TB is a leading cause of morbidity in developing countries and ranks as the eighth most frequent cause of all deaths worldwide. The World Health Organization (WHO) recently estimated that 8.8 million persons developed TB in the year 2010 and 1.4 million patients died from this disease that year . TB can develop following inhalation of Mycobacterium tuberculosis from the exhalate of patients with pulmonary TB, the predominant clinical manifestation of this disease. Mechanisms of innate immune defense involving the action of antimicrobial peptides and the activity of polymorphonuclear neutrophilic granulocytes may prevent persistent infection of the host [2, 3]. Once M. tuberculosis enters alveolar macrophages — the host cells for chronic pulmonary infection — the bacterium may evade killing, survive and replicate within these cells . However, even in cases of chronic infection, active TB is generally prevented by the recruitment of Ag-specific T cells to the site of infection. These T cells surround infected macrophages and form a granuloma to prevent further spread of the bacteria in the majority of cases .
Analyses of Ag-specific immune responses have played an important role in the diagnosis of TB for more than a century. In 1907, an “allergy test for tuberculosis” was proposed by the pediatrician Clemens von Pirquet from Vienna, who elicited delayed hypersensitivity skin reactions following the application of a sterile supernatant of liquid M. tuberculosis cultures (tuberculin) in skin lacerations of both children with TB and healthy controls . These observations provided the first evidence that immune responses by skin testing were unable to distinguish children with active TB from healthy children. Thus, healthy contacts with positive tuberculin skin test reactions were thought to be latently infected with M. tuberculosis. Based on information derived from the tuberculin skin test responses and the determination of the percentage of healthy individuals who subsequently developed TB, it was estimated that approximately 5–10% of persons with latent M. tuberculosis infection develop active TB during their lifetime , with the risk being highest in the first 2 years following primary infection (approximately 2% ). These observations formed the rationale for using tuberculin skin testing to estimate the risk for recent TB contacts and immunocompromised individuals of developing TB in the future. The tuberculin skin test was modified by Felix Mendel and Charles Mantoux for intradermal application [9, 10] and was used for almost one century in this format to diagnose latent infection with M. tuberculosis.
Apart from the inability to distinguish active from latent infection, another disadvantage of the skin test is its inability to distinguish individuals that are “truly” latently infected with M. tuberculosis from individuals who have been vaccinated with M. bovis BCG, the most commonly used vaccine worldwide ; however, specificity for infection with M. tuberculosis was able to be improved when bacterial virulence factors, such as early-secretory-antigenic target 6 and culture-filtrate-protein 10, were identified as being M. tuberculosis specific Ags that are not produced by M. bovis BCG vaccine strains . The use of these Ags was subsequently applied in tests that elicit IFN-γ responses from M. tuberculosis specific T cells in peripheral blood and two ex vivo IFN-γ release assays (IGRAs) are currently commercially available in ELISA (Quantiferon Gold in tube) or ELISPOT (T-SPOT.TB) formats.
While the detection of M. tuberculosis specific immune responses was improved by IGRAs, the positive predictive value to identify individuals at risk for the future development of TB remained considerably low . In addition, the standard use of IGRAs on cells from the peripheral blood has not been able to overcome one of the major limitations of the tuberculin skin test, namely to distinguish patients with active TB from individuals with latent M. tuberculosis infection  (Table 1). In general, the percentage of M. tuberculosis specific circulating cells identified by IGRAs is higher in patients with active TB as compared with those identified in healthy individuals with a positive IGRA response. Nevertheless, the test results of both populations overlap substantially, which precludes the correct assignment of the actual infection status on an individual basis. Therefore, new immune-based approaches have been explored to increase the specificity for the diagnosis of active TB (Table 1).
