MS Center ErasMS, Erasmus University Medical Center
Immunology

Author Of 1 Presentation

Neuromyelitis Optica and Anti-MOG Disease Oral Presentation

HT06.03 - Naive B cells in neuromyelitis optica spectrum disorders: impact of steroid use and relapse occurrence

Speakers
Presentation Number
HT06.03
Presentation Topic
Neuromyelitis Optica and Anti-MOG Disease
Lecture Time
10:39 - 10:51

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.

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Author Of 2 Presentations

Disease Modifying Therapies – Mechanism of Action Poster Presentation

P0403 - T-bet+ B-cell development in MS: Association with Bruton’s Tyrosine Kinase activity and targeting by evobrutinib (ID 1104)

Speakers
Presentation Number
P0403
Presentation Topic
Disease Modifying Therapies – Mechanism of Action

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.

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Pathogenesis – Immunology Poster Presentation

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.

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Presenter Of 1 Presentation

Disease Modifying Therapies – Mechanism of Action Poster Presentation

P0403 - T-bet+ B-cell development in MS: Association with Bruton’s Tyrosine Kinase activity and targeting by evobrutinib (ID 1104)

Speakers
Presentation Number
P0403
Presentation Topic
Disease Modifying Therapies – Mechanism of Action

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.

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