Cxcr3 pe

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CXCR3-PE is a fluorescently-labelled antibody that binds to the CXCR3 chemokine receptor. CXCR3 is a G protein-coupled receptor that plays a role in the trafficking and activation of T cells. The PE (Phycoerythrin) fluorescent label allows for the detection of CXCR3-expressing cells using flow cytometry.

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CXCR3-PE-Cy7 (3 citations)

12 protocols using cxcr3 pe

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).

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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Cross-presentation Experiments with Antibodies

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The following antibodies were used in the cross-presentation experiments: TruStain Fcblock anti-mouse CD16/32 (101320; BioLegend), FITC-CD11c (117306; Biolegend), APC-H2Kb-bound SIINFEKL (141606; Biolegend), APC/Cy7-CD40 (124637; Biolegend), PerCP/Cy5.5- ICAM_1 (116123; Biolegend), BV510-CD86 (563077; BD), PE/Cy7-MHC-II (107629; Biolegend), V450-CD80 (12519; BD). The following antibodies were used for the immunological analysis in the in vivo animal experiments: FITC-CD8 (553062; BD), PE-CD4 (100408; Biolegend), APC-CXCR3 (562266; BD), PerCP/Cy5.5-CD3 (100732; Biolegend), PE-CXCR3 (155903); FITC-CD8 (11083782); PerCP/Cy5.5-CD3 (100328);. Flow cytometric analyses were performed using Fortessa LSR Flow Cytometer (BD Biosciences) or BD Accuri C6 Plus (BD Bioscience). FlowJo software v.10 (FlowJo) was used for the data analysis.

Feola S., Russo S., Martins B., Lopes A., Vandermeulen G., Fluhler V., De Giorgi C., Fusciello M., Pesonen S., Ylösmäki E., Antignani G., Chiaro J., Hamdan F., Feodoroff M., Grönholm M, & Cerullo V. (2022). Peptides-Coated Oncolytic Vaccines for Cancer Personalized Medicine. Frontiers in Immunology, 13, 826164.

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Phenotypic Characterization of PBMCs

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Human PBMCs were resuspended with 200 μL PBS buffer and incubated with fluorochrome-conjugated antibodies, anti-human Krome Orange-CD4, Alexa Fluor 750-CD45RA, ECD-CD25 (Beckman Coulter Life Science, United States), Pacific blue™-CXCR5, PerCP/Cy5.5-PD-1, APC-TIM-3, Brilliant violet 650™-LAG-3, PE/Cy7-CTLA-4, FITC-CCR6, and PE-CXCR3 (BioLegend, United States), for 20 min at room temperature, and matched isotype controls were performed in this study according to the manufacturer’s instructions. The samples were detected by the CytoFLEX Flow Cytometer, and the data were analyzed by CyExpert software (Beckman Coulter, United States).

Cui D., Jiang D., Yan C., Liu X., Lv Y., Xie J, & Chen Y. (2022). Immune Checkpoint Molecules Expressed on CD4+ T Cell Subsets in Chronic Asymptomatic Hepatitis B Virus Carriers With Hepatitis B e Antigen-Negative. Frontiers in Microbiology, 13, 887408.

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Multiparametric Phenotyping of Human Blood Cells

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Cells were isolated from peripheral blood using Ficoll density gradient centrifugation and perioheral blood mononuclear cells (PBMC) were stained with the following anti-human antibody staining reagents: CD3-BV711 (clone: HIT3a, BD Biosciences); CD3-PE-Cy5.5 (clone: 7D6, Fisher Scientific); CD14-BV711 (clone: M5E2, BD Biosciences); CD14-PE-Cy5.5 (clone: TuK4, Thermo Fisher Scientific); IgD-FITC (IA6-2, BD Biosciences); CD19-PE-Cy7(clone: SJ25C1, BD Biosciences); CD27-APC-eFluor780 (clone: O323, Fisher Scientific); CD38-V450 (clone: HIT2, BD Biosciences); CD138-APC (clone: 44F9, Miltenyi Biotec); CXCR4-PE (clone: 12G5, BioLegend); CXCR4-BV711 (clone:12G5, BD Biosciences); CXCR3-PE(G025H7, BioLegend); Blimp1-PE (6D3, BD Biosciences); BCMA-PE (19F2, BioLegend), IL-6R-PE (M5, BD Biosciences). Approximately, 1 × 10³ to 5 × 10³ were collected for each cell population by using FACSAria II (BD Biosciences).

