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An abnormal phenotype of lung Vγ9Vδ2 T cells impairs their responsiveness in tuberculosis patients
Cellular Immunology 282 (2013) 106–112
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An abnormal phenotype of lung Vc9Vd2 T cells impairs their responsiveness in tuberculosis patients Sary El Daker a,d,⇑,1, Alessandra Sacchi a, Carla Montesano d, Alfonso Maria Altieri b, Giovanni Galluccio c, Vittorio Colizzi d, Federico Martini a, Angelo Martino a a
Laboratory of Cellular Immunology, National Institute for Infectious Diseases ‘‘Lazzaro Spallanzani’’, Rome, Italy Unità di Broncopneumologia e Tisiologia, Azienda Ospedaliera San Camillo-Forlanini, Rome, Italy Unità di Endoscopia Toracica, Azienda Ospedaliera San Camillo-Forlanini, Rome, Italy d Department of Biology, University of Rome Tor Vergata, Rome, Italy b c
a r t i c l e
i n f o
Article history: Received 5 March 2013 Accepted 2 May 2013 Available online 15 May 2013 Keywords: Mycobacterium cd T cells Primary infection Lung
a b s t r a c t Antigen-specific cd T cells represent an early innate defense known to play an important role in antimycobacterial immunity. We have investigated the immune functions of Vc9Vd2 T cells from BronchoAlveolar lavages (BAC) samples of active TB patients. We observed that BAC Vc9Vd2 T cells presented a strong down-modulation of CD3 expression compared with Vc9Vd2 T cells from peripheral blood. Furthermore, Vc9Vd2 T cells mainly showed a central memory phenotype, expressed high levels of NK inhibitory receptors and TEMRA cells showed low expression of CD16 compared to circulating Vc9Vd2 T cells. Interestingly, the ability of BAC Vc9Vd2 T cells to respond to antigen stimulation was dramatically reduced, differently from blood counterpart. These observations indicate that cd T cell functions are specifically impaired in situ by active TB, suggesting that the alveolar ambient during tuberculosis may affect resident cd T cells in comparison to circulating cells. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is one of the most prevalent and common serious infectious diseases worldwide, affecting almost 10 million people annually [1]. The disease, fuelled by human immunodeficiency virus (HIV) infection and poverty, is out of control in developing countries, and the emergence of drug resistant strains threatens TB control in several other regions of the world [2]. The current available vaccine, Bacillus Calmette-Guerin (BCG), as well as existing therapeutic interventions for TB, are at present almost suboptimal. Thus, new vaccines and immunotherapeutic strategies are urgently required in order to improve TB control efforts [3]. A better understanding of the immunopathogenesis of TB could facilitate the identification of correlates of immune protection, the design of more effective vaccines, the rational selection of immunotherapeutic agents, and the evaluation of new drug or adjuvant candidates. Immune response to MTB may play a major role in determining clinical course: specific cell mediated response is critical in host defense against mycobacteria, other mechanisms, including innate
⇑ Corresponding author. Address: National Institute for Infectious Diseases ‘‘Lazzaro Spallanzani,’’ Via Portuense 292, 00149 Rome, Italy. Fax: +39 06 55170962. E-mail address: [email protected] (S. El Daker). 1 Present address: Singapore Immunology Network, A⁄Star, 8A Biomedical Groove, #03-01 Biopolis, Singapore 138648, Singapore. 0008-8749/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellimm.2013.05.001
immunity, may play a role too [4]. Vc9Vd2 T cells, the major subset of circulating cd T cell pool, are known to be involved in immunity against microbial pathogens, including MTB [5–7]. This subset may be activated by two different mechanisms: (a) directly by non peptidic small phosphorylated compounds produced by mammalian cells, such as isopentenyl-pyrosphosphate (IPP), or by non mammalian cells, such as 4-hydroxy-3-dimethylallyl pyrophosphate; (b) indirectly by a group of non peptidic compounds, the aminobisphosphonates, as Zoledronic acid (Zol), that have been shown to activate Vc9Vd2T cells through the accumulation of mevalonate pathway metabolites, as IPP, subsequently recognized by Vc9Vd2TCR [8]. Similarly to CD4 and CD8 ab T cells, Vc9Vd2T cells are heterogeneous, and show distinct populations defined by surface marker expression and effector functions. Naïve (CD45RA+CD27+) and central memory (CD45RA CD27+) Vc9Vd2T cells abound in lymph nodes and lack immediate effector functions. Conversely, effector memory (CD45RA CD27 ) and terminally differentiated (CD45RA+CD27 ) Vc9Vd2T cells are poorly represented in the lymph nodes whereas abound at sites of inflammation, and display immediate effector functions [9]. The differentiation pathway for the generation of these subsets is uncertain, but circulating effector Vc9Vd2 T cells have been found significantly reduced in several diseases as pulmonary tuberculosis [10]. Studies done in humans and animal models have demonstrated complex patterns of Vc9Vd2 T cell immune responses during early mycobacterial infections and chronic TB. In effect, responses of Vc9Vd2 T cells
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to MTB were described as early as in 1989 [11]. A number of studies have attempted to determine the in vivo role of Vc9Vd2 T cells in the human immune response to MTB [12–16]. Barnes et al. established that patients with pulmonary or miliary tuberculosis had a diminished ability to expand Vc9Vd2 T cells in vitro in response to heat-killed MTB and IL-2, although there was quite a wide range of Vc9Vd2 T-cell expansion among the different groups. Studies of T-cell phenotype in bronchoalveolar cells from healthy PPD+ subjects and from lungs of patients with pulmonary TB found a T lymphocytic alveolitis in the lungs. Vc9Vd2 T cells were found among the lymphocytes in this alveolitis, but their proportion was not increased relative to ab T cells [17]. Thus, Vc9Vd2 T cells are present in situ in pulmonary TB but are not expanded compared to Vc9Vd2 T cells in peripheral blood or unaffected lung. It is already known, in the context of a natural infection, the consistent expansion of Vc9Vd2 T cells with a TCM phenotype in the peripheral blood of patients with active TB, which was accompanied by the dramatic reduction of the pool of Vc9Vd2 cells with immediate effector functions (TEM and TEMRA cells). However, this skewed representation of circulating Vc9Vd2 T cell phenotypes during active TB was transient and completely reversed after successful antimycobacterial therapy [18]. The mechanism causing the loss of Vc9Vd2 T cell effector functions during TB is unknown. One possibility is that sustained in vivo mycobacterial stimulation of Vc9Vd2 T cells causes their apoptosis. However, the analysis of Vc9Vd2 T cell functions in TB patients and especially in the site of infection needs further investigations. Here, we report the phenotypical and functional analysis of Vc9Vd2 T cells in the blood and BAC of TB patients. We observed that, in contrast to the circulating Vc9Vd2 T cells, lung Vc9Vd2 T cells responsiveness to antigen stimulation was dramatically compromised. Indeed, lung Vc9Vd2 T cells were not able to produce cytokines (INF-c, TNF-a) under specific phosphoantigen stimulation. This was found to be correlated to a dramatic down/modulation of CD3 molecule on lung Vc9Vd2 T cells, that could explain their inability to respond to antigenic stimulation. 2. Materials and methods 2.1. Patient population Thirty tuberculosis patients were enrolled at the respiratory disease wards in the ‘‘Azienda ospedaliera San Camillo Forlanini’’ in Rome. All study participants had newly diagnosed active tuberculosis with a positive culture for MTB from sputum. Patients with active TB were studied within 7 days of admission and before starting anti-TB therapy. In addition, healthy donors were recruited as controls at the ‘‘National Institute for Infectious Diseases, Lazzaro Spallanzani Rome, Italy. The ethics committee of both Institutions approved the study and all enrolled individuals provided written informed consent. Other criteria of inclusion were: negative results to HIV test and not receiving therapy with immune-suppressive drugs. Characteristics of all subjects are reported in Table 1. Table 1 Baseline characteristic of healthy donor and patients with active tuberculosis(TB). Parameter
