Antigen-dependent in vitro culture of protective T cells from BCG-primed mice

Induction of protective immunity against pathogenic mycobacteria depends on vaccination with live organisms such as Bacille Calmette-Guérin (BCG). However, it is not known how many and which antigens are involved in the protective host response. In this study, we developed a system of antigen-dependent in vitro culture which is suitable for the analysis of protective subunits, presented in a soluble form. Spleen cells from Mycobacterium bovis BCG-immune mice, enriched for T cells and depleted of adherent cells on a column of G-10 Sephadex, were cultured for periods varying between 3 and 14 days before transfer and challenge with M. tuberculosis in irradiated hosts. Following 10 days in culture, immune T cells sustained their capacity to transfer protection to tuberculous infection when incubated in the presence of either live BCG or a soluble extract from M. tuberculosis, but lost this ability when cultured in the absence of antigen, or in the presence of the polyclonal mitogen concanavalin A. One immunodominant antigen, represented by the recombinant 38-kD antigen, failed to sustain the adoptive protection, despite pronounced stimulation of lymphoproliferation in culture. Antigenic in vitro stimulation of protective T cells was accompanied by enhanced responsiveness to exogenous IL-2. The experimental system described may be generally suitable to test in vitro the protective potentials of soluble molecular subunits of mycobacteria.