CD4 + and CD8 + T cell functions are rapidly aborted during chronic infection, preventing viral clearance. CD4 + T cell help is required throughout chronic infection so as to sustain CD8 + T cell responses; however, the necessary factor(s) provided by CD4 + T cells are currently unknown. Using a mouse model of chronic viral infection, we demonstrated that interleukin-21 (IL-21) is an essential component of CD4 + T cell help. In the absence of IL-21 signaling, despite elevated CD4 + T cell responses, CD8 + T cell responses are severely impaired. CD8 + T cells directly require IL-21 to avoid deletion, maintain immunity, and resolve persistent infection. Thus, IL-21 specifically sustains CD8 + T cell effector activity and provides a mechanism of CD4 + T cell help during chronic viral infection.During chronic viral infections, antiviral CD4 + and CD8 + T cells become non-responsive to viral antigens and are either physically deleted or persist in a nonfunctional "exhausted" state, unable to produce important antiviral and immunostimulatory cytokines such as interleukin-2 (IL-2), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ); lyse virally infected cells; or proliferate (1-4). To date, the majority of work analyzing T cell exhaustion during chronic viral infection has focused on CD8 + T cells; however, CD4 + T cells also exhibit reduced function, thus further exacerbating viral persistence. CD4 + T cells are required throughout chronic viral infection in order to sustain CD8 + T cell function and control infection, prevent deletion of high-affinity antiviral CD8 + T cells, and eventually resolve the infection (5-7). The loss of CD4 + and CD8 + T cell function is associated with viruses that establish chronic viral infections in their hosts: HIV and hepatitis B and hepatitis C virus (HCV) in humans and lymphocytic chorio-meningitis virus (LCMV) in rodents (8)(9)(10)(11)(12). Chronic LCMV infection is eventually controlled in the periphery 60 to 80 days after infection in a CD4 + T cell-dependent manner, suggesting that exhausted CD4 + T cells retain helper activity. Expression of the key CD4 + T cell helper cytokine IL-2, however, is rapidly extinguished during viral persistence (4,13), indicating that an alternative helper mechanism is being implemented.To determine how CD4 + T cells help CD8 + T cells to clear a viral infection, we infected mice with the Armstrong strain of LCMV (Arm) that induces a robust T cell response, resulting in viral clearance within 8 to 10 days or with the Clone 13 strain of LCMV (Cl 13) that generates a chronic infection due to the up-regulation of immunosuppressive factors that induce a dramatic depletion and inactivation of virus-specific T cells (14-18). LCMV-Arm and -Cl 13 share identical CD4 + and CD8 + T cell epitopes, enabling direct comparison of

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Most cell types in multicellular eukaryotes exit from the mitotic cell cycle before terminal differentiation. We show that the dacapo gene is required to arrest the epidermal cell proliferation at the correct developmental stage during Drosophila embryogenesis. dacapo encodes an inhibitor of cyclin E/cdk2 complexes with similarity to the vertebrate Cip/Kip inhibitors. dacapo is transiently expressed beginning late in the G2 phase preceding the terminal division (mitosis 16). Mutants unable to express the inhibitor fail to arrest cell proliferation after mitosis 16 and progress through an extra division cycle. Conversely, premature dacapo expression in transgenic embryos results in a precocious G1 arrest.

