Reovirus infection induces stabilization and up-regulation of cellular transcripts that encode regulators of TGF-ß signaling.

Reovirus infection induces dramatic changes in host mRNA expression. We utilized oligonucleotide microarrays to measure cellular mRNA decay rates in mock- or reovirus-infected murine L929 cells to determine if changes in host mRNA expression are a consequence of reovirus-induced alterations in cellular mRNA stability. Our analysis detected a subset of cellular transcripts that were coordinately induced and stabilized following infection with the reovirus isolates c87 and c8, strains that led to an inhibition of cellular translation, but not following infection with Dearing, a reovirus isolate that did not negatively impact cellular translation. The induced and stabilized transcripts encode multiple regulators of TGF- ß signaling, including components of the Smad signaling network and apoptosis/survival pathways. The coordinate induction, through mRNA stabilization, of multiple genes that encode components of TGF-ß signaling pathways represents a novel mechanism by which the host cell responds to reovirus infection.

CCAAT/Enhancer-binding protein ß promotes pathogenesis of EAE.

The CCAAT/Enhancer Binding Protein ß (C/EBPß) transcription factor is activated by multiple inflammatory stimuli, including IL-17 and LPS, and C/EBPß itself regulates numerous genes involved in inflammation. However, the role of C/EBPß in driving autoimmunity is not well understood. Here, we demonstrate that Cebpb-/- mice are resistant to EAE. Cebpb-/- mice exhibited reduced lymphocyte and APC infiltration into CNS following EAE induction. Furthermore, MOG-induced Th17 cytokine production was impaired in draining LN, indicating defects in Th17 cell priming. In vitro Th17 polarization studies indicated that T cell responses are not inherently defective, instead supporting the known roles for C/EBPß in myeloid lineage cell activation as the likely mechanism for defective Th17 priming in vivo. However, we did uncover an unexpected role for C/EBPß in regulating ll23r expression in APCs. ChIP assays confirmed that C/EBPß binds directly to the Il23r gene promoter in dendritic cells and Th17 cells. These data establish C/EBPß as a key driver of autoimmune inflammation in EAE, and propose a novel role for C/EBPß in regulation of IL-23R expression.

C/EBPß Promotes Immunity to Oral Candidiasis through Regulation of ß-Defensins.

Humans or mice subjected to immunosuppression, such as corticosteroids or anti-cytokine biologic therapies, are susceptible to mucosal infections by the commensal fungus Candida albicans. Recently it has become evident that the Th17/IL-17 axis is essential for immunity to candidiasis, but the downstream events that control immunity to this fungus are poorly understood. The CCAAT/Enhancer Binding Protein-ß (C/EBPß) transcription factor is important for signaling by multiple inflammatory stimuli, including IL-17. C/EBPß is regulated in a variety of ways by IL-17, and controls several downstream IL-17 target genes. However, the role of C/EBPß in vivo is poorly understood, in part because C/EBPß-deficient mice are challenging to breed and work with. In this study, we sought to understand the role of C/EBPß in the context of an IL-17-dependent immune response, using C. albicans infection as a model system. Confirming prior findings, we found that C/EBPß is required for immunity to systemic candidiasis. In contrast, C/EBPß(-/-) mice were resistant to oropharyngeal candidiasis (OPC), in a manner indistinguishable from immunocompetent WT mice. However, C/EBPß(-/-) mice experienced more severe OPC than WT mice in the context of cortisone-induced immunosuppression. Expression of the antimicrobial peptide ß-defensin (BD)-3 correlated strongly with susceptibility in C/EBPß(-/-) mice, but no other IL-17-dependent genes were associated with susceptibility. Therefore, C/EBPß contributes to immunity to mucosal candidiasis during cortisone immunosuppression in a manner linked to ß-defensin 3 expression, but is apparently dispensable for the IL-17-dependent response.

Oral-resident natural Th17 cells and γδ T cells control opportunistic Candida albicans infections.

