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.     

Cryptococcus neoformans infection in mice previously infected with LP-BM5 MuLV, the agent of murine AIDS (MAIDS)

We studied susceptibility to experimental systemic cryptococcosis in mice previously infected with the retroviral complex LP-BM5 (responsible for murine AIDS). LP-BM5 was inoculated to C57B1/6 mice by intravenous (i.v.) injection 8 weeks before an i.v. challenge with 4×l0³ CFU of Cryptococcus neoformans. Uninfected and singly infected mice were used as controls. LP-BM5 infection did not result in a significant increase in fungal burdens in the lungs or brains of co-infected animals compared to mice infected with C. neoformans alone. However, mortality was enhanced in the co-infected animals. The kinetics of splenocyte subsets differed in co-infected mice and LP-BM5-infected mice; the increase in CD4⁺, CD8⁺ and Ly5⁺ cells was only moderate in the former. Cytokine production by concanavalin A (Con A)-stimulated splenocytes from co-infected mice showed a marked decrease in the Thl response (IFN-γ, IL-2) and an increase in the Th2 response (IL-4, IL-10). Furthermore, cryptococcosis altered the course of MAIDS, inhibiting splenomegaly. This effect was not related to a decrease in ecotropic virus titres in the spleen or to improved in vitro responsiveness of spleen cells to Con A. The marked decrease in IFN-γ production in co-infected animals could partly explain the inhibition of LP-BM5-induced splenomegaly. This model of murine retroviral infection does not seem to be suitable for studying cryptococcosis in immunosuppressed animals, but remains valuable for investigating in vivo interactions between two pathogens.     

Listeria monocytogenes-Infected Hepatocytes Are Targets of Major Histocompatibility Complex Class Ib-Restricted Antilisterial Cytotoxic T Lymphocytes

Subclinical infection of BALB/c mice with the intracellular bacterial pathogen Listeria monocytogenes results in the development of protective antilisterial immunity. L. monocytogenes can infect hepatocytes, and antilisterial cytotoxic T lymphocytes (CTL) lyse Listeria-infected hepatocytes in a major histocompatibility complex (MHC) class Ia-restricted manner. It remained to be determined whether L. monocytogenes-infected hepatocytes are susceptible to MHC class Ib-restricted cytolysis. In this study, we showed that hepatocytes express MHC class Ib molecule Qa-1^b mRNA and protein. We further showed that Listeria-infected hepatocytes are susceptible to MHC class Ib-restricted cytolysis, since C57BL/6-derived Listeria-infected hepatocytes were lysed by BALB/c-derived antilisterial CTL. These results establish that Listeria-infected hepatocytes are susceptible to cytolysis by MHC class Ib restricted Listeria-specific CTL.Injection of BALB/c mice with a sublethal dose of virulent Listeria monocytogenes leads to an initial wave of unhindered intracellular replication of the microorganism within the first 24 to 48 h (16). The spleen and liver are the principal target organs for the experimental infection. Hepatocytes are the principal cell type harboring the replicating bacterial pathogen within the liver (8, 18). By 72 h following injection, the numbers of CFU in these organs begin to decline, and by 6 days following injection, L. monocytogenes CFU