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Keywords:

  • CD4+ T cells;
  • Cytokines;
  • Mycobacterium tuberculosis infection;
  • Tuberculosis disease

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Th1 CD4+ T cells and their derived cytokines are crucial for protection against Mycobacterium tuberculosis. Using multiparametric flow cytometry, we have evaluated the distribution of seven distinct functional states (IFN-γ/IL-2/TNF-α triple expressors, IFN-γ/IL-2, IFN-γ/TNF-α or TNF-α/IL-2 double expressors or IFN-γ, IL-2 or TNF-α single expressors) of CD4+ T cells in individuals with latent M. tuberculosis infection (LTBI) and active tuberculosis (TB). We found that triple expressors, while detectable in 85–90%TB patients, were only present in 10–15% of LTBI subjects. On the contrary, LTBI subjects had significantly higher (12- to 15-fold) proportions of IL-2/IFN-γ double and IFN-γ single expressors as compared with the other CD4+ T-cell subsets. Proportions of the other double or single CD4+ T-cell expressors did not differ between TB and LTBI subjects. These distinct IFN-γ, IL-2 and TNF-α profiles of M. tuberculosis-specific CD4+ T cells seem to be associated with live bacterial loads, as indicated by the decrease in frequency of multifunctional T cells in TB-infected patients after completion of anti-mycobacterial therapy. Our results suggest that phenotypic and functional signatures of CD4+ T cells may serve as immunological correlates of protection and curative host responses, and be a useful tool to monitor the efficacy of anti-mycobacterial therapy.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Infections with Mycobacterium tuberculosis (M. tuberculosis) cause a global epidemic with almost 9 million new cases and over 1.6 million deaths per year 1, 2. Outcome of M. tuberculosis infection depends on early identification and proper treatment of individuals with active tuberculosis (TB), but the lack of accurate diagnostic techniques has contributed to the re-emergence of TB as a global health threat. More than 2 billion individuals are estimated to be latently infected with M. tuberculosis (LTBI). To date, however, there is no simple, rapid, sensitive and specific test that can differentiate patients with active TB from individuals with LTBI.

Th1-type CD4+ T cells and type-1 cytokines are crucial for protection against M. tuberculosis3, 4 and therefore the frequency of IFN-γ-producing cells has been widely used as a correlate of protection against M. tuberculosis. However, recent data from mice and cattle show that measurement of spleen or blood IFN-γ-producing CD4+ T cells does not correlate with protection 5–7 and that IFN-γ is necessary but not sufficient for protection against M. tuberculosis. Also in humans, although IFN-γ is necessary for protection against mycobacterial pathogens, it is not a correlate of protection by itself 8, 9. Thus, although CD4+ Th1 cells and IFN-γ are important components of the protective human response against M. tuberculosis, other essential immune mechanisms must contribute to protection.

A series of studies have recently investigated immune correlates of protective T-cell responses in various models of human viral infections 10. These studies have shown that IFN-γ and IL-2 production, and the proliferative capacities of CD4+ and CD8+ T cells are key functions that define different aspects of the protective response. In particular, immune responses associated with effective virus control or clearance were predominantly composed of multifunctional/polyfunctional CD4+ and CD8+ T cells. Multifunctional CD4+ T cells secreting IFN-γ, TNF-α and IL-2 have been proposed as a major component of such responses, and subsequently were also shown to correlate with protection in Leishmania major infection in mice 11. In the same study, a high frequency of purified protein derivative (PPD)-specific similarly multifunctional T cells was found in BCG vaccinated mice and humans. Although no direct evidence was provided that these cells actually conferred protection against M. tuberculosis in that study 11, the presence of large numbers of multifunctional T cells in the lungs of mice boosted with a recombinant adenovirus expressing M. tuberculosis Ag85A correlated with a reduction in mycobacterial burden in M. tuberculosis aerosol-challenged animals 12, 13. Multifunctional M. tuberculosis-specific CD4+ T cells have been detected in peripheral blood of children with active TB disease and children with LTBI 14, and are maintained in HIV-1-positive individuals in the absence of active disease 15, although their functional capacity is affected by HIV-1 disease status both in peripheral blood 15 and in the lungs 16. Moreover, it has been suggested that combined analyses of different cytokines coexpressed by multifunctional T cells can improve discrimination between TB patients and subjects with LTBI 17, 18. Therefore, quality rather than quantity of M. tuberculosis-specific T-cell responses has been assumed to indicate protection and the capacity to generate long-term memory.

