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Recombinant human il 15

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Recombinant human IL-15 is a cytokine that promotes the proliferation and activation of natural killer cells and CD8+ T cells. It is produced in a mammalian cell expression system.

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Lab products found in correlation

Recombinant human il 7, by R&D Systems (6 mentions) Golgistop, by BD (3 mentions) Fsl 1, by InvivoGen (3 mentions) Pam3csk4, by InvivoGen (3 mentions) Ficoll hypaque, by GE Healthcare (2 mentions) Anti cd107a efluor 660 antibody, by Thermo Fisher Scientific (2 mentions) Cpg dna tlr 9 ligand odn 2006, by InvivoGen (2 mentions) Interleukin 2 (il 2), by R&D Systems (2 mentions) Human cd8 t cell isolation kit, by Miltenyi Biotec (2 mentions) Human serum, by R&D Systems (2 mentions) Penicillin streptomycin, by R&D Systems (2 mentions) Rpmi 1640 medium, by Merck Group (2 mentions) Flagellin, by InvivoGen (2 mentions) Pd1 pecf594, by BD (2 mentions) Cd3 bv421, by BD (2 mentions) Cd8 apc h7, by BD (2 mentions) Facsaria 2 cell sorter, by BD (2 mentions) Mgp10025 33 egsrnqdwl, by GenScript (2 mentions) Mouse il 21, by R&D Systems (2 mentions) Pmel 1 tcr transgenic mice, by Jackson ImmunoResearch (2 mentions)

27 protocols using recombinant human il 15

1

Canine NK Cell Isolation and Culture

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Canine NK cells were selected from PBMCs with anti-caCD94 mAb (45 minutes at 4°C) and anti-mouse IgG microbeads (Miltenyi Biotec, Auburn, CA). The cells were positively selected using the Miltenyi magnetic column method. The cells were cultured in complete medium (45% Waymouth’s medium, 45% Iscove’s medium, 10% heat-inactivated dog serum, 10 ng/ml recombinant canine IL-2 (R&D Systems, Minneapolis, MN) 15 ng/ml recombinant human IL-15 (R&D Systems), penicillin/streptomycin, sodium pyruvate, glutamine) on a feeder layer of 5 × 105 lethally irradiated CTAC Cells were transferred to fresh wells containing irradiated CTAC cells and complete medium every other day.
Graves S.S., Gyurkocza B., Stone D.M., Parker M.H., Abrams K., Jochum C., Gallo S., Saad M., Johnson M.M., Rosinski S.L, & Storb R. (2019). Development and characterization of a canine-specific anti-CD94 (KLRD-1) monoclonal antibody. Veterinary immunology and immunopathology, 211, 10-18.
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2

PBMC Cytotoxicity Assay with IL-15 Stimulation

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Human PBMC were isolated from the blood of 8 healthy donors using Hypaque-Ficoll (GE Healthcare, Amersham, UK) density centrifugation, with informed consent and full ethical approval (NRES reference: 06/Q1701/120). 3 × 105 PBMCs were stimulated overnight with 1 ng/mL recombinant human IL-15 (R&D Systems). Peptide pulsed 721.221C*0304-ICP47 targets were prepared as for the stabilization assays. Target cells were resuspended with PBMCs at an effector-to-target (E:T) ratio of 5:1 in fresh R10 medium containing peptide and anti-CD107a-efluor-660 antibody (eBioscience, Hatfield, UK). Cells were incubated for 1 h at 26°C, then 6 μg/mL Golgi- Stop™ (BD Biosciences) was added, and incubated for a further 4 h at 26°C. Cells were washed, blocked with blocking buffer for 30 min and then stained with the following antibodies: anti-CD3-PerCP (Biolegend, San Diego, USA), anti-human CD56-PE, and anti-human KIR2DL2/L3/S2, CD158b-FITC (both BD Biosciences). Cells were fixed in 1% PFA and analyzed by flow cytometry. Individual assays for each donor were performed once in duplicate and the mean value used for subsequent analysis.
Mbiribindi B., Mukherjee S., Wellington D., Das J, & Khakoo S.I. (2019). Spatial Clustering of Receptors and Signaling Molecules Regulates NK Cell Response to Peptide Repertoire Changes. Frontiers in Immunology, 10, 605.
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3

