Journal: Cell Reports Medicine

Article Title: Immunogenic tumor cell death and T-cell-derived IFN-γ elicit tumoricidal macrophages to potentiate OX40 immunotherapy

doi: 10.1016/j.xcrm.2026.102699

Figure Lengend Snippet: NOS2-expressing macrophages is associated with response to αOX40 therapy (A) UMAP of monocytes/macrophages subclusters from scRNA-seq data in αOX40-treated MC38-bearing mice. (B) Representative marker genes in the monocyte/macrophage subclusters. (C) Pie chart showing the proportional distribution of monocyte/macrophage subsets of responders and nonresponders. (D) QuSAGE pathway analysis demonstrated enrichment of innate immune and phagocytic signaling pathways in distinct monocyte/macrophage subsets. (E) UMAP showing Mac_C1 signature genes and a heatmap of immune-related gene expression across TAM subclusters ( Z score normalized). (F) Violin plots comparing Nos2 expression levels in Mac_C1 subset between responsive and nonresponsive. (G) Flow cytometry analysis shows the percentage of M1-like (F4/80 + NOS2 + ) and M2-like (F4/80 + CD206 + ) macrophages in tumor tissues of control ( n = 5 mice), nonresponders (with minimal to no response, n = 4 mice), and responders (with a robust therapeutic response, n = 4 mice). (H and I) Comparison of Nos2 expression levels in responders versus nonresponders pre- or post-αOX40 treatment. Bilateral-MC38-bearing mice were treated with αOX40, and tumors from one side were analyzed by RNA-seq prior to (H) or following αOX40 treatment (I). The Nos2 expression was analyzed from RNA-seq data (left) and validated by RT-qPCR (right) ( n = 5 biological replicates). (J) NOS2 expression in tumor biopsies post-treatment determined by RNA-seq. Patients with advanced solid tumors and >1 prior therapy received HFB301001 monotherapy. Tumor biopsy samples were obtained on day 8 of cycle 2 for subsequent RNA-seq analysis. NOS2 expression were compared between patients achieving stable disease (SD, n = 3) and those with progressive disease (PD, n = 3). (K) GO enrichment analysis of upregulated genes in Mac_C1 of responders. (L) Calreticulin expression was quantified by flow cytometry in different response groups following αOX40 treatment ( n = 3 mice per group). (M) NOS2 expression in BMDMs was analyzed by flow cytometry after stimulation with CD8 + T cell supernatant and MC38 lysate, combined with TLR inhibition and IFN-γ blockade ( n = 5 biological replicates). (N) Quantification of Nos2 expression in BMDM by RT-qPCR after 24-h stimulation with MPLA (TLR4 agonist, 100 ng/mL), IFN-γ (20 ng/mL), or both. Data normalized to Gapdh and presented as fold-change relative to unstimulated controls ( n = 4 biological replicates). Data are shown as means ± SD from one of two independent experiments (G, H, I, L, M, and N). Statistical significance was determined using one-way ANOVA with multiple comparisons (G, L, M, and N) or using an unpaired two-tailed t test (H, I, and J). n.s., not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; VST, variance stabilized transformation; sup., supernatant; lys., tumor lysate; inh., inhibitor.

Article Snippet: To investigate whether macrophages tumoricidal effect is dependent on NOS2, 200 μM NOS2 inhibitor L-NIL (HY-12116, MCE) was added to the medium of cocultured cells.

Techniques: Expressing, Marker, Protein-Protein interactions, Gene Expression, Flow Cytometry, Control, Clinical Proteomics, Comparison, RNA Sequencing, Quantitative RT-PCR, Inhibition, Two Tailed Test, Transformation Assay

Journal: Cell Reports Medicine

Article Title: Immunogenic tumor cell death and T-cell-derived IFN-γ elicit tumoricidal macrophages to potentiate OX40 immunotherapy

doi: 10.1016/j.xcrm.2026.102699

Figure Lengend Snippet: NOS2-high macrophages are significantly associated with Combo treatment efficacy (A) UMAP of macrophage subclusters from scRNA-seq data of MC38-bearing mice treated with control, MPLA+IFN-γ, αOX40, or Combo. Cells are color-coded by annotated subtype. (B) Bubble chart showing the top variable marker genes for identified macrophage subclusters. (C) Pie chart shows the relative abundance of four macrophage subclusters in control, αOX40, MPLA+IFN-γ, or Combo. (D) GO pathway analysis identifying significantly enriched signaling pathways in the Mac_S2 subcluster compared to other macrophage subpopulations. (E) Violin plots showing Nos2 expression levels across macrophage subclusters. (F) Violin plots comparing Nos2 and Cd206 expression levels among different treatment groups. (G) Frequency of M1-like, M2-like, or the ratio of M1/M2-like macrophage cells in tumor tissues from control, αOX40, MPLA+IFN-γ, and Combo groups with two time points, as determined by flow cytometry ( n = 5–10 mice per group). (H) Multiple immunofluorescence signal intensities of NOS2 + F4/80 + and CD206 + F4/80 + cells in the TME of control, αOX40, MPLA+IFN-γ, and Combo groups. Scale bars, 20 μm. Data are shown as means ± SD from one of two independent experiments (G and H). Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test (G). n.s., not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: To investigate whether macrophages tumoricidal effect is dependent on NOS2, 200 μM NOS2 inhibitor L-NIL (HY-12116, MCE) was added to the medium of cocultured cells.

