Interleukin 2-induced proliferation of leukemic human B cells.

The proliferative responses of purified leukemic human B cells from nine B cell chronic lymphocytic leukemias to recombinant interleukin 2 (IL-2), spontaneously, and after preactivation by Staphylococcus aureus Cowan I (SAC) or anti-mu antibodies were studied. Three patterns of response were observed: (a) no response (three cases); (b) a moderate spontaneous response enhanced by anti-mu (one case); (c) a high proliferative response after preactivation by anti-mu and/or SAC (five cases). IL-2 could also trigger normal B cells, purified from spleen, to proliferative after preactivation by anti-mu or SAC. These results provide evidence that IL-2 is a lymphokine that acts physiologically on both B and T cells.

Secondary cell-mediated lympholysis: importance of H-2 LD and SD factors.

Lymphocytes stimulated in mixed leukocyte cultures and left for 13-17 days, i.e. beyond their peak proliferative and cytotoxic reactivities, can be restimulated to give a secondary-type rapid and strong proliferative and cytotoxic response when confronted with cells of the original sensitizing cell donor. We have concerned ourselves primarily with the requirements of restimulation for the presence of LD and/or SD stimuli on the restimulating cells. (a) The low level cell-mediated lympholysis (CML) associated with LD differences in a primary CML can be restimulated to give a secondary-type response by those same LD antigens. (b) If the original sensitizing cells differ from the responding cells by both LD and SD antigens, restimulation with only the LD antigens, or third-party cells presumably carrying cross-reactive LD antigens, can restimulate the secondary CML responses directed against the SD antigens on the original sensitizing cells. (c) The presence of SD antigens on the restimulating cells that are cross-reactive with the primary sensitizing SD antigens (as determined in a primary CML) leads to the preferential activation of cytotoxic T lymphocytes reactive to those antigens although maximum cytotoxicity is still directed at cells carrying the original sensitizing SD antigens. A model to explain these results is presented.

In vivo bioluminescence imaging for the study of intestinal colonization by Escherichia coli in mice.

Bioluminescence imaging (BLI) is emerging as a powerful tool for real-time monitoring of infections in living animals. However, since luciferases are oxygenases, it has been suggested that the requirement for oxygen may limit the use of BLI in anaerobic environments, such as the lumen of the gut. Strains of Escherichia coli harboring the genes for either the bacterial luciferase from Photorhabdus luminescens or the PpyRE-TS and PpyGR-TS firefly luciferase mutants of Photinus pyralis (red and green thermostable P. pyralis luciferase mutants, respectively) have been engineered and used to monitor intestinal colonization in the streptomycin-treated mouse model. There was excellent correlation between the bioluminescence signal measured in the feces (R2=0.98) or transcutaneously in the abdominal region of whole animals (R2=0.99) and the CFU counts in the feces of bacteria harboring the luxABCDE operon. Stability in vivo of the bioluminescence signal was achieved by constructing plasmid pAT881(pGB2OmegaPamiluxABCDE), which allowed long-term monitoring of intestinal colonization without the need for antibiotic selection for plasmid maintenance. Levels of intestinal colonization by various strains of E. coli could be compared directly by simple recording of the bioluminescence signal in living animals. The difference in spectra of light emission of the PpyRE-TS and PpyGR-TS firefly luciferase mutants and dual bioluminescence detection allowed direct in vitro and in vivo quantification of two bacterial populations by measurement of red and green emitted signals and thus monitoring of the two populations simultaneously. This system offers a simple and direct method to study in vitro and in vivo competition between mutants and the parental strain. BLI is a useful tool to study intestinal colonization.

Bactofection of lung epithelial cells in vitro and in vivo using a genetically modified Escherichia coli.

