Lyophilized peptides were dissolved in sterile water and used at 2 μg/mL in RPMI 1640 glutamax media (GIBCO) supplemented with 1% penicillin/streptomycin (GIBCO). Single peptides (185) were plated in duplicates in 96-well round-bottom TPP-treated culture plates. Peptide plates were then stored at −80°C until use. The day of the experiment, peptide plates were thawed at room temperature. Frozen PBMCs were thawed, washed, and resuspended in RPMI 1640 media (GIBCO). Viability and count were evaluated using a Vi-Cell XR Cell Counter (Beckman Coulter). PBMCs were then plated in RPMI 1640 glutamax media (GIBCO) supplemented with 1% penicillin/streptomycin (GIBCO), with 200 UI/mL rhIL2 (Miltenyi) and 200 UI/mL rhIL15 (Miltenyi) at a cell density of 10 × 103 cells and incubated with each peptide at 37°C in 5% CO2. PBMCs were stimulated with 60 ng/mL OKT-3 antibody (Thermo Fisher Scientific, clone OKT3) or with 10 μg/mL phytohemagglutinin as positive controls, and PBMCs alone served as negative controls. After 6 hours, 20 μL of human AB serum was added to each well and plates were incubated at 37°C in 5% CO2 for 6 additional days. On day 7, supernatants were harvested and frozen at −80°C. The concentration of IFNγ, IL9, IL5, and IL17A in the culture supernatant was determined using a commercial ELISA kit (ELISA Max Deluxe set human IFNγ, BioLegend).
Rhil 15
Manufactured by Miltenyi Biotec
Sourced in Germany
RhIL-15 is a recombinant human interleukin-15 protein produced in E. coli. Interleukin-15 is a cytokine that plays a role in the activation and proliferation of T cells and natural killer cells.
Automatically generated - may contain errors
Lab products found in correlation
Rhil 2, by Miltenyi Biotec (6 mentions)
Rhil 7, by Miltenyi Biotec (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Il 15, by Miltenyi Biotec (2 mentions)
Nk macs medium, by Miltenyi Biotec (2 mentions)
Nk cell isolation kit, by Miltenyi Biotec (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (1 mentions)
Elisa max deluxe set human ifn γ, by BioLegend (1 mentions)
Rpmi 1640 glutamax media, by Thermo Fisher Scientific (1 mentions)
Vi cell xr cell counter, by Beckman Coulter (1 mentions)
Robosep, by STEMCELL (1 mentions)
Ficoll paque, by GE Healthcare (1 mentions)
Prep9, by Thermo Fisher Scientific (1 mentions)
Hek blue il 12, by InvivoGen (1 mentions)
Il 12p70, by BioLegend (1 mentions)
Universal mycoplasma detection kit, by American Type Culture Collection (1 mentions)
Mycoalert plus mycoplasma detection kit, by Lonza (1 mentions)
Recombinant human il 15, by R&D Systems (1 mentions)
Celltracer cfse cell proliferation kit, by Thermo Fisher Scientific (1 mentions)
15 protocols using rhil 15
1
Peptide-Stimulated PBMC Cytokine Assay
2
Isolation and Culture of Human T Cells
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Human peripheral blood mononuclear cells (PBMCs) derived from blood discard kits of healthy donors (BloodworksNW) were isolated by Ficoll-Paque (Pharmacia Biotech). CD4+ and CD8+ cells were purified from the PBMC using RoboSep (Stemcell). In some instance, the CD4-, CD8- PBMC fraction was retained. CD4+ T cells were cultured in RPMI with 10% FBS, 5 ng/ml rhIL-7 (Miltenyi) and 0.5 ng/ml rhIL-15 (Miltenyi). CD8+ cells were cultured in RPMI with 10% FBS, 50 U/ml rhIL-2, (Chiron Corporation) and rhIL-15 (0.5 ng/ml). In some instances, CD4+ or CD8+ cells were cultured in 50 U/mL rhIL-2, 20 ng/mL IL-4 (Milteyni), 10 ng/mL IL-7 (Milteyni) and 20 ng/mL Il-21 (Milteyni). Medium was replaced twice a week.
3
Recombinant Cytokine Production and Cell Line Validation
4
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.
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.
5
Natural Killer Cell Activation Protocol
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NK cells were stimulated as previously described (20 (link)). Briefly, PBMCs and BM MNCs were incubated in RPMI 1640 with L-glutamine (Capricorn Scientific), supplemented with 10% Fetal Bovine Serum (FBS, Capricorn Scientific) and penicillin/streptomycin (Capricorn Scientific) with HSP90 inhibitor - geldanamycin (GA, 0.1µM) (Abcam) or DMSO in the presence or absence of the recombinant human (rh) IL-2 (100 IU/ml) (Miltenyi Biotec) and rhIL-15 (10 ng/ml) (Miltenyi Biotec) overnight at 37°C 5% CO2 prior to the addition of APC-Cy7 anti-CD107a (Miltenyi Biotec). Cells then were stimulated with anti-NKp46/anti-CD2 (human NK cell activation/expansion kit, Miltenyi Biotec) for 5 hours at 37°C, according to the manufacturer instruction. The incubation was done in complete RPMI medium, supplemented with Brefeldin A (Sony Biotechnology) at a final dilution of 1/1000. Cells were then stained for surface markers and intracellular Granzyme B (Miltenyi Biotec) or IFNγ (Sony Biotechnology) and analyzed by flow cytometry.
6
PBMC Isolation and NK Cell Conditioning
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Using Ficoll density gradient sedimentation, peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated using leukoreduction system chambers. PBMCs were activated in RPMI 1640 medium supplemented with 10% FBS and 1 ng/ml rhIL-15 (PeproTech, Rocky Hill, NJ, USA) for 5 days.
