In vitro–generated effector CD4 T cells exhibit effector function and an activated/effector phenotype.

Naive (CD45RA) and memory (CD45RO) subsets of CD4 T cells were purified by a 2-step immunomagnetic depletion (see “Materials and methods”). Effector CD4 T cells were generated by coculture of naive CD4 T cells with 2 μg/mL anti-CD3 antibody (OKT3) and twice the number of primary autologous monocytes for 3 days. (A) Functional analysis of naive, effector, and memory CD4 T cells. Freshly isolated ex vivo naive and memory CD4 T cells and in vitro–generated effector CD4 T cells purified by centrifugation through Ficoll and washing, were cultured alone or in wells to which anti-CD3 antibody was immobilized. After 24 hours, culture supernatants were collected and assayed for IFN-γ content by specific ELISA. These data are representative of 3 separate experiments. (B) Phenotypic analysis of effector CD4 T-cell differentiation. The expression of activation/differentiation markers on purified naive (CD45RA) CD4 T cells, naive cells activated for 24 to 72 hours with anti-CD3 and autologous monocytes to generate effectors, and naive cells incubated for 72 hours with monocytes alone as a control was assessed by specific staining with fluorescent-conjugated antibodies and analyzed by FACS. Forward scatter is shown at the bottom. These data from a single donor are representative of 3 separate experiments using T cells derived from 3 different donors.