p70(s6k) integrates phosphatidylinositol 3-kinase and rapamycin-regulated signals for E2F regulation in T lymphocytes.

In T lymphocytes, the hematopoietic cytokine interleukin-2 (IL-2) uses phosphatidylinositol 3-kinase (PI 3-kinase)-induced signaling pathways to regulate E2F transcriptional activity, a critical cell cycle checkpoint. PI 3-kinase also regulates the activity of p70(s6k), the 40S ribosomal protein S6 kinase, a response that is abrogated by the macrolide rapamycin. This immunosuppressive drug is known to prevent T-cell proliferation, but the precise point at which rapamycin regulates T-cell cycle progression has yet to be elucidated. Moreover, the effects of rapamycin on, and the role of p70(s6k) in, IL-2 and PI 3-kinase activation of E2Fs have not been characterized. Our present results show that IL-2- and PI 3-kinase-induced pathways for the regulation of E2F transcriptional activity include both rapamycin-resistant and rapamycin-sensitive components. Expression of a rapamycin-resistant mutant of p70(s6k) in T cells could restore rapamycin-suppressed E2F responses. Thus, the rapamycin-controlled processes involved in E2F regulation appear to be mediated by p70(s6k). However, the rapamycin-resistant p70(s6k) could not rescue rapamycin inhibition of T-cell cycle entry, consistent with the involvement of additional, rapamycin-sensitive pathways in the control of T-cell cycle progression. The present results thus show that p70(s6k) is able to regulate E2F transcriptional activity and provide direct evidence for the first time for a link between IL-2 receptors, PI 3-kinase, and p70(s6k) that regulates a crucial G1 checkpoint in T lymphocytes.

Phosphatidylinositol 3-kinase couples the interleukin-2 receptor to the cell cycle regulator E2F.

Cell cycle progression initiated by interleukin-2 (IL-2) in T cells is critical for lymphoproliferation and an immune response. Phosphatidyl inositol 3-kinase (PI3K) is activated by IL-2. However, nuclear targets for PI3K are not known. Here we identify the cell cycle regulator E2F as an IL-2 target in T lymphocytes and PI3K as the critical signaling pathway. We eliminate both Stat5 and Raf/MEK pathways from E2F regulation. Protein kinase B (PKB) is activated by IL-2 via PI3K. The expression of an active PKB is sufficient to induce E2F activity. Inhibition of PI3K inhibits phosphorylation of Rb, induction of cyclin D3, and degradation of p27kip1. These results establish a crucial PI3K/PKB-mediated link between the IL-2 teceptor and the cell cycle machinery.

Interleukin-2 activation of STAT5 requires the convergent action of tyrosine kinases and a serine/threonine kinase pathway distinct from the Raf1/ERK2 MAP kinase pathway.

Interleukin-2 (IL-2) induces DNA binding of STAT5, a member of the family of cytokine-regulated transcription factors termed 'signal transducers and activators of transcription'. IL-2-stimulated STAT5-DNA complexes include two tyrosine phosphoproteins which exhibit distinct mobilities in SDS-PAGE gels. Our studies have shown that IL-2 rapidly induces both tyrosine phosphorylation and serine phosphorylation of STAT5 and that the two STAT5 tyrosine phosphoproteins detected in IL-2-activated cells differ in their levels of phosphorylation on serine residues. The two different phosphoforms of STAT5 have identical in vitro DNA binding specificity and reactivity with tyrosine phosphopeptides, but differ in their cellular localization. As well, the present data indicate that the transcriptional activity of STAT5 is regulated by serine kinases in T lymphocytes. Two previously characterized serine kinases activated by IL-2, MAP kinase/ERK2 and p70 S6 kinase, do not appear to be involved in STAT5 regulation by this cytokine. Accordingly, STAT5 activation in T cells requires the convergent action of tyrosine kinases and a distinct serine/threonine kinase which has not previously been implicated in IL-2 signalling.

Defective in vitro antibody production in response to pokeweed mitogen and influenza antigen in patients with Hodgkin's disease.

Cultures of peripheral blood mononuclear cells (PBM) from 33 patients with Hodgkin's disease, were stimulated in vitro with pokeweed mitogen (PWM) or influenza antigen. Impaired production of immunoglobulin (Ig) of one or more of the three main classes (IgG, IgM and IgA) in PWM stimulated cultures was found in 22 patients and in 11 patients no Ig of any class was produced. Antibody to influenza virus was detected in PWM stimulated PBM cultures in 13 of 14 normal individuals, but in only four of 25 patients with treated Hodgkin's disease though IgG was produced in 16 of 25. Influenza antigen induced anti-influenza antibody production in 10 of 12 cultures from normal individuals but in only two of 22 from patients. The results confirm our earlier report of defective antibody production in vitro by PBM from patients with Hodgkin's disease and indicate that polyclonally activated production of immunoglobulins of several classes is defective, though in vivo humoral immunity is normal.

Increased therapeutic efficiency of a lipid-soluble alkylating agent incorporated in liposomes.

A highly hydrophobic alkylating agent, 1-N,N-bis(beta-bromoethyl) amino-3-methylnaphthalene, given as the free drug in oil, cured a substantial proportion of mice bearing the PC6 myeloma in the dose range 2-7 mg/kg. However, these doses were toxic, and the LD50 was 6-7 mg/kg. When incorporated in liposomes, similar curative effects were obtained at doses of 10-41 mg/kg without material toxicity, even at the highest dose. Liposome entrapment therefore greatly increases the therapeutic efficiency of this agent.

Thymus-dependent and thymus-independent antibody responses: contrasting patterns of immunosuppression.

The effects of mitoclomine, an anti-tumour agent, and of cortisone acetate on the antibody response of mice to thymus-dependent and thymus-independent antigens were compared. Both agents yielded patterns of immunosuppression which differed from that seen after X-rays, an agent which is likely to be active equally against T and B cells. Mitoclomine, at low doses, depressed thymus-independent responses the most; this effect can be explained if the drug is more active against B than T cells. Cortisone acetate at low doses, while depressing thymus-dependent responses, increased thymus-independent responses; this can be explained if the drug is more active against T than B cells. Thus three different patterns of immunosuppression (for X-rays, mitoclomine and cortisone acetate) can be interpreted in terms of effects simply on T or B cells.

A raft technique for chemotaxis: a versatile method suitable for clinical studies.

We describe a raft technique for the study of chemotaxis which possesses a number of advantages over the use of chambers. Membranes are laid on pads soaked in chemotactic agent and cells are contained in plastic cups, which are inverted on to the membrane. This method gives results which are similar to those obtained in conventional vessels. It would seem well suited to comparative measurements of chemotaxis in clinical series, for it avoids the use of special chambers, minimises the number of membranes and their handling and more than one cell suspension can be placed on a membrane. In addition, the method is experimentally versatile: simple manipulations allow work with cells adherent to other surfaces, studies of the effect of substituting a new agent or of reversing the gradient during an experiment, migration against gravity, and of the effect of non-adherent cells on migrating cells.

Antibody response by cells cultured on dialysis membrane in disposable plastic vessels.

Plastic vessels were used to culture mouse spleen cells by the technique of Marbrook (1967) and good primary antibody responses were obtained. These vessels some advantages over glassware, which may allow a wider use of this technique.