Differential Runx3, Eomes, and T-bet expression subdivides MS-associated CD4+ T cells with brain-homing capacity.

Multiple sclerosis (MS) is a common and devastating chronic inflammatory disease of the CNS. CD4+ T cells are assumed to be the first to cross the blood-central nervous system (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 a 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.

Impact of coding risk variant IFNGR2 on the B cell-intrinsic IFN-γ signaling pathway in multiple sclerosis.

B cells of people with multiple sclerosis (MS) are more responsive to IFN-γ, corresponding to their brain-homing potential. We studied how a coding single nucleotide polymorphism (SNP) in IFNGR2 (rs9808753) co-operates with Epstein-Barr virus (EBV) infection as MS risk factors to affect the IFN-γ signaling pathway in human B cells. In both cell lines and primary cells, EBV infection positively associated with IFN-γ receptor expression and STAT1 phosphorylation. The IFNGR2 risk SNP selectively promoted downstream signaling via STAT1, particularly in transitional B cells. Altogether, EBV and the IFNGR2 risk SNP independently amplify IFN-γ signaling, potentially driving B cells to enter the MS brain.

The association of Epstein-Barr virus infection with CXCR3+ B-cell development in multiple sclerosis: impact of immunotherapies.

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.

The Role of Autoimmunity-Related Gene CLEC16A in the B Cell Receptor-Mediated HLA Class II Pathway.

C-type lectin CLEC16A is located next to CIITA, the master transcription factor of HLA class II (HLA-II), at a susceptibility locus for several autoimmune diseases, including multiple sclerosis (MS). We previously found that CLEC16A promotes the biogenesis of HLA-II peptide-loading compartments (MIICs) in myeloid cells. Given the emerging role of B cells as APCs in these diseases, in this study, we addressed whether and how CLEC16A is involved in the BCR-dependent HLA-II pathway. CLEC16A was coexpressed with surface class II-associated invariant chain peptides (CLIP) in human EBV-positive and not EBV-negative B cell lines. Stable knockdown of CLEC16A in EBV-positive Raji B cells resulted in an upregulation of surface HLA-DR and CD74 (invariant chain), whereas CLIP was slightly but significantly reduced. In addition, IgM-mediated Salmonella uptake was decreased, and MIICs were less clustered in CLEC16A-silenced Raji cells, implying that CLEC16A controls both HLA-DR/CD74 and BCR/Ag processing in MIICs. In primary B cells, CLEC16A was only induced under CLIP-stimulating conditions in vitro and was predominantly expressed in CLIPhigh naive populations. Finally, CLIP-loaded HLA-DR molecules were abnormally enriched, and coregulation with CLEC16A was abolished in blood B cells of patients who rapidly develop MS. These findings demonstrate that CLEC16A participates in the BCR-dependent HLA-II pathway in human B cells and that this regulation is impaired during MS disease onset. The abundance of CLIP already on naive B cells of MS patients may point to a chronically induced stage and a new mechanism underlying B cell-mediated autoimmune diseases such as MS.

Induction of brain-infiltrating T-bet-expressing B cells in multiple sclerosis.

