Primary human B cells and monocytes were isolated using the RosetteSep Human B Cell and monocyte Enrichment Cocktails (StemCell Technologies) as per the manufacturer's instructions from leukocyte cones provided by UK National Health Service Blood and Transplant. Isolated cells were cultured in RPMI-1640 supplemented with 10% FCS, 4 mM L-glutamine, 10 mM HEPES, 1% non-essential amino acid solution (Gibco), and 1% penicillin-streptomycin solution (Gibco) at 37°C, 5% CO2. B cells were also cultured in the presence of 1 mM sodium pyruvate (Gibco), 50 ng/ml IL4 (PeproTech), 25 ng/ml IL2 (PeproTech), 100 ng/ml BAFF (BioLegend), and 100 ng/ml IL21 (BioLegend). Monocytes were differentiated into moDCs by culturing with 50 ng/ml IL4 (PeproTech) and 100 ng/ml GM-CSF (Immunotools) at 1 × 106/cm2 in adherent culture for 6 days. Twenty-four h before use in T cell stimulation assays, moDCs were activated by addition of 1 μM prostaglandin E2 (Sigma-Aldrich), 50 ng/ml TNFα (PeproTech), 10 ng/ml IL1β (Bio-Techne), and 20 ng/ml IFNγ (Bio-Techne). Differentiation was confirmed by assessing expression of CD11c and CD86 (see “Flow cytometry”).
Il 21
Manufactured by BioLegend
Sourced in United States
IL-21 is a recombinant human cytokine that belongs to the common gamma-chain cytokine family. It is involved in the regulation of immune responses.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 2 (il 2), by BioLegend (6 mentions)
Interleukin 6 (il 6), by BioLegend (5 mentions)
Il 23, by BioLegend (3 mentions)
Il 17a, by BioLegend (3 mentions)
Tnf α, by BioLegend (3 mentions)
Ifn α, by BioLegend (2 mentions)
Fix perm cell permeabilization kit, by Thermo Fisher Scientific (2 mentions)
Anti cd8 biotin ab, by Thermo Fisher Scientific (2 mentions)
Anti gfp ab, by Thermo Fisher Scientific (2 mentions)
Streptavidin allophycocyanin, by Thermo Fisher Scientific (2 mentions)
Cytoflex s, by Beckman Coulter (2 mentions)
Il 1β, by BioLegend (2 mentions)
Cd40l, by R&D Systems (2 mentions)
Nonessential amino acid solution, by Thermo Fisher Scientific (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Prostaglandin e2, by Merck Group (1 mentions)
21 protocols using il 21
1
Isolation and Differentiation of Primary Human B Cells and Monocytes
2
B Cell Culture and Activation
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Human B cells (preenriched, enriched, and depleted) from six donors were cultured in Iscove’ s modified Dulbecco’ s medium (IMDM; Gibco, Fisher Scientific) supplemented with 10% HI FBS (Gibco), 2:1,000 MycoZap (Lonza, Alpharetta, GA), 10,000 U/ml interleukin-2 (IL-2; Gibco), and 100 μg/ml IL-21 (BioLegend). Additionally, the medium was treated with antibody-cross-linked CD40L (Miltenyi Biotec), according to the manufacturer’s protocols, or 1 ng/ml CD40L homotrimer (BioLegend) in plates coated with 10 μg/ml anti-His antibody (BioLegend) or 3T3-msCD40L feeder cells obtained through the NIH AIDS Reagent Program (catalog no. 12535 3T3-msCD40L; Division of AIDS, NIAID, NIH, from Mark Connors, as described in reference 22 (link)). Although three different CD40L delivery methods were used, no statistical difference in IgGtot production was found between the three (not shown). B cells were plated with 3,000 to 5,000 cells/ml and incubated at 37°C in 5% CO2 for up to 12 days, after which the supernatant was collected and stored at –80°C for downstream analysis.
3
Cytokine-Induced STAT Phosphorylation Assay
4
Cytokine-Induced STAT Phosphorylation Assay
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Forty-eight hours after transduction, cells were first reseeded in IL-2–free media for 4 h, then cultured in RPMI medium with 10% FBS and anti–CD8-biotin Ab (53–6.7; eBioscience) in the presence or absence of rIL-2 (50 U/ml) for 10 min to induce STAT5 phosphorylation. For STAT1 and STAT3 phosphorylation, cells were cultured in the presence or absence of IFN-α (5000 U/ml; 30 min; BioLegend) or IL-21 (200 ng/ml; 30 min; BioLegend), respectively, and anti–CD8-biotin was added for the last 10 min. Cells were then fixed in 2% paraformaldehyde (5 min) and fixation reagent (15 min) (FIX & PERM Cell Permeabilization Kit; Thermo Fisher Scientific) and made permeable with ice-cold methanol (15 min) and permeabilization medium (FIX & PERM Cell Permeabilization Kit; Thermo Fisher Scientific), then stained with anti-GFP Ab (Thermo Fisher Scientific), anti–p-STAT1-PE (A15158B), anti–p-STAT3-AF647 (13A3–1; all from BioLegend), anti–p-STAT5 allophycocyanin (SRBCZX), streptavidin-PE (all from eBioscience), and streptavidin-allophycocyanin (Invitrogen). Cells were analyzed with a CytoFLEX S (Beckman Coulter) Flow Cytometer.
