We analyzed the cell composition of the T cells specifically activated by SARS-CoV-2 in the IFN-γ assay by subtracting the basal cytokine response from the background control. We stained the cell surface for 20 min at 4°C using the following fluorochrome-conjugated antibodies titrated to their optimal concentrations: CD45RA FITC (BD Pharmingen), CD27 APC (BD Pharmingen), CD3 VioGreen (Miltenyi Biotec), CD4 PECy7 (BD Pharmingen), CD8 APC Cy7 (BD Pharmingen), and 7AAD (BD Horizon). For the Treg panel CD25 BV421 (BD Horizon) and CD127 PE-CF594 (BD Horizon), were used. For the activation panel, HLA-DR BV 421 (BD Pharmingen), CD69 BV421 (Biolegend), and CD25 BV421 (BD Horizon), were used. For the exhaustion panel PD1 AF700 (Biolegend) and NKG2A BV421 (Biolegend) were used. For the chemokine panel, CD103 BV421 (BD Horizon) and CCR7 PE-CF594 (BD Horizon) were used. Cell acquisition was performed using a Navios cytometer (Beckman Coulter), acquiring an average of 200,000 cells. The analysis was performed using FlowJo 10.7.1 (FlowJo LLC).
Cd25 bv421
Manufactured by BD
Sourced in United States
CD25-BV421 is a fluorochrome-conjugated antibody that recognizes the CD25 antigen, also known as the interleukin-2 receptor alpha chain (IL-2Rα). CD25 is a cell surface marker expressed on activated T cells, regulatory T cells, and certain other cell types. The BV421 fluorochrome is used for detection and analysis of CD25-expressing cells by flow cytometry.
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Lab products found in correlation
Cd3 fitc, by BD (6 mentions)
Foxp3 pe, by BD (5 mentions)
Cd14 apc cy7, by BD (3 mentions)
Cd38 pe cy7, by BioLegend (3 mentions)
Cd19 bv510, by BioLegend (3 mentions)
Cd19 apc cy7, by BD (3 mentions)
Cd3 apc cy7, by BioLegend (3 mentions)
Cd10 pe dazzle594, by BioLegend (3 mentions)
Cd21 apc, by BioLegend (3 mentions)
Igd buv 737, by BD (3 mentions)
Igg percp, by BD (3 mentions)
Cxcr5 pe cy7, by BD (3 mentions)
Cd3 buv395, by BD (3 mentions)
Iga pe, by Miltenyi Biotec (3 mentions)
Cd27 bv650, by BioLegend (3 mentions)
Pd 1 bv711, by BioLegend (3 mentions)
Cd4 buv496, by BD (3 mentions)
Live dead fixable near ir dead cell stain kit, by Thermo Fisher Scientific (3 mentions)
Cd8 pe, by BD (3 mentions)
Cd127 pe cf594, by BD (3 mentions)
23 protocols using cd25 bv421
1
SARS-CoV-2-Specific T Cell Activation
2
Comprehensive Phenotyping of Immune Cells
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The phenotype assay was performed as previously described [25] . In summary, we stained the cell surface for 20 min at 4°C using the following fluorochrome-conjugated antihuman antibodies: CD45RA FITC, CD27 APC, CD3 VioGreen, CD4 PECy7, CD8 APC Cy7 and L/D 7AAD. We employed other antibodies for specific cell populations: CD25 BV421 (BD Horizon, Franklin Lakes, NJ, USA) and CD127 PE-CF594 (BD Horizon) for regulatory T cells (Treg); HLA-DR BV421 (BD Pharmingen, San Diego, CA, USA), CD69 PE (Miltenyi Biotec) and CD25 BV421 (BD Horizon) for activation makers; CD279 (PD1) AF700 (BioLegend, San Diego, CA, USA) and NKG2A BV421 (BD OptiBuild) for exhaustion markers; and, CD103 BV421 (BD Horizon) and CCR7 PE-CF594 (BD Horizon) for chemokine receptor and integrin markers.
The experiments with and without IL-15 incubation O/N and with different concentrations of dexamethasone were performed as described previously. After the staining, cell acquisition was performed using a Navios cytometer (Beckman Coulter), acquiring a mean of 200,000 cells. The analysis was performed using FlowJo 10.7.1 software (FlowJo LLC).
The experiments with and without IL-15 incubation O/N and with different concentrations of dexamethasone were performed as described previously. After the staining, cell acquisition was performed using a Navios cytometer (Beckman Coulter), acquiring a mean of 200,000 cells. The analysis was performed using FlowJo 10.7.1 software (FlowJo LLC).
