Generation and biochemical analysis of human effector CD4 T cells: alterations in tyrosine phosphorylation and loss of CD3 zeta expression

Human effector T cells have been difficult to Isolate and characterize due to their phenotypic and functional similarity to the memory subset, In this study, a biochemical approach was used to analyze human effector CD4 T cel ls generated in vitro by activation with anti-CD3 and autologous monocytes for 3 to 5 days. The resultant effector cells expressed the appropriate act ivation/differentiation markers and secreted high levels of interferon gamm a (IFN-gamma) when restimulated. Biochemically, effector CD4 T cells exhibi ted increases in total intracellular tyrosine phosphorylation and effector- associated phosphorylated species. Paradoxically, these alterations in tyro sine phosphorylation were concomitant with greatly reduced expression of CD 3 zeta and CD3 epsilon signaling subunits coincident with a reduction in su rface T-cell receptor (TCR) expression. Because loss of CD3 zeta has also b een detected in T cells isolated ex vivo from individuals with cancer, chro nic viral infection, and autoimmune diseases, the requirements and kinetics of CD3 zeta down-regulation were examined. The loss of CD3 zeta expression persisted throughout the course of effector T-cell differentiation, was re versible on removal from the activating stimulus, and was modulated by acti vation conditions. These biochemical changes occurred in effector T cells g enerated from naive or memory CD4 T-cell precursors and distinguished effec tor from memory T cells. The results suggest that human effector T-cell dif ferentiation is accompanied by alterations in the TCR signal transduction a nd that loss of CD3 zeta expression may be a feature of chronic T-cell acti vation and effector generation in vivo. (C) 2001 by The American Society of Hematology.