Both primary and memory effectors exhibit similar tyrosine phosphorylation and loss of CD3ζ expression, whereas memory CD4 T cells are more similar to the naive subset.
Purified naive (CD45RA) and memory (CD45RO) CD4 T cells were isolated from PBMCs from an individual donor. Primary effector cells were generated from the CD45RA subset and memory effectors were generated from the CD45RO subset by activation with OKT3 antibody and autologous monocytes for 3 days. T-cell subsets were left untreated (−) or cross-linked for 2 minutes with IgM anti-CD3 antibody (+) before lysis. Lysates from 106 cell equivalents of naive (lanes 1 and 2), primary effector (lanes 3 and 4), memory (lanes 5 and 6), and memory effector (lanes 7 and 8) CD4 T cells were resolved on 12.0% SDS-PAGE, blotted to nitrocellulose, and probed sequentially with antiphosphotyrosine (A), followed by stripping and reprobing with anti-CD3ζ antiserum (B). These blots are representative results from 12 different donors. Eff-spec indicates effector-specific bands.
