Ccr7 pe cy7

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CCR7-PE-Cy7 is a fluorescent-conjugated antibody used for the detection and analysis of CCR7 (C-C Chemokine Receptor Type 7) expression on cells. It is a useful tool for immunophenotyping and flow cytometry applications.

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CCR7-PE (26 citations) Anti-CCR7-PE-Cy7 (11 citations) CCR7-PE-Cy7 (G043H7, (3 citations) CCR7-PE-CY7 (clone G043H7, (2 citations) CD4 PE-Cy7 (SK3); CD8 PerCP-Cy5.5 (RPA-T8); CCR7 FITC (G043H7); CD45RA BV421 (HI100); HLA-DR PE (L243); CD38 BV605 (HIT2) (2 citations)

19 protocols using ccr7 pe cy7

HCV-Specific T-Cell Profiling Protocol

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Patient 355 (59-year old Male, infected with genotype 1a HCV, baseline viral load 467 000 IU/ml) received a prime vaccination of ChAd3-NSmut (2.5 × 10¹⁰ viral particles) and an MVA-NSmut (2 × 10⁸ plaque forming units) boost vaccination 8 weeks later. PBMC were collected 14 weeks post-boost vaccination for assessment of single cell gene expression (22 ). PBMC were thawed and prestained with a live/dead marker (Life Technologies, cat# L34976) and a PE-conjugated pentamer reagent (PE-labeled HCV NS31406–1415 (KLSALGINAV; HLA-A*0201)). The cells were then stained with the following antibodies: FITC 2B4 (BioLegend, cat# 329505), PerCP-eFluor 710 LAG3 (eBioscience, cat# 46–2239), PE-Cy7 CCR7 (BioLegend, cat# 329919), APC CD39 (BioLegend, cat# 328209), BV421 PD1 (BioLegend, cat# 329919), BV510 CD3 (BioLegend, cat# 317332), BV605 CD8A (BioLegend, cat# 301040), BV780 CD45RA (BioLegend, cat# 304140).
The relevant institutional review boards approved all human subject protocols, and all subjects provided written consent before enrollment.

Afik S., Yates K.B., Bi K., Darko S., Godec J., Gerdemann U., Swadling L., Douek D.C., Klenerman P., Barnes E.J., Sharpe A.H., Haining W.N, & Yosef N. (2017). Targeted reconstruction of T cell receptor sequence from single cell RNA-seq links CDR3 length to T cell differentiation state. Nucleic Acids Research, 45(16), e148.

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Phenotyping Yellow Fever-Specific CD8+ T Cells

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A healthy volunteer was vaccinated with a single dose (0.5 ml containing at least 10⁵ PFU) of 17D live-attenuated yellow fever vaccine strain administered subcutaneously. Seroconversion after vaccination was confirmed by assaying the neutralizing antibody titers for YF-17D (data not shown). A whole blood sample was obtained 9 months post-vaccination and lymphocytes were enriched from whole blood via Ficoll gradient centrifugation and a CD8 negative selection magnetic bead kit (Miltenyi Biotec). Cells were prestained with a live/dead marker (Life Technologies, cat# L34976) and an APC-labeled tetramer reagent (NS4B 214–222 LLWNGPMAV, kindly provided by Dr Rafi Ahmed). The cells were then stained with the following antibodies: FITC CD8A (BioLegend, cat# 300906), PE CXCR3 (BioLegend, cat# 353705), PE-Cy7 CCR7 (BioLegend, cat# 353226), BV421 IL2Rb (BioLegend, cat# 339009), BV510 CD3 (BioLegend, cat# 317332), BV605 CD95 (BioLegend, cat# 305627), BV780 CD45RA (BioLegend, cat# 304140).

