Immunomodulatory effects of ponesimod on human astrocytes

Ponesimod is a slective S1PR1 antagonist and is approved for the treatment of Multiple Sclerosis by FDA. To determine how ponesimod controls neuroinflammatory responses in astrocytes, single-cell RNA-sequencing (scRNA-seq) was conducted in cytokine mixture-stimulated human primary astrocytes in the presence or absence of ponesimod. Overall design: Human primary astrocytes (Lonza; Cat# CC-2565) were stimulated with or without cytokine mixture (25 ng/mL each; TNF-a, IFN-g, IL-17, IL-1b, and IL-6) in the presence or absence of ponesimod for 24 h or 48 hrs. Single-cell capture (target capture of 10,000 cells per sample) and library preparation was conducted using Chromium Single Cell 3' GEM, Library & Gel Bead Kit v3 (10x Genomics, Cat # PN-1000075) and Chromium Chip B Single Cell Kit (10X Genomics, Cat # PN-1000074) and according to manufacturer's instruction. Single-nucleus libraries were sequenced on the Illumina NovaSeq 6000 machine at GENEWIZ, Inc. (U.S.) Raw FASTQ files were input into the cellranger count pipeline (Cell Ranger V4.0.0, 10x Genomics) to align reads to the GRCh38 human genome.