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FEMS Immunology and Medical Microbiology 43 (2005) 339–350 www.fems-microbiology.org Gamma delta T cell responses associated with the development of tuberculosis in health care workers Diane J. Ordway a,b,c,*, Luisa Pinto c, Leonor Costa a, Marta Martins b, Clara Leandro b, Miguel Viveiros b, Leonard Amaral b, Maria J. Arroz a,c,d, Fernando A. Ventura a,c,d, Hazel M. Dockrell e a Centro de Malária e Outras Doenças Tropicais, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira 96, 1394-008 Lisbon, Portugal b Unit of Mycobacteriology, Instituto de Higiene e Medicina Tropical, Universidade de Lisboa, Rua da Junqueira 96, 1394-008 Lisbon, Portugal c Flow Cytometry Unit, Hospital Egas Moniz, Rua da Junqueira 126, 1349-019 Lisbon, Portugal d Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Campo Mártires da Pátria 130, 1169-056 Lisbon, Portugal e Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK Received 28 March 2004; received in revised form 28 June 2004; accepted 22 September 2004 First published online 13 October 2004 Abstract This study evaluated T cell immune responses to purified protein derivative (PPD) and Mycobacterium tuberculosis (Mtb) in health care workers who remained free of active tuberculosis (HCWs w/o TB), health care workers who went on to develop active TB (HCWs w/TB), non-health care workers who were TB free (Non-HCWs) and tuberculosis patients presenting with minimal (Min TB) or advanced (Adv TB) disease. Peripheral blood mononuclear cells (PBMC) were stimulated with Mtb and PPD and the expression of T cell activation markers CD25+ and HLA-DR+, intracellular IL-4 and IFN-c production and cytotoxic responses were evaluated. PBMC from HCWs who developed TB showed decreased percentages of cells expressing CD8+CD25+ in comparison to HCWs who remained healthy. HCWs who developed TB showed increased cd TCR+ cell cytotoxicity and decreased CD3+cd TCR cell cytotoxicity in comparison to HCWs who remained healthy. PBMC from TB patients with advanced disease showed decreased percentages of CD25+CD4+ and CD25+CD8+ T cells that were associated with increased IL-4 production in CD8+ and cd TCR+ phenotypes, in comparison with TB patients presenting minimal disease. TB patients with advanced disease showed increased cd TCR+ cytotoxicity and reduced CD3+cd TCR cell cytotoxicity. Our results suggest that HCWs who developed TB show an early compensatory mechanism involving an increase in lytic responses of cd TCR+ cells which did not prevent TB. 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: M. tuberculosis; cd T cells; Health care workers 1. Introduction * Corresponding author. Present address: Mycobacteria Research Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, 1682 Campus Delivery, 200 West St, Fort Collins, CO 80523-1682, USA. Tel.: +1 970 491 5777; fax: +1 970 491 1815. E-mail address: [email protected] (D.J. Ordway). In Portugal the incidence of pulmonary tuberculosis is the highest in Western Europe with 40 new cases per 100 thousand inhabitants [1]. In developing countries, nosocomial transmission of TB can be a threat to health care workers (HCWs) as there are increased numbers of 0928-8244/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsim.2004.09.005 340 D.J. Ordway et al. / FEMS Immunology and Medical Microbiology 43 (2005) 339–350 cases of TB on open wards, and minimal or absent TB infection control [1–4]. The majority of individuals infected with Mtb control primary infection and do not develop active disease [5]. The infection is controlled by activation of macrophages through Type 1 cytokine production by CD4+, CD8+ and cd+ T cells [6–12]. HCWs in Portugal are repeatedly exposed to aerosolized Mtb by TB patients not wearing masks on open hospital wards or in containment rooms. Furthermore sputum collection rooms, radiographic rooms and microbiological diagnosis of samples are not housed in biosafety level 3 facilities [1–4]. A small number of studies in developing countries suggest