International Immunology Advance Access originally published online on September 5, 2006
International Immunology 2006 18(11):1521-1529; doi:10.1093/intimm/dxl085
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Novel pan-DR-binding T cell epitopes of adenovirus induce pro-inflammatory cytokines and chemokines in healthy donors
1 Department of Pediatric Immunology and Hematology, Wilhelmina Children's Hospital, University Medical Center, KC.03.063.0, Lundlaan 6, Postbus 85090, 3508 AB, Utrecht, The Netherlands
2 IACOPO Institute, University of California, San Diego, CA, USA
3 Department of Hematology, University Medical Center, Utrecht, The Netherlands
4 Androclus Therapeutics, Milan, Italy
5 Department of Pediatrics, University of California, San Diego, CA, USA
6 La Jolla Institute of Allergy and Immunology, San Diego, CA, USA
Correspondence to: B. Prakken; E-mail: b.prakken{at}umcutrecht.nl
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Abstract |
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Adenovirus can cause fatal infections in the immunocompromised host. To date, no effective anti-viral therapy is available. Adoptive therapy with adenovirus-specific T cells could be a promising treatment, but requires the identification of such T cells. Aim of this study was to identify conserved adenoviral T cell epitopes recognized in a majority of healthy individuals. By using a computer algorithm designed to predict pan-HLA-DR-binding T cell epitopes, we selected 19 peptides of adenovirus serotype 5. PBMCs from 26 healthy subjects were isolated and incubated with these peptides to test epitope-specific T cell proliferation. Six epitopes derived from E1B protein, hexon protein (two epitopes), DNA polymerase, E3A glycoprotein and fiber protein induced a proliferative T cell response in the majority of healthy controls. In vitro MHC binding assays confirmed the potential capacity of the adenovirus epitopes to bind multiple MHC alleles. The cytokine and chemokine profile induced by these epitopes was determined with a multiplex immunoassay and revealed a predominant pro-inflammatory pattern. Based on the broad recognition and the induced cytokine and chemokine profile, the detected epitopes can be regarded as potential candidates to select adenovirus-specific T cells for immune intervention in the immunocompromised host.
Keywords: adenovirus, pan-DR binding T-cell epitopes
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Introduction |
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Adenoviruses are an important cause of morbidity and mortality in the immunocompromised host, especially after stem-cell transplantation (SCT). The incidence of adenovirus infection following SCT varies from 3 to 21% (1, 2). The highest frequencies of serious adenovirus infections are seen in patients receiving stem cells from matched unrelated donors and those receiving T cell-depleted grafts (2–4). Among pediatric transplant recipients, an even higher incidence of infection is found due to a higher rate of primary infection (4). The mortality rate in immunocompromised patients with adenoviral infections approaches up to 50% in adults and 83% in children (2–5). Currently, no specific anti-viral therapy is available for severe adenoviral infection. A promising novel form of treatment could be adoptive therapy with adenovirus-specific T cells from a graft donor. Adoptive therapy has already successfully been used for prevention and treatment of EBV and cytomegalovirus infections (6–8).
Adenovirus-specific immunity is predominantly mediated by a MHC class II-restricted CD4+ T cell response (9, 10). It is supposed that this response is mainly induced by the capsid subunits hexon, penton and fiber of adenovirus (10, 11). Recently, also MHC class I-restricted T cell epitopes of adenovirus hexon protein have been reported (12).
Although 51 serotypes of human adenovirus have been described, adenovirus serotype 5-specific CD4+ T cells can recognize different adenovirus serotypes (13). This cross-reactivity suggests that adenovirus epitopes are conserved among the different serotypes (14). Serotype 5 is frequently cultured both in the general population as well as in immunocompromised patients, making this serotype a suitable candidate for targeted immune intervention.
