Increased Intrathecal Activity of Follicular Helper T Cells in Patients With Relapsing-Remitting Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: Follicular helper T (Tfh) cells play a critical role in protective immunity helping B cells produce antibodies against foreign pathogens and are likely implicated in the pathogenesis of various autoimmune diseases. The purpose of this study was to investigate the role of Tfh cells in the pathogenesis of multiple sclerosis (MS). METHODS: Using flow cytometry, we investigated phenotype, prevalence, and function of Tfh cells in blood and CSF from controls and patients with relapsing-remitting MS (RRMS) and primary progressive MS (PPMS). In addition, an in vitro blood-brain barrier coculture assay of primary human astrocytes and brain microvascular endothelial cells grown in a Boyden chamber was used to assess the migratory capacity of peripheral Tfh cells. RESULTS: This study identified 2 phenotypically and functionally distinct Tfh cell populations: CD25- Tfh cells (Tfh1-like) and CD25int Tfh cells (Tfh17-like). Whereas minor differences in Tfh cell populations were found in blood between patients with MS and controls, we observed an increased frequency of CD25- Tfh cells in CSF of patients with RRMS and PPMS and CD25int Tfh cells in patients with RRMS, compared with controls. Increasing frequencies of CSF CD25- Tfh cells and the CD25- Tfh/Tfr ratio scaled with increasing IgG index in patients with RRMS. Despite an increased prevalence of intrathecal Tfh cells in patients with MS, no difference in the migratory capacity of circulating Tfh cells was observed between controls and patients with MS. Instead, CSF concentrations of CXCL13 scaled with total counts of Tfh and Tfr cell subsets in the CSF. DISCUSSION: Our study indicates substantial changes in intrathecal Tfh dynamics, particularly in patients with RRMS, and suggests that the intrathecal inflammatory environment in patients with RRMS promotes recruitment of peripheral Tfh cells rather than the Tfh cells having an increased capacity to migrate to CNS.

MCAM/CD146 Signaling via PLCγ1 Leads to Activation of ß1-Integrins in Memory T-Cells Resulting in Increased Brain Infiltration.

Multiple sclerosis is a chronic auto-inflammatory disease of the central nervous system affecting patients worldwide. Neuroinflammation in multiple sclerosis is mainly driven by peripheral immune cells which invade the central nervous system and cause neurodegenerative inflammation. To enter the target tissue, immune cells have to overcome the endothelium and transmigrate into the tissue. Numerous molecules mediate this process and, as they determine the tissue invasiveness of immune cells, display great therapeutic potential. Melanoma cell adhesion molecule (MCAM) is a membrane-anchored glycoprotein expressed by a subset of T-cells and MCAM+ T-cells have been shown to contribute to neuroinflammation in multiple sclerosis. The role of the MCAM molecule for brain invasion, however, remained largely unknown. In order to investigate the role of the MCAM molecule on T-cells, we used different in vitro and in vivo assays, including ex vivo flow chambers, biochemistry and microscopy experiments of the mouse brain. We demonstrate that MCAM directly mediates adhesion and that the engagement of MCAM induces intracellular signaling leading to ß1-integrin activation on human T-cells. Furthermore, we show that MCAM engagement triggers the phosphorylation of PLCγ1 which is required for integrin activation and thus amplification of the cellular adhesive potential. To confirm the physiological relevance of our findings in vivo, we demonstrate that MCAM plays an important role in T-cell recruitment into the mouse brain. In conclusion, our data demonstrate that MCAM expressed on T-cells acts as an adhesion molecule and a signaling receptor that may trigger ß1-integrin activation via PLCγ1 upon engagement.

CD8+ T cell-mediated endotheliopathy is a targetable mechanism of neuro-inflammation in Susac syndrome.

Neuroinflammation is often associated with blood-brain-barrier dysfunction, which contributes to neurological tissue damage. Here, we reveal the pathophysiology of Susac syndrome (SuS), an enigmatic neuroinflammatory disease with central nervous system (CNS) endotheliopathy. By investigating immune cells from the blood, cerebrospinal fluid, and CNS of SuS patients, we demonstrate oligoclonal expansion of terminally differentiated activated cytotoxic CD8+ T cells (CTLs). Neuropathological data derived from both SuS patients and a newly-developed transgenic mouse model recapitulating the disease indicate that CTLs adhere to CNS microvessels in distinct areas and polarize granzyme B, which most likely results in the observed endothelial cell injury and microhemorrhages. Blocking T-cell adhesion by anti-α4 integrin-intervention ameliorates the disease in the preclinical model. Similarly, disease severity decreases in four SuS patients treated with natalizumab along with other therapy. Our study identifies CD8+ T-cell-mediated endotheliopathy as a key disease mechanism in SuS and highlights therapeutic opportunities.

Human CCR5high effector memory cells perform CNS parenchymal immune surveillance via GZMK-mediated transendothelial diapedesis.

