Desferoxamine blocks IL 2 receptor expression on human T lymphocytes

Thymidine uptake by PHA-stimulated human lymphocytes is reduced in the presence of 100 microM or greater concentrations of the iron-chelating agent desferoxamine (DF). We assessed expression of IL 2 receptor, 4F2 and Ia antigens, IL 2 production, and cell cycle progression by blood mononuclear cells (MNC) stimulated by PHA in the presence or absence of DF to determine whether the lack of T cell proliferation was a manifestation of inhibition of an earlier activation event. Tac antigen expression on PHA-stimulated MNC was inhibited by DF throughout 8 days of culture, and those cells which were positive had a low density of Tac antigen as compared with controls without DF. Expression of other activation antigens, 4F2 and Ia, was not impaired by DF. The supernatants of the DF-containing and control cultures contained equivalent IL 2 activity, as measured on the HT-2 cell line. Cell cycle analysis of these cultures shows that the addition of DF at the beginning of culture blocks most cells from undergoing G0 to G1 transition, whereas later addition of DF arrests the progression of the T cell blasts through the cell cycle. Separation of cells cultured with PHA and DF into Tac+ and Tac- subsets showed that progression from G0 to G1 was restricted to the former subset. These results suggest that interference with IL 2 receptor expression might contribute to the block in mitogen-induced proliferation caused by DF.     

Interference of a C3-fragment preparation with IL 2-dependent proliferation

A C3-fragment preparation (C3-FP) was studied for its ability to regulate human peripheral blood lymphocyte activation. It was found that very low concentrations of this low m.w. fraction, which was free of C3a, inhibited the PHA-induced lymphocyte proliferation without any cytotoxicity. Cytofluorometric analysis showed that C3-FP did not influence the transition of T cells from the G0 to the G1a phase of the cell cycle. However, the IL 2-dependent transition from the G1a to the G1b phase of the cell cycle was effectively blocked. Addition of exogenous IL 2 did not release cells arrested in the G1a phase. Furthermore, neither IL 2 production nor IL 2 receptor formation was inhibited by C3-FP, and binding of IL 2 to its receptor was unaltered. It was found that only IL 2-dependent cell lines were inhibited in their proliferation; all other tested cell lines were unaffected by C3-FP. Our findings suggest that cleaved products of C3 may inhibit IL 2-dependent lymphocyte proliferation at a stage where the IL 2 signal is required for initiation of proliferation.     

Direct activation of human resting T cells by IL 2: the role of an IL 2 receptor distinct from the Tac protein

High concentrations of interleukin 2 (IL 2) were shown to produce a delayed but pronounced proliferation of purified resting T cells in the apparent absence of other activation signals. Because these stimulatory effects of IL 2 occurred in the absence of detectable Tac+ cells, the possibility that IL 2 might be initially interacting with an IL 2 binding protein distinct from the Tac protein was studied. Chemical cross-linking studies with 125I-IL 2 revealed the presence of an IL 2 binding protein distinct from the Tac protein on the surface of these unstimulated T cells. This second IL 2 receptor has an estimated molecular size of 70,000 daltons, lacks reactivity with the anti-Tac antibody, and appears to be identical to the p70 protein recently proposed as a component of the high affinity IL 2 receptor. Scatchard analysis of IL 2 binding assays performed with the unactivated T cells revealed approximately 600 to 700 p70 sites per cell and an apparent Kd of 340 pM. These data indicate that the p70 protein present on resting T cells binds IL 2 with an intermediate affinity compared with the previously recognized high and low affinity forms of the receptor and may account for the high concentration of IL 2 needed to induce resting T cell proliferation. To investigate the early biologic consequences of IL 2 binding to the p70 protein, potential changes in the expression of genes involved in T cell activation were examined. Northern blotting revealed the rapid induction of c-myc, c-myb, and Tac mRNA after stimulation of resting T cells with a high concentration of IL 2. The anti-Tac antibody did not inhibit IL 2 induced expression of these genes, suggesting that the p70 protein rather than the Tac antigen or the high affinity IL 2 receptor complex mediated this signal. However, in contrast to these early activation events, the anti-Tac antibody significantly inhibited IL 2 induced T cell proliferation. This finding implicates the high affinity form of the IL 2 receptor in the proliferative response of the IL 2 activated T cells. Thus these data support a two step model for the induction of resting T cell proliferation by high doses of IL 2 involving the initial generation of an activation or "competence" signal through the p70 protein and a subsequent proliferation or "progression" signal through the high affinity form of the receptor.     

