TEMPORAL AND SUBUNIT-SPECIFIC MODULATIONS OF THE REL NF-KAPPA-B TRANSCRIPTION FACTORS THROUGH CD28 COSTIMULATION/

Stimulation of highly purified primary T lymphocytes through CD2 and C D28 adhesion molecules induces a long-term proliferation, dependent on persistent autocrine secretion of interleukin 2 (IL-2), high and prol onged expression of inducible CD25/IL-2 receptor (alpha chain (IL-2R a lpha), and secretion of growth factors such as the granulocyte-macroph age colony-stimulating factor (GM-CSF). CD28 costimulation appears to activate cytokine gene expression through conserved KB-related CD28 re sponse (CD28RE) or cytokine 1 (CK-1) elements in addition to canonical NF-KB-binding sites. In this report, we assess: 1) the evolution of t he expression, over an 8-day time period, of the Rel/NF-KB family of p roteins in costimulated versus TcR/CD3 stimulated primary T cells; 2) the impact of changes on the in vitro occupancy of GM-CSF KB and CK-1, as well as IL-2R alpha KB sites; and 3) the differential regulation o f newly synthesized p65 and c-Rel by IKB proteins. We show that CD2 CD28 stimulation specifically induces, at maximal T cell proliferation phase, sustained nuclear overexpression of NFKB2 p52 and c-Rel subuni ts which might rely on long-lasting processing of p100 precursor for p 52 and increased neosynthesis of c-Rel. This up-regulation correlates with sustained occupancy of GM-CSF KB and CK-1 elements by both protei ns. Conversely, these subunits do not appear to bind to the IL-2R alph a KB site. Costimulation, but not TcR/CD3 stimulation, appears support ed by sustained down-regulation of both IKB alpha and -beta regulators . Furthermore, contrary to p65, c-Rel appears to display little affini ty for p105, p100 and IKB alpha regulators.