Cxcr3

Manufactured by BD

Sourced in United States

CXCR3 is a cell surface receptor that binds to the chemokine CXCL9, CXCL10, and CXCL11. It is expressed on various immune cells, including T cells, natural killer cells, and dendritic cells. CXCR3 plays a role in the regulation of immune cell trafficking and function.

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23 protocols using cxcr3

T Cell Subsets Profiling Protocol

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After 24 h (aCD3/28) or 6 days (CPE), lymphocytes were collected from the basolateral compartment. Cells were stained with a panel of antibodies [CD3, CXCR3, CRTH2, CD25 (all Biolegend), CD127, FoxP3, CD4 (all eBioscience)] and CXCR5 (BD Biosciences) after which T cell polarization of Th1 (CD3⁺CD4⁺CXCR3⁺), Th2 (CD3⁺CD4⁺CRTH2⁺), Tfh (follicular T helper) (CD3⁺CD4⁺CXCR5⁺), and Treg (CD3⁺CD4⁺CD25^highCD127⁻FoxP3⁺) was determined. FoxP3 staining was performed according to the manufacturer’s protocol (FoxP3 Transcription Factor Staining Buffer Set, Thermofisher, USA).

Hayen S.M., Otten H.G., Overbeek S.A., Knulst A.C., Garssen J, & Willemsen L.E. (2018). Exposure of Intestinal Epithelial Cells to Short- and Long-Chain Fructo-Oligosaccharides and CpG Oligodeoxynucleotides Enhances Peanut-Specific T Helper 1 Polarization. Frontiers in Immunology, 9, 923.

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Characterization of T cell subsets in Alzheimer's disease

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After obtaining informed consent from patients, whole blood was taken from AD patients (n = 6, Table 1). PBMCs were purified using CPT tubes (BD) and immediately frozen. Local ethics committees of the Heinrich-Heine University (Dusseldorf, Germany) approved the study. Healthy age- and gender-matched controls were also included in the study, and were processed as AD samples at the Heinrich-Heine University.
Total PBMCs from healthy donors and from AD patients (5 × 10⁶ each) were thawed and immediately stained for sorting. Cells were stained using CD4 (BD), CD45RO (BD), CXCR5 (R&D Systems), CXCR3 (BD), and CCR6 (BioLegend) for 30 min at 37°C.

Pattarini L., Trichot C., Bogiatzi S., Grandclaudon M., Meller S., Keuylian Z., Durand M., Volpe E., Madonna S., Cavani A., Chiricozzi A., Romanelli M., Hori T., Hovnanian A., Homey B, & Soumelis V. (2017). TSLP-activated dendritic cells induce human T follicular helper cell differentiation through OX40-ligand. The Journal of Experimental Medicine, 214(5), 1529-1546.

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Bladder Cancer TIL CXCR3 Expression

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Two human bladder cancer TIL samples were screened for CXCR3 (catalog# 562558, BD Biosciences) expression by flow cytometry as described above. Informed consent was obtained prior to surgical resection under an IRB-approved study (MCC 18142). Both samples were from patients with muscle-invasive bladder cancer undergoing radical cystectomy. One patient had neoadujvent cisplatin-based chemotherapy and one patient did not receive chemotherapy. TIL was expanded as previously described.26 (link)

Bunch B.L., Morse J., Asby S., Blauvelt J., Aydin A.M., Innamarato P., Hajiran A., Beatty M., Poch M, & Pilon-Thomas S. (2020). Systemic and intravesical adoptive cell therapy of tumor-reactive T cells can decrease bladder tumor growth in vivo. Journal for Immunotherapy of Cancer, 8(2), e001673.

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Characterization of P2X7R Expression in PBMC Subsets

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1×10⁶ PBMCs from patients were stained for surface markers CD3 (Biolegend, 344814), CD14 (Biolegend, 325614), CXCR3 (BD Bioscience, 550967), CCR4 (BD Bioscience, 561034) and CCR6 (BD Bioscience, 551773). Following surface staining, cells were fixed, permeabilized (Becton Dickinson, Lyse/Fix Buffer) and subsequently incubated with 1ug of P2X7R antibody (Aviva BioSystems, OASA05733) for 45 minutes on ice, followed by incubation with secondary antibody conjugated to Alexa488 (Invitrogen, A11006) for 30 minutes. Cells were washed three times and fixed in 2%PFA. All samples were acquired on an LSR-II flow cytometer and frequencies of T cell or monocyte populations positive for P2X7R, or corrected median flouresence intensity (MFI)[MFI of sample signal – MFI of isotype control] of P2X7R signal were determined with FlowJo analysis software (Treestar).

