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A Porgador

Publications and source records attributed to A Porgador.

At least 19 recordsLinked to original sources
PubMed2006

Pneumococcal 6-phosphogluconate-dehydrogenase, a putative adhesin, induces protective immune response in mice.

For most bacteria, adherence to human cells is achieved by bacterial lectins binding to mammalian surface glyconjugates. 6-Phosphogluconate dehydrogenase (6PGD) was identified by us as one of Streptococcus pneumoniae cell wall lectin proteins, which elicits an age-dependent immune response in humans. This study assesses the role of 6PGD in S. pneumoniae pathogenesis as an adhesin and its ability to elicit a protective immune response in mice. Recombinant 6PGD (r6PGD) was cloned from S. pneumoniae serotype 3 (strain WU2). r6PGD interference in adhesion of three genetically unrelated unencapsulated pneumococcal strains (3.8, 14.8 and R6) and two genetically unrelated encapsulated pneumococcal strains (WU2 and D39) to A549 type II lung carcinoma cell was tested. BALB/c mice were immunized with r6PGD and boosted after 3 weeks. Immunized mice were challenged intranasally with a lethal dose of S. pneumoniae. r6PGD inhibited 90% and 80% of pneumococcal adhesion to the A549 cells of three unencapsulated S. pneumoniae strains and two encapsulated S. pneumoniae strains, respectively, in a concentration-dependent manner (P < 0.05). Antibodies to r6PGD produced in mice significantly inhibited bacterial adhesion to A549 cell (P < 0.05). Immunization of mice with r6PGD protected 60% (P < 0.001) of mice for 5 days and 40% (P < 0.05) of the mice for 21 days following intranasal lethal challenge. We have identified 6PGD as a surface-located immunogenic lectin protein capable of acting as an adhesin. 6PGD importance to bacterial pathogenesis was demonstrated by the ability of r6PGD to elicit a protective immune response in mice.

Adhesins, Bacterial↗
PubMed2001

Recognition of haemagglutinins on virus-infected cells by NKp46 activates lysis by human NK cells.

Natural killer (NK) cells destroy virus-infected and tumour cells, apparently without the need for previous antigen stimulation. In part, target cells are recognized by their diminished expression of major histocompatibility complex (MHC) class I molecules, which normally interact with inhibitory receptors on the NK cell surface. NK cells also express triggering receptors that are specific for non-MHC ligands; but the nature of the ligands recognized on target cells is undefined. NKp46 is thought to be the main activating receptor for human NK cells. Here we show that a soluble NKp46-immunoglobulin fusion protein binds to both the haemagglutinin of influenza virus and the haemagglutinin-neuraminidase of parainfluenza virus. In a substantial subset of NK cells, recognition by NKp46 is required to lyse cells expressing the corresponding viral glycoproteins. The binding requires the sialylation of NKp46 oligosaccharides, which is consistent with the known sialic binding capacity of the viral glycoproteins. These findings indicate how NKp46-expressing NK cells may recognize target cells infected by influenza or parainfluenza without the decreased expression of target-cell MHC class I protein.

Animals↗
PubMed2001

Recognition of viral hemagglutinins by NKp44 but not by NKp30.

Natural killer (NK) cells destroy virus-infected and tumor cells without prior antigen stimulation. The NK cell cytotoxicity is regulated in large part by the expression of NK cell receptors that are able to bind major histocompatibility complex (MHC) class I glycoproteins. NK cells also express lysis triggering receptors specific for non-MHC ligands, including NKp30, NKp44, NKp46 and CD16. However, the nature of their ligands, recognized on target cells, is undefined. We have recently shown that the NKp46 protein, but not the CD16 protein, recognizes the hemagglutinin (HA) of influenza virus (IV) and the hemagglutinin-neuraminidase (HN) of Sendai virus (SV), and that the recognition of HA from IV requires the sialylation of NKp46 oligosaccharides. We have also demonstrated that binding of NKp46 to HA of IV is required for lysis of cells expressing the corresponding glycoproteins by a substantial subset of NK clones. Here we show that NKp44, but not NKp30, can also recognize the HA of both IV and SV and that the recognition of IV HA requires the sialylation of the NKp44 receptor in a similar way to that of NKp46. SV infection of 721.221 cells expressing MHC class I proteinsresulted in the abrogation of the inhibition by NK clones expressing high levels of NKp44. In addition, the binding of NKp44 to HA improves the ability of some NK clones to lyse IV infected cells.

