CRTAM expression on CD8⁺ T cells is tightly associated with TCR triggering. (A) A T-cell bulk culture was stimulated for 4 hours with PMA/ionomycin, and CRTAM expression was determined on unstimulated (unst, open histograms) and stimulated cells (shaded histograms) with Cr24.1. CD8 mAb was used as a counterstain to discriminate between CD4⁺ and CD8⁺ T cells. (B) CD8⁺ and CD4⁺ T cells were purified from PBMCs by positive selection (> 97% purity) and stimulated with plastic-bound CD3 mAb for the indicated time periods. CRTAM expression was detected as in Figure 2B. CRTAM was strongly up-regulated on CD8⁺ T cells 24 hours after stimulation and was expressed at high levels up to 72 hours after stimulation. At 96 hours, expression gradually decreased and completely disappeared 180 hours after stimulation (data not shown). CRTAM expression was detected only on a small percentage of CD4⁺ T cells 48 to 72 hours after stimulation. One of 2 similar experiments is shown. (C) Antigen recognition induces CRTAM expression in CD8⁺ T cells. CRTAM expression was determined over time on an alloreactive CD8⁺ T-cell clone, MLR-TF74, which recognizes HLA-B18 expressed by BM16, but not HLA-B7 expressed on BM14. The bar graph indicates proliferation of the T-cell clone stimulated with BM16 and BM14 as detected by standard ³H-Thymidine incorporation (cpm indicates counts per minute). CRTAM expression started to be detected as early as 5 hours after stimulation and peaked at 20 to 24 hours. Lack of expression of CRTAM in T cells incubated with the BM14 cell line, which does not induce MLR-TF74 activation, is shown as a control. CD8 was used as marker to distinguish between the responder T cells and the stimulator B cells.