Autologous moDCs Potentiate WT1-Specific CTL

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Mature WT1 mRNA-electroporated autologous moDCs were added in serial doses to triplicate wells containing 1 × 105 T cells in a 96 round-bottomed well plate (Corning Life Sciences). Final volume was 100 μL/well of RPMI-10% heat-inactivated, autologous serum, supplemented with recombinant human IL15 (10 ng/mL; R&D Systems). After 7 days of moDC-T cell culture, 5×103 target cells were added directly to each well, and cytolytic activity exerted by responder T lymphocytes was assessed after 4 to 6 hours with a colorimetric CTL assay (27 (link)). These data represented the total cytolytic activity generated in each culture according to the primary stimulation conditions, rather than per number of effector T cells irrespective of their frequency in the primary cultures. Target cells were 697 cells (HLA-A*0201+, WT1+ cell line). SKLY-16 cells (HLA-A*0201+, WT1neg cell line) served as a negative control.
Chung D.J., Pronschinske K.B., Shyer J.A., Sharma S., Leung S., Curran S.A., Lesokhin A.M., Devlin S.M., Giralt S.A, & Young J.W. (2015). T cell exhaustion in multiple myeloma relapse after autotransplant: Optimal timing of immunotherapy. Cancer immunology research, 4(1), 61-71.
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4

CFSE-labeled B-CLL Cell Culture

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CFSE-labeled B-CLL cells were cultured in an enriched medium used for normal B cell replication in long-term cultures (55 (link)) with added insulin/transferrin/selenium supplement (BioWhittaker cat# 17-8387). It should be noted that this medium contains the reducing agent, 2-ME (5×10−5 M). The latter replaces an important function of bone marrow stromal cells in converting cystine to cysteine which is needed for B-CLL uptake and use in the glutathione synthesis needed for retained viability (59 (link)). To minimize inter-experimental differences, medium was freshly prepared for each experiment, using stock additives whose expiration date was carefully monitored and, in the case of FCS and the insulin/transferrin/selenium supplement, kept frozen in aliquots until use. Cultures were routinely established in 96-well plates (Falcon, Costar) at 105 cells per 200 μl volume with triplicates for each culture condition. Recombinant human IL-15 (R&D Systems, Inc) and CpG DNA TLR-9 ligand (ODN-2006; InvivoGen, Inc) (frozen in aliquots until use) were added at final culture concentrations of 15 ng/ml and 0.2 μM (1.5 μg/ml), respectively. In designated cultures, the pan-caspase inhibitor, Z-VAD-FMK (Sigma-Aldrich, St. Louis, MO; 40 μM final) or DMSO vehicle were added
Mongini P.K., Gupta R., Boyle E., Nieto J., Lee H., Stein J., Bandovic J., Stankovic T., Barrientos J., Kolitz J.E., Allen S.L., Rai K., Chu C.C, & Chiorazzi N. (2015). TLR-9 and IL-15 Synergy Promotes the In Vitro Clonal Expansion of Chronic Lymphocytic Leukemia B Cells. The Journal of Immunology Author Choice, 195(3), 901-923.
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5

