Malin Lindstedt

Gene Family Clustering Identifies Functionally Associated Subsets of Human In Vivo Blood and Tonsillar Dendritic Cells

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.

A genomic biomarker signature can predict skin sensitizers using a cell-based in vitro alternative to animal tests

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.

Functional and transcriptional profiling of MUTZ-3, a myeloid cell line acting as a model for dendritic cells

The incidence of allergy is steadily increasing, but the molecular mechanisms involved in the allergic immune response are still not fully understood. In particular, further investigations focusing on dendritic cells, which are central in orchestrating the immune response, are needed. The objective of this study was to investigate the ability of myeloid leukaemia‐derived cell lines, such as KG‐1, THP‐1 and MUTZ‐3, to serve as in vitro models for dendritic cells. The ability of these cell lines to mature into functional dendritic cells, expressing costimulatory molecules, was assessed by functional and transcriptional profiling and compared with that of monocyte‐derived dendritic cells, which are now used as a standard source of dendritic cells. High‐density microarray analysis was utilized to study the transcriptional activity and kinetics of activation of the differentiated MUTZ‐3 cell line, in response to a cocktail of inflammatory cytokines. The data obtained clearly demonstrate that MUTZ‐3 cells have the ability to induce antigen‐independent proliferation in CD4+ CD45RA+ 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.

Transcriptional Profiling of Human Dendritic Cell Populations and Models - Unique Profiles of In Vitro Dendritic Cells and Implications on Functionality and Applicability

Background Dendritic 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. Objective To describe and assess functionality and applicability of the available in vitro DC models by using a genome-wide transcriptional approach. Methods Transcriptional 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). Results Principal 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. Conclusion The 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.

Immunomodulatory nanoparticles as adjuvants and allergen-delivery system to human dendritic cells: Implications for specific immunotherapy.

Novel adjuvants and antigen-delivery systems with immunomodulatory properties that shift the allergenic Th2 response towards a Th1 or regulatory T cell response are desired for allergen-specific immunotherapy. This study demonstrates that 200-nm sized biodegradable poly(gamma-glutamic acid) (gamma-PGA) nanoparticles (NPs) are activators of human monocyte-derived dendritic cells (MoDCs). Gamma-PGA NPs are efficiently internalized by immature MoDCs and strongly stimulate production of chemokines and inflammatory cytokines as well as up-regulation of co-stimulatory molecules and immunomodulatory mediators involved in efficient T cell priming. Furthermore, MoDCs from allergic subjects stimulated in vitro with a mixture of gamma-PGA NPs and extract of grass pollen allergen Phleum pratense (Phl p) augment allergen-specific IL-10 production and proliferation of autologous CD4(+) memory T cells. Thus, gamma-PGA NPs are promising as sophisticated adjuvants and allergen-delivery systems in allergen-specific immunotherapy.

The GARD assay for assessment of chemical skin sensitizers

Allergic contact dermatitis is a skin disease caused by an immunologic reaction to low molecular weight compounds, so called haptens. These substances are commonly present in products used by humans in daily life, such as in cosmetics and fragrances, as well as within chemical industry and in pharmaceuticals. The frequent usage of these compounds in different applications has led to increasing incidences of allergic contact dermatitis, which has become a substantial economic burden for society. As a consequence, chemicals are routinely tested for their ability to induce skin sensitization, using animal models such as the murine Local Lymph Node Assay. However, recent legislations regulate the use of animal models within chemical testing. Thus, there is an urgent need for in vitro alternatives to replace these assays for safety assessment of chemicals. Recently, we identified a signature of predictive genes, which are differentially regulated in the human myeloid cell-line MUTZ-3 when stimulated with sensitizing compounds compared to non-sensitizing compounds. Based on these findings, we have formulated a test strategy for assessment of sensitizing compounds, called Genomic Allergen Rapid Detection, GARD. In this paper, we present a detailed method description of how the assay should be performed.

Transcriptional profiling of human skin-resident Langerhans cells and CD1a dermal dendritic cells: differential activation states suggest distinct functions

