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Keywords:

  • δ T cells;
  • Mycobacterium tuberculosis;
  • tuberculin anergy

SUMMARY

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Earlier data suggest that γ/δ T cells may play an important role in the immune response to Mycobacterium tuberculosis. The aim of this study was to determine the percentage of different γ/δ subsets in peripheral blood of active tuberculosis patients with a positive or negative tuberculin reaction. Thirty-eight patients infected with M. tuberculosis and 22 healthy controls were included in the study. Venous blood was taken before starting antimycobacterial treatment. Lymphocytes were reacted with monoclonal antibodies specific for different γ/δ V chains (Vδ1, Vδ2, Vγ9 and Vγ4). The results were analysed in the context of tuberculin reactivity and X-ray findings. Our results revealed a selective loss of Vγ9/Vδ2 T cells in the peripheral blood of tuberculin-negative patients with active tuberculosis compared to healthy controls, while the ratio of Vγ9/Vδ2 T cells in the peripheral blood of patients with a positive skin test did not differ from that of healthy controls. These findings demonstrate a relationship between the loss of the major M. tuberculosis-reactive subset of γδ T cells and the absence of tuberculin reactivity. The data are consistent with the hypothesis that γδ T cells play a role in the protective immune response to M. tuberculosis infection.


INTRODUCTION

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Mycobacterium tuberculosis is the aetiological agent of tuberculosis and is a major cause of morbidity and mortality worldwide. Studies in humans and animal models suggest that γδ T cells may play an important role in the immune response to M. tuberculosis. In certain experimental conditions, mice lacking γδ T cells suffer a more severe form of tuberculosis and fail to control the infection [1]. The recent availability of monoclonal antibodies directed against distinct Vγ and Vδ gene products has substantially facilitated the analysis of human γ/δ subset distribution. At present, there are monoclonal antibodies available that recognize specifically Vγ9, Vδ2, Vδ1 and Vγ4. The application of these reagents has identified two mutually exclusive subsets of γ/δ T cells in peripheral blood. The majority of these cells express a T cell receptor (TCR) composed of a Vγ9 segment in association with Vδ2 [2,3]. A second subset of γ/δ+ cells bears Vδ1 associated with a Vγ 4. However, due to the dominance of Vγ9/Vδ2 cells, the Vδ1 subset usually accounts for only 10–30% of the peripheral blood γδ T cells. The preferential expression of certain TCR Vγ and Vδ elements in different anatomical sites is usually considered to reflect the importance of a particular γδ T cell subset in the local immune surveillance [4].

Vγ9/Vδ2 T cells recognize non-peptidic molecules. Antigen recognition by γδ TCRs resembles recognition by antibodies [5]. Whereas αβ TCRs recognize peptide antigens bound to MHC, γδ T cells have been shown to recognize intact protein antigens and small, phosphate- or amine-containing compounds. Small-molecule antigens include pyrophosphomonoesters from M. tuberculosis and alkylamines from several natural sources [6]. While MHC-presentation of these non-peptide antigens is not required, it has been shown that cell–cell contact is required for stimulation, suggesting either that non-MHC molecules may present small antigens to γδ TCRs or that co-stimulation from neighbouring cells is required.

In normal healthy individuals γδ T cells contain the highest frequency of M. tuberculosis-reactive T cells in the peripheral blood and the predominant subset of M. tuberculosis-reactive T cells express a TCR encoded by Vγ9 and Vδ2 gene segments [7]. T cells expressing a γ/δ TCR account for 1–10% of CD3+ peripheral blood lymphocytes [8,9]. Mature αβ T cells recognize antigens processed by antigen-presenting cells in the context of MHC class I or class II molecules. In contrast to mature αβ T cells, the majority of γδ T cells do not express CD4 or CD8, which is in agreement with the lack of MHC restriction of most γδ T cells. The dominant subset of peripheral blood γδ T cells, i.e. Vγ9/Vδ2-expressing cells, reacts strongly in a non-MHC-restricted fashion with inactivated M. tuberculosis[10,11]. The effector function of Vγ9/Vδ2 cells includes cytokine secretion (tumour necrosis factor-α, interferon-γ, CC chemokines, etc.) and cytotoxic activity against pathogen-infected macrophages [12,13]. γδ T cells expressing Vδ1 in association with various Vγ elements are localized preferentially in the intestine. In the adult intestine, γδ T cells comprise 30–40% of intraepithelial lymphocytes, of which up to 70% are Vδ1 + [14,15]. There is evidence that these cells are involved in the immune defence against infection. Vδ1 T cells are increased in the peripheral blood of HIV-infected individuals [16]. More recently, Vδ1 T cells have been implicated in the human immune response to CMV [17]. Additional evidence indicates that Vδ1-expressing γδ T cells are also involved in the immune defence against certain bacteria. Vincent et al. isolated Vδ1-expressing γδ T cells from the synovial fluid of Lyme arthritis patients [18].

