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author:("Kuka, mirena")
1.  A method for high purity sorting of rare cell subsets applied to TDC 
Journal of immunological methods  2013;0:10.1016/j.jim.2013.10.002.
TDC are a recently described subset of polyclonal αβ T-cells with dendritic cell properties. Because of their low number in peripheral immune compartments, isolation and characterization of TDC with existing purification methods is technically challenging. Here we describe a customized gating strategy and a flow cytometry-based cell sorting protocol for isolation of TDC. The protocol was developed because, despite very conservative gating for dead-cell and doublet exclusion, cells obtained with normal sorting procedures were enriched for TDC but not pure. Re-sorting the output of the first round of sorting results in highly pure TDC. Cells obtained with this method are viable and can be used for in vitro characterization. Moreover, this double-round sorting strategy can be universally applied to the isolation of other rare cell subsets.
doi:10.1016/j.jim.2013.10.002
PMCID: PMC3866963  PMID: 24120843
Flow cytometry; Sorting; TDC; Purification
2.  CD70 is downregulated by interaction with CD27 
Engagement of the receptor CD27 by CD70 affects the magnitude and quality of T cell responses in a variety of infection models, and exaggerated signaling via this pathway results in enhanced immune responses and autoimmunity. One means by which signaling is regulated is tight control of cell surface CD70, which is expressed on dendritic, T, and B cells only upon activation. Here we show that there is a second level of regulation. First, although undetectable on the cell surface by flow cytometry, immature dendritic cells (DC) have a small pool of CD70 that continuously recycles from the plasma membrane. In addition, surface levels of CD70 on DC and T cells were higher in mice deficient in CD27, or on DC for which the interaction between CD70 and CD27 was precluded by blocking antibodies. Binding of CD70 by its receptor resulted in downregulation of CD70 transcription and protein levels, suggesting that CD70-mediated “reverse signals” regulate its own levels. Therefore, the ability of CD70 to trigger costimulation is self-regulated when it binds its complementary receptor.
doi:10.4049/jimmunol.1300868
PMCID: PMC3750068  PMID: 23913967
3.  CD70-deficiency impairs effector CD8 T cell generation and viral clearance but is dispensable for the recall response to LCMV 
CD27 interactions with its ligand, CD70, are thought to be necessary for optimal primary and memory adaptive immune responses to a variety of pathogens. Thus far all studies addressing the function of the CD27-CD70 axis have been performed either in mice lacking CD27, overexpressing CD70, or in which these receptors were blocked or mimicked by antibodies or recombinant soluble CD70. Because these methods have in some cases led to divergent results, we generated CD70-deficient mice to directly assess its role in vivo. We find that lack of CD70-mediated stimulation during primary responses to LCMV lowered the magnitude of CD8 antigen-specific T cell response, resulting in impaired viral clearance, without affecting CD4 T cell responses. Unexpectedly, CD70-CD27 costimulation was not needed for memory CD8 T cell generation or the ability to mount a recall response to LCMV. Adoptive transfers of wild type (WT) memory T cells into CD70−/− or WT hosts also showed no need for CD70-mediated stimulation during the course of the recall response. Moreover, CD70-expression by CD8 T cells could not rescue endogenous CD70−/− cells from defective expansion, arguing against a role for CD70-mediated T:T help in this model. Therefore, CD70 appears to be an important factor in the initiation of a robust and effective primary response but dispensable for CD8 T cell memory responses.
doi:10.4049/jimmunol.1202353
PMCID: PMC3552005  PMID: 23269247
4.  Identification and characterization of polyclonal αβ T cells with dendritic cell properties 
Nature communications  2012;3:1223.
An efficient immune response requires coordination between innate and adaptive immunity, which act through cells different in origin and function. Here we report the identification of thymus-derived αβ TCR+ cells that express CD11c and MHC class II, and require FLT3L for development (TDC). TDC express genes heretofore found uniquely in T cells or DC, as well as a distinctive signature of cytotoxicity-related genes. Unlike other innate T cell subsets, TDC have a polyclonal TCR repertoire andrespond to cognate antigens. However, they differ from conventional T cells in that they do not require help from antigen-presenting cells, respond to TLR-mediated stimulation by producing IL-12 and process and present antigen. The physiologic relevance of TDC, found in mice and humans, is still under investigation, but the fact that they combine key features of T and DC cells suggests that they provide a bridge between the innate and adaptive immune systems.
doi:10.1038/ncomms2223
PMCID: PMC3528357  PMID: 23187623
5.  DC-ATLAS: a systems biology resource to dissect receptor specific signal transduction in dendritic cells 
Immunome Research  2010;6:10.
Background
The advent of Systems Biology has been accompanied by the blooming of pathway databases. Currently pathways are defined generically with respect to the organ or cell type where a reaction takes place. The cell type specificity of the reactions is the foundation of immunological research, and capturing this specificity is of paramount importance when using pathway-based analyses to decipher complex immunological datasets. Here, we present DC-ATLAS, a novel and versatile resource for the interpretation of high-throughput data generated perturbing the signaling network of dendritic cells (DCs).
Results
Pathways are annotated using a novel data model, the Biological Connection Markup Language (BCML), a SBGN-compliant data format developed to store the large amount of information collected. The application of DC-ATLAS to pathway-based analysis of the transcriptional program of DCs stimulated with agonists of the toll-like receptor family allows an integrated description of the flow of information from the cellular sensors to the functional outcome, capturing the temporal series of activation events by grouping sets of reactions that occur at different time points in well-defined functional modules.
Conclusions
The initiative significantly improves our understanding of DC biology and regulatory networks. Developing a systems biology approach for immune system holds the promise of translating knowledge on the immune system into more successful immunotherapy strategies.
doi:10.1186/1745-7580-6-10
PMCID: PMC3000836  PMID: 21092113
6.  Src Kinases Are Required for a Balanced Production of IL-12/IL-23 in Human Dendritic Cells Activated by Toll-Like Receptor Agonists 
PLoS ONE  2010;5(7):e11491.
Background
Pathogen recognition by dendritic cells (DC) is crucial for the initiation of both innate and adaptive immune responses. Activation of Toll-like Receptors (TLRs) by microbial molecular patterns leads to the maturation of DC, which present the antigen and activate T cells in secondary lymphoid tissues. Cytokine production by DC is critical for shaping the adaptive immune response by regulating T helper cell differentiation. It was previously shown by our group that Src kinases play a key role in cytokines production during TLR4 activation in human DC.
Principal Findings
In this work we investigated the role of Src kinases during different TLRs triggering in human monocyte-derived DC (MoDC). We found that Src family kinases are important for a balanced production of inflammatory cytokines by human MoDC upon stimulation of TLR3 and 8 with their respective agonists. Disruption of this equilibrium through pharmacological inhibition of Src kinases alters the DC maturation pattern. In particular, while expression of IL-12 and other inflammatory cytokines depend on Src kinases, the induction of IL-23 and co-stimulatory molecules do not. Accordingly, DC treated with Src inhibitors are not compromised in their ability to induce CD4 T cell proliferation and to promote the Th17 subset survival but are less efficient in inducing Th1 differentiation.
Conclusions
We suggest that the pharmacological modulation of DC maturation has the potential to shape the quality of the adaptive immune response and could be exploited for the treatment of inflammation-related diseases.
doi:10.1371/journal.pone.0011491
PMCID: PMC2901334  PMID: 20634889

Results 1-6 (6)