Protection against murine tuberculosis by an attenuated recombinant Salmonella typhimurium vaccine strain that secretes the 30-kDa antigen of Mycobacterium bovis BCG.

A recombinant (r-) Salmonella typhimurium aroA vaccine that secretes the naturally secreted protein of Mycobacterium bovis strain BCG, Ag85B, by means of the HlyB/HlyD/TolC export machinery (termed p30 in the following) was constructed. In contrast to r-S. typhimurium control, oral vaccination of mice with the r-S. typhimurium p30 construct induced partial protection against an intravenous challenge with the intracellular pathogen Mycobacterium tuberculosis, resulting in similar vaccine efficacy comparable to that of the systemically administered attenuated M. bovis BCG strain. The immune response induced by r-S. typhimurium p30 was accompanied by augmented interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) levels produced by restimulated splenocytes. These data suggest that the HlyB/HlyD/TolC-based antigen delivery system with attenuated r-S. typhimurium as carrier is capable of inducing an immune response against mycobacterial antigens.

Cytolytic T-cell responses to human dendritic cells and macrophages infected with Mycobacterium bovis BCG and recombinant BCG secreting listeriolysin.

Cytolytic T-cell responses from 63 normal blood donors were monitored in a Mycobacterium bovis BCG infection system in vitro. We wanted to know whether cultured dendritic cells were capable of potentiating the cytolytic T-cell responses to M. bovis BCG. Infected cultured dendritic cells were up to ten times more effective antigen-presenting cells than macrophages in proliferative assays, while cytolytic T-cell induction did not differ significantly between dendritic cells and macrophages. Separated CD4+ and CD8+ T-cell subsets contributed equally to lysis of infected targets. Experiments comparing wild-type M. bovis BCG strain with two new recombinant M. bovis BCG strains secreting listeriolysin revealed statistically significant higher maximal lysis values for recombinant M. bovis BCG. We conclude from our in vitro infection system with mycobacteria that dendritic cells are superior to macrophages in proliferative assays but equal to macrophages in their ability to induce cytolytic T-cell responses. Moreover, our data suggest that recombinant M. bovis BCG vaccine strains secreting listeriolysin improve cytolytic T-cell responses.

Impact of antigen-presenting cells on cytokine profiles of human Th clones established after stimulation with Mycobacterium tuberculosis antigens.

Human T cells reactive with mycobacterial antigens are generally considered to correlate with a Th1 cytokine profile. Our data show that, in addition, Th0 and Th2 clones develop in bulk culture with appropriate antigen-presenting cells before cloning. CD4+ blasts activated by mycobacterial antigens were cloned, and their mRNA patterns for the interleukins (IL) IL-2, IL-4, IL-5, IL-6, and IL-10 and gamma interferon were characterized by reverse-transcribed PCR. Nonadherent, nonrosetting, enriched peripheral blood mononuclear cells promoted development of Th0; after further depletion of monocytes and natural killer cells, Th2 clones were also found. Epstein-Barr virus-transformed B cells, with specificity for the stimulating antigen, increased the proportion of Th2 clones.

Listeria monocytogenes-induced gamma interferon secretion by intestinal intraepithelial gamma/delta T lymphocytes.

gamma/delta T cells represent a major proportion of intestinal intraepithelial lymphocytes (IEL), and it has been suggested that these IEL serve as a first immune barrier against microbial invasion and that they do so by destroying infected epithelial cells. In the present study, we confirm that both alpha/beta and gamma/delta IEL from naive mice express potent cytotoxicity and produce gamma interferon (IFN-gamma) after T-cell receptor (TCR) engagement by specific monoclonal antibodies (MAb). Intraperitoneal administration of the anti-gamma/delta TCR MAb GL3 caused downregulation of the gamma/delta TCR in IEL, and IEL from gamma/delta TCR-modulated mice failed to express cytotoxic activity and to secrete IFN-gamma after gamma/delta TCR engagement. In contrast, alpha/beta IEL from such mice were still cytolytic and secreted IFN-gamma. Mice were infected orally with virulent Listeria monocytogenes at doses which caused bacterial invasion through the intestinal epithelia. Although alpha/beta and gamma/delta IEL from these mice expressed high cytolytic activities in antibody-redirected killer assays, target cells pulsed with listerial antigens were not lysed. In contrast, IFN-gamma secretion by IEL from L. monocytogenes-infected mice was induced not only by anti-TCR MAb but also by target cells pulsed with listerial antigens, whereas irrelevant antigens, including heat shock protein 60, did not induce IFN-gamma secretion. Furthermore, the number of IFN-gamma-secreting IEL, as assessed by the enzyme-linked immunospot technique, was increased during listeriosis. gamma/delta TCR modulation by GL3 administration abrogated antigen-induced IFN-gamma secretion by IEL from infected mice. These findings suggest that L. monocytogenes induced IFN-gamma secretion by gamma/delta IEL from mice suffering from intestinal L. monocytogenes infection and invasion. Thus, the data provide evidence for a role of IFN-gamma-secreting IEL in local resistance against listeriosis and perhaps other food-borne diseases.

