Supporting the differential diagnosis of connective tissue diseases with neurological involvement by blood and cerebrospinal fluid flow cytometry.

OBJECTIVE: Neurological manifestations of autoimmune connective tissue diseases (CTD) are poorly understood and difficult to diagnose. We here aimed to address this shortcoming by studying immune cell compositions in CTD patients with and without neurological manifestation. METHODS: Using flow cytometry, we retrospectively investigated paired cerebrospinal fluid (CSF) and blood samples of 28 CTD patients without neurological manifestation, 38 CTD patients with neurological manifestation (N-CTD), 38 non-inflammatory controls, and 38 multiple sclerosis (MS) patients, a paradigmatic primary neuroinflammatory disease. RESULTS: We detected an expansion of plasma cells in the blood of both N-CTD and CTD compared to non-inflammatory controls and MS. Blood plasma cells alone distinguished the clinically similar entities N-CTD and MS with high discriminatory performance (AUC: 0.81). Classical blood monocytes indicated higher disease activity in systemic lupus erythematosus (SLE) patients. Surprisingly, immune cells in the CSF did not differ significantly between N-CTD and CTD, while CD4+ T cells and the CD4+/CD8+ ratio were elevated in the blood of N-CTD compared to CTD. Several B cell-associated parameters partially overlapped in the CSF in MS and N-CTD. We built a machine learning model that distinguished N-CTD from MS with high discriminatory power using either blood or CSF. CONCLUSION: We here find that blood flow cytometry alone surprisingly suffices to distinguish CTD with neurological manifestations from clinically similar entities, suggesting that a rapid blood test could support clinicians in the differential diagnosis of N-CTD.

A role for TASK2 channels in the human immunological synapse.

The immunological synapse is a transient junction that occurs when the plasma membrane of a T cell comes in close contact with an APC after recognizing a peptide from the antigen-MHC. The interaction starts when CRAC channels embedded in the T cell membrane open, flowing calcium ions into the cell. To counterbalance the ion influx and subsequent depolarization, Kv 1.3 and KCa3.1 channels are recruited to the immunological synapse, increasing the extracellular K+ concentration. These processes are crucial as they initiate gene expression that drives T cell activation and proliferation. The T cell-specific function of the K2P channel family member TASK2 channels and their role in autoimmune processes remains unclear. Using mass spectrometry analysis together with epifluorescence and super-resolution single-molecule localization microscopy, we identified TASK2 channels as novel players recruited to the immunological synapse upon stimulation. TASK2 localizes at the immunological synapse, upon stimulation with CD3 antibodies, likely interacting with these molecules. Our findings suggest that, together with Kv 1.3 and KCa3.1 channels, TASK2 channels contribute to the proper functioning of the immunological synapse, and represent an interesting treatment target for T cell-mediated autoimmune disorders.

Immunoadsorption versus double-dose methylprednisolone in refractory multiple sclerosis relapses.

OBJECTIVE: Intravenous methylprednisolone is the standard treatment for a multiple sclerosis relapse; however, this fails to improve symptoms in up to one quarter of patients. Immunoadsorption is an accepted treatment for refractory relapses, but prospective comparator-controlled studies are missing. METHODS: In this observational study, patients with steroid-refractory acute multiple sclerosis relapses receiving either six courses of tryptophan-immunoadsorption or double-dose methylprednisolone therapy were analysed. Outcomes were evaluated at discharge and three months later. Immune profiling of blood lymphocytes and proteomic analysis were performed by multi-parameter flow cytometry and Olink analysis, respectively (NCT04450030). RESULTS: 42 patients were enrolled (methylprednisolone: 26 patients; immunoadsorption: 16 patients). For determination of the primary outcome, treatment response was stratified according to relative function system score changes ("full/best" vs. "average" vs. "worse/none"). Upon discharge, the adjusted odds ratio for any treatment response ("full/best" + "average" vs. "worse/none") was 10.697 favouring immunoadsorption (p = 0.005 compared to methylprednisolone). At follow-up, the adjusted odds ratio for the best treatment response ("full/best" vs. "average" + "worse/none") was 103.236 favouring IA patients (p = 0.001 compared to methylprednisolone). Similar results were observed regarding evoked potentials and quality of life outcomes, as well as serum neurofilament light-chain levels. Flow cytometry revealed a profound reduction of B cell subsets following immunoadsorption, which was closely correlated to clinical outcomes, whereas methylprednisolone had a minimal effect on B cell populations. Immunoadsorption treatment skewed the blood cytokine network, reduced levels of B cell-related cytokines and reduced immunoglobulin levels as well as levels of certain coagulation factors. INTERPRETATION: Immunoadsorption demonstrated favourable outcomes compared to double-dose methylprednisolone. Outcome differences were significant at discharge and follow-up. Further analyses identified modulation of B cell function as a potential mechanism of action for immunoadsorption, as reduction of B cell subsets correlated with clinical improvement.

