A large fraction of human peripheral blood gamma/delta + T cells is activated by Mycobacterium tuberculosis but not by its 65-kD heat shock protein.

We report that M. tuberculosis organisms, but neither PHA nor allogeneic stimulator cells, preferentially activate gamma/delta+ cells within E rosette-purified peripheral blood T cells. gamma/delta+ T cells from purified protein derivative (PPD)-nonimmune healthy donors were enriched by depletion of CD4+ and CD8+ cells; double-negative (DN) cells contained 65-92% gamma/delta+ T cells. Limiting dilution (LD) analyses revealed that 1 of 2-19 purified DN cells proliferated in response to mycobacteria, while frequencies of DN cells proliferating in response to a recombinant 65-kD heat shock protein (hsp 65) of M. tuberculosis/M. bovis were 10-20-fold lower. Established clones of mycobacteria-reactive gamma/delta+ T cells specifically recognized mycobacteria, but neither PPD nor hsp 65. Restimulation of these clones required the presence of PBMC feeder cells; EBV-transformed lymphoblastoid cell lines could not substitute for PBMC. Mycobacteria-reactive gamma/delta+ clones proliferated equally well in the presence of autologous or allogeneic (HLA-DR-different) PBMC feeder cells and thus were not MHC class II restricted. Taken together, these results demonstrate that mycobacteria-reactive gamma/delta+ T cells are present in high frequency in the peripheral blood of healthy individuals, and suggest that hsp 65 of mycobacteria is not a major antigen for gamma/delta+ T cells of normal PPD-nonimmune blood donors.

Sequence homology of the diabetes-associated autoantigen glutamate decarboxylase with coxsackie B4-2C protein and heat shock protein 60 mediates no molecular mimicry of autoantibodies.

Molecular mimicry between viral antigens and host proteins was often suggested to be involved in induction of autoimmune diseases. In type 1 diabetes where pancreatic beta cells are destroyed by autoimmune phenomena, a linear sequence homology between a major autoantigen, glutamate decarboxylase (GAD), and the 2C protein of coxsackie B4 was identified. In addition, a sequence homology between GAD and the mycobacterial heat shock protein 60 was described and the suggestions were made that molecular mimicry between GAD, coxsackievirus B4-2C protein, and/or heat shock protein 60 (hsp60) may be actively involved in an autoimmune reaction towards the pancreatic beta-cells. Our group was the first to isolate human monoclonal autoantibodies to GAD (MICA 1-6) from a patient with newly diagnosed type 1 diabetes. The MICA allowed a detailed characterization of the diabetes associated self-epitopes in GAD and represent a set of GAD autoantibodies present in sera from patients with type 1 diabetes. Using deletion mutants of GAD we demonstrated that the regions of GAD covering the homology sequences to coxsackievirus B4 and to the hsp60 were absolutely required for binding of the MICA to GAD. We now designed an antibody-based analysis to ask whether molecular mimicry between GAD and coxsackie B4-2C or hsp60 is relevant in type 1 diabetes. Since part of the MICA recognize conformational epitopes, they allow to test for conformational molecular mimicry in viruses that have been incriminated in the development of type 1 diabetes. Our data reveal no crossreactivity between the diabetes associated GAD epitopes defined by the MICA and hsp60, rubellavirus, cytomegalovirus, and coxsackie B1-B6 virus antigens. Neither coxsackie B4-specific antibodies in sera from normal individuals nor GAD-positive sera from patients with type 1 diabetes indicated a crossreactivity between coxsackie B4-2C and GAD. Although the regions in GAD homologous to coxsackie B4-2C and hsp60 represented parts of GAD indispensible for binding of diabetes associated autoantibodies they did not mediate a crossreactivity of autoantibodies between GAD and these two proteins. No evidence for molecular mimicry between GAD and a whole panel of foreign antigens was detected by autoantibodies in type 1 diabetes.

T cells against a bacterial heat shock protein recognize stressed macrophages.

