Functional characterization of naturally occurring CD49b⁺CD200R3⁺ DC_regs. (A) The transcriptional expression of the selected genes in CD11c⁺ DCs, CD49b⁺CD200R3⁻ cells, CD49b⁺CD200R3⁺ cells, CD49b⁺FcϵRIα⁻ cells, and CD49b⁺FcϵRIα⁺ cells was measured by RT-PCR. The results of RT-PCR for Actb demonstrate the loading of equal amounts of DNA on the gel. (B) Morphology of CD11c⁺ DCs, CD49b⁺CD200R3⁻ cells, and CD49b⁺CD200R3⁺ cells was examined by phase-contrast microscopy (400×) after stimulation with or without CpG ODN (0.1 μM) for 24 hours. Images of micrographs were acquired using a 10 × 20 eyepiece (40 × 0.55 NA objective lenses with phosphate-buffered saline) on an Olympus model CKX41 microscope (Olympus, Yokohama, Japan). An Olympus model DP12-2 camera was used along with JPEG Preview 3.0.9 software (Apple, Cupertino, CA) and Adobe Photoshop Elements 2.0 (Adobe Systems, San Jose, CA) to capture and process the still images. Data are representative of 4 replicate experiments (A,B). (C) CD11c⁺ DCs, CD49b⁺CD200R3⁻ cells, or CD49b⁺CD200R3⁺ cells (5 × 10⁵) were stimulated or not stimulated with LPS (1 μg/mL) or CpG ODN (0.1 μM) for 24 hours, and the culture supernatants were analyzed for cytokine production. *P < .01 compared with CD11c⁺ DCs. (D) The cytotoxicity of CD11c⁺ DCs, CD49b⁺CD200R3⁻ cells, or CD49b⁺CD200R3⁺ cells (6.25 × 10³-5 × 10⁴) against YAC-1 cells (5 × 10³) was analyzed by a 4-hour ⁵¹Cr release assay. *P < .01 compared with CD11c⁺ DCs. (E) Rag2^−/−KJ1-26⁺ T cells (5 × 10⁴) were cultured with OVAp/CD11c⁺ DCs, OVAp/CD49b⁺CD200R3⁻ cells, or OVAp/CD49b⁺CD200R3⁺ cells (6.25 × 10²-5 × 10³) for 3 days, and the proliferative response was measured. *P < .01 compared with OVAp/CD11c⁺ DCs. Data are mean ± SEM, and the results are combined from 4 replicate experiments (C-E).