Cladribine treatment improves cortical network functionality in a mouse model of autoimmune encephalomyelitis.

BACKGROUND: Cladribine is a synthetic purine analogue that interferes with DNA synthesis and repair next to disrupting cellular proliferation in actively dividing lymphocytes. The compound is approved for the treatment of multiple sclerosis (MS). Cladribine can cross the blood-brain barrier, suggesting a potential effect on central nervous system (CNS) resident cells. Here, we explored compartment-specific immunosuppressive as well as potential direct neuroprotective effects of oral cladribine treatment in experimental autoimmune encephalomyelitis (EAE) mice. METHODS: In the current study, we compare immune cell frequencies and phenotypes in the periphery and CNS of EAE mice with distinct grey and white matter lesions (combined active and focal EAE) either orally treated with cladribine or vehicle, using flow cytometry. To evaluate potential direct neuroprotective effects, we assessed the integrity of the primary auditory cortex neuronal network by studying neuronal activity and spontaneous synaptic activity with electrophysiological techniques ex vivo. RESULTS: Oral cladribine treatment significantly attenuated clinical deficits in EAE mice. Ex vivo flow cytometry showed that cladribine administration led to peripheral immune cell depletion in a compartment-specific manner and reduced immune cell infiltration into the CNS. Histological evaluations revealed no significant differences for inflammatory lesion load following cladribine treatment compared to vehicle control. Single cell electrophysiology in acute brain slices was performed and showed an impact of cladribine treatment on intrinsic cellular firing patterns and spontaneous synaptic transmission in neurons of the primary auditory cortex. Here, cladribine administration in vivo partially restored cortical neuronal network function, reducing action potential firing. Both, the effect on immune cells and neuronal activity were transient. CONCLUSIONS: Our results indicate that cladribine exerts a neuroprotective effect after crossing the blood-brain barrier independently of its peripheral immunosuppressant action.

Immune response to SARS-CoV-2 vaccination in relation to peripheral immune cell profiles among patients with multiple sclerosis receiving ocrelizumab.

BACKGROUND: Vaccination has proven to be effective in preventing SARS-CoV-2 transmission and severe disease courses. However, immunocompromised patients have not been included in clinical trials and real-world clinical data point to an attenuated immune response to SARS-CoV-2 vaccines among patients with multiple sclerosis (MS) receiving immunomodulatory therapies. METHODS: We performed a retrospective study including 59 ocrelizumab (OCR)-treated patients with MS who received SARS-CoV-2 vaccination. Anti-SARS-CoV-2-antibody titres, routine blood parameters and peripheral immune cell profiles were measured prior to the first (baseline) and at a median of 4 weeks after the second vaccine dose (follow-up). Moreover, the SARS-CoV-2-specific T cell response and peripheral B cell subsets were analysed at follow-up. Finally, vaccination-related adverse events were assessed. RESULTS: After vaccination, we found anti-SARS-CoV-2(S) antibodies in 27.1% and a SARS-CoV-2-specific T cell response in 92.7% of MS cases. T cell-mediated interferon (IFN)-γ release was more pronounced in patients without anti-SARS-CoV-2(S) antibodies. Antibody titres positively correlated with peripheral B cell counts, time since last infusion and total IgM levels. They negatively correlated with the number of previous infusion cycles. Peripheral plasma cells were increased in antibody-positive patients. A positive correlation between T cell response and peripheral lymphocyte counts was observed. Moreover, IFN-γ release was negatively correlated with the time since the last infusion. CONCLUSION: In OCR-treated patients with MS, the humoral immune response to SARS-CoV-2 vaccination is attenuated while the T cell response is preserved. However, it is still unclear whether T or B cell-mediated immunity is required for effective clinical protection. Nonetheless, given the long-lasting clinical effects of OCR, monitoring of peripheral B cell counts could facilitate individualised treatment regimens and might be used to identify the optimal time to vaccinate.

