Single-cell transcriptomics combined with proteomics of intrathecal IgG reveal transcriptional heterogeneity of oligoclonal IgG-secreting cells in multiple sclerosis.

The phenotypes of B lineage cells that produce oligoclonal IgG in multiple sclerosis have not been unequivocally determined. Here, we utilized single-cell RNA-seq data of intrathecal B lineage cells in combination with mass spectrometry of intrathecally synthesized IgG to identify its cellular source. We found that the intrathecally produced IgG matched a larger fraction of clonally expanded antibody-secreting cells compared to singletons. The IgG was traced back to two clonally related clusters of antibody-secreting cells, one comprising highly proliferating cells, and the other consisting of more differentiated cells expressing genes associated with immunoglobulin synthesis. These findings suggest some degree of heterogeneity among cells that produce oligoclonal IgG in multiple sclerosis.

Stereotyped B-cell responses are linked to IgG constant region polymorphisms in multiple sclerosis.

Clonally related B cells infiltrate the brain, meninges, and cerebrospinal fluid of MS patients, but the mechanisms driving the B-cell response and shaping the immunoglobulin repertoires remain unclear. Here, we used single-cell full-length RNA-seq and BCR reconstruction to simultaneously assess the phenotypes, isotypes, constant region polymorphisms, and the paired heavy- and light-chain repertoires in intrathecal B cells. We detected extensive clonal connections between the memory B cell and antibody-secreting cell (ASC) compartments and observed clonally related cells of different isotypes including IgM/IgG1, IgG1/IgA1, IgG1/IgG2, and IgM/IgA1. There was a strong dominance of the G1m1 allotype constant region polymorphisms in ASCs, but not in memory B cells. Tightly linked to the G1m1 allotype, we found a preferential pairing of the immunoglobulin heavy-chain variable (IGHV)4 gene family with the κ variable (IGKV)1 gene family. The IGHV4-39 gene was most used and showed the highest frequency of pairing with IGKV1-5 and IGKV1(D)-33. These results link IgG constant region polymorphisms to stereotyped B-cell responses in MS and indicate that the intrathecal B-cell response in these patients could be directed against structurally similar epitopes.

Association of Body Mass Index in Adolescence and Young Adulthood and Long-term Risk of Multiple Sclerosis: A Population-Based Study.

BACKGROUND AND OBJECTIVES: To prospectively investigate the long-term relationship between body mass index (BMI) in adolescents and young adults and risk for multiple sclerosis (MS) at the population level. METHODS: We used data from the population-based compulsory Norwegian tuberculosis screening program during 1963 to 1975, including objectively measured height and weight from ≈85% of all eligible citizens. This was combined with data from the Norwegian MS registry and biobank up to November 2020. BMI was standardized according to age and sex, and risk for MS was calculated with Cox proportional hazard models. RESULTS: During 30,829,506 years of follow-up, we found 1,409 cases of MS among 648,734 participants in eligible age groups (14-34 years). Overall, obesity was associated with increased MS risk (hazard ratio [HR] 1.53 [95% confidence interval (CI) 1.25-1.88]), and the risk was similar in men (HR 1.4 [95% CI 0.95-2.06] and women (HR 1.59 [95% CI 1.25-2.02]). Risk was highest for the youngest age groups (age 14-16: HR 1.73 [95% CI 1.19-2.53]; 17-19: HR 1.61 [95% CI 1.08-2.39]; 20-24: HR 1.56 [95% CI 1.04-2.36]) and was no longer present for those >30 years of age. DISCUSSION: High BMI in individuals 14 to 24 years of age was associated with increased MS risk later in life in both male and female individuals.

CD4+ T Cells in the Blood of MS Patients Respond to Predicted Epitopes From B cell Receptors Found in Spinal Fluid.

B cells are important pathogenic players in multiple sclerosis (MS), but their exact role is not known. We have previously demonstrated that B cells from cerebrospinal fluid (CSF) of MS patients can activate T cells that specifically recognize antigenic determinants (idiotopes) from their B cell receptors (BCRs). The aim of this study was to evaluate whether in silico prediction models could identify antigenic idiotopes of immunoglobulin heavy-chain variable (IGHV) transcriptomes in MS patients. We utilized a previously assembled dataset of CSF IGHV repertoires from MS patients. To guide selection of potential antigenic idiotopes, we used in silico predicted HLA-DR affinity, endosomal processing, as well as transcript frequency from nine MS patients. Idiotopes with predicted low affinity and low likelihood of cathepsins cleavage were inert controls. Peripheral blood mononuclear cells from these patients were stimulated with the selected idiotope peptides in presence of anti-CD40 for 12 h. T cells were then labeled for activation status with anti-CD154 antibodies and CD3+CD4+ T cells phenotyped as memory (CD45RO+) or naïve (CD45RO-), with potential for brain migration (CXCR3 and/or CCR6 expression). Anti-CD14 and -CD8 were utilized to exclude monocytes and CD8+ T cells. Unstimulated cells or insulin peptides were negative controls, and EBNA-1 peptides or CD3/CD28 beads were positive controls. The mean proportion of responding memory CD4+ T cells from all nine MS patients was significantly higher for idiotope peptides with predicted high HLA-DR affinity and high likelihood of cathepsin cleavage, than toward predicted inert peptides. Responses were mainly observed toward peptides affiliated with the CDR3 region. Activated memory CD4+ T cells expressed the chemokine receptor CCR6, affiliated with a Th17 phenotype and allowing passage into the central nervous system (CNS). This in vitro study suggests that that antigenic properties of BCR idiotopes can be identified in silico using HLA affinity and endosomal processing predictions. It further indicates that MS patients have a memory T cell repertoire capable of recognizing frequent BCR idiotopes found in endogenous CSF, and that these T cells express chemokine receptors allowing them to reach the CSF B cells expressing these idiotopes.

