Monocytogenes-specific CD8 Research Articles

The role of commensal microbiota on CD8 T cell response to Listeria monocytogenes infection

Abstract Commensal microbiota is composed of trillions of microorganisms including fungi, protozoans, viruses and bacteria, which have co-evolved with the host immune system. In particular, the gut is the largest reservoir of microorganisms that are involved in dynamic interaction with the immune system, including maintaining intestinal homeostasis, induction of several metabolic pathways, and protection from pathogenic microbes. To better understand the mechanisms involved microbiota-mediated protection from the pathogenic bacteria, we have studied oral infection of germ-free (GF) mice with a strain of Listeria monocytogenes (L. monocytogenes) that has been engineered to efficiently infect the intestinal epithelial cells of the mouse. We found that GF mice are more susceptible to orally administrated L. monocytogenes than conventional SPF mice, with an increased Listeria burden in the mLN, small intestine and feces. In addition, in the infected GF mice, L. monocytogenes-specific CD8 T cells were vigorously activated and induced to differentiate into memory precursor effector cells (MPECs) and tissue resident memory (CD69+ CD103+) T cells in the intestinal epithelium at a faster rate than in infected SPF mice. L. monocytogenes-specific CD8 T cells in GF mice also secrete more effector cytokines, such as interferon-γ and tumor necrosis factor-α. The mechanisms by how commensal microbiota confers resistance to infection by L. moncoytogenes in SPF mice is currently under investigation.

The transcription factor Interferon Regulatory Factor 4 is required for the generation of protective effector CD8 + T cells

Robust cytotoxic CD8(+) T-cell response is important for immunity to intracellular pathogens. Here, we show that the transcription factor IFN Regulatory Factor 4 (IRF4) is crucial for the protective CD8(+) T-cell response to the intracellular bacterium Listeria monocytogenes. IRF4-deficient (Irf4(-/-)) mice could not clear L. monocytogenes infection and generated decreased numbers of L. monocytogenes-specific CD8(+) T cells with impaired effector phenotype and function. Transfer of wild-type CD8(+) T cells into Irf4(-/-) mice improved bacterial clearance, suggesting an intrinsic defect of CD8(+) T cells in Irf4(-/-) mice. Following transfer into wild-type recipients, Irf4(-/-) CD8(+) T cells became activated and showed initial proliferation upon L. monocytogenes infection. However, these cells could not sustain proliferation, produced reduced amounts of IFN-γ and TNF-α, and failed to acquire cytotoxic function. Forced IRF4 expression in Irf4(-/-) CD8(+) T cells rescued the defect. During acute infection, Irf4(-/-) CD8(+) T cells demonstrated diminished expression of B lymphocyte-induced maturation protein-1 (Blimp-1), inhibitor of DNA binding (Id)2, and T-box expressed in T cells (T-bet), transcription factors programming effector-cell generation. IRF4 was essential for expression of Blimp-1, suggesting that altered regulation of Blimp-1 contributes to the defects of Irf4(-/-) CD8(+) T cells. Despite increased levels of B-cell lymphoma 6 (BCL-6), Eomesodermin, and Id3, Irf4(-/-) CD8(+) T cells showed impaired memory-cell formation, indicating additional functions for IRF4 in this process. As IRF4 governs B-cell and CD4(+) T-cell differentiation, the identification of its decisive role in peripheral CD8(+) T-cell differentiation, suggests a common regulatory function for IRF4 in adaptive lymphocytes fate decision.