In the present study, a novel matrix-based computer algorithm predicting DR-binding epitopes on a given protein sequence is used to identify MHC class II-restricted T cell adenoviral serotype 5 epitopes. Using a computer algorithm has several advantages above conventional epitope-mapping techniques. It is less time and material consuming and theoretically the identified epitopes can be recognized in a large part of the population (15, 16). By using this computer algorithm, we recently showed that it was also possible to identify pan-DR-binding epitopes of heat shock protein 60 (17). In this study, the algorithm allowed to select six adenoviral epitopes that were recognized by PBMCs in a majority of healthy subjects and were naturally processed. The detected epitopes can be used to identify and select adenovirus-specific T cells for immune intervention in the immunocompromised host.
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Methods |
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Selection of adenovirus peptide sequences
Eleven proteins of adenovirus serotype were selected based on a search of the Entrez databases (www.ncbi.nlm.nih.gov), the Swiss-Prot knowledge base, a protein sequence database (www.ebi.ac.uk), and literature (10, 11). A HLA-DR1-binding matrix-based computer algorithm was used to predict pan-DR-binding epitopes (Sette, LIAI, la Jolla, CA, USA). Fifty-two potential pan-DR-binding epitopes were selected derived from different adenoviral proteins. By using the protein database (www.ncbi.nlm.nih.gov), BLAST database for sequence comparisons and Swiss Pdb-viewer (www.expasy.org/spdbv), the selection was narrowed to 19 epitopes, mainly based on high similarity with other adenovirus serotypes and the predicted binding scores. Peptides were synthesized as 15-mers containing the potential epitopes preferable with at least three flanking residues by automated simultaneous multiple peptide synthesis as described previously and checked by HPLC for a purity of at least 90% (Faculty of Veterinary medicine, University of Utrecht, The Netherlands) (18). In Tables 1 and 2, adenoviral proteins, sequences of 19 synthesized peptides, their origin and binding scores are shown. Six peptides were selected for further experiments, based on binding scores and the virus proteins from which they were derived (shown in bold, Table 1). The homology with other adenovirus strains is shown in Table 2.
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T cell proliferation assays
Peripheral blood was obtained from 26 healthy adults (mean age 31 years, range 22–47 years). PBMCs were isolated by Ficoll–Paque density gradient centrifugation (Amersham Pharmacia, Uppsala, Sweden). Cells were cultured in a 96-well round-bottomed plate (2 x 105 per well) in RPMI-1640 supplemented with 100 U ml–1 penicillin, 100 µg ml–1 streptomycin, 2 mM L-glutamine (Gibco BRL, Gaithersburg, MD, USA) and 10% human AB serum (Sanguin Blood Bank, Utrecht, The Netherlands). Cells were incubated at 37°C in 5% CO with either medium alone or with one of the 19 adenoviral peptides in a concentration of 10 µg ml–1. No co-stimulatory molecules or cytokines were added. Tetanus Toxoid (TT) (150 Lf ml–1, RIVM, Bilthoven, The Netherlands) was used as positive control; ovalbumin peptide (OVA) (wild type 323–339) was used as irrelevant control. After 96 h, cells were incubated with [3H]Thymidine ([3H]TdR) (Amersham, Buckinghamshire, UK). Incorporated radioactivity expressed in counts per minute (c.p.m.) was measured after 16 h by liquid scintillation counting. Stimulation index (SI) was defined as c.p.m. of cells after antigen-specific stimulation divided by c.p.m. of cells cultured with medium alone.
MHC restriction
To verify if the T cell response to the adenoviral peptides is MHC class II restricted, a HLA-blocking study was performed in five healthy persons. Peptides A2, A6, A14, A15 and A16 were used for this study. For these experiments (and the following experiments), the synthesis of peptide A9 yielded a purity of <90% and was therefore left out of the analysis. Cells were cultured as described above in the presence or absence of antibodies against HLA class I (clone G46-2.6; BD Biosciences, PharMingen, San Diego, CA, USA), HLA-DR (clone L243), HLA-DP (clone B7/21) or HLA-DQ (clone Ia3) or anti-mouse IgG as isotype control (Leinco Technologies, St Louis, MO, USA) in a concentration of 1.25 µg ml–1 per well.
HLA typing
In 21 subjects, HLA class II typing was performed to detect a possible relation between T cell response to the adenoviral peptides and HLA type. HLA-DR typing was performed using PCR-SSP according to the instructions of the manufacturer (GenoVision, Vienna, Austria).
