Okt 3

Tumor infiltrating lymphocytes (TILs) were isolated from breast cancer biopsy specimens by mincing the tissue into small pieces and then digesting them with collagenase type IV (0.1 mg/mL) (Sigma-Aldrich, Castle Hill, NSW, Australia) for 2 h, followed by culture in X-VIVO-15 medium (Lonza, Basel, Switzerland) containing 5% human AB serum and recombinant human IL-2 (150 IU/mL) in 24-well plates, followed by an expansion using a rapid expansion protocol (REP) [27 (link),28 (link)]. Once a sufficient number of T cells (>1 × 10⁷) was generated, they were cryopreserved for later expansion. A REP for “young TILs” that was previously used in melanoma patients was followed. Cryopreserved, pre-REP TILs from patients were thawed and then further expanded to treatment levels using an anti-CD3 antibody (OKT-3, 30 ng/mL, R&D Systems, Minneapolis, MN, USA), rhIL-2 (BD PharMingen, San Jose, CA, USA), and irradiated feeder cells, as previously described [28 (link)]. The expanded TILs were fixed and then labelled with human CD4 and CD8 antibody conjugates, followed by labelling human PD-1 antibody conjugate and being subjected to FACS analysis.

Tumor-associated MDSCs were generated from CD33⁺ cells isolated by anti-CD33 beads (Miltenyi Biotec Company) from peripheral blood mononuclear cells (PBMCs) of healthy donors in a co-culture Transwell system (0.4-μm pore, Corning) with the NPC cell lines TW03, CNE2, TW03-LMP1 or CNE2-LMP1 as previously described. Several parallel wells were then treated with siGLUT1, siNLRP3, 2-DG, the caspase-1 inhibitor VX-765 (Selleck, 50 μM) or metformin (Sigma-Aldrich, 40 mM). HLA-DR^-CD11b⁺CD33⁺ cells were defined as MDSCs and measured using a fluorescence-activated cell sorting (FACS) analysis. The suppression of MDSC on T cells was analyzed by co-culturing cells with carboxyfluorescein diacetate succinimidyl ester (CFSE, 10 μM, Molecular Probes)-labeled T cells obtained from healthy donors in 96-well plates that had been pre-coated with OKT-3 (1 μg/mL, R&D Systems) at a ratio of 1:1. T cells were harvested after 72 h and stained for the cell surface markers CD3, CD4, and CD8. Data were obtained and analyzed using FACS.

As an ex vivo model for RA, human PBMCs and SFMCs were cultured in RPMI supplemented with 10%FCS, 1% HEPES, 1% GlutaMAX, 1% penicillin/streptavidin 15 µg/ml, and gentamycin at a concentration of 1 × 10⁶ cells/ml, for 48 h, as previously described (n = 10) [29 (link)]. For the stimulation experiments, recombinant human LAG-3 (0.5 μg/ml, R&D Systems, Cat.2319-L3-050), neutralizing anti-LAG-3 antibody (10 μg/ml, 17B4, BPS Bioscience, Cat.71219), and/or Galectin-3 inhibitor (10 μM, GB0149, Galecto) were used. Pre-stimulation with plate-bound CD3 (0.5 μg/ml, OKT3, R&D Systems) and soluble CD28 (0.5 μg/ml, BD Bioscience) was conducted when applicable. Cells were cultured for 48 h at 37 °C without change of medium, and non-treated cultures or matching isotypes were used as controls. Supernatants were collected and kept at − 80 °C until cytokine measurements.