Additional file 1 of Abatacept downregulates Fcγ receptor I on circulating monocytes: a potential therapeutic mechanism in patients with rheumatoid arthritis
Creators: name: Fukue, Ryosuke
name: Okazaki, Yuka
name: Gono, Takahisa
name: Kuwana, Masataka
Publisher: name: figshare
Year: 2022
Subjects: subject: Immunology
subject: FOS: Clinical medicine

description: Additional file 1: Table S1. A list of cell surface molecules investigated and monoclonal antibodies used for flow cytometry. Table S2. Demographic and clinical features of RA patients and controls used in the series of experiments. Figure S1. Screening of monocyte-derived cell surface molecules that were differentially expressed after short-term culture with abatacept. Peripheral blood samples from 5 patients with rheumatoid arthritis (RA) and 5 controls were used for a derivation experiment. Circulating monocytes were cultured for 24 hours in the absence (mock) or presence of abatacept (ABT) or CD28-Ig. Cell surface expression was quantified by flow cytometry and is expressed as the mean fluorescence intensity (MFI) ratio. Statistical comparisons between two groups were made using the Mann–Whitney U test. Figure S2. Screening of monocyte-derived cytokines/chemokines that were differentially expressed after short-term culture with abatacept. Peripheral blood samples from 5 patients with rheumatoid arthritis (RA) and 5 controls were used for a derivation experiment. Circulating monocytes were cultured for 24 hours in the absence (mock) or presence of abatacept (ABT) or CD28-Ig. The concentrations of interleukin (IL)-1β, IL-6, IL-8, IL-10, C-C motif chemokine ligand 2 (CCL2), and tumor necrosis factor (TNF)-α were measured by multiplex bead arrays. Statistical comparisons between two groups were made using the Mann–Whitney U test. Figure S3. Time course of abatacept-induced downregulation of CD64/FcγRI on monocytes. Peripheral blood samples were obtained from 3 controls and cultured with or without abatacept for 6, 24, and 28 hours. The expression levels of CD64/FcγRI were quantified by flow cytometry and are expressed as the CD64/FcγRI expression ratio, which was calculated by dividing the mean fluorescence intensity ratio of monocytes cultured with abatacept by the mean fluorescence intensity ratio of monocytes cultured without abatacept. The results are shown as the mean and standard deviation. A representative result of 2 independent experiments is shown. Figure S4. Abatacept-induced downregulation of CD64/FcγRI on monocytes is independent of the IgG1-Fc portion of abatacept. Peripheral blood monocytes were obtained from 5 patients with rheumatoid arthritis (RA) and cultured without (mock) or with abatacept or intact human IgG1-Fc for 24 hours. The expression levels of CD64/FcγRI were quantified by flow cytometry and are expressed as the mean fluorescence intensity (MFI) ratio. The results are shown in boxplots, and statistical comparisons between two groups were made using the Mann–Whitney U test. A representative result of 3 independent experiments is shown. Figure S5. Confirmation of the peptidylarginine deiminase (PAD)-induced citrullination of fibrinogen by specific recognition of ACPA-positive IgG in enzyme-linked immunosorbent assay. PAD- or mock-treated fibrinogen was coated in duplicate on 96-well polyvinyl plates, and applied for enzyme-linked immunosorbent assay using purified ACPA-positive IgG, ACPA-negative IgG, serum from ACPA-positive RA patient or serum from ACPA-negative healthy control. All measurement was conducted in duplicate, and results are shown as mean ± standard deviation. A representative result of 3 independent experiments is shown.