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C V Harding

Publications and source records attributed to C V Harding.

At least 19 recordsLinked to original sources
PubMed1992

Recombinant Escherichia coli express a defined, cytoplasmic epitope that is efficiently processed in macrophage phagolysosomes for class II MHC presentation to T lymphocytes.

Although the processing of soluble Ag for presentation to T cells has been extensively studied in vitro, similar studies of phagocytic Ag processing have been limited. We have developed an in vitro model system to study the ability of macrophages to process recombinant Escherichia coli strain HB101 with cytoplasmic or surface expression of the well characterized T cell epitope of hen egg lysozyme (HEL) 52-61. This epitope was expressed within full length HEL or within a fusion protein containing the HEL epitope. Phagocytosis of E. coli with cytoplasmic expression of HEL or the HEL fusion protein resulted in strong presentation of HEL(52-61) to T cells. Surface-conjugated HEL was processed with even greater efficiency. Processing required viable macrophages, was inhibited by cytochalasin D, and was achieved within 20 min of bacterial contact with the macrophages. Within this time span, phagosomes containing bacteria fused with lysosomes, and the bacteria were extensively degraded. Uptake of as few as four bacteria per macrophage produced an Ag-specific T cell response. We conclude that bacterial compartmentalization of the antigenic epitope (cytoplasmic vs surface) had some effect on its processing, but that phagocytic Ag processing organelles contain extensive capacity to degrade internalized bacteria and liberate intracellular Ag epitopes for recycling and presentation, consistent with a central role for phagolysosomes. Thus, future recombinant bacterial vaccines may be effectively designed with T cell epitopes expressed either on the surface or within the bacterial cytoplasm.

Animals↗
PubMed1992

Processing of exogenous liposome-encapsulated antigens in vivo generates class I MHC-restricted T cell responses.

Acid-sensitive liposomes have been developed for cytosolic delivery of encapsulated substances. We now demonstrate delivery of liposome-encapsulated Ag into the class I MHC Ag processing pathway in peritoneal macrophages in vitro using several types of acid-sensitive liposomes, including those composed of dioleoylphosphatidylethanolamine (DOPE)/palmitoylhomocysteine, DOPE/cholesterol hemisuccinate, DOPE/dioleoylsuccinylglycerol, and DOPE/dipalmitoylsuccinylglycerol. Our previous studies showed that acid-resistant liposomes (dioleoylphosphatidylcholine/dioleoylphosphatidylserine) did not engender class I-mediated presentation in vitro. However, in vivo immunization with OVA encapsulated in acid-resistant as well as acid-sensitive liposomes generated class I MHC-restricted T cell responses, as determined by subsequent in vitro cytotoxicity assays using OVA-transfected target cells. Target lysis by these cells was OVA- and class I MHC (Kb)-specific. This response was not generated by immunization with equivalent amounts of soluble OVA. Thus, a pathway for in vivo class I processing of Ag encapsulated in acid-resistant liposomes has been missed in vitro, perhaps because it is dependent on specific populations of APC or interactions between cells that have not been reconstituted in vitro. This pathway may explain the ability of many exogenous particulate Ag (liposomes, bacteria, parasites, and mammalian cells) to generate class I MHC-restricted T cell responses.

Animals↗
PubMed1992

Electroporation of exogenous antigen into the cytosol for antigen processing and class I major histocompatibility complex (MHC) presentation: weak base amines and hypothermia (18 degrees C) inhibit the class I MHC processing pathway.

While endogenous antigens are presented by class I major histocompatibility complex (MHC) molecules, exogenous antigens generally require a means for penetration into the cytosol for processing prior to class I MHC presentation. We have optimized conditions for electroporation as a means to experimentally introduce exogenous antigens into the cytosol, providing a system with a number of advantages for dissecting the class I MHC processing pathway. Presentation was assessed by the response of class I or class II MHC-restricted T hybridoma cells. Essentially instantaneous antigen delivery by electroporation facilitated kinetic analysis of the class I pathway and investigation of the effects of various inhibitors or hypothermic conditions on class I MHC antigen processing. This pathway was inhibited by weak base amines (e.g. chloroquine and NH4Cl), cycloheximide, and hypothermia (18 degrees C, which inhibits certain intracellular vesicular processing pathways). The electroporation technique provides a simple, consistent approach for rapid cytosolic antigen delivery for analysis of class I MHC processing.

Ammonium Chloride↗
PubMed1992

Processing and presentation of intact hen egg-white lysozyme by dendritic cells.