OBJECTIVE: Results from anti-CD20 therapies demonstrate that B- and T-cell interaction is a major driver of multiple sclerosis (MS). The local presence of B-cell follicle-like structures and oligoclonal bands in MS patients indicates that certain B cells infiltrate the central nervous system (CNS) to mediate pathology. Which peripheral triggers underlie the development of CNS-infiltrating B cells is not fully understood. METHODS: Ex vivo flow cytometry was used to assess chemokine receptor profiles of B cells in blood, cerebrospinal fluid, meningeal, and brain tissues of MS patients (n = 10). Similar analyses were performed for distinct memory subsets in the blood of untreated and natalizumab-treated MS patients (n = 38). To assess T-bet(CXCR3)+ B-cell differentiation, we cultured B cells from MS patients (n = 21) and healthy individuals (n = 34) under T helper 1- and TLR9-inducing conditions. Their CNS transmigration capacity was confirmed using brain endothelial monolayers. RESULTS: CXC chemokine receptor 3 (CXCR3)-expressing B cells were enriched in different CNS compartments of MS patients. Treatment with the clinically effective drug natalizumab prevented the recruitment of CXCR3high IgG1+ subsets, corresponding to their increased ability to cross CNS barriers in vitro. Blocking of interferon-γ (IFNγ) reduced the transmigration potential and antigen-presenting function of these cells. IFNγ-induced B cells from MS patients showed increased T-bet expression and plasmablast development. Additional TLR9 triggering further upregulated T-bet and CXCR3, and was essential for IgG1 switching. INTERPRETATION: This study demonstrates that T-bethigh IgG1+ B cells are triggered by IFNγ and TLR9 signals, likely contributing to enhanced CXCR3-mediated recruitment and local reactivity in the CNS of MS patients. ANN NEUROL 2019;86:264-278.

Chemical and genetic control of IFNγ-induced MHCII expression.

The cytokine interferon-γ (IFNγ) can induce expression of MHC class II (MHCII) on many different cell types, leading to antigen presentation to CD4+ T cells and immune activation. This has also been linked to anti-tumour immunity and graft-versus-host disease. The extent of MHCII upregulation by IFNγ is cell type-dependent and under extensive control of epigenetic regulators and signalling pathways. Here, we identify novel genetic and chemical factors that control this form of MHCII expression. Loss of the oxidative stress sensor Keap1, autophagy adaptor p62/SQSTM1, ubiquitin E3-ligase Cullin-3 and chromatin remodeller BPTF impair IFNγ-mediated MHCII expression. A similar phenotype is observed for arsenite, an oxidative stressor. Effects of the latter can be reversed by the inhibition of HDAC1/2, linking oxidative stress conditions to epigenetic control of MHCII expression. Furthermore, dimethyl fumarate, an antioxidant used for the treatment of several autoimmune diseases, impairs the IFNγ response by manipulating transcriptional control of MHCII We describe novel pathways and drugs related to oxidative conditions in cells impacting on IFNγ-mediated MHCII expression, which provide a molecular basis for the understanding of MHCII-associated diseases.

The macrophage migration inhibitory factor pathway in human B cells is tightly controlled and dysregulated in multiple sclerosis.

In MS, B cells survive peripheral tolerance checkpoints to mediate local inflammation, but the underlying molecular mechanisms are relatively underexplored. In mice, the MIF pathway controls B-cell development and the induction of EAE. Here, we found that MIF and MIF receptor CD74 are downregulated, while MIF receptor CXCR4 is upregulated in B cells from early onset MS patients. B cells were identified as the main immune subset in blood expressing MIF. Blocking of MIF and CD74 signaling in B cells triggered CXCR4 expression, and vice versa, with separate effects on their proinflammatory activity, proliferation, and sensitivity to Fas-mediated apoptosis. This study reveals a new reciprocal negative regulation loop between CD74 and CXCR4 in human B cells. The disturbance of this loop during MS onset provides further insights into how pathogenic B cells survive peripheral tolerance checkpoints to mediate disease activity in MS.

High neurofilament levels are associated with clinically definite multiple sclerosis in children and adults with clinically isolated syndrome.

