SARS-CoV-2-Specific Immune Cytokine Profiles to mRNA, Viral Vector and Protein-Based Vaccines in Patients with Multiple Sclerosis: Beyond Interferon Gamma.

Disease-modifying therapies (DMTs) impact the cellular immune response to severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) vaccines in patients with multiple sclerosis (pwMS). In this study, we aim to elucidate the characteristics of the involved antigen-specific T cells via the measurement of broad cytokine profiles in pwMS on various DMTs. We examined SARS-CoV-2-specific T cell responses in whole blood cultures characterized by the release of interleukin (IL)-2, IL-4, IL-5, IL-10, IL-13, IL-17A, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α), as well as antibodies (AB) targeting the SARS-CoV-2 spike protein in pwMS following either two or three doses of mRNA or viral vector vaccines (VVV). For mRNA vaccination non-responders, the NVX-CoV2373 protein-based vaccine was administered, and immune responses were evaluated. Our findings indicate that immune responses to SARS-CoV-2 vaccines in pwMS are skewed towards a Th1 phenotype, characterized by IL-2 and IFN-γ. Additionally, a Th2 response characterized by IL-5, and to a lesser extent IL-4, IL-10, and IL-13, is observed. Therefore, the measurement of IL-2 and IL-5 levels could complement traditional IFN-γ assays to more comprehensively characterize the cellular responses to SARS-CoV-2 vaccines. Our results provide a comprehensive cytokine profile for pwMS receiving different DMTs and offer valuable insights for designing vaccination strategies in this patient population.

Effects of obstructive sleep apnea treatment on neurodegenerative biomarker neurofilament light chain and cognitive performance.

Obstructive sleep apnea is associated with cognitive impairment and increased risk for neurodegenerative diseases. Obstructive sleep apnea treatment with positive airway pressure therapy helps to improve cognitive symptoms and reduces long-term dementia risk. To test whether these treatment effects are due to a reduction in neuronal damage, we examined longitudinal changes in the neurodegenerative serum neurofilament light chain and cognitive performance of patients with obstructive sleep apnea. In this study, 17 patients with obstructive sleep apnea completed baseline and follow-up (9 month after starting PAP treatment) investigation of sleep, daytime symptoms, cognitive testing and serum neurofilament light chain measurements. Depending on treatment adherence and efficacy, participants were assigned either to the effective treatment (n = 10) or non-effective treatment group (n = 7). As results at baseline lower mean oxygen saturation during sleep was associated with higher serum neurofilament light chain. Patients in the non-effective treatment group showed a significant increase of age-adjusted percentile of serum neurofilament light chain levels at follow-up, whereas serum neurofilament light chain values remained constant in the effective treatment group. At a functional level, effective treatment leads to an improvement in processing speed, which was not the case in the non-effective treatment group. Longitudinal changes of age-adjusted serum neurofilament light chain levels were associated with changes in cognitive performance. To conclude, this longitudinal observational study showed that effective obstructive sleep apnea treatment positively affects the amount of neuronal damage as well as working memory performance. As cognitive symptoms might not only be attributed to obstructive sleep apnea-related sleep deficiency, but also neurodegeneration, our results underline the importance of treatment adherence and efficacy for the prevention of neuronal damage and cognitive consequences.

Associations of sNfL with clinico-radiological measures in a large MS population.

OBJECTIVE: Evaluation of serum neurofilament light chain (sNfL), measured using high-throughput assays on widely accessible platforms in large, real-world MS populations, is a critical step for sNfL to be utilized in clinical practice. METHODS: Multiple Sclerosis Partners Advancing Technology and Health Solutions (MS PATHS) is a network of healthcare institutions in the United States and Europe collecting standardized clinical/imaging data and biospecimens during routine clinic visits. sNfL was measured in 6974 MS and 201 healthy control (HC) participants, using a high-throughput, scalable immunoassay. RESULTS: Elevated sNfL levels for age (sNfL-E) were found in 1238 MS participants (17.8%). Factors associated with sNfL-E included male sex, younger age, progressive disease subtype, diabetes mellitus, impaired renal function, and active smoking. Higher body mass index (BMI) was associated with lower odds of elevated sNfL. Active treatment with disease-modifying therapy was associated with lower odds of sNfL-E. MS participants with sNfL-E exhibited worse neurological function (patient-reported disability, walking speed, manual dexterity, and cognitive processing speed), lower brain parenchymal fraction, and higher T2 lesion volume. Longitudinal analyses revealed accelerated short-term rates of whole brain atrophy in sNfL-E participants and higher odds of new T2 lesion development, although both MS participants with or without sNfL-E exhibited faster rates of whole brain atrophy compared to HC. Findings were consistent in analyses examining age-normative sNfL Z-scores as a continuous variable. INTERPRETATION: Elevated sNfL is associated with clinical disability, inflammatory disease activity, and whole brain atrophy in MS, but interpretation needs to account for comorbidities including impaired renal function, diabetes, and smoking.