In this study, we have analyzed multifunctional CD4+ T cells, expressing three cytokines simultaneously (IFN-γ, TNF-α and IL-2), in response to three M. tuberculosis antigens (ESAT-6, Ag85B and 16 kDa) in adults with active TB disease, and compared these with responses in LTBI subjects. Surprisingly, and in contrast to what has been assumed to be the hallmark of a protective CD4+ T-cell response, we found a significantly higher proportion of multifunctional CD4+ T cells simultaneously producing IFN-γ, IL-2 and TNF-α, in subjects with active TB disease, compared with LTBI subjects, while in the latter, IFN-γ-only secreting and IFN-γ/IL-2 dual secreting CD4+ T cells dominated the anti-mycobacterial response. Moreover, these distinct IFN-γ, IL-2 and TNF-α profiles of M. tuberculosis-specific CD4+ T cells may be associated with bacterial loads, as suggested by their decreased frequency in TB patients after completion of anti-TB chemotherapy.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

TB patients have a more diverse cytokine profile than LTBI subjects

It has been suggested that T cells simultaneously producing IFN-γ, IL-2 and TNF-α are associated with protective immunity and concomitant beneficial outcome, at least in chronic viral infectious diseases such as HIV 11, 19, 20. We therefore compared expression of IFN-γ, IL-2 and TNF-α in CD4+ T cells from patients with active TB and LTBI subjects, after short-term in vitro restimulation with the prominently recognized M. tuberculosis antigens ESAT-6, Ag85B and 16 kDa. Responding CD4+ T cells were classified as triple (3+), double (2+) or single (1+) cytokine-producing populations according to the expression profile of IFN-γ, IL-2 and TNF-α (Fig. 1).

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Figure 1. Cytokine profiles of M. tuberculosis-specific CD4+ T cells in one LTBI subject and in one TB-infected patient at the beginning of therapy. Flow cytometric detection of CD4+ T-cell cytokine expression in PBMC (106/mL) incubated with M. tuberculosis Ag85B protein antigen (10 μg/mL, final concentration) from a representative LTBI subject and a TB-infected patient before therapy (TB-0). The IFN-γ cutoff gate was determined using unstimulated T cells from PBMC incubated with medium only. IL-2/TNF-α subset gating was based on patterns of cytokine expression in SEB-stimulated PBMC. The data are representative of five different experiments with 18 LTBI subjects and 20 TB-0 patients (Italian cohort).

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Analysis of 15 normal, uninfected PPD-negative healthy donors revealed no detectable cytokine expressing CD4+ T cells after stimulation with the M. tuberculosis proteins, ESAT-6, Ag85B and 16 kDa (Table 1), thus confirming specificity of intracellular cytokine staining. Following stimulation with Staphylococcal enterotoxin fragment B (SEB), the proportion of 3+ CD4+ T cells, which produced IFN-γ, IL-2 and TNF-α simultaneously, was very low and did not differ statistically between TB patients and subjects with LTBI (data not shown). Similarly, there was no statistically significant difference in the proportions of 2+ CD4+ T cells (IFN-γ+IL-2+, IFN-γ+TNF-α+ and/or IL-2+TNF-α+) between TB patients and LTBI subjects, but the latter had a significantly lower proportion of 1+ TNF-α+ CD4+ T cells (data not shown).

Table 1. M. tuberculosis antigen Ag85B-, ESAT-6- and 16-kDa-specific, triple IFN-γ+, IL-2+ and TNF-α+ CD4+T cells in patients at different stages of M. tuberculosis infection/disease and in controls
GroupPositive over total (%)Triple CD4+ responses compared to
  Cured TBLTBIControl
  1. a

    *p values were calculated using the chi square test.

