Journal: Frontiers in Immunology

Article Title: Identification of a distinct cluster of GDF15 high macrophages induced by in vitro differentiation exhibiting anti-inflammatory activities

doi: 10.3389/fimmu.2024.1309739

Figure Lengend Snippet: GDF15 high macrophages exhibited reduced inflammatory activation in vitro . (A) Expression patterns of potential substitute cell surface markers for GDF15 based on the scRNA-seq data. (B) Flow cytometry results showing that rat BMMNC-derived macrophages contained a minor fraction of TNFSF9 high cells, whose expression level was correlated with that of GDF15 (from 3 independent experiments). (C) Flow cytometry verification of the correlation between TNFSF9 and GDF15 expressions in human PBMNC-derived macrophages (from 2 independent experiments). (D) Real-time PCR results showing that GDF15 high macrophages (H) exhibited reduced expressions of TNF-α, IL-1β and IL-6 in response to LPS stimulation (1 μg/mL for 6 hr), as compared to GDF15 low cells (L). Rat BMMNC-derived macrophages were FACS purified using TNFSF9 as a substitute marker for GDF15, and primed with IFN-γ (10 ng/mL for 12 hr). (E) Boyden chamber cell migration assay showing that GDF15 high macrophages (H) exhibited reduced migratory activity as compared to GDF15 low cells (L) in the absence and presence of LPS stimulation. (F) Representative fluorescent microscopic images and quantitative data showing that GDF15 high macrophages exhibited reduced phagocytic activity in the presence of LPS stimulation as compared to GDF15 low cells. Phagocytosis was assessed by internalization of fluorochrome-labeled latex beads (orange color). The macrophages were from CD68pro-GFP rats. Data were mean ± SEM. * P < 0.05, one-way ANOVA. NS, no significance.

Article Snippet: The following antibodies were used: anti-CD68 (#66231-2-Ig), anti-CD86 (#APC-65165), anti-CD80 (#PE-65083), anti-CD206 (#APC-65155) and anti-CD163 (#APC-65169) were from Proteintech; anti-GDF15 (#66231-2-Ig) was from ABclonal; anti-GDF15 (#BM5690) was from Boster (Wuhan); anti-TNFSF9 (tumor necrosis factor superfamily member 9) (#bs-3851R-APC) was from Bioss (Beijing, China); anti-interleukin (IL)-1β (#12-7018-41) was from Thermo Fisher; anti-IL-4 (#500808) was from BioLegend (San Diego, CA, USA).

Techniques: Activation Assay, In Vitro, Expressing, Flow Cytometry, Derivative Assay, Real-time Polymerase Chain Reaction, Purification, Marker, Cell Migration Assay, Activity Assay, Labeling

Journal: Translational Oncology

Article Title: Oncolytic vaccinia virus armed with 4–1BBL elicits potent and safe antitumor immunity and enhances the therapeutic efficiency of PD-1/PD-L1 blockade in a pancreatic cancer model

doi: 10.1016/j.tranon.2024.102151

Figure Lengend Snippet: VV-ΔTK-4L expressed 4–1BBL on the tumor cell surface and showed a similar cytotoxicity to the VV-ΔTK control virus. A: Schematic diagram of the construction of the 4–1BBL recombinant oncolytic virus. VV-ΔTK-4L was generated by the homologous recombination of murine 4–1BBL into the tk locus of the VV genome. B–D: Murine pancreatic cancer cell line Pan02 (1 × 10 5 cells/well) in a 24-well/plate was infected with VV-ΔTK-4L or mock-infected with VV-ΔTK at different MOIs. The cell pellets were harvested 24 h post-infection to detect A34R and 4–1BBL expression using RT-qPCR (B, C) and cell-surface staining and flow cytometric assay (D). E–H: Tumor cells were plated at 1.0 × 10 4 cells/well (except B16-F10 cells, which were plated at 5 × 10 3 cells/well) in 96-well plates and infected with VV-ΔTK-4L or VV-ΔTK viruses the next day at different MOIs. Six replicate wells were set up for each sample. Cell viability was measured using an MTT assay at 48 h post-infection. MOI, multiplicity of infection; ns, not significant.

