Engagement of CD3 and CD28 induces CD83L expression on the surface of T lymphocytes. Peripheral T cells were stimulated and tested for CD83L expression by staining with either dodCD83 (open curve) or control dodecameric protein (filled curve). (A) Purified CD4⁺ and CD8⁺ T cells demonstrated CD83L expression when stimulated by anti-CD3 and anti-CD28, but not when stimulated with anti-CD3 alone. (B) CD83L expression is demonstrated on CD3⁺ T cells stimulated with allogeneic mature DCs. Expression is abrogated by incubation with anti-CD80 and anti-CD86 mAbs but not with isotype controls. (C) Purified CD4⁺ and CD8⁺ T cells demonstrated CD83L expression when stimulated with allogeneic mature DCs, but not when stimulated with immature DCs. (D) Purified CD8⁺ T cells were optimally stimulated with anti-CD3 and anti-CD28 for the indicated time periods and analyzed for CD83 counterreceptor expression using dodCD83. The CD8 dim and CD8 bright populations were defined by electronic gating after staining with anti-CD8 mAb. (E) CD45RA and CD45RO CD8⁺ T cells were optimally stimulated with anti-CD3 and anti-CD28 for 48 hours and analyzed for CD83 counterreceptor expression using dodCD83. Between each experiment, flow cytometry settings were held constant to allow for direct comparison. Since DC-stimulated T cells have higher autofluorescence than mAb-activated T cells, the control staining was shifted slightly to the right for DC-stimulated T cells. Also, with increasing time of stimulation, greater autofluorescence of activated T cells was observed. Figures are representative of multiple donors: in panel A 1 of 4 is shown, and in panels B-E 1 of 2 is shown.