a new experimental approach it could be shown, that CD8 T cells did not induce the disease and had no effect on the clearly CD4-mediated disease. Interestingly CD8 T cells were present in high numbers in the CNS, but showed a distinctly different motility pattern from CD4 T cells. Also, CD8 T cells showed an activated effector phenotype, expressing high amounts of IFN-γ, but no IL-17 or regulatory markers. This is in line with the finding, that only IL-17-expressing MOG-specific CD4 Th17 cells, but not IFN-γ-expressing MOG- specific CD4 Th1 cells established long-lasting contacts with neurons, leading to neuronal cell death. Finally, it was examined if it is possible to generate or expand CD8 T cells with regulatory potential in vitro from EAE-recovered mice. It could be shown here, that co-culture with irradiated myelin-specific CD4 T cells generated CD8 T cells with the potential to specifically suppress proliferation and IL-17 production of myelin-specific CD4 Th17 cells in vitro in a contact-dependent way. This indicates that the generation/expansion of suppressor CD8 T cell is possible and an approach worthwhile following for the therapy of EAE.Multiple Sklerose (MS) ist die häufigste chronisch-entzündliche Erkrankung des zentralen Nervensystems in Europa und Nordamerika, mit unbekannter Ursache und beschränkten Therapiemöglichkeiten. Studien des Tiermodells der MS, der Experimentellen Autoimmunen Enzephalomyelitis (EAE), haben gezeigt, dass CD4 T-Zellen des Subtyps Th1 oder Th17 für die Krankheit verantwortlich sind, wohingegen die Rolle von CD8 T-Zellen umstritten ist. Neben der pathogenen Rolle von T-Zellen in der MS und der EAE ist aber auch eine regulatorische Rolle der T-Zellen bekannt. Das übergeordnete Ziel der vorliegenden Arbeit war es, den Phänotyp und die Rolle von regulatorischen T-Zellen des CD4 und CD8 Subtyps besser zu verstehen um sie als Therapie in der EAE einsetzen zu können. Dazu wurde der Phänotyp dieser Zellen in vitro untersucht und modifiziert, sowie das Verhalten der Zellen in vivo mittels 2 Photonenmikroskopie untersucht. Zuerst wurde untersucht, ob und wie das immunmodulatorische Medikament Atorvastatin T-Zellen in eine regulatorische Richtung lenken kann. Es konnte gezeigt werden, dass Atorvastatin die Proliferation von naiven CD4 T-Zellen direkt hemmt, diese aber nicht in eine regulatorische Richtung lenkt. Im Gegensatz dazu führte die Kokultur von CD4 T-Zellen mit unreifen dendritischen Zellen (DC), die in der Anwesenheit von Atorvastatin generiert worden waren, zu IL-10 Treg-Zellen mit höherem suppressorischem Potential. Die Behandlung von DC mit Atorvastatin und die Modulation von DC-T-Zell Interaktionen könnten deshalb vielversprechende Ansatzpunkte für neue Therapiemöglichkeiten sein Des Weiteren wurde die Rolle von CD8 T-Zellen in der MOG-induzierten EAE genauer untersucht. Mit Hilfe eines neuen experimentellen Ansatzes konnte gezeigt werden, dass CD8 T-Zellen die Krankheit nicht auslösten und auch keinen Einflauss auf die Krankheit hatten, welche klar CD4-vermittelt war. Viele CD8 T-Zellen wanderten ins Hirngewebe ein, zeigten aber ein anderes Verhaltensmuster als CD4 T- Zellen. Die CD8 T-Zellen zeigten ausserdem einen aktivierten Effektor-Phänotyp und exprimierten viel IFN-γ, aber kein IL-17 und keine regulatorischen Marker. Dies stimmt mit der Beobachtung überein, dass nur IL-17-produzierende MOG- spezifische CD4 Th17-Zellen aber nicht IFN-γ-produzierende MOG-specifische CD4 Th1-Zellen langanhaltende Kontakte mit Neuronen eingingen, die schliesslich zum neuronalen Zelltod führten. Abschliessend wurde untersucht, ob aus CD8 T-Zellen, die aus Mäusen in der Remissionsphase einer EAE isoliert wurden, in vitro regulatorische Zellen generiert oder expandiert werden können. Die Kokultur mit bestrahlten Myelin-spezifischen CD4 T-Zellen erzeugte regulatorische CD8 T-Zellen, welche die Proliferation und IL-17-Produktion von Myelin-spezifischen CD4 Th17-Zellen gezielt unterdrückten. Dies zeigt, dass es möglich ist regulatorische CD8 T-Zellen zu generieren/expandieren und dass dies einen vielversprechenden Therapieansatz in der EAE darstellen könnte

