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...

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.

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

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...

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 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...

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 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.

We obtained data on the influence of bLf, whose immunomodulant properties are now recognized, on MDDC differentiation/activation. We found that bLF interferes with the activation of MD-DC induced by different TLR agonists, but not by T cell mediated signals. Although bLF did not significantly influence monocyte differentiation in DCs, we found that it markedly reduced the up-modulation of CD83, co-stimulatory and MHC molecules (i.e. CD80, CD86, MHC class I and II), and cytokine secretion (IL-12, TNF-? and IL-23) induced by LPS or polyI-C. Consistently with an impa...

Our lab has obtained data on the role of p50 NF-kB in differentiation, survival and APC function in mice. Tumor growth is supported by tumor stroma, which is made by matrix and infiltrating cells, such as tumor associated macrophages (TAM) and tumor associated dendritic cells (TADC). We have recently reported that TAM display massive nuclear localization of the p50 NF-kB inhibitory homodimer, which correlates with impaired inflammatory functions (Saccani et al., 2006).The functional significance of this observation was demonstrated in p50 NF-kB deficient mice, whi...

Our laboratory has obtained data on the role of DC-derived cytokines in NK cell production of IFN-gamma.

Our lab has obtained data on the role of IL-10 and IDO in relation to Th1 and inflammatory responses in a colitis model tested on mice. IDO appears critical for the regulation of TNBS colitis upon CTLA-4 engagement, as the beneficial effect of anti-CTLA-4 treatment was lost in IDO-deficient mice. By contrast, the absence of IDO did not alter the course of inflammation in mice injected with TNBS only, an unexpected finding which suggests that IDO-dependent counter-regulation requires other factors/cells in addition to IDO production and T cell activation by TNBS. Ou...

Our lab has obtained data on the mechanism of how IL-10 regulates the immune response to MTb infection in mice. Control and clearance of intracellular pathogens such as MTb is dependent on the production of TNF and the induction of the T-helper 1 (Th1) cytokine IFN-gamma by IL-12. Conversely, IL-10 is a suppressive cytokine essential for dampening the immune response to a number of intracellular pathogens to limit host immune pathology. IL-10 has been shown to exert its suppressive effect by directly acting on the antigen presenting cell (APC), therefore functi...

Our lab has generated data on receptors on dendritic cells necessary for capture of exosomes. In 2007, we have finalized analysis of the receptors on DCs responsible for capture of exosomes secreted by mature DCs. We have shown that expression of LFA-1, but not of Mac-1, integrin, on DCs is required for capture of ICAM-1-bearing exosomes. Furthermore, the CD8+ subpopulation of DCs in lymph nodes expresses LFA-1 at higher levels than the CD8- subpopulation, and is responsible for in vivo capture of injected exosomes. We have thus proposed a new role for LFA-1 on DCs...

We have obtained valuable data on chemically well-defined TLR ligand conjugates and their effectiveness to induce a strong immune response via the cross-presentation pathway. We have studied long peptides which were covalently conjugated to either the TLR2 ligand or TLR9 ligand, Pam3CysSK4 or CpG, respectively have been used to study the uptake, antigen presentation, and induction of specific T-cells. A pronounced enhancement in antigen presentation by DC in vitro was facilitated by the conjugated peptides compared to peptide alone, or peptide mixed with the fre...

Preclinical data is available on the power of preventing disease and invoking immune response of our lead product for the prevention of infectious disease, ImmunoVexHSV2, a vaccine for genital herpes. ImmunoVex HSV2 is a novel live-attenuated vaccine candidate that expresses approximately 80 HSV-2 proteins intended to stimulate a broad and powerful immune response. In preclinical studies, ImmunoVex HSV2 completely prevents disease and invokes a powerful immune response. We intend to initiate a Phase I clinical trial for this product early in 2008.

Our lab has obtained data on maturation stimuli for GMP-grade DC.

Data has been generated by multiplex analysis on the response of human MoDC to yeast, spheroplasts, pseudohyphae and spores. Human blood contains myeloid (mDCs) and plasmacytoid (pDCs) dendritic cells that differ in cytokine secretion pattern and responsiveness to microbial stimuli. We found that LPS-stimulated mDCs are able to induce up-regulation of co-stimulatory molecules on co-cultured pDCs. Likewise, CpG-stimulated pDCs activate co-cultured mDCs. The cross talk between these two populations of DC is very efficient since it can be observed at mDC/pDC ratio ran...

Our lab obtained data on maturation of DC by TLR agonists.