Gamma interferon and interleukin 2, but not interleukin 4, are detectable in gamma/delta T-cell cultures after activation with bacteria.

Mycobacterium tuberculosis- or group A streptococcus-activated gamma/delta T cells from normal healthy individuals were negatively sorted and restimulated in vitro from 48 h. Significant amounts of gamma interferon were detected after restimulation with M. tuberculosis, group A streptococci, or Listeria monocytogenes. In contrast, interleukin 4 was undetectable in the culture supernatants. Our findings provide indirect evidence for the involvement of gamma/delta T cells in immunity against tubercle bacilli and probably other bacteria.

A novel form of CD4 (L3T4) mRNA in the murine fetal liver results in cell-surface expression of the L3T4 antigen.

The expression of the L3T4 antigen during ontogeny in the murine fetal liver has been investigated in parallel by northern blot analysis and cytofluorometry. The L3T4 gene is transcribed in the murine fetal liver in two polyadenylated mRNA species with the size of 3.5 kb and 3.7 kb. Whereas the 3.5-kb mRNA is expressed from days 13 to 18 of gestation, expression of the 3.7-kb mRNA is found only from days 16 to 18 of gestation and thus appears to be developmentally regulated. Immunofluorescent staining of fractionated fetal liver cells from days 12 to 18 of gestation with the anti-L3T4 antibody (GK1.5) provides evidence that cell-surface expression of the L3T4 antigen on a subset of lympho-haematopoietic cells in the murine fetal liver is the product of a novel form of L3T4 mRNA with the size of 3.5 kb.

CD3+ T cells in severe combined immunodeficiency (scid) mice. II. Transplantation of dm2 lymphoid cells into semi-allogeneic scid mice.

Intravenous injection of nonfractionated BALB/c-H-2dm2 (dm2) (Ld-) spleen cells into 4-week-old, semi-allogeneic (H-2d, Ld+) C.B-17 scid/scid severe combined immunodeficient (scid) mice (2 x 10(7) cells/mouse) reconstituted T lymphopoiesis in thymi and repopulated the lymphoid white pulp in spleens of these immunodeficient recipients. Transplantation of dm2 thymocytes into young scid mice (5 x 10(7) cells/mouse) established a donor-derived CD3+ T cell population in spleens of recipient scid mice, in which CD4+T cells predominated. This was demonstrated by marker analyses of thymocytes and splenocytes, and determinations of serum immunoglobulin levels in transplanted scid mice. Transfer of splenocytes from young primary scid recipients into young secondary or tertiary recipients (3 x 10(6) cells/mouse) engrafted preferentially dm2-derived CD3+CD4+CD8- T cells in spleens of scid mice despite the strong selective Ld-associated alloantigenic stimulus for CD8+ T cells. Intravenous injections of nonfractionated dm2 spleen cells (2 x 10(7) cells/mouse) or thymocytes 5 x 10(7) cells/mouse) into 10- to 12-month-old, "leaky" scid mice induced severe clinical signs of graft-vs.-host disease (GVHD) in all scid recipients. Lymphoid repopulation of spleen and thymus in old scid recipients was incomplete. This GVHD was not transferrable by injecting 3 x 10(6) spleen cells from old diseased primary scid recipients into secondary or tertiary young scid recipient mice. In these serial transfers, dm2-derived CD3+CD4+CD8- T cells were again preferentially engrafted in spleens of scid recipients. Transfer of purified CD4+ dm2 T cells into young scid mice (2 x 10(5) to 5 x 10(5) cells/mouse) engrafted this T cell subset into the spleen of semi-allogeneic scid recipients. This was revealed by histological examinations, surface marker analyses, in vitro isolation of donor-type CD3+CD4+ T cell lines from spleens of transplanted scid mice, and serial transfer experiments. These data indicated that the CD4+ T cell compartment of scid mice can be selectively repopulated by semi-allogeneic T cells. Injections of purified CD8+ dm2 T-cells into young scid mice (2 x 10(5) cells/mouse) did not establish a CD8+ T cell graft in spleens of recipients. It was necessary to inject transplanted scid mice biweekly with 10(4) units recombinant interleukin 2 to establish and/or maintain transferred dm2 CD8+ T cells in spleens of these recipients, dm2 CD8+ T cell-transplanted and interleukin 2-treated scid mice did not develop any evidence of GVHD over the 9-week observation period.

