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Departments of
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Surgery and
Pediatrics, University of Michigan Medical Center, Ann Arbor, MI 48109
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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12,000 genes and expressed sequence tags. Upon 24 h tumor lysate pulsing, the levels of 87 transcripts increased at least 3-fold while the levels of 121 transcripts were reduced by one-third or more, with accompanying p values <0.01. Most of these genes encoded proteins important for DC effector functions including cytokines, chemokines, and receptors, such as IL-12p40, macrophage inflammatory protein-2, and IL-6; Ag presentation, such as carboxypeptidase D and H2-DM; cell adhesion (e.g., EGF-like module containing, mucin-like, hormone receptor-like sequence 1, rhoB); and T cell activation. Interestingly, we observed a high level of expression of a novel member of the class A scavenger receptor family, macrophage receptor with collagenous structure (Marco). Marco is thought to play an important role in the immune response by mediating binding and phagocytosis, but also in the formation of lamellipodia-like structures and of dendritic processes. Therefore, we have identified a repertoire of genes that likely play a role in DC function. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Many strategies involving the use of DCs for inducing specific antitumor immune responses are being investigated. A number of phase I and phase II clinical trials using DCs pulsed with peptide, protein, RNA, and tumor lysate as sources of Ags have been performed in different types of cancers. These early studies have shown that specific T cell responses against tumors as well as some tumor regressions can be achieved with vaccines based on DCs (7, 8, 9). Efforts to optimize conditions for induction of therapeutic antitumor immunity have focused on the source and preparation of tumor-associated Ags for DC loading. We (10, 11, 12) and others (13, 14), have been studying killed tumor-pulsed DCs in vaccine strategies in experimental and clinical settings. DCs pulsed with tumor-associated Ags in the form of tumor cell lysates can elicit specific proliferation and CTL reactivity, and have shown efficacy in protecting naive mice from tumor challenge and in reducing the growth of established tumors in vivo. In human clinical trials, vaccines generated from tumor lysate-pulsed DCs (TP-DCs) have been shown in some cases to stimulate both CD4+ and CD8+ T cell activity in adult and pediatric cancer patients (15, 16).
In the past few years, there has been heightened interest in gene expression technologies. Gene microarray offers the first opportunity to examine global and subtle changes in the expression level of thousands of individual genes simultaneously in response to specific stimuli, and has provided new insights into the immune system (17). Several groups have used microarray approaches to investigate the differentiation of DCs (18, 19). DC maturation has also been investigated at the molecular level by oligonucleotide microarrays. A transcription profile of immature and LPS-matured human monocyte-derived DCs has been carried out, revealing 225 differentially expressed genes out of a total of 10,962 genes screened (20). These genes mainly consisted of those encoding chemokines and chemokine receptors. Moreover, microarrays have been used to measure gene expression of human monocyte-derived DCs in response to different types of stimuli, such as Gram-negative bacteria, yeast, and viruses. Immediately after contact with any of the three pathogens, a rapid down-regulation of genes associated with phagocytosis and pathogen recognition, and a transient increase in transcripts for cytokines, chemokines, and receptors that contribute to the recruitment of leukocytes at the site of infection has been documented (21). In the mouse, a kinetic study of gene expression following exposure to Gram-negative bacteria and LPS activation has revealed that during the process of maturation, DCs undergo a sequence of precise transitional stages (22). An unanticipated induction of IL-2 production by bacterially stimulated murine DCs at early time points after bacterial encounter was also noted, which could explain the capacity of activated DCs to prime CD8+ T cells in a CD4-independent manner (23).
To date, little has been reported on the effect(s) of phagocytic uptake of dead cells on DC function. In the current study, we generated TP-DCs and unpulsed dendritic cells (UP-DCs) and used Affymetrix oligonucleotide microarrays in an attempt to uncover distinct changes in gene expression patterns as a consequence of killed tumor cell loading. We have identified 208 RNAs modulated in DCs as a consequence of exposure to B16 melanoma lysates and describe functional groupings of these transcripts. The potential biologic significance of our findings is discussed.
