Freshly isolated skin mononuclear cells using the M.CoIV_6 h isolation protocol were labeled with Carboxyfluorescein succinimidyl ester (CFSE) at the final concentration of 2.5 μM. Briefly, cells were washed twice in PBS and the cell pellet was resuspended in PBS at density of 10 × 106 cells/mL, and then labeled with 2.5 μM CFSE at 37°C for 10 min. The reaction was stopped by adding 5 mL FCS and washed twice. Labeled skin cells were cultured in X-vivo 15 medium (Lonza) containing 10% human AB serum and 500 IU/mL Proleukin (IL-2; Novartis) as well as 1% Pen/Strep for 5 days. Fractions of skin cells were additionally stimulated with T activation/expansion beads (Miltenyi Biotec) at a bead-to-cell ratio of 1:1.
Il 2 proleukin
Manufactured by Novartis
Sourced in Switzerland, Germany
IL-2 (Proleukin) is a laboratory-produced protein that is similar to a natural human protein. It functions as an immune system regulator.
Automatically generated - may contain errors
Lab products found in correlation
X vivo 15, by Lonza (4 mentions)
Cd3 microbeads, by Miltenyi Biotec (4 mentions)
L glutamine, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
X vivo 15 medium, by Lonza (2 mentions)
Gm csf, by CellGenix (2 mentions)
Il 15, by Thermo Fisher Scientific (2 mentions)
Human cd8 microbeads, by Miltenyi Biotec (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
K562 erythroleukemia cells, by American Type Culture Collection (2 mentions)
Dmem medium, by Thermo Fisher Scientific (2 mentions)
X vivo 10 medium, by Lonza (2 mentions)
Brefeldin a, by Merck Group (2 mentions)
Tissue culture treated plate, by Corning (1 mentions)
Interleukin 6 (il 6), by CellGenix (1 mentions)
Il 15, by Novartis (1 mentions)
Interleukin 6 (il 6), by Novartis (1 mentions)
Human serum, by Novartis (1 mentions)
Gm csf, by Novartis (1 mentions)
25 protocols using il 2 proleukin
1
CFSE Labeling and Stimulation of Skin Mononuclear Cells
2
Expansion and Differentiation of NK Cells
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Hematopoietic stem cells, isolated from fresh umbilical cord blood (Anthony Nolan, United Kingdom), were expanded and differentiated into GTA002 NK cells as previously described (Spanholtz et al., 2010 (link)). In short, CD34+ cells were seeded at a concentration of 10,000 cells/ml in a six-well tissue culture–treated plate (Corning Incorporated) in Glycostem Basal Growth Medium (GBGM®, FertiPro) supplemented with 10% human serum (Sanquin, Netherlands), 25 ng/ml of TPO, IL-7, Flt-3L, and SCF (Cellgenix), 1 ng/ml GM-CSF, 0.05 ng/ml Il-6 (Cellgenix), and 0.25 ng/ml Neupogen (G-CSF; Amgen BV). After 9 days, TPO was replaced by 20 ng/ml IL-15 (Cellgenix). After 14 days of expansion, the differentiation phase was initiated by adding differentiation medium consisting of GBGM supplemented with 2% human serum, 20 ng/ml of IL-7, IL-15, and SCF, 1 ng/ml GM-CSF, 0.05 ng/ml Il-6, 0.25 ng/ml Neupogen, and 1000 U/ml Proleukin (IL-2; Novartis). The cells were cultured until day 28 and cryopreserved until further use. For usage in cytotoxicity assays, day 28 GTA002 cells were thawed and cultured for 1 week in differentiation medium.
3
Authentication and Culture of Urothelial and Control Cell Lines
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We used four UCC, namely, RT-112, BFTC905, VM-CUB-1, and UM-UC-3 (10 (link)). HBLAK was used as normal urothelial control cell line (20 (link)). All cell lines were regularly authenticated by STR profiling, checked for mycoplasma contamination, and cultured as described (21 (link)). Human embryonic kidney cells (HEK293T) were obtained from DSMZ (Braunschweig, Germany) and cultured in DMEM GlutaMAX supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) (all Thermo Fisher Scientific, Schwerte, Germany).
Human peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of healthy adult volunteers by density-gradient centrifugation (Cytiva, Marlborough, MA, USA). Blood donors gave informed consent according to the protocol (#2019-623) approved by the local ethics committee/IRB in Düsseldorf. Prior to transduction, T-cells were prestimulated with immobilized antibodies against CD3 (OKT3, Ortho Biotech, Neuss, Germany) and CD28 (BD Biosciences Pharmingen, San Diego, CA, USA), as well as 100 IU/ml Interleukin-2 (IL-2, Proleukin, Novartis, Basel, Switzerland) in IMDM (Sigma, MO, USA) containing 10% FBS, 1% P/S, 1% glutamine as previously described (22 (link), 23 (link)).
