T cell responses to Listeria monocytogenes

Owing to its unique intracellular biology that allows it to gain access to the host cell cytosol, Listeria monocytogenes induces potent, protective CD8 responses. The study of these responses has served as a paradigm to understand cell-mediated immunity to microbial pathogens. The availability of mutants specifically defective in unique aspects of the intracellular biology of this pathogen has greatly aided these studies. During the past few years, progress has been made to understand the contribution of the innate immune system and CD4 T cells in the generation of robust, long lasting CD8 responses to L. monocytogenes.     

Organ-specific CD4+ T cell response during Listeria monocytogenes infection

The immune response against the intracellular bacterium Listeria monocytogenes involves both CD4(+) and CD8(+) T cells. We used the MHC class II-presented peptide listeriolysin(189-201) to characterize the organ-specific CD4(+) T cell response during infection. Systemic listeriosis resulted in a strong peptide-specific CD4(+) T cell response with frequencies of 1/100 and 1/30 CD4(+) splenocytes at the peak of primary and secondary response, respectively. This response was not restricted to lymphoid organs, because we detected specific CD4(+) T cells in all tissues analyzed. However, the tissue distribution of the T cell response was dependent on the route of infection. After i.v. infection, the strongest CD4(+) T cell response and the highest levels of memory cells were observed in spleen and liver, the major sites of L. monocytogenes replication. After oral infection, we detected a strong response in the liver, the lamina propria, and the intestinal epithelium. These tissues also harbored the highest frequencies of listeriolysin(189-201)-specific CD4(+) memory T cells 5-8 wk post oral infection. Our results show that kinetics and magnitude of the CD4(+) T cell response and the accumulation of CD4(+) memory T cells depend on the route of infection and are regulated in a tissue-specific way.     

Organ-specific regulation of the CD8 T cell response to Listeria monocytogenes infection

The intestinal mucosal CD8 T cell response to infection with Listeria monocytogenes was measured using MHC class I tetramers and was compared with the response in peripheral blood, secondary lymphoid tissue, and liver. To assess the vaccination potential of Listeria and to analyze responses in C57BL/6 mouse strains, a recombinant Listeria expressing OVA (rLM-ova) was generated. The response peaked at 9 days postinfection with a much larger fraction of the intestinal mucosa and liver CD8 T cell pool OVA specific, as compared with the spleen. However, these differences were not linked to bacterial titers in each site. The higher responses in lamina propria and liver resulted in a larger CD8 memory population in these tissues. Furthermore, the level of memory induced was dependent on infectious dose and inversely correlated with the magnitude of the recall response after oral challenge. Recall responses in the tissues were most robust in the lamina propria and liver, and reactivated Ag-specific T cells produced IFN-gamma. Infection of CD40- or MHC class II-deficient mice induced poor CD8 T cell responses in the intestinal mucosa, but only partially reduced responses in the spleen and liver. Overall, the results point to novel pathways of tissue-specific regulation of primary and memory antimicrobial CD8 T cell responses.     

PDL-1 blockade impedes T cell expansion and protective immunity primed by attenuated Listeria monocytogenes

Infection with attenuated Listeria monocytogenes (Lm) is a robust in vivo model for examining how Ag-specific T cells are primed, and subsequent challenge with virulent Lm allows for the protective effects of T cell priming to be quantified. Herein, we investigated the role of programmed death ligand 1 (PDL-1) in T cell priming and immunity conferred after primary infection with Lm DeltaactA followed by virulent Lm challenge. In striking contrast to the inhibitory role of PDL-1 on T cell immunity in other infection models, marked reductions in the magnitude of T cell expansion and the kinetics of T cell proliferation were observed with PDL-1 blockade after primary Lm DeltaactA infection. More importantly, PDL-1 blockade beginning before primary infection and maintained throughout the experiment resulted in delayed bacterial clearance and T cell expansion after secondary challenge with virulent Lm. These results indicate that for immunity to intracellular bacterial infection, PDL-1 plays an important stimulatory role for priming and expansion of protective T cells.     