Heat shock proteins are evolutionarily highly conserved polypeptides that are produced under a variety of stress conditions to preserve cellular functions. A major antigen of tubercle bacilli of 65 kilodaltons is a heat shock protein that has significant sequence similarity and cross-reactivity with antigens of various other microbes. Monoclonal antibodies against this common bacterial heat shock protein were used to identify a molecule of similar size in murine macrophages. Macrophages subjected to various stress stimuli including interferon-gamma activation and viral infection were recognized by class I-restricted CD8 T cells raised against the bacterial heat shock protein. These data suggest that heat shock proteins are processed in stressed host cells and that epitopes shared by heat shock proteins of bacterial and host origin are presented in the context of class I molecules.

Analysis of primary T cell responses to intact and fractionated microbial pathogens.

Freshly isolated human T lymphocytes were tested for their response to mycobacteria, mycobacterial lysates, 2 dimensional (2D) PAGE separated mycobacterial lysates, leishmania and defined leishmanial antigen preparations. While gamma delta T cells proliferated vigorously in the presence of mycobacteria and mycobacteria derived lysates, a significant stimulation from 2 D gel separated lysates was not detected. In addition gamma delta T cells failed to respond towards leishmania or leishmanial components. In the alpha beta T cell compartment some donors, presumably according to their state of immunity against mycobacteria, responded to mycobacteria, mycobacterial lysates and 2 D gel separated mycobacterial lysates. Neither freshly isolated gamma delta T cells nor alpha beta T cells from naive donors did mount a significant immune response against leishmania.

Direct blotting with viable cells of protein mixtures separated by two-dimensional gel electrophoresis.

A procedure is described which combines the high resolution power of two-dimensional (2D) gel electrophoresis with the advantage of direct probing with viable cells. This device permits the transfer by electroelution of 480 distinct fractions from a 2D gel into soluble phase. Transferred fractions are virtually nontoxic, thus allowing direct probing with viable cells. Using this procedure it was shown that T cells from normal healthy individuals recognized a multitude of Mycobacterium tuberculosis antigens and that the fine antigen recognition pattern of T cells changed after short-term culture in vitro. The application of this procedure to the verification of antigen purity at the T cell level and to the identification of antigens within crude bacterial lysates which are recognized by cloned T cells is described. This approach should be applicable to the rapid identification and characterization of any interesting T cell antigen, for example from important pathogens against which a subunit vaccine is desirable. Moreover, it could be helpful for the analysis of interactions between soluble ligands and their target cells.

Rapid determination of gamma delta T-cell stimulation by microfluorimetry.

Activated T-cell express CD25, the p55 chain of the IL-2 receptor. Here we report a reliable procedure for rapid determination of human gamma delta T cell activation by microfluorimetric measurement of CD25. Three days after initiation of culture, CD25 expression was determined. The sensitivity of this detection method was compared with [3H]thymidine incorporation at day 8. This proliferation assay allowed 3-5-fold higher dilution of the stimulatory ligand. However, monitoring of CD25 expression speeded up screening by 5 days. Therefore, for rapid screening of gamma delta T cell stimulation, e.g. for identification of activating ligands, monitoring of CD25 at day 3 is superior to [3H]thymidine measurement.

Immunity to mycobacteria: possible role of alpha/beta and gamma/delta T lymphocytes.

Immunity to pathogenic mycobacteria is mediated by T lymphocytes. The possible contribution of CD4 alpha/beta T cells, CD8 alpha/beta T cells and gamma/delta T cells as well as the possible role of interleukin-mediated macrophage activation and target cell lysis through direct cell contact is discussed. Furthermore, attempts to define mycobacterial antigens for T lymphocytes with particular emphasis on heat shock proteins are described. The data currently available suggest complex interactions between different T-cell types in immunity to mycobacteria.

Hydrophobic interaction chromatography for the purification of cytolytic bacterial toxins.

