Antigen storage compartments in mature dendritic cells facilitate prolonged cytotoxic T lymphocyte cross-priming capacity.

Dendritic cells (DCs) are crucial for priming of naive CD8(+) T lymphocytes to exogenous antigens, so-called "cross-priming." We report that exogenous protein antigen can be conserved for several days in mature DCs, coinciding with strong cytotoxic T lymphocyte cross-priming potency in vivo. After MHC class I peptide elution, protein antigen-derived peptide presentation is efficiently restored, indicating the presence of an intracellular antigen depot. We characterized this depot as a lysosome-like organelle, distinct from MHC class II compartments and recently described early endosomal compartments that allow acute antigen presentation in MHC class I. The storage compartments we report here facilitate continuous supply of MHC class I ligands. This mechanism ensures sustained cross-presentation by DCs, despite the short-lived expression of MHC class I-peptide complexes at the cell surface.

Slow endocytosis of the LDL receptor-related protein 1B: implications for a novel cytoplasmic tail conformation.

The LDL receptor-related protein 1B (LRP1B) is a putative tumor suppressor homologous to LRP1. Both LRP1 and LRP1B contain cytoplasmic tails with several potential endocytosis motifs. Although the positions of these endocytic motifs are similar in both receptors, LRP1B is internalized at a 15-fold slower rate than LRP1. To determine whether the slow endocytosis of LRP1B is due to the utilization of an endocytosis motif other than the YATL motif used by LRP1, we tested minireceptors with mutations in each of the five potential motifs in the LRP1B tail. Only mutation of both NPXY motifs together abolished LRP1B endocytosis, suggesting that LRP1B can use either of these motifs for internalization. LRP1B contains a unique insertion of 33 amino acids not present in LRP1 that could lead to altered recognition of trafficking motifs. Surprisingly, deletion of this insertion had no effect on the endocytosis rate of LRP1B. However, replacing either half of the LRP1B tail with the corresponding LRP1 sequence markedly accelerated LRP1B endocytosis. From these data, we propose that both halves of the LRP1B cytoplasmic tail contribute to a unique global conformation, which results in less efficient recognition by endocytic adaptors and a slow endocytosis rate.

Innate immune recognition triggers secretion of lysosomal enzymes by macrophages.

Gamma interferon-induced lysosomal thiolreductase (GILT) is expressed constitutively in antigen-presenting cells, where it reduces disulfide bonds to facilitate antigen presentation. GILT is synthesized as an enzymatically active precursor protein and is processed in early endosomes to yield the mature enzyme. The exposure of the promonocytic cell line THP-1 to Escherichia coli causes a differentiation-dependent induction of GILT expression in which the majority of precursor GILT is secreted as active enzyme. We confirm this result in cultured primary monocytes and macrophages, and demonstrate, as an in vivo correlate of the phenomenon, upregulation of precursor GILT levels in the serum of mice injected with lipopolysaccharide. We show that macrophage differentiation is accompanied by a transcriptional downregulation of mannose-6-phosphorylation, which likely prevents the recognition and proper sorting of soluble lysosomal enzymes by the mannose-6-phosphate receptors. We provide evidence for a mechanism of generalized soluble lysosomal enzyme secretion through the constitutive secretory pathway.

Human mast cells produce and release the cytotoxic lymphocyte associated protease granzyme B upon activation.

Mast cells are widely distributed throughout the body and express effector functions in allergic reactions, inflammatory diseases, and host defense. Activation of mast cells results in exocytosis of preformed chemical mediators and leads to novel synthesis and secretion of lipid mediators and cytokines. Here, we show that human mast cells also express and release the cytotoxic lymphocyte-associated protease, granzyme B. Granzyme B was active and localized in cytoplasmic granules, morphologically resembling those present in cytotoxic lymphocytes. Expression and release of granzyme B by mast cell-lines HMC-1 and LAD 2 and by cord blood- and mature skin-derived human mast cells depended on the mode of activation of these cells. In mast cell lines and cord blood-derived mast cells, granzyme B expression was mainly induced by non-physiological stimuli (A23187/PMA, Compound 48/80) and substance P. In contrast, mature skin-derived mast cells only produced granzyme B upon IgE-dependent stimulation. We conclude that granzyme B is expressed and released by human mast cells upon physiologic stimulation. This suggests a role for granzyme B as a novel mediator in mast cell biology.

Characterization of the TGN exit signal of the human mannose 6-phosphate uncovering enzyme.

The human mannose 6-phosphate uncovering enzyme participates in the uncovering of the mannose 6-phosphate recognition tag on lysosomal enzymes, a process that facilitates recognition of those enzymes by mannose 6-phosphate receptors to ensure delivery to lysosomes. Uncovering enzyme has been identified on the trans-Golgi network at steady state. It has been shown to traffic to the plasma membrane from where it is rapidly internalized via endosomal structures, the process being mediated by a tyrosine-based internalization motif, Y488HPL, in its cytoplasmic tail. Using immunogold electron microscopy a GFP-uncovering enzyme fusion construct was found to be colocalized with the cation-dependent mannose 6-phosphate receptor in regions of the trans-Golgi network, suggesting that uncovering enzyme might follow a similar pathway of exit from the trans-Golgi network as that of the cation-dependent mannose 6-phosphate receptor. In this study, we identified the signal sequence in the cytoplasmic tail of uncovering enzyme responsible for its exit from the trans-Golgi network. Using GFP fusion constructs of the transmembrane and cytoplasmic domains of uncovering enzyme, we could show, by automated analysis of confocal immunofluorescence images, that residues Q492EMN in the cytoplasmic tail of uncovering enzyme are involved in its exit from the trans-Golgi network. Detailed characterization of the exit signal revealed that residue Q492 is the most important to the exit function while M494 and N495 also contribute. The cytoplasmic tail of the uncovering enzyme does not possess any of the known canonical signal sequences for interaction with Golgi-associated gamma ear-containing adaptor proteins. The identification of a trans-Golgi network exit signal in its cytoplasmic tail elucidates the trafficking pathway of uncovering enzyme, a crucial player in the process of lysosomal biogenesis.

