Cytometric detection of antigen-specific IFN-γ/IL-2 secreting cells in the diagnosis of tuberculosis

Tuberculosis (TB) is one of the most prevalent bacterial infections of humans worldwide [1], but the large majority of M. tuberculosis infected individuals are asymptomatic. In fact, healthy individuals who get infected by M. tuberculosis for the first time usually develop a mild disease: primary TB. These individuals clinically heal, but are unable to clear the bacterium that remains dormant within granulomas or cellular reservoirs [2‐4]. This latent TB infection may have a lifelong persistence, without clinical relevance [5]. In some subjects, due to concomitant infections, malnutrition or other factors, latent M. tuberculosis may reactivate causing a post-primary TB that usually develops chronically, unless treated with antibiotics [6, 7].

One of the main problems in the fight against TB is the correct diagnosis of its different stages. Tuberculin skin test (TST) is the most used screening method for the diagnosis of M. tuberculosis infection, even though a prior vaccination with Bacillus Calmette Guérin (BCG) can lead to false positive results using this test. Other widely recognized limits of TST are the false negative tests due to anergy or immune deficiency and its inability to distinguish latently infected individuals from patients with active TB. Moreover, the site of the skin test must be examined by a health professional within 48 – 72 hours to determine the result of the test [8, 9]. Newer assays based on the release of interferon (IFN)-γ upon specific antigen stimulation (IGRAs) have recently become available [10‐12], but their enhanced sensitivity and specificity compared to TST [8, 13] have not been demonstrated yet. These tests take advantage by the use of early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10 kDa (CFP-10), proteins that are encoded within the region of difference 1 (RD1) of M. tuberculosis but not BCG genome, and thus are not synthesized by BCG substrains or most environmental mycobacteria, with few exceptions [14‐16]. In addition, some of these tests are commercially available and approved for TB infection diagnosis. However, IGRAs can discriminate vaccinated from M. tuberculosis-infected individuals, but not patients affected by active TB disease from individuals with latent TB infection. Lastly, the instrumentation required to perform some of IGRAs is not available in all laboratories [17, 18].

A possible contribution to discriminate the different TB stages could be envisaged in a more accurate definition of the immunological response to selected M. tuberculosis antigens. In fact, the immune response should differ according to the antigen load, i.e. to active disease or latent infection. Since effector T cells (TEM) have a limited lifespan, it is possible to hypothesize that in the presence of active bacterial replication, i.e. active disease, their frequency should be increased in comparison to central memory T cells (CEM) in peripheral blood mononuclear cells (PBMC), whereas CEM should be predominant in latent infections. Beside phenotypic surface markers, memory cells can be distinguished according to their differential cytokine secretion: TEM secrete IFN-γ alone while CEM interleukin (IL)-2 or IFN-γ and IL-2. Moreover, they show different kinetics of stimulation: rapid (hours) for TEM and longer (days) for CEM [19, 20]. In this line, it has been recently shown that M. tuberculosis specific T cell response in patients with a non-replicating M. tuberculosis infection is mainly characterized by IL-2-secreting T cells [21].

In this study we investigate the possible contribution of active cytokine-secreting memory T cell enumeration as a diagnostic tool for TB infection and disease using a modified commercially available method [22, 23].

Tests based on IFN-γ release after PBMC stimulation with M. tuberculosis specific antigens have been recently introduced. These ELISA or ELISPOT tests contribute to the study of T cell responses in TB, and represent an important advance in diagnosis, since they may allow discrimination of BCG-vaccinated from infected individuals [10, 15, 16, 18]. However, a series of limitations to these tests call for a research aimed at ameliorating cytokine release detection [8, 13]. In this line, methods to enumerate cytokine-secreting lymphocytes by flow cytometry have been proposed [27, 28]. The most promising are based on cytokine detection by intracellular staining, which is possible only after cell fixation and permeabilization with cytokine release blocking substances. M. tuberculosis specific cells have low frequencies in PBMC and these procedures do not always guarantee the accumulation of an amount of cytokine high enough to be detected by intracellular antibodies. For this reason some of these methods use co-stimulatory antibodies to increase the cytokine secretion capacity of T cells [27].