Isolation and Culture of Human and Mouse NK Cells

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Human NK cells were isolated from discarded Leukopaks from healthy volunteer blood donors using the RosetteSep human NK cell enrichment protocol (StemCell Technologies), followed by Ficoll (GE Health-care) density gradient centrifugation (20 min, 800g) and culture for 12–18 h in X-VIVO 10 TM medium (Lonza) supplemented with gentamicin, 5% human AB serum (Corning) and 2.5 ng ml–1 recombinant human IL-15 (R&D Systems). Mouse NK cells were isolated from splenocytes obtained after mechanical dissociation of spleens and passage through a 40 μm sieve (BD Labware), using a NK cell magnetic purification kit (Miltenyi Biotec). Mouse NK cells were cultured in RPMI 1640 supplemented with 10% FCS, 1% penicillin/streptomycin, 1% l-glutamine (Gibco), 2.5 ng ml–1 recombinant mouse IL-15 and 100 units of IL-2 (R&D Systems).
Santara S.S., Lee D.J., Crespo Â., Hu J.J., Walker C., Ma X., Zhang Y., Chowdhury S., Meza-Sosa K.F., Lewandrowski M., Zhang H., Rowe M., McClelland A., Wu H., Junqueira C, & Lieberman J. (2023). The NK cell receptor NKp46 recognizes ecto-calreticulin on ER-stressed cells. Nature, 616(7956), 348-356.
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6

Sorting and Stimulating CD4 T-Cell Subsets

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CM, TM, and EM CD4 T-cell subsets were sorted from thawed PBMCs as explained above. Sorted cells were washed and resuspended (1 × 106 cells per ml) in R10 with 30 ng/ml recombinant human IL-15 (R&D systems), and 30 ng/ml recombinant human IL-7 (R&D systems). Before and after five days of stimulation, cells were washed and stained with the following combination of monoclonal antibodies: anti-CCR7-BB700 (clone 3D12), anti-Ki67-AL700 (B56), anti-CD3-BUV395 (clone SP34-2), anti-CD45RA-BUV737 (clone HI100), anti-CD27-BV605 (clone L128), anti-CD8-BUV496 (clone RPA-T8), anti-granzyme-B-TRPE (clone GB11) all from BD Biosciences; anti-CD4-BV650 (clone OKT4), anti-HLA-DR-BV750 (clone L243), anti-PD-1-BV785 (clone EH12.2H7) all from Biolegend; anti-CD38-APC (clone OKT10) from Ray Biotech; anti-CD127-PE-Cy5 (clone EBIORDR5) from Scientific Thermofisher; anti-Perforin-FITC (clone Pf344) from MAbtech; LIVE/ DEAD Fixable Near-IR Dead Cell Stain from Thermo Fisher Scientific. Samples were collected on a FACSymphony system (BD Biosciences) and analyzed in FlowJo (version 9.9.6, TreeStar).
Pino M., Pereira Ribeiro S., Pagliuzza A., Ghneim K., Khan A., Ryan E., Harper J.L., King C.T., Welbourn S., Micci L., Aldrete S., Delman K.A., Stuart T., Lowe M., Brenchley J.M., Derdeyn C.A., Easley K., Sekaly R.P., Chomont N., Paiardini M, & Marconi V.C. (2021). Increased homeostatic cytokines and stability of HIV-infected memory CD4 T-cells identify individuals with suboptimal CD4 T-cell recovery on-ART. PLoS Pathogens, 17(8), e1009825.
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7

Allogeneic DC-Based CD8+ T Cell Expansion

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CD8+ T cells were purified from PBMC by negative selection using a human CD8 T cell isolation kit (Miltenyi Biotech, Bergish Gladbach, Germany) according to the manufacturer’s instructions. T cell cultures were performed in X-VIVO 15 (Lonza, Verviers, Belgium) supplemented with 5% human serum (Sigma Aldrich, Burlington, Massachusetts, USA) and 100 U/mL penicillin/streptomycin (Lonza, Verviers, Belgium). T cells were cultured with 104 allogeneic DCIL-10, mDCIL-10, or mDCGFP at a 10:1 ratio. After 5 days of stimulation (short-term culture), T cells were collected, washed, and phenotypically and functionally analyzed. For long-term culture experiments, T cells were cultured with allogeneic mDCIL-10 (T(mDCIL-10)) or mDCGFP (T(mDCGFP)) at a 10:1 ratio. At day 3, 1 ng/mL of recombinant human IL-15 (R&D System, Minneapolis, MN, USA) was added. At day 14, cells were collected, washed, and analyzed. In some experiments, CD8+ T cells were labelled with Cell Proliferation Dye eFluor® 670 (eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions and analyzed by flow cytometry for their proliferation at the end of the T:DC co-culture.
Fortunato M., Amodio G, & Gregori S. (2023). IL-10-Engineered Dendritic Cells Modulate Allogeneic CD8+ T Cell Responses. International Journal of Molecular Sciences, 24(11), 9128.
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8

