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Six strains of Mycobacterium tuberculosis of different virulence in guinea-pigs were compared with regard to their resistance to low pH, to hydrogen peroxide (H202) at different pH values and to superoxide (-O,-). Low virulence was associated with susceptibility to H202 in native and isoniazid-resistant strains but not in laboratory-attenuated strain ~3 7~a . H,Oz resistance was only partly related to catalase content. Low virulence was not associated with susceptibility to an acid environment but the tuberculocidal effect of H202 was significantly increased at low pH. The strains were uniformly resistant to -02-and contained similar amounts of superoxide dismutase. The implications of these observations are discussed in the context of mechanisms of host defence in tuberculosis.

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As we seek to develop and evaluate new vaccines against tuberculosis, it is desirable that we understand the mechanisms of protective immunity in our models. Adoptive transfer of protection with hsp65-specific T-cell clones from infected or vaccinated mice into naïve mice had indicated that cytotoxic T cells can make a major contribution to protection. We characterized 28 CD4 ؉ CD8 ؊ and 28 CD4 ؊ CD8 ؉ hsp65-specific T-cell clones derived from infected or vaccinated mice. Half of the CD4 ؉ CD8 ؊ and 64% of the CD4 ؊ CD8 ؉ clones were cytotoxic. Cytotoxicity was associated with high expression of CD44 and gamma interferon production. Most (86%) of the cytotoxic CD4 ؉ CD8 ؊ clones lysed target cells via the Fas-FasL pathway, and most (83%) of the cytotoxic CD4 ؊ CD8 ؉ clones lysed target cells via cytotoxic granules. Only the clones using the granulemediated pathway caused substantial loss of viability of virulent Mycobacterium tuberculosis during lysis of infected macrophages, and the degree of killing closely correlated with the availability of granule marker enzyme activity. Granule-mediated cytotoxicity thus may have a key role in protection against tuberculosis by delivering mycobactericidal granule contents.New vaccines are needed in the fight against tuberculosis (1), but the designing and testing of new vaccines is hampered by our poor understanding of the mechanisms of acquired protective immunity. It is not simply that the key protective antigens have yet to be identified (15); we do not know with certainty what kinds of responses are needed. This knowledge would help both to design vaccines for the best balance of responses and to design clinical tests to monitor or predict vaccine efficacy in the field.Traditionally, protection against tuberculosis has been regarded as due to phagocytosis and killing of Mycobacterium tuberculosis by immunologically activated macrophages and monocytes (12). This is a result of a type 1 cellular response in which gamma interferon (IFN-␥) is produced by antigen-specific T lymphocytes, as distinct from a type 2 response, in which the cells produce interleukin (IL-4) (19). IFN-␥ is the main macrophage-activating factor, and it has been shown to be essential for protection (5,8). However, substantial killing by the activated macrophages or monocytes has been difficult to demonstrate in vitro (4,16,18,24), and there is increasing evidence for a protective role for antigen-specific cytotoxic T lymphocytes, in both murine and human tuberculoses (2, 17, 22). It is not clear how these cells are protective. One possibility is that the cytotoxic T cells are needed to release bacteria from safe havens inside ineffective macrophages so that they can be phagocytosed by fresh, fully activated monocytes or macrophages (7). Alternatively, studies with human peripheral blood cells in vitro have indicated that lysis of infected macrophages by antigen-specific T cells can directly result in death of the bacteria (17, 22). Mycobacterial death can be due to toxic enzymes discharged from lymph...

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Immunization by intramuscular injection of plasmid DNA expressing mycobacterial 65-kDa heat shock protein (hsp65) protects mice against challenge with virulent Mycobacterium tuberculosis H37Rv. During infection or after immunization, CD4 ؉ /CD8 ؊ and CD8 ؉ /CD4 ؊ hsp65-reactive T cells increased equally in spleens. During infection, the majority of these cells were weakly CD44 positive (CD44 lo ) and produced interleukin 4 (IL-4) whereas after immunization the majority were highly CD44 positive (CD44 hi ) and produced gamma interferon (IFN-␥). In adoptive transfer of protection to naive mice, the total CD8 ؉ /CD4 ؊ cell population purified from spleens of immunized mice was more protective than that from infected mice. When the cells were separated into CD4 ؉ /CD8 ؊ and CD8 ؉ /CD4 ؊ types and then into CD44 hi and CD44 lo types, CD44 lo cells were essentially unable to transfer protection, the most protective CD44 hi cells were CD8 ؉ /CD4 ؊ , and those from immunized mice were much more protective than those from infected mice. Thus, whereas the CD44 lo IL-4-producing phenotype prevailed during infection, protection was associated with the CD8 ؉ /CD44 hi IFN-␥-producing phenotype that predominated after immunization. This conclusion was confirmed and extended by analysis of 16 hsp65-reactive T-cell clones from infected mice and 16 from immunized mice; the most protective clones, in addition, displayed antigen-specific cytotoxicity.

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