BACKGROUND: A promising biomarker for axonal damage early in the disease course of multiple sclerosis (MS) is neurofilament light chain (NfL). It is unknown whether NfL has the same predictive value for MS diagnosis in children as in adults. OBJECTIVE: To explore the predictive value of NfL levels in cerebrospinal fluid (CSF) for MS diagnosis in paediatric and adult clinically isolated syndrome (CIS) patients. METHODS: A total of 88 adult and 65 paediatric patients with a first attack of demyelination were included and followed (mean follow up-time in adults: 62.8 months (standard deviation (SD) ±38.7 months) and 43.8 months (SD ±27.1 months) in children). Thirty control patients were also included. Lumbar puncture was done within 6 months after onset of symptoms. NfL was determined in CSF using enzyme-linked immunosorbent assay (ELISA). COX regression analyses were used to calculate hazard ratios (HR) for clinically definite multiple sclerosis (CDMS) diagnosis. RESULTS: After adjustments for age, oligoclonal bands (OCB), and asymptomatic T2 lesions on baseline magnetic resonance imaging (MRI), increased NfL levels in both paediatric and adult CIS patients were associated with a shorter time to CDMS diagnosis (children HR = 3.7; p = 0.007, adults HR = 2.1; p = 0.032). For CIS patients with a future CDMS diagnosis, children showed higher NfL levels than adults (geometric mean 4888 vs 2156 pg/mL; p = 0.007). CONCLUSION: CSF NfL levels are associated with CDMS diagnosis in children and adults with CIS. This makes NfL a promising predictive marker for disease course with potential value in clinical practice.

T helper 17.1 cells associate with multiple sclerosis disease activity: perspectives for early intervention.

Interleukin-17-expressing CD4+ T helper 17 (Th17) cells are considered as critical regulators of multiple sclerosis disease activity. However, depending on the species and pro-inflammatory milieu, Th17 cells are functionally heterogeneous, consisting of subpopulations that differentially produce interleukin-17, interferon-gamma and granulocyte macrophage colony-stimulating factor. In the current study, we studied distinct effector phenotypes of human Th17 cells and their correlation with disease activity in multiple sclerosis patients. T helper memory populations single- and double-positive for C-C chemokine receptor 6 (CCR6) and CXC chemokine receptor 3 (CXCR3) were functionally assessed in blood and/or cerebrospinal fluid from a total of 59 patients with clinically isolated syndrome, 35 untreated patients and 24 natalizumab-treated patients with relapsing-remitting multiple sclerosis, and nine patients with end-stage multiple sclerosis. Within the clinically isolated syndrome group, 23 patients had a second attack within 1 year and 26 patients did not experience subsequent attacks during a follow-up of >5 years. Low frequencies of T helper 1 (Th1)-like Th17 (CCR6+CXCR3+), and not Th17 (CCR6+CXCR3-) effector memory populations in blood strongly associated with a rapid diagnosis of clinically definite multiple sclerosis. In cerebrospinal fluid of clinically isolated syndrome and relapsing-remitting multiple sclerosis patients, Th1-like Th17 effector memory cells were abundant and showed increased production of interferon-gamma and granulocyte macrophage colony-stimulating factor compared to paired CCR6+ and CCR6-CD8+ T cell populations and their blood equivalents after short-term culturing. Their local enrichment was confirmed ex vivo using cerebrospinal fluid and brain single-cell suspensions. Across all pro-inflammatory T helper cells analysed in relapsing-remitting multiple sclerosis blood, Th1-like Th17 subpopulation T helper 17.1 (Th17.1; CCR6+CXCR3+CCR4-) expressed the highest very late antigen-4 levels and selectively accumulated in natalizumab-treated patients who remained free of clinical relapses. This was not found in patients who experienced relapses during natalizumab treatment. The enhanced potential of Th17.1 cells to infiltrate the central nervous system was supported by their predominance in cerebrospinal fluid of early multiple sclerosis patients and their preferential transmigration across human brain endothelial layers. These findings reveal a dominant contribution of Th1-like Th17 subpopulations, in particular Th17.1 cells, to clinical disease activity and provide a strong rationale for more specific and earlier use of T cell-targeted therapy in multiple sclerosis.

Multiple sclerosis-associated CLEC16A controls HLA class II expression via late endosome biogenesis.