In human skin, two main populations of dendritic cells (DC) can be discriminated: dermal DC (DDC) and epidermal Langerhans cells (LC). Although extensively studied, most of the knowledge about DDC and LC phenotype and function is obtained from studying DDC and LC cultured in vitro or DDC and LC migrated from skin explants. These studies have left the exact relationship between steady‐state human LC and DDC unclear: in particular, whether CD1a+ DDC represent migrated LC or whether they constitute a separate subset. To gain further insight in the kinship between skin‐resident CD1a+ DDC and LC, we analyzed CD1a+ DDC and LC, isolated from steady‐state skin samples, by high‐density microarray analysis. Results show that the CD1a+ DDC specifically express markers associated with DDC phenotype, such as the macrophage mannose receptor, DC‐specific ICAM‐grabbing nonintegrin, the scavenger receptor CD36, coagulation factor XIIIa, and chemokine receptor CCR5, whereas LC specifically express Langerin, membrane ATPase (CD39), and CCR6, all hallmarks of the LC lineage. In addition, under steady‐state conditions, both DC subsets display a strikingly different activation status, indicative of distinct functional properties. CD1a+ DDC exhibit a more activated, proinflammatory, migratory, and T cell‐stimulatory profile, as compared with LC, whereas LC mainly express molecules involved in cell adhesion and DC retention in the epidermis. In conclusion, transcriptional profiling is consistent with the notion that CD1a+ DDC and LC represent two distinct DC subsets but also that under steady‐state conditions, CD1a+ DDC and epidermal LC represent opposites of the DC activation spectrum.

Research resource: transcriptome profiling of genes regulated by RXR and its permissive and nonpermissive partners in differentiating monocyte-derived dendritic cells.

Retinoid X receptors (RXRs) are heterodimerization partners for many nuclear receptors and also act as homodimers. Heterodimers formed by RXR and a nonpermissive partner, e.g. retinoic acid receptor (RAR) and vitamin D receptor (VDR), can be activated only by the agonist of the partner receptor. In contrast, heterodimers that contain permissive partners, e.g. liver X receptor (LXR) and peroxisome proliferator-activated receptor (PPAR), can be activated by agonists for either the partner receptor or RXR, raising the possibility of pleiotropic RXR signaling. However, it is not known to what extent the receptor’s activation results in triggering mechanisms dependent or independent of permissive heterodimers. In this study, we systematically and quantitatively characterized all probable RXR-signaling pathways in differentiating human monocyte-derived dendritic cells (Mo-DCs). Using pharmacological, microarray and quantitative RT-PCR techniques, we identified and characterized gene sets regulated by RXR agonists (LG100268 and 9-cis retinoic acid) and agonists for LXRs, PPARs, RARα, and VDR. Our results demonstrated that permissiveness was partially impaired in Mo-DCs, because a large number of genes regulated by PPAR or LXR agonists was not affected by RXR-specific agonists or was regulated to a lesser extent. As expected, we found that RXR agonists regulated only small portions of RARα or VDR targets. Importantly, we could identify and characterize PPAR- and LXR-independent pathways in Mo-DCs most likely mediated by RXR homodimers. These data suggested that RXR signaling in Mo-DCs was mediated via multiple permissive heterodimers and also by mechanism(s) independent of permissive heterodimers, and it was controlled in a cell-type and gene-specific manner.

CD27(-) CD4(+) memory T cells define a differentiated memory population at both the functional and transcriptional levels

The memory T‐cell population is a heterogeneous population, including both effector cells, which exert a direct secondary immune response, and resting or intermediate cells, which serve as a reservoir and exert a possible regulatory role. To further dissect the T‐cell memory population residing in the CD4+ CD45RO+ T‐cell pool, we studied the functional properties of memory populations identified by the CD27 marker. This marker clearly divides the memory population into two groups. One group consists of effector cells lacking CD27 and displaying a high antigen recall response. The other group consists of an intermediate memory population, displaying CD27. This latter group lacks an antigen recall response and requires costimulation for T‐cell receptor triggering. To evaluate the function of the CD27+ memory pool, we analysed the transcriptional profile, using high‐density microarray technology. These gene data strongly support the different functional profiles of CD27+ and CD27– memory populations, in terms of protein expression and the capacity to respond to antigen.

Expression of CD137 (4-1BB) on Human Follicular Dendritic Cells

Follicular dendritic cells (FDCs) are the antigen (Ag)‐trapping accessory cells of the germinal centres (GCs), essential for the development of humoral immune responses and memory. FDCs reside in the microenvironment of secondary lymphoid tissue where Ag‐activated B cells expand, and undergo isotype switching and affinity maturation prior to becoming memory B cells. In addition to delivering Ag, FDCs also provide potent nonspecific accessory signals to the B cells, which are important for the GC reaction. In this report, we show that human tonsilar FDCs express the costimulatory molecule CD137. Surface expression of CD137 on FDCs was confirmed by immunofluorescent labelling and fluorescence‐activated cell sorter analysis. CD137 was also highly expressed by the human cell line HK, which displays many characteristics of in vivo FDCs. The interaction between B cells and FDCs is essential for the GC reactions, and our finding suggests that CD137 plays a role in FDC‐regulated B‐cell responses.