Techniques: Control, Marker, Protein-Protein interactions, Expressing, Flow Cytometry, Immunofluorescence

Journal: Cell Reports Medicine

Article Title: Immunogenic tumor cell death and T-cell-derived IFN-γ elicit tumoricidal macrophages to potentiate OX40 immunotherapy

doi: 10.1016/j.xcrm.2026.102699

Figure Lengend Snippet: NOS2-dependent direct tumor cell killing by macrophages in Combo therapy (A) Schematic of co-culture using CFSE-labeled MC38 cells with tumor- or spleen-derived macrophages, with/without NIL treatment. (B) Flow cytometry quantification of 7-AAD + MC38 cells after 48 h co-culture with tumor-(right) or spleen (left)-derived macrophages ( n = 3 biological replicates). (C) Quantification of 7-AAD + MC38 cells after 48 h in vitro co-culture with or without NIL treatment ( n = 3 biological replicates). (D) Treatment schedule for MC38- or B16-tumor-bearing Nos2 KOor WT mice treated with Combo ( n = 6 mice per group). (E and F) Survival curves of MC38-bearing (E) and B16-bearing mice (F) were analyzed using the log rank test. (G) Flow cytometry analysis of 7-AAD + MC38 cells after 48-h co-culture with CFSE-labeled MC38 cells and BMDMs from Nos2 KO mice ( n = 5 biological replicates). (H) Phagocytosis rate of MC38 cells engulfed by BMDMs was assessed by flow cytometry ( n = 3 biological replicates). (I) Surface expression of CRT on MC38 cells was assessed by flow cytometry after co-culture with MPLA- and IFN-γ-polarized BMDMs in vitro ( n = 3 biological replicates). (J) Analysis of CALR + MC38 cells from MC38-tumor-bearing mice following final treatment with control or Combo, assessed by flow cytometry ( n = 5 biological replicates). Data are shown as means ± SD from one of two independent experiments (B, C, E, F, G, H, I, and J). Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons (B, C, and I) or using unpaired Student’s t test (G, H, and J). Log rank tests (E and F) were also used for statistical analysis. n.s., not significant; ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Combo, MPLA, and IFN-γ combined with αOX86 (E and F).

Article Snippet: To investigate whether macrophages tumoricidal effect is dependent on NOS2, 200 μM NOS2 inhibitor L-NIL (HY-12116, MCE) was added to the medium of cocultured cells.

Techniques: Co-Culture Assay, Labeling, Derivative Assay, Flow Cytometry, In Vitro, Expressing, Control

Journal: Cell Reports Medicine

Article Title: Immunogenic tumor cell death and T-cell-derived IFN-γ elicit tumoricidal macrophages to potentiate OX40 immunotherapy

doi: 10.1016/j.xcrm.2026.102699

Figure Lengend Snippet: Foxp3 + Treg depletion and macrophage reprogramming are involved in the anti-tumor effect of Combo (A) Flow cytometry analysis of TME. MC38-tumor-bearing mice were treated with control, αOX40, MPLA+IFN-γ, or Combo for two and three doses, and tumors were analyzed by flow cytometry. (B) Frequency and absolute count of CD25 + FOXP3 + cells in tumor tissues from control, αOX40, MPLA+IFN-γ, and Combo groups with two time points, as determined by flow cytometry ( n = 5 mice per group). (C) Treatment schedule for MC38-tumor-bearing Fcer1g KO or FcγRIIb KO mice. Mice were treated with Control, αOX40, MPLA+IFN-γ, and Combo every 3 days for a total of four doses. (D and E) Survival curves of Fcgr1g KO (D) and FcgrIIb KO (E) mice following treatment ( n = 5–6 mice per group) were monitored. (F) Treatment schedule. MC38-tumor-bearing mice were treated with MPLA and IFN-γ in combination with either OX40-mIgG2a or OX40-hIgG1 agonist antibodies (top), and the corresponding survival curves are shown (bottom) ( n = 5–7 mice per group). (G) Schematic of the co-culture experiment involving BMDMs and Tregs at a ratio of 1:4 (BMDM:Treg); Nos2 expression was measured by RT-qPCR. (H) Relative expression of Nos2 following the co-culture ( n = 4 biological replicates). (I) Multiple immunofluorescence (mIF) staining of MC38 tumors from mice treated with control, αOX40, MPLA+IFN-γ, or Combo, showing FOXP3 and NOS2 expression in border or intra-tumoral. Scale bars, 50 μm. (J) Analysis of cell numbers of FOXP3 and NOS2 expression at the border and intra-tumoral. Representative images from five randomly chosen fields were quantified with ImageJ. Data are shown as means ± SD from one of two independent experiments (B, D, E, F, H, and I). Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons (B and H). Log rank test was also used (D–F). n.s., not significant; ∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Combo, MPLA, and IFN-γ combined with αOX86 (C–E).