HLA-DR peptide-binding assays
Selected Adv-peptides (A2, A6, A14, A15, A16) were tested for in vitro binding capacity to two common human HLA-DR molecules, namely DR1 and DR4. Human HLA-DR1 and HLA-DR4 molecules were purified from the lysate of EBV-transformed lymphoblastoid cell lines by immunoaffinity chromatography. Purified MHC molecules were solubilized in a Tris buffer containing 50 mM diethylamine and 2% b-octyl-glucopyranoside (Calbiochem, San Diego, CA, USA). DR molecules were incubated with fixed amounts (100 µM) of biotinylated probe [haemagglutinin (HA)] peptides and a dose range of 0–512 µM unlabeled Adv-peptides for 40 h at room temperature in the presence of a protease inhibitor cocktail. The used biotinylated probe in all assays was HA Y307–319, a strong MHC binder. All binding studies were performed at pH 7.0. The MHC–peptide mixtures were analyzed by SDS-PAGE under non-reducing conditions. The proteins were then blotted onto nitrocellulose (Hybond-ECL, Amersham, UK). After 1 h of blocking with 5% dried milk in PBS–0.05% Tween 20, the blot was incubated with biotinylated streptavidin–HRP complexes for 45 min in a 1:1000 dilution (Amersham Corp., Arlington Heights, IL, USA). Detection of the presence of biotinylated peptides was done by enhanced chemiluminescence using the western blot ECL kit (Amersham, UK). Blots were exposed on preflashed hyperfilm-ECL (Amersham, UK). Inhibition curves were generated from these results and the concentration of the competitor peptide at which 50% inhibition was achieved was extrapolated from these curves. This concentration in micrometers is indicated as IC50 value.
Cytokine and chemokine production
PBMCs from 10 subjects were cultured with medium alone, one of the selected adenoviral peptides or TT. After 96 h, cell supernatants were collected and stored at –80°C until further analysis. Cytokine and chemokine production was measured with multiplex immunoassay (MIA). This MIA combines the principle of a sandwich immunoassay with the luminex fluorescent-bead-based technology (LabMAP) (19). Others and we validated this method for the measurement of cytokines and chemokines in culture supernatants of human PBMCs. Comparing MIA with the regular ELISA technique values appear comparable in sensitivity, accuracy and reproducibility and is less time consuming (19, 20, 21). Antibody pairs used for the MIA were purchased from different commercial sources (Table 3) and coupled as previously described (20). Recombinant proteins were reconstituted in PBS, pH 7.4, containing 0.5% BSA (Sigma-Aldrich, Zwijndrecht, The Netherlands) to a concentration of 5 µg ml–1. Calibration curves from recombinant protein standards were prepared using 2-fold dilution steps in serum diluent (R&D Systems, Abingdon, UK). Samples were measured and blank values were subtracted from all readings. All samples were run undiluted and diluted 1:50 with high performance ELISA buffer (HPE-buffer; Sanquin, Amsterdam, the Netherlands). All assays were carried out directly in a 96-well 1.2-µm filter plate (Millipore, Amsterdam, The Netherlands) at room temperature and protected from light. A mix containing 1000 microspheres per mediator (total volume 10 µl per well) was incubated together with 50 µl standard, sample or blank for 60 min. Next 10 µl of a cocktail of biotinylated antibodies (16.5 µg ml–1 each) were added to each well and incubated for an additional 60 min. Beads were washed with PBS–1% BSA–0.5%-Tween 20 pH 7.4 in order to remove the sample and unbound antibodies. After incubation of 10 min with 50 ng per well streptavidin R–PE (BD Biosciences) and washing twice with PBS-1% BSA- 0.5%-Tween 20 pH 7.4, the fluorescence intensity of the beads was measured in a final volume of 100 µl HPE-buffer. Measurements and data analysis of all assays were performed using the Bio-Plex system in combination with the Bio-Plex Manager software version 3.0 using five parametric curve fitting (Bio-Rad Laboratories, Hercules, CA, USA). Inter-assay variance has proven to be <10%, whereas intra-assay variability ranged between 6 and 16%. The concentrations of the following soluble mediators were measured: IL-1
, IL-1ß, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, IL-18, tumor necrosis factor (TNF) , IFN
, Oncostatin M (OSM), CCL22 (macrophage-derived chemokine) and CXCL10 (IFN inducible protein).
