iNKT cells produce GM-CSF during Mtb infection in a CD1d-dependent manner and it is critical for controlling Mtb growth.

<p>(A, B) IFNγ^−/− iNKT cells added to uninfected or H37Rv-infected WT (A, B) and CD1d^−/− mϕ (B). Murine GM-CSF measured in supernatant harvested after 24 hours. (C) % CFU reduction calculated from CFU assays for H37Rv-infected WT mϕ with IFNγ^−/− iNKT cells added on d1 in the presence of anti-GM-CSF blocking antibody (10–50 µg/ml) or isotype control. (D) J3N.5 human iNKT cell clone added to uninfected or H37Rv-infected U937 cells in the presence of anti-human-CD1d blocking antibody or isotype control. Human GM-CSF measured in supernatant harvested after 24 hours. (E, F) Lung mononuclear cells from WT Mtb-infected mice were incubated with brefeldin A for four hours at 37°C and then stained. iNKT cells were identified as TCRβ⁺ and CD1d-tetramer⁺. Percentage (E) and number (F) of iNKT cells producing GM-CSF and IFNγ. (G) % GM-CSF⁺, % IFNγ⁺, and CD69 MFI for iNKT cells transferred iv into WT or CD1d^−/− Mtb-infected hosts and iNKT cells cultured for 20 hours in basic media. Lung mononuclear cells were treated and stained as in (E, F). Transferred iNKT cells were distinguished from host cells by eFluor 450 staining. Error bars indicate mean ± SEM. +P<.05, ++P<.01, +++P<.001 (unpaired Student's t-test, GM-CSF⁺ versus IFNγ⁺ for respective time points (E, F), WT versus CD1d^−/− hosts (B, G)). **P<0.01, ***p<0.001 (One-way ANOVA with Tukey post-test (C), and Dunnet's post-test (A, D), compared to uninfected mϕ). Data are representative of or compiled from four (A, C), two (B), and three (D, G) independent experiments with three or more replicates each or two independent experiments with 5 mice each (E, F). , MOI titration approximately 0.5∶1–3∶1 (A, B) and 2∶1–10∶1 (D).