Healthy donor (PBMC)
TB patients (PBMC/ BAL)
Number Gender Age (years) Smoker BCG vaccination BCG treatment HIV Immunosuppressive drugs
15 7M/8F – – No – No No
30 19M/11F 34 (16–62) 16Y/14N Yes No No No
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2.2. Cell preparation and stimulation Peripheral blood mononuclear cells (PBMC) from TB patients and healthy controls were isolated from whole blood using Ficoll density gradient centrifugation. After washing in PBS 1, PBMC at a concentration of 1 106 cells/ml were cultured for 24 h at 37 °C and 5% CO2 in a complete medium (RPMI-1640, 10% heatinactivated human serum, 10 mM HEPES, 2 mM L-glutamine and 10 U/ml penicillin–streptomycin, all from Euroclone Ltd, Weatherby, UK). Cultures were prepared in 48-well tissue culture plates (Costar, Corning Inc., NY, USA) in 1 ml/well. Bronchoalveolar cells (BAC) from TB patients were processed in a safety BSL3 laboratory. After filtering with BD Falcon cell strainer 100 lm, BAC were washed in PBS 1 and were cultured for 24 h at 37 °C and 5% CO2 in a complete medium (like PBMC). After 24 h to the separation, PBMC and BAC were stimulated in vitro in the presence of 80 lM isopentenyl pyrophosphate (IPP; Sigma–Aldrich, St. Louis, MO). 2.3. mAbs and flow cytometry Anti-human monoclonal Abs used in phenotype studies were the following: anti-CD27 APC-H7 (clone M-T271); anti-CD45RA CyChrome (clone HI100); anti-CD57 PE (clone NK1); anti-CD16 pacific blue (clone 3G8); anti-CD3 APC (clone UCHT1); anti-CCR7 PeCy7(clone 3D12); anti-CD62L APC (clone Dreg56). The anti-human Abs used in cytotoxic studies were the following: anti-perforin PE (clone dG9); anti-CD107 PerCp (clone H4A3). The anti-human Abs used in cytokines analyses were the following: anti-INFc Pe-Cy7 (clone B27) and anti-TNFa APC (clone 6401.1111). All the previously described mAbs were from BD Biosciences (Mountain View, CA). The anti-Vd2 mAb FITC (IgG1, clone IMMU1464) was purchased by Immunotech (Marseille, France). Isotype-matched control mAbs from BD Biosciences were used in all experiments. PBMC and BAC were incubated for 15 min at 4 °C with the conjugated mAb. Samples were washed in PBS 1, fixed in paraformaldehyde (PFA 1% for PBMC and 4% for BAC), suspended in FACSFlow (BD-Pharmagen) and immediately acquired with a FACS CANTO II flow cytometer (BD Biosciences). A total of 100,000 events was acquired for each sample and analyzed with CellQuest software (BD Biosciences). 2.4. Single-cell analysis of cytokine synthesis Cytokine production was detected by flow cytometry analysis as previously described (18). Human PBMC were stimulated for 12 h with IPP (80 lM;Sigma–Aldrich). Brefeldin A (10 lg/ml) was added 1 h after stimulation to block intracellular transport allowing cytokine accumulation in the Golgi. Cells were washed twice in PBS 1, 1% BSA, and 0.1% sodium azide and stained with mAb specific for the membrane Ags described above for 15 min at 4 °C. Samples were then fixed in 4% paraformaldehyde for 10 min at 4 °C, incubated with anti-cytokine mAbs in 1 PBS, 1% BSA, and 0.5% saponin. Cells were finally washed twice in 1 PBS, 1% BSA, 0.1% saponin, and acquired on a FACS CANTO II (BD Biosciences). Control for nonspecific staining was monitored with isotype-matched mAbs and nonspecific staining was always subtracted from specific results. 2.5. Detection of CD107a on cd T cells Considering that the degranulation of antigen-responding T cells is associated with acquisition of cell surface CD107a, PBMC and BAC (1 106 cells/ml) were incubated with or without stimulation for 18 h, and anti-CD107 PerCp (BD-Pharmigen) was added to the culture 1 h after stimulation. Thereafter, cells were washed
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and 100.000 events were acquired on Vc9Vd2 TCR+ lymphocytes and results are expressed as the percentage of CD107a+ cells among Vc9Vd2 T cells. 2.6. FITC-Annexin V staining For vitality assay we used the ‘‘Annexin V-FITC Apoptosis detection kit’’ produced by Bender MedSystem (BMS500FI20CE). After washing twice with PBS 1, cells were resuspended in binding buffer 1 (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mMCaCl,) at final concentration of 2–5 105/ml. FITC-Annexin V was added to a final concentration of 1 pg/ml. The mixture was incubated for 10 min in the dark at room temperature and after washing was acquired by using a FACS CANTO II (BD Biosciences). 2.7. Statistical analysis Statistical analysis to assess differences between the different groups was performed using Student’s t-test. Differences were considered significant when the P value was lower than 0.05. GraphPad Prism version 4.00 for Windows, (GraphPad Software) was used to perform the analysis.
3. Results 3.1. Phenotypic characterization of Vc9Vd2 T cells in BAC and PBMC of TB patients Vc9Vd2 T cells represent one of the first defense against MTB, in particular in the first site of infection. Thus we characterized Vc9Vd2 T cells from BAC of patients with active tuberculosis. First, we have observed that while the percentage of blood Vc9Vd2 T cells of healthy donor and TB patients is comparable (3.8% ± 0.11 and 3.6 ± 0.24%) the percentage of BAC Vc9Vd2 T cells is lower (1,75 ± 0.36) than their counterpart in the blood (Fig. 1). In order to better characterize BAC Vc9Vd2 T cells, CD45RA and CD27 were evaluated by flow citometry on Vc9Vd2 T cells derived from BAC and from blood of TB patients. Vc9Vd2 T cells can be devised in 4 subpopulations based on CD45RA and CD27 expression: naïve (NA, CD45+CD27+), central memory (CM, CD45RA CD27+), effector memory (EM, CD45RA CD27 ) and terminal differentiated (TEMRA, CD45RA+CD27 ). We observed that, as expected, blood Vc9Vd2 T cells from TB patients show mainly a CM phenotype (75 ± 17.5), while NA, EM and TEMRA were less represented (8 ± 13, 28.3 ± 13.8 and 18.3 ± 21 respectively). BAC Vc9Vd2 T cell subpopulations are comparable to blood Vc9Vd2 T cells, however, unlike blood Vc9Vd2 T cells,
CM cells were not significantly higher than EM and TEMRA Vc9Vd2 T cells (Fig. 2a). We also evaluated the expression of CCR7 (chemokine receptor), CD62L (homing receptor), CD16 (Fc receptor) and CD57 (terminal differentiated marker) on Vc9Vd2 T cells of TB patients. We observed that TEMRA Vc9Vd2 T cell from BAC of TB patients showed a down modulation of cytotoxic receptor CD16 compared to PBMC (Fig. 2e). No difference was observed for the other marker tested (Fig. 2b and e).
