Interleukin il 2

Tumor tissue was collected from patients with untreated HNSCC undergoing surgical resection. Tissue was minced and digested in 0.25% trypsin. Half of the tissue was plated onto irradiated T293 feeder cells to establish primary cell lines in keratinocyte media (DMEM/F12, 24.2 µg/mL Adenine Sigma, 1× non-essential amino acids, 100 µg/mL Primocin, 0,4 µg/mL hydrocortisone Sigma, 5% FBS, 1× sodium pyruvate, 8.3 ng/mL high Cholera toxin Sigma, 10 µM rock inhibitor Cayman chemical, 1× ITS Thermo Fisher and 0.05 µg/mL EGF Sigma)) and half was plated into 24-well plates in TIL media (Roswell Park Memorial Institute [RPMI], 1× Pen/Step, 1× sodium pyruvate, 2.5 mL of 7.5% stock solution sodium bicarbonate, 0.00025% 2-mercaptoethanal Fisher, 10% human serum Sigma, 6000 U/mL interleukin (IL)-2 (PeproTech), and 100 µg/mL Primocin) Invivogen. Cells were continuously cultured and split regularly to maintain growth.

Skin T cells from patients with L-CTCL and healthy donors were isolated from skin biopsies by short-term explant technique. Briefly, skin biopsies cut into small pieces were cultured on collagen-coated Cellfoam matrices in Iscove’s modification of Dulbecco’s media supplemented with 20% heat-inactivated FBS, 1% antibiotic-antimycotic (Gibco™), 2 mM L-glutamine, 100 IU/mL interleukin IL-2 and 10 ng/mL IL-15 (Peprotech) as previously reported.⁴⁵

PBMCs were isolated from fresh heparinized blood using Ficoll-Hypaque density gradient centrifugation at 800 × g for 20 min (HaoYang Biological Manufacture Co., Ltd., Tianjin, China) and were resuspended in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc.).
To assess the effects of blocking the PD-1:PD-L1 signaling pathway, PBMCs from HLA-A2⁺ individuals were cultured in a flat-bottomed 96-well plate (2–10×10⁵ cells/well) in the presence of 5 µg/ml HBcAg18-27 peptide, 5 µg/ml anti-PD-L1 or control IgG for PD-1:PD-L1 for 10 days, supplemented with 25 IU/ml interleukin (IL)-2 (PeproTech, Inc., Rocky Hill, NJ, USA) and 0.5 µg/ml anti-CD28 at 0 and 5 days.

Human peripheral blood mononuclear cells (PBMCs) were purified from buffy coats of three different donors, obtained from the Red Cross blood transfusion center, using density-gradient centrifugation (Lymphoprep; Axis-Shield). Primary CD4⁺ T cells were selectively enriched using bi-specific monoclonal antibody (mAb) CD3.8 (0.5 μg/mL, NIH AIDS Reagents Program; https://www.aidsreagent.org) for 5 days. CD4⁺ T cells were cultured in RPMI medium supplemented with 15% FBS, gentamycin, interleukin (IL)-2 (100 U/mL; Peprotech), and MEM Non-Essential Amino Acids Solution (MEM NEAA) (50 μg/mL; Gibco-BRL), referred to as T-cell medium (TCM). CD34⁺ HSCs were positively selected with anti-CD34-conjugated microbeads according to the manufacturer’s instructions (MACS; Miltenyi Biotec) and stimulated for 48 hr in StemSpan SFEMII medium containing CC100 Cytokine Cocktail (STEMCELL Technologies).

Bone marrow mononuclear cells (BMMC) and peripheral blood mononuclear cells (PBMC) were enriched by density-gradient centrifugation using Ficoll-Hypaque (Supplier, Country). Cells were then labeled with CD3 microbeads (Miltenyi Biotech, Germany) for 15 mins at 4°C–6°C. Immunomagnetic device VarioMACS (Miltenyi Biotech, Germany) was used for cell selection according to the manufacturer’s protocol. CD3+ cells were considered AML TIL, whereas CD3-cells were considered autologous AML cells.
BMMC and PBMC were seeded at 1×10⁶ cells/mL in X-VIVO 15 medium (Lonza, Switzerland) supplemented with 5% heat-inactivated human serum, 100 ng/mL anti-CD3, 1000 IU/mL of interleukin (IL)-2, 10 ng/mL of IL-7 and 10 ng/mL of IL-15 (PeproTech, USA) and co-cultured with 50 Gy-irradiated PBMC derived from unrelated donors as feeder cells at the ratio (10:1) of feeder cells to BMMC or PBMC for 14 days at 37°C. Fresh X-VIVO 15 medium with cytokines was added whenever the medium turned yellow.
AML TIL were cultured with autologous AML cells at the ratio of 1:1 in X-VIVO 15 medium with 5% heat-inactivated human serum, 10 ng/mL of IL-7 and 10 ng/mL of IL-15 overnight at 37°C. Cultured cells were washed with phosphate-buffered saline (PBS) and labeled with CD137 microbeads. Immunomagnetic sorting was conducted using the selection kit according to the manufacturer’s instructions (Miltenyi Biotech, Germany).

PBMCs from human leukocyte antigen A (HLA‐A)*02‐positive (+) donors with chronic HBV infection were isolated through a standard Ficoll gradient. Informed consent in writing was obtained from each patient. PBMCs were stimulated by 10 μM of HBV core_18‐27I peptide (FLPSDFFPSI; Sangon Biotech, Shanghai, China) for 10 days in X‐VIVO 15 (#04‐418QCN; Lonza) plus 10% human AB serum, 1% GlutMAX supplement (#35050061; Gibco), and penicillin‐streptomycin (#15140122; Gibco). We added 50 IU/mL interleukin (IL)‐2 (#200‐02; PEPROTECH), 10 ng/mL IL‐7 (#200‐07, PEPROTECH), and IL‐15 (#C016; Novoprotein) on day 1 and changed this solution every 2 days. T cells stained with R‐phycoerythrin (R‐PE)‐labeled Pro5 HLA‐A*02:01/FLPSDFFPSI pentamer (#F283‐2A‐G; Proimmune) were isolated by anti‐PE MicroBeads (#130‐105‐639; Miltenyi Biotec) according to the manufacturer’s protocol, followed by a rapid expansion protocol.