julilili42/papers huggingface dataset:
Cotransfection of Dendritic Cells with RNA Coding for a Tumor Antigen and Costimulatory Molecule 4-1BBL Increases the Induction of Her-2/neu Specific Cytotoxic T-Cells.
2004-11-16

Abstract Dendritic cells are professional antigen-presenting cells (APC) with the unique capacity to initiate primary immune responses. Recently, it was demonstrated that transfection of DC with RNA coding for specific antigens can elicit an effective T cell response in vitro and in vivo. 4-1BB (CD137) is a member of a family of receptors found on the surface of cells of the immune system. It plays an important role in the costimulation of T-cells: it enhances T-cell proliferation, cytokine production and cytotoxic effector functions and suppresses the induction of apoptosis. It is predominantly expressed on activated T-cells, and its ligand 4-1BBL is expressed on activated APC including DC. In the present study we analyzed the effects of 4-1BBL on the stimulatory capacity of DC. Monocyte derived DC were electroporated with in vitro transcribed RNA encoding the tumor-associated antigen Her-2/neu. Additionally these cells were cotransfected with in vitro transcribed RNA coding for the human 4-1BBL with the aim to enhance the costimulatory functions of the cells and therefore the induction of Her-2/neu specific cytotoxic T-cells (CTL). Standard 51Cr-release assays performed after two restimulations showed specific cytotoxic activity of the induced CTL against autologous DC transfected with Her-2/neu RNA and against the HLA-matched allogeneic Her-2/neu+/HLA-A2+ renal cell carcinoma cell line A498 whereas DC transfected with irrelevant RNA as well as Her-2/neu-/HLA-A2+ CROFT cells and the Her-2/neu+/HLA-A2- cell line SK-OV-3 were spared. There was no lysis of K562 cells, thus excluding a NK-cell mediated killing. In this experimental setting the specific lysis rate of target cells could nearly be doubled by the coadministration of Her-2/neu RNA and an equal amount of 4-1BBL RNA (50% vs 90% specific lysis at effector to target ratio of 30:1). Interestingly, overexpression of 4-1BBL had no effect on the stimulation of allogeneic T cells by electroporated DC in a mixed lymphocyte reaction, indicating that 4-1BBL increases the induction of antigen specific T cell responses rather than the unspecific stimulation of bulk T lymphocytes. Furthermore, FACS analysis of DC transfected with 4-1BBL plasmid DNA or in vitro transcribed 4-1BBL RNA showed higher expression of the costimulatory molecules CD80, CD86, CD40 and the T-cell adhesion molecule CD54 as compared to the controls indicating that 4-1BBL signaling can mediate activation of DC and thus increase their stimulatory capacity. In the context of immunotherapies for cancer patients the findings of our study could contribute to optimise the in vitro manipulation of DC for the induction of antigen-specific CTL responses.
Sensitivity of Childhood Acute Lymphoblastic Leukemia (ALL) to Idarubicin Is Significantly Higher Than to Daunorubicin Treatment Based on Ex Vivo Apoptosis Induction.
2004-11-16

Abstract Anthracyclines, especially daunorubicin, play a very important role in the treatment of acute lymphoblastic leukemia (ALL) and the relapsed ALL in childhood. In the present study, primary lymphoblasts isolated from 65 children with de novo ALL (median: 5.8 years; range: 1.9 – 16.9 years) and relapsed ALL (median: 12.7 years; range: 1.3 – 17.9 years) were treated with daunorubicin (10 mmol/l) or idarubicin (2 mmol/l) in vitro. We could show that both anthracylines induce apoptosis, as evidenced by measurement of genomic DNA fragmentation. Interestingly, daunorubicin only induced modest apoptosis, whereas idarubicin displayed a significantly stronger apoptosis inducing effect. Furthermore the treatment of daunorubicin-resistant lymphoblasts with idarubicin resulted in good response in most of the resistant cell populations. Out of the 65 patients analysed in this study 23 were female (13 de novo ALL, 10 relapsed ALL) and 42 were male (29 de novo ALL, 13 relapsed ALL). Primary lymphoblasts were obtained by bone marrow aspiration and separated by centrifugation over Ficoll. Within these cell populations following immunologic subgroups were found: 35 c-ALL, 10 pre-B-ALL, 7 pro-B-ALL, 10 T-ALL and 3 pre-T-ALL. Daunorubicin induced apoptosis in 33 out of 65 lymphoblast populations (response rate 50.8 %). Nevertheless, a far higher response rate was observed for idarubicin with 59/65 (90,8 %) (p &lt; 0.008), if response is defined as apoptosis induction higher than 1 %. Daunorubicin-resistance was found in 32/65 (49,2 %), resistance to both was observed in 6/65 (9,2 %). Treatment of daunorubicin-resistant lymphoblasts with idarubicin resulted in significant apoptosis induction in 26 out of 32 cell populations (81,3 %). We clearly demonstrated here that the in vitro treatment of lymphoblasts from children with de novo or relapsed ALL with idarubicin induces significantly higher response rates than daunorubicin treatment. The ex vivo sensitivity of daunorubicin-resistant lymphoblasts of childhood ALL to idarubicin treatment reflects the better potency of idarubicin to induce apoptosis and to overcome daunorubicin resistance. These data prompted us to study the clinical relevance of idarubicin in ongoing clinical trials to improve existing therapeutic regiments. First clinical data point to a good tolerability of idarubicin in the treatment of relapsed ALL in childhood.
NK Cell Recognition of Hepatocellular Carcinoma by Specific Induction of NKG2D Ligands Following Treatment with the Histone Deacetylase Inhibitor Sodium Valproate.
2004-11-16