Although the CNS is immune privileged, continuous search for pathogens and tumours by immune cells within the CNS is indispensable. Thus, distinct immune-cell populations also cross the blood-brain barrier independently of inflammation/under homeostatic conditions. It was previously shown that effector memory T cells populate healthy CNS parenchyma in humans and, independently, that CCR5-expressing lymphocytes as well as CCR5 ligands are enriched in the CNS of patients with multiple sclerosis. Apart from the recently described CD8+ CNS tissue-resident memory T cells, we identified a population of CD4+CCR5high effector memory cells as brain parenchyma-surveilling cells. These cells used their high levels of VLA-4 to arrest on scattered VCAM1, their open-conformation LFA-1 to crawl preferentially against the flow in search for sites permissive for extravasation, and their stored granzyme K (GZMK) to induce local ICAM1 aggregation and perform trans-, rather than paracellular diapedesis through unstimulated primary brain microvascular endothelial cells. This study included peripheral blood mononuclear cell samples from 175 healthy donors, 29 patients infected with HIV, with neurological symptoms in terms of cognitive impairment, 73 patients with relapsing-remitting multiple sclerosis in remission, either 1-4 weeks before (n = 29), or 18-60 months after the initiation of natalizumab therapy (n = 44), as well as white matter brain tissue of three patients suffering from epilepsy. We here provide ex vivo evidence that CCR5highGZMK+CD4+ effector memory T cells are involved in CNS immune surveillance during homeostasis, but could also play a role in CNS pathology. Among CD4+ T cells, this subset was found to dominate the CNS of patients without neurological inflammation ex vivo. The reduction in peripheral blood of HIV-positive patients with neurological symptoms correlated to their CD4 count as a measure of disease progression. Their peripheral enrichment in multiple sclerosis patients and specific peripheral entrapment through the CNS infiltration inhibiting drug natalizumab additionally suggests a contribution to CNS autoimmune pathology. Our transcriptome analysis revealed a migratory phenotype sharing many features with tissue-resident memory and Th17.1 cells, most notably the transcription factor eomesodermin. Knowledge on this cell subset should enable future studies to find ways to strengthen the host defence against CNS-resident pathogens and brain tumours or to prevent CNS autoimmunity.

VLA-2 blockade in vivo by vatelizumab induces CD4+FoxP3+ regulatory T cells.

Integrin α2ß1, also known as very late antigen (VLA)-2, is a collagen-binding molecule expressed constitutively on platelets. Vatelizumab, a monoclonal antibody targeting the α2 subunit (CD49b) of VLA-2, was recently investigated for its safety and efficacy during a Phase 2 clinical study in multiple sclerosis patients, as integrin-mediated collagen binding at the site of inflammation is central to a number of downstream pro-inflammatory events. In the course of this study, we could show that VLA-2 is expressed ex vivo on platelets, platelet-T-cell aggregates, as well as a small population of highly activated memory T cells. Even though the clinical trial did not meet its primary clinical end-point (reduction in the cumulative number of new contrast-enhancing lesions on magnetic resonance imaging (MRI)), we observed enhanced frequencies of regulatory T cells (TREG) following vatelizumab treatment. Elevated TREG frequencies might be explained by the inhibition of p38 mitogen-activated protein kinase (MAPK) signaling, which is critically involved in the polarization of T helper 17 (TH17) cells and is activated by the α2 integrin cytoplasmic domain. Our findings suggest that blockade of VLA-2 might be a way to safely shift the TH17/TREG balance by inducing TREGin vivo.

Blockade of MCAM/CD146 impedes CNS infiltration of T cells over the choroid plexus.

BACKGROUND: Very late antigen 4 (VLA-4; integrin α4ß1) is critical for transmigration of T helper (TH) 1 cells into the central nervous system (CNS) under inflammatory conditions such as multiple sclerosis (MS). We have previously shown that VLA-4 and melanoma cell adhesion molecule (MCAM) are important for trans-endothelial migration of human TH17 cells in vitro and here investigate their contribution to pathogenic CNS inflammation. METHODS: Antibody blockade of VLA-4 and MCAM is assessed in murine models of CNS inflammation in conjunction with conditional ablation of α4-integrin expression in T cells. Effects of VLA-4 and MCAM blockade on lymphocyte migration are further investigated in the human system via in vitro T cell transmigration assays. RESULTS: Compared to the broad effects of VLA-4 blockade on encephalitogenic T cell migration over endothelial barriers, MCAM blockade impeded encephalitogenic T cell migration in murine models of MS that especially depend on CNS migration across the choroid plexus (CP). In transgenic mice lacking T cell α4-integrin expression (CD4::Itga4-/-), MCAM blockade delayed disease onset. Migration of MCAM-expressing T cells through the CP into the CNS was restricted, where laminin 411 (composed of α4, ß1, γ1 chains), the proposed major ligand of MCAM, is detected in the endothelial basement membranes of murine CP tissue. This finding was translated to the human system; blockade of MCAM with a therapeutic antibody reduced in vitro transmigration of MCAM-expressing T cells across a human fibroblast-derived extracellular matrix layer and a brain-derived endothelial monolayer, both expressing laminin α4. Laminin α4 was further detected in situ in CP endothelial-basement membranes in MS patients' brain tissue. CONCLUSIONS: Our findings suggest that MCAM-laminin 411 interactions facilitate trans-endothelial migration of MCAM-expressing T cells into the CNS, which seems to be highly relevant to migration via the CP and to potential future clinical applications in neuroinflammatory disorders.