NLRC5 regulates MHC class I antigen presentation in host defense against intracellular pathogens

NOD-like receptors (NLRs) are a family of intracellular proteins that play critical roles in innate immunity against microbial infection. NLRC5, the largest member of the NLR family, has recently attracted much attention. However, in vitro studies have reported inconsistent results about the roles of NLRC5 in host defense and in regulating immune signaling pathways. The in vivo function of NLRC5 remains unknown. Here, we report that NLRC5 is a critical regulator of host defense against intracellular pathogens in vivo. NLRC5 was specifically required for the expression of genes involved in MHC class I antigen presentation. NLRC5-deficient mice showed a profound defect in the expression of MHC class I genes and a concomitant failure to activate L. monocytogenes-specific CD8(+) T cell responses, including activation, proliferation and cytotoxicity, and the mutant mice were more susceptible to the pathogen infection. NLRP3-mediated inflammasome activation was also partially impaired in NLRC5-deficient mice. However, NLRC5 was dispensable for pathogen-induced expression of NF-κB-dependent pro-inflammatory genes as well as type I interferon genes. Thus, NLRC5 critically regulates MHC class I antigen presentation to control intracellular pathogen infection.

Interleukin (IL)‐21‐independent pathogen‐specific CD8+ T‐cell expansion, and IL‐21‐dependent suppression of CD4+ T‐cell IL‐17 production

Although interleukin-21 (IL-21) potently activates and controls the differentiation of immune cells after stimulation in vitro, the role for this pleiotropic cytokine during in vivo infection remains poorly defined. Herein, the requirement for IL-21 in innate and adaptive host defence after Listeria monocytogenes infection was examined. In the innate phase, IL-21 deficiency did not cause significant defects in infection susceptibility, or in the early activation of natural killer and T cells. In the adaptive phase, L. monocytogenes-specific CD8(+) T cells expand to a similar magnitude in IL-21-deficient mice compared with control mice. Interestingly, the IL-21-independent expansion of L. monocytogenes-specific CD8(+) T cells was maintained even in the combined absence of IL-12 and type I interferon (IFN) receptor. Similarly, L. monocytogenes-specific CD4(+) T cells expanded and produced similar levels of IFN-γ regardless of IL-21 deficiency. Unexpectedly however, IL-21 deficiency caused significantly increased CD4(+) T-cell IL-17 production, and this effect became even more pronounced after L. monocytogenes infection in mice with combined defects in both IL-12 and type I IFN receptor that develop a T helper type 17-dominated CD4(+) T-cell response. Despite increased CD4(+) T-cell IL-17 production, L. monocytogenes-specific T cells re-expanded and conferred protection against secondary challenge with virulent L. monocytogenes regardless of IL-21 deficiency, or combined defects in IL-21, IL-12, and type I IFN receptor. Together, these results demonstrate non-essential individual and combined roles for IL-21, IL-12 and type I IFNs in priming pathogen-specific CD8(+) T cells, and reveal IL-21-dependent suppression of IL-17 production by CD4(+) T cells during in vivo infection.

Manipulating the Rate of Memory CD8+ T Cell Generation after Acute Infection

Infection with Listeria monocytogenes elicits expansion in numbers of Ag-specific CD8+ T cells, which then undergo programmed contraction. The remaining cells undergo further phenotypic and functional changes with time, eventually attaining the qualities of memory CD8+ T cells. In this study, we show that L. monocytogenes-specific CD8+ T cell populations primed in antibiotic-pretreated mice undergo brief effector phase, but rapidly develop phenotypic (CD127(high), CD43(low)) and functional (granzyme B(low), IL-2-producing) characteristics of memory CD8+ T cells. These early memory CD8+ T cells were capable of substantial secondary expansion in response to booster challenge at day 7 postinfection, resulting in significantly elevated numbers of secondary effector and memory CD8+ T cells and enhanced protective immunity compared with control-infected mice. Although early expansion in numbers is similar after L. monocytogenes infection of antibiotic-pretreated and control mice, the absence of sustained proliferation coupled with decreased killer cell lectin-like receptor G-1 up-regulation on responding CD8+ T cells may explain the rapid effector to memory CD8+ T cell transition. In addition, antibiotic treatment 2 days post-L. monocytogenes challenge accelerated the generation of CD8+ T cells with memory phenotype and function, and this accelerated memory generation was reversed in the presence of CpG-induced inflammation. Together, these data show that the rate at which Ag-specific CD8+ T cell populations acquire memory characteristics after infection is not fixed, but rather can be manipulated by limiting inflammation that will in turn modulate the timing and extent to which CD8+ T cells proliferate and up-regulate killer cell lectin-like receptor G-1 expression.

