Molecular Characterization of Antigen-Peptide Pulsed Dendritic Cells: Immature Dendritic Cells Develop a Distinct Molecular Profile when Pulsed with Antigen Peptide

As dendritic cells (DCs) are the most potent professional antigen-presenting cells, they are being tested as cancer vaccines for immunotherapy of established cancers. Although numerous studies have characterized DCs by their phenotype and function, few have identified potential molecular markers of antigen presentation prior to vaccination of host. In this study we generated pre-immature DC (piDC), immature DC (iDC), and mature DC (mDC) from human peripheral blood monocytes (PBMC) obtained from HLA-A2 healthy donors, and pulsed them with human papillomavirus E7 peptide (p11-20), a class I HLA-A2 binding antigen. We then characterized DCs for cell surface phenotype and gene expression profile by microarray technology. We identified a set of 59 genes that distinguished three differentiation stages of DCs (piDC, iDC and mDC). When piDC, iDC and mDC were pulsed with E7 peptide for 2 hrs, the surface phenotype did not change, however, iDCs rather than mDCs showed transcriptional response by up-regulation of a set of genes. A total of 52 genes were modulated in iDC upon antigen pulsing. Elongation of pulse time for iDCs to 10 and 24 hrs did not significantly bring further changes in gene expression. The E7 peptide up-modulated immune response (KPNA7, IGSF6, NCR3, TREM2, TUBAL3, IL8, NFKBIA), pro-apoptosis (BTG1, SEMA6A, IGFBP3 and SRGN), anti-apoptosis (NFKBIA), DNA repair (MRPS11, RAD21, TXNRD1), and cell adhesion and cell migration genes (EPHA1, PGF, IL8 and CYR61) in iDCs. We confirmed our results by Q-PCR analysis. The E7 peptide but not control peptide (PADRE) induced up-regulation of NFKB1A gene only in HLA-A2 positive iDCs and not in HLA-A2 negative iDCs. These results suggest that E7 up-regulation of genes is specific and HLA restricted and that these genes may represent markers of antigen presentation and help rapidly assess the quality of dendritic cells prior to administration to the host.


Introduction
Human dendritic cells (DCs) are the most potent antigen presenting cells. A relatively small number of DCs have a capacity to render a potent immunologic response against pathogens or other antigens, including tumors [1]. The most common classes of DCs are myeloid and plasmacytoid (or lymphoid). Myeloid DCs can be divided into at least two subsets: myeloid DC-1 which is a major stimulator of T-cells; and myeloid DC-2 which is a rare type and functions in fighting wound infection. Plasmacytoid DCs, having a plasma cell-like morphology, could play a major role in adaptive immunity as well as innate immunity [2]. A number of articles have been published on the characterization and use of DCs, peptide-or protein-pulsed DCs to treat patients with different types of cancers [3,4].
DCs can be generated by culture of peripheral blood monocytes (PBMC) or bone-marrow derived mononuclear cells in the presence of a variety of growth factors. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) have been most widely used to generate immature DCs (iDC) [5]. Mature DCs (mDC) are generated with different maturation cytokines, e.g., tumor necrosis factor alpha (TNFa), CD40 ligand (CD40L), lipopolysaccharide (LPS), interferon-c (INFc), and cytokine cocktail [6,7,8]. In addition, some clinical trials have also used antigen-pulsed PBMC that are short-term cultured only with GM-CSF gaining some phenotype of DCs [9]. These cells are termed pre-immature DCs (piDC). piDC, iDC and mDC are predominantly identified by phenotypic expression of CD11c, CD80, CD83, CD86 and HLA-DR and down modulation of CD14 expression. A large number of studies have reported on the gene expression changes during differentiation from monocytes to iDCs and mDCs using different stimulation and different microarray platforms [10,11,12,13,14,15,16]. However, few studies have examined the role of antigen pulse on gene expression changes in different activation states of DCs, and identified biomarkers of activation that can be rapidly assessed for quality testing of antigen pulsed dendritic cells.