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Polychromatic Flow Cytometry for Treg and Th Subsets

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Regulatory T cells (Treg) were defined as CD4⁺CD25⁺CD127^low/- cells. Other CD4⁺T subsets were identified by differential expression of CCR4, CXCR3, and CCR6 as previously reported (7 (link), 9 (link)): CXCR3⁺CCR4⁻CCR6⁻(Th1), CXCR3⁻CCR4⁺CCR6⁻ (Th2), CXCR3⁻CCR4⁺CCR6⁺(Th17), and CXCR3⁺CCR4⁻CCR6⁺ (Th1Th17) (Figure 1). In addition, Th17 cells were defined as the summation of CXCR3⁻CCR4⁺CCR6⁺ (Th17) and CXCR3⁺CCR4⁻CCR6⁺(Th1Th17).
The fluorochrome-conjugated antibodies used for polychromatic flow cytometry analysis were CD3-BV510 (Biolegend, USA, catalog no. 317332), CD4-APC-Cy7 (Biolegend, USA, catalog no. 317418), CCR4-APC (Biolegend, USA, catalog no. 359404), CXCR3-PE (Biolegend, USA, catalog no. 353706), CCR6-PE-Cy7 (Biolegend, USA, catalog no. 353418), CD25-BV421 (Biolegend, USA, catalog no. 302630), CD127-FITC (Biolegend, USA, catalog no. 351312), PE-Cy7-CCR7 (Biolegend, USA, catalog no. 353226) and CD62L-FITC (Biolegend, USA, catalog no. 304804). A viability dye, 7-AAD (Becton Dickinson, USA, catalog no. 559925), was used to exclude dead cells. Isotype antibodies were also used as negative controls for every detection to set proper gating for the receptor expression. Cells were analyzed by fluorescence-activated cell sorting (FACS), using the BD LSRII cytometer and FlowJo software.

Yu X., Wang M, & Cao Z. (2020). Reduced CD4+T Cell CXCR3 Expression in Patients With Allergic Rhinitis. Frontiers in Immunology, 11, 581180.

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Comprehensive Immune Cell Phenotyping

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Cell surface and intracellular molecular expressions were evaluated by flow cytometry using CytoFLEX (BeckmanCoulter, U.S.A.). Fluorescein-conjugated mouse anti-human antibodies were used, including CD3-Alexa Fluor 700, CD3-BV650, CD8-BV786, CD8-PerCP/Cy5.5, CD45RA-APC/CY7, CCR7-PE/CY7,CD27-PE, CD28-BV421, CD69-APC/CY7, CD103-BV605, CXCR3-BV510, HLA-DR-APC, CD39-BV421, PD-1-PE, CD127-PE/CY7, CD62L-PE/CY7, Perforin-APC, Granzyme B-PE, IFN-γ-PE, and IL-4-APC (Biolegend, UK). For cell-surface staining, single-cell suspensions were stained on ice for 30 min in PBS with 1% fetal bovine serum (FBS). For intracellular staining, cells were fixed and permeabilized using the Fix/Perm kit (BD Biosciences, U.S.A.). To detect intracellular cytokines, CD8⁺T cells were stimulated for 6 h with phorbol 12-myristate 13-acetate (PMA; 1 μg/mL; Sigma) and ionomycin (1 μg/mL; Sigma), and 4 h with GolgiStop (1 μL/mL; BD Biosciences) in a round-bottom 96-well plate. Thereafter, cells were harvested, stained for surface expression, and then fixed and permeabilized for intracellular staining. Flow cytometry data was analyzed using FlowJo software (BD, UK) and CytoExpert software (Beckman Coulter, U.S.A.).

Liu L., Huang X., Xu C., Chen C., Zhao W., Li D., Li L., Wang L, & Du M. (2020). Decidual CD8+T cells exhibit both residency and tolerance signatures modulated by decidual stromal cells. Journal of Translational Medicine, 18, 221.

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Multiparameter Flow Cytometry Staining

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The following antibodies were used for cell surface staining: CD3-eFluor 780-allophycocyanin (eF780-APC; clone UCHT1), TCR-allophycocyanin (APC; clone IP26), and IL-2–APC (clone MQ1-17H12) (all from eBioscience); CD4-BD Horizon R phycoerythrin-CF594 (PE-CF594; clone RPA-T4), CD27-PE-Cy7 (Pe-Cy7-clone M-T271), CXCR5-Alexa Fluor 488 (AF488; clone RF8B2), and CCR7-Alexa Fluor 647 (AF647; clone 3D12) (all from BD Biosciences); CD14-Viogreen (clone TÜK4) and CD20-Viogreen (clone LT29) (from Miltenyi Biotec); and CD38-Alexa Fluor 700 (AF700; clone HIT2), CD20-peridinin chlorophyll protein (PerCP)/Cy5.5 (clone 2H7), CD8-brilliant violet 785 (BV785; clone RPA-T8), CD45RA-brilliant violet 421 (BV421; clone HI100), CCR7-PE-Cy7 (clone G043H7), and CD3-APC (clone SK7) (all from BioLegend). The fixable viability dye eFluor 506 (eF506; eBioscience) was added to restrict the analysis to live cells.