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Materials and Methods |
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Seven- to 8-wk-old female C57BL/6 (denoted B6) mice were purchased from Harlan Laboratories (Indianapolis, IN) and housed at the Animal Maintenance Facility of the University of Michigan Medical Center (Ann Arbor, MI) for at least 1 wk prior to use. The animals were used between 8 and 9 wk of age. The B16-BL6 melanoma is of spontaneous origin in B6 mice and is poorly immunogenic. MCA 205 and MCA 207 are 3-methylcholanthrene-induced fibrosarcomas syngeneic to B6 mice.
Generation of bone marrow-derived DCs
Erythrocyte-depleted mouse bone marrow cells flushed from the marrow cavities of femurs and tibiae of B6 mice were cultured at 1 x 106 cells/ml in presence of 10 ng/ml GM-CSF and 10 ng/ml IL-4 in complete medium (CM) (RPMI 1640 containing 10% heat-inactivated FCS, 0.1 mM nonessential amino acids, 1 µM sodium pyruvate, 10 mM HEPES buffer, 2 mM glutamine, 50 µM 2-ME and antibiotics), as described previously (12, 24). After 4 days of culture, DCs were harvested by gentle pipetting, washed, resuspended at 5 x 106 cells/ml, and enriched by 14.5% (w/v) metrizamide (Sigma-Aldrich, St. Louis, MO) CM density gradient separation. Following centrifugation (15 min, 4°C, 2000 rpm), DCs were collected from the low density interface. The resulting DC population was 90% positive for coexpression of MHC class I and II, and CD11c. In each experiment, the UP-DCs act as an internal control, as the composition of our starting population does not change whether it is subsequently used as UP-DCs or B16 TP-DCs. In some experiments, CD11c+ purified DCs were obtained by positive selection of 4-day BM-DCs using MACS CD11c microbeads (Miltenyi Biotec, Auburn, CA). The purity of the fraction, assessed by flow cytometry using a PE-conjugated anti-CD11c (HL3 clone; BD PharMingen, San Diego, CA) for labeling, was usually >97%.
Preparation of tumor and normal tissue lysates
Viable cells from tumors or cell suspensions obtained from normal tissue were suspended at 3 x 107 cells/ml in CM. The cell suspension was frozen in liquid nitrogen for 2 min, then thawed in a 37°C water bath for 10 min. The freeze-thaw cycle was repeated three times in rapid succession and then centrifuged at low speed (500 rpm for 5 min). The supernatant (lysate) was collected and stored in liquid nitrogen for later use.
Preparation of RNA samples and gene chip hybridization
Day 4-purified DCs were either pulsed with B16 melanoma cell lysates at a ratio of 3:1 tumor cell to DC, or left unpulsed for 24 h. After another metrizamide gradient purification, total RNA was isolated using TRIzol reagent (Life Technologies, Carlsbad, CA), followed by clean-up on an RNeasy spin column (Qiagen, Valencia, CA), then used to generate cRNA probes. Preparation of cRNA, hybridization, and scanning of the mouse genome U74A arrays were performed according to the manufacturer’s protocol (Affymetrix, Santa Clara, CA).
Microarray analysis
Microarrays were scanned and probe intensities were extracted from the image (GeneArray scanner and Microarray Suite 4.0; Affymetrix). Each probe set on the MG-U74Av2 arrays typically consisted of 16 25-base oligonucleotides complementary to a specific cDNA called perfect match (PM) features, and 16 identical probes whose sequences had been altered at the central base, called mismatch (MM) features. Publicly available software was used to process the probe intensities in order to obtain normalized probe set intensities as follows (software and documentation at http://dot.ped.med.umich.edu:2000/pub/index.html). An unpulsed sample that gave a microarray with high signal and low background was selected as the standard. Probe pairs for which PM-MM <-100 on the standard were removed from the analysis, and the remaining PM-MM differences were averaged for each probe set on each microarray by discarding the 25% highest and lowest differences and averaging the remaining differences. The resulting intensities for each microarray were normalized to the standard using a piece-wise linear function that made 99 evenly spaced quantiles agree with the corresponding quantiles in the distribution of the standard. Normalized intensities were log-transformed by mapping x to log(max(x + 100) + 100). Fold changes were computed as the ratio of group means, after first replacing means that were <100 by 100. We classified a gene as detected by a probe set if a p value <0.01 was obtained for a one-sided signed-rank test of PM-MM >0. Algorithms for computing the Unigene Cluster IDs by homology of the probe set sequences to the sequences of the cluster members are also available and detailed at the Web page cited above.