C-type lectins are key players in immune regulation by driving distinct functions of antigen-presenting cells. The C-type lectin CLEC16A gene is located at 16p13, a susceptibility locus for several autoimmune diseases, including multiple sclerosis. However, the function of this gene and its potential contribution to these diseases in humans are poorly understood. In this study, we found a strong upregulation of CLEC16A expression in the white matter of multiple sclerosis patients (n = 14) compared to non-demented controls (n = 11), mainly in perivascular leukocyte infiltrates. Moreover, CLEC16A levels were significantly enhanced in peripheral blood mononuclear cells of multiple sclerosis patients (n = 69) versus healthy controls (n = 46). In peripheral blood mononuclear cells, CLEC16A was most abundant in monocyte-derived dendritic cells, in which it strongly co-localized with human leukocyte antigen class II. Treatment of these professional antigen-presenting cells with vitamin D, a key protective environmental factor in multiple sclerosis, downmodulated CLEC16A in parallel with human leukocyte antigen class II. Knockdown of CLEC16A in distinct types of model and primary antigen-presenting cells resulted in severely impaired cytoplasmic distribution and formation of human leucocyte antigen class II-positive late endosomes, as determined by immunofluorescence and electron microscopy. Mechanistically, CLEC16A participated in the molecular machinery of human leukocyte antigen class II-positive late endosome formation and trafficking to perinuclear regions, involving the dynein motor complex. By performing co-immunoprecipitations, we found that CLEC16A directly binds to two critical members of this complex, RILP and the HOPS complex. CLEC16A silencing in antigen-presenting cells disturbed RILP-mediated recruitment of human leukocyte antigen class II-positive late endosomes to perinuclear regions. Together, we identify CLEC16A as a pivotal gene in multiple sclerosis that serves as a direct regulator of the human leukocyte antigen class II pathway in antigen-presenting cells. These findings are a first step in coupling multiple sclerosis-associated genes to the regulation of the strongest genetic factor in multiple sclerosis, human leukocyte antigen class II.

The IL-7Rα pathway is quantitatively and functionally altered in CD8 T cells in multiple sclerosis.

The IL-7Rα single nucleotide polymorphism rs6897932 is associated with an increased risk for multiple sclerosis (MS). IL-7Rα is a promising candidate to be involved in autoimmunity, because it regulates T cell homeostasis, proliferation, and antiapoptotic signaling. However, the exact underlying mechanisms in the pathogenesis of MS are poorly understood. We investigated whether CD4 and CD8 lymphocyte subsets differed in IL-7Rα expression and functionality in 78 MS patients compared with 59 healthy controls (HC). A significantly higher frequency of IL-7Rα(+) CD8 effector memory (CD8EM) was found in MS. Moreover, IL-7Rα membrane expression was significantly increased in MS in naive and memory CD8 (all p < 0.05) with a similar trend in CD8EM (p = 0.055). No correlation was found between the expression level or frequency of IL-7Rα(+)CD8(+) and rs6897932 risk allele carriership. Upon IL-7 stimulation, MS patients had stronger STAT5 activation in CD8EM compared with HC. IL-7 stimulation had a differential effect on both mRNA and protein expression of granzyme A and granzyme B between MS and HC. Stainings of different lesions in postmortem MS brain material showed expression of IL-7 and CD8(+)IL-7Rα(+) in preactive, but not in active, demyelinating MS lesions, indicating involvement of IL-7Rα(+) lymphocytes in lesion development. The intralesional production of IL-7 in combination with the lower threshold for IL-7-induced cytotoxicity in MS may enhance the pathogenicity of these CD8 T cells. This is of special interest in light of the established demyelinating and cytotoxic actions of granzyme A.

Ocrelizumab associates with reduced cerebrospinal fluid B and CD20dim CD4+ T cells in primary progressive multiple sclerosis.

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.

Selective emergence of antibody-secreting cells in the multiple sclerosis brain.

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).

Distinct Effector Programs of Brain-Homing CD8+ T Cells in Multiple Sclerosis.

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.