Article Snippet: Single cells from tumor tissues, single splenocytes, or in vitro virus-infected cells were blocked with α-CD16/32 Ab (Cat#156603; Biolegend) and then stained with antibodies against mouse 4–1BBL (Cat#50067-R014-A; Sino Biological, Beijing, People's Republic of China; 1:2000), CD45 (BB700, Cat#566440; BD Biosciences, Franklin Lakes, NJ, USA; 1:500), CD11b (PE, Cat#101208, Boilegend; 1:500), CD11c (APC, Cat#117310, Biolegend; 1:500), CD103 (BV421, Cat#562771, BD Biosciences; 1:500), CD8a (APC—Cy7, Cat#100714, Biolegend; FITC, Cat#100706, Biolegend; 1:500), CD3 (PE, Cat#120031, eBioscience; FITC, Cat#11–0032–82, eBioscience; 1:500), CD4 (PE-eFluor 610, Cat#61–0041–82, eBioscience; FITC, Cat#100406, Biolegend; 1:500), NK1.1 (FITC, Cat#108706, Biolegend; 1:500), CD44 (FITC, Cat#103006, Biolegend; 1:500), CD62L (BV421, Cat#562910, BD Biosciences; 1:500), PD-1 (PE, Cat#135205, Biolegend; 1:500), 4–1BB (BV605, Cat#740364, BD Biosciences; 1:500), CD107a (BV786, Cat#564349, BD Biosciences; 1:500), CTLA-4 (PE-CF594, Cat#106318, Biolegend; 1:500), KLRG-1 (APC, 17–5893–82, eBioscience, San Diego, CA, USA; 1:500), TIGIT (BV421, Cat#565270, BD Biosciences; 1:500), Tim-3 (BV786, Cat#747621, BD Biosciences; 1:500), Perforin (APC, Cat#154304, Biolegend; 1:200), GZMB (PE, Cat#12–8898–82, eBioscience; 1:200), tumor necrosis factor (TNF)-α (FITC, Cat#563943, BD Biosciences; 1:200), interferon (IFN)-γ (PE, Cat#505808, Biolegend; 1:200), Foxp3 (PE, Cat#560408, BD Biosciences; 1:200), and Ki-67 (BV-786, Cat#417–5698–82, eBioscience; 1:200).

Techniques: Control, Virus, Recombinant, Generated, Homologous Recombination, Infection, Expressing, Quantitative RT-PCR, Staining, Flow Cytometry, MTT Assay

Journal: Translational Oncology

Article Title: Oncolytic vaccinia virus armed with 4–1BBL elicits potent and safe antitumor immunity and enhances the therapeutic efficiency of PD-1/PD-L1 blockade in a pancreatic cancer model

doi: 10.1016/j.tranon.2024.102151

Figure Lengend Snippet: VV-ΔTK-4L administration produces 4–1BBL in tumors and is effective in therapeutic tumor models. A–C: C57BL/6 mice were intraperitoneally injected with 1 × 10 6 Pan02 cells per mouse and treated with PBS, or VV-ΔTK-4L or VV-ΔTK viruses at 2 × 10 8 PFU/mouse 9 days post tumor inoculation ( n = 8/group). On day 14 post tumor inoculation, the mice were sacrificed (A). Tumor tissues (B) and spleens (C) were collected for quantitative assay for 4–1BBL expression by RT-qPCR. D–H: C57BL/6 mice were intraperitoneally injected with 1 × 10 6 Pan02 cells per mouse and treated with PBS, or VV-ΔTK-4L or VV-ΔTK viruses at 2 × 10 8 PFU/mouse 9 days post tumor inoculation ( n = 10/group; D). Mouse survival was observed daily (E, F), and the other cohort of mice was enrolled to repeat this experiment (G, H). A log-rank (Mantel–Cox) test was used to analyze survival rates. A T-test was applied to compare 4–1BBL expression levels and survival periods among mice. * P < 0.05; ** P < 0.01. ns: not significant.

Article Snippet: Single cells from tumor tissues, single splenocytes, or in vitro virus-infected cells were blocked with α-CD16/32 Ab (Cat#156603; Biolegend) and then stained with antibodies against mouse 4–1BBL (Cat#50067-R014-A; Sino Biological, Beijing, People's Republic of China; 1:2000), CD45 (BB700, Cat#566440; BD Biosciences, Franklin Lakes, NJ, USA; 1:500), CD11b (PE, Cat#101208, Boilegend; 1:500), CD11c (APC, Cat#117310, Biolegend; 1:500), CD103 (BV421, Cat#562771, BD Biosciences; 1:500), CD8a (APC—Cy7, Cat#100714, Biolegend; FITC, Cat#100706, Biolegend; 1:500), CD3 (PE, Cat#120031, eBioscience; FITC, Cat#11–0032–82, eBioscience; 1:500), CD4 (PE-eFluor 610, Cat#61–0041–82, eBioscience; FITC, Cat#100406, Biolegend; 1:500), NK1.1 (FITC, Cat#108706, Biolegend; 1:500), CD44 (FITC, Cat#103006, Biolegend; 1:500), CD62L (BV421, Cat#562910, BD Biosciences; 1:500), PD-1 (PE, Cat#135205, Biolegend; 1:500), 4–1BB (BV605, Cat#740364, BD Biosciences; 1:500), CD107a (BV786, Cat#564349, BD Biosciences; 1:500), CTLA-4 (PE-CF594, Cat#106318, Biolegend; 1:500), KLRG-1 (APC, 17–5893–82, eBioscience, San Diego, CA, USA; 1:500), TIGIT (BV421, Cat#565270, BD Biosciences; 1:500), Tim-3 (BV786, Cat#747621, BD Biosciences; 1:500), Perforin (APC, Cat#154304, Biolegend; 1:200), GZMB (PE, Cat#12–8898–82, eBioscience; 1:200), tumor necrosis factor (TNF)-α (FITC, Cat#563943, BD Biosciences; 1:200), interferon (IFN)-γ (PE, Cat#505808, Biolegend; 1:200), Foxp3 (PE, Cat#560408, BD Biosciences; 1:200), and Ki-67 (BV-786, Cat#417–5698–82, eBioscience; 1:200).