3.2. Cytokine production and cytotoxic assay It is know that Vc9Vd2 T cells release T helper type 1 (Th1) cytokines upon phosphoantigen stimulation, and this activity can be compromised in some pathologies. We analyzed the ability of BAC Vc9Vd2 T cells from TB patients to respond to specific antigen stimulation. To this aim we cultured cells derived from BAC in the presence of natural human phosphoantigen IPP and IL-2 for 12 h. INF-c+ and TNF-a+ Vc9Vd2 T cells were assessed by flow citometry and expressed as percentage of positive cells. Fig. 3a and b show that BAC Vc9Vd2 T cells from TB patients producing INF-c and TNF-a after TCR stimulation are significantly lower compared with Vc9Vd2 T cells from peripheral blood, suggesting that in the site of infection Vc9Vd2 T cells are impaired in their function. Activated Vc9Vd2 T cells have a potent cytotoxic activity mediated by release of perforin (cytolytic protein present in granules of cytotoxic cells), correlated with the increase expression of CD107 (marker of degranulation on lymphocytes). BAC and blood Vc9Vd2 T cells from TB patients were analyzed for the expression of CD107a and the presence of perforin upon phosphoantigen stimulation. Interestingly, we found that unstimulated BAC Vc9Vd2 T cells express high level of CD107a and lower level of perforin when comparated to blood Vc9Vd2 T cells. Moreover, differently from blood, stimulation with IPP of BAC Vc9Vd2 T cells did not induce perforin release or increase expression CD107a (Fig. 3c and d). 3.3. BAC Vc9Vd2 T cells show a strong down-modulation of CD3e T-cell Receptor (TCR) is a multisubunit complex in which the invariant subunit CD3 (CD3c-CD3e, CD3d-CD3e and CD3f-CD3f) couples antigen recognition with intracellular signal transduction. As for ab T cells, Vc9Vd2 TCR is associated with CD3 molecules. We found that Vc9Vd2 T cells from BAC of TB patients expressed significantly lower level of CD3e compared to Vc9Vd2 T cells from PBMC (Fig. 4a and b). However, CD3e expression on Vc9Vd2 T cells from PBMC of TB patients was comparable to that from healthy donors (data not show). These data suggest that in the site of infection, Vc9Vd2 T cells result impaired in the expression of one of the central molecule for T lymphocyte activation, thus explaining the inability of Vc9Vd2 T cells from lungs of TB patients to respond to antigen stimulation. 3.4. Apoptotic capacity of lung and blood Vc9Vd2 T cells of TB patients
Fig. 1. Vd2 TCR expression in PBMC derived from healthy donor and TB patients, and in BAC derived from lung of TB patients. ⁄P < 0.05, ⁄⁄P < 0.001.
Several studies have reported an increased apoptosis of cd T cells during MTB infection. To determine whether down modulation of CD3e on Vc9Vd2 T cells from BAC could be correlated to apoptosis, we evaluated the frequency of apoptotic Vc9Vd2 T cells derived from blood and BAC of TB patients. To this aim we evaluated the annexin V expression on Vc9Vd2 T cells from BAC and blood of TB patients by flowcytometry. We observed that the frequency of annexin V on Vc9Vd2 T cells was comparable between blood and BAC (Fig. 4c), suggesting that CD3e impairment was not consequence of cell death.
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Fig. 2. Phenotype analysis of Vc9Vd2 T cells presents in PBMC and BAL of TB patients. (a) Using flow cytometry, cell subsets were identified according to their expression of CD45RA and CD27 into naïve (NA), central memory (CM), effector memory (EM) and terminal differentiated (TEMRA) as show along x-axis. There is a significant difference (P < 0.05) between CM and effector phenotypes (EM and TEMRA) in blood cd T cells. This difference is not detected in lung cd T cells. (b–e) Each phenotype was characterized through CD57, CD16, CCR7 and CD62L expression. All data are expressed as mean of percentage of positive cell ± standard deviation (SD).
3.5. Different expression of iNK receptors on Vc9Vd2 T cells of TB patients Vc9Vd2 T cells express activating and inhibitory NK receptors, and their engagement coordinate the NK cell function of Vc9Vd2 T cells. It has been suggested that inhibitory receptor-mediated down-modulation of NK or cytotoxic T-lymphocyte (CTL) effector functions could account for the inefficient immune responses against certain pathogens as well as against cancer cells. To determinate whether inhibitor NK receptors are differently expressed on BAC and blood Vc9Vd2 T cells, we examined the surface distribution of NK inhibitor receptors CD158a, CD94, CD161 and ILT-2 on Vc9Vd2 T cells derived from BAC and PBMC of TB patients. We found that Vc9Vd2 T cells from BAC express higher level of CD158a and ILT-2 inhibitory receptor compared to Vc9Vd2 T cells from PBMC (Fig. 4d), suggesting that Vc9Vd2 T cells in lung of TB patients are also impaired in their NK function. 4. Discussion Vc9Vd2 T cells, a major human peripheral cd T-cell subset, exhibit several characteristics that place them at the border between innate and adaptative immunity. The ability to produce inflammatory cytokine against pathogens and tumors, and their cytolytic
and bactericidal activities suggest a direct involvement in immune control of cancer and infection [19–20]. Evidence from humans and animal models have demonstrated complex patterns of cd T cells immune responses during early mycobacterial infection and chronic TB [12–13]. The major protective functions are performed by macrophage activation by INF-c produced also by Vc9Vd2 T cells, typical of Th-1 response, and the modulation of DC functions [21]. Furthermore, Vc9Vd2 T cells can kill the mycobacterium through release a lethal combination of cytotoxic molecules (perforin, granzime and granulysin) [22]. Since pulmonary tuberculosis is an airway disease, bronchoalveolar T cells should be a better indicator of local immune reaction after TB infection than peripheral blood T cells. In this paper we propose a preliminary study on the characterization of Vc9Vd2 T cells from the lung, the first site of interaction with the pathogen, and from blood. There are many conflicting opinions about the distribution of Vc9Vd2 T cells during MTB infection: whereas some studies have reported an increase in Vc9Vd2 T cells in both blood and lung of tuberculosis patients [13,23], other studies have demonstrated that Vc9Vd2 T cell number remain constant in peripheral blood and in the lung [24]. These conflicting results, however, could depend on different stages of disease of the included patients that could affect the repertoire of Vc9Vd2 T cells during TB infection.
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Fig. 3. Interferon (INF)-c and TNF-a production in response to isopentenyl pyrophosphates (IPP) by Vc9Vd2 T cells derived from peripheral blood and lung of TB patients. (b) Vc9Vd2 T cells were stimulated for 12 h with IPP (80 lM) in vitro, and their cytokines production was assessed by intracellular FACS analysis. (a) The plots show (in representative experiment) percentage numbers of Vc9Vd2 T cells capable of producing TNF-a and INF-c in the blood (PBMC) and in the lung (BAL). Cells were gated on lymphocyte population. Data are expressed as mean of percentage of positive cell ± standard deviation (SD). ⁄P < 0.05. (c–d) Cytotoxic assay: CD107a (c) and Perforin (d) analysis of Vc9Vd2 T cells derived from BAL and PBMC of TB patients before (c/c) and after incubation for 12 h with IPP (80 lM). ⁄⁄P < 0.001.
Fig. 4. CD3 expression, apoptotic assay and NK inhibitors. (a) CD3 expression in Vc9Vd2 T cells derived from BAL and PBMC of TB patients ex-vivo. In the bar chart data are expressed as mean of percentage of CD3+ cell ± standard deviation (SD). ⁄P < 0.05. (b) The histogram show a representative experiment were CD3 expression in BAL (continues line) and in blood (dashed line) is shown as cell number counts, respect to control isotype (gray-filled histogram). (c) Annexin V expression in Vc9Vd2 T cells derived from BAL and PBMC of TB patients. ⁄P < 0.001 (d) Expression of NK inhibitors receptor in Vc9Vd2 T cells derived from BAL (gray bar) and blood (black bar) of TB patients. All data are expressed as mean of percentage of positive cell ± standard deviation (SD). ⁄P < 0.02.
