The discovery of the CD1 antigen presentation pathway has expanded the spectrum of T-cell antigens to include lipids, but the range of natural lipid antigens and functions of CD1-restricted T cells in vivo remain poorly understood. Here we show that the T-cell antigen receptor and the CD1c protein mediate recognition of an evolutionarily conserved family of isoprenoid glycolipids whose members include essential components of protein glycosylation and cell-wall synthesis pathways. A CD1c-restricted, mycobacteria-specific T-cell line recognized two previously unknown mycobacterial hexosyl-1-phosphoisoprenoids and structurally related mannosyl-beta1-phosphodolichols. Responses to mannosyl-beta1-phosphodolichols were common among CD1c-restricted T-cell lines and peripheral blood T lymphocytes of human subjects recently infected with M. tuberculosis, but were not seen in naive control subjects. These results define a new class of broadly distributed lipid antigens presented by the CD1 system during infection in vivo and suggest an immune mechanism for recognition of senescent or transformed cells that are known to have altered dolichol lipids.

The human tuberculous granuloma provides the morphological framework for local immune processes central to the outcome of tuberculosis. This review article describes investigations on human lung granulomas aimed at better understanding the regional host response and counter-measures to Mycobacterium tuberculosis. These findings lead to a revised view of the regional immune response in human tuberculosis. Novel insights into this dynamic cross-talk form the basis of novel intervention strategies.

The human tuberculous granuloma provides the morphological basis for local immune processes central to the outcome of tuberculosis. Because of the scarcity of information in human patients, the aim of the present study was to gain insights into the functional and structural properties of infiltrated tissue. To this end, the mycobacterial load in lesions and dissemination to different tissue locations were investigated, as well as distribution, biological functions, and interactions of host immune cells. Analysis of early granuloma formation in formerly healthy lung tissue revealed a spatio-temporal sequence of cellular infiltration to sites of mycobacterial infection. A general structure of the developing granuloma was identified, comprising an inner cell layer with few CD8(+) cells surrounding the necrotic centre and an outer area of lymphocyte infiltration harbouring mycobacteria-containing antigen-presenting cells as well as CD4(+), CD8(+), and B cells in active follicle-like centres resembling secondary lymphoid organs. It is concluded that the follicular structures in the peripheral rim of granulomas serve as a morphological substrate for the orchestration of the enduring host response in pulmonary tuberculosis.

Although tuberculosis remains a substantial global threat, the mechanisms that enable mycobacterial persistence and replication within the human host are ill defined. This study represents the first genome-wide expression analysis of Mycobacterium tuberculosis from clinical lung samples, which has enabled the identification of M. tuberculosis genes actively expressed during pulmonary tuberculosis. To obtain optimal information from our DNA array analyses, we analyzed the differentially expressed genes within the context of computationally inferred protein networks. Protein networks were constructed using functional linkages established by the Rosetta stone, phylogenetic profile, conserved gene neighbor, and operon computational methods. This combined approach revealed that during pulmonary tuberculosis, M. tuberculosis actively transcribes a number of genes involved in active fortification and evasion from host defense systems. These genes may provide targets for novel intervention strategies.

CD1-restricted presentation of lipid or glycolipid antigens derived fromMycobacterium tuberculosis is a human pathogen of enormous importance to global public health. According to the most recent data published by the World Health Organization, approximately 2.2 million deaths per year are attributable to M. tuberculosis, an additional 8 million people develop symptoms of tuberculosis (TB) every year, and every third human being on earth is infected with the bacterium (36). In spite of this devastating impact on human populations, it is also clear that the human immune system is capable of providing effective protection against disease due to M. tuberculosis infection, since the majority of immunocompetent people infected by this bacterium do not develop signs of serious illness. A major goal for future efforts to control tuberculosis is thus to understand how the immune system successfully recognizes and suppresses the growth of M. tuberculosis. Understanding the underlying mechanisms of the natural adaptive immune response to M. tuberculosis may allow development of novel vaccination strategies to control disease caused by this pathogen.A substantial body of clinical and experimental data indicate that antigen-specific T cells play a major role in maintaining solid and long-lived immunity to M. tuberculosis (reviewed in reference 4). It also has been shown for animal models and for humans that both CD4 ϩ and CD8 ϩ T cells are involved in the adaptive immune response to the pathogen (4, 24, 25). Thus, both classical pathways of antigen presentation, which depend on the peptide binding and presenting functions of the major histocompatibility complex (MHC) class I and class II molecules, have been shown to be involved in the protective immune response to M. tuberculosis. However, it has also become clear in recent years that CD1 molecules, a family of antigenpresenting molecules that bind lipids and present these to T cells, are also involved in the generation of cell-mediated immune responses to mycobacterial pathogens (6,21,33). The precise role and relative importance of this novel pathway for antigen recognition in generating protective immunity to M. tuberculosis remains poorly understood.Studies of the human CD1 system have identified this as a family of antigen-presenting molecules related in structure and evolution to MHC class I and class II molecules (21). CD1 is conserved in most or all mammals, although the size and number of CD1 genes and the variety of different CD1 isoforms vary widely among species. In humans, the CD1 family consists of five isoforms (CD1a, -b, -c, -d, and -e) encoded by a cluster of minimally polymorphic genes that map outside of the MHC. The current system of classification divides the human CD1 proteins into at least two distinct groups (group 1 and group 2) based on differences in structure, expression, and function. Group 1 CD1 proteins, which include CD1a, -b, and -c, are expressed predominantly on professional antigen-presenting

Vaccination against and diagnosis of tuberculosis are still insufficient. Proteins secreted by Mycobacterium tuberculosis induce strong immune responses in tuberculosis and constitute prime candidates for development of novel vaccines against tuberculosis as well as for immunodiagnostic assays. We investigated the role of the secreted proteins MPT63, MPT64 and ESAT6 from M. tuberculosis in healthy individuals and tuberculosis patients. None of the secreted proteins stimulated peripheral blood mononuclear cells from healthy donors. In contrast, CD4+ T cells from many tuberculosis patients were stimulated in an MHC class II-restricted fashion by ESAT6, but not by MPT63 or MPT64. T cell reactivities of tuberculosis patients were focused on the N-terminal region of ESAT6. The ESAT6 T cell epitopes were presented by different HLA-DR phenotypes. Cell cultures responding to either ESAT6 or synthetic peptides thereof showed mRNA transcripts for macrophage inflammatory protein (MIP)-1alpha, monocyte chemotactic protein (MCP)-1 or IL-8 and production of IFN-gamma and MIP-1alpha. Our results suggest that the secreted M. tuberculosis proteins MPT63, MPT64 or ESAT6 do not stimulate unprimed T cells, and that ESAT6 may be a potential candidate antigen for detection of clinical disease.

Ziehl-Neelsen (ZN) staining is the key technique for diagnosis of mycobacterial infections; however, a high percentage of patients exhibit positive signs of tuberculosis, as indicated by pathology, culture of mycobacteria, and polymerase chain-reaction analysis, and yet show negative results on ZN staining. In this report we present evidence that such ZN-negative specimens represent Mycobacterium tuberculosis bacilli in a dormant state with distinct cell-wall alterations: the classical cell-wall composition-dependent ZN staining of M. tuberculosis in lung sections gradually discontinued with persistence of infection, both in mice and in human patients; in contrast, detection of mycobacteria by cell-wall composition-independent staining using a polyclonal anti-M. bovis Bacille-Calmette-Guérin serum continued with persistence of infection. These findings have important implications for diagnosis, as well as for both chemotherapy and development of vaccine strategies.'

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