Il 15rα

Recombinant mouse IL-15 was purchased from BioLegend (catalog no. 566302). Mouse IL-15Rα subunit Fc chimera (IL-15 Rα) was purchased from R&D Systems (catalog no. 551-MR-100; Minneapolis, MN). Recent work has shown that the effects of IL-15 can be markedly enhanced by combining IL-15 with its IL-15Rα subunit (38 (link), 39 (link)). The IL-15/IL-15Rα complex was prepared as previously described (34 (link)). In brief, 20 μg IL-15 and 200 μg IL-15Rα-Fc were incubated in 430 μl of PBS at 37°C with 5% CO₂ for 20 min to IL-15/IL-15Rα complex. Samples were diluted 10-fold in PBS to a total volume of 4.3 ml, then aliquoted and frozen. For induction of CD8⁺ Tregs, aged mice were treated s.c. with 300 μl of IL-15/IL-15Rα complex solution (1 μg IL-15 and 7.0 μg IL-15Rα-Fc) at 6, 24, and 48 h after 1 mg/kg LPS administration. To obtain samples, we anesthetized and euthanized animals at the indicated times. The time of LPS administration was defined as 0 h in subsequent experiments. Survival rates and body weight were recorded for 7 consecutive days and analyzed.

For the allergic CHS model, mice were sensitized via topical application of 0.5% (v/v) 1-Fluoro-2,4-dinitrobenzene (DNFB) (Sigma-Aldrich) in 4:1 acetone/olive oil on their shaved back (50 µL) and were challenged 4 or 5 days later with 0.2% DNFB or vehicle control (5 µL on the dorsal and 5 µL on the ventral side of the ear). Ear thickness was measured using an engineer’s micrometer (Mitutoyo, Kawasaki, Kanagawa, Japan). The mice received neutralizing IL-27 antibody (nIL-27p28AB) (R&D Systems, Minneapolis, MN), IL-15 complex (cpx), or their respective IgG control (Ctrl). To make approximately 1 µg of the IL-15 cpx, 1 µg of IL-15 (PeproTech, Rocky Hill, NJ) and 4.5 µg of IL-15 Rα (R&D Systems) were incubated for 30 minutes at 37°C (34 (link)). Each mouse received 1.2 µg of IL-15 of the cpx.

PBMCs were cultured in RPMI1640 medium containing 8% human serum, 1 mM Sodium Pyruvate, 1 mM non-essential amino acids, 2 mM L-Glutamine, 50 μM β-mercaptoethanol and 1 μM of the respective peptide. After 3 days, fresh peptide and 20 ng/mL IL-15 and 500 ng/mL soluble, Fc-fused IL15-Rα (R&D Systems) was added. Cells were restimulated every 2 weeks with autologous, irradiated (40Gy), peptide-pulsed PBMCs. Cells were expanded every 4–5 days with fresh medium supplemented with IL-15 and receptor, as indicated above. Peptide specific expansion of T-cells was monitored by flow cytometric analysis using MHC-peptide pentamers on a regular basis. Cultures used for functional assays were >70% CD8+, >60% peptide-specific and 10–14 days after last restimulation.