Ryanodine receptor signaling is required for anti-CD3-induced T cell proliferation, interleukin-2 synthesis, and interleukin-2 receptor signaling

Ryanodine receptors (RyR) are involved in regulating intracellular Ca(++) mobilization in T lymphocytes. However, the importance of RyR signaling during T cell activation has not yet been determined. In this study, we have used the RyR-selective antagonists, ruthenium red and dantrolene, to determine the effect of RyR blockade on T cell receptor-mediated activation events and cytokine-dependent T cell proliferation. Both ruthenium red and dantrolene inhibited DNA synthesis and cell division, as well as the synthesis of interleukin (IL)-2 by T lymphocytes responding to mitogenic anti-CD3 antibody. Blockade of RyR at initiation of culture or as late as 24 h after T cell receptor stimulation inhibited T cell proliferation, suggesting a requirement for sustained RyR signaling during cell cycle progression. Although flow cytometry revealed that RyR blockade had little effect on activation-induced expression of the alpha chain (CD25) of the high affinity IL-2 receptor, the inhibitory effect of RyR antagonists could not be reversed by the addition of exogenous IL-2 at initiation of culture. In addition, both ruthenium red and dantrolene had a strong inhibitory effect on IL-2-dependent proliferation of CTLL-2 T cells. These data indicate that RyR are involved in regulating IL-2 receptor signaling that drives T cell progression through the cell cycle. We conclude that RyR-associated Ca(++) signaling regulates T cell proliferation by promoting both IL-2 synthesis and IL-2-dependent cell cycle progression.     

The role of interleukin 2 and T11 E rosette antigen in activation and proliferation of human NK clones

Although considerable data have recently been accumulated regarding the functional role of natural killer (NK) cells, relatively little is known about the factors that regulate NK cell activity. In these studies, we evaluated the role of interleukin 2 (IL 2) and the expression of the IL 2 receptor in the activation and proliferation of human NK cloned cell lines. By using a series of cloned cell lines, we were able to analyze homogeneous populations of NK cells that ordinarily comprise only a small fraction of peripheral blood lymphocytes and are extremely heterogeneous with respect to phenotypes and cytotoxic specificities. In comparison with several T cell clones, we found a much lower density of IL 2 receptors on NK clones, regardless of whether or not these cloned cells had a mature T cell phenotype. Correspondingly, NK clones needed a 10-fold higher concentration of recombinant IL 2 for maximal proliferation. Moreover, blocking studies with specific monoclonal IL 2 receptor antibodies indicated that IL 2 is both necessary and sufficient to induce the proliferation of NK clones. Because the majority of peripheral blood NK cells and NK clones express the T11 E rosette receptor antigen, which has been shown to be an antigen-independent activation pathway for T cells, we were able to study the role of monoclonal anti-T11 antibodies in the activation of various NK clones for which a specific target antigen is not known. In contrast to T cell clones, the induction of IL 2 receptor expression after T11 activation was possible only for some NK clones such as JT10 and JT3, but not for CNK5. Before activation, the IL 2 receptor expression of NK clones was confined to cells in the G2 - M phase, but after T11 activation the more pronounced IL 2 receptor expression became independent of the cell cycle. With respect to the direct proliferative effect of anti-T11 activation that has been noted with T cell clones, only the T3+ (JT10) and not the T3- NK clones could be directly stimulated. Nevertheless, IL 2 receptor expression could be triggered on some T3- clones such as JT3. Because T11-induced proliferation of T cells has been shown to be dependent on both the expression of the IL 2 receptor and on the interaction of this receptor with IL 2, it is proposed that the different responses of NK cells to T11 activation may reflect the ability of the individual clone to produce endogenous IL 2, as well as its ability to express the IL 2 receptor.     