Toll-like Receptor Agonist Stimulation

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Human TLR1-9 Agonist Kit, TLR1/TLR2 agonist Pam3CSK4, TLR5 agonist flagellin (FLA), and TLR2/TLR6 agonist FSL-1 were purchased from InvivoGen. Recombinant human IL-7 and recombinant human IL-15 were products of R&D Systems.
Qiu C., Wang J., Zhu L., Cheng X., Xia B., Jin Y., Qin R., Zhang L., Hu H., Yan J., Zhao C., Zhang X, & Xu J. (2022). Improving the ex vivo expansion of human tumor-reactive CD8 + T cells by targeting toll-like receptors. Frontiers in Bioengineering and Biotechnology, 10, 1027619.
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9

Cell Division Monitoring and Viability of Stat5 DKI NK Cells

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To monitor cell division, 20 million splenic or bone marrow cells from WT or Stat5 DKI mice were labelled in 1 ml of PBS with 2.5 μM of CFSE (CellTracer CFSE Cell Proliferation Kit, Invitrogen, Carlsbad, CA) at room temperature for 7 min, washed once with serum and twice with complete RPMI-1640 medium, 1.5 × 106 ml−1 CFSE-labelled cells were then cultured in the presence of 20 ng ml−1 recombinant human IL-15 (R&D Systems, Minneapolis MN or BioLegend), and fresh rhIL-15 was added every 2 days. NK cell division was monitored for CFSE dilution using flow cytometry on day 2, 3 and 4 by staining cells with fluorescent-labelled antibodies for CD3, CD122 and NK1.1.
To determine cell viability after IL-15 withdrawal, column purified (Miltenyi Biotec Inc., San Diego, CA) NK cells from WT or Stat5 DKI mice were cultured in complete RPMI-1640 medium supplemented with 20 ng ml−1 rhIL-15 for 6–7 days, fresh rhIL-15 was added every 2 days, cells were then washed three times with complete medium, cultured in complete medium without IL-15, stained at the indicated time points, and viable NK cells were determined as CD3−CD122+NK1.1+Annexin V−7AAD−. Annexin V+7AAD− cells were scored as apoptotic cells and Annexin V+7AAD+ as dead cells.
Lin J.X., Du N., Li P., Kazemian M., Gebregiorgis T., Spolski R, & Leonard W.J. (2017). Critical functions for STAT5 tetramers in the maturation and survival of natural killer cells. Nature Communications, 8, 1320.
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10

CLL Cell Proliferation Assay

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CLL primary cells (107 cells) were labeled with 0.5 µM carboxyfluoresceinsuccinimidyl ester (CFSE; Life Technologies) as reported previously50 (link). Briefly, 105 cells/200 μL were cultured for 6 days in an enriched RPMI-1640 supplemented with 15 ng/mL recombinant human IL-15 (R&D systems, Minneapolis, MN) to sustain survival and with 0.2 μM CpG DNA TLR-9 ligand (ODN-2006; Invivogen, San Diego, CA) to induce cell proliferation13 (link). The percentage of divided cells was determined as the percentage of CD19+ (PE)/Annexin-V-(Pacific Blue) cells showing a decrease in CFSE staining on flow cytometry. Fluorescence-minus-one (FMO) was used as a negative control. Data analysis was performed using FlowJow 10.0.7 software (FlowJo, Ashland, OR).
Gimenez N., Tripathi R., Giró A., Rosich L., López-Guerra M., López-Oreja I., Playa-Albinyana H., Arenas F., Mas J.M., Pérez-Galán P., Delgado J., Campo E., Farrés J, & Colomer D. (2020). Systems biology drug screening identifies statins as enhancers of current therapies in chronic lymphocytic leukemia. Scientific Reports, 10, 22153.
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