Icos pe cy7

Manufactured by Thermo Fisher Scientific

Sourced in United States

The ICOS-PE-Cy7 is a fluorochrome-conjugated antibody used in flow cytometry applications. It specifically binds to the Inducible T-cell COStimulator (ICOS) protein expressed on the surface of activated T cells. The PE-Cy7 fluorochrome attached to the antibody allows for detection and quantification of ICOS-positive cells.

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ICOS-PE (6 citations)

7 protocols using icos pe cy7

Comprehensive Immune Profiling of Pembrolizumab Therapy

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PBMC samples at the indicated visits pre- and post-pembrolizumab treatment were thawed and stained with a fixable Aqua viability dye (Invitrogen) and a cocktail of antibodies to the following surface markers: CD8–Qdot605 (Invitrogen, 3B5), CD4–Qdot655 (Invitrogen, S3.5), PD-1–PE (BD, MIH4), LAG-3–FITC (Enzo, 17B4), ICOS–PE-Cy7 (eBioscience, ISA-3), TIM-3–APC (R&D Systems, 344823). Cells were next fixed and permeabilized with the FOXP3/Ki67 Fixation/Permeabilization Concentrate and Diluent (eBioscience), and subsequently stained intracellularly with CD3–BV570 (Biolegend, UCHT1), Ki67–AlexaFluor700 (BD), FOXP3-eFluor450 (eBioscience), and CTLA-4–PerCP–eFluor710 (eBioscience). Stained cells were acquired on a BD Biosciences LSRFortessa and analysed using FlowJo software (FlowJo, LLC).

Huang A.C., Postow M.A., Orlowski R.J., Mick R., Bengsch B., Manne S., Xu W., Harmon S., Giles J.R., Wenz B., Adamow M., Kuk D., Panageas K.S., Carrera C., Wong P., Quagliarello F., Wubbenhorst B., D’Andrea K., Pauken K.E., Herati R.S., Staupe R.P., Schenkel J.M., McGettigan S., Kothari S., George S.M., Vonderheide R.H., Amaravadi R.K., Karakousis G.C., Schuchter L.M., Xu X., Nathanson K.L., Wolchok J.D., Gangadhar T.C, & Wherry E.J. (2017). T-cell invigoration to tumour burden ratio associated with anti-PD-1 response. Nature, 545(7652), 60-65.

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Multicolor Flow Cytometry for pDCs and Tregs

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Multicolor flow cytometry was carried out on fresh Ficoll-prepared (TBD Sciences, Tianjing, China) PBMCs. After suspension in PBS, PBMCs (100 μL) were separately added to two tubes (1 × 10⁶–2 × 10⁶ PBMCs per sample). The first tube was incubated with FITC-Lineage (CD2, CD3, CD14, CD16, CD19, CD235a, and CD56), APC-HLA-DR, PE-Cy7-CD11c, and Percp-Cy5.5-CD123 mAbs (all eBioscience, San Diego, CA, USA). The PBMCs defined as Lineage⁻HLA-DR⁺CD123^brightCD11c⁻ were pDCs. The other tube was used to identify CD4⁺CD25⁺Foxp3⁺ Tregs and analyze their ICOS expression. Briefly, after CD4-FITC (BD Pharmingen, San Diego, CA, USA), CD25-PE-Cy5 (BD Pharmingen) and ICOS-PE-Cy7 (eBioscience) surface staining, PBMCs were washed and fixed with fixation/permeabilization solution (eBioscience), then incubated with anti-Foxp3-PE mAbs (eBioscience). After washing, stained cells were analyzed on a flow cytometer (LSR-II; BD Biosciences) using BD FACSDiva Software. Lymphocytes (1–3 × 10⁴) were gated to analyze the Tregs. Considering the small proportion of pDCs, 100 000–200 000 events in the lymphocytes and monocytes were accumulated as the acquisition gates. The appropriate isotype was used.

Huang X.M., Liu X.S., Lin X.K., Yu H., Sun J.Y., Liu X.K., Chen C., Jin H.L., Zhang G.E., Shi X.X., Zhang Q, & Yu J.R. (2014). Role of plasmacytoid dendritic cells and inducible costimulator-positive regulatory T cells in the immunosuppression microenvironment of gastric cancer. Cancer Science, 105(2), 150-158.