Claireaux M., Galperin M., Benati D., Nouël A., Mukhopadhyay M., Klingler J., de Truchis P., Zucman D., Hendou S., Boufassa F., Moog C., Lambotte O, & Chakrabarti L.A. (2018). A High Frequency of HIV-Specific Circulating Follicular Helper T Cells Is Associated with Preserved Memory B Cell Responses in HIV Controllers. mBio, 9(3), e00317-18.

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Isolation and Sorting of CD4+ T Cell Subsets

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CD4⁺ T cells were isolated from whole blood by negative selection using RosetteSep^™ Human CD4⁺ T Cell Enrichment Cocktail (STEMCELL Technologies). To sort T cell subsets cells were stained with CD3-PacificBlue (BD Biosciences), CCR7-PE-Cy7 (BioLegend), CD45RA-PE-Cy5.5 (Invitrogen), CD27-PE (BD Biosciences), CD4-APC (BioLegend). T cell subsets were separated with FACSAria.

Michaels K.K., Natarajan M., Euler Z., Alter G., Viglianti G, & Henderson A.J. (2015). Blimp-1, an Intrinsic Factor that Represses HIV-1 Proviral Transcription in Memory CD4+ T Cells. Journal of immunology (Baltimore, Md. : 1950), 194(7), 3267-3274.

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Flow Cytometry of BAL and PBMC Cells

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BAL cells (200,000–500,000 cells) and PBMC (100,000–200,000) were resuspended in Facs Buffer (10%FBS+4mM EDTA +PBS) and stained for 20 minutes at room temperature with the following antibodies used per manufacturer’s instructions: CD3-FITC (Biolegend, #344804), CD4-APC-Cy7 (BD Biosciences, #BD557871), CD8-APC (Biolegend, #344722), CD56-BV421 (Biolegend, #362552), Gamma-delta-PE (Biolegend, #331210), CCR7-PECY7 (Biolegend, #353226) and CD45RA-PECY5 (Biolegend, #304110). Compensation was performed using antibody capture beads (anti-mouse k; BD Biosciences, #552843). Zombie Aqua fixable viability kit (BioLegend, #433101) was used as the live/dead discriminator. Cells were fixed, acquired and analyzed on a MacsQuant flow cytometer using MacsQuant software version 2.11.176.19438. Analysis was performed using Flow Jo version 10.6 (Oregon, USA). Cells where gated on lymphocyte sized, singlet, live cells. BAL and PBMC flow cytometric counts and percentages for each participant can be found in the S2 Dataset.

Thiel B.A., Worodria W., Nalukwago S., Nsereko M., Sanyu I., Rejani L., Zawedde J., Canaday D.H., Stein C.M., Chervenak K.A., Malone L.L., Kiyemba R., Silver R.F., Johnson J.L., Mayanja-Kizza H, & Boom W.H. (2021). Immune cells in bronchoalveolar lavage fluid of Ugandan adults who resist versus those who develop latent Mycobacterium tuberculosis infection. PLoS ONE, 16(4), e0249477.

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Comprehensive Immune Cell Profiling

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Fresh PBMCs were stained with anti-CD3-APC-Cy7, CD8-APC, CD45RA-PE-Cy5, CCR7-PE-Cy7, PD-1-Pacific Blue, IL-7Rα-FITC, CD27-PE, CD28-PE, CTLA4-PE, CX3CR1-PE antibodies or isotype control (all from BioLegend, San Diego, CA). PBMCs that had been stimulated with PMA/ionomycin were stained with anti-CD3-APC-Cy7, CD8-APC, CD45RAPE-Cy5 and CCR7-PE-Cy7 antibodies followed by fixation, permeabilization (Cytofix/Cytoperm Kit, BD Biosciences) and staining with anti-IFNγ-PE, TNF-α-FITC or IL-13-PE antibodies (BioLegend).^{14 (link)} Stained cells were analyzed on an LSRII® flow cytometer (BD Biosciences). Collected data were analyzed using FlowJo software (Tree Star, Ashland, OR).

