T cells were primed as previously described.25 (link) Briefly, naïve CD8+ T cells were isolated from the peripheral blood of healthy donor positive for HLA-B*35:02 by depletion of non-naive T cells and NK cells (using biotin-conjugated antibodies against CD45RO, CD56, CD57, and CD244), followed by positive selection using CD8 MicroBeads (Miltenyi Biotech, Bergisch Gladbach, Germany). Isolated naïve CD8+ T cells were incubated with IL-7 overnight. The next day, T cells were washed and mixed with matured autologous dendritic cells (DCs), pulsed with the investigational peptides (GL Biochem, Shanghai, China) as indicated at 1 µg/mL. Interleukin (IL)-21 (30 ng/mL, Peprotech, Rocky Hill, NJ) was added to the co-culture on day 0 the next morning. IL-7 and IL-15 (5 ng/mL, Peprotech) were added on days 3, 5, and 7. On day 10, cells were restimulated with the original peptide for 2 hours. On day 11, T cells were harvested and analyzed for OX40 and 4-1BB positivity, and media was analyzed for IFNγ secretion by ELISA (Biolegend, San Diego, California, USA).
Il 7 and il 15
Manufactured by Thermo Fisher Scientific
Sourced in United States
IL-7 and IL-15 are cytokines produced by various cell types that play important roles in the immune system. IL-7 is essential for the development and survival of T cells, while IL-15 is involved in the activation and proliferation of natural killer cells and memory T cells. These recombinant proteins can be used for cell culture applications and research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 2 (il 2), by Thermo Fisher Scientific (3 mentions)
Cd8 microbead, by Miltenyi Biotec (1 mentions)
Gentamycin, by Thermo Fisher Scientific (1 mentions)
Proleukin, by Novartis (1 mentions)
X vivo serum free medium, by Lonza (1 mentions)
Ab serum, by Merck Group (1 mentions)
Anti cd3 cd28 coated beads, by Thermo Fisher Scientific (1 mentions)
Recombinant human il 2, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Corning (1 mentions)
Anti cd3 cd28 dynabeads, by Thermo Fisher Scientific (1 mentions)
Polybrene infection transfection reagent, by Merck Group (1 mentions)
Anti cd3 cd28 beads, by Thermo Fisher Scientific (1 mentions)
Texmacs, by Miltenyi Biotec (1 mentions)
Celltrace violet cell proliferation dye, by Thermo Fisher Scientific (1 mentions)
Human serum, by Thermo Fisher Scientific (1 mentions)
X vivo 15 media, by Lonza (1 mentions)
24 well tissue culture plate, by Thermo Fisher Scientific (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Merck Group (1 mentions)
Facsaria 2, by BD (1 mentions)
9 protocols using il 7 and il 15
1
Priming Antigen-Specific CD8+ T Cells
2
Expansion of HBV-specific CD8+ T cells
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PBMC from HLA-A*02+ donors with an acute or resolved HBV infection were isolated via a standard Ficoll gradient. Informed consent in writing was obtained from each patient. T2 cells were pulsed with 1 nM or 1 μM of peptide (C18-27: FLPSDFFPSV, S20-28: FLLTRILTI, S172-180: WLSLLVPFV, JPT Peptide Technologies, Berlin, Germany) for 2 hours at 37°C. Then, they were irradiated with 35 Gy, washed 2 times and adjusted to the desired cell concentration. Complete PBMC or CD8+ T cells, purified with Dynabeads Untouched (Life Technologies, Darmstadt, Germany), were incubated with peptide-loaded T2 cells for 7–14 days and restimulated for another 7 days according to S1 Table . 10 ng/ml IL-7 and IL-15 (Peprotech, Hamburg, Germany) was added on day 0, and 50 U/ml Proleukin (Novartis Pharmaceuticals, West Sussex, UK) were added on days 1, 4, 8 and 11. PBMC were kept in T-cell medium (TCM): RPMI, 10% human serum (own production from male, healthy donors), 1% pen/strep, 1% glutamine, 1% sodium pyruvate, 1% non essential amino acids, 10 mM HEPES, 16,6 μg/ml Gentamycin (all from Life Technologies).
3
Expanded PD-1-deficient T-cell Stimulation
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Control T cells or PD-1 KO T-cells post transfection were cultured in AIM-V medium supplemented with IL-2 (300 U/ml, Peprotech) and half replaced by fresh complete medium containing IL-2 every 2 ~ 3 d until analyzes. For antigen stimulated T cells. Mature DCs were pulsed by peptide (25 μg/mL) for 2 ~ 3 h at 37°C, washed with pre-warmed PBS and then incubated with control T cells or PD-1 KO T-cells at a ratio of 1:10 in complete AIM-V medium supplemented with IL-2 (100 U/ml, Peprotech) in 6-well-plates (5 × 106 cells/well) on day 7 post electroporation. IL-7 and IL-15 (5 ng/mL, Peprotech) were added with fresh medium. For re-stimulation, autologous DCs were pulsed with peptide (25 μg/mL) for 2 h and added to the cultured cells for another 7 d. Fresh complete medium was added containing cytokines every 2 to 3 d until use for experiments.
