Anti cd16 cd32

Spleens were harvested on day 12 and splenocytes stained with the antibodies listed in online supplemental table 1. Non-specific binding was blocked using anti-CD16/CD32 (Miltenyi Biotec). After isolation, PBMCs from healthy donors or patients were stained with the antibodies listed in online supplemental table 2). Non-specific binding was blocked using FC block (BD bioscience). For intracellular staining, cells were fixed and permeabilized with fixation/permeabilization solution (ThermoFischer), according to the manufacturer’s instructions. Data acquired with a BD LSR-Fortessa flow cytometer were compensated and exported into FlowJo (version 10.0.8, TreeStar Inc.). An unbiased analysis was performed on splenocytes using the t-distributed stochastic neighbor embedding (t-SNE) algorithm tool (Flowjo software) to reduce the flow cytometry data to two dimensions. Live cells from mice in the same group were merged. Results are shown as a t-SNE map displaying the repartition of the expression of a given marker among randomly sampled cells from the indicated parent population.

Spleens were harvested on d12 and splenocytes stained with the antibodies listed in Online Supplementary Table S1. Non-specific binding was blocked using anti-CD16/ CD32 (Miltenyi Biotec). For intracellular cytokine staining, cells were stimulated for 5 hours with phorbol 12-myristate 13-acetate (PMA) 50 ng/mL and ionomycin 1 μg/mL (Sigma– Aldrich, Saint Louis, CA, USA,). Brefeldin A (BD Pharmingen, San Diego, CA, USA) was added for the last 4 hours. Then, cells were fixed and permeabilized with fixation/permeabilization solution (ThermoFischer), following the manufacturer’s instructions. Data acquired with a Canto 2 flow cytometer were compensated and exported into FlowJo (version 10.0.8, TreeStar Inc).