Expression of PD-1 on activated spleen DC. (A) Rag-1KO and Rag-1 PD-1 DKO mice were infected with 10⁵ LM by intravenous injection. Spleens were harvested 16 hours after infection. CD11c⁺ cells were purified using CD11c MACS beads and staining with MHC II (I-A^b), NK (DX5), PD-1, and DC (CD11c) markers. A total of 14.8% of CD11c⁺I-A^b+DX5⁻ DCs from Rag-1KO mice were PD-1⁺, whereas NK cells (CD11b⁺I-A^b− DX5⁺), neutrophils (I-A^b− Gr-1⁺ CD11b⁺), and peritoneal macrophages (I-A^b+CD11b⁺) from Rag-1KO mice stained negative for PD-1. (B) Splenic CD11c⁺ cells were purified using CD11c MACS beads from Rag-1KO and DKO mice, incubated in vitro for 48 hours with 5 × 10⁴ LM/mL, and stained for PD-1 expression. (C) Same procedure as in panel B. Conventional sDCs were further gated as CD11c^highI-A^{b high} population and stained for PD-1 and various other surface markers as indicated. (D) Semiquantitative RT-PCR analysis of PD-1 transcripts was performed with mRNA extracted from naive and anti-CD3–activated T cells from B6 WT and PD-1KO mice, followed by native and LM-activated splenic CD11c⁺ cells from B6 Rag-1KO and B6 DKO mice. RT-PCR specific for mouse β-actin was used as the loading control. (E) Splenic CD11c⁺ cells were purified using CD11c MACS beads from Rag-1KO and DKO mice, incubated in vitro for 48 hours with different TLR ligands and cytokines. Expression of PD-1 was measured as mean fluorescence intensity (MFI).