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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., (© 2023. The Author(s).)

After natalizumab (NAT) cessation, some multiple sclerosis (MS) patients experience a severe disease rebound. The rebound pathophysiology is still unclear; however, it has been linked to interleukin-17-producing T-helper (Th17) cells. We demonstrate that during NAT treatment, MCAM+CCR6+Th17 cells gradually acquire a pathogenic profile, including proinflammatory cytokine production, pathogenic transcriptional signatures, brain endothelial barrier impairment, and oligodendrocyte damage via induction of apoptotic pathways. This is accompanied by an increase in Th17 cell frequencies in the cerebrospinal fluid of NAT-treated patients. Notably, Th17 cells derived from NAT-treated patients, who later developed a disease rebound upon treatment cessation, displayed a distinct transcriptional pathogenicity profile associated with altered migratory properties. Accordingly, increased brain infiltration of patient Th17 cells was illustrated in a humanized mouse model and brain histology from a rebound patient. Therefore, peripheral blood-accumulated MCAM+CCR6+Th17 cells might be involved in rebound pathophysiology, and monitoring of changes in Th17 cell pathogenicity in patients before/during NAT treatment cessation might enable rebound risk assessment in the future.

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., (© 2022. The Author(s).)

The impact of distinct disease-modifying therapies (DMTs) on severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) vaccination efficacy in patients with multiple sclerosis (MS) is still enigmatic. In this prospective comparative study, we investigated humoral and cellular immune-responses in patients with MS receiving interferon beta, natalizumab, and ocrelizumab pre-vaccination and 6 weeks post second SARS-CoV-2 vaccination. Healthy individuals and interferon beta-treated patients generated robust humoral and cellular immune-responses. Although humoral immune responses were diminished in ocrelizumab-treated patients, cellular immune-responses were reduced in natalizumab-treated patients. Thus, both humoral and cellular immune responses should be closely monitored in patients on DMTs. Whereas patients with a poor cellular immune-response may benefit from additional vaccination cycles, patients with a diminished humoral immune-response may benefit from a treatment with SARS-CoV-2 antibodies in case of an infection., (© 2022 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Multiple sclerosis (MS) disease risk is associated with reduced sun-exposure. This study assessed the relationship between measures of sun exposure (vitamin D [vitD], latitude) and MS severity in the setting of two multicenter cohort studies ( n NationMS = 946, n variants was assessed. High serum vitD was associated with a reduced MS severity score (MSSS), reduced risk for relapses, and lower disability accumulation over time. Low latitude was associated with higher vitD, lower MSSS, fewer gadolinium-enhancing lesions, and lower disability accumulation. The association of latitude with disability was lacking in IFN-β-treated patients. In carriers of BIONAT :rs1805008(T), who reported increased sensitivity toward sunlight, lower latitude was associated with higher MRI activity, whereas for noncarriers there was less MRI activity at lower latitudes. In a further exploratory approach, the effect of ultraviolet (UV)-phototherapy on the transcriptome of immune cells of MS patients was assessed using samples from an earlier study. Phototherapy induced a vitD and type I IFN signature that was most apparent in monocytes but that could also be detected in B and T cells. In summary, our study suggests beneficial effects of sun exposure on established MS, as demonstrated by a correlative network between the three factors: Latitude, vitD, and disease severity. However, sun exposure might be detrimental for photosensitive patients. Furthermore, a direct induction of type I IFNs through sun exposure could be another mechanism of UV-mediated immune-modulation in MS.MC1R variants was assessed. High serum vitD was associated with a reduced MS severity score (MSSS), reduced risk for relapses, and lower disability accumulation over time. Low latitude was associated with higher vitD, lower MSSS, fewer gadolinium-enhancing lesions, and lower disability accumulation. The association of latitude with disability was lacking in IFN-β-treated patients. In carriers of MC1R :rs1805008(T), who reported increased sensitivity toward sunlight, lower latitude was associated with higher MRI activity, whereas for noncarriers there was less MRI activity at lower latitudes. In a further exploratory approach, the effect of ultraviolet (UV)-phototherapy on the transcriptome of immune cells of MS patients was