Human peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of healthy adult volunteers by density-gradient centrifugation (Cytiva, Marlborough, MA, USA). Blood donors gave informed consent according to the protocol (#2019-623) approved by the local ethics committee/IRB in Düsseldorf. Prior to transduction, T-cells were prestimulated with immobilized antibodies against CD3 (OKT3, Ortho Biotech, Neuss, Germany) and CD28 (BD Biosciences Pharmingen, San Diego, CA, USA), as well as 100 IU/ml Interleukin-2 (IL-2, Proleukin, Novartis, Basel, Switzerland) in IMDM (Sigma, MO, USA) containing 10% FBS, 1% P/S, 1% glutamine as previously described (22 (link), 23 (link)).
4
T-Cell Expansion and Transduction Protocol
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T cells were cultured in TCM consisting of IMDM (Lonza) supplemented with 5% fetal bovine serum (FBS; Gibco, Life Technologies), 5% human serum 1.5% glutamine (Lonza) and 1% penicillin/streptomycin (Lonza) and 100 IU/ml IL2 (Proleukin; Novartis Pharma). Expanded T-cell clones were cryopreserved or restimulated every 10–15 days with a feeder mix containing 1 × 10^6 PBMC’s, 0.1 × 10^6 Epstein–Barr virus-transformed lymphoblastoid cell line (EBV-LCL) JY cells and 0.8 mg/ml phytohemagglutinin (PHA; Oxoid Microbiology Products, Thermo Fisher Scientific). Cell lines were cultured in IMDM with 10% FBS, 1.5% glutamine and 1% penicillin/streptomycin. Cell lines negative for target HLA alleles were retrovirally transduced to express HLA-A1, -A24, -A3 or -A11. Viral vectors encoded murine CD19 or tNGFR as transduction markers. K562 cells were additionally transduced to express JCHAIN. Transduced cells were purified for transgene expression by MACS enrichment of transduction marker positive cells. PBMCs from healthy donors expressing one or two target HLA alleles were used to derive hematopoietic subsets as previously described [22 (link)]. Fibroblasts and keratinocytes were pre-treated for 48 h with 100 IU/ml IFN-γ (Immukine) prior to experiments. MM patient-derived BM samples were thawed and rested overnight in medium containing 10% human serum before use in cytotoxicity experiments.
5
CAR-T Cell Manufacturing Protocol
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CAR-T cells were manufactured as described in our previous study (23 (link)). The detailed methods were as follows: peripheral blood mononuclear cells (PBMCs) were collected and isolated by Ficoll density gradient centrifugation. CD3+ T cells were selected by CD3 microbeads (Miltenyi Biotec, Cambridge, MA, USA), stimulated by anti-CD3/anti-CD28 mAb-coated Human T-Expander beads (Cat.no. 11141D; Thermo Fisher Scientific, Waltham, MA, USA) and cultured in T-cell medium X-Vivo 15 (Lonza Group, Ltd., Basel, Switzerland) supplemented with 250 IU/ml interleukin-2 (IL-2; Proleukin; Novartis International AG, Basel, Switzerland). All the CD3+ T cells (3 × 106) were transduced with a lentiviral vector encoding humanized CD19 CAR constructs (10 μg, lenti-CD19-2rd-CAR; Shanghai Genbase Biotechnology Co., Ltd., Shanghai, China) and cultured in media containing recombinant human IL-2 (250 IU/ml). On the 12th day of cultivation, transduction efficiencies of anti-CD19-CAR were analyzed by flow cytometry (FCM) (BD Biosciences, San Jose, CA, USA).
6
CD8+ T Cell Expansion Protocol
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CD8+ cells were isolated from PBMCs using human CD8 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). CD8+ cells (2×106) were stimulated by peptide-pulsed irradiated autologous mature DCs (1×105). Autologous DCs were prepared from a limited supply; artificial antigen presenting cells (aAPCs) (K562/A2 or A24/CD80/CD83) were alternatively used for further examination. After 1 week, these cells were stimulated twice per week by peptide-pulsed irradiated artificial APC-A2 or artificial APC-A24 cells (1×105). Supplementation with 10 IU/ml IL-2 (Proleukin; Novartis Pharmaceuticals, Basel, Switzerland) and 10 ng/ml IL-15 (PeproTech) was performed every 3 to 4 days between stimulations (28 (link)).
7
Generating CD19 CAR-T Cells from B-ALL Patients
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We collected 50 mL leukocyte collection from the nine relapsed patients with B-ALL by leukapheresis, then PBMCs were isolated by Ficoll density gradient centrifugation. CD3+ T cells (1× 106) were selected using CD3 microbeads (MiltenyiBiotec, Inc., Cambridge, MA, USA), stimulated with anti-CD3/anti-CD28 mAb-coated Human T-Expander beads (the ratio of this bead to CD3+T cell was 1:1) (Cat. No. 11141D; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and cultured in T-cell medium X–Vivo 15 (Lonza Group, Ltd., Basel, Switzerland) supplemented with 250 IU/mL interleukin-2 (IL-2; Proleukin; Novartis International AG, Basel, Switzerland). Then, the T-cell medium X–Vivo 15 supplemented with 250 IU/mL interleukin-2 was added every two days or depending on the CD19 CAR-T cell growth. After 48 h of cell culture, at which point the CD19+ leukemic cells could not be detected in the culture by flow cytometry (FCM) (BD Biosciences, San Jose, CA, USA), T cells (3×106) were transduced with a lentiviral vector encoding CD19-CAR constructs (10µg; lenti-CD19-2rd-CAR; Shanghai Genbase Biotechnology Co., Ltd., Shanghai, China) and cultured in medium containing recombinant human IL-2 (250 U/mL). In addition, the CD3+ T cells from the PBMCs of the nine patients were cultured in T-cell medium X–Vivo 15 supplemented with 250 IU/mL IL-2 as control group.
