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H K Ziegler

Publications and source records attributed to H K Ziegler.

At least 19 recordsLinked to original sources
PubMed1995

Nonviable bacterial antigens administered with IL-12 generate antigen-specific T cell responses and protective immunity against Listeria monocytogenes.

The development of effective vaccine strategies for intracellular pathogens, including bacteria, viruses, and parasites, is one of the major frontiers of scientific research. For the studies described here, the murine model of Listeria infection was used to evaluate the adjuvant effects of IL-12 when used as an immunization component. These studies revealed that typically nonimmunogenic doses of heat-killed Listeria monocytogenes, or soluble listerial Ag preparations, elicit intense Th1-type Listeria-specific T cell responses when administered i.p. along with recombinant murine IL-12. In addition to the Ag-specific production of IL-2 by CD4+ peritoneal cells that was elicited, several other correlates of protective responses were noted, including dramatic induction of CD3+ and alpha beta TCR+ cell populations in the peritoneal cavity and increased expression of class II MHC and production of IL-12 (upon in vitro restimulation) by peritoneal macrophages. Protection studies demonstrated that the T cell responses elicited by a IL-12-potentiated, heat-killed L. monocytogenes vaccine were sufficient to effectively protect mice against challenge with a large dose of virulent Listeria.

Animals↗
PubMed1995

Mechanisms of T cell epitope immunodominance analyzed in murine listeriosis.

We demonstrate conclusively that the bacterial exotoxin listeriolysin O (LLO) is a target Ag for eliciting CD4+ T cell responses following infection with Listeria monocytogenes. The minimal I-Ek-restricted immunodominant CD4+ T cell epitope was identified as peptide 215-226 (p215-226). Most LLO-specific T cell hybridomas recognized p203-226, p208-226, p215-226, and p215-234, although each exhibited a characteristic pattern of preferential reactivity. One hybridoma (IIIC5) reacted to p203-226 but not to p208-226 or any other LLO peptide tested. With APCs from B10 congenic mice and cells transfected with either I-Ak or I-Ek, IIIC5 recognized p203-216 with I-Ak, while a different hybridoma (IB5) recognized p215-226 with I-Ek. Competitive binding studies demonstrated that of 15 LLO peptides tested, only p203-226, p215-226, and p215-234 had high affinity for I-Ek, while p203-226 could also bind to isolated I-Ak. Of nine LLO peptides tested, only p215-234 bound multiple class II MHC alleles. These findings suggest that the immunodominance of p203-226 may be due in part to the presence of multiple T cell epitopes with I-Ek- and I-Ak-binding capability. Many of the rules of immunodominance observed with model Ags are also operative in our murine model of bacterial infectious disease. Furthermore, a novel mechanism of immunodominance based on newly defined structural features of MHC molecules is implicated. This information is crucial for rational vaccine development.

Amino Acid Sequence↗
PubMed1995

Activation of gamma delta T cells for production of IFN-gamma is mediated by bacteria via macrophage-derived cytokines IL-1 and IL-12.

Gamma delta T cells are found at sites of microbial infection and have been reported to proliferate in response to bacterial Ags. We show here that although the response by Listeria-elicited peritoneal gamma delta T cells to heat-killed bacteria in the presence of macrophage accessory cells may be partially mediated via the TCR, it is predominantly mediated via cytokines produced by the macrophages. Macrophage cytokines IL-12 and IL-1 synergize to induce some proliferation and considerable IFN-gamma production by peritoneal gamma delta T cells. This cytokine synergy pattern differs from that reported for NK cells, in which IL-12 in combination with either IL-2 or TNF-alpha induces NK cells to produce IFN-gamma. The combination of IL-12 and IL-1 provides a strong stimulus for IFN-gamma production by gamma delta T cells, but a relatively weak signal for proliferation. This is in contrast to the strong proliferative signal from the combination of IL-7 and IL-1 and the relatively weak stimulation of IFN-gamma production by the IL-7/IL-1 combination. Thus, there is differential regulation of NK and gamma delta T cells by cytokines and differential regulation of activation functions within the gamma delta T cell population by combinations of cytokines. These data provide evidence for a potentially important pathway for augmentation of IFN-gamma secretion at sites of infection where gamma delta T cells are found in abundance and where IFN-gamma may play a major role in the control of the infection.

Animals↗
PubMed1995

Delivery of class I and class II MHC-restricted T-cell epitopes of listeriolysin of Listeria monocytogenes by attenuated Salmonella.

Using a Salmonella vaccine-Listeria infection model of intracellular infection, we studied the capacity of an attenuated strain of Salmonella carrying T-cell epitopes of listeriolysin (LLO) of L. monocytogenes to elicit epitope-specific T-cell responses. Class II (LLO 215-226) or class I (LLO 91-99) MHC-restricted T-cell epitopes of LLO were inserted within a central, hypervariable domain of the flagellin protein of an attenuated delta aroA Salmonella dublin strain. T cells from Listeria-immunized mice were activated by lysates or heat-killed preparations of Salmonella construct expressing the LLO 215-226 epitope, indicating that LLO 215-226 is processed and presented to T cells when offered to antigen-presenting cells as part of a flagellin-epitope fusion protein. The chimeric flagellin genes were integrated into the chromosome of the flagellin-negative S. dublin strain to obtain stable expression of the epitopes. Immunization with the living, chromosomally integrated Salmonella construct carrying LLO 215-226 epitope as part of the flagellin protein generated T cells reactive with the corresponding LLO peptide, indicating that this chimera can stimulate a class-specific immune response in vitro. The effect of flanking residues on the processing and presentation of MHC class I LLO 91-99 epitope was studied using Salmonella vaccine strains that express chimeric flagellins containing one of three LLO 91-99 inserts: 91-99 (normal flagellin amino acids as flanking residues); KK91-99KK (Lys-Lys flanking residues); and AAA91-99AAA (Ala-Ala-Ala flanking residues).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Induction of a cellular immune response to a defined T-cell epitope as an insert in the flagellin of a live vaccine strain of Salmonella.

Attenuated strains of Salmonella have been used as vaccines to deliver heterologous antigens mainly to generate a humoral immune response. However, little is known about their ability to induce a cell-mediated immune response to the T-cell epitopes of another infectious agent or how optimally to deliver these epitopes to the host immune system. In order to study this question, a well defined MHC class II-restricted epitope (residues 88-103) from moth cytochrome C (MCC) was inserted into the central hypervariable domain of the flagellin of an attenuated strain of Salmonella dublin. The resulting flagellin was exported to the bacterial surface and polymerized into flagellar filaments that contained multiple copies of the MCC epitope. When flanked by Lys-Lys cathepsin B cleavage sites to facilitate its proteolytic release within the endosomal compartment of antigen-presenting cells, the MCC-chimeric flagellin epitope was efficiently processed in vitro by mouse peritoneal macrophages and presented to 2B4 T-hybridoma cells (specific for the MCC epitope 88-103). Stable expression of the epitope and a higher immune response was obtained in H-2k mice by integrating the chimeric flagellin gene into the chromosome of the vaccine strain. Bacteria with MCC-chimeric flagellins that were expressed from a stable chromosomal locus and flanked by cathepsin B cleavage sites were cleared more rapidly from the livers and spleens of transgenic mice with T-cell receptor (TCR) alpha and beta chains specific for the MCC epitope than were bacteria lacking the epitope. Antigen processing and presentation of class II-restricted epitopes expressed as chimeric proteins by attenuated bacterial vaccine vectors may be facilitated by the presence of endosomal protease cleavage sites on each side of the epitope and by chromosomal integration of the coding sequence.

Amino Acid Sequence↗
PubMed1995

Human T-cell recognition of Listeria monocytogenes: recognition of listeriolysin O by TcR alpha beta + and TcR gamma delta + T cells.

