Identification and Characterization of Protective T Cells in hsp65 DNA-Vaccinated and Mycobacterium tuberculosis-Infected Mice

Immunization by intramuscular injection of plasmid DNA expressing mycobacterial 65-kDa heat shock protein (hsp65) protects mice against challenge with virulent Mycobacterium tuberculosis H37Rv. During infection or after immunization, CD4(+)/CD8(−) and CD8(+)/CD4(−) hsp65-reactive T cells increased equally in spleens. During infection, the majority of these cells were weakly CD44 positive (CD44(lo)) and produced interleukin 4 (IL-4) whereas after immunization the majority were highly CD44 positive (CD44(hi)) and produced gamma interferon (IFN-γ). In adoptive transfer of protection to naive mice, the total CD8(+)/CD4(−) cell population purified from spleens of immunized mice was more protective than that from infected mice. When the cells were separated into CD4(+)/CD8(−) and CD8(+)/CD4(−) types and then into CD44(hi) and CD44(lo) types, CD44(lo) cells were essentially unable to transfer protection, the most protective CD44(hi) cells were CD8(+)/CD4(−), and those from immunized mice were much more protective than those from infected mice. Thus, whereas the CD44(lo) IL-4-producing phenotype prevailed during infection, protection was associated with the CD8(+)/CD44(hi) IFN-γ-producing phenotype that predominated after immunization. This conclusion was confirmed and extended by analysis of 16 hsp65-reactive T-cell clones from infected mice and 16 from immunized mice; the most protective clones, in addition, displayed antigen-specific cytotoxicity.