We have used fluorescence microscopy to analyse the endocytic pathway in DC and to track C-type lectins that are internalised from the cell surface. From these analyses, there is data available. As part of this work, we have used microscopy to study the effect of Dectin-1 uptake on its ability to signal for downstream responses. Finally, we have shown that fluorescently-labelled antibodies against a novel C-type lectin, DNGR-1, can be used to mark DC upon injection into mice and might therefore be useful for in vivo imaging studies. Microscopy will continue to b...

Our group has obtained imaging data on the role of DALIS and related aggregates in antigen presentation and their interaction with the regulation of translation in response to pathogens. We have implemented in collaboration with lead coordinator Carl Figdor, the organization of the imaging core-facility of the DC-THERA NoE. Experimentally, we have been applying confocal microscopy to the study of DALIS and related aggregates in order to understand their role in antigen presentation and their interaction with the regulation of translation in response to pathogens....

The group has data available on a newly identified DC subset called IKDC producing IFNg and expressing TRAIL which can recognize and kill transformed tumor cells in vitro and in vivo. They performed a video-confocal microscopy overnight on cultures of tumor cells with IKDC. We visualized that IKDC generate long contacts and synapses with tumor cells and ultimately kill them within 4 hours.

We have obtained imaging data by confocal microscopy of translation activation in DC-activated T cells and have pursued our efforts on the definition of a novel way of monitoring DC and T cell activation as well as studying viral infections. We also have now applied the puromycin-based technology developed in our laboratory (SUnSET, surface sensing of translation) to monitor translation by FACs in individual or cell populations. We have demonstrated that translation activation is an excellent read-out for early antigen specific T cell activation by DCs. We hope tha...

Data was obtained on the infiltration and destruction of solid tumors by cytotoxic T lymphocytes (CTLs). Although the immune system evolved to fight infections, it may also attack and destroy solid tumors. In most cases, tumor rejection is initiated by CD8+ CTLs, which infiltrate solid tumors, recognize tumor antigens and kill tumor cells. We used a combination of two-photon intravital microscopy and immunofluorescence on ordered sequential sections to analyze the infiltration and destruction of solid tumors by CTLs. We show that in the periphery of a thymoma gr...

Data is available from an evaluation of different stimulation conditions for the cytometric assessment of antigen-specific Th-cells based on CD154 expression induced during short-term in vitro activation, a method established by us during previous DC-THERA activities. This is important and essential for implementation of the technology into clinical immune monitoring in e.g. vaccination trials. Moreover, the affect of stimulation length was determined. Differences with respect to the induced frequencies of antigen-specific, activated CD154+ Th-cells were minor. I...

Flow cytometric analysis data were obtained of translation activation in DC-activated T cells.

We have obtained data by fluorescence microscopy analysing DC distribution in spleen and how this is affected during inflammation driven by stromal cells or hematopoietic cells. One of our images of DC was selected as the cover for the June 2007 issue of Nature Immunology.

Our group has MRI data available on the migration of DCs in vivo in melanoma patients. From isolated lymphnodes obtained after surgery we could identify single DC after staining with Prussian blue for iron. We are in the process of analysing the T cell rosettes (activation stage) that surround these DC to get insight in the functional activity. It was also demonstrated that DCs transfected with RNA produce derived proteins in vivo in the lymph nodes. Also efforts were spent to get further information on the dynamics of immature and mature DCs. We discovered (van...

There is data available on translation during DC/T cell interaction in presence or absence of cognate antigenic peptides. Efforts on the definition of a novel way of monitoring DC and T cell activation as well as studying viral infections were finalized. The puromycin-based technology (SUnSET, surface sensing of translation) was applied to monitor translation by FACs during DC/T cell interaction in presence or absence of cognate antigenic peptides. It was demonstrated that translation activation is an excellent read-out for early antigen specific T cell activati...

Multi-photon imaging data on DC-CD8+ T cell interactions in intact lymph nodes (LNs) have been obtained. The initiation of cytotoxic immune responses requires the direct interaction between naïve CD8+ T lymphocytes and dendritic cells. Multi-photon imaging showed that during priming, naïve T cells and dendritic cells (DCs) establish sequentially brief (i.e. minutes) and long (hours) antigen-specific contacts. We observed that ICAM-1 expression by mature DCs is critical for long-lasting contacts with CD8+ T cells, but dispensable for short-lived antigen-specific i...

Our group has produced in-vivo multi-photon microscopy imaging data on the distribution and motility of NK cells by tracking migration of DC-primed NK cells in vivo. Since LPS-activated DC are potent inducers of NK cell priming and lymph nodes appear to be a key place in which DC and NK cell interactions occur, we have investigated the in vivo capacity of resting and DC-primed NK cells to reach the draining lymph nodes. DC –activated NK cells reach the draining lymph node within 24 hours after intravenous injection and persist there for at least 72 hours. NK cell...

Data was obtained on the regulatory T cells (Tregs) and their role in effective anti-tumor immune responses in FoxP3-DTR DEREG mice. Although multiple mechanisms of Treg action have been proposed, the actual mechanism involved in the suppression of anti tumor T cell responses are still unclear. Treg inactivation by anti-CD25 antibodies or by depletion in FoxP3-DTR DEREG mice resulted in effective rejection of the tumors upon adoptive transfer of antigen-specific CD8+ T cells. Treg inactivation or depletion induces a marked arrest in the migration of anti-tumor C...

DC-THERA applicant : Anita Sapoznikov Institution: The Weizmann Institute of Science, Israel Supervisor: Dr. Steffen Jung Data has been obtained on the cellular organization of the marrow of long bones. The bone marrow (BM) is a primary lymphoid organ, where diverse hematopoietic lineages arise from common multi-potential progenitors [1]. However, aside from this established role in hematopoiesis, the BM also serves as secondary lymphoid organ in the generation of adaptive T and B cell immune responses [2,3]. It has been shown that the BM is also populated by m...

Imaging data is available from MRI tracking of dendritic cells in melanoma patients for cellular therapy. In vivo magnetic resonance tracking of magnetically labeled cells is feasible in humans for detecting very low numbers of dendritic cells in conjunction with detailed anatomical information. MRI cell tracking using iron oxides appears clinically safe and well suited to monitor cellular therapy in humans.