Krn7000

The synthetic α-GalCer analog KRN7000 was purchased from Funakoshi (Tokyo, Japan). KRN was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 1 mg/ml at 80°C. It was then aliquotted and stored at −20°C until use. Prior to injection, the dissolved KRN was diluted in PBS to a final concentration of 25 µg/ml. Neonatal mice were randomized to receive KRN (0.2 µg/g BW) or vehicle (2.5% DMSO in PBS) in the same volume within 1 h after or 30 h prior to CS injection. Lyophilized carrier-free recombinant mouse TGF-β1 was purchased from R&D Systems (Minneapolis, MN, USA). TGF-β1 was reconstituted in 4 mM HCl at a concentration of 50 µg/ml. It was then aliquotted and stored at −20°C until use. Prior to injection the reconstituted TGF-β1 was diluted in PBS to a final concentration of 10 µg/ml. Neonatal mice were randomized to receive TGF-β1 (0.05 µg/g BW) or vehicle (PBS containing 0.8 mM HCl) in the same volume 1 h prior to CS injection.

All procedures were performed in a GMP-like cleaning room. iNKT cells were cultured in vitro following the method described in our previous work (38 (link)). Briefly, PBMCs (1 × 10⁷) were seeded into 6-well plates with 100 ng/mL KRN7000 (Funakoshi) in the X-VIVO (Lonza) culture medium and then cultured at 37°C in a humidified atmosphere containing 5% CO₂ for 1 week. We separately added recombinant human IL7 (20 ng/mL; R &D, GMP grade, 207-GMP-01M) and IL2 (100 IU/mL; R&D, GMP grade, 202-GMP-01M) to the culture medium on the sixth day. Autologous DCs loaded with KRN7000 were added to the culture medium every week. Recombinant human IL15 (20 ng/mL, R&D, GMP grade, 247-GMP-01M) was added 1 week before harvest, and IL12 (20 ng/mL, R&D, GMP grade, 219-GMP-01M) was added to the culture medium one day before harvest. All the cells were harvested, washed four times, resuspended in 250 mL of normal saline solution, and ready for infusion.