Journal: PloS one

Article Title: A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

doi: 10.1371/journal.pone.0043332

Figure Lengend Snippet: Figure 1. Binding and in vitro activity of murine 18V4F hybridoma antibody. (A) ELISA binding of original 18V4F hybridoma antibody to a panel of chemokines (determined in triplicate, shown as mean +/2 standard deviation). Directly coated chemokines were used here for direct comparisons, however in other experiments CCL3 showed a significantly enhanced signal when biotinylated and coated on streptavidin plates. (B) Chemotaxis inhibition by 18V4F hybridoma antibody of CCR5-transfected Ba/F3 cells to 5 ng/mL of CCL3, CCL4, and CCL5. Data are representative of at least three similar experiments. All chemotaxis data are represented as a percent of maximum migration in the absence of inhibitors and is fit using a standard four parameter dose-response model (GraphPad). doi:10.1371/journal.pone.0043332.g001

Article Snippet: Control inhibitors included vCCI-Fc (produced at VLST – the Fc is from human IgG1 with mutations to prevent interactions with Fc receptors [38]) and commercial antibodies against CCL3 (R&D Systems #MAB270), CCL4 (R&D Systems #MAB271), and CCL5 (R&D Systems #MAB278) as well as IgG controls.

Techniques: Binding Assay, In Vitro, Activity Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation, Chemotaxis Assay, Inhibition, Transfection, Migration

Journal: PloS one

Article Title: A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

doi: 10.1371/journal.pone.0043332

Figure Lengend Snippet: Figure 2. Diagram of phage display selection strategy. Individual CDR libraries were sequentially panned against CCL3, CCL4 and CCL5. In step 1, phage libraries were combined with biotinylated CCL3 and bound to streptavidin beads. Bound phage were eluted, amplified, and subjected to panning against biotinylated CCL4 and CCL5 in steps 2 and 3, respectively. This process was repeated 4–5 times with increasing stringency to yield sequences with improved affinities. doi:10.1371/journal.pone.0043332.g002

Article Snippet: Control inhibitors included vCCI-Fc (produced at VLST – the Fc is from human IgG1 with mutations to prevent interactions with Fc receptors [38]) and commercial antibodies against CCL3 (R&D Systems #MAB270), CCL4 (R&D Systems #MAB271), and CCL5 (R&D Systems #MAB278) as well as IgG controls.

Techniques: Selection, Amplification

Journal: PloS one

Article Title: A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

doi: 10.1371/journal.pone.0043332

Figure Lengend Snippet: Figure 3. Chemotaxis inhibition by affinity matured 18V4F variants. Chemotaxis inhibition by humanized 18V4F Fab, d5 variant, d7 variant, d5d7, and a negative control Fab of CCR5-transfected Ba/F3 cells to 5 ng/mL of (A) CCL3, (B) CCL4, and (C) CCL5. Data are representative of at least two similar experiments. A loss in potency of humanized 18V4F Fab was observed compared with the 18V4F hybridoma shown in Figure 1b and is likely a result of both the humanization process and loss in avidity caused by switching from full IgG to Fab fragment. doi:10.1371/journal.pone.0043332.g003

Article Snippet: Control inhibitors included vCCI-Fc (produced at VLST – the Fc is from human IgG1 with mutations to prevent interactions with Fc receptors [38]) and commercial antibodies against CCL3 (R&D Systems #MAB270), CCL4 (R&D Systems #MAB271), and CCL5 (R&D Systems #MAB278) as well as IgG controls.

Techniques: Chemotaxis Assay, Inhibition, Variant Assay, Negative Control, Transfection

Journal: PloS one

Article Title: A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

doi: 10.1371/journal.pone.0043332

Figure Lengend Snippet: Figure 4. Comparison of vCCI and d5d7 binding epitopes. Competitive binding ELISA examining molecules that can disrupt the d5d7-CCL3 binding interaction using d5d7 as a homologous competitor and vCCI-Fc, commercial anti-CCL3 antibody, and control IgG as heterologous competitors. Data are representative of at least two similar experiments. Competition experiments were also completed to analyze the d5d7-CCL4 and d5d7-CCL5 binding interactions and similar binding competition was observed between d5d7 and vCCI-Fc (data not shown). doi:10.1371/journal.pone.0043332.g004

Article Snippet: Control inhibitors included vCCI-Fc (produced at VLST – the Fc is from human IgG1 with mutations to prevent interactions with Fc receptors [38]) and commercial antibodies against CCL3 (R&D Systems #MAB270), CCL4 (R&D Systems #MAB271), and CCL5 (R&D Systems #MAB278) as well as IgG controls.

