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Multiple sclerosis (MS) is a common and devastating chronic inflammatory disease of the central nervous system (CNS). CD4+ T cells are assumed to be the first to cross the blood-CNS barrier and trigger local inflammation. Here, we explored how pathogenicity-associated effector programs define CD4+ T cell subsets with brain-homing ability in MS. Runx3- and Eomes-, but not T-bet-expressing CD4+ memory cells were diminished in the blood of MS patients. This decline reversed following natalizumab treatment and was supported by an Runx3+ Eomes+ T-bet- enrichment in cerebrospinal fluid samples of treatment-naïve MS patients. This transcription factor profile was associated with high granzyme K (GZMK) and CCR5 levels and was most prominent in Th17.1 cells (CCR6+ CXCR3+ CCR4-/dim ). Previously published CD28- CD4 T cells were characterized by a Runx3+ Eomes- T-bet+ phenotype that coincided with intermediate CCR5 and a higher granzyme B (GZMB) and perforin expression, indicating the presence of two separate subsets. Under steady-state conditions, granzyme Khigh Th17.1 cells spontaneously passed the blood-brain barrier in vitro. This was only found for other subsets including CD28- cells when using inflamed barriers. Altogether, CD4+ T cells contain small fractions with separate pathogenic features, of which Th17.1 seems to breach the blood-brain barrier as a possible early event in MS. This article is protected by copyright. All rights reserved.

The anti-CD20 monoclonal antibody ocrelizumab reduces disability progression in primary progressive multiple sclerosis. CD20 is a prototypical B-cell marker; however, subpopulations of CD4+ and CD8+ T cells in peripheral blood and cerebrospinal fluid also express low levels of CD20 (CD20dim). Therefore, direct targeting and depletion of these CD20dim T-cell subpopulations may contribute to the therapeutic effect of ocrelizumab. The aim of this observational cohort study was to compare CD20+ B-cell and CD20dim T-cell distributions between peripheral blood and cerebrospinal fluid of ocrelizumab-treated or ocrelizumab-untreated people with primary progressive multiple sclerosis. Ocrelizumab treatment was associated with depletion of circulating B cells and CD20dim CD4+ and CD20dim CD8+ T cells (P < 0.0001, P = 0.0016 and P = 0.0008, respectively) but, in cerebrospinal fluid, only with lower proportions of B cells and CD20dim memory CD4+ T cells (P < 0.0001 and P = 0.0043, respectively). The proportional prevalence of cerebrospinal fluid CD20dim memory CD8+ T cells was not significantly reduced (P = 0.1333). Only in cerebrospinal fluid, the proportions of CD20dim cells within CD4+ and not CD8+ T cells positive for CCR5, CCR6 and CXCR3 were reduced in ocrelizumab-treated participants. The proportion of CD20dim CD4+ T cells and abundance of CD4+ relative to CD8+ T cells in cerebrospinal fluid correlated positively with age (R = 0.6799, P = 0.0150) and Age-Related Multiple Sclerosis Severity score (R = 0.8087, P = 0.0014), respectively. We conclude that, in contrast to cerebrospinal fluid CD20dim CD8+ T cells, B cells and CD20dim CD4+ T cells are reduced in cerebrospinal fluid of people with primary progressive multiple sclerosis with an ocrelizumab-associated depletion of circulating B cells and CD20dim T cells. Therefore, these cells are likely to contribute to the therapeutic effects of ocrelizumab in people with primary progressive multiple sclerosis.

BACKGROUND: In multiple sclerosis (MS), B cells are considered main triggers of the disease, likely as the result of complex interaction between genetic and environmental risk factors. Studies on monozygotic twins discordant for MS offer a unique way to reduce this complexity and reveal discrepant subsets.METHODS: In this study, we analyzed B cell subsets in blood samples of monozygotic twins with and without MS using publicly available data. We verified functional characteristics by exploring the role of therapy and performed separate analyses in unrelated individuals.FINDINGS: The frequencies of CXCR3+ memory B cells were reduced in the blood of genetically identical twins with MS compared to their unaffected twin siblings. Natalizumab (anti-VLA-4 antibody) was the only treatment regimen under which these frequencies were reversed. The CNS-homing features of CXCR3+ memory B cells were supported by elevated CXCL10 levels in MS cerebrospinal fluid and their in vitro propensity to develop into antibody-secreting cells.CONCLUSIONS: Circulating CXCR3+ memory B cells are affected by non-heritable cues in people who develop MS. This underlines the requirement of environmental risk factors such as Epstein-Barr virus in triggering these B cells. We propose that after CXCL10-mediated entry into the CNS, CXCR3+ memory B cells mature into antibody-secreting cells to drive MS.FUNDING: This work was supported by Nationaal MS Fonds (OZ2021-016), Stichting MS Research (19-1057 MS, 20-490f MS, and 21-1142 MS), the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program grant agreement no. 882424, and the Swiss National Science Foundation (733 310030_170320, 310030_188450, and CRSII5_183478).

