5 op ru

For therapeutic vaccination, infected mice were anesthetized with isoflurane, suspended vertically, and their tongues were pulled aside to expose the pharynx^{43 (link)}, and then 1 μM 5-OP-RU or Ac6-FP (Cayman Chemical) diluted in 30 μl of PBS was administrated iph into the airway once in a week for 3 weeks. Alternatively, mice were treated with combination of 200 μg each of neutralizing antibody for cytokine blockade. In prophylactic vaccination experiments, iph inoculation with 5-OP-RU or CpG (10 μg of ODN1826, InvivoGen) alone or both in 30 μl of PBS was performed on naive mice. In some experiments, mice were inoculated iph with ~150 CFU of Mtb in combination with 20 μg of isotype, anti-MR1 (26.5, BioLegend), or anti-TGF-β antibody (1D11.16.8, Bio X cell) in 30 μl of PBS on day 0, and 100 μg each of antibody was given via intraperitoneally on day 5 p.i.

For infection experiments, mice were i.n. challenged with 5 × 10⁴ CFUs of K. pneumoniae in 30 μl of sterile PBS. For in vivo antibody-mediated blocking experiments, mice were intraperitoneally (i.p.) injected with the following antibodies 24–48 h before infection: αMR1 (150 μg/mouse; clone 26.5; Biolegend, Mouse IgG2a), αIFNAR1 (200 μg/mouse; clone MAR1-5A3; BioXCell, Mouse IgG1), αSiglecH (150 μg/mouse; clone 440c; BioXCell, Rat IgG2b), or their respective isotype controls: Mouse IgG2a (150 μg/mouse; clone MOPC-173; Biolegend), Mouse IgG1 (200 μg/mouse; clone MOPC-21; BioXCell), and Rat IgG2b (150 μg/mouse; clone LTF-2; BioXCell). When indicated, MAIT cell expansion in vivo was performed through a repeated i.n. inoculation (3×) of 5-OP-RU (100 μM) and LPS (17.4 μg/mouse; Invivogen).
Bacterial loads were determined by counting CFUs after plating 100-fold dilution series of tissue homogenates obtained from bacteria-infected mice. Colonies were counted at 24 h.