Dendritic cells in lymphoid tissues are of key importance as highly specialized antigen-presenting cells for the induction of T lymphocyte responses. Conflicting results have been published regarding antigen processing of intact proteins by dendritic cells. We now report that highly purified dendritic cells isolated from H-2k mouse spleens very efficiently generated immunogenic fragments of intact hen egg-white lysozyme (HEL) protein to present to an I-Ak-restricted T hybridoma cell line, specific for HEL peptide 46-61. Dendritic cells required 100 times less HEL protein than lipopolysaccharide-induced B cell blasts for effective presentation. Uptake of 125I-labeled HEL protein by dendritic cells and inhibition of presentation of HEL protein by chloroquine treatment was observed. This indicates an endocytotic process and the involvement of acidified compartments. Since the supernatant of dendritic cells, that were incubated with intact HEL protein, contained immunogenic fragments, further evidence for processing of HEL protein by dendritic cells was obtained. When HEL protein was covalently coupled to beads, dendritic cells were not able to ingest these beads, but could still process HEL protein for presentation. This suggests cell surface processing of HEL protein, although internalization of HEL protein released from the beads cannot be excluded. Taken together, these data show that H-2k dendritic cells are capable of processing and presenting intact HEL protein.

Animals↗
PubMed1992

Class II MHC molecules are present in macrophage lysosomes and phagolysosomes that function in the phagocytic processing of Listeria monocytogenes for presentation to T cells.

Phagocytic processing of heat-killed Listeria monocytogenes by peritoneal macrophages resulted in degradation of these bacteria in phagolysosomal compartments and processing of bacterial antigens for presentation to T cells by class II MHC molecules. Within 20 min of uptake by macrophages, Listeria peptide antigens were expressed on surface class II MHC molecules, capable of stimulating Listeria-specific T cells. Within this period, degradation of labeled bacteria to acid-soluble low molecular weight catabolites also commenced. Immunoelectron microscopy was used to evaluate the compartments involved in this processing. Upon uptake of the bacteria, phagosomes containing Listeria fused rapidly with both lysosomes and endosomes. Class II MHC molecules were present in a tubulo-vesicular lysosome compartment, which appeared to fuse with phagosomes, as well as in the resulting phagolysosomes containing internalized Listeria; these compartments were all positive for Lamp 1 and cathepsin D and lacked 46-kD mannose-6-phosphate receptors. In addition, class II MHC and Lamp 1 were co-localized in vesicles of the trans Golgi reticulum, where they were segregated from 46-kD mannose-6-phosphate receptors. Vesicles containing both Listeria-derived components and class II MHC molecules were also observed; some of these may represent vesicles recycling from phagolysosomes, potentially bearing processed immunogenic peptides complexed with class II MHC. These results support a central role for lysosomes and phagolysosomes in the processing of bacterial antigens for presentation to T cells. Tubulo-vesicular lysosomes appear to represent an important convergence of endocytic, phagocytic and biosynthetic pathways, where antigens may be processed to allow binding to class II MHC molecules and recycling to the cell surface.

Animals↗
PubMed1991

Reduction of disulfide bonds within lysosomes is a key step in antigen processing.

Reduction of disulfide bonds is a key step in antigen processing both to allow the unfolding of protein antigens, increasing the access of proteolytic processing enzymes, and to expose free Cys residues within linear peptide epitopes recognized by T cells. We show here that reduction and alkylation of Ag (hen egg lysozyme and ribonuclease A) vastly increased their proteolysis (by specific enzymes or lysosomal fractions) and the production of specific immunogenic peptides that bound to class II MHC molecules recognized by T hybridoma cells. We also show that the lysosome is the vesicular compartment that mediates protein disulfide reduction. We coupled [125I]tyrosine to 131I-alpha 2-macroglobulin or [131I] transferrin via a reducible disulfide linker. Removal of [125I]tyrosine from the alpha 2-macroglobulin conjugate was initiated only after 15 to 20 min of uptake by macrophages, suggesting that reduction occurred late in the endocytic pathway. No reduction of transferrin conjugates was seen, indicating that early, recycling endosomes did not contain reducing activity. Subcellular fractionation showed that the disulfide bonds were reduced only in heavy density (lysosome) fractions and remained intact in fractions of light density (endosomes and plasma membrane). These results indicate the importance of lysosomes in the biochemical processing of protein Ag presented to T cells.

Animals↗
PubMed1991

Liposome-encapsulated antigens engender lysosomal processing for class II MHC presentation and cytosolic processing for class I presentation.

Liposome-encapsulated protein Ag were used to dissect the roles of various subcellular compartments in Ag processing for class I and class II MHC-restricted presentation. Macrophages exhibited efficient processing of Ag encapsulated in acid-resistant dioleoylphosphatidylcholine/dioleoylphosphatidylserine liposomes, which sequester their contents from potential endosomal processing events and release them only after delivery to lysosomes. Lysosomal processing was demonstrated for all four Ag studied (OVA, murine hemoglobin, bovine ribonuclease A, and hen egg lysozyme), establishing the recycling of immunogenic peptides from lysosomes after Ag processing. These acid-resistant liposomes did not engender class I processing. Ag encapsulated within acid-sensitive dioleoylphosphatidylethanolamine/palmitoylhomocysteine liposomes were also processed via the class II pathway. Of the four Ag encapsulated in liposomes, one, OVA, was tested for ability to stimulate a class I-specific response. OVA in acid-resistant liposomes did not engender a class I-specific response. In contrast, OVA encapsulated in acid-sensitive liposomes was presented by class I molecules, albeit less efficiently than it was presented by class II molecules. We interpret this to be the result of the release of a minor portion of the encapsulated Ag into the cytosol.

