Listeria monocytogenes mediated CFTR transgene transfer to mammalian cells

Background

Several approaches for gene therapy of cystic fibrosis using viral and non‐viral vectors are currently being undertaken. Nevertheless, the present data suggest that vectors currently being used will either have to be further modified or, alternatively, novel vector systems need to be developed. Recently, bacteria have been proven as suitable vehicles for DNA transfer to a wide variety of eukaryotic cells. In this study, we assessed the ability of the facultative intracellular pathogen Listeria monocytogenes to deliver a cDNA encoding the human cystic fibrosis transmembrane conductance regulator (CFTR) to CHO‐K1 cells, since these cells have been extensively used for heterologous CFTR expression.

Methods

An established in vitro gene transfer system based on antibiotic‐mediated lysis of intracellular L. monocytogenes was exploited to transfer eukaryotic expression plasmids. Transient as well as stable CFTR transgene expression was analyzed by microscopical and biochemical methods; functionality was tested by whole‐cell patch‐clamp recordings.

Results

L. monocytogenes mediated gene transfer to CHO‐K1 cells was facilitated by an improved transfection protocol. In addition, the use of the isogenic mutant L. monocytogenes hlyW491A, engineered to produce a hemolysin variant with low toxigenic activity, greatly enhanced the efficiency of gene transfer. This strain allowed the transfer of functional CFTR to CHO‐K1 cells.

Conclusions

This is the first demonstration of L. monoyctogenes mediated CFTR transgene transfer. The successful in vitro transfer suggests that L. monocytogenes might be a potential vector for cystic fibrosis gene therapy or alternative applications and deserves further investigation in vitro as well as in vivo. Copyright © 2002 John Wiley & Sons, Ltd.

     

Generation of alloreactive cytotoxic T lymphocytes: production of T cell and macrophage helper factors in addition to IL 1 and IL 2 by peritoneal cells from mice immunized to Listeria monocytogenes

We investigate the production and biological activity of soluble helper factors produced by peritoneal T cells and macrophage derived from mice primed in vivo with Listeria monocytogenes. Supernatant fluids from co-cultures of these immune T cells and activated macrophages contained Interleukin 1 (IL 1) and Interleukin 2 (IL 2), and had the ability to assist the generation of cytotoxic T lymphocytes (CTL) from a population of nylon wool nonadherent spleen cells sensitized to allogeneic heat-treated thymocytes. The ability to assist CTL development involved T cell and macrophage factors in addition to IL 1 and IL 2. Immune T cells cultured alone produced a factor, devoid of significant IL 2 activity, that assisted CTL development only if adherent cells were present in the responding population. Activated macrophage produced a 38,000 dalton component, distinct from IL 1 on the basis of m.w., that assisted the development of CTL from nylon wool nonadherent splenic cells. Supernatants fluids from co-cultures of immune T cells and allogeneic, nonactivated macrophage contained a CTL helper factor but did not contain IL 1 or IL 2 activities. In contrast, supernatant fluids from co-cultures of immune T cells and syngeneic, nonactivated macrophage contained all 3 activities. This suggests a genetic restriction for the production of IL 1 and IL 2 that does not restrict the production of a CTL helper factor. These results demonstrate that T cell- and macrophage-derived helper factors distinct from IL 1 and IL 2 participate in the development of CTL.     

T cell-mediated cytotoxicity induced by Listeria monocytogenes. I. Activation of Listeria antigen-responsive T cells

Soon after rats are infected with Listeria monocytogenes (LM), Listeria antigen- (LMA) responsive lymphocytes are delivered to the animal's thoracic duct. These LM-responsive lymphocytes can be restimulated in vitro by LMA to generate cells that have a potent cytolytic capability. The activation of LMA responsive lymphocytes is immunologically specific and dependent upon the activity of histocompatible accessory cells. Neither cell-free LMA nor LMA-pulsed allogeneic accessory cells can promote a significant cytotoxic response by negatively selected responder lymphocytes. LM-dependent cytolytic lymphocytes differ from natural killer (NK) cells inasmuch as their activation is not facilitated by interferon (IF). Likewise, supernatants from cultures containing specifically sensitized thymus-derived (T) lymphocytes and histocompatible LMA-pulsed accessory cells fail to augment (day 2 and day 3 supernatants actually inhibit) the activation process. The results imply that the successful activation of LM dependent cytolytic lymphocytes requires the cooperative interplay of responder T cells and specific-antigen-pulsed accessory cells.     

