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

Journal: Journal of viral hepatitis

Article Title: A class C CpG toll-like receptor 9 agonist successfully induces robust interferon-alpha production by plasmacytoid dendritic cells from patients chronically infected with hepatitis C.

doi: 10.1111/j.1365-2893.2008.01011.x

Figure Lengend Snippet: Fig. 4 Immunophenotypic maturation of pDCs is not impaired in cHCV infection. After 20–24 h stimulation with TLR agonists, pDCs were processed for flow cytometry to identify immunophenotypic changes consistent with maturation. FACS histograms from a representative NHD and cHCV subject show (a) the rise in CD86 expression and (b) fall in BDCA-2 expression following stimulation with CpG-C (solid histogram) compared to unstimulated cells (open histogram overlay). The bar graphs show the average fold change in mean fluorescence intensity (MFI) ± SEM of (a) CD86 and (b) BDCA-2 on pDCs from 9 NHD (solid bars) and eight cHCV (open bars) subjects (seven NHD for TLR7 agonists) induced by the four TLR agonists. For each TLR agonist, the degree of (a) upregulation of CD86 and (b) downregulation of BDCA-2 did not differ significantly between NHD–pDC and cHCV–pDCs (n.s.). CpG-C was a significantly stronger stimulus for CD86 upregulation than CpG-A (NHD–pDCs, P < 0.001; cHCV–pDCs, P < 0.01) and for cHCV–pDCs loxoribine was also significantly more potent than CpG-A (P < 0.05). CpG-C caused significantly stronger downregulation of BDCA-2 than CpG-A (NHD–pDCs, P < 0.01; cHVC–pDCs, P < 0.01).

Article Snippet: Cells in PBS containing 0.1% NaN3, 1% BSA and 100 lg ⁄ mL human IgG (Jackson Immunoresearch, West Grove, PA, USA) were blocked for 15 min on ice then labelled with monoclonal antibodies against HLA-DR FITC (clone L243), CD86-FITC (clone FUN-1), CD83-APC (clone HB15e) (all from BD Biosciences) or CD303-APC (BDCA2, clone AC144; Miltenyi Biotec) or with isotype controls [murine IgG1 (clone MOPC 31, APC or FITC conjugated) or IgG2a FITC (clone G155-178) (all from BD Biosciences)].

Techniques: Infection, Cytometry, Expressing

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: Immunity

Article Title: PD-L1:CD80 Cis -Heterodimer Triggers the Co-stimulatory Receptor CD28 While Repressing the Inhibitory PD-1 and CTLA-4 Pathways

doi: 10.1016/j.immuni.2019.11.003

Figure Lengend Snippet: (A) Representative flow-cytometry histograms of CD28-huFc staining of the indicated types of Raji cells. Bound CD28-huFc was labeled by AF647 anti-human IgG Fc, the MFI of which was plotted against (CD28-huFc. Shown in gray are Raji (CD80+CD86−) cells stained by isolated huFc domain. Means ± SEM, n = 3.

Article Snippet: The SLBs were then overlaid with 200 μL of either 3 nM human PD-1-His (Sino Biological, 10377-H08H), 3 nM human CD80–His (Sino Biological, 10698-H08H), or 3 nM human CD86–His (Sino Biological, 10699-H08H).

Techniques: Flow Cytometry, Staining, Labeling, Isolation

Journal: Immunity

Article Title: PD-L1:CD80 Cis -Heterodimer Triggers the Co-stimulatory Receptor CD28 While Repressing the Inhibitory PD-1 and CTLA-4 Pathways

doi: 10.1016/j.immuni.2019.11.003

Figure Lengend Snippet: (A) Representative TIRF images of PD-L1 LUVs captured by PD-1 SLB, CD80 SLB, or CD86 SLB; each LUV is registered as a green spot. Bar graph summarizes the fluorescence intensity (FI) of the LUV channel under indicated conditions, normalized to the intensity of the condition with PD-1 SLBs. Data are means ± SEM, n = 3. Scale bars, 5 μm.

Article Snippet: The SLBs were then overlaid with 200 μL of either 3 nM human PD-1-His (Sino Biological, 10377-H08H), 3 nM human CD80–His (Sino Biological, 10698-H08H), or 3 nM human CD86–His (Sino Biological, 10699-H08H).

Techniques: Fluorescence

Journal: Immunity

Article Title: PD-L1:CD80 Cis -Heterodimer Triggers the Co-stimulatory Receptor CD28 While Repressing the Inhibitory PD-1 and CTLA-4 Pathways

doi: 10.1016/j.immuni.2019.11.003

Figure Lengend Snippet: (A) Representative flow-cytometry histograms of CTLA-4-huFc staining of the indicated types of Raji cells. Bound CTLA-4-huFc was labeled by AF647 anti-human IgG Fc, the MFI of which was plotted against (CTLA-4-huFc). Shown in gray are Raji (CD80+CD86−) cells stained by isolated huFc domain. Means ± SEM, n ≥ 3.

