Journal: bioRxiv

Article Title: IL-12-expressing highly immunogenic recombinant modified vaccinia virus Ankara reprograms tumor-infiltrating myeloid cells to overcome immune resistance

doi: 10.1101/2022.09.25.509429

Figure Lengend Snippet: a, Schematic diagram of the experimental schedule. 5 x10 5 of B16-F10 cells were intradermally implanted into the right flanks of WT C57BL/6J mice. IT MQ833 injections were given twice a week once the tumors were established. I.p. αCD8, αCD4, αNK1.1, and αLy6G antibodies were given one day prior to every virus injection. I.p. αCSF1 were given every 5 days starting at one day prior to the first virus injection. Tumor size and mice survival were monitored. b, Kaplan-Meier survival curve of mice in each treatment group. PBS was used as a control. Survival data were analyzed by log-rank (Mantel-Cox) test ( n=10-20; *P < 0.05, **P < 0.01, ****P < 0.0001 ). c , Tumor volumes of mice separated by group over days post treatment. ( d ,) Schematic diagram of the flow cytometry analysis experiment. e , f , Contour plots ( e ) and bar graphs ( f ) showing percentages of iNOS + cells among CD45 + CD3 - CD11b + Ly6G + neutrophils (top row), CD45 + CD3 - CD68 + CD11b + macrophages (middle row), and percentages of CD206 + cells among macrophages (bottom row) in MQ833 or PBS treated tumors. g,h , Survival study for B16-F10 tumor-bearing mice treated with MQ833 in a Nos2 -/- mouse model. 5x10 5 of B16-F10 cells were intradermally implanted into the right flank of Nos2 -/- mice. IT MQ833 injections were given twice a week once the tumors were established. Tumor size and mice survival were monitored. g , Kaplan-Meier survival curve of mice in each treatment group. PBS was used as a control. (n=10) Survival data were analyzed by log-rank (Mantel-Cox) test ( n=5-10; *P < 0.05, ***P < 0.001, ****P < 0.0001 ). h , Tumor volumes of mice separated by group over days post treatment. i,j , scRNA-seq analysis of CD45 + cells isolated from rMVAΔE3L or MQ833 treated tumors.. i , UMAP display showing nos2 expression (right panel) in highlighted clusters (left panel) j , Heat maps showing average expression of selected antitumor marker genes in all the CD45 + cells (first panel), neutrophil (second panel), M1-like macrophage (third panel), and monocyte (fourth panel) clusters divided by treatment groups.

Article Snippet: The following antibodies were used for flow cytometric staining: CD45 Pacific blue (clone 30-F11, Biolegend), CD3 BUV395 (clone 145-2C11, BD), CD4 BUV737 (clone RM4-5, BD), CD8Alexa Fluor 700 (clone 53-6.7, Biolegend), OX40 BV605 (clone OX-86, Biolegend), TIM3 BV711 (clone RMT3-23, Biolegend), PD-1 AF647 (clone RMPI-30, BD), KLRG1 APC-Cy7 (clone 2F1/KLRG1, Biolegend), Foxp3 PerCP-Cy5.5 (clone FJK-16s, Invitrogen), Ki67 BV786 (clone B56, BD), TOX PE (clone REA473, Miltenyi Biotec), Granzyme B PE-Texas red (clone GB11, Invitrogen), CD11b APC-eFluor780 (clone M1/70, eBioscience), Ly6G PE-Cy7 (clone 1A8, BD), F4/80 APC (clone BM8, Biolegend), CD68 BV421 (clone FA-11, Biolegend), iNOS PE (clone W16030C, Biolegend).

Techniques: Injection, Flow Cytometry, Isolation, Expressing, Marker

Journal: bioRxiv

Article Title: IL-12-expressing highly immunogenic recombinant modified vaccinia virus Ankara reprograms tumor-infiltrating myeloid cells to overcome immune resistance

doi: 10.1101/2022.09.25.509429

Figure Lengend Snippet: a, Schematic diagram of the survival and flow cytometry experimental design. 5 x10 5 of B16-F10 cells were intradermally implanted into the right flank of Rag1 -/- mice. IT MQ833 injections were given twice weekly once the tumors were established. For survival study, tumor size and mice survival were monitored. For flow cytometric analysis, tumors were harvested at day 14 post implantation and tumor infiltrating immune cells were stained by fluorescent antibodies and analyzed by flow cytometry. b, Kaplan-Meier survival curve of mice in each treatment group. PBS was used as a control. Survival data were analyzed by log-rank (Mantel-Cox) test ( n=5-10; ****P < 0.0001 ). c , Tumor volumes of mice separated by group over days post treatment. d , e , Dot plos ( d ) and bar graphs ( e ) showing percentage of CD45 + CD3 - CD11b + Ly6G + neutrophils among total CD45 + cells (top row) and iNOS + cells among neutrophils (bottom row) in MQ833 or PBS treated tumors. f,g , 1 x10 6 of B2m -/- B16-F10 cells were intradermally implanted into the right flank of WT C57BL/6J mice. IT MQ833 injections were given twice weekly once the tumors were established. tumor sizes and mice survival were monitored. f , Kaplan-Meier survival curve of mice in each treatment group. PBS was used as a control. Survival data were analyzed by log-rank (Mantel-Cox) test ( n=10; **P < 0.01, ****P < 0.0001 ). g , Tumor volumes of mice separated by group over days post treatment. h , Mice rejected primary B16-F10 or B2m -/- B16-F10 tumors were rechallenged with a lethal dose of B16-F10 tumor cells. Kaplan-Meier survival curve of the rechallenged mice. i , Working model illustrating the mechanisms of action MQ833.

