Tofacitinib citrate

Msl transgenic mice, which have knock-in of human mesothelin ortholog into the murine mesothelin locus such that human mesothelin is expressed in the native distribution, were engineered and bred as described in (35 (link)). Mice (Msln^ki/+) were treated with tofacitinib (25 mg/kg, 200 µL volume, twice daily by OG, days 1-6) plus LMB-100 (3.5 mg/kg IV, 200 µL volume, Days 4 and 6), oral vehicle (200 µL OG, days 1-6) plus LMB-100, or oral vehicle plus IV PBS (200 µL volume IV, Days 4 and 6). Tofacitinib citrate was purchased from SelleckChem (product S5001), dissolved in DMSO to 250 mg/mL for storage, then diluted into 0.5% methylcellulose, followed by 0.05% Tween20 and PBS just before use. LMB-100 iTox was manufactured by Roche and provided for these studies through a Collaborative Research and Development Agreement. Animals were euthanized 24 hours after last treatment. Tissue specimens were fixed in 10% neutral buffered formalin solution for 48-72 h at RT (HT501128, Sigma-Aldrich, USA), then sent to Molecular Histotechnology Laboratory (MHL) Core facility for all histologic studies. Analysis and quantification were performed by a trained veterinary pathologist affiliated with the MHL Core using criteria described in Supplementary Methods.

SFMCs were seeded in a density of 2 million cells/mL. SFMCs were cultured with pembrolizumab at 5 μg/mL with or without the TNFα inhibitor adalimumab (Humira™, Abbvie, North Chicago, IL, USA) at 5 μg/mL, the IL-6R inhibitor tocilizumab (RoActemra™, Roche, Hvidovre, Denmark) at 5 μg/mL or the Janus kinase (JAK) inhibitors tofacitinib citrate at 200 nM (Selleckchem, Munich, Germany), or baricitinib citrate at 200 nM (Selleckchem) as described previously [25 (link), 26 (link)]. In all experiments, negative controls included untreated cells or the vehicle dimethyl sulfoxide (DMSO). Cell cultures treated with LPS at 100 ng/mL were included as positive controls for cytokine expression. After 48 h of incubation, cell suspensions were transferred to Eppendorf tubes and centrifuged at 300 × g for 10 min at RT. The supernatants were collected and stored until later analysis for MCP-1 production.