High antigen load in chronic viral infections has been associated with impairment of antigen-specific T cell responses; however, the relationship between antigen load in chronic Mycobacterium tuberculosis (Mtb) infection and functional capacity of Mtb-specific T cells in humans is not clear. We compared Mtb-specific T cell-associated cytokine production and proliferative capacity in peripheral blood from adults with progressively higher mycobacterial loads, i.e., persons with latent Mtb infection (LTBI), with smear − pulmonary tuberculosis (TB), and with smear+ TB. Patients with smear+ TB had decreased polyfunctional IFN-γ+IL-2+TNF-α+ and IL-2-producing specific CD4 T cells and increased TNF-α-single positive cells, when compared with smear − TB and LTBI. TB patients also had increased frequencies of Mtb-specific CD8 T cells, compared with LTBI. Mtb-specific CD4 and CD8 T cell proliferative capacity was profoundly impaired in individuals with smear+ TB, and correlated positively with ex vivo IFN-γ+IL-2+TNF-α+ CD4 T cells, and inversely with TNF-α single-positive CD4 T cells. During 6 months of anti-TB treatment, specific IFN-γ+IL-2+TNF-α+ CD4 and CD8 T cells increased, whereas TNF-α- and IFN-γ-single positive T cells decreased. These results suggest progressive impairment of Mtb-specific T cell responses with increasing mycobacterial load, and recovery of responses during therapy. Furthermore, these data provide a link between specific cytokine-producing subsets and functional capacity of Mtb-specific T cells, and between the presence of specific CD8 T cells ex vivo and active TB disease. Taken together, these data have potentially significant applications for diagnosis of TB and for identification of T cell correlates of TB disease progression.

One third of the global population is estimated to be latently infected with Mycobacterium tuberculosis (M.tb). We performed a phase 1 randomized, controlled trial of isoniazid preventive therapy (IPT) before re-vaccination with Bacille Calmette-Guerin (BCG) in healthy, tuberculin skin test positive (≥15mm induration), HIV-negative, South African adults. We hypothesised that pre-clearance of latent bacilli with IPT modulates BCG immunogenicity following re-vaccination. Frequencies and co-expression of IFNγ, TNFα, IL-2, IL-17, and/or IL-22 in CD4, and IFNγ-expressing CD8, γδ T, CD3+CD56+ NKT-like and NK cells in response to BCG were measured using whole blood intracellular cytokine staining and flow cytometry. We analyzed 72 participants who were BCG re-vaccinated after IPT (n=33) or without prior IPT (n=39). IPT had little effect on frequencies or cytokine co-expression patterns of M.tb- or BCG-specific responses. Re-vaccination transiently boosted BCG-specific Th1 cytokine-expressing CD4, CD8 and γδ T cells. Despite high frequencies of IFNγ-expressing BCG-reactive CD3+CD56+ NKT-like, CD3−CD56dim and CD3−CD56hi NK cells at baseline, BCG re-vaccination boosted these responses, which remained elevated up to one year after re-vaccination. Such BCG-reactive memory NK cells were induced by BCG vaccination in infants, while in vitro IFN-γ expression by NK cells upon BCG stimulation was dependent on IL-12 and IL-18. Our data suggest that isoniazid pre-clearance of M.tb bacilli has little effect on the magnitude, persistence or functional attributes of lymphocyte responses boosted by BCG re-vaccination. Our study highlights surprising durability of BCG-boosted memory NKT-like and NK cells expressing anti-mycobacterial effector molecules, which may be novel targets for TB vaccines.

Antigen-specific proliferation is a critical function of memory T cells that is often utilised to measure vaccine immunogenicity and T cell function. We proposed that measurement of intracellular expression of the nuclear protein, Ki67, could reliably assess specific T cell proliferation in vitro.Ki67 was expressed in CD4+ and CD8+ T cells that had undergone in vitro proliferation after 6-day culture of human whole blood or PBMC with antigens. T cells cultured with no antigen did not express Ki67. When compared to current flow cytometry based proliferation assays, Ki67 detected proliferating cells with greater sensitivity than BrdU incorporation, whereas its sensitivity was similar to dye dilution of Oregon Green (OG), a CFSE derivative. Overall, the magnitude and cytokine expression profile of proliferating T cells detected by Ki67 expression correlated strongly with T cells detected with BrdU or OG. The intra-assay variability of Ki67 proliferation was 2–3% for CD4+ T cells, and 10–16% for CD8+ T cells. Finally, we demonstrate that the Ki67 assay detects tetanus toxoid-specific CD4+ T cell proliferation after infant vaccination with tetanus toxoid (TT).Overall our data suggest that intracellular Ki67 expression provides a specific, quantitative and reproducible measure of antigen-specific T cell proliferation in vitro.