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Profiling T Cell Activation and Cytokines

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Human peripheral blood mononuclear cell (PBMC) samples were thawed and stained with a fixable Aqua viability dye (Invitrogen) and a cocktail of cell surface antibodies. For measurement of T cell activation/exhaustion status, fluorescent activated cell sorting (FACS) analysis was performed using the following cell surface markers: CD8-Qdot605 (Invitrogen, 3B5), CD4-Qdot 655 (Invitrogen, S3.5), PD-1-PE (BD, MIH4), LAG-3-FITC (Enzo, 17B4), ICOS-PE-Cy7 (eBioscience, ISA-3), and TIM-3-APC (R&D Systems, 344823). For multiplex cytokine measurements, validated V-PLEX Proinflammatory Panel 10-plex (human) kits (for IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IFNγ, TNFα, IL-1β, and IL-13) were used (MSD, Cat #K15049D-1). See Supplementary Materials and Methods for all FACS and cytokine assay details.

Autio K.A., Klebanoff C.A., Schaer D., Kauh J.S., Slovin S.F., Adamow M., Blinder V.S., Brahmachary M., Carlsen M., Comen E., Danila D.C., Doman T.N., Durack J.C., Fox J.J., Gluskin J.S., Hoffman D.M., Kang S., Kang P., Landa J., McAndrew P.F., Modi S., Morris M.J., Novosiadly R., Rathkopf D.E., Sanford R., Chapman S.C., Tate C.M., Yu D., Wong P, & McArthur H.L. (2020). Immunomodulatory Activity of a Colony-Stimulating Factor-1 Receptor Inhibitor in Patients With Advanced Refractory Breast or Prostate Cancer: A Phase 1 Study. Clinical cancer research : an official journal of the American Association for Cancer Research, 26(21), 5609-5620.

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Isolation and Analysis of Skin Lymphocytes and DCs

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Isolation and analysis of skin lymphocytes [18 (link), 27 (link)] were carried out as described. Briefly, after removing fur with an electric groomer, trunk skin was separated from subcutaneous fat tissue, cut to small pieces and, for lymphocyte analysis, digested for 45 min. at 37°C in RPMI 1640 containing collagenase XI (4000 U/ml), hyaluronidase (260 U/ml) and DNase (0.1mg/ml), all from Sigma-Aldrich. Cells were harvested by filtration, washed and stained, first for surface markers with mAbs CD45.2-PerCP, CD3-APC-eFluor780, CD4-FITC, CD25-PE, CTLA4-APC, ICOS-PE-Cy7, and live/dead-eFluor506, then fixed and permeabilized and stained with FoxP3-eFluor450, all from eBioscience. For DC analysis, digestion was with 150μg/ml Liberase and 120μg/ml in HBSS and cells were stained with mAbs CD11c-PerCPCy5.5 and CD103-PE (both BioLegend) and, after fixation and permeabilization, with anti-langerin-Alexa488 (clone 929F3.01, Dendritics).

Weber C.S., Hainz K., Deressa T., Strandt H., Florindo Pinheiro D., Mittermair R., Pizarro Pesado J., Thalhamer J., Hammerl P, & Stoecklinger A. (2015). Immune Reactions against Gene Gun Vaccines Are Differentially Modulated by Distinct Dendritic Cell Subsets in the Skin. PLoS ONE, 10(6), e0128722.

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Immunophenotyping of T Cell Subsets

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PBMCs were separated by a gradient centrifugation procedure on a lymphocyte separation medium (Secoll Separation Media, Mannheim, Germany). After separation, freshly isolated cells were stained with the following fluorophore-conjugated antibodies: CD4 APC-Cy7 (clone: RFT-4g), ICOS PE-Cy7 (clone: ISA-3, eBioscience, ThermoFisher), CXCR5 FITC (CD185, clone: REA303), CXCR3 APC (CD183, clone: REA232), PD-1 PE-Vio 770 (clone: PD-1.3.1.3, Miltenyi Biotec), CD45RA PE-Cy5 (clone: HI100, BioLegend), and CCR6 PE (CD196, clone: 11A9, BD Bioscience).
The freshly isolated thymocytes and the PBMCs obtained at the time of thymectomy were stained with the following fluorochrome-conjugated antibodies: CD4 FITC (clone: RPA-T4, Beckman Coulter), CD8 APC (clone: RPA-T8), CXCR3 eFluor660 (CD183, clone: CEW33D, eBioscience), CCR6 PE (CD196, clone: 11A9), PD-1 PE (CD279, clone: MIH4, BD Bioscience), ICOS PE (clone: C398.4A, BioLegend), and CXCR5 PE (CD185, clone: 51505, R&D Systems). The samples were analyzed on an Attune Flow Cytometry (Thermofisher, USA). Fluorescence minus one control, which contains all flurochromes in a panel except for the target markers measured, was used to identify and to gate the cells.