Although together the results of these studies suggest strongly that γδ T cells play a role in the immune response to M. tuberculosis, to date a limited number of studies have produced contradictory results. Whereas some studies have reported an increase in γδ T cells in the peripheral blood of tuberculosis patients [19,20], other studies have demonstrated that γδ T cell number remain constant in both the peripheral blood and granulomatous lymph node lesions of tuberculosis patients [21,22]. These conflicting results may be a consequence of analysing γδ T cells from patients at different stages of disease progression or at different Mantoux status. In addition, these studies did not address the possibility that the repertoire of γδ T cells, in particular the M. tuberculosis-reactive Vγ9/Vδ2 subset, changes with infection. Long-standing clinical observations have established that certain diseases that do not induce a generalized immunosuppressive state may induce impaired DTH reaction to specific antigens, a state defined clinically as ‘anergy’. Anergy in the setting of tuberculosis refers to the paradoxical absence of dermal reactivity to intradermal injection with tuberculin purified protein derivative in infected persons. This occurs in about 15–35% of patients with active pulmonary disease and is associated with absence of granuloma formation and all other manifestations of cellular hypersensitivity [23].

The aim of this study was to measure the percentage of different γ/δ subsets in the peripheral blood of patients with active tuberculosis who had a positive or negative tuberculin reaction.

METHODS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Study population

Thirty-eight patients (28 men, 10 women) of mean age 51·7 years (15–87) infected with M. tuberculosis were included in the study (Table 1). All of them had a positive sputum culture for M. tuberculosis, nine of whom were also sputum smear-positive. Tuberculin testing was performed according to the original Mantoux test − i.e. 5 TU PPD (Human Rt, Godollo, Hungary) were given intradermally in the forearm and the results were evaluated 72 h later. The skin test was considered positive if there was an induration of 10 mm or more and negative if there was no reaction. Positive reactions of more than 10 mm in diameter were seen in 27 cases (the size of the reaction ranged between 11 and 30 mm in diameter, median 20 mm) and 11 patients had no tuberculin skin reaction at all. Twenty-two volunteers (five men, 17 women) of mean age 33 years (20–53) acted as healthy controls. Each had been vaccinated with BCG as part of the required Hungarian national vaccination programme and all were tuberculin-positive. The members of the control group were selected on the basis of negative X-ray findings during the annual tuberculin screening programme. Therefore no smear and culture were taken from these individuals. Permission for the study was obtained from the Clinical Ethics Committee of the Pecs University, and all subjects gave their consent to participate. All the patients and healthy volunteers were HIV-negative. The incidence of HIV infection in Hungary is still very low. Negative tuberculin reaction occurred in 29% of our patients, which is in line with the data found in the literature [23].

Table 1.  Comparison of results for patients and healthy volunteers
 MaleFemaleAgeTuberculin skin testSputum
NegativePositiveSmear+Culture+
  • *

    Among the tuberculin-negative patients only 1 patient was older than 70 years.

Patients281051·7 (15–87)*1127938
Healthy volunteers 51733 (20–53) 22  

Patients were classified according to the extent and type of X-ray findings into three stages following the classification of Dlugovitzky et al. [24]: stage I, mild cases (n = 13), patients with a single lobe involved and without visible cavities; stage II, moderate cases (n = 15), patients presenting unilateral involvement of two or more lobes with cavities, if present, reaching a total diameter no greater than 4 cm; and stage III, advanced cases (n = 10), bilateral disease with massive involvement and multiple cavities (Table 2).

Table 2.  Classification of the radiologic manifestation of patients with pulmonary tuberculosis and tuberculin skin test reactivity
 Stage IStage IIStage III
Patients131510
Tuberculin skin test
 Negative 2 3 6
 Positive1112 4

Monoclonal antibodies

The following monoclonal antibodies were used: mouse antihuman TCR Vγ4 (clone 4A11, Serotec, Oxford, UK), mouse antihuman TCR Vδ2 (clone 15D1), Vγ9 (clone 7A51), Vδ1 (clone TS8·21) (T cell Diagnostic Inc., Woburn, MA, USA).

γ/δ T cell counts

Ten ml of venous blood was taken before starting antimycobacterial treatment. Lymphocytes were separated from heparinized venous blood on a Ficoll-Paque gradient. The cells were washed in medium RPMI-1640 (Gibco, Grand Island, NY, USA); the cell count was adjusted to 1 × 106/ml and 100 µl of this suspension was centrifuged and transferred to microscope slides. After air-drying the cells were fixed with acetone at 4°C for 10 min. Immunocytochemistry was carried out as described previously [25]. The primary antibodies for detection of different γ/δ subpopulations were Vδ1, Vδ2, Vγ9 and Vγ4 (also known as Vγ1.4) monoclonal antibodies. The reaction was developed by diaminobenzidine and intensified with silver staining. Nuclei were counterstained with haematoxylin and the slides were mounted with glycerol-gelatin.

