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One third of the global population is estimated to be latently infected with Mycobacterium tuberculosis. We performed a phase I randomized controlled trial of isoniazid preventive therapy (IPT) before revaccination with bacillus Calmette–Guérin (BCG) in healthy, tuberculin skin test–positive (≥15-mm induration), HIV-negative South African adults. We hypothesized that preclearance of latent bacilli with IPT modulates BCG immunogenicity following revaccination. Frequencies and coexpression of IFN-γ, TNF-α, IL-2, IL-17, and/or IL-22 in CD4 T cells and IFN-γ–expressing CD8 T, 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 revaccinated with BCG after IPT (n = 33) or without prior IPT (n = 39). IPT had little effect on frequencies or cytokine coexpression patterns of M. tuberculosis– or BCG-specific responses. Revaccination transiently boosted BCG-specific Th1 cytokine-expressing CD4, CD8, and γδ T cells. Despite high frequencies of IFN-γ–expressing BCG-reactive CD3+CD56+ NKT-like cells and CD3−CD56dim and CD3−CD56hi NK cells at baseline, BCG revaccination boosted these responses, which remained elevated up to 1 y after revaccination. Such BCG-reactive memory NK cells were induced by BCG vaccination in infants, whereas in vitro IFN-γ expression by NK cells upon BCG stimulation was dependent on IL-12 and IL-18. Our data suggest that isoniazid preclearance of M. tuberculosis bacilli has little effect on the magnitude, persistence, or functional attributes of lymphocyte responses boosted by BCG revaccination. Our study highlights the surprising durability of BCG-boosted memory NKT-like and NK cells expressing antimycobacterial effector molecules, which may be novel targets for tuberculosis vaccines.

Recent estimates of the global tuberculosis burden indicate reductions in the number of persons with tuberculosis, the incidence of tuberculosis, and deaths due to tuberculosis [1]. Despite this, approximately 8.8 million new tuberculosis cases were diagnosed in 2010; 1.45 million individuals died from tuberculosis [1]. BCG vaccination in infancy is routine in tuberculosis-endemic countries. However, BCG confers reliable protection against severe forms of tuberculosis in infancy only [2]. Protection against pulmonary disease at all ages is variable and mostly poor. Epidemiological modeling suggests that a new tuberculosis vaccine or vaccination strategy that has greater protective efficacy than the current BCG strategy is critical for tuberculosis elimination [3]. Most investigators agree that future vaccination against tuberculosis would involve a heterologous prime-boost regimen, with BCG or an improved live mycobacterial vaccine as a prime, followed by an adjuvanted subunit or viral vectored boost [4, 5]. A major hurdle to designing novel vaccination strategies is our limited knowledge of key T-cell response characteristics induced by BCG. The kinetics of T cells induced by neonatal BCG vaccination have not been studied in humans; in particular, we do not know when this immune response peaks. This is a critical knowledge gap, because studies of CD8+ [6, 7] and CD4+ [8, 9] responses in viral infection models suggest that the best time for boosting is after the peak effector phase, when effector T cells have transitioned into an established memory population. Vaccine boosting during the primary effector phase, when cells are activated, may lead to T-cell exhaustion or activation-induced cell death [6, 9, 10]. Another unresolved question is whether BCG vaccination consistently induces long-lived central memory cells. Cross-sectional studies in infants have suggested that BCG may induce primarily effector T cells [11–13]. This is supported by studies of murine BCG vaccination, which induces a predominant effector T-cell response that fails to establish a long-lived central memory response [14, 15]. These data led to the hypothesis that the short-lived nature of BCG-induced effector T cells underlies the limited duration of BCG-induced protection against tuberculosis [15]. However, BCG vaccination of infants and adults induces immunological memory [16–18], and a recent retrospective analysis of long-term efficacy of BCG given between 1935–1938 suggests that BCG induces long-lived protection [19]. A comprehensive longitudinal study of memory phenotype following human newborn BCG vaccination, particularly a study that yields clarity about the induction of central memory T cells, is required. Therefore, we characterized the kinetics, function, and phenotype of BCG-specific T cells in a longitudinal study of infants routinely vaccinated with BCG at birth. Expansion and contraction of the primary BCG-specific T-cell response occurred within 3 months of vaccination, and the magnitude of specific CD4+ T cells peaked 6–10 weeks after vaccination. After peak, BCG-specific CD4+ T cells displayed a phenotype of classic central memory T cells but retained functional characteristics more consistent with effector T cells.

High Ag load in chronic viral infections has been associated with impairment of Ag-specific T cell responses; however, the relationship between Ag load in chronic Mycobacterium tuberculosis infection and functional capacity of M. tuberculosis-specific T cells in humans is not clear. We compared M. tuberculosis-specific T cell-associated cytokine production and proliferative capacity in peripheral blood from adults with progressively higher mycobacterial loads—that is, persons with latent M. tuberculosis infection (LTBI), with smear-negative pulmonary tuberculosis (TB), and smear-positive TB. Patients with smear-positive 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-negative TB and LTBI. TB patients also had increased frequencies of M. tuberculosis-specific CD8 T cells, compared with LTBI. M. tuberculosis-specific CD4 and CD8 T cell proliferative capacity was profoundly impaired in individuals with smear-positive 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 mo 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 M. tuberculosis-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 M. tuberculosis-specific T cells, and between the presence of specific CD8 T cells ex vivo and active TB disease. These data have potentially significant applications for the diagnosis of TB and for the identification of T cell correlates of TB disease progression.