In this study, we have investigated transcriptional biomarkers of differentiated DCs and gene expression changes in piDC, iDC and mDC after pulsing with a Major Histocompatibility Complex (MHC) class 1 antigen peptide. We used a 10-mer peptide (human papillomavirus16 E7p11-20), which is frequently used in clinical trials [17]. We discovered that when different types of DCs were pulsed with the antigen-peptide, iDCs showed molecular changes but not mDCs. These results suggest that iDCs may efficiently uptake and process peptide antigen when pulsed in vitro, and that the identified genes could be used for assessment the quality of antigen pulsed DCs. Furthermore, the information obtained from such studies may provide insights in the understanding of the process of peptide-antigen uptake and presentation by dendritic cells.

Isolation of monocytes and generation of dendritic cells in vitro
Human monocytes from nine human leukocyte antigen (HLA) -A2, and two non-HLA-A2 healthy donors were obtained from the NIH Blood Bank. The monocytes were cryopreserved in liquid N2 until use. To generate dendritic cells, the cryopreserved monocytes were thawed, put in tissue culture plate for 4 hrs at 37uC. The non-adherent cells were removed by washing with phosphate buffered saline (PBS). The adherent cells were cultured in complete RPMI 1640 medium (Mediatech, Inc. Manassas, VA) supplemented with human GM-CSF (1000 U/ml, BD Biosciences, San Jose, CA) for two days to generate pre-immature dendritic cells (piDC) or with human GM-CSF (1000 U/ml) and IL-4 (50 ng/ml, BD Biosciences) for 4 days to generate immature dendritic cells (iDC), or with human GM-CSF (1000 U/ml) and IL-4 (50 ng/ml) for 4 days and additional 2 days with TNFa (20 ng/ml, BD Biosciences) and CD40 ligand (200 ng/ml, InvivoGen, San Diego, CA) to generate mature dendritic cells (mDC) ( Table 1). Two donors (D1 and D2) were also matured by culturing 2 days with a cytokine cocktail, which contained IL-1b (10 ng/ml, BD Biosciences), IL6 (10 ng/ml, InvivoGen), TNFa (10 ng/ml) and PGE 2 (10 27 M, Sigma-Aldrich, St. Louis, MO).
Human papillomavirus (HPV) E7 peptide (p11-20) pulsing A ten amino acid (p11-p20) HPV oncogene E7 peptide (YMLDLQPETT), and a 13 aa synthetic, non-natural pan HLA-DR-binding Epitope (PADRE) [(D)alanine -K-(L)cyclohexylalanine -VAAWTLKAA-(D)alanine], were obtained from NIH Pharmacy Department. Endotoxin assay was done using Pierce LAL Chromogenic Endotoxin Quantitation Kit (ThermoScientific Cat# 88282). There were no detectable endotoxins in the peptide solution (,0.1 EU/ml as the limit of detection of the kit is 0.1 EU/ml). To investigate the effect of peptide pulsing on gene expression of human dendritic cells, piDC, iDC and mDC generated from monocytes of four HLA-A2 donors (D1-D4) were incubated with 5 mg/ml peptide at 37uC for 2 hrs. For the time course experiment, iDCs generated from monocytes of additional 3 HLA-A2 donors (D5-D7) were incubated with the same E7 peptide for 2 hrs, 10 hrs and 24 hrs. To investigate whether the E7p11-20 stimulation is HLA-A2 specific, iDCs derived from two HLA-A2 donors and two non-HLA-A2 donors (D8-11) were pulsed with E7 peptide and PADRE peptide.

Flow cytometry analysis of DC
To evaluate the phenotype of three stages of dendritic cells, fluorescein isothiocyanate (FITC) labeled antibodies specific for CD11c (eBioscience, San Diego, CA), CD14 (BD Biosciences, San Jose, CA), CD80 (BD), CD83 (BD), CD86 (BD) and HLA-DR (BD), as well as isotype control mouse IgG1 (eBioscience) and IgG2a (eBioscience) were used to measure the surface markers as described by the Manufacturer (BD). In brief, the cells in flow cytometry staining buffer (PBS pH 7.4 containing 0.5% BSA and 0.02% Azide (eBioscience) were incubated with antibody on ice for 30 mins and washed with DPBS buffer twice. The stained cells were analyzed by BD FACScan (BD, Franklin Lakes, NJ) and the population of cells was recorded.