Serum neurofilament indicates that DBS surgery can cause neuronal damage whereas stimulation itself does not.

Deep brain stimulation (DBS) is a potent symptomatic therapy for Parkinson's disease, but it is debated whether it causes or prevents neurodegeneration. We used serum neurofilament light chain (NFL) as a reporter for neuronal damage and found no difference between 92 patients with chronic STN-DBS and 57 patients on best medical treatment. Serum NFL transiently increased after DBS surgery whereas the initiation of STN stimulation did not affect NFL levels, suggesting that DBS surgery can be associated with neuronal damage whereas stimulation itself is not.

Cladribine Alters Immune Cell Surface Molecules for Adhesion and Costimulation: Further Insights to the Mode of Action in Multiple Sclerosis.

Cladribine (CLAD) is a deoxyadenosine analogue prodrug which is given in multiple sclerosis (MS) as two short oral treatment courses 12 months apart. Reconstitution of adaptive immune function following selective immune cell depletion is the presumed mode of action. In this exploratory study, we investigated the impact of CLAD tablets on immune cell surface molecules for adhesion (CAMs) and costimulation (CoSs) in people with MS (pwMS). We studied 18 pwMS who started treatment with CLAD and 10 healthy controls (HCs). Peripheral blood mononuclear cells were collected at baseline and every 3 months throughout a 24-month period. We analysed ICAM-1, LFA-1, CD28, HLADR, CD154, CD44, VLA-4 (CD49d/CD29), PSGL-1 and PD-1 with regard to their expression on B and T cells (T helper (Th) and cytotoxic T cells (cT)) and surface density (mean fluorescence intensity, MFI) by flow cytometry. The targeted analysis of CAM and CoS on the surface of immune cells in pwMS revealed a higher percentage of ICAM-1 (B cells, Th, cT), LFA-1 (B cells, cT), HLADR (B cells, cT), CD28 (cT) and CD154 (Th). In pwMS, we found lower frequencies of Th and cT cells expressing PSGL-1 and B cells for the inhibitory signal PD-1, whereas the surface expression of LFA-1 on cT and of HLADR on B cells was denser. Twenty-four months after the first CLAD cycle, the frequencies of B cells expressing CD44, CD29 and CD49d were lower compared with the baseline, together with decreased densities of ICAM-1, CD44 and HLADR. The rate of CD154 expressing Th cells dropped at 12 months. For cT, no changes were seen for frequency or density. Immune reconstitution by oral CLAD was associated with modification of the pro-migratory and -inflammatory surface patterns of CAMs and CoSs in immune cell subsets. This observation pertains primarily to B cells, which are key cells underlying MS pathogenesis.

Interleukin-17 and Th17 Lymphocytes Directly Impair Motoneuron Survival of Wildtype and FUS-ALS Mutant Human iPSCs.

Amyotrophic lateral sclerosis (ALS) is a progressive disease leading to the degeneration of motor neurons (MNs). Neuroinflammation is involved in the pathogenesis of ALS; however, interactions of specific immune cell types and MNs are not well studied. We recently found a shift toward T helper (Th)1/Th17 cell-mediated, pro-inflammatory immune responses in the peripheral immune system of ALS patients, which positively correlated with disease severity and progression. Whether Th17 cells or their central mediator, Interleukin-17 (IL-17), directly affects human motor neuron survival is currently unknown. Here, we evaluated the contribution of Th17 cells and IL-17 on MN degeneration using the co-culture of iPSC-derived MNs of fused in sarcoma (FUS)-ALS patients and isogenic controls with Th17 lymphocytes derived from ALS patients, healthy controls, and multiple sclerosis (MS) patients (positive control). Only Th17 cells from MS patients induced severe MN degeneration in FUS-ALS as well as in wildtype MNs. Their main effector, IL-17A, yielded in a dose-dependent decline of the viability and neurite length of MNs. Surprisingly, IL-17F did not influence MNs. Importantly, neutralizing IL-17A and anti-IL-17 receptor A treatment reverted all effects of IL-17A. Our results offer compelling evidence that Th17 cells and IL-17A do directly contribute to MN degeneration.