Improving Glucocorticoid Sensitivity of Brain-Homing CD4+ T Helper Cells by Steroid Hormone Crosstalk.

In early multiple sclerosis (MS), an IFN-γhighGM-CSFhighIL-17low CD4+ T-cell subset termed T helper 17.1 (Th17.1) reveals enhanced capacity to infiltrate the central nervous system. Th17.1 cells express high levels of multidrug resistance protein 1 (MDR1), which contributes to their poor glucocorticoid responsiveness. In this study, we explored whether glucocorticoid sensitivity of Th17.1 cells can generically be improved through synergy between steroid hormones, including calcitriol (1,25(OH)2D3), estradiol (E2) and progesterone (P4). We showed that human blood Th17.1 cells were less sensitive to 1,25(OH)2D3 than Th17 cells, as reflected by lower vitamin D receptor (VDR) levels and reduced modulation of MDR1, IFN-γ and GM-CSF expression after 1,25(OH)2D3 exposure. Upon T-cell activation, VDR levels were increased, but still lower in Th17.1 versus Th17 cells, which was accompanied by a 1,25(OH)2D3-mediated decline in MDR1 surface expression as well as secretion of IFN-γ and GM-CSF. In activated Th17.1 cells, 1,25(OH)2D3 amplified the suppressive effects of methylprednisolone (MP) on proliferation, MDR1 surface levels, secretion of IFN-γ and granzyme B, as well as expression of brain-homing markers CCR6 and VLA-4. The addition of P4 to 1,25(OH)2D3 further enhanced MP-mediated reduction in proliferation, CD25, CCR6 and CXCR3. Overall, this study indicates that glucocorticoid sensitivity of Th17.1 cells can be enhanced by treatment with 1,25(OH)2D3 and further improved with P4. Our observations implicate steroid hormone crosstalk as a therapeutic avenue in Th17.1-associated inflammatory diseases including MS.

Human T-bet+ B cell development is associated with BTK activity and suppressed by evobrutinib.

Recent clinical trials have shown promising results for the next-generation Bruton's tyrosine kinase (BTK) inhibitor evobrutinib in the treatment of multiple sclerosis (MS). BTK has a central role in signaling pathways that govern the development of B cells. Whether and how BTK activity shapes B cells as key drivers of MS is currently unclear. Compared with levels of BTK protein, we found higher levels of phospho-BTK in ex vivo blood memory B cells from patients with relapsing-remitting MS and secondary progressive MS compared with controls. In these MS groups, BTK activity was induced to a lesser extent after anti-IgM stimulation. BTK positively correlated with CXCR3 expression, both of which were increased in blood B cells from clinical responders to natalizumab (anti-VLA-4 antibody) treatment. Under in vitro T follicular helper-like conditions, BTK phosphorylation was enhanced by T-bet-inducing stimuli, IFN-γ and CpG-ODN, while the expression of T-bet and T-bet-associated molecules CXCR3, CD21, and CD11c was affected by evobrutinib. Furthermore, evobrutinib interfered with in vitro class switching, as well as memory recall responses, and disturbed CXCL10-mediated migration of CXCR3+ switched B cells through human brain endothelial monolayers. These findings demonstrate a functional link between BTK activity and disease-relevant B cells and offer valuable insights into how next-generation BTK inhibitors could modulate the clinical course of patients with MS.

The monocyte transcriptome during pregnancy in multiple sclerosis: prominent expression of the Fc-receptor CD64.