The usefulness of hydrophobic interaction chromatography for the simple purification of cytolytic bacterial toxins was studied. Conditions are described for different hydrophobic interaction chromatographic media for purifying with high yields two different kinds of such haemolysins, the thiol-activated toxin listeriolysin O from Listeria monocytogenes and alpha-toxin from Staphylococcus aureus. For listeriolysin O, purification on butyl-Sepharose was followed by gel filtration chromatography. From butyl-Sepharose the recovery of 22%. Alpha-toxin was obtained by a single purification step from alkyl-Superose with 80% recovery and a specific activity of 29,000 U/mg. On sodium dodecyl sulphate polyacrylamide gel electrophoresis purified listeriolysin O and alpha-toxin showed a single band. Another thiol-activated toxin, streptolysin O from group A streptococci, showed a recovery of 38% from butyl-Sepharose. The results suggest the feasibility of using hydrophobic interaction chromatography, particularly with columns of weak hydrophobicity, for the purification of bacterial haemolysins in high yield.

Tuberculosis and leprosy: attempts to identify T-cell antigens of potential value for vaccine design.

Tuberculosis and leprosy are chronic bacterial infectious diseases which represent major health problems worldwide. It is generally accepted that, on the one hand, effective vaccination strategies are required for satisfactory control of these diseases and, on the other hand, that currently available vaccination measures are insufficient for this purpose. Ideally, a subunit vaccine should be designed which is composed of one or a few protective antigens. In this brief treatise our approach towards the identification of antigens with potential value for vaccine design is described. It comprises high resolution fractionation by two-dimensional gel electrophoresis, transfer of separated fractions by electroelution and testing of separated fractions with viable T cells and accessory cells. Using this approach we find: (1) multiple antigens are recognized by T cells from leprosy and tuberculosis patients as well as healthy contacts; (2) apparently, suppressive antigens exist in leprosy; (3) an antigen cluster which is apparently indicative for immunity against M. tuberculosis is present among secreted proteins. We hope that further improvement of this methodology will help in the rational design of subunit vaccines against tuberculosis and leprosy.

Quantification of protein in dilute and complex samples: modification of the bicinchoninic acid assay.

The colorimetric assay using bicinchoninic acid (BCA) as test reagent is useful for quantitative protein determinations due to its high sensitivity, ease, and tolerance to various contaminations present in biological samples or added during purification. For removal of interfering substances, protein precipitations have been described. Yet, obstructions became apparent with diluted and complex samples. Therefore we tested different solvents for removal of such interfering contaminants from the protein precipitate, and 1 M HCl was identified as the useful washing agent. The protocol described allows simple and accurate microdetermination in microtiter plates of proteins from complex samples.

Absence of detectable transgenes in local landraces of maize in Oaxaca, Mexico (2003-2004).

In 2000, transgenes were detected in local maize varieties (landraces) in the mountains of Oaxaca, Mexico [Quist, D. & Chapela, I. H. (2001) Nature 414, 541-543]. This region is part of the Mesoamerican center of origin for maize (Zea mays L.), and the genetic diversity that is maintained in open-pollinated landraces is recognized as an important genetic resource of great cultural value. The presence of transgenes in landraces was significant because transgenic maize has never been approved for cultivation in Mexico. Here we provide a systematic survey of the frequency of transgenes in currently grown landraces. We sampled maize seeds from 870 plants in 125 fields and 18 localities in the state of Oaxaca during 2003 and 2004. We then screened 153,746 sampled seeds for the presence of two transgene elements from the 35S promoter of the cauliflower mosaic virus and the nopaline synthase gene (nopaline synthase terminator) from Agrobacterium tumefaciens. One or both of these transgene elements are present in all transgenic commercial varieties of maize. No transgenic sequences were detected with highly sensitive PCR-based markers, appropriate positive and negative controls, and duplicate samples for DNA extraction. We conclude that transgenic maize seeds were absent or extremely rare in the sampled fields. This study provides a much-needed preliminary baseline for understanding the biological, socioeconomic, and ethical implications of the inadvertent dispersal of transgenes from the United States and elsewhere to local landraces of maize in Mexico.

The amount of Tamm-Horsfall-protein in the human kidney, related to its daily excretion.

1. The amount of Tamm-Horsfall-protein in the human kidney was determined using zonal-immunoelectrophoresis. 2. A value of 115 micrograms Tamm-Horsfall-protein per gram kidney-tissue was found (1.3 micrograms Tamm-Horsfall-protein per mg protein), representing a total amount of about 40 mg Tamm-Horsfall-protein in both kidneys. 3. As the well documented daily excretion of Tamm-Horsfall-protein is 40 mg, i.e. in the range of its content in the kidneys, it is inferred that the total amount of Tamm-Horsfall-protein must be synthesized de novo each day.