Immunoproteasomes Are Essential for Clearance ofListeria monocytogenesin Nonlymphoid Tissues but Not for Induction of Bacteria-Specific CD8+ T Cells

Microbial infections induce the replacement of constitutive proteasomes by immunoproteasomes (I-proteasomes). I-proteasomes support efficient generation of MHC class I epitopes and influence immunodominance hierarchies of CD8(+) T cells. Recently, the function of I-proteasomes in antimicrobial responses was challenged by showing that the lack of I-proteasomes has no effect on induction and function of lymphocytic choriomeningitis virus-specific CD8(+) T cells. Here, we show that infection with Listeria monocytogenes rapidly induces I-proteasomes in nonlymphoid tissues, which leads to enhanced generation of protection relevant CD8(+) T cell epitopes. I-proteasome-deficient mice (beta5i(-/-) mice) exhibited normal frequencies of L. monocytogenes-specific CD8(+) T cells. However, clearance of L. monocytogenes in liver but not spleen was significantly impaired in I-proteasome-deficient mice. In summary, our studies demonstrate that induction of I-proteasomes is required for CD8(+) T cell-mediated elimination of L. monocytogenes from nonlymphoid but not lymphoid tissues.

Gliotoxin-mediated suppression of innate and adaptive immune functions directed againstListeria monocytogenes

Gliotoxin is an immunosuppressive apoptogenic mycotoxin produced by a number of fungi including important human pathogens as Aspergillus fumigatus. In order to elucidate the potential role of gliotoxin as immunoevasive fungal virulence factor we studied the effects of gliotoxin on the innate and adaptive T cell-mediated immune response against the facultatively intracellular bacterium Listeria monocytogenes. Gliotoxin induced apoptosis of bone marrow-derived macrophages, dendritic cells and CD8 T cells in a dose- and cell type-dependent manner. In vitro the apoptogenic effect of gliotoxin correlated with a strong reduction of TNF-alpha and interleukin (IL)-12 production by dendritic cells and bone marrow-derived macrophages infected with L. monocytogenes and in the case of infected macrophages also in reduced NO-production and recognition by L. monocytogenes-specific CD8 T cells. Further gliotoxin pre-treatment of CD8 T cells reduced target cell lysis. In vivo, treatment of mice with gliotoxin increased the bacterial burden during the innate and the adaptive phase of primary L. monocytogenes infection. Taken together, these results demonstrate the suppressive effects of gliotoxin on the innate and also on the adaptive T cell-mediated antilisterial immunity.

T Cells Undergo Rapid ON/OFF but Not ON/OFF/ON Cycling of Cytokine Production in Response to Antigen

Inflammatory cytokines such as IFN-gamma and TNF produced by Ag-stimulated CD4(+) and CD8(+) T cells are important in defense against microbial infection. However, production of these cytokines must be tightly regulated to prevent immunopathology. Previous studies, conducted with BALB/c mice, have suggested that 1) CD8(+) T cells maintain IFN-gamma production but transiently produce TNF in the continued presence of Ag and 2) lymphocytic choriomeningitis virus-specific and in vitro-propagated effector CD8(+) T cells could rapidly cycle IFN-gamma production ON/OFF/ON in response to Ag exposure, removal, and re-exposure. In contrast with CD8(+) T cells, our results show that Listeria monocytogenes-specific CD4(+) T cells from C57BL/6 mice rapidly initiate (ON cycling) and maintain production of both IFN-gamma and TNF in the continued presence of Ag. Upon Ag removal, production of both cytokines rapidly ceases (OFF cycling). However, if the initial stimulation was maximal, Ag-specific CD4(+) T cells were unable to reinitiate cytokine production after a second Ag exposure. Furthermore, L. monocytogenes-specific CD8(+) T cells in the same mice and lymphocytic choriomeningitis virus-specific CD8(+) T cells in BALB/c mice also underwent ON/OFF cycling, but if the initial Ag stimulus was maximal, they could not produce IFN-gamma after Ag re-exposure. As the initial Ag dose was reduced, the number of cells producing cytokine in response to the second Ag exposure exhibited a corresponding increase. However, T cells that were marked for IFN-gamma secretion during the first stimulation did not contribute cytokine production during the second stimulation. Thus, T cells are not able to undergo rapid ON/OFF/ON cytokine cycling in vitro in response to Ag.

