HIV Nef, paxillin, and Pak1/2 regulate activation and secretion of TACE/ADAM10 proteases.

The HIV Nef protein recruits the polycomb protein Eed and mimics an integrin receptor signal for reasons that are not entirely clear. Here we demonstrate that Nef and Eed complex with the integrin effector paxillin to recruit and activate TNFα converting enzyme (TACE alias ADAM 17) and its close relative ADAM10. The activated proteases cleaved proTNFα and were shuttled into extracellular vesicles (EVs). Peripheral blood mononuclear cells that ingested these EVs released TNFα. Analyzing the mechanism, we found that Pak2, an established host cell effector of Nef, phosphorylated paxillin on Ser272/274 to induce TACE-paxillin association and shuttling into EVs via lipid rafts. Conversely, Pak1 phosphorylated paxillin on Ser258, which inhibited TACE association and lipid raft transfer. Interestingly, melanoma cells used an identical mechanism to shuttle predominantly ADAM10 into EVs. We conclude that HIV-1 and cancer cells exploit a paxillin/integrin-controlled mechanism to release TACE/ADAM10-containing vesicles, ensuring better proliferation/growth conditions in their microenvironment.

Perforin-2 Breaches the Envelope of Phagocytosed Bacteria Allowing Antimicrobial Effectors Access to Intracellular Targets.

Perforin-2, the product of the MPEG1 gene, limits the spread and dissemination of bacterial pathogens in vivo. It is highly expressed in murine and human phagocytes, and macrophages lacking Perforin-2 are compromised in their ability to kill phagocytosed bacteria. In this study, we used Salmonella enterica serovar Typhimurium as a model intracellular pathogen to elucidate the mechanism of Perforin-2's bactericidal activity. In vitro Perforin-2 was found to facilitate the degradation of Ags contained within the envelope of phagocytosed bacteria. In contrast, degradation of a representative surface Ag was found to be independent of Perforin-2. Consistent with our in vitro results, a protease-sensitive, periplasmic superoxide dismutase (SodCII) contributed to the virulence of S. Typhimurium in Perforin-2 knockout but not wild-type mice. In aggregate, our studies indicate that Perforin-2 breaches the envelope of phagocytosed bacteria, facilitating the delivery of proteases and other antimicrobial effectors to sites within the bacterial cell.

Interleukin-2 and inflammation induce distinct transcriptional programs that promote the differentiation of effector cytolytic T cells.

Interleukin(IL)-2 and inflammation regulate effector and memory cytolytic T-lymphocyte (CTL) generation during infection. We demonstrate a complex interplay between IL-2 and inflammatory signals during CTL differentiation. IL-2 stimulation induced the transcription factor eomesodermin (Eomes), upregulated perforin (Prf1) transcription, and repressed re-expression of memory CTL markers Bcl6 and IL-7Ralpha. Binding of Eomes and STAT5 to Prf1 cis-regulatory regions correlated with transcriptional initiation (increased recruitment of RNA polymerase II to the Prf1 promoter). Inflammation (CpG, IL-12) enhanced expression of IL-2Ralpha and the transcription factor T-bet, but countered late Eomes and perforin induction while preventing IL-7Ralpha repression by IL-2. After infection of mice with lymphocytic choriomeningitis virus, IL-2Ralpha-deficient effector CD8(+) T cells expressed more Bcl6 but less perforin and granzyme B, formed fewer KLRG-1(+) and T-bet-expressing CTL, and killed poorly. Thus, inflammation influences both effector and memory CTL differentiation, whereas persistent IL-2 stimulation promotes effector at the expense of memory CTL development.

The transcriptional control of the perforin locus.