Bacteria-mediated gene transfer ('bactofection') has emerged as an alternative approach for genetic vaccination and gene therapy. Here, we assessed bactofection of airway epithelial cells in vitro and in vivo using an attenuated Escherichia coli genetically engineered to invade non-phagocytic cells. Invasive E. coli expressing green fluorescent protein (GFP) under the control of a prokaryotic promoter was efficiently taken up into the cytoplasm of cystic fibrosis tracheal epithelial (CFTE29o-) cells and led to dose-related reporter gene expression. In vivo experiments showed that following nasal instillation the vast majority of GFP-positive bacteria pooled in the alveoli. Further, bactofection was assessed in vivo. Mice receiving 5 x 10(8) E. coli carrying pCIKLux, in which luciferase (lux) expression is under control of the eukaryotic cytomegalovirus (CMV) promoter, showed a significant increase (P<0.01) in lux activity in lung homogenates compared to untransfected mice. Surprisingly, similar level of lux activity was observed for the non-invasive control strain indicating that the eukaryotic CMV promoter might be active in E. coli. Insertion of prokaryotic transcription termination sequences into pCIKLux significantly reduced prokaryotic expression from the CMV promoter allowing bactofection to be detected in vitro and in vivo. However, bacteria-mediated gene transfer leads to a significantly lower lux expression than cationic lipid GL67-mediated gene transfer. In conclusion, although proof-of-principle for lung bactofection has been demonstrated, levels were low and further modification to the bacterial vector, vector administration and the plasmids will be required.

Functional gene transfer from intracellular bacteria to mammalian cells.

We provide evidence of direct transfer of functional DNA from bacteria to mammalian cells. An Escherichia coli K12 diaminopimelate auxotroph made invasive by cloning the invasin gene from Yersinia pseudotuberculosis transfers DNA after simple co-incubation, into a variety of mammalian cell lines. Transfer efficiency was enhanced in some cells by coexpression of the gene for listeriolysin from Listeria monocytogenes. Expression of the acquired genes occurs in both dividing and quiescent cells. The only requirement for bacteria to transfer genetic material into nonprofessional phagocytic cells and macrophages is the ability to invade the host cell.

Recombinant Escherichia coli as a gene delivery vector into airway epithelial cells.

To transfer genes into airway epithelial cells, we have generated auxotrophic dap Escherichia coli BM2710 mutant that expresses the invasin of Yersinia pseudotuberculosis and the listeriolysin of Listeria monocytogenes. E. coli BM2710 harboring a plasmid carrying the gfp gene was incubated with immortalized normal or cystic fibrosis (CF) airway epithelial cells or with primary bronchial epithelial cells grown as an explant-outgrowth cell culture model. Approximately 2% of immortalized cells expressed GFP. Few primary cells were transfected that were always poorly differentiated and located at the edge of the outgrowth. This was consistent with the expression of beta1-integrins only on these cells and with the required interaction for cell entry of E. coli expressing the invasin with beta1-integrins. The subsequent intracellular trafficking of E. coli BM2710 studied by confocal and electronic microscopy showed that the E. coli-containing phagosomes rapidly matured into phagolysosomes. This is the first demonstration that recombinant bacteria are able to transfer genes into primary airway epithelial cells, provided that they are able to invade the cells.

Engineered E. coli delivers therapeutic genes to the colonic mucosa.

Taking advantage of the proximity of bowel mucosa to luminal bacteria, we have attempted to deliver a therapeutic gene to the colonic mucosa by oral administration of an invasive and non-pathogenic Escherichia coli. E. coli diamenopimelate (dap) auxotroph, harboring plasmid pGB2Omegainv-hly, express the inv gene from Yersinia pseudotubercolosis that confers the ability to invade nonprofessional phagocytic cells and the hly gene from Listeria monocytogenes that allows expression of lystreriolysin O, a perforin cytolysin able to perfore phagosomal membranes. This bacterial vector invades and transfers functional DNA to epithelial cells in vitro. We have shown that this strain carrying a therapeutic gene (pC1OmegaTGF-beta1) can significantly reduce the severity of experimental colitis in mice. However, as a consequence of mucosal barrier disruption during colitis, vector-specific mRNA transcripts could be recovered from the colon and also from extra-colonic tissues. We therefore replaced the constitutive CMV promoter in pC1OmegaTGF-beta1 by the inflammation-inducible interleukin-8 promoter generating plasmid pC1OmegaTGF-beta1IND. Plasmid-specific TGF-beta1 mRNA transcripts were detectable in mouse CMT-93 epithelial cells incubated with E. coli BM2710/pGB2Omegainv-hly carrying pC1OmegaTGF-beta1IND following exposure to inflammatory cytokines. Furthermore, the transcripts were detectable only within inflamed tissues and the therapeutic effects were comparable to those in animals treated with E. coli BM2710/pGB2Omegainv-hly+pC1OmegaTGF-beta1. In summary, engineered enteric bacteria can efficiently deliver in vivo therapeutic genes to the intact intestinal mucosa and regulation expression of the therapeutic gene by an inflammation-inducible promoter prevents its dissemination during colitis.

