In vitro measurement of protective mycobacterial immunity: Antigen-specific expansion of T cells capable of inhibiting intracellular growth of bacille Calmette-Guerin

We investigated the ability of T cells expanded with mycobacterial antigens from healthy purified protein derivative-reactive donors and bacille Calmette-Guerin (BCG)-vaccinated volunteers to inhibit intracellular growth of BCG. Peripheral blood mononuclear cells were incubated for 7 days with mycobacterial whole lysate, live BCG, tetanus toroid as control antigen, or medium alone. Autologous monocytes were separated by plastic adherence, allowed to mature for 6 days, and infected with BCG before serving as target cells. Expanded effector cells were cocultured with target cells for 72 h, Cocultures were then treated with 0.2% saponin to lyse infected monocytes and release intracellular BCG. Quantities of viable BCG present in these lysates were studied by colony-forming unit counting and radiometric labeling. We reproducibly found that lymphocytes expanded with mycobacterial whole lysate or live BCG significantly inhibited the intracellular growth of BCG, compared with lymphocytes expanded with tetanus toroid or rested in medium. In addition, BCG vaccination enhanced the ability of T cells to inhibit intracellular mycobacterial growth in 3 of 5 volunteers. This assay may be useful for estimates of protective immunity induced by tuberculosis vaccines in human trials.