T cell reactivity to Listeria monocytogenes amongst us

In the last two decades, listeriosis, caused by the intracellular pathogen Listeria monocytogenes, became one of the most concerning food-born infections. Although it had been known before as an infectious disease of limited importance, listeriosis was brought into attention of scientists due to the frequent outbreaks recently reported. Despite the major progress made towards understanding the mechanisms of virulence of L. monocytogenes, our current knowledge into the process of Listeria-associated pathogenesis and virulence is still partial and fragmentary. In this study we demonstrate that T lymphocytes with reactivity to L. monocytogenes are frequently present in healthy individuals (73%), most probably as a consequence of subclinical infections. Host resistance to infection by L. monocytogenes involves a series of interactions between cells of the immune system, of which the antigen presenting cell/T lymphocyte partnership is essential. The ability of memory T cells to respond when exposed to their target antigen is traditionally assessed by measuring uptake of [3H] - thymidine. Our study has been carried out by means of an alternative methodology based on flow-cytometry, an approach which has several advantages on [3H] - thymidine incorporation technique: allows targeted analysis of particular cell types, simultaneous assessment of various cellular markers, and circumvents handling of radioisotopes.     

The interplay between classical and alternative isoprenoid biosynthesis controls gammadelta T cell bioactivity of Listeria monocytogenes

Isoprenoids are synthesised either through the classical, mevalonate pathway, or the alternative, non-mevalonate, 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. The latter is found in many microbial pathogens and proceeds via (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), a potent activator of human Vgamma9/Vdelta2 T cells. Listeria monocytogenes is the only pathogenic bacterium known to contain both pathways concurrently. Strategic gene knockouts demonstrate that either pathway is functional but dispensable for viability. Yet, disrupting the mevalonate pathway results in a complementary upregulation of the MEP pathway. Vgamma9/Vdelta2 T cell bioactivity is increased in DeltalytB mutants where HMB-PP accumulation is expected, and lost in DeltagcpE mutants which fail to produce HMB-PP.     

CD8 T cell immunome analysis of Listeria monocytogenes

The identification of T cell epitopes is crucial for the understanding of the host response during infections with pathogenic microorganisms. Generally, the identification of relevant T cell responses is based on the analysis of T cell lines propagated in vitro. We used an ex vivo approach for the analysis of the CD8 T cell response against Listeria monocytogenes that is based upon the fractionation of naturally processed antigenic peptides and subsequent analysis with T cells in an enzyme-linked immunospot (ELISPOT) assay. Our data indicate that the direct ex vivo ELISPOT analysis of peptides extracted from infected tissues represents a versatile and potent test system for the analysis of the CD8 T cell immunome of microorganisms that furthermore requires neither the knowledge of the microbial genome nor of the specificity of responding T cells.     

Class Ia MHC-deficient BALB/c mice generate CD8+ T cell-mediated protective immunity against Listeria monocytogenes infection

CD8(+) T cells are required for protective immunity against intracellular pathogens such as Listeria monocytogenes. In this study, we used class Ia MHC-deficient mice, which have a severe reduction in circulating CD8(+) T cells, to determine the protective capacity of class Ib MHC-restricted T cells during L. monocytogenes infection. The K(b-/-)D(b-/-) mutation was backcrossed onto a C.B10 (BALB/c congenic at H-2 locus with C57BL/10) background, because BALB/c mice are more susceptible to Listeria infection than other commonly studied mouse strains such as C57BL/6. C.B10 K(b-/-)D(b-/-) mice immunized with a sublethal dose of L. monocytogenes were fully protected against a subsequent lethal infection. Adoptive transfer of Listeria-immune splenocyte subsets into naive K(b-/-)D(b-/-) mice indicated that CD8(+) T cells were the major component of this protective immune response. A CD8(+) T cell line isolated from the spleen of a Listeria-infected class Ia MHC-deficient mouse was shown to specifically recognize Listeria-infected cells in vitro, as determined by IFN-gamma secretion and cytotoxicity assays. Adoptive transfer of this T cell line alone resulted in significant protection against L. monocytogenes challenge. These results suggest that even a limited number of class Ib MHC-restricted T cells are sufficient to generate the rapid recall response required for protection against secondary infection with L. monocytogenes.     