Ag85B responses
Active TB (TB-0)17/20 (85%)*p<0.001p<0.001p<0.001
Cured TB (TB-6)3/20 (15%)NsNs
LTBI3/18 (17%)Ns
Control0/15 (0%)
ESAT-6
Active TB (TB-0)18/20 (85%)p<0.001p<0.001p<0.001
Cured TB (TB-6)3/20 (15%)NsNs
LTBI3/18 (17%)Ns
Control0/15 (0%)
16-kDa
Active TB (TB-0)17/20 (85%)p<0.001p<0.001p<0.001
Cured TB (TB-6)2/20 (10%)NsNs
LTBI2/18 (11%)Ns
Control0/15 (0%)

There were a number of differences between TB patients and subjects with LTBI following stimulation with ESAT-6, Ag85B and the 16-kDa antigen (Fig. 2). Most notably, and in contrast with the previously reported results in chronic viral infections, we found a significantly higher proportion of 3+ CD4+ T cells simultaneously secreting IFN-γ, IL-2 and TNF-α in patients with TB, as compared with LTBI subjects, upon stimulation with any of the three tested M. tuberculosis antigens (Fig. 2). Using a threshold of 0.01% to avoid systematic biases incurred by zeroing negative values (frequency values <0.01% were set to zero), we found that 3+ CD4+ T cells were detectable in very few LTBI subjects (3/18, 3/18 and 2/18 in response to Ag85B, ESAT-6 and 16 kDa, respectively), but were frequently detected in most TB patients (17/20, 18/20 and 17/20, in response to Ag85B, ESAT-6 and 16 kDa, respectively; see also Table 1 for comparison).

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Figure 2. Polyfunctional cytokine production analysis of M. tuberculosis-specific CD4+ T cells. PBMC (106/mL) were stimulated overnight in vitro with Ag85B, ESAT-6 or 16-kDa antigens (10 μg/mL, final concentration) and were stained with mAb to CD4+, IFN-γ, IL-2 and TNF-α, or with isotype-control mAb. Summary cumulative data of the IFN-γ, IL-2 and TNF-α capability of antigen-specific CD4+ T cells in LTBI subjects (n=18, white bars) and TB-infected patients with active disease before therapy (n=20, TB-0, grey bars) are shown (Italian cohort). The data are expressed as the percentage of CD4+ T cells that secrete each possible combination of the three tested cytokines and are representative of at least three independent experiments performed in duplicate. The boxes represent interquartile ranges; the line in the middle of the box represents the median, and minimum/maximum values are shown. *p<0.01 and **p<0.001, as calculated by Mann–Whitney U-test.

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In contrast, LTBI subjects had significantly higher (12- to 15-fold) proportions of 2+ CD4+ T cells that produced IL-2 and IFN-γ (IFN-γ+IL-2+) in response to Ag85B, ESAT-6 and 16 kDa, compared with TB patients (Fig. 2). Moreover, LTBI subjects also had higher proportions of 1+ CD4+ T cells that produced IFN-γ only (IFN-γ+), compared with TB patients, although this difference attained statistical significance only in response to Ag85B. Proportions of any other 2+ or 1+ cytokine secreting CD4+ T-cell subsets did not differ between TB patients and subjects with LTBI after short-term antigen stimulation (Fig. 2). This suggests that the type of response is not determined by the type of antigen, but is rather homogenous against the whole pathogen.

It has been previously reported that LTBI individuals with a negative short-term (24 h) IFN-γ release test (IGRA) may turn to a positive response after long-term (6 days) stimulation 21. The highly distinct proportions of multifunctional CD4+ T cells in TB patients and their absence in LTBI subjects prompted us to examine further differences that might exist between them. Therefore, a set of long-term stimulation assays was undertaken, of human PBMC stimulated for 6 days in vitro with combined ESAT-6/CFP-10 peptide pool, and cytokine production was analysed at day 6. These long-term stimulation assays confirmed the presence of a significantly higher proportion of 3+ CD4+ T cells simultaneously secreting IFN-γ, IL-2 and TNF-α in Dutch and Italian TB patients, as compared with LTBI subjects (Fig. 3). Briefly, 3+ cells were detected (at least two times medium values) in 3/3 TB patients, in 1/8 LTBI subjects and in none of the tested healthy controls. Additionally, and contrasting to the short-term assay, the percentage of 2+ CD4+ cells producing IFN-γ and IL-2 was significantly increased in TB-infected patients versus LTBI subjects (Fig. 3).

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Figure 3. Polyfunctional cytokine producing cells in LTBI subjects and cured TB-infected patients after prolonged in vitro stimulation. PBMC (106/mL) from LTBI subjects (n=8, white bars) and cured TB patients (n=4, grey bars) (Duch cohort) were stimulated in vitro for 6 days with ESAT-6/CFP-10 peptides (in pools containing 1 μg/mL per peptide) and stained with mAb to CD3, CD4+, CD8+, IFN-γ, TNF-α and IL-2. Summary cumulative data of IFN-γ, IL-2 and TNF-α capability by CD4+ T cells. Cells were gated on CD3+ lymphocytes, followed by gating on CD4+CD8+ cells and then analysed for cytokine production using Boolean gating. Bars represent mean+SEM triplicates of one experiment and are representative of five independent experiments. *p<0.005, as calculated by the Mann–Whitney U-test.

