Secreted proteins from Mycobacterium tuberculosis gain access to the cytosolic MHC class-I antigen-processing pathway.

CD8+ T cells play an important role in the host response to infection with Mycobacterium tuberculosis (Mtb). Mtb resides in an arrested phagosome that is phenotypically similar to an early endosome. The mechanisms by which Mtb-derived Ags gain access to the HLA-I-processing pathway are incompletely characterized. Studies with CD8+ T cell lines have suggested that Mtb Ags gain access to the HLA-I pathway in an alternate vacuolar pathway that is both brefeldin A (BFA) and TAP independent. To define the requirements of entry of Ag into the HLA-I pathway, we have used human CD8+ T cell clones specific for the secreted Mtb Ag CFP10. Human monocyte-derived dendritic cells were pulsed with CFP10 expressed in a recombinant adenovirus, surface adsorbed to microspheres, or in its native form by Mtb. When delivered by adenovirus, processing and presentation of CFP10 were blocked by both BFA and the proteasomal blocker lactacystin. In contrast, processing of CFP10 adsorbed to the surface of microspheres was not affected by either of these Ag-processing inhibitors. BFA, lactacystin, and TAP inhibition blocked the recognition of Mtb-infected dendritic cells, suggesting that processing was via a cytosolic pathway for this secreted protein Ag. We conclude that secreted proteins from Mtb can be processed in a BFA- and proteasome-dependent manner, consistent with egress of Ag into the cytosol and subsequent loading of proteasomally derived peptides.

Mycobacterium tuberculosis-specific CD8+ T cells preferentially recognize heavily infected cells.

Both CD4+ and CD8+ T cells are important for successful immunity to tuberculosis and have redundant effector functions, such as cytolysis and release of potent antimycobacterial cytokines such as interferon-gamma and tumor necrosis factor-alpha. We hypothesized that CD8+ T cells play a unique role in host defense to Mycobacterium tuberculosis infection as well. Possibilities include preferential and/or enhanced release of granular constituents and/or preferential recognition of heavily infected cells. Utilizing human, Mycobacterium tuberculosis-specific, CD4+ and CD8+ T cell clones, we demonstrate that, after recognition of antigen-presenting cells displaying peptide antigen, CD4+ T cells preferentially release interferon-gamma, whereas CD8+ T cells preferentially lyse antigen-presenting cells. Furthermore, utilizing dendritic cells infected with Mycobacterium tuberculosis expressing green fluorescent protein, we show that CD8+ T cells preferentially recognize heavily infected cells that constitute the minority of infected cells. These data support the hypothesis that the central role of CD8+ T cells in the control of infection with Mycobacterium tuberculosis may be that of surveillance; in essence, recognition of cells in which the containment of Mycobacterium tuberculosis is no longer effective.

HLA-E-dependent presentation of Mtb-derived antigen to human CD8+ T cells.

Previous studies in mice and humans have suggested an important role for CD8+ T cells in host defense to Mtb. Recently, we have described human, Mtb-specific CD8+ cells that are neither HLA-A, B, or C nor group 1 CD1 restricted, and have found that these cells comprise the dominant CD8+ T cell response in latently infected individuals. In this report, three independent methods are used to demonstrate the ability of these cells to recognize Mtb-derived antigen in the context of the monomorphic HLA-E molecule. This is the first demonstration of the ability of HLA-E to present pathogen-derived antigen. Further definition of the HLA-E specific response may aid development of an effective vaccine against tuberculosis.