Effectiveness and safety of cladribine in MS: Real-world experience from two tertiary centres.

BACKGROUND: Oral cladribine has been approved for the treatment of relapsing multiple sclerosis (MS) yet real-world evidence regarding its effectiveness and safety remains scarce. OBJECTIVE: To evaluate efficacy and safety outcomes of MS patients following induction of cladribine. METHODS: We evaluated our prospective cohort of cladribine-treated MS patients from two tertiary centres in Germany. Relapses, disability worsening and occurrence of new or enlarging T2-hyperintense magnetic resonance imaging (MRI) lesions were assessed as well as lymphocyte counts and herpes virus infections. RESULTS: Among 270 patients treated with cladribine, we observed a profound reduction of both relapses and new or enlarging MRI lesions. Treatment appeared more efficacious, especially in patients without previous therapy or following platform substances. Patients switching from natalizumab were prone to re-emerging disease activity. Among patients following dimethyl fumarate pre-treatment, severe lymphopenia was common and associated with increased rates of herpes virus manifestations. CONCLUSION: Overall, we observed an efficacy and safety profile of cladribine consistent with data from the phase 3 clinical trial. However, patients switching from natalizumab experienced suboptimal disease control beyond rebound activity following cessation of natalizumab. Furthermore, dimethyl fumarate pre-treatment was associated with a profound risk of developing severe lymphopenia and subsequent herpes virus infections.

Classification of neurological diseases using multi-dimensional CSF analysis.

Although CSF analysis routinely enables the diagnosis of neurological diseases, it is mainly used for the gross distinction between infectious, autoimmune inflammatory, and degenerative disorders of the CNS. To investigate, whether a multi-dimensional cellular blood and CSF characterization can support the diagnosis of clinically similar neurological diseases, we analysed 546 patients with autoimmune neuroinflammatory, degenerative, or vascular conditions in a cross-sectional retrospective study. By combining feature selection with dimensionality reduction and machine learning approaches we identified pan-disease parameters that were altered across all autoimmune neuroinflammatory CNS diseases and differentiated them from other neurological conditions and inter-autoimmunity classifiers that subdifferentiate variants of CNS-directed autoimmunity. Pan-disease as well as diseases-specific changes formed a continuum, reflecting clinical disease evolution. A validation cohort of 231 independent patients confirmed that combining multiple parameters into composite scores can assist the classification of neurological patients. Overall, we showed that the integrated analysis of blood and CSF parameters improves the differential diagnosis of neurological diseases, thereby facilitating early treatment decisions.

The Innate Immune Response Characterizes Posterior Reversible Encephalopathy Syndrome.

While posterior reversible encephalopathy syndrome (PRES) is often characterized by an inflammatory cerebrospinal-fluid (CSF) profile, knowledge of immune cell patterns in PRES is lacking. Thus, we retrospectively characterized CSF and peripheral blood (PB) from 15 PRES patients, which we analyzed by multidimensional flow cytometry (FC). Results were compared to 72 controls, as well as to 9 patients with progressive multifocal leukoencephalopathy (PML, as a relevant differential diagnosis) and 15 multiple sclerosis patients (MS, as a classical neuroinflammatory disorder), respectively. Total protein level in CSF from PRES patients was elevated compared to that in controls, but not to MS and PML. In-depth FC analysis revealed no differences for adaptive immune cells (B cells, plasma cells, CD4+, and CD8+ T cells) in PB or CSF of PRES compared to controls. In contrast, we observed alterations of the adaptive immune response in CSF of PML and MS compared to PRES, indicating that the adaptive immune response is not a driver of disease in PRES. Indeed, PRES was characterized by an innate immune response with CD14++/CD16+ (intermediate) monocytes elevated in PB and CSF, while CD14++/CD16- (classical) monocytes were decreased in PB from PRES patients as compared to controls. Levels of CD14++/CD16+ monocytes correlated with the duration of hospital stay as a surrogate marker for disease severity in PRES patients. Our findings argue for a role of innate rather than adaptive immunity in the pathophysiology of PRES. The observed shift in monocyte subsets might provide valuable diagnostic clues for the clinical management of these patients.