Article Snippet: The SLBs were then overlaid with 200 μL of either 3 nM human PD-1-His (Sino Biological, 10377-H08H), 3 nM human CD80–His (Sino Biological, 10698-H08H), or 3 nM human CD86–His (Sino Biological, 10699-H08H).

Techniques: Flow Cytometry, Staining, Labeling, Isolation

Journal: Immunity

Article Title: PD-L1:CD80 Cis -Heterodimer Triggers the Co-stimulatory Receptor CD28 While Repressing the Inhibitory PD-1 and CTLA-4 Pathways

doi: 10.1016/j.immuni.2019.11.003

Figure Lengend Snippet: (A) On the left are flow-cytometry histograms of CD80 and CD86 surface levels on DCs and macrophages (Macs) isolated from tumor tissues of 4T1 implanted BALB/C female mice treated with anti-PD-L1 (magenta traces), anti-PD-1 (cyan traces), or control IgG (black traces). On the right are bar graphs summarizing the MFI of CD80 and CD86 staining under the indicated conditions. Data are shown as mean ± SEM, n ≥ 3 mice.

Article Snippet: The SLBs were then overlaid with 200 μL of either 3 nM human PD-1-His (Sino Biological, 10377-H08H), 3 nM human CD80–His (Sino Biological, 10698-H08H), or 3 nM human CD86–His (Sino Biological, 10699-H08H).

Techniques: Flow Cytometry, Isolation, Staining

Journal: Immunity

Article Title: PD-L1:CD80 Cis -Heterodimer Triggers the Co-stimulatory Receptor CD28 While Repressing the Inhibitory PD-1 and CTLA-4 Pathways

doi: 10.1016/j.immuni.2019.11.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The SLBs were then overlaid with 200 μL of either 3 nM human PD-1-His (Sino Biological, 10377-H08H), 3 nM human CD80–His (Sino Biological, 10698-H08H), or 3 nM human CD86–His (Sino Biological, 10699-H08H).

Techniques: Recombinant, Isolation, Enzyme-linked Immunosorbent Assay, Cell Isolation, Software, Imaging

Journal: Journal for Immunotherapy of Cancer

Article Title: Circulating CD14 high CD16 low intermediate blood monocytes as a biomarker of ascites immune status and ovarian cancer progression

doi: 10.1136/jitc-2019-000472

Figure Lengend Snippet: Correlation between CD14 high CD16 low IBMs and ascites immune status and cancer development in patients with ovarian cancer. (A–C) Analysis of immune cell population and soluble mediators of blood and ascitic fluid samples from 28 patients with ovarian cancer. (A) Correlation of circulating IBMs with the proportion in ascites of T-cell populations, B cells, NK cells and with the CD8/regulatory T-cell ratio. (B) Correlation of circulating IBMs with the geomean of several markers (CCR2, CD163, CD206 and CD86) at the surface of CD14+ cells of ascites. (C) Correlation of circulating IBMs with the concentration of several soluble mediators of ascites involved in immunity (IFN-γ, CCL3, CXCL10 and granzyme B), tolerance (IL-6 and IL-10) and tumor progression (VEGF). (D) Correlation of circulating IBMs with the PCI. (E) Correlation of circulating IBMs with the platelet count. P values were determined using the Spearman rank correlation. CCL3, C-C Motif Chemokine Ligand 3; CXCL10, C-X-C Motif Chemokine Ligand 10; IBM, intermediate blood monocyte; IFN-γ, interferon-γ; IL, interleukin; NK, natural killer; PCI, peritoneal cancer index; VEGF, vascular endothelial growth factor.

Article Snippet: To study the phenotype of blood monocytes and macrophages from ascitic fluid, cells were labelled with the following antibodies: CD14-PerCPVio700, CCR2-PEVio770, CD163-PEVio770, CD86- Vioblue, TLR-2-APC, CD36-FITC, MHC2-APCVio700 (Myltenyi Biotec) and CD206-APC (BD Biosciences).

Techniques: Concentration Assay

Journal: Biomolecules

Article Title: Palmitic Acid Esterification Boosts Epigallocatechin Gallate’s Immunomodulatory Effects in Intestinal Inflammation

doi: 10.3390/biom15081208

Figure Lengend Snippet: Dot plots of representative pro-inflammatory (CD86-Coralite ® Plus 647, M1) and propidium iodide double stains of M0 macrophages (differentiated from THP-1 monocytic cells treated with Phorbol-Myristate Acetate) exposed to pro-inflammatory growth media of Caco-2 cells in the presence/absence of epigallocatechin gallate and palmitoyl epigallocatechin gallate. Distinct lowercase letters indicate significantly different values at p < 0.05 according to one-way analyses of variance (ANOVA) and Duncan’s multiple range test ( n = 3).

Article Snippet: CoraLite ® Plus 647 Anti-Human CD86 and CD206 antibodies were obtained from Proteintech ® (ThermoFisher Scientific, Madrid, Spain; Catalog #CL647-65165 and #CL647-65155, respectively).

Techniques: