Michael F. Radonovich

Synergy of IL-21 and IL-15 in regulating CD8+ T cell expansion and function

Interleukin (IL)-21 is the most recently recognized of the cytokines that share the common cytokine receptor γ chain (γc), which is mutated in humans with X-linked severe combined immunodeficiency. We now report that IL-21 synergistically acts with IL-15 to potently promote the proliferation of both memory (CD44high) and naive (CD44low) phenotype CD8+ T cells and augment interferon-γ production in vitro. IL-21 also cooperated, albeit more weakly, with IL-7, but not with IL-2. Correspondingly, the expansion and cytotoxicity of CD8+ T cells were impaired in IL-21R−/− mice. Moreover, in vivo administration of IL-21 in combination with IL-15 boosted antigen-specific CD8+ T cell numbers and resulted in a cooperative effect on tumor regression, with apparent cures of large, established B16 melanomas. Thus, our studies reveal that IL-21 potently regulates CD8+ T cell expansion and effector function, primarily in a synergistic context with IL-15.

Characterization and isolation of stem cell–enriched human hair follicle bulge cells

The human hair follicle bulge is an important niche for keratinocyte stem cells (KSCs). Elucidation of human bulge cell biology could be facilitated by analysis of global gene expression profiles and identification of unique cell-surface markers. The lack of distinctive bulge morphology in human hair follicles has hampered studies of bulge cells and KSCs. In this study, we determined the distribution of label-retaining cells to define the human anagen bulge. Using navigated laser capture microdissection, bulge cells and outer root sheath cells from other follicle regions were obtained and analyzed with cDNA microarrays. Gene transcripts encoding inhibitors of WNT and activin/bone morphogenic protein signaling were overrepresented in the bulge, while genes responsible for cell proliferation were underrepresented, consistent with the existence of quiescent noncycling KSCs in anagen follicles. Positive markers for bulge cells included CD200, PHLDA1, follistatin, and frizzled homolog 1, while CD24, CD34, CD71, and CD146 were preferentially expressed by non-bulge keratinocytes. Importantly, CD200+ cells (CD200hiCD24loCD34loCD71loCD146lo) obtained from hair follicle suspensions demonstrated high colony-forming efficiency in clonogenic assays, indicating successful enrichment of living human bulge stem cells. The stem cell behavior of enriched bulge cells and their utility for gene therapy and hair regeneration will need to be assessed in in vivo assays.

Tat modifies the activity of CDK9 to phosphorylate serine 5 of the RNA polymerase II carboxyl-terminal domain during human immunodeficiency virus type 1 transcription.

ABSTRACT Tat stimulates human immunodeficiency virus type 1 (HIV-1) transcriptional elongation by recruitment of carboxyl-terminal domain (CTD) kinases to the HIV-1 promoter. Using an immobilized DNA template assay, we have analyzed the effect of Tat on kinase activity during the initiation and elongation phases of HIV-1 transcription. Our results demonstrate that cyclin-dependent kinase 7 (CDK7) (TFIIH) and CDK9 (P-TEFb) both associate with the HIV-1 preinitiation complex. Hyperphosphorylation of the RNA polymerase II (RNAP II) CTD in the HIV-1 preinitiation complex, in the absence of Tat, takes place at CTD serine 2 and serine 5. Analysis of preinitiation complexes formed in immunodepleted extracts suggests that CDK9 phosphorylates serine 2, while CDK7 phosphorylates serine 5. Remarkably, in the presence of Tat, the substrate specificity of CDK9 is altered, such that the kinase phosphorylates both serine 2 and serine 5. Tat-induced CTD phosphorylation by CDK9 is strongly inhibited by low concentrations of 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole, an inhibitor of transcription elongation by RNAP II. Analysis of stalled transcription elongation complexes demonstrates that CDK7 is released from the transcription complex between positions +14 and +36, prior to the synthesis of transactivation response (TAR) RNA. In contrast, CDK9 stays associated with the complex through +79. Analysis of CTD phosphorylation indicates a biphasic modification pattern, one in the preinitiation complex and the other between +36 and +79. The second phase of CTD phosphorylation is Tat-dependent and TAR-dependent. These studies suggest that the ability of Tat to increase transcriptional elongation may be due to its ability to modify the substrate specificity of the CDK9 complex.

Activated AKT regulates NF-kappaB activation, p53 inhibition and cell survival in HTLV-1-transformed cells.