Impact of previous disease-modifying treatment on effectiveness and safety outcomes, among patients with multiple sclerosis treated with alemtuzumab.

OBJECTIVES: Alemtuzumab is effective in patients with active multiple sclerosis but has a complex safety profile, including the development of secondary autoimmunity. Most of patients enrolled in randomised clinical trials with alemtuzumab were either treatment naïve or pretreated with injectable substances. Other previous disease-modifying treatments (DMTs) were not used in the study cohorts, and therefore, associated risks might yet remain unidentified. METHODS: We retrospectively evaluated a prospective dual-centre alemtuzumab cohort of 170 patients. We examined the baseline characteristics as well as safety and effectiveness outcomes, including the time to first relapse, the time to 3 months confirmed disability worsening and the time to secondary autoimmunity. RESULTS: The regression analysis showed that, among all previously used DMTs, the pretreatment with fingolimod (n=33 HRs for the time to first relapse (HR 5.420, 95% CI 2.520 to 11.660; p<0.001)) and for the time to worsening of disability (HR 7.676, 95% CI 2.870 to 20.534; p<0.001). Additionally, patients pretreated with fingolimod were more likely to experience spinal relapses (55% vs 10% among previously naïve patients; p<0.001) and had an increased risk of secondary autoimmunity (HR 5.875, 95% CI 2.126 to 16.27; p<0.001). CONCLUSION: In the real-world setting, we demonstrated suboptimal disease control and increased risk of secondary autoimmunity following alemtuzumab, among patients previously treated with fingolimod. These data can provide guidance for improving MS therapeutic management.

Neutrophil granulocytes promote flow stagnation due to dynamic capillary stalls following experimental stroke.

Flow stagnation of peri-ischemic capillaries due to dynamic leukocyte stalls has been described to be a contributor to ongoing penumbral injury in transient brain ischemia, but has not been investigated in permanent experimental stroke so far. Moreover, it is discussed that obstructing neutrophils are involved in this process; however, their contribution has not yet been proven. Here, we characterize the dynamics of neutrophil granulocytes in two models of permanent stroke (photothrombosis and permanent middle cerebral artery occlusion) using intravital two-photon fluorescence microscopy. Different to previous studies on LysM-eGFP+ cells we additionally apply a transgenic mouse model with tdTomato-expressing neutrophils to avoid interference from additional immune cell subsets. We identify repetitively occurring capillary stalls of varying duration promoted by neutrophils in both models of permanent cerebral ischemia, validating the suitability of our new transgenic mouse model in determining neutrophil occlusion formation in vivo. Flow cytometric analysis of peripheral blood (PB) and brain tissue from mice subjected to photothrombosis reveal an increase in the total proportion of neutrophils, with selective upregulation of endothelial adherence markers in the PB. In conclusion, the dynamic microcirculatory stall phenomenon that is described after transient ischemia followed by reperfusion also occurs after permanent small- or large-vessel stroke and is clearly attributable to neutrophils.

Dysphagia in neuromyelitis optica spectrum disorder and myelin oligodendrocyte glycoprotein antibody disease as a surrogate of brain involvement?

BACKGROUND AND PURPOSE: Neuromyelitis optica spectrum disorder (NMOSD) and myelin oligodendrocyte glycoprotein antibody disease (MOGAD) are demyelinating disorders that typically affect the optic nerves and the spinal cord. However, recent studies have demonstrated various forms of brain involvement indicating encephalitic syndromes, which consequently are included in the diagnostic criteria for both. Swallowing is processed in a distributed brain network and is therefore disturbed in many neurological diseases. The aim of this study was to investigate the occurrence of oropharyngeal dysphagia in NMOSD and MOGAD using flexible endoscopic evaluation of swallowing (FEES) as a surrogate parameter of brain involvement. METHODS: Thirteen patients with NMOSD and MOGAD (mean age 54.2 ± 18.6 years, six men) who received FEES during clinical routine were retrospectively reviewed. Their extent of oropharyngeal dysphagia was rated using an ordinal dysphagia severity scale. FEES results were compared to a control group of healthy individuals. Dysphagia severity was correlated with the presence of clinical and radiological signs of brain involvement, the Expanded Disability Status Scale (EDSS) and the occurrence of pneumonia. RESULTS: Oropharyngeal dysphagia was present in 8/13 patients, including six patients without other clinical indication of brain involvement. Clinical or subclinical swallowing impairment was significantly more severe in patients with NMOSD and MOGAD compared to the healthy individuals (p = 0.009) and correlated with clinical signs of brain involvement (p = 0.038), higher EDSS (p = 0.006) and pneumonia (p = 0.038). CONCLUSION: Oropharyngeal dysphagia can occur in NMOSD and MOGAD and might be associated with pneumonia and disability. FEES may help to detect subclinical brain involvement.