Chen W., Hong S.H., Jenks S.A., Anam F.A., Tipton C.M., Woodruff M.C., Hom J.R., Cashman K.S., Faliti C.E., Wang X., Kyu S., Wei C., Scharer C.D., Mi T., Hicks S., Hartson L., Nguyen D.C., Khosroshahi A., Lee S., Wang Y., Bugrovsky R., Ishii Y., Lee F.E, & Sanz I. (2023). SLE Antibody-Secreting Cells Are Characterized by Enhanced Peripheral Maturation and Survival Programs. Research Square.

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Multicolor Flow Cytometry for T and B Cell Subsets

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PBMC were isolated from peripheral blood by Ficoll gradient and frozen for batched analysis. We designed six multicolor flow cytometry panels to quantify 60 T cell subsets along with two B cell subsets, and calculated the CD4⁺/CD8⁺ T cell ratio (Supplementary Table 1). The following fluorochrome-conjugated anti-human antibodies were used from BD Biosciences (San Jose, CA): CD3-FITC, CD3-PerCP-Cy5.5, CD4-APC, CD4-APC-Cy7, CD8-BV510, CD45RO-FITC, CD45RA-APC, CD45RA-APC-Cy7, CCR4-PE, CD27-PE, CD28-BV421, CD138-BV421, CCR6-BV421, CXCR3-PE, CCR7-A700, IL-17-BV786, IFN-γ-PE-Cy7, iso IgG1k-FITC, iso IgG1k-PE-Cy7, iso IgG2bk-APC, iso IgG1k-APC-Cy7, and iso IgG1k-BV510; from Biolegend (San Diego, CA): CD127-FITC, CD27-APC, CD57-PerCp-Cy5.5, CD19-BV510, PD-1-APC-Cy7, CXCR5-FITC, and TNFα-FITC; from eBioscience (San Diego, CA): CD4-PE-Cy7, and IL-2-PE; from Miltenyi Biotec (San Diego, CA): CD25-APC and KLRG1-PE; from Beckman Coulter (Brea, CA): CD38-PE-Cy7.

Hartzell S., Bin S., Cantarelli C., Haverly M., Manrique J., Angeletti A., Manna G.L., Murphy B., Zhang W., Levitsky J., Gallon L., Yu S.M, & Cravedi P. (2020). Kidney Failure Associates With T Cell Exhaustion and Imbalanced Follicular Helper T Cells. Frontiers in Immunology, 11, 583702.

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Multicolor Flow Cytometry of Skin Immune Cells

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Protocols were conducted as previously described [19 (link)]. CD8α-AF700 (BioLegend), CXCR3-PE (BioLegend), CD4-PE-Cy7 (eBioscience), Foxp3-FITC (eBioscience) and CD45-PB (BioLegend) were used for these studies. Skin samples were prepared as previously described [22 (link)]. Briefly, single cell suspensions (1 million cells) were first incubated with Fc Block (BD Pharmingen. San Diego, CA) for 10 mins, then co-incubated with antibodies for 20 min at 4°C followed by washing with staining buffer (PBS + 1% FBS). Foxp3 intracellular staining was performed using an eBioscience kit (Cat#00-5523-00) according to the manufacturer’s protocol. Flow cytometry was performed on a LSR Fortessa and data were analyzed by FlowJo software (TreeStar).

Pai C.C., Hsiao H.H., Sun K., Chen M., Hagino T., Tellez J., Mall C., Blazar B.R., Monjazeb A., Abedi M, & Murphy W.J. (2014). Therapeutic benefit of bortezomib on acute GVHD is tissue specific and is associated with IL-6 levels. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation, 20(12), 1899-1904.

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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 Phenotyping of Tumor-Infiltrating Lymphocytes

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Single cell suspensions of TILs were stained with different antibodies: Zombie NIR^TM (423105; Biolegend) for dead cell preclusion, TruStain FcX^TM (anti-mouse CD16/32; Biolegend) for the removal of nonspecific adsorption, CD45-BV421 (103134; Biolegend), CD45-BV510 (103138; Biolegend), CD4-AF488 (557667; BD Biosciences, San Jose, CA), CD4-PE/Dazzle^TM 594 (100456; Biolegend), CD8-FITC (100706; Biolegend), CD8-BV421 (100738; Biolegend) and CXCR3-PE (155904; Biolegend) to mark different TILs.

