Figure 2 from OPG-Producing B Cells and RANKL-Expressing T Cells Define Immune Signatures Predictive of Bone Metastases in Breast Cancer

Adoptive transfer of 67NR-derived CD19+ B cells reduces RANKL production by 4T1 tumor–specific CD3+ T cells, inhibits bone resorption, and prevents bone metastases in BALB/c nude recipient mice. A, Experimental scheme: CD3+ T cells were magnetically isolated from the iliac BM of 4T1 tumor-bearing BALB/c mice on day 11 after implantation, and CD19+ B cells were isolated from the BM of 67NR tumor–bearing mice at the same time point. The cells were transferred intravenously into BALB/c nude mice either alone (T 4T1) or in combination (T 4T1 + B 67NR), followed by in vivo restimulation with 4T1 tumor sAgs. Naive mice were used as controls (Nv). B, RANKL levels were quantified by ELISA in culture supernatants of total spleen and BM cell suspensions obtained from recipient mice 14 days after transfer, following in vitro restimulation with 4T1 sAg. Each value reflects total RANKL concentration normalized to cell number (1 × 106 cells/mL), representing the overall functional output of the transferred T cell populations within the respective tissue compartments. T 4T1 means mice that received only 4T1+ CD3+ T cells with sAg, and T 4T1 + B 67NR means mice that received both 4T1+ CD3+ T cells and 67NR+ CD19+ B cells with sAg. Naive mice were used as experimental controls (Nv). C, Histomorphometric analysis of iliac bones was performed 14 days after cell transference. Sagittal sections from demineralized iliac bones were made following conventional methods and stained with H&E. All microscope slides were scanned with a ScanScope GL equipped with a 40× objective. Trabecular bone volume was expressed as a percentage of total tissue volume (% BV/TV). D, Experimental design for the metastatic setting: BALB/c nude mice were injected orthotopically with 4T1 tumor cells and subsequently received the adoptive transfer of CD3+ T cells from 4T1-bearing mice with or without CD19+ B cells from 67NR-bearing donors on day 11 after tumor injection. E, Histomorphometric analysis of iliac bones was performed on days 12 and 28 after cell transference, as described in C. F, Metastatic colonization was assessed on days 12 and 28 by clonogenic metastatic assays in draining LNs and BM. G, RANKL quantification by ELISA in culture supernatants of in vitro restimulated LN-derived cells with sAg, collected from the same experimental groups at endpoint. All data represent mean ± SD of five mice per group and are representative of at least two independent experiments. Statistical comparisons between more than two groups were performed using one-way ANOVA followed by Tukey post hoc test for pairwise comparisons when data were normally distributed. Statistical significance was considered when P < 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001.