Journal: Cell reports

Article Title: VISTA promotes the metabolism and differentiation of myeloid-derived suppressor cells by STAT3 and polyamine-dependent mechanisms

doi: 10.1016/j.celrep.2023.113661

Figure Lengend Snippet: (A and B) WT and Vsir −/− BM-derived MDSCs were cultured with GM-CSF and IL-6 for 4 days either in normoxia or exposed to hypoxia during the last 24 h. Total cell lysates were extracted and examined for ODC1 expression (A). Intracellular putrescine levels were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (B) (n = 4 for normoxia replicates, n = 3 for hypoxia replicates). (c) Hypoxic WT and Vsir −/− MDSCs were cultured as in (A) in the presence of putrescine (800 μM) or vehicle control. Cells were rested for 8 h before being restimulated with IL-6. Total cell lysates were examined by western blotting. The ratios of phosphorylated-STAT3 Y705 and STAT3 S727 vs. total STAT3 were quantified using ImageJ. (D) WT BM-derived MDSCs were cultured as in (A) in the presence of DFMO (400 μM) or vehicle control for 4 days. Total cell lysates were generated, and levels of pSTAT3 Y705 and total STAT3 were examined by western blotting. (E) WT BM progenitors (n = 3) were cultured for 4 days as in (A). On day +1, CK2 inhibitor (20 μM), putrescine (800 μM), vehicle control, or combined drugs were added to the culture medium and continuously cultured for another 3 days. During the last 24 h, cells were treated with hypoxia before flow analysis to detect Arg1 expression. (F) WT and Vsir −/− BM-MDSCs (n = 3) were cultured for 4 days as in (A). On day +1, STAT3 inhibitor (2 mM), putrescine (1 mM), vehicle control, or combined drugs were added to the culture medium. Cells were treated with hypoxia during the last 24 h before flow analysis. All experiments were repeated at least three times, and representative results are shown. (G and H) WT and Vsir −/− BM-MDSCs were cultured for 4 days under normoxia or exposed to hypoxia during the last 24 h. Mitochondrial respiration of normoxic (G) and hypoxic MDSCs (H) was examined using the Seahorse XF Cell Mito stress test. The OCR and ECAR were recorded simultaneously. (I) Basal OCR, maximal OCR, SRC, and ECAR are summarized. (J) The bioenergetic profiles of WT and Vsir −/− MDSCs were plotted based on basal OCR and ECAR levels. (K and L) To rescue the defective mitochondrial function in Vsir −/− MDSCs, putrescine (800 μM) was added to the culture medium on day +1. Cells were cultured for a total of 4 days and treated with hypoxia during the last 24 h. M-MDSCs were purified and examined using the Mito stress test. OCR was recorded (K). Basal OCR, maximal OCR, and SRC are summarized (L). (M and N) Total cell lysates were extracted, and the expression of ETC complex V and Arg1 was examined. β-Actin was used as the loading control. The ratios of each protein vs. β-actin were quantified using ImageJ. All experiments were repeated at least three times, and representative results are shown. Statistical analysis: n = 8 (WT, normoxia), 9 (KO, normoxia), 8 (WT, hypoxia), and 10 (KO, hypoxia) replicates (A–C); n = 5 (WT control), 4 (WT putrescine), 14 (KO, control), and 8 (KO putrescine) replicates (D). All data were presented as mean ± SEM. * p < 0.05; ** p < 0.025; *** p < 0.005; **** p < 0.0001.

Article Snippet: For ex vivo treatment with inhibitors or chemicals, DFMO (400 μM) (Tocris Bioscience, Fisher Scientific, Cat no. 27-611-0), or putrescine (800 μM) (Fisher Scientific, Cat no. ICN10044105), or STAT3 inhibitor (2 μM), or CK2 inhibitor (20 μM), or combinations when indicated, were added to BM cell culture on day 2.

Techniques: Derivative Assay, Cell Culture, Expressing, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Control, Western Blot, Generated, Purification

Journal: Cell reports

Article Title: VISTA promotes the metabolism and differentiation of myeloid-derived suppressor cells by STAT3 and polyamine-dependent mechanisms

