Determinants of T cell reactivity to the Mycobacterium leprae GroES homologue.

The 10-kDa protein Ag of Mycobacterium leprae, a human GroES hsp10 cognate, is a major T cell Ag in human leprosy infection. We investigated the mechanism for T cell responsiveness to this Ag according to the trimolecular interaction between T cell, peptide, and Ag-presenting element. This research was accomplished by mapping T cell epitopes in leprosy patients and correlating these responses with peptide-MHC binding affinities. We found that the majority of tuberculoid leprosy patients responded to peptides corresponding to residues 25-39 and 28-42. Truncation analysis of these peptides mapped the exact epitope to be within the overlapping region comprising residues 28-39. Responsiveness was correlated with the HLA-DRB5*0101 allele, which bound the peptides with moderate affinity. This allele is linked to HLA-DR2, which is associated with the resistant form of leprosy. Therefore, T cell responsiveness in tuberculoid leprosy may be mediated by the ability of HLA-DRB5*0101 to bind and present peptides of the immunodominant 10-kDa Ag.

An integrated map of the genome of the tubercle bacillus, Mycobacterium tuberculosis H37Rv, and comparison with Mycobacterium leprae.

An integrated map of the genome of the tubercle bacillus, Mycobacterium tuberculosis, was constructed by using a twin-pronged approach. Pulsed-field gel electrophoretic analysis enabled cleavage sites for Asn I and Dra I to be positioned on the 4.4-Mb circular chromosome, while, in parallel, clones from two cosmid libraries were ordered into contigs by means of fingerprinting and hybridization mapping. The resultant contig map was readily correlated with the physical map of the genome via the landmarked restriction sites. Over 165 genes and markers were localized on the integrated map, thus enabling comparisons with the leprosy bacillus, Mycobacterium leprae, to be undertaken. Mycobacterial genomes appear to have evolved as mosaic structures since extended segments with conserved gene order and organization are interspersed with different flanking regions. Repetitive sequences and insertion elements are highly abundant in M. tuberculosis, but the distribution of IS6110 is apparently nonrandom.

Structure of the heat shock protein chaperonin-10 of Mycobacterium leprae.

Members of the chaperonin-10 (cpn10) protein family, also called heat shock protein 10 and in Escherichia coli GroES, play an important role in ensuring the proper folding of many proteins. The crystal structure of the Mycobacterium leprae cpn10 (Ml-cpn10) oligomer has been elucidated at a resolution of 3.5 angstroms. The architecture of the Ml-cpn10 heptamer resembles a dome with an oculus in its roof. The inner surface of the dome is hydrophilic and highly charged. A flexible region, known to interact with cpn60, extends from the lower rim of the dome. With the structure of a cpn10 heptamer now revealed and the structure of the E. coli GroEL previously known, models of cpn10:cpn60 and GroEL:GroES complexes are proposed.

CD1-restricted T cell recognition of microbial lipoglycan antigens.

It has long been the paradigm that T cells recognize peptide antigens presented by major histocompatibility complex (MHC) molecules. However, nonpeptide antigens can be presented to T cells by human CD1b molecules, which are not encoded by the MHC. A major class of microbial antigens associated with pathogenicity are lipoglycans. It is shown here that human CD1b presents the defined mycobacterial lipoglycan lipoarabinomannan (LAM) to alpha beta T cell receptor-bearing lymphocytes. Presentation of these lipoglycan antigens required internalization and endosomal acidification. The T cell recognition required mannosides with alpha(1-->2) linkages and a phosphotidylinositol unit. T cells activated by LAM produced interferon gamma and were cytolytic. Thus, an important class of microbial molecules, the lipoglycans, is a part of the universe of foreign antigens recognized by human T cells.

Isolation and characterization of the aspartokinase and aspartate semialdehyde dehydrogenase operon from mycobacteria.

Diaminopimelic acid (DAP) is a major component of the peptidoglycan layer of the mycobacterial cell wall. The mycobacterial cell wall has been implicated as a potential virulence factor and is highly immunogenic. The pathway for biosynthesis of DAP may serve as a target in the design of antimycobacterial agents and construction of in vivo selection systems. Despite its significance, this biosynthetic pathway is poorly understood in mycobacteria. In order to develop a better understanding of mycobacterial DAP biosynthesis, the aspartate semialdehyde dehydrogenase (asd) genes of Mycobacterium smegmatis, bacille Calmette-Guerin (BCG), Mycobacterium avium, Mycobacterium leprae, and Mycobacterium tuberculosis were isolated. The M. smegmatis asd gene was isolated by complementation in Escherichia coli. This gene was then used to isolate the asd genes from other mycobacteria. The asd-complementing fragments from BCG and M. smegmatis were sequenced. An open reading frame upstream of the mycobacterial asd gene was identified as the mycobacterial aspartokinase gene (ask). Primer extension analysis revealed that the only transcriptional start in this region is found 5' of the ask gene. This observation indicates that the mycobacterial ask and asd genes are in an operon.

