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Toll-like receptors (TLRs) can recognize microbial patterns and utilize adaptor molecules, such as-MyD88 or (TRIF TIR-domain-containing adapter-inducing interferon-β), to initiate downstream signaling that ultimately affects the initiation of adaptive immunity. In addition to this inflammatory role, TLR5 expression on dendritic cells can favor antigen presentation of flagellin peptides and thus increase the sensitivity of flagellin-specific T-cell responses in vitro and in vivo. Here, we examined the role of alternative signaling pathways that might regulate flagellin antigen presentation in addition to MyD88. These studies suggest a requirement for spleen tyrosine kinase, a noncanonical TLR-signaling adaptor molecule, and its downstream molecule CARD9 in regulating the sensitivity of flagellin-specific CD4(+) T-cell responses in vitro and in vivo. Thus, a previously unappreciated signaling pathway plays an important role in regulating the dominance of flagellin-specific T-cell responses., (© 2014 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Peptides presented by MHC class I molecules are mostly derived from proteins synthesized by the antigen-presenting cell itself, while peptides presented by MHC class II molecules are predominantly from materials acquired by endocytosis. External antigens can also be presented by MHC class I molecules in a process referred to as cross-presentation. Here, we report that mouse dendritic cell (DC) engagement to a phagocytic target alters endocytic processing and inhibits the proteolytic activities. During phagocytosis, endosome maturation is delayed, shows less progression toward the lysosome, and the endocytosed soluble antigen is targeted for MHC class I cross-presentation. The antigen processing in these arrested endosomes is under the control of NAPDH oxidase associated ROS. We also show that cathepsin S is responsible for the generation of the MHC class I epitope. Taken together, our results suggest that in addition to solid structure uptake, DC phagocytosis simultaneously modifies the kinetics of endosomal trafficking and maturation. As a consequence, external soluble antigens are targeted into the MHC class I cross-presentation pathway., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Phospholipases catalyze the cleavage of membrane phospholipids into smaller bioactive molecules. The lysosomal phospholipase A2 (LPLA2 ) is specifically expressed in macrophages. LPLA2 gene deletion in mice causes lysosomal phospholipid accumulation in tissue macrophages leading to phospholipidosis. This phenotype becomes most prominent in alveolar macrophages where LPLA2 contributes to surfactant phospholipid degradation. High expression of LPLA2 in alveolar macrophages prompted us to investigate its role in host immunity against the respiratory pathogen Mycobacterium tuberculosis, the causative agent of tuberculosis. Here we report that adaptive immune responses to M. tuberculosis were impaired in LPLA2 deficient mice. Upon aerosol infection with M. tuberculosis, LPLA2 deficient mice showed enhanced mycobacterial counts but less lung immunopathology and pulmonary inflammatory responses. Compromised T-cell priming in the lymph nodes was associated with impaired pulmonary T-cell recruitment and activation. Together with reduced Th1 type cytokine production, these results indicate that LPLA2 is indispensable for the induction of adaptive T-cell immunity to M. tuberculosis. Taken together, we identified an unexpected and novel function of a lysosomal phospholipid-degrading enzyme., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Integrin trafficking, including internalization, recycling, and lysosomal degradation, is crucial for the regulation of cellular functions. Exosomes, nano-sized extracellular vesicles, are believed to play important roles in intercellular communications. This study demonstrates that exosomes released from human macrophages negatively regulate endothelial cell migration through control of integrin trafficking. Macrophage-derived exosomes promote internalization of integrin β1 in primary HUVECs. The internalized integrin β1 persistently accumulates in the perinuclear region and is not recycled back to the plasma membrane. Experimental results indicate that macrophage-derived exosomes stimulate trafficking of internalized integrin β1 to lysosomal compartments with a corresponding decrease in the integrin destined for recycling endosomes, resulting in proteolytic degradation of the integrin. Moreover, ubiquitination of HUVEC integrin β1 is enhanced by the exosomes, and exosome-mediated integrin degradation is blocked by bafilomycin A, a lysosomal degradation inhibitor. Macrophage-derived exosomes were also shown to effectively suppress collagen-induced activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway and HUVEC migration, which are both dependent on integrin β1. These observations provide new insight into the functional significance of exosomes in the regulation of integrin trafficking., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this study, we report that cytoplasmic granules from in vivo and in vitro derived mouse mast cells (MCs) contain active granzyme B (gzmB) and caspase-3, which is consistent with recent findings. Studying WT and gzmB-deficient mice, we observed that BM-derived MCs (BMMCs) from both strains contain cytosolic pro-caspase-3, but only WT BMMCs expressed active caspase-3 limited to their secretory lysosomes. Confocal microscopy revealed colocalization of active caspase-3 and gzmB in these cytoplasmic granules. The combined data demonstrate that the generation and storage of active caspase-3 is gzmB-dependent. The finding that BMMCs secrete caspase-3 and gzmB after Ag stimulation suggests that both proteases contribute to extracellular MC-mediated proteolytic events. Although the extracellular function of MC-derived caspase-3 remains unclear, we show that BMMC-secreted caspase-3 cleaves IL-33, a cytokine that contributes to the development of asthma and arthritis. We also show that an in vitro propagated cytolytic T-lymphocyte line constitutively expresses gzmB together with active caspase-3, suggesting a novel interaction of these proteases in the execution of multiple innate and adaptive immune responses., (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.)