Signals involved in T cell activation. II. Distinct roles of intact accessory cells, phorbol esters, and interleukin 1 in activation and cell cycle progression of resting T lymphocytes

The signals involved in the initiation of mitogen-induced activation of resting guinea pig T cells were examined. The combination of phytohemagglutinin (PHA) and 4 beta-phorbol 12-myristate 13-acetate (PMA) stimulated DNA synthesis by accessory cell (AC)-depleted T cells cultured at high density, but the use of low density cultures indicated that intact AC were absolutely necessary for PHA-stimulated T cell DNA synthesis even in the presence of PMA, interleukin 1 (IL 1), or interleukin 2 (IL 2). In contrast, AC-depleted T cells were able to respond to the combination of the calcium ionophore, ionomycin, and PMA regardless of the cell density at which they were cultured. Cell cycle analysis by acridine orange staining indicated that neither PHA nor ionomycin, in the absence of AC, activated resting T cells. PMA in the absence of all AC, supported cell cycle entry and progression to the DNA synthetic phase of the majority of ionomycin-stimulated T cells, but permitted only a small number of PHA-triggered T cells to enter the initial stage of the cell cycle (G1a) characterized by a modest increase in cellular RNA content. Although PMA permitted some PHA-stimulated T cells to enter the cell cycle, most required intact AC to enter G1, and all required intact AC to progress through G1 and synthesize maximal amounts of RNA. No PHA-stimulated cells reached the S phase without intact AC. In PHA-stimulated cultures containing intact AC, PMA increased the number of cells entering the cell cycle and increased the rate of their progress to the DNA synthetic phase. IL 1 also augmented PHA-stimulated AC-dependent T cell DNA synthesis in the presence or absence of PMA, but appeared to be most active during the later stage of the first cell cycle, augmenting the number of activated cells that entered the S phase of the cell cycle. These results support the conclusion that intact AC, IL 1, and a PMA-like signal play distinct roles in the progression of mitogen-stimulated T cells through the first round of the cell cycle.     

Relative roles of T‐cell receptor ligands and interleukin‐2 in driving T‐cell proliferation

Stimulation of T cells by the T‐cell receptor (TCR)/CD3 complex results in interleukin‐2 (IL‐2) synthesis and surface expression of the IL‐2 receptor (IL‐2R), which in turn drive T‐cell proliferation. However, the significance of the requirement of IL‐2 in driving T‐cell proliferation, when TCR stimulation itself delivers potential mitogenic signals, is unclear. We show that blocking of IL‐2 synthesis by Cyclosporin A (CsA) suppressed both the Concanavalin A (Con A)‐ and phorbol myristate acetate (PMA)/ionomycin‐induced proliferation of T cells. The latter is also inhibited by anti‐IL‐2R. Kinetic studies showed that T‐cell proliferation begins to become resistant to CsA inhibition by about 12 h and became largely resistant by 18 h of stimulation. PMA, the protein kinase C activator, enhanced Con A‐induced T‐cell proliferation if added only within first 12 h of stimulation, and not after that. Given the fact that, in the present study, TCR is downregulated within 2 h of Con A stimulation and T cells entered the S phase of cell cycle by about 18 h of stimulation, the above results suggest that TCR stimulation provides the initial trigger to the resting T cells, which allows the cells to traverse the first two third portions of G1 phase of cell cycle and become proliferation competent. IL‐2 action begins afterward, delivering the actual proliferation signal(s), allowing the cells to traverse the rest of G1 phase and enter the S phase of the cell cycle. J. Cell. Biochem. 76:37–43, 1999. © 1999 Wiley‐Liss, Inc.     

Resumption of cell cycle in BALB/c-3T3 fibroblasts arrested by polyamine depletion: relation with "competence" gene expression