Immune regulation: learning from leprosy.

The specific anergy seen in severe, disseminated leprosy appears to represent multifaceted actions of macrophages and suppressor T lymphocytes. Identification of cytokine mediation in these events helps to account for the immune system's "choice" of cellular versus humoral defenses--and points toward manipulation of cytokine switches for immunotherapy against infectious and autoimmune disease.

Immunosuppressive roles for IL-10 and IL-4 in human infection. In vitro modulation of T cell responses in leprosy.

IL-10 and IL-4 have been shown to exert an inhibitory effect on cell-mediated immune responses. Our previous studies of leprosy demonstrated that IL-10 and IL-4 mRNA were preferentially expressed in lesions from lepromatous patients, those immunologically unresponsive individuals that manifest widespread infection. To define more precisely the regulatory roles of these two cytokines in the immune response to infection, we studied in vitro responses to Mycobacterium leprae. M. leprae triggered IL-10 release from PBMC of patients and healthy donors; the predominant source of the IL-10 was found to be monocytes/macrophages. Stimulation of PBMC in the presence of neutralizing anti-IL-10 mAb indicated that endogenous IL-10 production inhibits PBMC proliferation and release of TNF-alpha, GM-CSF, and IFN-gamma. Paradoxically, studies using neutralizing anti-IL-4 mAb indicated that endogenous IL-4 production enhances PBMC proliferative responses most strikingly in lepromatous patients. We found that rIL-4 expanded CD8+ T cells from lepromatous patients in vitro. CD8+ T cells from lepromatous patients have been shown to suppress CD4+ T cell responses, in part by the release of IL-4. Our study indicated that endogenous IL-4 production inhibited IL-10 secretion and, concomitantly, increased TNF-alpha and GM-CSF release. The present data suggest that, on balance, IL-4 and IL-10 contribute to immunosuppression in human infectious disease.

Cytokine patterns of immunologically mediated tissue damage.

Reactional states in leprosy are produced by different immunologic mechanisms and are responsible for a major component of tissue damage of the disease. Reversal reactions exhibit increased CD4 T cell infiltration in lesions and augmented cell-mediated immune reactivity to Ag of Mycobacterium leprae that can rapidly produce nerve damage. Erythema nodosum leprosum (ENL) reactions also have CD4 T cell infiltration but appear to be associated with the formation of immune complexes that are responsible for panniculitis, arthritis, vasculitis, and nerve injury. Because these reactional states may serve as paradigms for other types of human immunologically mediated tissue damage, this study sought to characterize the dynamic changes in cytokines associated with these reactions. Expression of cytokine mRNA in lesions of leprosy reactional states were measured by PCR. In reversal reactions, IL-1 beta, TNF-alpha, IL-2, and IFN-gamma mRNA were prominent and found to increase during the reaction, concomitant with decreases in expression of mRNA for IL-4, IL-5, and IL-10. In ENL, selective increases in the expression of IL-6, IL-8, and IL-10 mRNA was observed, with persistent expression of IL-4 and IL-5 mRNA. Reversal reactions represent naturally occurring delayed-type hypersensitivity reactions that favor macrophage activation and protective immunity, but which can engender concomitant cell injury. In contrast, ENL lesions represent immediate-type hypersensitivity reactions reflecting the selective stimulation of cytokines that attract neutrophils, stimulate antibody production, and down-regulate macrophage activation. The analysis of cytokine dynamics within different inflammatory responses can provide insights into immune mechanisms of tissue damage, and provide a useful framework for developing strategies for therapeutic intervention.

Immunoprophylactic trial with combined Mycobacterium leprae/BCG vaccine against leprosy: preliminary results.