assessed using samples from an earlier study. Phototherapy induced a vitD and type I IFN signature that was most apparent in monocytes but that could also be detected in B and T cells. In summary, our study suggests beneficial effects of sun exposure on established MS, as demonstrated by a correlative network between the three factors: Latitude, vitD, and disease severity. However, sun exposure might be detrimental for photosensitive patients. Furthermore, a direct induction of type I IFNs through sun exposure could be another mechanism of UV-mediated immune-modulation in MS., Competing Interests: Competing interest statement: H.W. received honoraria and consultation fees from Bayer Healthcare, Biogen, Fresenius Medical Care, GlaxoSmithKline, GW Pharmaceuticals, Merck Serono, Novartis, Sanofi Genzyme, and Teva Pharma. K.L. is coinventor on patent no. US 2016/0158322 A1 entitled “NDP-MSH for treatment of inflammatory and/or neurodegenerative disorders of the CNS.” A.S. received speaker honoraria and/or travel compensation for activities with Almirall Hermal GmbH, Biogen, Merck, Novartis, Roche and Sanofi Genzyme, none related to this work. R.G. serves on scientific advisory boards for Teva Pharmaceutical Industries Ltd. (Teva), Biogen Idec, Bayer Schering Pharma, and Novartis; has received speaker honoraria from Biogen Idec, Teva, Bayer Schering Pharma, and Novartis; serves as editor for Therapeutic Advances in Neurological Diseases, and is on the editorial boards of Experimental Neurology and the Journal of Neuroimmunology; and receives research support from Teva, Biogen Idec, Bayer Schering Pharma, Genzyme, Merck Serono, and Novartis, none related to this work. L.K. receives honoraria for lecturing and travel expenses for attending meetings from Biogen, Merck, Sanofi Genzyme, Novartis and Roche; her research is funded by the German Ministry for Education and Research (BMBF), Deutsche Forschungsgemeinschaft (DFG), Interdisciplinary Center for Clinical Studies (IZKF) Muenster, Biogen, Merck, and Novartis. M.A.F. received honoraria for consultation and travel expenses from Biogen, Merck KGaA, Novartis, and Roche; his research is funded by the BMBF, DFG, Landesforschungsförderung Hamburg, Gemeinnützige Hertie-Stiftung, and Else Kröner-Fresenius-Stiftung. S.G.M. receives honoraria for lecturing, and travel expenses for attending meetings from Almirall, Amicus Therapeutics Germany, Bayer Health Care, Biogen, Celgene, Diamed, Genzyme, MedDay Pharmaceuticals, Merck Serono, Novartis, Novo Nordisk, ONO Pharma, Roche, Sanofi-Aventis, Chugai Pharma, QuintilesIMS, and Teva; his research is funded by the BMBF, DFG, Else Kröner Fresenius Foundation, German Academic Exchange Service, Hertie Foundation, IZKF Muenster, German Foundation Neurology and Almirall, Amicus Therapeutics Germany, Biogen, Diamed, Fresenius Medical Care, Genzyme, Merck Serono, Novartis, ONO Pharma, Roche, and Teva. R.H. has received grant support from Biogen, Genzyme-Sanofi, Merck Serono, Novartis, Teva, and personal fees from Actelion, Biogen, Genzyme-Sanofi, Medday, Merck Serono, Novartis, Roche, and Teva. T.K. has received travel expenses and personal compensation from Bayer Healthcare, Teva Pharma, Merck, Novartis Pharma, Sanofi-Aventis/Genzyme, Roche, and Biogen, as well as grant support from Bayer Schering AG, Novartis, and Chugai Pharma. U.Z. has received grants from DFG, BMBF, the European Research Council, Bristol Myers Squibb, Janssen Pharmaceuticals NV, Servier, and Biogen Idec, and personal consultation fees from Pfizer GmbH, Bayer Vital GmbH, and CorTec GmbH. F.Z. received funds for scientific consultation or research by DFG, BMBF, the Pharmaceutical Management Science Association, Novartis, Octapharma, Merck Serono, ONO Pharma, Biogen, Genzyme, Celgene, Roche, Sanofi Aventis. A.S.-M. received travel expenses and research support from Novartis. C.C.G. received speaker honoraria and travel expenses for attending meetings from Biogen, Euroimmun, Genzyme, MyLan, Novartis Pharma GmbH, and Bayer Health Care, none related to this study; her work is funded by Biogen, Novartis, the BMBF (01Gl1603A), the DFG (GR3946/3-1, SFB Transregio 128 A09), the European Union (Horizon2020, ReSToRE), the IZKF, and the Innovative Medizinische Forschung. D.B. receives lectures, congress invitation, board participation with Bayer, Biogen, Medday, Merck, Novartis, Roche, Sanofi Genzyme, and Teva. B.T. received personal speaker honoraria and consultancy fees as a speaker and advisor from Alexion, Bayer Healthcare, Biogen, CSL Behring, GILEAD, GRIFOLS, Merck Serono, Novartis, Octapharma, Roche, Sanofi Genzyme, Teva, and UCB Pharma; his university received unrestricted research grants from Biogen-Idec, Novartis, Teva, Bayer Healthcare, CSL-Behring, GRIFOLS, Octapharma, Sanofi Genzyme, and UCB Pharma. F.T.B. has received, through his institution, grant support and/or travel reimbursement to attend scientific meetings from Actelion, Bayer, Biogen, Merck, Novartis, Sanofi Genzyme, and Teva; he has received personal honoraria for speaking or advisory board activities from Actelion, Bayer, Biogen, Merck, Novartis, Roche, and Sanofi Genzyme; his research is funded, in part, by the DFG and the BMBF. C.W. has received institutional support from Novartis, Sanofi Genzyme, Biogen, and Roche. H.