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Regulation of effector T cells is an important mechanism to control organ-specific inflammation. Thereby regulatory T cells (Treg cells) are essential for maintaining peripheral immune tolerance and for establishing parenchyma immune homeostasis. A novel population of natural human Treg characterized by the constitutive expression of the immune-tolerogenic human HLA-G molecule has been identified. In the first part of the study, we elucidated the mechanism(s) by which CD4+ HLA-Gpos Treg modulates their cellular targets namely autologous HLA-G negative responder T cells (HLAGneg Tresp). Using a suppression system free of antigen-presenting cells (APC), we demonstrate a T-T cell interaction resulting in suppression of HLA-Gneg Tresp. We could also show that this suppression was independent of cell-cell contact. Importantly, stimulus of T cell receptor (TCR) on HLA-Gpos Treg facilitated their suppressive capacity. We also observed that removal of HLA-Gpos Treg from the established co-cultures could restore the ability of HLA-Gneg Tresp to proliferate upon TCR re-stimulation, indicating that the suppression was reversible. Further, HLA-Gpos Treg–mediated suppression was critically depending on the secretion of IL-10 but not TGF-β. Taken together, this part of the work provides an in-depth characterization of the mechanisms of how HLA-Gpos Treg suppresses T responder cells in direct T-T interactions. Understanding the suppressive mechanism used by HLA-Gpos Treg may help to develop therapeutic strategies to modulate regulatory arms of T-cell suppression. In the second part of this study, the potential role of HLA-Gpos Treg in the pathophysiological process of Multiple Sclerosis (MS), a prototypic autoimmune inflammatory central nervous system (CNS), has been investigated. We found that HLA-Gpos Treg are enriched in the cerebrospinal fluid (CSF) from MS patients, but not in non-inflammatory controls. CSFderived HLA-Gpos Treg showed predominance of central memory (CD45RA-CD27+) phenotype, exhibited markers of activation (ICOS), and had significantly higher expression of the inflammatory chemokine receptor CCR5. Importantly, these cells demonstrated as potent suppressors to autologous CD4+ T-cell proliferation. Using an in vitro model of human blood brain barrier, we showed that HLA-Gpos Treg have a strong propensity to migrate, which could be facilitated by MIP1α and RANTES (ligands of CCR5) but not MIP3β (a ligand of CCR7). The HLA-Gpos Treg migration triggered by chemokines was also associated with a gain of suppressive capacity upon cellular transmigration. In contrast to CD4+CD25+ naturally occurring FoxP3-expressing Treg, HLA-Gpos Treg from patients with MS did not exhibit impaired function, suggesting that HLA-Gpos Treg are selectively recruited to the sites of CNS inflammation in an effort to combat destructive inflammation during MS. Our results contribute to the understanding of the role and function of HLA-Gpos Treg and provide an important example of “beneficial” T-cell inflammation in CNS autoimmunity- interesting both from a patho/-physiological and a therapeutically point of view.
