Basophil activation assessed by CD203/CD63 in cat allergy patients

To evaluate antibody-mediated inhibition of basophil activation, a 20 pM final constant concentration of Fel d 1 (Indoor Biotechnologies) was pre-incubated for 30 min at 37 °C with REGN1908–1909 combination or IgG4^P isotype control antibody at final concentrations ranging from 0.8 fM to 1.0 μM. Concurrently with the Fel d 1 and antibody pre-incubation, PBMCs (BioreclamationIVT) were purified from fresh whole blood from allergic donors by Ficoll density gradient centrifugation. The purified PBMCs were washed, resuspended in X-VIVO 15 media (Lonza), and plated in duplicate columns in a v-bottom, polypropylene, 96-well plate (approximately 5 × 10⁵ cells/well). To prime the basophils contained in the overall PBMC population for activation, hIL-3 (R&D Systems, 0.3 nM) was added to the cell suspension and the plate was incubated at 37 °C for 10 min. The pre-incubated antibodies and Fel d 1 were then added to the primed PBMC. Cells without the addition of antibody were included as negative control samples and a dose response of Fel d 1 ranging from final concentrations of 7.8 aM to 10.0 nM was included as a positive control. The cells were then incubated at 37 °C for 20 min to facilitate basophil activation. Activation was subsequently stopped by incubation at 4 °C for 5 min. Basophil activation was then evaluated by flow cytometry. The cells were stained at 4 °C for 20 min with either an antibody cocktail containing anti-HLA-DR-FITC (Beckman Coulter, Catalog #IM0463U, clone B8.12.2), anti-CD123-APC (BD, clone 7G3, Catalog #560087), and anti-CD203c-PE (Beckman Coulter, Catalog #IM3575, clone 97A6) or a cocktail containing anti-HLA-DR-PE-Cy7 (Biolegend, Catalog #307616, clone L243), anti-CD123-APC (BD, Catalog #560087,clone 7G3), and anti-CD63-FITC (Beckman Coulter Catalog #IM1165U clone CLBGran/12). After staining, the cells were washed in a 1:20 dilution of MACS BSA Stock Solution (Miltenyi Biotec) in autoMACS Rinsing Solution (Miltenyi Biotec), fixed in Cytofix (BD) diluted 1:4 in Dulbecco’s phosphate-buffered saline (Gibco), and analyzed on a flow cytometer (BD LSRFortessa X-20). The gating strategy (Supplementary Fig. ^6c) was used to identify basophils within the larger population of PBMC and to determine levels of basophil activation. Samples were run to collect approximately 1000 events determined to be basophils. Basophils were defined as singlet, lymphoid, HLA-DR-, and CD123+. Activated basophils were further defined as CD203c^Hi or CD63^Hi. To specify a baseline level of activation, gates were set so that 10% of basophil events from hIL-3-primed, unstimulated samples (no Fel d 1) were positive for activation (CD203c^Hi or CD63^hi). These gates were then applied to all other experimental conditions to determine the relative level of basophil activation.