Data have been obtained defining the differential roles of intestinal lymph DC subsets in activation of naïve and memory T cells in rats. We have shown that two of the three DC subsets are highly potent activators of naïve CD4+ T cells, and do not appear to be constitutively suppressed in any way. We are currently examining the phenotype of the activated cells and determining if Tregs are induced. We have initiated (in collaboration with Pfizer) a study of nano-particles as potential deliverers of intestinal vaccines.

We have data available on some functional immune responses in a small animal model to study the development and function of the human hematopoietic and immune system as well as human specific infections in vivo. We established “human hemato-lymphoid-system mice” by transplanting human CD34+ cord blood cells into irradiated newborn Rag2-/-gc-/- mice, leading to de novo development of human B, T, and dendritic cells; formation of structured primary and secondary lymphoid organs; and production of some limited functional immune responses.

We obtained data from an evaluation whether antigens could be rerouted from the endosomal into the cytosolic compartment by combining antigen with cell penetrating peptides to further enhance cross-presentation. We made significant progress using biodegradable polymers to enhance cross-presentation of loaded antigens, and the best results were obtained by maturing DCs with a combination of the TLR ligands R848 and polyI:C. Furthermore, we have shown that DC electroporated with melanoma antigen RNA express the proteins ex vivo and in vivo.

Our laboratory obtained data from studies of DC phagosomes and cross-presentation using K/O and mutant mouse strains. Cross presentation is the process by which Dendritic cells (DC) phagocytose pathogens or dying cell fragments, and present proteolytic peptides derived from these antigens in association with MHC class I molecules. The reasons why DCs are the only antigen presenting cells that efficiently cross present antigens are not well understood. We have demonstrated that the NADPH oxidase NOX2 is recruited to DC early phagosomes mediating a sustained produc...

We obtained data on immune tolerance in mice breast-fed by antigen-exposed lactating mothers. We have previously generated a monoclonal antibody, 2C44, that reacted to a peptide derived from the Leishmania LACK protein bound to I-Ad MHC class II molecules. We have successfully used the 2C44 mAb to visualize LACK-presenting DCs in mice infected with the L. major. In 2007, we have attempted to identify the APCs that are responsible for the development of immune tolerance in mice that have been breast-fed by antigen-exposed lactating mothers. Recent experiments perf...

We have obtained data on the immuno-physiology of rat and mouse intestinal dendritic cells. We have used a known adjuvant, E. coli heat-labile toxin (Etx) and a potential adjuvant R-848, a small TLR7/8 ligand. We have shown that R-848 causes massive changes in DC migration and leads to their activation in lymph nodes but not lymph. These changes are due to TNF-alpha (migration) and Type 1 interferons (activation), but are not accompanied by obvious oral adjuvant effects. In contrast Etx causes only minor changes in migration and activation but is a potent oral adju...

Our lab gathered data on the responses to EBV and HIV in hu-SCID mice.

Data were obtained on responses to viral infections in TLR9 -/- mice, C57Bl/6 mice and IFNalpha receptor -/- mice. Toll receptor 9 (TLR9) is an important pattern recognition receptor. It is stimulated by foreign DNA or CpG motivs. We found that TLR9 -/- mice were very susceptible to infection with the mouse pox virus Ectromelia. In contrast to DC from C57Bl/6 mice, DC from TLR9 -/- produced little if any interferon alpha. However, MVA-BN induced IFNalpha in presence or absence of TLR9 -/-. Moreover, coinfection with MVA-BN and Ectromilia protected TLR9 -/- mice ...

Our lab obtained data on functional homogeneity from a gene expression analysis at single-cell level for the expression of 8 effector genes from normal donors. Until 2006 we subdivided CD8 PBL from normal donors in 14 different cell sets based in the expression levels and the co-expression of 5 different cell surface markers and studied functional homogeneity by screening cells from each subset at single-cell level for the expression of 8 effector genes (Blood, 2007). Although these studies allowed us isolate some homogeneous cell types, we yet found heterogenei...

We obtained data from a study of the correlation between CD8 multiple cell surface markers and functional profiles studied at single cell level. For that purpose, we subdivided peripheral CD8 T cells into eleven different cell subtypes based on the association of multiple cell surface markers. In each subtype, we isolated single-cells. In each single cell, we quantified the expression of multiple genes. Moreover, we isolated and studied cells from different normal donors. These results were published in Blood. We showed that - this strategy allowed the identifi...

We obtained data using specific inhibitors on the role of src-family tyrosine kinases in the maturation of human monocytes derived dendritic cells upon stimulation with several toll like receptor (TLR) agonists. The effect of these kinase inhibitors on the capacity of DC to be activated by a TLR2 (PAM3CSK4), TLR3 (Poly I:C), TLR5 (Flagellin), and TLR8 (Poly U) agonist was evaluated. Src kinases were found to be required for the initiation of some maturation characteristics but not of others, since their inhibition was able to dissociate cytokines and chemokines ...

Data has been obtained on the BRAF/NRAS mutation status and expression of COX-2 and iNOS comparing their prognostic value for overall survival in stage III malignant melanoma. New prognostic markers for malignant melanoma are urgently needed. Inducible nitric oxide synthase (iNOS) and cyclooxygenase type 2 (COX-2) have been described to correlate with progression of melanoma. Moreover, activating mutations in BRAF/NRAS oncogenes are often detected in melanoma. We have examined the BRAF/NRAS mutation status and expression of COX-2 and iNOS to compare their progno...

Collaborative work with P34 (Sica) has been started to study the effects of Japanin on DC exposed to hypoxia (below) and to perform transcriptional profiling of the treated cells. This work, which could not have been undertaken at this stage without support from DC-THERA, has now led to a joint publication in 2008 (Mancino et al.). Transcriptional data are available.

We obtained data on the effect of pro-inflammatory stroma-derived cytokines on DC in vivo. Our investigation has shown that, in the absence of additional stimuli, such inflammatory signals are ineffectual in promoting DC activation and cannot substitute for engagement of innate receptors directly on DC.

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...

We have obtained data from our work on biodegradable poly (D, L-lactide-co-glycolide) (PLGS) micropsheres (MS) coencapsulating ligands for endosomally expressed TLRs plus exogeneous Antigen (Ag) to deliver its cargo to endosomes of murine Dendritic cells (DCs), thus initiating TLR mediated DC maturation as well as processing of MHC class I and class II restricted epitopes. In these studies we used as Ag either Ovalbumin, or rec. Prion-protein. In the latter system, a CD4 T cell response could be initiated. We also found that the DNA sugar backbone determines TLR9...

Just a test

Among the battery of cell surface receptors expressed by DCs, the receptor for the Fc portion of IgG (FcgammaRs) recognizes the antibodies coupled to their specific antigens, called immune complexes (IC). We have data available from our demonstration that the target of FcgammaRs with the antigen induces the internalization of the IC, leading to the maturation of CD11c+CD11b+ conventional DCs. These DCs become thus efficient to present antigenic peptides to helper CD4 T cells and cytotoxic CD8 T cells via cross-presentation pathway. This capability might then enhan...

Concerning the pathway based analysis and database we have started the curation of the TLR4-CD14 pathway in DCs and we have described the events occurring at the DC membrane level. We have also started global gene expression analyses to better difine the role of NFAT in DCs.

During 2008, we extended all the data regarding TLR3 signaling to TLR8 signaling. Cytokines assays and immunoblotting studies suggest that Src kinases play a crucial role in the control of both MyD88- and TRIF-dependent pathways. We also extended microarray analysis on human MoDC stimulated with R848 (TLR8 agonist), pretreated or not with PP2. In summary, the new generated data on TLR8 triggered gene expression are similar to previous data obtained with TLR3 stimulation, and confirmed that src kinases inhibition is associated to inhibition of key genes in the infl...