8
MART-1 TCR CD8 T Cell Generation
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MART-1 TCR CD8 T cells were generated as previously described.21 (link) In short, primary CD8 T cells were isolated from healthy male or female donor buffy coats (Sanquin, Amsterdam, the Netherlands), activated with plate-coated CD3 and CD28 antibodies (both 5 μg/ 2x106 cells/ 24-well, 16-0037-85 and 16-0289-85, Thermo Fisher Scientific) for 48 h in primary human CD8 T cell medium (RPMI Medium (GIBCO) containing 10% human serum (H3667, Sigma-Aldrich), 100 U/ml of Penicillin-Streptomycin (GIBCO), 100 U/ml IL-2 (Proleukin, Novartis), 10 ng/ml IL-7 (11340077, ImmunoTools) and 10 ng/ml IL-15 (11340157, ImmunoTools)). Right after 48 h activation, T cells were removed from activation plates and spinfected with MART-1 TCR retrovirus on Retronectin-coated (25 μg/ 24-well, TB T100B, Takara) non-tissue culture-treated plates. Cells were harvested and maintained in primary human CD8 T cell medium 24 h after transduction. 1 week after transduction, MART-1 TCR expression was checked by flow cytometry (α-mouse TCR β chain, 553172, BD Pharmingen) and cells were cultured in human CD8 T cell medium (RPMI containing 10% fetal bovine serum (Sigma), 100 U/ml of Penicillin-Streptomycin (GIBCO) and 100 U/ ml IL-2 (Proleukin, Novartis)).
9
Generation of Anti-CD19 CAR-T Cells
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PBMCs from 9 patients were isolated by Ficoll density gradient centrifugation. CD3+ T cells were separated from the PBMCs by magnetic cell sorting (MACS) using CD3 microbeads (Miltenyi Biotec, Inc). CD3+ T cells were stimulated with anti‐CD3/anti‐CD28 mAb‐coated Human T‐Expander beads (cat. no. 11141D; Thermo Fisher Scientific, Inc) and cultured in T‐cell medium X‐Vivo 15 (Lonza Group, Ltd.) supplemented with 250 IU/mL interleukin‐2 (IL‐2; Proleukin; Novartis International AG). On the fourth day of culture, T cells (3 × 106) were transduced with a lentiviral vector encoding anti‐CD19 CAR constructs (5 µg; lenti‐EF1a‐CD19‐2rd‐CAR; Creative Biolabs, Inc) and cultured in medium containing recombinant human IL‐2 (250 IU/mL). At the same time, CD3+ T cells were cultured in T‐cell medium X‐Vivo 15 supplemented with 250 IU/mL interleukin‐2. On the 12th day of culture, the transduction efficiency of CAR 19 was analyzed by flow cytometry (FCM) to detect the ratio of CAR‐T 19 cells in CD3+ T cells.
10
Culture and Maintenance of Cell Lines
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Human MDA-MB453 breast carcinoma and HEK 293T cells (both ATCC, Manassas, VA) were propagated in DMEM (Lonza, Cologne, Germany). Human Raji and WSU-NHL lymphoma, BV173 leukemia and C1R-neo B-cell lymphoblastoid cells (all ATCC) were cultured in RPMI 1640 medium (Lonza). Media were supplemented with 10% heatinactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin (Life Technologies, Darmstadt, Germany). MDA-MB453/CD19 breast carcinoma cells were generated by transduction with GALV pseudotyped retroviral vector encoding truncated human CD19 (kindly provided by Malcolm Brenner and Gianpietro Dotti, Baylor College of Medicine, Houston, TX) following standard protocols [30] . Primary human pre-B-ALL blasts were grown in Iscove's modified Dulbecco's medium (IMDM; Lonza) supplemented with 1 µg/mL bovine insulin (Sigma-Aldrich, Taufkirchen, Germany), 50 M b-mercaptoethanol, 200 µg/mL Fe 3+ -saturated human apo-transferrin (Invitrogen, Karlsruhe, Germany), 0.6% human serum albumin (Sanquin, Amsterdam, The Netherlands), 2.0 mM L-glutamine, and 20 µg/mL cholesterol [31] . Human NK-92 cells (kindly provided by NantKwest, Inc., Culver City, CA) were cultured in X-VIVO 10 (Lonza) containing 5% heat-inactivated human plasma (German Red Cross Blood Donation Service Baden-Württemberg -Hessen) and 100 IU/mL IL-2 (Proleukin; Novartis Pharma).
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