Oropharyngeal candidiasis (OPC) is an opportunistic fungal infection caused by Candida albicans. OPC is frequent in HIV/AIDS, implicating adaptive immunity. Mice are naive to Candida, yet IL-17 is induced within 24 h of infection, and susceptibility is strongly dependent on IL-17R signaling. We sought to identify the source of IL-17 during the early innate response to candidiasis. We show that innate responses to Candida require an intact TCR, as SCID, IL-7Rα(-/-), and Rag1(-/-) mice were susceptible to OPC, and blockade of TCR signaling by cyclosporine induced susceptibility. Using fate-tracking IL-17 reporter mice, we found that IL-17 is produced within 1-2 d by tongue-resident populations of γδ T cells and CD3(+)CD4(+)CD44(hi)TCRß(+)CCR6(+) natural Th17 (nTh17) cells, but not by TCR-deficient innate lymphoid cells (ILCs) or NK cells. These cells function redundantly, as TCR-ß(-/-) and TCR-δ(-/-) mice were both resistant to OPC. Whereas γδ T cells were previously shown to produce IL-17 during dermal candidiasis and are known to mediate host defense at mucosal surfaces, nTh17 cells are poorly understood. The oral nTh17 population expanded rapidly after OPC, exhibited high TCR-ß clonal diversity, and was absent in Rag1(-/-), IL-7Rα(-/-), and germ-free mice. These findings indicate that nTh17 and γδ T cells, but not ILCs, are key mucosal sentinels that control oral pathogens.

The adaptor CARD9 is required for adaptive but not innate immunity to oral mucosal Candida albicans infections.

Oropharyngeal candidiasis (OPC [thrush]) is an opportunistic infection caused by the commensal fungus Candida albicans. OPC is common in individuals with HIV/AIDS, infants, patients on chemotherapy, and individuals with congenital immune defects. Immunity to OPC is strongly dependent on the interleukin-23 (IL-23)/IL-17R axis, as mice and humans with defects in IL-17R signaling (IL17F, ACT1, IL-17RA) or in genes that direct Th17 differentiation (STAT3, STAT1, CARD9) are prone to mucocutaneous candidiasis. Conventional Th17 cells are induced in response to C. albicans infection via signals from C-type lectin receptors, which signal through the adaptor CARD9, leading to production of Th17-inducing cytokines such as IL-6, IL-1ß, and IL-23. Recent data indicate that IL-17 can also be made by numerous innate cell subsets. These innate "type 17" cells resemble conventional Th17 cells, but they can be activated without need for prior antigen exposure. Because C. albicans is not a commensal organism in rodents and mice are thus naive to this fungus, we had the opportunity to assess the role of CARD9 in innate versus adaptive responses using an OPC infection model. As expected, CARD9(-/-) mice failed to mount an adaptive Th17 response following oral Candida infection. Surprisingly, however, CARD9(-/-) mice had preserved innate IL-17-dependent responses to Candida and were almost fully resistant to OPC. Thus, CARD9 is important primarily for adaptive immunity to C. albicans, whereas alternate recognition systems appear to be needed for effective innate responses.

The anaphase-promoting complex protein 5 (AnapC5) associates with A20 and inhibits IL-17-mediated signal transduction.

IL-17 is the founding member of a family of cytokines and receptors with unique structures and signaling properties. IL-17 is the signature cytokine of Th17 cells, a relatively new T cell population that promotes inflammation in settings of infection and autoimmunity. Despite advances in understanding Th17 cells, mechanisms of IL-17-mediated signal transduction are less well defined. IL-17 signaling requires contributions from two receptor subunits, IL-17RA and IL-17RC. Mutants of IL-17RC lacking the cytoplasmic domain are nonfunctional, indicating that IL-17RC provides essential but poorly understood signaling contributions to IL-17-mediated signaling. To better understand the role of IL-17RC in signaling, we performed a yeast 2-hybrid screen to identify novel proteins associated with the IL-17RC cytoplasmic tail. One of the most frequent candidates was the anaphase promoting complex protein 7 (APC7 or AnapC7), which interacted with both IL-17RC and IL-17RA. Knockdown of AnapC7 by siRNA silencing exerted no detectable impact on IL-17 signaling. However, AnapC5, which associates with AnapC7, was also able to bind IL-17RA and IL-17RC. Moreover, AnapC5 silencing enhanced IL-17-induced gene expression, suggesting an inhibitory activity. Strikingly, AnapC5 also associated with A20 (TNFAIP3), a recently-identified negative feedback regulator of IL-17 signal transduction. IL-17 signaling was not impacted by knockdown of Itch or TAXBP1, scaffolding proteins that mediate A20 inhibition in the TNFα and IL-1 signaling pathways. These data suggest a model in which AnapC5, rather than TAX1BP1 and Itch, is a novel adaptor and negative regulator of IL-17 signaling pathways.

Human ovarian tumor ascites fluids rapidly and reversibly inhibit T cell receptor-induced NF-κB and NFAT signaling in tumor-associated T cells.

Human memory T cells present in ovarian tumor ascites fluids fail to respond normally to stimulation via the T cell receptor (TCR). This immunosuppression is manifested by decreases in NF-κB and NFAT activation, IFN-γ production, and cell proliferation in response to TCR stimulation with immobilized antibodies to CD3 and CD28. The anergy of the tumor-associated T cells (TATs) is mediated by soluble factors present in ovarian tumor ascites fluids. The non-responsiveness of the T cells is quickly reversed when the cells are assayed in the absence of the ascites fluid, and is rapidly reestablished when a cell-free ascites fluid is added back to the T cells. Based upon the observed normal phosphorylation patterns of the TCR proximal signaling molecules, the inhibition of NF-κB, and NFAT activation in response to TCR stimulation, as well as the ability of the diacylglycerol analog PMA and the ionophore ionomycin to bypass the ascites fluid-induced TCR signaling arrest, the site of the arrest in the activation cascade appears to be at or just upstream of PLC-γ. An identical TCR signaling arrest pattern was observed when T cells derived from normal donor peripheral blood were incubated with either malignant or nonmalignant (cirrhotic) ascites fluids. The immunosuppressive activity of ascites fluids reported here suggests that soluble factors acting directly or indirectly upon T cells present within tumors contribute to the anergy that has previously been observed in T cells derived from malignant and nonmalignant inflammatory microenvironments. The soluble immunosuppressive factors represent potential therapeutic targets for ovarian cancer.

Memory T cells in the chronic inflammatory microenvironment of nasal polyposis are hyporesponsive to signaling through the T cell receptor.

A majority of T cells from chronic inflammatory tissues derived from patients with nasal polyposis were found to express an effector memory phenotype. We report here that these memory T cells failed to activate NF-κB in response to TCR stimulation but responded normally when the proximal TCR signaling molecules were bypassed with PMA and ionomycin. The dysfunction of these cells was associated with a decrease in the phosphorylation of several TCR proximal signaling molecules including ZAP70, Lck and SLP-76. In addition to the disruption in the TCR signaling pathway, the nasal polyp-associated T cells were shown to have a defect in their ability to translocate LAMP-1 to the cell surface. The results presented here establish that the phenotype and anergy of the T cells in the nasal polyp are similar to those which is seen in memory T cells derived from human tumors and other sites of chronic inflammation.

Humanized mouse model of ovarian cancer recapitulates patient solid tumor progression, ascites formation, and metastasis.