Legionella pneumophila (L. pneumophila) is an intracellular bacterium of human alveolar macrophages that causes Legionnaires' disease. In contrast to humans, most inbred mouse strains are restrictive to L. pneumophila replication. We demonstrate that autophagy targets L. pneumophila vacuoles to lysosomes and that this process requires ubiquitination of L. pneumophila vacuoles and the subsequent binding of the autophagic adaptor p62/SQSTM1 to ubiquitinated vacuoles. The L. pneumophila legA9 encodes for an ankyrin-containing protein with unknown role. We show that the legA9 mutant replicate in WT mice and their bone marrow-derived macrophages. This is the first L. pneumophila mutant to be found to replicate in WT bone marrow-derived macrophages other than the Fla mutant. Less legA9 mutant-containing vacuoles acquired ubiquitin labeling and p62/SQSTM1 staining, evading autophagy uptake and avoiding lysosomal fusion. Thus, we describe a bacterial protein that targets the L. pneumophila-containing vacuole for autophagy uptake., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Macrophages provide a first line of defense against bacterial infection by engulfing and killing invading bacteria, but intracellular bacteria such as Listeria monocytogenes (LM) can survive in macrophages by various mechanisms of evasion. Complement receptor of the immunoglobulin (CRIg), a C3b receptor, binds to C3b on opsonized bacteria and facilitates clearance of the bacteria by promoting their uptake. We found that CRIg signaling induced by agonistic anti-CRIg mAb enhanced the killing of intracellular LM by macrophages, and that this occurred in LM-containing phagosomes. Chloride intra-cellular channel 3 CLIC3, an intracellular chloride channel protein, was essential for CRIg-mediated LM killing by directly interacting with the cytoplasmic domain of CRIg, and the two proteins colocalized on the membranes of LM-containing vacuoles. CLIC3(-/-) mice were as susceptible to LM as CRIg(-/-) mice. These findings identify a mechanism embedded in the process by which macrophages take up opsonized bacteria that prevents the bacteria from evading cell-mediated killing., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Invariant natural killer T (iNKT) cells are a specialised subset of T cells that are restricted to the MHC class I like molecule, CD1d. The ligands for iNKT cells are lipids, with the canonical superagonist being α-galactosylceramide, a non-mammalian glycosphingolipid. Trafficking of CD1d through the lysosome is required for the development of murine iNKT cells. Niemann-Pick type C (NPC) disease is a lysosomal storage disorder caused by dysfunction in either of two lysosomal proteins, NPC1 or NPC2, resulting in the storage of multiple lipids, including glycosphingolipids. In the NPC1 mouse model, iNKT cells are virtually undetectable, which is likely due to the inability of CD1d to be loaded with the selecting ligand due to defective lysosomal function and/or CD1d trafficking. However, in this study we have found that in NPC1 patients iNKT cells are present at normal frequencies, with no phenotypic or functional differences. In addi-tion, antigen-presenting cells derived from NPC1 patients are functionally competent to present several different CD1d/iNKT-cell ligands. This further supports the hypothesis that there are different trafficking requirements for the development of murine and human iNKT cells, and a functional lysosomal/late-endosomal compartment is not required for human iNKT-cell development., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Intracellular pathogen-specific antibodies (Abs) can contribute to host protection by a number of different mechanisms. Ab opsonization of pathogens residing outside a host cell can prevent infection of target cells either via neutralization of the critical surface epitopes required for host cell entry, complement-mediated degradation, or via subsequent intracellular degradation. In the case of intracellular localization, Abs can bind to infected cells and thus mark them for destruction by Fc receptor (FcR)-bearing effector cells. This review focuses on the protective role of Abs against intracellular bacteria and parasites involving FcR interactions that modulate the intracellular trafficking of the pathogen, the ability of FcRs to interfere with the establishment of an intracellular replicative niche and the involvement of FcRs to modulate pathogen-specific T-cell responses., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The essential roles of the endovacuolar system in health and disease call for the development of new tools allowing a better understanding of the complex molecular machinery involved in endocytic processes. We took advantage of the floating properties of small latex beads (sLB) on a discontinuous sucrose gradient to isolate highly purified endosomes following internalization of small latex beads in J774 macrophages and bone marrow-derived dendritic cells (DC). We particularly focused on the isolation of macrophages early endosomes and late endosomes/lysosomes (LE/LYS) as well as the isolation of LE/LYS from immature and lipopolysaccharide-activated (mature) DC. We subsequently performed a comparative analysis of their respective protein contents by MS. As expected, proteins already known to localize to the early endosomes were enriched in the earliest fraction of J774 endosomes, while proteins known to accumulate later in the process, such as hydrolases, were significantly enriched in the LE/LYS preparations. We next compared the LE/LYS protein contents of immature DC and mature DC, which are known to undergo massive reorganization leading to potent immune activation. The differences between the protein contents of endocytic organelles from macrophages and DC were underlined by focusing on previously poorly characterized biochemical pathways, which could have an unexpected but important role in the endosomal functions of these highly relevant immune cell types., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Inflammasomes are cytosolic multi-protein complexes that form in response to infectious or injurious challenges. Inflammasomes control the activity of caspase-1, which is essential for the maturation and release of IL-1beta family cytokines. The NLRP1, IPAF and AIM2 inflammasomes recognize specific substances, while the NLRP3 inflammasome responds to many structurally and chemically diverse triggers. Here, we discuss the critical roles of priming and lysosomal damage in NLRP3 inflammasome activation.

