Journal: NPJ Vaccines

Article Title: Rational design of innate defense regulator peptides as tumor vaccine adjuvants

doi: 10.1038/s41541-021-00334-3

Figure Lengend Snippet: a NY-ESO-1 was labeled with Alexa Fluor® 594. BMDCs were stimulated with labeled-NY-ESO-1, labeled-NY-ESO-1/CpG, labeled-NY-ESO-1/DP7, or labeled-NY-ESO-1/CpG/DP7 complex for 1 h. Cell membranes tagged with green fluorescent dye. Cells were fixed and stained with DAPI followed by observation with confocal microscopy. b–d BMDCs were incubated with CpG, DP7, or CpG/DP7 for 16 h. Then, the surface maturation markers of CD40 ( b ), CD80 ( c ) and CD86 ( d ) in BMDCs were analyzed by flow cytometry. e BMDCs were stimulated with CpG, DP7, or the CpG/DP7 complex for 30 or 60 min and then collected to detect the expression of p-Erk1/2 and p-p65 by western blotting.

Article Snippet: To study the effect of the CpG/DP7 complex on BMDCs, BMDCs were incubated with DP7 (40 µg/ml), CpG (20 µg/ml) or the CpG/DP7 combination for 16 h, stained with APC-anti-Mouse CD11c (Clone HL3; BD Biosciences Cat# 550261), FITC-anti-mouse-CD40 (Clone 3/23; BD Biosciences Cat# 561845), PerCP-Cy5.5-anti-mouse-CD80(clone 16-10A1; BD Biosciences Cat# 560526) or FITC-anti-mouse-CD86 (clone GL1; BD Biosciences Cat# 561962).

Techniques: Labeling, Staining, Confocal Microscopy, Incubation, Flow Cytometry, Expressing, Western Blot

Journal: Journal of Inflammation Research

Article Title: Tfh Exosomes Derived from Allergic Rhinitis Promote DC Maturation Through miR-142-5p/CDK5/STAT3 Pathway

doi: 10.2147/jir.s365217

Figure Lengend Snippet: Figure 1 AR-derived Tfhs in a DC co-culture system promote DC maturation. (A) Gating strategy and flow plot for Tfhs sorting according to Materials and Methods. (B) A schematic of Tfhs/DCs Transwell co-culture system. (C) Relative mRNA expression levels of CD80 or CD86 in treated or untreated DCs are expressed as mean ± standard error from at least three independent experiments. *** P < 0.001; **** P < 0.0001. (D) Flow cytometry results of DC cell surface expression of the co-stimulatory molecules CD80, CD86 and MHCII in control (no treatment), WT mice-derived Tfhs, AR mice-derived Tfhs, or LPS treated group. Flow cytometry results demonstrate that the ratio of CD80, CD86 and MHC II of DCs was significantly increased in the AR-derived Tfhs/DCs co-culture group. Dot plots with the label for the proportion of CD80+ CD86+ or MHCII+ cells are representative of at least three independent experiments.

Article Snippet: Rabbit polyclonal anti-STAT3 antibody (Cat# A19566, 1:1000 diluted for WB), anti-phospho-STAT3-Y705 antibody (Cat# AP0705, 1:1000 diluted for Western blot), rabbit anti-CD80 antibody (Cat# A16039, 1:100 diluted for immunofluorescence, 1:2000 diluted for Western blot) and rabbit anti-CD86 antibody (Cat# A1199, 1:100 diluted for immunofluorescence, 1:1000 diluted for Western blot) were purchased from Abclonal (Wuhan, Hubei, China).

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

Journal: Journal of Inflammation Research

Article Title: Tfh Exosomes Derived from Allergic Rhinitis Promote DC Maturation Through miR-142-5p/CDK5/STAT3 Pathway

doi: 10.2147/jir.s365217

Figure Lengend Snippet: Figure 2 Expression of CD80 and CD86 of DCs under four different conditions. Immunofluorescence staining was performed to determine the expression level of CD80 and CD86 on DC cell surface. Scale bar at the bottom right stands for 50 μm. Data shown are representative of 3 independent experiments. Green represents CD80 staining, red CD86 staining, and blue DAPI staining for cell nuclei.

Article Snippet: Rabbit polyclonal anti-STAT3 antibody (Cat# A19566, 1:1000 diluted for WB), anti-phospho-STAT3-Y705 antibody (Cat# AP0705, 1:1000 diluted for Western blot), rabbit anti-CD80 antibody (Cat# A16039, 1:100 diluted for immunofluorescence, 1:2000 diluted for Western blot) and rabbit anti-CD86 antibody (Cat# A1199, 1:100 diluted for immunofluorescence, 1:1000 diluted for Western blot) were purchased from Abclonal (Wuhan, Hubei, China).