In the present work we adapted a commercially available cytokine-secretion method at single living-cell level [22, 23] to study T cell responses in M. tuberculosis infection. The use of this approach allows the identification and enumeration of living cells actively secreting the cytokines of interest upon specific stimulation, and also their characterization on the basis of membrane expression of selected markers. The frequency of cytokine secreting cells after 72 hours was higher than after 18 hours and sufficient to discriminate with high efficiency M. tuberculosis-infected individuals from controls. Due to the characteristics of the method, the increased frequency of secreting cells after 72 hours was not due to an additive effect of cytokines secreted during the three day culture, but it consisted of an increased number of cells effectively secreting cytokines. TEM are terminal cells exerting their functions, such as cytokine secretion, upon encounter with antigen presenting cells (APC). They subsequently are programmed to die [19, 29]. Thus, the increased frequency found after a three-day culture is likely to represent the contribution of CEM cells that, upon stimulation, undergo cell division and cytokine secretion [29]. Probably due to the cumulative analysis of both M. tuberculosis specific CEM and TEM populations, the specificity and the sensitivity of our test reached high levels. The enumeration of the sole IFN-γ secreting cells upon ESAT-6/CFP-10 stimulation was sufficient to discriminate M. tuberculosis-infected individuals from controls. In fact, the contribution of IL-10 and IL-12 secreting cells was irrelevant. Some papers reported levels of IL-10 in TB patients higher than in healthy controls [30]. IL-10 observed in other publications might be dependent on IL-10 release by natural regulatory T cells, that are not stimulated in an antigen specific assay or by using co-stimulating antibodies to increase the cytokine secretion [28]. On the other hand, we observed a trend for IL-2-secreting cells to be detected at frequencies that are higher in individuals with latent TB infection than in those with active disease. The difference was statistically significant when PPD and BCG were used as stimuli, but not significant upon ESAT-6/CFP-10 stimulation. Since the number of epitope specific CEM is relatively small, it is not surprising that responses of increased magnitude are obtained using antigen mixtures such as PPD or the whole mycobacterium (BCG), and not with single antigens such as ESAT-6/CFP-10. These data are likely to reflect the increased number of IL-2 or IL-2/IFN-γ-secreting CEM and the reduced number of IFN-γ-secreting TEM in individuals with latent TB infection in comparison to active TB patients, due to the absence of M. tuberculosis replication and, consequently, antigen load [16, 21]. However, a larger study is required to confirm these data and to exclude any possible role of environmental mycobacteria.

Our data are in agreement with the recent observation by Millington et al. describing the increase of M. tuberculosis specific IL-2 and IL-2/IFN-γ-secreting T cells in the follow-up of active TB treated patients [21]. The emergence of IL-2 secreting cells in patients after antibiotic treatment could be considered as the consequence of the expansion of a new population of cells that can be considered CEM, caused by the reduced mycobacterial antigen load.

Recently, M. tuberculosis-specific IL-17 secreting T cells have been described [31, 32] and a role of these T helper (Th)17 cells in the pathogenesis of TB hypothesized [33]. Since IL-17 secretion has been associated to chronic inflammations [34, 35], we sought to determine its specific secretion in the attempt to identify a surrogate marker of active disease. However, ESAT-6/CFP-10 stimulated PBMC from active TB patients, similarly to control donors, did not secrete IL-17. Only 3 out of 14 patients showed a specific IL-17 secretion upon stimulation with PPD, but these patients showed no clinical signs correlating with the increased IL-17 secretion. Our data confirm that PPD-specific Th17 cells are detected in some active TB patients, but analyses on a larger population would be required to verify whether circulating PPD-specific Th17 cells play any role in disease pathogenesis.

In our assay we noticed the contribution of CD8⁺ T cells to the cytokine secretion upon M. tuberculosis specific stimulation. Although previous papers reported the participation of CD8⁺ T cells in cytokine secretion [36‐38], we were surprised to observe a CD8⁺ T cell response using PPD and ESAT-6/CFP-10, which are soluble antigens and therefore processed in late-endosomes [39] for presentation to CD4⁺ T cells. It is possible to speculate that ESAT-6 can be cross-presented [40] by APC after phagocytosis of apoptotic cells, since recent data indicate the capacity of ESAT-6 to induce apoptosis [41]. Moreover, ESAT-6 and other RD1 associated proteins could possibly be directly involved in the endoplasmic reticulum migration from the cytoplasm in a lysteriolysin-like mechanism, as suggested by recent observations [42, 43]. Beyond the possible added value for TB diagnosis, the observation of cytokine secretion by CD8⁺ T cells corroborates the hypothesis of their contribution to the anti-M. tuberculosis immune response [44, 45] and encourages further studies aimed at correlating CD8⁺ T cell activation with different clinical stages or prognostic indexes of TB [46].

We have developed a simple and reliable flow cytometric method to study the antigen specific T cell response in TB. Our assay presents several advantages as compared to other previously described methods based on cytokine release. Moreover, its excellent performance would make it suitable for diagnostic purposes even in field studies [47]. In fact, our preliminary data indicate that detection of IL-2 in IFN-γ positive subjects, after stimulation with M. tuberculosis antigens, may discriminate individuals with latent infection from patients affected by active TB. Lastly, since this method allows the contemporaneous evaluation of at least two cytokines it could represent a reference method to track IL-2-secreting T cells and to monitor the efficacy of anti-TB therapy.