Ovarian cancer is the most common cause of death from gynecological cancer. Understanding the biology of this disease, particularly how tumor-associated lymphocytes and fibroblasts contribute to the progression and metastasis of the tumor, has been impeded by the lack of a suitable tumor xenograft model. We report a simple and reproducible system in which the tumor and tumor stroma are successfully engrafted into NOD-scid IL2Rγ(null) (NSG) mice. This is achieved by injecting tumor cell aggregates derived from fresh ovarian tumor biopsy tissues (including tumor cells, and tumor-associated lymphocytes and fibroblasts) i.p. into NSG mice. Tumor progression in these mice closely parallels many of the events that are observed in ovarian cancer patients. Tumors establish in the omentum, ovaries, liver, spleen, uterus, and pancreas. Tumor growth is initially very slow and progressive within the peritoneal cavity with an ultimate development of tumor ascites, spontaneous metastasis to the lung, increasing serum and ascites levels of CA125, and the retention of tumor-associated human fibroblasts and lymphocytes that remain functional and responsive to cytokines for prolonged periods. With this model one will be able to determine how fibroblasts and lymphocytes within the tumor microenvironment may contribute to tumor growth and metastasis, and will make it possible to evaluate the efficacy of therapies that are designed to target these cells in the tumor stroma.

Reciprocal functional modulation of the activation of T lymphocytes and fibroblasts derived from human solid tumors.

Fibroblasts are a dominant cell type in most human solid tumors. The possibility that fibroblasts have the capacity to interact with and modulate the function of tumor-associated T lymphocytes makes them a potential therapeutic target. To address this question, primary cultures of fibroblasts derived from human lung tumors were established and cultured with T cells derived from the same tumor. The tumor fibroblasts significantly enhance the production of IFN-gamma and IL-17A by the tumor-associated T cells following a CD3/CD28-induced activation of the T cells. This enhancement was fibroblast cell dose-dependent and did not require direct contact between the two cell types. Tumor-associated fibroblast-conditioned media similarly enhanced both IFN-gamma and IL-17A in activated T cells, and this enhancement was significantly reduced by Abs to IL-6. Conditioned media derived from activated lymphocyte cultures significantly enhanced IL-6 production by tumor fibroblasts. A similar enhancement of IFN-gamma and IL-17A was observed when activated T cells from a normal donor were cultivated with skin fibroblasts derived from the same donor. These results establish that fibroblasts and autologous lymphocytes, whether derived from the tumor microenvironment or from nonmalignant tissues, have the capacity to reciprocally interact and modulate function. In contrast to other reports, fibroblasts are shown to have an immunostimulatory effect upon activated T lymphocytes. The ability of fibroblasts to enhance two T cell cytokines known to have an impact upon tumor progression suggests that fibroblasts play an important role in tumor pathogenesis that could be exploited therapeutically.

T cells and stromal fibroblasts in human tumor microenvironments represent potential therapeutic targets.

The immune system of cancer patients recognizes tumor-associated antigens expressed on solid tumors and these antigens are able to induce tumor-specific humoral and cellular immune responses. Diverse immunotherapeutic strategies have been used in an attempt to enhance both antibody and T cell responses to tumors. While several tumor vaccination strategies significantly increase the number of tumor-specific lymphocytes in the blood of cancer patients, most vaccinated patients ultimately experience tumor progression. CD4+ and CD8+ T cells with an effector memory phenotype infiltrate human tumor microenvironments, but most are hyporesponsive to stimulation via the T cell receptor (TCR) and CD28 under conditions that activate memory T cells derived from the peripheral blood of the cancer patients or normal donors. Attempts to identify cells and molecules responsible for the TCR signaling arrest of tumor-infiltrating T cells have focused largely upon the immunosuppressive effects of tumor cells, tolerogenic dendritic cells and regulatory T cells. Here we review potential mechanisms by which human T cell function is arrested in the tumor microenvironment with a focus on the immunomodulatory effects of stromal fibroblasts. Determining in vivo which cells and molecules are responsible for the TCR arrest in human tumor-infiltrating T cells will be necessary to formulate and test strategies to prevent or reverse the signaling arrest of the human T cells in situ for a more effective design of tumor vaccines. These questions are now addressable using novel human xenograft models of tumor microenvironments.

IL-12 delivered intratumorally by multilamellar liposomes reactivates memory T cells in human tumor microenvironments.

