Human CCR7 Antibody

Chemotaxis Induced by CCL19/MIP‑3 beta and Neutral-ization by Human CCR7 Antibody.

Recombinant Human CCL19/MIP-3 beta (Catalog # 361-MI) chemoattracts the BaF3 mouse pro-B cell line transfected with human CCR7 in a dose-dependent manner (orange line). The amount of cells that migrated through to the lower chemotaxis chamber was measured by Resazurin (Catalog # AR002). Chemotaxis elicited by Recombinant Human CCL19/MIP-3 beta (50 ng/mL) is neutralized (green line) by increasing concentrations of Mouse Anti-Human CCR7 Monoclonal Antibody (Catalog # MAB197). The ND₅₀ is typically 1-5 µg/mL.

Detection of Human CCR7 by Flow Cytometry

HCMV strain-dependent variability of UL4015-23 sequences and HCMV strain-specific HLA-EUL40 T-cell response in hosts.(A, B) Genomic DNAs isolated from HCMV positive blood samples in HCMV+ transplant recipients (n = 25) were sequenced for the identification of UL40 protein (amino acids 1–221) provided by the circulating HCMV strains. (A) Amino acid variability, expressed as a number of amino acid variants, within the HLA-E-binding peptide (UL4015-23, shown in red) among the sequence for HCMV UL40 signal peptide (UL401-37, shown in grey). A total of 32 UL40 sequences from 25 hosts were analysed. UL40 protein sequence from the Merlin HCMV clinical strain was used as reference. Positions 1 to 9 of residues in the HLA-E-binding peptide (UL4015-23) are indicated. (B) Sequence LOGO of the UL4015-23 HLA-E-binding peptide from 25 transplanted hosts. The height of the letter is proportional to the frequency of each amino acid in a given position (P1 to P9). Major anchor residues for binding in the HLA-E peptide groove are indicated in blue. Red letters highlight the important variability observed in position 8 of the HLA-E-binding peptide. Grey boxes correspond to a constitutive deletion of the corresponding amino acid in the UL40 sequence from the infecting viral strain. (C) Representative dot plot analyses showing the detection of strain-specific anti-UL40 HLA-E-restricted CD8 T-cell responses in 4 KTRs (KTR#026, #105, #108 and #109). Frequencies (%) of the HLA-EUL40-specific T cells among total circulating alpha beta CD8 T cells are indicated. Image collected and cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.ppat.1007041), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human CCR7 by Proximity Ligation Assay

CXCR4 ligand binding facilitates CXCR4/CCR7 hetero-oligomer formation.(A) CCR7 homo-oligomer formation in H9 cells after treatment with or without 1 μg/ml recombinant gp120 was examined by in situ PLA. The number of in situ PLA signals per cell was counted by using the Duolink Image Tool software. The result shown is a representative result from three independent experiments showing the mean number of the signals plotted on the vertical axis. *, p < 0.05 by Mann-Whitney’s U test. (B) CCR7 homo-oligomer formation in H9 cells after treatment with 100 ng/ml CXCL12 was examined, as described in (A). Quantification of CCR7 homo-oligomers after CXCL12 pretreatment in the presence or absence of 1 or 5 μg/ml AMD3100. The result shown is a representative result from five independent experiments. *, p < 0.05 by Mann-Whitney’s U test. (C) CXCR4/CCR7 or CXCR4/CCR1 hetero-oligomer formation in H9 cells by in situ PLA using the indicated combinations of antibodies. The z-stack images derived from sections covering 10 μm with a 0.5-μm step are shown. Scale bar represents 50 μm. (D) Quantification of the detected in situ PLA signals per cell was performed. The result shown is a representative result from three independent experiments. *, p < 0.05 by Mann-Whitney’s U test. (E) CXCR4/CCR7 hetero-oligomer formation in H9 cells after treatment with 1 μg/ml gp120 in a native form (N) or control heat-denatured form (D) was examined by the PLA using the indicated combinations of antibodies. (F) CXCR4/CCR7 hetero-oligomer formation was examined with 100 ng/ml CXCL12 as described in (E). Image collected and cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pone.0117454), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Rhesus Macaque CCR7 by Flow Cytometry