Serum deprivation arrests BALB/c-3T3 fibroblasts (clone A31) in G0 phase, where resumption of the cell division cycle can be induced by addition of serum or of specific growth factors in a defined sequence: PDGF (inducer of a state of "competence," characterized by the expression of a family of genes including c-myc), epidermal growth factor EGF and IGF1 (Leof et al., 1982, 1983). When exponentially growing A31 cells are placed for greater than or equal to 2 days in a medium containing the alpha-difluoromethylornithine (alpha DFMO), an irreversible inhibitor of ornithine decarboxylase, they become arrested in G1 phase as a consequence of polyamine depletion (Medrano et al., 1983). In the alpha DFMO-arrested cells, addition of putrescine (60 microM) in a culture medium containing 6% fetal calf serum (FCS), but not in serum-free medium, is sufficient to induce G1 progression and entry into S phase (as determined by 3H-thymidine incorporation). The level of "competence" mRNAs is high in alpha DFMO-arrested cells. After addition of putrescine in FCS-containing medium, these mRNAs continue to be present for at least 3 h. A large proportion of alpha DFMO-arrested cells can be induced to progress to S phase by insulin (1 microM, acting via IGF1 receptor) plus putrescine in a serum-free medium (greater than or equal to 50% of FCS effect). In this case, the levels of "competence" mRNAs become low or undetectable within 3 h, EGF (10 nM) plus insulin had only slightly greater effect than insulin alone on the progression of alpha DFMO-arrested cells. When the alpha DFMO-arrested cells are subsequently incubated during 3 days in a low-serum-containing medium (0.25% FCS), they do not replenish their supply of polyamines, and then continue to express the c-myc gene. The recruitment of the polyamine-depleted, serum-deprived cells into the cell division cycle does not require PDGF and can be induced by addition of EGF and insulin plus putrescine. These data indicate that alpha DFMO arrests majority of the cells at a point situated beyond the PDGF- and EGF-dependent portion of G1 phase. During the subsequent serum deprivation, the alpha DFMO-arrested cells remain "competent" (PDGF-independent), probably as a consequence of their continued expression of c-myc (and possibly other PDGF-inducible genes).     

Requirements for T cell activation by OKT3 monoclonal antibody: role of modulation of T3 molecules and interleukin 1

The requirements for activation of human peripheral blood T cells by the mitogenic monoclonal antibody OKT3 were examined. OKT3 binds to a T cell molecule, T3, associated with the T cell antigen receptor and involved in T cell activation. Activation of T cells by OKT3 requires signals provided by accessory cells and is IL 2 dependent. In the presence of accessory cells, OKT3 induces loss of T3 molecules from the cell surface, production of IL 2, expression of IL 2 receptors, and proliferation. Modulation of T3 molecules by OKT3 can be induced in the absence of accessory cells with anti-mouse IgG. These T cells, however, are not induced to express IL 2 receptors or secrete IL 2. The addition of IL 1 induces expression of IL 2 receptors, but does not induce IL 2 secretion or proliferation. Thus, peripheral blood T cells appear to have different requirements for activation compared with antigen-specific T cell clones that can be induced to produce IL 2 when stimulated with OKT3 and IL 1. Expression of IL 2 receptors does not require modulation of T3 molecules, because the binding of OKT3 to T cells in the presence of IL 1 alone is sufficient to induce IL 2 receptor expression. The results suggest that IL 2 secretion depends on cross-linking and modulation of T3 molecules, and additional, as yet undefined, accessory cell signals. The expression of IL 2 receptors and proliferation of T cells can be induced in the absence of these signals when exogenous IL 2 is provided.     

Lymphokine regulation of activated (G1) lymphocytes. II. Glucocorticoid and anti-Tac-induced inhibition of human T lymphocyte proliferation

The regulation of the first cell cycle of human, activated (G1) PBL was analyzed by flow cytometry and [3H]thymidine incorporation. Endogenous IL 2 production was blocked in situ by pharmacologic concentration of DEX (100 to 1000 nM), resulting in an 80 to 90% reduction of thymidine uptake. Although T lymphocyte activation (G0-G1a transition) by PHA was unaltered, cells remained in the G1a phase of the cell cycle due to insufficient RNA synthesis for proliferation. The addition of IL 2-containing supernatants reversed this inhibitory effect of DEX by allowing the cells to synthesize more RNA (G1a-G1b transition). Such cells could enter the S phase and proliferate. Similar studies were performed on cells treated with a monoclonal antibody (anti-Tac) against the IL 2 receptor. In these studies, IL 2-induced RNA synthesis, and subsequent proliferation of DEX-treated and PHA-stimulated cells was inhibited by anti-Tac. Anti-Tac did not, however, inhibit the effect of endogenous IL 2 (PHA-stimulated PBL without DEX treatment), although it did bind equally well to such cells. Thus, IL 2 directly or indirectly regulates human T cell proliferation at the level of RNA synthesis. Furthermore, anti-Tac can inhibit the mitogenic signal given by endogenous IL 2, but not by in situ produced IL 2, an observation of importance to further investigations of the mechanisms by which IL 2 interacts with specific receptors to elicit proliferation.     

