Proleukin

The tumor samples from metastatic lesions isolated during palliative surgery at MD Anderson Cancer Center were obtained using an Institutional Review Board (IRB) approved laboratory protocol (LAB06-0755). The tumor samples were cut into 3–5 mm² fragments and placed in TIL culture media (TIL-CM) and 6,000 IU/ml IL-2 (Proleukin™) in 24-well plates for a period of 4–5 weeks (1 fragment per well). The TIL-CM contained RPMI 1640 with Glutamax (Gibco/Invitrogen; Carlsbad, CA), 1 mM pyruvate (Gibco/Invitrogen; Carlsbad, CA), 20 µg/ml Gentamicin (Gibco/Invitrogen; Carlsbad, CA), 50 µM 2-mercaptoethanol (Gibco/Invitrogen; Carlsbad, CA), 10% human AB serum (Sigma-Aldrich, St. Louis, MO) and 1X Pen-Strep (Gibco/Invitrogen; Carlsbad, CA). The TIL-CM was used for the rest of our experiments. The TIL were split 1∶1 in new TIL-CM and IL-2 across the plate from well-to-well after reaching confluence. After 4–5 weeks, the cells were harvested and designated as “pre-rapid expansion protocol TIL” (pre-REP TIL). Pre-REP TIL that were not expanded immediately further expanded using the REP were cryopreserved in 10% DMSO, 90% human AB serum and stored in liquid nitrogen.

PBMCs were isolated from healthy donors by leukapheresis. CD3⁺ T cells were depleted by VarioMACS (Miltenyi Biotec, Germany). T cell-depleted PBMCs were expanded at a seeding concentration of 2×10⁵ cells/mL in CellGro SCGM serum-free medium (CellGenix, Germany) with 1% auto-plasma, 1×10⁶ cells/mL irradiated (2,000 rad) autologous PBMCs, 10 ng/mL anti-CD3 monoclonal antibody OKT3 (Orthoclon, Switzerland) and 500 IU/mL IL-2 (Proleukin, Switzerland) in a A-350N culture bag (NIPRO, Japan). OKT3 was supplemented just once at the beginning of the expansion to stimulate T cell population in the irradiated feeder cells. NK cells were fed fresh medium with 500 IU/mL IL-2 every two days without removal of preexisting culture medium to maintain cellular concentration at 1∼2×10⁶ cells/mL for 14 days. The viability of expanded NK cells was evaluated by staining of propidium iodide. The NK cell expansion was performed under the conditions of GMP at Green Cross LabCell Corp. (Korea).

TCR gene transfer was carried out as described before (17 (link), 23 (link)). In brief, for retrovirus generation, 293GP-GLV cells were transfected with MP71 vector carrying the respective TCR cassettes using Lipofectamine 3000 (ThermoFisher Scientific). On the same day, PBMCs from healthy donors were seeded on plates coated with 5 μg/ml anti-CD3 (OKT3, Invitrogen) and 1 μg/ml anti-CD28 antibodies (CD28.2, Invitrogen) in TCM supplemented with 100 IU/ml IL-2 (Peprotech). 48 hours later, the virus supernatant was harvested, filtered and supplemented with 8 μg/ml protamine sulfate (Sigma-Aldrich) and 100 IU/ml IL-2, before spinoculation with the activated T cells at 800g for 90 minutes at 32°C was performed. The next day, a second supernatant was harvested from the same 293GP-GLV cells, transferred to a RetroNectin (Takara Bio) coated plate and centrifuged at 3200g for 90 minutes at 4°C. The PBMCs were harvested, supplemented with 100 IU/ml IL-2 and 8 μg/ml protamine sulfate and spinoculated with the virus-containing plates at 800g for 30 minutes at 32°C. After the second transduction, T cells were expanded for 10 days, before being transferred to low IL-2 (10 IU/ml). After 48 hours, transduced T cells were harvested, analyzed for TCR expression by flow cytometry and frozen for future experiments. To detect the transduction rate of the TCRs transduced into primary T cells, the following antibodies were used in a 1:100 dilution at 4°C for 30 min: anti-human CD3-PerCP (UCHT1, Biolegend), anti-human CD8-APC (HIT8a, Biolegend) and anti-mouse TCR β chain-PE (H57-597, Biolegend).
For mouse transductions, Plat-E cells were transfected with MP71 vector carrying the respective TCR cassettes using Lipofectamine 3000 (ThermoFisher Scientific). On the following day, spleen cells were isolated from HHD mice (24 (link)) and erythrocytes were lysed by ammonium chloride treatment. 2x10⁶/ml cells were incubated in mTCM supplemented with 1 μg/ml anti-mouse CD3, 0.1 μg/ml antimouse CD28 antibodies (BD Biosciences (BD), Franklin Lakes, NJ, USA) and 10 IU/ml human IL-2 (Proleukin S, Novartis, Basel, Switzerland). On the next day, 1x10⁶ cells were transduced by spinoculation in 24-well non-tissue culture-treated plates pre-coated with 12.5 μg/ml RetroNectin (TaKaRa, Otsu, Japan) and virus particles (3200 x g, 90 min, 4°C) in 1ml mTCM supplemented with 10 IU/ml IL-2 and 4x10⁵ mouse T-Activator beads (Life Technologies). A second transduction was performed on the following day by spinoculation with 1 ml virus supernatant (+ 10 IU/ml IL-2). T cells were expanded in mTCM (+ 50 ng/ml IL-15 (Miltenyi Biotec) for 10 days. TCR transduction rate was measured by flow cytometry using the following antibodies in a 1:100 dilution at 4°C for 30 min: anti-mouse CD3-BV421 (UCHT1, Biolegend), anti-mouse CD8-APC (HIT8a, Biolegend) and FITC-labeled anti-human TCR Vβ22 (IMMU 546, Beckman Coulter), Vβ9 (MKB1, Biolegend) and Vβ1 (BL37.2, Beckman Coulter) for TCRs 22894, 5934 and 14/35, respectively. These antibody combinations were also used to stain blood from adoptively transferred HHDxRag^-/- mice [ (21 (link)); see below].

PBMC from patients and haploidentical donors were isolated by Lympholyte-H (Cedarlane Laboratories) density-gradient centrifugation. To obtain PHA-blasts, PBMC were stimulated with the mitogen phytohemagglutinin-M (PHA-M, 1μg/mL; Sigma-Aldrich) and cultured in RPMI 1640 medium (Lonza) supplemented with 10% FBS (Euroclone), 2 mM L-glutamine (Lonza), 100 U/mL penicillin-streptomycin (Lonza) and Interleukin (IL)-2 (300IU/ml) (Proleukin, Clinigen Healthcare Ltd.).