Techniques: Expressing, Immunofluorescence, Staining

Journal: Journal of Inflammation Research

Article Title: Tfh Exosomes Derived from Allergic Rhinitis Promote DC Maturation Through miR-142-5p/CDK5/STAT3 Pathway

doi: 10.2147/jir.s365217

Figure Lengend Snippet: Figure 3 Tfhs secrete exosomes and AR-derived Tfh exosomes promote DC maturation. (A) Morphology of exosomes isolated from WTor AR mice by scanning electron microscopy. Scale bar represents 100 nm. (B) Comparison of relative mRNA expression level (mean ± standard error of at least three independent experiments) of CD80 and CD86 in the DCs treated with control (no treatment), WT mice-derived Tfh exosome, AR mice-derived Tfh exosome, LPS, or WT mice-derived Tfh exosomes with the addition of GW4869. *** P < 0.001; **** P < 0.0001. (C) Flow cytometry results of DC cell surface expression of the co-stimulatory molecules CD80, CD86 and MHCII in control (no treatment), WT mice-derived Tfhs exosome, AR mice-derived Tfhs exosome group, LPS, or WT mice-derived Tfh exosomes with the addition of GW4869 group. Dot plots with the label for the proportion of CD80+ CD86+ or MHCII+ cells are representative of at least three independent experiments.

Article Snippet: Rabbit polyclonal anti-STAT3 antibody (Cat# A19566, 1:1000 diluted for WB), anti-phospho-STAT3-Y705 antibody (Cat# AP0705, 1:1000 diluted for Western blot), rabbit anti-CD80 antibody (Cat# A16039, 1:100 diluted for immunofluorescence, 1:2000 diluted for Western blot) and rabbit anti-CD86 antibody (Cat# A1199, 1:100 diluted for immunofluorescence, 1:1000 diluted for Western blot) were purchased from Abclonal (Wuhan, Hubei, China).

Techniques: Derivative Assay, Isolation, Electron Microscopy, Comparison, Expressing, Control, Flow Cytometry

Journal: Journal of Inflammation Research

Article Title: Tfh Exosomes Derived from Allergic Rhinitis Promote DC Maturation Through miR-142-5p/CDK5/STAT3 Pathway

doi: 10.2147/jir.s365217

Figure Lengend Snippet: Figure 4 Expression of CD80 and CD86 on DC cells under five different conditions. Sections of DCs were stained Immunofluorescence dyes for CD80 (green) and CD86 (red). DAPI was used to stain nuclei (blue). Representative images are shown from at least three independent experiments. Scale bar at the bottom right stands for 50 μm.

Article Snippet: Rabbit polyclonal anti-STAT3 antibody (Cat# A19566, 1:1000 diluted for WB), anti-phospho-STAT3-Y705 antibody (Cat# AP0705, 1:1000 diluted for Western blot), rabbit anti-CD80 antibody (Cat# A16039, 1:100 diluted for immunofluorescence, 1:2000 diluted for Western blot) and rabbit anti-CD86 antibody (Cat# A1199, 1:100 diluted for immunofluorescence, 1:1000 diluted for Western blot) were purchased from Abclonal (Wuhan, Hubei, China).

Techniques: Expressing, Staining, Immunofluorescence

Journal: Journal of Inflammation Research

Article Title: Tfh Exosomes Derived from Allergic Rhinitis Promote DC Maturation Through miR-142-5p/CDK5/STAT3 Pathway

doi: 10.2147/jir.s365217

Figure Lengend Snippet: Figure 5 miR-142-5p in AR mouse-derived exosomes regulated DC maturation. (A) Heat map of differentially expressed miRNAs. The heat map was divided into two columns (left: AR; right: Control). Up- or down-regulation of the miRNAs was represented with red or green color, respectively. A specific miRNA, miR-142b, was down- regulated in AR, as indicated with a yellow arrow. (B) The expression level of miR-142-5p in exosomes of Tfhs from AR vs control mice. (C) Detection of the expression level of CD80 and CD86 in different groups with overexpression or inhibition of miR-142-5p. *** P < 0.001; **** P < 0.0001. (D) Flow cytometry analysis of co-stimulatory molecules CD80, CD86 and MHCII on surface of DCs transfected with miR-142-5p mimics or inhibitors. Dot plots with the label for the proportion of CD80+ CD86+ or MHCII+ cells are representative of at least three independent experiments.

