CD28 is the primary T cell costimulatory receptor, and upon ligation with its ligands, it enhances T cell proliferation and IL-2 synthesis. In this study we examined the role of CD28 in the initial proliferative response and cell cycle entry of T lymphocytes. Stimulation through CD3 alone resulted in a poor proliferative response, while in the presence of CD28 costimulation a strong increase in the number of cells in S-phase could be detected after 48 h of stimulation. CD28 costimulation enhanced expression of cyclin D3 and induced down-regulation of p27kip1 expression. Cross-linking CD28 was much more effective in inducing cyclin D3 expression and in down-regulating p27kip1 expression than addition of IL-2. Blocking experiments, using antibodies that neutralize IL-2 or the IL-2 receptor, showed that the effects induced by CD28 are independent of endogenous IL-2. Moreover, using a variety of immunosuppressants that interfere with IL-2 signaling pathways, we were able to show that IL-2 is not required for cell cycle entry induced by CD28 costimulation. From these experiments it can be concluded that CD28 and IL-2 use different signaling pathways for down-regulation of p27kip1 expression. We hypothesize that costimulation through CD28 is responsible for initial cell cycle entry of T lymphocytes, while IL-2, which is produced after costimulation, might be involved in sustaining proliferation.

Activation of the T cell receptor in leukemic T cell lines or T cell hybridomas causes growth inhibition.A similar growth inhibition is seen when protein kinase C is activated through addition of phorbol myristate acetate. This inhibition is due to an arrest of cell cycle progression in G 1 combined with an induction of apoptosis. Here we have investigated the mechanism by which these stimuli induce inhibition of proliferation in Jurkat and H9 leukemic T cell lines. We show that expression of cyclin D3 is reduced by each of these stimuli, resulting in a concomitant reduction in cyclin D-associated kinase activity. This reduction in cyclin D3-expression is crucial to the observed G 1 arrest, since ectopic expression of cyclin D3 can abrogate the G 1 arrest seen with each of these stimuli. Moreover, ectopic expression of cyclin D3 also prevents the induction of programmed cell death by phorbol myristate acetate and T-cell receptor activation, leading us to conclude that cyclin D3 not only plays a crucial role in progression through the G 1 phase, but is also involved in regulating apoptosis of T cells. Activation of resting T lymphocytes requires ligation of the T cell receptor (TCR)1 and a co-stimulatory signal, provided by IL-2 or co-ligation of CD28 (reviewed in Ref. 1). The combination of these stimuli enables the resting lymphocytes to exit G 0 and proliferate. Remarkably, stimulation of the same TCR in T cells that are actively proliferating results in cessation of proliferation and subsequent apoptosis (2-5). This particular response is evident in thymocytes, but has also been described in leukemic T cells and T cell hybridomas (2, 6 -8). This indicates that the signals from the activated TCR complex can affect cell proliferation very differently, depending on the proliferative state of the T cell.Cell cycle progression from G 0 to S phase in normal T cells requires the induction of the D-type cyclins (9). These cyclins are not expressed in resting T cells, but are induced upon activation of the TCR complex (10). Three D-type cyclins have been identified, of which cyclins D2 and D3 are predominantly expressed in T cells (11,12). Cyclin D2 and D3 can form an active kinase complex with the cyclin-dependent kinase (cdk) 4 or cdk6, and the resulting kinase complexes are involved in phosphorylation of the retinoblastoma protein (pRb) (11, 13). Phosphorylation of pRb results in its functional inactivation and allows progression of the cell through the late G 1 restriction point and subsequent entry into S phase (reviewed in Ref. 14). However, cyclin D-cdk complexes are able to phosphorylate pRb only partially and complete phosphorylation and functional inactivation of pRb requires additional phosphorylation by cyclin E-cdk2 complexes (15). Although cyclin D-and cyclin E-cdk complexes seem to phosphorylate overlapping sites in pRb, it has been demonstrated that some sites, such as Ser 780 , are only phosphorylated by cyclin D-cdk complexes (16).Induction of cyclin D expression alone is not sufficient to drive rest...