The effects of 1,25-dihydroxyvitamin D3 on human T lymphocyte activation and proliferation: a cell cycle analysis

Recent studies have demonstrated that 1,25-dihydroxyvitamin D3 (calcitriol), the most biologically active metabolite of vitamin D, is a potent inhibitor of both lectin- and antigen-driven human T lymphocyte proliferation. To better characterize this effect, we performed cell cycle analysis of both untreated and calcitriol-treated peripheral blood mononuclear cells after PHA stimulation. By using the metachromatic dye acridine orange and flow cytometry, we found that calcitriol blocks the transition from the early, low RNA compartment of G1 (G1A) to the late, higher RNA compartment of G1 (G1B). Consistent with this observation was the inability of exogenous IL 1 or phorbol myristic acetate to overcome calcitriol's suppression of DNA synthesis. Indomethacin slightly reversed calcitriol's inhibition of transition from early to late G1, suggesting a minor, prostaglandin-dependent component to calcitriol's antiproliferative activity. Finally, by using the monoclonal antibodies anti-Tac and OKT9, we found that calcitriol had no effect on IL 2 receptor expression, an early G1 event, but markedly inhibited transferrin receptor expression, an IL 2-dependent, late G1 event. Thus, analysis of calcitriol's effects on the expression of these T cell activation antigens provides further evidence of the cell cycle specificity of calcitriol's action in regulating human T lymphocyte proliferation.     

Activation of the Na+/H+ antiport is not required for lectin-induced proliferation of human T lymphocytes

Interaction of some mitogenic lectins and growth factors with the cell surface leads to activation of the Na+/H+ antiport and a resultant cytoplasmic alkalinization. Because amiloride inhibits both Na+/H+ exchange and cell proliferation, it has been hypothesized that activation of the antiport is an obligatory requirement and may, perhaps, be the "trigger" for proliferation. However, concentrations of amiloride which inhibit the antiport also inhibit several other intracellular processes, including protein synthesis and phosphorylation. To determine whether activation of the Na+/H+ antiport is necessary for lectin-induced proliferation, we examined the inhibitory activity of a series of potent amiloride analogs by measuring [3H]thymidine incorporation, cell cycle progression, and induction of the interleukin 2 (IL 2) receptor on human lymphocytes. In medium containing bicarbonate, and at concentrations at least 10 times higher than required to inhibit the antiport, these drugs did not inhibit the proliferative response of human peripheral blood T cells to the mitogen phytohemagglutinin. The amiloride analogs also failed to inhibit induction of the IL 2 receptor. Similarly, with human thymocytes, the amiloride analogs did not inhibit the co-mitogenic effects of the lectins phytohemagglutinin and concanavalin A together with IL 2 or the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. This finding suggests that Na+/H+ exchange through the antiport is not an obligatory requirement for activation or proliferation of human lymphocytes or thymocytes.     

Retinoblastoma susceptibility gene product (pRb) and p107 functionally separate the requirements for serum and anchorage in the cell cycle G1-phase

Growth factors and cell anchorage are both required for cell cycle G(1)-phase progression, but it is unclear whether their function is mediated through the same set of cell cycle components and whether they are both required during the same periods of time. We separately analyzed the requirements of serum and anchorage during G(1)-phase progression and found that human dermal fibroblasts as well as wild type, pRb(-/-), and p107(-/-) mouse embryonic fibroblasts needed serum (growth factors) until mid-G(1)-phase but required cell anchorage until late G(1)-phase to be competent for S-phase entry. Importantly, however, pRb/p107 double-null mouse embryonic fibroblasts lacked serum requirement in mid-G(1)-phase but still required cell anchorage until late G(1)-phase to enter S-phase. Our results indicate that pRb and p107 do not constitute the last control point for extracellular factors during G(1)-phase progression, and they functionally separate the requirements for serum and cell anchorage in terms of involved cell cycle components.     

