Journal: iScience

Article Title: Macrophage repolarization by immune checkpoint blockade drives T cell engagement in the tumor microenvironment

doi: 10.1016/j.isci.2025.113538

Figure Lengend Snippet: TAMs within checkpoint antibody-treated tumors make wide contact interfaces with infiltrating CD8 + T cells (A) Experimental schematic of subcutaneous B16-F10 melanoma tumor mouse model treated with 3 doses of 200 μg of IgG2A isotype control or combination 200 μg anti-PD-1 and 200 μg anti-PD-L1 antibody intraperitoneally and harvested on day 16. (B) Tumor growth curves of isotype control and combo-treated mice. Data shown represent combined data from 3 experiments with n = 3 mice per experimental group. (C) Volcano plot of transcriptomic differences of total tumor cells isolated from isotype control and combo-treated B16-F10 tumors. Gray indicates no significant change, and red indicates differentially upregulated genes (148 genes). (D) Representative images of isotype control and combo-treated B16-F10 tumor cryosections stained with antibodies against TCRβ (green), F4/80 (orange), and DAPI (blue). Scale bars represent 50 μm; arrows denote T cells. (E) Quantification of mean T cell count. (F) TAM area in fields of view. Data shown represent combined data from 3 to 4. experiments, 2 tumors per experimental group; number of cells: Iso = 29, Combo = 28. Data displayed as means ± SEMs. (G) Representative images of CD8α (green)-, F4/80 (orange)-, and DAPI (blue)-stained tumor cryosections from isotype control and combo-treated tumors. Scale bars represent 5 μm. (H) Quantification of mean CD8 + T cell to TAM distance. Direct interaction characterized by < 3 μm distance. Data shown represent combined data from 3 experiments, 2 tumors per experimental group; number of cells: Iso = 23, Combo = 25. Data displayed as means ± SEMs. (I) Live two-photon imaging of isotype control and combo-treated LLC tumors from MacBlue; CD11c -mCherry mice with adoptively transferred EGFP-CD8 + T cells (yellow). CD8 + T cell and TAM interactions denoted by magenta arrows. Scale bars represent 50 μm. (J) Zoomed in two-photon imaging of (I) with 2 different angles. Scale bars represent 5 μm. (K) Quantification of CD8 + T cell to TAM overlapped volume ratio over time in isotype control and combo-treated tumors at 15 days post-inoculation (dpi). Data shown represent combined data from 3 experiments, 2 tumors per experimental group; number of cells: Iso = 7, Combo = 7, at 40 time points. (L) Live two-photon imaging of isotype control and combo-treated LLC tumors from MacBlue or MAFIA mice with adoptively transferred dsRed-CD8 + T cells (yellow). Macrophages and second harmonic generation displayed in blue. T cell migration tracks displayed as multicolored lines. Scale bars represent 50 μm. (M) Quantification of CD8 + T cell track speed mean in isotype control and combo-treated tumors at 15 dpi. Data shown represent combined data from 3 to 4 experiments, 2 tumors per experimental group; number of cells: Iso = 96, Combo = 184. Data displayed as means ± SEMs. (N) Quantification of CD8 + T cell track straightness in isotype control and combo-treated tumors at 15 dpi. Data displayed as means ± SEMs. Analyzed by Student’s t test, one- or two-way ANOVA; ∗∗ p <0.01, ∗∗∗∗ p <0.0001.

Article Snippet: Anti-mouse IgG2a Isotype Control (clone C1.18.4) , BioXCell , Cat#BE0085.

Techniques: Control, Isolation, Staining, Cell Counting, Imaging, Migration

Journal: iScience

Article Title: Macrophage repolarization by immune checkpoint blockade drives T cell engagement in the tumor microenvironment

