Author Of 3 Presentations
P0330 - Effects of cladribine on proliferation, survival and cytokine release of human astrocytes (ID 1581)
Abstract
Background
Cladribine is a synthetic purine nucleoside analogue with immunosuppressive functions that has demonstrated beneficial effects in patients with relapsing-remitting multiple sclerosis (MS) and that may also regulate the immune function as an analogue of adenosine receptors. Although the effect of cladribine is well studied on immune cells, it remains unveiled how it affects the immune function of glial populations of the central nervous system.
Objectives
In the context of MS, we aimed to test the effect of cladribine on proliferation, survival and cytokine release of human astrocytes.
Methods
To assess the effect of cladribine on cell survival and proliferation, primary human astrocytes were cultured with cladribine at high concentrations (2µM, 0.2µM), at the mean estimated brain exposure of the drug (0.02µM) and at a low concentration (0.002µM) for 72h. The percentages of dead and proliferating cells were determined by flow cytometry. To assess the effect of cladribine on cytokine release, human astrocytes were stimulated for 6h with 20ng/ml IL1-β and TNF-α. The stimulus was withdrawn and cells were cultured for additional 18h. Cladribine was added for the whole 24h of culture. Supernatants were harvested to quantify IL1-β, IL6, TNF-α and GM-CSF release by Luminex. To assess the effect of cladribine independently of deoxycytidine kinase (DCK), deoxycytidine was also added to human astrocytes.
Results
Only high concentrations of cladribine induced death on human astrocytes (2µM: 35.89%±7.62 or 0.2µM: 7.27%±3.12 vs control: 3.17%1.84±; p<0.0001 and p=0.156, respectively) and inhibited their proliferative capacity (2µM: 0.96%±1.14 or 0.2µM: 14.06%±5.44 vs control: 33.06%±1.42; both p-values<0.0001). Additionally, the percentage of proliferating cells in the 2µM and 0.2µM conditions presented a limited capacity of proliferation (measured as the Relative Intensity of Proliferation Staining respect to basal; 2µM: 0.24±0.04 and 0.2µM: 0.55±0.22, both p-values<0.0001). When DCK activity was blocked by deoxycytidine, cell death and proliferation were reversed to control condition values. We are currently determining the effect of cladribine on pro-inflammatory cytokine release by human astrocytes.
Conclusions
The mean estimated brain exposure to cladribine does not influence cell survival or proliferation of human astrocytes neither in a DCK-dependent nor in a DCK-independent manner, suggesting that cladribine does not affect the normal astrocyte function in MS patients.
P0334 - Evobrutinib, a highly selective BTK inhibitor, prevents antigen-activation of B cells and ameliorates experimental autoimmune encephalomyelitis (ID 1125)
Abstract
Background
Background: B cells are key mediators of inflammatory processes in multiple sclerosis, a notion substantiated by the success of B-cell depletion therapies; however, overall depletion does not only target pathogenic B cells but can also affect regulatory B-cell properties. An alternative strategy may be the specific inhibition of Bruton’s tyrosine kinase (BTK), which is centrally involved in B-cell receptor (BCR) signaling and subsequently mediates B-cell activation and differentiation. BTK inhibitors therefore hold the promise to control pathogenic functions such as antigen presentation and cytokine release.
Objectives
Objectives: To evaluate the BTK inhibitor evobrutinib in a mouse model of experimental autoimmune encephalomyelitis (EAE).
Methods
Methods: C57Bl/6 mice received oral evobrutinib or vehicle starting 7 days before immunization with conformational MOG1-117 protein (a B cell–mediated model of EAE). EAE severity was assessed for 60 days using a standard scale. B-cell maturation and activation markers on B and T cells were analyzed by flow cytometry on day 12 post immunization. T cell proliferation and differentiation were assessed after a 3-day co-culture with BTKi-treated B cells. Intracellular calcium flux was analyzed using calcium-sensitive dyes and BCR or T cell receptor (TCR) stimulation. BTK expression and phosphorylation as well as cytokine production were assessed on healthy human B cells via PhosFlow protocols or ELISA, respectively.
Results
Results: Evobrutinib showed a dose-dependent amelioration of EAE severity throughout the 60-day observation period. Evobrutinib led to an accumulation of follicular type (FO) II B cells and a corresponding reduction in FO I B cells, a BTK-dependent transition. Expression of CD86, CD69, and major histocompatibility complex class II on B cells, and CD25 and CD69 on T cells, was reduced. Evobrutinib inhibited the B cell-mediated proliferation and proinflammatory differentiation of T cells. BCR-mediated mobilization of excitatory calcium was reduced by evobrutinib, while TCR signaling remained unaffected. In human B cells, BTK expression and phosphorylation were depending on the maturation of B cells, while the overall cytokine release was inhibited by evobrutinib.
Conclusions
Conclusion: Evobrutinib efficiently reduces BTK-dependent signaling after BCR stimulation, preventing B-cell activation, proinflammatory differentiation, and function. This translates into reduced CNS inflammation and clinical amelioration in a B cell–mediated EAE model.
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.