Susann Pankratz

Blockade of the kinin receptor B1 protects from autoimmune CNS disease by reducing leukocyte trafficking.

Disruption of the blood brain barrier (BBB) and transendothelial trafficking of immune cells into the central nervous system (CNS) are pathophysiological hallmarks of Multiple Sclerosis (MS) and its animal model, Experimental Autoimmune Encephalomyelitis (EAE). Kinins are proinflammatory peptides which are released during tissue injury including EAE. They increase vascular permeability and enhance inflammation by acting on distinct bradykinin receptors, B1R and B2R. We studied the expression of B1R and B2R and the effect of their inhibition on the disease course, BBB integrity and T cell migration following myelin oligodendrocyte glycoprotein (MOG(35-55))-induced EAE. B1R, but not B2R expression was markedly enhanced in inflammatory CNS lesions in mice and humans. Brain endothelial cells could be identified as major source of B1R protein. The severity of EAE was significantly alleviated in B1R(-/-) mice compared with wild-type (WT) controls (P<0.05). Treatment of WT mice with the B1R antagonist R715 before and after disease onset was equally effective (P<0.05) while B1R activation by R838 promoted EAE (P<0.05). B1R inhibition was accompanied by a remarkable reduction of BBB disruption and tissue inflammation. In vitro analyses revealed that B1R suppression reverses the upregulation of ICAM-I and VCAM-I at the inflamed BBB thereby limiting T cell transmigration. In contrast, blocking B2R had no significant impact on EAE. We conclude that B1R inhibition can reduce BBB damage and cell invasion during autoimmune CNS disease and may offer a novel anti-inflammatory strategy for the treatment of MS.

Blood coagulation factor XII drives adaptive immunity during neuroinflammation via CD87-mediated modulation of dendritic cells

Aberrant immune responses represent the underlying cause of central nervous system (CNS) autoimmunity, including multiple sclerosis (MS). Recent evidence implicated the crosstalk between coagulation and immunity in CNS autoimmunity. Here we identify coagulation factor XII (FXII), the initiator of the intrinsic coagulation cascade and the kallikrein–kinin system, as a specific immune cell modulator. High levels of FXII activity are present in the plasma of MS patients during relapse. Deficiency or pharmacologic blockade of FXII renders mice less susceptible to experimental autoimmune encephalomyelitis (a model of MS) and is accompanied by reduced numbers of interleukin-17A-producing T cells. Immune activation by FXII is mediated by dendritic cells in a CD87-dependent manner and involves alterations in intracellular cyclic AMP formation. Our study demonstrates that a member of the plasmatic coagulation cascade is a key mediator of autoimmunity. FXII inhibition may provide a strategy to combat MS and other immune-related disorders.

Human CD4+HLA-G+ regulatory T cells are potent suppressors of graft-versus-host disease in vivo

CD4+ T cells expressing the immunotolerizing molecule HLA‐G have been described as a unique human thymus‐derived regulatory T (tTreg) cell subset involved in immunoregulation and parenchymal homeostasis during infectious and autoimmune inflammation. We compared properties and molecular characteristics of human CD4+HLA‐G+ with those of CD4+CD25+FoxP3‐expressing tTreg cells using in vitro studies of T‐cell receptor (TCR) signaling, single‐cell electrophysiology, and functional in vivo studies. Both tTreg populations are characterized by alterations in proximal‐signaling pathways on TCR stimulation and a hyperpolarization of the plasma membrane when compared to conventional CD4+ T cells. However, both clearly differ in phenotype and pattern of secreted cytokines, which results in distinct mechanisms of suppression: While CD4+HLA‐G+ cells secrete high levels of inhibitory molecules (IL‐10, soluble HLA‐G, IL‐35), CD4+CD25+FoxP3+ cells express these molecules at significantly lower levels and seem to exert their function mainly in a contact‐dependent manner via cyclic adenosine‐monophosphate. Finally we demonstrate that human CD4+HLA‐G+ tTreg cells significantly ameliorated graft‐versus‐host disease in a humanized mouse model as a first proof of their in vivo relevance. Our data further characterize and establish CD4+HLA‐G+ cells as a potent human tTreg population that can modulate polyclonal adaptive immune responses in vivo and thus being a promising candidate for potential clinical applications in the future.—Pankratz, S., Bittner, S., Herrmann, A. M., Schuhmann, M. K., Ruck, T., Meuth, S. G., Wiendl, H. Human CD4+HLA‐G+ regulatory T cells are potent suppressors of graft‐versus‐host disease in vivo. FASEB J. 28, 3435‐3445 (2014). www.fasebj.org

CD4+ CD25+ FoxP3+ regulatory T cells suppress cytotoxicity of CD8+ effector T cells: implications for their capacity to limit inflammatory central nervous system damage at the parenchymal level