In our studies a low frequency of lung Vc9Vd2 T cells was seen in patient with active TB when compared with PBMC. Other found no reduction in size of Vc9Vd2 T cells in blood and BAC samples from patients with sarcoidosis or berillosis, or in samples from patients with mycobacterial-induced tuberculous pleuritis
[15] or tuberculoid leprosy [25], than we may speculate that the lost of Vc9Vd2 T cells from BAC could be associated with the failure of the protective immune response against Mycobacterium tuberculosis; however, this issue needs further investigations.
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Vc9Vd2 T cells respond to antigen challenge by secreting large quantities of TNF-a and IFN-c [26], which contribute to the activation of both specific and non-specific immune responses. We found, that unlike blood Vc9Vd2 T cells, Vc9Vd2 T cells present in lung of TB patients are not able to produce IFN-c or TNF-a after phosphoantigen stimulation. Adequate amounts of functional T cell receptor (TCR)/CD3 complexes on the cell surface are critical for mounting a successful antigen-dependent T cell-mediated immune response [27] and defects in TCR/CD3 expression and function have been reported in a variety of clinical conditions. The down modulation of these complexes abolishes signaling in T-APC conjugates and affects the responsiveness of T cells to further antigenic stimulation [28]. In our studies we have observed a strong down modulation of CD3 receptor in lung Vc9Vd2 T cells, that can explain the functional inability of these cells, and we hypothesized that this could be a consequence of a iper-activaction of Vc9Vd2 T cells in lung microenvironment. Indeed is it know that TCR complex activation can induce a its down modulation, probably by preventing their recycling on cell surface [29]. Previous studies in human and non-human primates [18,30] observed that Vc9Vd2 T cells show mainly a CM phenotype (CD45RA-CD27+) after TB or phosphoantigen stimulations, with a transient phenotypic change (in effector phenotype) after each stimulation [31]. In our study, through analysis ex-vivo of Vc9Vd2 T cells present in BAC and in PBMC of TB patients, we have not observed differences between peripheral blood and BAC in Vc9Vd2 T cells phenotype; however, while there was a significant prevalence of the CM phenotype in the blood of TB patients, in the lung the difference with CM cells was not significant. This could be a consequence of a greater activation of Vc9Vd2 T cells present in the first site of infection, most likely due to a prolonged exposure to the pathogen in respect to their counterpart in the blood. It is know that, to evade the host of immune response, some pathogens can induce immunosuppression through direct interaction of the host immune effector mechanism. It was shown that in TB patients there is a decreased expression of NK activating receptors (CD314 and CD160) and increased expression of NK inhibitory receptors (CD158a, CD158b1 and CD158b2), compared to healthy controls [32]. The relationship between iNK receptor and the activation of Vc9Vd2 T cells has been observed also in many different diseases not correlated with MTB. For example, Marthe C.D’Ombrain et all. [33] observed that non responsive Vc9Vd2 T cells express more frequently the KIR receptor CD158a than malariaresponsive Vc9Vd2 T cells, and that the expression of CD94 and CD161 is positively correlated with the total amount of INF-c produced by human PBMC. Emilie Lesport et al. [34], studying the antitumor activity of HLA-G on Vc9Vd2 T cells, observed that in Vc9Vd2 T cell populations expressing elevated levels of ILT2, soluble HLA-G5 induced a markedly decreased IFNc production. We also found an increase of some iNK receptors (CD158a, ILT2, CD94 and CD161) in PBMC of TB patients compared to healthy control (data not shown), but more interestingly we observed a greater expression of CD158a and ILT-2 on lung Vc9Vd2 T cells compared to blood. This could contribute to the inability of these cells to respond to phosphoantigens stimulation. Under normal conditions, the effector function of mature circulating Vc9Vd2 T cells include a potent cytolytic activity mediated by the release of perforin [14], correlated with the increase expression of CD107a. In agreement with Yokobori N. [35], in our study we observed that ex vivo lung Vc9Vd2 T cells express high level of CD107a and a lower level of perforin. After phosphoantigens stimulation, the percentage of blood Vc9Vd2 T cells CD107a+ increased significantly, while perforin was released. In the lung, after phosphoantigen stimulation, we did notobserved any change in
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the percentage of CD107a+ and perforin+ cd T cells, suggesting an exhaustion of Vc9Vd2 T cells cytotoxicity potential. Moreover, we observed that, unlike blood Vc9Vd2 T cells, lung TEMRA Vc9Vd2 T cells show a down modulation of CD16 receptor. CD16 is known to be involved in Ab-dependent cellular cytotoxicity and its cross-linking on NK cells increase intracellular calcium levels and activates a cascade of biochemical events similar to those activated in lymphocyte via TCR [36,37]. Some studies analyzed the expression of CD16 on Vc9Vd2 T cells according to their activation state [38]. It has been proposed that once stimulated through the TCR-CD3 complex by phosphoantigen, Vc9Vd2 T cells acquire, through long-lasting CD16 expression, the ability to be reactivated by Abs produced during the acquire immune response, with consequent release of cytokines. Therefore we can conclude that during TB infection, while blood Vc9Vd2 T cells showed a potential cytotoxic activity, lung Vc9Vd2 T cells seem to have lost this ability, showing a phenotype similar to already stimulated T cells. One of possible causes of this ‘‘anergic state’’ could be the activation-induced cell death. Recent studies [32] have shown that in Vc9Vd2 T cells derived from blood of TB patients, surface FasL expression was significantly higher than in healthy controls. In our studies, through annexin expression analysis, we don’t observe a significantly difference between lung and blood Vc9Vd2 T cells. So, the loss of CD3e expression and than the inability of lung Vc9Vd2 T cells to respond to phosphoantigen stimulation does not appear to be correlated with activation- induced cell death. An alternative possibility that could contribute to the impaired phenotype and function of Vc9Vd2 T cells present in the BAC is the dysfunction of other immune cell populations that normally interact with Vc9Vd2 T cells. For example the APC activity of mononuclear phagocytic cells and the cytokines produced by CD4+ ab T cells both correlated with the activity of Vc9Vd2 T cells [39]. Alternatively, Vc9Vd2 T cells may be more susceptible to cytokines or some different products secreted by macrophages and/or cells of lung compartment after infection with Mtb. These issues need further investigations. To understand the mechanisms of immunomodulation in the first site of interaction with the pathogen could be the key to propose new therapies aimed to neutralize the negative regulatory factors rather than accentuating an already intense immune response. Acknowledgments We wish to dedicate this work in memory of Angelo Martino, teacher and friend, who enjoyed making science too briefly. This work was supported by Ricerca Corrente Grants from Italian Ministry of Health. References [1] V.M. Vashishtha, WHO global tuberculosis Control Report 2009: tuberculosis elimination is a distant dream, Indian Pediatr. 46 (2009) (2009) 401–402. [2] A. Wright et al., Epidemiology of antituberculosis drug resistance 2002–07: an updated analysis of the global project on anti-tuberculosis drug resistance surveillance, Lancet 373 (2009) 1861–1873. [3] L.F. Barker, M.J. Brennan, P.K. Rosenstein, J.C. Sadoff, Tuberculosis vaccine research: the impact of immunology, Curr. Opin. Immunol. 21 (2009) 331–338. [4] D.S. Korbel, B.E. Schneider, U.E. Schaible, Innate immunity in tuberculosis: myths and truth, Microbes Infect. 10 (2008) 995–1004. [5] A. Martino, Mycobacteria and innate cells: critical encounter for immunogenicity, J. Biosci. 33 (2008) 137–144. [6] R. Casetti, A. Martino, The plasticity of gamma delta T cells: innate immunity, antigen presentation and new immunotherapy, Cell. Mol. Immunol. 5 (2008) 161–170. [7] S. Chiplunkar, S. Dhar, D. Wesch, D. Kabelitz, Gammadelta T cells in cancer immunotherapy: current status and future prospects, Immunotherapy 1 (2009) 663–678. [8] Y.H. Chien, Y. Konigshofer, Antigen recognition by gammadelta T cells, Immunol. Rev. 215 (2007) 46–58.