Semiquantitative RT-PCR
Single-stranded cDNA was synthesized from the same RNA samples used for microarray analysis, using SuperScript II reverse transcriptase (Life Technologies). Preliminary experiments were performed to determine the conditions in which cDNAs were amplified in the linear region of the PCR curve. The reaction mixture was composed of 2 or 4 µl of cDNA template obtained from 1 µg of extracted RNA; 25 pmol of primers; 25 nmol of each dNTP; 2.5 U of Taq DNA polymerase; 10 µl of 10 x PCR buffer in a final volume of 50 µl. PCR amplification conditions were as follows: denaturation at 94°C for 5 min, amplification composed of denaturation at 94°C for 30 s, annealing at 57°C for 30 s, and extension at 72°C for 1 min, followed by a final extension at 72°C for 10 min. Cycle numbers were 50 cycles for CCR2, and 35 cycles for the other mRNA. The nucleotide sequence of primers used are described in Table I. For 
-actin, samples were negatively tested for possible genomic DNA contamination by performing PCR on RNAs, which was not reverse-transcribed (data not shown). The expression level for each transcript was evaluated after ethidium bromide staining on a 1.5% agarose gel. Each PCR was replicated three times per sample.
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To quantify cell surface coexpression of macrophage receptor with collagenous structure (Marco) and CD11c molecules on B16 TP-DCs and UP-DCs, 5 x 105 cells were stained for 30 min at 4°C with a rat anti-mouse Marco (ED31; Serotec, Raleigh, NC) or IgG control Ab (5 µg/ml), washed twice with 2% FCS and 0.1% sodium azide in PBS, and then incubated with FITC-conjugated goat anti-rat IgG Ab (2 µg/ml) and PE-conjugated anti-CD11c mAb for 30 min at 4°C. Labeled cells were then washed, fixed in 1% paraformaldehyde in PBS, and analyzed for fluorescence. Data analysis was based on examination of 10,000 cells/sample.
SDS-PAGE and Western blotting
Cells were rinsed twice with ice-cold PBS and lysed by successive freeze-thaw cycles, in 20 mM Tris-HCl, pH 7.5, buffer containing 5 mM EDTA and 100 mM KCl. The homogenate was centrifugated at 6000 x g for 10 min and the supernatant was collected. To analyze for Marco, 30 µg of protein were loaded onto a 10% polyacrylamide gel, and then transferred onto a PVDF membrane. For Western blotting analysis, membranes were incubated with a blocking buffer consisting of TBS, 1.8% nonfat dry milk, and 0.01% Tween 20 for 2 h, followed by incubation for 2 h with rat anti-mouse Ab against Marco and actin (ICN Biomedicals, Aurora, OH) at dilutions of 1/500 and 1/2500, respectively. After three washes with washing buffer (TBS containing 0.01% Tween 20), the membranes were incubated with a HRP-labeled conjugate secondary Ab at a 1/1000 dilution for 1 h at room temperature, washed, and briefly incubated in ECL (Amersham Pharmacia Biotech, Piscataway, NJ).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Day 4 bone marrow-derived DCs were either pulsed with B16 melanoma cell lysates or left unpulsed for 24 h, and RNA transcript levels for different genes were measured using Affymetrix MG-U74Av2 microarrays that contained 12,488 probe sets (genes). Three independent experiments were performed to assess the reproducibility of observed changes. We determined that from the entire set of genes represented, 5,890 genes (47%) were judged as detectable, on average. B16 TP-DC samples had an average correlation of 0.974 using all transcripts, and UP-DC samples had an average correlation of 0.959. We compared the mean intensity values for each gene in DCs during pulsing with B16 tumor lysate to those obtained from UP-DCs to determine differences in the measured expression levels. A two-sample t test on the log-transformed data comparing B16 TP-DCs and UP-DCs gave 818 probe sets with p < 0.01, whereas only 125 were expected by chance. When combined with the additional criterion that the fold change (FC) should be 
3 in either direction, 219 probe sets which represented 208 transcripts were retained.