For the 51 chromium (Cr) release assay and preparation of NK cell conditioned medium (CdM), NK cells were isolated from PBMCs using MACS columns and an NK cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Puri ed NK cells were cultured in NK MACS® Medium (Miltenyi Biotec) supplemented with 10% FBS, 500 IU/ml rhIL-2, and 10 ng rhIL-15. The purity of isolated NK cells was con rmed by ow cytometry. Puri ed NK cells were cultured in RPMI with 500 IU/ml rhIL-2 for 3 days to generate CdM. Cell culture supernatants were harvested by centrifugation and passed through a 0.45-µm Acrodisc syringe lter (Pall Corporation, Port Washington, NY, USA). IFN-γ mAb (NIB42; Thermo Fisher Scienti c, Waltham, MA, USA) was used to neutralize IFN-γ in CdM.
For the 51 chromium (Cr) release assay and preparation of NK cell conditioned medium (CdM), NK cells were isolated from PBMCs using MACS columns and an NK cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Puri ed NK cells were cultured in NK MACS® Medium (Miltenyi Biotec) supplemented with 10% FBS, 500 IU/ml rhIL-2, and 10 ng rhIL-15. The purity of isolated NK cells was con rmed by ow cytometry. Puri ed NK cells were cultured in RPMI with 500 IU/ml rhIL-2 for 3 days to generate CdM. Cell culture supernatants were harvested by centrifugation and passed through a 0.45-µm Acrodisc syringe lter (Pall Corporation, Port Washington, NY, USA). IFN-γ mAb (NIB42; Thermo Fisher Scienti c, Waltham, MA, USA) was used to neutralize IFN-γ in CdM.
7
Expansion and Transduction of CD19-CAR T Cells
8
Generating CAR T Cells from PBMCs
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For CAR T production, CD8+ and CD4+ T cells were isolated from PBMCs by either a custom sequential positive selection program using a RoboSep-S and EasySep CD8 and CD4 Positive Selection Kits (Stemcell Technologies) or manually by sequential positive selection using CD4 and CD8 MicroBeads (Miltenyi Biotec). The negative cell fraction was cryopreserved in FBS/10% DMSO for subsequent CD14+ isolation. T cells were stimulated with Dynabeads and cultured in RPMI-1640 (Gibco) with 10% FBS (VWR Seradigm), 2 mM l-glutamine (Gibco), 0.5 ng/mL rhIL-15 (Miltenyi Biotec) and either 50 U/mL rhIL-2 (CD8+ T cells; Chiron Corporation) or 5 ng/mL rhIL-7 (CD4+ T cells; Miltenyi Biotec). T cells were transduced with CD19 CAR-HER2tG lentivirus via spinoculation with protamine sulfate (APP Pharma), expanded, enriched for the HER2tG+ subset using biotinylated Trastuzumab antibody (online supplemental table 1 ) and anti-Biotin Microbeads (Miltenyi Biotec), and further expanded prior to cryopreservation or use in luciferase killing assays. Briefly, CAR T cells were added to eGFP:ffluc GEMs or eGFP:ffluc Raji cells and killing was measured by luminescence 10 min after luciferin addition, using lysis in 1% SDS as a positive control.
9
Generation of GT-00AxIL15, its Parental Anti-TA-MUC1 mAb, and Isotype Control
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GT-00AxIL15, its parental anti-TA-MUC1 mAb GT-00A and an untargeted MOPCxIL15 isotype control lacking TA-MUC1 binding were generated by Glycotope as described in Supplemental methods. rh-IL15 (Miltenyi, Bergisch Gladbach, Germany), human IgG1 (Sigma (St. Louis, MO, USA) or Biolegend (San Diego, CA, USA)), αEGFR mAb cetuximab (Erbitux, Eli Lilly and Merck KGaA, Darmstadt, Germany), αPD-L1 mAb avelumab (Bavencio, Merck Serono and Pfizer) and αPD-L1 clone 10F.9G2 (ratIgG2bκ, BioXCell, Lebanon, NH, USA) were purchased from commercial sources. For in vitro binding studies using primary human cells, GT-00AxIL15 was labelled with Alexa Fluor 647 according to manufacturer´s instructions (Molecular Probes, Invitrogen, Waltham, MA, USA). For in vivo biodistribution studies, GT-00AxIL15 and MOPCxIL15 were radiolabeled with the radionuclide zirconium-89 (89Zr) by conjugation of p-SCN-Bn-Deferoxamine to lysine amino acid residues on the antibodies via a thiourea linkage and purified by size-exclusion chromatography. Radiopurity and specific activity was comparable for both samples (>99% and 60 MBq/mg).
10
Expansion of Autologous Vδ2 T Cells
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PBMCs from ALL patients and HV (healthy volunteers) were isolated by density gradient (Lymphoprep) and were then frozen until use. To expand autologous Vδ2 T cells, frozen PBMCs from ALL patients were stimulated with ZOL 1µM (Sigma-Aldrich, Saint Louis, MO) and rhIL-2 (Miltenyi Biotec, Bergisch Gladbach, Germany) at day 0. From day (d) 5, rhIL-2 was renewed every 2 days and the cells were maintained at 1.5 × 106/mL until d14. rhIL-15 (10 ng/mL; Miltenyi Biotec) was also added starting at d2 and was renewed every 2 days, as IL-15 has been shown to enhance the proliferative capacity of Vδ2 T cells [55 (link),56 (link),57 (link)]. Fresh autologous expanded Vδ2 T cells were then used for functional assays, depending on the quantity of Vδ2 T cells. The fold increase in viable Vδ2 T cells was calculated using the formula: (d14 %Vδ2 × d14 total cell number/(d0 % Vδ2 × d0 total cell number).
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