Isolation of recombinant mycobacterial antigens by an automatic and generally applicable purification method for beta-galactosidase fusion proteins.

An automated two-dimensional chromatographic method has been developed for the isolation and concentration of recombinant fusion proteins with beta-galactosidase. The system consists of an immunoaffinity column with anti-beta-galactosidase antibodies as ligand, followed by an anion-exchange column. It was used for the purification and concentration of recombinant fusion proteins from Mycobacterium tuberculosis and M. leprae. Small amounts of crude lysates of Escherichia coli were loaded stepwise onto the immunoaffinity column with intermittent washing, elution and re-equilibration. After several cycles the eluate was passed through the anion-exchanger. Using an immunoaffinity gel of 5-ml volume and the anion-exchanger Mono Q HR 5/5, from 10 ml of crude E. coli lysate (containing up to 50 mg of protein) up to 100 micrograms of recombinant protein in a 2-ml volume could be isolated overnight.

Influence of mycobacterial virulence and culture condition on gamma delta T cell activation.

Activation of human gamma delta T cells by culture supernatants of virulent and avirulent mycobacteria was examined. The stimulatory potential of mycobacteria was influenced by the type of culture media and independent from their virulence. Activation of gamma delta T cells by phagocytes infected with viable virulent Mycobacterium tuberculosis H37Rv and avirulent M. bovis BCG was comparable. We conclude that gamma delta T cell stimulation occurs in response to infection with mycobacteria independent from their virulence.

Hydrophobic interaction chromatography for the purification of a mycobacterial heat shock protein of relative molecular mass 60,000.

A recombinant mycobacterial heat shock protein of relative molecular mass 60,000 was purified by hydrophobic interaction chromatography. Chromatographic media with ligands of medium hydrophobicity, such as phenyl-Sepharose, bound too strongly to be used for the purification of this heat shock protein. Butyl-Sepharose, with weak hydrophobicity, allowed binding and elution with decreasing concentrations of ammonium sulphate, but only alkyl-Superose allowed the separation of two similar proteins from the Escherichia coli clone expressing the recombinant heat shock protein (relative molecular mass 60,000) of Mycobacterium bovis BCG. The binding parameters of recombinant human heat shock proteins of relative molecular mass 60,000 and 70,000 indicate that phenyl-Sepharose also binds too strongly for the separation of these two heat shock proteins.

Elongated peptides, not the predicted nonapeptide stimulate a major histocompatibility complex class I-restricted cytotoxic T lymphocyte clone with specificity for a bacterial heat shock protein.

The peptides recognized by an H-2Db-restricted CD8 cytotoxic T lymphocyte (CTL) clone which is specific for the 60-kDa mycobacterial heat shock protein (hsp) and cross-reacts with stressed host cells were characterized. None of the nonapeptides from hsp60 conforming to the H-2Db binding motif were able to sensitize target cells for lysis by this CTL clone. Sequence analysis of the stimulatory fraction from a trypsin digest of hsp60, together with synthetic peptide studies, defined a cluster of overlapping epitopes. Carboxy-terminal extension by at least one amino acid of the nonamer predicted to bind best to H-2Db was essential for CTL recognition. Two such elongated peptides, a 10-mer and a 12-mer stimulated the clone at similarly low concentrations in the 100 pM range. We assume that these two peptides comply best with the natural epitope. In contrast, the 11-mer was inactive. The stimulatory 10-mer bound to H-2Db with an efficacy similar to that of the nonapeptide corresponding to the H-2Db motif, as revealed by peptide induced major histocompatibility complex (MHC) surface expression on RMA-S cells and competitive blocking of epitope recognition by the nonamer. Binding of these carboxy-terminally extended peptides to the MHC groove can be explained by anchoring through the amino acid residue Asn in position 5 of the peptide and by intrusion of the hydrophobic carboxy-terminal Ala (10-mer) or Leu (12-mer), but not Gly (11-mer), into the hydrophobic pocket of the H-2Db cleft. Because the carboxy-terminal part is thus larger than predicted, this region of the peptide may arch up from the binding groove. We assume that recognition of steric components of the MHC/peptide complex broaden the range of epitope specificity for a single T cell receptor. This flexibility not only promotes recognition of several overlapping peptides from a single antigen, but may also increase the chance of cross-reaction with similar peptides from unrelated proteins, including autoantigens. Consistent with this latter assumption, the T cell clone cross-recognizes mycobacterial hsp60 and stressed host cells.