Bacterial transfer of large functional genomic DNA into human cells.

Efficient transfer of chromosome-based vectors into mammalian cells is difficult, mostly due to their large size. Using a genetically engineered invasive Escherichia coli vector, alpha satellite DNA cloned in P1-based artificial chromosome was stably delivered into the HT1080 cell line and efficiently generated human artificial chromosomes de novo. Similarly, a large genomic cystic fibrosis transmembrane conductance regulator (CFTR) construct of 160 kb containing a portion of the CFTR gene was stably propagated in the bacterial vector and transferred into HT1080 cells where it was transcribed, and correctly spliced, indicating transfer of an intact and functional locus of at least 80 kb. These results demonstrate that bacteria allow the cloning, propagation and transfer of large intact and functional genomic DNA fragments and their subsequent direct delivery into cells for functional analysis. Such an approach opens new perspectives for gene therapy.

Gene transfer from bacteria to mammalian cells.

Transfer of genetic information between phylogenetically remote bacterial genera [1], from bacteria to yeast [2] and from bacteria to plants [3] by plasmid conjugation has been described. However, direct DNA transfer from prokaryotes to mammalian cells has not yet been demonstrated. Certain bacterial species have evolved the ability to enter mammalian cells by inducing their own internalization [4]. We show that invasive strains of Shigella flexneri and Escherichia coli, that undergo lysis upon entry into mammalian cells because of impaired cell wall synthesis, can act as stable DNA delivery systems to their host. This direct gene transfer is efficient, of broad host cell range and the replicative or integrative vectors so delivered are stably inherited and expressed by the cell progeny. DNA delivery by abortive invasion of eukaryotic cells by bacteria is of potential interest for stimulation of mucosal immunity and for in vivo or ex vivo gene therapy of human diseases.

Differentiation by interleukin 2 of a subpopulation of human B cells.

We demonstrate here that purified recombinant interleukin 2 (IL-2) induces a sizeable population of highly purified spleen B cells, by both negative and positive (cell-sorting) selection to differentiate polyclonally into plasma cells. This effect is seen at concentrations of IL-2 that are optimal for T-cell growth (1-5 U/ml) and is inhibitable by anti-Tac monoclonal antibody. In contrast, peripheral blood B cells show no or minimal terminal differentiation. When spleen B cells were separated into small and large cells, only the latter responded to IL-2; since this state of activation is achieved in vivo, these data support the possibility that IL-2 may be a physiological mediator in B-cell differentiation.

Cross-linking of membrane IgM on B CLL cells.

We report here the study of early events following surface Ig cross-linking such as Ca2+ mobilization and IP3 generation, in chronic lymphocytic leukemia (CLL) B cells. Those leukemic populations were previously shown to be frozen at different stages of activation with unique requirements for proliferation. The study reported here disclosed 3 patterns of response in terms of Ca2+ mobilization and proliferation. These results are interpreted in terms of differential anti-mu signalling on B cells at different stages of activation.

Induction of suppressive cells within the T4 subset by a human T-T hybridoma-produced factor.