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Short-term cell line
PBMCs from healthy subjects were cultured (2 x 105 per well) with selected Adv-peptides (10 µg ml–1) supplemented with IL-2 (40 U ml–1). OVA was used as irrelevant control peptide. After 7 days, cells were re-stimulated with the same peptides. Subsequently, cells were cultured with methylene blue visible light inactivated adenovirus as described earlier (22) at a multiplicity of infection of 10. After 96 h, cells were incubated with [3H]TdR for 16 h. Percentage of proliferation was determined in comparison with the response to OVA pre-stimulated cells.
Similarly, PBMCs were pre-stimulated with whole inactivated adenovirus for 96 h, followed by re-stimulation with the different selected Adv-peptides for 5 days. Subsequently, IFN production was measured in supernatant with MIA and compared with the IFN production in medium.
Statistics
Basic descriptive statistics were used to describe the proliferative responses. Data analysis of detected soluble mediators was done with Bio-Plex Manager 3.0 (Bio-Rad Laboratories, Hercules, CA, USA) using a five parametric curve fitting. The average production of cytokines and chemokines in reaction to adenoviral peptides and TT was compared with the production in medium with the Wilcoxon signed ranks test ( 
0.05).
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Results |
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T cell proliferation assays
PBMCs of 26 healthy persons were stimulated for 112 h with the 19 adenoviral peptides, TT or medium alone. Figure 1 shows the percentage of responders to epitopes of adenovirus serotype 5. Six of the 19 peptides induced proliferative responses in PBMCs in a majority of healthy subjects. Most proliferative responses to these epitopes were in the lower range (SI 1.7–2.5) probably reflecting that these were directly peptide-stimulated PBMCs without any co- or prestimulation. The highest response rate of subjects tested was found in response to peptide A15 derived from hexon protein (73%), followed by peptides A6 derived from early 1B protein (65%), A14 derived from hexon protein (58%) and A16 derived from DNA polymerase (57%). These peptides together with peptides A2 derived from fiber protein (34%) and A9 derived from early 3A 10.5 kDa glycoprotein (46%) were used in the following experiments. Adv-peptide A19, although very promising in the initial T cell proliferative responses, was left out in further experiments as technical problems precluded a large-scale peptide production with satisfactory purity.
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MHC restriction
Cells were cultured as described in Methods with Adv-peptides in the presence or absence of antibodies against HLA class I, HLA-DR, HLA-DP or HLA-DQ or anti-mouse IgG as isotype control. The induced proliferation to Adv-peptides in the presence of the isotype control was set at 100%. Figure 2 shows the results of blocking in five healthy controls. Proliferation of T cells in response to the pan-DR binding all adenoviral peptides except A16 decreased after addition of antibodies against HLA-DR, but not, or to a much lesser extent, after adding antibodies against HLA class I, HLA-DP and HLA-DQ (Fig. 2). Based on this experiment (in which four of the five tested persons did not show clear proliferation to peptide A16), the predicted HLA-DR restriction of A16 could not be confirmed. These results confirm that the selected adenovirus epitopes are mediated by a MHC class II T cell response.
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HLA typing
In 21 subjects, HLA-DR typing was performed. The results of the typing together with the response to the six selected adenoviral peptides are shown in Table 4. No correlation between the proliferative response to the peptides and HLA-DR type could be found (data not shown).
