PD-1 negatively regulates DC IL-12 and TNF-α production. (A) Total splenocytes from Rag-1KO or DKO mice were incubated with PBS control or 10⁶ CFU/mL HKLM for 4 hours in the presence of Golgi blocker brefeldin A. Intracellular staining of TNF-α and IL-12p70 revealed that CD11b⁺ myeloid cells were the main producer of both cytokines on LM stimulation. Cytokine-producing cells were further gated and stained with MHC II (I-A^b) and CD11c markers. (B) Rag-1KO or DKO mice were intravenously infected with 10⁵ CFU LM. At 24 hours later, splenocytes were purified and restimulated with PBS or HKLM in the presence of brefeldin A for 4 hours. DCs were purified using CD11c MACS beads. I-Ab⁺CD11c⁺ gated DC population was stained for intracellular IL-12p70 and TNF-α. (C) Same condition as in panel B: anti-B7-H1 mAb 10B5 was added during in vitro restimulation. (D) Same condition as in panel B: CD11c⁺ gated Rag-1KO sDCs population was costained for cell-surface PD-1 and intracellular IL-12p70 and TNF-α. Percentage of quadrants was marked on the plots.