anti cd3e antibody  (Cell Signaling Technology Inc)

Journal: Frontiers in Cell and Developmental Biology

Article Title: Fucoidan-Supplemented Diet Potentiates Immune Checkpoint Blockage by Enhancing Antitumor Immunity

doi: 10.3389/fcell.2021.733246

Figure Lengend Snippet: Fucoidan diet enhances the antitumor efficacy of PD-1 antibodies. (A) Experimental scheme for fucoidan-supplemented diet combined with PD-1 immunotherapy in the B16 melanoma model. C57BL/6 mice were subcutaneously inoculated with B16 cells, fed with fucoidan A or F from the inoculation day (day 0) to the end point (day 20). Each mouse was administered with 200 μg PD-1 antibody at day 7, 10, and 13. i.p., intraperitoneally. (B) Tumor volumes recorded at indicated times are shown. (C) Tumor images and (D) weights of harvested tumors at day 20 are shown. (E) Flow cytometry analysis of spleen resident and tumor infiltrating lymphocytes (TILs). Spleens, tumor-draining lymph nodes, and tumor tissues were collected respectively for flow cytometry analysis. Top: Representative FCM plots (left) and analysis (right) of CD8 + and CD4 + T cells in spleen. Middle: Representative FCM plots (left) and analysis (right) of NK cells in spleen. Bottom: FCM plots (left) and analysis (right) of CD45 + CD3 + TILs. A, B, C, D ( n = 8 mice per group) and E ( n = 6 mice per group) are representative of two independent experiments. Fuco, fucoidan. Error bars, mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. n.s., non-significant.

Article Snippet: CD3E was detected with CD3E (CD3-12) rat mAb (CST Signaling, cat. #4443) at 1:1000 dilution and goat anti-rat IgG (H + L) HRP conjugates (Proteintech, cat. #SA00001) at 1:2000 dilution.

Techniques: Flow Cytometry

Journal: Frontiers in Cell and Developmental Biology

Article Title: Fucoidan-Supplemented Diet Potentiates Immune Checkpoint Blockage by Enhancing Antitumor Immunity

doi: 10.3389/fcell.2021.733246

Figure Lengend Snippet: Fucoidan alone is insufficient to inhibit melanoma cell growth. (A) B16 cells were cultured with fucoidan A or F with indicated concentration for 48 h in 96-well plates. CCK8 assay was used to monitor cell proliferation. (B) B16 cells were cultured with 100 μg/ml fucoidan A or F for different durations. Cell apoptosis (C) was assessed by Annexin V/PI staining and cell cycle (D) was measured using PI staining. B16 cells were incubated with 100 μg/ml fucoidan A or F for 48 hours. Growth (E) and weights (F) of B16 tumors harvested from control mice, or mice with dietary fucoidan A or F treatments. The flow cytometry analysis of splenic CD8 + /CD4 + T cells (G) , CD3 + TILs (H) , or splenic NK cells (I) , in fucoidan A treated or untreated B16-bearing mice. In vitro experiments were repeated at least three times. For in vivo experiments, data in E-F ( n = 7 mice per group) and G-I ( n = 6 mice per group) are representative of two independent replicates. Error bars, mean ± SEM. * P < 0.05, non-significant.

Article Snippet: CD3E was detected with CD3E (CD3-12) rat mAb (CST Signaling, cat. #4443) at 1:1000 dilution and goat anti-rat IgG (H + L) HRP conjugates (Proteintech, cat. #SA00001) at 1:2000 dilution.