BackgroundCD4+ CD25+ forkhead box P3 (FoxP3)+ regulatory T cells (T reg cells) are known to suppress adaptive immune responses, key control tolerance and autoimmunity.MethodsWe challenged the role of CD4+ T reg cells in suppressing established CD8+ T effector cell responses by using the OT-I/II system in vitro and an OT-I-mediated, oligodendrocyte directed ex vivo model (ODC-OVA model).ResultsCD4+ T reg cells dampened cytotoxicity of an ongoing CD8+ T effector cell attack in vitro and within intact central nervous system tissue ex vivo. However, their suppressive effect was limited by the strength of the antigen signal delivered to the CD8+ T effector cells and the ratio of regulatory to effector T cells. CD8+ T effector cell suppression required T cell receptor-mediated activation together with costimulation of CD4+ T reg cells, but following activation, suppression did not require restimulation and was antigen non-specific.ConclusionsOur results suggest that CD4+ T reg cells are capable of suppressing CD8+ T effector cell responses at the parenchymal site, that is, limiting parenchymal damage in autoimmune central nervous system inflammation.

CD4(+)HLA-G(+) regulatory T cells: Molecular signature and pathophysiological relevance.

The regulation of potentially harmful immune responses by regulatory T (Treg) cells is essential for maintaining peripheral immune tolerance and homeostasis. Especially CD4(+) Treg cells have been regarded as pivotal regulators of autoreactive and inflammatory responses as well as inducers of immune tolerance by using a variety of immune suppressive mechanisms. Besides the well-known classical CD4(+)CD25(+)FoxP3(+) Treg cells, CD4(+) T cells expressing the immune tolerizing molecule human leukocyte antigen G (HLA-G) have been recently described as another potent thymus-derived Treg (tTreg) cell subset. Albeit both tTreg subsets share common molecular characteristics, the mechanisms of their immunosuppressive function differ fundamentally. Dysfunction and numerical abnormalities of classical CD4(+) tTreg cells have been implicated in the pathogenesis of several immune-mediated diseases such as multiple sclerosis (MS). Clearly, a deeper understanding of the various CD4(+) tTreg subsets and also the underlying mechanisms of impaired immune tolerance in these disorders are essential for the development of potential therapeutic strategies. This review focuses on the current knowledge on defining features and functioning of HLA-G(+)CD4(+) tTreg cells as well as their emerging role in various pathologies with special emphasis on the pathogenesis of MS. Furthermore, future research possibilities together with potential therapeutic applications are discussed.

CD4+NKG2D+ T cells exhibit enhanced migratory and encephalitogenic properties in neuroinflammation.

Migration of encephalitogenic CD4+ T lymphocytes across the blood-brain barrier is an essential step in the pathogenesis of multiple sclerosis (MS). We here demonstrate that expression of the co-stimulatory receptor NKG2D defines a subpopulation of CD4+ T cells with elevated levels of markers for migration, activation, and cytolytic capacity especially when derived from MS patients. Furthermore, CD4+NKG2D+ cells produce high levels of proinflammatory IFN-γ and IL-17 upon stimulation. NKG2D promotes the capacity of CD4+NKG2D+ cells to migrate across endothelial cells in an in vitro model of the blood-brain barrier. CD4+NKG2D+ T cells are enriched in the cerebrospinal fluid of MS patients, and a significant number of CD4+ T cells in MS lesions coexpress NKG2D. We further elucidated the role of CD4+NKG2D+ T cells in the mouse system. NKG2D blockade restricted central nervous system migration of T lymphocytes in vivo, leading to a significant decrease in the clinical and pathologic severity of experimental autoimmune encephalomyelitis, an animal model of MS. Blockade of NKG2D reduced killing of cultivated mouse oligodendrocytes by activated CD4+ T cells. Taken together, we identify CD4+NKG2D+ cells as a subpopulation of T helper cells with enhanced migratory, encephalitogenic and cytotoxic properties involved in inflammatory CNS lesion development.

4-Aminopyridine ameliorates mobility but not disease course in an animal model of multiple sclerosis ☆