SUMMARY: Natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) use cytotoxic granules containing perforin and granzymes to lyse infected or malignant host cells, thereby providing immunity to intracellular microbes and tumors. Perforin is essential for cytotoxic granule-mediated killing. Perforin expression is regulated transcriptionally and correlates tightly with the development of cells that can exhibit cytotoxic activity. Although a number of genes transcribed by T cells and NK cells have been studied, the cell-specificity of perforin gene expression makes it an ideal model system in which to clarify the transcriptional mechanisms that guide the development and activation of cytotoxic lymphocytes. In this review, we discuss what is known about perforin expression and its regulation, then elaborate on recent studies that utilized chromosome transfer and bacterial artificial chromosome transgenics to define a comprehensive set of cis-regulatory regions that control transcription of the human PRF1 gene in a near-physiologic context. In addition, we compare the human and murine Prf1 loci and discuss how transcription factors known to be important for driving CTL differentiation might also directly regulate the cis-acting domains that control Prf1. Our review emphasizes how studies of PRF1/Prf1 gene transcription can illuminate not only the mechanisms of cytotoxic lymphocyte differentiation but also some basic principles of transcriptional regulation.

The Fallacy of Teaching and the Illusion of Learning: Improving Articulation of Basic Science in the Medical School Curriculum.

Despite calls from educators to re-engineer how faculty deliver medical student curricula with integrated basic science concepts, this content is still frequently disarticulated from other curricular components. We renewed our curriculum using evidence-based pedagogical and cognitive learning strategies to interleave basic science across the 4-year curriculum.

A reliable method to display authentic DNase I hypersensitive sites at long-ranges in single-copy genes from large genomes.

The study of eukaryotic gene transcription depends on methods to discover distal cis-acting control sequences. Comparative bioinformatics is one powerful strategy to reveal these domains, but still requires conventional wet-bench techniques to elucidate their specificity and function. The DNase I hypersensitivity assay (DHA) is also a method to identify regulatory domains, but can also suggest their function. Technically however, the classical DHA is constrained to mapping gene loci in small increments of approximately 20 kb. This limitation hinders efficient and comprehensive analysis of distal gene regions. Here, we report an improved method termed mega-DHA that extends the range of existing DHAs to facilitate assaying intervals that approach 100 kb. We demonstrate its feasibility for efficient analysis of single-copy genes within a large and complex genome by assaying 230 kb of the human ADAMTS14-perforin-paladin gene cluster in four experiments. The results identify distinct networks of regulatory domains specific to expression of perforin and its two neighboring genes.

Characterization of a R115777-resistant human multiple myeloma cell line with cross-resistance to PS-341.

The farnesyl transferase inhibitor R115777 has been found to have clinical activity in diverse hematopoietic tumors. Clinical efficacy, however, does not correlate with Ras mutation status or inhibition of farnesyl transferase. To further elucidate the mechanisms by which R115777 induces apoptosis and to investigate drug resistance, we have identified and characterized a R115777-resistant human myeloma cell line. 8226/R5 cells were found to be at least 50 times more resistant to R115777 compared with the parent cell line 8226/S. K-Ras remained prenylated in both resistant and sensitive cells after R115777 treatment; however, HDJ-2 farnesylation was inhibited in both lines, implying that farnesyl transferase (the drug target) has not been mutated. Whereas many 8226 lines that acquire drug resistance have elevated expression of P-glycoprotein, we found that P-glycoprotein expression is not increased in the 8226/R5 line and intracellular accumulation of R115777 was not reduced. In fact, 8226/R5 cells were insensitive to a diverse group of antitumor agents including PS-341, and multidrug resistance did not correlate with the expression of heat shock proteins. Comparison of gene expression profiles between resistant and sensitive cells revealed expression changes in several genes involved in myeloma survival and drug resistance. Future experiments will attempt to identify genes that are directly linked to the resistant phenotype. Identification of molecules associated with R115777 and PS-341 resistance is clinically relevant because both compounds are being tested in solid tumors and hematopoietic malignancies.

R115777 induces Ras-independent apoptosis of myeloma cells via multiple intrinsic pathways.

