Identification and Characterization of Murine Cytotoxic T Cells That KillMycobacterium tuberculosis

Abstract

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...

Mycobacterium tuberculosis-Specific CD8+ T Cells and Their Role in Immunity

“…Indeed, CD1b-restricted CD8 + T cell clones dependent upon granule exocytosis for cytolytic activity, still retained their bactericidal effectiveness even when target cell apoptosis was blocked by caspase inhibitors (221). In addition, an analysis of hsp65-specific CD8 + T cell clones grown from infected mice correlated bactericidal activity with granule exocytosis-dependent cytolysis of infected macrophages (205). Thus, the granule contents have a direct bactericidal effect independent of the induction of apoptosis.…”

“…Two of the peptides (Ag85B [143][144][145][146][147][148][149][150][151][152] and Ag85B [199][200][201][202][203][204][205][206][207] ) were able to stimulate short-term antigenspecific CD8 + T cell lines from BCG-vaccinated individuals. In the case of Ag85B [199][200][201][202][203][204][205][206][207] , tetramers were made and CD8 + T cells that stained with the tetramers were enriched in the short-term cell lines. Disappointingly, tetramer + cells could not be detected directly in the peripheral blood of BCG-responsive donors suggesting that their frequency in the circulation is <1/1000.…”

“…Similarly, Stenger et al analyzed mycobacterial lipid specific group 1 CD1-restricted human T cell clones and found that the CD8 + T cell clones exclusively used granule exocytosis for target cell lysis, whereas the CD8 − CD4 − T cell clones relied on the CD95/CD95L pathway (15). Additionally, Silva and Lowrie cloned hsp65-specific CD8 + T cells from M. tuberculosis infected mice that were dependent upon each the granule release and the CD95/CD95L pathways to kill infected macrophages (205). In contrast, the cytolytic activity of short term CD8 + T cell lines established from PPD + patients was inhibited by blocking of both CD95/CD95L ligation and granule release/ perforin polymerization, suggesting that these pathways are simultaneously activated during target cell recognition (206).…”

“…Using computer algorithms, Klein et al identified three epitopes from the Ag85 complex that were recognized by BCG-vaccinated HLA-B*3501 individuals: a) an epitope shared by Ag85ABC (Ag85 110-118 ) b) Ag85C 268-276 ; and Ag85B [204][205][206][207][208][209][210][211][212] (110). An interesting strategy for the identification of mycobacterial epitopes that are presented by human class I MHC is to use transgenic mice that express HLA-A*0201 (109).…”

Perforin level in CD4+ T cells from patients with systemic lupus erythematosus

“…The PRF1 gene is expressed primarily in NK cells and effector CD8 + T cells [31,32]. PRF1 is also expressed in a subpopulation of cytotoxic CD4 + T cells, which may combat various pathogens (33)(34)(35)(36)(37)(38)(39). An increased number of cytotoxic CD4 + T cells has been observed in patients with multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, and acute coronary syndromes [20][21][22][23].…”