Contributions
Abstract: EP1633
Type: ePoster
Abstract Category: Therapy - disease modifying - 26 Immunomodulation/Immunosuppression
Objective: To investigate the effect of teriflunomide, an approved therapy for RRMS, on the activation and proliferation of B-cells.
Background: Teriflunomide is a high affinity inhibitor of dihydroorotate dehydrogenase (DHODH), a key enzyme in pyrimidine synthesis. De-novo pyrimidine synthesis is required by rapidly proliferating cells, such as activated lymphocytes to progress through the cell cycle.
Methods: Peripheral blood mononuclear cells (PBMC) were isolated from 20 untreated RRMS patients. Teriflunomide effects on B-cells were studied using in-vitro assays. PBMC were stimulated with anti-IgG/IgM and rCD40L in the absence or presence of 100 µM teriflunomide and exogenous uridine. Proliferation and surface markers expressions of CD19+ B-cells were measured using flow cytometry and teriflunomide effect on the signalling pathways in B-cells were analysed using western blotting.
Results: In proliferation assay, anti-IgG/IgM and rCD40L treatment for 4 days induced a significant proliferation of CD19+ B-cells (p< 0.001, fold change: 1.83), which was inhibited by teriflunomide treatment (p< 0.001, fold change: 0.87). The addition of exogenous uridine reversed inhibition of CD19+ B-cell proliferation. Stimulation of CD19+ B-cells increased the expression of CD25, OX40, CD80, CD5, CD24, and CD20. Teriflunomide treatment down-regulated the expressions of activation markers CD25
(p< 0.05) and IgD (p< 0.05), B-cell chemokine receptor CCR2 (p< 0.01), costimulatory molecule CD80 (p< 0.001), and CD5 (p< 0.05), which are all reversed in the presence of exogenous uridine. Teriflunomide treatment of activated B-cells inhibited the phosphorylation of p65 (p< 0.001), IKKα (p< 0.001), pERK 1/2 (p< 0.001), pSTAT1 (p< 0.001) and pSTAT3 (p< 0.001) .
Conclusion: Teriflunomide inhibits activation and proliferation of B-cells derived from RRMS patients. Inhibition of pSTAT1, pSTAT3, and NF-kappa B signalling, and CD5 and CD80 costimulatory molecule expression may inhibit B-cell cytokine secretion and antigen presenting capacity, while inhibition of CCR2 may decrease B-cell migration to the CNS.
Disclosure:
Madhan Thamilarasan: nothing to disclose
Xin Zhang: nothing to disclose
Silva Markovic-Plese: research support from Chugai Inc., Genzyme Inc., Novartis, Biogen Idec, consultant compensation from Genzyme Inc. and Serono
Abstract: EP1633
Type: ePoster
Abstract Category: Therapy - disease modifying - 26 Immunomodulation/Immunosuppression
Objective: To investigate the effect of teriflunomide, an approved therapy for RRMS, on the activation and proliferation of B-cells.
Background: Teriflunomide is a high affinity inhibitor of dihydroorotate dehydrogenase (DHODH), a key enzyme in pyrimidine synthesis. De-novo pyrimidine synthesis is required by rapidly proliferating cells, such as activated lymphocytes to progress through the cell cycle.
Methods: Peripheral blood mononuclear cells (PBMC) were isolated from 20 untreated RRMS patients. Teriflunomide effects on B-cells were studied using in-vitro assays. PBMC were stimulated with anti-IgG/IgM and rCD40L in the absence or presence of 100 µM teriflunomide and exogenous uridine. Proliferation and surface markers expressions of CD19+ B-cells were measured using flow cytometry and teriflunomide effect on the signalling pathways in B-cells were analysed using western blotting.
Results: In proliferation assay, anti-IgG/IgM and rCD40L treatment for 4 days induced a significant proliferation of CD19+ B-cells (p< 0.001, fold change: 1.83), which was inhibited by teriflunomide treatment (p< 0.001, fold change: 0.87). The addition of exogenous uridine reversed inhibition of CD19+ B-cell proliferation. Stimulation of CD19+ B-cells increased the expression of CD25, OX40, CD80, CD5, CD24, and CD20. Teriflunomide treatment down-regulated the expressions of activation markers CD25
(p< 0.05) and IgD (p< 0.05), B-cell chemokine receptor CCR2 (p< 0.01), costimulatory molecule CD80 (p< 0.001), and CD5 (p< 0.05), which are all reversed in the presence of exogenous uridine. Teriflunomide treatment of activated B-cells inhibited the phosphorylation of p65 (p< 0.001), IKKα (p< 0.001), pERK 1/2 (p< 0.001), pSTAT1 (p< 0.001) and pSTAT3 (p< 0.001) .
Conclusion: Teriflunomide inhibits activation and proliferation of B-cells derived from RRMS patients. Inhibition of pSTAT1, pSTAT3, and NF-kappa B signalling, and CD5 and CD80 costimulatory molecule expression may inhibit B-cell cytokine secretion and antigen presenting capacity, while inhibition of CCR2 may decrease B-cell migration to the CNS.
Disclosure:
Madhan Thamilarasan: nothing to disclose
Xin Zhang: nothing to disclose
Silva Markovic-Plese: research support from Chugai Inc., Genzyme Inc., Novartis, Biogen Idec, consultant compensation from Genzyme Inc. and Serono