Among those 208 transcripts, 149 were known genes, and 59 were expressed sequence tags. Tables II and III list the known genes that are increased and decreased, respectively, in DCs after 24 h of pulsing with B16 tumor lysate, including the FC and p value of the t test. Most of the genes modulated in B16 TP-DCs encoded proteins important for effector function. Expression of many genes with inflammatory and/or chemotactic activities was strongly increased. These included: macrophage inflammatory protein (MIP)-2, growth-related oncogene (Gro) 1, IL-12p40, TNF-
, IL-6, monocyte chemoattractant protein (MCP)-1, IL-10, and the small inducible cytokine subfamily D1 transcripts. Expression of B7.1 (CD80), a costimulatory signal molecule for T cell activation, and several cell surface molecules related to cell adhesion such as Marco, Gap junction membrane channel protein 1, EGF-like module containing, mucin-like, hormone receptor-like sequence 1, CD38, and MYD1 mRNAs were increased as well. Transcript levels for carboxypeptidase D, and the lysozomal acid phosphatases 2 and 5, were also increased. Transcript levels for all probed H-2 class I and class II molecules were largely unaffected by tumor lysate pulsing. One exception was pulsing-associated reduction of transcript levels for the histocompatibility 2, class II, locus DMa (H2-DM). We also observed an increase in several transcripts involved in eicosanoid and glutathione metabolism, including platelet-activating factor (PAF) acetylhydrolase, PGE synthase, cyclooxygenase (COX)2 and glutathione S-transferase, µ1, 2, and 3 mRNAs. This is of particular interest as eicosanoid and PAF are known to play an important role in processes such as leukocyte migration, NK cell activation, and type 2 Th cell differentiation (25). Regulation of a large group of genes involved in cell cycle control and components of the mitotic spindle assembly checkpoint, which prevents the cell from entering anaphase until all chromosomes are properly aligned, was observed. Levels of nephroblastoma overexpressed gene, vaccinia-related kinase 1, serine/threonine kinase 6, cyclin B1 and B2, survivin, bub1a, kinesin-like 1, polo-like kinase, Ttk protein kinase, MAD2-like 1, cell division cycles 2, 25, 45, and 28 protein kinase 1 mRNAs were strongly decreased. Concomitantly, a decrease of DNA repair/replication/maintenance proteins such as replication protein A2, Rad51 homolog, apurinic/apyrimidinic endonuclease nuclease, nuclear protein 95, and mini-chromosome maintenance proteins 5 and 4 was detected as well. Finally, some members of the Kallikrein family encoding for secreted serine proteases were also observed to be down-regulated in DCs after tumor lysate pulsing.
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Expression of select genes identified as differentially expressed by microarray analysis was examined by RT-PCR. Total RNA from B16 TP-DCs and UP-DCs was analyzed for the expression of 19 genes relative to the level of -actin mRNA in each sample (Fig. 1). -actin was chosen as a reference because its mRNA levels, measured by microarray analysis, were comparable between B16 TP-DCs and UP-DCs (FC = 1.1). Semi-quantitative PCR results correlated well with the differential gene expression data produced using Affymetrix genechips, although there was no possible comparison with the FC values detected by the cDNA arrays. This result gave us confidence that the gene expression data derived from the gene arrays were reliable. We also examined the kinetics of expression of those transcripts at early time points after tumor lysate pulsing, and determined differences in the induction of their expression level. For example, an optimal decrease in CCR2, nephroblastoma overexpressed gene and COX1 mRNA, as well as an optimal increase in OX40 ligand (OX40L), CD14, and serum amyloid A3 mRNA were observed as soon as after 6 h following exposure to tumor lysate, whereas PGE synthase, MCP-1, and MIP-2 mRNA were not modulated until a later time point.