In an attempt to find a vaccine that gives greater and more consistent protection against leprosy than BCG vaccine, we compared BCG with and without killed Mycobacterium leprae in Venezuela. Close contacts of prevalent leprosy cases were selected as the trial population since they are at greatest risk of leprosy. Since 1983, 29,113 contacts have been randomly allocated vaccination with BCG alone or BCG plus 6 x 10(8) irradiated, autoclaved M leprae purified from the tissues of infected armadillos. We excluded contacts with signs of leprosy at screening and a proportion of those whose skin-test responses to M leprae soluble antigen (MLSA) were 10 mm or more (positive reactions). By July, 1991, 59 postvaccination cases of leprosy had been confirmed in 150,026 person-years of follow-up through annual clinical examinations of the trial population (31 BCG, 28 BCG/M leprae). In the subgroup for which we thought an effect of vaccination was most likely (onset more than a year after vaccination, negative MLSA skin-test response before vaccination), leprosy developed in 11 BCG recipients and 9 BCG/M leprae recipients; there were 18% fewer cases (upper 95% confidence limit [CL] 70%) in the BCG/M leprae than in the BCG alone group. For all cases with onset more than a year after vaccination irrespective of MLSA reaction the relative efficacy was 0% (upper 95% CL 54%; 15 cases in each vaccine group). Retrospective analysis of data on the number of BCG scars found on each contact screened suggested that BCG alone confers substantial protection against leprosy (vaccine efficacy 56%, 95% CL 27-74%) and there was a suggestion that several doses of BCG offered additional protection. There is no evidence in the first 5 years of follow-up of this trial that BCG plus M leprae offers substantially better protection against leprosy than does BCG alone, but the confidence interval on the relative efficacy estimate is wide.

A major T cell antigen of Mycobacterium leprae is a 10-kD heat-shock cognate protein.

Several mycobacterial antigens, identified by monoclonal antibodies and patient sera, have been found to be homologous to stress or heat-shock proteins (hsp) defined in Escherichia coli and yeast. A major antigen recognized by most Mycobacterium leprae-reactive human T cell lines and cell wall-reactive T cell clones is a 10-kD protein that has now been cloned and sequenced. The predicted amino acid sequence of this protein is 44% homologous to the hsp 10 (GroES) of E. coli. The purified native and recombinant 10-kD protein was found to be a stronger stimulator of peripheral blood T cell proliferation than other native and recombinant M. leprae proteins tested. The degree of reactivity paralleled the response to intact M. leprae throughout the spectrum of leprosy. Limiting-dilution analysis of peripheral blood lymphocytes from a patient contact and a tuberculoid patient indicated that approximately one third of M. leprae-reactive T cell precursors responded to the 10-kD antigen. T cell lines derived from lepromin skin tests were strongly responsive to the 10-kD protein. T cell clones reactive to both the purified native and recombinant 10-kD antigens recognized M. leprae-specific epitopes as well as epitopes crossreactive with the cognate antigen of M. tuberculosis. Further, the purified hsp 10 elicited strong delayed-type hypersensitivity reactions in guinea pigs sensitized to M. leprae. The strong T cell responses against the M. leprae 10-kD protein suggest a role for this heat-shock cognate protein in the protective/resistant responses to infection.

Stigma variations: observations on suppressor T cells and leprosy.

Few areas of immunology have been so controversial as that of suppressor T cells. Studies of T cell clones derived from patients with infectious diseases, including leprosy, and allergies have allowed the delineation of functional human T cell subsets. Both CD4 and CD8 cells can be discriminated into subsets that are differentiated by their functions and patterns of lymphokines. Type 1 CD4 cells reactive with lepromin and PPD produce IFN-gamma and IL-2 predominantly, while Type 2 CD4 clones, specific for tetanus toxoid, produce IL-4 and IL-5. Type 1 CD8 cytotoxic T lymphocytes produce predominantly IFN-gamma and IL-2. T suppressor clones derived from immunologically unresponsive lepromatous leprosy patients are antigen-specific, CD8 cells, HLA-DQ restricted, and produce predominantly IL-4, and were designated Type 2 CD8 cells. Several models for peripheral tolerance based on distinct functional T cell subsets are discussed. Previous models of T cell suppression in the mouse and the reciprocal relationship between humoral and cell-mediated immunity in general are reinterpreted in light of such T cell subset interactions.

Expression of adhesion molecules in leprosy lesions.

Leprosy presents as a clinical spectrum that is precisely paralleled by a spectrum of immunological reactivity. The disease provides a useful and accessible model, in this case in the skin, in which to study the dynamics of cellular immune responses to an infectious pathogen, including the role of adhesion molecules in those responses. In lesions characterized by strong delayed-type hypersensitivity against Mycobacterium leprae (tuberculoid, reversal reaction, and Mitsuda reaction), the overlying epidermis exhibited pronounced keratinocyte intracellular adhesion molecule 1 (ICAM-1) expression and contained lymphocytes expressing the ICAM-1 ligand, LFA-1. Conversely, in lesions in which delayed-type hypersensitivity was lacking (lepromatous), keratinocyte ICAM-1 expression was low and LFA-1+ lymphocytes were rare. Expression of these adhesion molecules on the cells within the dermal granulomas was equivalent throughout the spectrum of leprosy. The percentage of lymphocytes in these granulomas containing mRNA coding for gamma interferon and tumor necrosis factor alpha, synergistic regulators of ICAM-1 expression, paralleled epidermal ICAM-1 expression. In lesions of erythema nodosum leprosum, a reactional state of lepromatous leprosy thought to be due to immune complex deposition, keratinocyte ICAM-1 expression and gamma interferon mRNA+ cells were both prominent. Antibodies to LFA-1 and ICAM-1 blocked the response of both alpha beta and gamma delta T-cell clones in vitro to mycobacteria. Overall, the expression of adhesion molecules by immunocompetent epidermal cells, as well as the cytokines which regulate such expression, correlates with the outcome of the host response to infection.