CNS inflammation after natalizumab therapy for multiple sclerosis: A retrospective histopathological and CSF cohort study.

Natalizumab, a recombinant humanized monoclonal antibody directed against the α4 subunit of the integrins α4ß1 and α4ß7, has been approved for the treatment of active relapsing-remitting MS. Although natalizumab is a highly beneficial drug that effectively reduces the risk of sustained disability progression and the rate of clinical relapses, some patients do not respond to it, and some are at higher risk of developing progressive multifocal leukoencephalopathy (PML). The histopathological effects after natalizumab therapy are still unknown. We, therefore, performed a detailed histological characterization of the CNS inflammatory cell infiltrate of 24 brain specimens from natalizumab treated patients, consisting of 20 biopsies and 4 autopsies and 21 MS controls. To complement the analysis, immune cells in blood and cerebrospinal fluid (CSF) of 30 natalizumab-treated patients and 42 MS controls were quantified by flow cytometry. Inflammatory infiltrates within lesions were mainly composed of T cells and macrophages, some B cells, plasma cells, and dendritic cells. There was no significant difference in the numbers of T cells or macrophages and microglial cells in lesions of natalizumab-treated patients as compared to controls. A shift towards cytotoxic T cells of a memory phenotype was observed in the CSF. Plasma cells were significantly increased in active demyelinating lesions of natalizumab-treated patients, but no correlation to clinical disability was observed. Dendritic cells within lesions were found to be reduced with longer ongoing therapy duration. Our findings suggest that natalizumab does not completely prevent immune cells from entering the CNS and is associated with an accumulation of plasma cells, the pathogenic and clinical significance of which is not known. As B cells are considered to serve as a reservoir of the JC virus, the observed plasma cell accumulation and reduction in dendritic cells in the CNS of natalizumab-treated patients may potentially play a role in PML development.

Long-term peripheral immune cell profiling reveals further targets of oral cladribine in MS.

OBJECTIVES: To expand the knowledge about the immunological consequences of cladribine (CLAD), a pulsed immune reconstitution therapy approved for active multiple sclerosis (MS), beyond the known short-term effects on peripheral immune cell subsets. METHODS: In this study, we characterized depletion and restitution kinetics as well as cytokine profiles of peripheral immune cell subsets in 18 patients with MS following treatment with oral CLAD. The methods involved blood collection prior to CLAD and every three months over a period of 24 months, and extensive characterization of various immune cells subsets by multiparametric flow cytometry. RESULTS: We found a selectivity of CLAD towards central memory T cells and memory B cells and detected a hyper-repopulation of maturing B cells. Counts of classical (-65%) and various nonclassical TH17 cells (-84% to -87%) were markedly reduced 24 months after treatment start, and were comparable with depletion rates of class-switched memory B-cell phenotypes (-87% to -95%). The nadir of TH cells was more pronounced in the second treatment year. We observed a proportional surge of CD20 T-cell subsets and an expansion of regulatory T, B and NK cells. Natural killer T cells (NKT) were only depleted in year two and did not recover. INTERPRETATION: Peripheral immune cell profiling revealed more differentiated insights into the immunological effects of CLAD. While some immune cell subsets expanded, we also observed additive depleting effects after the second treatment course. Further studies are required to elucidate whether these changes are paramount for the consistent and prolonged disease-modifying effect of CLAD.

The role of TH17 cells in multiple sclerosis: Therapeutic implications.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) where immunopathology is thought to be mediated by myelin-reactive CD4+ T helper (TH) cells. The TH cells most commonly implicated in the pathogenesis of the disease are of TH1 and TH17 lineage, which are defined by the production of interferon-γ and interleukin-17, respectively. Moreover, there is emerging evidence for the involvement of TH17.1 cells, which share the hallmarks of TH1 and TH17 subsets. In this review, we summarise current knowledge about the potential role of TH17 subsets in the initiation and progression of the disease and put a focus on their response to approved immunomodulatory MS drugs. In this regard, TH17 cells are abundant in peripheral blood, cerebrospinal fluid and brain lesions of MS patients, and their counts and inflammatory mediators are further increased during relapses. Fingolimod and alemtuzumab induce a paramount decrease in central memory T cells, which harbour the majority of peripheral TH17 cells, while the efficacy of natalizumab, dimethyl fumarate and importantly hematopoietic stem cell therapy correlates with TH17.1 cell inhibition. Interestingly, also CD20 antibodies target highly inflammatory TH cells and hamper TH17 differentiation by IL-6 reductions. Moreover, recovery rates of TH cells best correlate with long-term efficacy after therapeutical immunodepletion. We conclude that central memory TH17.1 cells play a pivotal role in MS pathogenesis and they represent a major target of MS therapeutics.