RNA isolation
Total RNA from monocyte, piDC, iDC and mDC, and E7 peptide pulsed piDC, iDC and mDC from each donor was isolated, using the Qiagen RNeasy Mini Kit (Qiagen, Inc. Valencia, CA) following the manufacture's instructions. The concentration and quality of the RNA were accessed by NanoDrop spectrophotometer (NanoDrop Technologies, Inc. Wilmington, DE) and Agilent 2100 Bioanalyzer (Agilent Technologies, Inc. Germantown, MD) following the manufacture's instructions. RNA purity is a critical factor for the reliability of
For study of gene expression profiles of piDC, iDC and mDC as well as their genomic response to E7 peptide stimulation, total RNA isolated from piDC, iDC and mDC and corresponding E7 peptide pulsed piDC, iDC and mDC from four donors (D1-D4) were used as targets, and their mixture of equal amount of monocyte RNA served as a reference (monoRef). 16K arrays were hybridized for this set of experiments. For study of gene expression kinetics in iDCs pulsed with E7 peptide for 2, 10 and 24 hrs, pulsed iDCs were used as targets and non-pulsed served as references. 35K arrays were hybridized for the experiment. Total RNA of 5 mg in 10 ml molecular H 2 O was reverse-transcribed by incubating with 1 ml poly-dT 18 (1 mg/ml, synthesized by our core facility of CBER/FDA) at 70uC for 2 min, then incubated with 2 ml of 106 first-strand buffer, 2 ml of 206 dNTP (including 10 mM of dATP, dCTP and dGTP, 6 mM of dTTP, and 4 mM of aminoallyl-dUTP), 2 ml of 0.1 M dithiothreitol (DTT), and 1 ml of Affinity Script Multi-Temp Reverse Transcriptase (Stratagene, La Jolla, CA) in a total volume of 20 ml reaction at 42uC for 1 hr as previously described [18,19]. The cDNA generated was purified with Qiagen MinElute column (Qiagen) according to manufacture's instructions. Reference cDNA and the cDNA of DC samples were swapped labeling with Cy3 and Cy5 (Amersham Biosciences/GE HealthCare, Piscataway, NJ) at room temperature in dark for 1 hr. After incubation, labeled cDNA was purified again with MinElute column. The Cy3-cDNA reference and Cy5-cDNA samples were combined (24 ml) and mixed with 24 ml of SlideHyb Glass Array Hybridization Buffer (Applied Biosystems, Foster City, CA), and hybridized with human arrays in MAUI FL mixer (MAUI Hybridization System Inc., BioMicroSystems, Salt Lake City, UT) overnight (16 hrs). After washing once with 16 SSC (206, Invitrogen, Grand Island, NY) and 0.05% SDS (10%, Sigma-Aldrich, St. Louis, MO) for 4 min, and twice with 0.16 SSC for 4 min, the arrays were scanned to acquire images.

Microarray Image Acquisition and Data Analysis
(http://www.fda.gov/nctr/science/centers/toxicoinformatics/ ArrayTrack). After filtering out flagged spots, we normalized the ratio data two ways, a) the Loess fit that minimized local nonlinear biases between the two fluorescence channels, and b) a linear normalization that made the distribution of the log2 intensity ratios to have a median of zero on each slide. Some normalization conditions were set, such as smoothing factor as 0.2, robustness iterations as 3, scaling using geometric mean, and target value as 1000, and the flagged spots were excluded.
Analysis of Variance (ANOVA) was used to search for the significant genes during transition from piDC to iDC and to mDC differentiation stages, the genes/transcripts with an F-value.4 were subjected further pairwise t-tests between stages. Criteria were set as p-value,0.05, excluding bad flagged spots, absolute fold change $1.5. Two-class paired Significance Analysis of Microarrays (SAM) was used to compare the gene expression profiles of E7 peptide pulsed piDC, iDC and mDC with corresponding non-pulsed DC samples. Criteria were set as Target False Discovery Rate (FDR),0.05, excluding bad Flagged spots, absolute fold change $1.5. For time course experiments, gene expression was directly compared between E7 pulsed and non-pulsed iDCs from 3 donors (D5-D7). For this purpose, we used mAdb statistical tool available at NCI. We set the criteria as absolute fold change of $1.5 fold in all 3 arrays.