Antibodies, Viral↗
PubMed2001

NKp46.

Combating diseases such as cancer and virus infection is one of the major goals of the next millennium. Remarkably, our immune system has developed a unique cell type called Natural Killer (NK) cells, able to kill cancer and virus-infected cells. The nature of the lysis/killer receptors expressed on NK cells, mediating this killing, and their ligands, is only little understood. Four lysis receptors for human NK cells were recently identified, including the NKp30, NKp44, NKp46 and CD16, all of which are capable of mediating direct killing of virus-infected and tumor cells. Among these, the NKp46 receptor is considered to be the major lysis receptor for NK cells. The viral hemagglutinin protein was recently identified as the ligand for the NKp46 receptor. However, the cellular (non-viral) ligands recognized by all of the lysis receptors (NKp30, NKp44, NKp46 and CD16) are currently unknown.

Animals↗
PubMed1999

The induction of virus-specific CTL as a function of increasing epitope expression: responses rise steadily until excessively high levels of epitope are attained.

The role of epitope expression levels in CD8+ T cell priming has been controversial. Yet this parameter is of great importance in the design of rational approaches to optimize CTL responses to a variety of pathogens. In this paper we examine the influence of epitope production on CD8+ T cell priming by exploiting a system that allows a 200-fold range of cell surface epitope expression in vitro with a fixed dose of vaccinia virus. Our results demonstrate that, with the exception of a notable decline at the highest level of epitope, the magnitude of the responding CTL population generated in vivo following equivalent viral infections is essentially proportional to epitope density.

Animals↗
PubMed1999

Peptide-receptive class I major histocompatibility complex molecules on TAP-deficient and wild-type cells and their roles in the processing of exogenous antigens.

These studies addressed the nature and origin of peptide-receptive class I major histocompatibility complex (MHC-I) molecules used to present exogenous antigens. Peptide-receptive Kb molecules in transporter for antigen presentation (TAP)1-/- and TAP1+/+ macrophages were quantitated by exposing cells to exogenous ovalbumin (OVA)(257-264) peptide and then measuring OVA(257-264):Kb complexes with a T hybridoma assay or flow cytometry (using a complex-specific antibody). Relative to TAP1+/+ cells, TAP1-/- cells had decreased levels of pre-existing cell-surface peptide-receptive MHC-I molecules at 37 degrees. With continued exposure of viable cells to peptide, however, TAP1-/- and TAP1+/+ cells formed similar levels of OVA(257-264):Kb complexes, suggesting that nascent labile MHC-I molecules were captured and stabilized by exogenous peptide. Brefeldin A inhibited generation of OVA(257-264):Kb complexes on TAP1-/- (but not TAP1+/+) cells at 37 degrees, confirming the importance of a flux of unstable nascent MHC-I molecules in TAP1-/- cells at 37 degrees. In contrast, at 26 degrees both TAP1-/- and TAP1+/+ cells expressed brefeldin A-resistant, peptide-receptive MHC-I molecules at similar levels. Alternate MHC-I processing of exogenous particulate antigen correlated with ability to present exogenous peptide. Thus, processing was brefeldin A-sensitive with TAP1-/- macrophages at 37 degrees, but brefeldin A-resistant with TAP1+/+ cells at 37 degrees, as well as with TAP1+/+ or TAP1-/- cells at 26 degrees. We conclude that alternate MHC-I antigen processing normally utilizes pre-existing MHC-I molecules, but TAP1-/- cells at 37 degrees mainly use nascent MHC-I molecules, because of a lack of pre-existing, stable, peptide-receptive MHC-I molecules. The results support a vacuolar processing mechanism with binding of peptides to MHC-I molecules in post-Golgi compartments or on the cell surface.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Predominant role for directly transfected dendritic cells in antigen presentation to CD8+ T cells after gene gun immunization.