Secretion of colony-stimulating factors by T cell clones. Role in adoptive protection against Listeria monocytogenes

CSF have been postulated to be important mediators of host defenses. The current studies were undertaken to investigate the production of CSF by Listeria-specific, T cell clones and to assess the participation of CSF in anti-listerial host resistance. Listeria-specific L3T4+, Lyt-2- T cell clones were isolated and expanded by standard techniques. The clones themselves protected mice from listerial challenge when injected intravenously, and supernatants generated from Ag-stimulated clones were protective. In order to define factors important in the protection, supernatants from the clones were assayed for CSF by several in vitro assays. Total colony-stimulating activity was measured with a bone marrow colony-forming assay. T cell clones secreted 1000 to 2000 U/ml of colony-stimulating activity after 48 hours of stimulation with specific antigen. The relative amounts of the various CSF were determined by the capacity of supernatants to support proliferation of the factor-dependent cell lines FDCP-1 and 32D cl 3 in the presence and absence of specific anti-CSF antibodies. Results showed that most of the CSF activity was due to granulocyte-macrophage (GM)-CSF and IL-3. The role of GM-CSF in anti-listerial host resistance was assessed in two types of experiments. In one set of experiments GM-CSF activity was neutralized in the supernatants by addition of specific rabbit anti-GM-CSF antibodies. Treated and untreated supernatants were then tested for their capacity to protect nonimmune mice against listerial challenge. Neutralization of GM-CSF in the supernatants decreased the protective capacity of the supernatants by approximately 23%. In a second set of studies, the administration of recombinant murine GM-CSF was shown to protect mice from challenges of L. monocytogenes. Taken together, these experiments provide evidence that CSF are important mediators of immune T cell mediated host defenses.     

ISA 61 VG佐剂增强单核细胞增生李斯特氏菌灭活疫苗的保护性免疫应答

单核细胞增生李斯特氏菌(Listeria monocytogenes,Lm)是重要的人兽共患李斯特氏菌病的致病菌,疫苗免疫是预防该病原菌感染的有效手段之一。本研究研制了添加矿物油佐剂MontanideTM ISA61VG的新型灭活细菌疫苗,并对其安全性和免疫应答特性进行了研究。结果表明,ISA 61 VG佐剂疫苗具有较好的安全性;诱导小鼠产生的抗李斯特氏菌溶血素O抗体滴度以及IgG2a/IgG1比值显著高于无佐剂免疫组;在致死剂量Lm攻毒下,能对小鼠提供100%的免疫保护。因此,ISA 61VG佐剂能显著增强灭活疫苗诱导宿主产生体液免疫和细胞免疫应答的能力,从而提高灭活疫苗的保护性免疫应答作用,是预防人和动物Lm感染的潜在疫苗候选株。     

The metalloprotease of Listeria monocytogenes controls cell wall translocation of the broad-range phospholipase C

Listeria monocytogenes is a gram-positive bacterial pathogen that multiplies in the cytosol of host cells and spreads directly from cell to cell. During cell-to-cell spread, bacteria become temporarily confined to secondary vacuoles. The broad-range phospholipase C (PC-PLC) of L. monocytogenes contributes to bacterial escape from secondary vacuoles. PC-PLC requires cleavage of an N-terminal propeptide for activation, and Mpl, a metalloprotease of Listeria, is involved in the proteolytic activation of PC-PLC. Previously, we showed that cell wall translocation of PC-PLC is inefficient, resulting in accumulation of PC-PLC at the membrane-cell wall interface. In infected cells, rapid cell wall translocation of PC-PLC is triggered by a decrease in pH and correlates with cleavage of the propeptide in an Mpl-dependent manner. To address the role of the propeptide and of Mpl in cell wall translocation of PC-PLC, we generated a cleavage site mutant and a propeptide deletion mutant. The intracellular behavior of these mutants was assessed in pulse-chase experiments. We observed efficient translocation of the proform of the PC-PLC cleavage site mutant in a manner that was pH sensitive and Mpl dependent. However, the propeptide deletion mutant was efficiently translocated into host cells independent of Mpl and pH. Overall, these results suggest that Mpl regulates PC-PLC translocation across the bacterial cell wall in a manner that is dependent on the presence of the propeptide but independent of propeptide cleavage. In addition, similarly to Mpl-mediated cleavage of PC-PLC propeptide, Mpl-mediated translocation of PC-PLC across the bacterial cell wall is pH sensitive.     