The bidirectional influence of parenchymal cells and cells of the immune system, especially of antigen-presenting and CD8\(^+\) T cells, in situations of putative auto- immune pathogenicity and degeneration was the main topic of this thesis. In the first part, the influence of human muscle cells on antigen-presenting cells was investigated. In inflammatory myopathies prominent infiltrates of immune cells containing T cells and antigen-presenting cells like macrophages and dendritic cells are present. The hypothesis was that human myoblasts have an inhibiting influence on these antigen-presenting cells under homeostatic conditions. A dysfunction or impairment under inflammatory circumstances might contribute to the development of myopathic conditions. The surface analysis of dendritic cells cocultured with myoblasts showed that immature dendritic cells could be driven into a reversible semi- mature state with significantly elevated levels of CD80. These dendritic cells were additionally characterized by their inhibiting function on T-cell proliferation. It was also shown that the lysates of healthy myoblasts could strongly enhance the phagocytic ability of macrophages, which could help with muscle regeneration and which might be disturbed in myositis patients. The second part of this thesis was about the clonal specificity of CD8\(^+\) T cells in a mouse model with genetically induced over-expression of PLP in oligodendrocytes. Here, we could show that the cytotoxic T lymphocytes, which had previously been shown to be pathogenic, were clonally expanded in the CNS of the transgenic mice. The amino acid sequences of the corresponding receptor chains were not identical, yet showed some similarities, which could mean that these clones recognize similar antigens (or epitopes of the same antigen). The knockout of PD-1 in this setting allowed for an analysis of the importance of tissue immune regulation. It became evident that the absence of PD-1 induced a larger number of clonal expansions in the CNS, hinting towards a reduced threshold for clonal disturbance and activation in these T cells. The expansions were, however, not pathogenic by themselves. Only in the presence of tissue damage and an antigenic stimulus (in our case the overexpression of PLP), the PD-1 limitation exacerbated the immune pathogenicity. Therefore, only in the presence of a “tissue damage signal”, the dyshomeostasis of T cells lacking PD-1 achieved high pathogenetic relevance. Finally, we investigated the pathogenetic role of CD8 T cells in Rasmussen encephalitis, a rare and chronic neurological disease mainly affecting children. The analysis of the T-cell receptor repertoire in Rasmussen encephalitis patients in the peripheral CD4\(^+\) and CD8\(^+\) T-cell compartments as well as the brain revealed the involvement of T cells in the pathogenicity of this disease. Many clonal expansions in the brain matched CD8\(^+\) T-cell expansions in the periphery on the sequence level. These putatively pathogenic clones could be visualized by immunohistochemistry in the brain and were found in close proximity to astrocytes and neurons. Additionally, the expanded clones could be found in the periphery of patients for at least one year.
Aim of Investigation: The neurological manifestations of Fabry’s disease, a rare, X-linked, multisystem disorder caused by alpha-galactosidase A deficiency and globotriosylceramide (Gb3) accumulation, include both peripheral and central nervous system symptoms. Here we evaluated a prospectively recruited cohort of patients with Fabry’s disease for pain, small nerve fiber function, and skin innervation. Methods: 66 patients (31 male and 35 female) were enrolled,31 patients were on ERT. All patients underwent quantitative sensory testing (QST), electrophysiological examination, and extra- and transcranial Doppler sonography. For pain and mood assessment standardized questionnaires were used. Skin biopsies were performed at the left distal leg in 38 patients for intraepidermal nerve fiber density (IENFD) assessment. Results: Age at examination did not differ significantly between women (40.2+/-16.2 years) and men (38.9+/-13.8; n.s.). 29/31 male and 19/35 female patients complained of acroparesthesias or neuropathic pain. QST abnormalities indicative of small fiber impairment were found in 26/31 male and 28/35 female patients. Electrophysiological examination of large fibers and autonomic fibers revealed pathological findings in 11/31 male and 3/35 female patients. All patients had normal Doppler sonography results. Indicators for depression were present in 14/31 male and 10/35 female patients. 20/31 male and 18/35 female patients had a skin biopsy, the IENFD was significantly reduced in male (2.0+/-2.8 fibers/mm) compared with female patients (6.7 +/- 4.4 fibers/mm). In 10 patients free from neurological symptoms, QST and IENFD abnormalities were still detected. Follow up examination after one year in 12 patients under ERT (2.1+/-1.7 years) showed improvement in some symptoms and in QST and neurophysiology in six patients with normal renal function. 20/35 female patients older than 40 y had concomitant diseases, while none of the 18 younger female patients did. The corresponding radio in male patients was 5/19 (>=40y) and 2/13 (<40y) respectively. Conclusions: Neuropathic pain and sensory deficits of the distal extremities are common in patients with Fabry’s disease. QST and IENFD analysis are important for early diagnosis of nerve involvement in Fabry’s disease. Small fiber function may improve under ERT in patients without severe renal impairment.
