We report that M. tuberculosis organisms, but neither PHA nor allogeneic stimulator cells, preferentially activate gamma/delta+ cells within E rosette-purified peripheral blood T cells. gamma/delta+ T cells from purified protein derivative (PPD)-nonimmune healthy donors were enriched by depletion of CD4+ and CD8+ cells; double-negative (DN) cells contained 65-92% gamma/delta+ T cells. Limiting dilution (LD) analyses revealed that 1 of 2-19 purified DN cells proliferated in response to mycobacteria, while frequencies of DN cells proliferating in response to a recombinant 65-kD heat shock protein (hsp 65) of M. tuberculosis/M. bovis were 10-20-fold lower. Established clones of mycobacteria-reactive gamma/delta+ T cells specifically recognized mycobacteria, but neither PPD nor hsp 65. Restimulation of these clones required the presence of PBMC feeder cells; EBV-transformed lymphoblastoid cell lines could not substitute for PBMC. Mycobacteria-reactive gamma/delta+ clones proliferated equally well in the presence of autologous or allogeneic (HLA-DR-different) PBMC feeder cells and thus were not MHC class II restricted. Taken together, these results demonstrate that mycobacteria-reactive gamma/delta+ T cells are present in high frequency in the peripheral blood of healthy individuals, and suggest that hsp 65 of mycobacteria is not a major antigen for gamma/delta+ T cells of normal PPD-nonimmune blood donors.

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