Naïve human cells were isolated by using Human Naïve CD4+ T Cell isolation kit (Stemcell Technologies, 17555) from peripheral blood mononuclear cells (PBMCs) after Ficoll-Paque gradient (GE Healthcare Systems). Naïve human CD4+ T cells were incubated with Dynabeads™ Human T-Activator CD3/CD28 (ThermoFisher, 11161D) for 3 days in R10 under the following skewing conditions: a) rhIL-23 (25 ng/ml, PeproTech); b) rhIL-23 (25 ng/ml, PeproTech) plus rhIL-10 (10 ng/ml PeproTech); c) rhIL-12 p70 (1 ng/ml, PeproTech) and d) rhIL-12 p70 (1 ng/ml, PeproTech) plus rhIL-10 (10 ng/ml PeproTech). The former conditions were cultured in the presence/absence of hTGF-β (5 ng/ml) (R&D Systems).
Rhil 10
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
RhIL-10 is a recombinant human interleukin-10 protein. Interleukin-10 is a cytokine that plays a key role in the regulation of the immune response.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by MP Biomedicals (2 mentions)
Fsl 1, by InvivoGen (1 mentions)
Pam3csk4, by InvivoGen (1 mentions)
Rpmi 1640 medium, by Lonza (1 mentions)
Lipopolysaccharides (lps), by InvivoGen (1 mentions)
Penicillin, by Lonza (1 mentions)
Streptomycin, by Lonza (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Rhil 23, by R&D Systems (1 mentions)
Rhil 12, by R&D Systems (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Roswell park memorial institute (rpmi), by Roche (1 mentions)
Rhil 2, by Roche (1 mentions)
Panserin 401, by PAN Biotech (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Rhil 6, by Thermo Fisher Scientific (1 mentions)
9 protocols using rhil 10
1
Naïve Human T Cell Differentiation under Cytokine Skewing
2
Establishment and Maintenance of HTLV-1-Infected T-Cell Lines
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ILTs were established by long-term culture of PBMCs of HTLV-1-infected patients in the presence of rhIL-2 (Shionogi, Osaka, Japan) for at least 6 months, following stimulation with phytohemagglutinin or Dynabeads Human T-Expander CD3/CD28 (Invitrogen, Carlsbad, CA). ILT-22, ILT-227, and ILT-H2 are derived from patients with ATL, while ILT-294, ILT-439, and ILT-441 are derived from patients with HAM/TSP. ILTs were maintained in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) containing 10% FBS (Sigma-Aldrich), 100 U/mL penicillin, 100 μg/mL streptomycin (Wako, Osaka, Japan), supplemented with 30–50 U/mL (ILT-22, -227, -H2) or 50–100 U/mL (ILT-294, -439 and -441) of rhIL-2. As the growth speed of HAM/TSP-derived ILTs was extremely slow, rhIL-10 (20 ng/mL, PeproTech, London, United Kingdom) was added to cultures to expand these cells, and then cultured in rhIL-2-containing medium without rhIL-10 at least for 1 week before use in most of the experiments. PBMCs from a healthy individual that had been activated by Dynabeads Human T-Expander CD3/CD28 (Invitrogen) were also used. HTLV-1-infected cell lines MT-2 [72 (link)] and TL-OmI [73 (link)], and HTLV-1-negative Jurkat [74 (link)] and MOLT4 [75 (link)] cells were cultured in RPMI 1640 medium containing 10% FBS.
3
Isolation and Characterization of EN-MSCs
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The procedure used to isolate EN‐MSCs from human uterine eutopic endometrium and ectopic endometriotic lesions has been described previously 23 and is summarised in supplementary material, Supplementary materials and methods. For CM collection, human EN‐MSCs were treated with medium alone, recombinant human IL‐10 (rhIL‐10; Pepro Tech, Rocky Hill, NJ, USA), or mAb against the human IL‐10 receptor (hIL‐10R; eBioscience, San Diego, CA, USA) for 24 h; fresh medium without any treatments was then incubated with the cells for an additional 24 h. Cell‐free CM was then used to culture HUVECs for assessing their angiogenic activity.