that there is a high risk of acquiring TB infection among HCWs in health care settings [14–18]. Most of these studies identified contact with TB patients and duration of employment as the main risk factors for occupational TB infection in HCWs. A University Hospital in Lima, Peru, documented very high rates of active pulmonary TB among HCWs and occupational acquisition in the laboratory was associated with HCW-to-HCW transmission [19]. Gamma delta TCR+ cells, mainly the mycobacterium reactive subset that expresses a T cell receptor (TCR) encoded by Vc9 and Vd2 gene segments, are involved in the protective human immune response against tuberculosis infection as shown by expansion in response to non-peptide antigens of Mtb [20,21] and their accumulation in BCG-vaccinated and purified protein derivative skin test positive (PPD+) individuals with latent tuberculosis infection [22–26]. Gamma delta TCR+ cells effector functions such as production of TNF-a and IFN-c, capacity to mediate cytolysis, and ability to reduce the viability of extracellular and intracellular Mtb have been associated with protective immunity [26–29]. Our studies have shown that despite HCWs having an increased absolute number of cd TCR+ cells, Mantoux skin reactions of 11–21 mm and moderate to high IFN-c production in CD4+, CD8+ and cd+ TCR+ cells stimulated with Mtb, active TB still developed [30]. The HCWs also showed low levels of IL-4 in CD8+ and cd+ TCR+ cells and this profile predicted the subsequent development of TB. Gamma delta T cell involvement during TB infection has been demonstrated by the presence of specific Vc9/ Vd2 T cells in children with TB and this was associated with increased proliferation of these cells, reduced IFNc production and granulysin expression [31]. After successful chemotherapy the childrenÕs responses were reversed [31]. In healthy Mantoux positive donors and TB patients with pleuritis, the number of Mtb reactive cd TCR+ cells increased and was higher than those from patients presenting with advanced pulmonary or miliary TB, suggesting that these cells may be involved in immune resistance [32]. A recent study shows that nonMHC restricted lytic activity was mainly mediated by cd TCR+CD56+ and/or CD16+ cells in TB patients [33]. The higher the non-MHC restricted lytic activity, the greater the loss in CD4+ and CD8+ mediated T cell cytotoxicity and this was associated with severity of pulmonary involvement [33]. In the current study, we stimulated PBMC from fifteen HCWs, fifteen Non-HCWs and twenty TB patients with Mtb and assayed expression of the T cell activation markers CD25+ and HLA-DR+, cytotoxicity and production of IFN-c and IL-4 by flow cytometry. These individuals were studied longitudinally for five years after the assays were completed, and during this time six HCWs developed active TB. 2. Materials and methods 2.1. Subjects Thirty healthy Portuguese HCWs (n = 15) and NonHCWs (n = 15) were recruited from Lisbon, Portugal. Participants in this study had been vaccinated with M. bovis bacille Calmette–Guérin (BCG) at 1 year of age, again on school entry at 5–6 years and at 11–13 years of age for those whose Mantoux tests were negative (induration <5 mm) [34]. All the donors were HIV-1 and HIV-2 seronegative. The subjects did not present with any clinical symptoms of atopic disorders or parasitic/helminth infestation. The HCWs and Non-HCWs at the time of assay were all deemed healthy and free of clinical TB by clinical and laboratory evaluation performed by the Serviço de Patologia Clı́nica, Hospital Egas Moniz, Lisbon, Portugal. The subjects were studied longitudinally for five years after the time of assay for TB presentation. During the study time period of five years there was no hospital or university staff turnover within the recruited groups of HCWs or NonHCWs, allowing follow up of all the studied subjects. Table 1 describes individual clinical characteristics of the HCWs (n = 15), Non-HCWs (n = 15) and TB patients (n = 20) who participated in the study. The HCWs were