Human plasmacytoid dendritic cells (PDC) are believed to link innate and adaptive immunity by producing type I interferon (IFN-I) and triggering adaptive T cell-mediated immunity. However, it remains elusive to which degree both PDC functions are linked. Here we show that CMV antigen targeted to PDC using a CD303 (blood dendritic cell antigen 2, BDCA-2) mAb is rapidly endocytosed and traffics via early sorting endosomes to emerging MHC-enriched compartments. Both processes occur independently of TLR ligand stimulation. Restimulation of CMV-specific CD4(+) effector-memory T helper cells by autologous PDC and induction of IFN-I production in PDC are dependent on appropriate stimulation. Type B CpG oligonucleotide (CpG-B)-stimulated PDC efficiently process and present CMV antigen and are thus capable of stimulating CMV-specific effector-memory T helper cells. CpG-A-stimulated PDC produce large amounts of IFN-I and express programmed death receptor-1 ligand 1. CpG-A plus CpG-B-co-stimulated PDC behave like CpG-B-stimulated PDC, suggesting that antigen processing and presentation in PDC is dependent on stimulation that concurrently inhibits IFN-I production. In vivo targeting of antigens to PDC via CD303 combined with appropriate PDC stimulation may allow induction of specific T cell activation.

Peptide loading onto MHC class II molecules takes place in endosomal compartments along the endocytic pathway. There, loading is facilitated by the catalytic function of the accessory molecule H2-M, which helps to exchange the invariant chain-derived CLIP peptide in the groove of class II molecules for antigenic peptide. H2-O is another accessory molecule specific to the class II pathway, which is found tightly associated with H2-M and selectively expressed in B cells. Using stable H2-O ribozyme-antisense transfectants, H2-O overexpressing murine B cell lines, and H2-O-transgenic mice, we investigated the effects of H2-O on antigen presentation. The results show that presentation of a variety of exogenous protein antigens to a panel of T cell hybridomas depended on the levels of H2-O in the antigen-presenting B cells. Thus, increased H2-O expression downmodulated, whereas reduced H2-O levels, enhanced presentation. Presentation of endogenous antigen was also diminished by H2-O. Despite the pronounced effects on antigen presentation, the mass spectrometric profiles of peptides eluted from Ab molecules were very similar in cells expressing different H2-O levels. The intracellular location of H2-O inhibitory activity was investigated with the drug chloroquine, which prevents acidification of the endocytic pathway. The observations indicate that H2-O predominantly inhibits antigen presentation in early endosomal compartments. Thus, H2-O appears to skew peptide loading to late endosomal/lysosomal compartments. This may favor presentation of antigens taken up by the B cell receptor.

