Author Of 1 Presentation
HT06.03 - Naive B cells in neuromyelitis optica spectrum disorders: impact of steroid use and relapse occurrence
Abstract
Background
Neuromyelitis optica spectrum disorders (NMOSD) are a group of rare, but severe autoimmune diseases characterized by antibody-driven inflammation of mainly the ocular nerves and spinal cord. Although naive B cells are considered key players, it remains unknown whether their composition and outgrowth differ between serological NMOSD subgroups.
Objectives
We examined how ex vivo proportions and germinal center-like development of naive B cells are related to AQP4- and MOG-IgG serostatus, steroid treatment and relapse occurrence in NMOSD.
Methods
The presence of blood transitional, naive mature and both switched and unswitched memory B cells was determined in 10 AQP4- and 8 MOG-IgG-positive NMOSD patients without any form of previous immune suppressive treatment. Results were compared to 9 steroid-treated AQP4- or MOG-IgG-positive patients, 20 age- and gender-matched healthy controls (HCs) and 10 treatment-naive RRMS patients. Furthermore, naive B cells were cultured under T-bet-inducing, germinal center-like conditions for 11 days to address plasmablast formation in vitro.
Results
Under complete treatment-naive circumstances, naive mature/transitional B-cell ratios were significantly reduced in AQP4-IgG-positive NMOSD vs MOG-IgG-positive NMOSD, MS and HC groups. This was caused by increased proportions of transitional B cells, which were the highest in 2 AQP4-IgG-positive cases with relapsing disease (35% and 44% of the total B-cell pool). In steroid-treated patients, transitional B-cell proportions were strongly diminished and correlated negatively with time since start of treatment. For naive B cells from 7 relapsing NMOSD patients, TLR9 ligand CpG synergized with IFN-γ to enhance plasmablast formation in IL-21/CD40L-containing cultures. This was not seen for 11 non-relapsing patients or 9 HCs. IFN-γ- and CpG-induced naive B cells showed increased secretion of total IgG in the AQP4-IgG-positive group, and especially for patients with relapsing disease. In vitro-induced AQP4-IgG secretion was found for 3 relapsing but not for 6 non-relapsing patients, which was enhanced by IFN-γ and CpG (2 out of 3 relapsing patients). MOG-IgG was not detected in any of the naive B-cell culture supernatants of 4 relapsing and 4 non-relapsing MOG-IgG-positive patients.
Conclusions
These findings reveal that naive B-cell homeostasis is different and affected by steroid treatment amongst NMOSD subgroups, and offers a first rationale for exploring TLR9-dependent in vitro platforms to predict NMOSD activity.
Presenter Of 1 Presentation
HT06.03 - Naive B cells in neuromyelitis optica spectrum disorders: impact of steroid use and relapse occurrence
Abstract
Background
Neuromyelitis optica spectrum disorders (NMOSD) are a group of rare, but severe autoimmune diseases characterized by antibody-driven inflammation of mainly the ocular nerves and spinal cord. Although naive B cells are considered key players, it remains unknown whether their composition and outgrowth differ between serological NMOSD subgroups.
Objectives
We examined how ex vivo proportions and germinal center-like development of naive B cells are related to AQP4- and MOG-IgG serostatus, steroid treatment and relapse occurrence in NMOSD.
Methods
The presence of blood transitional, naive mature and both switched and unswitched memory B cells was determined in 10 AQP4- and 8 MOG-IgG-positive NMOSD patients without any form of previous immune suppressive treatment. Results were compared to 9 steroid-treated AQP4- or MOG-IgG-positive patients, 20 age- and gender-matched healthy controls (HCs) and 10 treatment-naive RRMS patients. Furthermore, naive B cells were cultured under T-bet-inducing, germinal center-like conditions for 11 days to address plasmablast formation in vitro.
Results
Under complete treatment-naive circumstances, naive mature/transitional B-cell ratios were significantly reduced in AQP4-IgG-positive NMOSD vs MOG-IgG-positive NMOSD, MS and HC groups. This was caused by increased proportions of transitional B cells, which were the highest in 2 AQP4-IgG-positive cases with relapsing disease (35% and 44% of the total B-cell pool). In steroid-treated patients, transitional B-cell proportions were strongly diminished and correlated negatively with time since start of treatment. For naive B cells from 7 relapsing NMOSD patients, TLR9 ligand CpG synergized with IFN-γ to enhance plasmablast formation in IL-21/CD40L-containing cultures. This was not seen for 11 non-relapsing patients or 9 HCs. IFN-γ- and CpG-induced naive B cells showed increased secretion of total IgG in the AQP4-IgG-positive group, and especially for patients with relapsing disease. In vitro-induced AQP4-IgG secretion was found for 3 relapsing but not for 6 non-relapsing patients, which was enhanced by IFN-γ and CpG (2 out of 3 relapsing patients). MOG-IgG was not detected in any of the naive B-cell culture supernatants of 4 relapsing and 4 non-relapsing MOG-IgG-positive patients.