Cathepsin S controls MHC class II-mediated antigen presentation by epithelial cells in vivo.

Epithelial cells at environmental interfaces provide protection from potentially harmful agents, including pathogens. In addition to serving as a physical barrier and producing soluble mediators of immunity, such as cytokines or antimicrobial peptides, these cells are thought to function as nonprofessional APCs. In this regard, intestinal epithelial cells are particularly prominent because they express MHC class II molecules at the site of massive antigenic exposure. However, unlike bone marrow-derived professional APC, such as dendritic cells or B cells, little is known about the mechanisms of MHC class II presentation by the nonprofessional APC in vivo. The former use the lysosomal cysteine protease cathepsin S (Cat S), whereas thymic cortical epithelial cells use cathepsin L (Cat L) for invariant chain degradation and MHC class II maturation. Unexpectedly, we found that murine Cat S plays a critical role in invariant chain degradation in intestinal epithelial cells. Furthermore, we report that nonprofessional APC present a class II-bound endogenous peptide to naive CD4 T cells in vivo in a Cat S-dependent fashion. These results suggest that in vivo, both professional and nonprofessional MHC class II-expressing APC use Cat S, but not Cat L, for MHC class II-mediated Ag presentation.

3-D Structure of multilaminar lysosomes in antigen presenting cells reveals trapping of MHC II on the internal membranes.

In late endosomes and lysosomes of antigen presenting cells major histocompatibility complex class II (MHC II) molecules bind peptides from degraded internalized pathogens. These compartments are called MHC class II compartments (MIICs), and from here peptide-loaded MHC II is transported to the cell surface for presentation to helper T-lymphocytes to generate an immune response. Recent studies from our group in mouse dendritic cells indicate that the MHC class II on internal vesicles of multivesicular late endosomes or multivesicular bodies is the main source of MHC II at the plasma membrane. We showed that dendritic cell activation triggers a back fusion mechanism whereby MHC II from the inner membranes is delivered to the multivesicular bodies' outer membrane. Another type of MIIC in B-lymphocytes and dendritic cells is more related to lysosomes and often appears as a multilaminar organelle with abundant MHC II-enriched internal membrane sheets. These multilaminar lysosomes have a functioning peptide-loading machinery, but to date it is not clear whether peptide-loaded MHC II molecules from the internal membranes can make their way to the cell surface and contribute to T cell activation. To obtain detailed information on the membrane organization of multilaminar lysosomes and investigate possible escape routes from the lumen of this organelle, we performed electron tomography on cryo-immobilized B-lymphocytes and dendritic cells. Our high-resolution 3-D reconstructions of multilaminar lysosomes indicate that their membranes are organized in such a way that MHC class II may be trapped on the inner membranes, without the possibility to escape to the cell surface.

Involvement of the endoplasmic reticulum in peroxisome formation.

The traditional view holds that peroxisomes are autonomous organelles multiplying by growth and division. More recently, new observations have challenged this concept. Herein, we present evidence supporting the involvement of the endoplasmic reticulum (ER) in peroxisome formation by electron microscopy, immunocytochemistry and three-dimensional image reconstruction of peroxisomes and associated compartments in mouse dendritic cells. We found the peroxisomal membrane protein Pex13p and the ATP-binding cassette transporter protein PMP70 present in specialized subdomains of the ER that were continuous with a peroxisomal reticulum from which mature peroxisomes arose. The matrix proteins catalase and thiolase were only detectable in the reticula and peroxisomes. Our results suggest the existence of a maturation pathway from the ER to peroxisomes and implicate the ER as a major source from which the peroxisomal membrane is derived.

A reliable and convenient method to store ultrathin thawed cryosections prior to immunolabeling.

Ultracryotomy of fixed specimens in combination with immunogold labeling is widely used for ultrastructural localization of many interesting molecules. Since the introduction of this technique, vast improvements in techniques and machinery have been established and the entire process has been made easier and more accessible. Normally, sections are cut and labeled within 1 day to prevent possible loss or redistribution of soluble antigens within the sections. An increasing demand for more sections and multiple labeling protocols prompted us to investigate the extent to which ultrathin cryosections can be stored. This would render the time spent behind an ultracryomicrotome more efficient and would allow immunogold labeling at a later stage. We investigated whether gelatin plates, 2.3 M sucrose, or 1.0% methyl cellulose/1.2 M sucrose can be used to store thawed frozen sections for a longer period of time. Ultrathin sections of mildly fixed tissue and cultured cells were stored for up to 6 months before immunogold labeling. The preservation of the ultrastructure of stored sections was excellent and was similar to that of immediately processed sections. Importantly, prolonged storage did not affect the labeling intensity.