Patients with a relapsing course of steroid-responsive encephalopathy associated with autoimmune thyroiditis exhibit persistent intrathecal CD4+ T-cell activation.

BACKGROUND AND PURPOSE: Steroid-responsive encephalopathy associated with autoimmune thyroiditis (SREAT) is a rare condition defined by encephalopathy with acute or subacute onset, the presence of serum anti-thyroid antibodies, and reasonable exclusion of alternative causes. Despite having strong response towards corticosteroid treatment, some patients exhibit a chronic-relapsing course and require long-term immunosuppression. Markers for early identification of those patients are still absent. Thus, we aimed to characterise clinical as well as laboratory parameters of our local SREAT cohort. METHODS: We retrospectively evaluated a cohort of 22 SREAT patients treated in our hospital from January 2014. RESULTS: A total of 14 patients with a monophasic disease course and eight patients with multiple relapses were identified. Neither baseline characteristics nor routine cerebrospinal fluid (CSF) parameters were able to distinguish between those patient groups. Flow cytometry following initial relapse therapy showed treatment-resistant sequestration of activated CD4+ T cells in patients with a relapsing disease course, whereas other lymphocyte subsets showed uniform changes. Such changes were also present in long-term follow-up CSF examination. CONCLUSION: Our findings indicate a potential biomarker for risk stratification in patients with SREAT. Currently, it remains unclear whether the observed two phenotypes are different spectra of SREAT or represent separate diseases in terms of pathophysiology.

Generation of a Model to Predict Differentiation and Migration of Lymphocyte Subsets under Homeostatic and CNS Autoinflammatory Conditions.

The central nervous system (CNS) is an immune-privileged compartment that is separated from the circulating blood and the peripheral organs by the blood-brain and the blood-cerebrospinal fluid (CSF) barriers. Transmigration of lymphocyte subsets across these barriers and their activation/differentiation within the periphery and intrathecal compartments in health and autoinflammatory CNS disease are complex. Mathematical models are warranted that qualitatively and quantitatively predict the distribution and differentiation stages of lymphocyte subsets in the blood and CSF. Here, we propose a probabilistic mathematical model that (i) correctly reproduces acquired data on location and differentiation states of distinct lymphocyte subsets under homeostatic and neuroinflammatory conditions, (ii) provides a quantitative assessment of differentiation and transmigration rates under these conditions, (iii) correctly predicts the qualitative behavior of immune-modulating therapies, (iv) and enables simulation-based prediction of distribution and differentiation stages of lymphocyte subsets in the case of limited access to biomaterial. Taken together, this model might reduce future measurements in the CSF compartment and allows for the assessment of the effectiveness of different immune-modulating therapies.

Fulminant MS Reactivation Following Combined Fingolimod Cessation and Yellow Fever Vaccination.

A major concern caused by the discontinuation of disease modifying treatment for multiple sclerosis (MS) is a rebound of disease activity. Hypotheses about the underlying mechanism of fingolimod (FTY) induced exaggerated inflammatory responses are diverse. So far, vaccinations as a trigger for rebound activity following FTY suspension have not been described. However, several reports have highlighted the occurrence of neurological and autoimmune side effects after single or combined multi-vaccination procedures. Here, we describe the case of a highly active female MS patient demonstrating recurrent, severe MS relapses accompanied by extensive MRI activity, subsequent to yellow fever vaccination two months following FTY withdrawal. Blood and cerebrospinal fluid immunophenotyping indicated a B cell/plasma cell autoreactivity. Following a therapy with natalizumab the clinical, laboratory, MRI, and disease course improved significantly. This case hints towards a combined immunological mechanism characterized by molecular mimicry, bystander activation, and lymphocyte re-egress, resulting in extensive neurological impairment and shows that natalizumab represents a therapeutic option to counteract B cell mediated autoreactivity. Especially, the diagnostic and therapeutic management of this complex scenario might be instructive for clinical practice.