Enhanced conjugative transfer of plasmid DNA from Escherichia coli to Staphylococcus aureus and Listeria monocytogenes

Abstract Transfer of mobilizable shuttle cloning vectors by conjugation from Escherichia coli to Staphylococcus aureus occurred at a very low frequency (10⁻⁹ transconjugants per donor colony-forming unit after the mating period). It was observed that subinhibitory concentrations of penicillins (oxacillin or penicillin G) in the mating medium resulted in increased transfer frequency by conjugation of the shuttle vector pAT18 from E. coli SM10 to S. aureus 80CR5 Str (54-fold) and to Listeria monocytogenes LO17RF (45-fold). These results were interpreted as indicating that the cell wall of Gram-positive bacteria constitutes an important barrier for conjugative transfer of genetic information demonstrated that presence of a restriction system(s) in S. aureus recipients represented a major barrier to introduction of foreign DNA.     

Protection by dexamethasone from a lethal infection with Listeria monocytogenes in mice

Abstract The effects of dexamethasone (DEX) on a lethal infection with Listeria monocytogenes were studied in mice. Mice were completely protected against the lethal infection when treated with 3.3 mg per kg of DEX. The effect was observed only when DEX was injected before infection. The control mice died from day 3 to day 5 of infection, whereas DEX-treated mice could eliminate L. monocytogenes cells from the organs by day 11 of infection. High titres of endogenous tumour necrosis factor (TNF), interleukin-6 (IL-6) and gamma interferon (IFN-γ) were induced in the bloodstreams and organs of the drug-free mice. DEX suppressed IL-6 production, but augmented TNF and IFN-γ production within 24 h of infection, whereas production of all three endogenous cytokines was suppressed in the DEX-treated mice on day 3 of infection when the control mice began to die. These results suggest that DEX shows a protective effect on a lethal infection with L. monocytogenes in mice and that regulation of production of endogenous cytokines might be involved in the effect of DEX.     

Differing contribution of polymorphonuclear cells and macrophages to protection of mice against Listeria monocytogenes and Pseudomonas aeruginosa.

Bacterial growth and lethality of Listeria monocytogenes in mice were augmented by carrageenan-treatment and X-irradiation (8 J kg-1), whereas growth and lethality of Pseudomonas aeruginosa were augmented by X-irradiation but not by carrageenan-treatment. Protection against L. monocytogenes, at least in the early phases, appears to depend mainly on macrophages, since carrageenan depletes macrophages but not polymorphonuclear cells (PMN), whereas protection against P. aeruginosa appears to depend mainly on PMN. Ineffectiveness of PMN in elimination of L. monocytogenes is supported by histological examination and observation of intracellular killing in vitro.     

In vitro heterologous cytotoxicity by T effector cells from mice immunized with Sindbis virus.

An in vitro correlate of cell-mediated cross-protection among alpha-viruses was demonstrated by cytotoxicity of Sindbis-immune spleen cells from mice to both Sindbis and Semliki Forest virus (SFV)-infected target cells. This cytotoxicity was shown to be mediated by the T cell population of the spleen and was independent of the presence of macrophages or B cells. The time when the level of the lymphocyte-mediated cytotoxicity (LMC) to SFV-infected cells was maximal coincides with the time when immunity to SFV is maximal in vivo, as reported previously, and when adoptive immunity to SFV can be transferred. After one i.p. injection of Sindbis virus, the level of homologous LMC was higher than the level of heterologous LMC. However, following a second injection of Sindbis virus as immunogen, at a time when the mice are cross-protected to SFV, the heterologous LMC was considerably higher than homologous LMC. We propose that there is suppression of the effector T cells specific for Sindbis-infected cells after the second immunizing injection, probably by homologous antibody. In contrast, there appears to be an anamnestic cell-mediated response to SFV.     

