Expression of and immune responses to leukemia antigens in patients with hematological malignancies

Leukemia cells are characterized by differentially expressed leukemia-associated antigens (LAAs). We wondered whether the expression of the LAAs WT1 and RHAMM as well as the T cellular immune response to these LAAs correlated with the clinical outcome of patients suffering from leukemia. We investigated the expression of WT1 and RHAMM at RNA level using qPCR before and after treatment. We concluded that WT1 is a suitable marker for MRD after allogeneic SCT and that a WT1-specific T cell response might contribute to the maintenance of CR

Immune Responses to RHAMM in Patients with Acute Myeloid Leukemia after Chemotherapy and Allogeneic Stem Cell Transplantation

Leukemic blasts overexpress immunogenic antigens, so-called leukemia-associated antigens like the receptor for hyaluronan acid-mediated motility (RHAMM). Persistent RHAMM expression and decreasing CD8+ T-cell responses to RHAMM in the framework of allogeneic stem cell transplantation or chemotherapy alone might indicate the immune escape of leukemia cells. In the present study, we analyzed the expression of RHAMM in 48 patients suffering from acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Furthermore, we correlated transcripts with the clinical course of the disease before and after treatment. Real-time quantitative reverse transcriptase polymerase chain reaction was performed from RNA of peripheral blood mononuclear cells. T cell responses against RHAMM were assessed by tetramer staining (flow cytometry) and enzyme-linked immunospot (ELISPOT) assays. Results were correlated with the clinical outcome of patients. The results of the present study showed that almost 60% of the patients were RHAMM positive; specific T-cells recognizing RHAMM could be detected, but they were nonfunctional in terms of interferon gamma or granzyme B release as demonstrated by ELISPOT assays. Immunotherapies like peptide vaccination or adoptive transfer of RHAMM-specific T cells might improve the immune response and the outcome of AML/MDS patients

Block of death-receptor apoptosis protects mouse cytomegalovirus from macrophages and is a determinant of virulence in immunodeficient hosts.

The inhibition of death-receptor apoptosis is a conserved viral function. The murine cytomegalovirus (MCMV) gene M36 is a sequence and functional homologue of the human cytomegalovirus gene UL36, and it encodes an inhibitor of apoptosis that binds to caspase-8, blocks downstream signaling and thus contributes to viral fitness in macrophages and in vivo. Here we show a direct link between the inability of mutants lacking the M36 gene (ΔM36) to inhibit apoptosis, poor viral growth in macrophage cell cultures and viral in vivo fitness and virulence. ΔM36 grew poorly in RAG1 knockout mice and in RAG/IL-2-receptor common gamma chain double knockout mice (RAGγC(-/-)), but the depletion of macrophages in either mouse strain rescued the growth of ΔM36 to almost wild-type levels. This was consistent with the observation that activated macrophages were sufficient to impair ΔM36 growth in vitro. Namely, spiking fibroblast cell cultures with activated macrophages had a suppressive effect on ΔM36 growth, which could be reverted by z-VAD-fmk, a chemical apoptosis inhibitor. TNFα from activated macrophages synergized with IFNγ in target cells to inhibit ΔM36 growth. Hence, our data show that poor ΔM36 growth in macrophages does not reflect a defect in tropism, but rather a defect in the suppression of antiviral mediators secreted by macrophages. To the best of our knowledge, this shows for the first time an immune evasion mechanism that protects MCMV selectively from the antiviral activity of macrophages, and thus critically contributes to viral pathogenicity in the immunocompromised host devoid of the adaptive immune system

Predictions versus high-throughput experiments in T-cell epitope discovery: competition or synergy?

Prediction methods as well as experimental methods for T-cell epitope discovery have developed significantly in recent years. High-throughput experimental methods have made it possible to perform full-length protein scans for epitopes restricted to a limited number of MHC alleles. The high costs and limitations regarding the number of proteins and MHC alleles that are feasibly handled by such experimental methods have made in silico prediction models of high interest. MHC binding prediction methods are today of a very high quality and can predict MHC binding peptides with high accuracy. This is possible for a large range of MHC alleles and relevant length of binding peptides. The predictions can easily be performed for complete proteomes of any size. Prediction methods are still, however, dependent on good experimental methods for validation, and should merely be used as a guide for rational epitope discovery. We expect prediction methods as well as experimental validation methods to continue to develop and that we will soon see clinical trials of products whose development has been guided by prediction methods

Fischer-Tropsch Catalysts for Jet Fuel Production

Diverse β-zeolite and β-zeolite/Fischer Tropsch catalysts were prepared under hydrothermal conditions to obtain a tailored jet fuel catalyst. The Fischer Tropsch catalyst that was employed is cobalt-based and it was provided by Statoil Research Center at Rotvoll. A sample of pure β-zeolite was synthesized to obtain a reference product. The hydrothermal synthesis of the β-zeolite and β-zeolite/Fischer catalyst was performed at 155 °C. It was decided to elaborate the bifunctional catalyst to obtain jet fuel products with β-zeolite because this zeolite type posses larger micropores than other zeolites. The initial chemical components of the β-zeolite/Fischer catalyst were: silicon source, aluminum source, organic template, water, and Fischer Tropsch cobalt based catalyst. Especial attention was paid to the silicon source because is well known that a few ppm s of sodium could affect negatively the performance of the Fischer Tropsch cobalt-based catalyst. It was concluded that fumed silica possess the best trade-off qualities between purity and price to perform large a number of hydrothermal synthesis. Different procedures were used to modify the final hydrothermal β-zeolite/Fischer catalyst. The most successful was the impregnation of the internal pores of the Fischer Tropsch cobalt-based catalyst alumina support with the organic template. Prior to the Fischer Tropsch synthesis test, all the produced β-zeolite/Fischer catalyst were characterized. The BET method was utilized to measure the surface area, pore volume, and pore size. XRD powder patterns were used to verify the presence of the β-zeolite and also to prove that the calcination at 550°C did not damage the Fischer Tropsch cobalt based catalyst. SEM was employed as a qualitative technique to confirm the presence of the β-zeolite in the Fischer Tropsch catalyst surface. Chemisorption was used to determine the metal dispersion and the cobalt crystal size. Several bifunctional catalyst were tested in the Fischer Tropsch synthesis. The results show that a higher quantity of β-zeolite in the β-zeolite/Fischer Tropsch catalyst produces elevated amounts of methane and decreases the production of C₅+ products. Comparing the resulting products of the pure Fischer Tropsch catalyst with the ones of the β-zeolite/Fischer catalyst, the final amount of C₃ to C₆ products remain in the same values. However, the remarkable change is the olefin/paraffin ratio; which is affected by the β-zeolite presence, decreasing the olefin percentage. A linear correlation between methane and C₅+ was found