Animals↗
PubMed1991

Modulation of antigen presentation and peptide-MHC-specific, LFA-1-dependent T cell-macrophage adhesion.

Incubation of peritoneal macrophages in vitro before fixation increased their ability to present exogenous peptides to 3A9 T hybridoma cells. The enhanced level of presentation correlated with a greatly increased, peptide-specific adhesion of 3A9 cells to the macrophages, whereas peptide-independent adhesion was minimal and essentially unaltered. 3A9 cells exhibited rapid peptide-specific adhesion (plateau by 5 to 10 min) and deadhesion (complete reversal by 5 min). Peptide-specific adhesion was blocked by anti-I-Ak and anti-LFA-1. Interaction of T cell receptors and CD-4 with peptide-I-Ak complexes appeared to provide little direct contribution to the avidity of T cell-macrophage adhesion, but activated a LFA-1-mediated adhesion mechanism. In addition, anti-T cell receptor, anti-CD3, and anti-CD4 antibodies themselves activated LFA-1-dependent adhesion in the absence of peptide. Unlike the peptide-induced adhesion, this adhesion was similar for macrophages whether or not they were incubated in vitro before fixation. We conclude that the different macrophage populations supported LFA-1-mediated adhesion equally. Therefore, the enhancement of T cell stimulation observed after in vitro incubation of macrophages was due to increased peptide presentation and consequently increased triggering of LFA-1-mediated adhesion. Mechanisms may exist to regulate the effectiveness with which peptide-class II MHC complexes are displayed for T cell recognition.

Animals↗
PubMed1991

Effects of pH and polysaccharides on peptide binding to class II major histocompatibility complex molecules.

The binding of immunogenic peptides to class II major histocompatibility molecules was examined at various pH values. We studied binding of peptides containing residues 52-61 from hen egg lysozyme (HEL) to I-Ak on fixed peritoneal macrophages or to solubilized affinity-purified I-Ak. Optimum binding occurred at pH 5.5-6.0 with accelerated kinetics relative to pH 7.4; equilibrium binding was also higher at pH 5.5-6.0 than at 7.4. Similar enhancement at pH 5-6 was observed for the binding of hemoglobin-(64-76) to I-Ek and of ribonuclease-(41-61) to I-Ak. In contrast, the binding of HEL-(34-45) to I-Ak was minimally enhanced at acid pH. Dissociation of cell-associated or purified peptide-I-Ak complexes was minimal between pH 5.5 and 7.4, with increased dissociation only at or below pH 4.0 [HEL-(46-61)] or pH 5.0 [HEL-(34-45)]. Thus, optimum peptide binding occurs at pH values similar to the endosomal environment, where the complexes appear to be formed during antigen processing. In addition, we examined the effect of a number of polysaccharides on the binding of peptide to I-Ak. None of these competed with the HEL peptide 125I-labeled YE52-61 for binding to I-Ak. [3H]Dextran also failed to bind purified I-Ak. Polysaccharides do not appear to bind to class II major histocompatibility complex molecules, which explains the T-cell independence of polysaccharide antigens.

Animals↗
PubMed1991

Liposome-encapsulated antigens are processed in lysosomes, recycled, and presented to T cells.

Antigen processing requires intracellular antigen catabolism to generate immunogenic peptides that bind to class II MHC molecules (MHC-II) for presentation to T-cells. We now provide direct evidence that these peptides are produced within dense lysosomes, as opposed to earlier endocytic compartments. The protein antigen hen egg lysozyme was targeted to endosomes or lysosomes by encapsulating it in liposomes of different membrane composition. Acid-sensitive liposomes released their contents in early endosomes, whereas acid-resistant liposomes sequestered their contents from potential endosomal processing events and released their contents only after delivery to lysosomes. Antigen encapsulated in acid-resistant liposomes was processed in a chloroquine-sensitive manner and presented more efficiently than soluble antigen or antigen encapsulated in acid-sensitive liposomes. Thus, peptides may be recycled from lysosomes, transported to endosomes to bind MHC-II, and then expressed at the cell surface.

Animals↗
PubMed1991

Pathways of antigen processing.

Separate pathways exist for the processing of antigens to be presented by MHC class I and class II molecules. We are beginning to determine the subcellular location of certain events in both pathways.