Article Snippet: Rabbit polyclonal anti-STAT3 antibody (Cat# A19566, 1:1000 diluted for WB), anti-phospho-STAT3-Y705 antibody (Cat# AP0705, 1:1000 diluted for Western blot), rabbit anti-CD80 antibody (Cat# A16039, 1:100 diluted for immunofluorescence, 1:2000 diluted for Western blot) and rabbit anti-CD86 antibody (Cat# A1199, 1:100 diluted for immunofluorescence, 1:1000 diluted for Western blot) were purchased from Abclonal (Wuhan, Hubei, China).

Techniques: Derivative Assay, Control, Expressing, Over Expression, Inhibition, Flow Cytometry, Transfection

Journal: Journal of Inflammation Research

Article Title: Tfh Exosomes Derived from Allergic Rhinitis Promote DC Maturation Through miR-142-5p/CDK5/STAT3 Pathway

doi: 10.2147/jir.s365217

Figure Lengend Snippet: Figure 6 miR-142-5p targets CDK5 to mediate the regulation of DC maturation by Tfhs derived from AR mice. (A) The potential miR-142-5p target sites in CDK5 transcript. (B) Levels of relative luciferase activity in HEK293T cells following transfection with PGL-3-control-WT-miR-142-5p mimics, PGL-3-CDK5-WT-miR-142-5p mimics and PGL-3-CDK5-MT-miR-142-5p mimics respectively (1 μg). The relative luciferase activity refers to the ratio of firefly luminescence/renilla luminescence. (C) Relative expression of miR-142-5p after CDK5 knockdown. (D) CDK5 expression level under overexpression or inhibition of miR-142-5p by RT-qPCR. The values shown are mean ± SD of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. (E) CDK5 expression level under overexpression or inhibition of miR-142-5p by Western Blot. (F) Flow cytometry assesses the CD80, CD86 and MHCII expression on the surface of DCs by inhibiting CDK5. Dot plots with the label for the proportion of CD80+ CD86+ or MHCII+ cells are representative of at least three independent experiments.

Article Snippet: Rabbit polyclonal anti-STAT3 antibody (Cat# A19566, 1:1000 diluted for WB), anti-phospho-STAT3-Y705 antibody (Cat# AP0705, 1:1000 diluted for Western blot), rabbit anti-CD80 antibody (Cat# A16039, 1:100 diluted for immunofluorescence, 1:2000 diluted for Western blot) and rabbit anti-CD86 antibody (Cat# A1199, 1:100 diluted for immunofluorescence, 1:1000 diluted for Western blot) were purchased from Abclonal (Wuhan, Hubei, China).

Techniques: Derivative Assay, Luciferase, Activity Assay, Transfection, Control, Expressing, Knockdown, Over Expression, Inhibition, Quantitative RT-PCR, Western Blot, Flow Cytometry

Journal: Journal of Inflammation Research

Article Title: Tfh Exosomes Derived from Allergic Rhinitis Promote DC Maturation Through miR-142-5p/CDK5/STAT3 Pathway

doi: 10.2147/jir.s365217

Figure Lengend Snippet: Figure 7 Tfh-derived exosomes promote DC cell maturation through miR-142-5p/CDK5/STAT3 pathway. (A) Relative expression of miR-142-5p and CDK5 in DCs upon LPS stimulation at different time points. The values shown are mean ± SD of three independent experiments. ** P < 0.01; **** P < 0.0001. (B) CDK5 expression level changed with prolonged LPS stimulation. (C) Detection of STAT3 phosphorylation in overexpression and inhibition of miR-142-5p. (D) Detection of STAT3 phosphorylation in inhibition of miR-142-5p with or without si-CDK5. (E) Expression of CD80 and CD86 on DCs with STAT activator or STAT inhibitor and LPS stimulation for different times. Dot plots with the label for the proportion of CD80+ CD86+ or MHCII+ cells are representative of at least three independent experiments.

Article Snippet: Rabbit polyclonal anti-STAT3 antibody (Cat# A19566, 1:1000 diluted for WB), anti-phospho-STAT3-Y705 antibody (Cat# AP0705, 1:1000 diluted for Western blot), rabbit anti-CD80 antibody (Cat# A16039, 1:100 diluted for immunofluorescence, 1:2000 diluted for Western blot) and rabbit anti-CD86 antibody (Cat# A1199, 1:100 diluted for immunofluorescence, 1:1000 diluted for Western blot) were purchased from Abclonal (Wuhan, Hubei, China).

