Protective immunity against mycobacteria is dependent on antigen-specific Th lymphocytes. It has been assumed that these Th upon recognition of antigen/MHC secrete macrophage-activating factors that enable effector macrophages to eliminate the intracellular organism (1).Recently, however, we have found that mycobacteria are very potent inducers of antigen-specific CD4+ and CD8+ cytotoxic T (Tc) cells as well as antigen-nonspecific killer cells that lyse human monocytes (2). Also, in experimental animal models, evidence has recently accumulated in favor of an important role for Tc cells in the immune response to mycobacteria (reviewed in reference 3). In addition, Mycobacterium bovis Bacillus Calmette-Guerin (BCG)-reactive human CD4+ T cell clones have been reported to lyse PPD-pulsed target (antigen-presenting) cells (4). The identification ofthe cytotoxic effector cells as well as the antigens that they recognize are of obvious importance for the design of mycobacterial subunit vaccines .Here we report that the recombinant 65-kD heat shock protein (HSP) (5) ofM. bovis BCG, which is identical to the 65-kD protein ofM. tuberculosis, shares >95% sequence homology with the M. leprae 65-kD gene (6) and shows extensive crossreactivity with the latter (reference 7) is an immunodominant antigen for CD4+ BCG-specific Tc cells in a significant number (t 20%) of BCG-responsive individuals. In addition, recombinant 65-kD HSP-stimulated effector cells also efficiently lysed autologous monocytes in the absence of antigen. This observation may shed new light on the role of the 65-kD HSP in autoimmunity.

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Acquired cell-mediated immunity to intracellular parasites like mycobacteria is dependent on antigen-specific T lymphocytes. We have recently found that mycobacteria not only induce helper T cells but also cytotoxic CD4+ and/or CD8+ T cells as well as nonspecific killer cells that lyse human macrophages in vitro. In addition, we have described that the recombinant heat-shock protein (hsp) 65 of Mycobacterium bovis BCG/M, tuberculosis is an important target antigen for CD4+CD8- cytotoxic T cells. We have now further investigated the cytotoxic effector cells that are induced by the hsp65 of BCG. Purified protein derivative of tuberculin (PPD)- or hsp65-specific cytotoxic T cells specifically lysed PPD, hsp65 of BCG and hsp65 of M. leprae-pulsed macrophages in an HLA-DR-restricted manner. Nonpulsed macrophages were lysed to a much lower but still significant extent. hsp65-induced effector cells expressed CD3, CD5, CD4, CD8 and CD56 markers. Depletion experiments showed that the antigen-specific HLA-DR-restricted killer cell was of the CD5+CD4+CD8-CD56- phenotype. Experiments using N-terminal truncated hsp65 fusion (cro-lacZ) proteins suggested that the N-terminal 65 amino acid residues of the 540 amino acid molecule are critical for the expression of the cytotoxic target epitope(s) in two individuals tested. In addition to inducing antigen-specific cytotoxic effector cells, the hsp65 also triggered nonspecific nonrestricted effector cells with lytic activity against nonpulsed autologous or allogeneic macrophages as well as K-562 and Daudi tumor cells. hsp65-stimulated effector cells produced both interferon and tumor necrosis factor-alpha. An important finding was that hsp65-stimulated effector cells strongly inhibited colony-forming unit formation from live BCG-infected autologous macrophages.