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Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
An abnormal phenotype of lung Vc9Vd2 T cells impairs their responsiveness in tuberculosis patients Sary El Daker a,d,⇑,1, Alessandra Sacchi a, Carla Montesano d, Alfonso Maria Altieri b, Giovanni Galluccio c, Vittorio Colizzi d, Federico Martini a, Angelo Martino a a
Laboratory of Cellular Immunology, National Institute for Infectious Diseases ‘‘Lazzaro Spallanzani’’, Rome, Italy Unità di Broncopneumologia e Tisiologia, Azienda Ospedaliera San Camillo-Forlanini, Rome, Italy Unità di Endoscopia Toracica, Azienda Ospedaliera San Camillo-Forlanini, Rome, Italy d Department of Biology, University of Rome Tor Vergata, Rome, Italy b c
a r t i c l e
i n f o
Article history: Received 5 March 2013 Accepted 2 May 2013 Available online 15 May 2013 Keywords: Mycobacterium cd T cells Primary infection Lung
a b s t r a c t Antigen-specific cd T cells represent an early innate defense known to play an important role in antimycobacterial immunity. We have investigated the immune functions of Vc9Vd2 T cells from BronchoAlveolar lavages (BAC) samples of active TB patients. We observed that BAC Vc9Vd2 T cells presented a strong down-modulation of CD3 expression compared with Vc9Vd2 T cells from peripheral blood. Furthermore, Vc9Vd2 T cells mainly showed a central memory phenotype, expressed high levels of NK inhibitory receptors and TEMRA cells showed low expression of CD16 compared to circulating Vc9Vd2 T cells. Interestingly, the ability of BAC Vc9Vd2 T cells to respond to antigen stimulation was dramatically reduced, differently from blood counterpart. These observations indicate that cd T cell functions are specifically impaired in situ by active TB, suggesting that the alveolar ambient during tuberculosis may affect resident cd T cells in comparison to circulating cells. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is one of the most prevalent and common serious infectious diseases worldwide, affecting almost 10 million people annually [1]. The disease, fuelled by human immunodeficiency virus (HIV) infection and poverty, is out of control in developing countries, and the emergence of drug resistant strains threatens TB control in several other regions of the world [2]. The current available vaccine, Bacillus Calmette-Guerin (BCG), as well as existing therapeutic interventions for TB, are at present almost suboptimal. Thus, new vaccines and immunotherapeutic strategies are urgently required in order to improve TB control efforts [3]. A better understanding of the immunopathogenesis of TB could facilitate the identification of correlates of immune protection, the design of more effective vaccines, the rational selection of immunotherapeutic agents, and the evaluation of new drug or adjuvant candidates. Immune response to MTB may play a major role in determining clinical course: specific cell mediated response is critical in host defense against mycobacteria, other mechanisms, including innate
⇑ Corresponding author. Address: National Institute for Infectious Diseases ‘‘Lazzaro Spallanzani,’’ Via Portuense 292, 00149 Rome, Italy. Fax: +39 06 55170962. E-mail address: [email protected] (S. El Daker). 1 Present address: Singapore Immunology Network, A⁄Star, 8A Biomedical Groove, #03-01 Biopolis, Singapore 138648, Singapore. 0008-8749/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellimm.2013.05.001
immunity, may play a role too [4]. Vc9Vd2 T cells, the major subset of circulating cd T cell pool, are known to be involved in immunity against microbial pathogens, including MTB [5–7]. This subset may be activated by two different mechanisms: (a) directly by non peptidic small phosphorylated compounds produced by mammalian cells, such as isopentenyl-pyrosphosphate (IPP), or by non mammalian cells, such as 4-hydroxy-3-dimethylallyl pyrophosphate; (b) indirectly by a group of non peptidic compounds, the aminobisphosphonates, as Zoledronic acid (Zol), that have been shown to activate Vc9Vd2T cells through the accumulation of mevalonate pathway metabolites, as IPP, subsequently recognized by Vc9Vd2TCR [8]. Similarly to CD4 and CD8 ab T cells, Vc9Vd2T cells are heterogeneous, and show distinct populations defined by surface marker expression and effector functions. Naïve (CD45RA+CD27+) and central memory (CD45RA CD27+) Vc9Vd2T cells abound in lymph nodes and lack immediate effector functions. Conversely, effector memory (CD45RA CD27 ) and terminally differentiated (CD45RA+CD27 ) Vc9Vd2T cells are poorly represented in the lymph nodes whereas abound at sites of inflammation, and display immediate effector functions [9]. The differentiation pathway for the generation of these subsets is uncertain, but circulating effector Vc9Vd2 T cells have been found significantly reduced in several diseases as pulmonary tuberculosis [10]. Studies done in humans and animal models have demonstrated complex patterns of Vc9Vd2 T cell immune responses during early mycobacterial infections and chronic TB. In effect, responses of Vc9Vd2 T cells
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to MTB were described as early as in 1989 [11]. A number of studies have attempted to determine the in vivo role of Vc9Vd2 T cells in the human immune response to MTB [12–16]. Barnes et al. established that patients with pulmonary or miliary tuberculosis had a diminished ability to expand Vc9Vd2 T cells in vitro in response to heat-killed MTB and IL-2, although there was quite a wide range of Vc9Vd2 T-cell expansion among the different groups. Studies of T-cell phenotype in bronchoalveolar cells from healthy PPD+ subjects and from lungs of patients with pulmonary TB found a T lymphocytic alveolitis in the lungs. Vc9Vd2 T cells were found among the lymphocytes in this alveolitis, but their proportion was not increased relative to ab T cells [17]. Thus, Vc9Vd2 T cells are present in situ in pulmonary TB but are not expanded compared to Vc9Vd2 T cells in peripheral blood or unaffected lung. It is already known, in the context of a natural infection, the consistent expansion of Vc9Vd2 T cells with a TCM phenotype in the peripheral blood of patients with active TB, which was accompanied by the dramatic reduction of the pool of Vc9Vd2 cells with immediate effector functions (TEM and TEMRA cells). However, this skewed representation of circulating Vc9Vd2 T cell phenotypes during active TB was transient and completely reversed after successful antimycobacterial therapy [18]. The mechanism causing the loss of Vc9Vd2 T cell effector functions during TB is unknown. One possibility is that sustained in vivo mycobacterial stimulation of Vc9Vd2 T cells causes their apoptosis. However, the analysis of Vc9Vd2 T cell functions in TB patients and especially in the site of infection needs further investigations. Here, we report the phenotypical and functional analysis of Vc9Vd2 T cells in the blood and BAC of TB patients. We observed that, in contrast to the circulating Vc9Vd2 T cells, lung Vc9Vd2 T cells responsiveness to antigen stimulation was dramatically compromised. Indeed, lung Vc9Vd2 T cells were not able to produce cytokines (INF-c, TNF-a) under specific phosphoantigen stimulation. This was found to be correlated to a dramatic down/modulation of CD3 molecule on lung Vc9Vd2 T cells, that could explain their inability to respond to antigenic stimulation. 2. Materials and methods 2.1. Patient population Thirty tuberculosis patients were enrolled at the respiratory disease wards in the ‘‘Azienda ospedaliera San Camillo Forlanini’’ in Rome. All study participants had newly diagnosed active tuberculosis with a positive culture for MTB from sputum. Patients with active TB were studied within 7 days of admission and before starting anti-TB therapy. In addition, healthy donors were recruited as controls at the ‘‘National Institute for Infectious Diseases, Lazzaro Spallanzani Rome, Italy. The ethics committee of both Institutions approved the study and all enrolled individuals provided written informed consent. Other criteria of inclusion were: negative results to HIV test and not receiving therapy with immune-suppressive drugs. Characteristics of all subjects are reported in Table 1. Table 1 Baseline characteristic of healthy donor and patients with active tuberculosis(TB). Parameter