Distinct immunologic specificity of tumor regression mediated by effector cells isolated from immunized and tumor-bearing mice

Sensitized T lymphocytes can mediate potent antitumor effects when transferred to tumor-bearing animals. Employing the MCA 105 and MCA 106 sarcomas, we were able to generate antitumor effector cells by immunization of syngeneic mice with tumor cells admixed with Corynebacterium parvum. These immune splenocytes could be further sensitized and expanded in culture by the in vitro sensitization (IVS) method utilizing tumor stimulator cells and IL-2. Adoptive immunotherapy of pulmonary metastases mediated by noncultured splenocytes from immunized mice or immune IVS cells showed exquisite specificity between the two sarcomas. These results demonstrate the presence of tumor-specific antigens on MCA 105 and MCA 106 tumor cells which can serve as target molecules for immunotherapy. Recently, we have generated therapeutic T lymphocytes from mice bearing progressively growing tumors by the IVS method. However, IVS cells from tumor-bearing mice showed cross-reactivity between the MCA 105 and 106 sarcomas in adoptive immunotherapy experiments. Since these IVS cells did not affect other control tumors, the limited cross-reactivity suggests the presence of common tumor-associated antigens on MCA 105 and MCA 106 tumor cells which can also serve as the target for tumor rejection. Therefore, immune responses to progressive tumor growth and to immunization are distinct with respect to antigen recognition by T lymphocytes.     

Antigen-specific Lyt-2+ cytolytic T lymphocytes from mice infected with the intracellular bacterium Listeria monocytogenes

In vitro expanded T cell lines were used to determine whether antigen-specific cytolytic T lymphocytes are generated after infection with the intracellular bacterium, Listeria monocytogenes. Spleen cells from infected mice were cultured in the presence of syngeneic accessory cells, listerial antigen, and interleukin 2 containing supernatants. Cell lines were greater than 98% Thy-1+, L3T4-, Lyt-2+. Bone-marrow macrophages were used as target cells in two in vitro cytolytic assay systems. The Lyt-2+ T cells killed bone marrow macrophages only when infected with L. monocytogenes as assessed in a 4-hr 51Cr release assay and in an 18-hr neutral red uptake assay. Cytolysis was blocked by anti-LFA-1 and anti-Lyt-2 monoclonal antibodies. These cytolytic T cells produced interferon-gamma after co-stimulation with antigen, accessory cells, and recombinant interleukin 2. Bone marrow macrophages infected with Mycobacterium bovis were not killed by T cells from L. monocytogenes-infected mice but by T cell lines from M. bovis-infected mice, indicating that cytolysis was antigen specific. L. monocytogenes-infected target cells of different haplotype were lysed by the Lyt-2+ T cells. By using a low cell density split culture system, antigen-specific, H-2-restricted cytolytic T cells could be identified. These findings demonstrate that during infection with intracellular bacteria, Lyt-2+ T cells with cytolytic activity are generated that may be involved in antibacterial protection.     

Differential susceptibility of bone marrow-derived dendritic cells and macrophages to productive infection with Listeria monocytogenes

Dendritic cells (DC) are required for the immune response against Listeria monocytogenes and are permissive for infection in vivo and in vitro. However, it is unclear if DC provide a desirable intracellular niche for bacterial growth. To address this issue, we have compared the behaviour of L. monocytogenes in murine bone marrow-derived DC and macrophages (BMM). Similar to BMM, bacteria escaped to the cytosol in DC, replicated, and spread to adjacent cells. However, DC infection was less robust in terms of intracellular doubling time and total increase in bacterial numbers. Immunofluorescence analysis using a strain of L. monocytogenes that expresses green fluorescent protein upon bacterial entry into the cytosol suggested that a subpopulation of DC restricted bacteria to vacuoles, a finding that was confirmed by electron microscopy. In unstimulated DC cultures, L. monocytogenes replicated preferentially in phenotypically immature cells. Furthermore, DC that were induced to mature prior to infection were poor hosts for bacterial growth. We conclude that DC provide a suboptimal niche for L. monocytogenes growth, and this is at least in part a function of the DC maturation state. Therefore, the generation of an effective T cell response may be a net effect of both productive and non-productive infection of DC.     