Sullivan J., Jankowska-Gan E., Shi L., Roenneburg D., Hegde S., Greenspan D., Wilkes D., Denlinger L, & Burlingham W. (2014). Differential Requirement for P2X7R Function in IL-17 Dependent vs IL-17 Independent Cellular Immune Responses. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 14(7), 1512-1522.

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Immunophenotyping of Renal Transplant Patients

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The peripheral blood of control, chronic renal allograft dysfunction (CRAD) with AMR, CRAD without AMR patients after renal transplantation was collected, mononuclear cells were separated by the lymphocytes separation medium (TBD), then labeled by the flow type antibodies CD4, CXCR5, Foxp3, ICOS, IL-17, IL-4, IFN-γ, CD127, CXCR3 and CCR6 (All purchased from BD), and sent to the flow cytometry after three washes using PBS.
Renal biopsies of all groups of patients as mentioned above were selected and cut into slices in 4 µm thickness after the formalin fixation and paraffin embedding. The primary antibodies CD4, CXCR5 and Foxp3 (All purchased from Abcam), were added and stay overnight at 4 °C. After three washes using PBS, fluorescent secondary antibody was added and incubated in 37 °C thermostat for 30 min, and observed under the fluorescent microscope after three washes using PBS. We randomly selected 7 horizons in each biopsy and counted positive cell number.

Chen W., Bai J., Huang H., Bi L., Kong X., Gao Y., Han Y., Xiao L, & Shi B. (2017). Low proportion of follicular regulatory T cell in renal transplant patients with chronic antibody-mediated rejection. Scientific Reports, 7, 1322.

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Flow Cytometry Analysis of Tumor-Specific T Cells

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Tumors and spleens were processed into single cell suspensions as described above. Cells (1×10⁶) were washed with 1 mL PBS. Zombie NIR viability dye (Biolegend, 1:1000) was added to each sample and incubated at room temperature for 30 min in the dark. Samples were washed with flow buffer (1.0 L PBS, 5% heat-inactivated fetal bovine serum, 1 mM EDTA, 0.1% sodium azide) and stained for OVA tetramer (MBL International, Woburn, Massachusetts, catalog# TB5001-1) at 4°C for 20 min in the dark followed by cell surface markers CD3 (catalog# 565643), CD8 (catalog# 558106), CD4 (catalog# 550954), CXCR3 (catalog# 562152) and CD45.1 (catalog# 560578, all from BD Biosciences, San Jose, California, USA) at 4°C for 20 min in the dark. Samples were run on a FACSCelesta flow cytometer (BD Biosciences) and analyzed using FlowJo V.10 software.

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Multiparameter Analysis of T Cell Responses

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Recombinant-human cytokines and chemokines were from R&D systems. Antibodies for flow cytometry were CCR7, CD3, CD4, CD8, CD25, CD31, CD45RA, CD45RO, CD62L, CD69, CD154, CXCR2, CXCR3, CXCL8, IFN-γ, IL-4, IL-10, IL-17, and IL-22 (BD), CD57, CD122, Foxp3 (eBiosciences/ThermoFischer), KLRG-1 (BioLegend). Antibodies for T cell stimulation were CD3, CD28 (BD or Affymetrix). Small-molecule CXCR1/2 chemical inhibitor Reparixin (RPX) was purchased from MedChem Express.

Crespo J., Wu K., Li W., Kryczek I., Maj T., Vatan L., Wei S., Opipari A.W, & Zou W. (2018). Human naïve T cells express functional CXCL8 and promote tumorigenesis. Journal of immunology (Baltimore, Md. : 1950), 201(2), 814-820.

