Distinct IFN-gamma and IL-2 profiles of antigen-specific CD4+ T cells have recently been associated with different clinical disease states and antigen loads in viral infections. We assessed the kinetics and functional profile of Mycobacterium tuberculosis antigen-specific T cells secreting IFN-gamma and IL-2 in 23 patients with untreated active tuberculosis, when bacterial and antigen load are high, and after curative treatment when antigen load is reduced. Frequencies of M. tuberculosis antigen-specific IFN-gamma-secreting T cells declined during 28 months of follow up with an average percentage decline of 5.8% per year (P=0.005), whilst frequencies of antigen-specific IL-2-secreting T cells increased during treatment (P=0.02). These contrasting dynamics for the two cytokines led to a progressive convergence of the frequencies of IFN-gamma and IL-2-secreting cells over 28 months. Simultaneous measurement of IFN-gamma and IL-2 secretion at the single cell level revealed a co-dominance of IFN-gamma-only and IFN-gamma/IL-2 dual-secreting CD4+ T cells in active disease which shifted to dominance of IFN-gamma/IL-2-secreting CD4+ T cells and newly detectable IL-2-only-secreting CD4+ T cells during and after treatment. These distinct T cell functional signatures before and after treatment suggest a novel immunological marker of mycobacterial load and clinical status in tuberculosis, which now requires validation in larger prospective studies.

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Rationale: The kinetics of Mycobacterium tuberculosis-specific Th1type T-cell responses after M. tuberculosis infection are likely to be important in determining clinical outcome. Objective: To investigate the kinetics of T-cell responses, in the context of a point-source school tuberculosis outbreak, in three groups of contacts who differed by preventive treatment status and tuberculin skin test (TST) results: 38 treated TST-positive students, 11 untreated TST-positive staff, and 14 untreated students with negative or borderline TST results. Methods: We used the ex vivo IFN-␥ enzyme-linked immunospot assay (ELISpot) to track T cells specific for two region of difference 1 (RD1) antigens, early secretory antigenic target 6 and culture filtrate protein 10, for 18 mo after cessation of tuberculosis exposure. Main Results: The treated TST-positive students had an average 68% decline in frequencies of RD1-specific IFN-␥-secreting T cells per year (p Ͻ 0.0001) and 6 of 38 students had no detectable RD1specific T cells by 18 mo. No change in frequencies of these cells was observed in the untreated TST-positive staff (p ϭ 0.38) and none were ELISpot-negative at 18 mo. Of the 14 untreated students, 7 were persistently ELISpot-positive (all of whom had borderline TST results), and 7 became ELISpot-negative (all but one had negative TST results) during follow-up. Conclusions: The decrease in M. tuberculosis-specific T cells and their disappearance in a proportion of treated students likely reflect declining antigenic and bacterial load in vivo induced by antibiotic treatment. The observed disappearance of M. tuberculosis-specific T cells in the untreated TST-negative contacts suggests that an acute resolving infection may occur in some contacts.

Positive ELISpot results predict subsequent development of active tuberculosis in recent tuberculosis contacts. Although tuberculosis contacts with positive ELISpot results have an incidence rate of tuberculosis similar to that of contacts with positive tuberculin skin test results, ELISpot testing could allow more focused targeting of preventive therapy to fewer contacts.

The 6-kDa early secretory antigenic target of Mycobacterium tuberculosis (ESAT-6) and the 10-kDa culture filtrate antigen (CFP-10), encoded in region of difference 1 (RD1) and secreted by the ESAT-6 system 1 (Esx-1) secretion system, are the most immunodominant and highly M. tuberculosis (MTB)-specific antigens. These attributes are responsible for their primary importance in tuberculosis (TB) immunodiagnosis and vaccine development. Rv3615c [Esx-1 substrate protein C (EspC)], encoded outside RD1, is similar in size and sequence homology to CFP-10 and ESAT-6, suggesting it might be a target of cellular immunity in TB. Using ex vivo enzyme-linked immunospot-and flow cytometry-based cytokine-secretion assay, we comprehensively assessed cellular immune responses to EspC in patients with active TB, latently infected persons, and uninfected bacillus CalmetteGuérin (BCG)-vaccinated controls. EspC was at least as immunodominant as ESAT-6 and CFP-10 in both active and latent TB infection. EspC contained broadly recognized CD4 + and CD8 + epitopes, inducing a predominantly CD4 + T-cell response that comprised functional T-cell subsets secreting both IFN-γ and IL-2 as well as functional T-cell subsets secreting only IFN-γ. Surprisingly, T-cell responses to EspC were as highly specific (93%) for MTB infection as responses to ESAT-6 and CFP-10, with only 2 of 27 BCG-vaccinated controls responding to each antigen. Using quantitative proteomics and metabolically labeled mutant and genetically complemented MTB strains, we identified the mechanism of the specificity of anti-EspC immunity as the Esx-1 dependence of EspC secretion. The high immunodominance of EspC, equivalent to that of ESAT-6 and CFP-10, makes it a TB vaccine candidate, and its high specificity confers strong potential for T-cell-based immunodiagnosis.

The ELISpot(PLUS) assay is more sensitive than standard ELISpot and, when used in combination with tuberculin skin testing, enables rapid exclusion of active infection in patients with moderate to high pretest probability of tuberculosis.

T-cell interferon-gamma release assays (IGRAs) are more specific and probably more sensitive than the tuberculin skin test (TST) for the diagnosis of latent tuberculosis infection (LTBI). Patients with immune-mediated inflammatory diseases (IMID) and suspected LTBI who are candidates for anti-TNF therapy are at a significant risk of TB reactivation yet are prone to false-negative TST results because they are already on immunosuppressive medications. The role of these new blood tests in this patient population is therefore of considerable interest but is currently unclear. The limited published evidence-base shows that agreement between IGRA and TST results is poor in patients with IMID compared to patients without IMID, due to lower proportions of TST-positive results in patients with IMID. Discordant TST-positive, IGRA-negative results are associated with prior BCG vaccination and discordant TST-negative, IGRA-positive results are associated with steroid therapy. Notably, positive IGRA results are more closely associated with the presence of risk factors for LTBI than TST. The percentage of indeterminate IGRAs can be up to 12%. IGRA results in patients already taking anti-TNF agents currently remain uninterpretable. Given the clinical imperative to prevent reactivation of TB in patients starting anti-TNF therapy, screening algorithms should maximise diagnostic sensitivity for detection of LTBI. Therefore, a positive result to either an IGRA or TST, in addition to currently recommended clinical screening for risk factors for LTBI, should prompt consideration of preventive treatment of LTBI in this population.

Although longitudinal studies quantifying risk of progression to tuberculosis in tuberculosis-exposed children with positive TIGRA results are required urgently, the small but rapidly expanding evidence-base since the first application of TIGRAs to childhood tuberculosis in 2003 combined with recent national guidelines makes a strong case for judicious use of TIGRAs in clinical management of paediatric tuberculosis.