Journal: Frontiers in Immunology

Article Title: Targeting immunosuppressive Ly6C+ classical monocytes reverses anti-PD-1/CTLA-4 immunotherapy resistance

doi: 10.3389/fimmu.2023.1161869

Figure Lengend Snippet: Anti-PD-1/CTLA-4/Ly6C treatment reduces tumor growth and increases CD8 T cells/dendritic cells. (A) 344SQ tumor bearing 129/Sv mice were weekly treated with anti-PD-1_CTLA-4 (200 µg of anti-PD-1 plus 200 µg of anti-CTLA-4 per mouse), anti-Ly6C alone (200 µg of anti-Ly6C per mouse), anti-PD-1_CTLA-4_Ly6C (200 µg of anti-PD-1 plus 200 µg of anti-CTLA-4 plus 200 µg of anti-Ly6C per mouse), or their IgG control mixture (IgG control) beginning on week 2 after a subcutaneous cancer cell injection (1 x 10 6 cells per mouse; n = 5) for 4 weeks. Mice received total of 4 treatments starting at week 2 post tumor cell implantation. (B) tSNE CD45 plots from 344SQ tumors treated with IgG control, anti-PD-1_CTLA-4, or anti-PD-1_CTLA-4_Ly6C from (A) . Tumors from (A) were harvested to prepare single cell suspensions for FACS analysis. (C) Percentages of CD8 T cells (left), Effector/memory CD8 T cells (middle), and dendritic cells (right). (D) Percentage of Ly6C- (left) and Ly6C+ (right) CD14+CD115+ monocytes. Kras LSL-G12D /p53 fl/fl mice generated through intratracheal administration of adenovirus expressing Cre recombinase were treated with either anti-PD-1/CTLA-4 or anti-PD-1/CTLA-4/Ly6C for 8 weeks (E) Micro-CT images shown at week 0 (baseline) and week 8 (endpoint) for anti-PD-1/CTLA-4 or anti-PD-1/CTLA-4/Ly6C. Dashed yellow circles indicate lung tumors. H indicates of heart. (F) Percentage change of tumor area was calculated taking into account prior time point and normalized to the baseline measurement. (G) H&E stained lung sections at week 8 from anti-PD-1/CTLA-4 or anti-PD-1/CTLA-4/Ly6C treated mice from (E) . bar= 5 mm. (H) Number of lung tumors on H&E sections from (G) treated with anti-PD-1/CTLA-4 or anti-PD-1/CTLA-4/Ly6C weekly for 8 weeks. (I) IHC of CD8 stained lung tumors from (F) week 8. Percentage of CD8 T cells found in the lung tumors from Kras LSL-G12D /p53 fl/fl mice treated for 8 weeks with anti-PD-1/CTLA-4 or anti-PD-1/CTLA-4/Ly6C. (J) The dendritic cells from CD11c-DTR mice were transferred into C57BL/6 mice to generate the chimerical mice, after 8-week stabilization, the mice were treated with diphtheria toxin twice a week to maintain the depletion of dendritic cells. The B16 melanoma-bearing mice were treated weekly with anti-PD-1_CTLA-4_Ly6C for 3 weeks staring on week 1 after tumor cells inoculation. (K) 344SQ tumor bearing 129/Sv mice were treated weekly with blockade of PD-1, CTLA-4, and Ly6C (200 µg of anti-PD-1 plus 200 µg of anti-CTLA-4 plus 200 µg of anti-Ly6C per mouse) or their IgG control mixture (IgG control) beginning on day 7 after a subcutaneous cancer cell injection (0.1 x 10 6 cells per mouse; n = 5) for 4 weeks. For blocking B7 signal, antibodies (anti-B7: 300 μg of anti-CD80 and 300 μg of anti-CD86 per mouse) were intraperitoneally administered 1 day before the first dose of therapy, and then once a week to maintain the blockade. ANOVA test was used to analyze the data. n.s., not significance; *p < 0.05; **p < 0.01; ***p< 0.001; ****p < 0.0001.

Article Snippet: Anti-mouse PD-1 (clone RMP1-14), anti-mouse CTLA-4 (clone 9D9), anti-mouse CD80 (clone 16-10A1), anti-mouse CD86 (clone GL-1), anti-mouse Ly6C (clone Monts 1), anti-mouse CCL2 (clone 2H5), and the isotype-matched IgG controls were purchased from BioXCell.

Techniques: Control, Injection, Generated, Expressing, Micro-CT, Staining, Blocking Assay

Journal: bioRxiv

Article Title: Adjuvant Discovery via a High Throughput Screen using Human Primary Mononuclear Cells

doi: 10.1101/2022.06.17.496630

Figure Lengend Snippet: Key resource table

Article Snippet: CD86 Antibody, anti-human, PE, REAfinityTM (Clone- REA968) , Miltenyi Biotec , Order Number: 130-116-160.