AKT activation enhances resistance to apoptosis and induces cell survival signaling through multiple downstream pathways. We now present evidence that AKT is activated in HTLV-1-transformed cells and that Tax activation of AKT is linked to NF-κB activation, p53 inhibition and cell survival. Overexpression of AKT wild type (WT), but not a kinase dead (KD) mutant, resulted in increased Tax-mediated NF-κB activation. Blocking AKT with the PI3K/AKT inhibitor LY294002 or AKT SiRNA prevented NF-κB activation and inhibition of p53. Treatment of C81 cells with LY294002 resulted in an increase in the p53-responsive gene MDM2, suggesting a role for AKT in the Tax-mediated regulation of p53 transcriptional activity. Further, we show that LY294002 treatment of C81 cells abrogates in vitro IKKβ phosphorylation of p65 and causes a reduction of p65 Ser-536 phosphorylation in vivo, steps critical to p53 inhibition. Interestingly, blockage of AKT function did not affect IKKβ phosphorylation of IκBα in vitro suggesting selective activity of AKT on the IKKβ complex. Finally, AKT prosurvival function in HTLV-1-transformed cells is linked to expression of Bcl-xL. We suggest that AKT plays a role in the activation of prosurvival pathways in HTLV-1-transformed cells, possibly through NF-κB activation and inhibition of p53 transcription activity.

Inactivation of p53 by Human T-Cell Lymphotropic Virus Type 1 Tax Requires Activation of the NF-κB Pathway and Is Dependent on p53 Phosphorylation

ABSTRACT p53 plays a key role in guarding cells against DNA damage and transformation. We previously demonstrated that the human T-cell lymphotropic virus type 1 (HTLV-1) Tax can inactivate p53 transactivation function in lymphocytes. The present study demonstrates that in T cells, Tax-induced p53 inactivation is dependent upon NF-κB activation. Analysis of Tax mutants demonstrated that Tax inactivation of p53 function correlates with the ability of Tax to induce NF-κB but not p300 binding or CREB transactivation. The Tax-induced p53 inactivation can be overcome by overexpression of a dominant IκB mutant. Tax-NF-κB-induced p53 inactivation is not due to p300 squelching, since overexpression of p300 does not recover p53 activity in the presence of Tax. Further, using wild-type and p65 knockout mouse embryo fibroblasts (MEFs), we demonstrate that the p65 subunit of NF-κB is critical for Tax-induced p53 inactivation. While Tax can inactivate endogenous p53 function in wild-type MEFs, it fails to inactivate p53 function in p65 knockout MEFs. Importantly, Tax-induced p53 inactivation can be restored by expression of p65 in the knockout MEFs. Finally, we present evidence that phosphorylation of serines 15 and 392 correlates with inactivation of p53 by Tax in T cells. This study provides evidence that the divergent NF-κB proliferative and p53 cell cycle arrest pathways may be cross-regulated at several levels, including posttranslational modification of p53.

A Novel NF-κB Pathway Involving IKKβ and p65/RelA Ser-536 Phosphorylation Results in p53 Inhibition in the Absence of NF-κB Transcriptional Activity

Nuclear factor κB (NF-κB) plays an important role in regulating cellular transformation and apoptosis. The human T-cell lymphotropic virus type I protein, Tax, which is critical for viral transformation, modulates the transcription of several cellular genes through activation of NF-κB. We have demonstrated previously that Tax inhibits p53 activity through the p65/RelA subunit of NF-κB. We now present evidence that suggests that the upstream kinase IKKβ plays an important role in Tax-induced p53 inhibition through phosphorylation of p65/RelA at Ser-536. First, mouse embryo fibroblast (MEF) IKKβ–/–cells did not support Tax-mediated p53 inhibition, whereas MEFs lacking IKKα allowed Tax inhibition of p53. Second, transfection of IKKβ wild type (WT), but not a kinase-dead mutant, into IKKβ–/–cells rescued p53 inhibition by Tax. Third, the IKKβ-specific inhibitor SC-514 decreased the ability of Tax to inhibit p53. Fourth, we show that phosphorylation of p65/RelA at Ser-536 is important for Tax inhibition of p53 using MEF p65/RelA–/–cells transfected with p65/RelA WT or mutant plasmids. Moreover, Tax induced p65/RelA Ser-536 phosphorylation in WT or IKKα–/– cells but failed to induce the phosphorylation of p65/RelA Ser-536 in IKKβ–/–cells, suggesting a link between IKKβ and p65/RelA phosphorylation. Consistent with this observation, blocking IKKβ kinase activity by SC-514 decreases the phosphorylation of p65/RelA at Ser-536 in the presence of Tax in human T-cell lymphotropic virus type I-transformed cells. Finally, the ability of Tax to inhibit p53 is distinguished from the NF-κB transcription activation pathway. Our work, therefore, describes a novel Tax-NF-κB p65/RelA pathway that functions to inhibit p53 but does not require NF-κB transcription activity.