The cell-mediated immune response to Listeria monocytogenes has been well characterized in the mouse. Listeriolysin O (LLO) is a major antigen in murine T-cell recognition of L. monocytogenes. In this study, we show that LLO is also recognized by human TcR alpha beta T cells and TcR gamma delta T cells. Human peripheral blood mononuclear cells (PBMC) cultured in vitro with live listeriae and then expanded with interleukin 2 were shown to respond to purified LLO. The generation of LLO-responsive T cells was dependent on the use of live bacteria during the initial in vitro challenge. LLO-induced proliferation of T cells expanded by exposure of PBMC to live listeriae was major histocompatibility complex restricted. PBMC cultured with formalin-fixed listeriae and subsequently expanded by interleukin 2 gave high proliferative responses to fixed bacteria but failed to respond to LLO. PBMC stimulated in vitro with fixed listeriae contained predominantly TcR alpha beta + T cells. In contrast, PBMC obtained from 85% of the donors studied generated high numbers of TcR gamma delta + T cells following in vitro culture with live listeriae. Using a panel of synthetic amphipathic LLO peptides, we found that LLO-specific T cells from different individuals recognized both common and unique peptides. LLO 470-508 was recognized by three of five individuals, while LLO 203-226 and LLO 107-126 were recognized by two of six individuals. A TcR gamma delta + T-cell line was established from PBMC stimulated with live listeriae and was shown to recognize LLO 470-508. Proliferative responses could be induced in this cell line by peptide-pulsed autologous PBMC but not by peptide-pulsed allogeneic PBMC. Our results establish the importance of LLO in human T-cell recognition of listeriae and show that both TcR alpha beta + T cells and TcR gamma delta + T cells recognize this antigen. Finally, since LLO 470-508 has a high degree of homology with other gram-positive bacterial toxins, the recognition of this peptide by TcR gamma delta + T cells suggests that an important role of these T cells in host defense is the recognition of bacterium-derived toxins.

Amino Acid Sequence↗
PubMed1993

Induction of murine peritoneal gamma/delta T cells and their role in resistance to bacterial infection.

Previous studies have reported an association of gamma/delta T cells with microbial infection in both human lesions and murine infectious disease models. In this study we provide a comprehensive analysis of the conditions under which the induction of gamma/delta T cells occurs at a site of infection. We found a site-specific induction of gamma/delta T cells after the injection of Listeria monocytogenes in the peritoneal cavity of C3H mice. No changes were seen in the splenic or lymph node populations after these injections. Both the proportion and the absolute number of gamma/delta T cells increased in the peritoneal cavity. Additionally, when peritoneal T cells from Listeria-immune mice were restimulated in vitro, the induced gamma/delta T cells exhibited a greater expansion potential than the alpha/beta T cells. Neither the induced gamma/delta T cells nor those from normal mice expressed CD4 or CD8 on the cell surface. Thy-1 was expressed on only 29% of normal peritoneal gamma/delta T cells, but after intraperitoneal Listeria injection 65% of induced gamma/delta T cells expressed. Thy-1, Pgp-1 and CD45R expression on both normal and induced gamma/delta T cells was consistent with an activation phenotype. Significant increases in peritoneal gamma/delta T cells were not seen until 5-7 d after Listeria injection. The proportion of the CD3+ population expressing the gamma/delta T cell receptor remained elevated for 6-7 wk, while the absolute numbers of peritoneal gamma/delta T cells declined gradually over this time period, reflecting a decrease in both the number of lymphocytes and the percentage of these that were CD3+. Peak numbers of gamma/delta T cells were seen at day 10 with live microbes such as Listeria. A variety of microbes, toxins, mitogens, antigens, cytokines, and nonspecific inflammatory agents were evaluated for their ability to induce gamma/delta T cells in the peritoneal cavity. Both Gram-positive and Gram-negative bacteria as well as Mycobacteria were able to induce gamma/delta T cells that showed increased in vitro expansion potential. An exotoxin from a Gram-positive organism, listeriolysin-o, and the lipopolysaccharide (LPS) endotoxin from a Gram-negative organism were also effective. gamma/delta T cell responses to LPS were under lps gene control. Peak numbers of gamma/delta T cells were observed at day 3 after injection with exotoxins and endotoxins. Modifications that abrogated the virulence of a bacterial strain also eliminated the inductive effect for gamma/delta T cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Intercellular interactions and cytokine responsiveness of peritoneal alpha/beta and gamma/delta T cells from Listeria-infected mice: synergistic effects of interleukin 1 and 7 on gamma/delta T cells.