Using a novel loading technique, IL-12 is reported here to be efficiently encapsulated within large multilamellar liposomes. The preclinical efficacy of the cytokine loaded liposomes to deliver IL-12 into human tumors and to reactive tumor-associated T cells in situ is tested using a human tumor xenograft model. IL-12 is released in vivo from these liposomes in a biologically active form when injected into tumor xenografts that are established by the subcutaneous implantation of non-disrupted pieces of human lung, breast or ovarian tumors into immunodeficient mice. The histological architecture of the original tumor tissue, including tumor-associated leukocytes, tumor cells and stromal cells is preserved anatomically and the cells remain functionally responsive to cytokines in these xenografts. The local and sustained release of IL-12 into the tumor microenvironment reactivates tumor-associated quiescent effector memory T cells to proliferate, produce and release IFN-gamma resulting in the killing of tumor cells in situ. Very little IL-12 is detected in the serum of mice for up to 5 days after an intratumoral injection of the IL-12 liposomes. We conclude that IL-12 loaded large multilamellar liposomes provide a safe method for the local and sustained delivery of IL-12 to tumors and a therapeutically effective way of reactivating existing tumor-associated T cells in human solid tumor microenvironments. The potential of this local in situ T cell re-stimulation to induce a systemic anti-tumor immunity is discussed.

Long-term engraftment and expansion of tumor-derived memory T cells following the implantation of non-disrupted pieces of human lung tumor into NOD-scid IL2Rgamma(null) mice.

Non-disrupted pieces of primary human lung tumor implanted into NOD-scid IL2Rgamma(null) mice consistently result in successful xenografts in which tissue architecture, including tumor-associated leukocytes, stromal fibroblasts, and tumor cells are preserved for prolonged periods with limited host-vs-graft interference. Human CD45(+) tumor-associated leukocytes within the xenograft are predominantly CD3(+) T cells with fewer CD138(+) plasma cells. The effector memory T cells that had been shown to be quiescent in human lung tumor microenvironments can be activated in situ as determined by the production of human IFN-gamma in response to exogenous IL-12. Plasma cells remain functional as evidenced by production of human Ig. Significant levels of human IFN-gamma and Ig were detected in sera from xenograft-bearing mice for up to 9 wk postengraftment. Tumor-associated T cells were found to migrate from the microenvironment of the xenograft to the lung, liver, and primarily the spleen. At 8 wk postengraftment, a significant portion of cells isolated from the mouse spleens were found to be human CD45(+) cells. The majority of CD45(+) cells were CD3(+) and expressed a phenotype consistent with an effector memory T cell, consisting of CD4(+) or CD8(+) T cells that were CD45RO(+), CD44(+), CD62L(-), and CD25(-). Following adoptive transfer into non-tumor bearing NOD-scid IL2Rgamma(null) mice, these human T cells were found to expand in the spleen, produce IFN-gamma, and maintain an effector memory phenotype. We conclude that the NOD-scid IL2Rgamma(null) tumor xenograft model provides an opportunity to study tumor and tumor-stromal cell interactions in situ for prolonged periods.

Targeting the TCR signaling checkpoint: a therapeutic strategy to reactivate memory T cells in the tumor microenvironment.

BACKGROUND: One of the major conundrums in cancer immunotherapy is why human tumors are not rejected, and progress despite the presence of inflammatory leukocytes in the tumor microenvironment. While studies addressing the mechanisms responsible for the failure of immunocompetent cells to control tumor progression have shed considerable light upon this issue, not enough is known about the mechanisms contributing to the regulation of tumor-associated T cells in the microenvironment of human tumors. OBJECTIVE: A persistent and robust response is likely to be required for the complete eradication of tumors. Such a durable immune response will require the development and persistence of functional tumor-reactive memory T cells. METHODS: Various studies have investigated the mechanisms of suppression in the tumor microenvironment. However, very few studies have investigated the hyporesponsiveness of tumor-associated T cells at the molecular level. This review focuses on the hyporesponsiveness of tumor-associated T cells and how this relates to the T cell receptor signaling cascade. RESULTS/CONCLUSIONS: We postulate that this hyporesponsiveness results from a normal regulatory mechanism or TCR signaling checkpoint that is initiated by the persistence of antigen. If the TCR checkpoint is defined, it will be possible to design therapeutic strategies that reverse the TCR arrest. Essentially, this will reactivate the T cells in situ, leading to the killing and lysis of tumors locally, the release of tumor antigens and the generation of a systemic antitumor immunity.