typically are not detectable (14, 15). The decline and eventual clearance of L. monocytogenes have been attributed to stimulation of Listeria-specific CD4⁺ and CD8⁺ T-cell subsets (11, 16). Adoptive transfer studies have shown that the Listeria-specific CD4⁺ T-cell subset mediates delayed-type hypersensitivity reactivity while the Listeria-specific CD8⁺ T-cell subset is responsible for protective immunity against a lethal challenge dose of virulent L. monocytogenes (1, 2).In vitro studies have shown that the CD8⁺ T-cell subset is cytolytic, and targets cells such as J774 macrophage-like cells, bone marrow-derived macrophages, and fibroblasts that have been infected with viable, hemolysin-secreting strains of L. monocytogenes are lysed by Listeria-immune CD8⁺ T cells (3). In addition, gamma interferon is released from Listeria-specific CD8⁺ T cells only following interaction with target cells infected with viable, hemolysin-secreting strains of L. monocytogenes (7). Target cells infected with non-hemolysin-secreting strains of L. monocytogenes or pulsed with nonviable bacteria such as heat-killed L. monocytogenes are not lysed by CD8⁺ Listeria-immune cells (4). The CD8⁺ T-cell subset is major histocompatibility complex (MHC) class I restricted, and both classical (MHC class Ia) and nonclassical (MHC class Ib) elements have been described as restriction molecules (3). In BALB/c mice, MHC class Ia responses are restricted to H2-K^d-presented peptides. MHC class Ib molecules that are restricting elements for antilisterial cytotoxic T lymphocytes (CTL) include Qa-1^b, which is T region encoded (6), and M3, which is M region encoded (12, 17).A role for MHC class Ib-restricted CD8⁺ T cells in antilisterial immunity is indicated from recent studies which showed that short-lived antilisterial protection can be adoptively transferred to MHC class Ia-disparate, MHC class Ib-syngeneic recipients (10, 13). MHC class Ib-restricted CTL appear to be biologically relevant, since MHC class Ib-restricted CTL activity can be detected in vivo when CFU are declining and the disappearance of effector function of this population correlates with clearance of L. monocytogenes from the immunized animals (5). It has been clearly established that L. monocytogenes-infected macrophages present MHC class Ia- and Ib-associated peptide targets to antilisterial CTL (3). It has recently been established that Listeria-infected hepatocytes are susceptible to MHC class Ia-restricted lysis by Listeria-specific CD8⁺ T cells obtained from mice immunized 11 days previously (9). This same CD8⁺ T-cell population failed to exhibit MHC class Ib-restricted cytolytic activity. This finding suggests that Listeria-infected hepatocytes may not present MHC class Ib-associated targets to Listeria-specific CTL. The study presented in this report was initiated to address specifically whether hepatocytes express MHC class Ib molecules and to determine if Listeria-infected hepatocytes are susceptible to MHC class Ib-restricted cytolysis by utilizing a population of Listeria-specific effector cells with known MHC class Ib-restricted CTL activity (3). The data presented show that hepatocytes express MHC class Ib molecules and that L. monocytogenes-infected hepatocytes are lysed by MHC class Ib-restricted Listeria-specific CTL.     