The percentage of γ/δ T cells was determined by microscopic counting of 300 lymphocytes.

Statistics

Student's t-test was used for statistical evaluation of the data. Differences were considered significant if P-value was equal or less than 0·05.

RESULTS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Distribution of γδ T cell subsets in peripheral blood of patients with active tuberculosis in the context of tuberculin reactivity

Immunocytochemistry was used to detect the subsets of circulating γδ T cells in peripheral blood samples obtained from tuberculin-positive (n = 27) and -negative patients (n = 11) with active tuberculosis and from tuberculin-positive healthy control subjects (n = 22). Data were analysed in the context of tuberculin reactivity.

The percentage of Vγ4+ cells was significantly higher in patients with negative tuberculin reaction than in those with positive tuberculin reaction or in healthy volunteers. The percentage of Vδ1+ cells was significantly higher in patients with negative tuberculin reaction than in healthy volunteers. There was no significant difference in the ratio of Vδ1 T cells between tuberculin-negative and -positive patients. (Fig. 1).

image

Figure 1. Percentage of γ/δ subsets in peripheral blood of tuberculin-positive and tuberculin-negative patients and healthy individuals. The bars represent the mean ± s.e.m. of 22 (healthy volunteers), 27 (tuberculin-positive patients) and 11 (tuberculin-negative patients) determinations (*significantly different from healthy volunteers: *P < 0·05, **P < 0·02 and ***P < 0·01). ▪, Healthy volunteers; □, tuberculin-positive patients; bsl00039 , tuberculin-negative patients.

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The ratio of Vδ2+ and Vγ9+ cells was significantly lower in patients with negative tuberculin reaction than in healthy volunteers. There was no significant difference in the ratio of Vδ2+ and Vγ9+ cells between healthy controls and patients with positive tuberculin reaction (Fig. 1).

Based on the available haematology data of our patients, we calculated absolute counts of γ/δ subsets. Changes in absolute cell counts correlated with those in the ratio of the cells.

Modification of peripheral γδ T cell subsets in patients at different stages of the infection

To compare the proportion of γδ T cell subsets in the peripheral blood of control donors and M. tuberculosis-infected patients at different stages of the disease, immunocytochemistry was performed with different anti-V chain monoclonal antibodies. Patients were classified according to the extent and type of X-ray findings into three stages following the classification of Dlugovitzky et al. [24]

Compared to healthy controls we observed a significant increase in γδ T cells expressing the Vδ1 or Vγ4 chain as early as in radiological stage I of tuberculosis. We observed similar increase in radiological stages II and III, but these changes were not significant (Fig. 2).

image

Figure 2. The proportion of γ/δ T cell subsets (Vδ1+ and Vγ4+ T cells) in the peripheral blood of healthy volunteers and Mycobacterium tuberculosis-infected patients at different stages of the disease (*significantly different from healthy volunteers: *P < 0·05). ▪, Vδ1+ T cells; ◆, Vγ4+ T cells.

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In radiological stage I of the infection, we observed a slight decrease in γδ T cells expressing the Vδ2 or Vγ9 chain compared to healthy controls. These findings became more pronounced at stage III of the disease (Fig. 3).

image

Figure 3. The proportion of γ/δ T cell subsets (Vδ2+ and Vγ9+ T cells) in the peripheral blood of healthy volunteers and Mycobacterium tuberculosis infected patients at different stages of the disease (*significantly different from healthy volunteers: *P < 0·05). ▪, Vδ2+ T cells; ◆, Vγ9+ T cells.

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Following classification of patients into three stages according to the extent and type of X-ray findings, we found that among the 11 tuberculin anergic patients, six had an advanced pulmonary manifestation (stage III), three belonged to stage II and only two to stage I. Among 27 patients with positive tuberculin skin reaction four were classified as stage III, 12 as stage II and 11 as stage I (Table 2).

DISCUSSION

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

The generation of an effective immune response depends on the cross-talk between adaptive and innate pathways. Among natural effector cells, Vγ9/Vδ2 T lymphocytes are believed to play a major role in promoting the cell-mediated response against intracellular pathogens as M. tuberculosis[26]. We observed in this study that γδ T cell distribution is particularly affected in tuberculin-negative patients.

