(A) C57BL/6 WT mice (n = 3) were immunized s.c. with CFA/CFA or were left untreated (without CFA) and challenged with LPS/IFN-γ, M. tuberculosis/IFN-γ, or vehicle (0h). Spleens were harvested after 6 hours, 24 hours, and 48 hours after the mice received the challenge injections, and the absolute cDC, pDC, and CD4⁺ and CD8⁺ T cell numbers were calculated from the frequencies of the indicated surface marker stainings among the whole spleen cell counts. (B) C57BL/6 WT (n = 3) and Nos2^–/– mice (n = 3) were used for immunization as above but only challenged with LPS/IFN-γ. The individual cell populations were stained using the indicated markers and analyzed by flow cytometry after 6 hours and 24 hours for the frequency of apoptotic cells using annexin V or (C) their live-cell frequencies were calculated. (D) DC in vitro killing assay. In vitro–generated BM-DCs were matured overnight with LPS and then cocultured at 1:1 ratio with M. tuberculosis–activated BM-MDSCs that were generated from WT or Nos2^–/– mice and sorted into CD11b⁺Ly6C^hiLy 6G^– monocytic or CD11b⁺Ly6C^loLy6G⁺ granulocytic cell subsets (n = 4 independent experiments). Apoptosis of DCs was determined by annexin V FACS staining and normalized to DC cultures without BM-MDSCs. (E) Cryosections of spleens from CFA/CFA plus LPS/IFN-γ–immunized mice were analyzed by confocal microscopy to assess apoptosis of CD11c⁺ DCs via TUNEL or FVF staining. B cell areas were identified by B220 staining. Scale bars: 60 μm. (F) Quantification of E at 24 hours. CD11c⁺TUNEL⁺ cells within the T cell zone of WT (n = 16) and Nos2^–/– (n = 20) spleen cryosections were analyzed with StrataQuest software. (G) Cytospin preparations of LPS-matured BM-DC and M. tuberculosis–activated BM-MDSC cocultures to demonstrate CD11c⁺TUNEL⁺ DC killing in vitro. Statistics shown in black were performed using an unpaired 2-tailed t test. *P < 0.05, **P < 0.01, ***P < 0.001. Mean and SD are shown. Statistical P values shown in red were performed using 2-way ANOVA testing for changes over time, while no differences were found between the differentially treated groups. Statistical P values shown in blue were performed using 2-way ANOVA testing for changes over time and between the WT and Nos2^–/– mice. ANOVA analyses were performed without the group that received only LPS/IFN-γ (violet bars).