We have followed Sp1 expression in primary human T lymphocytes induced, via CD2 plus CD28 costimulation, to sustained proliferation and subsequent return to quiescence. Binding of Sp1 to wheat germ agglutinin lectin was not modified following activation, indicating that the overall glycosylation of the protein was unchanged. Sp1 underwent, instead, a major dephosphorylation that correlated with cyclin A expression and, thus, with cell cycle progression. A similar change was observed in T cells that re-entered cell cycle following secondary interleukin-2 stimulation, as well as in serum-induced proliferating NIH/3T3 fibroblasts. Phosphatase 2A (PP2A) appears involved because 1) treatment of dividing cells with okadaic acid or cantharidin inhibited Sp1 dephosphorylation and 2) PP2A dephosphorylated Sp1 in vitro and strongly interacted with Sp1 in vivo. Sp1 dephosphorylation is likely to increase its transcriptional activity because PP2A overexpression potentiated Sp1 site-driven chloramphenicol acetyltransferase expression in dividing Kit225 T cells and okadaic acid reversed this effect. This increase might be mediated by a stronger affinity of dephosphorylated Sp1 for DNA, as illustrated by the reduced DNA occupancy by hyperphosphorylated Sp factors from cantharidin-or nocodazole-treated cells. Finally, Sp1 dephosphorylation appears to occur throughout cell cycle except for mitosis, a likely common feature to all cycling cells.Sp1 is the founding member of a multigene family of transcription factors including Sp1, Sp2, Sp3, Sp4 (see Refs. 1-3 for reviews), and Sp5 proteins (4, 5). Both Sp1 and Sp3 are abundant and ubiquitous, whereas Sp4 is mainly expressed in neuronal tissues and Sp5 exhibits a dynamic and highly restricted expression pattern during embryogenesis. This protein family shares three highly conserved zinc finger DNA binding motifs that recognize GC or GT/CACC boxes present in many promoters. Sp1 was first viewed as a constitutive transcriptional activator regulating basal expression of many cellular and viral genes. However, it is now established that Sp1 activity is modulated in response to numerous signals and that this factor plays a critical role in cell growth and differentiation. Sp1 mediates the induction of dihydrofolate reductase (6) and thymidine kinase (7) genes associated with DNA synthesis and is therefore intricately linked to growth/cell cycle progression. In line with this, the ectopic expression of truncated Sp1 prolongs the S phase and reduces the growth rate (8). Conversely, Sp1 mediates cell division arrest by up-regulating the expression of genes coding for negative regulators of the cell cycle such as p2lWaf1/Cip1 , in p53-dependent growth control (9) or p53-independent pathway of terminal differentiation (10 -12).Sp1's manifold roles can be reconciled if one integrates the multiple time-ordered regulations to which this factor is itself submitted. First, Sp1 is a direct target of numerous cell cycle regulators, which activate or repress its activity. For instance, cyclin A (13...

Stimulation of highly purified primary T lymphocytes through CD2 and CD28 adhesion molecules induces a long-term proliferation, dependent on persistent autocrine secretion of interleukin 2 (IL-2), high and prolonged expression of inducible CD25/IL-2 receptor ␣ chain (IL-2R␣), and secretion of growth factors such as the granulocyte-macrophage colony-stimulating factor (GM-CSF). CD28 costimulation appears to activate cytokine gene expression through conserved B-related CD28 response (CD28RE) or cytokine 1 (CK-1) elements in addition to canonical NF-Bbinding sites. In this report, we assess: 1) the evolution of the expression, over an 8-day time period, of the Rel/NF-B family of proteins in costimulated versus TcR/CD3-stimulated primary T cells; 2) the impact of changes on the in vitro occupancy of GM-CSF B and CK-1, as well as IL-2R␣ B sites; and 3) the differential regulation of newly synthesized p65 and c-Rel by IB proteins. We show that CD2؉CD28 stimulation specifically induces, at maximal T cell proliferation phase, sustained nuclear overexpression of NFKB2 p52 and c-Rel subunits which might rely on long-lasting processing of p100 precursor for p52 and increased neosynthesis of c-Rel. This up-regulation correlates with sustained occupancy of GM-CSF B and CK-1 elements by both proteins. Conversely, these subunits do not appear to bind to the IL-2R␣ B site. Costimulation, but not TcR/CD3 stimulation, appears supported by sustained down-regulation of both IB␣ and -␤ regulators. Furthermore, contrary to p65, c-Rel appears to display little affinity for p105, p100 and IB␣ regulators.

Stimulation of the human T-cell line, Jurkat, by a monoclonal antibody (mAb) directed against the CD28 molecule leads to sustained increases in intracellular levels of Ca2+ ([Ca2+]i); the initial rise in Ca2+ comes from internal stores, followed by Ca2+ entry into the cells. The CD28 molecule also appears to activate polyphosphoinositide (InsPL)-specific phospholipase C (PLC) activity in Jurkat cells, as demonstrated by PtdInsP2 breakdown, InsP3 and 1,2-diacylglycerol generation and PtdIns resynthesis. We also observed that interleukin-2 (IL2) production induced via CD28 triggering was sensitive to a selective protein kinase C inhibitor. Of the four other anti-CD28 mAbs (CD28.2, CD28.4, CD28.5, CD28.6) tested, only one (CD28.5) was unable to generate any InsPL-specific PLC or IL2 secretion. However, the cross-linking of cell-bound CD28.5 with anti-mouse Ig antibodies led to an increase in [Ca2+]i. CD28-molecule clustering in itself appears to be a sufficient signal for induction of PLC activity.

We have identified a new T cell activation pathway mediated by the lymphocyte homing receptor/CD44 molecule, 8B2.5, a local monoclonal antibody (mAb), which recognizes two glycoproteins of 85 and 220 kDa with wide tissue distribution, is shown by sequential immunoprecipitations and competitive antibody-binding inhibition experiments with several CD44 reference mAb to recognize the CD44 molecule. The 8B2.5 mAb, but not reference CD44 mAb, is able to induce resting peripheral blood lymphocytes to proliferate in the presence of phorbol esters. This proliferation is monocyte dependent but Fc independent and results from 8B2.5 mAb binding to CD44 molecules both expressed by both T cells and monocytes. In the absence of monocytes, proliferation can be restored by solid-phase 8B2.5 mAb, or, to a lesser extent, by adding interleukin 2. Although CD3 and CD44 surface molecules are found physically independent, T cell activation via the CD44 pathway is inhibited by CD3 modulation. In addition to the direct role of CD44 molecules in T cell proliferation, CD44 mAb can up- or- down-regulate the CD3 and CD28 pathways, depending on the presence of monocytes. These results suggest that T cell and monocyte binding to high endothelial venule or extracellular matrix proteins could further promote clonal expansion of resting T cells migrating in certain specific anatomic sites.

R. Costello : poste d' accueil lnsenn. P. Lecine : n.llocatrâre de recl!l'rche MESR. B. Kahn-Perlès : chmgée de 1•echerrhe au Cnrs. M. Algané : al/1) w.tmre de re cherche MESR. C. Lipcey : ingé nieur à 1'/nsenn. D. Olive : directeur de recherrhe à l'Inserm. J. 1 rn bert : chargé de 1• echerche à l'fn senn. Laboratoire d'immunologie fo nction nelle el moléculaire, Inserm U. l l9, 27, bou levard Lei-Roure, 13009 Marseille, France. Voir aussi la. mini-sy nthès e d'A. lsmil, jmge 101 7 de ce numéro.