BACKGROUND: Although distinct brain-homing B cells have been identified in multiple sclerosis (MS), it is unknown how these further evolve to contribute to local pathology. We explored B-cell maturation in the central nervous system (CNS) of MS patients and determined their association with immunoglobulin (Ig) production, T-cell presence, and lesion formation.METHODS: Ex vivo flow cytometry was performed on post-mortem blood, cerebrospinal fluid (CSF), meninges and white matter from 28 MS and 10 control brain donors to characterize B cells and antibody-secreting cells (ASCs). MS brain tissue sections were analysed with immunostainings and microarrays. IgG index and CSF oligoclonal bands were measured with nephelometry, isoelectric focusing, and immunoblotting. Blood-derived B cells were cocultured under T follicular helper-like conditions to evaluate their ASC-differentiating capacity in vitro.FINDINGS: ASC versus B-cell ratios were increased in post-mortem CNS compartments of MS but not control donors. Local presence of ASCs associated with a mature CD45low phenotype, focal MS lesional activity, lesional Ig gene expression, and CSF IgG levels as well as clonality. In vitro B-cell maturation into ASCs did not differ between MS and control donors. Notably, lesional CD4+ memory T cells positively correlated with ASC presence, reflected by local interplay with T cells.INTERPRETATION: These findings provide evidence that local B cells at least in late-stage MS preferentially mature into ASCs, which are largely responsible for intrathecal and local Ig production. This is especially seen in active MS white matter lesions and likely depends on the interaction with CD4+ memory T cells.FUNDING: Stichting MS Research (19-1057 MS; 20-490f MS), National MS Fonds (OZ2018-003).

Background and purpose: Multiple sclerosis (MS) is associated with abnormal B-cell function, and MS genetic risk alleles affect multiple genes that are expressed in B cells. However, how these genetic variants impact the B-cell compartment in early childhood is unclear. In the current study, we aim to assess whether polygenic risk scores (PRSs) for MS are associated with changes in the blood B-cell compartment in children from the general population. Methods: Six-year-old children from the population-based Generation R Study were included. Genotype data were used to calculate MS-PRSs and B-cell subset-enriched MS-PRSs, established by designating risk loci based on expression and function. Analyses of variance were performed to examine the effect of MS-PRSs on total B-cell numbers (n = 1261) as well as naive and memory subsets (n = 675). Results: After correction for multiple testing, no significant associations were observed between MS-PRSs and total B-cell numbers and frequencies of subsets therein. A naive B-cell-MS-PRS (n = 26 variants) was significantly associated with lower relative, but not absolute, naive B-cell numbers (p = 1.03 × 10−4 and p = 0.82, respectively), and higher frequencies and absolute numbers of CD27+ memory B cells (p = 8.83 × 10−4 and p = 4.89 × 10−3, respectively). These associations remained significant after adjustment for Epstein–Barr virus seropositivity and the HLA-DRB1*15:01 genotype. Conclusions: The composition of the blood B-cell compartment is associated with specific naive B-cell-associated MS risk variants during childhood, possibly contributing to MS pathophysiology later in life. Cell subset-specific PRSs may offer a more sensitive tool to define the impact of genetic risk on the immune system in diseases such as MS.

The effector programs of CD8+ memory T cells are influenced by the transcription factors RUNX3, EOMES and T-bet. How these factors define brain-homing CD8+ memory T cells in multiple sclerosis (MS) remains unknown. To address this, we analyzed blood, CSF and brain tissues from MS patients for the impact of differential RUNX3, EOMES and T-bet expression on CD8+ T cell effector phenotypes. The frequencies of RUNX3- and EOMES-, but not T-bet-expressing CD8+ memory T cells were reduced in the blood of treatment-naïve MS patients as compared to healthy controls. Such reductions were not seen in MS patients treated with natalizumab (anti-VLA-4 Ab). We found an additional loss of T-bet in RUNX3-expressing cells, which was associated with the presence of MS risk SNP rs6672420 (RUNX3). RUNX3+EOMES+T-bet- CD8+ memory T cells were enriched for the brain residency-associated markers CCR5, granzyme K, CD20 and CD69 and selectively dominated the MS CSF. In MS brain tissues, T-bet coexpression was recovered in CD20dim and CD69+ CD8+ T cells, and was accompanied by increased coproduction of granzyme K and B. These results indicate that coexpression of RUNX3 and EOMES, but not T-bet, defines CD8+ memory T cells with a pre-existing brain residency-associated phenotype such that they are prone to enter the CNS in MS.