Ras activation is frequently observed in multiple myeloma either by mutation or through interleukin-6 receptor signaling. Recently, drugs designed to inhibit Ras have shown promise in preclinical myeloma models and in clinical trials. In this report, we characterize the pathways by which the clinically tested farnesyl transferase inhibitor (FTI) R115777 induces apoptosis in multiple myeloma cells. Contrary to the proposed mechanistic action of FTIs, we found that R115777 induces cell death despite Ras prenylation implying participation of Ras-independent mechanism(s). Apoptosis proceeded via an intrinsic cascade and was associated with an increase in the expression and activity of Bax. Bax activation correlated with a loss of mitochondrial membrane integrity and activation of the endoplasmic reticulum (ER) stress response. These pathways activate caspase-9 and consistent with this, cell death was prevented by caspase-9 blockade. Interestingly, cells overexpressing Bcl-X(L) remained partially sensitive to R115777 despite suppression of mitochondrial membrane dysfunction and ER-related stress. Taken together, these results indicate that R115777 induces apoptosis in a Ras-independent fashion via multiple intrinsic pathways.

Perforin-2 is essential for intracellular defense of parenchymal cells and phagocytes against pathogenic bacteria.

Perforin-2 (MPEG1) is a pore-forming, antibacterial protein with broad-spectrum activity. Perforin-2 is expressed constitutively in phagocytes and inducibly in parenchymal, tissue-forming cells. In vitro, Perforin-2 prevents the intracellular replication and proliferation of bacterial pathogens in these cells. Perforin-2 knockout mice are unable to control the systemic dissemination of methicillin-resistant Staphylococcus aureus (MRSA) or Salmonella typhimurium and perish shortly after epicutaneous or orogastric infection respectively. In contrast, Perforin-2-sufficient littermates clear the infection. Perforin-2 is a transmembrane protein of cytosolic vesicles -derived from multiple organelles- that translocate to and fuse with bacterium containing vesicles. Subsequently, Perforin-2 polymerizes and forms large clusters of 100 Å pores in the bacterial surface with Perforin-2 cleavage products present in bacteria. Perforin-2 is also required for the bactericidal activity of reactive oxygen and nitrogen species and hydrolytic enzymes. Perforin-2 constitutes a novel and apparently essential bactericidal effector molecule of the innate immune system.

Farnesyl transferase inhibitors enhance death receptor signals and induce apoptosis in multiple myeloma cells.

Multiple myeloma is an incurable plasma cell malignancy in which Ras may be constitutively active either via interleukin-6 (IL-6) receptor signaling or by mutation. Inactivation of Ras may be achieved with farnesyl transferase (FTase) inhibitors a class of drugs which have shown promise in clinical trials particularly in patients with acute leukemia. This report investigates the efficacy of two distinct classes of FTase inhibitors in diverse myeloma cell lines and primary isolates. While Ras signaling has traditionally been linked to myeloma cell growth, we found that these compounds also potently triggered cell death. Death induced by perillic acid (PA) was caspase dependent without evidence of death receptor activation. Apoptosis was associated with mitochondrial membrane depolarization and activation of caspase-9 and 3 but proceeded despite over-expression of Bcl-XL a known correlate of relapsed and chemorefractory myeloma. In addition, Fas ligand and TRAIL mediated apoptosis was potentiated in death receptor resistant (U266) and sensitive (RPMI 8226/S) cell lines. Of clinical relevance, the FTase inhibitor R115777 induced cell death in myeloma lines at doses observed in clinical trials. Furthermore, both R115777 and PA induced cell death in primary isolates with relative specificity. Taken together these preclinical data provide evidence that FTase inhibitors may be an effective therapeutic modality for the treatment of multiple myeloma.

Understanding the biology of ex vivo-expanded CD8 T cells for adoptive cell therapy: role of CD62L.