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We observed a high level expression (FC = 141.9; p < 0.0001) of Marco, a new member of the gene family that encodes class A scavenger receptor molecules, 24 h after pulsing of DCs with B16 melanoma lysate. In mice, Marco is expressed constitutively by macrophages of the spleen and lymph nodes. RT-PCR, Western blot, and FACS analysis experiments were conducted on purified, sorted CD11c+ cells, as well as on metrizamide gradient-purified DCs. Marco expression by DCs was confirmed by semi-quantitative PCR analysis of UP-DCs vs B16 TP-DCs (Fig. 2A). Marco mRNA was already increased at early time points after tumor lysate pulsing, as shown in Fig. 2A for 6 and 12 h. In agreement with data obtained from experiments with metrizamide gradient-purified DCs, an increase in the expression level of Marco transcript was also observed in CD11c+ TP-DCs by RT-PCR (data not shown). Western blot analysis showed a strong increase in Marco protein expression by TP-DCs as well (Fig. 2B). Total protein extracts from adherent splenocytes, obtained from C57BL/6 mice, were used as a positive control. Double staining FACS results for CD11c and Marco cell surface coexpression by DCs are shown in Table IV. No cell surface expression of Marco was detected on CD11c+ DCs 6 h after tumor lysate pulsing. However, at longer time points of pulsing, a CD11c+ Marco+ DC population was observed. An increase in the mean fluorescence intensity (MFI) was also noted. In contrast, no cell surface expression of Marco was observed in the CD11c+ UP-DCs population. This finding indicated that after B16 melanoma lysate pulsing, Marco was selectively expressed on the cell surface of DCs.
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Discussion |
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An important function of DCs is Ag uptake, processing, and presentation. Collectively, the microarray analysis showed that the Ag/processing machiney is mostly unchanged 24-h exposure to tumor lysate; this observation is not entirely unexpected because by 24 h of our pulsing conditions, all tumor cells have already been processed by DCs. Using microarrays, we have indeed observed that most of the transcripts involved in Ag processing and presentation are increased 12 h after tumor lysate loading, but have returned to levels similar to those of UP-DCs by 24 h (data not shown). Previous studies have reported an early modulation of genes associated with Ag processing and presentation in DCs following exposure to a variety of pathogens and Ags (21, 22). Identification of protein processing-induced genes following early exposure to tumor lysate should allow us to further dissect the Ag processing and presentation pathway(s) in DCs.
For productive immunity to occur, DCs must present to T cells not only peptide-MHC complexes but also additional costimulatory signals. Up-regulation of gene expression for CD80, a member of the B7 family, and OX40L, a member of the TNF family, was observed in DCs following tumor lysate pulsing. These costimulatory molecules play a critical role as activation and/or polarization signals to T cells. For example, the OX40L-OX40 interaction induces the production of cytokines, including IL-4, and promotes the differentiation of Th2 CD4 T cells (26). Tumor lysate loading can also affect activation and function of DCs through increased expression of secreted molecules with inflammatory and chemotactic activities, including MYD1, a surface Ag specifically associated with DCs, which also plays a role in CD4+ T lymphocyte activation (27).
DCs remodel their gene profile of chemokine receptors following tumor lysate loading. We found the levels of transcripts for CCR5, CXCR2, and CCR2 at least five times higher in UP-DCs than in B16 TP-DCs. We did not detect transcripts for CCR7, CCR6, or CXCR1. Simultaneously, B16 TP-DCs experienced down-regulated expression of a few cytokine transcripts as well. Small inducible cytokine A6 and A17 mRNA levels were markedly reduced, while the expression of small inducible cytokine B subfamily, member 9 and cytokine inducible Src homology (SH)2-containing protein were modestly affected. Following tumor lysate pulsing, DCs also produced high levels of particular chemoattractant cytokines. For instance, MIP-2, Gro-1, and MCP-1 transcripts were strongly up-regulated. Significantly modulated expression of some of these cytokines and chemokines and their respective receptors by B16 TP-DCs would be expected to have important immunoregulatory effects on their function, in particular by affecting the magnitude and cytokine polarity of the T cell response, as well as in the ability of the DCs to respond to inflammatory signals.
