Recombinant il 33

Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by Ficoll centrifugation. CD4⁺ and CD8⁺ T cells were purified using negative selection beads (Stem Cell Technologies). The sorted CD4+T and CD8+T cells were seeded with a 96-well U plate and were stimulated with Gag pool (2 μg/mL, Sigma) or CEF (Cytomegalovirus, Epstein-Barr virus, and Influenza A virus) peptide pools (0.03 μg/mL, Miltenyi Biotec). Recombinant IL-33 (R&D Systems) was added to the culture at concentrations of 0, 0.1, 1, 10, and 100 ng/mL (19 (link), 20 (link)), respectively for 3 days at 37°C. Cultured cells were kept in a 5% CO2 incubator. Golgistop (500 μg/mL, BD Biosciences) was added during the final 5 h.

Murine experiments were approved by the Ethics Committee of Tongji University. Female B6 mice between 8 and 10 weeks old were purchased from Shanghai Laboratory Animal Center and housed in specific pathogen free conditions. As previously described [1 (link)], induction of AIHA murine model was achieved by immunizing B6 mice with 2 × 10⁸ rat RBCs on a weekly basis for 10 weeks. For transfusion study, mouse RBCs obtained from naive female B6 mice were labeled with PKH-26 (Sigma) and injected into control mice or those that had developed AIHA through tail-vein. Destruction of these syngeneic RBCs was represented by the clearance of fluorescent RBCs measured by flow cytometry. To show the clearance kinetics, injected RBCs at 1 min after injection were taken as 100 %, and the remaining RBCs were calculated at different time points as the average for each group of mice.
For regulation of IL-33, B6 mice were injected with IL-33 neutralizing antibody or recombinant IL-33 respectively with isotype controls (R&D Systems). Briefly, 1 day before rat RBCs immunization, mice were intraperitoneally injected with 100 μg neutralizing antibodies per mouse or 2 μg IL-33 per mouse respectively. Injections were repeated every 3 days during mice immunization.