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Biomedical subjects

Ira Mellman

Publications and source records attributed to Ira Mellman.

At least 37 records · Page 2Linked to original sources
PubMed2004

IFN-gamma enables cross-presentation of exogenous protein antigen in human Langerhans cells by potentiating maturation.

We compared monocyte-derived dendritic cells and transforming growth factor-beta1-induced Langerhans-like cells (LCs) for their capacity to cross-present exogenous NY-ESO-1 protein/antibody immune complexes to an NY-ESO-1-specific CD8+ T cell clone. In contrast to dendritic cells, LCs were not able to cross-present NY-ESO-1 to the T cell clone constitutively but did so after treatment with IFN-gamma. Remarkably, this IFN-gamma-inducible characteristic was due neither to enhanced antigen uptake nor to facilitated antigen processing in LCs. Rather, IFN-gamma acted at least in part by potentiating the maturation of otherwise refractory LCs, enabling in turn exogenous antigen to reach the processing machinery. This model of conditional cross-presentation establishes an original level of action for IFN-gamma as an effective immune modulator and supports the use of IFN-gamma in protein vaccination strategies targeting LCs.

Amino Acid Sequence↗
PubMed2004

Immunotherapy: bewitched, bothered, and bewildered no more.

The field of immunotherapy holds clear promise not only for the development of new approaches to cancer and other diseases, but also for providing fundamental insight into the human immune response. In order for this promise to be realized, however, the scientific community must overcome an array of challenges. These challenges reflect not only the difficulties inherent in conducting investigations in human patients, but also difficulties created by the culture and practice of our own institutions, reward structure, and funding mechanisms. We suggest steps to be taken to reinvigorate basic research in human subjects as part of the mainstream of science.

Animals↗
PubMed2004

The J-domain protein Rme-8 interacts with Hsc70 to control clathrin-dependent endocytosis in Drosophila.

By screening for mutants exhibiting interactions with a dominant-negative dynamin, we have identified the Drosophila homologue of receptor-mediated endocytosis (Rme) 8, a J-domain-containing protein previously shown to be required for endocytosis in Caenorhabditis elegans. Analysis of Drosophila Rme-8 mutants showed that internalization of Bride of sevenless and the uptake of tracers were blocked. In addition, endosomal organization and the distribution of clathrin were greatly disrupted in Rme-8 cells, suggesting that Rme-8 participates in a clathrin-dependent process. The phenotypes of Rme-8 mutants bear a strong resemblance to those of Hsc70-4, suggesting that these two genes act in a common pathway. Indeed, biochemical and genetic data demonstrated that Rme-8 interacts specifically with Hsc70-4 via its J-domain. Thus, Rme-8 appears to function as an unexpected but critical cochaperone with Hsc70 in endocytosis. Because Hsc70 is known to act in clathrin uncoating along with auxilin, another J-protein, its interaction with Rme-8 indicates that Hsc70 can act with multiple cofactors, possibly explaining its pleiotropic effects on the endocytic pathway.

Animals↗
PubMed2004

Protein kinase D regulates basolateral membrane protein exit from trans-Golgi network.

Protein kinase D (PKD) binds to diacylglycerol (DAG) in the trans-Golgi network (TGN) and is activated by trimeric G-protein subunits beta gamma. This complex then regulates the formation of transport carriers in the TGN that traffic to the plasma membrane in non-polarized cells. Here we report specificity of different PKD isoforms in regulating protein trafficking from the TGN. Kinase-inactive forms of PKD1, PKD2 and PKD3 localize to the TGN in polarized and non-polarized cells. PKD activity is required only for the transport of proteins containing basolateral sorting information, and seems to be cargo specific.

Animals↗
PubMed2004

Sorting of H,K-ATPase beta-subunit in MDCK and LLC-PK cells is independent of mu 1B adaptin expression.