Techniques: Derivative Assay, Expressing, Phospho-proteomics, Over Expression, Inhibition

Journal: Journal of Inflammation Research

Article Title: Tfh Exosomes Derived from Allergic Rhinitis Promote DC Maturation Through miR-142-5p/CDK5/STAT3 Pathway

doi: 10.2147/jir.s365217

Figure Lengend Snippet: Figure 8 Results from an in vivo experiment showing the effect of AR-derived Tfh exosomes on occurrence of AR. (A) Hematoxylin and eosin staining of nasal mucosal from different group of mice. Scale bars at the bottom right of each image represent 50 µm. The area of mucosal thickening and inflammation appeared as dark blue area in the image, with one area indicated with a yellow arrow. (B) The relative mRNA expression of CD80 and CD86 in mouse nasal mucosa of various groups. The data shown are mean ± standard error of at least 3 independent experiments. (C) Flow cytometry results of the co-stimulatory molecules CD80, CD86 and MHCII in DCs. Dot plots with the label for the proportion of CD80+ CD86+ or MHCII+ cells are representative of at least three independent experiments. (D) Inflammatory factors including IFN-γ, IgE, IL-13 and IL-17 are studied in the serum of mice of various group. ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Rabbit polyclonal anti-STAT3 antibody (Cat# A19566, 1:1000 diluted for WB), anti-phospho-STAT3-Y705 antibody (Cat# AP0705, 1:1000 diluted for Western blot), rabbit anti-CD80 antibody (Cat# A16039, 1:100 diluted for immunofluorescence, 1:2000 diluted for Western blot) and rabbit anti-CD86 antibody (Cat# A1199, 1:100 diluted for immunofluorescence, 1:1000 diluted for Western blot) were purchased from Abclonal (Wuhan, Hubei, China).

Techniques: In Vivo, Derivative Assay, Staining, Expressing, Flow Cytometry

Journal: Theranostics

Article Title: ILT4 inhibition prevents TAM- and dysfunctional T cell-mediated immunosuppression and enhances the efficacy of anti-PD-L1 therapy in NSCLC with EGFR activation

doi: 10.7150/thno.52435

Figure Lengend Snippet: ILT4 in EGFR-activated tumor cells promoted TAM recruitment and M2-like polarization. (A-B) Patients with high ILT4 expression in tumor cells of NSCLC tissues showed markedly elevated infiltration of CD68 + TAMs by IHC analysis. (A) Representative images of ILT4 expression and TAM infiltration, brown granules represent positive staining. (B) Average results from 80 patients. ILT4-low and -high were defined by IHC score < 6 (median) and ≥ 6. Scale bar: 20 µm. (C-D) TAMs induced by PC9 and H1975 cells displayed increased migration ability compared with parental macrophages, whereas ILT4 knockdown in PC9 and H1975 cells restricted tumor-induced TAM migration. Tumor cells were first transfected with lentivirus carrying ILT4 shRNA for 48 h, and CM was collected for macrophage culture and TAM induction. The migration ability of TAMs was evaluated by the Transwell migration assay. (C) Images of migrated cells and (D) Average results from 3 independent experiments. Scale bar: 50 µm. *, p < 0.05; **, p < 0.01 compared with the medium group; ***, p < 0.001. ### , p < 0.001 compared with the LV-shNC group. (E-F) ILT4 knockdown in PC9 and H1975 cells decreased the secretion of CCL2 and CCL5. (E) showed ELISA results of CCL2, (F) showed CCL5. (G-H) Recombinant human CCL2 or CCL5 reversed ILT4 knockdown and decreased macrophage migration. 100 ng/mL recombinant human CCL2 or 200 ng/mL CCL5 were added to the CM of ILT4 knockdown PC9 and H1975 cells to induce TAM migration. The migration ability of TAMs was evaluated by the Transwell migration assay. (G) Images of migrated cells. (H) Average results from 3 independent experiments. Scale bar: 50 µm. *, p < 0.05; **, p < 0.01 compared with the medium group; ***, p < 0.001. compared with LV-shNC group. (I-J) Patients with high ILT4 expression in tumor cells displayed more frequent CD206 + but less CD86 + TAM accumulation in tumor tissues by IHC analysis. (I) Representative images of CD68 + TAMs, CD206 + M2-like TAMs and CD86+ M1-like TAMs frequencies; brown granules represent positive staining. (J) Average results from 80 patients. ILT4-low and -high were defined by IHC score < 6 and ≥ 6 respectively. Scale bar: 20 µm. (K-L) ILT4 knockdown in PC9 and H1975 cells decreased M2-like markers (CD163, CD206, IL-10 and Arg1) and increased M1-like markers (CD80, CD86, IL-12 and TNFα) in TAMs by real-time PCR (K) and flow cytometry (L). TAMs were induced by CM as in (D) for 24 h. (M-N) TAMs induced by ILT4-downregulated PC9 and H1975 cells promoted the proliferation (M) and IFN-γ expression (N) of T cells. For T cell proliferation assay, CD3 + T cells separated from fresh PBMCs were first pre-activated by anti-CD3 for 24 h and stained with CFSE (1:1000), and then co-cultured for 4 days with TAMs induced by CM of ILT4-downregulated PC9 and H1975 cells. Flow cytometry was performed to determine CFSE strength. For IFN-γ expression, pre-activated CD3 + T cells were co-cultured with TAMs as described above for 48 h and IFN-γ levels were analyzed by flow cytometry. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Arg1: arginase 1; CM, conditioned medium; LV-shILT4: lentivirus carrying ILT4 shRNA; LV-shNC: lentivirus carrying control shRNA.