Çebi M., Durmus H., Aysal F., Özkan B., Gül G.E., Çakar A., Hocaoglu M., Mercan M., Yentür S.P., Tütüncü M., Yayla V., Akan O., Dogan Ö., Parman Y, & Saruhan-Direskeneli G. (2020). CD4+ T Cells of Myasthenia Gravis Patients Are Characterized by Increased IL-21, IL-4, and IL-17A Productions and Higher Presence of PD-1 and ICOS. Frontiers in Immunology, 11, 809.

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Comprehensive Treg Immunophenotyping by Flow Cytometry

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Expression of markers associated with Treg phenotype was evaluated by flow cytometry. Briefly, PBMCs were first stained with antihuman CD4-fluorescein isothiocyanate (FITC) and CD25-phycoerythrin (PE) (eBioscience, San Diego, CA). As a next step, intracellular staining for FOXP3 detection was performed using antihuman FOXP3-allophycocyanin (APC; PCH101FOXP3) commercial kit according to manufacturer’s instructions (eBioscience). Additional markers were analyzed, including CD3-FITC, CD8-PE-CY7, CD69-PE, CD95-FITC, GITR-PE, ICOS-PE-CY7, PD-1-FITC, CD55-APC, CD58-APC, CD59-APC (purchased from eBioscience, Biolegnd or TONBO). Finally, cells were analyzed on the FACSCalibur (Becton Dickinson) using the CellQuest software (BD Biosciences) and FlowJo software.

Beider K., Naor D., Voevoda V., Ostrovsky O., Bitner H., Rosenberg E., Varda-Bloom N., Marcu-Malina V., Canaani J., Danilesko I., Shimoni A, & Nagler A. (2017). Dissecting the mechanisms involved in anti-human T-lymphocyte immunoglobulin (ATG)-induced tolerance in the setting of allogeneic stem cell transplantation - potential implications for graft versus host disease. Oncotarget, 8(53), 90748-90765.

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Comprehensive Flow Cytometry Immunophenotyping

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Different immune cell subpopulations were immunophenotyped with flow cytometry from fresh PB samples with 6 panels of different cell-surface markers, including the following immune checkpoint receptors and cytotoxicity and migration markers: CD3-PerCP-Cy5.5 (BD, catalog 332771), CD4-PE-Cy7 (BD, catalog 560649), CD45-APC-H7 (BD, catalog 560178), CD8-BV510 (BD, 563919), CD56-BV421 (BD, 562751), CXCR1-FITC (BioLegend, catalog 341606), CD16-PE (BD, 561313), TCR γδ-APC (BD, catalog 555718), PD1-FITC (BD, catalog 557860), LAG3-PE (BD, catalog 125209), ICOS-PE-Cy7 (eBioscience, catalog 25-9948-42), CTLA-4–APC (BD, catalog 560938), HLA-DR-BB515 (BD, catalog 560938), CD27-PE (BD, catalog 555441), CD25-PE-Cy7 (BD, catalog 561405), CD11b-APC (BD, catalog 550019), NKG2C-AF488 (R&D Systems, catalog FAB138G), CD161-PE (BD, catalog 556081), NKG2D-PE-Cy7 (BD, catalog 562365), NKG2A-APC (R&D Systems, catalog FAB1059A), DNAM-BB515 (BD, catalog 565152), CD57-PE (BD, catalog 560844), NKp46-PE-Cy7 (BD, catalog 562101), NKp30-AF647 (BD, 558408), CXCR3-AF488 (BD, catalog 561730), CCR7-PE (R&D Systems, catalog FAB197P), CD45RO-PE-Cy7 (BD, catalog 560608), and CXCR4-APC (BD, catalog 560936). CD45⁺ lymphocytes were acquired with the BD FACS Verse, and the data were analyzed with FlowJo, version 10.4. The results are shown in Supplemental Table 1.

Huuhtanen J., Kasanen H., Peltola K., Lönnberg T., Glumoff V., Brück O., Dufva O., Peltonen K., Vikkula J., Jokinen E., Ilander M., Lee M.H., Mäkelä S., Nyakas M., Li B., Hernberg M., Bono P., Lähdesmäki H., Kreutzman A, & Mustjoki S. (2023). Single-cell characterization of anti–LAG-3 and anti–PD-1 combination treatment in patients with melanoma. The Journal of Clinical Investigation, 133(6), e164809.

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