Listeriolysin O-deficient Listeria monocytogenes as a vaccine delivery vehicle: antigen-specific CD8 T cell priming and protective immunity

Strains of Listeria monocytogenes (LM) that are deficient in the virulence factor listeriolysin O (LLO) are highly attenuated and are thought not to elicit protective immunity. This failure has been attributed to the inability of the bacterium to enter the host cell cytosol and access MHC class I Ag processing machinery. We reexamined this issue using recombinant strains of LM that are deficient in LLO but express an additional CD8 T cell epitope derived from lymphocytic choriomeningitis virus. After infection with LLO-deficient strains, we find sizable priming of epitope-specific CD8 T cells and the development of a functional memory cell population. Mice primed with the LLO-deficient LM strain are equally resistant against high-dose challenge with virulent LM as mice primed with wild-type virulent bacteria and also resist heterologous challenge with lymphocytic choriomeningitis virus. Interestingly, priming with a low dose of LLO-deficient LM, which occurred in environment of reduced inflammation (IFN-gamma), allowed rapid amplification of Ag-specific CD8 T cells by booster immunization, despite an undetectable primary response. We conclude that the generation of protective immunity by LLO-deficient strains of LM does in fact occur and that this highly attenuated LM strain may be a useful platform for vaccine delivery.     

Cloned Listeria monocytogenes specific non-MHC-restricted Lyt-2+ T cells with cytolytic and protective activity

Mice were infected with Listeria monocytogenes and Lyt-2+ T cell clones capable of lysing Ag-primed bone marrow macrophages were established. In accordance with earlier findings obtained at the population level, some T cell clones were identified which lysed bone marrow macrophages of different MHC type provided the relevant Ag was present. This unusual target cell recognition was further analyzed using a T3+, L3T4-, Lyt-2+, F23+, KJ16+ T cell clone, designated L-28. Target cell lysis by this clone was Ag specific, apparently non-MHC restricted. In contrast, YAC cells and P815 cells were not lysed by clone L-28. However, lysis of irrelevant targets could be induced by anti-T3, F23, or KJ16 mAb. Furthermore, Ag-specific lysis was blocked by anti-Lyt-2 mAb and by F(ab)2 fragments of F23 mAb. In addition to its cytolytic activity, clone L-28 produced IFN-gamma after co-stimulation with accessory cells, Ag, and rIL-2 and conferred significant protection on recipient mice when given together with rIL-2. These data suggest that non-MHC-restricted Lyt-2+ killer cells generated during listeriosis are cytolytic T lymphocytes that interact with their target Ag via the T cell receptor/T3 complex and the Lyt-2 molecule and, furthermore, that these cells play a role in anti-listerial resistance. The possible relevance of IFN-gamma secretion and target cell lysis for antibacterial protection is discussed.     

Induction of protective immunity to Listeria monocytogenes in neonates

Neonates suffer unduly from infections and also respond suboptimally to most commonly used vaccines. However, a CD8 T cell response can be elicited in neonates if the Ag is introduced into the cytoplasm of APCs. Listeria monocytogenes (Lm) targets the cytoplasm of APC and is a strong CD8 and CD4 Th1-promoting vaccine vehicle in adult mice. We hypothesized that an attenuated strain of Lm would be safe and induce long-lasting protective immunity, even in neonates. We found that neonatal mice immunized only once with the attenuated strain DeltaactA-Lm developed robust primary and secondary CD8 and CD4 Th1 responses and were fully protected from lethal challenge with virulent wild-type Lm without the need for a booster immunization. Furthermore, DeltaactA-Lm expressing a heterologous recombinant Ag induced a strong CD8 and Th1 memory response to that Ag. Based on these data, we propose that DeltaactA-Lm or derivatives thereof might serve as a vaccine vehicle for neonatal immunization.     

Listeria monocytogenes exploits ERM protein functions to efficiently spread from cell to cell

Cell-to-cell spread is a fundamental step in the infection cycle of Listeria monocytogenes that strictly depends on the formation of bacteria-induced protrusions. Since Listeria actin tails in the protrusions are tightly associated with the plasma membrane, we hypothesised that membrane-cytoskeleton linkers would be required for initiating and sustaining their formation and the subsequent cell-to-cell spread. We have found that ezrin, a member of the ezrin, radixin and moesin (ERM) family that functions as a key membrane-cytoskeleton linker, accumulates at Listeria protrusions. The ability of Listeria to induce protrusions and effectively spread between adjacent cells depends on the interaction of ERM proteins with both a membrane component such as CD44 and actin filaments. Interfering with either of these interactions or with ERM proteins phosphorylation not only reduces the number of protrusions but also alters their morphology, resulting in the formation of short and collapsed protrusions. As a consequence, Listeria cell-to-cell spread is severely impaired. Thus, ERM proteins are exploited by Listeria to escape the host immune response and to succeed in the development of the infection.