Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation (Pharmacia LKB Biotechnology, Piscataway, NJ). Naïve T cells from PBMCs were isolated using a Miltenyi Biotec (Alburn, CA) negative selection kit. Purified naïve CD4+ T cells were activated with plate-bound anti-CD3 (5 μg/ml, BD Bioscience, San Jose CA), soluble anti-human CD28 (1 μg/ml, BD Bioscience), and IL-2 (20 ng/ml, R&D Systems) with or without FTY720 (100 ng/ml, Novartis). After 6 days, cell-free culture supernatants were collected for cytokine analysis by Luminex assay (Miltenyi Biotec), and cells were harvested for RNA extraction and intracellular staining. Naïve T cells were stimulated with PMA (Sigma), ionomycin (Sigma), and Golgistop for 4 h. Cells were stained for anti-human CD4 APC (BD Bioscience) and violet fluorescent reactive dye VVD (Life Technologies), and then cells were fixed and permeabilized with BD fixation and permeabilization buffer and stained for IFN-γ FITC and GZMB FITC (BD Bioscience). For surface staining, the following antibodies were used: anti-human CD4 pacific blue, anti-human CCR7 PE, and anti-human CD45RA APC, IgG2a PE isotype control, IgG2b, and k APC isotype control (all from BD Bioscience). All antibodies were titrated for flow cytometry, which was performed on a BD LSR II (BD Bioscience) and analyzed using Flowjo software.
Anti human cd4 apc
Manufactured by BD
Sourced in United States
Anti-human CD4-APC is a monoclonal antibody conjugated with allophycocyanin (APC) that binds to the CD4 receptor on human T helper cells. It is used for the identification and quantification of CD4+ T cells in flow cytometric analysis.
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Lab products found in correlation
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Ionomycin, by Merck Group (2 mentions)
Facscalibur, by BD (2 mentions)
Anti human cd8 pe, by BD (2 mentions)
Ifn γ fitc, by BD (1 mentions)
Lsr 2, by BD (1 mentions)
Anti cd3, by BD (1 mentions)
Soluble anti human cd28, by BD (1 mentions)
Luminex assay, by Miltenyi Biotec (1 mentions)
Violet fluorescent reactive dye vvd, by Thermo Fisher Scientific (1 mentions)
Anti human cd4 pacific blue, by BD (1 mentions)
Anti human cd45ra apc, by BD (1 mentions)
Igg2a pe isotype control, by BD (1 mentions)
Interleukin 2 (il 2), by R&D Systems (1 mentions)
Fty720, by Novartis (1 mentions)
Anti human il 17a pe, by Thermo Fisher Scientific (1 mentions)
Monensin, by BD (1 mentions)
Anti human ifnγ alexa fluor 488, by BD (1 mentions)
Anti human foxp3 fitc, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
12 protocols using anti human cd4 apc
1
Isolation and Activation of Naïve T Cells
2
Polarized Subsets Cytokine Profiling
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All the polarized subsets were induced with 20 ng/ml PMA (Sigma Aldrich), 1 μM ionomycin (Sigma Aldrich) and monensin (BD Biosciences) for 4 h after 6 days. A fraction of cells was preserved in Trizol for RNA isolation and other set was used for flow cytometry staining. Cell surface staining was done with anti-human CD4-APC (BD), anti-human CD45RA-FITC (BD) for naïve CD4+ T cells. The cells were later permeabilized and intracellular staining was done for anti-human IL4-PE-Cy7 (eBioscience), anti-human IFNγ-Alexa Fluor 488 (BD), anti-human IL17A-PE (eBioscience), anti-human IL17F-Alexa Fluor 647 (eBioscience), anti-human Foxp3-FITC (BD). Flow cytometry was performed on BD FACS calibur and data were analyzed using CellQuest Pro and FlowJo software.
3
Quantifying IL-36α+ CD4+ T Cells
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PBMCs were stimulated with 2 μL mixture (BD Bioscience, USA) containing phorbol myristate acetate, ionomycin and monensin at 37°C and 5% CO2 for 4 h. Then, the cells were stained with anti-human CD4-APC at 4°C under light-free environment for 30 min. Fixation and permeabilization were conducted with a Cytofix/Cytoperm kit (BD Biosciences, USA). Then, the cells were incubated with primary antibody anti-human IL-36α (Lifespan Bioscience, USA) at 4°C for 30 min under light-free environment. The cells were sequentially washed with 1 mL BD Perm/Wash™ buffer (1 ×) (BD Bioscience, USA), and stained with secondary antibody goat anti-rabbit IgG-FITC (Santa, USA) at room temperature for 30 min. Finally, FACScalibur Flow cytometer (Beckman coulter) was used to analyze the stained cells (CD4+IL-36α+T cells) immediately.
