Generation and biochemical analysis of human effector CD4 T cells: alterations in tyrosine phosphorylation and loss of CD3zeta 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 cells generated in vitro by activation with anti-CD3 and autologous monocytes for 3 to 5 days. The resultant effector cells expressed the appropriate activation/differentiation markers and secreted high levels of interferon gamma (IFN-gamma) when restimulated. Biochemically, effector CD4 T cells exhibited increases in total intracellular tyrosine phosphorylation and effector-associated phosphorylated species. Paradoxically, these alterations in tyrosine phosphorylation were concomitant with greatly reduced expression of CD3zeta and CD3epsilon signaling subunits coincident with a reduction in surface T-cell receptor (TCR) expression. Because loss of CD3zeta has also been detected in T cells isolated ex vivo from individuals with cancer, chronic viral infection, and autoimmune diseases, the requirements and kinetics of CD3zeta down-regulation were examined. The loss of CD3zeta expression persisted throughout the course of effector T-cell differentiation, was reversible on removal from the activating stimulus, and was modulated by activation conditions. These biochemical changes occurred in effector T cells generated from naive or memory CD4 T-cell precursors and distinguished effector from memory T cells. The results suggest that human effector T-cell differentiation is accompanied by alterations in the TCR signal transduction and that loss of CD3zeta expression may be a feature of chronic T-cell activation and effector generation in vivo. (Blood. 2001;97:3851-3859)
Krishnan
S
S
Department of Surgery, University of Maryland Baltimore, MSTF Bldg., 685 W. Baltimore St., Baltimore, MD 21202, USA.
Warke
V G
VG
Nambiar
M P
MP
Wong
H K
HK
Tsokos
G C
GC
Farber
D L
DL
D017252:CD3 Complex / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000276:immunology; D002478:Cells, Cultured; D015536:Down-Regulation; D006801:Humans; D016130:Immunophenotyping; D008213:Lymphocyte Activation / Q000187:drug effects; D016853:Muromonab-CD3 / Q000494:pharmacology; D010766:Phosphorylation; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D016176:T-Lymphocyte Subsets; D014443:Tyrosine / Q000378:metabolism
D017252:CD3 Complex; C121704:CD3 antigen, zeta chain; C115335:CD3E protein, human; D016853:Muromonab-CD3; D011948:Receptors, Antigen, T-Cell; D014443:Tyrosine