This study examines the target cell and the mode of action of a factor (TsF), produced by a human T-T hybridoma, which suppresses pokeweed mitogen-induced B cell differentiation as previously reported (J. Immunol. 1982. 129: 1008.). The suppressive effect still exists when peripheral blood leukocytes are depleted of T8+ cells or when TsF is added to a mixture of purified T4+ and B cells. This suppressive effect disappears when the T4 cells are irradiated. The suppression, however, is restored when a large number of T8 cells are added to the irradiated T4 cells. Lastly, a 24 h preincubation of T4 but not T8 cells, with TsF is able to generate suppressive effectors. These results suggest that the target cell for TsF is a radioresistant T4 cell which, upon activation, is able to induce radiosensitive T4 or T8 cells to suppress B cell differentiation.

Antigenic requirements for the generation of secondary cytotoxicity.

The in vitro role of products of the H-2 complex in restimulation of secondary proliferative and cytotoxic responses has been studied. In this paper we have studied in particular the role of CD antigens, the target antigens for cytotoxic T lymphocytes. We have used UV-treated cells that express their CD antigens but no LD. Like others, we demonstrate that CD antigens alone are able to generate a secondary cytotoxic response and furthermore that these CD antigens must be the same as those used for primary sensitization. However, in contrast to the results of others, in some cases UV-treated cells were unable to restimulate such a response even when appropriate controls showed that the CD antigens on UV-treated cells were functional. Furthermore, in these cases the addition of an LD stimulus in the presence of UV-treated cells results in a secondary cytotoxic response that is significantly greater than that elicited by LD restimulation alone. Such potentiation is not observed if the CD antigens used for restimulation are different from those used for the primary sensitization. In addition, a non-H 2 differences(s), presumably the M1s locus, appears to be able to generate a secondary response. The implication of these results are discussed in terms of "memory" T lymphocytes in cell-mediated immunity.

A role for MHC class II antigens in B-cell activation.

Class II antigens of the major histocompatibility complex (MHC) have a well-defined role in restricting cellular interactions and presenting processed antigen to T cells. In addition, a fundamental role for Class II antigens in cellular activation has been suggested, following studies demonstrating that Class II antigen binding alters the proliferation of various cell types. This is further supported by biochemical evidence of signal transduction by second messengers after ligation of the Class II antigens. We have investigated the role of HLA Class II antigens in the activation of B cells. Both activated and resting B cells proliferate in the presence of Sepharose--conjugated anti-Class II antibodies. This proliferation was not epitope-restricted and was unaffected by low m.w. BCGF. Intracellular free calcium elevation was also examined as a marker of cellular activation. (Ca2+)i was increased after the binding and cross-linking of an anti-DR antibody. The above results further support the role of Class II antigens as signal-transducing molecules.

Regulation by interleukin 2 of CD23 expression of leukemic and normal B cells: comparison with interleukin 4.

Recombinant interleukin (IL) 4 has been shown to be able to up-regulate low-affinity Fc receptor for IgE (Fc epsilon RII)-CD23 expression on B cells as well as on other human mononuclear cells. We demonstrate here that, in opposition with previous reports, recombinant IL2 also can up-regulate CD23 expression on B cells. This was first observed on CLL cells which represent a monoclonal proliferation of B cells arrested at an intermediate stage of activation. Cells of 5 out of 12 B-CLL studied display such an increase and all 5 proliferated, in vitro, directly in response to IL2. Similarly, upon triggering with anti-mu and IL2, normal tonsillar B lymphocytes also demonstrate an increase of CD23 expression but this was observed only on day 3. Interferon-gamma was able to inhibit this IL2-mediated up-regulation of CD23 on normal B cells but not on CLL-B cells; on those cells interferon-gamma was similarly unable to inhibit the IL4-mediated CD23 up-regulation. These results suggest that CD23 regulation is complex and related to the stage of activation and the cell type.

Heterogeneity of responsiveness of chronic lymphocytic leukemic B cells to B cell growth factor or interleukin 2.