Dendritic cells tip the balance towards induction of regulatory T cells upon priming in experimental autoimmune encephalomyelitis

Counter-balancing regulatory mechanisms, such as the induction of regulatory T cells (Treg), limit the effects of autoimmune attack in neuroinflammation. However, the role of dendritic cells (DCs) as the most powerful antigen-presenting cells, which are intriguing therapeutic targets in this context, is not fully understood. Here, we demonstrate that conditional ablation of DCs during the priming phase of myelin-specific T cells in experimental autoimmune encephalomyelitis (EAE) selectively aborts inducible Treg (iTreg) induction, whereas generation of T helper (Th)1/17\ua0cells is unaltered. DCs facilitate iTreg induction by creating a milieu with high levels of interleukin (IL)-2 due to a strong proliferative response. In the absence of DCs, B220 B cells take over priming of Th17\ua0cells in the place of antigen-presenting cells (APCs), but not the induction of iTreg, thus leading to unregulated, severe autoimmunity

Cross-recognition of a myelin peptide by CD8+ T cells in the CNS is not sufficient to promote neuronal damage

Multiple sclerosis (MS) is an inflammatory disease of the CNS thought to be driven by CNS-specific T lymphocytes. Although CD8 T cells are frequently found in multiple sclerosis lesions, their distinct role remains controversial because direct signs of cytotoxicity have not been confirmed in vivo. In the present work, we determined that murine ovalbumin-transgenic (OT-1) CD8 T cells recognize the myelin peptide myelin oligodendrocyte glycoprotein 40–54 (MOG) both in vitro and in vivo. The aim of this study was to investigate whether such cross-recognizing CD8 T cells are capable of inducing CNS damage in vivo. Using intravital two-photon microscopy in the mouse model of multiple sclerosis, we detected antigen recognition motility of the OT-1 CD8 T cells within the CNS leading to a selective enrichment in inflammatory lesions. However, this cross-reactivity of OT-1 CD8 T cells with MOG peptide in the CNS did not result in clinically or subclinically significant damage, which is different from myelin-specific CD4 Th17-mediated autoimmune pathology. Therefore, intravital imaging demonstrates that local myelin recognition by autoreactive CD8 T cells in inflammatory CNS lesions alone is not sufficient to induce disability or increase axonal injury

Modulation of Dendritic Cell Immunobiology via Inhibition of 3-Hydroxy-3-Methylglutaryl-CoA (HMG-CoA) Reductase

<div><p>The maturation status of dendritic cells determines whether interacting T cells are activated or if they become tolerant. Previously we could induce T cell tolerance by applying a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor (HMGCRI) atorvastatin, which also modulates MHC class II expression and has therapeutic potential in autoimmune disease. Here, we aimed at elucidating the impact of this therapeutic strategy on T cell differentiation as a consequence of alterations in dendritic cell function. We investigated the effect of HMGCRI during differentiation of peripheral human monocytes and murine bone marrow precursors to immature DC <i>in vitro</i> and assessed their phenotype. To examine the stimulatory and tolerogenic capacity of these modulated immature dendritic cells, we measured proliferation and suppressive function of CD4+ T cells after stimulation with the modulated immature dendritic cells. We found that an HMGCRI, atorvastatin, prevents dendrite formation during the generation of immature dendritic cells. The modulated immature dendritic cells had a diminished capacity to take up and present antigen as well as to induce an immune response. Of note, the consequence was an increased capacity to differentiate naïve T cells towards a suppressor phenotype that is less sensitive to proinflammatory stimuli and can effectively inhibit the proliferation of T effector cells <i>in vitro</i>. Thus, manipulation of antigen-presenting cells by HMGCRI contributes to an attenuated immune response as shown by promotion of T cells with suppressive capacities.</p></div