Induction of antigen‐specific CD4⁺ HLA‐DR‐restricted cytotoxic T lymphocytes as well as nonspecific nonrestricted killer cells by the recombinant mycobacterial 65‐kDa heat‐shock protein

Abstract

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.

Lack of cytotoxic activity against Mycobacterium leprae 65-kD heat shock protein (hsp) in multibacillary leprosy patients

“…On the other hand, 16/20 MB patients were unable to develop specific cytotoxic T cells; only those patients undergoing an ENL episode elicited cytotoxic responses against hsp65-pulsed macrophages (Table 3). Our results thus further confirm and extend the findings of Kale Ab et al [14] that Myco. leprae hsp65 does not trigger cytotoxic effector cells in most MB patients.…”

“…leprae recombinant proteins [11][12][13] and induction of cytotoxic T lymphocytes by the recombinant 65-kD heat shock protein (hsp65) of Myco. bovis BCG have been demonstrated [14][15][16]. These cytotoxic T lymphocytes react with macrophages pulsed with BCG-hsp or Myco.…”

Interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α) are necessary in the early stages of induction of CD4 and CD8 cytotoxic T cells byMycobacterium lepraeheat shock protein (hsp) 65 kD

“…Although mycobacteria induce preferentially CD4 CTL [16,21], we [8] as well as others [5] have demonstrated that both CD4 and CD8 cytotoxic effector cells can be generated from PBMC. An important role has been recently suggested for class I-restricted CD8 T cells in TB [22].…”

“…Several studies have suggested that CTL could play an important role in the immune response to mycobacterial infections such as leprosy and tuberculosis (TB) [5,7,16]. In a previous work we have demonstrated that M. leprae hsp65 could induce cytotoxic effector T cells from PBMC of PB patients and normal controls; in MB patients a marked deficiency in developing hsp65-specific CTL activity was found [10].…”

“…Mycobacterium leprae-specific cytotoxic T cell activity has been identified in leprosy patients [5][6][7]. Cytotoxic T cells may be beneficial for the host by elimination of M. leprae within Schwann cells and macrophages, but at the same time they participate in some of the adverse responses observed in leprosy patients.…”