PD-L2 on AAM is required and sufficient to mediate inhibition. (A) Untreated (black line) or anti-TCR/anti-CD28 stimulated (filled) CFSE-labeled wild-type splenocytes were cultured with IL-4–treated (10 ng/mL) BMDM from wild-type mice in the absence (control) or presence of anti-PD-L2 (5 μg/mL) or isotype control (5 μg/mL) and analyzed after 4 days. Histograms are gated on CD4⁺ T cells. The experiment has been repeated with similar results. (B) BMDM from Stat6^−/− mice were transduced with a bicistronic retroviral PD-L2/GFP expression vector (PD-L2 Mϕ) or empty GFP control vector (control Mϕ). Cells were stained for F4/80 and PD-L2. Dot plots are gated on F4/80⁺ cells. F4/80⁺GFP⁺ macrophages were sorted with > 94% purity. (C) CFSE-labeled wild-type splenocytes were left untreated (black line) or were stimulated with plate-bound anti-TCR/anti-CD28 (filled), cultured in the presence of GFP⁺-sorted macrophages and analyzed after 4 days. Histograms are gated on CD4⁺ or CD8⁺ cells as indicated. The data are representative of 3 independent experiments.