The effector programs of CD8+ memory T cells are influenced by the transcription factors RUNX3, EOMES and T-bet. How these factors define brain-homing CD8+ memory T cells in multiple sclerosis (MS) remains unknown. To address this, we analyzed blood, CSF and brain tissues from MS patients for the impact of differential RUNX3, EOMES and T-bet expression on CD8+ T cell effector phenotypes. The frequencies of RUNX3-and EOMES-, but not T-bet-expressing CD8+ memory T cells were reduced in the blood of treatment-naïve MS patients as compared to healthy controls. Such reductions were not seen in MS patients treated with natalizumab (anti-VLA-4 Ab). We found an additional loss of T-bet in RUNX3-expressing cells, which was associated with the presence of MS risk SNP rs6672420 (RUNX3). RUNX3+EOMES+T-bet− CD8+ memory T cells were enriched for the brain residency-associated markers CCR5, granzyme K, CD20 and CD69 and selectively dominated the MS CSF. In MS brain tissues, T-bet coexpression was recovered in CD20dim and CD69+ CD8+ T cells, and was accompanied by increased coproduction of granzyme K and B. These results indicate that coexpression of RUNX3 and EOMES, but not T-bet, defines CD8+ memory T cells with a pre-existing brain residency-associated phenotype such that they are prone to enter the CNS in MS.

Background: Multiple sclerosis (MS) patients are protected from relapses during pregnancy and have an increased relapse risk after delivery. It is unknown how pregnancy controls disease-contributing CD4+ T helper (Th) cells and whether this differs in MS patients who experience a postpartum relapse. Here, we studied the effector phenotype of Th cells in relation to pregnancy and postpartum relapse occurrence in MS. Methods: Memory skewing and activation of effector Th subsets were analyzed in paired third trimester and postpartum blood of 19 MS patients with and without a postpartum relapse and 12 healthy controls. Ex vivo results were associated with circulating levels of pregnancy-induced hormones and mirrored in vitro by exposing proliferating Th cells to corresponding serum samples. Results: Based on HSNE-guided analyses, we found that effector memory proportions of Th cells were increased in postpartum vs. third trimester samples from MS patients without a postpartum relapse. This was not seen for relapsing patients or healthy controls. CXCR3 was upregulated on postpartum memory Th cells, except for relapsing patients. These changes were verified by adding sera from the same individuals to proliferating Th cells, but did not associate with third trimester cortisol, estradiol or progesterone levels. For relapsing patients, activated memory Th cells of both third trimester and postpartum samples produced higher levels of pro-inflammatory cytokines. Conclusion: Effector Th cells are differentially regulated during pregnancy in MS patients, likely via serum-related factors beyond the studied hormones. The pro-inflammatory state of memory Th cells during pregnancy may predict a postpartum relapse.

Epstein-Barr virus (EBV) infection of B cells is associated with increased multiple sclerosis (MS) susceptibility. Recently, we found that CXCR3-expressing B cells preferentially infiltrate the central nervous system of MS patients. In chronic virus-infected mice, these types of B cells are sustained and show increased antiviral responsiveness. How EBV persistence in B cells influences their development remains unclear. First, we analyzed ex vivo B-cell subsets from MS patients who received autologous bone marrow transplantation (n=9), which is often accompanied by EBV reactivation. The frequencies of non-class-switched and class-switched memory B cells were reduced at 3-7 months, while only class-switched B cells returned back to baseline at 24-36 months post-transplantation. At these time points, EBV DNA load positively correlated to the frequency of CXCR3+ , and not CXCR4+ or CXCR5+ , class-switched B cells. Second, for CXCR3+ memory B cells trapped within the blood of MS patients treated with natalizumab (anti-VLA-4 antibody n=15), latent EBV infection corresponded to enhanced in vitro formation of anti-EBNA1 IgG-secreting plasma cells under germinal center-like conditions. These findings imply that EBV persistence in B cells potentiates brain-homing and antibody-producing CXCR3+ subsets in MS. This article is protected by copyright. All rights reserved.

