Dendritic cells (DCs) are professional antigen presenting cells with the unique capacity to induce primary and secondary immune responses in vivo. Here, we show that DCs secrete antigen presenting vesicles, called exosomes, which express functional Major Histocompatibility Complex class I and class II, and T-cell costimulatory molecules. Tumor peptide-pulsed DC-derived exosomes prime specific cytotoxic T lymphocytes in vivo and eradicate or suppress growth of established murine tumors in a T cell-dependent manner. Exosome-based cell-free vaccines represent an alternative to DC adoptive therapy for suppressing tumor growth.

Toll-like receptor (TLR)3 recognizes dsRNA and transduces signals to activate NF-kappaB and IFN-beta promoter. Type I IFNs (IFN-alpha/beta) function as key cytokines in anti-viral host defense. Human fibroblasts express TLR3 on the cell surface, and anti-TLR3 mAb inhibits dsRNA-induced IFN-beta secretion by fibroblasts, suggesting that TLR3 acts on the cell surface to sense viral infection. In this study, we examined the expression and localization of human TLR3 in various DC subsets using anti-TLR3 mAb. In monocyte-derived immature dendritic cells (iDCs), TLR3 predominantly resided inside the cells but not on the cell surface. iDCs produced IL-12p70 and IFN-alpha and -beta in response to poly(I:C). Similar response was observed in iDCs treated with rotavirus-derived dsRNA. These responses could not be blocked by pretreatment of the cells with anti-TLR3 mAb. In CD11c(+) blood DCs, cytoplasmic retention of TLR3 was also observed as in monocyte-derived iDCs, again endorsing a different TLR3 distribution profile from fibroblasts. In precursor DC2, however, TLR3 could not be detected inside or outside the cells. Of note, there was a putative centrosomal protein that shared an epitope with TLR3 in myeloid DCs and precursor DC2, but not peripheral blood monocytes. Immunoelectron microscopic analysis revealed that TLR3, when stably expressed in the murine B cell line Ba/F3, was specifically accumulated in multivesicular bodies, a subcellular compartment situated in endocytic trafficking pathways. Thus, regulation and localization of TLR3 are different in each cell type, which may reflect participation of cell type-specific multiple pathways in antiviral IFN induction via TLR3.

Antigen processing in B lymphocytes entails initial binding of antigen to the surface Ig and internalization of the antigen into acidic compartments where the antigen is degraded, releasing peptides for binding to major histocompatibility complex class II molecules. Using subcellular fractionation techniques we show that functional, processed antigen-class II complexes capable of activating antigen-specific T cells in vitro are first formed in dense vesicles cosedimenting with lysosomes which are distinct from early endosomes and the bulk of late endosomes. With time, processed antigen-class II complexes appear in vesicles sedimenting with early endosomes and finally cofractionate with plasma membrane. A separate compartment is identified which contains major histocompatibility complex class II receptive to peptide binding but which does not have access to processed antigen in the B cell. These class II molecules are in the so-called "floppy" form in contrast to the class II molecules in the very dense vesicles which are in the "compact" form. These results demonstrate a correlation between the floppy and compact forms of class II molecules and their association with processed antigen and show that floppy and compact forms of class II reside in distinct and physically separable subcellular compartments.

The use of bacteria and bacterial extracts for immunotherapy has a checkered past. Recent developments in immunology reveal that these nonspecific immune activators actually work by triggering specific receptors that are expressed by subsets of immune cells. Identification of these receptors and the molecular signaling pathways that they activate has enabled a new era of specific targeted immunotherapy using chemically synthesized mimics of pathogen molecules.