Association of Clinical Relapses With Disease Outcomes in Multiple Sclerosis Patients Older Than 50 Years.

BACKGROUND AND OBJECTIVES: Relapse and MRI activity usually decline with aging but are replaced by progression independent of relapse activity (PIRA) in patients with multiple sclerosis (PwMS). However, several older PwMS continue to experience clinical relapses, and the impact on their disease remains undetermined. We aimed to determine the impact of an index relapse on disease outcomes in patients older than 50 years and to identify risk factors of disadvantageous outcomes. METHODS: We performed a secondary analysis from 3 prospective cohorts in Germany. We evaluated all PwMS 50 years and older with a relapse ≤60 days before a baseline visit and at least 18 months of follow-up compared with a control cohort of PwMS without a relapse. Patients were stratified according to age ("50-54" vs "55-59" vs "60+") or disease outcomes ("stable" vs "active" vs "progressive," according to the Lublin criteria). We analyzed relapses, MRI activity, relapse-associated worsening, and PIRA. Regression analysis was performed to evaluate the association of specific baseline risk factors and treatment regimen changes with disease outcomes at month 18. RESULTS: A total of 681 patients were included in the "relapse cohort" (50+: 361; 55+: 220; 60+: 100). The "control cohort" comprised 232 patients (50+: 117; 55+: 71; 60+: 44). Baseline epidemiologic parameters were balanced among cohorts and subgroups. We observed increased abundance of inflammatory activity and relapse-independent disability progression in the "relapse" vs "control" cohort. In the "relapse" cohort, we identified 273 patients as "stable" (59.7%), 114 patients as "active" (24.9%), and 70 patients as "progressive" (15.3%) during follow-up. Cardiovascular risk factors (CVRFs) and older age at baseline were identified as risk factors of progressive, whereas disease-modifying treatment (DMT) administration at baseline favored stable disease. DMT during follow-up was associated with stable over active, but not over progressive disease. DISCUSSION: A relapse-suggesting underlying active disease-in PwMS older than 50 years was associated with continued disease activity and increased risk of PIRA. Presence of CVRF and absence of DMT at baseline appeared as risk factors of disadvantageous disease courses. An escalation of DMT switch was associated with stable over active but not progressive disease.

Immune Response to Seasonal Influenza Vaccination in Multiple Sclerosis Patients Receiving Cladribine.

Cladribine has been approved for the treatment of multiple sclerosis (MS) and its administration results in a long-lasting depletion of lymphocytes. As lymphopenia is known to hamper immune responses to vaccination, we evaluated the immunogenicity of the influenza vaccine in patients undergoing cladribine treatment at different stages vs. controls. The antibody response in 90 cladribine-treated MS patients was prospectively compared with 10 control subjects receiving platform immunotherapy (NCT05019248). Serum samples were collected before and six months after vaccination. Response to vaccination was determined by the hemagglutination-inhibition test. Postvaccination seroprotection rates against influenza A were comparable in cladribine-treated patients and controls (H1N1: 94.4% vs. 100%; H3N2: 92.2% vs. 90.0%). Influenza B response was lower in the cladribine cohort (61.1% vs. 80%). The increase in geometric mean titers was lower in the cladribine group vs. controls (H1N1: +98.5 vs. +188.1; H3N2: +225.3 vs. +300.0; influenza B: +40.0 vs. +78.4); however, titers increased in both groups for all strains. Seroprotection was achieved irrespective of vaccination timing and lymphocyte subset counts at the time of vaccination in the cladribine cohort. To conclude, cladribine-treated MS patients can mount an adequate immune response to influenza independently of treatment duration and time interval to the last cladribine administration.

Effect of Previous Disease-Modifying Therapy on Treatment Effectiveness for Patients Treated With Ocrelizumab.

