Cpg odn

CY5 labeled CpG-ODN (CpG-ODN, Sangon Biotech, Shanghai) was used to produce Man-EG7/CH@CpG and EG7/CH@CpG. C57BL/6 mice were subcutaneously injected with Man-EG7/CH@CpG (2 × 10⁶) or EG7/CH@CpG (2 × 10⁶). Mice without immunization were used as controls. Mice were sacrificed to detect the targeting efficiency at different time points (3 h and 6 h). In lymph node uptake detection, lymph nodes from the subcutaneous model were minced into small pieces to release immune cells; the obtained cells were next analyzed by flow cytometry. For DCs or macrophage uptake detection, the collected immune cells were first stained with FITC-conjugated CD11c or FITC-conjugated F4/80 antibody for 30 min at 4 °C, then analyzed by flow cytometry (NovoCyte Flow Cytometer, ACEA Biosciences, USA).

The CpG-ODN 1668 (5ʹ-TCC ATG ACG TTC CTG ATG CT-3′) was synthesized and supplied by Sangon Biotech (Shanghai, China), CpG-ODN (30 μg/mice) was administrated by i.p. injection.

B10 cells were purified from stimulated B cells using a Breg cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, B cells from mouse spleen were pre-enriched and stimulated using Porphyromonas gingivalis LPS (5 µg/mL; InvivoGen, San Diego, CA, USA) and CpG-ODN (1 µM; Sangon Biotech, Shanghai, China) for 24 h. Phorbol myristate acetate (PMA, 50 ng/mL; Sigma-Aldrich, St. Louis, MO, USA) and ionomycin (500 ng/mL, Sigma-Aldrich) were then added to the enriched B cell culture medium for the last 5 h of stimulation. B10 cells were isolated using a B10 isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and were then transferred intravenously into OVX and control mice.