Peritoneal gamma/delta T cells from Listeria-immune mice show an enhanced potential to expand when restimulated with antigens or mitogens in vitro (see companion paper [Skeen, M. J., and H. K. Ziegler. 1993. J. Exp. Med. 178:971]). When cocultured with peritoneal alpha/beta T cells, the gamma/delta T cell population expanded preferentially even when the in vitro stimulus was specific for the alpha/beta T cell population. Purified gamma/delta T cells did not respond to alpha/beta T cell-specific stimuli. If isolated T cell subsets were recombined in cell mixing experiments, the resulting proliferative response was greater than additive. Irradiated alpha/beta T cells could enhance the proliferation of responding gamma/delta T cells, but the effect was unidirectional; i.e., irradiated gamma/delta T cells did not stimulate responding gamma/delta T cells. This effect appeared to be cytokine mediated and did not require cell-cell contact. Both recombinant interleukin 2 (rIL-2) and rIL-7 could support the expansion of the gamma/delta T cells, while rIL-7 was only minimally stimulatory for the alpha/beta T cells. The magnitude of the response by gamma/delta T cells to rIL-7 exceeded the response to other in vitro stimuli, including immobilized anti-T cell receptor monoclonal antibody, and was 50-100-fold greater than the alpha/beta T cell response to IL-7. This unique sensitivity of gamma/delta T cells to IL-7 was strongly enhanced by the presence of accessory cells. These cells could be replaced by rIL-1, establishing a synergy for IL-1 and IL-7 as factors that could uniquely stimulate this gamma/delta T cell population. Isolated peritoneal gamma/delta T cells from Listeria-immune mice react to heat-killed Listeria preparations in the presence of macrophages accessory cells in a non-H-2-restricted manner. Considered collectively, these results suggest a potential mechanism by which gamma/delta T cells can predominate in epithelial tissues and at sites of infection.

Animals↗
PubMed1993

Mechanisms of processing and presentation of the antigens of Listeria monocytogenes.

Antigen processing is the series of events through which protein antigens become degraded and are sent to the cell surface for recognition by T cells. These events within the cell have been studied extensively using the model system of infection with Listeria monocytogenes. This bacteria resides primarily intracellularly; immunity to it is mediated by cellular responses. Upon phagocytosis by a macrophage, engulfed Listeria express a hemolytic molecule, listeriolysin O (LLO), and can disrupt the membrane of the endosome and escape into the cellular cytoplasm. This allows the organism to escape the hostile environment of the endosome and also gives it access to the processing machinery of the cytoplasm. Therefore, Listeria monocytogenes can be processed within an endosome and be presented by class II MHC, or can escape into the cytoplasm and be processed there and presented by class I MHC. This LLO molecule is not only an important virulence factor, it is also a dominant antigen in the cellular immune response to Listeria. The outcome of antigen processing and presentation can be influenced by the expression of LLO, by the state of activation of the macrophage, and by the cytokines involved in the immune response.

Animals↗
PubMed1992

Listeria monocytogenes activation of human peripheral blood lymphocytes: induction of non-major histocompatibility complex-restricted cytotoxic activity and cytokine production.

Gram-negative bacteria have been shown to activate human natural killer (NK) cells. In this report, we show that the gram-positive bacterium Listeria monocytogenes can also activate human NK cells with regard to non-major histocompatibility complex (MHC)-restricted killing and the production of cytokines. Overnight incubation of peripheral blood mononuclear (PBM) cells or enriched NK cell populations with live or Formalin-fixed L. monocytogenes resulted in high levels of non-MHC-restricted cytotoxic activity. Listeria-stimulated non-MHC-restricted cytotoxic activity could be achieved with pathogenic as well as nonpathogenic Listeria strains. PBM cells also produced tumor necrosis factor alpha and different interferons (IFNs) after incubation with Listeria strains. Optimal cytokine production appeared to be dependent on nylon wool- and plastic-adherent cells. Different IFNs were produced by Listeria-stimulated PBM cells obtained from different donors. IFN-gamma was always produced but was sometimes associated with IFN-alpha and/or IFN-beta. Interleukin-2 (IL-2) activity was never detected in culture supernatants obtained from Listeria-stimulated PBM cell cultures. However, IL-2 appeared to be produced by Listeria-stimulated PBM cells, since antibody to IL-2 inhibited Listeria-stimulated NK cell cytotoxic activity. Listeria activation of NK cell cytotoxic activity was also dependent on tumor necrosis factor alpha production. Antibody to IFN-gamma, IFN-beta, or IFN-alpha had no effect on Listeria-stimulated NK cell cytotoxic activity. These results demonstrate that NK cells can be activated by Listeria strains and add further evidence that NK cells may play an important role in host defense against bacterial infections.

