TARP is an immunotherapeutic target in acute myeloid leukemia expressed in the leukemic stem cell compartment.

Immunotherapeutic strategies targeting the rare leukemic stem cell compartment might provide salvage to the high relapse rates currently observed in acute myeloid leukemia (AML). We applied gene expression profiling for comparison of leukemic blasts and leukemic stem cells with their normal counterparts. Here, we show that the T-cell receptor γ chain alternate reading frame protein (TARP) is over-expressed in de novo pediatric (n=13) and adult (n=17) AML sorted leukemic stem cells and blasts compared to hematopoietic stem cells and normal myeloblasts (15 healthy controls). Moreover, TARP expression was significantly associated with a fms-like tyrosine kinase receptor-3 internal tandem duplication in pediatric AML. TARP overexpression was confirmed in AML cell lines (n=9), and was found to be absent in B-cell acute lymphocytic leukemia (n=5) and chronic myeloid leukemia (n=1). Sequencing revealed that both a classical TARP transcript, as described in breast and prostate adenocarcinoma, and an AML-specific alternative TARP transcript, were present. Protein expression levels mostly matched transcript levels. TARP was shown to reside in the cytoplasmic compartment and showed sporadic endoplasmic reticulum co-localization. TARP-T-cell receptor engineered cytotoxic T-cells in vitro killed AML cell lines and patient leukemic cells co-expressing TARP and HLA-A*0201. In conclusion, TARP qualifies as a relevant target for immunotherapeutic T-cell therapy in AML.

Evolution of self-organisation in Dictyostelia by adaptation of a non-selective phosphodiesterase and a matrix component for regulated cAMP degradation.

Dictyostelium discoideum amoebas coordinate aggregation and morphogenesis by secreting cyclic adenosine monophosphate (cAMP) pulses that propagate as waves through fields of cells and multicellular structures. To retrace how this mechanism for self-organisation evolved, we studied the origin of the cAMP phosphodiesterase PdsA and its inhibitor PdiA, which are essential for cAMP wave propagation. D. discoideum and other species that use cAMP to aggregate reside in group 4 of the four major groups of Dictyostelia. We found that groups 1-3 express a non-specific, low affinity orthologue of PdsA, which gained cAMP selectivity and increased 200-fold in affinity in group 4. A low affinity group 3 PdsA only partially restored aggregation of a D. discoideum pdsA-null mutant, but was more effective at restoring fruiting body morphogenesis. Deletion of a group 2 PdsA gene resulted in disruption of fruiting body morphogenesis, but left aggregation unaffected. Together, these results show that groups 1-3 use a low affinity PdsA for morphogenesis that is neither suited nor required for aggregation. PdiA belongs to a family of matrix proteins that are present in all Dictyostelia and consist mainly of cysteine-rich repeats. However, in its current form with several extensively modified repeats, PdiA is only present in group 4. PdiA is essential for initiating spiral cAMP waves, which, by organising large territories, generate the large fruiting structures that characterise group 4. We conclude that efficient cAMP-mediated aggregation in group 4 evolved by recruitment and adaptation of a non-selective phosphodiesterase and a matrix component into a system for regulated cAMP degradation.

Pharmacological profiling of the Dictyostelium adenylate cyclases ACA, ACB and ACG.

Intracellular and secreted cAMPs play crucial roles in controlling cell movement and gene regulation throughout development of the social amoeba Dictyostelium discoideum. cAMP is produced by three structurally distinct ACs (adenylate cyclases), ACA, ACG and ACB, which have distinctive but overlapping patterns of expression and, as concluded from gene disruption studies, seemingly overlapping functions. In addition to gene disruption, acute pharmacological abrogation of protein activity can be a powerful tool to identify the protein's role in the biology of the organism. We analysed the effects of a range of compounds on the activity of ACA, ACB and ACG to identify enzyme-specific modulators. Caffeine, which was previously used to specifically block ACA function, also inhibited cAMP accumulation by ACB and ACG. IPA (2',3'-O-isopropylidene adenosine) specifically inhibits ACA when measured in intact cells, without affecting ACB or ACG. All three enzymes are inhibited by the P-site inhibitor DDA (2',5'-dideoxyadenosine) when assayed in cell lysates, but not in intact cells. Tyrphostin A25 [alpha-cyano-(3,4,5-trihydroxy)cinnamonitrile] and SQ22536 [9-(tetrahydro-2'-furyl)adenine] proved to be effective and specific inhibitors for ACG and ACA respectively. Both compounds acted directly on enzyme activity assayed in cell lysates, but only SQ22536 was also a specific inhibitor when added to intact cells.

A novel neutralizing human monoclonal antibody broadly abrogates hepatitis C virus infection in vitro and in vivo.