Clonal anergy is maintained independently of T cell proliferation

Ag encounter in the absence of proliferation results in the establishment of T cell unresponsiveness, also known as T cell clonal anergy. Anergic T cells fail to proliferate upon restimulation because of the inability to produce IL-2 and to properly regulate the G(1) cell cycle checkpoint. Because optimal TCR and CD28 engagement can elicit IL-2-independent cell cycle progression, we investigated whether CD3/CD28-mediated activation of anergic T cells could overcome G(1) cell cycle block, drive T cell proliferation, and thus reverse clonal anergy. We show here that although antigenic stimulation fails to elicit G(1)-to-S transition, anti-CD3/CD28 mAbs allow proper cell cycle progression and proliferation of anergic T cells. However, CD3/CD28-mediated cell division does not restore Ag responsiveness. Our data instead indicate that reversal of clonal anergy specifically requires an IL-2-dependent, rapamycin-sensitive signal, which is delivered independently of cell proliferation. Thus, by tracing proliferation and Ag responsiveness of individual cells, we show that whereas both TCR/CD28 and IL-2-generated signals can drive T cell proliferation, only IL-2/IL-2R interaction regulates Ag responsiveness, indicating that proliferation and clonal anergy can be independently regulated.     

Effect of serum on heat response of synchronized mouse neuroblastoma cells: protection of cell cycle progression, protein synthesis and survival

The effect of serum and temperature elevation on proliferation has been studied in synchronized mouse neuroblastoma (Neuro-2A) cells. The effects of serum were studied on the induction of (a) mitotic delay due to a non-lethal heat treatment (30 min at 42.7 degrees C) and (b) the loss of colony-forming capacity after a more extensive heat treatment (45 min at 44 degrees C or a continuous 42.7 degrees C heat treatment). The following results were obtained. Under conditions of serum depletion, cell cycle extension of heated G1 phase cells was more than that of heated G2 phase cells. Serum protected against heat-induced alterations of cell cycle progression in G1- but not in G2 phase cells. This effect of serum could be mimicked by a supplement to the medium of human transferrin, bovine pancreas insulin and selenium, and was correlated with protection of protein synthesis. Serum also affected heat-induced cell killing. Under conditions of serum depletion, G1 phase cells were more resistant to heat compared to G2 cells. The presence of serum during heat treatment further increased the thermoresistance of G1 phase cells, but did not affect sensitivity of G2 phase cells. This effect of serum could not be mimicked by a supplement of transferrin, insulin and selenium. These results indicate that serum protects G1 phase cells for heat-induced changes of cell cycle progression as well as on cell survival, but the mechanisms involved in both phenomena seem to be different.     

Elevated titers of cell-free interleukin 2 receptor in serum of lupus mice

Activation/proliferation of mouse and human T and B cells is associated with expression and subsequent release of interleukin 2 receptors (IL 2R) into the milieu. The soluble form of IL 2R, at least in part, retains its ability to bind to IL 2 and to anti-receptor antibodies, but its exact structure remains unknown. Because systemic lupus erythematosus (SLE) is associated with T and B cell activation, we have used monoclonal anti-IL 2R antibodies in an ELISA to measure levels of IL 2R in sera of various lupus strains. High levels of the released receptor were found at an active clinical stage in sera of four autoimmune strains of mice homozygous for the lpr (lymphoproliferation) gene that causes T cell expansion, massive lymphoid organ enlargement, and promotes the autoimmune process. High levels were also found in lupus mice characterized primarily by B cell proliferation (BXSB males) and in (NZB X W)F1 mice characterized by T and B cell activation. Similarly high IL 2R serum levels could be induced experimentally in normal mice injected with immunostimulants such as bacterial lipopolysaccharide or Freund's complete adjuvant. The results indicate that IL 2R serum levels may provide a good marker of ongoing lymphoid cell activation/proliferation, and thus might be useful in the follow-up of patients with systemic autoimmune or other lymphoproliferative disorders. The biologic roles, if any, of the soluble form of IL 2R and its effects in normal and abnormal conditions remain to be determined.     