Kim J.W., Shin M.S., Kang Y., Kang I, & Petrylak D.P. (2017). Immune Analysis of Radium-223 in Patients with Metastatic Prostate Cancer. Clinical genitourinary cancer, 16(2), e469-e476.

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Multicolor Flow Cytometry to Characterize Tfh Cells

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Immunophenotyping of lymphocytes was performed by multicolor flow cytometry. Heparinized whole blood samples were immediately stained for 15 minutes with the following antibodies: CD3-APC/Cy7 (UCHT, BioLegend; San Diego, CA, USA), CD4-HorizonV500 (RPA-T4, BD Biosciences; San Jose, CA, USA), CD45RA-BV421 (HI-100, BioLegend), CXCR5-PerCP/Cy5.5 (J252D4, BioLegend), CXCR3-APC (1C6, BD Biosciences), CCR6-PE (11A9, BD Biosciences), CCR7-PE/Cy7 (G043H7, BioLegend), PD-1-FITC (EH12.2H7, BioLegend), CD19-BV421 (HIB19, BioLegend), CD20-APC (2H7, BioLegend), CD27-APC/Cy7 (O323, BioLegend), and CD38-FITC (HIT2, BioLegend). Circulating Tfh cells were defined as CD3⁺CD4⁺CD45RA^-CXCR5⁺ cells, and Tfh1, Tfh2, or Tfh17 cell subsets as CXCR3⁺CCR6^- cells, CXCR3^-CCR6^- cells, or CXCR3^-CCR6⁺ cells among Tfh cells, respectively [19 (link), 20 (link)]. Activated Tfh cells were defined as the CCR7^lowPD-1^high cells among Tfh cells [26 (link)]. CD19⁺CD20^-CD27⁺CD38⁺ cells were defined as plasmablasts. Red blood cells were lysed with FACS Lysing Solution (BD Biosciences). All samples were analyzed with a FACS Aria III (BD Biosciences), and data were analyzed with FlowJo v.7.6.4 Software (Tree Star, Stanford University, CA, USA). Proportions of lymphocyte subsets were determined by the combination of surface marker staining, with exclusion of doublets by forward and side scatter.

Akiyama M., Yasuoka H., Yamaoka K., Suzuki K., Kaneko Y., Kondo H., Kassai Y., Koga K., Miyazaki T., Morita R., Yoshimura A, & Takeuchi T. (2016). Enhanced IgG4 production by follicular helper 2 T cells and the involvement of follicular helper 1 T cells in the pathogenesis of IgG4-related disease. Arthritis Research & Therapy, 18, 167.

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Detailed Immunophenotyping of T-cell Subsets

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The following antibodies were used for T-cell phenotyping: CD8-BV650 (BD), CD4-BV605 (BD Biosciences), CD3-Alexa Fluor700 (BD Biosciences), CD95-BV711 (BD Biosciences), CD45RO-PerCPCy5.5 (BD Biosciences), CD25-APC-Cy7 (BD Biosciences), CD127-BV421 (BioLegend), CCR7-PE-Cy7 (BioLegend), CD45RA-ECD (Beckman Coulter), PD-1-BV785 (BioLegend), LAG3-FITC (R&D Systems), and TIM3-APC (R&D Systems). The dead cell exclusion stain (Live/Dead Aqua) was purchased from Invitrogen. To detect transduced NY-ESO-1^c259TCR-expressing cells, PE-conjugated pentamer reagents specific for the HLA-A*02:01 SLLMWITQC complex (ProImmune) were used at the manufacturer’s recommended concentrations.

D'Angelo S.P., Melchiori L., Merchant M.S., Bernstein D., Glod J., Kaplan R., Grupp S., Tap W.D., Chagin K., Binder G.K., Basu S., Lowther D.E., Wang R., Bath N., Tipping A., Betts G., Ramachandran I., Navenot J.M., Zhang H., Wells D.K., Winkle E.V., Kari G., Trivedi T., Holdich T., Pandite L., Amado R, & Mackall C.L. (2018). Antitumor Activity Associated with Prolonged Persistence of Adoptively Transferred NY-ESO-1c259T Cells in Synovial Sarcoma. Cancer discovery, 8(8), 944-957.

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