recruited from a hospital in Lisbon, Portugal. The HCWs had a history of five to nine years of working in hospital wards with patients infected with variety of infectious diseases, including active TB and HIV/TB of varying cavitatory status. The HCWs had never been given preventive therapy for TB infection. At the time of assay, active TB had been excluded in these individuals by yearly screening using chest radiography. Six HCWs developed active TB disease 2–4 years after the time of assay. Clinically active class 3 pulmonary TB was diagnosed by chest radiograph and confirmed by positive acid-fast bacillus (AFB) smear or positive sputum culture (SCP) [35]. An active TB infection was diagnosed by the existence of middle or lower lung zone infiltrate with ipsilateral hilar adenopathy D.J. Ordway et al. / FEMS Immunology and Medical Microbiology 43 (2005) 339–350 341 Table 1 Clinical characteristics of HCWs (n = 15), Non-HCWs (n = 15) and TB patients (n = 20) who participated in the study Subjectsa Age Sexb Mantouxc Occupation/wardd TB/diagnosise Time of diagnosisf HCWs HCWs HCWs HCWs HCWs HCWs HCWs HCWs HCWs HCWs HCWs HCWs HCWs HCWs HCWs Non-HCWs (n = 15) Minimal TB patients (n = 10) Advanced TB patients (n = 10) 29 33 42 41 40 38 33 39 28 40 39 38 44 47 41 28.3 ± 5.0 33. ± 7.25 37.0. ± 4.12 F F F F M M M M F M F M M M M 7F/8M 5F/5M 3F/7M 21 11 12 15 13 20 30 25 21 22 9 10 11 15 11 7.4 ± 1.54 6.6 ± 1.07 4.4 ± 0.96 N/TBHIV N/TBHIV N/TBHIV N/TBHIV C/TBHIV C/TB C/TBHIV C/TB N/TB C/TB N/TB C/TB C/TB C/TBHIV C/TBHIV U P P TB/AFB TB/AFB TB/SCP TB/AFB TB/SCP TB/AFB NTB/– NTB/– NTB/– NTB/– NTB/– NTB/– NTB/– NTB/– NTB/– NTB/– TB/AFB/SCP TB/AFB/SCP 24 28 28 26 34 48 – – – – – – – – – – – – a Individual ages and time of diagnosis is provided for all the health care workers (HCWs). The mean and ±SD are provided for the non-health care workers (Non-HCWs) and the minimal and advanced tuberculosis (TB) patients. b Sex of donors female (F) or male (M). c Mantoux skin test (mm), that uses soluble antigens from M. tuberculosis, PPD, induces the classic delayed type hypersensitivity (DTH) response and is used clinically to identify infection with M. tuberculosis in persons who do not have tuberculosis disease. d Occupation of clinician (C), nurse (N), university personnel (U) or patient (P)/infectious disease wards with TB/HIV patients (TBHIV) or TB (TB) patients only (TB). e Developed TB (TB) or no TB (NTB). Method of TB diagnosis, positive acid-fast bacillus (AFB) smear or sputum culture positive (SCP). TB patients with acid fast bacilli in sputum smears, were classified as mild (few 2+) or advanced (numerous 4+) TB. f Time in months between time of assay and diagnosis of TB. being present [35]. The HCWs that developed disease were seronegative for HIV-1 and HIV-2 infection. Following a clinical diagnosis of TB, the HCWs were started on chemotherapy according to WHO/American Thoracic Society recommendations [35] (daily therapy with isoniazid, rifampicin, ethambutol, and pyrazinamide) which resulted in resolution of disease. The Non-HCWs were recruited from university personnel in Lisbon, Portugal. The Non-HCWs had no previous history of contact with TB patients; there was no history of TB in family members, and these individuals had never been admitted to hospital or presented with any physical symptoms associated with TB disease. Chest radiography could not be performed on the Non-HCWs. Five years after the time of assay none of the fifteen subjects had been diagnosed with clinical TB. Portuguese patients with active pulmonary TB (n = 20) were recruited from Serviço de Doenças Infecciosas e Parasitárias, Serviço de Pneumologia and Serviço de Medicina II of the Hospital Egas Moniz, Lisbon, Portugal, Pneumologia IV of the Pulido Valente Hospital, Lisbon, Portugal and from Hospital Dr. José Maria Antunes Junior, Torres Vedras, Portugal. Clinically active class 3 pulmonary TB patients [35], who were seronegative for HIV-1 and HIV-2 infection were diagnosed clinically, by chest radiograph and confirmed