Subjects
14 subjects with mild-to-moderate allergic asthma were enrolled in this study. Inclusion criteria included a physician diagnosis of allergic asthma and serum Der p 1 specific-IgE>0.35kUA/L. Exclusion criteria included upper respiratory infection within 4 weeks, prednisone use within 3 months, pregnancy, and other lung diseases or autoimmune diseases. All patients were taking low or medium dose ICS-LABA as their daily treatment and were well-controlled. Detailed patient information is shown in Table S1. The study was approved by the Ethic Committee of Ruijin Hospital (No.2019-YK061). All participants signed an informed consent form prior to the study.
In vitro eosinophils stimulation assays andco-culture of eosinophils and autologous naïve CD4+ T cells
Eosinophils and naïve CD4+ T cells from allergic asthmatics were separated from peripheral blood by magnetic cell separation system (MACS) using Eosinophil Isolation Kit (#130-092-010, Miltenyi) and Naïve CD4+ T Cell Isolation Kit II (#130-094-131, Miltenyi) as previously described17. Purity of isolated cells was checked by flowcytometry and was always >95%. Purified eosinophils were resuspended at 1.0x106 /mL in RPMI 1640(Hyclone) supplemented with 10% FBS, 1% penicillin/streptomycin (Servicebio), and 10ng/ml IL-5 (Peprotech). Eosinophils were stimulated with HDM (100μg/ml, Greer Lab, low endotoxin) and/or IL-25(1ng/ml, Peprotech) at 37°C in a humidified 5% CO2 incubator for 18 h, and then cells were harvested for co-culture or direct flowcytometry analysis.
After 18 h stimulation with HDM and/or IL-25, eosinophils (1.0x106 /ml) were resuspended with autologous naïve CD4+ T cells (106 /ml) in 1:1(v:v) RPMI 1640 (Hyclone) with ImunoCult-XF T Cell Expansion Medium (Stemcell) supplemented with 10ng/ml IL-2 (Peprotech), 10ng/ml IL-5 (Peprotech) and 25μl/ml ImmunoCult Human CD3/CD28 T Cell Activator (Stem Cell). Cells were cultured at 37°C in a humidified 5% CO2 incubator for 48h. Individual naïve CD4+ T cells (1.0x106 /ml) groups were served as control, culturing in the same condition medium above. 4 h before the end of culture, 1:1000 Monensin Solution (Biolegend) were added for intracellular cytokines analysis.
The flow cytometry analyses were performed with a Cytoflex LX instrument (Beckman). Dead cells were excluded by Zombie UV (Biolegend). For eosinophil surface marker analysis, cells were incubated with Fc Receptor Blocking Solution (Biolegend) to eliminate Fc receptor-mediated antibody binding, and then followed with staining of APC/Cy7 anti-human CD16 antibody(Clone:3G8, BD sciences) , APC anti-human Siglec-8 antibody(Clone:7C9), PE anti-human CD40 antiobody(Clone:HB14), PE/Cyanine7 anti-human HLA-DR antibody(Clone:L243), Brilliant Violet 421 anti-human CD80 antibody(Clone:L307.4, BD sciences), BB515 anti-human CD86 antibody (Clone:FUN-1, BD sciences), PE/Cyanine7 anti-human PD-L1 antibody (Clone:29E.2A3), PE anti-human OX-40L antibody(Clone:11C3.1) for 20 min at 20°C in the dark, then cells were washed, fixed with Fixation Buffer (Biolgend) and resuspended in Cyto-last Buffer (Biolegend) until analysis.
To measure cytokines production of T cells after co-culture, following surface staining of FITC anti-human CD3 antibody (Clone: UCHT1), PE/Cyanine7 anti-human CD4 antibody (Clone: RPA-T4), cells were fixed by Fixation Buffer (Biolgend) for 20 min at 20°C avoided from light, and then washed twice with Intracellular Permeabilization Buffer. Cells were stained with APC/Fire 750 anti-human IFN-γ antibody (Clone:4S.B3), Brilliant Violet 421 anti-human IL-4 antibody (Clone:G077F6), PE anti-human IL-9 antibody (Clone:MH9A4), APC anti-human IL-17A antibody (Clone:BL168) in remaining permeabilization buffer for 30 min at 20°C in the dark, and then washed and resuspended in FACS buffer for analysis. All antibodies were purchased from Biolegend unless otherwise specified.