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Intracellular Cytokine Staining and Tetramer Analysis

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TCC ICS (intracellular cytokine staining) combined with tetramer staining was performed as previously described [25 (link)]. Cells were stained with a panel of antibodies directed against cytokines of interest, IL-4 (eBiosciences), IL-5 (Biolegend), IL-13 (Biolegend), IL-17A (Biolegend), IFN-γ (Biolegend) and IL-10 (BD Biosciences) for 20 minutes at room temperature. For phenotype analysis TCC were stained with a panel of antibodies directed against markers of interest, CCR4 (Biolegend), CRTH2 (BD Biosciences), CCR6 (Biolegend), CXCR3 (BD Biosciences) and CD27 (BD Biosciences). Two profiles (T_H2 and T_H2/T_H17) of TCCs were arbitrarily defined as follows (Figure 6): T_H2 profile was exemplified by CCR4⁺ with or without CRTH2 expression (data not shown) and production of IL-4 (≥10%), IL-5(≥10%), and IL-13 (≥10%); T_H2/T_H17 profile was characterized by co-expression CCR4 and CCR6 (data not shown) and co-production of both IL-4 and IL17A (≥10%) but no IFN-γ and IL-5 by individual cells within the clone.

Archila L.D., Chow I.T., McGinty J.W., Renand A., Jeong D., Robinson D., Farrington M.L, & Kwok W.W. (2016). Ana o 1 and Ana o 2 cashew allergens share cross-reactive CD4+ T-cell epitopes with other tree nuts. Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology, 46(6), 871-883.

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Characterization of HSPC Surface Markers

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Flow cytometry was performed to analyze surface expression markers of PGE2-modulated HSPCs and dmPGE2 (16, 16-dimethyl PGE2)-modulated HSPCs. Anti-mouse PD-L1, PD-L2, PD-1, CD40, CD80, CD86, CD4, CD8, Ly-6G (Gr-1), B220, CD3, CXCR4, CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CXCR3, IL-4, IL-10, and IFN-γ were purchased from BD Biosciences, eBioscience (San Diego, CA, USA) and BioLegend. The following antibodies corresponded to isotype controls for the murine antibodies above: PE mouse IgG1, κ isotype ctrl, Armenian hamster IgG; APC mouse IgG2b, κ isotype ctrl, Armenian hamster IgG. Cells were subjected to FACS analysis and were run on a FACSCalibur™ (Becton Dickinson). Data were analyzed using FlowJo software version 8.7.3 (Treestar).

Ben Nasr M., D’Addio F., Malvandi A.M., Faravelli S., Castillo-Leon E., Usuelli V., Rocchio F., Letizia T., El Essawy A.B., Assi E., Mameli C., Giani E., Macedoni M., Maestroni A., Dassano A., Loretelli C., Paroni M., Cannalire G., Biasucci G., Sala M., Biffi A., Zuccotti G.V, & Fiorina P. (2018). Prostaglandin E2 Stimulates the Expansion of Regulatory Hematopoietic Stem and Progenitor Cells in Type 1 Diabetes. Frontiers in Immunology, 9, 1387.

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Multiparameter Immune Profiling of PBMCs

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The frozen PBMCs were resuscitated, washed and labeled with fluorochrome-conjugated antibodies, including anti-mouse TSPAN1, CD19, CD11c, CD38, CD80, CD86, HLADR, CXCR3, CXCR4 (BD Bioscience, San Diego, CA, USA). In intracellular cytokine staining, the cells were stimulated with lipopolysaccharide for 1 hour, and then Leukocyte Activation Cocktail (BD Biosciences, San Diego, CA, USA) for 4 hours at 37°C. Specific fluorochrome-conjugated antibodies were first added to stain surface markers at 4°C for 30 minutes. Subsequently, cells were fixed and permeabilized with Cytofix/Cytoperm solution (BD Biosciences, San Diego, CA, USA) at 4°C for 20 minutes. Ultimately, intracellular markers including granzyme B, interferon-γ (IFN-γ), TNF-α, and TGF-β (BD Biosciences, San Diego, CA, USA) were stained at 4°C for 1 hour. All cells were detected by LSR Fortessa X-20 analyzers (BD Biosciences). Statistics and t-SNE (t-distributed stochastic neighbor embedding) analysis were performed by FlowJo.

Ou Y., Chen R., Qian Q., Cui N., Miao Q., Tang R., You Z., Ma X, & Wang Q. (2022). The immunological characteristics of TSPAN1 expressing B cells in autoimmune hepatitis. Frontiers in Immunology, 13, 1076594.

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