-P.H. has received fees for consulting, speaking, and serving on steering committees from Bayer Healthcare, Biogen, GeNeuro, MedImmune, Merck, Novartis, Opexa, Receptos Celgene, Roche, Sanofi Genzyme, CSL Behring, Octapharma, and Teva, with approval by the Rector of Heinrich-Heine University. H.T. received speaker honoraria from Bayer, Biogen, Fresenius, Genzyme, Merck, Novartis, Roche, Siemens, and Teva; he serves as section editor for the Journal of Neurology, Psychiatry, and Brain Research; H.T. and his institution receives research support from the BMBF, DMSG, Fresenius, Genzyme, Merck, and Novartis, none related to this work. A.B. has received personal compensation from Merck, Biogen, Bayer Vital, Novartis, Teva, Roche, Celgene, Alexion, and Sanofi Genzyme, and grants for congress trips and participation from Biogen, Teva, Novartis, Sanofi Genzyme, Celgene, and Merck, none related to this work. C.L. received an endowed professorship supported by the Novartis foundation, has received consulting and speaker’s honoraria from Biogen-Idec, Bayer Schering, Novartis, Sanofi Genzyme, and Teva; and has received research scientific grant support from Merck-Serono and Novartis. B.W. received research grants and/or honoria from Merck Serono, Biogen, Teva, Novartis, Sanofi Genzyme, Bayer Healthcare, Roche, and research grants from the Dietmar Hopp Foundation, the Klaus Tschira Foundation, the BMBF, and the DFG. U.K.Z. received honoraria for lecturing and/or travel expenses for attending meetings from Almirall, Alexion, Bayer, Biogen, Celgene, Merck Serono, Novartis, Octapharma, Roche, Sanofi Genzyme, and Teva, as well as grant support from the BMBF and European Union, none related to this study; his research is funded by the BMBF, German Ministry of Economy, DFG, and European Union. S.B. has received honoria and compensation for travel from Biogen Idec, Merck Serono, Novartis, Sanofi Genzyme, and Roche. M.S. has received honoraria for scientific lectures or consultancy from Alexion, Bayer Healthcare, Biogen, CSL Behring, Grifols, MedDay, Merck-Serono, Novartis, Roche, Sanofi Genzyme, Takeda, and Teva; his institution received research support from Biogen, Sanofi-Genzyme, and Merck-Serono. R.A.L. received personal compensation from Celgene, Bayer Healthcare, Teva Pharma, Merck, Novartis Pharma, Sanofi-Aventis/Genzyme, Roche, and Biogen, as well as grant support from Novartis and Merck. F.W. served on the scientific advisory board of Genzyme, Merck Serono, and Novartis; he has received travel funding and/or speaker honoraria from Bayer, Biogen, Teva, Merck Serono, Novartis, and Pfizer; he is employed by Sana and received research support from the BMBF, Merck Serono, and Novartis. F.P. reported other support from Bayer, Novartis, Biogen-Idec, Teva, Sanofi-Aventis/Genzyme, Merck Serono, Alexion, Chugai, MedImmune, Roche, and Shire outside the submitted work; he has a post as academic editor for PLoS One and is associate editor for Neurology: Neuroimmunology & Neuroinflammation; he has scientific advisory board membership for Novartis and consulting fees from Sanofi Genzyme, Biogen-Idec, MedImmune, Shire, and Alexion; and research support from Bayer, Novartis, Biogen Idec, Teva, Sanofi-Aventis/Genzyme, Alexion, Merck Serono, the German Research Council, Werth Stiftung, German Ministry of Education and Research, Arthur Arnstein Stiftung Berlin, EU FP7 Framework Program, Arthur Arnstein Foundation Berlin, Guthy Jackson Charitable Foundation, and National Multiple Sclerosis of the United States. B.H. has served on scientific advisory boards for Novartis; he has served as data monitoring and safety committee member for AllergyCare and TG therapeutics; he or his institution have received speaker honoraria from Desitin; he holds part of two patents, one for the detection of antibodies against KIR4.1 in a subpopulation of MS patients and one for genetic determinants of neutralizing antibodies to interferon. All conflicts are not relevant to the topic of the study. F.L. has served on the advisory board of Roche and received travel funding from Teva. M.M. received research support from Merck Serono and Novartis as well as travel expenses for attending meetings from Merck Serono. K.L.-H. has received research support (to Technische Universität München) from Novartis, honoraria from Novartis and F. Hoffmann-La Roche, and compensation for travel expenses from Merck Serono., (Copyright © 2021 the Author(s). Published by PNAS.)