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Therefore, irrespective of the tested population (Italian versus Dutch), the duration of the assay (short term versus long term) and the nature of the antigen used for in vitro stimulation (protein versus peptides), M. tuberculosis antigen-specific 3+ CD4+ T cells simultaneously producing IFN-γ, IL-2 and TNF-α can only be detected in patients with (a history of) TB disease.

Comparison of functional CD4+ T-cell subsets before and after anti-mycobacterial therapy

We next studied the relative proportions and frequencies of cytokine-secreting CD4+ T cells in relation to the curative response to treatment, in samples from 20 patients with active TB before the initiation of therapy (TB-0) compared with blood samples from the same patients taken 6 months later, i.e. at the end of therapy (TB-6).

As shown in Fig. 4, the frequencies of Ag85B-, ESAT-6- and 16-kDa antigen-specific 3+ CD4+ T cells, which simultaneously produced IFN-γ, IL-2 and TNF-α, were significantly decreased further after 6 months of treatment, compared with untreated patients with active TB (Fig. 4). In contrast, the relative proportion of antigen-specific 2+ CD4+ T cells, secreting IL-2 and IFN-γ and that of 1+ CD4+ T cells secreting IFN-γ only, was both significantly higher after treatment compared with pretreatment. The relative proportions and frequencies of other 2+ and 1+ cytokine secreting, antigen-specific CD4+ T cells did not change significantly between untreated TB patients and after therapy (data not shown).

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Figure 4. Polyfunctional cytokine production analysis of M. tuberculosis-specific CD4+ T cells in TB-infected patients before and after therapy. PBMC (106/mL) from 20 TB-infected patients before (TB-0) and 6 months after therapy (TB-6) (Italian cohort) were stimulated overnight in vitro with Ag85B, ESAT-6 or 16-kDa antigens (10 μg/mL, final concentration) and were stained with mAb to CD4+, IFN-γ, IL-2 and TNF-α, or with isotype-control mAb. Summary cumulative data of the frequency of 3+(IFN-γ, IL-2 and TNF-α), 2+(IFN-γ and IL-2) and 1+(IFN-γ), antigen-specific CD4+ T cells in 20 TB-infected patients with active disease before therapy (TB-0, grey bars) and 6 months after therapy (TB-6, white bars). The boxes represent interquartile ranges; the line in the middle of the box represents the median, and minimum/maximum values are shown. Data are from representative of three independent experiments performed in triplicate. p-Values were calculated by Mann–Whitney U-test.

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It is worth noting that the distribution of 3+, 2+ and 1+ CD4+ T cells secreting IFN-γ, IL-2 and TNF-α in response to all three tested M. tuberculosis antigens, Ag85B, ESAT-6 and the 16-kDa antigen, was comparable and did not differ between TB-infected patients after treatment and LTBI subjects (compared with Fig. 2). However, 3+ CD4+ T cells were detectable in TB-infected patients after therapy, but not LTBI subjects, upon long-term stimulation in vitro (Fig. 3).

Figure 5 shows the relative proportions of M. tuberculosis-specific +3, +2 and +1 CD4+ T-cell subsets in untreated patients with active TB (TB-0), cured TB patients 6 months after treatment (TB-6), and LTBI subjects, in the short- and in the long-term assays: each portion of a pie chart indicates the percentages of specific CD4+ T cells that responded with one, two or three functions. Finally, we analysed the observed frequencies of cytokine-producing CD4+ T cells by scoring the results as negative (responses <0.01%) versus positive and compared the 3+ CD4+ T cells statistically in the different groups of individuals. As summarized in Table 1, the highest proportion of positive responses was found among patients with active TB, followed by those patients with cured TB (at the end of anti-mycobacterial treatment). Lower proportions of 3+ CD4+ T cells positive responses were found in individuals with LTBI, whereas all of the controls were negative (data not shown). Pair-wise comparisons of the positivity rates for 3+ CD4+ T cells in the four groups of individuals are summarized in Table 1: the proportion of positive responses among active TB-infected patients was significantly higher than that recorded among patients with cured TB, individuals with LTBI and control subjects.

