Abstract
Replicative senescence of human T cells is characterized by the loss of CD28 expression, exemplified by the clonal expansion of CD28(null) T cells during repeated stimulation in vitro as well as in chronic inflammatory and infectious diseases and in the normal course of aging. Because CD28 is the major costimulatory receptor for the induction of T cell-mediated immunity, the mechanism(s) underlying CD28 loss is of paramount interest. Current models of replicative senescence involve protracted procedures to generate CD28(null) cells from CD28(+) precursors; hence, a T-cell line model was used to examine the dynamics of CD28 expression. Here, we show the versatility of the JT and Jtag cell lines in tracking CD28(null) <--> CD28(hi) phenotypic transitions. JT and Jtag cells were CD28(null) and CD28(lo), respectively, but expressed high levels of CD28 when exposed to phorbol 12-myristate 13-acetate. This was a result of the reconstitution of the CD28 gene transcriptional initiator (INR). Tumor necrosis factor-alpha reduced CD28 expression because of the inhibition of INR-driven transcription. Ligation of CD28 by an antibody or by CD80 also down-regulated CD28 transcription through the same mechanism, providing evidence that CD28 can generate a T cell receptor-independent signal with a unique biological outcome. Collectively, these data unequivocally demonstrate the critical role of the INR in the regulation of CD28 expression. T cell lines with transient expression of CD28 are invaluable in the dissection of the biochemical processes involved in the transactivation of the CD28 INR, the silencing of which is a key event in the ontogenesis of senescent T cells.
Highlights
The CD28 molecule is a membrane glycoprotein with nearly restricted expression to T lymphocytes
The notion that CD28null T cells have advanced senescent features comes from observations that they are highly oligoclonal [2, 5, 16] and have significantly shortened telomeres compared with their CD28ϩ counterparts [9, 17]
In transcription assays (Fig. 5B), nuclear extracts from phorbol 12-myristate 13acetate (PMA)-stimulated JT cells supported the transcription of CD28 INR-driven DNA templates, but similar extracts derived from unstimulated JT cells or those incubated in tumor necrosis factor (TNF)-␣ alone or with TNF-␣ during PMA exposure showed little or no transcriptional activity
Summary
The CD28 molecule is a membrane glycoprotein with nearly restricted expression to T lymphocytes. In EMSAs, nuclear extracts from unstimulated JT cells consistently showed complete absence of DNA-binding complexes specific to the CD28 INR, which were found at high levels in similar extracts from PMA-stimulated cells (Fig. 2A). Nuclear extracts from PMA-stimulated, but not unstimulated, JT cells were highly capable of activating transcription of DNA templates driven by the CD28 INR (Fig. 2C).
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