CD62L governs the circulation of CD8(+) T cells between lymph nodes and peripheral tissues, whereby the expression of CD62L by CD8(+) T cells promotes their recirculation through lymph nodes. As such, CD62L participates in the fate of adoptively transferred CD8(+) T cells and may control their effectiveness for cancer immunotherapy, including settings in which host preconditioning results in the acute lymphopenia-induced proliferation of the transferred cells. Indeed, previous studies correlated CD62L expression by donor CD8(+) cells with the success rate of adoptive cell therapy (ACT). Here, we analyzed the functions and fate of ex vivo-activated, tumor-specific CD62L(-/-) CD8(+) T cells in a mouse melanoma model for ACT. Unexpectedly, we observed that CD62L(-/-) CD8(+) T cells were functionally indistinguishable from CD62L(+/+) CD8(+) T cells, i.e., both greatly expanded in cyclophosphamide preconditioned animals, controlled subcutaneously and hematogenously spreading tumors, and generated anti-tumor-specific CD8(+) T cell memory. Moreover, even in hosts with rudimentary secondary lymphoid organs (LT(-/-) animals), CD8(+) T cells with and without CD62L expanded equivalently to those adoptively transferred into wild-type animals. These results put into question the utility of CD62L as a predictive biomarker for the efficacy of ex vivo-expanded T cells after ACT in lymphopenic conditions and also offer new insights into the homing, engraftment, and memory generation of adoptively transferred ex vivo-activated CD8(+) T cells.

Interleukin-21 and cellular activation concurrently induce potent cytotoxic function and promote antiviral activity in human CD8 T cells.

Infection with human immunodeficiency virus (HIV)-1 induces a progressive deterioration of the immune system that ultimately leads to acquired immune deficiency syndrome (AIDS). Murine models indicate that the common γ-chain (γ(c))-sharing cytokine interleukin (IL)-21 and its receptor (IL-21R) play a crucial role in maintaining polyfunctional T cell responses during chronic viral infections. Therefore, we analyzed the ability of this cytokine to modulate the properties of human CD8 T cells in comparison with other γ(c)-sharing cytokines (IL-2, IL-7, and IL-15). CD8 T cells from healthy volunteers were stimulated in vitro via T cell receptor signals to mimic the heightened status of immune activation of HIV-infected patients. The administration of IL-21 upregulated cytotoxic effector function and the expression of the costimulatory molecule CD28. Notably, this outcome was not accompanied by increased cellular proliferation or activation. Moreover, IL-21 promoted antiviral activity while not inducing HIV-1 replication in vitro. Thus, IL-21 may be a favorable molecule for immunotherapy and a suitable vaccine adjuvant in HIV-infected individuals.

Synergy of brief activation of CD8 T-cells in the presence of IL-12 and adoptive transfer into lymphopenic hosts promotes tumor clearance and anti-tumor memory.

Adoptive T-cell therapy holds great promise for the treatment of metastatic melanoma. However, prohibitive costs associated with current technology required for culture and expansion of tumor-reactive T-cells, the need for intense preconditioning regimens to induce lymphopenia, and the unpredictable anti-tumor effect of adoptively transferred T-cells remain significant impediments for its clinical implementation. Here we report a simplified combinatorial approach that involves short activation of CD8(+) T cells in the presence of IL-12 followed by adoptive transfer into tumor bearing animals after a single injection of cyclophosphamide. This approach resulted in complete eradication of B16 melanoma, and the establishment of long term immunological memory capable of fully protecting mice after a second B16 melanoma challenge. The activated donor cells were unique because they simultaneously exhibited traits for cytotoxic effector function, central memory-like, homing, and senescence. After tumor eradication and within three months after transfer, CD8+ cells exhibited a conventional memory CTL phenotype. Moreover, these memory CTLs acquired functional attributes characteristic of memory stem cells, including the ability to resist chemotherapy-induced toxicity. Our results suggest that short-term T-cell receptor signaling in the presence of IL-12 promotes promiscuous qualities in naïve CTL which - upon transfer into lymphopenic hosts- are sufficient to eradicate tumors and generate life-long tumor-specific memory.

Runx3 and T-box proteins cooperate to establish the transcriptional program of effector CTLs.

Activation of naive CD8(+) T cells with antigen induces their differentiation into effector cytolytic T lymphocytes (CTLs). CTLs lyse infected or aberrant target cells by exocytosis of lytic granules containing the pore-forming protein perforin and a family of proteases termed granzymes. We show that effector CTL differentiation occurs in two sequential phases in vitro, characterized by early induction of T-bet and late induction of Eomesodermin (Eomes), T-box transcription factors that regulate the early and late phases of interferon (IFN) gamma expression, respectively. In addition, we demonstrate a critical role for the transcription factor Runx3 in CTL differentiation. Runx3 regulates Eomes expression as well as expression of three cardinal markers of the effector CTL program: IFN-gamma, perforin, and granzyme B. Our data point to the existence of an elaborate transcriptional network in which Runx3 initially induces and then cooperates with T-box transcription factors to regulate gene transcription in differentiating CTLs.