Effector T‐cell differentiation during viral and bacterial infections: Role of direct IL‐12 signals for cell fate decision of CD8+ T cells

To study the role of IL‐12 as a third signal for T‐cell activation and differentiation in vivo, direct IL‐12 signaling to CD8⁺ T cells was analyzed in bacterial and viral infections using the P14 T‐cell adoptive transfer model with CD8⁺ T cells that lack the IL‐12 receptor. Results indicate that CD8⁺ T cells deficient in IL‐12 signaling were impaired in clonal expansion after Listeria monocytogenes infection but not after infection with lymphocytic choriomeningitis virus, vaccinia virus or vesicular stomatitis virus. Although limited in clonal expansion after Listeria infection, CD8⁺ T cells deficient in IL‐12 signaling exhibited normal degranulation activity, cytolytic functions, and secretion of IFN‐γ and TNF‐α. However, CD8⁺ T cells lacking IL‐12 signaling failed to up‐regulate KLRG1 and to down‐regulate CD127 in the context of Listeria but not viral infections. Thus, direct IL‐12 signaling to CD8⁺ T cells determines the cell fate decision between short‐lived effector cells and memory precursor effector cells, which is dependent on pathogen‐induced local cytokine milieu.     

Sequential appearance of γ/δ- and α/β-bearing T cells in the peritoneal cavity during an i.p. infection with Listeria monocytogenes

To search for a potential role of T cell antigen receptor (TcR) γ/β-bearing cells in host-defense against Listeria monocytogenes, we analyzed the sequential appearance of γ/δ and α/β T cell in the peritoneal exudate cells (PEC) during an i.p. infection with sublethal dose (2 × 10³) of viable Listeria organisms in mice. The PEC on day 1 after the infection consisted of 48% macrophages and 50% lymphocytes, most of which were surface IgM⁺ (B) cells. The number of PEC increased to the maximal level by day 3. The PEC at this stage contained an appreciable number of CD3⁺ T cells in addition to a large number of macrophages. Of the CD3⁺ cells, the proportion of CD4^?CD8^? cells, most of which expressed no TcR α/β, increased to the maximal level on day 3 after the infection. In correlation with an increased number of CD3⁺CD4^?CD8^?TcR α/β^? cells, high level of TcR γ/δ chain gene messages was detected in the nonadherent population of the PEC on this stage. On the other hand, the PEC on day 8 contained an increased number of CD4⁺CD8^? and CD4^?CD8⁺ cells which expressed TcR α/β chain on their surface. These results suggest that the γ/δ T cells precede the α/β T cells in appearance during listerial infection. The γ/β T cells may be involved at the first line of the host-defense against Listeria.     

Homeostatic regulation of CD8+ T cells after antigen challenge in the absence of Fas (CD95)

The role of Fas in the homeostatic regulation of CD8⁺ T cells after antigen challenge was analyzed in the murine model of lymphocytic choriomeningitis virus (LCMV) infection. Mice homozygous for the lpr mutation and carrying T cell receptor (TCR) αβ transgenes specific for the LCMV glycoprotein peptide aa 33–41 in the context of H-2D^b were used. Five main results emerged: first, development of lymphadenopathy and of CD4⁻CD8⁻ double-negative B220⁺ T cells in lpr mice was not inhibited by the αβ TCR transgenes; second, tolerance induction and peripheral deletion of CD8⁺ T cells induced by LCMV glycoprotein peptide injection was independent of Fas expression; third, clonal down-regulation of Fas-deficient TCR-transgenic CD8⁺ T cells after acute LCM virus infection was identical to the decline of transgenic T cells expressing Fas; fourth, in vivo activated CD8⁺ effector T cells from TCR transgenic and TCR-lpr/lpr mice were equally susceptible to activation-induced cell death in vitro; and fifth, transgenic effector T cells from lpr/lpr mice were cleared in the declining phase of the immune response in vivo without giving rise to CD4⁻CD8⁻ double-negative T cells. Taken together, these data suggest that the homeostatic regulation of CD8⁺ T cells after antigen challenge in vivo is regulated by mechanisms that do not require Fas.     

A six-hour in vitro virulence assay for Listeria monocytogenes using myeloma and hybridoma cells from murine and human sources

An in vitro cell culture assay using myeloma cells and hybrid lymphocytes was developed which detected pathogenic Listeria strains in just 6 h. Three separate hybridoma cell lines, murine Ped-2E9 and EM-7G1 and human RI.37 and murine myeloma NS1 cells, proved equally sensitive in responding to virulent Listeria species. Listeria monocytogenes along with other Listeria spp., collected from food and clinical sources, were inoculated at 10⁸ cfu/ml into a suspension of Ped-2E9 (10⁶/ml). Pathogenic Listeria spp. killed 80% of hybridoma cells by 4 h, as determined by trypan blue exclusion test, Conversely, none of all nonpathogenic Listeria spp. killed the hybridoma cells. Ped-2E9 cells exposed to three strains of L. monocytogenes strains showed 96-97.5% death in 6 h measured by trypan blue staining and release of 91-97% of lactate dehydrogenase (LDH) enzyme. RI.37 cells showed similar results. A multiplicity of exposure (MOE) of 100 L. monocytogenes to 1 hybridoma cell or of 10:1 killed about 80% of the hybridoma cells in 4 or 6 h respectively. The in vitro virulence assay of L. monocytogenes with hybridoma cells compared favorably with the immunocompromised mouse model, yielding results in 6 h instead of 3 days. Intracellular L. monocytogenes and L. innocua were not recovered from Ped-2E9 hybridoma cells after 2 or 4 h of exposure. However, attachment of both L. monocytogenes and L. innocua cells on Ped-2E9 cell surfaces were observed under epifluorescence microscopy. Direct contact of hemolysin positive L. monocytogenes with hybridoma cells is essential to cause death, since hybridoma cells were not killed when they were separated from the growing bacteria by a 0.45 μm filter.     