3
SARS-CoV-2-specific CD4 T-cell assay
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The AIM assay was also performed with whole blood antigen-specific stimulation by using heparinized antigen tubes from the QuantiFERON SARS-CoV-2 kit (Qiagen).
Whole blood samples were collected directly into the assay collection tubes, shaken, and incubated for 16–24 h, as described above. Whole-blood aliquots (120 μL) were withdrawn from Nil (negative control) and from the two Ag tubes (60 μL each, mixed together) of the QuantiFERON SARS-CoV-2 kit before centrifugation, and stained for 15 min at room temperature with the following combination of anti-human fluorescent monoclonal antibodies: CD3 BV786, CD4 BV750, CD134 BB700 (3μL, Clone L106), and CD25 BV421 (all Becton Dickinson, San Jose, CA, USA). Incubation in 2 mL of FacsLyse solution (Becton Dickinson, San Jose, CA, USA) for 10 min was used to remove red blood cells. After centrifugation at 300× g for 5 min and washing with 2 mL of stain buffer, 0.5 mL samples were acquired on the FACSymhony A3 flow cytometer and data was analyzed using FlowJo software (Tree Star) and FacsDiva software (Becton Dickinson, San Jose, CA, USA). Cells were gated on the forward scatter/side scatter cell gate, FSC-A vs. FSC-H, to exclude doublets and then on the CD3+CD4+ gate for the quantification of CD25+OX-40+ SARS-CoV-2-specific CD4 T-cells.
Whole blood samples were collected directly into the assay collection tubes, shaken, and incubated for 16–24 h, as described above. Whole-blood aliquots (120 μL) were withdrawn from Nil (negative control) and from the two Ag tubes (60 μL each, mixed together) of the QuantiFERON SARS-CoV-2 kit before centrifugation, and stained for 15 min at room temperature with the following combination of anti-human fluorescent monoclonal antibodies: CD3 BV786, CD4 BV750, CD134 BB700 (3μL, Clone L106), and CD25 BV421 (all Becton Dickinson, San Jose, CA, USA). Incubation in 2 mL of FacsLyse solution (Becton Dickinson, San Jose, CA, USA) for 10 min was used to remove red blood cells. After centrifugation at 300× g for 5 min and washing with 2 mL of stain buffer, 0.5 mL samples were acquired on the FACSymhony A3 flow cytometer and data was analyzed using FlowJo software (Tree Star) and FacsDiva software (Becton Dickinson, San Jose, CA, USA). Cells were gated on the forward scatter/side scatter cell gate, FSC-A vs. FSC-H, to exclude doublets and then on the CD3+CD4+ gate for the quantification of CD25+OX-40+ SARS-CoV-2-specific CD4 T-cells.
4
Comprehensive B-cell Phenotyping by Flow Cytometry
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Anonymised heparinised venous blood samples were collected and cells were labelled for 1 h with mouse anti-human antibody–fluorophore conjugates: CD3-PE (T cells), CD10-PECy7, CD19-PECy5, CD25-BV421, CD27-BV205, CD38-APC, IgD-BB515 and IgG1k-BV421 isotype (Becton-Dickinson, Oxford, UK) and cells were analysed by flow cytometry. Memory B cells were identified as CD19+/CD27+ lymphocytes. The presence of class-switched (CD19+/CD27+/IgD−), unswitched (CD19+/CD27+/IgD+) and interleukin-2 receptor expressing (CD19+/CD25+/CD27+) memory B cell subsets was assessed. The number of immature (CD10+/CD19+/CD27−/CD38+); naïve/mature (CD10−/CD19+/CD27−/CD38+) and plasmablasts (CD19+/CD27+/CD38+) were estimated. The absolute number of cell subsets was calculated based on automated full blood counts (Sysmex XE-2100, Kobe).
5
Quantifying Immune Cell Subsets
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For cell phenotype analysis, monoclonal antibodies were directly added to 100 μL aliquots of heparinized whole blood and incubated at room temperature for 15 min. The antibodies used were: CD3 PECy7, CD14 APC, CD56 PE-Cy5 and CD19 PerCp (Biolegend, San Diego, CA, USA), CD4 Viogreen (Miltenyi Biotec GmBh, Bergisch Gladbach, Germany), CD39 Fitc, CD73 PE, CD25 BV421 and CD127 AlexaFluor 647 (BD Biosciences, San Jose, CA, USA). Red blood cells were lysed and white cells were fixed using BD FACS lysing solution (BD Bioscience). Negative populations were determined using respective anti-human isotype controls. Samples were acquired using a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec GmBh) and the data were analyzed using FlowJo software v. 10.0 (TreeStar Inc., Ashland, OR, USA).