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Figure 5. Functional composition of M. tuberculosis-specific CD4+ T-cell responses stimulated for (A) short (16 h) and (B) long (6 days) time periods with proteins and peptides, respectively, in LTBI subjects, in TB-infected patients before (TB-0) and after 6 months of therapy (TB-6) (Italian cohort). Responses are grouped and colour-coded according to the number of functions. The pie charts summarize the fractions of single (1+, green), double (2+, blue), and triple (3+, red) producers of IFN-γ, IL-2 and TNF-α.

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Taken together, these data suggest that 3+ CD4+ T cells simultaneously secreting IFN-γ, IL-2 and TNF-α to three antigens of M. tuberculosis, Ag85B, ESAT-6 and the 16-kDa antigen, are more frequently found in patients with current or historic TB disease compared with LTBI which are able to control M. tuberculosis replication.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

This study provides a detailed analysis of the frequency and quality of cytokine-producing CD4+ T cells in patients with active TB disease, cured TB and in subjects with LTBI. Importantly, we show here that the frequency of CD4+ T lymphocytes that produce multiple cytokines (IFN-γ, IL-2 and TNF-α) is significantly higher in subjects with active TB disease, not supporting current beliefs that such responses may be associated with protection. In contrast, CD4+ T cells that produced IL-2 and IFN-γ, or IFN-γ alone, were lower in active TB-infected patients compared with cured TB patients or individuals who controlled infection naturally (LTBI). Lending further support to our results is the observation that this pattern of distribution of cytokine-producing CD4+ T cells was consistently observed in response to three different M. tuberculosis antigens, Ag85B, ESAT-6 and 16 kDa antigen.

Data from HIV and other chronic viral infections have associated CD4+ and/or CD8+ T cells that simultaneously produce the three cytokines IFN-γ, IL-2 and TNF-α, with non-disease progression and efficient control of infection 20, 22, 23. Such “multifunctional” cell profiles have subsequently also been used to define correlates of vaccine-mediated protection against Leishmania11 and M. tuberculosis12, 24, 25 in mouse models of vaccination. Moreover, a similar multifunctional profile of antigen-specific T cells has been reported in adults 11 and in 10-wk-old infants routinely vaccinated with BCG at birth 8, as well as after prime-boost vaccination with BCG and modified vaccinia virus Ankara-expressing Ag85A 12, although in none of these studies direct evidence could be presented that these 3+ CD4+ T-cell responses are directly relevant to protection against M. tuberculosis, nor they were evaluated in patients with active or cured TB. Our starting hypothesis was to find increased proportions of multifunctional T cells in LTBI subjects, since they are, to a certain level, protected against disease development, and a decreased frequency in those that developed disease. However, our data show the opposite pattern, namely, an increased frequency of multifunctional T cells in patients with current or historic-active TB disease and almost undetectable levels in LTBI subjects. In line with our observations, a very recent study by Ota and colleagues in Gambia 26 also showed that TB cases had significantly higher levels of 3+ CD4+ T cells secreting simultaneously IFN-γ, IL-2 and TNF-α, compared with exposed household contacts. Collectively, the results from two different ethnic populations are in agreement, and together suggest that this particular 3+ “multifunctional” CD4+ T-cell population may be the hallmark of active TB disease.

Furthermore, and not shown previously, our results suggest that the bacterial load is related to the functional patterns of the CD4+ T-cell response as shown in Fig. 4, the frequencies of Ag85B-, ESAT-6- and 16-kDa antigen-specific 3+ CD4+ T cells, which simultaneously produce IFN-γ, IL-2 and TNF-α, were significantly increased during active disease, but decreased after 6 months of curative TB treatment to undetectable levels. In contrast, the relative proportion of antigen-specific 2+ CD4+ T cells, secreting IL-2 and IFN-γ and that of 1+ CD4+ T cells secreting IFN-γ only were significantly higher after treatment compared with pretreatment, mimicking the pattern observed in LTBI subjects. Our data are in agreement with those of Millington et al. 18 who showed that functional CD4+ T-cell heterogeneity is associated with changes in M. tuberculosis bacterial load induced by therapy. However, to our knowledge, our study provides the first evidence for pre/postchemotherapy changes of “multifunctional” CD4+ T cells, simultaneously secreting three different cytokines, IFN-γ, IL-2 and TNF-α.