4
Longitudinal Immune Cell Profiling in aHSCT
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We collected whole blood samples from patients before aHSCT (pre) with varying timepoints before mobilization chemotherapy and on the following timepoints after HSCT (in months): 1, 3, 6, 9, 12, 18, 24, 30, 36, 42, 48, 54, 60. Samples collected after 5 years after aHSCT were summarized (post).
Lymphocyte immunophenotyping of samples was performed with BD FACSCalibur™ using conjugated antibodies from BD Biosciences™: anti-human CD3 FITC (SK7), anti-human CD45 PerCP (2D1), anti-human CD8 PE (SK1), anti-human CD4 APC (SK3), anti-human CD19 APC (2SJ25C1), anti-human CD16 PE (B73.1), anti-human CD56 PE (NCAM16.2) and FITC Mouse IgG1, κ Isotype Control.
Lymphocyte immunophenotyping of samples was performed with BD FACSCalibur™ using conjugated antibodies from BD Biosciences™: anti-human CD3 FITC (SK7), anti-human CD45 PerCP (2D1), anti-human CD8 PE (SK1), anti-human CD4 APC (SK3), anti-human CD19 APC (2SJ25C1), anti-human CD16 PE (B73.1), anti-human CD56 PE (NCAM16.2) and FITC Mouse IgG1, κ Isotype Control.
5
Evaluating CD4+IL-10+ T Cells and PD-1 in GVHD
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The percentage of CD4+IL-10+ T cells in PBMCs and PD-1 expression in samples obtained from GVHD patients and healthy donors and in the mononuclear cells in the liver and spleen of the GVHD mouse model were evaluated using the FACSCanto II flow cytometer (BD Biosciences). Briefly, cells were incubated with antibodies against surface markers at 4°C for 25 min in the dark. To perform intracellular staining, the cells were fixed, permeabilized, and stained with fluorescently labeled antibodies for an additional 25 min at 4°C in the dark.
The following antibodies were used: anti-mouse CD3-PerCP-Cy5.5 (BD Biosciences, CA, USA), anti-mouse CD4-APC (BD Biosciences), anti-mouse IL-10-FITC (BD Biosciences), anti-mouse PD-1-PE (BD Biosciences), anti-human CD4-APC (BD Biosciences), anti-human IL-10-FITC (BD Biosciences), and anti-human PD-1-PE (BD Biosciences). An APC Annexin V apoptosis detection kit with 7-AAD (BioLegend, San Diego, CA) was also used.
The following antibodies were used: anti-mouse CD3-PerCP-Cy5.5 (BD Biosciences, CA, USA), anti-mouse CD4-APC (BD Biosciences), anti-mouse IL-10-FITC (BD Biosciences), anti-mouse PD-1-PE (BD Biosciences), anti-human CD4-APC (BD Biosciences), anti-human IL-10-FITC (BD Biosciences), and anti-human PD-1-PE (BD Biosciences). An APC Annexin V apoptosis detection kit with 7-AAD (BioLegend, San Diego, CA) was also used.
6
Tetramer-based Identification of Antigen-specific T Cells
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IGRP247–259 and PPI76–90, 88S tetramers were produced by the Tetramer Core Laboratory, Benaroya Research Institute at Virginia Mason, using methods previously described (17 (link)). Fourteen days after generation, 5 × 106 antigen-pulsed cells were stained with tetramer (1-μL tetramer/105 cells) in AIM V medium plus 2% human serum for 2.5 h at a concentration of 1,000 cells/μL. Cells were then washed and stained with anti-human CD25-fluorescein isothiocyanate and anti-human CD4-APC (BD Biosciences) for an additional 45 min. Cells were analyzed/sorted on the BD FACS Vantage SE using the following guidelines: CD4+ cells were first gated, and cells were then examined using CD25 and tetramer staining. Cells staining tetramer positive and CD25mid were then collected as the final target population.