Antigens such as MUC1 coupled to oxidized mannan lead to rapid and efficient MHC class I presentation to CD8+ cells and a preferential T1 response; after reduction there is class II presentation and a T2 immune response. We now show that the selective advantage of the oxidized mannan-MUC1 is due to the presence of aldehydes and not Schiff bases, and that oxidized mannan-MUC1 binds to the mannose and not scavenger receptors and is internalized and presented by MHC class I molecules 1,000 times more efficiently than when reduced. After internalization there is rapid access to the class I pathway via endosomes but not lysosomes, proteasomal processing and transport to the endoplasmic reticulum, Golgi apparatus and cell surface. Aldehydes cause rapid entry into the class I pathway, and can therefore direct the subsequent immune response.

Langerhans cells (LC) represent the dendritic cell (DC) lineage in the epidermis. They capture and process antigens in the skin and subsequently migrate to the draining lymph nodes to activate naive T cells. Efficient uptake and processing of protein antigens by LC would, therefore, seem a prerequisite. We have now compared the capacity of human epidermal LC, blood-derived DC and peripheral blood mononuclear cells to endocytose and present (mannosylated) antigens to antigen-specific T cells. Moreover, we have determined the expression of mannose receptors, and the composition of the intracellular endosomal/lysosomal MHC class II-positive compartment. The results indicate that LC have poor endocytic capacity and do not exploit mannose receptor-mediated endocytosis pathways. Furthermore, the composition of the class II compartment in LC is distinct from that in other antigen-presenting cells and is characterized by the presence of relatively low levels of lysosomal markers. These results underscore the unique properties of LC and indicate that LC are relatively inefficient in antigen uptake, processing and presentation. This may serve to avoid hyper-responsiveness to harmless protein antigens that are likely to be frequently encountered in the skin due to (mechanical) skin damage.

The strict segregation of the major histocompatibility complex (MHC) class I and class II loading pathways has been challenged by recent reports indicating that MHC class I molecules can acquire antigen in the phagocytic pathway. We now show that this alternative peptide loading pathway can be used efficiently to generate macrophages able to activate unprimed antigen-specific cytotoxic T cells in vitro. Short peptides (8-11 residues), administered in the phagocytic pathway at nanomolar concentrations, were found to be effective in specifically activating naïve cytotoxic T lymphocytes (CTL) in vitro, but longer peptides or whole protein antigen were not. Whole protein antigen coated on beads did, however, render macrophages susceptible to lysis by an antigen-specific CTL clone. This indicates that proteolysis in the phagocytic pathway has limited capability for class I-restricted presentation. We propose a model for class I loading in the phagocytic pathway consisting of direct trafficking of nascent MHC class I from the trans-Golgi network to the phagosome, prior to appearance at the cell surface, and the use of the narrow cavity between bead and phagosomal membrane as a peptide exchange/loading compartment. Targeting immunogenic class I-binding peptide to the phagocytic pathway of macrophages facilitates presentation in association with class I. This is a useful tool for CTL response induction in vitro.

