More than 2 billion individuals are latently infected with Mycobacterium tuberculosis (Mtb). Knowledge of the key Mtb antigens and responding T-cell subsets mediating protection against Mtb is critical for developing improved tuberculosis (TB) vaccines. We previously reported that Mtb DosR-regulon-encoded antigens are recognized well by human T cells in association with control of Mtb infection. The characteristics of the responding T-cell subsets, however, remained unidentified. We have therefore studied the cytokine production and memory phenotypes of Mtb DosR-regulon-encoded antigen-specific T cells from individuals who had been infected with Mtb decades ago, yet never developed TB (long-term latent Mtb-infected individuals). Using multi-parameter flow cytometry and intracellular cytokine staining for IFN-γ, TNF-α and IL-2, we found double and single cytokine-producing CD4 + as well as CD8 + T cells to be the most prominent subsets, particularly IFN-γ + TNF-α + CD8 + T cells. The majority of these T cells comprised effector memory and effector T cells. Furthermore, CFSE labeling revealed strong CD4 + and CD8 + T-cell proliferative responses induced by several "immunodominant" Mtb DosR antigens and their specific peptide epitopes. These findings demonstrate the prominent presence of double- and monofunctional CD4 + and CD8 + T-cell responses in naturally protected individuals and support the possibility of designing Mtb DosR antigen-based TB vaccines.