Oxidative and phagocytic functions of macrophages during infections induced in mice by Mycobacterium intracellulare and Listeria monocytogenes

The oxidative metabolism (chemiluminescence and H2O2 release) and phagocytic activity of mouse peritoneal macrophages during chronic infections induced by Mycobacterium intracellulare and more acute infections due to Listeria monocytogenes were studied. In M. intracellulare infections, macrophage chemiluminescence in response to phorbol myristate acetate (PMA) was greatest at around 2 weeks, with a 1 week lag phase after infection, while the PMA-triggered H2O2 release was markedly enhanced even 1 d after challenge, and remained high thereafter for up to 10 weeks. The pattern of changes in the phagocytic activity of host macrophages in response to latex beads during this infection resembled the pattern seen with macrophage H2O2 release. In the L. monocytogenes infections, the PMA-triggered chemiluminescence of the host macrophages increased 4 d (in a sublethal infection) and 2 d (in a lethal infection) after bacterial challenge, whereas the PMA-triggered H2O2 release was markedly enhanced as early as 1 d after infection and the elevated level persisted until either the bacteria were eliminated or the animals died. The patterns of changes in phagocytic activity of the host macrophages during L. monocytogenes infection at sublethal and lethal doses differed. In the former, phagocytosis was most active in the early phase of infection, with a peak around day 2, followed by a rapid decrease; in the latter, the phagocytic ability increased more slowly, and remained elevated until the animals died. The results suggest that the macrophages induced by M. intracellulare are in a more activated state than are those induced by L. monocytogenes.     

Eukaryotic expression plasmid transfer from the intracellular bacterium Listeria monocytogenes to host cells

The facultative intracellular, Gram-positive bacterium Listeria monocytogenes invades phagocytic and non-phagocytic cells from the tissues and organs of a wide variety of animals and humans. Here, we report the use of these bacteria as vehicles for gene transfer. Eukaryotic expression plasmids were introduced into the nucleus of host cells following lysis of the intracytosolic, plasmid-carrying bacteria with antibiotics. Cell lines of different tissues and species could be transfected in this way. We examined bacterial properties required for delivery of the expression plasmids and found that this was strictly dependent on the ability of these bacteria to both invade eukaryotic cells and egress from the vacuole into the cytosol of the infected host cells. Macrophage-like cell lines or primary, peritoneal macrophages proved to be almost refractory to Listeria ‐mediated gene transfer. Thus, attenuated L. monocytogenes represents a serious candidate for consideration as a DNA-transfer vehicle for in vivo somatic gene therapy. The potential for oral administration of L. monocytogenes and the ease in producing and cultivating recombinant strains are further attributes that make its use as a gene transfer vehicle attractive.     

Relative contributions of NK and CD8 T cells to IFN-gamma mediated innate immune protection against Listeria monocytogenes

During the innate immune response to Listeria monocytogenes (LM), the secretion of IFN-gamma is crucial in controlling bacterial numbers. We have shown recently that CD8 T cells have the ability to rapidly secrete IFN-gamma independent of Ag, in response to IL-12 and IL-18, during a LM infection. In the current study, we compared the relative abilities of NK and CD8 T cells to provide innate immune protection. Upon transfer of either NK or memory OT-I T cells (specific for the OVA protein) into IFN-gamma-deficient hosts that were infected subsequently with wild-type LM, both cell types were found in the spleen and had the ability to secrete IFN-gamma. However, the OT-I T cells were more effective at providing innate immune protection as determined by spleen and liver LM burdens. We used immunocytochemistry to demonstrate that upon infection with LM, marginal zone macrophages were localized to the T cell area of the splenic follicle. Transferred memory OT-I T cells were also found in the T cell area of the spleen, co-localizing with the LM and macrophages. In sharp contrast, NK cells were found predominantly in the red pulp region of the spleen. In addition, memory OT-I T cells were also found to be associated with LM lesions in the liver. These results highlight the importance of CD8 T cells in innate immune responses to LM and suggest that their increased protective ability compared with NK cells is the result of their colocalization with LM and macrophages.     

