Monocyte NOTCH2 expression predicts IFN-β immunogenicity in multiple sclerosis patients
Marsilio Adriani, … , Elizabeth C. Jury, The ABIRISK Consortium
Marsilio Adriani, … , Elizabeth C. Jury, The ABIRISK Consortium
Published June 7, 2018
Citation Information: JCI Insight. 2018;3(11):e99274. https://doi.org/10.1172/jci.insight.99274.
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Research Article Immunology Neuroscience

Monocyte NOTCH2 expression predicts IFN-β immunogenicity in multiple sclerosis patients

Abstract

Multiple sclerosis (MS) is an autoimmune disease characterized by CNS inflammation leading to demyelination and axonal damage. IFN-β is an established treatment for MS; however, up to 30% of IFN-β–treated MS patients develop neutralizing antidrug antibodies (nADA), leading to reduced drug bioactivity and efficacy. Mechanisms driving antidrug immunogenicity remain uncertain, and reliable biomarkers to predict immunogenicity development are lacking. Using high-throughput flow cytometry, NOTCH2 expression on CD14+ monocytes and increased frequency of proinflammatory monocyte subsets were identified as baseline predictors of nADA development in MS patients treated with IFN-β. The association of this monocyte profile with nADA development was validated in 2 independent cross-sectional MS patient cohorts and a prospective cohort followed before and after IFN-β administration. Reduced monocyte NOTCH2 expression in nADA+ MS patients was associated with NOTCH2 activation measured by increased expression of Notch-responsive genes, polarization of monocytes toward a nonclassical phenotype, and increased proinflammatory IL-6 production. NOTCH2 activation was T cell dependent and was only triggered in the presence of serum from nADA+ patients. Thus, nADA development was driven by a proinflammatory environment that triggered activation of the NOTCH2 signaling pathway prior to first IFN-β administration.

Authors

Marsilio Adriani, Petra Nytrova, Cyprien Mbogning, Signe Hässler, Karel Medek, Poul Erik H. Jensen, Paul Creeke, Clemens Warnke, Kathleen Ingenhoven, Bernhard Hemmer, Claudia Sievers, Raija L.P. Lindberg Gasser, Nicolas Fissolo, Florian Deisenhammer, Zsolt Bocskei, Vincent Mikol, Anna Fogdell-Hahn, Eva Kubala Havrdova, Philippe Broët, Pierre Dönnes, Claudia Mauri, Elizabeth C. Jury, The ABIRISK Consortium

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Figure 5

Serum factors drive increased NOTCH2 activation in neutralizing antidrug antibody–positive (nADA+) multiple sclerosis (MS) patients.

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Serum factors drive increased NOTCH2 activation in neutralizing antidrug...
(A–D) mRNA isolated from ex vivo FACS-sorted CD14+ monocytes from healthy donors (HCs, n = 8), nADA– (n = 8)/nADA+ (n = 8) IFN-β–treated (MS-T) patients and nADAp+ (n = 6)/nADAp– (n = 6) treatment-naive (MS-N) patients. NOTCH target genes HES1 and DTX1 (A and B) and cytokine genes IL6 and IL10 (C and D) were analyzed by qPCR relative to cyclophilin. Results are expressed as fold change from HCs. Mann Whitney U test, *P ≤ 0.05, **P ≤ 0.01. (E–G) Ex vivo peripheral blood mononuclear cells (PBMCs) from nADA– (n = 4)/nADA+ (n = 4) MS-T patients and nADAp+ (n = 6)/nADAp– (n = 6) MS-N patients were cultured for 6 hours in presence LPS (10μg/ml). PMA (50ng/ml), ionomycin (250ng/ml), and Brefeldin A (1μg/ml) were added to the cultures for the last 2 hours before harvesting. Intracellular IL-6 expression was evaluated by flow cytometry. (E) Representative histograms and cumulative data (percentage positive and mean fluorescence intensity; MFI) showing IL-6 expression in monocytes from (F) MS-T and (G) MS-N patients. Mann Whitney U test, *P < 0.05. (H) Experimental strategy for in vitro experiments. HC PBMCs were cultured with 10% serum from MS-T nADA+ or nADA– patients. Following 18-hour culture, mRNA was isolated from purified monocytes for qPCR analysis. (I) Cumulative expression of NOTCH target genes HES1 and DTX1 relative to cyclophilin A in nADA– (n = 6) or nADA+ (n = 5) MS-T patients evaluated by qPCR. Mann Whitney U test, *P < 0.05. (J) PBMCs were precultured for 16 hours ± N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT) or vehicle (DMSO) before 18-hour culture with 10% serum from nADA+/nADA– patients (n = 4). Cumulative expression of HES1 and DTX1 relative to cyclophilin A. Mann Whitney U test, *P < 0.05, **P ≤ 0.01, ***P ≤ 0.001. (K) Cumulative expression of genes previously reported to be associated to classical (CD163) or nonclassical (STAT1) monocyte subsets (32). (L) HES1 expression in sorted CD14+ monocyte cultures and cocultures of CD14+ monocytes and CD19+ B cells or CD14+ monocytes and CD4+ T cells (1:1 ratio) after culture in presence of serum from MS-T nADA+ or nADA– patients (n = 7). Mann Whitney U test. *P < 0.05. Box plots show the median and 25th and 75th percentiles; whiskers represent minimum and maximum values.