BACKGROUND AND OBJECTIVES: B cell-depleting antibodies were proven as effective strategy for the treatment of relapsing multiple sclerosis (RMS). The monoclonal antibody ocrelizumab was approved in 2017 in the United States and in 2018 in the European Union, but despite proven efficacy in randomized, controlled clinical trials, its effectiveness in the real-world setting remains to be fully elucidated. In particular, most study patients were treatment naive or switched from injectable therapies, whereas oral substances or monoclonal antibodies made up >1% of previous treatments. METHODS: We evaluated ocrelizumab-treated patients with RMS enrolled in the prospective cohorts at the University Hospitals Duesseldorf and Essen, Germany. Epidemiologic data at baseline were compared, and Cox proportional hazard models were applied to evaluate outcomes. RESULTS: Two hundred eighty patients were included (median age: 37 years, 35% male patients). Compared with using ocrelizumab as a first-line treatment, its use as a third-line therapy increased hazard ratios (HRs) for relapse and disability progression, whereas differences between first- vs second-line and second- vs third-line remained smaller. We stratified patients according to their last previous disease-modifying treatment and here identified fingolimod (FTY) (45 patients, median age 40 years, 33% male patients) as a relevant risk factor for ongoing relapse activity despite 2nd-line (HR: 3.417 [1.007-11.600]) or 3rd-line (HR: 5.903 [2.489-13.999]) ocrelizumab treatment, disability worsening (2nd line: HR: 3.571 [1.013-12.589]; 3rd line: HR: 4.502 [1.728-11.729]), and occurrence of new/enlarging MRI lesions (2nd line: HR: 1.939 [0.604-6.228]; 3rd line: HR: 4.627 [1.982-10.802]). Effects were persistent throughout the whole follow-up. Neither peripheral B-cell repopulation nor immunoglobulin G levels were associated with rekindling disease activity. DISCUSSION: Our prospectively collected observational data suggest suboptimal effectiveness of ocrelizumab in patients switching from FTY compared with those switching from other substances or having been treatment naive. These findings support previous studies indicating abated effectiveness of immune cell-depleting therapies following FTY treatment in patients with RMS. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that for patients with RMS, previous treatment with FTY compared with previous treatment with other immunomodulating therapies decreases the effectiveness of ocrelizumab.

Impact of NKG2D Signaling on Natural Killer and T-Cell Function in Cerebral Ischemia.

Background Typically defined as a thromboinflammatory disease, ischemic stroke features early and delayed inflammatory responses, which determine the extent of ischemia-related brain damage. T and natural killer cells have been implicated in neuronal cytotoxicity and inflammation, but the precise mechanisms of immune cell-mediated stroke progression remain poorly understood. The activating immunoreceptor NKG2D is expressed on both natural killer and T cells and may be critically involved. Methods and Results An anti-NKG2D blocking antibody alleviated stroke outcome in terms of infarct volume and functional deficits, coinciding with reduced immune cell infiltration into the brain and improved survival in the animal model of cerebral ischemia. Using transgenic knockout models devoid of certain immune cell types and immunodeficient mice supplemented with different immune cell subsets, we dissected the functional contribution of NKG2D signaling by different NKG2D-expressing cells in stroke pathophysiology. The observed effect of NKG2D signaling in stroke progression was shown to be predominantly mediated by natural killer and CD8+ T cells. Transfer of T cells with monovariant T-cell receptors into immunodeficient mice with and without pharmacological blockade of NKG2D revealed activation of CD8+ T cells irrespective of antigen specificity. Detection of the NKG2D receptor and its ligands in brain samples of patients with stroke strengthens the relevance of preclinical observations in human disease. Conclusions Our findings provide a mechanistic insight into NKG2D-dependent natural killer- and T-cell-mediated effects in stroke pathophysiology.

Translational imaging of TSPO reveals pronounced innate inflammation in human and murine CD8 T cell-mediated limbic encephalitis.

Autoimmune limbic encephalitis (ALE) presents with new-onset mesial temporal lobe seizures, progressive memory disturbance, and other behavioral and cognitive changes. CD8 T cells are considered to play a key role in those cases where autoantibodies (ABs) target intracellular antigens or no ABs were found. Assessment of such patients presents a clinical challenge, and novel noninvasive imaging biomarkers are urgently needed. Here, we demonstrate that visualization of the translocator protein (TSPO) with [18F]DPA-714-PET-MRI reveals pronounced microglia activation and reactive gliosis in the hippocampus and amygdala of patients suspected with CD8 T cell ALE, which correlates with FLAIR-MRI and EEG alterations. Back-translation into a preclinical mouse model of neuronal antigen-specific CD8 T cell-mediated ALE allowed us to corroborate our preliminary clinical findings. These translational data underline the potential of [18F]DPA-714-PET-MRI as a clinical molecular imaging method for the direct assessment of innate immunity in CD8 T cell-mediated ALE.