Characterization of human lung tumor-associated fibroblasts and their ability to modulate the activation of tumor-associated T cells.

The tumor microenvironment of human non-small cell lung cancer (NSCLC) is composed largely of stromal cells, including fibroblasts, yet these cells have been the focus of few studies. In this study, we established stromal cell cultures from primary NSCLC through isolation of adherent cells. Characterization of these cells by flow cytometry demonstrated a population which expressed a human fibroblast-specific 112-kDa surface molecule, Thy1, alpha-smooth muscle actin, and fibroblast activation protein, but failed to express CD45 and CD11b, a phenotype consistent with that of an activated myofibroblast. A subset of the tumor-associated fibroblasts (TAF) was found to express B7H1 (PD-L1) and B7DC (PD-L2) constitutively, and this expression was up-regulated by IFN-gamma. Production of cytokines and chemokines, including IFN-gamma, monokine induced by IFN-gamma, IFN-gamma-inducible protein-10, RANTES, and TGF-beta1 was also demonstrated in these cells. Together, these characteristics provide multiple opportunities for the TAF to influence cellular interactions within the tumor microenvironment. To evaluate the ability of TAF to modulate tumor-associated T cell (TAT) activation, we conducted coculture experiments between autologous TAF and TAT. In five of eight tumors, TAF elicited a contact-dependent enhancement of TAT activation, even in the presence of a TGF-beta1-mediated suppressive effect. In the three other tumors, TAF had a net suppressive effect upon TAT activation, and, in one of these cases, blockade of B7H1 or B7DC was able to completely abrogate the TAF-mediated suppression. We conclude that TAF in human NSCLC are functionally and phenotypically heterogeneous and provide multiple complex regulatory signals that have the potential to enhance or suppress TAT function in the tumor microenvironment.

Reovirus induces and benefits from an integrated cellular stress response.

Following infection with most reovirus strains, viral protein synthesis is robust, even when cellular translation is inhibited. To gain further insight into pathways that regulate translation in reovirus-infected cells, we performed a comparative microarray analysis of cellular gene expression following infection with two strains of reovirus that inhibit host translation (clone 8 and clone 87) and one strain that does not (Dearing). Infection with clone 8 and clone 87 significantly increased the expression of cellular genes characteristic of stress responses, including the integrated stress response. Infection with these same strains decreased transcript and protein levels of P58(IPK), the cellular inhibitor of the eukaryotic initiation factor 2alpha (eIF2alpha) kinases PKR and PERK. Since infection with host shutoff-inducing strains of reovirus impacted cellular pathways that control eIF2alpha phosphorylation and unphosphorylated eIF2alpha is required for translation initiation, we examined reovirus replication in a variety of cell lines with mutations that impact eIF2alpha phosphorylation. Our results revealed that reovirus replication is more efficient in the presence of eIF2alpha kinases and phosphorylatable eIF2alpha. When eIF2alpha is phosphorylated, it promotes the synthesis of ATF4, a transcription factor that controls cellular recovery from stress. We found that the presence of this transcription factor increased reovirus yields 10- to 100-fold. eIF2alpha phosphorylation also led to the formation of stress granules in reovirus-infected cells. Based on these results, we hypothesize that eIF2alpha phosphorylation facilitates reovirus replication in two ways-first, by inducing ATF4 synthesis, and second, by creating an environment that places abundant reovirus transcripts at a competitive advantage for limited translational components.

Tristetraprolin down-regulates IL-2 gene expression through AU-rich element-mediated mRNA decay.