The cytoplasmic tail of the H,K-ATPase beta-subunit contains a putative tyrosine-based motif that directs the beta-subunit's basolateral sorting when it is expressed in Madin-Darby Canine Kidney (MDCK) cells. When expressed in LLC-PK(1) cells, however, the beta-subunit is localized to the apical membrane. Several proteins that contain tyrosine-based motifs, including the low-density lipoprotein and transferrin receptors, show a similar sorting 'defect' when expressed in LLC-PK(1) cells. For low-density lipoprotein and transferrin receptors, this behavior is due to the differential expression of the mu 1B subunit of the AP-1B clathrin adaptor complex. mu 1B is expressed by MDCK cells, but not LLC-PK(1) cells, and transfection of mu 1B into LLC-PK(1) cells restores basolateral localization of low-density lipoprotein and transferrin receptors. For the beta-subunit, however, mu B expression in LLC-PK(1) cells does not induce its basolateral expression. We found that the beta-subunit interacts with both mu 1B and mu 1A in vitro and in vivo. The capacity to participate in a mu 1B interaction therefore is not sufficient to program the beta-subunit's basolateral localization in MDCK cells. Our data suggest that the H,K-ATPase beta-subunit's basolateral sorting signal is either masked in certain epithelial cells, or requires an interaction with sorting machinery other than AP-1B for delivery to the basolateral plasma membrane.

Adaptor Protein Complex mu Subunits↗
PubMed2003

The Rab8 GTPase selectively regulates AP-1B-dependent basolateral transport in polarized Madin-Darby canine kidney cells.

The AP-1B clathrin adaptor complex plays a key role in the recognition and intracellular transport of many membrane proteins destined for the basolateral surface of epithelial cells. However, little is known about other components that act in conjunction with AP-1B. We found that the Rab8 GTPase is one such component. Expression of a constitutively activated GTP hydrolysis mutant selectively inhibited basolateral (but not apical) transport of newly synthesized membrane proteins. Moreover, the effects were limited to AP-1B-dependent basolateral cargo; basolateral transport of proteins containing dileucine targeting motifs that do not interact with AP-1B were targeted normally despite overexpression of mutant Rab8. Similar results were obtained for a dominant-negative allele of the Rho GTPase Cdc42, previously implicated in basolateral transport but now shown to be selective for the AP-1B pathway. Rab8-GFP was localized to membranes in the TGN-recycling endosome, together with AP-1B complexes and the closely related but ubiquitously expressed AP-1A complex. However, expression of active Rab8 caused a selective dissociation of AP-1B complexes, reflecting the specificity of Rab8 for AP-1B-dependent transport.

Adaptor Protein Complex 1↗
PubMed2003

The AP-1A and AP-1B clathrin adaptor complexes define biochemically and functionally distinct membrane domains.

Most epithelial cells contain two AP-1 clathrin adaptor complexes. AP-1A is ubiquitously expressed and involved in transport between the TGN and endosomes. AP-1B is expressed only in epithelia and mediates the polarized targeting of membrane proteins to the basolateral surface. Both AP-1 complexes are heterotetramers and differ only in their 50-kD mu1A or mu1B subunits. Here, we show that AP-1A and AP-1B, together with their respective cargoes, define physically and functionally distinct membrane domains in the perinuclear region. Expression of AP-1B (but not AP-1A) enhanced the recruitment of at least two subunits of the exocyst complex (Sec8 and Exo70) required for basolateral transport. By immunofluorescence and cell fractionation, the exocyst subunits were found to selectively associate with AP-1B-containing membranes that were both distinct from AP-1A-positive TGN elements and more closely apposed to transferrin receptor-positive recycling endosomes. Thus, despite the similarity of the two AP-1 complexes, AP-1A and AP-1B exhibit great specificity for endosomal transport versus cell polarity.

Adaptor Protein Complex 1↗
PubMed2003

Presentation of exogenous antigens on major histocompatibility complex (MHC) class I and MHC class II molecules is differentially regulated during dendritic cell maturation.

During maturation, dendritic cells (DCs) regulate their capacity to process and present major histocompatibility complex (MHC) II-restricted antigens. Here we show that presentation of exogenous antigens by MHC I is also subject to developmental control, but in a fashion strikingly distinct from MHC II. Immature mouse bone marrow-derived DCs internalize soluble ovalbumin and sequester the antigen intracellularly until they receive an appropriate signal that induces cross presentation. At that time, peptides are generated in a proteasome-dependent fashion and used to form peptide-MHC I complexes that appear at the plasma membrane. Unlike MHC II, these events do not involve a marked redistribution of preexisting MHC I molecules from intracellular compartments to the DC surface. Moreover, out of nine stimuli well known to induce the phenotypic maturation of DCs and to promote MHC II presentation, only two (CD40 ligation, disruption of cell-cell contacts) activated cross presentation on MHC I. In contrast, formation of peptide-MHC I complexes from endogenous cytosolic antigens occurs even in unstimulated, immature DCs. Thus, the MHC I and MHC II pathways of antigen presentation are differentially regulated during DC maturation.

