Detection and characterization of lipopeptide-specific T cells in guinea pigs sensitized with bacteria of the Mycobacterium tuberculosis complex
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The only available vaccine against TB the Bacille Calmette-Guérin (BCG) vaccine provides reliable protection against severe manifestations of childhood TB, but not against pulmonary tuberculosis in adults. Despite many attempts, no improved alternative TB vaccine has been fully licensed so far. It may be crucial for future vaccine development to gain further knowledge on the stimulatory antigens of this vaccine strain and their elicited immune responses. The aim of the current study was to investigate the BCG-mediated adaptive immune responses directed against lipid preparations (chloroform-methanol extracts, CMEs) of bacteria of the Mycobacterium tuberculosis complex (MTC). To this end, the BCG-sensitized guinea pig was employed as a model for human BCG vaccination. In particular, the present study addressed whether lipopeptides in the CME preparations constitute relevant stimulatory antigens for adaptive cellular immune responses upon BCG sensitization and whether the expression of stimulatory lipid antigens varies across the genus Mycobacterium. It was further attempted to identify the molecular nature of the stimulatory antigens present in CME. Outbred Dunkin-Hartley guinea pigs were sensitized with BCG or, for comparison purposes, with heat-inactivated wet mass of the virulent M. bovis strain AN5. Ex vivo, the proliferation of peripheral blood mononuclear cells (PBMCs) was investigated in a CFSE-based proliferation assay which was subsequently analyzed by flow cytometry. In this assay, CMEs of M. bovis BCG and M. tuberculosis H37Rv and, in addition, a Koch s Old Tuberculin standard were used as antigenic stimuli. The molecular nature of the relevant stimulatory antigens within these preparations was investigated by means of delipidation and protease treatment. Furthermore, the stimulatory potentials of a lipopeptide-enriched subfraction of CME(H37Rv) (LppEL), of a defined mycobacterial protein antigen (AG85A), and of different BCG preparations (live or heat-inactivated bacteria, bacterial lysates derived from BCG cultures in the exponential as well as in the static growth phase, and culture supernatant) were tested for their stimulatory potentials in the ex vivo lymphocyte proliferation assay. A restimulation assay based on the antigen-specific expansion of ex vivo lymphocytes, the sorting for expanded lymphocytes and the restimulation of these cells, was established in this study and served subsequently for determination of the antigen-specificity of the stimulated lymphocytes as well as for investigating the distribution of the stimulatory antigens among the tested preparations (Tuberculin, CME(BCG), CME(H37Rv), LppEL). The distribution of the stimulatory antigens was further analyzed among the genus Mycobacterium using CMEs of several MTC strains, including clinical ones, (M. bovis AN5, M. tuberculosis CDC 1551, Haarlem 2336, HN 878, Beijing 1934, East African-Indian strains 1797 and 91/0079, M. canetti) and of non-tuberculous mycobacteria (NTM) (M. marinum, M. avium ssp. avium and M. avium ssp. paratuberculosis, M. leprae, M. fortuitum). It was attempted to identify the molecular nature of the stimulatory antigens in the lipid preparations by two complementary approaches: the purification of lipopeptides from enriched antigen preparations by means of reversed phase chromatography and the investigation of the stimulatory potentials of synthetic, N-terminal peptides of M. tuberculosis lipoproteins. Flowcytometric analysis of the ex vivo lymphocyte proliferation assay revealed that guinea pigs mount strong, adaptive lymphocyte responses upon sensitization with both BCG and inactivated, virulent M. bovis AN5. The lymphocytes proliferated upon stimulation with CME preparations of M. bovis BCG and M. tuberculosis H37Rv. The CME(BCG)-stimulated proliferation manifested in a similar range as lymphocyte responses elicited by tuberculin with the median proliferation being represented by ~ 45 % CFSE-low lymphocytes in BCG-sensitized guinea pigs. This proliferation was significantly stronger than the lymphocyte responses to the defined mycobacterial protein antigen AG85A (p & #8804; 0.0001, student s t test). The above-mentioned preparations of BCG bacteria, however, stimulated strong proliferation as well (significantly above 20 % CFSElow cells, p & #8804; 0.001, one-sample t test). The CME(BCG)-responding lymphocytes were predominantly (~ 83 %) CD4-positive T cells. Delipidation as well as protease treatment of CME(BCG) decreased the stimulatory capacities significantly (p & #8804; 0.0001, student s t test) to median levels below 20 % CFSE-low cells. The same effect on the ex vivo lymphocyte proliferation was observed upon delipidation as well as proteinase K treatment of tuberculin. Furthermore, when stimulating the ex vivo PBMCs with a lipopeptide-enriched subfraction of CME(H37Rv) (LppEL), the median lymphocyte proliferation manifested similar to the lymphocyte response to the parental CME(H37Rv) preparation and both proliferative responses were positively correlated (r = 0.64, Spearman correlation). Using the restimulation assay, it could be confirmed that the proliferated T cells responded specifically to antigens present in tuberculin, CME(BCG) and CME(H37Rv), as well as in LppEL. Strong proliferative responses (on average significantly above 20 % CFSE-low cells, p & #8804; 0.05, one-sample t test) were, however, only observed in response to CME preparations of MTC bacteria. In contrast, the average proliferative response to CME preparations of NTM did not exceed the 20 % cut-off significantly (p > 0.05). So far, it was not possible to identify single lipopeptides which accounted for the observed, strong lymphocyte proliferation. However, it is remarkable that some synthetic N-terminal peptides of M. tuberculosis lipoproteins (LprG, PstS3, LppW, LprL, Pitlp, and LppD) stimulated stronger lymphocyte proliferation on average in the group or in individual guinea pigs. The results demonstrate that the guinea pig is well-suited to investigate adaptive immune responses elicited by MTC bacteria and, in particular, it is a good model to investigate BCG vaccination in a defined experimental setting. All findings were in full accordance with the observations in M. tuberculosis-infected humans. Strong evidence for the stimulatory potential and the relevance of lipopeptides in CME preparations was provided by three independent immunological approaches. Moreover, it could be demonstrated that lipopeptides account for a majority of the stimulatory capacities not only of CME, but also of Koch s Old Tuberculin. The extensive expression and the release of strong antigens, such as the putative lipopeptides, by BCG is supported by the data. Furthermore, the putative stimulatory lipopeptides must be specifically expressed or cross-recognized within the Mycobacterium tuberculosis complex, but only rarely in non-tuberculous mycobacteria. However, the identification of single strong lipopeptide antigens requires further investigation. In this line, it must be considered that antigen mixtures of several single, strong lipopeptide antigens, whose existence has been suggested by the present results, might be necessary to elicit similar strong lymphocyte responses as observed upon stimulation with complex, lipopeptide-containing antigen mixtures such as CME.Verknüpfung zu Publikationen oder weiteren Datensätzen
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Giessen : VVB Laufersweiler Verlag