Although multifunctional 3+ CD4+ T cells were undetectable in LTBI individuals, in a short-term in vitro stimulation assay, they could be detected, although at a very low frequency after long-term in vitro stimulation. Moreover, using the long-term stimulation assay, we were also able to detect significant proportion of 3+ cells in cured TB patients. It has been hypothesized that in the short-term assay only the recently primed CD4+ T cells, the product of residual antigen would be detected, but a major reservoir of tuberculosis-specific CD4+ T cells that returned to the resting state 27, 28 would be missed. Consequently, in individuals who have been infected with M. tuberculosis in the past, multifunctional CD4+ T cells may persist but in a resting state, and hence causing negative results in a short-term incubation assay, but positive responses after a prolonged incubation. In accordance with this line of thought are findings from a recent study of hepatitis C virus showing that short-term cytokine responses were not influenced by depletion of CCR7+ T cells (most likely representing central memory cells), whereas the depletion of CCR7+ T cells decreased cytokine response after prolonged culture 29.

From these results, we speculate that the functional signatures of CD4+ T-cell subsets during anti-mycobacterial response could be detected using different times of in vitro stimulation (short versus long term) irrespective of the use of mycobacterial peptides versus proteins, because of the presence of different subsets of CD4+ T cells that need more time to rescue from the resting state 30.

According to the scheme proposed by Seder et al.31, CD4+ T-cell differentiation can be modelled as a linear process, in which cells progressively gain functionality with further differentiation, until they reach the stage that is optimized for their effector function. Continued antigenic stimulation can lead to the generation of central memory multifunctional cells (which produce simultaneously IFN-γ, IL-2 and TNF-α) and then to the progressive loss of memory potential as well as cytokine production (effector memory 2+ cells producing IL-2 and IFN-γ), resulting in terminally differentiated CD4+ T cells that only produce IFN-γ and are short lived. According to Seder, the amount of initial antigen exposure will govern the extent of differentiation, with high-antigen stimulation leading to completion of this proposed differentiation pathway. How do our results fit with this differentiation pathway? The finding that multifunctional 3+ cells are detected in patients with active disease, but not in LTBI subject or cured TB patients, almost suggests that the LTBI cases or patients with cured TB disease, have passed the stage of multifunctional 3+ T cells already and are now effector memory cells. This implies that it is rather the presence of 2+ effector memory cells which is associated with the lack of TB disease or successful control of M. tuberculosis infection by the immune system.

Alternatively, or in addition to, it has been proposed 28 that multifunctional CD4+ T cells represent a population of antigen-primed T cells which return to a resting state by default in the absence of antigen contact. This possibility should explain why we failed to detect multifunctional T cells in LTBI subjects and cured TB patients in the short-term stimulation assay which measure only the recently primed CD4+ T cells, but no T cells that returned to a resting state 27, 28.

Finally, multifunctional activity of CD4+ T cells in TB patients may be suppressed by simultaneous presence of Treg cells or by monocytes/macrophages/DC products as TGF-β or IL-10. Sustained secretion of IL-10 and TGF-β has been reported to result in a long-lasting hyporesponsive (anergic state) to specific antigens 32, 33. Treg cells have been implicated in infectious diseases, particularly in chronic or persistent infections 34, 35, but discordant results were found ex vivo in terms of Treg expansion during active TB disease, with some authors reporting an increase of CD4+ CD25+FoxP3+ T cells, and other reported the absence of modulation of this T-cell subset 36–40. Moreover, a recent study found that depletion of CD4+ CD25highCD39+ increased M. tuberculosis-specific responses, as well as other recall antigens responses, indicating that Treg broadly modulate antigen-specific immunity 41.

In conclusion, this study shows that active TB disease is associated with an increase in the proportion of 3+ “multifunctional” CD4+ T lymphocytes capable of simultaneously producing IFN-γ, IL-2 and TNF-α, but a relative paucity of CD4+ T cells that produce either both IFN-γ and IL-2, or IFN-γ alone, when compared with the pattern of cytokine produced by CD4+ T cells from LTBI subjects. Strikingly, this pattern of cytokine production seems to be associated with bacterial loads and disease activity as it reverses 6 months after therapy.