Article Snippet: Following antigen retrieval, the sections were incubated with 3% hydrogen peroxide for 20 min. Tissue slides were then incubated overnight at 4 °C with following primary antibodies: anti-ILT4 antibody (1:50; Affinity; Cat No. DF9604), anti-pEGFR antibody (1:200; Abcam; Cat No. ab40815), anti-PD-L1 antibody (1:100; CST; Cat No.13684), anti-CD68 antibody (1:100; Abcam; Cat No. ab125212), anti-CD206 antibody (1:100; Abcam; Cat No. ab64693), anti-CD163 antibody (1:100; Abcam; Cat No. ab182422), anti-CD3 antibody (1:200; Abcam; Cat No. ab5690; 1:100; CST; Cat No. 85061), anti-IFN-γ antibody (1:50; Affinity; Cat No. DF6045), and anti-F4/80 antibody (1:200; Abcam; Cat No. ab111101), anti-CD80 antibody (1:500; Boster; Cat No. BM4121), anti-CD86 antibody (1:500; Proteintech; Cat No. 66406-1-Ig), anti-PIRB antibody (1:100; Immunoway; Cat No. YN1914).

Techniques: Expressing, Staining, Migration, Knockdown, Transfection, shRNA, Transwell Migration Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Real-time Polymerase Chain Reaction, Flow Cytometry, Proliferation Assay, Cell Culture, Control

Journal: Frontiers in immunology

Article Title: Impact of CTLA-4 checkpoint antibodies on ligand binding and Transendocytosis.

doi: 10.3389/fimmu.2022.871802

Figure Lengend Snippet: FIGURE 1 Pre-engagement of Ipilimumab and Tremelimumab with soluble CTLA-4-Ig prevents CTLA4 binding to cell bound CD80 or CD86 ligand. (A) Schematic of experimental set-up. A fixed dose (2µg/ml) of soluble APC conjugated Abatacept was incubated on ice with a titration of soluble anti- CTLA-4 Abs to pre-engage CTLA-4 with anti-CTLA-4. This was added to DG75 B cells expressing either CD80 or CD86-GFP and incubated on ice for 30 minutes. Cells were washed and analysed for Abatacept-ligand interaction by flow cytometry. All reagents were chilled on ice before use. (B) Representative concatenated FACS plots show impact of Abatacept-ligand engagement as the dose of anti-CTLA-4 Abs increased. (C) Anti-CTLA-4 doses were Log(x) transformed and dose response curves fitted using Prism v6 to obtain Log IC50 for ipilimumab(Ipi) (Black line) and tremelimumab (Treme) (Blue line). (D) Graphs show the mean IC50 with 95% confidence interval calculated using Prism v6. Data for ipilimumab and tremelimumab were acquired in separate 96-well plates and data presented as mean +/- SD from 3 independent experiments.

Article Snippet: CD80 (Cat# 10133-B1) and CD86 (Cat# 141-B2) -Ig expressing human IgG1 Fc were purchased from R&D Systems.