Animals↗
PubMed2003

Activation of lysosomal function during dendritic cell maturation.

In response to a variety of stimuli, dendritic cells (DCs) transform from immature cells specialized for antigen capture into mature cells specialized for T cell stimulation. During maturation, the DCs acquire an enhanced capacity to form and accumulate peptide-MHC (major histocompatibility complex) class II complexes. Here we show that a key mechanism responsible for this alteration was the generalized activation of lysosomal function. In immature DCs, internalized antigens were slowly degraded and inefficiently used for peptide loading. Maturation induced activation of the vacuolar proton pump that enhanced lysosomal acidification and antigen proteolysis, facilitating efficient formation of peptide-MHC class II complexes. Lysosomal function in DCs thus appears to be specialized for the developmentally regulated processing of internalized antigens.

Animals↗
PubMed2002

Hsc70 is required for endocytosis and clathrin function in Drosophila.

By screening for Drosophila mutants exhibiting aberrant bride of sevenless (Boss) staining patterns on eye imaginal disc epithelia, we have recovered a point mutation in Hsc70-4, the closest homologue to bovine clathrin uncoating ATPase. Although the mutant allele was lethal, analysis of mutant clones generated by FLP/FRT recombination demonstrated that the Sevenless-mediated internalization of Boss was blocked in mutant Hsc70-4 eye disc epithelial cells. Endocytosis of other probes was also greatly inhibited in larval Garland cells. Immunostaining and EM analysis of the mutant cells revealed disruptions in the organization of endosomal/lysosomal compartments, including a substantial reduction in the number of clathrin-coated structures in Garland cells. The Hsc70-4 mutation also interacted genetically with a dominant-negative mutant of dynamin, a gene required for the budding of clathrin-coated vesicles (CCVs). Consistent with these phenotypes, recombinant mutant Hsc70 proteins exhibited diminished clathrin uncoating activity in vitro. Together, these data provide genetic support for the long-suspected role of Hsc70 in clathrin-mediated endocytosis, at least in part by inhibiting the uncoating of CCVs.

Animals↗
PubMed2002

The immunosuppressive agent 15-deoxyspergualin functions by inhibiting cell cycle progression and cytokine production following naive T cell activation.

Immunosuppressive agents are commonly used in the prevention of graft rejection following transplantation and in the treatment of autoimmunity. In this study, we examined the immunosuppressive mechanism of the drug 15-deoxyspergualin (DSG), which has shown efficacy in the enhancement of graft survival and in the treatment of autoimmunity. Using a murine model of chronic relapsing and remitting experimental autoimmune encephalomyelitis, we were able to demonstrate that DSG both delayed and reduced the severity of experimental autoimmune encephalomyelitis. Subsequent in vitro studies to examine the mechanism of immune suppression showed that DSG was not able to inhibit early activation of naive CD4 T cells, but DSG did effectively inhibit the growth of naive CD4 T cells after activation. An analysis of cell proliferation and cell cycle showed that DSG treatment led to a block in cell cycle progression 2-3 days following Ag stimulation. In addition, DSG treatment inhibited the production of IFN-gamma by Th1 effector T cells. These studies suggest that CD4 T cells are a predominant target for DSG and the immunosuppressive effects of the drug may result from reduced CD4 T cell expansion and decreased polarization into IFN-gamma-secreting Th1 effector T cells in the induction of certain autoimmune disorders.

Animals↗
PubMed2002

Dendritic cell maturation triggers retrograde MHC class II transport from lysosomes to the plasma membrane.

Central to the initiation of immune responses is recognition of peptide antigen by T lymphocytes. The cell biology of dendritic cells makes them ideally suited for the essential process of antigen presentation. Their life cycle includes several stages characterized by distinct functions and mechanisms of regulation. Immature dendritic cells synthesize large amounts of major histocompatibility complex class II molecules (MHC II), but the alpha beta-dimers are targeted to late endosomes and lysosomes (often referred to as MHC class II compartments) where they reside unproductively with internalized antigens. After exposure to microbial products or inflammatory mediators, endocytosis is downregulated, the expression of co-stimulatory molecules is enhanced, and newly formed immunogenic MHC II-peptide complexes are transported to the cell surface. That these MHC II molecules reach the surface is surprising, as the lysosomes comprise the terminal degradative compartment of the endocytic pathway from which exogenous components generally cannot be recovered intact. Here we have visualized this pathway in live dendritic cells by video microscopy, using cells expressing MHC II tagged with green fluorescent protein (GFP). We show that on stimulation, dendritic cells generate tubules from lysosomal compartments that go on to fuse directly with the plasma membrane.

