α galcer

Mice were i.v. or i.p. injected with vehicle or 2 μg α-GalCer (Funakoshi, CA, USA) in 200 μl. Con A (Sigma-Aldrich, MO, USA) was dissolved in PBS and intravenously (i.v.) injected into mice at a sub-lethal concentration (15 mg/kg). For survival studies, a 30 mg/kg lethal dose was applied. For depletion of CD8⁺ T cells, mice were intraperitoneally injected with anti-CD8 mAb (clone: YTS 169.4) 24 h before Con A injection. For blocking of BTLA, mice were intravenously injected with anti-BTLA (clone: pj196, BioXcell, NH, USA) 2 h before α-GalCer (2 μg) injection. For neutralizing of CD1d, mice were intraperitoneally administrated 200 μg of anti-CD1d antibodies (clone: 20H2, BioXcell) on day 1 before α-GalCer (2 μg) injection.

Female BALB/c mice, 6–8 weeks old, were obtained from the National Laboratory Animal Center and housed at the in-house animal care facility of the Animal Center of the College of Medicine, National Taiwan University under a 12 hour day-night-cycle and standardized environment. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of National Taiwan University, College of Medicine and College of Public Health. Mice were intranasally administered with 2μg α-GalCer (0.2 mg/ml in 0.5% polysorbate 20 in PBS)(Funakoshi, Tokyo, Japan) once a week for 6 weeks. A vehicle control solution was prepared from a solution of 0.5% polysorbate 20 in PBS. Two weeks after the last α-GalCer administration, mice were sacrificed by pentobarbitol (50mg/kg) administration and then cervical dislocation and examined for pathological changes. For IL-4 neutralization, 150 μg of anti-IL-4 antibodies (clone 11B11, BioXcell, Lebanon, NH, USA) were intraperitoneally injected at 1 hour prior to every α-GalCer administration. BALB/c mice, but not C57BL/6 mice, were used in this study because the features of acute and chronic airway inflammation by α-GalCer administration were much higher in BALB/c mice than in C57BL/6 mice.