The CXCR5-CXCL13 axis influences the proliferation of B-ALL cells in a young more than in an old BMM. (A) Number of primary GFP⁺ (BCR-ABL1⁺) BP-1⁺ cells plated on 5 × 10⁴ macrophages (MΦ) from young vs old mice in the presence of 2.5 μg of isotype control or anti-CXCL13 antibody; 2 × 10⁴ leukemia cells had been plated and were counted 12 and 24 hours after addition of the antibodies. The P values are as indicated (2-way ANOVA, Tukey test, n = 3). (B) Representative confocal images (scale bar, 20 μm) of BCR-ABL1⁺ BA/F3 cells plated on macrophages (MΦ) from young or old mice stained with an antibody to pAKT (red) after treatment with 2.5 μg of isotype control or anti-CXCL13 antibody for 5 days (n = 3). Nuclei are stained with DAPI (blue); 3 × 10⁵ BCR-ABL1⁺ BA/F3 cells had been plated and then cultured for 5 days before staining. (C) Corrected total cell fluorescence (CTCF) × 10⁶ for nuclear pAKT, as in panel B, in BCR-ABL1⁺ BA/F3 cells plated on macrophages from young or old mice stained with an antibody to pAKT after treatment with 2.5 μg of isotype control or anti-CXCL13 antibody (2-way ANOVA, Tukey test, n = 3). The antibodies had been added at the start of the coculture. (D) Number of primary live GFP⁺ (BCR-ABL1⁺) BP-1⁺ cells cultured in the presence of 1% BSA or 100 ng/mL recombinant CXCL13 on young (left) vs old (right) macrophages (MΦ); 4 × 10⁴ leukemia cells had been plated. Cells were enumerated after exclusion of trypan blue⁺ dead cells. The P values for the timepoint at 48 hours are as indicated (2-way ANOVA, Tukey test, n = 3). (E) Number of sorted primary GFP⁺ (BCR-ABL1⁺) BP-1⁺ cells from young (left) or old (right) mice with established B-ALL cultured in the presence of 1% BSA or 100 ng/mL recombinant CXCL13; 2 × 10⁵ cells had been plated. The P values are P < .0001 on day 3 and 4, P = .005 on day 5 (left) and P = .004 on day 3 and P = .012 on day 4 (right) (2-way ANOVA, Tukey test) for the indicated timepoints. The data are representative of 3 independent experiments. (F) Kaplan-Meier–style survival curve of nonirradiated BALB/c recipient mice transplanted with 2 × 10⁶ transduced BM cells pretreated with 1% of BSA or 100 ng/mL recombinant CXCL13 for 18 hours before transplantation (P = .002; log-rank test, n = 5). (G) Number of primary GFP⁺ (BCR-ABL1⁺) BP-1⁺ cells that had migrated toward 1% BSA or 100 ng/mL recombinant CXCL13 or young vs old macrophages in the presence of 2.5 μg of isotype control or anti-CXCL13 antibody after 24 hours; 2 × 10⁴ GFP⁺ (BCR-ABL1⁺) BP-1⁺ cells had been plated. The P values are as indicated (one-way ANOVA, Tukey test, n = 3). Percentage of GFP⁺ (BCR-ABL1⁺) BP-1⁺ cells in peripheral blood (P < .0001; Student t test, n = 4-8) (day 21 after transplantation) (H) and Kaplan-Meier–style survival curve (P = .05; log-rank test, n = 5-8) (I) of young, nonirradiated C57BL/6J recipient mice, treated with control (blue) or clodronate (red) liposomes, after transplantation of 2 × 10⁶ BCR-ABL1–transduced BM in the B-ALL model. Control liposomes or clodronate liposomes were administered weekly at 5 mg/10 g of animal weight starting from day 12 after transplantation. Concentration of CXCL13 in pg/mL in the plasma on day 21 after transplantation (J) and flushed bone at the time of death (K) of young C57BL/6J recipient mice transplanted with BCR-ABL1–transduced BM and treated with control liposomes or clodronate liposomes as in panel I. The P values are as indicated (Student t test, n = 3-4).