CD3+ T cells in severe combined immune deficiency (scid) mice. I. Transferred purified CD4+ T cells, but not CD8+ T cells are engrafted in the spleen of congenic scid mice.

Young (less than 3 months of age) and old (greater than 1 year of age) C.B-17 scid/scid mice were tested for the presence of immunoglobulin in serum and CD3+ T cells in spleen and peritoneal cavity. In all old severe combined immune deficiency (scid) mice tested we found detectable, but very variable levels of serum immunoglobulin as well as splenic and peritoneal CD3+ T cells comprising 3% to 10% of the nonfractionated cell populations of these organs (n = 10). In contrast, none of the analyzed young scid mice showed any evidence of peripheral lymphocytes. Low numbers (2 x 10(5) to 5 x 10(5) cells/mouse) of highly purified CD4+ cells from congenic C.B-17 or BALB/c donor mice were injected intravenously into young scid recipient mice. A CD4+ T cell population was clearly engrafted when transplanted scid mice were analyzed 8 to 13 weeks after T cell transfer: (a) a CD3+CD4+CD8- T cell population was detectable in the spleens of all recipient scid mice by flow microfluorometry analyses; (b) CD3+CD4+CD8 T cell lines could be grown out of these spleens in vitro; (c) the histological examination revealed evidence of lymphoid cell repopulation in the spleens of all transplanted scid mice and (d) transplanted CD4+ T cell populations could be serially transferred into secondary and tertiary recipient scid mice. These data indicate that scid mice can be constructed in which only the CD4+ T cell compartment is selectively reconstituted. In contrast to the successful engraftment of CD4+ T cell, highly purified congenic CD8+ T cells could not be engrafted into the spleen of scid mice.

A novel subset of CD2-, CD3/T cell receptor alpha/beta+ human peripheral blood T cells. Phenotypic and functional characterization of interleukin 2-dependent CD2-CD3+ T cell clones.

It is generally believed that CD2 (T11, sheep erythrocyte receptor) is expressed on all human T cells. In the present study we have identified and characterized a minor subset of CD2- CD3/TCR alpha/beta+ T cells in the peripheral blood of healthy individuals. CD2-CD3+ T cells were enriched in PBMC depleted of plastic-adherent macrophages, E-rosetting (i.e., CD2+) T cells and surface Ig+ B cells. CD2-CD3+ T cells accounted for 0.1-0.8% of PBMC in six individuals. IL-2-dependent long-term clones of CD2-CD3+ T cells neither reacted with a panel of anti-CD2 mAbs nor expressed detectable levels of CD2 mRNA by Northern blot analysis. These clones, however, expressed a full-length TCR C beta mRNA and reacted with mAbs against TCR-alpha/beta, CD3, and CD4, and thus were mature T cells. CD2-CD3/TCR+ T cell clones could be triggered into proliferation, IL-2 production, and cytotoxic effector activity by anti-CD3 and anti-TCR mAbs. We conclude that (a) a minor subset of CD2-, CD3/TCR-alpha/beta+ T cells is present in normal peripheral blood; and (b) expression of CD2 at the level of protein and/or mRNA is not required for T cell signaling via the CD3/TCR molecular complex.

Myxothiazol: a reversible blocker of the cell cycle.

Myxothiazol, a potent inhibitor of the cytochrome bc1 oxidoreductase, was shown by the use of flow cytometry to block reversibly the late G1/S phase of the cell cycle of human lymphoblastic T-cell line Jurkat (clone 886) at concentrations of 0.5 microgram/ml. These observations are compared to those of other drugs, such as antimycin, which effect the respiratory chain, and with O2-deficiency.

T lymphocyte activation by staphylococcal enterotoxins: role of class II molecules and T cell surface structures.