Chemokine receptor 5 is dispensable for innate and adaptive immune responses to Listeria monocytogenes infection.

Chemokine receptor 5 (CCR5) binds macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, RANTES, and members of the monocyte chemotactic protein family and is also a receptor for human immunodeficiency virus (HIV). CCR5 ligands can suppress HIV-1 entry into cells. In humans, homozygous mutations of the ccr5 gene confer resistance to HIV-1 infection. The role of CCR5 in defense against microbial infection is unclear. In this study we examined the innate and adaptive immune responses of CCR5-deficient mice to the intracellular bacterial pathogen Listeria monocytogenes. We found that migration of monocytic cells, formation of L. monocytogenes-containing lesions, and bacterial clearance occurred normally in the spleens and livers of CCR5-deficient animals. Activation of macrophages and dendritic cells during the first 3 days postinfection was normal in the absence of CCR5, as demonstrated by intact expression of inducible nitric oxide synthase (iNOS) and production of the cytokines tumor necrosis factor alpha, gamma interferon, and interleukin-12. Priming of L. monocytogenes-specific CD8 T cells also occured independently of CCR5 expression. Previously immunized, CCR5-deficient animals mounted normal secondary CD8 T-cell responses and cleared bacteria from infected organs similarly to wild-type controls, suggesting that CCR5 is dispensable for migration and activation of memory CD8 T cells. Our data indicate that CCR5-mediated chemotaxis is not required for defense against infection with L. monocytogenes.

Promiscuity of MHC class Ib-restricted T cell responses.

Murine infection with the Gram-positive intracellular bacterium Listeria monocytogenes activates CD8(+) T cells that recognize bacterially derived N-formyl methionine peptides in the context of H2-M3 MHC class Ib molecules. Three peptides, fMIGWII, fMIVIL, and fMIVTLF, are targets of L. monocytogenes-specific CD8(+) T cells. To investigate epitope cross-recognition by H2-M3-restricted CD8(+) T cells, we deleted the sequence encoding fMIGWII from a virulent strain of L. monocytogenes. Infection with fMIGWII-deficient L. monocytogenes unexpectedly primed CD8(+) T cells that stain with fMIGWII/H2-M3 tetramers and lyse fMIGWII-coated target cells in vivo. Because the fMIGWII sequence is nonredundant, we speculated that other bacterially derived Ags are priming these responses. HPLC peptide fractionation of bacterial culture supernatants revealed several distinct L. monocytogenes-derived peptides that are recognized by fMIGWII-specific T cells. Our results demonstrate that the dominant H2-M3-restricted CD8(+) T cell population, although reactive with fMIGWII, is primed by other, non-fMIGWII peptides derived from L. monocytogenes. Although this degree of Ag receptor promiscuity is unusual for the adaptive immune system, it may be a more common feature of T cell responses restricted by nonpolymorphic MHC class Ib molecules.

Rescue of CD8 T cell-mediated antimicrobial immunity with a nonspecific inflammatory stimulus.

Reconstitution of protective immunity by adoptive transfer of pathogen-specific T cells has been successful in patients with compromised cellular immunity. The in vivo effectiveness of in vitro-expanded CD8 CTLs is variable, however. For example, adoptively transferred Listeria monocytogenes-specific CD8 CTLs only confer protective immunity if challenge infection occurs within 48 hours of T cell infusion. Herein we show that transferred CTLs persist in lymphoid compartments for many weeks, but that their response to bacterial challenge decreases during the first week following transfer. While T cells transferred less than 48 hours before infection proliferate, those transferred 7 days before infection die. Remarkably, treatment of mice with anti-CD40 at the time of T cell infusion reprograms transferred T cells, allowing them to proliferate and confer protective immunity upon bacterial challenge 7 days later. Our study demonstrates, for the first time to our knowledge that CD40-mediated stimuli can influence CD8 T cell activation independent of concurrent antigen exposure. The ability to modulate long-term responsiveness of CD8 T cells with a transient, nonspecific inflammatory stimulus has importation implications for adoptive immunotherapy.