Dynamics of CCR7 expression on CD1c+ mDC and pDC during primary SIVmac239 infection.(A) The dynamics of CCR7 expression on CD1c+ mDC; (B) The dynamics of CCR7 expression on pDC. Image collected and cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pone.0029036), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human CCR7 by Immunohistochemistry

CCR7 homodimers are dissociated by the CCR7 TM4-derived peptide. (A) The levels of bioluminescence signals are presented for HEK293T cells transfected with the indicated combinations of CCR7-CGLuc, CCR7-NGLuc, CGLuc and NGLuc plasmids (1.5 μg each). A representative experiment from at least three independent experiments is shown. Data represent mean ± SD of triplicate samples. *p < 0.05 by one-way ANOVA. (B) The levels of bioluminescence signals are shown for cells transfected with combinations of CCR7-CGLuc and CCR7-NGLuc in the presence of the CCR7 TM4 peptide or the shuffled peptide (15 μg/ml). Relative luminescence was obtained by normalizing the values against untransfected cells. A representative experiment from at least three independent experiments is shown. Data represent mean ± SD of triplicate samples. *p < 0.05 by Student’s t test. (C) The levels of CCR7 homodimer in parental H9 cells were measured by in situ PLA. CCR7-CCR1 heterodimer formation detected by anti-CCR7 mAb-PLAPlus and anti-CCR1 mAb -PLAMinus (left), and CCR7 homodimer formation by anti-CCR7 mAb-PLAPlus and -PLAMinus (right). A representative experiment from at least three independent experiments is shown with the mean number of PLA signals plotted on the vertical axis. *p < 0.05 by Mann-Whitney’s U test. (D) CCR7 (top) or CCR1 (bottom) homodimer formation after treatment with the CCR7 TM4 peptide (right panel) or the shuffled peptide (left panel). The number of PLA signals per cell was counted using the Duolink Image Tool software. A representative experiment from three independent experiments is shown with the mean number of PLA signals plotted on the vertical axis. *p < 0.05 by Mann-Whitney’s U test; NS, not significant. (E) CCR7 expression levels were evaluated in the presence (red open histogram) or absence (blue open histogram) of the CCR7 TM4 peptide (15 μg/ml) by flow cytometry using anti-human CCR7 antibody or isotype control antibody (gray-filled histogram). MFI is indicated on the histograms. A representative experiment from three independent experiments is shown. Image collected and cropped by CiteAb from the following publication (https://www.nature.com/articles/s41598-017-09113-4), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Rhesus Macaque CCR7 by Flow Cytometry

Two-color strategy for identifying CD1c+ mDCs and pDCs.(A) PBMCs (R1) were first gated on Forward-scatter/side-scatter (FSC/SSC) scattergram. Then, CD1c+ mDCs (CD1c+CD14−CD20−) were selected in the R2 region and pDCs (CD123brightHLA-DR+) were selected in the R3 region. (B) The expressions of immunophenotypes, including CD4, CCR5, CD80, CD83, CD86, and CCR7 on DCs subsets were analyzed. Open histograms correspond to isotype controls and filled gray histograms correspond to specific mAbs staining. Image collected and cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pone.0029036), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of CCR7 in Human Tonsil.

Formalin-fixed paraffin-embedded tissue sections of human tonsil were probed for CCR7 mRNA (ACD RNAScope Probe, catalog # 410728; Fast Red chromogen, ACD catalog # 322750). Adjacent tissue section was processed for immunohistochemistry using mouse anti-human CCR7 monoclonal antibody (R&D Systems catalog # MAB197) at 15ug/mL with overnight incubation at 4 degrees Celsius followed by incubation with anti-mouse IgG VisUCyte HRP Polymer Antibody (Catalog # VC001) and DAB chromogen (yellow-brown). Tissue was counterstained with hematoxylin (blue). Specific staining was localized to lymphocytes.