HIV Nef enhances Tat-mediated viral transcription through a hnRNP-K-nucleated signaling complex.

Although dispensable in vitro, HIV Nef enables high-level viral replication in infected hosts by an as yet unexplained mechanism. Previously, we proposed that Nef functionally cooperates with the viral transactivator Tat by derepressing the viral promoter via a Nef-associated kinase complex (NAKC). Here, we demonstrate that hnRNP-K, a host factor thought to facilitate crosstalk between kinases and gene expression, interacts with Nef and, as part of NAKC, nucleates Nef-interacting kinases, including Lck, PKCdelta, and PI-3 kinase, leading to Lck and Erk1/2 activation. This strongly increased HIV transcription, which depended on Tat and the NF-kB motif in the viral promoter, but not on NF-kB activation. Depletion of hnRNP-K in a Jurkat model of HIV latency increased Erk1/2 activity and greatly augmented HIV reactivating stimuli. We conclude that hnRNP-K coordinates membrane signaling with transcriptional derepression through Erk1/2 and is targeted by HIV to enable Tat-mediated transcription.

IL-21 augments natural killer effector functions in chronically HIV-infected individuals.

OBJECTIVE: This study addresses the interleukin (IL)-21 effects on resting peripheral blood natural killer (NK) cells in chronically HIV-infected individuals. DESIGN: The effects of IL-21 on perforin expression, proliferation, degranulation, interferon (IFN)-gamma production, cytotoxicity and induction of STAT phosphorylation in NK cells were determined in vitro. METHODS: Peripheral blood mononuclear cells from HIV-infected and healthy individuals were incubated in vitro for 6 h, 24 h or 5 days with IL-21 or IL-15. Percentages of perforin, IFN-gamma, CD107a, NKG2D and STAT3-5 positive cells were determined within NK cell populations. K562 cells were used as target cells in NK cytotoxicity assay. RESULTS: Frequency of CD56 cells in chronically HIV-infected individuals was diminished. Perforin expression in CD56 and CD56 was comparable in healthy and HIV-infected individuals. IL-15 upregulated perforin expression primarily in CD56 NK cells, whereas IL-21 upregulated perforin in both NK subsets. IL-21 and IL-15 upregulated CD107a and IFN-gamma, as well as NK cytotoxicity. IL-15 predominantly activated STAT5, whereas IL-21 activated STAT5 and STAT3. IL-15, but not IL-21 increased NK cell proliferation in uninfected and HIV-infected individuals. CONCLUSION: IL-21 augments NK effector functions in chronically HIV-infected individuals and due to its perforin enhancing properties it has potential for immunotherapy or as a vaccine adjuvant.

Murine neonatal CD4+ cells are poised for rapid Th2 effector-like function.

Murine neonates typically mount Th2-biased immune responses. This entails a cell-intrinsic component whose molecular basis is unknown. We found that neonatal CD4(+) T cells are uniquely poised for rapid Th2 function. Within 24 h of activation, neonatal CD4(+) cells made high levels of IL-4 and IL-13 mRNA and protein. The rapid high-level IL-4 production arose from a small subpopulation of cells, did not require cell cycle entry, and was unaffected by pharmacologic DNA demethylation. CpG methylation analyses in resting neonatal cells revealed pre-existing hypomethylation at a key Th2 cytokine regulatory region, termed conserved noncoding sequence 1 (CNS-1). Robust Th2 function and increased CNS-1 demethylation was a stable property that persisted in neonatal Th2 effectors. The transcription factor STAT6 was not required for CNS-1 demethylation and this state was already established in neonatal CD4 single-positive thymocytes. CNS-1 demethylation levels were much greater in IL-4-expressing CD4 single-positive thymocytes compared with unactivated cells. Together, these results indicate that neonatal CD4+ T cells possess distinct qualities that could predispose them toward rapid, effector-like Th2 function.