Techniques: Binding Assay, Incubation, Titration, Expressing, Cytometry, Transformation Assay

Journal: Frontiers in immunology

Article Title: Impact of CTLA-4 checkpoint antibodies on ligand binding and Transendocytosis.

doi: 10.3389/fimmu.2022.871802

Figure Lengend Snippet: FIGURE 2 Ipilimumab and Tremelimumab outcompete cell bound CD80 or CD86 for engagement with soluble CTLA-4-Ig. (A) Schematic of experimental set-up. DG75 B cells expressing either CD80 or CD86-GFP were mixed with a titration of soluble anti-CTLA-4 Abs. A fixed dose (2µg/ml) of soluble APC conjugated Abatacept was added to assess direct competition between soluble anti-CTLA-4 and cell bound ligand for Abatacept binding. Cells were incubated on ice, washed and Abatacept-ligand engagement analysed by flow cytometry. All reagents/cells were chilled on ice before use. (B) Concatenated FACS plots show reduced Abatacept-ligand engagement as the dose of anti-CTLA-4 Abs increased. (C) Anti- CTLA-4 doses were Log(x) transformed and dose response curves fitted using Prism v6 to obtain Log IC50 for ipilimumab (Black line) and tremelimumab (Blue line). (D) Graphs show the mean IC50 with 95% confidence interval calculated using Prism v6. Data for ipilimumab and tremelimumab were acquired in separate 96-well plates and data presented as mean +/- SD from 3 independent experiments.

Article Snippet: CD80 (Cat# 10133-B1) and CD86 (Cat# 141-B2) -Ig expressing human IgG1 Fc were purchased from R&D Systems.

Techniques: Expressing, Titration, Binding Assay, Incubation, Cytometry, Transformation Assay

Journal: Frontiers in immunology

Article Title: Impact of CTLA-4 checkpoint antibodies on ligand binding and Transendocytosis.

doi: 10.3389/fimmu.2022.871802

Figure Lengend Snippet: FIGURE 3 Ipilimumab and Tremelimumab are unable to displace pre-engaged CTLA-4-Ig from cell bound CD80 or CD86. (A) Schematic of experimental set-up. DG75 B cells expressing either CD80 or CD86-GFP were pre-incubated on ice with a fixed dose (2µg/ml) of soluble APC conjugated Abatacept to pre-engage soluble CTLA-4 with ligand. Cells were washed, treated with a titration of soluble anti-CTLA-4 Abs and incubated on ice or at 37°C. Cells were washed and analysed for Abatacept-ligand engagement by flow cytometry. All reagents/cells were chilled on ice before use. (B) Concatenated FACS plots show Abatacept-ligand binding at different doses of anti-CTLA-4 Abs when incubated on ice or when incubated at 37°C. (C) Dose response curves for ipilimumab (Black line) and tremelimumab (Blue line) presented as MFI CTLA-4-Ig binding from data shown in (B) Data presented are mean +/- SD from 3-6 independent experiments. Data for ipilimumab and tremelimumab were acquired in separate 96-well plates (n=3-6).

Article Snippet: CD80 (Cat# 10133-B1) and CD86 (Cat# 141-B2) -Ig expressing human IgG1 Fc were purchased from R&D Systems.

Techniques: Expressing, Incubation, Titration, Cytometry, Ligand Binding Assay, Binding Assay

Journal: Frontiers in immunology

Article Title: Impact of CTLA-4 checkpoint antibodies on ligand binding and Transendocytosis.

doi: 10.3389/fimmu.2022.871802

Figure Lengend Snippet: FIGURE 4 Pre-engagement of cell bound CTLA-4 with soluble CD80 and CD86Ig inhibits Ipilimumab and Tremelimumab from binding. (A) Schematic of experimental set-up. Jurkat cells expressing CTLA-4 were pre-incubated on ice with either soluble CD80 or CD86Ig to pre-engage CTLA-4 with ligand. Cells were washed, incubated with a fixed dose (2µg/ml) of soluble PE conjugated anti-CTLA4 Abs and incubated on ice. Cells were washed and analysed for anti-CTLA-4-CTLA-4 binding by flow cytometry. All reagents/cells were chilled on ice before use. (B) Concatenated FACS plots show reduced anti-CTLA-4 engagement as the dose of CD80 or CD86Ig was increased. CD80 or CD86Ig doses were Log(x) transformed and dose response curves fit using Prism v6 to obtain Log IC50 for ipilimumab (Black line) and tremelimumab (Blue line). (C) CD80 or CD86Ig doses were Log(x) transformed and dose response curves fitted using Prism v6 to obtain Log IC50 for ipilimumab (Black line) and tremelimumab (Blue line). (D) Graphs show the mean IC50 with 95% confidence interval calculated using Prism v6. Data for ipilimumab and tremelimumab were acquired in separate 96-well plates (n=3).

Article Snippet: CD80 (Cat# 10133-B1) and CD86 (Cat# 141-B2) -Ig expressing human IgG1 Fc were purchased from R&D Systems.