Infections with hepatitis C virus (HCV) represent a worldwide health burden and a prophylactic vaccine is still not available. Liver transplantation (LT) is often the only option for patients with HCV-induced end-stage liver disease. However, immediately after transplantation, the liver graft becomes infected by circulating virus, resulting in accelerated progression of liver disease. Although the efficacy of HCV treatment using direct-acting antivirals has improved significantly, immune compromised LT-patients and patients with advanced liver disease remain difficult to treat. As an alternative approach, interfering with viral entry could prevent infection of the donor liver. We generated a human monoclonal antibody (mAb), designated 2A5, which targets the HCV envelope. The neutralizing activity of mAb 2A5 was assessed using multiple prototype and patient-derived HCV pseudoparticles (HCVpp), cell culture produced HCV (HCVcc), and a human-liver chimeric mouse model. Neutralization levels observed for mAb 2A5 were generally high and mostly superior to those obtained with AP33, a well-characterized HCV-neutralizing monoclonal antibody. Using humanized mice, complete protection was observed after genotype 1a and 4a HCV challenge, while only partial protection was achieved using gt1b and 6a isolates. Epitope mapping revealed that mAb 2A5 binding is conformation-dependent and identified the E2-region spanning amino acids 434 to 446 (epitope II) as the predominant contact domain. CONCLUSION: mAb 2A5 shows potent anti-HCV neutralizing activity both in vitro and in vivo and could hence represent a valuable candidate to prevent HCV recurrence in LT-patients. In addition, the detailed identification of the neutralizing epitope can be applied for the design of prophylactic HCV vaccines.

HIV-1 Vpr N-terminal tagging affects alternative splicing of the viral genome.

To facilitate studies on Vpr function in replicating HIV-1, we aimed to tag the protein in an infectious virus. First we showed that N-, but not C-terminal HA/FLAG tagging of Vpr protein preserves Vpr cytopathicity. Cloning the tags into proviral DNA however ablated viral production and replication. By construction of additional viral variants we could show this defect was not protein- but RNA-dependent and sequence specific, and characterized by oversplicing of the genomic RNA. Simulation of genomic RNA folding suggested that introduction of the tag sequence induced an alternative folding structure in a region enriched in splice sites and splicing regulatory sequences. In silico predictions identified the HA/His6-Vpr tagging in HIV-1 to affect mRNA folding less than HA/FLAG-Vpr tagging. In vitro infectivity and mRNA splice pattern improved but did not reach wild-type values. Thus, sequence-specific insertions may interfere with mRNA splicing, possibly due to altered RNA folding. Our results point to the complexity of viral RNA genome sequence interactions. This should be taken into consideration when designing viral manipulation strategies, for both research as for biological interventions.

Vaccine-induced monoclonal antibodies targeting circumsporozoite protein prevent Plasmodium falciparum infection.

Malaria, which is the result of Plasmodium falciparum infection, is a global health threat that resulted in 655,000 deaths and 216 million clinical cases in 2010 alone. Recent phase 3 trials with malaria vaccine candidate RTS,S/AS01 (RTS,S) in children has demonstrated modest efficacy against clinical and severe malaria. RTS,S targets the pre-erythrocytic phase of the disease and induces high antibody titers against the P. falciparum circumsporozoite protein (CSP) and a moderate CD4(+) T cell response. The individual contribution of these adaptive immune responses to protection from infection remains unknown. Here, we found that prophylactic administration of anti-CSP mAbs derived from an RTS,S-vaccinated recipient fully protected mice with humanized livers from i.v.- and mosquito bite­delivered P. falciparum sporozoite challenge. Titers of anti-CSP that conveyed full protection were within the range observed in human RTS,S vaccine recipients. Increasing anti-CSP titers resulted in a dose-dependent reduction of the liver parasite burden. These data indicate that RTS,S-induced antibodies are protective and provide sterilizing immunity against P. falciparum infection when reaching or exceeding a critical plasma concentration.

Genetic control of lithium sensitivity and regulation of inositol biosynthetic genes.

Lithium (Li(+)) is a common treatment for bipolar mood disorder, a major psychiatric illness with a lifetime prevalence of more than 1%. Risk of bipolar disorder is heavily influenced by genetic predisposition, but is a complex genetic trait and, to date, genetic studies have provided little insight into its molecular origins. An alternative approach is to investigate the genetics of Li(+) sensitivity. Using the social amoeba Dictyostelium, we previously identified prolyl oligopeptidase (PO) as a modulator of Li(+) sensitivity. In a link to the clinic, PO enzyme activity is altered in bipolar disorder patients. Further studies demonstrated that PO is a negative regulator of inositol(1,4,5)trisphosphate (IP(3)) synthesis, a Li(+) sensitive intracellular signal. However, it was unclear how PO could influence either Li(+) sensitivity or risk of bipolar disorder. Here we show that in both Dictyostelium and cultured human cells PO acts via Multiple Inositol Polyphosphate Phosphatase (Mipp1) to control gene expression. This reveals a novel, gene regulatory network that modulates inositol metabolism and Li(+) sensitivity. Among its targets is the inositol monophosphatase gene IMPA2, which has also been associated with risk of bipolar disorder in some family studies, and our observations offer a cellular signalling pathway in which PO activity and IMPA2 gene expression converge.

