Enhancement of the human T cell response to culture filtrate fractions of Mycobacterium tuberculosis by microspheres.

An improved method of assessment of the human immune response against Mycobacterium tuberculosis antigens will assist the development of new vaccines or diagnostic reagents. In this study, we have analyzed human T cell responses to culture filtrate fractions (CFF) of actively replicating M. tuberculosis strain H37Rv using peripheral blood mononuclear cells from healthy PPD skin test positive and negative individuals. Adsorption of CFF onto polystyrene microspheres, that were approximately the size of the M. tuberculosis (bead-adsorbed antigens, BAA) significantly enhanced IFN-gamma production compared to soluble antigens (SA) in PPD skin test positive individuals in an antigen-specific manner. Further, BAA induced activation of both CD4(+) and CD8(+) T cell subsets. However, CD4(+) responses in general were higher and their antigenic repertoire was wider than the CD8(+) responses. By contrast, CD8(+) responses were strongest to the lower molecular weight BAA. When CFF were chemically coupled to carboxyl modified microspheres (bead-coupled antigens, BCA), induction of IFN-gamma was similar to BAA. Enhancement of T cell responses to particulate M. tuberculosis antigens may prove useful in vaccine design strategies.

Screening of a cosmid library of Mycobacterium bovis BCG in Mycobacterium smegmatis for novel T-cell stimulatory antigens.

We have developed a novel method for screening a Mycobacterium bovis (BCG) cosmid library in Mycobacterium smegmatis for the detection of immunostimulatory T-cell antigens (Ag). Distinctive protein banding patterns were demonstrated in culture filtrates of three of 30 recombinant M. smegmatis clones: pBCCS13 (41 and 73 kDa); pBCCS221 (30, 50 and 68 kDa); pBCCS223 (100 kDa). Western immunoblots indicated that monoclonal antibodies (mAb) directed to the previously characterized 19-, 30-, 38-, 65- and 71-kDa mycobacterial Ag were not reactive with the distinctive recombinant proteins. Furthermore, T-cell Western blots demonstrated that fractions containing the distinctive proteins were immunostimulatory. A given tuberculin-positive donor expressed unique patterns of blastogenic reactivity to protein fractions isolated from each of the three recombinant clones. Restriction enzyme digests of the three recombinant BCG inserts revealed distinctive DNA-banding patterns. The immunostimulatory Ag, therefore, are most likely encoded within different regions of the BCG genome, as contained within three distinct inserts. T-cell Western blots further indicated a heterogeneity in the repertoire of BCG-responsive T cells since tuberculin-positive donors varied in the pattern of reactivity to protein fractions isolated from the same recombinant filtrate. Most likely, immunity to M. tuberculosis results from activation of a heterogeneous array of T cells targeted to multiple immunostimulatory Ag. The method we describe should greatly enhance our ability to define the full spectrum of T-cell Ag encoded by mycobacteria, particularly those which are secreted proteins.

Role of the mononuclear phagocyte as an antigen-presenting cell for human gamma delta T cells activated by live Mycobacterium tuberculosis.

gamma delta T cells, both human and murine, have been found to be highly responsive to mycobacterial antigens. However, the role and function of gamma delta T cells in the immune response to Mycobacterium tuberculosis remain largely unknown. In earlier studies, we demonstrated that monocytes infected with live M. tuberculosis were particularly effective inducers of human peripheral blood gamma delta T cells. The present studies were performed to further characterize the interaction between human mononuclear phagocytes, gamma delta T cells, and live M. tuberculosis, in comparison with CD4+ T cells. First, we found that resting gamma delta T cells expanded in vitro by live M. tuberculosis were specific for M. tuberculosis, and that heat killing and washing the mycobacteria removed the antigen(s) for gamma delta T cells. In contrast, the heat-killed mycobacteria retained significant antigenicity for CD4+ T cells. Second, live M. tuberculosis-expanded gamma delta T cells from healthy tuberculin-positive donors did not respond significantly to the antigens in M. tuberculosis culture filtrate, including the 65- and 71-kDa mycobacterial heat shock proteins. Third, the activation of gamma delta T cells by live mycobacteria was dependent on antigen-presenting cells, and mononuclear phagocytes were found to be very efficient antigen-presenting cells both for resting peripheral blood gamma delta T cells and for activated expanded gamma delta T cells. The mononuclear phagocyte carried the necessary costimulatory factors necessary for gamma delta T-cell proliferation. Fourth, the antigen repertoire and HLA requirements for CD4+ memory T cells and those for gamma delta T cells appear to be quite distinct from each other. CD4+ T cells recognized both soluble protein antigens and whole organisms in a class II major histocompatibility complex-restricted manner, whereas gamma delta T cells appeared to recognize only constituents associated with the whole organism and were not restricted by class I or class II major histocompatibility complex molecules. Finally, the assay system described to expand and purify responding CD4+ and gamma delta T cells after stimulation with live M. tuberculosis represented a simple approach to the direct comparison of these two T-cell populations in the interaction with mononuclear phagocytes infected with M. tuberculosis. Such studies provide insight not only into the relative roles of human CD4+ and gamma delta T cells in the human immune response to intracellular bacterial pathogens such as M. tuberculosis but also into the basic biologic role of human gamma delta T cells in antimicrobial immunity.