Techniques: Binding Assay, Expressing, Incubation, Cytometry, Transformation Assay

Journal: Frontiers in immunology

Article Title: Impact of CTLA-4 checkpoint antibodies on ligand binding and Transendocytosis.

doi: 10.3389/fimmu.2022.871802

Figure Lengend Snippet: FIGURE 5 Soluble CD80 and CD86Ig outcompete Ipilimumab and Tremelimumab for engagement with cell bound CTLA4. (A) Schematic of experimental set-up. A fixed dose (2µg/ml) of soluble PE conjugated anti-CTLA4 Abs was pre-incubated with a titration of either soluble CD80 or CD86Ig on ice. Jurkat expressing CTLA-4 cells were added to facilitate direct competition between anti-CTLA4 Abs and CD80 or CD86Ig for cell bound CTLA-4 occupancy. Cells were incubated on ice, washed and anti-CTLA4-CTLA4 engagement analysed by flow cytometry. All reagents/cells were chilled on ice before use. (B) Concatenated FACS plots show reduced anti-CTLA-4-CTLA-4 engagement as the dose of CD80 or CD86Ig was increased. (C) CD80 or CD86Ig doses were Log(x) transformed and dose response curves fitted using Prism v6 to obtain Log IC50 for ipilimumab (Black line) and tremelimumab (Blue line). (D) Graphs show the mean IC50 with 95% confidence interval calculated using Prism v6. Data for ipilimumab and tremelimumab were acquired in separate 96-well plates (n=3).

Article Snippet: CD80 (Cat# 10133-B1) and CD86 (Cat# 141-B2) -Ig expressing human IgG1 Fc were purchased from R&D Systems.

Techniques: Incubation, Titration, Expressing, Cytometry, Transformation Assay

Journal: Frontiers in immunology

Article Title: Impact of CTLA-4 checkpoint antibodies on ligand binding and Transendocytosis.

doi: 10.3389/fimmu.2022.871802

Figure Lengend Snippet: FIGURE 6 Soluble CD80 and CD86Ig are unable to displace pre-engaged Ipilimumab and Tremelimumab from cell bound CTLA-4. (A) Schematic of experimental set-up. Jurkat cells expressing CTLA-4 were pre-treated with a fixed dose (2µg/ml) of soluble PE conjugated anti-CTLA-4 Abs on ice to pre-engage anti-CTLA-4-CTLA-4. Cells were washed and a titration of either soluble CD80 or CD86Ig was added. Cells expressing WT CTLA-4 were incubated on ice and cells expressing a non-internalising CTLA-4 mutant (Del 36) were incubated at either on ice or at 37°C. Cells were washed and analysed for anti-CTLA-4-CTLA-4 staining by flow cytometry. (B) Concatenated FACS plots showinganti-CTLA-4 binding at various doses of CD80 or CD86Ig (C) Dose response curves for ipilimumab (Black line) and tremelimumab (Blue line) presented as MFI anti- CTLA-4 binding from data in (B) Data for ipilimumab and tremelimumab were acquired in separate 96-well plates (n=3-6).

Article Snippet: CD80 (Cat# 10133-B1) and CD86 (Cat# 141-B2) -Ig expressing human IgG1 Fc were purchased from R&D Systems.

Techniques: Expressing, Titration, Incubation, Mutagenesis, Staining, Cytometry, Binding Assay

Journal: Frontiers in immunology

Article Title: Impact of CTLA-4 checkpoint antibodies on ligand binding and Transendocytosis.

doi: 10.3389/fimmu.2022.871802

Figure Lengend Snippet: FIGURE 7 Ipilimumab and Tremelimumab block CD80 and CD86 ligand loss for APC by transendocytosis. (A) CTV stained CHO cells expressing either CD80 or CD86-GFP were cultured in the presence of CHO cells expressing CTLA-4 at a 1:1 ratio for 5 hours at 37°C. A titration of soluble ipilimumab (Black line) or tremelimumab (Blue line) were added at 0h. Percentage of CD80 or CD86-GFP ligand loss from CTV+ CHO cells by transendocytosis was determined by making the GFP MFI of CTV+ CHO cells relative to a negative control where no CTLA-4 was present. (B) Anti-CTLA-4 doses were Log(x) transformed and dose response curves fit using Prism v6 to obtain Log EC50 for Iipilimumab (Black line) and tremelimumab (Blue line). (C) Graphs show the mean EC50 with 95% confidence interval calculated using Prism v6. (D-F) As in A, B & C except with CTLA-4 expressing Jurkat and CTV stained DG75 B cells expressing CD80 or CD86-GFP. Data for ipilimumab and tremelimumab were acquired in separate 96-well plates, data presented as mean +/- SD from 3 independent experiments.

Article Snippet: CD80 (Cat# 10133-B1) and CD86 (Cat# 141-B2) -Ig expressing human IgG1 Fc were purchased from R&D Systems.