Animals↗
PubMed1991

Blastomycosis and opportunistic infections in patients with acquired immunodeficiency syndrome. An autopsy study.

Patients with acquired immunodeficiency syndrome (AIDS) are subject to a host of opportunistic infections, but to our knowledge a predisposition to blastomycosis has not previously been established. Autopsies of two patients with AIDS revealed disseminated blastomycosis with massive pulmonary involvement, Blastomyces meningoencephalitis, and widespread dissemination. The massive systemic involvement and rapid terminal course in both cases may reflect the state of acquired immunodeficiency. An analysis of an autopsy series showed that the incidence of blastomycosis was increased in patients with AIDS, although some other opportunistic organisms were more common (eg, Pneumocystis carinii, Mycobacterium avium-intracellulare, and Candida species). Thus, the diagnosis and treatment of blastomycosis must be pursued in patients with AIDS. Additional data are needed to further determine the incidence of blastomycosis in the population of patients with AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Quantitation of antigen-presenting cell MHC class II/peptide complexes necessary for T-cell stimulation.

The number of specific complexes formed between peptide and the class II major histocompatibility complex (MHC) molecules expressed by an antigen-presenting cell (APC) after exposure to protein antigens is unknown, as is the number that activates T cells. Presentation of foreign peptides by APC takes place when many class II molecules may be occupied by autologous peptides. We have now estimated the number of specific peptide/class II complexes per APC by quantitative immunoprecipitation of I-Ak after pulsing the APC with stimulatory levels of a radioactive immunogenic peptide derived from hen egg-white lysozyme protein. T cells were activated by APC that expressed as few as 210-340 specific peptide/class II complexes (0.1% of the I-Ak molecules). These figures were confirmed using anti-CD3 antibody bound to latex beads as an alternative activating ligand. This low number explains the simultaneous presentation of multiple foreign antigens, even in the face of peptide competition.

Antigen-Presenting Cells↗
PubMed1990

Low-temperature inhibition of antigen processing and iron uptake from transferrin: deficits in endosome functions at 18 degrees C.

Antigen processing involves endocytosis, proteolysis and denaturation of antigens to generate peptides that bind to major histocompatibility complex class II molecules (Ia) in a complex recognized by CD4+ T cells. Ia and antigen are internalized and processed intracellularly, but the exact subcellular site of antigen degradation and formation of the Ia-peptide complex remains unclear. The present studies utilized low-temperature incubation in an attempt to functionally block certain steps in the processing of the antigen hen egg white lysozyme (HEL) by peritoneal exudate cells (PEC) and TA3 B lymphoma cells. Ia endocytosis and uptake of HEL by PEC persisted at 18 degrees C, albeit at somewhat slower rates, but delivery of ligands to lysosomes was blocked. Under these conditions HEL catabolism and antigen processing were effectively blocked, although enough catabolism and antigen processing were effectively blocked, although enough HEL was internalized at 18 degrees C to provide effective presentation during a subsequent incubation at 37 degrees C. In TA3 cells transferrin endocytosis and recycling were notably slowed at 18 degrees C, and iron uptake from transferrin by TA3 cells was completely blocked, indicating that certain specifically endosomal functions were inhibited at 18 degrees C. Thus, intracellular steps in antigen processing were blocked at 18 degrees C, corresponding to deficits in endosomal processing and targeting. These results demonstrate that antigen endocytosis and certain temperature-sensitive endosomal and lysosomal processes are essential for antigen processing.

Animals↗
PubMed1990

Functional and ultrastructural evidence for intracellular formation of major histocompatibility complex class II-peptide complexes during antigen processing.

Antigen presentation requires intracellular processing of native antigens to produce immunogenic peptides that bind to major histocompatibility complex class II (MHC-II) molecules. In functional studies of antigen processing by elicited peritoneal macrophages, MHC-II-peptide complexes were formed intracellularly. Immunogenic peptides were not released to bind surface MHC-II molecules. Ultrastructural studies employing immunogold staining in ultrathin cryosections of these macrophages showed large amounts of MHC-II molecules in intracellular sac-like vacuoles in the peripheral cytoplasm; most of these were negative for the lamp 1 lysosomal/endosomal membrane protein and cathepsin D. MHC-II molecules were also present in endosomes containing cathepsin D and lamp 1 as well as previously internalized gold-transferrin. The intracellular pool of MHC-II molecules was only slightly decreased by treatment with cycloheximide for 3 hr, indicating that it consisted mainly of endocytosed, recycling molecules, as opposed to nascent ones. These ultrastructural studies support the notion that there is endocytosis of MHC-II molecules into endocytic compartments, consistent with our earlier biochemical data. Furthermore, we have defined the distinct endocytic compartments that must mediate important functions in antigen processing, including the formation of MHC-II-peptide complexes.

Animals↗