Techniques: Cell Culture, Concentration Assay, CCK-8 Assay, Staining, Incubation, Control, Flow Cytometry, In Vitro, In Vivo

Journal: Frontiers in Cell and Developmental Biology

Article Title: Fucoidan-Supplemented Diet Potentiates Immune Checkpoint Blockage by Enhancing Antitumor Immunity

doi: 10.3389/fcell.2021.733246

Figure Lengend Snippet: Fucoidan activates the JAK-STAT pathway and promotes T cell proliferation and activation. (A) IFNγ and TNFα expression in primary CD8 + T cells cultured with anti-CD3/CD28 beads, with or without fucoidan (25 μg/ml) treatments. Numbers adjacent to the outlined areas indicate the frequency of cells expressing IFNγ and TNFα. All data are quantified on the right. (B) IFNγ expression levels in CD8 + T cells treated by indicated concentrations of fucoidan. (C) CFSE assays quantifying the proliferation of CD8 + T cells stimulated by indicated concentrations of fucoidan. (D) Gene set enrichment analysis (GSEA) of the RNAseq data of fucoidan-treated (10 μg/ml) and control CD8 + T cells. (E) Heatmap of fucoidan-regulated genes involved in the JAK-STAT pathway. Ctrl, control. A1, A2, two biological replicates of fucoidan A-treated samples. F1, F2, two biological replicates of fucoidan F-treated samples. (F) Volcano plots of differentially expressed genes from RNAseq data of fucoidan-treated and control CD8 + T cells. The x axis represents log2 of fold changes (FC) of gene expression levels in fucoidan-treated cells relative to control cells, and the y axis represents log10 of corresponding P values. Genes within the JAK-STAT pathway were highlighted. (G) qRT-PCR analysis of the mRNA expression of indicated genes in fucoidan-treated and control CD8 + T cells. (H,I) IFNγ expression in CD8 + T cells activated by anti-CD3/CD28 beads, treated with fucoidan (10 μg/ml) and/or AZD1480 (50 nM). Data are representative of three independent experiments. Error bars, mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: CD3E was detected with CD3E (CD3-12) rat mAb (CST Signaling, cat. #4443) at 1:1000 dilution and goat anti-rat IgG (H + L) HRP conjugates (Proteintech, cat. #SA00001) at 1:2000 dilution.

Techniques: Activation Assay, Expressing, Cell Culture, Control, Gene Expression, Quantitative RT-PCR

Journal: Frontiers in Cell and Developmental Biology

Article Title: Fucoidan-Supplemented Diet Potentiates Immune Checkpoint Blockage by Enhancing Antitumor Immunity

doi: 10.3389/fcell.2021.733246

Figure Lengend Snippet: Fucoidan interacts with the T cell receptor complex to enhance T cell activity. (A) Genes Ontology (GO) analysis showing the enrichment of specific pathways regulated by indicated transcription factors, based on upregulated hallmark genes in fucoidan-treated CD8 + T cells. (B) Flow cytometry analysis of CD8 + T cells unstained or stained with FITC-labeled Ulex Europaeus Agglutinin I (FITC-UEA-I), supplemented with or without 100 μg/ml fucoidan. (C) Representative fluorescent images of Jurkat cells overexpressing RFP-CD3E (red) stained with FITC-UEA-I (green), supplemented with or without 100 μg/ml fucoidan. Nuclei were stained with DAPI (blue). Scale bar, 10 μm. (D) Lysates from primary CD8 + T and Jurkat cells treated with or without fucoidan were subjected to pull-down assays with Ulex Europaeus Agglutinin I (UEA I) conjugated agarose, followed by immunoblotting analysis with CD3E antibodies. (E,F) Granzyme B expression levels in Jurkat cells activated by anti-human CD3/CD28 supplemented with fucoidan (10 μg/ml) and/or AZD1480 (40 μM). (G) Knockdown efficiencies of CD3E mRNA and protein using indicated shRNAs in Jurkat cells. (H) qRT-PCR analysis of GZMB and IL2 mRNA expression in Jurkat cells transfected with CD3E shRNA#2 or shNC. GZMB, Granzyme B. NC, non-targeted control. Data are representative of two independent experiments. Error bars, mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, and n.s., non-significant.

Article Snippet: CD3E was detected with CD3E (CD3-12) rat mAb (CST Signaling, cat. #4443) at 1:1000 dilution and goat anti-rat IgG (H + L) HRP conjugates (Proteintech, cat. #SA00001) at 1:2000 dilution.

Techniques: Activity Assay, Flow Cytometry, Staining, Labeling, Western Blot, Expressing, Knockdown, Quantitative RT-PCR, Transfection, shRNA, Control