Cell isolation and purification
Splenic mononuclear cells used for flow cytometry (FCM) were separated from red blood cells using a Ficoll-Hypaque solution (17-1440-02, GE healthcare, Boston, MA, USA). The mononuclear cells were collected from the upper layer following centrifugation at 2000 rpm for 20 min at room temperature (RT). After washing, cells were suspended in ice-cold PBS containing 2% fetal bovine serum (2% PBS-FBS) (10437028, Thermo Fisher, Waltham, MA, USA). Splenic B cells used for other assays were further purified from mononuclear cells by removing T cells with anti-CD90.2 mAb (105310; BioLegend, San Diego, CA, USA) and guinea pig complement (C300-0500; Rockland Immunochemicals, Gilbertsville, PA, USA), followed by incubation for 1 h in a cell culture flask to remove mononuclear cells. For BM isolation cells from the femoral and tibial cavities were flushed with 5 ml ice-cold 2% PBS-FBS. Following centrifugation for 5 min at 2000 rpm (4 °C), cell pellets were lysed with Red Cell Lysis Buffer (RT122-02, Tiagen, Beijing, China), resuspended and subsequently passed through a cell strainer. For peritoneal cells isolation, euthanize mice were injected 5 ml of ice-cold PBS into the peritoneal cavity and then the abdomen was massaged gently for 1-2 min to dislodge attached cells before collection. This was repeated twice to get as many cells as possible. Next, the peritoneum was cut and the liquid in the cavity was collected. The cell suspension was centrifuged at 2000 rpm for 5 min (4 °C), the supernatant was removed, and the cells were resuspended in 2 ml 2% PBS-FBS.