Induction and regulation of cytotoxic T cells by microbial antigens and recombinant interleukin 2.

The proliferation and development of cytotoxic T cells was investigated in human peripheral blood mononuclear cell (PBMC) cultures stimulated with an antigenic extract from Candida albicans (MPPS), or with the purified protein derivative from Mycobacterium tuberculosis (PPD), or with human recombinant interleukin 2 (rIL-2). Microbial antigen- and rIL-2-induced cytotoxic T cells were able to lyse both natural killer (NK) sensitive and resistant targets. No correlation was observed between the development of T cell cytotoxicity and interferon (IFN) production in vitro. The addition of anti-class II monoclonal antibodies at the beginning of MPPS/PPD-stimulated cultures inhibited the cell proliferation, IFN production and T cell cytotoxicity, while all these cellular activities were not inhibited by anti-class II antibodies in rIL-2-stimulated cultures. Finally, antibodies to class I determinants inhibit T cell cytotoxicity, suggesting a role of such determinants in the development of the non-adaptive immunity to microbial infections.

The combined treatment of human peripheral blood mononuclear cells with thymolymphotropin and interleukin 2 increases PPD-driven T-cell proliferation and IL-2 induced cellular cytotoxicity against HIV-infected cells.

Two in vitro systems (the DNA synthetic response to mycobacterial antigens and cytotoxicity against lymphoid cells) were used to analyse the effect of thymolymphotropin (TLT) on peripheral blood mononuclear cells (PBMC). Purified protein derivative of mycobacteria (PPD)-driven T-cell proliferation in low-responder donors was increased by the combined treatment with TLT and suboptimal doses of recombinant interleukin 2 (IL-2). Similarly, the activities of natural killer (NK) cells and lymphokine-activated killer (LAK) cells have been enhanced in PBMC cultures pretreated with TLT. Also, TLT showed an enhancing effect on the development of LAK cells capable of lysing Epstein-Barr virus (EBV)-transformed B-lymphocytes infected or uninfected with the human immunodeficiency virus (HIV).

Identification of a 35-kilodalton Mycobacterium tuberculosis protein containing B- and T-cell epitopes.

Screening of a Mycobacterium tuberculosis genomic DNA library in the lambda gt11 expression vector was carried out by using, as probes, sera from tuberculous patients and murine monoclonal antibody H61.3 recognizing a mycobacterial 35-kilodalton protein present only on the M. tuberculosis complex. The recombinant beta-galactosidase-fused protein present in the crude lysate induced the proliferation of T lymphocytes from patients with tuberculous pleuritis. As the recombinant insert contains an internal EcoRI restriction site, it was possible to identify two fragments, one proximal to the lacZ gene and 1.7 kilobases (kb) in size and the other distal to the lacZ gene and 2.2 kb in size. Southern blot analysis showed that both of them hybridized with the genomic DNA from M. tuberculosis and M. bovis but not with the DNA from other mycobacterial species. To perform extensive immunological studies, the amount of beta-galactosidase-fused protein being very low, we fused the 1.7-kb fragment to the N-terminal part of the gene coding for the DNA polymerase of bacteriophage MS2 in the expression vector pEx34. The fusion protein was partially purified, and subsequent Western blotting (immunoblotting) and T-cell proliferation experiments confirmed the presence of B- and T-cell mycobacterial epitopes. Furthermore, to isolate the chromosomal region containing the 35-kilodalton gene, we constructed another mycobacterial genomic library in the lambda 2001 vector by cloning 15 to 20 kb of foreign DNA. Screening of this library was carried out by using 1.7- and 2.2-kb recombinant fragments as probes. Restriction maps of some clones isolated were determined.