Differentiation of distinct long-lived memory CD4 T cells in intestinal tissues after oral Listeria monocytogenes infection

Mucosal antigen-specific CD4 T-cell responses to intestinal pathogens remain incompletely understood. Here we examined the CD4 T-cell response after oral infection with an internalin A ‘murinized’ Listeria monocytogenes (Lm). Oral Lm infection induced a robust endogenous listeriolysin O (LLO)-specific CD4 T-cell response with distinct phenotypic and functional characteristics in the intestine. Circulating LLO-specific CD4 T cells transiently expressed the ‘gut-homing’ integrin α₄β₇ and accumulated in the intestinal lamina propria and epithelium where they were maintained independent of interleukin (IL)-15. The majority of intestinal LLO-specific CD4 T cells were CD27⁻ Ly6C⁻ and CD69⁺ CD103⁻ while the lymphoid LLO-specific CD4 T cells were heterogeneous based on CD27 and Ly6C expression and predominately CD69⁻. LLO-specific effector CD4 T cells transitioned into a long-lived memory population that phenotypically resembled their parent effectors and displayed hallmarks of residency. In addition, intestinal effector and memory CD4 T cells showed a predominant polyfunctional Th1 profile producing IFNγ, TNFα, and IL-2 at high levels with minimal but detectable levels of IL-17A. Depletion of CD4 T cells in immunized mice led to elevated bacterial burden after challenge infection highlighting a critical role for memory CD4 T cells in controlling intestinal intracellular pathogens.     

Treatment with Interleukin-7 Restores Host Defense against Pneumocystis in CD4+ T-Lymphocyte-Depleted Mice

Pneumocystis pneumonia (PCP) is a major cause of morbidity and mortality in patients with HIV infection. CD4⁺ T lymphocytes are critical for host defense against this infection, but in the absence of CD4⁺ T lymphocytes, CD8⁺ T lymphocytes may provide limited host defense. The cytokine interleukin-7 (IL-7) functions to enhance lymphocyte proliferation, survival, and recruitment of immune cells to sites of infection. However, there is little known about the role of IL-7 in PCP or its potential use as an immunotherapeutic agent. We hypothesized that treatment with recombinant human IL-7 (rhIL-7) would augment host defense against Pneumocystis and accelerate pathogen clearance in CD4-depleted mice. Control and CD4-depleted mice were infected with Pneumocystis, and rhIL-7 was administered via intraperitoneal injection. Our studies indicate that endogenous murine IL-7 is part of the normal host response to Pneumocystis murina and that administration of rhIL-7 markedly enhanced clearance of Pneumocystis in CD4-depleted mice. Additionally, we observed increased recruitment of CD8⁺ T lymphocytes to the lungs and decreased apoptosis of pulmonary CD8⁺ T lymphocytes in rhIL-7-treated animals compared to those in untreated mice. The antiapoptotic effect of rhIL-7 was associated with increased levels of Bcl-2 protein in T lymphocytes. rhIL-7 immunotherapy in CD4-depleted mice also increased the number of gamma interferon (IFN-γ)-positive CD8⁺ central memory T lymphocytes in the lungs. We conclude that rhIL-7 has a potent therapeutic effect in the treatment of murine Pneumocystis pneumonia in CD4-depleted mice. This therapeutic effect is mediated through enhanced recruitment of CD8⁺ T cells and decreased apoptosis of lung T lymphocytes, with a preferential action on central memory CD8⁺ T lymphocytes.     