Our study demonstrated that many genes linked to or regulated through the NF-
B pathway of gene transcription were induced by tumor lysate pulsing, including those encoding for MIP-2, Gro-1, MCP-1, superoxide dismutase, TNF-, TNF-induced protein 3, TNFR, molecule possessing ankyrin-repeats induced by LPS, G-CSF, and IL-6. NF-B plays a pivotal role in the regulation of immunologic processes, by regulating expression of cellular genes particularly involved in immune, acute phase, and inflammatory responses, and it promotes the transcription of a variety of genes mediating maturation of DCs (28). NF-B also regulates expression of multiple genes important in immunologic and inflammatory responses, such as those encoding for MHC molecules, cytokines and growth factors and their receptors, cell adhesion molecules, transcription factors, and the Rel/NF-B and IB proteins themselves (29). Recently, investigators have reported differential expression of Rel/NF-B family genes during differentiation and maturation of DCs (30). The increase of numerous regulators of the NF-B activation cascade upon tumor lysate pulsing may be suggestive of a role of these proteins in the functional phenotypes of DCs, but additional experiments will be required to establish this possibility directly and to examine the relative importance of each of these genes.
Of interest, 15 genes involved in regulating cell cycle and mitotic spindle function were expressed in B16 TP-DCs at distinctly lower levels than in UP-DCs. For example, cdc2 associates with cyclin B1 and cyclin B2 to form a protein kinase complex known as M-phase promoting factor (MPF), which is essential for G1/S and G2/M transitions of the eukaryotic cell cycle (31). Cdc2, the B-type cyclin, as well as cdc25C, the phosphatase responsible for triggering activation of cyclin B/cdc2 complexes (32), are transcriptionally down-regulated in B16 TP-DCs as well. The expression of six genes that are involved in DNA synthesis and replication and, thus, necessary for the S phase of cell cycle was also higher in UP-DCs. Replication protein A2 transcript, for instance, was down-regulated in B16 TP-DCs. Minichromosome maintenance proteins also crucially take part in DNA replication (33). Collectively, these gene expression patterns indicate a lower cycling activity of B16 TP-DCs compared to UP-DCs, suggesting that B16 TP-DCs are arrested in G0 phase, which could serve as a molecular explanation for the finding that tumor lysate pulsing promotes survival of DCs in a growth-arrested state (e.g., after deprivation of IL-4 and GM-CSF). A similar observation has been reported during activation of DCs by LPS after deprivation of growth factors (34).
The transcript that was most highly expressed in DCs following tumor lysate pulsing encodes for Marco, a recently identified class A scavenger receptor (35, 36). Marco is an integral membrane composed of three 52-kDa monomeres mediating the uptake of chemically modified low density lipoproteins, bacteria but not yeast. Marco is constitutively expressed in a subset of macrophages in the marginal zone of the spleen and in the medullary cord of lymph nodes, i.e., regions where macrophages are believed to be actively engaged in the removal of pathogens and other foreign substances. Marco can be up-regulated on other macrophages that normally do not express it, for example, after bacterial infection (35, 36). This has implied a direct role for Marco in the removal of pathogens, and recent studies have shown that Marco participates not only in the host defense against microorganisms but also serves as a major receptor on alveolar macrophages for the binding of unopsonized environmental particles (37, 38). Interestingly, it has been reported that transfection of Marco in many different cell lines induces dramatic cell shape changes (39). Typically these changes include formation of large lamellipodia-like structures and of long dendritic processes. The Marco-induced morphologic changes are accompanied by rearrangement of the actin skeleton and are dependent on cell adhesion (39). In the present study we have demonstrated that DCs transcribe and specifically express Marco on their cell surface in response to different tumor lysate pulsing. The fact that Marco expression also increases when using lysates from fresh normal tissues to pulse DCs suggests that Marco up-regulation is an event associated with the phagocytic capacity of the DCs. Based on previously described functions of Marco, this receptor may likely play an important role in the adhesion and shape changes observed in TP-DCs and may be necessary for the trapping and removal of pathogens. Indeed, we have observed that when DCs were lysate pulsed overnight in presence of a specific Ab against Marco, they remain round and do not undergo the formation of long plasma membrane processes, which are observed in absence of anti-marco Ab (data not shown). Marco may also participate in the initial response induced by TP-DCs when used in immunization protocols in vivo. Further studies will be necessary to unravel the precise role of Marco in DC biology and in innate immunity.