The tremendous heterogeneity of the human population presents a major obstacle in understanding how autoimmune diseases like multiple sclerosis (MS) contribute to variations in human peripheral immune signatures. To minimize heterogeneity, we made use of a unique cohort of 43 monozygotic twin pairs clinically discordant for MS and searched for disease-related peripheral immune signatures in a systems biology approach covering a broad range of adaptive and innate immune populations on the protein level. Despite disease discordance, the immune signatures of MS-affected and unaffected cotwins were remarkably similar. Twinship alone contributed 56% of the immune variation, whereas MS explained 1 to 2% of the immune variance. Notably, distinct traits in CD4 + effector T cell subsets emerged when we focused on a subgroup of twins with signs of subclinical, prodromal MS in the clinically healthy cotwin. Some of these early-disease immune traits were confirmed in a second independent cohort of untreated early relapsing-remitting MS patients. Early involvement of effector T cell subsets thus points to a key role of T cells in MS disease initiation., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Cerebrospinal fluid (CSF) protects the central nervous system (CNS) and analyzing CSF aids the diagnosis of CNS diseases, but our understanding of CSF leukocytes remains superficial. Here, using single cell transcriptomics, we identify a specific location-associated composition and transcriptome of CSF leukocytes. Multiple sclerosis (MS) - an autoimmune disease of the CNS - increases transcriptional diversity in blood, but increases cell type diversity in CSF including a higher abundance of cytotoxic phenotype T helper cells. An analytical approach, named cell set enrichment analysis (CSEA) identifies a cluster-independent increase of follicular (TFH) cells potentially driving the known expansion of B lineage cells in the CSF in MS. In mice, TFH cells accordingly promote B cell infiltration into the CNS and the severity of MS animal models. Immune mechanisms in MS are thus highly compartmentalized and indicate ongoing local T/B cell interaction.

