Mycobacterium tuberculosis (Mtb)-specific IFN-gamma secretion plays important roles in anti-tuberculosis (TB) immunity. Mtb-specific IFN-gamma response can be induced in HIV/TB co-infected patients with a low CD4 lymphocyte count; this suggests that the source of Mtb-specific IFN-gamma production is not limited in CD4(+) T lymphocytes. Currently, the major sources of Mtb-specific IFN-gamma production and the function and phenotype of Mtb-specific IFN-gamma-producing cells still remain unclear. Thirty-nine participants (24 active TB patients, 10 HIV/TB co-infected patients, and 5 healthy volunteers) were recruited according to conventional tests and Mtb-specific IFN-gamma ELISPOT assay. Multicolor flow cytometry was used to investigate the production of intracellular IFN-gamma in peripheral blood mononuclear cells (PBMCs) after Mtb-specific antigen stimulation. Our results showed that CD4(+), CD8(+) T cells and NK cells are all major sources of Mtb-specific IFN-gamma production in PBMCs of TB patients. Moreover, CD8(+) T cells are the highest number of Mtb-specific IFN-gamma-producing cells in HIV/TB co-infected patients. Although the activity of NK cells is significantly reduced in TB patients when compared with healthy controls, Mtb-specific antigen stimulation induces a significant increase in NK cell activity. We also showed that CD45RO is the characteristic marker of Mtb-specific IFN-gamma-producing T cells but not that of Mtb-specific IFN-gamma-producing NK cells in peripheral blood. High expression of CD11a may be the characteristic feature of Mtb-specific IFN-gamma-producing NK cells. This study put forward a new insight on the source of antigen-specific IFN-gamma-production in PBMCs of TB patients. (C) 2016 Elsevier B.V. All rights reserved.