Techniques: Comparison, Binding Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: PloS one

Article Title: A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

doi: 10.1371/journal.pone.0043332

Figure Lengend Snippet: Figure 5. Inhibition of chemotaxis induced with mixtures of chemokines by MAb d5d7. Inhibition of chemotaxis of (A) CCR5 transfectants to a pool of recombinant CCL3, CCL4, and CCL5 and (B) CCR1 transfectants to a pool of CCL3 and CCL5, by MAb d5d7 antibody, vCCI-Fc, individual commercial anti-chemokine antibodies (anti-CCL3, anti-CCL4, and anti-CCL5), and IgG controls. Chosen chemokine concentrations were those that produced 50% maximal chemotaxis when tested individually (a pool of 3 ng/mL CCL3, 10 ng/mL CCL4, and 3 ng/mL CCL5 was used in CCR5 experiments and a mixture of 20 ng/ mL CCL3 and 5 ng/mL CCL5 was used in CCR1 experiments). doi:10.1371/journal.pone.0043332.g005

Article Snippet: Control inhibitors included vCCI-Fc (produced at VLST – the Fc is from human IgG1 with mutations to prevent interactions with Fc receptors [38]) and commercial antibodies against CCL3 (R&D Systems #MAB270), CCL4 (R&D Systems #MAB271), and CCL5 (R&D Systems #MAB278) as well as IgG controls.

Techniques: Inhibition, Chemotaxis Assay, Recombinant, Produced

Journal: PloS one

Article Title: A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

doi: 10.1371/journal.pone.0043332

Figure Lengend Snippet: Figure 8. SCID-hu mouse model of leukocyte migration. (A) NSG (NOD/SCID/IL2r-cnull) mice were injected i.v. with human PBMC and allowed to engraft for 10 d. MAb d5d7 was administered i.v. just before chemokines were injected s.c. in Matrigel. After 7 d the skin sites were harvested and single cell suspensions were generated. Human leukocytes were tagged with specific antibodies and analyzed by flow cytometry. (B) Inhibition by MAb d5d7 of skin leukocyte migration into chemokine-embedded Matrigel plugs in NSG mice engrafted with human PBMC. The negative control group consisted of animals treated with s.c. injection of Matrigel + PBS and i.v. administration of control IgG. All other groups had s.c. injections of Matrigel containing CCL3, CCL4, and CCL5 (400 ng each) with i.v. administration of PBS, control IgG, or MAb d5d7 antibody. Data were analyzed using a student t test. doi:10.1371/journal.pone.0043332.g008

Article Snippet: Control inhibitors included vCCI-Fc (produced at VLST – the Fc is from human IgG1 with mutations to prevent interactions with Fc receptors [38]) and commercial antibodies against CCL3 (R&D Systems #MAB270), CCL4 (R&D Systems #MAB271), and CCL5 (R&D Systems #MAB278) as well as IgG controls.

Techniques: Migration, Injection, Generated, Flow Cytometry, Inhibition, Negative Control, Control

Journal: Hepatology (Baltimore, Md.)

Article Title: Intratumoral γδ T-CellInfiltrates, Chemokine (C-C Motif) Ligand 4/Chemokine (C-C Motif) Ligand 5 Protein Expression and Survival in Patients With Hepatocellular Carcinoma

doi: 10.1002/hep.31412

Figure Lengend Snippet: Gene expression profiles of HCC tumor tissues stratified by γδ T-cell gene signature predicted low- and high-risk subgroups. (A) Hierarchical clustering of the 974 differentially expressed genes (P < 0.001) between γδ T-cell-signature–predicted subgroups. Pseudocolors indicate transcript levels above (blue), below (yellow), or equal to (black) the mean, respectively. Genes were ordered by centered correlation and complete linkage. The scale represents gene expression level from −3.0 to 3.0 in a log2 scale. (B) Result of GSEA enrichment of chemokine signal pathway based on the genes highly expressed in the γδ T high-tumor subgroup. (C) Pearson’s correlation analysis between CCL4, CCL5, and CCR5 expression level and γδ T-cell weights in HCC tumor tissues from the LCI cohorts. (D) Comparison of expression of CCL4, CCL5, and CCR5 in the subgroups defined by γδ T-cell signature in tumor tissues from the LCI cohorts. Abbreviation: KEGG, Kyoto Encyclopedia of Genes and Genomes. ***P < 0.001.

