Journal: Journal of Inflammation Research

Article Title: Tumor-Infiltrating PD-L1+ Neutrophils Induced by GM-CSF Suppress T Cell Function in Laryngeal Squamous Cell Carcinoma and Predict Unfavorable Prognosis

doi: 10.2147/JIR.S347777

Figure Lengend Snippet: LSCC tumor microenvironment remodels and regulates the phenotype and function of neutrophils. ( A ) TCCS or cultural medium from AMC-HN-8 cells upregulated the level of immunosuppressive molecule PD-L1 on neutrophils. ( B ) ELISA detected the concentration of GM-CSF, G-CSF and TGF-β1 in the plasma, TCCS and NTCCS of LSCC patients. ( C ) Representative images were shown for GM-CSF IHC staining (magnification, X 50) of tumor tissues and normal tissues from LSCC patients. Bars = 500μm. ( D ) Cytokine GM-CSF most significantly upregulated the level of immunosuppressive molecule PD-L1 on neutrophils. ( E-G ) Representative histograms and statistics analysis of the proliferative capacity of CD4+T cells and CD8+ T cells following the coculture of PANs from LSCC patients with PBMCs at different stimuli. ( H and I ) IFN-γ and TNF-α production of T cells following the coculture of PANs from LSCC patients with PBMCs at different stimuli. ns = p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Commercial ELISA kits GM-CSF (70-EK1632, MultiSciences, Hangzhou, China), G-CSF (70-EK1692, MultiSciences, Hangzhou, China) and TGF-β1 (70-EK1812, MultiSciences, Hangzhou, China) were performed to assess the cytokines concentrations in the plasma of healthy controls and LSCC patients, and tissue supernatant of TCCS and NTCCS according to the manufacturers’ instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Immunohistochemistry

Journal: Journal of Inflammation Research

Article Title: Tumor-Infiltrating PD-L1+ Neutrophils Induced by GM-CSF Suppress T Cell Function in Laryngeal Squamous Cell Carcinoma and Predict Unfavorable Prognosis

doi: 10.2147/JIR.S347777

Figure Lengend Snippet: Schematic representation of the proposed mechanism was created with from BioRender.com that tumor-derived GM-CSF may upregulate the expression of PD-L1 on tumor-infiltrating neutrophils recruited by CXCL5 release accumulation, which reprograms the phenotype and function of neutrophils and ultimately results in suppressing T cell proliferation and activation in the inflammatory microenvironment of LSCC and fostering tumor progression.

Article Snippet: Commercial ELISA kits GM-CSF (70-EK1632, MultiSciences, Hangzhou, China), G-CSF (70-EK1692, MultiSciences, Hangzhou, China) and TGF-β1 (70-EK1812, MultiSciences, Hangzhou, China) were performed to assess the cytokines concentrations in the plasma of healthy controls and LSCC patients, and tissue supernatant of TCCS and NTCCS according to the manufacturers’ instructions.

Techniques: Derivative Assay, Expressing, Activation Assay

Journal: Frontiers in Immunology

Article Title: Optimized Intracellular Staining Reveals Heterogeneous Cytokine Production Ability of Murine and Human Hematopoietic Stem and Progenitor Cells

doi: 10.3389/fimmu.2021.654094

Figure Lengend Snippet: Cytokine production and secretion by PB- or CB-derived HSPCs. (A, B) Adult PB- (A) or neonatal CB-derived (B) CD34 + HSPCs were either left untreated or stimulated with LPS and Pam3CSK4 for 12 h. The percentages of cell expressing GM-CSF, IL-6 and TNF-α were determined by flow cytometry. MFI of the cytokine signals in the corresponding positive cells are shown. The secretion of cytokines in the supernatant was quantified by ELISA, with 20,000 CD34 + cells in 200 µl medium. Data are shown as the mean ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001; ns, not significant, by 2-way ANOVA followed by Bonferroni’s test or Student’s t test. (C) The number of cytokine-expressing HSPCs were measured by ELISPOT. CD34 + CD38 - cells (4,800 cells/well) or CD34 + CD38 - cells (45,000 cells/well) were incubated for 20 h in ELISPOT plates in the presence or absence of LPS and Pam3CSK4. The data refer to a typical experiment out of three that generated similar results. (D) Summary of two different regulatory modes of cytokine production in HSPCs.

Article Snippet: Levels of GM-CSF, IL-6 or TNF-α in conditioned medium were determined with commercial high-sensitivity ELISA Kits (human GM-CSF, Cat. No. EK163HS, Multi-Science, China; human IL-6, Cat. No.88-7066, Invitrogen; human TNF-α, Cat. No. 88-7346, Invitrogen; mouse GM-CSF, Cat. No. EK263HS, Multi-Science; mouse IL-6, Cat. No. EK206HS, Multi-Science; and mouse TNF-α, Cat. No. EK282HS, Multi-Science) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Incubation, Generated

Journal: Frontiers in Immunology

Article Title: Optimized Intracellular Staining Reveals Heterogeneous Cytokine Production Ability of Murine and Human Hematopoietic Stem and Progenitor Cells

doi: 10.3389/fimmu.2021.654094

Figure Lengend Snippet: Cytokine production and secretion by PB- or CB-derived HSPCs. (A, B) Adult PB- (A) or neonatal CB-derived (B) CD34 + HSPCs were either left untreated or stimulated with LPS and Pam3CSK4 for 12 h. The percentages of cell expressing GM-CSF, IL-6 and TNF-α were determined by flow cytometry. MFI of the cytokine signals in the corresponding positive cells are shown. The secretion of cytokines in the supernatant was quantified by ELISA, with 20,000 CD34 + cells in 200 µl medium. Data are shown as the mean ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001; ns, not significant, by 2-way ANOVA followed by Bonferroni’s test or Student’s t test. (C) The number of cytokine-expressing HSPCs were measured by ELISPOT. CD34 + CD38 - cells (4,800 cells/well) or CD34 + CD38 - cells (45,000 cells/well) were incubated for 20 h in ELISPOT plates in the presence or absence of LPS and Pam3CSK4. The data refer to a typical experiment out of three that generated similar results. (D) Summary of two different regulatory modes of cytokine production in HSPCs.

Article Snippet: Levels of GM-CSF, IL-6 or TNF-α in conditioned medium were determined with commercial high-sensitivity ELISA Kits (human GM-CSF, Cat. No. EK163HS, Multi-Science, China; human IL-6, Cat. No.88-7066, Invitrogen; human TNF-α, Cat. No. 88-7346, Invitrogen; mouse GM-CSF, Cat. No. EK263HS, Multi-Science; mouse IL-6, Cat. No. EK206HS, Multi-Science; and mouse TNF-α, Cat. No. EK282HS, Multi-Science) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Incubation, Generated