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Background: Dendritic cells (DCs) are specialized antigen presenting cells that play a pivotal role in bridging innate and adaptive immune responses. Given the scarcity of peripheral blood myeloid dendritic cells (mDCs) investigators have used different model systems for studying DC biology. Monocyte-derived dendritic cells (moDCs) and KG-1 cells are routinely used as mDC models, but a thorough comparison of these cells has not yet been carried out, particularly in relation to their proteomes. We therefore sought to run a comparative study of the proteomes and functional properties of these cells. Results: Despite general similarities between mDCs and the model systems, moDCs and KG-1 cells, our findings identified some significant differences in the proteomes of these cells, and the findings were confirmed by ELISA detection of a selection of proteins. This was particularly noticeable with proteins involved in cell growth and maintenance (for example, fibrinogen γ chain (FGG) and ubiquinol cytochrome c) and cell-cell interaction and integrity (for example, fascin and actin). We then examined the surface phenotype, cytokine profile, endocytic and T-cell-activation ability of these cells in support of the proteomic data, and obtained confirmatory evidence for differences in the maturation status and functional attributes between mDCs and the two DC models. Conclusion: We have identified important proteomic and functional differences between mDCs and two DC model systems. These differences could have major functional implications, particularly in relation to DC-T cell interactions, the so-called immunological synapse, and, therefore, need to be considered when interpreting data obtained from model DC systems. Published: 1 March 2007 Genome Biology 2007, 8:R30 (doi:10.1186/gb-2007-8-3-r30) Received: 2 August 2006 Revised: 1 December 2006 Accepted: 1 March 2007 The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2007/8/3/R30 Genome Biology 2007, 8:R30 R30.2 Genome Biology 2007, Volume 8, Issue 3, Article R30 Horlock et al. http://genomebiology.com/2007/8/3/R30 Background Dendritic cells (DCs) are highly specialized antigen presenting cells that originate from bone marrow progenitor cells. They represent a major cellular component of the innate immune system and their interaction with cells of the adaptive immune system (for example, T cells) is critical for initiating immune responses and maintaining tolerance [1]. DCs exist in two stages of maturation. Immature cells are found throughout the body where they act as sentinels, continuously taking up antigen and undergoing activation [2]. Activation leads to the secretion of pro-inflammatory cytokines, resulting in up-regulation of co-stimulatory molecules and migration to the lymph nodes. During their maturation, DCs lose their antigen-capturing capacity and become mature immuno-stimulatory cells that have the ability to activate naïve T cells. There are two main DC types in human peripheral blood, known as myeloid DCs (mDCs) and plasmacytoid DCs (pDCs). mDCs are the major subset, representing around 80% of blood DCs [3]. For ex vivo studies, mDCs can be isolated from peripheral blood using immunomagnetic cell separation [3]. However, the main obstacle here is that DCs represent only around 1% to 3% of peripheral blood mononuclear cells (PBMCs). This has, therefore, prompted researchers to use other model systems for studying mDC biology. For instance, DCs can be generated in vitro from peripheral blood monocytes by culturing them for six days in the presence of interleukin (IL)-4 and granulocyte-macrophage colony stimulating factor (GM-CSF). Under such culture conditions, cells acquire an immature DC morphology and express DC differentiation antigens [4]. These monocyte-derived DCs (moDCs) are routinely used as an mDC model in DC research. Several human monocytic cell lines are also available, including U937, THP-1, MUTZ-3, HL-60, KG1 and MM6, and some of these have been shown to be able to differentiate into DClike cells [5-9]. KG1 cells, which acquire a DC-like phenotype after stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin [6], are probably the most widely used in DC research. PMAand ionomycin-stimulated KG1 cells show typical DC morphology and become adherent with long neurite processes. They also show up-regulation of major histocompatibility (MHC) class I and II molecules, co-stimulatory molecules and DC-specific markers [6]. Furthermore, they are able to stimulate allogeneic T cell proliferation at levels similar to PBMC-derived DCs [6]. It has also been shown that KG1 cells are able to cross-present exogenous antigen to CD8+ T cells and display similar regulation of MHC class II trafficking to DCs [8]. Therefore, KG1 cells are considered to be a good model system to study human DC biology. Despite the extensive use of both moDC and KG1 cells as mDC models, their similarity to peripheral blood DCs is yet to be properly defined. This study aims to assess the suitability of both moDC and KG1 cells as model cells for peripheral blood DCs by comparing their proteomes in relation to their surface phenotypes, cytokine profiles and T cell activation ability. Results and discussion DCs are sentinels of the immune system and play a pivotal role in bridging innate immunity with the adaptive immune response. Given the scarcity of peripheral blood DCs and the ethical and technical difficulties involved in obtaining tissuederived DCs from human sources, investigators have resorted to using different model systems for studying DC biology. Although moDC and KG-1 cells are routinely used as mDC models [8,10,11], a thorough comparison of these cells has not yet been carried out. A number of phenotypic and functional comparisons have previously been made between mDCs and moDCs [12] and moDCs and KG-1 cells [13], but no studies have compared the proteomes of all three cell types. In this study we compare the proteomes of mDCs, moDCs and KG-1 cells, and then attempt to relate this to the functional properties of these cells. Figure 1 shows the workflow and the way in which each cell population was generated or separated. Dendritic cell proteomes Proteomic data are scarce in relation to DC biology, and where available they only focus on moDCs [14-16]. Others have focused on gene expression, as well as obtaining some proteomic data, in monocytes and moDCs [17-19]. The present study compared the whole cell proteome of immature mDCs, moDCs and KG-1 cells. Clearly, a major challenge in proteomic studies of DCs is obtaining enough protein for performing two-dimensional electrophoresis. This limitation was partly overcome by using a large volume of blood (approximately 120 ml) for cell separation. We also pooled whole cell lysates of DCs from seven individuals to obtain sufficient quantities of protein and to eliminate inter-individual variations. We found that peripheral blood mDCs have sixand five-fold lower protein content per cell than moDCs and KG-1 cells, respectively (data not shown). Unfortunately, the low numbers of mDCs in peripheral blood (approximately 1% of PBMCs), together with their lower protein content, meant that, despite pooling samples, we were able to run only duplicate gels for mDCs. Figures 2 and 3 show three representative two-dimensional gel images of the different cell types. Gel images were analyzed using PDQuest software and all images were normalized before any comparisons between gels were made. The total number of spots in the gels were 661, 619, and 770 for mDCs, moDCs and KG-1 cells, respectively. To analyze the comparability of gels, the densities of spots matched in all three gels were plotted and a correlation coefficient value was calculated. The proteome of mDCs showed different levels of similarity compared with those of moDCs and KG-1 cells (correlation coefficient 0.68 and 0.62, respectively) (Figure 4). Duplicate gels of mDCs were reproducible (correlation coefficient >0.90), as were triplicate gels of moDC and KG-1 Genome Biology 2007, 8:R30 http://genomebiology.com/2007/8/3/R30 Genome Biology 2007, Volume 8, Issue 3, Article R30 Horlock et al. R30.3