The enterotoxins produced by Staphylococcus aureus (SE) are the most potent mitogens known. Triggering of proliferation or cytotoxicity by SE requires the presence of MHC class II molecules on accessory or target cells. In this study we have investigated the role of HLA class II molecules in the activation of human T cells by SE and the nature of the target structure on the responding T lymphocyte for SE. This dependence on class II molecules is not due to an immunological "recognition" of SE since there is no restriction by polymorphic determinants of HLA molecules and since even xenogeneic class II molecules can reconstitute the human T cell response to SE. Furthermore, HLA class II-positive but not -negative cells absorb the mitogenic activity from SE solutions and significant binding of 125I-labeled SE can be demonstrated to class II-positive but not to class II-negative cells. Enterotoxin molecules react directly with T cells since they cause an increase in cytosolic Ca2+ concentration similar to anti-CD3 mAb. This increase is abrogated by prior modulation of the TCR/CD3 complex. Antibodies to CD2, CD3 and the TCR that block antigen-specific activation also block T cell activation by SE. Moreover, preincubation of purified resting accessory cell-free T cells with SE leads to modulation of the TCR/CD3 complex. Taken together these data indicate that SE interact selectively with HLA class II molecules on accessory or target cells and with a TCR-associated structure on the T cell.

Adoptive transfer of human peripheral blood lymphocytes (PBL) in scid mice.

Two protocols were examined for the ability to transfer a human T cell system into SCID mice. Upon intraperitoneal injection (i.p.) of human peripheral blood lymphocytes (PBL) into SCID mice the injected cells could be recovered over weeks from the peritoneal cavity, yet human T cells did not seed into secondary lymphoid organs such as the spleen, lymph nodes or bone marrow. In contrast, SCID mice grafted with human embryonal thymus tissue contained high numbers of CD4+CD8- and CD8+CD4- human T cells in their lymph nodes and spleen when they had been injected i.p. with human PBL.

Identification of CD2-/CD3+ T cells in fetal human tissue.

From 1 to 23% of fetal human spleen or thymus cells (from the 20th to 24th week of gestation) were found to display a previously unrecognized CD2-/CD3+ phenotype. IL-2-dependent, long-term clones of CD2-/3+ T cells did not react with a panel of anti-CD2 mAbs and did not form rosettes with sheep erythrocytes. These results show that (a) significant numbers of CD2-/3+ T cells are present in fetal human spleen and/or thymus; and (b) in contrast to the widely accepted view, expression of CD2 is not a prerequisite for the expression of the CD3 molecular complex on human T cells.

Development of autoreactive L3T4+ T cells from double-negative (L3T4-/Ly-2-) Thy-1+ spleen cells of normal mice.

Thy-1+/L3T4-/Ly-2- spleen cells were purified from normal C57BL/6 (B6) and C,B-17 mice. Cells within this subset expressed the T cell receptor (TcR) for antigen: the majority of cells in this subset were CD3+; a fraction of the cells was stained with the monoclonal antibody (mAb) F23.1; and the TcR molecule was immunoprecipitable with mAb F23.1 from cells within this subset. In limiting dilution analyses, about 1/30 cells within this subset were growth inducible in vitro by stimulation with phorbol myristate acetate (PMA) plus ionomycin; conditioned media containing interleukin (IL) 1, IL2, IL3 or IL4 activity neither triggered nor promoted in vitro growth of these cells. The in vitro generated T cells displayed the Thy-1+/L3T4+/Ly-2- surface phenotype and were self-reactive, i.e., proliferated preferentially in response to syngeneic stimulator cells, and secreted IL2 and IL3 only in response to syngeneic but not allogeneic stimulator cells. The proliferative response of these cells to syngeneic stimulator cells was blocked by anti-self Ia mAb. This autoreactive helper T cell subset was not inducible in purified Thy-1+ spleen cell subsets from athymic nude mice or scid mice. Autoreactive helper T cells did not express detectable levels of the IL2 receptor (IL2R), and their proliferative response was not blocked by anti-IL2R mAb. From PMA plus ionomycin-stimulated double-negative Thy-1+ spleen cells, 14 T cell clones were established in long-term culture which displayed the CD3+CD4+CD8- surface phenotype and were self-reactive.

An improved semi-quantitative enzyme immunostaining procedure for glycosphingolipid antigens on high performance thin layer chromatograms.

