Ril 4

Femoral and tibial bone marrow cells were harvested from mice, and subsequently cultured in RPMI-1640 medium (R8758, Sigma-Aldrich) supplemented with 10% FBS, sodium pyruvate (Thermo Fisher Scientific), MEM NEAA (Thermo Fisher Scientific), and recombinant (r) M-CSF (40 ng/ml, BioLegend) for 6 days, with medium changes every 2 days. The differentiated BMDMs were treated with rIFN-γ (100 ng/ml; BioLegend) + LPS (50 ng/ml; L5273, Sigma-Aldrich) or rIL-4 (20 ng/ml; BioLegend) for 16 to 24 hours to achieve M1 or M2 polarisation, respectively. In order to evaluate M2 polarisation, we stained BMDMs treated with rIL-4 with PE-conjugated anti-CD11b (M1/70, BioLegend,), FITC-conjugated anti-F4/80 (BM8, eBioscience), and Alexa Fluor® 647-conjugated anti-CD206 (MR5D3, AbD Serotec), and analysed on a FACSCalibur™ cytometer (BD Biosciences).

As described above, spleen naive B cells were cultured with pam3CSK4 (1 μg ml⁻¹, Invivogen, USA), heat-killed E. coli (10⁷ CFU ml⁻¹), CpG (20 μg ml⁻¹, GeneDsign Inc, Japan) or anti-IgM-Ab (15 μg ml⁻¹; Jackson Immuno Research Laboratories, USA) for 3 days in vitro, separately. Then, 1 × 10⁵ B220⁺ B cells were sorted from the simulated cells and subsequently seeded on 40LB feeder cells with rIL-4 (1 ng ml⁻¹; Biolegend, USA) to induce iGB cells. Non-stimulated spleen naive B cells were seeded on 40LB feeder cells with rIL-4 (1 ng ml⁻¹; Biolegend, USA) as a control group.
After 4-day culturing, 3 × 10⁵ iGB cells (B220⁺) of naive B-40LB cells, pam3CSK4-40LB cells, E. coli-40LB, IgM-40LB and CpG-40LB cells were sorted and labeled with CellTracker Orange CMTMR fluorescent dye (Invitrogen, USA) and then separately injected intravenously to a mouse (Balb/c, 8–12 weeks old) together with 10 μg of AF488 anti-mouse MAdCAM-1 (Biolegend mAb MECA-367, USA) to identify high endothelial venules (HEVs). Under anesthesia, to stain the IgA⁺ cells for GC identification, 1 μg of AF647 anti-mouse IgA (Southern Biotech, USA) was directly injected to a PP of the same mouse transferred with iGB cells. The localization of transferred labeled iGB cells was observed under an LSM880 microscope (Carl Zeiss, Germany) and analyzed with ZEN2009 software (Carl Zeiss, Germany).