The Correlation Matrix function was used to compute between two data sets. Hierarchical Clustering Analysis (HCA) was used to view the correlation of biological repeats based on gene expression. Ingenuity Pathway Analysis (IPA) was used to network the gene biological function.
The Basic Local Alignment Search Tool (BLAST) was used to query features without gene description according to the database of National Center for Biotechnology Information (NCBI, http:// blast.ncbi.nlm.nih.gov/Blast.cgi). Blastx was used to query nucleotides in a protein database, and blastp to query translated nucleotides (nt) in proteins database.

Real-Time Quantitative Polymerase Chain Reaction (qPCR) Assay
Total RNA (1 mg) from iDCs, iDCs pulsed with E7 and control PADRE peptide from two HLA-A2 and two non-HLA-A2 donors was reverse transcribed to cDNA using iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad Life Science Research, Hercules, CA). The quantification of the expression of NFKBIA was carried out using PrimePCR SYBR Green Assay for human NFKBIA and b-actin using SsoAdvanced SYBR Green Supermix, and CFX96 Touch Deep Well TM Real-Time PCR Detection System (Bio-Rad). The manufacturer's protocols were followed. The level of NFKBIA expression in each sample was normalized to its internal control b-actin. The specificity of each PCR reaction was confirmed by melting curve analyses by CFX manager V3.0 (Bio-Rad) software. The relative quantification of NFKBIA expression in iDCs pulsed with two peptides separately was compared to iDCs.

Phenotype of HPV16 E7 class I peptide pulsed human monocytes-derived dendritic cells
Dendritic cells-piDCs, iDCs and mDCs were generated from human PBMC with different cytokines and under different conditions ( Table 1). The resultant cells were characterized by fluorescence-activated cell analysis using flow cytometry. As shown in Fig. 1, from a representative donor, monocytes cultured for 48 hrs with GM-CSF alone (piDCs) showed a small shift in CD11c expression but expressed similar levels of CD14, CD80, CD83, CD86 and HLA-DR as monocytes. When monocytes were cultured with GM-CSF and IL-4 for 4 days, the resultant cells displayed iDC phenotype by down-regulating monocyte marker CD14 and up-regulating dendritic cell marker CD11c, costimulatory molecules CD80 and CD86. When the iDCs were cultured further with CD40 ligand/TNFa or cytokine cocktail for 2 additional days, they began to display mature phenotype by dramatically up-regulating activation marker CD83. The MHC class II molecule (HLA-DR) was also up-regulated in immature and mature DC groups. Interestingly, piDCs, iDCs or mDCs when pulsed with HPV E7-peptide for 2 hrs displayed almost identical phenotype as their non-pulsed states (Fig. 1). These results indicate short-term peptide pulsing of DCs does not modulate phenotype that can be appreciated by FACS analysis.

Gene Expression profiling of DCs
We evaluated gene expression profiles of piDCs, iDCs and mDCs from four donors (D1-D4) and found a set of 59 genes that distinguished the three differentiation stages of DCs. The supervised HCA of gene expression showed that piDCs, iDCs and mDCs were grouped together within donors but different between stages of differentiation (Fig. 2). Significant gene expression variations between differentiation stages and their description were presented in Table 2. Compared to piDCs, iDCs over expressed 26 genes including CD86 and HLA antigens (HLA-DPA1 and -DQA2), and under expressed 7 genes including IL-8 and CD14, which were dominant in monocytes. Compared to iDCs, mDCs over expressed 12 genes including mDC marker genes CD83, CD86 and HLA-B, and under expressed 9 genes including further decrease of CD14. Compared to monocytes, piDCs modulated 32 genes, 17 genes were under expressed representing cytokines IL-6, IL-8, IL-1B, CCL3, CCL14, CCL20, CXCL1. IPA revealed that the 59 genes were involved in immunological and inflammatory diseases, immune cell trafficking, hematological system development and function, and lipid metabolism ( Table 3).