by positive acid-fast bacillus (AFB) smear or positive sputum culture (SCP). The majority of patients were new cases of TB who had not received any therapy (n = 16) or who had not received any therapy or had a maximum of seven days of chemotherapy (n = 4) according to WHO recommendations (daily therapy with isoniazid, rifampicin, ethambutol, and pyrazinamide). There were no statistical differences in the capacity of the patientsÕ PBMC to proliferate or produce IFN-c or IL-5 in response to Mtb before treatment or after five days of treatment (data not shown). TB patients were further subdivided into those according to the extent of pulmonary TB with minimal (Min TB) (n = 10) and with advanced disease (Adv TB) (n = 10). The division of the subgroups were based on acid fast bacilli in sputum smears (few 2+ or numerous 4+) and radiographic features of minimal cavitation of apical or posterior segments of the right and left upper lobes and advanced involvement of lung parenchymal with presence of patchy bronchopneumonia indicative of progressive disease [35]. Informed consent was obtained from all study volunteers or their parents or guardians. Human experimentation guidelines of the US Department of Health and Human Services and of Instituto de Higiene and Medicina Tropical, Hospital Egas Moniz and London School of Hygiene and Tropical Medicine review boards were followed. 342 D.J. Ordway et al. / FEMS Immunology and Medical Microbiology 43 (2005) 339–350 2.2. Blood collection Seventy millilitres of intravenous blood sample was obtained from each subject and twenty millilitres transferred into 50 U of preservative-free sodium heparin (Monoparin, CP Pharmaceuticals Ltd, Wrexham, UK) and used in flow cytometry assays. The remaining fifty millilitres of blood was defibrinated and used in cytotoxic lymphocyte assays. Blood samples were processed as soon as possible after phlebotomy. Mantoux skin tests (2TU) were performed immediately afterwards; all the subjects were Mantoux positive (P5 mm induration). 2.3. Antigens PPD batch RT44 was purchased from the Statens Serum Institute, Copenhagen, Denmark and used at a final concentration of 10 lg/ml. The antigens were all tested to determine the optimal concentration for use. The mitogen phytohemagglutinin (PHA) (Difco, Detroit, MI, USA) was used as a positive control at a final concentration of 8 lg/ml (data not shown). The nonmycobacterial antigen streptokinase–streptodornase (SK/SD (‘‘Varidase’’), Wyeth Laboratories, Maidenhead, UK) was used at 250 U/ml. 2.4. Bacteria Mycobacterium tuberculosis H37Rv (ATCC 25618) was grown in Middlebrook 7H9 broth (Difco) containing 0.05% w/v Tween 80 and 10% v/v OADC supplement (Becton Dickinson, UK), or on Middlebrook 7H10 agar (Difco) plus 10% v/v OADC supplement. Stocks of mycobacteria to be used for in vitro stimulation were first grown in 10 ml of liquid medium until an OD600 between 0.5 and 1.0 was reached, washed once in phosphate buffered saline (PBS), resuspended in 5 ml of sterile PBS and stored at 70 C in aliquots until required, and then, added to PBMC cultures at a concentration estimated to give 1 colony forming unit (CFU) per monocyte. Individual donors cell counting by the use of the trypan blue and napthanol blue black method (for adherent cell counting) is cited as described by Ordway et al. [36,37]. Infection of 1 bacteria per monocyte was verified in healthy subjects and TB patients by lysis of Mtb infected macrophages and the plating on agar to obtain CFU of phagocytosed, viable bacteria. The number of phagocytosed bacteria in healthy donors and TB patients was further quantified by adherence of monocyte-derived macrophages using chamber slides and intracellular staining of bacteria with a Bacto TB stain set K for acid fast bacteria. 