4
Elucidating Monocyte Responses to TLR Agonists
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PBMC or purified monocytes were cultured (1×106 cells/ml) in complete RPMI 1640 medium (BioWhittaker, Verviers, Belgium) including 10% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (Biowhittaker) and with TLRs agonists: TLR2/TLR6 agonist, FSL-1 (0.01 μg/ml), TLR2/TLR1 agonist, Pam3CSK4 (0.1 μg/ml) and TLR4 agonist, LPS (0.2 μg/ml) (Invivogen, San Diego, CA). To analyze the effect of exogenous IL-19, PBMC were treated with rhIL-19 (400 ng/ml) (ImmunoTools, Friesoythe, Germany). To study the effect of IL-10 on IL-19 production, rhIL-10 (200 ng/ml) (Preprotech, USA) and blocking anti-IL-10 (500 ng/ml) (BD, Biosciences, USA) were added to PBMC cultures.
5
Cell Culture Conditions and Stimulation Assays
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HEK293T cells were cultured in DMEM (Gibco) supplemented with 10% FBS (Invitrogen). EBV-B cells were cultured in RPMI (Gibco) supplemented with 10% FBS. HVS-T cells were cultured in a 1:1 mixture (by volume) of RPMI and Panserin 401 (PAN Biotech) supplemented with 10% FBS, GlutaMAX (350 µg/ml; Gibco), gentamicin (0.1 mg/ml; Gibco), and rhIL-2 (20 IU/ml; Roche).
The cells were starved for 2 h by incubation in serum-free RPMI. The cells were then left unstimulated or were stimulated with rhIFN-α 2b (105 IU/ml; Schering), rhIL-23 (100 ng/ml; R&D Systems), rhIL-12 (20 ng/ml; R&D Systems), or rhIL-10 (50 ng/ml; PeproTech) for 5 min to assess the phosphorylation of JAKs, and for 30 min to assess the phosphorylation of STATs. For quantitative RT-PCR, cells were stimulated for 6 h with IL-10 (50 ng/ml) or IFN-α (105 IU/ml). For RNA-seq experiments, cells were starved for 1.5 h in RPMI containing 1% FCS and were stimulated for 2 h with IFN-α (105 IU/ml) and IL-21 (100 ng/ml). RNA was extracted with the Zymoresearch kit. For scRNA-seq experiments, PBMCs were either left unstimulated or were stimulated with 100 ng/ml of IL-23 or 103 IU/ml IFN-α2b for 6 h.
The cells were starved for 2 h by incubation in serum-free RPMI. The cells were then left unstimulated or were stimulated with rhIFN-α 2b (105 IU/ml; Schering), rhIL-23 (100 ng/ml; R&D Systems), rhIL-12 (20 ng/ml; R&D Systems), or rhIL-10 (50 ng/ml; PeproTech) for 5 min to assess the phosphorylation of JAKs, and for 30 min to assess the phosphorylation of STATs. For quantitative RT-PCR, cells were stimulated for 6 h with IL-10 (50 ng/ml) or IFN-α (105 IU/ml). For RNA-seq experiments, cells were starved for 1.5 h in RPMI containing 1% FCS and were stimulated for 2 h with IFN-α (105 IU/ml) and IL-21 (100 ng/ml). RNA was extracted with the Zymoresearch kit. For scRNA-seq experiments, PBMCs were either left unstimulated or were stimulated with 100 ng/ml of IL-23 or 103 IU/ml IFN-α2b for 6 h.
6
Cytokine-Mediated Immune Modulation in PBMC
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PBMC from healthy donors were cultured in complete medium (RPMI 1640 supplemented with 10% heat-inactivated FBS, 100 µ/mL penicillin and 100 µg/mL streptomycin [Life Technologies]) at a concentration of 1 × 106 cells/mL for 3, 5, and 7 days with the plasma of HBV-ACLF and NC, or various concentrations (10–50 ng/mL) of recombinant human (rh) interleukin rhIL-1β (PeproTech), rhIL-6 (PeproTech), rhIL-10 (PeproTech), rhIL-22 (PeproTech), rhIL-37 (R&D), and tumor necrosis factor (rhTNF-α, PeproTech), or 10 μg/mL neutralizing antibody of anti-IL-6 (PeproTech), anti-TNF-α (PeproTech), and anti- IL-1β (PeproTech). The medium and cytokines were refreshed every other day. The expression of BTLA in CD4+ T cells was analyzed using flow cytometry.