5
Naive B cell activation and plasma cell differentiation
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Naive B cells from healthy donors and BENTA patients were cultured in Iscove’s modified Dulbecco medium (IMDM) plus 10% fetal bovine serum supplemented with penicillin/streptomycin (Sigma). Purified B cells were plated at 1 × 106 cells/mL in 24-well flat-bottom plates (1 mL/well) or 96-well round bottom plates (0.2 mL/well). Cells were activated using (a) 100 ng/mL of dextran-conjugated anti-IgD antibodies (αIgD-DEX) (Fina Biosolutions), (b) SAC (Protein A from Staphylococcus aureus Cowan I) + 200 U/mL of rIL-2 (Peprotech), or (c) 10 µg/mL of Affinipure F(ab′2) fragment-specific goat anti-human IgM (Jackson Immunoresearch Laboratories) plus 1 µg/mL anti-CD40 Ab (AF632, R&D Biosystems) plus IL-21 (100 ng/mL, Biolegend) and IL-2 (20 U/mL, Biolegend). For (c), additional combinations of cytokines (Biolegend) IL-4 (100 ng/mL) + IL-13 (50 U/mL), or IL-10 (25 ng/mL) were concomitantly used to induce isotype-specific antibody responses. For long-lived plasma cell generation, IL-6 (10 ng/mL), IFN-α (100 U/mL), and IL-21 (100 ng/mL) along with Lipid Mixture 1, chemically defined (Sigma, 1:500 dilution) and MEM amino acids solution (Sigma, 1:100 dilution) were added on day 6 to cells that were previously stimulated with α-IgM Ab/α-CD40 Ab/IL-21/IL-2. Culture media was changed every 4 days.
6
B cell activation and gene expression
7
Evaluating iTreg Stability Under Stress
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For evaluation of iTreg stability, cells were (1) re‐stimulated without iTreg differentiation components and (2) challenged with Th17‐polarising cytokines. For (1), cells were re‐stimulated using 500 U mL−1 of IL‐2 and a 1:1 expander beads without addition of TGF‐β, ATRA and rapamycin, for 7 days at 1 × 106 cells mL−1 then rested with 500 U mL−1 of IL‐2 for 3 days at 2 × 106 cells mL−1, after an initial 7‐day stimulation and 3‐day rest. On day 3 of rest, cells were used for flow cytometric analysis and in vitro suppression assay. For (2), cells were challenged for 3 days with Th17‐polarising cytokines, IL‐1β, IL‐6, IL‐21 and IL‐23 (10 ng mL−1 for all, Biolegend, San Diego, CA, USA), in the presence of 25 U mL−1 of IL‐2 and a 1:10 expander beads at 1 × 106 cells mL‐1, after an initial 7‐day stimulation and 3‐day rest. Cells were stimulated with 25 U mL−1 of IL‐2 and a 1:10 expander beads at 1 × 106 cells mL−1 as unchallenged controls. On day 3 of challenge, cells were washed three times with PBS (+2% FCS). After washing, expander beads were magnetically removed. Cells were used for flow cytometric analysis, cytokine production assays and reverse transcription quantitative polymerase chain reaction.
8
Cytokine Stimulation of CD8+ T Cells
9
Comprehensive Immune Cell Profiling
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Antibodies: CD279 (PD1) (Clone PD1.3 Beckman Coulter), CD45RA (Clone ALB11 Beckman Coulter), CD185 (CXCR5) (Clone J252D4 Biolegend), CD4 (Clone 13B8.2 Beckman Coulter), CD20 (Clone B9E9 Beckman Coulter), CD19 (Clone J4.119 Beckman Coulter), CD38 (Clone HB-7 Biolegend), CD27 (Clone M-T271 Biolegend), IGD (Clone IA6-2 Biolegend), CD11c (Clone BU15 Beckman Coulter), CD14 (Clone RMO52 Beckman Coulter), CD16 (Clone 3G8 Beckman Coulter), CD24 (Clone ALB9 Beckman Coulter), CD45 (Clone J.33 Beckman Coulter), CD3 (Clone UCHT1 Beckman Coulter), CD8 (Clone B9.11 Beckman Coulter), CD56 (Clone N901 Beckman Coulter), CD57 (Clone NC1 Beckman Coulter), CD25 (Clone B1.49.9 Beckman Coulter), CD127 (Clone R34.34 Beckman Coulter), CD159a (NKG2A)(Clone S19004C Biolegend), CD337 (NKp30) (Clone P30-15 Biolegend), IFN-γ (Clone 45.15 Beckman Coulter), IL-21 (Clone 3A3-N2 Biolegend), IL-17 (Clone BL168 Beckman Coulter), granzyme (Clone QA16A02 Biolegend), perforin (Clone B-D48 Biolegend)
Flow Cytometer: Navios, Beckman Coulter
Cytometric Bead Array: RAISECARE; Analysis Software: Kaluza, LEGEND plex.
Flow Cytometer: Navios, Beckman Coulter
Cytometric Bead Array: RAISECARE; Analysis Software: Kaluza, LEGEND plex.
10
Quantifying IRF4 and IRF8 Expression
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To test IRF4 and IRF8 expression, 14,000 to 15,000 sorted B cells were incubated in complete RPMI at 37°C with 5% CO2 for 24 hours in chambers without any stimulations, or with BCR stimulation with either PLBs containing biotinylated goat F(ab′)2 anti-human λ + κ (SouthernBiotech) for membrane Ag engagement or 10 nM biotinylated goat F(ab′)2 anti-human λ + κ (SouthernBiotech) for soluble antigen engagement. The cells stimulated for BCR were supplemented with CpG (ODN2006) (1.25 μg/ml, Invivogen), IL-2 (50 ng/ml, BioLegend), IL-21 (100 ng/ml, BioLegend), IL-10 (250 ng/ml, BioLegend), and BAFF (100 ng/ml, R&D Systems). Following incubation, cells were lysed, and RNA was extracted using Lysis Solution from the Cells-to-CT 1-Step TaqMan Kit (Invitrogen) following the manufacturer’s protocol. Expression of IRF4 and IRF8 in the cell lysates was analyzed by reverse transcription and qRT-PCR.
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