Stat5 Synergizes with T Cell Receptor/Antigen Stimulation in the Development of Lymphoblastic Lymphoma

Signal transducer and activator of transcription (STAT) proteins are latent transcription factors that mediate a wide range of actions induced by cytokines, interferons, and growth factors. We now report the development of thymic T cell lymphoblastic lymphomas in transgenic mice in which Stat5a or Stat5b is overexpressed within the lymphoid compartment. The rate of lymphoma induction was markedly enhanced by immunization or by the introduction of TCR transgenes. Remarkably, the Stat5 transgene potently induced development of CD8+ T cells, even in mice expressing a class II–restricted TCR transgene, with resulting CD8+ T cell lymphomas. These data demonstrate the oncogenic potential of dysregulated expression of a STAT protein that is not constitutively activated, and that TCR stimulation can contribute to this process.

Human T-lymphotropic Virus Type I Tax Protein Utilizes Distinct Pathways for p53 Inhibition That Are Cell Type-dependent

p53 plays a pivotal role in transmitting signals from many forms of genotoxic stress to genes and factors that control the cell cycle and apoptosis. We have previously shown that the human T-lymphotropic virus type I Tax protein can inhibit p53 function. Recently we reported that Tax inhibits p53 function in Jurkat cells and mouse embryo fibroblasts through a mechanism involving the nuclear factor κB pathway and correlates with phosphorylation on serines 15 and 392 of p53. However, several groups have also observed a mechanism that correlates with p300 binding of Tax. To address this controversy and to determine the mechanism by which Tax inhibits p53 function, we examined the activation functions of Tax required for p53 inhibition. In HeLa and H1299 cells the cAMP-response element-binding protein/activating transcription factor activation function is essential, as demonstrated by the Tax mutants M47 and K88A. In addition, expression of exogenous p300 in H1299 cells allows full recovery of p53 transactivation in the presence of Tax. Consistent with p300 being a limiting factor in H1299, Saos-2, and HeLa cells, we found that the level of endogenous p300 is relatively low in these cells compared with Jurkat cells or the human T-lymphotropic virus type I-infected C81 and MT2 cells. Thus our data suggests that Tax utilizes distinct mechanisms to inhibit p53 function that are cell type-dependent.

Acetylation of Nucleosomal Histones by p300 Facilitates Transcription from Tax-Responsive Human T-Cell Leukemia Virus Type 1 Chromatin Template

ABSTRACT Expression of human T-cell leukemia virus type 1 (HTLV-1) is regulated by the viral transcriptional activator Tax. Tax activates viral transcription through interaction with the cellular transcription factor CREB and the coactivators CBP/p300. One key property of the coactivators is the presence of histone acetyltransferase (HAT) activity, which enables p300/CBP to modify nucleosome structure. The data presented in this manuscript demonstrate that full-length p300 and CBP facilitate transcription of a reconstituted chromatin template in the presence of Tax and CREB. The ability of p300 and CBP to activate transcription from the chromatin template is dependent upon the HAT activity. Moreover, the coactivator HAT activity must be tethered to the template by Tax and CREB, since a p300 mutant that fails to interact with Tax did not facilitate transcription or acetylate histones. p300 acetylates histones H3 and H4 within nucleosomes located in the promoter and 5′ proximal regions of the template. Nucleosome acetylation is accompanied by an increase in the level of binding of RNA polymerase II transcription factor TFIID and RNA polymerase II to the promoter. Interestingly, we found distinct transcriptional activities between CBP and p300. CBP, but not p300, possesses an N-terminal activation domain which directly activates Tax-mediated HTLV-1 transcription from a naked DNA template. Finally, using the chromatin immunoprecipitation assay, we provide the first direct experimental evidence that p300 and CBP are associated with the HTLV-1 long terminal repeat in vivo.

A 36-kilodalton cellular transcription factor mediates an indirect interaction of human T-cell leukemia/lymphoma virus type I TAX1 with a responsive element in the viral long terminal repeat.

The human T-cell leukemia/lymphoma virus type I (HTLV-I) trans activator, TAX1, interacts indirectly with a TAX1-responsive element, TRE-2, located at positions -117 to -163 in the viral long terminal repeat. This report describes the characterization of a 36-kilodalton (kDa) protein identified in HeLa nuclear extract which mediates the interaction of TAX1 with TRE-2. Purification of the protein was achieved by zinc chelate chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The renatured 36-kDa protein bound specifically to a TRE-2 oligonucleotide but not to nonfunctional base substitution mutant probes in a gel retardation assay. Renatured proteins of differing molecular weights were unable to form this complex. In addition, the 36-kDa protein specifically activated transcription from the HTLV-I promoter in vitro. Purified TAX1 protein formed a complex with the TRE-2 oligonucleotide in the presence of the 36-kDa protein, suggesting that indirect interaction of TAX1 with the viral long terminal repeat may be one of the mechanisms by which HTLV-I transcription is regulated.