Charcot-Marie-Tooth 1B (CMT1B) is a progressive inherited demyelinating disease of human peripheral nervous system leading to sensory and/or motor function disability and is caused by mutations in the P0 gene. Mice heterozygously deficient for P0 (P0+/-) are an adequate model of this human disorder showing myelin degeneration, formation of onion bulbs, remyelination and a reduced motor conduction velocity of around 30m/s similar to patients. Previously, it had been shown that T-lymphocytes and macrophages play a crucial role during pathogenesis in peripheral nerves of P0+/- mice. Both, T-lymphocytes and macrophages increase in number in the endoneurium and deletion of T-lymphocytes or deletion of a macrophage-directed cytokine ameliorates the disease. In this study the monocyte chemoattractant protein-1 (MCP-1) was identified as an early regulated cytokine before onset of disease is visible at the age of six months. MCP-1 mRNA and protein expression could be detected in femoral quadriceps and sciatic nerves of P0+/- mice already at the age of one month but not in cutaneous saphenous nerves which are never affected by the disease. MCP-1 was shown to be expressed by Schwann cells and to mediate the immigration of immune cells into peripheral nerves. Deletion of MCP-1 in P0+/- mice accomplished by crossbreeding P0 and MCP-1 deficient mice revealed a substantial reduction of immune cells in peripheral nerves of P0+/-/MCP-1+/- and P0+/-/MCP-1-/- mice at the age of six months. In twelve months old mice reduction of immune cells in peripheral nerves is accompanied by amelioration of demyelinating disease in P0+/-/MCP-1+/- and aggravation of demyelinating disease in lumbar ventral roots of P0+/ /MCP-1-/- mice in comparison to P0+/ /MCP 1+/+ mice. Furthermore, activation of the MEK1/2-ERK1/2 signalling cascade could be demonstrated to take place in Schwann cells of affected peripheral nerves of P0+/- mice overlapping temporarily and spatially with MCP-1 expression. An animal experiment using a MEK1/2-inhibitor in vivo, CI-1040, revealed that upon reduction of ERK1/2 phosphorylation MCP-1 mRNA expression is diminished suggesting that the activation of the MEK1/2-ERK1/2 signalling cascade is necessary for MCP-1 expression. Additionally, peripheral nerves of P0+/- mice showing reduced ERK1/2 phosphorylation and MCP-1 mRNA expression also show reduced numbers of macrophages in the endoneurium. This study shows a molecular link between a Schwann cell based mutation and immune cell function. Inhibition of the identified signalling cascade might be a putative target for therapeutic approaches.
Myelinmutationen des zentralen und peripheren Nervensystems verursachen erheblich behindernde und bislang nicht heilbare Erkrankungen. In dieser Arbeit verwendeten wir transgene PLP überexprimierende Mäuse (PLPtg) als Modell für zentrale Myelinopathien und heterozygot P0 defiziente (P0+/-) Mäuse als Modell für hereditäre Neuropathien des peripheren Nervensystems. Beide Modelle zeigen eine niedriggradige Inflammation des Nervengewebes. Durch Verpaarung mit immundefizienten Mausstämmen konnten wir die Relevanz von Makrophagen und T- Lymphozyten in der Entstehung der Myelinpathologie zeigen. Nachdem wir beweisen konnten, dass CD8+ T- Lymphozyten maßgeblich zur Pathologie in PLPtg Mäusen beitragen untersuchten wir den Einfluss eines wichtigen zytotoxischen Moleküls, Granzym B, auf den neuralen Schaden. Durch Generierung von Granzym B defizienten PLPtg Knochenmarkschimären konnten wir eine deutliche Reduktion des glialen Schadens und der Oligodendrozytenapoptose nachweisen. Granzym B ist also zumindest teilweise verantwortlich für die Schädigung, die durch T- Lymphozyten hervorgerufen wird. Um die zusätzliche Informationen über die Rolle der Immunmodulation in unseren Modellen zu gewinnen, untersuchten wir das koinhibitorische Molekül PD-1, einen CD-28 verwandten Rezeptor, der auf B- und T- Lymphozyten exprimiert wird. Bei der Untersuchung von Myelinmutanten des ZNS und