Immunological screening of a genomic M. bovis BCG library expressed in M. smegmatis and identification of the M. bovis BCG analog of ClpB.

This work describes the screening of a M. bovis BCG cosmid library in M. smegmatis with a hyperimmune rabbit anti-BCG serum. Cross-reactive antibodies interfere with the detection of BCG specific antigens in M. smegmatis culture filtrates. We, therefore, screened parallel western blots with serum adsorbed with a M. smegmatis cell lysate and unadsorbed serum. Comparison of the western blots allowed distinction between BCG specific and cross-reactive M. smegmatis antigens. Thirty-one cosmids expressed BCG specific antigens. One of them, a hitherto undescribed 100 kDa antigen was subcloned, sequenced and expressed in E. coli. It shows a high degree of homology to ClpB, a member of the Clp family of proteases and was immunologically reactive with the rabbit hyperimmune serum against M. bovis BCG. A positive signal was also obtained with sera of patients with tuberculosis. This antigen is a previously unrecognized target of the human immune response to mycobacteria.

Modulation of IL-12 by transforming growth factor-beta (TGF-beta) in Mycobacterium tuberculosis-infected mononuclear phagocytes and in patients with active tuberculosis.

In humans, tuberculosis is associated with suppression of T-cell responses to antigens of Mycobacterium tuberculosis. Recently, the macrophage product, transforming growth factor-beta (TGF-beta) has been implicated in suppression of T-cell proliferation and cytokine production during tuberculosis. We studied the effect of TGF-beta on production of IL-12, and on the augmentation of M. tuberculosis-induced IFN gamma production by IL-12, in patients with pulmonary tuberculosis and by M. tuberculosis. Induction of IL-12 p35, but not IL-12 p40, by M. tuberculosis in monocytes was dependent on prior priming of the cells with IFN gamma. Expression of both IL-12 p40 and p35, however, was suppressed by TGF-beta. Further, TGF-beta interfered with the bioactivity of IL-12 in the enhancement of M. tuberculosis-induced IFN gamma mRNA expression and cytokine production. However, in mononuclear cells from patients with tuberculosis the main effect of TGF-beta on IL-12 appeared to be counter action to IL-12 induced IFN gamma production in response to M. tuberculosis.

Accessory function and properties of monocytes from healthy elderly humans for T lymphocyte responses to mitogen and antigen.

The waning of cell-mediated immunity during aging has been attributed primarily to defects in T lymphocyte properties and functions. We assessed the potential contribution of accessory dysfunction of monocytes from the elderly on responses of T cells to phytohemagglutinin (PHA) and to tetanus toxoid after in vivo boosting. Accessory function of monocytes from the elderly subjects for T lymphocyte responses to tetanus toxoid was comparable to the young. Expression of the cytokines interleukin-1, interleukin-6 and tumor necrosis factor, the cell adhesion molecules ICAM-1 and LFA-3 and the class II major histocompatibility molecule HLA-DR by monocytes from the elderly and young subjects was similar. T lymphocytes from the elderly responded poorly to PHA. Monocytes from the elderly had a decreased accessory function for PHA-stimulated T cells from young, third donors. Thus, although many accessory properties of monocytes from the elderly are normal, the monocyte and T lymphocyte defects in the elderly for mitogen may represent interactive factors in cell-mediated immunity during aging.

Transient normalization of lymphocyte blastogenic and specific antibody responses following boosting of healthy elderly subjects with tetanus toxoid.

The diminished in vitro blastogenic response of lymphocytes from the elderly to mitogenic stimuli is cited as evidence of immunosenescence, but the response to specific microbial antigens has not been well characterized. We measured the response to tetanus toxoid before and after boosting in young and elderly subjects. Elderly subjects (age > or = 70) and young controls (age < 35) were subjected to clinical, laboratory, and nutritional evaluation to ensure a cohort of healthy subjects. Responses of lymphocytes from the elderly to the mitogens phytohemagglutinin and concanavalin A were markedly diminished compared to those from the young. For all subjects, the average in vitro blastogenic response to tetanus toxoid of lymphocytes from elderly subjects (n = 23) was significantly diminished compared to young controls (n = 23; 31,985 +/- 4502 vs 14,411 +/- 3714 cpm, p < .01). Following boosting with tetanus in those subjects in whom boosting with tetanus toxoid was indicated, blastogenesis was comparable between elderly (n = 17) and young subjects (n = 7; 38,078 +/- 11,451 vs 42,103 +/- 9247 cpm). The boosted response to tetanus apparently was not sustained, since in the subset of subjects with a history of tetanus immunization in the past 10 years, the response of the elderly was much less than that of the young. Thus, a cohort of healthy elderly with diminished blastogenic responses to mitogens was capable of at least a transiently normal response to tetanus post boosting.