Impairment of T cell-mediated immunity to Listeria monocytogenes in pregnant mice

In order to study pregnancy-induced changes in cell-mediated immunity to Listeria monocytogenes, acquired resistance and T cell functions in pregnant mice were compared with those in nonpregnant mice after immunization with viable listerial cells. Impaired generation of acquired resistance was evident in pregnant mice from the impaired elimination of bacteria and poor survival after secondary challenge. Delayed footpad reactivity to listerial antigen was also lower in the pregnant mice. When immune spleen cells were examined for their ability to produce macrophage activating factor in vitro, culture supernatants from pregnant-mouse spleen cells with listerial antigen showed far less ability to render macrophages cytostatic for P815 mastocytoma cells. To elucidate further the impairment of listeria-immune T cell generation in pregnant mice, a local transfer experiment was carried out. When a given number of immune spleen cells was transferred locally into the footpads of naive mice, both delayed footpad reaction and local protection were much lower in the pregnant mice. This local transferability of the reactions was abrogated after treatment of cells with anti-Thy 1 antibody plus complement. These findings indicate that pregnancy impairs the generation of specific T cells capable of contributing to acquired resistance to L. monocytogenes. Possible mechanisms for this impairment and the relationship to macrophage functions are discussed.     

Cloned L3T4+ T lymphocytes protect mice against Listeria monocytogenes by secreting IFN-gamma

Murine T lymphocyte clones sensitized to Listeria monocytogenes were developed to investigate specific mechanisms of T cell-mediated immunity. The clones were of the Thy-1.2+, L3T4+, Lyt-2- phenotype and proliferated in a dose response fashion to heat-killed Listeria. Cloned T lymphocytes injected intravenously protected nonimmune mice against L. monocytogenes challenge as determined by spleen and liver bacterial numbers. Supernatants, produced by stimulating clones with heat-killed Listeria for 48 h, also afforded protection against L. monocytogenes. The clonal supernatants contained significant quantities of IFN-gamma and CSF. IFN-gamma production was Ag specific and occurred within 24 h of stimulation. CSF production by clones was increased four- to sixfold over baseline as determined by a bone marrow colony-forming assay and was Ag specific. When the IFN-gamma in supernatants was neutralized with a specific mAb, protection afforded by the supernatants was lost. These data indicate that one mechanism for Ag-specific, T lymphocyte-mediated protection against L. monocytogenes is the release of IFN-gamma.     

T cell-mediated cytotoxicity induced by Listeria monocytogenes. III. Phenotypic characteristics of mediator T cells

Cytotoxic thymus-derived lymphocytes (CTL) generated in vitro by restimulating rat cells with Listeria antigen- (LMA) pulsed syngeneic accessory cells were characterized in respect to their surface membrane markers. LM-dependent CTL were devoid of detectable surface immunoglobulin (Ig) and receptors for the Fc region of rabbit IgG. Experiments with monoclonal antibodies to rat T cell markers revealed that these cytotoxic cells have the phenotypic profile W3/13+, W3/25-, MRC OX 8+. LM-dependent CTL also bind the monoclonal antibody, MRC OX 3, which recognizes an Ia-antigen-like determinant on rat cells. Although LM-dependent CTL lack the W3/25 marker, their generation depends on the cooperative interplay of W3/25+ and W3/25- T cells.     