doi: 10.1016/j.celrep.2023.113661

Figure Lengend Snippet: (A) WT and Vsir −/− BM progenitor cells (n = 3) were cultured with GM-CSF and IL-6 for 5 days and exposed to hypoxia during the last 24 h. Nonadherent and adherent cells were harvested and pooled. MoDCs (CD11b + CD11c + MHCII + ), M-MDSCs (CD11b + CD11c neg Ly6C + Ly6G neg ), and PMN-MDSCs (CD11b + CD11c neg Ly6C + Ly6G + ) were examined by flow cytometry. (B) WT BM progenitor cells (n = 3) were cultured as in (A). On day +1, putrescine (800 μM), DFMO (400 μM), or combined drugs were added to the culture medium. Cells were exposed to hypoxia during the last 24 h before being harvested and examined by flow cytometry. (C–E) WT (CD45.1) and Vsir −/− (CD45.2) mixed BM chimera mice were inoculated with MC38 colon cancer cells (1 × 10 6 ). Tumor tissues were harvested when tumors reached 8–9 mm in diameter (around day 18). Tumor-associated M-MDSCs (CD11b + Ly6C hi CD11c neg Ly6G neg ), PMN-MDSCs (CD11b + Ly6C lo CD11c neg Ly6G + ), and moDCs (CD11b + CD11c hi MHC class II + ) were detected by flow cytometry. Percentages of each subset among the total CD11b + myeloid cells are shown (C) (n = 6). Gene expression of Arg1 , nos2 , and Odc1 in purified M-MDSCs was analyzed by RT-qPCR (D) (n = 3). Protein expression of Arg1 and iNOS in M-MDSCs was examined by flow cytometry (E) (n = 6). (F and G) Transcriptomic analyses of WT and Vsir −/− tumor-associated M-MDSCs. Numbers of DEGs are illustrated in volcano plots (F). GSEAs revealed molecular pathways that were significantly upregulated or downregulated in Vsir −/− tumor M-MDSCs. Normalized enrichment scores (NESs) and a graphic view of the leading edge enrichment plot are shown (G). (H) Tumor-associated M-MDSCs from the BM chimera mice were stained with MitoTracker Green and Deep Red and examined by flow cytometry (n = 5). All data were presented as mean ± SEM. * p < 0.05; ** p < 0.025; *** p < 0.005; **** p < 0.0001. All experiments were repeated at least two or three times, and representative results are shown.

Article Snippet: For ex vivo treatment with inhibitors or chemicals, DFMO (400 μM) (Tocris Bioscience, Fisher Scientific, Cat no. 27-611-0), or putrescine (800 μM) (Fisher Scientific, Cat no. ICN10044105), or STAT3 inhibitor (2 μM), or CK2 inhibitor (20 μM), or combinations when indicated, were added to BM cell culture on day 2.

Techniques: Cell Culture, Flow Cytometry, Gene Expression, Purification, Quantitative RT-PCR, Expressing, Staining

Journal: Cell reports

Article Title: VISTA promotes the metabolism and differentiation of myeloid-derived suppressor cells by STAT3 and polyamine-dependent mechanisms

doi: 10.1016/j.celrep.2023.113661

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For ex vivo treatment with inhibitors or chemicals, DFMO (400 μM) (Tocris Bioscience, Fisher Scientific, Cat no. 27-611-0), or putrescine (800 μM) (Fisher Scientific, Cat no. ICN10044105), or STAT3 inhibitor (2 μM), or CK2 inhibitor (20 μM), or combinations when indicated, were added to BM cell culture on day 2.

Techniques: Virus, Western Blot, Recombinant, Staining, cDNA Synthesis, SYBR Green Assay, Isolation, Mutagenesis

Journal: Science advances

Article Title: Tumor-derived semaphorin 4A improves PD-1-blocking antibody efficacy by enhancing CD8 + T cell cytotoxicity and proliferation.

doi: 10.1126/sciadv.ade0718

Figure Lengend Snippet: Fig. 6. The Sema4A–plexin B axis induces CD8+ T cell proliferation by promoting the polyamine biosynthesis pathway. (A) mRNA expression levels of polyamine biosynthesis pathway components in CD8+ T cells were compared after anti-CD3 antibody stimulation versus anti-CD3 antibody plus rSema4A stimulation (n = 3). (B) Summary of an analysis of metabolites involved in the polyamine biosynthesis pathway of CD8+ T cells simulated with anti-CD3 antibody (n = 3) versus anti-CD3 antibody plus rSema4A stimulation (n = 4). ND, not detected, *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test. (C) Percentage of IFN-γ+ and mean fluorescence intensity (MFI) of Ki- 67 in CD8+ T cells after anti-CD3 antibody stimulation versus anti-CD3 antibody plus rSema4A stimulation with or without sardomozide dihydrochloride (10 μM) and eflornithine hydrochloride hydrate (500 μM). n = 3 per group, *P < 0.05, **P < 0.01, one-way ANOVA. SAM, S-adenosylmethionine; dcSAM, decarboxylated S-adenosyl- methionine; MTA, methylthioadenosine. Results are expressed as the mean ± SEM.

Article Snippet: In the inhibition experiments, rapamycin (25 ng/ml, Sigma), sardomozide dihydrochloride (10 μM, Medchemexpress), and eflornithine hydrochloride hydrate (500 μM, Medchemexpress) were added.

Techniques: Expressing, Fluorescence