Necl-2–CRTAM interaction leads to strong IFN-γ secretion by CD8⁺ T cells. (A) BM16 cells were sorted into the BM16.1 and BM16.2 cell lines expressing high levels and very low levels of CRTAM ligand, respectively (open histograms are control; shaded histograms are CRTAM-hFc labeling). Necl-2 expression in BM16.1 was confirmed by immunoblot with a rabbit anti–Necl-2 antiserum (data not shown). (B-D) BM16.1 and BM16.2 were separately used to stimulate the MLR-TF74 alloreactive T-cell clone (B-C), and Mart1-specific HLA-A0201–restricted CD8⁺ T cells (D). INF-γ secretion was measured after 48 hours (B) or 20 hours (D) of stimulation in cell culture supernatants. Proliferation (C) was measured by a standard ³H-thymidine incorporation assay after 72 hours of stimulation. Graded numbers of BM16 were used (B-C) to generate a dose-response curve of antigen (APC, antigen-presenting cell). Serial dilutions of the MART-1 26-35 peptide were used (D) to pulse BM16 cells. Experiments were performed in the presence of CRTAM-hFc to specifically block CRTAM–Necl-2 interaction or hIgG1 as control. One of 3 representative experiments is shown.