These different functional signatures of CD4+ T cells could be used as immunological markers of mycobacterial load to monitor the response to treatment, to evaluate new therapies for active tuberculosis and the efficacy of new vaccines in clinical trials where new biomarkers are needed. Moreover, phenotypic and functional signatures of CD4+ T cells could also be used to monitor individuals LTBI at a high risk of progression to active TB, such as those with HIV coinfection or on anti-TNF therapy.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Human subjects

Peripheral blood was obtained from 20 adults with TB disease (11 men, 9 women, age range 46–55 years) from the Dipartimento di Medicina Clinica e delle Patologie Emergenti, University Hospital, Palermo, and Monaldi Hospital, Naples, Italy, 18 LTBI subjects (10 men, 8 women, age range 38–52 years) and 15 tuberculin (PPD)-negative healthy subjects (8 men and 7 women, age range 41–55 years).

TB-infected patients had clinical and radiological findings consistent with active pulmonary TB 42. Diagnosis was confirmed by bacteriological isolation of M. tuberculosis in 18 patients. Two further patients were classified as having highly probable pulmonary TB on the basis of clinical and radiological features that were highly suggestive of TB and unlikely to be caused by any other disease; the decision was made by the attending physician to initiate anti-TB chemotherapy, which resulted in an appropriate response to therapy. All patients were treated in accordance with Italian guidelines and received therapy for 6 months. Treatment was successful in all participants all of whom completed the full course of anti-TB chemotherapy, as evidenced by the absence of any clinical or radiographic evidence of recurrent disease and sterile mycobacterial cultures. Peripheral blood was collected before (TB-0) and after completion of chemotherapy (TB-6). None of the TB patients had been vaccinated with BCG, or had evidence of HIV infection, or was being treated with steroid or other immunosuppressive or anti-tubercular drugs at the time of their first sampling. Tuberculin (PPD) skin tests were considered positive when the induration diameter was larger than 10 mm at 72 h since injection of 5 U of PPD (Statens Seruminstitut, Copenhagen, Denmark). The study was approved by the Ethical Committee of the Dipartimento di Medicina Clinica e delle Patologie Emergenti, University Hospital, Palermo, and Monaldi Hospital, Naples, Italy, where the patients were recruited. Informed consent was written by all participants.

For the identification of LTBI subjects, in the absence of a gold standard, the most widely used diagnostic test remains the tuberculin skin test, based on the delayed-type hypersensitivity reaction that develops in M. tuberculosis-infected individuals upon intradermal injection of PPD. Individuals with LTBI were defined as healthy people with a positive tuberculin skin test and no symptoms and signs of active TB. However, because the PPD skin test suffers from many limitations 43, the QuantiFERON-TB Gold test (Cellestis, Victoria, Australia) was also performed and showed that among PPD+ LTBI subjects the response to QuantiFERON-TB Gold test was found in 74% (18/24), whereas this test was negative in all PPD skin test-negative healthy donors 44, 45; therefore, only those subjects positive to GFT-G were considered as being latently infected and were included in the study. All of the LTBI subjects were health care workers, and thus very likely to be close contacts of TB index cases. Moreover, none of the LTBI subjects included in this study had been vaccinated with BCG.

Additional patients and controls were recruited at the Department of Infectious Diseases at the Leiden University Medical Center, Leiden, The Netherlands, including four cured TB patients (2 men, 2 women, age range 42–77 years); eight LTBI subjects (5 men, 3 women, age range 26–56 years) and four healthy subjects (PPD negative) (1 man, 3 women, age range 25–39 years). TB-infected patients were successfully treated and completed their therapy more than 2 years prior to study participation. LTBI subjects were recruited from a previous study 46. All subjects were HIV negative; none of them received BCG vaccination. All individuals volunteered to participate in the study and signed informed consent, as approved by the local ethics committee.