Cutaneous gene (DNA) bombardment results in substantial expression of the encoded antigen in the epidermal layer as well as detectable expression in dendritic cells (DC) in draining lymph nodes (LNs). Under these conditions, two possible modes of DC antigen presentation to naive CD8+ T cells might exist: (a) presentation directly by gene-transfected DC trafficking to local lymph nodes, and (b) cross-presentation by untransfected DC of antigen released from or associated with transfected epidermal cells. The relative contributions of these distinct modes of antigen presentation to priming for cytotoxic T cell (CTL) responses have not been clearly established. Here we show that LN cells directly expressing the DNA-encoded antigen are rare; 24 h after five abdominal skin bombardments, the number of these cells does not exceed 50-100 cells in an individual draining LN. However, over this same time period, the total number of CD11c+ DC increases more than twofold, by an average of 20,000-30,000 DC per major draining node. This augmentation is due to gold bombardment and is independent of the presence of plasmid DNA. Most antigen-bearing cells in the LNs draining the site of DNA delivery appear to be DC and can be depleted by antibodies to an intact surface protein encoded by cotransfected DNA. This finding of predominant antigen presentation by directly transfected cells is also consistent with data from studies on cotransfection with antigen and CD86-encoding DNA, showing that priming of anti-mutant influenza nucleoprotein CTLs with a single immunization is dependent upon coexpression of the DNAs encoding nucleoprotein and B7.2 in the same cells. These observations provide insight into the relative roles of direct gene expression and cross-presentation in CD8+ T cell priming using gene gun immunization, and indicate that augmentation of direct DC gene expression may enhance such priming.

Animals↗
PubMed1998

Assembly of MHC class I molecules with biosynthesized endoplasmic reticulum-targeted peptides is inefficient in insect cells and can be enhanced by protease inhibitors.

To study the requirements for assembly of MHC class I molecules with antigenic peptides in the endoplasmic reticulum (ER), we studied Ag processing in insect cells. Insects lack a class I recognition system, and their cells therefore provide a "blank slate" for identifying the proteins that have evolved to facilitate assembly of class I molecules in vertebrate cells. H-2Kb heavy chain, mouse beta 2-microglobulin, and an ER-targeted version of a peptide corresponding to Ova(257-264) were expressed in insect cells using recombinant vaccinia viruses. Cell surface expression of Kb-OVA(257-264) complexes was quantitated using a recently described complex-specific mAb (25-D1.16). Relative to TAP-deficient human cells, insect cells expressed comparable levels of native, peptide-receptive cell surface Kb molecules, but generated cell surface Kb-OVA(257-264) complexes at least 20-fold less efficiently from ER-targeted peptides. The inefficient assembly of Kb-OVA(257-264) complexes in the ER of insect cells cannot be attributed solely to a requirement for human tapasin, since first, human cells lacking tapasin expressed endogenously synthesized Kb-OVA(257-264) complexes at levels comparable to tapasin-expressing cells, and second, vaccinia virus-mediated expression of human tapasin in insect cells did not detectably enhance the expression of Kb-OVA(257-264) complexes. The assembly of Kb-OVA(257-264) complexes could be greatly enhanced in insect but not human cells by a nonproteasomal protease inhibitor. These findings indicate that insect cells lack one or more factors required for the efficient assembly of class I-peptide complexes in vertebrate cells and are consistent with the idea that the missing component acts to protect antigenic peptides or their immediate precursors from degradation.

Aedes↗
PubMed1998

Dissociation of proteasomal degradation of biosynthesized viral proteins from generation of MHC class I-associated antigenic peptides.