We also compared gene expression profiles of mDCs that were maturated by CD40L/TNFa and a cytokine cocktail containing IL1b, IL6, TNFa and PGE 2 . Results showed that CD40L/TNFa (G5) stimulated expression of 52 genes, while the cytokine cocktail (G7) stimulate 108 significant genes in mDC (Table S1 and  Table S2 in File S1). The increased gene number by cytokine cocktail indicated higher transcription activity of mDCs compared to TNFa and CD40L activated mDCs. Some of these genes overlapped and some were different, indicating different maturation stimuli have different effect on DCs.
As presented in Table 4 Extending the peptide pulsing time to 10 hrs, the gene expression profiles were similar to 2 hrs pulsed iDCs without any overall statistically significant difference. Continuing pulsing to 24 hrs, there was no further change on overall gene expression except the expression of the NPY2R gene was significantly lower than the other two time points at p-value,0.05. These results indicate that 2 hrs pulsing was sufficient to induce iDC full response at transcription level.
To confirm microarray results and determine the specificity of gene regulation, iDCs generated from HLA-A2 positive and negative donor cells were pulsed with E7 and control PADRE peptides and gene expression of NFKB1A was assessed by Q-PCR. Our results show that E7-peptide pulsing of HLA-A2 positive donor derived iDCs significantly up-regulated NFKBIA gene compared to non-pulsed iDCs (p-value,0.0003 and fold change $1.5). In sharp contrast, the E7 peptide pulsing of non-HLA-A2 iDCs showed no regulation of NFKB1A as well as PADRE peptide pulsed iDCs showed no modulation (Fig. 4).
These results indicate that iDCs response to the E7 stimulation is specific and HLA restricted.

Bioinformatics of novel genes
We identified six up-regulated features responding to antigenpeptide pulsing but they lacked gene information ( Table S3 in File S1). Therefore, we queried their potential biological function in the NCBI protein database by using translated BLAST (blastx). The query showed 4 sequences which share similarities with existing proteins and 2 with no significant similarity found. Feature opHsV0400003515 covered 94% with 'golgi auto-antigen', opHsV0400005414 covered 68% with 'cDNA clones with function of inhibiting cancer cell growth', and H300003055 covered 68% with 'stAR-related lipid transfer protein' at ,0.001 E-value. We further translated the 2 remaining feature's nucleotide (nt) sequences into amino acid (aa) sequences, and queried their translated residues with NCBI protein database by using protein BLAST (blastp). Within the translated 6 hypothetical peptides of feature opHsV0400007698, 5939frame +2 and +3 exhibited partial similarities with 'trans-membrane and ubiquitinlike domain-containing protein', 'multi-drug and toxin extrusion protein', and 'ATP-binding cassette sub-family G and C isoform'. Other frames did not bring output or have stop codon in residues. Feature opHsV0400002236 displayed 4 hypothetical proteins, like 'lysine-specific demethylase', low-density lipoprotein receptor', protein bicaudal D', 'probable ATP-dependent RNA helicase', and 'ADP-ribosyltransferase' by 4 frames.

Discussion
By comparing DCs at different stages of differentiation, we identified a set of 59 gene markers that distinguish piDCs, iDCs and mDCs that have been used in cancer vaccine clinical trials. Conventionally, flow cytometry analysis of several surface markers including CD14, CD11c, CD80, CD83, CD86 and HLA-DR are used to characterize DCs. It is known that as PBMCs (as well as piDCs) differentiate to iDC phenotype, iDC cells show downregulation of CD14 and up-regulation of CD11c, CD86 and HLA-DR. Similarly, as DCs differentiate from iDCs to mDCs, they began to show further up-regulation of CD83, CD86 and HLA-DR [1]. In our study, we also observed these surface markers expressed in DCs. At transcription level, we identified a set of 59 genes that were modulated during DCs differentiation, including CD14 and CD83. These 59 genes may constitute genetic biomarkers of DC differentiation, that can be potentially used to rapidly assess the quality of dendritic cells using nucleic acid based multiplex technology, which is cost effective, efficient and quantitative. Interestingly, we found that despite the fact that piDCs showed no significant changes in the conventional surface markers phenotype when compared to PBMCs, there were significant changes at the gene expression level. These changes may indicate the state of differentiation currently not appreciated by standard cell surface markers.