We compared the proliferative responses of purified human leukemic B cells from 12 cases of chronic lymphocytic leukemia to highly purified B cell growth factor (BCGF) and recombinant interleukin 2 (rIL 2) spontaneously, and in a coactivation assay using anti-mu monoclonal antibodies. Heterogeneity of response to one or the other lymphokine was observed from case to case. Spontaneous responses were observed to BCGF in one case, to rIL 2 in 3 cases and to both lymphokines in one other case. Costimulation with anti-mu monoclonal antibody induced a proliferative response to BCGF in 3 additional cases and to rIL 2 in 4 cases. Interestingly, cells from BCGF-responsive cases also proliferated in response to rIL 2. The leukemic B cells from 4 chronic lymphocytic leukemia patients were unresponsive. Cells from 5 cases expressed IL 2 receptors, although rIL 2 induced a direct proliferative response in only 3 of these cases. Expression of the B cell activation antigen B5 was associated to BCGF responsiveness.

Cross-linking of membrane IgM on B CLL cells: dissociation between intracellular free Ca2+ mobilization and cell proliferation.

It has been shown previously that chronic lymphocytic leukemia (CLL) B cells are frozen at different stages of activation with unique requirements for proliferation. Although most B CLL cells express surface IgM, anti-mu antibodies are able to trigger only some of them to proliferate and/or respond to B cell growth factor (BCGF) or interleukin 2 (IL2), as normal B cells. In this report we extend these observations using three different monoclonal antibodies (mAb) to human mu chain (one mitogenic in soluble form for normal B cells, the two others mitogenic only when coupled on Sepharose 4B beads). Cells from only 3 out of 11 B CLL patients proliferated in the presence of either mitogenic anti-mu. When the early events following surface Ig cross-linking, such as calcium mobilization (by flow cytometry on indo-1-labeled cells), were studied all three mAb in soluble form were able to induce a similar increase in the intracellular free calcium concentration ([Ca2+]i); analogous to [Ca2+]i rise after the mitogenic F(ab')2 anti-mu stimulation. This response was seen only in 8 out of the 12 CLL B cells studied. All B CLL cells, however, proliferate in response to a combination of phorbol ester 12,13-dibutyrate (PBt2) and ionomycin. Therefore, three patterns of response to sIg cross-linking by anti-mu could be distinguished: cells from 4 out of 12 cases proliferate and mobilize Ca2+ upon anti-mu triggering (behaving like resting B lymphocytes); in 4 other cases anti-mu lead to Ca2+ mobilization without cell proliferation; in the last 4 cases neither Ca2+ mobilization, IP3 generation (in the one case studied) nor cell proliferation are observed although these cells do proliferate directly in response to growth factors. Moreover, anti-mu stimulation in this group leads to increased proliferation in response to BCGF and IL2 suggesting an anti-Ig signaling independent of inositol phosphate metabolism. These results are interpreted in terms of differential anti-mu signaling on B cells at different stages of activation.

Signal transduction via MHC class II antigens on B lymphocytes.

The role of the MHC class II antigens in the activation of resting human B lymphocytes (B-Go) was examined with respect to both early and late events in the activation process. The (Ca2+)i induced by anti-IgM was enhanced in the presence of, or following pre-incubation with, an anti-MHC class II DR antibody (D1.12). Pre-incubation with a sepharose conjugated antibody (Seph.-D1.12) augmented the proliferation of B-Go in response to a sub-optimal concentration of anti-IgM. The 2D PAGE profile of B-Go differed from that of in vivo activated B lymphocytes. The 2D PAGE profile of B-Go activated by Seph.-D1.12 was not identical to the profile of B-Go activated by either anti-IgM or PMA. These data suggest that the activation of B-Go via the class II antigens shares part of the pathway of anti-IgM induced activation but does not follow an identical pathway.

Characterization of a suppressive factor produced by a human T hybridoma.

The mode of action and the target cell of a suppressive factor continuously and spontaneously produced by a human hybrid T cell line constructed recently are reported. This factor was shown to suppress pokeweed mitogen-induced B cell differentiation when added within the first 48 hr of culture. It does not suppress B cell differentiation induced by Nocardia extracts, a relatively T-independent mitogen. The target cell for this factor seems to be a T lymphocyte in that it can be absorbed by E-rosetting cells but not by B lymphocytes or monocytes. This factor does not impair mitogen or allogeneic-induced lymphocyte proliferation and has an apparent m.w. of approximately 45,000 as estimated by Sephacryl S200 gel chromatography.