Healthy donor (PBMC)
TB patients (PBMC/ BAL)
Number Gender Age (years) Smoker BCG vaccination BCG treatment HIV Immunosuppressive drugs
15 7M/8F – – No – No No
30 19M/11F 34 (16–62) 16Y/14N Yes No No No
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2.2. Cell preparation and stimulation Peripheral blood mononuclear cells (PBMC) from TB patients and healthy controls were isolated from whole blood using Ficoll density gradient centrifugation. After washing in PBS 1, PBMC at a concentration of 1 106 cells/ml were cultured for 24 h at 37 °C and 5% CO2 in a complete medium (RPMI-1640, 10% heatinactivated human serum, 10 mM HEPES, 2 mM L-glutamine and 10 U/ml penicillin–streptomycin, all from Euroclone Ltd, Weatherby, UK). Cultures were prepared in 48-well tissue culture plates (Costar, Corning Inc., NY, USA) in 1 ml/well. Bronchoalveolar cells (BAC) from TB patients were processed in a safety BSL3 laboratory. After filtering with BD Falcon cell strainer 100 lm, BAC were washed in PBS 1 and were cultured for 24 h at 37 °C and 5% CO2 in a complete medium (like PBMC). After 24 h to the separation, PBMC and BAC were stimulated in vitro in the presence of 80 lM isopentenyl pyrophosphate (IPP; Sigma–Aldrich, St. Louis, MO). 2.3. mAbs and flow cytometry Anti-human monoclonal Abs used in phenotype studies were the following: anti-CD27 APC-H7 (clone M-T271); anti-CD45RA CyChrome (clone HI100); anti-CD57 PE (clone NK1); anti-CD16 pacific blue (clone 3G8); anti-CD3 APC (clone UCHT1); anti-CCR7 PeCy7(clone 3D12); anti-CD62L APC (clone Dreg56). The anti-human Abs used in cytotoxic studies were the following: anti-perforin PE (clone dG9); anti-CD107 PerCp (clone H4A3). The anti-human Abs used in cytokines analyses were the following: anti-INFc Pe-Cy7 (clone B27) and anti-TNFa APC (clone 6401.1111). All the previously described mAbs were from BD Biosciences (Mountain View, CA). The anti-Vd2 mAb FITC (IgG1, clone IMMU1464) was purchased by Immunotech (Marseille, France). Isotype-matched control mAbs from BD Biosciences were used in all experiments. PBMC and BAC were incubated for 15 min at 4 °C with the conjugated mAb. Samples were washed in PBS 1, fixed in paraformaldehyde (PFA 1% for PBMC and 4% for BAC), suspended in FACSFlow (BD-Pharmagen) and immediately acquired with a FACS CANTO II flow cytometer (BD Biosciences). A total of 100,000 events was acquired for each sample and analyzed with CellQuest software (BD Biosciences). 2.4. Single-cell analysis of cytokine synthesis Cytokine production was detected by flow cytometry analysis as previously described (18). Human PBMC were stimulated for 12 h with IPP (80 lM;Sigma–Aldrich). Brefeldin A (10 lg/ml) was added 1 h after stimulation to block intracellular transport allowing cytokine accumulation in the Golgi. Cells were washed twice in PBS 1, 1% BSA, and 0.1% sodium azide and stained with mAb specific for the membrane Ags described above for 15 min at 4 °C. Samples were then fixed in 4% paraformaldehyde for 10 min at 4 °C, incubated with anti-cytokine mAbs in 1 PBS, 1% BSA, and 0.5% saponin. Cells were finally washed twice in 1 PBS, 1% BSA, 0.1% saponin, and acquired on a FACS CANTO II (BD Biosciences). Control for nonspecific staining was monitored with isotype-matched mAbs and nonspecific staining was always subtracted from specific results. 2.5. Detection of CD107a on cd T cells Considering that the degranulation of antigen-responding T cells is associated with acquisition of cell surface CD107a, PBMC and BAC (1 106 cells/ml) were incubated with or without stimulation for 18 h, and anti-CD107 PerCp (BD-Pharmigen) was added to the culture 1 h after stimulation. Thereafter, cells were washed
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and 100.000 events were acquired on Vc9Vd2 TCR+ lymphocytes and results are expressed as the percentage of CD107a+ cells among Vc9Vd2 T cells. 2.6. FITC-Annexin V staining For vitality assay we used the ‘‘Annexin V-FITC Apoptosis detection kit’’ produced by Bender MedSystem (BMS500FI20CE). After washing twice with PBS 1, cells were resuspended in binding buffer 1 (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mMCaCl,) at final concentration of 2–5 105/ml. FITC-Annexin V was added to a final concentration of 1 pg/ml. The mixture was incubated for 10 min in the dark at room temperature and after washing was acquired by using a FACS CANTO II (BD Biosciences). 2.7. Statistical analysis Statistical analysis to assess differences between the different groups was performed using Student’s t-test. Differences were considered significant when the P value was lower than 0.05. GraphPad Prism version 4.00 for Windows, (GraphPad Software) was used to perform the analysis.
3. Results 3.1. Phenotypic characterization of Vc9Vd2 T cells in BAC and PBMC of TB patients Vc9Vd2 T cells represent one of the first defense against MTB, in particular in the first site of infection. Thus we characterized Vc9Vd2 T cells from BAC of patients with active tuberculosis. First, we have observed that while the percentage of blood Vc9Vd2 T cells of healthy donor and TB patients is comparable (3.8% ± 0.11 and 3.6 ± 0.24%) the percentage of BAC Vc9Vd2 T cells is lower (1,75 ± 0.36) than their counterpart in the blood (Fig. 1). In order to better characterize BAC Vc9Vd2 T cells, CD45RA and CD27 were evaluated by flow citometry on Vc9Vd2 T cells derived from BAC and from blood of TB patients. Vc9Vd2 T cells can be devised in 4 subpopulations based on CD45RA and CD27 expression: naïve (NA, CD45+CD27+), central memory (CM, CD45RA CD27+), effector memory (EM, CD45RA CD27 ) and terminal differentiated (TEMRA, CD45RA+CD27 ). We observed that, as expected, blood Vc9Vd2 T cells from TB patients show mainly a CM phenotype (75 ± 17.5), while NA, EM and TEMRA were less represented (8 ± 13, 28.3 ± 13.8 and 18.3 ± 21 respectively). BAC Vc9Vd2 T cell subpopulations are comparable to blood Vc9Vd2 T cells, however, unlike blood Vc9Vd2 T cells,
CM cells were not significantly higher than EM and TEMRA Vc9Vd2 T cells (Fig. 2a). We also evaluated the expression of CCR7 (chemokine receptor), CD62L (homing receptor), CD16 (Fc receptor) and CD57 (terminal differentiated marker) on Vc9Vd2 T cells of TB patients. We observed that TEMRA Vc9Vd2 T cell from BAC of TB patients showed a down modulation of cytotoxic receptor CD16 compared to PBMC (Fig. 2e). No difference was observed for the other marker tested (Fig. 2b and e).
