Anti human cd3 ucht1

Human mo-DCs were prepared as described previously [90 (link)]. Briefly, peripheral blood mononuclear cells (PBMCs) were obtained from healthy donor blood by Ficoll-Hypaque (GE Healthcare) density centrifugation. CD14⁺ cells (purity > 95%) were isolated using the EasySep™ Human CD14 Positive Selection Kit (STEMCELL) and cultured in RPMI-1640 medium supplemented with penicillin/streptomycin, 10% FBS, recombinant hGM-CSF (100 ng/ml) and hIL-4 (100 ng/ml; both from R&D) for 6 days. Human mo-DCs were treated with 10 µM SB203580 and HDM for 24 h, washed extensively and cocultured with human blood naïve CD4⁺ T cells isolated using the Naïve CD4⁺ T Cell Isolation Kit II (Miltenyi Biotec) at a ratio of 1:10. After 7 days of coculture, live T cells were purified and stimulated with plate-bound anti-human CD3 (UCHT1; BioLegend) for 5 h and then harvested for mRNA analysis. PBMCs were collected from allergic rhinitis (AR) patients and stimulated with the p38 inhibitor SB203580 or DMSO (vehicle) for 8 h, with GolgiStop added to the culture medium for the last 5 h. Cells were harvested, and IL-12p40 (C8.6, eBioscience) expression in DCs was detected by ICS. This study was approved by the Ethics Committee of the Eye & ENT Hospital of Fudan University (2017-0301). Informed consent was obtained from all volunteers.

Live immune cells from peripheral blood, spleen, and BM were determined by staining with live/dead fixable blue dead cell stain kit (Life Technologies) for 30 min prior to cell-specific marker labeling. Cells were labeled with anti-human CD34 (clone 581; BD Biosciences), anti-human CD3 (UCHT1; Biolegend), anti-human CD56 (MEM-188, Biolegend), anti-human CD14 (63D3; Biolegend), anti-human CD19 (SJ25C1; BD Biosciences), anti-human CD117 (104D2; Biolegend), anti-human CD38 (HB-7; Biolegend), anti-human CD33 (WM53; BD Biosciences), mouse CD45.1 (A20; BD Biosciences), anti-human CD8 (SK1; Biolegend), anti-human CD4 (SK3; BD Biosciences), and anti-human CD45 (HI30; Biolegend) monoclonal antibodies for 30 min at room temperature. After incubation, cells were washed and resuspended in FACs buffer containing phosphate buffered saline (PBS), 0.2% bovine serum albumin (GE Healthcare Life Sciences), and 0.05% sodium azide (Merck) for flow cytometry data acquisition. Data was acquired using a LSR II flow cytometer (BD Biosciences) with FACSDiva software, and analysis was performed using FlowJo software (version 10; Tree Star Inc). Absolute count of cells in peripheral blood was determined using CountBright™ Absolute Counting Beads (Thermo Fisher Scientific).

Immediately after perfusion, renal tissues were fixed in 4% paraformaldehyde and embedded in paraffin after 3 days. Tissue slices were stained with hematoxylin and eosin for analyses of renal structure. For immunohistochemistry, tissue slices were stained with anti-human CD3 (UCHT1; BioLegend, San Diego, USA) or CD274 Polyclonal Antibody (Bioss Antibodies, Woburn, Massachusetts, USA) by using the Zytochem Plus HRP Polymer System (Zytomed Systems, Berlin, Germany). Counterstaining was performed using Papanicolaou’s solution and samples were fixed with DPX Mountant (Sigma-Aldrich, St. Louis, Missouri, USA). Afterward, visualization was performed using a Keyence microscope (Keyence, Itasca, Illinois, USA) and samples were quantified via QuPath v0.3.0 bioimage analysis software (open source; https://qupath.github.io/).