Neuropathological changes following demyelination in multiple sclerosis (MS) lead to a reorganization of axolemmal channels that causes conduction changes including conduction failure. Pharmacological modulation of voltage-sensitive potassium channels (K(V)) has been found to improve conduction in experimentally induced demyelination and produces symptomatic improvement in MS patients. Here we used an animal model of autoimmune inflammatory neurodegeneration, namely experimental autoimmune encephalomyelitis (EAE), to test the influence of the K(V)-inhibitor 4-aminopyridine (4-AP) on various disease and immune parameters as well as mobility in MOG₃₅₋₅₅ immunized C57Bl/6 mice. We challenged the hypothesis that 4-AP exerts relevant immunomodulatory or neuroprotective properties. Neither prophylactic nor therapeutic treatment with 4-AP altered disease incidence or disease course of EAE. Histopathological signs of demyelination and neuronal damage as well as MRI imaging of brain volume changes were unaltered. While application of 4-AP significantly reduced the standing outward current of stimulated CD4(+) T cells compared to controls, it failed to impact intracellular calcium concentrations in these cells. Compatibly, KV channel inhibition neither influenced CD4(+) T cell effector functions (proliferation, IL17 or IFNγ production). Importantly however, despite equal disease severity scores 4-AP treated animals showed improved mobility as assessed by 2 independent methods, 1) foot print and 2) rotarod analysis (0.332 ± 0.03, n=7 versus 0.399 ± 0.08, n=14, p<0.001, respectively). Our data suggest that 4-AP while having no apparent immunomodulatory or direct neuroprotective effects, significantly ameliorates conduction abnormalities thereby improving gait and coordination. Improvement of mobility in this experimental model supports trial data and clinical experience with 4-AP in the symptomatic treatment of MS.

Targeting Ewing sarcoma with activated and GD2-specific chimeric antigen receptor-engineered human NK cells induces upregulation of immune-inhibitory HLA-G

ABSTRACT Activated and in vitro expanded natural killer (NK) cells have substantial cytotoxicity against many tumor cells, but their in vivo efficacy to eliminate solid cancers is limited. Here, we used chimeric antigen receptors (CARs) to enhance the activity of NK cells against Ewing sarcomas (EwS) in a tumor antigen-specific manner. Expression of CARs directed against the ganglioside antigen GD2 in activated NK cells increased their responses to GD2+ allogeneic EwS cells in vitro and overcame resistance of individual cell lines to NK cell lysis. Second-generation CARs with 4-1BB and 2B4 co-stimulatory signaling and third-generation CARs combining both co-stimulatory domains were all equally effective. By contrast, adoptive transfer of GD2-specific CAR gene-modified NK cells both by intratumoral and intraperitoneal delivery failed to eliminate GD2-expressing EwS xenografts. Histopathology review revealed upregulation of the immunosuppressive ligand HLA-G in tumor autopsies from mice treated with NK cells compared to untreated control mice. Supporting the relevance of this finding, in vitro co-incubation of NK cells with allogeneic EwS cells induced upregulation of the HLA-G receptor CD85j, and HLA-G1 expressed by EwS cells suppressed the activity of NK cells from three of five allogeneic donors against the tumor cells in vitro. We conclude that HLA-G is a candidate immune checkpoint in EwS where it can contribute to resistance to NK cell therapy. HLA-G deserves evaluation as a potential target for more effective immunotherapeutic combination regimens in this and other cancers.

Prothrombin and factor X are elevated in multiple sclerosis patients

Animal models have implicated an integral role for coagulation factors in neuroinflammatory diseases such as multiple sclerosis (MS) beyond their role in hemostasis. However, their relevance in humans requires further elucidation. This study aimed to determine whether levels of coagulation factors differ between patients with neuroimmunological disorders and respective controls. Individuals suffering from relapsing–remitting and secondary progressive MS had significantly higher prothrombin and factor X levels than healthy donors, whereas levels were unchanged in primary progressive MS and neuromyelitis optica patients. Our study demonstrates that coagulation factors may be key mediators in neuroinflammation and may therefore provide future targets for therapeutic strategies. Ann Neurol 2016;80:946–951

Phospholipase D1 mediates lymphocyte adhesion and migration in experimental autoimmune encephalomyelitis.

Lymphocyte adhesion and subsequent trafficking across endothelial barriers are essential steps in various immune‐mediated disorders of the CNS, including MS. The molecular mechanisms underlying these processes, however, are still unknown. Phospholipase D1 (PLD1), an enzyme that generates phosphatidic acid through hydrolysis of phosphatidylcholine and additionally yields choline as a product, has been described as regulator of the cell mobility. By using PLD1‐deficient mice, we investigated the functional significance of PLD1 for lymphocyte adhesion and migration in vitro and after myelin oligodendrocyte glycoprotein (MOG)35–55‐induced EAE, a model of human MS. The lack of PLD1 reduced chemokine‐mediated static adhesion of lymphocytes to the endothelial adhesion molecules vascular cell adhesion molecule 1 (VCAM‐1) and intercellular adhesion molecule 1 (ICAM‐1) in vitro, and was accompanied by a decreased migratory capacity in both blood brain barrier and cell migration models. Importantly, PLD1 is also relevant for the recruitment of immune cells into the CNS in vivo since disease severity after EAE was significantly attenuated in PLD1‐deficient mice. Furthermore, PLD1 expression could be detected on lymphocytes in MS patients. Our findings suggest a critical function of PLD1‐dependent intracellular signaling cascades in regulating lymphocyte trafficking during autoimmune CNS inflammation.