Exaggerated proinflammatory and Th1 responses in the absence of gamma/delta T cells after infection with Listeria monocytogenes.

While gamma/delta T cells are involved in host defense and immunopathology in a variety of infectious diseases, their precise role is not yet clearly defined. In the absence of gamma/delta T cells, mice die after infection with a dose of Listeria monocytogenes that is not lethal in immunologically intact animals. Morbidity might result from insufficient levels of cytokines normally produced by gamma/delta T cells or conversely from an excess of cytokines due to a lack of down-regulation of the inflammatory response in the absence of gamma/delta T cells. Consistent with a regulatory role, we found that systemic levels of proinflammatory cytokines (interleukin-6 [IL-6], IL-12, and gamma interferon [IFN-gamma]) were significantly higher in the absence of gamma/delta T cells during the innate phase of the response. Using combinations of genetically altered and immunodepleted mice, we found evidence for gamma/delta T-cell-mediated regulation of IFN-gamma production by multiple cell types of both lymphoid and myeloid lineages. The antigen-specific alpha/beta T-cell response that followed the exaggerated innate response was also increased in gamma/delta T-cell-deficient mice. These findings are consistent with an emerging picture from a variety of immune response models of a critical role for gamma/delta T cells in down-modulation of the immune response.

The polysaccharide portion of lipopolysaccharide regulates antigen-specific T-cell activation via effects on macrophage-mediated antigen processing.

The lipopolysaccharide (LPS) structure of Salmonella typhimurium has been correlated with the virulence of wild-type strain LT2. Mutants of LT2 with truncated polysaccharide portions of LPS are less virulent than strains with a complete LPS structure. Polyclonal T cells and monoclonal T-cell hybridomas were more reactive to heat-killed rough mutants than to heat-killed smooth strains, as measured by interleukin-2 (IL-2) production. Using a large panel of strains with truncated LPS molecules, we found that T-cell reactivity decreased with certain lengths of polysaccharide. The decreased response was not due to differential phagocytic uptake, IL-12 production, or major histocompatibility complex class II surface expression by macrophages. Also, LT2 did not mediate any global suppression since addition of LT2 did not diminish the response of T cells specific for antigens unrelated to Salmonella. In an experiment in which processing times were varied, we found that antigens from rough strains were processed and presented more quickly than those associated with smooth strains. At longer processing times, epitopes from LT2 were presented well. We hypothesize that the slower antigen processing and presentation of wild-type Salmonella may be caused by masking of surface antigens by the longer polysaccharide portion of smooth LPS. This blocking of effective antigen presentation may contribute to the virulence of Salmonella.

Long-lived protective immunity to Listeria is conferred by immunization with particulate or soluble listerial antigen preparations coadministered with IL-12.

The ability of IL-12 to promote the development of Th1-type immune responses, and thus promote cellular immunity, has been well documented. In a previous report, we showed that coadministration of IL-12 with heat-killed Listeria monocytogenes elicited intense antigen-specific T cell responses that conferred protective listerial immunity. Herein, we have extended those studies by demonstrating that multiple injections of heat-killed L. monocytogenes and IL-12 elicit memory responses that confer long-lived (> or = 3 months) protective immunity and that immunity can be transferred adoptively with cells from immunized mice injected into naive mice. These studies have also demonstrated that the powerful adjuvanticity of IL-12 is observed with soluble as well as particulate immunogens and is operative in mouse strains that have different MHC haplotypes. These findings suggest that IL-12 may be a useful adjuvant component of vaccines for a wide variety of pathogens in animal and human systems.

A synthetic peptide administered with IL-12 elicits immunity to Listeria monocytogenes.

IL-12 is a pivotal cytokine signal for the development of Th1-type cellular responses that are required for control of intracellular pathogens. We previously demonstrated that coinjection of IL-12 with heat-killed Listeria monocytogenes, which was not immunogenic when injected alone, elicited intense Ag-specific T cell responses that conferred protection against subsequent challenge with Listeria. Herein we describe the remarkable finding that a nonimmunogenic synthetic peptide corresponding to a dominant MHC class II (H-2k)-restricted listerial determinant, when coinjected i.p. with murine IL-12, elicited potent Ag-specific immune responses that conferred protective immunity against Listeria.

