Human dendritic cells (DCs) are a distinct but heterogeneous lineage of APCs operating as the link between innate and adaptive immune responses, with the function to either maintain tolerance or trigger immunity. The DC lineage consists of several subpopulations with unique phenotypes; however, their functional characteristics and transcriptional similarities remain largely unknown. To further characterize the phenotypes and transcriptomes of the subsets, we purified myeloid CD16+, blood DC Ag 1+ (BDCA1+), and BDCA3+ DC populations, as well as plasmacytoid CD123+ DCs, from tonsillar tissue and peripheral blood. Transcriptional profiling and hierarchical clustering visualized that BDCA1+ DCs clustered with BDCA3+ DCs, whereas CD16+ DCs and CD123+ DCs clustered as distinct populations in blood. Differential expression levels of chemokines, ILs, and pattern recognition receptors were demonstrated, which emphasize innate DC subset specialization. Even though highly BDCA1+ and BDCA3+ DC-specific gene expression was identified in blood, the BDCA1+ DCs and BDCA3+ DCs from tonsils displayed similar transcriptional activity, most likely due to the pathogenic or inflammatory maturational signals present in tonsillar tissues. Of note, plasmacytoid DCs displayed less plasticity in their transcriptional activity compared with myeloid DCs. The data demonstrated a functionally distinct association of each of the seven subsets based on their signatures, involving regulatory genes in adaptive and innate immunity.

BackgroundAllergic contact dermatitis is an inflammatory skin disease that affects a significant proportion of the population. This disease is caused by an adverse immune response towards chemical haptens, and leads to a substantial economic burden for society. Current test of sensitizing chemicals rely on animal experimentation. New legislations on the registration and use of chemicals within pharmaceutical and cosmetic industries have stimulated significant research efforts to develop alternative, human cell-based assays for the prediction of sensitization. The aim is to replace animal experiments with in vitro tests displaying a higher predictive power.ResultsWe have developed a novel cell-based assay for the prediction of sensitizing chemicals. By analyzing the transcriptome of the human cell line MUTZ-3 after 24 h stimulation, using 20 different sensitizing chemicals, 20 non-sensitizing chemicals and vehicle controls, we have identified a biomarker signature of 200 genes with potent discriminatory ability. Using a Support Vector Machine for supervised classification, the prediction performance of the assay revealed an area under the ROC curve of 0.98. In addition, categorizing the chemicals according to the LLNA assay, this gene signature could also predict sensitizing potency. The identified markers are involved in biological pathways with immunological relevant functions, which can shed light on the process of human sensitization.ConclusionsA gene signature predicting sensitization, using a human cell line in vitro, has been identified. This simple and robust cell-based assay has the potential to completely replace or drastically reduce the utilization of test systems based on experimental animals. Being based on human biology, the assay is proposed to be more accurate for predicting sensitization in humans, than the traditional animal-based tests.

T cells, whereas KG-1 and THP-1 only induced a marginal response. Furthermore, MUTZ-3 displayed the phenotypic and transcriptional profiles of immature dendritic cells, after differentiation with granulocyte-macrophage colony-stimulating factor and interleukin-4. Upon activation with inflammatory cytokines, MUTZ-3 matured phenotypically and exhibited a gene induction similar to that of monocyte-derived dendritic cells. This delineation of the cellular and transcriptional activity of MUTZ-3, in response to maturational stimuli, demonstrates the significance of this cell line as a model for functional studies of inflammatory responses.

BackgroundDendritic cells (DCs) comprise heterogeneous populations of cells, which act as central orchestrators of the immune response. Applicability of primary DCs is restricted due to their scarcity and therefore DC models are commonly employed in DC-based immunotherapy strategies and in vitro tests assessing DC function. However, the interrelationship between the individual in vitro DC models and their relative resemblance to specific primary DC populations remain elusive.ObjectiveTo describe and assess functionality and applicability of the available in vitro DC models by using a genome-wide transcriptional approach.MethodsTranscriptional profiling was performed with four commonly used in vitro DC models (MUTZ-3-DCs, monocyte-derived DCs, CD34-derived DCs and Langerhans cells (LCs)) and nine primary DC populations (dermal DCs, LCs, blood and tonsillar CD123+, CD1c+ and CD141+ DCs, and blood CD16+ DCs).ResultsPrincipal Component Analysis showed that transcriptional profiles of each in vitro DC model most closely resembled CD1c+ and CD141+ tonsillar myeloid DCs (mDCs) among primary DC populations. Thus, additional differentiation factors may be required to generate model DCs that more closely resemble other primary DC populations. Also, no model DC stood out in terms of primary DC resemblance. Nevertheless, hierarchical clustering showed clusters of differentially expressed genes among individual DC models as well as primary DC populations. Furthermore, model DCs were shown to differentially express immunologically relevant transcripts and transcriptional signatures identified for each model DC included several immune-associated transcripts.ConclusionThe unique transcriptional profiles of in vitro DC models suggest distinct functionality in immune applications. The presented results will aid in the selection of an appropriate DC model for in vitro assays and assist development of DC-based immunotherapy.

The need for non-animal data to assess skin sensitisation properties of substances, especially cosmetics ingredients, has spawned the development of many in vitro methods. As it is widely believed that no single method can provide a solution, the Cosmetics Europe Skin Tolerance Task Force has defined a three-phase framework for the development of a non-animal testing strategy for skin sensitization potency prediction. The results of the first phase – systematic evaluation of 16 test methods – are presented here. This evaluation involved generation of data on a common set of ten substances in all methods and systematic collation of information including the level of standardisation, existing test data,potential for throughput, transferability and accessibility in cooperation with the test method developers.A workshop was held with the test method developers to review the outcome of this evaluation and to discuss the results. The evaluation informed the prioritisation of test methods for the next phase of the non-animal testing strategy development framework. Ultimately, the testing strategy – combined with bioavailability and skin metabolism data and exposure consideration – is envisaged to allow establishment of a data integration approach for skin sensitisation safety assessment of cosmetic ingredients.