We compared the frequencies of four subsets based on CD39 and CD73 expression (CD39−CD73+, CD39+CD73+, CD39+CD73−, and CD39−CD73−) within each studied population (B cells as CD3−CD19+, CD4+ T cells as CD3+CD4+, CD8+ T cells as CD3+CD4−, NK cells as CD3−CD4−CD19−CD56+, Tregs as CD3+CD4+CD25++CD127−, and monocytes as CD14+). The percentage of these four subsets was related to their corresponding precursor populations (CD4+ T, CD8+ T, B, NK, and monocytes). In the case of Treg, the percentage was related to CD4+ T cells.
We compared the frequencies of four subsets based on CD39 and CD73 expression (CD39−CD73+, CD39+CD73+, CD39+CD73−, and CD39−CD73−) within each studied population (B cells as CD3−CD19+, CD4+ T cells as CD3+CD4+, CD8+ T cells as CD3+CD4−, NK cells as CD3−CD4−CD19−CD56+, Tregs as CD3+CD4+CD25++CD127−, and monocytes as CD14+). The percentage of these four subsets was related to their corresponding precursor populations (CD4+ T, CD8+ T, B, NK, and monocytes). In the case of Treg, the percentage was related to CD4+ T cells.
6
Splenic T Cell Subsets by FACS
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For splenic T cell sorting, magnetic bead-mediated positively selected CD4+ cells were stained with combinations of antibodies (0.5μg/mL per) to CD62L-PE (BioLegend, San Diego, CA) or CD62L-BB515 (BD Bioscience), CD44-APC (BioLegend, San Diego, CA), CD45Rb-APC/Cy7 (BioLegend, San Diego, CA), CD124 (1μg/mL, BD Biosciences, San Jose, CA), and CD25-BV421 (BD Biosciences, San Jose, CA). For Tregs, FoxP3-RFP reporter signal was used for FACS. Cells were washed twice in MACS buffer (Miltenyi Biotec, San Diego, CA) before sterile cell sorting using a FACSAria (BD Biosciences, San Jose, CA) with the support of the Cytometry & Imaging Microscopy Core Facility of the Case Comprehensive Cancer Center. Analysis of all FACS data was performed using FlowJo v10 (Tree Star, Inc., Ashland, OR).
7
Comprehensive B and T Cell Profiling
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Donor PBMCs was stained with live-dead marker (1:1000; Invitrogen, LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit) to gate out the dead cells for 20 min in 4 °C. After washing with FACS buffer, the cells were stained with respective B, T cell marker panel antibodies diluted in FACS buffer (1xPBS, 2% FBS, 0.1% sodium azide) at 4 °C for 30 min. B cell panel: CD19-BV510 (1:40; Biolegend, Cat. No. 363020), CD14-APC-Cy7 (1:50; BD, Cat. No. 557831), CD3-APC-Cy7 (1:125; Biolegend, Cat. No. 317342), CD10-PE-Dazzle594 (1:50; Biolegend, Cat. No. 312228), CD21-APC (1:50; Biolegend, Cat. No. 354906), CD27-BV650 (1:40; Biolegend, Cat. No. 302828), CD38-PE-Cy7 (1:40; Biolegend, Cat. No. 356608), PD-1-BV711 (1:20; Biolegend, Cat. No. 329928), IgD-BUV 737 (1:40; BD, Cat. No. 612798), IgG-PerCP (1:40; BD), IgA-PE (1:40; Miltenyi, Cat. No. 130-114-002). T cell panel: CD14-APC-Cy7 (1:50; BD, Cat. No. 557831), CD19-APC-Cy7 (1:50; BD, Cat. No. 557791), CD3-BUV395 (1:40; BD, Cat. No. 563546), CD4-BUV496 (1:40; BD, Cat. No. 612936), CD8-PE (1:40; BD, Cat. No. 555367), CXCR5-PE-Cy7 (1:100; BD, Cat. No. 624052), CD25-BV421 (1:40; BD, Cat. No. 562442), CD127-FITC (1:20; BD, Cat. No. 557938). The samples were then washed, resuspended in FACS buffer and analysed on BD LSRFortessa flow cytometer (Data availability—Figshare 10.6084/m9.figshare.23549964).
