Fig 2: PDVC57 tumors recruit more tumor-associated macrophages and fewer T cells and dendritic cells than PDV tumors. (A) Percentage of intratumoral CD45+ leukocytes that are CD11b+ F4/80+, indicating tumor-associated macrophages; significantly higher in PDVC57 tumors. (B) Percentage of intratumoral CD45 + leukocytes that are CD3+ CD4+ or CD3+ CD8+, indicating tumor-infiltrating T cells; CD3+ CD4+, CD3+ CD8+ cells significantly lower in PDVC57 tumors. (C) Percentage of intratumoral CD45+ leukocytes that are CD11c+ MHCII+, indicating tumor-infiltrating dendritic cells; significantly lower in PDVC57 tumors. (D) Arginase activity in units of catalytic activity (U) as assessed by enzymatic fluorometric assay; significantly higher in PDVC57 tumors. *P < 0.05; **P < 0.01; ***P < 0.001 using the unpaired t-test adjusted for multiple comparisons. Two separate experiments with n > 5 per group.

Fig 3: Transcutaneous arginase inhibition is associated with significantly more intratumoral T cells and dendritic cells in PDVC57 tumors. (A) Percentage of intratumoral CD45+ leukocytes that are CD3+ CD4+, CD3+ CD8+, or CD3+ PD-1+ indicating tumor-infiltrating T cells; CD3+ CD4+, CD3+ CD8+, CD3+ PD-1+ cells significantly higher in nor-NOHA-treated tumors. (B) Percentage of intratumoral CD45+ leukocytes that are CD11c+ MHCII+, indicating tumor-infiltrating dendritic cells; significantly higher in nor-NOHA-treated tumors. **P < 0.01; ***P < 0.001 using the unpaired t-test adjusted for multiple comparisons. Two separate experiments with n > 5 per group.

Fig 4: Blood cell counts from flow cytometry analysis in an independent mouse cohort. (A) Leucocytes (CD45 + cells)/µL of blood, (B) B cells (CD19 +)/µL of blood, (C) T cells (CD3e +)/µL of blood; and (D) Myeloid cells/µL of blood are plotted with dose as the independent variable. All doses of irradiation produce a significant change of T and B cell numbers compared to control levels; however, B cells are more sensitive. 5 males and 5 females were used for this analysis and data is combined, as there were no differences by sex. (p values are indicated in the figure as *p < 0.05).

Fig 5: Becn1 deficiency reduces HSC reconstitution. (A–C) Freshly isolated 50 HSCs from WT or Becn1vKO mice were transplanted into lethally irradiated recipients together with 2.5 × 105 competitor cells. Chimerism in peripheral blood was evaluated every month until the fourth month post transplantation (Tx). (A) The schematic diagram showing the experimental design for HSC competitive transplantation. (B) The line plots showing donor chimerism in overall (CD45.2+), T (CD3+), B (B220+) and myeloid (CD11b+) cell every month after HSC transplantation (HSCT) (n = 6 for WT and 5 for Becn1vKO group). The gating strategy to generate these line plots is presented in sup. Fig. 2A. (C) This histogram displays the lineage distribution of donor-derived peripheral blood at the fourth month after transplantation (n = 6 for WT and 5 for Becn1vKO group). Data are shown as mean ± SD. The gating strategy for lineage distribution is presented in sup. Fig. 2B. (D and E) Representative flow cytometry plots (D) and scatter plot (E) showing donor-derived LSK and HSC engraftment in recipient bone marrow at the 4th month post-HSC transplantation (n = 6 for WT and 5 for Becn1vKO group). Data are shown as mean ± SD. The gating strategy of LSK and HSC engraftment is presented in sup. Fig. 2C. (F and G) The histogram (F) and representative flow cytometry plots (G) display the cell cycle analysis of WT and Becn1vKO HSCs. n = 5 mice per group, data are shown as mean ± SD.