Epstein–Barr virus (EBV) infection of B cells is associated with increased multiple sclerosis (MS) susceptibility. Recently, we found that CXCR3-expressing B cells preferentially infiltrate the CNS of MS patients. In chronic virus-infected mice, these types of B cells are sustained and show increased antiviral responsiveness. How EBV persistence in B cells influences their development remains unclear. First, we analyzed ex vivo B-cell subsets from MS patients who received autologous bone marrow transplantation (n = 9), which is often accompanied by EBV reactivation. The frequencies of nonclass-switched and class-switched memory B cells were reduced at 3–7 months, while only class-switched B cells returned back to baseline at 24–36 months posttransplantation. At these time points, EBV DNA load positively correlated to the frequency of CXCR3+, and not CXCR4+ or CXCR5+, class-switched B cells. Second, for CXCR3+ memory B cells trapped within the blood of MS patients treated with natalizumab (anti-VLA-4 antibody n = 15), latent EBV infection corresponded to enhanced in vitro formation of anti-EBNA1 IgG-secreting plasma cells under GC-like conditions. These findings imply that EBV persistence in B cells potentiates brain-homing and antibody-producing CXCR3+ subsets in MS.

Background and purpose: Patients with multiple sclerosis (MS) have altered T cell function and composition. Common genetic risk variants for MS affect proteins that function in the immune system. It is currently unclear to what extent T cell composition is affected by genetic risk factors for MS, and how this may precede a possible disease onset. Here, we aim to assess whether an MS polygenic risk score (PRS) is associated with an altered T cell composition in a large cohort of children from the general population. Methods: We included genotyped participants from the population-based Generation R study in whom immunophenotyping of blood T cells was performed at the age of 6 years. Analyses of variance were used to determine the impact of MS-PRSs on total T cell numbers (n = 1261), CD4+ and CD8+ lineages, and subsets therein (n= 675). In addition, T-cell-specific PRSs were constructed based on functional pathway data. Results: The MS-PRS negatively correlated with CD8+ T cell frequencies (p = 2.92 × 10−3), which resulted in a positive association with CD4+/CD8+ T cell ratios (p = 8.27 × 10−9). These associations were mainly driven by two of 195 genome-wide significant MS risk variants: the main genetic risk variant for MS, HLA-DRB1*15:01 and an HLA-B risk variant. We observed no significant associations for the T-cell-specific PRSs. Conclusions: Our results suggest that MS-associated genetic variants affect T cell composition during childhood in the general population.

Historically, multiple sclerosis (MS) has been viewed as being primarily driven by T cells. However, the effective use of anti-CD20 treatment now also reveals an important role for B cells in MS patients. The results from this treatment put forward T-cell activation rather than antibody production by B cells as a driving force behind MS. The main question of how their interaction provokes both B and T cells to infiltrate the CNS and cause local pathology remains to be answered. In this review, we highlight key pathogenic events involving B and T cells that most likely contribute to the pathogenesis of MS. These include (1) peripheral escape of B cells from T cell-mediated control, (2) interaction of pathogenic B and T cells in secondary lymph nodes, and (3) reactivation of B and T cells accumulating in the CNS. We will focus on the functional programs of CNS-infiltrating lymphocyte subsets in MS patients and discuss how these are defined by mechanisms such as antigen presentation, co-stimulation and cytokine production in the periphery. Furthermore, the potential impact of genetic variants and viral triggers on candidate subsets will be debated in the context of MS.

Historically, multiple sclerosis (MS) has been viewed as being primarily driven by T cells. However, the effective use of anti-CD20 treatment now also reveals an important role for B cells in MS patients. The results from this treatment put forward T-cell activation rather than antibody production by B cells as a driving force behind MS. The main question of how their interaction provokes both B and T cells to infiltrate the CNS and cause local pathology remains to be answered. In this review, we highlight key pathogenic events involving B and T cells that most likely contribute to the pathogenesis of MS. These include (1) peripheral escape of B cells from T cell-mediated control, (2) interaction of pathogenic B and T cells in secondary lymph nodes, and (3) reactivation of B and T cells accumulating in the CNS. We will focus on the functional programs of CNS-infiltrating lymphocyte subsets in MS patients and discuss how these are defined by mechanisms such as antigen presentation, co-stimulation and cytokine production in the periphery. Furthermore, the potential impact of genetic variants and viral triggers on candidate subsets will be debated in the context of MS.