A young woman with seizures, visual impairment, and paralysis. / En ung kvinne med krampeanfall, synsforstyrrelser og lammelser.

BACKGROUND: Anti-MOG encephalomyelitis is a recently described demyelinating, autoimmune disease of the central nervous system, identified by antibodies against myelin oligodendrocyte glycoprotein (MOG). CASE PRESENTATION: A previously healthy 20-year-old woman was admitted to hospital after a seizure. MRI showed leptomeningeal enhancement and lumbar puncture revealed moderate pleocytosis but no evidence of infection. Over the following months, she experienced a series of neurological deficits including bladder dysfunction, loss of sensation in the lower extremities and genital area, impaired motor function of the legs and episodes of visual loss. All symptoms had MRI correlates in the medulla, brainstem, optic tract, thalami and corpus callosum. She responded excellently to corticosteroid treatment, but experienced relapses shortly after discontinuation of treatment. Repeated lumbar puncture revealed pleocytosis up to 475 ∙ 106 cells/l but there were no signs of intrathecal IgG synthesis or infection. Serum anti-MOG antibodies were detected two months after the initial episode. She has been treated with low dose corticosteroids in combination with rituximab for two years, without clinical or radiological relapse. INTERPRETATION: Symptoms and signs mimicking acute demyelinating encephalomyelitis and neuromyelitis optica are typical for anti-MOG encephalomyelitis. This case illustrates that the response to corticosteroids may be excellent but transient, and that the disease can be controlled with moderate immunosuppression.

B-cell composition in the blood and cerebrospinal fluid of multiple sclerosis patients treated with dimethyl fumarate.

BACKGROUND: B cells may contribute to the immunopathogenesis of multiple sclerosis (MS). Dimethyl fumarate (DMF) has recently been shown to reduce the frequency of memory B cells in blood, but it is not known whether the drug influences the cellular composition in the cerebrospinal fluid (CSF). METHODS: A cross-sectional study examining the cellular composition in blood and cerebrospinal fluid (CSF) from 10 patients treated with DMF and 18 patients receiving other disease modifying drugs or no treatment. RESULTS: Patients treated with DMF had reduced proportions of memory B cells in blood compared to other MS patients (p = 0.0007), and the reduction correlated with treatment duration (rs = -0.75, p = 0.021). In the CSF, the absolute number of mononuclear cells were significantly lower in DMF-treated patients compared to the other patients (p = 0.023), and there was a disproportionate decrease of plasmablasts (p = 0.031). CONCLUSION: The results of this exploratory study support a B-cell mediated mechanism of action for DMF in both blood and CSF.

In Silico Prediction Analysis of Idiotope-Driven T-B Cell Collaboration in Multiple Sclerosis.

Memory B cells acting as antigen-presenting cells are believed to be important in multiple sclerosis (MS), but the antigen they present remains unknown. We hypothesized that B cells may activate CD4+ T cells in the central nervous system of MS patients by presenting idiotopes from their own immunoglobulin variable regions on human leukocyte antigen (HLA) class II molecules. Here, we use bioinformatics prediction analysis of B cell immunoglobulin variable regions from 11 MS patients and 6 controls with other inflammatory neurological disorders (OINDs), to assess whether the prerequisites for such idiotope-driven T-B cell collaboration are present. Our findings indicate that idiotopes from the complementarity determining region (CDR) 3 of MS patients on average have high predicted affinities for disease associated HLA-DRB1*15:01 molecules and are predicted to be endosomally processed by cathepsin S and L in positions that allows such HLA binding to occur. Additionally, complementarity determining region 3 sequences from cerebrospinal fluid (CSF) B cells from MS patients contain on average more rare T cell-exposed motifs that could potentially escape tolerance and stimulate CD4+ T cells than CSF B cells from OIND patients. Many of these features were associated with preferential use of the IGHV4 gene family by CSF B cells from MS patients. This is the first study to combine high-throughput sequencing of patient immune repertoires with large-scale prediction analysis and provides key indicators for future in vitro and in vivo analyses.

