BLM induces in vivo Treg accumulation throught TGFb production.

<p>A: Spleens from CT26 tumor-bearing mice, treated with PBS or BLM, were harvested and the cells were analyzed through flow cytometry for Foxp3⁺CD4⁺ Treg detection. B: Treg from PBS- or BLM- treated CT26-bearing mice were isolated from spleen and co-cultured with OT-I in presence of SIINFEKL (SII). After 3 days of culture, IFNγ was titrated in the supernatant using ELISA method. C: Schematic representation for D and E panel experiments. D: GFP⁺ CD4⁺ cells were sorted from CD45.2 FOXP3-EGFP mice, then injected i.v. in CD45.1 mice bearing CT26 tumor. The mice then received PBS, IL-2 or BLM treatment. All mice received EdU injection. One day after treatment, spleens and tumors were collected and the proliferation status of transferred cells was assessed by revealing EdU by flow cytometry. E: CD4⁺ CD62L⁺ GFP⁻ naive T cells were sorted from CD45.2 FOXP3-EGFP mice, and injected i.v. in CD45.1 mice bearing CT26 tumor. The mice then received PBS or BLM treatment. Spleens were harvested and the cells were analyzed through flow cytometry for Treg detection. F: Mice were injected with 1.10⁶ CT26 cells i.p. Ten days later mice received PBS or BLM injection. The following day ascites were collected, and TGFβ was assessed using ELISA method. G: CT26 cells were treated with PBS or BLM <i>in vitro</i> for 24 h, then the supernatant was collected and assessed for TGFβ using ELISA method. H: Mice bearing CT26 tumor cells were treated with PBS or BLM. The day after, CT26 tumors were collected and tumor cells were separated from the tumor infiltrating lymphocytes. Tumor cells were stained for LAP and analyzed by flow cytometry. I: GFP⁺ CD4⁺ cells were sorted from CD45.2 FOXP3-EGFP mice, then cultured <i>in vitro</i> under TCR-stimulating conditions. We added culture supernatant of CT26 treated for 24 h with PBS of BLM. In some wells, blocking anti-TGFβ antibody was added. After three days of culture, the cells were incubated with BrdU for 3 h, and then proceeded to BrdU detection by flow cytometry. J: Same as A, and mice received injection of blocking anti-TGFβ antibody. All data presented are representative of one out of two (panels B, D, E, G and I) or three experiments (panels A, F, H and J). Graphs show mean +/− SEM.</p

BLM in vivo antitumor effect through immune-based mechanisms.

<p>A: Footpad injection of different vaccines was performed: PBS-, DOX- or BLM-treated B16-OVA, or ovalbumin protein. 5 days later, the popliteal lymph nodes were harvested and cells were rechallenged with OVA peptide SIINFEKL. After 3 days of culture, IFNγ was titrated in the supernatant using ELISA method. B: shControl CT26 (left panel) or shCRT CT26 (right panel) were injected in the flank of mice. When the tumor reached about 25 mm², mice were treated with PBS or BLM and tumor growth was monitored with a caliper over time. C and D: CT26 cells were injected in the flank of mice. When the tumor reached about 25 mm², mice were treated with PBS or BLM. Some of the mice received injection of isotype control or depleting anti-CD8 (C) or anti-IFNγ (D). We monitored tumor growth with a caliper over time. All data presented are representative of one out of two experiments. Graphs show mean +/− SEM.</p

BLM treatment triggers characteristic events of immunogenic cell death.

<p>A: Western blotting analysis of phosphorylated, total eIF2α and β-actin in CT26 treated 24 h with the indicated drugs. B: indicated tumor cells were treated <i>in vitro</i> with different chemotherapeutic drugs for 24 h, then CRT (left panel) or ERp57 (right panel) exposure was detected by flow cytometry. C: tumor cells were treated with BLM, with or without N-acetylcystein (NAC) for 24 h, then CRT (left panel) or ERp57 (right panel) exposure was detected by flow cytometry. C: HMGB1 was titrated with ELISA method in supernatant of CT26 cells treated 24 h with indicated drugs. E: CT26 cells were cultured on glass slides and treated with indicated drugs for 24 h. The cells were then submitted to anti-LC3 (green) and DAPI (blue) labeling and observed under an epifluorescence microscope. F: CT26 cells were treated with indicated drugs for 24 h, then stained using Cyto-ID autophagy detection kit and analysed by flow cytometry. G: ATP was titrated in supernatant of CT26 treated with indicated drugs for 24 h. All data presented are representative of one out of three experiments. B-D, E and F show mean +/− SEM.</p

BLM anti-tumor effect is enhanced by Treg or TGFβ depletion.

<p>Five hundred thousand CT26 cells were injected in the flank of mice. When the tumor reached about 25 mm², mice were treated with PBS or BLM. Some of the mice received injections of isotype control antibody or depleting anti-CD4 (A), anti-CD25 (B) or blocking anti-TFGβ (C) antibodies. Tumor growth was monitored with a caliper over time. All data presented are representative of one out of two experiments. Graphs show mean +/− SEM.</p

BLM has no obvious effect on innate immune cells.

<p>A: Spleens from CT26 tumor-bearing mice, treated with PBS or BLM, were harvested and the cells were analyzed through flow cytometry. The graph shows proportion of different splenocyte populations identified by immunostaining. B: Mice received a single injection of PBS, LPS or BLM. One day later, spleens were collected and the maturation status of dendritic cells defined as CD11c^hi and MHC-II⁺ was assessed by flow cytometry, with CD40 (left panel) and CD86 (right panel) staining. C: We injected CT26 cells in the thigh of mice. When the tumor reached about 25 mm², we treated them with PBS, single or five BLM injections. One day after last injection, inguineal lymph nodes were collected and NK cell activation was analysed through CD69 immunostaining. D: Same as A with further identification of MDSC sub-populations with Ly6G and Ly6C immunostaining. The cells are gated as CD11b^hi among the live cells. E: MDSC from PBS- or BLM- treated CT26-bearing mice were isolated from spleen and co-cultured with OT-I in presence of SIINFEKL. After 3 days of culture, IFNγ was titrated in the supernatant using ELISA method. All data presented are representative of one out of three experiments. Graphs show mean +/− SEM.</p