Intracellular cytokine staining

Short-term in vitro stimulation

Recombinant M. tuberculosis proteins, ESAT-6, Ag85B and 16 kDa, were expressed in Escherichia coli and purified as described previously 21, PBMC (106/mL) were stimulated with M. tuberculosis protein antigens at a final concentration of 10 μg/mL or SEB (Sigma, St. Louis, MO, USA, 5 μg/mL final concentration), for 16 h at 37°C in 5% CO2. Unstimulated PBMC were used to assess nonspecific/background cytokine production. Monensin (Sigma, 10 μg/mL final concentration) was added after 2 h. Following stimulation, PBMC were harvested, washed in PBS containing 1% FCS and 0.1% sodium azide, and then stained with the amine-reactive LIVE/DEAD fixable violet dead cell stain kit (Molecular Probes, Invitrogen) 47 and with allophycocyanin (APC)-conjugated anti-CD4+ mAb (BD Pharmingen, San Josè, CA, USA) in incubation buffer (PBS-1% FCS-0.1% Na azide) for 30 min at 4°C. Subsequently, PBMC were washed, permeabilized (Cytofix/Cytoperm Kit, BD Pharmingen) according to the manufacturer's instructions and stained for intracellular cytokines with anti-IFN-γ-PE, anti-IL-2-FITC and TNF-α-PECy7, or isotype-matched control mAb. All mAb were from BD Pharmingen. Cells were washed, fixed in 1% paraformaldehyde and at least 250 000 lymphocytes were acquired using a modified FACS Aria (BD Biosciences), following gating according to forward and side scatter plots. FACS plots were analysed using FlowJo software (version 6.1.1; Tree Star, Ashland, OR, USA). Nonviable cells were excluded using a dump channel versus CD4+. Percent frequencies of the different combinations of IFN-γ, IL-2 and TNF-α-positive cells following antigenic stimulation were calculated within the total population of CD4+ T cells and background values subtracted (as determined from the medium alone control). Nonspecific background was extremely low when more than one cytokine was examined. A cutoff of 0.01% was used as described previously 48; values below this were set to zero.

Long-term in vitro stimulation

PBMC were stimulated in IMDM (Invitrogen, Breda, The Netherlands) containing 10% pooled human serum and ESAT-6+CFP-10 peptides, tested in pools containing 1 μg/mL per peptide. Cells were cultured in a humidified incubator at 37°C with 5% CO2 for 6 days, the last 18 h in the presence of 5 μg/mL Brefeldin A (Sigma, Zwijndrecht, The Netherlands). Intracellular staining was performed using intrastain reagents (Dako cytomation, Heverlee, Belgium). Ab used were CD3APC-Cy7, CD4+-PE-Cy7, CD8+-Am Cyan, IFN-γ-Alexa 700, IL-2-PE and TNF-α-APC (all from BD Biosciences, Alphen aan den Rijn, The Netherlands). Data were acquired on a BD LSRII flow cytometer using FACSDiva software (BD Biosciences) and analysed using FlowJo software (Tree Star). Graphical representations were made using Pestle and Spice software, software provided free of charge by the National Institute of Allergy & Infectious Disease (Bethesda, MD, USA), written in collaboration with Dr. Mario Roederer, Senior Investigator of the ImmunoTechnology section of the Vaccine Research Center at the National Institute of Allergy and Infectious Diseases.

Statistical analysis

Median and interquartile range of data were calculated and Mann–Whitney U-test was used to compare medians. Chi-square testing was used for dichotomous (positive/negative) measures. Values of p<0.05 were considered significant. Data were analyzed using statistical software SYSTAT 11 (Systat Software) or Graph Pad Prism (4.02) (Graph Pad Software).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

The authors acknowledge Dr. Sandra Arend (Department of Infectious Diseases, Leiden University Medical Center, The Netherlands) for selection and recruitment of patients and Kees CLM Franken (Department of Infectious Diseases, Leiden University Medical Center, The Netherlands) for production of recombinant proteins. This work was supported by grants from the European Commission within the 6th Framework Programme, TB-VAC contract no. LSHP-CT-2003-503367 and the 7th Framework Programme, NEWTBVAC contract no. HEALTH-F3-2009-241745 (The text represents the authors' views and does not necessarily represent a position of the Commission who will not be liable for the use made of such information), the Bill and Melinda Gates Foundation, Grand Challenges in Global Health (GC6♯74, GC12♯82), the Italian Ministry for Instruction, University and Research (MIUR-PRIN to FD) and the University of Palermo (60% to F. D. and N. C.). Moreover, the authors gratefully acknowledge funding by The Netherlands Organization for Scientific Research (VENI grant 916.86.115), the Gisela Thier Foundation of the Leiden University Medical Center and University of Leiden and the Netherlands Leprosy Relief foundation (grants ILEP 702.02.68 and 702.02.70).

Conflict of interest: The authors declare no financial or commercial conflict of interest.

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  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

See accompanying article: http://dx.doi.org/10.1002/eji.201040731

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