Cytokines↗
PubMed1991

Role of bacterial hemolysin production in induction of macrophage Ia expression during infection with Listeria monocytogenes.

The production of a hemolytic exotoxin (Hly) termed listeriolysin O (LLO) is a major determinant of the virulence of the Gram-positive bacterium Listeria monocytogenes. As determined by lethal inoculum size, LLO- strains of L. monocytogenes generally are several orders of magnitude less virulent than their LLO+ counterparts. The generation of protective anti-Listeria T cell immunity also has been shown to depend on the LLO phenotype of the bacteria present during primary infection, although the cellular basis of this observation is not known. The experiments described here address the role of LLO in regulation of the expression of class II MHC (Ia) molecules by murine macrophages. Because Ia expression by macrophages and other APC is thought to be a central factor in the generation of T cells specific for bacterial Ag, we have tested the hypothesis that the failure of LLO- strains to elicit anti-Listeria T cell responses might be secondary to an inability of these strains to stimulate increases in macrophage Ia levels. Our results show that the macrophage Ia response after i.p. injection of L. monocytogenes correlates strongly with the LLO phenotype of the bacteria. The presence of LLO+ organisms, even at very small numbers (as few as 10), elicits a striking increase in Ia expression by peritoneal macrophages. In contrast, even at very high numbers (up to 10(6) per mouse), LLO- bacteria fail to stimulate a strong Ia response. We also have analyzed macrophage Ia expression after injection of lysates of Escherichia coli expressing recombinant LLO protein. Similar to the results obtained with LLO+ and LLO- L. monocytogenes, we have observed Ia induction only with LLO+ lysates. Ia induction by this crude recombinant LLO preparation can be inhibited by cholesterol or heat. Furthermore, supernatants derived from cultures of LLO+ (but not LLO-) L. monocytogenes can cause Ia induction when administered via i.p. injection. Taken together, these findings suggest that the failure of macrophages to respond to LLO- organisms with an increase in Ia expression may be a major underlying cause of the failure of these bacteria to induce Listeria-specific protective T cell immunity. Furthermore, we propose that the induction of macrophage Ia expression in response to bacterial toxins such as Hly may represent one component of a set of early, innate immune mechanisms, and that this induction may provide a critical "bridge" to later, acquired, Ag-specific immune processes.

Animals↗
PubMed1991

Lipopolysaccharide responsiveness is an important factor in the generation of optimal antigen-specific T cell responses during infection with gram-negative bacteria.

We previously have found that the endotoxin (LPS) of Gram-negative bacteria is a major determinant of macrophage Ia induction during infection with these organisms. Specifically, i.p. injection of Gram-negative bacteria elicits a striking macrophage Ia response in LPS-responder mice but virtually no response in LPS-low-responder mice. As an extension of these findings, in this report we have tested the hypothesis that the inability of LPS-low responder mice to mount an Ia response during Gram-negative infection may in turn impair their capacity for generation of appropriate antibacterial T cell responses. Our results demonstrate that for a variety Gram-negative organisms (Salmonella typhimurium, Salmonella minnesota, and Escherichia coli), both macrophage Ia induction and the generation of Ag-specific T cell responses are controlled by the lps gene. We also have asked whether the expression of additional toxins (other than LPS) by infecting Gram-negative organisms can "override" this lps gene control of macrophage and T cell responses. We have found that infection of LPS-low-responder mice with an E. coli strain that expresses a hemolytic exotoxin (Hly) leads to the induction of macrophage Ia expression as well as the generation of T cell responses to both the Hly molecule and to other E. coli-associated Ag, whereas no responses are generated during infection with a Hly- strain. This result suggests that LPS-low responder mice have no inherent defect in T cell responsiveness to Gram-negative bacterial Ag but rather that these mice fail to receive an LPS-mediated signal required for the induction of Ia expression and subsequent generation of peritoneal T cell immunity. These findings, when taken together with results presented in the accompanying paper, strengthen the argument that bacterial toxin production (and the ability of the host to respond to the toxin) can represent a critical determinant of the induction of macrophage Ia expression and in turn, of Ag-specific T cell responses during bacterial infection.