Antigen-shift in varicella-zoster virus-specific T-cell immunity over the course of Fingolimod-treatment in relapse-remitting multiple sclerosis patients.

BACKGROUND: Fingolimod (FTY) applied as treatment regimen of relapsing-remitting multiple sclerosis (RRMS) induces downregulation of sphingosine-1-phosphate receptors on the lymphocytes. As a result CC chemokine receptor type 7 (CCR7) expressing lymphocytes are retained within the peripheral lymph nodes thus suppressing their accumulation into the cerebrospinal fluid of multiple sclerosis (MS) patients and hampering disease progress. Unfortunately, MS patients treated with FTY suffer from an increased incidence of varicella-zoster virus (VZV) infections which has been associated with a decrease of VZV immediate early 63 (IE63)-specific T-cell immunity. To elucidate VZV-specific T-cell immunity over the course of FTY-treatment, we analyzed T-cell immunity for immediate early, early and late VZV-antigens. METHODS: T-cell immune responses were detected via intracellular IFN-γ staining after stimulation with VZV-specific peptide mixes for IE62 and IE63 and recombinant proteins for open reading frame 26 (ORF26), ORF9 and glycoprotein E (gE) using flow cytometry. Analyzed samples comprised of different groups including 18 patients with RRMS at baseline (BL), 6 and 12 months after FTY-treatment start, 12 patients with long-term (LT) FTY-treatment, one FTY-treated patient, before and after VZV-reactivation. In addition, VZV-specific IgG and IgM titers were assessed by ELISA. RESULTS: After FTY-treatment start, absolute numbers of CCR7 expressing CD4+ T cells and CD8+T cells dropped rapidly. However, VZV-specific immunity could be detected in the majority of RRMS patients throughout FTY-treatment with increasing prevalence after 6 months of treatment. We found an increase in the prevalence of VZV-specific IFN-γ+CD8+ T-cell immunity in FTY-treated patients after six months of therapy, while in parallel VZV-specific IFN-γ+CD4+ T cells declined dramatically. Additionally, a strong correlation between VZV-specific IgG serum titers and the percentage of RRMS patients with detectable VZV-specific T cells was observed (r = 0.985). Most remarkably, FTY-treated RRMS patients presented a shift in the predominant CD8+ T cell-mediated antigen-response from immediate early (IE62) to early virus antigens (ORF26) six months after treatment in parallel to a decrease of VZV-specific CD4+ T-cell immunity. ORF26-specific CD8+ T cells still dominated the VZV-specific cellular immunity at month 12 after FTY-treatment start and in LT FTY-treated MS patients. In a RRMS patient an increase of VZV-specific CD4+ T cells at VZV-reactivation accompanied with a four-fold increase of a VZV-specific IgG titer was detected which might indicate an important role in cellular immune control of VZV-infections. CONCLUSION: Monitoring VZV-specific T-cell immunity might provide a valuable tool to RRMS patient risk management during FTY-treatment.

Engrafting human regulatory T cells with a flexible modular chimeric antigen receptor technology.

As regulatory T cells (Tregs) play a fundamental role in immune homeostasis their adoptive transfer emerged as a promising treatment strategy for inflammation-related diseases. Preclinical animal models underline the superiority of antigen-specific Tregs compared to polyclonal cells. Here, we applied a modular chimeric antigen receptor (CAR) technology called UniCAR for generation of antigen-specific human Tregs. In contrast to conventional CARs, UniCAR-endowed Tregs are indirectly linked to their target cells via a separate targeting module (TM). Thus, transduced Tregs can be applied universally as their antigen-specificity is easily adjusted by TM exchange. Activation of UniCAR-engrafted Tregs occurred in strict dependence on the TM, facilitating a precise control over Treg activity. In order to augment efficacy and safety, different intracellular signaling domains were tested. Both 4-1BB (CD137) and CD28 costimulation induced strong suppressive function of genetically modified Tregs. However, in light of safety issues, UniCARs comprising a CD137-CD3ζ signaling domain emerged as constructs of choice for a clinical application of redirected Tregs. In that regard, Tregs isolated from patients suffering from autoimmune or inflammatory diseases were, for the first time, successfully engineered with UniCAR 137/ζ and efficiently suppressed patient-derived effector cells. Overall, the UniCAR platform represents a promising approach to improve Treg-based immunotherapies for tolerance induction.