To study the role of proteasomes in Ag presentation, we analyzed the effects of proteasome inhibitors Cbz-Leu-Leu-Leucinal and lactacystin on the ability of mouse fibroblast cells to present recombinant vaccinia virus gene products to MHC class I-restricted T cells. The effects of the inhibitors depended on the determinant analyzed. For influenza virus nucleoprotein (NP), presentation of the immunodominant Kk-restricted determinant (NP(50-57)) was marginally inhibited, whereas presentation of the immunodominant Kd-restricted determinant (NP(147-155)) was enhanced, particularly by lactacystin. Biochemical purification of peptides confirmed that lactacystin enhanced the generation of Kd-NP(147-155) complexes fourfold. Lactacystin also enhanced the recovery of one Kd-restricted vaccinia virus determinant from HPLC fractions, while inhibiting recovery of another. The inhibitors were used at sufficient concentrations to block presentation of biosynthesized full-length OVA and to completely stabilize a rapidly degraded chimeric ubiquitin-NP fusion protein. Strikingly, presentation of antigenic peptides from this protein was unaffected by proteasome inhibitors. We also observed that proteasome inhibitors induced expression of cytosolic and endoplasmic reticulum stress-responsive proteins. These data demonstrate first that the processes of protein degradation and generation of antigenic peptides from cytosolic proteins can be dissociated, and second that effects of proteasome inhibitors on Ag presentation may reflect secondary effects on cellular metabolism.

Acetylcysteine↗
PubMed1998

Calnexin expression does not enhance the generation of MHC class I-peptide complexes.

We investigated the requirement for calnexin in the biogenesis of MHC class I molecules. Mutant human cells lacking calnexin were infected with recombinant vaccinia viruses encoding mouse MHC class I molecules, Kd, Kb, Kk, Dd, Db, and Ld. Flow cytometry indicated that each of the six MHC class I allomorphs was transported to the cell surface at similar rates in calnexin-deficient cells and transfectants expressing calnexin. For Kb and Kd, the calnexin-independent biogenesis occurred regardless of whether the MHC class I molecules contained human or mouse beta 2-microglobulin. Also addressed was the effect of calnexin on the surface expression of Kb molecules bearing the immunodominant peptide from ovalbumin (OVA257-264). This was detected with a recently described monoclonal antibody specific for the Kb/peptide complex. Calnexin expression had no significant effect on the formation of Kb/peptide complexes generated from full-length OVA, cytosolic OVA257-264, or endoplasmic reticulum-targeted OVA257-264, which was expressed in the presence of the herpes simplex virus ICP47 protein to ensure detection of TAP-independent peptide-MHC class I complexes. Complementary results were obtained with TAP-independent formation of Kd/ peptide complexes. These findings indicate that calnexin is not required for the efficient assembly of MHC class I molecules with TAP-dependent or independent peptides.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Intranasal immunization with cytotoxic T-lymphocyte epitope peptide and mucosal adjuvant cholera toxin: selective augmentation of peptide-presenting dendritic cells in nasal mucosa-associated lymphoid tissue.

We previously reported that cholera toxin (CT) was required as a mucosal adjuvant for the induction of peptide-specific cytotoxic T lymphocytes (CTL) following intranasal immunization with CTL epitope peptides (A. Porgador et al., J. Immunol. 158:834-841, 1997). The present study was performed to identify the site and the antigen-presenting cell (APC) population responsible for the presentation of intranasally administered CTL epitope peptide immunogens and to determine whether CT directly affects antigen presentation by these APCs. For these experiments, C57BL/6 mice were intranasally immunized with the ovalbumin H-2Kb-restricted CTL epitope SIINFEKL with or without CT. Cells were then isolated from the cervical lymph nodes (CLN) and the nasal mucosa-associated lymphoid tissue (NALT) and tested for the ability to stimulate the B3Z T-cell hybridoma, which recognizes SIINFEKL in association with H-2Kb. Dendritic cell (DC)-enriched CLN cells from mice immunized with peptide and CT or peptide only could stimulate B3Z cells, while DC-depleted CLN cells from either group were unable to stimulate B3Z cells. NALT cells of mice immunized with peptide and CT, but not with peptide alone, were able to efficiently stimulate B3Z hybridomas. Depletion of N418-positive DC from these NALT cells resulted in significant reduction of B3Z activation. Our results indicate that DC are the APC responsible for the presentation of CTL epitope peptides following intranasal immunization and that CT augments the ability of dendritic cells in the NALT, but not in the draining CLN, to present CLT epitope peptides. This finding suggests that CT acts locally as a mucosal adjuvant and that NALT DC are the predominant APC involved with the induction of immunity after intranasal immunization with peptide immunogens and CT.