Phosphate is essential for stimulation of V gamma 9V delta 2 T lymphocytes by mycobacterial low molecular weight ligand.

T lymphocytes are divided into two subsets which express different T cell receptor heterodimers. In the peripheral blood of healthy individuals, the majority of T cells express the alpha/beta T cell receptor (> 90%) while a minority have the gamma/delta T cell receptor (< 10%). The gamma/delta T cells of adults use preferentially the V gamma 9V delta 2 chain combination. Although the stimulation requirements for gamma/delta T lymphocytes are still undetermined, it has been reported that gamma/delta T cells are not only stimulated, like alpha/beta T cells, by conventional protein antigens and superantigens, but also by unusual ligands. Mycobacteria selectively stimulate V gamma 9V delta 2 T cells, and a nonproteinacious low molecular weight fraction of 1-3 kDa has been identified as the tentative active component. Here, we confirm the nonproteinacious nature of this ligand, and show that it is comprised of unusual carbohydrate and phosphate. Importantly, cleavage of the terminal phosphate by alkaline phosphatase completely abrogates the stimulatory activity of the low molecular weight ligand for V gamma 9V delta 2 T cells. Even mycobacterial whole lysate loses its stimulatory activity, for this T cell subset, after dephosphorylation with alkaline phosphatase. These findings identify phosphocarbohydrates as a novel molecular entity with selective stimulatory activity for a defined T cell subset.

Heterogeneity of the repertoire of T cells of tuberculosis patients and healthy contacts to Mycobacterium tuberculosis antigens separated by high-resolution techniques.

In this report, we describe studies to examine the repertoire of freshly isolated human T lymphocytes to 400 distinct antigen fractions of Mycobacterium tuberculosis separated by a novel method involving two-dimensional gel electrophoresis. Separated antigens were probed with T cells from tuberculosis patients and purified protein derivative (PPD)-positive (PPD+) and PPD-negative (PPD-) contacts as well as normal healthy donors. T cells from all donors tested responded to separated antigens. Stimulation profiles for tuberculosis patients and PPD+ contacts were extremely heterogeneous, formally demonstrating that an enormous number of different antigens serve as targets of the cellular immune response to M. tuberculosis. Stimulation profiles for tuberculosis patients and PPD+ contacts were indistinguishable. However, stimulation profiles for tuberculosis patients and PPD+ contacts were easily distinguishable from those for PPD- contacts. Normal healthy donors showed T cell responses similar to those of either PPD+ or PPD- contacts.

Secreted antigens of Mycobacterium tuberculosis: characterization with T lymphocytes from patients and contacts after two-dimensional separation.

Little is known about T cell antigens involved in immunity against Mycobacterium tuberculosis. Most model systems use in vitro culture of human T lymphocytes with bacterial lysates or secreted proteins as antigens. In this study, proteins from 3-week-old M. tuberculosis culture filtrates were separated by two-dimensional PAGE and subsequently transferred into soluble phase. The resulting 480 fractions were screened with T lymphocytes from tuberculosis patients and healthy contacts. T cells from all 9 patients and from 8 of 10 tuberculin-positive contacts preferentially responded to a cluster of acidic proteins (pI 4-5) with molecular masses of 30-100 kDa, although they also recognized a number of other fractions. In contrast, of 7 tuberculin-negative contacts, 4 were not and 3 were only weakly stimulated by this cluster region. Therefore, this distinct cluster of secreted proteins seems to comprise dominant T cell antigens of M. tuberculosis.