FoxM1 is a forkhead box transcription factor and a known master regulator required for different phases of the cell cycle. In cell lines, FoxM1 deficient cells exhibit delayed S phase entry, aneuploidy, polyploidy and can't complete mitosis. In vivo, FoxM1 is expressed mostly in proliferating cells but is surprisingly also found in non-proliferating CD4+CD8+ double positive thymocytes. Here, we addressed the role of FoxM1 in T cell development by generating and analyzing two different lines of T-cell specific FoxM1 deficient mice. As expected, FoxM1 is required for proliferation of early thymocytes and activated mature T cells. Defective expression of many cell cycle proteins was detected, including cyclin A, cyclin B1, cdc2, cdk2, p27 and the Rb family members p107 and p130 but surprisingly not survivin. Unexpectedly, loss of FoxM1 only affects a few cell cycle proteins in CD4+CD8+ thymocytes and has little effect on their sensitivity to apoptosis and the subsequent steps of T cell differentiation. Thus, regulation of cell cycle genes by FoxM1 is stage- and context-dependent.

The PI3K-AKT pathway can mediate diverse biological responses and is crucial for optimal immune responses and lymphocyte development. Deletion of PI3K subunits or AKT leads to blockage of T-cell development at the TCR-b checkpoint. Studies with over-expression of constitutively activated AKT have implicated this pathway in anti-apoptosis of developing thymocytes and in development of regulatory T cells. However, the role of endogenous PI3K-AKT in T-cell development beyond the TCR-b checkpoint remains unclear. Here, we inhibited the endogenous PI3K-AKT pathway in thymocytes after double negative stages by expressing the negative regulator, PTEN. These mice exhibit normal early T-cell development, but the transition from intermediate single positive to double positive (DP) thymocytes is inhibited, leading to a significantly decreased number of DP, single positive thymocytes and peripheral T cells. Proliferation of peripheral T cells is reduced but apoptosis of DP cells and subsequent T-cell maturation, including regulatory T cells, are normal. AKT phosphorylation can be readily observed in most WT T-cell compartments but not DP thymocytes in response to TCR activation. Thus, the PI3K-AKT pathway is crucial for the transition of intermediate single positive to DP thymocytes but is dispensable for apoptosis and maturation of developing thymocytes.Key words: AKT . Apoptosis . Proliferation . PTEN . Thymocytes IntroductionActivation of the PI3K pathway by growth factor receptors is one of the major signaling events leading to growth, proliferation and anti-apoptosis. PI3K activation results in an increase of the lipid phosphatidylinositol-3,4,5-triphosphate, which recruits a lipidbinding domain containing kinases PDK1 and AKT to the membrane [1][2][3]. PDK1 activates AKT [4] and SGK, a serum and glucocorticoid-induced kinase with a similar structure as AKT. AKT has many direct substrates, including the cell cycle inhibitor p27, Foxo family of transcription factors, the proapoptotic Bcl-2 family protein Bad and TSC1, a protein that inhibits the TOR pathway [2,4]. Phosphorylation of these substrates by AKT leads to cell cycle entry, resistance to apoptosis, increase in cell size and other growth-related events. Unlike AKT, SGK has more restricted substrates but it also phosphorylates the Foxo family of transcription factors to promote cell survival and cell cycle entry [5].Regulation of the PI3K signaling pathway is important for normal differentiation; its dysregulation can lead to cancer. PTEN is a lipid phosphatase and a major negative regulator of the PI3K pathway [2,6,7]. Loss of PTEN in both alleles is frequently found in many human cancer cells. Germline heterozygous mutation of PTEN is present in patients with the Cowden syndrome [8,9], who develop hyperplastic lesions in multiple organs with increased risks of cancer. In mice, heterozygous mutation of PTEN eventually leads to development of malignancy in different organs, including thymus, prostate, thyroid, liver and intestines [10][11][12]. In T cells, condi...

Proliferation and apoptosis are two opposing processes. In the first instance, cells overcoming the G 1 checkpoint will enter the S phase, replicate their DNA and undergo mitosis, resulting in separation of their daughter cells. Different cell cycle phases have specific cell cycle protein expression profiles (1, 2). Cyclindependent kinases (CDK) 2 are master regulators of cell proliferation. Their activities are in turn regulated by CDK activating kinases, cyclins, and the CDK inhibitors. CDK2 is crucial for cell cycle progression in cell lines and functions in the G 1 /S transition and the S and G 2 phases of the cell cycle. In most cells, mitogenic stimulation leads to increased transcription of the first G 1 kinases, CDK4 or CDK6 and cyclin D. The increased protein levels of CDK4⅐CDK6⅐cyclin D titrate p27 away from the inactive CDK2⅐cyclin E⅐p27 complex, resulting in activation of the CDK2⅐cyclin E complex. CDK2 mediates its functions through phosphorylation of many substrates, including Rb and p27. Rb phosphorylation releases E2F from its pocket, allowing E2F to activate transcription of S phase-related genes (3, 4). p27 phosphorylation initiates its own eventual degradation through the Skp2 ubiquitin E3 ligase (1, 2

Humanized monoclonal antibody KD-247 targets the Gly(312)-Pro(313)-Gly(314)-Arg(315) arch of the third hypervariable (V3) loop of the HIV-1 surface glycoprotein. It potently neutralizes many HIV-1 clade B isolates, but not of other clades. To understand the molecular basis of this specificity, we solved a high-resolution (1.55 Å) crystal structure of the KD-247 antigen binding fragment and examined the potential interactions with various V3 loop targets. Unlike most antibodies, KD-247 appears to interact with its target primarily through light chain residues. Several of these interactions involve Arg(315) of the V3 loop. To evaluate the role of light chain residues in the recognition of the V3 loop, we generated 20 variants of KD-247 single-chain variable fragments with mutations in the antigen-binding site. Purified proteins were assessed for V3 loop binding using AlphaScreen technology and for HIV-1 neutralization. Our data revealed that recognition of the clade-specificity defining residue Arg(315) of the V3 loop is based on a network of interactions that involve Tyr(L32), Tyr(L92), and Asn(L27d) that directly interact with Arg(315), thus elucidating the molecular interactions of KD-247 with its V3 loop target.