|Tuberculin skin tests, commercial IGRAs||Enumeration of M. tuberculosis specific T cells from blood and extrasanguinous fluids||Cytokine profiling and/or phenotyping of M. tuberculosis specific T cells from whole blood||Phenotyping of bulk CD4+ T cells from blood and extrasanguinous fluids|
|Diagnosis of active tuberculosis||No||Yes||Yes||Yes|
|Test format||e.g. RT 23 SSIa T.SPOT.TB; QuantiFERON TB Gold in tube||ELISPOT assay||Flow cytometry||Flow cytometry|
|Material required||Skin, blood||Blood and extrasanguinous fluids||Blood||Blood and extrasanguinous fluids|
|Time required for the result||48–72 h (TST) 24 h (IGRAs)||20-h incubation and 3 h for the final results||6- to 20-h incubation and 2 h for the final results||1 h|
|Read-out||Diameter of dermal induration (mm) IFN-γ secretion/IFN-γ producing cells||Accumulation of cytokine-producing cells in extrasanguinous samples versus blood||Shift in cytokine profile/change in phenotype||Accumulation of CD27−CD4+ T cells in extrasanguinous samples versus blood|
The most promising of these approaches have made use of the selective recruitment of M. tuberculosis specific T cells from the peripheral blood to the site of infection , and of the differential phenotype and functionality of Ag-specific T cells in association with pathogen load. Indeed, when comparatively analyzing peripheral blood and extrasanguinous fluids from patients with active TB, an increased percentage of Ag-specific T cells was found at the site of the infection, which was not found in individuals with latent infection; this has convincingly been shown for bronchoalveolar lavage fluid in pulmonary TB , pleural fluid in tuberculous pleurisy [17, 18], cerebrospinal fluid in tuberculous meningitis [19, 20], and ascites in tuberculous peritonitis , with the concentration of Ag-specific T cells being five- to 17-fold higher at the site of the infection when compared with that in the peripheral blood [16, 17, 22]. In a recent meta-analysis by the Tuberculosis European Network Trials Group (TBNET; www.tb-net.org), the IGRA ELISPOT technique performed on cells from extrasanguinous fluids was the best immunodiagnostic method for identifying patients with active TB, giving a pooled diagnostic sensitivity of 88% and a pooled diagnostic specificity of 82% on cases that predominantly had an acid fast bacilli smear negative manifestation of the disease . Therefore, commercial blood-based assays may be modified to give novel methods for the immunodiagnosis of acid fast bacilli smear negative pulmonary and extrapulmonary TB.
An alternative approach to identify patients with active disease has made use of the fact that the functionality [23, 24] and phenotype  of Ag-specific T cells may critically determine the immune control of pathogen replication (Table 1). In this respect, a shift in the cytokine profile from multifunctionality toward cells expressing single cytokines has been associated with active TB [23, 24]. In addition, cytokine-producing cells in patients with active disease typically exhibit a terminally differentiated phenotype that is characterized as predominantly CD27− , whereas the Ag-specific immune response in individuals with nonactive states generally consists of equal percentages of CD27+ and CD27− CD4+ T cells [26, 27]. Both phenotypic and functional analyses may be elegantly performed using multiparameter flow cytometry directly on whole blood, but Ag-specific stimulation and cytokine induction are required to assign key characteristics of functionally anergic cells to cells specific for mycobacterial Ags.
In this issue of the European Journal of Immunology, Nemeth et al.  have used flow cytometry to combine a compartmentalized analysis of T-cell subpopulations with a differential assessment of the phenotypic characteristics of the Ag-specific T cells that are typical for patients with active TB. The phenotype and function of T cells from the peripheral blood and from the site of the infection were analyzed in patients with extrapulmonary TB (mainly tuberculous pleurisy) and in patients with other lung diseases unrelated to TB. The most striking finding was the fact that in TB patients the percentage of CD27−CD4+ T cells at the site of infection was significantly lower compared with that in peripheral blood. Of note, this finding was irrespective of Ag-specificity and no such difference was found when similar analyses were performed in patients with diagnoses other than TB (Table 1). When the T-cell subtypes in both compartments were analyzed for specificity toward M. tuberculosis by concomitant assessment of IFN-γ, cytokine-producing cells in peripheral blood were found among both CD27− and CD27+ populations, whereas M. tuberculosis specific CD4+ T cells at the site of infection were predominantly restricted to the CD27− subset. Together the data indicate that M. tuberculosis specific T cells that accumulate at the site of infection are mainly confined to the CD27−CD4+ T-cell subpopulation. As an increase in the percentage of CD4+ T cells with this phenotype is already evident from bulk CD4+ T cells in the absence of specific stimulation, this offers the possibility to use the selective accumulation of CD27−CD4+ T cells in samples from the site of infection as surrogates for M. tuberculosis specific cells and thus as an indicator for active TB. This analysis may be performed from small sample volumes without the need for specific stimulation as highlighted in Table 1. However, given that even phenotypic bulk analyses were performed after an 18 h incubation period in the presence of brefeldin A, the diagnostic potential of a direct cell surface staining procedure still needs to be formally proven.
The study has some limitations in that only 13 of 178 suspects for active TB were actually included, of which seven served as controls with diagnoses other than TB. Thus, the total number of patients with active disease was very small and not representative for pulmonary TB or different forms of extrapulmonary TB. Moreover, control groups of individuals with BCG vaccination or clear evidence of latent infection with M. tuberculosis should have been recruited to prove whether a high percentage of CD27− T cells is specifically confined to patients with active TB. Likewise, patients with other types of inflammatory, granulomatous, and/or infectious diseases should have been evaluated as controls. If confirmed in larger studies with larger sample sizes, direct immunophenotyping of cells from the site of the infection (e.g. bronchoalveolar lavage, ascites, cerebrospinal fluid, pleural effusion, pericardial fluid) using flow cytometry may become an option to improve the difficult identification of TB cases as the diagnostic accuracy of nucleic acid amplification for M. tuberculosis DNA is still inadequate to rule out active disease in patients with acid fast bacilli negative smears  or in patients with extrapulmonary TB .