3.2. Cytokine production and cytotoxic assay It is know that Vc9Vd2 T cells release T helper type 1 (Th1) cytokines upon phosphoantigen stimulation, and this activity can be compromised in some pathologies. We analyzed the ability of BAC Vc9Vd2 T cells from TB patients to respond to specific antigen stimulation. To this aim we cultured cells derived from BAC in the presence of natural human phosphoantigen IPP and IL-2 for 12 h. INF-c+ and TNF-a+ Vc9Vd2 T cells were assessed by flow citometry and expressed as percentage of positive cells. Fig. 3a and b show that BAC Vc9Vd2 T cells from TB patients producing INF-c and TNF-a after TCR stimulation are significantly lower compared with Vc9Vd2 T cells from peripheral blood, suggesting that in the site of infection Vc9Vd2 T cells are impaired in their function. Activated Vc9Vd2 T cells have a potent cytotoxic activity mediated by release of perforin (cytolytic protein present in granules of cytotoxic cells), correlated with the increase expression of CD107 (marker of degranulation on lymphocytes). BAC and blood Vc9Vd2 T cells from TB patients were analyzed for the expression of CD107a and the presence of perforin upon phosphoantigen stimulation. Interestingly, we found that unstimulated BAC Vc9Vd2 T cells express high level of CD107a and lower level of perforin when comparated to blood Vc9Vd2 T cells. Moreover, differently from blood, stimulation with IPP of BAC Vc9Vd2 T cells did not induce perforin release or increase expression CD107a (Fig. 3c and d). 3.3. BAC Vc9Vd2 T cells show a strong down-modulation of CD3e T-cell Receptor (TCR) is a multisubunit complex in which the invariant subunit CD3 (CD3c-CD3e, CD3d-CD3e and CD3f-CD3f) couples antigen recognition with intracellular signal transduction. As for ab T cells, Vc9Vd2 TCR is associated with CD3 molecules. We found that Vc9Vd2 T cells from BAC of TB patients expressed significantly lower level of CD3e compared to Vc9Vd2 T cells from PBMC (Fig. 4a and b). However, CD3e expression on Vc9Vd2 T cells from PBMC of TB patients was comparable to that from healthy donors (data not show). These data suggest that in the site of infection, Vc9Vd2 T cells result impaired in the expression of one of the central molecule for T lymphocyte activation, thus explaining the inability of Vc9Vd2 T cells from lungs of TB patients to respond to antigen stimulation. 3.4. Apoptotic capacity of lung and blood Vc9Vd2 T cells of TB patients
Fig. 1. Vd2 TCR expression in PBMC derived from healthy donor and TB patients, and in BAC derived from lung of TB patients. ⁄P < 0.05, ⁄⁄P < 0.001.
Several studies have reported an increased apoptosis of cd T cells during MTB infection. To determine whether down modulation of CD3e on Vc9Vd2 T cells from BAC could be correlated to apoptosis, we evaluated the frequency of apoptotic Vc9Vd2 T cells derived from blood and BAC of TB patients. To this aim we evaluated the annexin V expression on Vc9Vd2 T cells from BAC and blood of TB patients by flowcytometry. We observed that the frequency of annexin V on Vc9Vd2 T cells was comparable between blood and BAC (Fig. 4c), suggesting that CD3e impairment was not consequence of cell death.
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Fig. 2. Phenotype analysis of Vc9Vd2 T cells presents in PBMC and BAL of TB patients. (a) Using flow cytometry, cell subsets were identified according to their expression of CD45RA and CD27 into naïve (NA), central memory (CM), effector memory (EM) and terminal differentiated (TEMRA) as show along x-axis. There is a significant difference (P < 0.05) between CM and effector phenotypes (EM and TEMRA) in blood cd T cells. This difference is not detected in lung cd T cells. (b–e) Each phenotype was characterized through CD57, CD16, CCR7 and CD62L expression. All data are expressed as mean of percentage of positive cell ± standard deviation (SD).
3.5. Different expression of iNK receptors on Vc9Vd2 T cells of TB patients Vc9Vd2 T cells express activating and inhibitory NK receptors, and their engagement coordinate the NK cell function of Vc9Vd2 T cells. It has been suggested that inhibitory receptor-mediated down-modulation of NK or cytotoxic T-lymphocyte (CTL) effector functions could account for the inefficient immune responses against certain pathogens as well as against cancer cells. To determinate whether inhibitor NK receptors are differently expressed on BAC and blood Vc9Vd2 T cells, we examined the surface distribution of NK inhibitor receptors CD158a, CD94, CD161 and ILT-2 on Vc9Vd2 T cells derived from BAC and PBMC of TB patients. We found that Vc9Vd2 T cells from BAC express higher level of CD158a and ILT-2 inhibitory receptor compared to Vc9Vd2 T cells from PBMC (Fig. 4d), suggesting that Vc9Vd2 T cells in lung of TB patients are also impaired in their NK function. 4. Discussion Vc9Vd2 T cells, a major human peripheral cd T-cell subset, exhibit several characteristics that place them at the border between innate and adaptative immunity. The ability to produce inflammatory cytokine against pathogens and tumors, and their cytolytic
and bactericidal activities suggest a direct involvement in immune control of cancer and infection [19–20]. Evidence from humans and animal models have demonstrated complex patterns of cd T cells immune responses during early mycobacterial infection and chronic TB [12–13]. The major protective functions are performed by macrophage activation by INF-c produced also by Vc9Vd2 T cells, typical of Th-1 response, and the modulation of DC functions [21]. Furthermore, Vc9Vd2 T cells can kill the mycobacterium through release a lethal combination of cytotoxic molecules (perforin, granzime and granulysin) [22]. Since pulmonary tuberculosis is an airway disease, bronchoalveolar T cells should be a better indicator of local immune reaction after TB infection than peripheral blood T cells. In this paper we propose a preliminary study on the characterization of Vc9Vd2 T cells from the lung, the first site of interaction with the pathogen, and from blood. There are many conflicting opinions about the distribution of Vc9Vd2 T cells during MTB infection: whereas some studies have reported an increase in Vc9Vd2 T cells in both blood and lung of tuberculosis patients [13,23], other studies have demonstrated that Vc9Vd2 T cell number remain constant in peripheral blood and in the lung [24]. These conflicting results, however, could depend on different stages of disease of the included patients that could affect the repertoire of Vc9Vd2 T cells during TB infection.
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Fig. 3. Interferon (INF)-c and TNF-a production in response to isopentenyl pyrophosphates (IPP) by Vc9Vd2 T cells derived from peripheral blood and lung of TB patients. (b) Vc9Vd2 T cells were stimulated for 12 h with IPP (80 lM) in vitro, and their cytokines production was assessed by intracellular FACS analysis. (a) The plots show (in representative experiment) percentage numbers of Vc9Vd2 T cells capable of producing TNF-a and INF-c in the blood (PBMC) and in the lung (BAL). Cells were gated on lymphocyte population. Data are expressed as mean of percentage of positive cell ± standard deviation (SD). ⁄P < 0.05. (c–d) Cytotoxic assay: CD107a (c) and Perforin (d) analysis of Vc9Vd2 T cells derived from BAL and PBMC of TB patients before (c/c) and after incubation for 12 h with IPP (80 lM). ⁄⁄P < 0.001.
Fig. 4. CD3 expression, apoptotic assay and NK inhibitors. (a) CD3 expression in Vc9Vd2 T cells derived from BAL and PBMC of TB patients ex-vivo. In the bar chart data are expressed as mean of percentage of CD3+ cell ± standard deviation (SD). ⁄P < 0.05. (b) The histogram show a representative experiment were CD3 expression in BAL (continues line) and in blood (dashed line) is shown as cell number counts, respect to control isotype (gray-filled histogram). (c) Annexin V expression in Vc9Vd2 T cells derived from BAL and PBMC of TB patients. ⁄P < 0.001 (d) Expression of NK inhibitors receptor in Vc9Vd2 T cells derived from BAL (gray bar) and blood (black bar) of TB patients. All data are expressed as mean of percentage of positive cell ± standard deviation (SD). ⁄P < 0.02.
In our studies a low frequency of lung Vc9Vd2 T cells was seen in patient with active TB when compared with PBMC. Other found no reduction in size of Vc9Vd2 T cells in blood and BAC samples from patients with sarcoidosis or berillosis, or in samples from patients with mycobacterial-induced tuberculous pleuritis
[15] or tuberculoid leprosy [25], than we may speculate that the lost of Vc9Vd2 T cells from BAC could be associated with the failure of the protective immune response against Mycobacterium tuberculosis; however, this issue needs further investigations.