Administration of superantigens protects mice from lethal Listeria monocytogenes infection by enhancing cytotoxic T cells.

Superantigens stimulate T-cell-receptor Vbeta-selective T-cell proliferation accompanying the release of cytokines, which may eventually protect the host from microbial infections. We investigated here whether superantigens can rescue the host from lethal bacterial infection. Mice were pretreated with Staphylococcus aureus enterotoxin B (SEB) 1 and 2 days before bacterial infection, and the mortality of infected mice was assessed. SEB pretreatment protected mice from lethal infection with Listeria monocytogenes but not from lethal infection with Streptococcus pyogenes. This enhanced protection was also observed upon pretreatment with recombinant streptococcal pyrogenic exotoxin A. Furthermore, L. monocytogenes-specific delayed-type hypersensitivity (DTH) due to type 1 helper T (Th1) cells and the cytotoxicity of CD8(+) T cells were significantly enhanced after SEB administration and bacterial infection. Depletion of either CD4(+) T cells or CD8(+) T cells in SEB-pretreated mice completely abolished this protection. This phenomenon was ascribed to the elimination of L. monocytogenes-specific CD8(+) cytotoxic T lymphocytes (CTL). It was found that CD4(+) T cells contributed to the induction of the CTL populations. Furthermore, SEB pretreatment of heat-killed L. monocytogenes-immunized mice enhanced the protection from challenge of L. monocytogenes. Taken together, these results indicated that administrations of superantigens protected mice from infection with L. monocytogenes, which was dependent on the enhanced L. monocytogenes-specific CTL activity in the presence of CD4(+) T cells, and superantigens exhibited adjuvant activity in the immunization against intracellular pathogens.

Endogenous interleukin-10 is required for prevention of a hyperinflammatory intracerebral immune response in Listeria monocytogenes meningoencephalitis.

To analyze the role of interleukin-10 (IL-10) in bacterial cerebral infections, we studied cerebral listeriosis in IL-10-deficient (IL-10(-/-)) and wild-type (WT) mice, the latter of which express high levels of IL-10 in both primary and secondary cerebral listeriosis. IL-10(-/-) mice succumbed to primary as well as secondary listeriosis, whereas WT mice were significantly protected from secondary listeriosis by prior intraperitoneal immunization with Listeria monocytogenes. Meningoencephalitis developed in both strains; however, in IL-10(-/-) mice the inflammation was more severe and associated with increased brain edema and multiple intracerebral hemorrhages. IL-10(-/-) mice recruited significantly increased numbers of leukocytes, in particular granulocytes, to the brain, and the intracerebral cytokine (tumor necrosis factor, IL-1, IL-12, gamma interferon, and inducible nitric oxide synthase) and chemokine (crg2/IP-10, RANTES, MuMig, macrophage inflammatory protein 1alpha [MIP-1alpha], and MIP-1beta) transcription was enhanced compared to that in WT mice. Despite this prominent hyperinflammation, the frequencies of intracerebral L. monocytogenes-specific CD8(+) T cells were reduced and the intracerebral bacterial load was not reduced in IL-10(-/-) mice compared to WT mice. Following intraperitoneal infection, IL-10(-/-) mice exhibited hepatic hyperinflammation without better bacterial clearance; however, in contrast to the mice with cerebral listeriosis, they did not succumb, illustrating that intrinsic factors of the target organ have a strong impact on the course and outcome of the infection.