Animals↗
PubMed1991

Role of listeriolysin-O (LLO) in the T lymphocyte response to infection with Listeria monocytogenes. Identification of T cell epitopes of LLO.

Using a murine model, we investigated the role of the bacterial exotoxin listeriolysin O (LLO) in cellular immunity to Listeria monocytogenes. A correlation between LLO production by infecting bacteria and generation of protective immunity to virulent LLO-producing bacteria was noted. Using isogeneic hemolysin (Hly+ or Hly-) strains of L. monocytogenes, we demonstrated that LLO production by infecting bacteria is required to elicit T cells reactive both to bacteria-associated Ag and to the secreted LLO molecule as measured by IL-2 production in vitro. Distinct sets of T cells specific for largely nonoverlapping pools of antigenic determinants represented by LLO and cell-associated Ag (heat-killed L. monocytogenes) are generated after infection. We have used models for prediction of T cell epitopes based on primary structure of LLO, and synthetic amphipathic LLO peptides were evaluated as Ag in vitro or as immunogenes in vivo. Infection of several strains of mice (H-2k and H-2d) with LLO-producing L. monocytogenes resulted in the generation of T cells that could respond consistently to two peptides, LLO 215-234 and LLO 354-371. Mouse strains lacking expression of I-E molecules (e.g., B10.A(4R) and C57BL/6) responded to LLO but not to the peptides tested. With C3HeB/FeJ mice, antibodies to I-Ek blocked the presentation of LLO 215-234. The importance of the N-terminal portion of LLO 215-234 was evidenced by the drastic reduction in antigenic activity of truncated peptides (e.g., LLO 221-234 and LLO 224-234). LLO 215-234, the strongest and most consistent activator of T cells from L. monocytogenes-immune mice, fit well some models for antigenic peptides in several ways. It could be predicted to form an amphipathic alpha-helix, it contained multiple "Rothbard motifs" (charged residue or glycine, two or three hydrophobic amino acids and then a glycine or polar residue), it had a net charge of +2, and it contained the correct spacing of amino acids (five to six residues between a hydrophobic and basic amino acid) that is characteristic of I-Ek-binding peptides. Immunization with 8 of 10 synthetic LLO peptides generated T cells that recognized the immunizing peptide in vitro, but such T cells were only poorly reactive with LLO. Our results indicate that LLO is an important target Ag for stimulation of CD4+ L. monocytogenes-specific T cells, and that LLO 215-234 is antigenically dominant in C3HeB/FeJ mice.

Animals↗
PubMed1990

Induction of macrophage Ia expression in vivo by a synthetic block copolymer, L81.

Certain synthetic, nonionic, block copolymers of polyoxypropylene and polyoxyethylene are potent immunoadjuvants. While investigating their mechanisms of action, we found that one of these copolymers, L81, induced the expression of macrophage Ia in vivo. L81 is a 2750-Da, linear copolymer with a 43-unit core of hydrophobic polyoxypropylene flanked on each side by 3 U of hydrophilic polyoxyethylene. It induced threefold to sevenfold increases in the proportion of peritoneal macrophages expressing I-A from 5 to 7 days after an i.p. injection of 5 mg. As little as 1 mg caused a twofold increase. I-A density increased with time after injection of L81. Macrophages induced by L81 actively synthesized I-A, showing an 18-fold increase in biosynthetic capacity. Ia induction did not require the presence of mature T lymphocytes, because similar increases in I-A expression were seen in athymic and euthymic mice. L81-induced macrophages were 10-fold more effective than normal macrophages in presenting Ag to a T cell hybridoma. Other functional studies showed that they were primed for the secretion of superoxide ion and could be stimulated in vitro by IFN-gamma and LPS to lyse tumor target cells. These results suggest that the induction of macrophage Ia expression by L81 may play a role in its activity as an immunoadjuvant.