Posttranscriptional regulation of IL-2 gene expression at the level of mRNA decay is mediated by an AU-rich element (ARE) found in the 3'-untranslated region. We hypothesized that the ARE-binding protein tristetraprolin (TTP) regulates T lymphocyte IL-2 mRNA decay by interacting with the IL-2 ARE and targeting the transcript for decay. rTTP protein expressed in HeLa cells bound specifically to the IL-2 ARE with high affinity in a gel shift assay. In primary human T lymphocytes, TTP mRNA and protein expression were induced by TCR and CD28 coreceptor stimulation. Using a gel shift assay, we identified a cytoplasmic RNA-binding activity that was induced by TCR and CD28 coreceptor stimulation and bound specifically to the IL-2 ARE sequence. Using anti-TTP Abs, we showed by supershift that this inducible activity contained TTP. We also showed that insertion of the IL-2 ARE sequence into the 3'-untranslated region of a beta-globin reporter construct conferred TTP-dependent mRNA destabilization on the beta-globin reporter. To determine whether TTP also regulates IL-2 gene expression in vivo, we examined IL-2 expression in primary cells from wild-type and TTP knockout mice. Compared with their wild-type counterparts, TCR- and CD28-activated splenocytes and T cells from TTP knockout mice overexpressed IL-2 mRNA and protein. Also, IL-2 mRNA was more stable in activated splenocytes from TTP knockout mice compared with wild-type mice. Taken together, these data suggest that TTP functions to down-regulate IL-2 gene expression through ARE-mediated mRNA decay.

Characterization of the caprine arthritis encephalitis virus (CAEV) rev N-terminal elements required for efficient interaction with the RRE.

The Caprine Arthritis Encephalitis Virus (CAEV) genome encodes three structural (gag, pol, and env) and three accessory (rev, tat, and vif) genes. The Rev-C protein regulates Gag, Pol and Env expression by transporting their mRNAs to the cytoplasm. Rev trans-activation requires binding of Rev to an RNA structure called the Rev Response Element (RRE-C). Previous mutational analyses have shown that two domains of Rev are required for its function. The basic domain mediates RRE binding and multimer formation, and the nuclear export signal (NES) mediates trans-activation. Preliminary experiments demonstrate that Rev-C N-terminal deletion mutants bind the RRE less avidly than does wildtype Rev. As a result, it was hypothesized that an additional domain located in the N-terminal exon of Rev-C was required for optimal RRE binding. To test this hypothesis, Rev-C alanine scanning mutants were generated and in vitro RRE binding assays were performed. Alteration of Rev-C amino acids K13, E14, N15, V19, T20, M21 and R27 dramatically decreased affinity for RRE-C. These data demonstrate that Rev-C N-terminal amino acids are required for optimal RRE-C binding and suggest that a third functional domain exists within the N-terminus of Rev-C.

Genome-wide analysis of mRNA decay in resting and activated primary human T lymphocytes.

We used microarray technology to measure mRNA decay rates in resting and activated T lymphocytes in order to better understand the role of mRNA decay in regulating gene expression. Purified human T lymphocytes were stimulated for 3 h with medium alone, with an anti-CD3 antibody, or with a combination of anti-CD3 and anti-CD28 antibodies. Actinomycin D was added to arrest transcription, and total cellular RNA was collected at discrete time points over a 2 h period. RNA from each point was analyzed using Affymetrix oligonucleotide arrays and a first order decay model was used to determine the half-lives of approximately 6000 expressed transcripts. We identified hundreds of short-lived transcripts encoding important regulatory proteins including cytokines, cell surface receptors, signal transduction regulators, transcription factors, cell cycle regulators and regulators of apoptosis. Approximately 100 of these short-lived transcripts contained ARE-like sequences. We also identified numerous transcripts that exhibited stimulus-dependent changes in mRNA decay. In particular, we identified hundreds of transcripts whose steady-state levels were repressed following T cell activation and were either unstable in the resting state or destabilized following cellular activation. Thus, rapid mRNA degradation appears to be an important mechanism for turning gene expression off in an activation-dependent manner.