4
Generating CAR T cells from PBMCs
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Peripheral blood mononuclear cells (PBMCs) were harvested from healthy donors or patients and isolated by density gradient centrifugation. T cells were enriched and activated by anti-CD3/CD28 coated beads (Invitrogen) and cultured in X-VIVO serum-free medium (Lonza, 04-744Q) supplemented with 5% AB serum (Sigma), 10% nonessential amino acids (Corning), 0.01% recombinant human IL-2, and 0.05% IL-7 and IL-15 (PeproTech). Transduction was performed with CAR-encoding lentiviral vector after 24 h of stimulation, and lentiviral transduction efficiency was assessed 7 days after transduction by flow cytometry.
5
In Vitro T-Cell Expansion and Activation
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Label‐free sorted SSClow and SSChigh cells were either expanded alone or reconstituted at a ratio of 1:1 (equal to total lymphocytes within the initial cell sample) for co‐expansion. The starting cell numbers for each condition were 0.06–0.08 million (experiment dependent) per well in 96‐well plates. After 2–4 hours’ incubation at 37 °C, all cell groups were activated using anti‐CD3/CD28 Dynabeads (3:1 bead‐to‐cell ratio) (Gibco) with 5 ng mL−1 IL‐2 (Gibco, PHC0021) supplemented. In some experiments, the expanded T cells were re‐stimulated by anti‐CD3/CD28 microbeads at a 1:1 bead‐to‐cell ratio under 5 ng mL−1 IL‐2, or 5 ng mL−1 each of IL‐7 and IL‐15 (PeproTech). Half of the culture medium was replaced with fresh medium containing 5 ng mL−1 IL‐2 every two days during initial activation/expansion stages (day 3 to day 7) and daily during late expansion phases (day 7 to day 11 or more where indicated). Cells were counted by trypan blue exclusion and split into ≈0.1 million per well. Compositional changes of T‐cell differentiation subsets and functionality as well were determined at indicated time points post‐activation.
6
Functional Evaluation of Engineered CAR-T Cells
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Untouched CD8+ T cells were isolated (Miltenyi Biotec) from PBMCs from HLA-B*35-positive donors and activated with anti-CD3/CD28 beads (Thermo Fisher Scientific) at a ratio of 1:1 in TexMACS (Miltenyi Biotec) with 3% human serum (c.c.pro; CTL medium) supplemented with 12.5 ng/mL IL-7 and IL-15 (PeproTech). After 1 day, T cells were transduced with lentiviral particles by spinoculation using an multiplicity of infection (MOI) of 3 and addition of 5 µg/mL Polybrene Infection/Transfection Reagent (Merck). Untransduced T cells were treated equally and served as controls. The anti-CD3/CD28 beads were removed on the following day. On day 7 or 9, EGFRt+ T cells were enriched as described30 (link) or via fluorescence-activated cell sorting (FACS). After a total expansion time of 9 to 17 days, functionality of the transduced T cells was assessed by co-culturing them with target cells. For this, 5×104 target cells and effector cells according to the specified effector-to-target (E:T) ratios were seeded in 200 µL CTL medium. Since they do not express IL-12, iEGFP-expressing TÜ165 CAR-Ts (TÜ165 EGFP-TRUCKs) were expected to show the same functionality as TÜ165 CAR-Ts and were both used to compare functionality with iIL-12-secreting TÜ165 CAR-Ts (TÜ165 TRUCKs).
7
Anticancer Drug Screening on T Cells
8
Thawing and Culturing PBMCs
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PBMCs were thawed using X-VIVO 15 media (LONZA, Switzerland) supplemented with 5% human serum (Gibco) and 200 U/mL IL-7 and IL-15 (PeproTech, United States) and washed once in media before being cultured. Cells were cultured in 24-well tissue culture plates (NUNC, Denmark) and maintained at concentrations of 1x106 cells/mL.
9
Allogeneic T cell expansion for immunotherapy
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Allogeneic PBLs were co-cultured with the E007 for raising alloreactive TSCM cells. PBLs were stained with 5 μΜ carboxyfluorescein diacetate succinimidyl ester (CFSE, sigma) for 8 min at 37 °C and washed with RPMI1640 supplemented with 10% FBS three times. E007 were inactivated by irradiating (2.0 Gy). PBLs were mixed with E007 on day 0 at a ratio of 5:1 with 5 μM TWS119 in the co-culture for the first week. PBLs cultured alone used as control. TWS119 and medium were replaced on day 4, and cells were counted by Trypan blue dye exclusion. As the allo-specific T cells showed proliferation in the co-culture, CFSEdim cells were sorted by FACS with BD FACS AriaII on day 7. Sorted cells were expanded by cytokines IL-7 and IL-15 (Peprotech, USA) of 25 ng/ml each for a further week.
T cell subsets in the co-culture bulks were identified by their surface markers, CD3+ CD8+ CD45RA+ CD62L+ CCR7+ CD95+ CD28+ for TSCM, CD3+ CD8+ CD45 RA- CD62L+ for TCM, CD3+ CD8+ CD45RA- CD62L- for TEM, and CD3+ CD8+ CD45 RA+ CD62L- for TEF.
T cell subsets in the co-culture bulks were identified by their surface markers, CD3+ CD8+ CD45RA+ CD62L+ CCR7+ CD95+ CD28+ for TSCM, CD3+ CD8+ CD45 RA- CD62L+ for TCM, CD3+ CD8+ CD45RA- CD62L- for TEM, and CD3+ CD8+ CD45 RA+ CD62L- for TEF.
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