Conclusions
These findings reveal that naive B-cell homeostasis is different and affected by steroid treatment amongst NMOSD subgroups, and offers a first rationale for exploring TLR9-dependent in vitro platforms to predict NMOSD activity.
Author Of 4 Presentations
P0403 - T-bet+ B-cell development in MS: Association with Bruton’s Tyrosine Kinase activity and targeting by evobrutinib (ID 1104)
Abstract
Background
B-cell depletion is an efficacious treatment in relapsing and progressive multiple sclerosis (MS). A phase II trial (NCT02975349) showed promising results for Bruton’s tyrosine kinase (BTK) inhibitor evobrutinib in the treatment of MS. Previously, we found that T-bet+ B cells preferentially infiltrate the central nervous system and are induced under IFN-γ- and TLR9-stimulating, germinal center-like conditions in MS.
Objectives
We aimed to elucidate how BTK is expressed and activated in distinct ex vivo B-cell subsets during the course of MS. Moreover, the relation between BTK activity and T-bet+ B-cell differentiation was assessed both ex vivo and in vitro.
Methods
We determined BTK and phosphorylated BTK (pBTK) levels in transitional, naive mature, class-switched and non class-switched B cells in blood from both treatment-naive patients with CIS, RRMS, SPMS and PPMS and healthy controls (HC; n=30 per group), as well as clinical MS responders and non-responders to natalizumab (pre- vs 1y post-treatment) using flow cytometry. Purified naive mature and memory B cells were cultured under several IL-21/CD40L-inducing conditions with and without evobrutinib.
Results
BTK was mainly expressed in non-class-switched memory B cells, while pBTK levels were high in both class-switched and unswitched memory B cells. In contrast to BTK, pBTK was significantly higher in ex vivo memory B cells of RRMS and SPMS compared to CIS, PPMS and HC groups. In both RRMS and SPMS, pBTK was also less induced after a-IgM stimulation. BTK and pBTK levels were elevated in blood B cells from clinical responders, but not in non-responders to natalizumab. These levels correlated positively with CXCR3 and VLA-4 expression. No correlation was seen for CXCR4, CXCR5, CD40 and HLA-DR. In vitro experiments revealed that pBTK in B cells was particularly triggered by IFN-γ and TLR9 induction. Evobrutinib attenuated class-switching during in vitro cultures of naive B cells, while it interfered with plasmablast formation in memory B-cell cultures. T-bet and T-bet-related markers (CD21, CD11c) were only affected by evobrutinib in IFN-γ- and TLR9-stimulating naive B-cell cultures.
Conclusions
These data demonstrate that BTK is more activated in memory B cells from RRMS and SPMS patients and functionally related to pathogenic T-bet+ B-cell development. This study provides new mechanistic insights into how evobrutinib intervenes in human B-cell differentiation and can modulate the clinical course of MS.
P0528 - T cell composition and polygenic multiple sclerosis risk: a population-based study in children (ID 943)
Abstract
Background
Multiple sclerosis (MS) is associated with extensive immunological alterations in adult patients. MS patients show changes in T cell composition, including increased CD4+/CD8+ ratios. However, it is unclear to which extent these changes in T cell composition are influenced by genetic risk for MS, and how this may precede a possible disease onset.
Objectives
In the current study we investigate the association between polygenic risk scores (PRSs) for MS and T cell subsets in a large population-based pediatric sample, to provide new understanding about the link between genetic risk for MS and disease pathophysiology.
Methods
We included participants from the population-based Generation R study who had genetic- and immunological data available. Children were sampled for immunological data around the age of 6 years (IQR: 5.9-6.2). Linear regression analyses were used to analyze the impact of MS-PRSs on absolute T cell numbers (n=1,261) and CD4+ and CD8+ T cell fractions (n=675) adjusted for important child- (age and sex) and environmental confounding factors (serum vitamin D levels and cytomegalovirus positivity).
Results
The MS-PRS showed a negative correlation with CD8+ T cell frequencies (β=-0.05, SE=0.015, ΔR2=0.020, p=2.88 × 10-4), which resulted in a positive association with CD4+/CD8+ ratios (β=0.07, SE=0.011, ΔR2=0.054, p=9.20 × 10-10). Interestingly, the latter was driven by 2 out of 196 genome-wide significant MS risk variants. Both from within the HLA class II region, risk variants rs3135388 and rs9271366 were positively associated with the CD4+/CD8+ ratio. No association was found with absolute total T cell numbers.
Conclusions
This study shows that higher genetic risk for MS is associated with T cell alterations at an early age. Our results show a possibility that MS genetics affect the T cell composition during childhood, which may contribute to increased risk of MS disease later in life.