8
Comprehensive Immune Cell Profiling
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A panel of monoclonal antibodies was developed to enable phenotypic characterization of B and T lymphocyte subpopulations: CD3-FITC, CD4-APC, CD25-BV421, FoxP3-PE, CD19-PeCy7, CD9-BB700 (BD Bioscience, France), CD24-BV510, and CD38-APC-Cy7 (Sony Biotechnology, UK); hematopoietic stem and progenitor cells: CD34-BV421 (Biolegend, France), cKit-APC-H7, CD135-PE, lineage-FITC, Sca-1-BV510, CD16/32-PerCPCy5.5, CD127-APC (BD Bioscience), and TLR-4-PeCy7 (Biolegend); and dendritic cells: CD11c-PeCy7 (eBioscience, France), CMH-II-FITC, Siglec-H-BV510, CD11b-APC-H7, and CD103-PerCP5. For intracellular staining, cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD Biosciences). For in vitro human B cell differentiation experiments, cells were stained with Fixable Viability Dye eFluor 450 to identify dead cells, followed by staining with CD25-BV605 (Biolegend), CD19-BUV395, CD9-FITC, CD27-BUV737, CD38-BV711 and intracellular IL-10-PE (BD Bioscience). Cells were analyzed on a Canto II flow cytometer (BD Biosciences). Data were acquired using Diva 8.0 software and analyzed with FlowJo X (TreeStar, Williamson Way, Ashland, USA). Fluorescence minus one staining controls were used for all panels, and dead cells were removed using viability staining.
9
Comprehensive B and T Cell Profiling
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Donor PBMCs was stained with live-dead marker (1:1000; Invitrogen, LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit) to gate out the dead cells for 20 min in 4 °C. After washing with FACS buffer, the cells were stained with respective B, T cell marker panel antibodies diluted in FACS buffer (1xPBS, 2% FBS, 0.1% sodium azide) at 4 °C for 30 min. B cell panel: CD19-BV510 (1:40; Biolegend, Cat. No. 363020), CD14-APC-Cy7 (1:50; BD, Cat. No. 557831), CD3-APC-Cy7 (1:125; Biolegend, Cat. No. 317342), CD10-PE-Dazzle594 (1:50; Biolegend, Cat. No. 312228), CD21-APC (1:50; Biolegend, Cat. No. 354906), CD27-BV650 (1:40; Biolegend, Cat. No. 302828), CD38-PE-Cy7 (1:40; Biolegend, Cat. No. 356608), PD-1-BV711 (1:20; Biolegend, Cat. No. 329928), IgD-BUV 737 (1:40; BD, Cat. No. 612798), IgG-PerCP (1:40; BD), IgA-PE (1:40; Miltenyi, Cat. No. 130-114-002). T cell panel: CD14-APC-Cy7 (1:50; BD, Cat. No. 557831), CD19-APC-Cy7 (1:50; BD, Cat. No. 557791), CD3-BUV395 (1:40; BD, Cat. No. 563546), CD4-BUV496 (1:40; BD, Cat. No. 612936), CD8-PE (1:40; BD, Cat. No. 555367), CXCR5-PE-Cy7 (1:100; BD, Cat. No. 624052), CD25-BV421 (1:40; BD, Cat. No. 562442), CD127-FITC (1:20; BD, Cat. No. 557938). The samples were then washed, resuspended in FACS buffer and analysed on BD LSRFortessa flow cytometer (Data availability—Figshare 10.6084/m9.figshare.23549964).
10
Comprehensive Immune Phenotyping of Cryopreserved PBMCs
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Cryopreserved PBMC were thawed at 37 °C, then washed in RPMI 1640 supplemented with 10% FCS and HEPES to remove DMSO. 5 × 105 cells were stained for each patient at each time-point. Cells were stained with Fixable Viability Dye ef780 (ThermoFisher), plus antibodies against CD3-BV510 (BD, UCHT1, 563109), CD4-BV711 (BioLegend, OKT4, 317440), CD8a-eVolve605 (ThermoFisher, RPA-T8, 83–0088-42), CD14-APC-eF780 (ThermoFisher, 61D3, 47-0149-42), CD19-APC-eF780 (ThermoFisher, HIB19, 47-0199-), CD25-BV421 (BD, M-A251, 562442), CD45RA-BV785 (BioLegend, HI100, 304140), CD127-PE-CF594 (BD, HIL-7R-M21, 562397), PD-1-PECy7 (BioLegend, EH12.2H7, 329918), PD-L1-BUV395 (BD, HI30, 740320), ICOS-PerCP-eF710 (ThermoFisher, ISA-3, 46-9948-42). Cells were permeabilised with FoxP3 Fixation/Permeabilisation Kit (ThermoFisher), and stained for FoxP3-PE (ThermoFisher, 263A/E7, 12-4777-42) and Ki67-FITC (BD, B56, 51-36524X). Fixation was performed with Stabilising Fixative (BD) diluted in water. Data was acquired on a BD SORP Fortessa (BD) and analysed (Supplementary Fig. S1 ) with FlowJo software40 (BD). Fluorescence minus one controls were used to determine gates.
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