Fig. 2: Agonist CD40 induces CD11b⁺ regulatory B cells in melanoma.

A 1014 cells were injected in C57BL/6 female mice. Treatment of C57BL/6 mice with aCD40 (30 μg/mouse every 3 days, intraperitoneal) started at Day 10 post tumor cell inoculation. Analysis of CD40 levels by flow cytometry of CD45⁺CD19⁺ B cells prepared from tumor samples at day 22 post-treatment (n = 5 per group) using a one-sided Mann–Whitney U test. B Splenocytes were isolated from C57BL/6 mice by magnetic separation, serum-free starved for 4hrs, and stimulated with or without 12.5 μg/ml aCD40. After 4 days of culture, splenic B cells were isolated by magnetic separation and stained for flow cytometric analysis (n = 6 per group). C, D Tumor volume of 1014 melanoma in C57BL/6 female mice. Treatment with 200 μg/mouse αPD1 (every 3 days, intraperitoneal), 100 μg/mouse αCTLA4 (every 3 days, intraperitoneal), 30 μg/mouse agonist CD40 (aCD40, every 3 days, intraperitoneal), or IgG control antibody, starts at day 10 post tumor cell inoculation. E Frequency of CD19⁺CD11b⁺ regulatory B cells in 1014 melanoma tumors or tumor draining lymph node (TDLN) after 22 days of aCD40 treatment (every 3 days, intraperitoneal) TDLN IgG, n = 5; TDLN aCD40, n = 5; Tumor IgG, n = 4; Tumor aCD40, n = 5. F Flow cytometric analysis of CD45⁺CD19⁺ B cells in the tumor microenvironment of 1014 melanoma after 3 weeks treatment of aCD40. Data were replicates from one experiment (n = 5 mice per group). G C57BL/6 mice were intravenously injected with formalin-fixed 2 × 10⁶ metastatic B16F10-Luc2 cells via the tail vein. Splenic CD8⁺ T and total B cells were isolated by magnetic separation after 2 days of tumor antigen priming. Total B cells were stimulated with 12.5 μg/ml aCD40 for 2 days and CD11b⁺ B cells were isolated by magnetic separation. CD8⁺ cytotoxic T cell killing assay were performed via co-culturing CD11b⁺ B cells, CD8⁺ T cells and B16F10-Luc2 cells in the conditioned RPMI complete medium containing 0.5 μg/ml of IL-7, 30U/ml of IL-2, and CD3/CD28 Dynabeads at a bead-to-cell ratio 2:1. Dead cells were removed and the luciferase activity of remaining live tumor cells was quantified to calculate % inhibition of CTL killing capacity. (n = 3 independent experiments). Created in BioRender. Yan, C. (2026) https://BioRender.com/83jrya2. Exact p values are provided as a Source Data file. H Splenic B cells were stimulated with 12.5 μg/ml aCD40 for 4 days. The CD11b^- and CD11b⁺ B cells were isolated by magnetic separation and restimulated with 12.5 μg/ml aCD40 for 30 min. Whole-cell extracts were harvested and immunoblotted. β-actin was used as a loading control for densitometry quantification. n = 3 independent experiments. One-way analysis of variance (ANOVA) with post hoc test. Source data are provided as a Source Data file.