Techniques: Blocking Assay, Staining, Expressing, Cell Culture, Titration, Negative Control, Transformation Assay

Journal: Frontiers in immunology

Article Title: Impact of CTLA-4 checkpoint antibodies on ligand binding and Transendocytosis.

doi: 10.3389/fimmu.2022.871802

Figure Lengend Snippet: FIGURE 8 Ipilimumab and Tremelimumab interfere with pre-established transendocytosis of CD80 and CD86 to prevent further ligand loss from APC. (A) CTV stained CHO cells expressing either CD80 or CD86-GFP were cultured in the presence of CHO cells expressing CTLA-4 at a 1:1 ratio for 0, 3 or 6 hours at 37°C. Transendocytosis was established for 3 hours in the absence of anti-CTLA-4 Abs at which point soluble ipilimumab (dotted black line) or tremelimumab (dotted blue line) were spiked into culture. Transendocytosis was continued for a further 3 hours to observe the effect of anti-CTLA4 spike. Soluble anti-CTLA-4 antibodies were also added at timepoint 0h to block transendocytosis from the start of the assay (solid black – ipilimumab or blue – tremelimumab lines). Transendocytosis was also performed in the absence of anti-CTLA-4 treatment (Purple line). (B) % of CD80 or CD86-GFP ligand loss from CTV+ CHO cells by transendocytosis was determined by making the GFP MFI CTV+ CHO cells relative to a negative control where CTLA4 was not expressed (n=3). (C, D) As in A & B except with CTLA-4 expressing Jurkat and CTV stained DG75 B cells expressing CD80 or CD86-GFP cultured at a Jurkat:DG75 ratio of 2:1. Also an additional spike was performed at 6h and the assay was run for a total of 20h. Data for ipilimumab and tremelimumab were acquired in separate 96-well plates (n=2).

Article Snippet: CD80 (Cat# 10133-B1) and CD86 (Cat# 141-B2) -Ig expressing human IgG1 Fc were purchased from R&D Systems.

Techniques: Staining, Expressing, Cell Culture, Blocking Assay, Negative Control

Journal: Nature Communications

Article Title: Gut commensal Bifidobacterium -derived extracellular vesicles modulate the therapeutic effects of anti-PD-1 in lung cancer

doi: 10.1038/s41467-025-58553-4

Figure Lengend Snippet: a Immunofluorescent detection of uptake of PKH67 (green) stained Bif .BEVs (5 µg/ml) by LL/2 cells after 24 h post treatment with endocytosis inhibitors. DAPI (blue) was used to visualize nuclei. The scale bar is 20 µm. b Quantification of fluorescence intensity of images from 2a by ImageJ analysis using 17 different areas. Statistical comparisons of medians were conducted using the Kruskal-Wallis test. For all box plots, the central line inside each box represents the median (50th percentile). The box bounds correspond to the first quartile (Q1, 25th percentile) and third quartile (Q3, 75th percentile). The whiskers extend to the minimum and maximum values. Exact p values are indicated in the figure. c Western blot analysis of anti-LTA in LL/2 cells after pre-treatment with endocytosis inhibitor and Bif .BEVs (5 ug/ml). GAPDH was used as internal control. d Western blot analysis of PDL-1, CD80, PVR/CD155 in lung cancer cells (A549, H460, LL/2) along with immortal noncancerous lung cells BEAS-2B after treatment with either Bif .BEVs (5 µg/ml) or cell lysates (5 µg/ml). GAPDH and actin were used as internal controls. e Western blot analysis of TLR-4, TLR-7 and NF-κB after treatment with Bif .BEVs (5 µg/ml). Actin was used as internal loading control. f Western blot analysis of NF-κB and PD-L1 after pre-treatment with NF-κB inhibitor for 2 h and Bif .BEVs (5 µg/ml) for 48 h. All Western blots except using BEAS-2B (due to slow growth) have been repeated at least twice in independent experiments. Source data are provided as a Source Data file.

Article Snippet: The primary antibodies used included anti-PD-L1 [E1L3N(R)], Cat # 13684S) at 1:1000, CD80 (Cat #15416), PVR/CD155 (Cat #81254), TLR-2 (Cat #13744), TLR-4 (Cat # 14358), TLR-6 (Cat # 12717), TLR-7 (Cat # 82658), NF-kB (Cat# 8242) and anti-GAPDH (14C10, Cat # 2118S) at 1:1000 were purchased from Cell Signaling Technology.

Techniques: Staining, Fluorescence, Western Blot, Control