Article Snippet: To investigate whether macrophages tumoricidal effect is dependent on NOS2, 200 μM NOS2 inhibitor L-NIL (HY-12116, MCE) was added to the medium of cocultured cells.

Techniques: Flow Cytometry, Control, Co-Culture Assay, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: Virology

Article Title: Myeloid-Derived Suppressor Cells in Murine AIDS Inhibit B-Cell Responses in Part via Soluble Mediators including Reactive Oxygen and Nitrogen Species, and TGF-β

doi: 10.1016/j.virol.2016.08.031

Figure Lengend Snippet: (A) Suppression of B-cell proliferation by MDSCs from LP-BM5-infected wild-type or VISTA/iNOS double knockout MDSCs. (B) Supernate transfer suppression assays (as in Fig. 3) left untreated or treated with superoxide dismutase (SOD) showed that soluble suppression by double knockout MDSCs accounted for about 2/3rds of total suppression, as opposed to with WT M-MDSCs, where soluble mediators account for approximately 55% the suppression (see Fig. 3a). Data shown are from a representative experiments. A similar pattern of results was observed in at least 3 experiments for each panel. Significance levels: **, p<0.01; NS, not significant.

Article Snippet: To test for iNOS-dependence, the iNOS/NOS2 inhibitor L-Nil (Enzo Life Sciences, Farmingdale, NY) was added to suppressor cells or control media before culture at 100μM.

Techniques: Infection, Double Knockout

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Reoxygenation‐Derived Toxic Reactive Oxygen/Nitrogen Species Modulate the Contribution of Bone Marrow Progenitor Cells to Remodeling After Myocardial Infarction

doi: 10.1161/JAHA.113.000471

Figure Lengend Snippet: Time course and source of superoxide generation in heart tissue during chronic ischemia. A, Representative fluorescence photomicrographs of confocal optical sections of left ventricle myocardium labeled with DHE and counterstained with DAPI for nuclei. Each panel shows infarcted regions with infiltrated cells and adjacent myocardium. Original magnification: ×20; bar, 100 μm for all images. B, Effect of in vivo treatment of mice with the SOD mimetic MnTMPyP (SODm). Representative images of frozen sections of LV myocardium from SODm‐treated and untreated mice after 7 days of coronary ligation, labeled with DHE. Each panel shows infarcted regions with infiltrated cells and adjacent myocardium. Bars, 100 μm. C, Intensity of DHE fluorescence measured from the corresponding left ventricle sections of controls and MI at 1, 3, 5, and 7 days, (DHE) and after pretreatment of sections with the NADPH oxidase inhibitor VF244 (+VF244) or the NOS2 inhibitor L‐NIL (+L‐NIL). Displayed results are based on subtraction from all values of the residual background fluorescence after treatment of the sections with the SODm MnTBAP (background column in A). Values are means±SD per each condition (arbitrary fluorescence units), n=3 to 5 images per section from 3 sections, each from a different heart. † P <0.05 VF244 vs untreated (DHE) MI groups; * P <0.01, L‐NIL vs untreated (DHE) MI groups. D, Total intensity of DHE fluorescence measured from the corresponding left ventricle sections of MI at 7 days from controls (PBS treated) and SODm‐treated mice. Values are means±SDs (in arbitrary fluorescence units per microscopic field, n=3 to 5 images per section, from 3 sections, each from a different heart); * P <0.001. DHE indicates dihydroethidine; L‐NIL, N6‐(1‐iminoethyl)‐L‐lysine, dihydrochloride; LV, left ventricular; MI, myocardial infarction; MnTBAP, Mn(III)tetrakis(4‐benzoic acid)porphyrin chloride; MnTMPyP, manganese (III) tetrakis (1‐methyl‐4‐pyridyl) porphyrin NADPH, nicotine adenine dinucleotide phosphate, reduced; NOS2, nitric oxide synthase, inducible; PBS, phosphate buffered saline; SOD, superoxide dismutase.

Article Snippet: To identify the origin of superoxide, we incubated 3 additional sections from the same hearts, each with DHE as described but in the presence of either the SOD mimetic Mn(III)tetrakis(4‐benzoic acid)porphyrin chloride (Alexis Biochemicals, San Diego, CA), the NADPH oxidase inhibitor VF244 (Molecules for Health, Richmond, VA), or the NOS2 inhibitor L‐NIL (Caymen, Ann Arbor, MI) at 100 and 50 μmol/L.

Techniques: Fluorescence, Labeling, In Vivo, Ligation