Adjuvants, Immunologic↗
PubMed1997

Natural killer cell lines kill autologous beta2-microglobulin-deficient melanoma cells: implications for cancer immunotherapy.

Cancer vaccines used to generate specific cytotoxic T lymphocytes are not effective against tumor cells that have lost or suppressed expression of their class I major histocompatibility complex proteins. This loss is common in some cancers and particularly in metastatic lesions. We show that beta2-microglobulin-deficient class I-negative melanoma variants derived from patients undergoing specific T cell therapy are lysed by heterologous as well as autologous natural killer (NK) lines and clones, but not by specific T cells. Moreover, the minor NK cell fraction but not the major T cell fraction derived from heterologous lymphokine activated killer cells kills those tumor cell lines. ICAM-1 expression by the different class I protein deficient tumors was correlated with their sensitivity to lysis by NK cells. Adoptive autologous NK therapy may be an important supplement to consider in the design of new cancer immunotherapies.

Cancer Vaccines↗
PubMed1997

Production of a specific major histocompatibility complex class I-restricted epitope by ubiquitin-dependent degradation of modified ovalbumin in lymphocyte lysate.

Peptide epitopes presented through class I major histocompatability complex (MHC class I) on the cell surface, are generated by proteolytic processing of protein-antigens in the cytoplasm. The length and amino acid sequence determine whether a given peptide can fit into the peptide binding groove of class I heavy chain molecules and subsequently be presented to the immune system. The mode of action of the processing pathway is therefore of great interest. To study the processing mechanism of MHC class I-restricted intracellular antigens, we reconstituted the proteolytic processing of a model antigen in a cell-free system. Incubation of oxidized and urea-treated OVA in lymphocyte lysate resulted in partial degradation of the antigen. Degradation of the antigen depended on the presence of ATP. Addition of methylated ubiquitin abolished the reaction which was then restored by addition of an excess of native ubiquitin, indicating that the breakdown of the antigen in lymphocyte lysate is mediated by the ubiquitin proteolytic system. Upon incubation of modified OVA in lymphocyte lysate, a specific antigenic peptide was generated. The peptide was recognized by cytotoxic T lymphocytes directed against OVA-derived, H-2Kb-restricted peptide (SIINFEKL), and by a monoclonal antibody that recognizes cell-bound Kb-SIINFEKL complexes. Formation of the peptide epitope depended on the presence of ATP and ubiquitin. These results indicate that proteolytic processing of modified OVA is carried out by the ubiquitin-mediated degradation system. The experimental system described provides a tool to analyze the molecular mechanisms underlying the generation of specific, MHC class I-restricted peptide epitopes.

Animals↗
PubMed1997

Direct delivery of exogenous MHC class I molecule-binding oligopeptides to the endoplasmic reticulum of viable cells.

After brief incubation of cells with fluorescein-conjugated peptides that bind major histocompatibility complex (MHC) class I molecules, peptides were detected within the endoplasmic reticulum (ER) by microscopy or by binding to radiolabeled class I molecules. ER delivery of a nonfluorescent peptide was demonstrated using a mAb highly specific for the peptide-class I molecule complex. ER localization of peptides: (i) required expression of appropriate class I molecules in the ER but not on the cell surface, (ii) was diminished by expression of TAP, the MHC-encoded cytosol to ER peptide transporter, and (iii) was blocked by pinocytosis inhibitors but not by brefeldin A. These findings demonstrate the existence of a pathway, likely vesicular in nature, that conveys small extracellular substances to the ER without traversing the Golgi complex or the cytosol. This pathway contributes to the loading of exogenous peptides to MHC class I molecules, but its evolutionary significance may lie in other cellular processes, such as maintaining ER homeostasis or signaling by extracellular substances.