PNS (PLPtg und P0+/-), die zusätzlich PD-1 defizient waren, konnten wir einen signifikanten Anstieg von CD8+ T- Lymphozyten und eine deutliche Verschlechterung des glialen Schadens beobachten. In PLPtg Mäusen induzierte die Abwesenheit von PD-1 verstärkte Oligodendrozytenapoptose und klonale Expansion. Außerdem neigen ZNS- Lymphozyten aber nicht periphere CD8+ T- Zellen zur verstärkten Sekretion von proinflammatorischen Zytokinen. In P0+/- Mäusen führt Abwesenheit von PD-1 zu moderaten motorischen und sensorischen Störungen, was die wichtige Rolle von PD-1 in immunologischen Regulationsmechanismen unterstreicht. Zusammenfassend kann man festhalten, daß Granzym B ein wichtiges Effektormolekül zytotoxischer T- Zellen in PLPtg Mäusen ist. PD-1 spielt eine wichtige Rolle in der Regulation von Effektorzellen in unseren Modellen für zentrale und periphere Myelinopathien. Veränderungen dieser Regulation können deutliche Neuroinflammation mit starker Myelinpathologie hervorrufen. Diese Ergebnisse können dazu beitragen, die starke klinische Variabilität von polygenen und sogar monogenen neurologischen Erkrankungen zu erklären.
Summary Myelin protein zero (P0) is a key myelin component in maintaining the integrity and functionality of the peripheral nervous system. Mutated variants are the cause for several disabilitating peripheral neuropathies such as Charcot-Marie-Tooth disease or Dejerine –Sotas syndrome. Using P0 knockout mice - a mouse model for these diseases - together with their wt counterparts on C57BL/6 background we studied the shaping of the T-cell repertoire specific for P0 in the presence and in the absence of this protein during the ontogeny of T-cells. Our approach was to use a series of overlapping 20-mer peptides covering the entire amino acid sequence of P0. This series of P0 peptides was employed for epitope mapping of the H2-Ab restricted T cell response. Thus, P0 peptide 5 (P0 41-60) in the extracellular domain of P0 was identified as the main immunogenic peptide. The immunogenic peptide containing the core immunodominant determinant in the P0 sequence was employed in studies of tolerance, revealing a highly reactive P0 specific T-cell repertoire in P0 ko mice while in wt mice the high avidity repertoire was inactivated in order to ensure self tolerance. In wild type and heterozygous P0 mice tolerance is not dependent on gene dosage. P0 is a tissue specific antigen whose expression is limited to myelinating Schwann cells. The classical view on tolerance to tissue specific antigens attributed this role to peripheral mechanisms. Driven by the finding that intrathymic expression of tissue-specific antigens is a common occurrence, we confirmed that “promiscuous” expression on thymic stroma holds true also for myelin P0. In addition, using bone marrow chimeras we investigated the capacity of bone marrow derived cells versus nonhematopoietic cells to induce tolerance towards P0. Our findings show that bone marrow derived cells although tolerogenic to some degree are not sufficient to mediate complete tolerance. P0 expression on cells with origin other than bone marrow showed to be sufficient and necessary to induce sound tolerance. We identified one cryptic (P0 peptide 8) and two subdominant epitopes (P0 petides 1, and 3). P0 peptide 8 was reactive in both wt and P0 ko mice. Peptides 1 and 3 were immunogenic in P0 ko but not in wt mice. Several P0 peptides including the immunogenic peptide 5 were involved in direct and adoptive transfer EAN studies. None of them induced clinical signs of EAN. Immunization with P0 peptide 3 did induce inflammation of the peripheral nerves reflected by the infiltration of macrophages and CD3 positive cells. More studies involving highly P0 specific T-cell lines are needed to characterize the P0 induced EAN. Our findings may have direct implications for secondary autoimmunity and inflammation in peripheral nerves developing after correcting the P0 genetic defect by gene therapy in aforementioned diseases.