Abstract Histone deacetylase inhibitors (HDAC-I) are currently being evaluated as novel treatment for various malignancies. Recently, the HDAC-I sodium valproate (VPA) was shown to exert anti-neoplastic effects on human hepatoma cells while being well tolerated by primary human hepatocytes (PHH). However, the mechanisms underlying this differential mode of action are unclear. In this study, we used microarray analysis to unravel alterations of genes in hepatoma cells upon treatment with VPA. VPA induced the transcription of MICA and MICB, the ligands of the activating immunoreceptor NKG2D which is expressed on cytotoxic lymphocytes and has been shown to play a central role in the immune surveillance of tumors. Changes in MIC mRNA were mirrored by an increase of cell surface, soluble and total MIC protein as determined by immunofluorescense, flow cytometry and ELISA techniques. No significant changes of the NKG2D ligands (NKG2DL) ULBP1-3 were observed. Most importantly, VPA treatment did not induce MIC protein expression in PHH. The VPA-induced MIC expression profoundly triggered lysis of hepatoma cells by NK92 NK cells, which was abrogated by addition of a NKG2D antibody. Since the strength of an anti-tumor response by NK cells and CD8 T cells is critically depending on NKG2DL expression levels and the HDAC-I VPA mediates lysis of hepatoma cells via specific induction of NKG2DL, our results suggest a potential use of VPA in priming tumor cells for innate immune effector mechanisms.
Transfection of Dendritic Cells with In Vitro Transcribed RNA Induces Polyclonal CMV-Specific CD8+ and CD4+ Mediated T Cell Responses.
2004-11-16

Abstract Transfection of dendritic cells (DC) with in vitro transcribed RNA was shown to be a highly efficient method to generate antigen specific T cells, probably due to the induction of a polyclonal T cell response directed against multiple antigens presented on different HLA allels. However, the experimental evidence of this assumption remains to be demonstrated. To accomplish this, we used monocyte derived DC that were electroporated with RNA coding for the CMV pp65 antigen. The induction and expansion of antigen specific CD8+ and CD4+ T cells was assessed using a pannel of peptides derived from this antigen and presented on HLA-A2, -A1, -A11, -A24, -B35 and -B7 in IFN-g ELISPOT, 51Cr-release and proliferation assays. Autologous DC generated from CMV positive healthy donors were pulsed with peptides or transfected with pp65 RNA and utilized as stimulators. Autologous purified CD8+ and CD4+ lymphocytes were used as effector cells. By applying this approach we found that transfection of DC with pp65 RNA induces an expansion of polyclonal CD8+ mediated T cell responses that recognized peptide antigens presented on different HLA molecules. These in vitro generated cytotoxic T cells were able to efficiently lyse DC pulsed with pp65 derived peptides or transfected with the cognate RNA in an antigen specific manner after several in vitro restimulations. Furthermore, this experimental approach allowed the identification of the immunodominace of T cell epitopes presented upon RNA transfection. The HLA-2 directed responses were more pronounced as compared to the HLA-A1, -A11, -A24 or -B35 allels. In contrast, in 7 out of 7 HLA-A2 and HLA-B7 positive donors B7-peptides elicited a stronger T cell response than the A2-peptide, indicating the immunodominance of HLA-B7 epitopes. Interestingly, transfection of DC with pp65 RNA resulted in the induction of CD4+ antigen specific T cells that produced IFN-g and proliferated upon stimulation with transfected DC. In the next set of experiments we analyzed the possible induction of CMV specific T cells that recognize epitopes deduced from different antigens. Co-transfection of DC with a mixture of RNAs coding for the CMV pp65 and IE1 antigens elicited polyclonal T lymphocytes specific for peptides derived from both antigens. More importantly, polyclonal cytotoxic T cells could be elicited in peripheral blood of 2 out of 3 CMV negative donors demonstrating the efficiency of this approach. Our results demonstrate that DC transfected with RNA can elicit polyclonal T cell responses and have implications for the development of immunotherapeutic strategies to target viral or tumor associated antigens.
Identification of BCR-ABL Dependent Gene Regulation by Using a Phosphoproteomics Approach.
2004-11-16

