The mammalian target of rapamycin (mTOR) integrates nutrient and mitogen signals to regulate cell growth (increased cell mass and cell size) and cell division. The immunosuppressive drug rapamycin inhibits cell cycle progression via inhibition of mTOR; however, the signaling pathways by which mTOR regulates cell cycle progression have remained poorly defined.

Activation of phosphatidylinositol 3-kinase (PI3K) and activation of the 70/85-kDa S6 protein kinases (␣II and ␣I isoforms, referred to collectively as pp70 S6k ) have been independently linked to the regulation of cell proliferation. We demonstrate that these kinases lie on the same signalling pathway and that PI3K mediates the activation of pp70 by the cytokine interleukin-2 (IL-2). We also show that the activation of pp70 S6k can be blocked at different points along the signalling pathway by using specific inhibitors of T-cell proliferation. Inhibition of PI3K activity with structurally unrelated but highly specific PI3K inhibitors (wortmannin or LY294002) results in inhibition of IL-2-dependent but not phorbol ester (conventional protein kinase C T cells are activated, representing G 0 -to-G 1 transition, by antigen presentation to the multimeric T-cell receptor. This results in the transcription, production, and secretion of the 15-kDa glycoprotein lymphokine, interleukin-2 (IL-2). Antigen stimulation also induces expression of the IL-2 receptor (IL-2R) ␣ subunit (p55) and increases the level of IL-2R ␤ subunit (p75). Together with the ␥ subunit, they form the high-affinity IL-2R (reviewed in references 75 and 103). IL-2 then stimulates activated T cells in an autocrine/paracrine fashion, driving G 1 -S transition and cell proliferation. The IL-2R has no intrinsic kinase activity, yet ligand binding increases tyrosine phosphorylation of many proteins, including the IL-2R ␤ chain. IL-2-dependent signalling also results in activation of c-Ras and phosphatidylinositol 3-kinase (PI3K) and increased serine/ threonine protein phosphorylation. Although much is known about Ras-regulated signal transduction (see references 12, 39, and 68 for reviews), the identities of the signalling proteins lying downstream of PI3K remain to be established.PI3K is a novel signal transducer composed of an 85-kDa SH2-and SH3-domain-containing regulatory subunit and a 110-kDa catalytic subunit with specificity toward the D3 hydroxyl in the inositol ring of phosphatidylinositol (37,54,81,98). Numerous studies provide evidence that PI3K, in association with various mitogenically active receptor and nonreceptor protein tyrosine kinases, mediates the transmission of growth-regulatory information within cells (reviewed in references 18, 21, and 82). These studies suggest that the activation of PI3K contributes a positive, but undefined, cell proliferation signal.The activity of the 70/85-kDa S6 protein kinases (␣I and ␣II isoforms, referred to collectively as pp70 S6k ) is also stimulated by IL-2 in IL-2-responsive cells (16,63,106) as well as in other cell types by many growth factors and oncogenes (reviewed in reference 36). However, the cytosolic mediators involved in its signal cascade have been previously unknown. pp70S6k was identified on the basis of its ability to phosphorylate the 40S ribosomal protein S6 in vitro. A number of other kinases, including the growth-regulated 90-kDa ribosomal S6 kinases (RSKs), can also phosphoryl...

The pp7O/85-kDa S6 kinases, collectively referred to as pp7OS6k, are thought to participate in transit through the GI phase of the cell cycle. pp7Os6k regulates the phosphorylation of the 40S ribosomal protein S6 and the transcription factor CREMT. pp7oS6k is regulated by serine/threonine phosphorylation, and although 1-phosphatidylinositol 3-kinase and phospholipase C have been implicated as upstream regulators, the mechanism of activation and identity of the upstream pp7QS6k kinases remain unknown. To improve our understanding of how this mitogenstimulated protein kinase is regulated by growth factors and the immunosuppressant rapamycin, we have initiated a structure/function analysis of pp79S6k. Our results indicate that both the N and C termini participate in the complex regulation of pp7OS6k activity.Two families of growth factor-regulated serine/threonine kinases that phosphorylate the 40S ribosomal protein S6 in vitro have been previously identified and partially characterized. One of these, the family of 85-92 kDa S6 kinases, referred to collectively as ppgorsk, has been shown to participate in Rasmediated signal transduction and is a direct target of erkencoded MAP kinases (MAPK) (1, 2). The other family, referred to as pp7os6k, consists of two enzymes termed p85a1-and p70aII-S6 kinase. The p85ac isoform is nuclear, while the p70aII isoform is both cytoplasmic and nuclear (ref. 3; M.M.C., unpublished data). Like ppgorsk, pp70S6k is regulated by serine/threonine phosphorylation (4, 5). In contrast to pp9orsk, a pp70S6k kinase has not been identified.

• Cord blood content of ALDH br cells correlates well with CFUs and may act as a surrogate potency assay for cord blood units.• ALDH br cells in segments are assayed rapidly, allowing potency results to be used for release of the unit from a public cord blood bank.Banked, unrelated umbilical cord blood provides access to hematopoietic stem cell transplantation for patients lacking matched bone marrow donors, yet 10% to 15% of patients experience graft failure or delayed engraftment. This may be due, at least in part, to inadequate potency of the selected cord blood unit (CBU). CBU potency is typically assessed before cryopreservation, neglecting changes in potency occurring during freezing and thawing. Colony-forming units (CFUs) have been previously shown to predict CBU potency, defined as the ability to engraft in patients by day 42 posttransplant. However, the CFU assay is difficult to standardize and requires 2 weeks to perform. Consequently, we developed a rapid multiparameter flow cytometric CBU potency assay that enumerates cells expressing high levels of the enzyme aldehyde dehydroge- is a reliable assessment of potency that allows rapid selection and release of CBUs from the cord blood bank to the transplant center for transplantation.

The maintenance and propagation of complex mixtures of cells in vitro in the form of native organs or engineered organoids has contributed to understanding mechanisms of cell and organ development and function which can be translated into therapeutic benefits. For example, allogeneic cultured postnatal human thymus tissue has been shown to support production of naïve recipient T cells when transplanted into patients with complete DiGeorge anomaly and other genetic defects that result in congenital lack of a thymus. Patients receiving such transplants typically exhibit reversal of their immunodeficiency and normalization of their peripheral blood T cell receptor V-beta repertoire, with long-term survival. This study was designed to assess the histopathologic changes that occur in postnatal human thymus slices when cultured according to protocols used for transplanted tissues. Results showed that as thymic organ cultures progressed from days 0 through 21, slices developed increasing amounts of necrosis, increasing condensation of thymic epithelium, and decreasing numbers of residual T cells. The architecture of the thymic epithelial network remained generally well-preserved throughout the 21 days of culture, with focal expression of cytokeratin 14, a putative biomarker of thymic epithelial cells with long-term organ-repopulating potential. All organ slices derived from the same donor thymus closely resembled one another, with minor differences in size, shape, and relative content of cortex versus medulla. Similarly, slices derived from different donors showed similar histopathologic characteristics when examined at the same culture time point. Taken together, these results demonstrate that diagnostic criteria based on structural features of the tissue identifiable via hematoxylin and eosin staining and cytokeratin immunohistochemistry can be used to evaluate the quality of slices transplanted into patients with congenital athymia.