Kinetic analysis of the development of pancreatic lesions in mice infected with a murine retrovirus

Sjögren's syndrome (SjS)-like sialoadenitis and exocrine pancreatitis were induced in mice infected with LP-BM5 murine leukemia virus, which induces a severe immunodeficiency termed murine AIDS (MAIDS). All mice with MAIDS showed advancing cellular infiltration around the pancreatic ducts as well as systemic exocrinopathy. The primary target tissue of the pancreas was acinar cells, and the pancreatic islets were well preserved until a late phase of the disease. Immunofluorescence and flow cytometry demonstrated that CD4⁺ T cells, Mac-1⁺ cells, and B220⁺ cells were major inflammatory components, and IFN-γ and IL-10 were mainly detected on CD4⁺ T and Mac-1⁺ cells in the pancreas. Both Th1 and Th2 cells were found. TUNEL⁺ apoptotic cells were mostly detected among pancreas-infiltrating cells. Fas ligand and TNF-α were also detected among pancreas-infiltrating cells, whereas Fas was rarely expressed in the pancreatic acinar cells. Thus, MAIDS mice could be valuable for analyzing the pathogenesis of autoimmune-related pancreatitis associated with SjS.     

Increased Fas antigen expression in murine retrovirus-induced immunodeficiency syndrome,MAIDS

The Fas antigen (Fas), which is a cell surface protein belonging to the tumor necrosis factor receptor family, mediates apoptosis. To assess the contribution of Fas to the pathogenesis of retrovirus-induced immunodeficiency, we examined the kinetics of Fas expression on the lymphocytes during the course of murine acquired immunodeficiency syndrome (MAIDS) induced by a defective LP-BM5 murine leukemia virus. The Fas-positive cells were increased in proportion both in αβ T cells and B cells with the progression of MAIDS. The appearance of Fas-positive cells in αβ T cells preceeded those in B cells during the course of MAIDS. Among αβ T cells, about half of the Thy1.2⁺ αβ T cells were positive for Fas, while almost all of Thy1.2^? CD4⁺ αβ T cells were of the Fas-positive phenotype. The Fas-positive cells in MAIDS mice, especially unique Thy1.2^?CD4⁺ αβ T cells, were easily rendered apoptotic by stimulation via Fas, indicating that Fas expressed on the lymphocytes is functional. Furthermore, concomitant infection with Mycobacterium avium in MAIDS mice caused a marked increase in Fas-positive cells accompanied by a severely impaired T cell reactivity to polyclonal stimuli. Taken together, these results suggest the possible participation of the Fas system in the pathogenesis of retrovirus-induced immunodeficiency.     

Recombinant interleukin-12 enhances resistance of mice to Listeria monocytogenes infection

The effect of recombinant murine IL-12 (rIL-12) or anti-IL-12 antibody administration on resistance to murine listeriosis was investigated. Mice given a single 0.5 μg dose of rIL-12 had 1.5 log₁₀ fewer listeriae in their spleens and livers as compared with control infected mice 3 days after L. monocytogenes challenge. Conversely, administration of anti-IL-12 IgG caused an equivalent increase in the cfu of L. monocytogenes recovered from the spleens and livers as compared to control mice. This is the first report of such a protective effect from a single dose of rIL-12. Treatment of uninfected mice with rIL-12 induced IFN-γ mRNA production in their livers. Infection of mice with L. monocytogenes caused a similar increase in IFN-γ mRNA levels that was not increased further by concurrent treatment with rIL-12. Treatment of mice with an anti-IFN-γ MAb eliminated the protective effect of IL-12 on Listeria infection. Expression of TNF-γ, IL-10 and IL-12p40 mRNA in L. monocytogenes-infected mice were not significantly altered by administration of either anti-IL-12 IgG or rIL-12. rIL-12 administration was associated with increased serum AST levels, a measure of liver damage, 1 day after treatment in L. monocytogenes-infected mice. In addition, rIL-12 administration was associated with the increased presence of small inflammatory foci and necrotic hepatocytes in both infected and uninfected mice, suggesting a proinflammatory role for IL-12 in the liver.     