Abstract Introduction: The selective tyrosine kinase inhibitor imatinib (formerly STI571, Glivecâ) has been shown to block phosphorylation of tyrosine residues by occupying the ATP binding site of the Abl tyrosine kinases Bcr-Abl, c-Abl, v-Abl and Abl-related gene (ARG), as well as platelet-derived growth factor receptor (PDGF) alpha and beta and of the receptor for human stem cell factor (SCF) c-kit. We chose a large scale phospho-proteomics approach to identify novel downstream targets of imatinib which could possibly be utilized for combined treatment strategies. Material and Methods: Phospho-proteomics was performed by comparison of large scale phosphotyrosine-immunoprecipitation of imatinib (10 mM/2 hours) versus DMSO treated K562 cells separated by one-dimensional polyacrylamide gelelectrophoresis. In addition enriched CD34+ cells (&amp;gt;70%) of a newly diagnosed Bcr-Abl positive CML patient were used immediately after purification and treated in the same way as described above. Resulting differentially immuno-precipitated proteins were analyzed using matrix-assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF) and nano electrospray ionization tandem mass spectrometry (ESI-MS/MS). Protein identification via peptide mass-fingerprinting and peptide sequencing was performed using Mascot search tool and NCBI nr database. Differential phosphorylation was confirmed by combined immunoprecipitation and western blot analysis of selected candidate proteins. Results: Phospho-proteomics of K562 cells revealed 8 differentially phosphorylated proteins after a two hour treatment with imatinib including the recently identified c-cbl, and Bcr-Abl itself, the latter confirming autophosphorylation. Ship2 which was originally identified as beeing constitutively phosphorylated in chronic myelogenous leukemia progenitor cells showed reduced, imatinib sensitive phosphorylation. Remaining candidates could be classified as being involved in protein folding or in ATPase activities associated with a variety of functions (type II AAA). The analysis of primary CD34+ cells from a CML patient showed a predominance of the nonmuscular myosin heavy chain protein in different molecular weight forms. Discussion: We detected significant imatinib-dependent differences in protein phosphotyrosine-immunoreactivity of the Bcr-Abl -dependent cell line K562. Previously identified down-stream targets of Bcr-Abl could be confirmed and novel candidate proteins were identified. Phosphorylation of Ship2, a previously identified down-stream target of Bcr-Abl, was found to be inhibited by imatinib treatment. Ongoing studies are aimed at the characterization of the role of the identified phospho-proteins, particularly type II AAAs for Bcr-Abl induced signal transduction as well as for the development of resistance to imatinib.
Proaerolysin Multi Color Flow-Fish Reveals Shortened Telomere Length in GPI− as Compared to GPI+ Granulocytes from Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH).
2004-11-16