Dual action by fumaric acid esters synergistically reduces adhesion to human endothelium.

OBJECTIVE: Dimethyl fumarate (DMF) is prescribed against relapsing-remitting multiple sclerosis (MS). Here, we investigated the effects of DMF and monomethyl fumarate (MMF), its metabolite in vivo, at the (inflamed) blood-brain barrier (BBB). METHODS: Effects of fumaric acid esters were analyzed using primary human brain-derived microvascular endothelial cells (HBMECs) in combination with peripheral blood mononuclear cells (PBMCs) derived from DMF-treated MS patients. RESULTS: MMF-binding to brain endothelium cells leads to activation of nuclear factor (erythroid-derived 2)-related factor 2 (Nrf2)-induced downregulation of vascular cell adhesion molecule 1 (VCAM-1). This might be mediated via the G-protein-coupled receptor (GPCR) hydroxycarboxylic acid receptor 2 (HCA2), a known molecular target of MMF, as we could demonstrate its expression and regulation on HBMECs. DMF treatment in vivo led to a strongly reduced expression of VCAM-1's ligand very late antigen 4 (VLA-4) by selectively reducing integrin high-expressing memory T cells of MS patients, potentially due to inhibition of their maturation by reduced trans-localization of NFκB. CONCLUSION: DMF-mediated VCAM-1 downregulation on the endothelial side and reduction in T cells with a migratory phenotype on the lymphocyte side result in a synergistic reduction in T-cell adhesion to activated endothelium and, therefore, to reduced BBB transmigration in the setting of MS.

Impaired NK-mediated regulation of T-cell activity in multiple sclerosis is reconstituted by IL-2 receptor modulation.

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS) resulting from a breakdown in peripheral immune tolerance. Although a beneficial role of natural killer (NK)-cell immune-regulatory function has been proposed, it still needs to be elucidated whether NK cells are functionally impaired as part of the disease. We observed NK cells in active MS lesions in close proximity to T cells. In accordance with a higher migratory capacity across the blood-brain barrier, CD56(bright) NK cells represent the major intrathecal NK-cell subset in both MS patients and healthy individuals. Investigating the peripheral blood and cerebrospinal fluid of MS patients treated with natalizumab revealed that transmigration of this subset depends on the α4ß1 integrin very late antigen (VLA)-4. Although no MS-related changes in the migratory capacity of NK cells were observed, NK cells derived from patients with MS exhibit a reduced cytolytic activity in response to antigen-activated CD4(+) T cells. Defective NK-mediated immune regulation in MS is mainly attributable to a CD4(+) T-cell evasion caused by an impaired DNAX accessory molecule (DNAM)-1/CD155 interaction. Both the expression of the activating NK-cell receptor DNAM-1, a genetic alteration consistently found in MS-association studies, and up-regulation of the receptor's ligand CD155 on CD4(+) T cells are reduced in MS. Therapeutic immune modulation of IL-2 receptor restores impaired immune regulation in MS by increasing the proportion of CD155-expressing CD4(+) T cells and the cytolytic activity of NK cells.

CD8(+) T-cell pathogenicity in Rasmussen encephalitis elucidated by large-scale T-cell receptor sequencing.

Rasmussen encephalitis (RE) is a rare paediatric epilepsy with uni-hemispheric inflammation and progressive neurological deficits. To elucidate RE immunopathology, we applied T-cell receptor (TCR) sequencing to blood (n=23), cerebrospinal fluid (n=2) and brain biopsies (n=5) of RE patients, and paediatric controls. RE patients present with peripheral CD8(+) T-cell expansion and its strength correlates with disease severity. In addition, RE is the only paediatric epilepsy with prominent T-cell expansions in the CNS. Consistently, common clones are shared between RE patients, who also share MHC-I alleles. Public RE clones share Vß genes and length of the CDR3. Rituximab/natalizumab/basiliximab treatment does not change TCR diversity, stem cell transplantation replaces the TCR repertoire with minimal overlap between donor and recipient, as observed in individual cases. Our study supports the hypothesis of an antigen-specific attack of peripherally expanded CD8(+) lymphocytes against CNS structures in RE, which might be ameliorated by restricting access to the CNS.