Unique functions of splenic CD8alpha+ dendritic cells during infection with intracellular pathogens

Deciphering the prerequisites for the induction of protective cytotoxic T cell responses is essential for future development of more effective CD8⁺ T cell-based vaccines against infectious diseases and cancer. Since crucial events for CD8⁺ T cell priming and differentiation occur during the first contacts of naïve T cells with distinct antigen-presenting cells (APCs), the identification and therapeutic targeting of these ‘master’ APCs has become a major quest in the field.A decade ago, dendritic cells (DCs) were discovered as potent APCs, as they combine all major features for the initiation of T cell responses: (1) naïve DCs demonstrate high endocytic activity and scan continuously their environment in strategic positions throughout the whole body; (2) after activation (e.g. during pathogen invasion), DCs migrate into T cell zones of their draining lymphatic compartments, meanwhile processing captured antigen and maturing in order to stimulate encountered antigen-specific T cells.During the last years, different subsets of DCs that can be distinguished by specific surface marker expression and effector functions have been identified in mice. Their distinct functional capabilities have led to the concept of work-sharing; “migrating” DCs primarily transport antigens to the lymph node, where a specialized subset of “resident” DCs, defined by the expression of the CD8αα homodimer (CD8α⁺ DCs), primes CD8⁺ T cells upon antigen cross-presentation. Accordingly, CD8α⁺ DCs have been found to prime CD8⁺ T cells against different viruses as well as intracellular bacteria such as Listeria monocytogenes (L.m.).Recently, L.m. was found to survive specifically in splenic CD8α⁺ DCs shortly after intravenous infection. Further experiments revealed a more generalized sampling activity of CD8α⁺ DCs for blood-borne particles. These findings indicate that splenic CD8α⁺ DCs might unite efficacious antigen-trapping with the licence to prime CD8⁺ T cells. This new aspect of DC function could have evolved to guarantee a more rapid antigen-specific response against generalized infections.     

Modulation of specific T cell responses by concurrent infection with Leishmania major and LP-BM5 murine leukemia viruses

C57BL/6 mice infected with a murine leukemia virus (MuLV) mixturedesignated LP-BM5 develop an immunodeficiency syndrome termedMAIDS, characterized by a variety of T and B cell abnormalities,including elevated levels of IgE, suggesting that IL-4 expressionis increased in these animals. It has been suggested that theimmunodeficiency associated with MAIDS is caused by a conversionof immune responses normally characterized by T_h1 developmenttowards a T_h2- dominated response. Mice of the same strain,infected with Leishmania major, mount a protective T_h1 responsewith the induction of high levels of IFN- and undetectable IL-4.We therefore infected mice with L. major at differing time pointsbefore and after virus infection and assessed the effects onT cell responsiveness, cytokine production and survival to L.major, as well as the effect on MAIDS-associated pathology.We have also immunized C57BL/6 mice with trinitrophenol-keyholelimpet haemocyanln (TNP-KLH), which leads to a predominantlyT_h2 response, and compared the effects of MAIDS on the responseto TNP-KLH with the effect of MAIDS on L. major infection. Ourresults show that significant immunodeficiency with regard toinfection by L. major is only apparent after 8 weeks of LP-BM5MuLV infection, by which time T and B cell defects are welladvanced. Further, we have found that the strongly polarizedT_h1 response stimulated by L. major infection can modulate theeffect of MAIDS on T cells, leading to the survival of antigen-specificT cells. Our results suggest that the impairment of immune responsesto either TNP–KLH or L. major is due not to an alterationof the balance of T_h1/T_h2 subsets but to a general loss of reactivityin antigen-specific CD4⁺ cells. However, prior activation ofT_h1 but not T_h2 cells can inhibit the development of lymphoproliferationand immunodeficiency caused by MAIDS.     

Interleukin-22-Induced Antimicrobial Phospholipase A2 Group IIA Mediates Protective Innate Immunity of Nonhematopoietic Cells against Listeria monocytogenes