Gamma delta T lymphocytes regulate the induction and maintenance of oral tolerance.

Oral administration of Ag, over a period of several days, induces a state of tolerance that is associated with activation of CD8+ T cells that can transfer unresponsiveness to naive syngeneic hosts. We previously demonstrated that these T cells are not CTL precursors and that they inhibit responses by CD8+ CTL, as well as Ab and CD4+ T cell responses. Activation of noncytolytic, CD8+ suppressor T cells by oral Ag is a process that is not understood. In these studies, we asked whether depletion of the gamma delta T cells altered induction of oral tolerance. Injection of the anti-delta-chain Ab (GL3) down-modulated the expression of gamma delta TCR and inhibited the induction of oral tolerance to OVA, as measured by Ab, CD4+, and CD8+ T cell responses. GL3 did not activate IL-2 secretion that could be detected in the serum, nor did it induce IL-2R expression by intraepithelial lymphocytes, suggesting that GL3 inhibited the function of gamma delta T cells rather than activating them. This interpretation is supported by our observation that oral administration of Ag did not induce tolerance in TCR-delta knockout mice. These data suggest that gamma delta T cells play a critical, active role in tolerance induced by orally administered Ag.

Protective immunity to Listeria monocytogenes elicited by immunization with heat-killed Listeria and IL-12. Potential mechanism of IL-12 adjuvanticity.

The results presented here demonstrate the striking potentiating effects of IL-12 when it is combined with listerial immunogens. Although HKLM alone does not elicit strong T-cell responses, the results presented here demonstrate that the combination of HKLM and IL-12 elicited vigorous Listeria-specific Th1-type T-cell responses when administered intraperitoneally. The intensity of these responses, as well as the cytokine profiles of the Listeria-specific peritoneal T cells and macrophages, was remarkably similar to that of Listeria-infected/immune mice. These studies also revealed that typically nonimmunogenic forms of soluble listerial antigen preparations (cLLO, SLP) and LLO peptide homologs (M. A. Miller et al., manuscript in preparation) elicited intense Listeria-specific T-cell responses when administered with IL-12. In conjunction with the generation of specific T-cell responses following injection of IL-12 in combination with either killed Listeria or soluble listerial antigen preparations, macrophages from these mice expressed upregulated quantities of class II MHC and produced increased amounts of IL-12 following restimulation in vitro. Protection studies established that the Listeria-specific T-cell responses elicited by the HKLM + IL-12 mixture conferred protective immunity of mice to a lethal dose of viable L. monocytogenes. Studies designed to investigate the regulation of IL-12 production by peritoneal macrophages revealed that activated macrophages are particularly sensitive to bacterial products. However, nonviable or replication-incompetent bacteria or bacterial products injected alone were unable to influence the ability of macrophages to produce IL-12. The ability of activated macrophages to respond to HKLM was dramatically upregulated upon addition of IFN-gamma and markedly downregulated in the presence of the Th2 cytokines, IL-4 and IL-10. In light of what is known about the ability of IL-12 to induce IFN-gamma production by NK cells and gamma delta T cells, these results suggest that the exogenous addition of IL-12 may help initiate a cytokine cascade which enables the immune system to interact productively with an antigen that is typically nonimmunogenic when administered alone. These findings demonstrate that IL-12 may prove to be a powerful and broadly useful adjuvant component of particulate and soluble antigen-based vaccines directed towards many types of intracellular pathogenic microorganisms. Studies aimed at determining the generality of these findings in other infectious disease models as well as experiments designed to further elucidate the mechanism(s) of IL-12 adjuvanticity are continuing.

The presentation of class I and class II epitopes of listeriolysin O is regulated by intracellular localization and by intercellular spread of Listeria monocytogenes.