Spontaneous rejection of intradermally transplanted non-engineered tumor cells by neutrophils and macrophages from syngeneic strains of mice

It is not surprising that tumors arising spontaneously are rarely rejected by T cells, because in general they lack molecules to elicit a primary T-cell response. In fact, cytokine-engineered tumors can induce granulocyte infiltration leading to tumor rejection. In the present study, we i.d. injected seven kinds of non-engineered tumor cells into syngeneic strains of mice. Three of them (i.e. B16, KLN205, and 3LL cells) continued to grow, whereas four of them (i.e. Meth A, I-10, CL-S1, and FM3A cells) were spontaneously rejected after transient growth or without growth. In contrast to the i.d. injection of B16 cells into C57BL/6 mice, which induces infiltration of TAMs into the tumors, the i.d. injection of Meth A cells into BALB/c mice induced the invasion of cytotoxic inflammatory cells, but not of TAMs, into or around the tumors leading to an IFN-γ-dependent rejection. On day 5, the cytotoxic activity against the tumor cells reached a peak; and the effector cells were found to be neutrophils and macrophages. The i.d. Meth A or I-10 cell-immunized, but not non-immunized, mice rejected i.p.- or i.m.-transplanted Meth A or I-10 cells without growth, respectively. The main effector cells were CTLs; and there was no cross-sensitization between these two kinds of tumor cells, suggesting specific rejection of tumor cells by CTLs from i.d. immunized mice. These results indicate that infiltration of cytotoxic myeloid cells (i.e. neutrophils and macrophages, but not TAMs) into or around tumors is essential for their IFN-γ-dependent spontaneous rejection.     

Cytolysin-dependent delay of vacuole maturation in macrophages infected with Listeria monocytogenes

The bacterial pathogen Listeria monocytogenes (Lm) evades the antimicrobial mechanisms of macrophages by escaping from vacuoles to the cytosol, through the action of the cytolysin listeriolysin O (LLO). Because of heterogeneities in the timing and efficiency of escape, important questions about the contributions of LLO to Lm vacuole identity and trafficking have been inaccessible. Expression of cyan fluorescent protein (CFP)-labelled endocytic membrane markers in macrophages along with a yellow fluorescent protein (YFP)-labelled indicator of Lm entry to the cytosol identified compartments lysed by bacteria. Lm escaped from Rab5a-negative, lysosome-associated membrane protein-1 (LAMP1)-negative, Rab7-positive, phosphatidylinositol 3-phosphate [PI(3)P]-positive vacuoles. Lm vacuoles did not label with YFP-Rab5a unless the bacteria were first opsonized with IgG. Wild-type Lm delayed vacuole fusion with LAMP1-positive lysosomes, relative to LLO-deficient Lm. Bacteria prevented from expressing LLO until their arrival into LAMP1-positive lysosomes escaped inefficiently. Thus, the LLO-dependent delay of Lm vacuole fusion with lysosomes affords Lm a competitive edge against macrophage defences by providing bacteria more time in organelles they can penetrate.     

Upregulation of vascular endothelial growth factor by heat-killed Listeria monocytogenes in macrophages

We investigated the effect of heat-killed Listeria monocytogenes (HKLM) on the expression of vascular endothelial growth factor (VEGF) in RAW264.7 macrophage-like cells. The expression of VEGF was induced in RAW264.7 cells treated with HKLM. Pretreatment of cells with cycloheximide, a protein synthesis inhibitor, inhibited the induction of VEGF mRNA by HKLM. Induction of VEGF by HKLM was partially inhibited by treatment of cells with SB203580, a p38 mitogen-activated protein kinase (MAPK) inhibitor, or a neutralizing antibody against tumor necrosis factor-alpha (TNF-alpha). In addition, HKLM induced phosphorylation of p38 MAPK. These results suggest that p38 MAPK and TNF-alpha are involved in the VEGF expression induced by HKLM in RAW264.7 cells. We confirmed that increased VEGF expression is immunohistochemically detected in splenic macrophages of mice infected with L. monocytogenes (L. monocytogenes). VEGF is thought to be involved in inflammatory reactions induced by L. monocytogenes infection.