Animals↗
PubMed2002

Differential presentation of a soluble exogenous tumor antigen, NY-ESO-1, by distinct human dendritic cell populations.

Dendritic cells (DCs) play a critical role in initiating antigen-specific immune responses, because they are able to capture exogenous antigens for presentation to naive T cells on both MHC class I and II molecules. As such, DCs represent important elements in the development of vaccine therapy for cancer. Although DCs are known to present antigens from phagocytosed tumor cells or preprocessed peptides, we explored whether they might also present soluble recombinant NY-ESO-1, a well characterized cancer antigen. We compared the abilities of human monocyte-derived DCs and DCs derived in vitro from CD34-positive stem cells to present NY-ESO-1 epitopes to MHC class I-restricted cytotoxic T cells. Although monocyte-derived DCs did not efficiently crosspresent free NY-ESO-1 protein, IgG-immune complexes containing NY-ESO-1 were avidly presented after uptake by Fcgamma receptors (FcgammaRII). In contrast, CD34-derived DCs were unable to process either soluble or immune complexed NY-ESO-1, although they efficiently presented preprocessed NY-ESO-1 peptides. This difference did not necessarily correlate with endocytic capacity. Although monocyte-derived DCs exhibited greater fluid-phase uptake than CD34-derived DCs, the two populations did not differ with respect to their surprisingly limited capacity for Fcgamma receptor-mediated endocytosis. These results indicate that monocyte-derived DCs will be easier to load by using protein antigen in vitro than CD34-derived DCs, and that the latter population exhibits a restricted ability to crosspresent soluble exogenous antigens.

Antigen Presentation↗
PubMed2002

Transferrin receptor recycling in the absence of perinuclear recycling endosomes.

In mammalian cells, internalized receptors such as transferrin (Tfn) receptor are presumed to pass sequentially through early endosomes (EEs) and perinuclear recycling endosomes (REs) before returning to the plasma membrane. Whether passage through RE is obligatory, however, remains unclear. Kinetic analysis of endocytosis in CHO cells suggested that the majority of internalized Tfn bypassed REs returning to the surface from EEs. To determine directly if REs are dispensable for recycling, we studied Tfn recycling in cytoplasts microsurgically created to contain peripheral EEs but to exclude perinuclear REs. The cytoplasts actively internalized and recycled Tfn. Surprisingly, they also exhibited spatially and temporally distinct endosome populations. The first appeared to correspond to EEs, labeling initially with Tfn, being positive for early endosomal antigen 1 (EEA-1) and containing only small amounts of Rab11, an RE marker. The second was EEA-1 negative and with time recruited Rab11, suggesting that cytoplasts assembled functional REs. These results suggest that although perinuclear REs are not essential components of the Tfn recycling pathway, they are dynamic structures which preexist in the peripheral cytoplasm or can be regenerated from EE- and cytosol-derived components such as Rab11.

Animals↗
PubMed2002

Actin dependence of polarized receptor recycling in Madin-Darby canine kidney cell endosomes.

Mammalian epithelial cell plasma membrane domains are separated by junctional complexes supported by actin. The extent to which actin acts elsewhere to maintain cell polarity remains poorly understood. Using latrunculin B (Lat B) to depolymerize actin filaments, several basolateral plasma membrane proteins were found to lose their polarized distribution. This loss of polarity did not reflect lateral diffusion through junctional complexes because a low-density lipoprotein receptor mutant lacking a functional endocytosis signal remained basolateral after Lat B treatment. Furthermore, Lat B treatment did not facilitate membrane diffusion across the tight junction as observed with ethylenediaminetetraacetic acid or dimethyl sulfoxide treatment. Detailed analysis of transferrin recycling confirmed Lat B depolarized recycling of transferrin from endosomes to the basolateral surface. Kinetic analysis suggested sorting was compromised at both basolateral early endosomes and perinuclear recycling endosomes. Despite loss of function, these two endosome populations remained distinct from each other and from early endosomes labeled by apically internalized ligand. Furthermore, apical and basolateral early endosomes were functionally distinct populations that directed traffic to a single common recycling endosomal compartment even after Lat B treatment. Thus, filamentous actin may help to guide receptor traffic from endosomes to the basolateral plasma membrane.

Actin Cytoskeleton↗