John N. Brady

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.

Expression Profiling of Serous Low Malignant Potential, Low-Grade, and High-Grade Tumors of the Ovary

Papillary serous low malignant potential (LMP) tumors are characterized by malignant features and metastatic potential yet display a benign clinical course. The role of LMP tumors in the development of invasive epithelial cancer of the ovary is not clearly defined. The aim of this study is to determine the relationships among LMP tumors and invasive ovarian cancers and identify genes contributing to their phenotypes. Affymetrix U133 Plus 2.0 microarrays (Santa Clara, CA) were used to interrogate 80 microdissected serous LMP tumors and invasive ovarian malignancies along with 10 ovarian surface epithelium (OSE) brushings. Gene expression profiles for each tumor class were used to complete unsupervised hierarchical clustering analyses and identify differentially expressed genes contributing to these associations. Unsupervised hierarchical clustering analysis revealed a distinct separation between clusters containing borderline and high-grade lesions. The majority of low-grade tumors clustered with LMP tumors. Comparing OSE with high-grade and LMP expression profiles revealed enhanced expression of genes linked to cell proliferation, chromosomal instability, and epigenetic silencing in high-grade cancers, whereas LMP tumors displayed activated p53 signaling. The expression profiles of LMP, low-grade, and high-grade papillary serous ovarian carcinomas suggest that LMP tumors are distinct from high-grade cancers; however, they are remarkably similar to low-grade cancers. Prominent expression of p53 pathway members may play an important role in the LMP tumor phenotype.

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.

A gene signature predicting for survival in suboptimally debulked patients with ovarian cancer.

Despite the existence of morphologically indistinguishable disease, patients with advanced ovarian tumors display a broad range of survival end points. We hypothesize that gene expression profiling can identify a prognostic signature accounting for these distinct clinical outcomes. To resolve survival-associated loci, gene expression profiling was completed for an extensive set of 185 (90 optimal/95 suboptimal) primary ovarian tumors using the Affymetrix human U133A microarray. Cox regression analysis identified probe sets associated with survival in optimally and suboptimally debulked tumor sets at a P value of <0.01. Leave-one-out cross-validation was applied to each tumor cohort and confirmed by a permutation test. External validation was conducted by applying the gene signature to a publicly available array database of expression profiles of advanced stage suboptimally debulked tumors. The prognostic signature successfully classified the tumors according to survival for suboptimally (P = 0.0179) but not optimally debulked (P = 0.144) patients. The suboptimal gene signature was validated using the independent set of tumors (odds ratio, 8.75; P = 0.0146). To elucidate signaling events amenable to therapeutic intervention in suboptimally debulked patients, pathway analysis was completed for the top 57 survival-associated probe sets. For suboptimally debulked patients, confirmation of the predictive gene signature supports the existence of a clinically relevant predictor, as well as the possibility of novel therapeutic opportunities. Ultimately, the prognostic classifier defined for suboptimally debulked tumors may aid in the classification and enhancement of patient outcome for this high-risk population.

IL-2 negatively regulates IL-7 receptor α chain expression in activated T lymphocytes

Interleukin (IL)-2 is a type I four-α-helical bundle cytokine that plays vital roles in antigen-mediated proliferation of peripheral blood T cells and also is critical for activation-induced cell death. We now demonstrate that IL-2 potently decreases expression of IL-7 receptor α chain (IL-7Rα) mRNA and protein. The fact that IL-7Rα is a component of the receptors for both IL-7 and thymic stromal lymphopoietin (TSLP) suggests that IL-2 can negatively regulate signals by each of these cytokines. Previously it was known that the IL-2 and IL-7 receptors shared the common cytokine receptor γ chain, γc, which suggested a possible competition between these cytokines for a receptor component. Our findings now suggest a previously unknown type of cross-talk between IL-2 and IL-7 signaling by showing that IL-2 signaling can diminish IL-7Rα expression via a phosphatidylinositol 3-kinase/Akt-dependent mechanism.

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.

Tat gets the "green" light on transcription initiation

Human immunodeficiency virus type 1 (HIV-1) Tat transactivation is an essential step in the viral life cycle. Over the past several years, it has become widely accepted that Tat exerts its transcriptional effect by binding the transactivation-responsive region (TAR) and enhancing transcriptional elongation. Consistent with this hypothesis, it has been shown that Tat promotes the binding of P-TEFb, a transcription elongation factor composed of cyclin T1 and cdk9, and the interaction of Tat with P-TEFb and TAR leads to hyperphosphorylation of the C-terminal domain (CTD) of RNA Pol II and increased processivity of RNA Pol II. A recent report, however, has generated renewed interest that Tat may also play a critical role in transcription complex (TC) assembly at the preinitiation step. Using in vivo chromatin immunoprecipitation assays, the authors reported that the HIV TC contains TBP but not TBP-associated factors. The stimulatory effect involved the direct interaction of Tat and P-TEFb and was evident at the earliest step of TC assembly, the TBP-TATA box interaction. In this article, we will review this data in context of earlier data which also support Tats involvement in transcriptional complex assembly. Specifically, we will discuss experiments which demonstrated that Tat interacted with TBP and increased transcription initiation complex stability in cell free assays. We will also discuss studies which demonstrated that over expression of TBP alone was sufficient to obtain Tat activated transcription in vitro and in vivo. Finally, studies using self-cleaving ribozymes which suggested that Tat transactivation was not compatible with pausing of the RNA Pol II at the TAR site will be discussed.

PCAF Interacts with Tax and Stimulates Tax Transactivation in a Histone Acetyltransferase-Independent Manner

ABSTRACT Recent studies have shown that the p300/CREB binding protein (CBP)-associated factor (PCAF) is involved in transcriptional activation. PCAF activity has been shown strongly associated with histone acetyltransferase (HAT) activity. In this report, we present evidence for a HAT-independent transcription function that is activated in the presence of the human T-cell leukemia virus type 1 (HTLV-1) Tax protein. In vitro and in vivo GST-Tax pull-down and coimmunoprecipitation experiments demonstrate that there is a direct interaction between Tax and PCAF, independent of p300/CBP. PCAF can be recruited to the HTLV-1 Tax responsive element in the presence of Tax, and PCAF cooperates with Tax in vivo to activate transcription from the HTLV-1 LTR over 10-fold. Point mutations at Tax amino acid 318 (TaxS318A) or 319 to 320 (Tax M47), which have decreased or no activity on the HTLV-1 promoter, are defective for PCAF binding. Strikingly, the ability of PCAF to stimulate Tax transactivation is not solely dependent on the PCAF HAT domain. Two independent PCAF HAT mutants, which knock out acetyltransferase enzyme activity, activate Tax transactivation to approximately the same level as wild-type PCAF. In contrast, p300 stimulation of Tax transactivation is HAT dependent. These studies provide experimental evidence that PCAF contains a coactivator transcription function independent of the HAT activity on the viral long terminal repeat.

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.