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Vc9Vd2 T cells respond to antigen challenge by secreting large quantities of TNF-a and IFN-c [26], which contribute to the activation of both specific and non-specific immune responses. We found, that unlike blood Vc9Vd2 T cells, Vc9Vd2 T cells present in lung of TB patients are not able to produce IFN-c or TNF-a after phosphoantigen stimulation. Adequate amounts of functional T cell receptor (TCR)/CD3 complexes on the cell surface are critical for mounting a successful antigen-dependent T cell-mediated immune response [27] and defects in TCR/CD3 expression and function have been reported in a variety of clinical conditions. The down modulation of these complexes abolishes signaling in T-APC conjugates and affects the responsiveness of T cells to further antigenic stimulation [28]. In our studies we have observed a strong down modulation of CD3 receptor in lung Vc9Vd2 T cells, that can explain the functional inability of these cells, and we hypothesized that this could be a consequence of a iper-activaction of Vc9Vd2 T cells in lung microenvironment. Indeed is it know that TCR complex activation can induce a its down modulation, probably by preventing their recycling on cell surface [29]. Previous studies in human and non-human primates [18,30] observed that Vc9Vd2 T cells show mainly a CM phenotype (CD45RA-CD27+) after TB or phosphoantigen stimulations, with a transient phenotypic change (in effector phenotype) after each stimulation [31]. In our study, through analysis ex-vivo of Vc9Vd2 T cells present in BAC and in PBMC of TB patients, we have not observed differences between peripheral blood and BAC in Vc9Vd2 T cells phenotype; however, while there was a significant prevalence of the CM phenotype in the blood of TB patients, in the lung the difference with CM cells was not significant. This could be a consequence of a greater activation of Vc9Vd2 T cells present in the first site of infection, most likely due to a prolonged exposure to the pathogen in respect to their counterpart in the blood. It is know that, to evade the host of immune response, some pathogens can induce immunosuppression through direct interaction of the host immune effector mechanism. It was shown that in TB patients there is a decreased expression of NK activating receptors (CD314 and CD160) and increased expression of NK inhibitory receptors (CD158a, CD158b1 and CD158b2), compared to healthy controls [32]. The relationship between iNK receptor and the activation of Vc9Vd2 T cells has been observed also in many different diseases not correlated with MTB. For example, Marthe C.D’Ombrain et all. [33] observed that non responsive Vc9Vd2 T cells express more frequently the KIR receptor CD158a than malariaresponsive Vc9Vd2 T cells, and that the expression of CD94 and CD161 is positively correlated with the total amount of INF-c produced by human PBMC. Emilie Lesport et al. [34], studying the antitumor activity of HLA-G on Vc9Vd2 T cells, observed that in Vc9Vd2 T cell populations expressing elevated levels of ILT2, soluble HLA-G5 induced a markedly decreased IFNc production. We also found an increase of some iNK receptors (CD158a, ILT2, CD94 and CD161) in PBMC of TB patients compared to healthy control (data not shown), but more interestingly we observed a greater expression of CD158a and ILT-2 on lung Vc9Vd2 T cells compared to blood. This could contribute to the inability of these cells to respond to phosphoantigens stimulation. Under normal conditions, the effector function of mature circulating Vc9Vd2 T cells include a potent cytolytic activity mediated by the release of perforin [14], correlated with the increase expression of CD107a. In agreement with Yokobori N. [35], in our study we observed that ex vivo lung Vc9Vd2 T cells express high level of CD107a and a lower level of perforin. After phosphoantigens stimulation, the percentage of blood Vc9Vd2 T cells CD107a+ increased significantly, while perforin was released. In the lung, after phosphoantigen stimulation, we did notobserved any change in
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the percentage of CD107a+ and perforin+ cd T cells, suggesting an exhaustion of Vc9Vd2 T cells cytotoxicity potential. Moreover, we observed that, unlike blood Vc9Vd2 T cells, lung TEMRA Vc9Vd2 T cells show a down modulation of CD16 receptor. CD16 is known to be involved in Ab-dependent cellular cytotoxicity and its cross-linking on NK cells increase intracellular calcium levels and activates a cascade of biochemical events similar to those activated in lymphocyte via TCR [36,37]. Some studies analyzed the expression of CD16 on Vc9Vd2 T cells according to their activation state [38]. It has been proposed that once stimulated through the TCR-CD3 complex by phosphoantigen, Vc9Vd2 T cells acquire, through long-lasting CD16 expression, the ability to be reactivated by Abs produced during the acquire immune response, with consequent release of cytokines. Therefore we can conclude that during TB infection, while blood Vc9Vd2 T cells showed a potential cytotoxic activity, lung Vc9Vd2 T cells seem to have lost this ability, showing a phenotype similar to already stimulated T cells. One of possible causes of this ‘‘anergic state’’ could be the activation-induced cell death. Recent studies [32] have shown that in Vc9Vd2 T cells derived from blood of TB patients, surface FasL expression was significantly higher than in healthy controls. In our studies, through annexin expression analysis, we don’t observe a significantly difference between lung and blood Vc9Vd2 T cells. So, the loss of CD3e expression and than the inability of lung Vc9Vd2 T cells to respond to phosphoantigen stimulation does not appear to be correlated with activation- induced cell death. An alternative possibility that could contribute to the impaired phenotype and function of Vc9Vd2 T cells present in the BAC is the dysfunction of other immune cell populations that normally interact with Vc9Vd2 T cells. For example the APC activity of mononuclear phagocytic cells and the cytokines produced by CD4+ ab T cells both correlated with the activity of Vc9Vd2 T cells [39]. Alternatively, Vc9Vd2 T cells may be more susceptible to cytokines or some different products secreted by macrophages and/or cells of lung compartment after infection with Mtb. These issues need further investigations. To understand the mechanisms of immunomodulation in the first site of interaction with the pathogen could be the key to propose new therapies aimed to neutralize the negative regulatory factors rather than accentuating an already intense immune response. Acknowledgments We wish to dedicate this work in memory of Angelo Martino, teacher and friend, who enjoyed making science too briefly. This work was supported by Ricerca Corrente Grants from Italian Ministry of Health. References [1] V.M. Vashishtha, WHO global tuberculosis Control Report 2009: tuberculosis elimination is a distant dream, Indian Pediatr. 46 (2009) (2009) 401–402. [2] A. Wright et al., Epidemiology of antituberculosis drug resistance 2002–07: an updated analysis of the global project on anti-tuberculosis drug resistance surveillance, Lancet 373 (2009) 1861–1873. [3] L.F. Barker, M.J. Brennan, P.K. Rosenstein, J.C. Sadoff, Tuberculosis vaccine research: the impact of immunology, Curr. Opin. Immunol. 21 (2009) 331–338. [4] D.S. Korbel, B.E. Schneider, U.E. Schaible, Innate immunity in tuberculosis: myths and truth, Microbes Infect. 10 (2008) 995–1004. [5] A. Martino, Mycobacteria and innate cells: critical encounter for immunogenicity, J. Biosci. 33 (2008) 137–144. [6] R. Casetti, A. Martino, The plasticity of gamma delta T cells: innate immunity, antigen presentation and new immunotherapy, Cell. Mol. Immunol. 5 (2008) 161–170. [7] S. Chiplunkar, S. Dhar, D. Wesch, D. Kabelitz, Gammadelta T cells in cancer immunotherapy: current status and future prospects, Immunotherapy 1 (2009) 663–678. [8] Y.H. Chien, Y. Konigshofer, Antigen recognition by gammadelta T cells, Immunol. Rev. 215 (2007) 46–58.
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