Article Snippet: The IHC score of CCL5 was calculated by multiplying the percentage of cells positively stained (0, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%) by the intensity of staining (0 = negative, 1 = weak, 2 = intermediate, and 3 = strong) with a maximum of 300. ( 18 ) γδ T CELLS MIGRATION ASSAY Chemotaxis of γδ T cells was analyzed in transwell migration assays. γδ T cells were expanded and sorted from splenocytes as described. ( 19 ) Next, 5 × 10 5 γδ T cells were taken up in RPMI 1640 and placed into 5-mm pore-size transwell inserts (Corning, NY), which were placed into 24 wells of a tissue-culture plate containing 5 × 10 5 Hepa1–6 cells with 0.5 μg/mL of anti-CCL5 (R&D MAB478; R&D Systems, Minneapolis, MN) or 3 μg/mL of anti-CCL4 (R&D MAB451).

Techniques: Expressing

Journal: Hepatology (Baltimore, Md.)

Article Title: Intratumoral γδ T-CellInfiltrates, Chemokine (C-C Motif) Ligand 4/Chemokine (C-C Motif) Ligand 5 Protein Expression and Survival in Patients With Hepatocellular Carcinoma

doi: 10.1002/hep.31412

Figure Lengend Snippet: Roles of tumor-cell–derived CCL4/5 on γδ T cells. (A) Spearman’s correlation analysis between CCL5 expression level and TCRγδ-positive staining cells number in the 182 FFPE HCC tissues. (B) Comparison of CCL5 expression levels between the high and low γδ T-cell-infiltrating groups. (C) Results of transwell migration assay, reflected by the proportion of chemo-attracted γδ T cells by murine hepatoma cell line Hepa1–6 with or without neutralized anti-CCL4/5. (D) Comparison of IFN-γ expression levels in transmigrated and unmigrated γδ T cells in the coculture model with Hepa1–6. (E,F) B6 mice were treated with intrahepatic trasplantation of either CCL5 shRNA-transduced or control oligo-transduced Hepa 1–6. At day 14, tumor-bearing livers were excised and used for the analysis of γδ T-cell proportion by flow cytometry (E) or analysis of expression levels of IFN-γ and perforin by real-time RT-PCR (F). Abbreviation: shCCL5, chemokine (C-C motif) ligand 5 with short hairpin RNA. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The IHC score of CCL5 was calculated by multiplying the percentage of cells positively stained (0, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%) by the intensity of staining (0 = negative, 1 = weak, 2 = intermediate, and 3 = strong) with a maximum of 300. ( 18 ) γδ T CELLS MIGRATION ASSAY Chemotaxis of γδ T cells was analyzed in transwell migration assays. γδ T cells were expanded and sorted from splenocytes as described. ( 19 ) Next, 5 × 10 5 γδ T cells were taken up in RPMI 1640 and placed into 5-mm pore-size transwell inserts (Corning, NY), which were placed into 24 wells of a tissue-culture plate containing 5 × 10 5 Hepa1–6 cells with 0.5 μg/mL of anti-CCL5 (R&D MAB478; R&D Systems, Minneapolis, MN) or 3 μg/mL of anti-CCL4 (R&D MAB451).

Techniques: Derivative Assay, Expressing, Staining, Transwell Migration Assay, shRNA, Flow Cytometry, Quantitative RT-PCR

Journal: Blood

Article Title: Inhibition of interleukin-1 signaling enhances elimination of tyrosine kinase inhibitor–treated CML stem cells

doi: 10.1182/blood-2015-11-679928

Figure Lengend Snippet: Gene expression assays used for qRT-PCR

Article Snippet: CCL4 (MIP-1-β) , Hs99999148_m1.

Techniques: Gene Expression

Journal: Blood

Article Title: Inhibition of interleukin-1 signaling enhances elimination of tyrosine kinase inhibitor–treated CML stem cells

doi: 10.1182/blood-2015-11-679928

Figure Lengend Snippet: IL-1RA in combination with NIL inhibits NF-κB target gene and cytokine gene expression in CML LSC. Expression of the NF-κB target genes, NFKB1A (A), BCL2L1 (B), and BIRC3 (C) and the inflammatory cytokines, IL-1α (D), IL-1β (E), IL-6 (F), CXCL1 (G), CXCL2 (H), CCL2 (I), CCL3 (J), CCL4 (K), and TNF-α (L) in CML CD34+CD38−CD90+ cells treated with IL-1RA, NIL, or the combination of NIL and IL-1RA, was measured by qRT-PCR. Results represent mean ± SEM. Significance values: *P < .05; **P < .01, ***P < .001, paired Student t test.

Article Snippet: CCL4 (MIP-1-β) , Hs99999148_m1.