Abstract Objective: Replication-dependent telomere shortening can result in cellular senescence or genetic instability. Telomere length both reflects and limits the replicative potential of normal somatic cells. Shortened telomeres have been linked to disease stage, degree of cytopenia as well as to response to treatment in patients with bone marrow failure syndromes. Paroxysmal nocturnal haemoglobinuria (PNH) is caused by a somatic mutation in the X-linked PIG-A gene resulting in a deficiency of GPI-linked proteins on the cell surface. Pancytopenia in PNH is thought to result from an auto-immune mediated depletion of the GPI+ stem cell compartment. In the current study, we aimed to develop a methodology that allows the selective analysis of telomere length in granulocytes from patients with PNH dependent on GPI expression. The aim was to find out whether replicative stress posed on the residual GPI− HSC is reflected in shortened telomere length compared to their GPI+ counterparts. Methods: The telomere length of GPI+ and GPI− granulocytes of 12 patients with PNH and 22 healthy individuals was analysed. For this purpose, we developed Proearolysine Multi-color flow FISH which is based on the crosslinking of a fluorescence-labeled non-toxic derivative of the bacterial toxine Aerolysine (that selectively binds to the GPI anchor of cells) in combination with fluorescence in situ hybridization and flow cytometry. Results: We found significantly (p&lt;0.05) shortened telomeres in GPI− granulocytes (6.68±0.3 telomere fluorescence unit (TFU), n=12) compared to age-matched healthy individuals (mean±S.E.: 7.73±0.8 TFU, n=22), but no significant shortening in GPI+ (7.06±0.3 TFU, n=12) granulocytes from PNH patients. Telomere length in GPI− granulocytes was found to be significantly shorter as compared to their GPI+ counterparts (deltaTEL: 0.38±0.1, p=0.002). These results were confirmed by Multi-color flow FISH using an anti-CD59 antibodies. Conclusion: Autoimmune-mediated damage to the GPI+ HSC compartment leads to compensatory hyperproliferation of predominantly residual GPI− HSC. In line with this hypothesis, coexisting GPI-negative HSC clones showed even more accelerated telomere shortening as compared to their GPI+ counterparts. Potentially, replicative exhaustion of individual HSC clones could contribute to disease progression in PNH. Whether acquired genetic instability due to progressive telomere shortening also accounts for an increased incidence of secondary clonal disorders will need to be evaluated in future prospective studies.
Impact of Immunosuppressive Drugs on Adaptive and Innate Immune Responses to Aspergillus fumigatus Antigens.
2004-11-16

Abstract Appropriate activation of the innate and adaptive immune system is crucial for the successful control of invasive aspergillosis (IA). Acute and chronic graft-versus-host disease as well as corticosteroids were described as major risk factors for the development of IA. In this study, we assessed the impact of immunosuppressive agents (dexamethasone, rapamycin, Cyclosporin A, FK506) on the A. fumigatus induced activation of monocyte-derived immature dendritic cells (iDC) and A. fumigatus-specific T-cell responses in well established cell culture models. Immature DCs were found to be activated and to differentiate into mature DCs in response to A. fumigatus antigens. The upregulation of CD86 was inhibited by dexamethasone (D) in 3 out of 3 experiments, and of CD40 and CD80 in 2/3. CSA and FK506 had a variable impact on the upregulation of CD86, but not on CD40 and CD80, whereas the expression of co-stimulatory molecules was found unchanged upon incubation with rapamycin. Autologous DCs were found to restore A. fumigatus-specific T-cell responses. T-cell proliferation to A. fumigatus hyphae and a cellular extract of the culture filtrate were found to be strongly inhibited by rapamycin and dexamethasone (n=3), whereas the effect of CSA and FK506 (n=3) at the concentrations analysed was variable. The release of IFN-g in culture supernatants upon stimulation with A. fumigatus antigens was strongly reduced in the presence of rapamycin (n=3), whereas the release of IL-4 was found to be increased in the majority of experiments (n=3). Comparable results were observed upon stimulation with tetanus toxoid and a CMV lysate (n=3). These data indicate, that A. fumigatus-spec. T-cell responses may be directed towards a TH2 phenotype in the presence of immunosuppressive agents. In summary, immunosuppressive agents were found to exert differential effects on adaptive and innate immune responses directed against A. fumigatus. Whereas dexamethasone was found to modulate the expression of co-stimulatory molecules on A. fumigatus activated iDCs and to suppress A. fumigatus-specific lymphoproliferation, rapamycin exerted only minor effects on DC-activation but had a strong impact on A. fumigatus-induced T-cell responses. These results may help to tailor immunosuppressive regimens in patients at high risk for invasive aspergillosis.
Generation of Leukemia-Derived Dendritic Cells (DCleu) as a Basis for Specific Immunotherapy in Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS).
2004-11-16