Li X., Lu M., Yuan M., Ye J., Zhang W., Xu L., Wu X., Hui B., Yang Y., Wei B., Guo C., Wei M., Dong J., Wu X, & Gu Y. (2022). CXCL10-armed oncolytic adenovirus promotes tumor-infiltrating T-cell chemotaxis to enhance anti-PD-1 therapy. Oncoimmunology, 11(1), 2118210.

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Comprehensive Immune Cell Profiling

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Vehicle-, nivolumab- or ipilimumab-expanded lymphocytes were stained with zombie NIR viability dye, CD8-BV421, CD4-BV510, CD3-FITC, CXCR3-PE, CCR6-APC, CTLA-4-PerCPCy5.5, TIGIT-PE/Cy7, PD-L1-PerCPCy5.5, PD-1-PECy/7, CD25-FITC and FOXP3-BV421 (Biolegend, USA). Intracellular FoxP3 permeabilization and fixation buffer was used for FoxP3 staining (Biolegend, USA). Cells were acquired using the BD FACs Canto II (BD Biosciences) using Diva software and analysed using FlowJoTM v10.7.

Davern M., Gaughan C., O’ Connell F., Moran B., Mylod E., Sheppard A.D., Ramjit S., Yun-Tong Kung J., Phelan J.J., Davey M.G., Ryan E.J., Butler C., Quinn L., Howard C., Tone E., Phoenix E., Butt W.T., Lynam-Lennon N., Maher S.G., Ravi N., Donohoe C.L., Reynolds J.V., Lysaght J, & Donlon N.E. (2023). PD-1 blockade attenuates surgery-mediated immunosuppression and boosts Th1 immunity perioperatively in oesophagogastric junctional adenocarcinoma. Frontiers in Immunology, 14, 1150754.

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Multicolor Flow Cytometry of PBMC

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We isolated peripheral blood mononuclear cells (PBMC) from peripheral blood by Ficoll gradient and we stored them in liquid nitrogen for batched analysis. For multicolor flow cytometry analyses, we used the following monoclonal antibodies: from Becton Dickinson (BD, Franklin Lakes, NJ), CD3-FITC, CD3-PerCP-Cy5.5, CD4-APC-Cy7, CD8-BV510, CD45RO-FITC, CD45RA-APC, CD45RA-APC-Cy7, CD71-PE, IgD-PerCP-Cy5.5, CD25-APC-Cy7, CD138-BV421, CCR4-PE, CD27-PE, CD95-BV421, CD28-BV421, TIM3-BV421, CCR6-BV421, CCR7-AF700, CXCR3-PE, IL-2-PE, IL-17-BV786, and IFN-γ-PE-Cy7; from Biolegend (San Diego, CA), CD19-BV510, CD56-FITC, CD27-APC, CD127-FITC, CD57-PerCp-Cy5.5, PD-1-APC-Cy7, CXCR5-FITC, LAG3-APC, IL-10-PerCP-Cy5.5, and TNF-α-FITC. From eBioscience (San Diego, CA), CD4-PE-Cy7, CD21-PE, and CD24-APC-Cy7 were used. From Miltenyi Biotec (Auburn, CA), CD25-APC and anti-KLRG1-PE were used. From Beckman Coulter (Indianapolis, IN), CD38-PE-Cy7 was used.
We performed intracellular staining for IL-2, IL-4, IL-17, IFN-g, and TNF-a together with extracellular markers for CD4⁺, CD8⁺, and CD19⁺. Cells were fixed and permeabilized using Intracellular Fixation and Permeabilization Buffer Set (eBioscience) according to the manufacturer’s instructions.
Data were acquired (> 1 × 10⁶ events) on a 3-laser FACSLyric flow cytometer (BD Biosciences) and analyzed with FlowJo^® software.

Tawhari I., Hallak P., Bin S., Yamani F., Safar-Boueri M., Irshad A., Leventhal J., Ansari M.J., Cravedi P, & Gallon L. (2022). Early calcineurin-inhibitor to belatacept conversion in steroid-free kidney transplant recipients. Frontiers in Immunology, 13, 1096881.

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