UL36 Rescues Apoptosis Inhibition and In vivo Replication of a Chimeric MCMV Lacking the M36 Gene.

Apoptosis is an important defense mechanism mounted by the immune system to control virus replication. Hence, cytomegaloviruses (CMV) evolved and acquired numerous anti-apoptotic genes. The product of the human CMV (HCMV) UL36 gene, pUL36 (also known as vICA), binds to pro-caspase-8, thus inhibiting death-receptor apoptosis and enabling viral replication in differentiated THP-1 cells. In vivo studies of the function of HCMV genes are severely limited due to the strict host specificity of cytomegaloviruses, but CMV orthologues that co-evolved with other species allow the experimental study of CMV biology in vivo. The mouse CMV (MCMV) homolog of the UL36 gene is called M36, and its protein product (pM36) is a functional homolog of vICA that binds to murine caspase-8 and inhibits its activation. M36-deficient MCMV is severely growth impaired in macrophages and in vivo. Here we show that pUL36 binds to the murine pro-caspase-8, and that UL36 expression inhibits death-receptor apoptosis in murine cells and can replace M36 to allow MCMV growth in vitro and in vivo. We generated a chimeric MCMV expressing the UL36 ORF sequence instead of the M36 one. The newly generated MCMV(UL36) inhibited apoptosis in macrophage lines RAW 264.7, J774A.1, and IC-21 and its growth was rescued to wild type levels. Similarly, growth was rescued in vivo in the liver and spleen, but only partially in the salivary glands of BALB/c and C57BL/6 mice. In conclusion, we determined that an immune-evasive HCMV gene is conserved enough to functionally replace its MCMV counterpart and thus allow its study in an in vivo setting. As UL36 and M36 proteins engage the same molecular host target, our newly developed model can facilitate studies of anti-viral compounds targeting pUL36 in vivo

ΔM36 grows poorly in the presence of macrophages.

<p>(A) CD11b positive cells were removed from MEF cell preparations by monoclonal antibodies coupled to magnetic beads, upon which the cells were infected with ΔM36 (white bars) or M36rev (grey bars), alone or in the presence of Zymosan (30 µg/ml) or IFNγ (100 ng/ml). Virus titer in the supernatant of cells depleted of macrophages was compared to macrophage-undepleted MEF preparations at day 4 post infection. (B) Upon macrophage depletion, primary fibroblasts were cultured with indicated amounts of ANA-I macrophages (MΦ), in the presence or absence of Zymosan (30 µg/ml) and IFNγ (100 ng/ml). Infectious virus titer in supernatants was established at day 4 post infection. Histograms indicate mean values from three separate experiments, error bars show standard deviation, * p<0.05.</p

Apoptosis inhibition is required for viral dissemination to distant organs.

<p>RAG1^−/− mice were (A) i.p. or (B) s.c. infected with 10⁵ PFU of indicated virus and monitored for survival (n = 4–6/group). Mortality also includes mice that were sacrificed because they had lost more than 20% of body weight. (C) Infectious virus was determined by plaque assay on MEF cells in spleen (top panel), lungs (middle panel), and salivary glands (SG, bottom panel) of i.p. infected mice on day 13 after infection with 10⁵ PFU of indicated virus. Each symbol represents an individual mouse. Differences in median values are highlighted by grey shading. The dashed line shows the limit of detection.</p

Diagram of the proposed mechanism of action.

<p>Activated macrophages secrete TNFα (and possibly additional cytokines) which synergize with IFNγ in fibroblasts to block virus growth by a mechanism that is dependent on caspase signaling. M36 blocks the caspase-dependent signaling pathway and thus prevents apoptosis and rescues the virus growth.</p

Macrophage, but not NK cell, depletion rescues ΔM36 MCMV <i>in vivo</i>.

<p>In a combined experiment to elucidate the role of (A) NK cells and (B) macrophages in the control of ΔM36 MCMV growth, RAG1^−/− and RAGγC^−/− mice received injections of 200 µl liposome encapsulated (A) PBS or (B) clodronate 48 hours (i.v.) and 24 hours (i.p.) prior to viral infection. Following liposome injection mice were i.p. injected with 10⁵ PFU ΔM36 (○) or M36rev (•) MCMV (n = 4–5/group). At day 3 post infection infectious virus was determined by plaque assay on MEF cells in spleen (top panels), lungs (middle panels) and liver (bottom panels). Each symbol represents an individual mouse. Differences in median values are highlighted by grey shading. The dashed line shows the limit of detection. *p<0.05; **p<0.01.</p