Chromosome transfer activates and delineates a locus control region for perforin.

Perforin gene (PRF1) transcription regulates perforin expression in NK cells and CTL. Here we identified the locus-wide ensemble of cis-acting sequences that drives PRF1 transcription physiologically. By using chromosome transfer, we revealed that de novo activation of a silent PRF1 locus was controlled by a 150 kb domain comprised of 16 DNase I hypersensitive sites (DHSs). These cis-acting sequences included a locus control region (LCR) and conferred developmentally appropriate and lineage-specific expression of human perforin from BAC transgenes. The LCR included four distal DHSs that were required for perforin expression from its natural locus, and their engineered deletion from the PRF1 BAC transgene abolished LCR function and led to rapid gene silencing. Thus, LCR function is central for regulating the developmental and activation-specific PRF1 promoter activity characteristic of NK cells and CTL.

Differential effects of IL-21 and IL-15 on perforin expression, lysosomal degranulation, and proliferation in CD8 T cells of patients with human immunodeficiency virus-1 (HIV).

An urgent need exists to devise strategies to augment antiviral immune responses in patients with HIV who are virologically well controlled and immunologically stable on highly active antiretroviral therapy (HAART). The objective of this study was to compare the immunomodulatory effects of the cytokines interleukin (IL)-21 with IL-15 on CD8 T cells in patients with HIV RNA of less than 50 copies/mL and CD4 counts greater than 200 cells/mm.(3) Patient CD8 T cells displayed skewed maturation and decreased perforin expression compared with healthy controls. Culture of freshly isolated patient peripheral-blood mononuclear cells (PBMCs) for 5 hours to 5 days with IL-21 resulted in up-regulation of perforin in CD8 T cells, including memory and effector subsets and virus-specific T cells. IL-21 did not induce T-cell activation or proliferation, nor did it augment T-cell receptor (TCR)-induced degranulation. Treatment of patient PBMCs with IL-15 resulted in induction of perforin in association with lymphocyte proliferation and augmentation of TCR-induced degranulation. Patient CD8 T cells were more responsive to cytokine effects than the cells of healthy volunteers. We conclude that CD8 T cells of patients with HIV can be modulated by IL-21 to increase perforin expression without undergoing overt cellular activation. IL-21 could potentially be useful for its perforin-enhancing properties in anti-HIV immunotherapy.

Farnesyltransferase inhibitor R115777 (Zarnestra, Tipifarnib) synergizes with paclitaxel to induce apoptosis and mitotic arrest and to inhibit tumor growth of multiple myeloma cells.

Despite major advances, multiple myeloma (MM) remains an incurable malignancy. Recently we have found that disease stabilization was achieved in 64% of patients with advanced MM treated with the farnesyltransferase inhibitor R115777 (Zarnestra) in a phase 2 clinical trial. In order to enhance R115777 antitumor activity in MM, we examined the combination of this novel agent with other anticancer drugs in MM cell lines. In this study, R115777 was found to synergize with paclitaxel and docetaxel, but not with other chemotherapy agents, including doxorubicin, 5-fluorouracil, cisplastin, melphalan, mitoxantrone, and dexamethasone. R115777 synergized with paclitaxel to inhibit MM cell proliferation and to induce apoptosis. Synergism in the induction of apoptosis was accompanied by increase in cytochrome c release and caspase-3 activation. Furthermore, flow cytometry analysis also showed that paclitaxel and R115777 synergized to induce G(2)/M cell-cycle arrest. Importantly, synergism was observed in taxane- and R115777-resistant MM cells. In the human severe combined immunodeficient (SCID-hu) bone model of myeloma growth, the ability of paclitaxel to inhibit tumor growth in vivo was enhanced by R115777. Combination of paclitaxel or docetaxel with R115777 in the treatment of MM cells from patients with multiple myeloma was more beneficial than treatment with single agents. Our results provide the basis for combination therapy clinical trials with paclitaxel or docetaxel with R115777 in MM patients.