Article Snippet: The following antibodies were used for flow cytometric staining: CD45 Pacific blue (clone 30-F11, Biolegend), CD3 BUV395 (clone 145-2C11, BD), CD4 BUV737 (clone RM4-5, BD), CD8Alexa Fluor 700 (clone 53-6.7, Biolegend), OX40 BV605 (clone OX-86, Biolegend), TIM3 BV711 (clone RMT3-23, Biolegend), PD-1 AF647 (clone RMPI-30, BD), KLRG1 APC-Cy7 (clone 2F1/KLRG1, Biolegend), Foxp3 PerCP-Cy5.5 (clone FJK-16s, Invitrogen), Ki67 BV786 (clone B56, BD), TOX PE (clone REA473, Miltenyi Biotec), Granzyme B PE-Texas red (clone GB11, Invitrogen), CD11b APC-eFluor780 (clone M1/70, eBioscience), Ly6G PE-Cy7 (clone 1A8, BD), F4/80 APC (clone BM8, Biolegend), CD68 BV421 (clone FA-11, Biolegend), iNOS PE (clone W16030C, Biolegend).

Techniques: Flow Cytometry, Staining

Journal: Frontiers in Immunology

Article Title: Stroke Exacerbates Cancer Progression by Upregulating LCN2 in PMN-MDSC

doi: 10.3389/fimmu.2020.00299

Figure Lengend Snippet: PMN-MDSC may be the major source of the increased LCN2 in the tumor after stroke. (A) Gating strategy of the MDSC populations in the tumor 3 days after sham or stroke surgery. (B,C) LCN2 expression in M-MDSC (CD45 + CD11b + Ly6G low Ly6C hi ) and PMN-MDSC (CD45 + CD11b + Ly6G hi Ly6C low ) in the tumor tissues after stroke ( n = 6/group. * P ≤ 0.05, ** P ≤ 0.01).

Article Snippet: Antibodies specific for the mouse cell surface markers CD45-PE-A (catalog: 25-0451-82, ebioscience, USA), CD11b-APC (catalog: 101212, BD), Ly-6G-BV421 (catalog: 1127628C, BD, USA), Ly6C-PE-Cy7 (catalog: 128018, BD, USA), LCN2-Alexfluo488 (catalog: ab63929, Abcam, Britain), NK1.1-PE-A (catalog: 12-5941-81, ebioscience, USA), CD3-APC-A (catalog: 17-0032-80, ebioscience, USA), CD11c-APC-A (catalog: 17-0114-82, ebioscience, USA), F4/80 (catalog: 4341619, Invitrogen, USA).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Stroke Exacerbates Cancer Progression by Upregulating LCN2 in PMN-MDSC

doi: 10.3389/fimmu.2020.00299

Figure Lengend Snippet: Flow cytometry of LCN2 expression in M-MDSCs and PMN-MDSCs from blood. (A) Gating strategy of the MDSC populations in blood 3 days after sham or stroke surgery. (B,C) LCN2 expression in M-MDSC (CD45 + CD11b + Ly6G low Ly6C hi ) and PMN-MDSC (CD45 + CD11b + Ly6G hi Ly6C low ) in the blood after stroke ( n = 6/group. * P ≤ 0.05).

Article Snippet: Antibodies specific for the mouse cell surface markers CD45-PE-A (catalog: 25-0451-82, ebioscience, USA), CD11b-APC (catalog: 101212, BD), Ly-6G-BV421 (catalog: 1127628C, BD, USA), Ly6C-PE-Cy7 (catalog: 128018, BD, USA), LCN2-Alexfluo488 (catalog: ab63929, Abcam, Britain), NK1.1-PE-A (catalog: 12-5941-81, ebioscience, USA), CD3-APC-A (catalog: 17-0032-80, ebioscience, USA), CD11c-APC-A (catalog: 17-0114-82, ebioscience, USA), F4/80 (catalog: 4341619, Invitrogen, USA).

Techniques: Flow Cytometry, Expressing