Techniques: Gene Expression, Expressing, Quantitative RT-PCR

Journal: International Journal of Biological Sciences

Article Title: Changes in Chemokines and Chemokine Receptors Expression in a Mouse Model of Alzheimer's Disease

doi: 10.7150/ijbs.26703

Figure Lengend Snippet: mRNA expression of A: CCL3, B: CCL4 and C: CCL5 and D: CCR5 by Western-blot in cortex of APP/PS1 and WT mice. A representative immunoblot is shown in the panel. Data are mean ± SD of four independent experiments. *p<0.05 vs . WT.

Article Snippet: Ready-to-use primers and probes from the assay-on-demand service of Applied Biosystems were used for the quantification of selected target gene: CCL1 (Mm99999220_Mh), CCL3 (Mm00441259_g1), CCL4 (Mm00443111_m1), CCL5 (Mm01302428_m1), CCR5 (Mm01963251_s1), CCR8 (Mm99999115_s1), ABCF1 (Mm01275245_m1), IL-3 (Mm00439631_m1), and endogenous reference gene β-actin (Mm00607939_s1).

Techniques: Expressing, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Changes in Chemokines and Chemokine Receptors Expression in a Mouse Model of Alzheimer's Disease

doi: 10.7150/ijbs.26703

Figure Lengend Snippet: Inflammatory protein changes in APP/PS1 mice. The schema shows the up-regulation or down-regulation of different proteins. Pro-inflammatory mediators, such as CCL3 and CCL4, are increased. By contrast, CCR5 is down-regulated. The protein ABCF1 (a member of the superfamily of ATP-binding cassette (ABC) transporters) is increased. IL-3 (involved in the immune system) is highly expressed. Also CCR8 is increased (important for the migration of various cell types into the inflammatory sites).

Article Snippet: Ready-to-use primers and probes from the assay-on-demand service of Applied Biosystems were used for the quantification of selected target gene: CCL1 (Mm99999220_Mh), CCL3 (Mm00441259_g1), CCL4 (Mm00443111_m1), CCL5 (Mm01302428_m1), CCR5 (Mm01963251_s1), CCR8 (Mm99999115_s1), ABCF1 (Mm01275245_m1), IL-3 (Mm00439631_m1), and endogenous reference gene β-actin (Mm00607939_s1).

Techniques: Binding Assay, Migration

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 4D electron microscopy of T cell activation

doi: 10.1073/pnas.1914078116

Figure Lengend Snippet: Non-antigen-specific T cell activation. (A) Plot of PINEM intensity of ionomycin-treated Jurkat T cells with increasing concentration of ionomycin. Error bars represent ± SEM (n = 8 to 12 cells). (B) Bar plot of IL-2 secretion by ionomycin-treated Jurkat cells measured by ELISA. Top error bars represent s.d. (n = 3). (C) Corresponding confocal fluorescence micrographs of filamentous actin. (Scale bar: 20 μm.) (D) Plot of zeta potential of ionomycin-treated Jurkat cells. Error bars represent ± SEM (n = 3). (E) Correlation analysis of PINEM intensity (A) and zeta potential (D) (R2 = 0.62). Unless otherwise specified, PMA concentration is fixed at 50 ng⋅mL−1.

Article Snippet: CD28 monoclonal antibody (MAB342), human CCL4 ELISA kit (DMB00), and human IL-2 ELISA kit (D2050) were purchased from R & D Systems.

Techniques: Activation Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Fluorescence, Zeta Potential Analyzer

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 4D electron microscopy of T cell activation

doi: 10.1073/pnas.1914078116

Figure Lengend Snippet: Antigen-specific T cell stimulation. (A) Plot of PINEM intensity (red) and MHC binding (blue) of Jurkat cells treated with increasing concentration of pMHC tetramer (nanomolar concentrations). PINEM intensity is shown as mean ± SEM, collected from 4 to 10 individual cells. MHC binding (surface fluorescence intensity per cell) is shown as mean±SEM (n = 2); each measurement is from 28,750 to 90,000 cells in individual wells. (B) Correlation analysis of A shows that PINEM intensity vs. MHC binding represents a strong correlation (R2 = 0.88). (C) Bar plot of IL-2 secretion by pMHC-treated Jurkat cells measured by ELISA. Top error bars represent SD (n = 3). (D) Corresponding confocal fluorescence micrographs of filamentous actin. (Scale bar: 20 μm.) Unless otherwise specified, anti-CD28 antibody concentration is fixed at 1 μg⋅mL−1.

Article Snippet: CD28 monoclonal antibody (MAB342), human CCL4 ELISA kit (DMB00), and human IL-2 ELISA kit (D2050) were purchased from R & D Systems.

Techniques: Cell Stimulation, Binding Assay, Concentration Assay, Fluorescence, Enzyme-linked Immunosorbent Assay