Cell Line↗
PubMed1997

Intranasal immunization with CTL epitope peptides from HIV-1 or ovalbumin and the mucosal adjuvant cholera toxin induces peptide-specific CTLs and protection against tumor development in vivo.

To evaluate the ability of mucosal immunization protocols using peptide immunogens to induce CTL responses, BALB/c and C57BL/6 mice were immunized intranasally (i.n.) with peptides corresponding to a known CTL epitope in HIV-1 glycoprotein 120 or OVA, respectively, and the mucosal adjuvant cholera toxin (CT). Intranasal immunization of BALB/c mice with a 10- or 15-amino acid peptide corresponding to a CTL determinant in HIV-1 glycoprotein 120 and CT induced peptide-specific CTLs in spleen cells that persisted through 35 days after the last immunization. Intranasal immunization of C57BL/6 mice with the octameric OVA peptide and CT produced similar results with detectable peptide-specific CTL in both the cervical lymph node and spleen. To test whether CTL induced by i.n. immunization with OVA peptide and CT were functional in vivo, groups of C57BL/6 mice were injected with E.G7-OVA tumor cells that express the OVA protein and monitored for tumor growth. Animals immunized i.n. with OVA and CT were protected against tumor development as efficiently as animals immunized by the potent CTL induction protocol of i.v. injection with OVA-pulsed dendritic cells. Intranasal immunization with peptides corresponding to known CTL epitopes and CT provides a noninvasive route of immunization for the induction of CTL responses in vivo.

Adjuvants, Immunologic↗
PubMed1997

Localization, quantitation, and in situ detection of specific peptide-MHC class I complexes using a monoclonal antibody.

CD8+ T lymphocytes recognize antigens as short peptides bound to MHC class I molecules. Available methods cannot determine the number and distribution of these ligands on individual cells or detect antigen-presenting cells in tissues. Here we describe a method for eliciting and identifying monoclonal antibodies specific for a particular peptide-MHC class I combination. One such antibody can identify antigen complexes with a limit of detection approaching that of T cells. We used this antibody to determine the number of peptide-class I complexes generated upon viral infection, to identify antigen-presenting cells in cell mixtures, to determine the site of peptide-MHC class I interaction inside cells, and to visualize cells bearing specific peptide-MHC class I complexes after in vivo infection. Similar antibodies may prove useful for diagnostic or therapeutic purposes in cancer, infectious diseases, and autoimmune disorders.

Animals↗
PubMed1996

Induction of antitumor immunity using bone marrow-generated dendritic cells.

We have previously shown that bone marrow-generated dendritic cells (DC) pulsed with a class I-restricted peptide are potent inducers of CD8+ CTL. In the present study we have investigated whether bone marrow-generated DC are capable of inducing antitumor immunity. We show that a single immunization with DC pulsed with OVA peptide was highly effective in eliciting a protective immune response against a challenge with tumor cells expressing the OVA gene (E.G7-OVA), more so than immunization with irradiated E.G7-OVA cells, OVA peptide-pulsed RMA-S cells, or free OVA peptide mixed with adjuvant. The addition of free OVA protein to day 4 or day 7 bone marrow cultures, but not to day 9 mature DC, was also effective in eliciting CTL and engendering antitumor immunity, but was less effective than peptide-pulsed DC. Induction of CTL and antitumor immunity by bone marrow-generated DC pulsed with the class I-restricted OVA peptide correlated with the expression of syngeneic MHC class II molecules on the DC. This and the fact that induction of tumor immunity was dependent on CD4+ T cells suggest that in vivo priming of CTL and induction of antitumor immunity by bone marrow-generated DC also require the presentation of MHC class II-restricted epitopes and activation of CD4+ T cells. This observation has potentially important implications to the use of peptide-pulsed DC in clinical immunotherapy.

Animals↗