Differing lymphokine profiles of functional subsets of human CD4 and CD8 T cell clones.

Functional subsets of human T cells were delineated by analyzing patterns of lymphokines produced by clones from individuals with leprosy and by T cell clones of known function. CD4 clones from individuals with strong cell-mediated immunity produced predominantly interferon-gamma, whereas those clones that enhanced antibody formation produced interleukin-4. CD8 cytotoxic T cells secreted interferon-gamma. Interleukin-4 was produced by CD8 T suppressor clones from immunologically unresponsive individuals with leprosy and was found to be necessary for suppression in vitro. Both the classic reciprocal relation between antibody formation and cell-mediated immunity and resistance or susceptibility to certain infections may be explained by T cell subsets differing in patterns of lymphokine production.

Defining protective responses to pathogens: cytokine profiles in leprosy lesions.

The immunological mechanisms required to engender resistance have been defined in few infectious diseases of man, and the role of specific cytokines is unclear. Leprosy presents clinically as a spectrum in which resistance correlates with cell-mediated immunity to the pathogen. To assess in situ cytokine patterns, messenger RNA extracted from leprosy skin biopsy specimens was amplified by the polymerase chain reaction with 14 cytokine-specific primers. In lesions of the resistant form of the disease, messenger RNAs coding for interleukin-2 and interferon-gamma were most evident. In contrast, messenger RNAs for interleukin-4, interleukin-5, and interleukin-10 predominated in the multibacillary form. Thus, resistance and susceptibility were correlated with distinct patterns of cytokine production.

Insertional mutagenesis and illegitimate recombination in mycobacteria.

Mycobacteria, particularly Mycobacterium tuberculosis, Mycobacterium leprae, and Mycobacterium avium, are major pathogens of man. Although insertional mutagenesis has been an invaluable genetic tool for analyzing the mechanisms of microbial pathogenesis, it has not yet been possible to apply it to the mycobacteria. To overcome intrinsic difficulties in directly manipulating the genetics of slow-growing mycobacteria, including M. tuberculosis and bacille Calmette-Guérin (BCG) vaccine strains, we developed a system for random shuttle mutagenesis. A genomic library of Mycobacterium smegmatis was subjected to transposon mutagenesis with Tn5 seq1, a derivative of Tn5, in Escherichia coli and these transposon-containing recombinant plasmids were reintroduced into mycobacterial chromosomes by homologous recombination. This system has allowed us to isolate several random auxotrophic mutants of M. smegmatis. To extend this strategy to M. tuberculosis and BCG, targeted mutagenesis was performed using a cloned BCG methionine gene that was subjected to Tn5 seq1 mutagenesis in E. coli and reintroduced into the mycobacteria. Surprisingly for prokaryotes, both BCG and M. tuberculosis were found to incorporate linear DNA fragments into illegitimate sites throughout the mycobacterial genomes at a frequency of 10(-5) to 10(-4) relative to the number of transformants obtained with autonomously replicating vectors. Thus the efficient illegitimate recombination of linear DNA fragments provides the basis for an insertional mutagenesis system for M. tuberculosis and BCG.

Lipoarabinomannan, a possible virulence factor involved in persistence of Mycobacterium tuberculosis within macrophages.

Mycobacterium tuberculosis and Mycobacterium leprae, the causative agents of tuberculosis and leprosy, respectively, produce large quantities of lipoarabinomannan (LAM), a highly immunogenic, cell wall-associated glycolipid. This molecule has been previously reported to be a potent inhibitor of gamma interferon-mediated activation of murine macrophages. Studies of the mechanism by which this mycobacterial glycolipid down-regulates macrophage effector functions provide evidence that LAM acts at several levels and that it can (i) scavenge potentially cytotoxic oxygen free radicals, (ii) inhibit protein kinase C activity, and (iii) block the transcriptional activation of gamma interferon-inducible genes in human macrophage-like cell lines. These results suggest that LAM can inhibit macrophage activation and triggering and cytocidal activity and that it may represent a chemically defined virulence factor contributing to the persistence of mycobacteria within mononuclear phagocytes.