IFN-gamma activates macrophages to kill diverse intracellular pathogens, but does not activate human macrophages to kill virulent Mycobacterium tuberculosis. We tested the hypothesis that this is due to inhibition of IFN-gamma signaling by M. tuberculosis and found that M. tuberculosis infection of human macrophages blocks several responses to IFN-gamma, including killing of Toxoplasma gondii and induction of FcgammaRI. The inhibitory effect of M. tuberculosis is directed at transcription of IFN-gamma-responsive genes, but does not affect proximal steps in the Janus kinase-STAT pathway, as STAT1alpha tyrosine and serine phosphorylation, dimerization, nuclear translocation, and DNA binding are intact in M. tuberculosis-infected cells. In contrast, there is a marked decrease in IFN-gamma-induced association of STAT1 with the transcriptional coactivators CREB binding protein and p300 in M. tuberculosis-infected macrophages, indicating that M. tuberculosis directly or indirectly disrupts this protein-protein interaction that is essential for transcriptional responses to IFN-gamma. Gamma-irradiated M. tuberculosis and isolated cell walls reproduce the effects of live bacteria, indicating that the bacterial component(s) that initiates inhibition of IFN-gamma responses is constitutively expressed. Although lipoarabinomannan has been found to exert effects on macrophages, it does not account for the inhibitory effects of cell walls. These results indicate that one mechanism for M. tuberculosis to evade the human immune response is to inhibit the IFN-gamma signaling pathway, and that the mechanism of inhibition is distinct from that reported for Leishmania donovani or CMV, in that it targets the interaction of STAT1 with the basal transcriptional apparatus.

Induction of apoptosis in cells by TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF family, is believed to be regulated by expression of two death-inducing and two inhibitory (decoy) receptors on the cell surface. In previous studies we found no correlation between expression of decoy receptors and susceptibility of human melanoma cells to TRAIL-induced apoptosis. In view of this, we studied the localization of the receptors in melanoma cells by confocal microscopy to better understand their function. We show that the death receptors TRAIL-R1 and R2 are located in the trans-Golgi network, whereas the inhibitory receptors TRAIL-R3 and -R4 are located in the nucleus. After exposure to TRAIL, TRAIL-R1 and -R2 are internalized into endosomes, whereas TRAIL-R3 and -R4 undergo relocation from the nucleus to the cytoplasm and cell membranes. This movement of decoy receptors was dependent on signals from TRAIL-R1 and -R2, as shown by blocking experiments with Abs to TRAIL-R1 and -R2. The location of TRAIL-R1,-R3, and -R4 in melanoma cells transfected with cDNA for these receptors was similar to that in nontransfected cells. Transfection of TRAIL-R3 and -R4 increased resistance of the melanoma lines to TRAIL-induced apoptosis even in melanoma lines that naturally expressed these receptors. These results indicate that abnormalities in "decoy" receptor location or function may contribute to sensitivity of melanoma to TRAIL-induced apoptosis and suggest that further studies are needed on the functional significance of their nuclear location and TRAIL-induced movement within cells.

A well conserved 83-kDa apical membrane antigen of Plasmodium falciparum, PF83/AMA-1, is the analogue of PK66/AMA-1, a 66-kDa P. knowlesi protective merozoite protein. PK66/AMA-1 is expressed in late-stage schizonts; is localized within the merozoite apex; and is processed to a 44/42-kDa doublet at, or around, the time of schizont rupture. The processed forms can associate with the merozoite surface. We were interested to further analyze the timing of synthesis and processing, and subcellular localization of PF83/AMA-1, a malaria vaccine candidate, using monoclonal antibodies (mAbs) developed against PF83/AMA-1. Using [35S]methionine metabolically labeled asexual blood stage parasites, in combination with indirect single and dual immunofluorescence, we have determined that, in similar fashion to PK66/AMA-1, protein expression of PF83/AMA-1 is restricted to late-stage schizonts with greater than 8 nuclei. PF83/AMA-1 is post-synthetically processed rapidly by cleavage of an N-terminal peptide to a 66-kDa molecule. Both the 83- and the 66-kDa molecules are initially localized at the merozoite apex. In P. falciparum (7G8 strain and CVD-1 clone) the full-length 83-kDa molecule remains apically restricted following merozoite release. However, the processed 66-kDa form can become circumferentially associated with the merozoite surface at or around the time of schizont rupture and merozoite release. After merozoite invasion a processed form of PF83/AMA-1 is present in early ring stage parasites. Comparative analysis of a rhoptry associated protein RAP-1, shows a co-ordinated and compartmentalized release of rhoptry components.