Kinetics of CD3 and TCR down-regulation during effector CD4 T cell differentiation.

CD4 T cells were cultured with anti-CD3/monocytes, harvested 24 to 120 hours, and either lysed for immunoblotting or stained and analyzed by FACS. (A) Kinetics of differentiation-induced CD3ζ and CD3ε down-regulation. Anti–ZAP-70, actin, CD3ε, and CD3ζ immunoblot of lysates derived from CD4 T cells before activation (lane 1), CD4 T cells harvested and purified 24 to 72 hours after culture with anti-CD3/monocytes (lanes 2-4, 24, 48, 72), 72 hours purified effector CD4 T cells rested in media alone for 2 days (lane 5, 72+rst), or purified CD4 T cells cultured with monocytes alone for 72 hours (lane 6, T+mc). Molecular weight markers are indicated at left. (B) Change in CD3ζ expression over time of activation. A compilation of the densitometric analysis of 5 separate kinetic experiments (Exp 1-5) performed as in panel A with purified T cells and monocytes from 5 different donors. Relative CD3ζ expression is defined as in Figure 4. In Exp 5 (shown in panel A), the 120-hour time point was not done. (C) TCR/CD3 and surface marker expression during effector differentiation. Histograms show surface expression of αβ TCR and CD3ε compared to control staining.