Monomethyl fumarate augments NK cell lysis of tumor cells through degranulation and the upregulation of NKp46 and CD107a.

Dimethyl fumarate (DMF) is a new drug used to treat multiple sclerosis (MS) patients. Here, we examined the effects of DMF and the DMF metabolite monomethyl fumarate (MMF) on various activities of natural killer (NK) cells. We demonstrated that MMF augments the primary CD56(+), but not CD56(-), NK cell lysis of K562 and RAJI tumor cells. MMF induced NKp46 expression on the surface of CD56(+), but not CD56(-), NK cells after incubation for 24 h. This effect was closely correlated with the upregulation of CD107a expression on the surface of CD56(+) NK cells and the induction of Granzyme B release from these cells through this metabolite. An anti-NKp46 antibody inhibited the MMF-induced upregulation of CD107a and the lysis of tumor cells through CD56(+) NK cells. Thus, these results are the first to show that MMF augments CD56(+) NK cell lysis of tumor target cells, an effect mediated through NKp46. This novel effect suggests the use of MMF for therapeutic and/or preventive protocols in cancer.

Multiple sclerosis and the role of immune cells.

Multiple sclerosis (MS) is a complex disease with many different immune cells involved in its pathogenesis, and in particular T cells as the most recognized cell type. Recently, the innate immune system has also been researched for its effect on the disease. Hence, cells of the immune system play vital roles in either ameliorating or exacerbating the disease. The genetic and environmental factors, as well as the etiology and pathogenesis are of utmost importance for the development of MS. An insight into the roles play by T cells, B cells, natural killer cells, and dendritic cells in MS and the animal model experimental autoimmune encephalomyelitis, will be presented. Understanding the mechanisms of action for current therapeutic modalities should help developing new therapeutic tools to treat this disease and other autoimmune diseases.

A one year follow-up study of natural killer and dendritic cells activities in multiple sclerosis patients receiving glatiramer acetate (GA).

BACKGROUND: Multiple sclerosis (MS) is a chronic inflammatory, demyelinating and neurodegenerative disease. It is thought to be mediated by CD4(+) Th1/Th17 cells. More recently, cells of the innate immune system such as dendritic cells (DCs) and natural killer (NK) cells have been in focus. Glatiramer acetate (GA) is an approved drug for treating MS patients. METHODOLOGY/PRINCIPAL FINDINGS: In the current study we examined the activities of NK and DCs in nine relapsing remitting MS patients for up to one year after initiation of GA treatment. We observed that NK cells isolated from most of these patients have increased cytotoxic activity against K562 cells. Further analysis showed that the same NK cells lysed both autologous immature (i) and mature (m) DCs. In most patients this increased activity was correlated with increased NK cell activating cytotoxicity receptors such as NKp30, NKp44, NKp46 and NKG2D, and reduced expression of the inhibitory molecule CD158 on the surface of these NK cells. The expression of HLA-DR was increased on iDCs and mDCs in the majority of the patients, but no consistency was observed for the expression of HLA-I or HLA-E. Also, the co-stimulatory receptors CD80, CD83 or CD86 expression was down-regulated on iDCs and mDCs in most cases. Further, the expression of CCR6 was increased on mDCs at later time points of therapy (between 32-48 weeks). CONCLUSIONS/SIGNIFICANCE: Our results are the first showing the effects of GA treatment on NK cells in MS patients, which may impact future use of this and other drugs to treat this disease.

Identification of human NK17/NK1 cells.

BACKGROUND: Natural killer (NK) cells have both cytolytic and immunoregulatory functions. We recently described that these cells release the inflammatory cytokines IL-17 and IFN-γ. However, the precise identity of the NK cell subset(s) that secrete these cytokines is not known. METHODOLOGY/PRINCIPAL FINDINGS: To isolate the cells secreting IL-17 and IFN-γ, we took advantage of the findings that Th17/Th1 cells express chemokine receptors. Therefore, CD56(+)NK cells were stained with antibodies against various chemokine receptors and intracellularly with antibodies toward IL-17 and IFN-γ. Consequently, we identified previously unrecognized subset of NK cells generated from normal human peripheral blood after activation with IL-2 but not PMA plus ionomycin. The cells are characterized by the expression of CD56(+) and CCR4(+), produce IL-17 and IFN-γ and are consequently named NK17/NK1 cells. They also express CD161, NKp30, NKp44, NKp46, NKG2D, CD158, CCL22, IL-2Rß and the common γ chain but not CD127 or IL-23R. Further, they possess T-bet and RORγt transcription factors. Antibodies to IL-1ß, IL-6, IL-21, or TGF-ß1 do not inhibit IL-2-induced generation of NK17/NK1 cells, suggesting that IL-2 has the capacity to polarize these cells. Notably, NK17/NK1 cells are abundant in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) without activation, and are generated from the peripheral blood of these patients after activation with IL-2. CONCLUSIONS/SIGNIFICANCE: NK17/NK1 cells identified here have not been previously described in healthy or MS patients.