We performed immunoelectron microscopic studies to investigate the expression of HLA class II molecules in Langerhans cells (LC). In the epidermis, LC expressed class II molecules on the plasma membrane of the dendrites, resulting in a class II positive reticulo-epithelial network, but not on the surface of the cell body. In contrast, isolated LC as well as activated LC in situ displayed an even and strong expression of class II molecules on their entire cell surface. Therefore, isolation and/or activation results in redistribution and up-regulation of class II molecules on the cell surface. In addition, double-labeling experiments were carried out with monoclonal antibodies to class II antigens and to LAMP-1, CD63 and alpha-glucosidase, specific markers for organelles of the endosomal/lysosomal system. The results show expression of class II molecules on intracellular, electron-dense vesicular structures, and co-localization of class II molecules and the markers for late endosomes and early lysosomes in human LC in situ. Expression of these markers was not found on Birbeck granules, an LC-specific organelle.

We have developed an in vitro assay for tetanus toxin (tt) C fragment (C-fr) degradation. Purified endosomes (abbreviated endosomes 1101 or 1104) and lysosomes (abbreviated lysosomes 1101 or 1104) from the DRB1*1101 (Gly 86) and DRB1*1104 (Val 86) B cell lines were used to degrade 125I-labeled C-fr in vitro. Using three distinct methods of analysis, we show that the capacity of endosomes and lysosomes to degrade the tt C-fr or tt synthetic Y-P30 peptide differed. Using sodium dodecylsulfate-polyacrylamide gel electrophoresis, 125I-labeled C-fr degradation patterns observed either with endosomes 1101/1104 or lysosomes 1101/1104 are distinct both in terms of the number of fragments and the kinetics of generation of the fragments. These results were confirmed by high-performance liquid chromatography analysis, where we observed that the elution profiles of the 125I-labeled Y-P30 peptide digested by endosomes 1101/1104 were different compared to those obtained with lysosomes 1101/1104. Furthermore, the kinetics of degradation of 125I-labeled Y-P30 were faster with lysosomes 1104 than with lysosomes 1101. This difference in activity of the 1101 and 1104 organelles was also found in a functional assay where we showed that the activation capacity of the P30 peptide was diminished when digested by lysosome 1104, regardless of the antigen-presenting cell (APC) used, whereas endosomes 1101 or lysosomes 1101 modified P30 peptide in a form that discriminated between presentation by 1101 or 1104 APC. Taken together, these results suggest that the differential processing and presentation displayed by the DRB1*1101 and DRB1*1104 APC is due partly to a different enzymatic content and partly to the dimorphism at position DR beta 86.

A BCL1 immunoglobulin (Ig) transfectant, expressing wild-type surface (s)IgM with the TEPC-15 idiotype (T15-Id) and anti-phosphorylcholine (PC) specificity, was previously shown to present PC-conjugated hen egg-white lysozyme (PC-HEL) to a HEL-specific T cell hybridoma at a lower antigen (Ag) concentration than that required for native HEL. Two variant Ig transfectants, expressing T15-Id sIgM with substitutions either in the entire spacer, transmembrane (TM) domain and cytoplasmic tail (B186 variant) or in the NH2-terminal third of TM domain only (TM2 variant), failed to display this sIgM-mediated, enhanced presentation of PC-HEL at low concentrations. However, prolonged treatment with anti-T15-Id monoclonal antibody (mAb) led to a reduction of surface expression of the T15-Id sIgM in the wild-type and TM2 variant, but not in the B186 variant sIgM transfectants. Treatment with anti-T15-Id mAb also resulted in an increased intracellular accumulation of T15-Id sIgM in the wild-type transfectant, but not in the B186 variant. Subcellular fractionation analysis revealed that the ligands bound to the T15-Id sIgM are not efficiently transported to the dense lysosomal compartments in both B186 and TM2 transfectants, as compared to the wild-type sIgM transfectant. A significant increase in tyrosine phosphorylation after cross-linking of the T15-Id sIgM was observed only in the wild-type sIgM transfectant. These results suggest that, while the NH2-terminal third of the TM region is not involved in the process responsible for the ligand-induced reduction of surface expression of sIgM, it appears to be essential for subsequent transport of sIgM/ligand complexes to the lysosomal compartments, as well as efficient activation of tyrosine kinases. These results strongly suggest that sIg-mediated enhancement of specific antigen presentation reflects the ability of sIg to efficiently transport antigen to the lysosomal compartments, and possibly the activation of protein tyrosine kinases.