The hemolysin, listeriolysin 0 (LLO), produced by Listeria monocytogenes is both a virulence factor and an immunodominant Ag. In this study, we investigated how the lytic activity of LLO effects the context of presentation of two known LLO epitopes by either class I or class II MHC molecules. T cell hybridomas were used to monitor each peptide/MHC ligand. APCs infected with strains of Listeria expressing hemolytic LLO strongly presented the class I MHC epitope; however, this ligand was not well presented by cells infected with nonhemolytic Listeria. In contrast, there was almost no presentation of the class II-binding LLO epitope in cells infected with fully hemolytic Listeria. Only hemolysin-deficient Listeria were presented by class II MHC. Listeria expressing wild-type LLO but deficient in other virulence factors showed a presentation pattern equivalent to that of hemolytic Listeria. To address further the divergence of presentation, we used an intercellular spread assay to detect Ag presentation by cells neighboring the primarily infected one. We found that hemolytic Listeria were presented by both class I and II MHC on cells adjacent to the initially infected one(s). Finally, our kinetic analysis of presentation revealed that the class II ligand is presented over 4 h before the class I ligand. We have demonstrated that LLO's lytic activity potentiates presentation of listerial Ags by class I MHC and inhibits presentation via class II MHC. LLO-mediated intracellular localization (cytoplasmic vs endosomal) of bacteria corresponds to the operative presentation pathway.

Interferon-gamma and interleukin-10 have cross-regulatory roles in modulating the class I and class II MHC-mediated presentation of epitopes of Listeria monocytogenes by infected macrophages.

IFN-gamma is an important cytokine in resistance to infection with Listeria monocytogenes, and interleukin 10 is known to exacerbate infection with Listeria and other intracellular pathogens. We examined the effects of these cytokines on antigen presentation by macrophages infected with live Listeria. Listeriolysin O, a hemolysin secreted by Listeria, is an immunodominant antigen presented by both class I and class II MHC on infected cells. Thioglycollate-elicited macrophages were pretreated with exogenous IFN-gamma, IL-10, or both cytokines overnight, infected with bacteria, and then fixed. Epitope-specific, MHC-restricted, T cell hybridomas were then added to detect the presentation of the class I or class II ligand. We found that IFN-gamma enhanced the presentation of both the class I and class II epitopes and IL-10 strongly inhibited the presentation of both ligands. The degree of inhibition of presentation caused by IL-10 was dose dependent. IL-10 was also able to inhibit the presentation of exogenously added class II-binding peptide but had a less dramatic effect on the presentation of the added class I-binding polypeptide epitope. Flow cytometric analysis of expression of class I and class II on treated macrophages demonstrated that the inhibitory effect of IL-10 on antigen presentation was not due to significant downregulation of MHC expression. This loss of antigen presentation was also not due to downregulation of the costimulatory molecule, B7-2. We have found that IFN-gamma and IL-10 have opposing immunoregulatory effects on the presentation of antigens derived from an intracellular pathogen and that the class I vs. class II-mediated presentation of antigens is differentially regulated by IL-10.

Regulation of murine macrophage IL-12 production. Activation of macrophages in vivo, restimulation in vitro, and modulation by other cytokines.

IL-12 is important in the host response to a variety of pathogens. It plays an adjuvant-like role in an initial immune response as well as a therapeutic role in established infections. Despite its well documented importance, comparatively little is known about the regulation of IL-12 production. In this study, we examined IL-12 production by cultured murine peritoneal macrophages from two perspectives: 1) macrophage activation in vivo, and 2) stimulation of IL-12 secretion in vitro. Macrophages were maximally activated within 48 h in vivo during infection with Listeria. Interestingly, although avirulent or heat-killed Listeria induced only minimal production of IL-12 by macrophages, the immunogenic combination of heat-killed bacteria and rIL-12 was highly stimulatory for IL-12 production. LPS and peritoneal inflammatory agents were also stimulatory, but latex beads were ineffective, indicating that microbial components were essential and phagocytosis alone was insufficient. Restimulation in vitro revealed similar patterns, in that infection and LPS were stimulatory but latex beads were not. A systematic survey of potential stimulatory agents showed that microbial heat shock proteins, crude bacterial extracts, bacterial superantigens, a yeast extract, and dsRNA induced IL-12 in vitro. Other cytokines also influenced IL-12 induction. IFN-gamma, which is up-regulated during infection, acted in synergy with other stimuli, suggesting an amplification loop for IL-12 production, whereas IL-4, IL-10, IL-13, and TGF-beta were inhibitory. The existence of a broad range of stimuli from a wide variety of pathogenic organisms underscores the fundamental importance of IL-12 in host defense.

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.

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.

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.

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.

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)

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.

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.

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)