Li et al. [27] reported that γδ T cell subset distribution is altered in some M. tuberculosis-infected patients. Our results show that this is due to a decrease in Vδ2 and Vγ9 T cell number. The Vδ2 and Vγ9 T cell deletion was more evident in patients with negative tuberculin reaction. In contrast to these findings, a selective increase of the Vγ4 and Vδ1 T cells was found in the peripheral blood of anergic patients with active tuberculosis compared to healthy volunteers. Earlier data from our laboratory revealed a significantly higher rate of γ/δ-positive T cells in the peripheral blood of tuberculin anergic patients compared with that of tuberculin-positive patients or healthy individuals [28]. The present study revealed that within the increased γ/δ population the ratio of the Vδ1/Vγ4 subpopulation to the Vδ2/Vγ9 subpopulation is 4·5 times higher in peripheral blood of patients with negative tuberculin reaction than in patients with a positive tuberculin reaction explaining the selective loss of the Vγ9/Vδ2 T cells.

Although little is known about the function of the antigens recognized by Vδ1 and Vγ4 cells, their specific expansion has been observed in several different diseases. Accumulation of Vδ1 T cells was reported in the lungs of patients with pulmonary sarcoidosis and in leprotic lesions [29, 30]. There is no conclusive evidence suggesting that the expansion of Vδ1 T cells is due to the recognition of specific antigens. Vγ4 and Vδ1 T cell expansion in the peripheral blood of tuberculin-negative patients with active tuberculosis may be the consequence of cell migration from tissues to the bloodstream under the influence of cytokine as a result of chronic inflammation.

A protective response to intracellular pathogens requires potent activation of cell-mediated immunity, promoted by a Th1 cytokine profile. A major role as a Th1-inducing factor is played by IFN-γ, stimulating the activation of natural killer cells and recruiting cytotoxic T lymphocytes. Under normal conditions, Vγ9/Vδ2 T cells respond to antigen challenge by secreting large quantities of TNF-α and IFN-γ[31], which contributes to the activation of both specific and non-specific immune responses. Moreover, γδ T cells have been shown to be important IFN-γ producers during tuberculosis infection, and knockout mice lacking γδ T cells develop a more exacerbated disease [32].

The presence of Th2 response or of type 2 cytokines IL-4 and IL-10 is associated with progressive disease [33]. IL-4 blocks IL-2 and IFN-γ secretion by polyclonally stimulated human T cells and exerts a selective potentiating effect on the proliferation and cytokine synthesis of Th2 clones, while IL-10 might be involved in damaging ongoing antigen driven immune responses rather than in the selective regulation of the Th1 function. Thus IL-4 and IL-10 could be associated with diminished resistance to infection by mycobacteria. Earlier data from our laboratory showed that patients with tuberculin anergy had a significantly higher ratio of IL-4- and IL-10-positive lymphocytes in the peripheral blood than either those with a positive tuberculin skin test or healthy volunteers. On the other hand, patients with tuberculin anergy had a significantly lower ratio of IL-12 positive lymphocytes than those with a positive tuberculin skin test or healthy volunteers [34].

We suggest that the decreased ratio of Vδ2 and Vγ9 T cells, together with the increased ratio of IL-4- and IL-10-positive lymphocytes and the decreased ratio of IL-12-positive lymphocytes contributes to the tuberculin skin anergy and, in part, also for the progression of the disease. It should be emphasized that only one of our patients was over 70 years; thus we believe that the age of those studied had no effect on the observed tuberculin anergy.

As noted, patients with tuberculin anergy usually have a more advanced disease than those with a positive tuberculin reaction; therefore, it was important to evaluate whether the results simply correlate with the degree of pulmonary involvement of the disease or the tuberculin reaction has its own impact on the result independently. Following classification of patients into stages according to the extent and type of X-ray findings we found that among the 11 tuberculin anergic cases, six were in stage III (54%); among 27 tuberculin positive cases only four were classified as stage III (14·8%) (Table 2). In radiological stage III of the infection we observed a significant decrease in γδ T cells expressing the Vδ2 or Vγ9 chain compared to healthy controls. We also found that the ratio of Vδ2 and Vγ9 cells was significantly lower in patients with negative tuberculin reaction that in healthy volunteers.

Dieli et al. found that compartmentalization of CD4 T cells at the site of the disease could be one possible explanation for the negative skin test reaction to PPD in vivo[35]. Li et al. found no evidence of an accumulation of Vγ9/Vδ2 T cells in the lungs of patients with pulmonary tuberculosis [27]. One possible explanation for the reduced number of Vγ9/Vδ2 T cells in the peripheral blood of patients with active tuberculosis is that mycobacterial antigens induce the up-regulation of FasL on activated γδ T cells, resulting in activation-induced cell death [36]. Based on these findings, we suggest that the level of Vδ2 and Vγ9 T cells, together with the Th2-biased immune response, correlates with the degree of the tuberculin skin test and consequently with the pulmonary manifestation of the disease.

ACKNOWLEDGEMENTS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

This study was supported by OTKA T031737 and ETT T05-347, Hungary.

REFERENCES

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES
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