An immunoassay is described which allows the detection of glycosphingolipid (GSL) antigens on high performance thin layer chromatograms (HPTLC). The method involves: (1) the separation of GSL on HPTLCs; (2) incubation with specific antibodies against carbohydrate structures of GSL, and (3) the detection of specifically bound antibodies with alkaline phosphatase-conjugated second antibodies and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) as substrate. Using a monoclonal rat IgG2c antibody against Forssman GSL, a BALB/c monoclonal antibody against asialo GM2, and polyclonal rabbit antibodies against asialo GM1, it was shown that as little as 3 ng GSL antigen could be detected in a procedure taking detected in a procedure taking only 4 h to perform. The assay should be useful for screening mono- and polyclonal antibodies with potential specificity for GSL antigens, for the detection and quantification of GSL-antigens in tissue extracts, and for defining the specificity of anti-GSL antibodies.

Effects of pyocyanine, a blue pigment from Pseudomonas aeruginosa, on separate steps of T cell activation: interleukin 2 (IL 2) production, IL 2 receptor formation, proliferation and induction of cytolytic activity.

Pyocyanine was isolated by chloroform extraction of cultures of Pseudomonas aeruginosa, and purified by thin layer chromatography. The effects of pyocyanine on the various stages of T cell activation were studied with concanavalin A-stimulated CBA/J mouse splenocytes. At 12.5 microM concentration pyocyanine totally inhibited Con A-dependent proliferation and development of cytotoxic effector cells. Protein and RNA synthesis was only 50% inhibited at this concentration. Inhibitory doses of pyocyanine were nontoxic, in that cell viability was maintained, and the inhibitory effects were reversible after removal of the drug. Pyocyanine did not interfere with interleukin 2 synthesis, nor did it affect the lytic stage of cytotoxic effector T cells. However, T blasts generated by Con A in the presence of pyocyanine did not grow in response to IL2 even in the absence of pyocyanine, and IL2 receptors, detected by indirect immunofluorescence with the receptor-specific monoclonal antibody AMT-13, were diminished in pyocyanine-treated cells. Pyocyanine also inhibited IL2-dependent proliferation of T blasts with fully developed IL2 receptors. The substance thus interferes with several discrete stages of T cell activation.

The influence of caffeine on tumour incidence in Sprague-Dawley rats.

Food-grade natural caffeine was given in the drinking-water (available ad lib.) to barrier-maintained specified-pathogen free Sprague-Dawley rats for 2 yr. Groups of 50 animals per sex received levels of 200, 430, 930 and 2000 mg caffeine/litre, while two control groups, each of 50 animals per sex, received plain water. No unusual tumours or sites of origin for neoplastic growth were found in any animal receiving caffeine. Neoplasms found in various organs showed incidences not exceeding those seen in controls. Thus, exposure to caffeine for 2 yr did not enhance or induce neoplasia in the Sprague-Dawley rats.

Effect of diazepam on microsomal diethylnitrosamine metabolism in gerbils.

Dealkylation of diethylnitrosamine (DEN) by a liver microsomal fraction was measured after treatment of the Mongolian gerbil with DEN, Valium, or DEN plus Valium. Valium-dosed male and female animals showed strongly stimulated deethylase levels compared with the controls. This was also true of the DEN-treated females. Combined treatment (DEN + Valium) resulted in a significant loss of activity compared with treatment with DEN only, but was not significantly lower than that in the controls. Significant differences were also found between the experimental groups in microsomal enzymes. Metabolism of 14C-DEN to 14CO2 by gerbil liver slices in vitro showed dose-dependent inhibition by Valium. Kinetic analysis of DEN-dealkylation by purified gerbil microsomes revealed at least two Km values. In the microsomal system, diazepam inhibited DEN-dealkylation significantly at low substrate levels.

Transformation-dependent quantitative changes in glycopeptide binding to concanavalin A-sepharose.

Differentially L-fucose-labelled glycopeptides from the surface of a Syrian golden hamster (SGH) fetal lung control cell line were compared with those from chemically-transformed and tumour cell lines derived from the control line by cochromatography on Concanavalin A-Sepharose (Con A-Sepharose) and Sephadex G-50. Quantitative differences were found both in the unbound and specifically-bound fractions between control and transformed cells upon Con A-Sepharose chromatography. In the glycopeptides from transformed and tumour cells, the unretarded fraction was concomitantly decreased compared to the controls. When the ratio of unbound to specifically-bound fractions was used, a statistically significant difference could be calculated between the values of control versus transformed or tumour cells. In all transformed and tumour cell lines investigated, the quantitative change in Concanavalin A binding, expressed as an increase of the ratio of unretarded to specifically-bound glycopeptides, was paralleled by a shift of transformed or tumour glycopeptides to higher apparent molecular weight compared to the control in gel chromatography.