Abstract The presentation of leukemic antigens can be improved in AML and MDS by in vitro generation of dendritic cells of leukemic origin (DCleu), thereby creating a platform for the generation of leukemia-specific cytotoxic lymphocytes (CTL). To further investigate this approach, we developed a serum-free culture system using MCM-Mimic medium (X-vivo + GM-GSF + IL-4 + FL + IL1β + IL-6 + TNFα + PGE2) for differentiation of malignant myeloid blasts to DCleu. Periperal blood mononuclear cells (PB-MNC) were obtained from 100 AML and 55 MDS-patients. Samples contained a mean of 59%/and 6% of blasts, respectively. After 14 days, cultures contained on average 34% (AML) and 20%(MDS) DC. DC yields were best in monocytic subtypes (AML M4, M5 and CMML). Cytogenetic aberrations of the leukemia had no influence. The leukemic origin of cultured DC was demonstrated using a acombined FISH/immune phenotyping assay (FISH/IPA): In cells showing a characteristic DC morphology and immune phenotype, FISH was used to detect specific cytogenetic aberrations identified in the leukemic population at time of diagnosis. Alternativly, DCleu were identified by detecting coexpression of DC markers and a leukemia-specific immune phenotype (as defined by aberrant expression of lineage markers or a characteristic CD34+/CD117+ phenotype in MDS). However, in most cases not all leukemic blasts in a given sample could be differentiated, since on average 47% of clonal cells did not acquire a DC-like immunophenotype. Vice versa, not all DC identified at the end of the culture period were DCleu. The capacity of DCleu to elicit a specific T-cell response was demonstrated by upregulation of contact-molecules, responsible for DC/T-cell contact, by DC-activated T-cell proliferation and by the capacity of DC-activated T-cells to specifically lyse naive blasts. In 6%/31% of AML/MDS-cases, &amp;lt;10% DC could be generated. Therefore, other DC-culture-assays were compared with respect to DC harvest, to identify the best method for DC-generation in each individual patient. Compared to `MCM-Mimic`, harvest of DC could be improved by `CA-Ionophore-media`(A23187 + Il4) in 4 of 7 cases, whereas in 3 cases, MCM led to higher DC-yields. In conclusion, DCleu can be generated in the majority of patients with AML and MDS. To optimize DC harvest for in vitro and in vivo use, different culture assays should be compared in each individual case. DCleu are able to elicit a specific T-cell response in vitro. Nevertheless, cultures containing DCleu also contain relevant numbers of undifferentiated blasts and DC of non-leukemic origin. These cells may represent an obstacle for the clinical use of DCleu, since they may cause specific anergy or unspecific stimulation of effector T-cells. Improvement of culture conditions for generation of DCleu, and methods to separate DCleu before stimulation of effector cells will be required, before clinical trials are feasible.
The Effects of Imatinib on the Function of Monocyte Derived Dendritic Cells Are Mediated Via the Inhibition of Akt and NF-kappaB Signal Transduction Pathways.
2004-11-16

Abstract Imatinib is a novel tyrosine kinase inhibitor effective against Abl kinases, c-Kit and platelet-derived growth-factor receptor (PDGF-R) and is currently used for the treatment of patients with CML and GIST. However, little is known about the effects of imatinib on function and differentiation of non-transformed normal cells. Using this compound, we show that human monocyte derived DC generated in the presence of therapeutic concentrations of imatinib show a concentration dependent reduced expression of CD1a, HLA and co-stimulatory molecules as well as decreased activation-induced secretion of chemokines and cytokines involved in T cell activation. Moreover, exposure to imatinib reduces the capacity of DC to prime T cell responses that cannot be restored by the addition of IL-12 and which is not due to induction of apoptosis or IL-10 secretion. Using Western blot analyses we found that these effects are mediated by a pronounced downregulation of nuclear localized protein levels of NF-kB family members RelB, RelA and NF-kB p50. Furthermore, imatinib treatment inhibited the phosphorylation of Akt, indicating the involvement of the PI3 kinase pathways while not affecting the phosphorylation state of p38 and ERK1 MAP kinase. In line with these results, incubation of monocytes with PI3 kinase inhibitors resulted in a similar phenotype of DC as described above. Gene expression profiling utilizing DNA microarrays revealed upregulation of lysosomal genes and molecules preferentially expressed in monocytes/macrophages. However, in contrast to these observations, imatinib treatment had no effect on the incorporation of latex beads by DC and resulted in a reduced FITC-labeled dextran uptake. Importantly, utilizing blocking antibodies and tyrosine kinase inhibitors we demonstrate that the inhibitory effects of imatinib on DC differentiation are not mediated by PDGF-R and c-Kit but most likely via c-Abl tyrosine kinase. These results demonstrate that imatinib affects the antigen presenting function of DC on several levels: their phenotype, antigen uptake and processing as well as production of cytokines and chemokines.