Our study has uncovered a large number of genes with distinctive expression patterns between B16 TP-DCs and UP-DCs, and demonstrated that tumor lysate loading of DCs resulted in the increased expression of an array of genes previously associated with DC activation of T cells. In addition, the pattern of expression induced upon tumor lysate loading of DCs shows some similarities to that seen in maturation-induced DCs. For example, decreased expression of genes encoding for growth-modulatory cell division cycle proteins (19), or chemokines receptors (40), and increased expression of genes involved in inflammation (20) or cell surface molecules and receptors (40) have been implicated frequently in LPS-induced maturation. Thus, tumor lysate pulsing may be sufficient to drive DC toward a stage of maturation appropriate for immune response activation, and could explain our findings showing that tumor lysate-pulsed DCs generated tumor-specific proliferative, cytokine release, and cytolytic reactivities in vitro (10). Examination of the function of several of the preferentially expressed genes identified from this study is underway in animal models, and this effort may provide information for further designing DC-based immunotherapies for cancer.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. James J. Mulé, Department of Surgery, University of Michigan Medical Center, 1520 Medical Sciences Research Building, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0666. E-mail address: jimmule{at}umich.edu
3 Abbreviations used in this paper: DC, dendritic cell(s); TP-DC, tumor lysate-pulsed DC; UP-DC, unpulsed DC; CM, complete medium; PM, perfect match; MM, mismatch; FC, fold change; MIP, macrophage inflammatory protein; Gro, growth-related oncogene; MCP, monocyte chemoattractant protein; PAF, platelet-activating factor; COX, cyclooxygenase; OX40L, OX40 ligand; MFI, mean fluorescent intensity; SH, Src homology; Marco, macrophage receptor with collagenous structure.
Received for publication December 9, 2002. Accepted for publication July 9, 2003.
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References |
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  A. Grolleau-Julius, E. K. Harning, L. M. Abernathy, and R. L. Yung Impaired Dendritic Cell Function in Aging Leads to Defective Antitumor Immunity Cancer Res., August 1, 2008; 68(15): 6341 - 6349. [Abstract] [Full Text] [PDF]
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 M. P. Hubner, B. Pasche, S. Kalaydjiev, P. T. Soboslay, A. Lengeling, H. Schulz-Key, E. Mitre, and W. H. Hoffmann Microfilariae of the Filarial Nematode Litomosoides sigmodontis Exacerbate the Course of Lipopolysaccharide-Induced Sepsis in Mice Infect. Immun., April 1, 2008; 76(4): 1668 - 1677. [Abstract] [Full Text] [PDF]
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 S. A. Shell, C. Hesse, S. M. Morris Jr., and C. Milcarek Elevated Levels of the 64-kDa Cleavage Stimulatory Factor (CstF-64) in Lipopolysaccharide-stimulated Macrophages Influence Gene Expression and Induce Alternative Poly(A) Site Selection J. Biol. Chem., December 2, 2005; 280(48): 39950 - 39961. [Abstract] [Full Text] [PDF]
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A. Lehtonen, V. Veckman, T. Nikula, R. Lahesmaa, L. Kinnunen, S. Matikainen, and I. Julkunen Differential Expression of IFN Regulatory Factor 4 Gene in Human Monocyte-Derived Dendritic Cells and Macrophages J. Immunol., November 15, 2005; 175(10): 6570 - 6579. [Abstract] [Full Text] [PDF]
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F. Ito, Q. Li, A. B. Shreiner, R. Okuyama, M. N. Jure-Kunkel, S. Teitz-Tennenbaum, and A. E. Chang Anti-CD137 Monoclonal Antibody Administration Augments the Antitumor Efficacy of Dendritic Cell-Based Vaccines Cancer Res., November 15, 2004; 64(22): 8411 - 8419. [Abstract] [Full Text] [PDF]
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