The reversible phosphorylation of tyrosine residues is an important mechanism for modulating biological processes such as cellular signaling, differentiation, and growth, and if deregulated, can result in various types of cancer. Therefore, an understanding of these dynamic cellular processes at the molecular level requires the ability to assess changes in the sites of tyrosine phosphorylation across numerous proteins simultaneously as well as over time. Here we describe a sensitive approach based on multidimensional liquid chromatography͞ mass spectrometry that enables the rapid identification of numerous sites of tyrosine phosphorylation on a number of different proteins from human whole cell lysates. We used this methodology to follow changes in tyrosine phosphorylation patterns that occur over time during either the activation of human T cells or the inhibition of the oncogenic BCR-ABL fusion product in chronic myelogenous leukemia cells in response to treatment with STI571 (Gleevec). Together, these experiments rapidly identified 64 unique sites of tyrosine phosphorylation on 32 different proteins. Half of these sites have been documented in the literature, validating the merits of our approach, whereas motif analysis suggests that a number of the undocumented sites are also potentially involved in biological pathways. This methodology should enable the rapid generation of new insights into signaling pathways as they occur in states of health and disease.M any cellular processes are directly controlled through the reversible phosphorylation of protein tyrosine residues. These regulatory functions are ultimately affected through the coordinated phosphorylation of numerous tyrosine residues across multiple proteins over time. Clearly, there are benefits to individually characterizing specific components of a particular pathway, such as identifying a site of phosphorylation on a given protein, the kinase responsible for the modification, or the identity of subsequently interacting proteins. Ultimately, though, a thorough understanding of these signaling pathways at the molecular level requires the wide-scale, simultaneous evaluation of these phosphorylation events as they occur over time.To date, two-dimensional gel electrophoresis (2D-GE) remains the most common methodology for assessing wide-scale changes in phosphorylation (1). However, this methodology is relatively slow, and suffers from a number of well documented operational limitations. For example, 2D-GE has been shown to be poorly suited for the direct detection and analysis of medium to low abundance proteins from whole cell lysates, a particular concern in the case of regulatory proteins such as kinases, which often exist at very low copy numbers per cell (2). Even with the improved dynamic range afforded by multiple 2D-GE runs of prefractionated samples, the individual gelisolated proteins still require further characterization by using methods such as two-dimensional tryptic phosphopeptide mapping (3), Edman degradation (4), or precursor ion scan...

SUMMARY Although deficient CD8 T cell responses have long been associated with chronic viral infections, the underlying mechanisms are still unclear. Here we report that enhanced and sustained TGF-β/Smad signaling is a distinctive feature of virus-specific CD8 T cells during chronic versus acute viral infections in vivo. The result is TGF-β-dependent up-regulation of the pro-apoptotic protein Bim that relates to cell-intrinsic apoptosis and significantly reduced numbers of virus-specific CD8 T cells. Moreover, selective attenuation of TGF-β signaling on T cells increases the function of CD8 T cells in an indirect fashion, rapidly eradicates the persistence-prone virus and enables the generation of an effective memory response. Our findings reveal persisting TGF-β/Smad signaling as a hallmark and key regulator of virus-specific CD8 T cell responses during chronic viral infections in vivo.

Pleckstrin homology (PH) domain-mediated protein recruitment to cellular membranes is of paramount importance for signal transduction. The recruitment of many PH domains is controlled through production and turnover of their membrane ligand, phosphatidylinositol 3,4,5-trisphosphate (PIP3). We show that phosphorylation of the second messenger inositol 1,4,5-trisphosphate (IP3) into inositol 1,3,4,5-tetrakisphosphate (IP4) establishes another mode of PH domain regulation through a soluble ligand. At physiological concentrations, IP4 promoted PH domain binding to PIP3. In primary mouse CD4+CD8+ thymocytes, this was required for full activation of the protein tyrosine kinase Itk after T cell receptor engagement. Our data suggest that IP4 establishes a feedback loop of phospholipase C-gamma1 activation through Itk that is essential for T cell development.

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Themis (Thymocyte expressed molecule involved in selection), a member of a family of proteins with unknown functions, is highly conserved among vertebrates. Here we found that Themis is expressed in high amounts in thymocytes between the pre-T cell receptor (TCR) and positive selection checkpoints, and in low amounts in mature T cells. Themis-deficient thymocytes exhibit defective positive selection, which results in reduced numbers of mature thymocytes. Negative selection is also impaired in Themis-deficient mice. A higher percentage of Themis-deficient T cells exhibit CD4+CD25+Foxp3+ regulatory and CD62LloCD44hi memory phenotypes than in wild-type mice. Supporting a role for Themis in TCR signaling, this protein is phosphorylated quickly after TCR stimulation, and is needed for optimal TCR-driven Ca2+ mobilization and Erk activation.