Anti il 10 monoclonal antibody

Human PBMCs from patients with AR or buffy coat from “anonymous donors” were isolated by density centrifugation with Ficoll‐Paque Plus (MP Biomedicals, Santa Ana, CA, USA). CD4⁺ T cells were sorted from PBMCs of buffy coat of human volunteers using the CD4 Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). sEV‐mDCs were harvested and washed twice, and then were co‐cultured with allogeneic PBMCs isolated from patients with AR or freshly sorted CD4⁺ T cells (1:10) for 5 days in RPMI 1640 (Hyclone, Pittsburgh, PA, USA) supplemented with 10% FBS and IL‐2 (100 U/mL). Cell counting was performed manually using a hemocytometer and the cell viability was over 95%. In some experiments, CD4⁺ T cells were treated with the rhIL‐10 (10 ng/mL; PeproTech Inc.) together with mDC, or incubated with anti‐IL‐10 monoclonal antibody (75 ng/mL) or IL‐10Rα blocking antibody (5 μg/mL; both from R&D systems, Minneapolis, MN, USA) for 30 min before the co‐culture. The PBMCs or the T cells were collected for the analysis of intracellular Th2 cytokines using flow cytometry, and the supernatants were used for the evaluation of IL‐13, IL‐9, IL‐10, or IFN‐γ. T cells were also evaluated for IL‐10⁺Foxp3⁺ T cells or Treg cells.

To evaluate the soluble factors produced by iPSC-MSCs, the monocytes were removed from the plates of co-culture (on day 5) and then iPSC-MSCs were cultured further in a fresh medium for an additional 24 h. Prostaglandin (PG)E2, IL-10, IL-6, and tumor necrosis factor-stimulated gene 6 (TSG-6) levels were determined in the supernatants of iPSC-MSCs cultured with or without monocytes. To evaluate the cytokines produced by DCs, DCs were collected from the co-cultures on day 7 and then cultured in the new plates for an additional 12 h. IL-10 or IL-12p70 levels were determined in the supernatants of DCs cultured with or without iPSC-MSCs. The factor levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA).
To investigate the role of soluble factors on the immunomodulatory effects of iPSC-MSCs, the following reagents were used for the co-culture systems: neutralizing anti-IL-6 (0.25 μg/mL; R&D Systems Europe, Abingdon, UK), anti-IL-10 monoclonal antibody (0.075 μg/mL; R&D Systems Europe), human recombinant IL-10 (0.5μg/mL; R&D Systems Europe), or NS-398 (5 μM; Cayman Chemical, Ann Arbor, MI, USA), an inhibitor of PGE2 synthesis.

mDCs or sEV-mDCs were harvested and washed twice, and then co-cultured with PBMCs from patients with AR or purified ILC2s (1:10) for 3 days. In some experiments, MF63 (0.1 μM, 1 μM), ONO-AE3-208/PF-04418948 (1 μM) (MCE, Shanghai, China) or anti-IL-10 monoclonal antibody (2 μg/mL; R&D Systems, Minneapolis, MN, USA) was added into co-cultures. Supernatants were collected for the analysis of the cytokine levels using enzyme linked immunosorbent assay (ELISA), and the cells were analyzed for IL-13⁺ILC2s and GATA3⁺ILC2s by flow cytometry.