Vectors for expression of proteins with single or combinatorial fluorescent protein and tandem affinity purification tags in Dictyostelium.

We constructed a series of expression vectors for purification of native proteins and protein complexes in Dictyostelium. Protein purification is achieved by either a C-terminal or N-terminal fusion of the protein of choice to the tandem affinity purification (TAP) tag. The TAP tag consists of a protein A tag and a calmodulin binding peptide (CBP) and has been successfully used for purification of native protein complexes from yeast and animal cells. Protein expression is driven by the constitutive actin 15 promoter and the vectors optionally carry additional green- or yellow fluorescent protein (GFP or YFP) tags for fusion at either a C- or N-terminal location. Tandem affinity purification of native Dictyostelium protein complexes was tested by using pArc-34, one of the members of the well characterized Dictyostelium Arp2/3 complex, as bait. After denaturation and SDS-PAGE separation of the pArc-34 associated proteins all members of the Arp2/3 complex could be identified.

Characterization of a cAMP-stimulated cAMP phosphodiesterase in Dictyostelium discoideum.

A cyclic nucleotide phosphodiesterase, PdeE, that harbors two cyclic nucleotide binding motifs and a binuclear Zn(2+)-binding domain was characterized in Dictyostelium. In other eukaryotes, the Dictyostelium domain shows greatest homology to the 73-kDa subunit of the pre-mRNA cleavage and polyadenylation specificity factor. The Dictyostelium PdeE gene is expressed at its highest levels during aggregation, and its disruption causes the loss of a cAMP-phosphodiesterase activity. The pdeE null mutants show a normal cAMP-induced cGMP response and a 1.5-fold increase of cAMP-induced cAMP relay. Overexpression of a PdeE-yellow fluorescent protein (YFP) fusion construct causes inhibition of aggregation and loss of the cAMP relay response, but the cells can aggregate in synergy with wild-type cells. The PdeE-YFP fusion protein was partially purified by immunoprecipitation and biochemically characterized. PdeE and its Dictyostelium ortholog, PdeD, are both maximally active at pH 7.0. Both enzymes require bivalent cations for activity. The common cofactors Zn(2+) and Mg(2+) activated PdeE and PdeD maximally at 10 mm, whereas Mn(2+) activated the enzymes to 4-fold higher levels, with half-maximal activation between 10 and 100 microm. PdeE is an allosteric enzyme, which is approximately 4-fold activated by cAMP, with half-maximal activation occurring at about 10 microm and an apparent K(m) of approximately 1 mm. cGMP is degraded at a 6-fold lower rate than cAMP. Neither cGMP nor 8-Br-cAMP are efficient activators of PdeE activity.

Contrasting activities of the aggregative and late PDSA promoters in Dictyostelium development.

Expression of the Dictyostelium PdsA gene from the aggregative (PdA) and late (PdL) promoter is essential for aggregation and slug morphogenesis, respectively. We studied the regulation of the PdA and PdL promoters in slugs using labile beta-galactosidase (gal) reporter enzymes. PdL was active in prestalk cells as was also found with stable gal. PdA activity decreased strongly in slugs from all cells, except those at the rear. This is almost opposite to PdA activity traced with stable gal, where slugs showed sustained activity with highest levels at the front. PdA was down-regulated after aggregation irrespective of stimulation with any of the factors known to control gene expression. PdL activity was induced in cell suspension by cAMP and DIF acting in synergy. However, a DIF-less mutant showed normal PdL activity during development, suggesting that DIF does not control PdL in vivo. Dissection of the PdL promoter showed that all sequences essential for correct spatiotemporal control of promoter activity are downstream of the transcription start site in a region between -383 and -19 nucleotides relative to the start codon. Removal of nucleotides to position -364 eliminated responsiveness to DIF and cAMP, but normal PdL activity in prestalk cells in slugs was retained. Further 5' deletions abolished all promoter activity. This result also indicates that the induction by DIF and cAMP as seen in cell suspensions is not essential for PdL activity in normal development.

cAMP signaling in Dictyostelium. Complexity of cAMP synthesis, degradation and detection.

cAMP plays a pivotal role in control of cell movement, differentiation and response to stress in all phases of the Dictyostelium life cycle. The multitudinous functions of cAMP require precise spatial and temporal control of its production, degradation and detection. Many novel proteins have recently been identified that critically modulate the cAMP signal. We focus in this review on the properties and functions of the three adenylyl cyclases and the three cAMP-phosphodiesterases that are present in Dictyostelium, and the network of proteins that regulate the activity of these enzymes. We also briefly discuss the two modes of detection of cAMP.