doi: 10.1016/j.isci.2025.113538

Figure Lengend Snippet: Anti-PD-L1 treatment increases phagocytic activity of TCMs (A) Experimental schematic of subcutaneous B16-OVA-GFP melanoma tumor mouse model treated with 3 doses of 200 μg of IgG2A isotype control or combination 200 μg anti-PD-1 and 200 μg anti-PD-L1 antibody intraperitoneally and harvested on day 16. (B) Tumor growth curves of isotype control and combo-treated mice. Data shown represent combined data from 2 experiments with n = 3 mice per experimental group. (C) Flow cytometric quantification of CD45 + immune cell infiltrate from isotype control and combo-treated tumors at 16 dpi. Each symbol represents a single tumor, and data are displayed as means ± SEMs. (D) Frequencies of TAMs from isotype control and combo-treated tumors at 16 dpi. (E) Frequencies of GFP+SIINFEKL-MHC1+ TAMs from isotype control and combo-treated tumors at 16 dpi (left panel). Representative flow cytometry plots of GFP and SIINFEKL-MHC1 within F4/80+CD11b+ TAM population (right panel). (F) Representative fluorescence curves of red pHrodo Zymosan particle intake by wild-type TCMs analyzed by Incucyte (top panel). Area under curve (AUC) fold change of red pHrodo signal normalized to isotype control wells (bottom panel). (G) Fluorescence curves of red pHrodo Zymosan particle intake by TCMs from LysM -Cre+ Cd274 fl/fl mice analyzed by Incucyte (top panel). AUC fold change of red pHrodo signal normalized to isotype control wells (bottom panel). Data shown represents combined data from 3 independent experiments with duplicate or triplicate wells and displayed as means ± SEMs. Analyzed by Student’s t test, one-way or two-way ANOVA; ∗ p <0.05.

Article Snippet: Anti-mouse IgG2a Isotype Control (clone C1.18.4) , BioXCell , Cat#BE0085.

Techniques: Activity Assay, Control, Flow Cytometry, Fluorescence

Journal: iScience

Article Title: Macrophage repolarization by immune checkpoint blockade drives T cell engagement in the tumor microenvironment

doi: 10.1016/j.isci.2025.113538

Figure Lengend Snippet: Macrophage repolarization and tumor control by dual PD-1/PD-L1 blockade is dependent on myeloid-specific PD-L1 (A) Tumor growth curves of isotype control, anti-PD-1, anti-PD-L1, and combo treatment of B16-F10 tumors. Data shown represent combined data from 3 experiments with n = 3 mice per experimental group. (B) Flow cytometric quantification of T cell subsets at 16 dpi. Each symbol represents a single tumor, and data are displayed as means ± SEMs. (C) Frequency of TAMs at 16 dpi. (D) Representative flow cytometry plots (left panel), ARG1-/ARG1+ ratio of TAMs (middle panel), and frequency of ARG1-MHC2+ TAMs (right panel) at 16 dpi. (E) Experimental schematic of subcutaneous B16-F10 melanoma tumor model in Pdcd1 −/− (PD-1 KO) mice treated with 3 doses of 200 μg IgG2A isotype control or combination 200 μg anti-PD-1 and 200 μg anti-PDL1 antibody intraperitoneally and harvested on day 16. (F) Tumor growth curves of WT and PD-1 KO mice. Data shown represent combined data from 3 experiments with n = 3 mice per experimental group. (G) Flow cytometric quantification of T cell subsets at 16 dpi. Each symbol represents a single tumor, and data are displayed as means ± SEMs. (H) Frequency of TAMs at 16 dpi. (I) Quantification of ARG1−/ARG1+ ratio of TAMs at 16 dpi (left panel). Representative flow cytometry plots of ARG1+ TAMs (left panel). (J) Experimental schematic of subcutaneous B16-F10 melanoma tumor model in LysM-Cre; Cd274 fl/fl mice treated with 3 doses of 200 μg of IgG2A isotype control or combination 200 μg anti-PD-1 and 200 μg anti-PD-L1 antibody intraperitoneally and harvested on day 16. (K) Tumor growth curves of WT and LysM-Cre; Cd274 fl/fl mice. Data shown represent combined data from 3 experiments with n = 2–3 mice per experimental group. (L) Flow cytometric quantification of T cell subsets at 16 dpi. Each symbol represents a single tumor, and data are displayed as means ± SEMs. (M) Frequency of TAMs at 16 dpi. (N) Frequency of ARG1−/ARG1+ ratio of TAMs at 16 dpi. Analyzed by Student’s t test, one-way or two-way ANOVA; ∗ p <0.05, ∗∗ p <0.01, ∗∗∗ p <0.001, ∗∗∗∗ p <0.0001.

Article Snippet: Anti-mouse IgG2a Isotype Control (clone C1.18.4) , BioXCell , Cat#BE0085.

Techniques: Control, Flow Cytometry