PBMC from healthy donors were cultured at a concentration of 1 × 106 cells/mL in complete medium and were treated for 3 days in the following groupings: (a) control; (b) stimulant alone: rhIL-6 alone (50 ng/mL), or rhTNF-α (50 ng/mL); (c) inhibitor alone: anti-stat3 (SH-4-54, 50 μmol/L, Selleck) or the anti-NF- kappa (QNZ, 20 μmol/L, Selleck) or Polymyxin B (PXB, 10 μg/mL, Sigma-Aldrich); and (d) stimulant plus inhibitors. All experiments were performed at least in duplicate.
PBMC from healthy donors were cultured at a concentration of 1 × 106 cells/mL in complete medium and were treated for 3 days in the following groupings: (a) control; (b) stimulant alone: rhIL-6 alone (50 ng/mL), or rhTNF-α (50 ng/mL); (c) inhibitor alone: anti-stat3 (SH-4-54, 50 μmol/L, Selleck) or the anti-NF- kappa (QNZ, 20 μmol/L, Selleck) or Polymyxin B (PXB, 10 μg/mL, Sigma-Aldrich); and (d) stimulant plus inhibitors. All experiments were performed at least in duplicate.
7
Generation and Activation of Human Macrophages
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Human monocytes were isolated from buffy coats or fresh blood from healthy donors by serial density‐gradient centrifugation as previously described,23 or using the CD14 MicroBead Kit (Miltenyi Biotec, Bergisch Gladbach, DE) accordingly to the manufacturer's instructions. Human blood cell isolation was approved by the institutional Ethical Committee of the Humanitas Clinical and Research Center (Authorization n° 2502, 09/04/2020 and Approval for the use of buffy coats issued on 28/01/2016). Monocytes were treated for 7 days with 25 ng/ml rhM‐CSF (Peprotech, London, GB) to obtain Mϕs, in RPMI 1640 (Lonza, Basel, CH) with 10% FBS (Lonza, Basel, CH) and 1% l ‐glutamine (Lonza, Basel, CH). Mϕs were activated in vitro by 18 h incubation with 100 ng/ml 055:B5 LPS (Sigma–Aldrich, St Louis, MO, USA), 20 ng/ml rhIFN‐γ (Peprotech, London, GB), a combination of 100 ng/ml LPS and 20 ng/ml rhIFN‐γ, 20 ng/ml rhTNF (Peprotech, London, GB), 20 ng/ml rhIL‐10 (Peprotech, London, GB), 20 ng/ml rh TGF‐β (rhTGF‐β) (Peprotech, London, GB), 10−6 M Dexamethasone (Dex) (MP‐Biomedicals, Illkirch, FR), 20 ng/ml rhIL‐4, or 10−6 M Dex in combination with 20 ng/ml IL‐4.
8
Generation and Characterization of Dendritic Cells
9
Modulation of Th2 Responses by mDC-derived sEVs
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Human PBMCs from patients with AR or buffy coat from “anonymous donors” were isolated by density centrifugation with Ficoll‐Paque Plus (MP Biomedicals, Santa Ana, CA, USA). CD4+ T cells were sorted from PBMCs of buffy coat of human volunteers using the CD4 Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). sEV‐mDCs were harvested and washed twice, and then were co‐cultured with allogeneic PBMCs isolated from patients with AR or freshly sorted CD4+ T cells (1:10) for 5 days in RPMI 1640 (Hyclone, Pittsburgh, PA, USA) supplemented with 10% FBS and IL‐2 (100 U/mL). Cell counting was performed manually using a hemocytometer and the cell viability was over 95%. In some experiments, CD4+ T cells were treated with the rhIL‐10 (10 ng/mL; PeproTech Inc.) together with mDC, or incubated with anti‐IL‐10 monoclonal antibody (75 ng/mL) or IL‐10Rα blocking antibody (5 μg/mL; both from R&D systems, Minneapolis, MN, USA) for 30 min before the co‐culture. The PBMCs or the T cells were collected for the analysis of intracellular Th2 cytokines using flow cytometry, and the supernatants were used for the evaluation of IL‐13, IL‐9, IL‐10, or IFN‐γ. T cells were also evaluated for IL‐10+Foxp3+ T cells or Treg cells.
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