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 α4β1 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.

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.

Abstract 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.

Background: Cladribine is a highly effective immunotherapy that is applied in two short-term courses over 2 years and reduces relapse rate and disease progression in patients with relapsing multiple sclerosis (MS). Despite the short treatment period, cladribine has a long-lasting effect on disease activity even after recovery of lymphocyte counts, suggesting a yet undefined long-term immune modulating effect. Objectives: Our aim was to provide a more profound understanding of the detailed effects of cladribine, also with regard to the patients' therapy response. Design: We performed an open-labeled, explorative, prospective, single-arm study, in which we examined the detailed lymphocyte subset development of MS patients who received cladribine treatment over 2 years. Methods: We performed in-depth profiling of the effects of cladribine on peripheral blood lymphocytes by flow cytometry, bulk RNA sequencing of sorted CD4+ T cells, CD8+ T cells, and CD19+ B cells as well as single-cell RNA sequencing of peripheral blood mononuclear cells in a total of 23 MS patients before and at different time points up to 24 months after cladribine treatment. Data were correlated with clinical and cranial magnetic resonance imaging (MRI) disease activity. Results: Flow cytometry revealed a predominant and sustained reduction of memory B cells compared to other B cell subsets after cladribine treatment, whereas T cell subsets were slightly reduced in a more uniform pattern. The overall transcriptional profile of total blood B cells exhibited reduced expression of proinflammatory and T cell activating genes, while single-cell transcriptomics revealed that gene expression within each B cell cluster did not change over time. Stable patients displayed stronger reductions of selected memory B cell clusters as compared to patients with clinical or cerebral MRI disease activity. Conclusion: We describe a pronounced and sustained effect of cladribine on the memory B cell compartment, and the resulting change in B cell subset composition causes a significant alteration of B cell transcriptional profiles resulting in reduced proinflammatory and T cell activating capacities. The extent of reduction in selected memory B cell clusters by cladribine may predict treatment response. [ABSTRACT FROM AUTHOR]

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.

Leukocyte sequestration is an established therapeutic concept in multiple sclerosis (MS) as represented by the trafficking drugs natalizumab (NAT) and fingolimod (FTY). However, the precise consequences of targeting immune cell trafficking for immunoregulatory network functions are only incompletely understood. In the present study, we performed an in-depth longitudinal characterization of functional and phenotypic immune signatures in peripheral blood (PB) and cerebrospinal fluid (CSF) of 15 MS patients during switching from long-term NAT to FTY treatment after a defined 8-week washout period within a clinical trial (ToFingo successor study; ClinicalTrials.gov: NCT02325440). Unbiased visualization and analysis of high-dimensional single cell flow-cytometry data revealed that switching resulted in a profound alteration of more than 80% of investigated innate and adaptive immune cell subpopulations in the PB, revealing an unexpectedly broad effect of trafficking drugs on peripheral immune signatures. Longitudinal CSF analysis demonstrated that NAT and FTY both reduced T cell subset counts and proportions in the CSF of MS patients with equal potency; NAT however was superior with regard to sequestering non-T cell populations out of the CSF, including B cells, natural killer cells and inflammatory monocytes, suggesting that disease exacerbation in the context of switching might be driven by non-T cell populations. Finally, correlation of our immunological data with signs of disease exacerbation in this small cohort suggested that both (i) CD49d expression levels under NAT at the time of treatment cessation and (ii) swiftness of FTY-mediated effects on immune cell subsets in the PB together may predict stability during switching later on.

There is increasing evidence that natural killer (NK) cells exhibit regulatory features. Among them, CD56bright natural killer cells have been suggested to play a major role in controlling T-cell responses and maintaining homeostasis. Dysfunction in NK cell-mediated regulatory features has been recently described in untreated multiple sclerosis (MS), suggesting a contribution to MS pathogenesis. Moreover, biological disease-modifying treatments effective in MS apparently enhance the frequencies and/or regulatory function of NK cells, further pointing toward an immuno-protective role of NK cells in MS. Here we summarize the current knowledge on the regulatory functions of NK cells, based on their interactions with other cells belonging to the innate compartment, as well as with adaptive effector cells. We review the more recent data reporting disruption of NK-cell/T-cell interactions in MS, and discuss how disease-modifying treatments for MS affect NK cells.