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HLA-DR peptide-binding assays
The capacity of five Adv-peptides (A2, A6, A14, A15 and A16) to bind both HLA-DR1 and HLA-DR4 was tested in an in vitro MHC binding assay as described in Methods. Figure 3 shows the competitive inhibition of MHC binding of biotinylated peptide HA307–319 by competitor peptides. Competition studies for binding to 3 µM DR1 and DR4 MHC molecules were performed with different concentrations of non-labeled peptide A2, A6, A14, A15 or A16 and 100 µM of biotinylated peptide HA307–319, a well-known excellent pan-DR-binder peptide. This concentration in micrometers of the competitor peptide at which 50% inhibition was achieved is indicated as IC50 value (dotted line). The figure shows that the five Adv-peptides tested can efficiently bind both HLA-DR1 and HLA-DR4 with IC50 values ranging from 10 to 500 µM, with the sole exception being the lack of binding capacity of Adv-peptide A16 to bind HLA-DR1.
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Cytokine and chemokine production
The cytokine and chemokine production of PBMCs in response to the six selected adenoviral peptides, the medium and the TT was measured by MIA. In order to visualize the complete spectrum of cytokine and chemokine production, data are digitized in a portrait (Fig. 4) (20). The mean concentration (pg ml–1) of tetanus stimulation is plotted at two-thirds of a logarithmic scale. Differences in profiles after stimulation with the different peptides can be observed. In addition, the median values for each cytokine and chemokine are shown in Table 5. In response to all six peptides, significant differences (P < 0.05) with medium were seen in the production of the pro-inflammatory cytokines IL-1ß and IL-6 and chemokine OSM. IL-17 production was significantly increased after culture with all peptides except peptide A6 (derived from E1B protein). Significant production of IFN
and the anti-inflammatory cytokines IL-10 and IL-13 was seen in response to peptide A15 derived from hexon protein. PBMCs cultured with peptide A16 derived from hexon protein also showed significant production of IL-10.
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Short-term cell line
Cells were cultured with different adenoviral peptides (A2, A6, A14, A15, A16) or OVA followed by activation with whole inactivated adenovirus. As measured by thymidine incorporation, Adv-peptide pre-stimulated cells had increased proliferative responses to whole inactivated adenovirus compared with OVA pre-stimulated cells (increase in adenovirus-specific proliferation for A2: 18 ± 15%, A6: 61 ± 19%, A14: 79 ± 12%, A15: 65 ± 29%, A16: 60 ± 45%).
Similarly, culturing PBMCs with whole inactive adenovirus followed by activation with the selected Adv-peptides or OVA led to significant higher IFN production in response to almost all peptides (Fig. 5). These results suggest that the Adv-peptides are naturally processed.
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Discussion |
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The aim of this study was to find conserved adenoviral T cell epitopes recognized in a majority of healthy individuals to identify and characterize adenovirus-specific CD4+ cells in healthy individuals. Such adenoviral-specific T cells can be used for the study of adenovirus-specific immunity and ultimately for adoptive therapy. Recently, by using adenovirus lysate, adenovirus-specific T cells were detected in children with adenovirus infection after allogeneic SCT, and the presence of these cells was correlated with a more favorable prognosis (23). Selecting adenovirus-specific T cells based on specific T cell epitopes would allow to study the virus-specific immune response at the molecular way.
A major difference with previous attempts to identify MHC class II restricted T cell epitopes is the use of a computer algorithm to predict and select potential epitopes. Based on the computerized prediction, initially 19 peptides were selected and tested for their capacity to induce T cell proliferation in PBMCs from healthy individuals. Next, based on peptide-specific T cell proliferation six peptides were selected for subsequent studies.