Techniques: Recombinant, Sterility, Injection, Modification, Staining, Saline, Enzyme-linked Immunosorbent Assay, Multiplex Assay, Software

Journal: Central European Journal of Immunology

Article Title: Macrophage M2 polarization induced by ANKRD22 in lung adenocarcinoma facilitates tumor angiogenesis

doi: 10.5114/ceji.2025.149372

Figure Lengend Snippet: Fig. 2. Cont. F-H) Flow cytometry was used to detect the effects of ANKRD22 overexpression or knockdown on the proportions of CD68+CD86+ and CD68+CD206+ macrophages. All experiments were conducted with 3 independent replicates. The data are presented as mean ± standard deviation. *p < 0.05

Article Snippet: The cells were incubated with PerCP Anti-Human CD68 Antibody (1 : 100; 333813, BioLegend, USA), PE Anti-Human CD86 Antibody (1 : 100; E-AB-F1012D, Elabscience, China), and/or FITC AntiHuman CD206/MMR Antibody (1 : 100; E-AB-F1161C, Elabscience, China) for 15 min. After washing with PBS, the cells were resuspended, and the flow cytometry data were collected and analyzed using Agilent’s flow analysis software.

Techniques: Flow Cytometry, Over Expression, Knockdown, Standard Deviation

Journal: Central European Journal of Immunology

Article Title: Macrophage M2 polarization induced by ANKRD22 in lung adenocarcinoma facilitates tumor angiogenesis

doi: 10.5114/ceji.2025.149372

Figure Lengend Snippet: Fig. 2. Cont. I) Flow cytometry was used to detect the ef- fects of ANKRD22 overexpression or knockdown on the pro- portions of CD68+CD86+ and CD68+CD206+ macrophages. J) WB was used to detect the effects of ANKRD22 over- expression or knockdown on the protein expression levels of CD80 and CD163. All experiments were conducted with 3 independent replicates. The data are presented as mean ± standard deviation. *p < 0.05

Article Snippet: The cells were incubated with PerCP Anti-Human CD68 Antibody (1 : 100; 333813, BioLegend, USA), PE Anti-Human CD86 Antibody (1 : 100; E-AB-F1012D, Elabscience, China), and/or FITC AntiHuman CD206/MMR Antibody (1 : 100; E-AB-F1161C, Elabscience, China) for 15 min. After washing with PBS, the cells were resuspended, and the flow cytometry data were collected and analyzed using Agilent’s flow analysis software.

Techniques: Flow Cytometry, Over Expression, Knockdown, Expressing, Standard Deviation

Journal: Journal for Immunotherapy of Cancer

Article Title: SP140 inhibits STAT1 signaling, induces IFN-γ in tumor-associated macrophages, and is a predictive biomarker of immunotherapy response

doi: 10.1136/jitc-2022-005088

Figure Lengend Snippet: Overexpression of SP140 induces the production of inflammatory cytokines and chemokines and reprogram macrophages to induce TAM-mediated tumor cytotoxicity. (A, B) Control CRISPR/dCAS9 (control) or SP140 CRISPR/dCAS9 (SP140 overexpression) was transfected in naïve macrophages, and the expression of CD80 and CD86 was quantified by flow cytometry. (C–E) SP140 CRISPR/dCAS9 (SP140 OE) or scramble control (control CRISPR/dCAS9) were introduced to the naïve macrophages, and a multiplex fluorescence BioLegend assay was used to identify the levels of CXCL10, IFN-γ, and IL-12 (P70) in the supernatant after 48 hours. LPS (100 nM) was administered after 24 hours. (F–I) Correlation of SP140 expression and IFNG, CXCL10, IL-12B, and IL-12A levels in over 500 HNSCCs in the TCGA dataset. (J) Control CRISPR/dCAS9 or SP140 CRISPR/dCAS9 was introduced in TAMs isolated from syngeneic HNSCC tumor and cocultured with murine SCC7 cells. Cell viability after 48 hours of coculture was quantified with a quantitative viability assay kit. Data are presented based on the fold change of non-treated control. *P<0.05, ***P<0.001, ****P<0.0001. HNSCC, head and neck squamous cell carcinoma; IFN-γ, interferon gamma; IL, interleukin; SP140, speckled protein 140; TAM, tumor-associated macrophage; TCGA, The Cancer Genome Atlas.

Article Snippet: A flow cytometry panel consisting of human CD80 (PE, BioLegend), human CD86 (BV711, BioLegend), and human CD206 (PerCP-Cy5.5, BioLegend) was used for the characterization of macrophages.

Techniques: Over Expression, Control, CRISPR, Transfection, Expressing, Flow Cytometry, Multiplex Assay, Fluorescence, Isolation, Viability Assay