Abstract. Widespread experience indicates that application of suboptimal concentrations of stimulating ligands (secretagogues) to secretory cells elicits submaximal extents of secretion. Similarly, for permeabilized secretory cells, the extent of secretion is related to the concentration of applied intracellular effectors. We investigated the relationship between the extent of secretion from mast cells (assessed as the release of hexosaminidase) and the degranulation (exocytosis) responses of individual cells. For permeabilized mast cells stimulated by the effector combination Ca 2+ plus GTP-3,-S and for intact cells stimulated by the Ca 2÷ ionophore ionomycin, we found that exocytosis has the characteristics of an all-or-none process at the level of the individual cell. With a suboptimal stimulus, the population comprised only totally degranulated cells and fully replete cells. In contrast, a suboptimal concentration of compound 48/80 applied to intact cells induced a partial degree of degranulation. This was determined by observing the morphological changes accompanying degranulation by light and electron microscopy and also as a reduction in the intensity of light scattered at 90 °, indicative of a change in the cell-refractive index. These results may be explained by the existence of a threshold sensitivity to the cornbined effectors that is set at the level of individual cells and not at the granule level.We used flow cytometry to establish the relationship between the extent of degranulation in individual rat peritoneal mast cells and the extent of secretion in the population (measured as the percentage release of total hexosaminidase). For comparison, secretion was also elicited by applying the Ca 2÷ ionophore ionomycin or compound 48/80 to intact cells. For permeabilized cells and also for intact cells stimulated with the ionophore, levels of stimulation that generate partial secretion gave rise to bimodal frequency distributions of 90 ° light scatter. In contrast, a partial stimulus to secretion by compound 48/80 resulted in a single population of partially degranulated cells, the degree of degranulation varying across the cell population. The difference between the all-or-none responses of the permeabilized or ionophore-treated cells and the graded responses of cells activated by compound 48/80 is likely to stem from differences in the effective calcium stimulus. Whereas cells stimulated with receptor-directed agonists can undergo transient and localized Ca 2÷ changes, a homogeneous and persistent stimulus is sensed at every potential exocytotic site in the permeabilized cells.UR understanding of stimulated secretion in terms of intracellular signals and their target proteins leaves an important question unanswered. How can cells that are able to release close to 100% of their granule contents, such as mast cells, give rise to partial secretion? Does this reflect a difference in the susceptibility of individual secretory granules to undergo exocytosis, or is it a consequence of inhomogeneities in the i...

No abstract

CD28 is the primary T cell costimulatory receptor, and upon ligation with its ligands, it enhances T cell proliferation and IL-2 synthesis. In this study we examined the role of CD28 in the initial proliferative response and cell cycle entry of T lymphocytes. Stimulation through CD3 alone resulted in a poor proliferative response, while in the presence of CD28 costimulation a strong increase in the number of cells in S-phase could be detected after 48 h of stimulation. CD28 costimulation enhanced expression of cyclin D3 and induced down-regulation of p27kip1 expression. Cross-linking CD28 was much more effective in inducing cyclin D3 expression and in down-regulating p27kip1 expression than addition of IL-2. Blocking experiments, using antibodies that neutralize IL-2 or the IL-2 receptor, showed that the effects induced by CD28 are independent of endogenous IL-2. Moreover, using a variety of immunosuppressants that interfere with IL-2 signaling pathways, we were able to show that IL-2 is not required for cell cycle entry induced by CD28 costimulation. From these experiments it can be concluded that CD28 and IL-2 use different signaling pathways for down-regulation of p27kip1 expression. We hypothesize that costimulation through CD28 is responsible for initial cell cycle entry of T lymphocytes, while IL-2, which is produced after costimulation, might be involved in sustaining proliferation.