7
Isolation of Human CD4+ and CD8+ T Cells
8
Quantification of Murine Leukocyte Subsets
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Leukocytes from mice blood, spleen, and bone marrow (BM) were quantified using flow cytometry. Briefly, 100 μL peripheral blood was collected in EDTA anticoagulant tubes. Single-cell suspensions of the spleen were obtained through mechanical disaggregation. Single-cell suspensions of the BM were flushed from femurs and tibias. These single-cell suspensions were then filtered through 70 μm cell strainers (15-1070, Biologix, West Hollywood, CA, USA). The erythrocytes were lysed using RBC lysis buffer (R1010, Solarbio, Beijing, China). After 2 washes with staining buffer, cells were preincubated with Human TruStain FcX™ (422302, Biolegend, San Diego, CA, USA) for 10 min at room temperature, followed by staining with fluorochrome-labeled antibodies (1:50) in the dark at 4 °C for 45 min, and detection with an LSRfortessa flow cytometer (BD Biosciences, San Jose, CA, USA). The following fluorochrome-conjugated antibodies were used: PE-Cy™5 anti-human CD45 (555490, BD Biosciences), PE anti-mouse CD45 (561087, BD Biosciences), APC R700 anti-human CD3 (659119 BD Biosciences), APC anti-human CD4 (565994, BD Biosciences), FITC anti-human CD8 (555634, BD Biosciences), BV421 anti-human CD19 (562440, BD Biosciences). The data were analyzed employing FlowJo software.
9
Generating Constitutively Expressed A3 Cell Lines
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In order to create constitutively expressed A3 cell lines, the Sleeping Beauty transposase system [39 (link)] was adapted to electroporate Jurkat cells using the Lonza SE Cell Line kit (Lonza, V4SC-1960). The pSBbi-RP plasmid was a gift from Eric Kowarz (Addgene plasmid #60513) [71 (link)]. A3G-3XFLAG was cloned into the pSBbi-RP vector using the NcoI/XbaI restriction sites. A3C-A3Hhap II-3XFLAG was cloned into the pSBbi-RP vector using the EcoRV/XbaI restriction sites. Using the Lonza 4D-Nucleofactor (program ‘Jurkat E6.1(NEW)’ and pulse code ‘CK116’), Jurkat cells were electroporated with the pSBbi-RP-A3 and pCMV(CAT)T7-SB100 (gift from Zsuzsanna Izsvak, Addgene plasmid #34879) [72 (link)]. Post electroporation, cells were recovered in RPMI and 24 hours later, transferred into RPMI supplemented with 0.4μg/mL puromycin for selection. To further ensure that only electroporated cells survived, cells were flow sorted for dTomato positive cells and maintained in RPMI media supplemented with 0.2μg/mL puromycin (Sigma, #P8833) selection. Additionally, cells were sorted for similar CD4 levels, using APC anti-human CD4 (PharMingen, #555349), based on the CD4 levels of the A3C-A3Hhap II expressing cells.
10
Quantifying Peripheral Blood T Cell Subsets
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Peripheral blood was diluted with PBS to the same volume, and PBMCs in diluted peripheral blood were isolated with lymphocyte separation fluid (lymphocyte separation fluid volume:diluted peripheral blood volume = 1:1). The finally about 0.5-1×106 cells per tube were used for flow cytometry analysis. PBMCs were labeled with APC-anti-human CD4, Perp-anti-human CD8, PE-anti-human CD25 and FITC-anti-human CD127 antibodies (all flow cytometry reagents came from BD Biosciences by following the manufacturer’s protocols). The percentages of CD4+, CD8+, CD4+ CD25+ and CD4+ CD25+CD127-/low cells were assessed by flow cytometry. The specificity of immunostaining was ascertained by the background fluorescence of cells incubated with CD25+ and CD127+ Ig isotype controls. FACSCaliber (BD Biosciences, Mississauga, Canada) was used to quantify the percentage of cells in all groups. CD8+Tregs had a low abundance (0.2- 2% of CD8+ T cells) in both the circulation and periphery in healthy controls. In comparison, the well-studied CD4+ Tregs comprise approximately 5-12% of the CD4+ T cell population. In our study, Tregs indicate CD4+ Treg cells.
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