BACKGROUND: During the third trimester of pregnancy multiple sclerosis (MS) disease activity is reduced. It is not fully understood which factors mediate this disease amelioration. OBJECTIVE: To study alterations of the monocyte transcriptome during pregnancy in MS patients, using a genomewide approach to identify differentially regulated genes. METHODS: Women with MS and healthy controls were longitudinally studied, including a visit before pregnancy. RESULTS: RNA-microarray analysis was performed in six patients. We found a significant increase of CD64 (Fc gamma receptor 1a, FcgR1a) during the third trimester compared with baseline, confirmed by RT-PCR in a group of ten patients. Analysis with Ingenuity software was performed using all genes expression of which was altered at least 1.5-fold in at least five out of six patients. Major networks that were altered during MS pregnancy were: cell-to-cell signalling and interaction, immune response, and cell signalling. From the genes selected for Ingenuity analysis, seven additional candidate genes, selected for their biological interest, were tested using RT-PCR in ten patients with MS and nine controls. We found an increased expression of JAK2 and STAT1 directly postpartum in patients with MS and in controls. CONCLUSION: The increased CD64 expression during pregnancy is indicative of enhanced innate immune functions.

Pregnancy-induced effects on memory B-cell development in multiple sclerosis.

In MS, pathogenic memory B cells infiltrate the brain and develop into antibody-secreting cells. Chemokine receptors not only define their brain-infiltrating capacity, but also assist in their maturation in germinal centers. How this corresponds to pregnancy, as a naturally occurring modifier of MS, is underexplored. Here, we aimed to study the impact of pregnancy on both ex vivo and in vitro B-cell differentiation in MS. The composition and outgrowth of peripheral B cells were compared between 19 MS pregnant patients and 12 healthy controls during the third trimester of pregnancy (low relapse risk) and postpartum (high relapse risk). Transitional, and not naive mature, B-cell frequencies were found to drop in the third trimester, which was most prominent in patients who experienced a pre-pregnancy relapse. Early after delivery, these frequencies raised again, while memory B -cell frequencies modestly declined. CXCR4 was downregulated and CXCR5, CXCR3 and CCR6 were upregulated on postpartum memory B cells, implying enhanced recruitment into germinal center light zones for interaction with T follicular helper (TFH) cells. Postpartum memory B cells of MS patients expressed higher levels of CCR6 and preferentially developed into plasma cells under TFH-like in vitro conditions. These findings imply that memory B- cell differentiation contributes to postpartum relapse risk in MS.

Effector T Helper Cells Are Selectively Controlled During Pregnancy and Related to a Postpartum Relapse in Multiple Sclerosis.

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.

Pregnancy-induced fluctuations in functional T-cell subsets in multiple sclerosis patients.

BACKGROUND: During pregnancy, especially during the third trimester, multiple sclerosis (MS) disease activity is reduced. It is not known which factors mediate this disease amelioration. OBJECTIVE: To study whether the frequency of two important T-cell subsets, T-helper 17 (Th17) and regulatory T-cells (Treg), is altered in relation to pregnancy-induced MS disease amelioration. METHODS: Each individual was tested longitudinally, after sampling of blood at timepoints before pregnancy, during the first and third trimester, and in the early post-partum period. Frequencies of Th17 cells were assessed after short (4 hours) re-stimulation of peripheral blood lymphocytes with PMA and ionomycin, followed by flow cytometry using CD4, CD45RO and IL-17A antibodies. To assess peripheral blood Treg frequencies, we used six-colour flow cytometry with antibodies against CD3, CD4, CD25, CD127, FoxP3 and HLA-DR, to specifically identify Treg. RESULTS: Both MS patients (n = 9) and controls (n = 8) displayed unaltered Th17 frequencies during pregnancy. In contrast, circulating Treg frequency significantly decreased in MS patients (n = 15) during the first and third (p < 0.001) trimesters compared with the period before pregnancy. In the post-partum period, the frequency of circulating Treg again resurged back to near pre-pregnancy levels. In controls (n = 15) comparable frequency kinetics were observed in that post-partum a significant increase in circulating Treg frequency was detected compared with the first (p < 0.001) and third (p = 0.012) trimester. CONCLUSIONS: Third trimester amelioration is not related to the fluctuation of circulating Th17 cells. Furthermore, a paradoxical decrease of immunosuppressive circulating Tregs can be observed during this phase, both in MS patients and controls.