Listeria monocytogenes is a bacterial pathogen which establishes intracellular parasitism in various cells, including macrophages and nonhematopoietic cells, such as hepatocytes. It has been reported that several proinflammatory cytokines have pivotal roles in innate protection against L. monocytogenes infection. We found that a proinflammatory cytokine, interleukin 22 (IL-22), was expressed by CD3⁺ CD4⁺ T cells at an early stage of L. monocytogenes infection in mice. To assess the influence of IL-22 on L. monocytogenes infection in hepatocytes, cells of a human hepatocellular carcinoma line, HepG2, were treated with IL-22 before L. monocytogenes infection in vitro. Gene expression analysis of the IL-22-treated HepG2 cells identified phospholipase A2 group IIA (PLA2G2A) as an upregulated antimicrobial molecule. Addition of recombinant PLA2G2A to the HepG2 culture significantly suppressed L. monocytogenes infection. Culture supernatant of the IL-22-treated HepG2 cells contained bactericidal activity against L. monocytogenes, and the activity was abrogated by a specific PLA2G2A inhibitor, demonstrating that HepG2 cells secreted PLA2G2A, which killed extracellular L. monocytogenes. Furthermore, colocalization of PLA2G2A and L. monocytogenes was detected in the IL-22-treated infected HepG2 cells, which suggests involvement of PLA2G2A in the mechanism of intracellular killing of L. monocytogenes by HepG2 cells. These results suggest that IL-22 induced at an early stage of L. monocytogenes infection enhances innate immunity against L. monocytogenes in the liver by stimulating hepatocytes to produce an antimicrobial molecule, PLA2G2A.     

Th0-like CD4+ T cells protect mice with murine retrovirus-induced immunodeficiency syndrome (MAIDS) against co-infection with Listeria monocytogenes.

We examined the host defence mechanism against infection with Listeria monocytogenes, a facultative intracellular bacterium, in mice with murine acquired immunodeficiency syndrome (MAIDS) caused by LP-BM5 murine leukaemia virus (MuLv) infection. Although LP-BM5 MuLV infection in C57BL/6 mice leads to a stage of immunodeficiency characterized by severe compromise of cell-mediated immunity, the mice with established MAIDS infected with LP-BM5 8 weeks previously, showed resistance to an intraperitoneal infection with Listeria monocytogenes. These MAIDS mice also showed resistance to a lethal dose of secondary listerial challenge, while the delayed-type hypersensitivity response to heat-killed Listeria (HKL.) was severely impaired in MAIDS mice. The resistance of MAIDS mice to listerial infection was mediated by CD4+ alpha beta T cells but neither by gamma delta T cells nor natural killer (NK) cells. Interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) were produced by CD4+ T cells from Listeria-infected MAIDS mice in response to the in vitro stimulation with HKL, whereas IFN-gamma but not IL-10 were produced by those from Listeria-infected control mice. These results suggest that T-helper 0 (Th0)-like immune responses of CD4+ T cells occur and participate in host defence mechanisms against listerial infection in MAIDS mice.     

The interaction of intestinal epithelial cells and intraepithelial lymphocytes in host defense

Intestinal intraepithelial lymphocytes (i-IEL) are located at the basolateral surfaces of intestinal epithelial cells (i-EC) and play important roles in the homeostasis of intestinal microenvironment. i-IEL comprise unique T cell populations including CD4^-CD8αα⁺ T cells expressing T cell receptor (TCR)αΒ or TCRγδ and CD4⁺ CD8αα⁺ T cells expressing TCR αΒ. We show here that CD4⁺ CD8αα⁺ i-IEL belongs to Th1 type T cells capable of responding to self-MHC class I on i-EC and that a significant fraction of i-IEL expressed Fas ligand (Fas-L) and induced apoptosis in the i-EC via Fas-dependent pathway. i-IEL may recognize and eliminate the effete i-EC for homeostatic regulation of intestinal epithelia. The interaction of i-EC and i-IEL through E-cadherin/α_EΒ₇ integrin is important for homing and maintenance of i-IEL in intestine.Listeria monocytogenes are also known to interact with E-cadherin on i-EC and invade into the epithelial cells. Invasion ofL. monocytogenes into i-EC activated NFk-B and subsequently up-regulated the expression of IL-15 gene, which has a NFk-B binding site at the promoter region. i-IEL, especially γδ T cells, were significantly activated to produce Th1 type cytokines at the early stage after oral infection withL. monocytogenes in mice and rats. The activation of i-IEL coincided with a peak response of IL-15 production by i-EC after infection. Taken together, mutual interaction of i-IEL and i-EC may be important not only for homeostatic regulation but also host defense against microbial infection in intestine.