P0973 - Impact of Epstein-Barr virus infection on CXCR3+ B-cell development: lessons learned from immunotherapies in MS (ID 751)
Abstract
Background
Epstein-Barr virus (EBV) infection of B cells is strongly associated with the onset of several chronic inflammatory and autoimmune diseases such as multiple sclerosis (MS). In MS, a subset of memory B cells infiltrates the central nervous system (CNS) and further differentiates into antibody-secreting cells to mediate local pathology.
Objectives
Here, we aimed to decipher whether and how EBV impacts the development of such CNS-homing memory B cells in MS patients.
Methods
Chemokine receptor profiles were analyzed for ex vivo B cells in single-cell suspensions obtained from paired CNS compartments of 10 MS patients (NBB) using multicolor flow cytometry. The CNS infiltration capacity of memory B-cell subsets was confirmed using confluent brain endothelial monolayers. Similar analyses were performed for distinct memory subsets in the blood from 16 untreated, 32 natalizumab-treated and 9 bone-marrow transplant (BMT)-treated MS patients as well as matched healthy controls. An IL-21-/CD40L-based germinal center-like culture system was used to compare naive and memory B-cell differentiation. EBV copy numbers were determined in total and memory B cells using a multiplex BALF5-related qPCR assay.
Results
CXCR3-expressing B cells were selectively enriched in paired CSF, meningeal and brain tissues versus blood of MS patients. Treatment of patients with natalizumab resulted in an accumulation of CXCR3high IgG+ B cells in the blood, corresponding to their increased ability to cross CNS barriers in vitro. Naive B cells developed into plasmablasts under IFN-γ-mediated germinal center-like conditions and required additional TLR9 signaling for differentiation into CXCR3+ switched memory cells. In 3-7 months post- vs pre-BMT blood samples, EBV DNA load was elevated and positively correlated to the frequency of CXCR3+, and not CXCR4+ or CXCR5+, switched memory B cells. High EBV load in memory B cells from natalizumab-treated MS patients corresponded to an increased potential to develop into anti-EBNA1 IgG-producing CXCR3+ plasma cells (CD38++CD27++CD138+) during IFN-γ-mediated germinal center-like cultures.
Conclusions
This study implicates that persistent viral infections such as EBV potentiate brain-homing and antibody-producing CXCR3(T-bet)+ B cells in MS patients. These findings may mechanistically link EBV infection to anti-EBNA1 IgG production as being a predictor of disease onset and to the massive B-cell influx into the CNS after natalizumab discontinuation in MS.
P1121 - Effector T cells are selectively controlled and related to postpartum relapses during pregnancy in MS (ID 1610)
Abstract
Background
Women with multiple sclerosis (MS) experience limited relapses during pregnancy, while an increased relapse risk is observed within the first period after delivery. How extensive pregnancy-associated fluctuations of serum proteins correspond to brain-homing effector T cells and whether this differs between pregnant MS patients and healthy controls is currently unknown.
Objectives
Here, we aimed to assess the effector phenotype and outgrowth of T-cell subsets in relation to pregnancy, pregnancy-related serum factors and postpartum relapses in MS.
Methods
Paired third trimester and 4-8 weeks postpartum blood samples from pregnant MS patients with and without a postpartum relapse (n=21) were compared to age-matched pregnant healthy controls (n=12) for memory phenotype, skewing and activation of T cells. Memory T cells were sorted, activated and analyzed for production of inflammatory cytokines (multiplex assay). Third trimester and postpartum sera were added to CFSE-labeled, proliferating T cells for 72 hours. Serum cortisol, estradiol and progesterone levels were determined using liquid chromatography-mass spectrometry.
Results
Frequencies of effector memory (CCR7-CD45RA-) CD4+ and not CD8+ T cells were selectively increased in postpartum versus third trimester blood of 15 MS patients without a postpartum relapse. This was not seen for 6 patients experiencing a postpartum relapse or in pregnant healthy controls. Brain-homing markers CCR6 and CXCR3 were enriched on memory CD4+ T cells derived from postpartum samples, except for patients with a postpartum relapse. These differences in effector T-cell phenotypes were also found in vitro using paired third trimester and postpartum sera from pregnant MS patients and controls, suggesting that this is at least partly regulated by soluble factors. Serum cortisol, estradiol and progesterone levels did not differ or correlate to outcome measures in third trimester samples. In sharp contrast to non-relapsing patients, memory CD4+ T cells were activated (HLA-DR+) and showed an overall increase in inflammatory cytokine production in third trimester samples from MS patients who experienced a postpartum relapse.
Conclusions
These data demonstrate that effector CD4+ T cells are alternatively controlled in pregnant MS patients, which is influenced by serum-derived factors. Moreover, we found first indications that the activation state of these cells during pregnancy may be used to predict a postpartum relapse in MS.