A large number of studies have reported on the gene expression changes during DC maturation stimulated with different factors. These factors included CD40L [13,14,15], TNFa [11,13,16], INFc [13], LPS [8,10,15], mixture of INFc and IL-1b, mixture of LPS and INFc, mixture of TNFa, IL-1b, LPS and INFc [8,20], and cocktails containing IL6, TNFa, IL-1b and PGE 2 [12,15]. Simultaneously, various microarray platforms have also been used to profile DCs including cDNA arrays [12,13], Affymetrix arrays [10,11,14,15], and in-house fabricated oligonucleotide microarrays [8,20]. Consequently, a large data set has been accumulated in the literature. It has been shown that different stimuli stimulated different gene expression patterns in mDCs. A single factor stimulated a relatively fewer number of genes compared to cytokine cocktails, e.g., CD40L stimulated 40 genes, LPS 619 genes and the combination cocktail 1116 genes after 48 hr culture of mDCs [15]. Recently, some studies have identified as much as 9576 genes that were modulated in mDCs [8,20]. Moschella et al., used a single cytokine TNFa, CD40L or INFc as stimuli and found that they induced different gene expression patterns in mDCs, suggesting important functional differences among the DC populations [13]. Han et al., used three combinations, LPS plus INFc, INFc plus IL-1b, and a cocktail that contained LPS, INFc, IL-1b and TNFa as DC maturation stimuli, and found that these 3 combinations had no difference in terms of expression of costimulatory molecules CD80, CD83 and CD86, HLA-DR, chemokine receptor CCR7, and interleukin IL-12p70and IL-10 [8]. Jin et al., recommended CCL5, CCR7 and CD83 as particularly good potency biomarker candidates that were selected by evaluating 2370 significant modulated genes for maturation [20]. Although the modulated gene number varied, in most publications, the activated pathways remained the same. These common pathways are cell signaling, cell adhesion and lipid metabolism [8,11,20].
In our study, we also compared gene expression profiles of mDCs stimulated by a cytokine cocktail (IL-1b, IL6, TNFa and PGE 2 ) with mDCs that stimulated by CD40L and TNFa. The cytokine cocktail induced 108 genes in mDCs vs. 52 genes induced with CD40L and TNFa. Consistent with previous studies [11,12,14,15], the results show that the cytokine cocktail supplies stronger stimulation that induced more modulated gene during the process of DC maturation than CD40L/TNFa. The modulated genes were categorized into cell signaling, immune response and lipid metabolism that are very similar with previous publications [11]. As CD83, CD86, LAMP3 and DUPS6 up-modulated, and CD14 and CCR2 down-modulated, our results agree with other reports in which CD40L or TNFa were used as stimuli [10,11,13]. Further studies are needed to determine the significance of these stimuli in DC function.
The HPV-16 E7 HLA-A2 (11)(12)(13)(14)(15)(16)(17)(18)(19)(20) peptide has been used in clinical trials for the treatment of patients with cervical neoplasms. The vaccination with this peptide, albeit administered directly or pulsed on DCs in these clinical trials has shown series of immune and clinical benefits [21,22,23]. However, the molecular profile of peptide pulsed DCs were not known. Hence, we tested the effect of the peptide pulsing on the gene expression profile of DCs; whether differentiation stages of DCs affect the effect; and whether peptideantigen pulsed DC can rapidly be assessed by gene expression profiling. In our experiments, when piDC, iDC and mDC were pulsed with E7 peptide for 2 hrs, no phenotypic changes were  observed by flow cytometry analysis. However, at transcription level, iDCs, but not mDCs, responded to the peptide-pulsing by up-regulating a set of genes. This set of genes we identified is apparently related to iDC antigen uptake, processing and presentation (ADAM33, KIF26B, MRPS11, NPY2R, SLC25A10, TNKS1BP1, TREM2, and ZNF791). Thus, our study may be the first evaluation of gene expression of E7-peptide pulsing of DC for immunotherapy. Gene NFKBIA attracted a special attention since it was highly expressed at all three time points after antigen pulsing (at 2, 10 and 24 hrs). NFKBIA functions as a 'nuclear factor of kappa light polypeptide gene enhancer' promoting IL-6 and TNF-a synthesis. It has been shown that human tumor antigen MUC1, when pulsed into iDC, produced inflammatory cytokines IL-6 and TNF-a [24,25], which in turn induced a strong MUC1-specific CD8+ Tcell response [26]. Therefore, it is likely that the E7 peptide pulsed DCs will generate a strong CD8+ T cell response. Interestingly, E7 up-regulated four pro-apoptosis genes (BTG1, SEMA6A, IGFBP3 and SRGN) in iDCs indicating that antigen pulse selectively depletes some population of iDCs. However, NFKBIA, a strong negative regulator of apoptosis, and 3 other genes (MRPS11, RAD21 and TXNRD1) involved in DNA damage repair were upregulated that supported cell survival. In addition, another gene DNAJA3 also involved in cell survival was up-regulated at all three time points. These counteracting mechanisms may allow iDC to survive and present antigen. Furthermore, 4 genes (EPHA1, PGF, IL8 and CYR61) involved in cell adhesion were also up regulated at all three time points indicating significance of these genes in iDC antigen presentation, interaction with T cells and biology.