T cell receptor triggering induces responsiveness to interleukin 1 and interleukin 2 but does not lead to T cell proliferation

The antigen-like activity of monoclonal antibodies directed at the T3-Ti antigen receptor complex of human T lymphocytes was employed to study activation requirements of resting T cells. Efficient antigen recognition (signal 1) by T lymphocytes requires multimeric antigen receptor triggering because under appropriate experimental conditions soluble ligands do not produce this initial signal for T cell activation. The latter leads to receptiveness for both interleukin 1 (IL 1) and interleukin 2 (IL 2). Importantly, induction of proliferation requires an additional signal (signal 2), namely IL 1, which appears to be required to enable optimal secretion of IL 2. In contrast, presensitized T lymphocytes do not require IL 1 for IL 2 production. In this case, antigen receptor oligomerization is in itself sufficient to induce IL 2 receptor expression, and IL 2 secretion as well.     

Inactivation of a Cdk2 inhibitor during interleukin 2-induced proliferation of human T lymphocytes.

Peripheral blood T lymphocytes require two sequential mitogenic signals to reenter the cell cycle from their natural, quiescent state. One signal is provided by stimulation of the T-cell antigen receptor, and this induces the synthesis of both cyclins and cyclin-dependent kinases (CDKs) that are necessary for progression through G1. Antigen receptor stimulation alone, however, is insufficient to promote activation of G1 cyclin-Cdk2 complexes. This is because quiescent lymphocytes contain an inhibitor of Cdk2 that binds directly to this kinase and prevents its activation by cyclins. The second mitogenic signal, which can be provided by the cytokine interleukin 2, leads to inactivation of this inhibitor, thereby allowing Cdk2 activation and progression into S phase. Enrichment of the Cdk2 inhibitor from G1 lymphocytes by cyclin-CDK affinity chromatography indicates that it may be p27Kip1. These observations show how sequentially acting mitogenic signals can combine to promote activation of cell cycle proteins and thereby cause cell proliferation to start. CDK inhibitors have been shown previously to be induced by signals that negatively regulate cell proliferation. Our new observations show that similar proteins are down-regulated by positively acting signals, such as interleukin 2. This finding suggests that both positive and negative growth signals converge on common targets which are regulators of G1 cyclin-CDK complexes. Inactivation of G1 cyclin-CDK inhibitors by mitogenic growth factors may be one biochemical pathway underlying cell cycle commitment at the restriction point in G1.     

CD3 pathway of T-cell activation. II. Role of HLA-class I molecules in early events

The role of distinct regions of HLA class I molecules in regulating T-cell activation via the CD3-antigen receptor complex was investigated. Monoclonal antibodies (MoAbs) which recognize monomorphic and polymorphic epitopes on HLA Class I molecules were shown to inhibit T-cell proliferation to OKT3. These MoAbs have differential effects on the synthesis of interleukin-2 (IL-2) and IL-2 receptor expression. Cell cycle analysis demonstrated that these MoAbs function both in inhibiting cell cycle entry (G0-G1 shift) and in blocking cell cycle progression (G1-S shift) of activated T cells. Furthermore, these MoAbs have regulatory effects on the alternate pathway of T-cell activation via the CD2 molecule, T-cell activation induced by PHA, and activation induced by the phorbol ester PMA in conjunction with the calcium ionophore Ionomycin. Thus these MoAbs have different effects depending upon the pathway of T-cell activation. The results indicate that HLA class I molecules are selectively involved in the sequence of intracellular events leading to T-cell activation and proliferation.     

The EGF receptor defines domains of cell cycle progression and survival to regulate cell number in the developing Drosophila eye

The number of cells in developing organs must be controlled spatially by extracellular signals. Our results show how cell number can be regulated by cell interactions controlling proliferation and survival in local neighborhoods in the case of the Drosophila compound eye. Intercellular signals act during the second mitotic wave, a cell cycle that generates a pool of uncommitted cells used for most ommatidial fates. We find that G1/S progression to start the cell cycle requires EGF receptor inactivity. EGF receptor activation is then required for progression from G2 to M phase of the same cells, and also prevents apoptosis. EGF receptor activation depends on short-range signals from five-cell preclusters of photoreceptor neurons not participating in the second mitotic wave. Through proliferation and survival control, such signals couple the total number of uncommitted cells being generated to the neural patterning of the retina.