The six selected peptides were derived from hexon protein, one of the structural proteins in the viral coat (peptide A15, 73% recognition and A14, 58%), E1B protein (A6, 65%), DNA polymerase (A16, 57%), E3A glycoprotein (A9, 46%) and fiber protein (A2, 34%). Thus, besides the expected MHC class II-restricted CD4+ T cell response induced by the capsid subunits hexon, penton and fiber subunits of adenovirus, we also found epitopes derived from other proteins to be immunogenic. Early proteins 3A and 1B are both non-structural proteins expressed in infected cells. Interestingly, epitope A15 derived from hexon protein, one of the structural proteins in the viral coat, has an overlapping amino acid sequence with an earlier reported highly conserved HLA-DP4-restricted T cell epitope of adenovirus identified with conventional technology (10, 24). HLA-blocking studies showed that the response towards these peptides was MHC class II restricted. The restriction was mainly HLA-DR, because of the weak inhibition DR is a possible restriction element but does not exclude others. Tang et al. (24) found that an epitope derived from hexon protein with overlapping sequences with our peptide A15 was mainly HLA-DP restricted. It is well-known that just a slight difference in amino acid sequence can result in complete different binding and processing. The fact that epitopes selected on a pan-DR-binding motif to a certain extent may also be capable of binding to HLA-DP and HLA-DQ may reflect overlap in binding requirements for the different alleles. Whether during adenovirus infections certain epitopes can be presented by both alleles and whether this may have functional consequences remain to be seen. The presence of a proliferative response towards the epitopes was, as expected, not correlated to certain HLA-DR alleles, underlining the true pan-DR-binding capacity of the epitopes. MHC–peptide binding is the first prerequisite for inducing a peptide-specific T cell response. However, other factors are also essential for determining the outcome, such as the TCR affinity for a given MHC-peptide, and the timing, quality and quantity of antigen priming (in this case adenovirus). This is underlined in this study by the observation that donors with same HLA-DR alleles can exhibit different proliferative responses to the Adv-peptides (Table 4).
The binding study shows that the Adv-peptides can efficiently bind to HLA-DR1 and HLA-DR4, only peptide A16 lack binding to HLA-DR1 and only minimal inhibition for HLA-DR4. In the blocking experiments, peptide A16 did not show clear HLA-DR restriction. Indeed, the binding prediction was also the lowest among the selected Adv-peptides (Tables 1 and 2). Thus, the predicted binding capacity to multiple MHC alleles of the Adv-peptides tested in the binding assays could be confirmed by MHC class II-binding studies. This study, however, was not set up to analyze the relationship between predicted and actual binding and thus these data cannot be generalized to other peptides not tested here (see also http://www.immuneepitope.org).
The cytokine and chemokine production of PBMCs in response to selected six peptides showed significant production of IL-1ß, IL-6, IL-17 and OSM. Peptide A15 also induced production of IFN and TNF in PBMCs. These results indicate a pro-inflammatory cytokine and chemokine response, fitting with an anti-viral cytokine and chemokine profile. Also the PBMCs of two healthy subjects without proliferative response to one of the peptides showed a pro-inflammatory cytokine response after peptide stimulation. With MIA it is possible to detect multiple cytokines and chemokines at the same time in one single sample. A drawback is that all cytokines and chemokines have to be measured at one time point. This reflects the relatively low levels of IFN and TNF production in response to selected peptides, both important cytokines for the induction of an anti-viral response. We measured cytokine and chemokine production after 112 h of culturing, whereas highest IFN production can be expected within 48 h. Indeed, we could detect IFN production to all six peptides at other time points (data not shown, Fig. 5).
In conclusion, by using a computer algorithm, a technique to predict epitopes, we identified novel MHC class II-restricted T cell epitopes of adenovirus. This can be an important step towards adoptive immunotherapy. In allogeneic SCT, a major concern of adoptive therapy with non-selected donor T cells is the potential for inducing graft-versus-host disease. This can be overcome by using antigen-specific memory T cells (25). As the selected epitopes are recognized in a majority of healthy donors and induce a predominantly pro-inflammatory cytokine and chemokine profile, they can be considered as excellent candidates to select adenovirus-specific T cells to be used for immune intervention.
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Acknowledgements |
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This work was supported by grants from the Dutch Cancer Society (KWF) and the European Commission (Grant no. QLK2-CT-2002-01432). Prakken is supported by a VIDI innovation grant from the Dutch Society for Scientific Research.
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Abbreviations |
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| c.p.m., counts per minute |
| HA, haemagglutinin |
| HPE, high performance ELISA |
| [3H]TdR, [3H]Thymidine |
| MIA, multiplex immunoassay |
| OSM, Oncostatin M |
| OVA, ovalbumin peptide |
| SCT, stem-cell transplantation |
| SI, stimulation index |
| TNF, tumor necrosis factor |
| TT, Tetanus Toxoid |
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Notes |
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Transmitting editor: G. Hammerling
Received 2 March 2005, accepted 4 August 2006.
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