The mere fact that only iDC showed changes in the gene expression profile after E7 pulsing indicates that these changes are probably secondary to the antigen uptake process, since iDC is the only DC type amongst the three that is capable of antigen uptake in addition to presentation. We confirmed the response of iDCs by Q-PCR, which also demonstrated that E7 induced NFKB1A gene expression is HLA restricted since iDCs derived from non-HLA-A2 donors did not respond to E7 stimulation. Furthermore, the upregulation of NFKBIA expression in iDCs was specific as PADRE-peptide pulse did not induce gene expression. Further studies are needed to determine whether the gene expression markers identified by the HPV16 E7 antigen-peptide are unique to that sequence or similar when pulsed with different antigens.
It is important to note that we identified 6 potential novel genes that were induced by antigen pulsing of iDC, as gene information and description of these putative genes is unknown in the current NCBI nucleotide database. When their potential functions were queried by BCBI Blastx, we found that three of these genes have high similarities with 'golgi auto-antigen', 'cDNA clones with function of inhibiting cancer cell growth', and 'stAR-related lipid transfer protein' with very low e-values and high max scores. The other 2 feature's lacking similarities with the nucleotide (nt) database were further translated into amino acid (aa) sequences, and their translated residues were queried with the NCBI protein database by blastp. These analyses showed similarities with 'transmembrane and ubiquitin-like domain-containing protein', ATP-binding cassette sub-family G and C isoforms', 'lysine- Table 3. Functional network analysis of significant genes involved in DC differentiation*.  specific demethylase', and low-density lipoprotein receptor'. Although the exact identity and significance of these novel genes are still not known, they may have an important role in antigen presentation and immune response. Taken together, our results indicate that peptide-pulsing induces changes in iDC which may be important for optimal functioning of DCs in vivo. In addition, we have identified novel gene markers of antigen uptake and presentation that can be rapidly assessed and when correlated with function may serve as a surrogate marker of potency. Further studies will examine other peptide antigens and analyze similarity in gene expression profile.

Supporting Information
File S1 Contents include: Table S1. Significant genes in mDC induced by CD40L/TNFa compared with piDC.  35K arrays were used for these experiments. iDCs generated from the monocytes of donor D5-D7 were pulsed with or without E7-peptide for 2, 10 and 24 hrs. Comparison was made between E7 peptide pulsed and non-pulsed iDCs. b The Fold change represents mean of intensity ratio of E7-pulsed to non-pulsed iDC samples D5-D7, $1.5 meaning up-regulation, and #0.6 meaning down-regulation. SE is the standard errors. ANOVA showed no significant difference among 3 pulsing time points at p-value,0.5, therefore, significant genes pulsed for 2 hr were presented. doi:10.1371/journal.pone.0086306.t004 Figure 4. Quantification of NFKBIA gene expression after E7 peptide pulsing of iDCs derived from HLA-A2 disparate donors. iDCs derived from monocytes of two HLA-A2 and two non-HLA-A2 donors (D8-D11) were pulsed with the HLA-A2 binding E7 peptide and a non-natural peptide (PADRE) for 2 hrs. RNA was extracted and reverse transcribed to cDNA. b-actin was used as an internal gene control. Expression of NFKBIA in E7 and PADRE-pulsed iDCs from four different donors were compared to that of native iDCs. doi:10.1371/journal.pone.0086306.g004