Fig 2: Absence of circulating B cells results in rapid expansion of granulocytic MDSCs.C57/Bl6 mice bearing E0771 tumors were treated with either an anti-CD20 mAB or isotype mAB, followed by treatment with saline, FEC, oHSV-1, CP, FEC + oHSV-1, or FEC + oHSV-1 + CP. Blood was drawn on days 6, 10, and 15 and analyzed via flow cytometry. a Heat map showing selected MDSC-related genes and their expression across all oHSV-1 and FEC + oHSV-1 samples, as determined by whole tumor RNA sequencing analysis. b Representative flow plot showing the gating strategy for B cells (CD19+B220+ cells) in mice treated with the isotype mAB (left) and an anti-CD20 mAB (right). c Representative flow plot showing the gating strategy for MDSCs (Ly6GhiLy6Cint cells) in mice treated with the isotype mAB (left) and the anti-CD20 mAB (right). d Bar plots showing the frequency of B cells in circulation across all timepoints. e Bar plots showing the frequency of MDSCs in circulation across all timepoints. f Kaplan–Meier survival plot for TNBC patients OS (n = 144) based on mean expression levels of a signature comprised of all available genes from Table 1. High versus low expression defined as above or below median expression. Logrank p value and hazard ratio (95% confidence interval) are displayed. g Kaplan–Meier survival plot for breast cancer patients OS (n = 943) based on mean expression levels of a signature comprised of all available genes from Table 1. High versus low expression defined as above or below median expression. Logrank p value and hazard ratio (95% confidence interval) are displayed. *Two-tailed unpaired t test was used for statistical analyses. Error bars are representative of standard deviation.

Fig 3: The effect of anti-CD20 and zoledronate treatment on tumor growth. (A) Schematic views of in vivo experiment. Animals were treatment with 100 µg/mouse anti-CD20 antibody after tumor cells were inoculated. Zoledronate was given at a dose of 6µg/animal for six times after tumors were formed. The number of circulating CD19-positive cells was analyzed throughout the experiment by Flow cytometry 3 times. (B) sh-NT and (C) sh-CD73 tumor growth upon treatment shown as a function of time. Tumor dimensions were measured with a caliper once a week. (D) Tumor volume and (E) fold-change of tumor volume at the sacrifice. (F) Representative images of H&E staining of lungs. Scale bar 200 μm. The number (G) and size (H) of lung metastases from 4T1 sh-NT and sh-CD73 cells. Data is expressed as mean ± SEM, by one-way ANOVA with a Sidak post-test. * P < 0.05, ** P < 0.01 and **** P < 0.0001, comparing within the same group upon different treatment. # P < 0.05, comparing sh-CD73 treated tumors vs. sh-NT cells treated tumors.

Fig 4: The effect of anti-CD20 treatment and zoledronate on circulating and tumor-infiltrating immune cells. The number of (A) B220-positive cells, (B) CD8-positive cells and (C) CD4-positive cells from 4T1 sh-NT and sh-CD73 tumors. The percentage of circulating (D) CD19-positive and (E) CD8-positive cells from 4T1 sh-NT and sh-CD73 tumor-bearing mice. The percentage of (F) CD19-positive and (G) CD8-positive cells in lungs from 4T1 sh-NT and sh-CD73 tumor-bearing mice. Data is expressed as mean ± SEM, by one-way ANOVA with a Sidak post-test. * P < 0.05, ** P < 0.01, comparing within the same group upon different treatment.

Fig 5: Confirmation of B cell depletion.a, Gating strategy to identify B220+CD19+ cells in Isotype control, and Anti-CD20 injected WT and Acly KO mice. Briefly, cells were gated as follows: SSC vs FSC plot of lymphocytes population, FSC-H vs FSC-A to identify single cell population, FSC vs 7-AAD to identify live cells population, B220 vs CD19 to identify B220+CD19+ B cell population. b. B220+CD19+ cells in Iso (Isotype control) and Anti-CD20 (B cell-depleted)-injected WT or Acly KO mice. Data are presented as mean ± SEM, Significant reductions were observed in WT-Anti-CD20 (n = 4; P = 2.37 × 10–6) and in Acly KO-Anti-CD20 (n = 7; P = 9.76 × 10–8), compared to WT-Isotype control (n = 11)-injected mice. The Acly KO-Isotype control group included n = 8 mice. Statistical analysis was performed using one-way ANOVA followed by Fisher’s LSD test. Source data