Biochemical analysis of effector CD4 T cells reveals alterations in tyrosine phosphorylation and loss of CD3ζ and CD3ε protein expression.
Purified CD4 T cells from peripheral blood were used to generate effector cells using OKT3 antibody and autologous monocytes (see “Materials and methods”). CD4 cells and effectors were then left untreated (0) or cross-linked for 2 minutes with IgM anti-CD3 antibody (CD3) before lysis. Lysates from 106 CD4 T cells (lanes 1 and 2), and equivalent numbers of effector CD4 T cells (designated as ce, for cell equivalents, lanes 3 and 4), or equivalent levels of total effector cell protein (designated as pe, for 20μg protein equivalents, lanes 5 and 6) were resolved on reducing 12.0% SDS-PAGE, blotted to nitrocellulose, and probed sequentially with antiphosphotyrosine (A), followed by stripping and reprobing with anti-CD3ζ and anti–ZAP-70 antisera (B). These blots are representative results from 19 individual donors. In panel A, the effector-associated phosphorylated species of molecular weights 120/121, 76, 72, 60, 40/41, 37, and 10 kd are indicated on the right side of the blot. Bands of 135 kd and the phosphorylated CD3ζ band present in CD4 T-cell lysates, but not in effector T cells are indicated on the left side of the blot. (C) RT-PCR analysis of CD3ζ, ZAP-70, and actin transcript amplified from equal amounts of RNA isolated from resting CD4 T cells (CD4), effector CD4 T cells (Eff), and monocytes (mc). This gel is representative of 5 experiments. (D) Expression of ZAP-70, actin, CD3ε, and CD3ζ protein products in lysates of resting and effector CD4 T cells. Protein equivalents (4 μg/lane) from lysates of CD4 T cells or 5-day activated effector CD4 T cells purified as above were resolved by 4% to 20% gradient reducing SDS-PAGE, blotted to nitrocellulose, and blots were cut into strips and hybridized with polyclonal antiserum directed against ZAP-70, actin, CD3ε, and CD3ζ. IB indicates immunoblot.