Dimethyl fumarate, an approved treatment for relapsing-remitting multiple sclerosis, exerts pleiotropic effects on immune cells as well as CNS resident cells. Here, we show that dimethyl fumarate exerts a profound alteration of the metabolic profile of human CD4+ as well as CD8+ T cells and restricts their antioxidative capacities by decreasing intracellular levels of the reactive oxygen species scavenger glutathione. This causes an increase in mitochondrial reactive oxygen species levels accompanied by an enhanced mitochondrial stress response, ultimately leading to impaired mitochondrial function. Enhanced mitochondrial reactive oxygen species levels not only result in enhanced T-cell apoptosis in vitro as well as in dimethyl fumarate-treated patients, but are key for the well-known immunomodulatory effects of dimethyl fumarate both in vitro and in an animal model of multiple sclerosis, i.e. experimental autoimmune encephalomyelitis. Indeed, dimethyl fumarate immune-modulatory effects on T cells were completely abrogated by pharmacological interference of mitochondrial reactive oxygen species production. These data shed new light on dimethyl fumarate as bona fide immune-metabolic drug that targets the intracellular stress response in activated T cells, thereby restricting mitochondrial function and energetic capacity, providing novel insight into the role of oxidative stress in modulating cellular immune responses and T cell-mediated autoimmunity. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objective: Utilize immune cell profiles in the cerebrospinal fluid (CSF) to advance the understanding and potentially support the diagnosis of inflammatory neuropathies. Methods: We analyzed CSF cell flow cytometry data of patients with definite Guillain-Barré syndrome (GBS, n = 26) and chronic inflammatory demyelinating polyneuropathy (CIDP, n = 32) based on established diagnostic criteria in comparison to controls with relapsing-remitting multiple sclerosis (RRMS, n = 49) and idiopathic intracranial hypertension (IIH, n = 63). Results: Flow cytometry revealed disease-specific changes of CSF cell composition with a significant increase of NKT cells and CD8+ T cells in CIDP, NK cells in GBS, and B cells and plasma cells in MS in comparison to IIH controls. Principal component analysis demonstrated distinct CSF immune cells pattern in inflammatory neuropathies vs. RRMS. Systematic receiver operator curve (ROC) analysis identified NKT cells as the best parameter to distinguish GBS from CIDP. Composite scores combing several of the CSF parameters differentiated inflammatory neuropathies from IIH and GBS from CIDP with high confidence. Applying a novel dimension reduction technique, we observed an intra-disease heterogeneity of inflammatory neuropathies. Conclusion: Inflammatory neuropathies display disease- and subtype-specific alterations of CSF cell composition. The increase of NKT cells and CD8+ T cells in CIDP and NK cells in GBS, suggests a central role of cytotoxic cell types in inflammatory neuropathies varying between acute and chronic subtypes. Composite scores constructed from multi-dimensional CSF parameters establish potential novel diagnostic tools. Intra-disease heterogeneity suggests distinct disease mechanisms in subgroups of inflammatory neuropathies. [ABSTRACT FROM AUTHOR]

Leukocyte sequestration is an established therapeutic concept in multiple sclerosis (MS) as represented by the trafficking drugs natalizumab (NAT) and fingolimod (FTY). However, the precise consequences of targeting immune cell trafficking for immunoregulatory network functions are only incompletely understood. In the present study, we performed an in-depth longitudinal characterization of functional and phenotypic immune signatures in peripheral blood (PB) and cerebrospinal fluid (CSF) of 15 MS patients during switching from long-term NAT to FTY treatment after a defined 8-week washout period within a clinical trial (ToFingo successor study; ClinicalTrials.gov : NCT02325440). Unbiased visualization and analysis of high-dimensional single cell flow-cytometry data revealed that switching resulted in a profound alteration of more than 80% of investigated innate and adaptive immune cell subpopulations in the PB, revealing an unexpectedly broad effect of trafficking drugs on peripheral immune signatures. Longitudinal CSF analysis demonstrated that NAT and FTY both reduced T cell subset counts and proportions in the CSF of MS patients with equal potency; NAT however was superior with regard to sequestering non-T cell populations out of the CSF, including B cells, natural killer cells and inflammatory monocytes, suggesting that disease exacerbation in the context of switching might be driven by non-T cell populations. Finally, correlation of our immunological data with signs of disease exacerbation in this small cohort suggested that both (i) CD49d expression levels under NAT at the time of treatment cessation and (ii) swiftness of FTY-mediated effects on immune cell subsets in the PB together may predict stability during switching later on. [ABSTRACT FROM AUTHOR]

There is increasing evidence that natural killer (NK) cells exhibit regulatory features. Among them, CD56bright NK cells have been suggested to play a major role in controlling T cell responses and maintaining homeostasis. Dysfunction in NK cell-mediated regulatory features has been recently described in untreated multiple sclerosis (MS), suggesting a contribution to MS pathogenesis. Moreover, biological disease-modifying treatments effective in MS apparently enhance the frequencies and/or regulatory function of NK cells, further pointing toward an immunoprotective role of NK cells in MS. Here, we summarize the current knowledge on the regulatory functions of NK cells, based on their interactions with other cells belonging to the innate compartment, as well as with adaptive effector cells. We review the more recent data reporting disruption of NK cell/T cell interactions in MS and discuss how disease-modifying treatments for MS affect NK cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]