Adjuvants, Immunologic↗
PubMed1990

Intracellular hemolysin-producing Listeria monocytogenes strains inhibit macrophage-mediated antigen processing.

We found that virulent hemolysin-producing (Hly+) Listeria monocytogenes strains inhibit antigen processing and presentation when added to macrophages in vitro. A virulent Hly- bacteria caused little or no inhibition. Live Hly+ bacteria inhibited presentation of both heat-killed L. monocytogenes and ovalbumin. Several observations indicate that hemolysin produced by intracellular bacteria was responsible for the inhibition. First, inhibition was observed even when extracellular bacteria were removed after a brief 10-min bacterial uptake period. Second, inhibition was not prevented by the addition of cholesterol, a substance which inactivates soluble hemolysin. Third, only very high concentrations of soluble hemolysin were inhibitory. Under conditions which inhibit antigen presentation (10(5) per well), macrophages retained normal levels of Ia, maintained normal morphology, and were not permeable when assayed by chromium release. The uptake and catabolism of 35S-labeled live bacteria by macrophages were similar for both Hyl+ and Hly- bacteria. Only a small decrease in uptake and catabolism of surface-iodinated heat-killed L. monocytogenes by macrophages pretreated with inhibitory numbers of live Hly+ bacteria was observed. Additionally, macrophages pretreated with live Hly+ bacteria and fixed 1.5 h later were able to effectively present an ovalbumin peptide (amino acids 323 to 339) to the T-cell hybridoma DO11.10. Hemolysin-producing bacteria inhibited the presentation of antigens that need processing better than they did of antigens that do not require a processing event. Thus, we have demonstrated inhibition of an intracellular antigen processing pathway by hemolysin-producing L. monocytogenes, which may contribute to the virulence of this pathogen.

Animals↗
PubMed1990

Cloning and characterization of T-cell-reactive protein antigens from Listeria monocytogenes.

To explore the molecular basis of the T-cell-mediated immune response to Listeria monocytogenes, we cloned and expressed listerial antigens in Escherichia coli using the lambda-ZAP bacteriophage and Bluescript plasmid vectors. A two-stage screening strategy was implemented to identify T-cell-reactive antigens; the first stage involved antibodies or oligonucleotide probes and the second stage was based on assays for T-cell activation. A library of genomic DNA from L. monocytogenes was generated in lambda-ZAP, and then antigens, were detected in infected cells with a polyclonal rabbit anti-L. monocytogenes antiserum and an L. monocytogenes-specific monoclonal antibody. Also, synthetic oligonucleotide probes corresponding to the structural gene for listeriolysin O (LLO) were used to screen the recombinant DNA library. In each case, positive isolates were evaluated for T-cell antigenicity by measuring antigen-induced interleukin-2 production by polyclonal T cells taken from L. monocytogenes-immune mice. Phage clones were subcloned and expressed in the Bluescript plasmid and tested further for antigenic activity and LLO expression. Using this screening strategy, we successfully identified bacterial clones producing recombinant listerial antigens which activate L. monocytogenes-immune T cells in vitro. Antigens operative in the T-cell response during infection with L. monocytogenes include LLO, 62- and 39-kilodalton proteins, and other poorly defined bacterial surface components. We also found that high concentrations of recombinant LLO inhibited macrophage-mediated antigen presentation. These results are discussed in terms of the multiple functions of LLO as a virulence factor, inhibitor of antigen presentation, and potent antigen in the T-cell response to L. monocytogenes. These studies represent the first step toward a genetic definition of the antigens recognized in immune defense to L. monocytogenes.

Animals↗