Techniques: Injection, Expressing, Quantitative RT-PCR

Journal: Translational Oncology

Article Title: Oncolytic vaccinia virus armed with 4–1BBL elicits potent and safe antitumor immunity and enhances the therapeutic efficiency of PD-1/PD-L1 blockade in a pancreatic cancer model

doi: 10.1016/j.tranon.2024.102151

Figure Lengend Snippet: VV-ΔTK-4L administration did not produce obvious hepatoxicity in therapeutic tumor models. A: Schematic diagram of the hepatotoxicity assay for α4–1BB mAb and VV-ΔTK-4L treatment in a pancreatic mouse model. B–E: C57BL/6 mice were intraperitoneally injected with 1 × 10 6 of Pan02 cells and treated with agonist α4–1BB mAb, rat IgG2a isotype control, PBS, VV-ΔTK-4L, or VV-ΔTK 9 days post tumor inoculation. The treated mice and healthy mice ( n = 5/group) were sacrificed 21 days post tumor inoculation to harvest their livers, which were then photographed (B), weighed (C), and underwent blood collection to measure ALT (D) and AST (E) levels in sera. Liver tissues were collected to perform RT-qPCR to analyze 4–1BBL expression (F). * P < 0.05; ** P < 0.01. ns: not significant.

Article Snippet: Single cells from tumor tissues, single splenocytes, or in vitro virus-infected cells were blocked with α-CD16/32 Ab (Cat#156603; Biolegend) and then stained with antibodies against mouse 4–1BBL (Cat#50067-R014-A; Sino Biological, Beijing, People's Republic of China; 1:2000), CD45 (BB700, Cat#566440; BD Biosciences, Franklin Lakes, NJ, USA; 1:500), CD11b (PE, Cat#101208, Boilegend; 1:500), CD11c (APC, Cat#117310, Biolegend; 1:500), CD103 (BV421, Cat#562771, BD Biosciences; 1:500), CD8a (APC—Cy7, Cat#100714, Biolegend; FITC, Cat#100706, Biolegend; 1:500), CD3 (PE, Cat#120031, eBioscience; FITC, Cat#11–0032–82, eBioscience; 1:500), CD4 (PE-eFluor 610, Cat#61–0041–82, eBioscience; FITC, Cat#100406, Biolegend; 1:500), NK1.1 (FITC, Cat#108706, Biolegend; 1:500), CD44 (FITC, Cat#103006, Biolegend; 1:500), CD62L (BV421, Cat#562910, BD Biosciences; 1:500), PD-1 (PE, Cat#135205, Biolegend; 1:500), 4–1BB (BV605, Cat#740364, BD Biosciences; 1:500), CD107a (BV786, Cat#564349, BD Biosciences; 1:500), CTLA-4 (PE-CF594, Cat#106318, Biolegend; 1:500), KLRG-1 (APC, 17–5893–82, eBioscience, San Diego, CA, USA; 1:500), TIGIT (BV421, Cat#565270, BD Biosciences; 1:500), Tim-3 (BV786, Cat#747621, BD Biosciences; 1:500), Perforin (APC, Cat#154304, Biolegend; 1:200), GZMB (PE, Cat#12–8898–82, eBioscience; 1:200), tumor necrosis factor (TNF)-α (FITC, Cat#563943, BD Biosciences; 1:200), interferon (IFN)-γ (PE, Cat#505808, Biolegend; 1:200), Foxp3 (PE, Cat#560408, BD Biosciences; 1:200), and Ki-67 (BV-786, Cat#417–5698–82, eBioscience; 1:200).

Techniques: Injection, Control, Quantitative RT-PCR, Expressing