Intestinal and Splenic T Cell Responses to EntericListeria monocytogenesInfection: Distinct Repertoires of Responding CD8 T Lymphocytes

Listeria monocytogenes is an intracellular bacterium that causes systemic infections after traversing the intestinal mucosa. Clearance of infection and long term protective immunity are mediated by L. monocytogenes-specific CD8 T lymphocytes. In this report, we characterize the murine CD8 T cell response in the lamina propria and intestinal epithelium after enteric L. monocytogenes infection. We find that the frequency of MHC class Ia-restricted, L. monocytogenes-specific T cells is approximately 4- to 5-fold greater in the lamina propria than in the spleen of mice after oral or i.v. infection. Although the kinetics of T cell expansion and contraction are similar in spleen, lamina propria, and intestinal epithelium, high frequencies of Ag-specific T cells are detected only in the lamina propria 1 mo after infection. In contrast to MHC class Ia-restricted T cells, the frequency of H2-M3-restricted, L. monocytogenes-specific T cells is decreased in the intestinal mucosa relative to that found in the spleen. In addition to this disparity, we find that MHC class Ia-restricted CD8 T cells specific for a dominant L. monocytogenes epitope have different TCR V beta repertoires in the spleen and intestinal mucosa of individual mice. These findings indicate that the intestinal mucosa is a depot where L. monocytogenes-specific effector CD8 T cells accumulate during and after infection irrespective of immunization route. Furthermore, our results demonstrate that CD8 T cell populations in these two sites, although overlapping in Ag specificity, are distinct in terms of their repertoire.

Suppression of acquired immunity against Listeria monocytogenes by amphotericin B-mediated inhibition of CD8 T cell function.

Amphotericin B is frequently used for the treatment of fungal infections of immunocompromised individuals. Whereas immunomodulatory side effects of this agent are known, the influence of amphotericin B was studied in the model of murine Listeria monocytogenes infection. Treatment of L. monocytogenes-immune mice with a nontoxic dose of amphotericin B (0.75 mg/kg) reduced antilisterial protection by 4-5 orders of magnitude, while it had no significant effect on natural immunity against L. monocytogenes in naive mice. Treatment of mice with amphotericin B also abolished the protection mediated by transfer of an L. monocytogenes-specific CD8 T cell line. Furthermore, in vitro analysis showed that amphotericin B impaired target cell lysis and interferon-gamma production by peptide-specific CD8 T cell lines and antigen presentation by L. monocytogenes-infected macrophagelike cells. These data indicate that amphotericin B has a strong suppressive effect on the function of CD8 T cells in vitro and in vivo.

Acquired immunity to an intracellular pathogen: immunologic recognition of L. monocytogenes-infected cells.

Listeria monocytogenes (L. monocytogenes) is a pathogenic bacterium, and subclinical infection in mice is utilized as a prototypic model to investigate the development and expression of acquired resistance to facultative intracellular organisms. A key virulence factor of L. monocytogenes is the hemolysin listeriolysin O (LLO), and BALB/c mice immunized with hemolysin-secreting strains of L. monocytogenes develop specific acquired resistance, while mice immunized with hemolysin-negative strains or non-viable preparations of L. monocytogenes do not develop a protective immune response. Adoptive transfer studies show that L. monocytogenes-immune CD8+ T cells mediate acquired resistance. The L. monocytogenes-immune CD8+ population is cytotoxic, and target cells infected with hemolysin-secreting strains of L. monocytogenes are lysed, while target cells infected with hemolysin-negative strains or non-viable preparations of L. monocytogenes are not lysed. MHC class Ia and Ib molecules present L. monocytogenes-derived peptides, and we have identified Qa-Ib, a T-region-encoded MHC class Ib molecule, as a restriction element for L. monocytogenes-specific CD8+ CTL. MHC class Ib-restricted CTL are stimulated following infection with L. monocytogenes and are a significant component of the total MHC class I-restricted CTL population. These findings support the observation that cytoplasmic L. monocytogenes-derived antigens are endogenously processed and presented in association with MHC class Ia and Ib molecules to CD8+ effector cells, and that both populations of effector cells contribute to the immune response to this intracellular pathogen.