2.5. Absolute lymphocyte counts, expression of activation markers CD25 and HLA-DR on T cells and intracellular cytokine staining Aliquots of blood (100 ll) were incubated with 10 ll of fluorescent labelled antibody (Becton Dickinson, Oxford, UK) in 6 ml tubes (Greiner) for 30 min at 4 C in the dark. Red blood cells were lysed with lysis solution and washed once in cold PBS. These blood samples were processed for absolute lymphocyte counts. Activation markers or intracellular cytokine staining was performed by isolation of PBMC which were resuspended at 1 · 106 cells/ml in growth medium and 2 ml of this suspension incubated with antigen or without antigen and incubated at 37 C in the presence of 5% CO2. The presence of intracellular IFN-c and IL-4 was analysed using a commercial IS Ultra Cell-Fix Perm Kit (Immune Source, CA, USA). Assays were performed as described by Ordway et al. [36,37]. Fluorescence was analysed by gating on lymphocytes according to their characteristic forward and side scatter profile and measuring intensity of fluorscence emited by fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP) and allophycocyanin (APC). 2.6. Cytotoxic lymphocyte assays Preparation of the blood sample prior to PBMC isolation was carried out by defibrination. Isolated PBMC were resuspended at 2 · 106 cells/ml in growth medium. Effector cells were prepared by incubating 2 · 106 cell/ml with PPD (10 lg/ml), M. tuberculosis H37Rv (1:1, bacteria:monocyte ratio), Listeria monocytogenes (1:1, bacteria:monocyte ratio) or no antigen, in 24 well tissue culture plates (Nunc, Roskilde, Denmark) for 7 days at 37 C in an atmosphere of 5% CO2. Target cells were prepared by incubating 150 ll of the cell suspension in 96 well plates at 37 C, in 5% CO2. Approximately 10% of the cells are monocytes and adhere to the plate. On day 1, non-adherent cells were removed by gently pipetting up and down 5 times and 100 ll of the supernatant was removed and replaced with 100 ll fresh growth medium. On day 6, 100 ll of growth medium was removed from each well and 25 ll of growth medium containing 2 lCi (74 Bq) 51Cr (Amersham International Plc, Buckinghamshire, UK) and 25 ll antigen or no antigen was added to each well. On day 7 the excess 51 Cr and antigen was discarded by washing the cells 3 times with warm growth medium and then targets were left with 50 ll growth medium. On day 7 the effector cells were isolated and washed once in HankÕs balanced salt solution (HBSS) (Sigma–Aldrich, Madrid, Spain) and resuspended in growth medium at the correct concentration for 20:1 effector:target ratios, and added in 100 ll volumes to the target cells. The plate was then D.J. Ordway et al. / FEMS Immunology and Medical Microbiology 43 (2005) 339–350 incubated at 37 C, in 5% CO2 for 6 h. On day 8, the supernatant (150 ll) was removed from each well and placed into Beckman Ready Caps. To lyse the remaining cells 150 ll of 5% SDS (Sigma–Aldrich, Madrid, Spain) was added to each well and this ‘‘pellet’’ was then transferred to a second set of Beckman Ready Caps. Freshly prepared 2% paraformaldehyde (Sigma–Aldrich, Madrid, Spain) was added in 50 ll aliquots to each Ready Caps to inactivate any infectious material for 2–3 h. The activity of each individual tube was read in counts per minute (cpm) using a gamma counter (Beckman Coulter, Miami, FL, USA) and the specific activity calculated as % isotope releaseðIRÞ ¼ cpm supernatant cpm supernatant þ cpm pellet 100 Specific isotope release ¼ % IR test wells % IR medium control wells 2.7. Depletion of cd T cells from PBMC Positive selection of cd TCR+ cells was carried out using the MACS (Miltenyi Biotech, Surrey, UK) separation technique. PBMC which had been incubated with or without antigen for 6 days were harvested and resuspended in PBS containing 1% bovine serum albumin (PBS-BSA) (Sigma–Aldrich) at a concentration of 1 · 107 cells/80 ll PBS-BSA. The cell suspension was mixed with 20 ll of MACS anti-human cd TCR+ antibody coated micro-beads (Miltenyi Biotech, Surrey, UK) and incubated for 4 C for 15 min, washed once in 10 ml of PBS-BSA and resuspended in 500 ll of PBS-BSA. A sterile MACS A1 column was placed in the Midi/Mini magnetic separator (Miltenyi Biotech, Surrey, UK) and prepared by passing 2–3 volumes of ice cold PBS-BSA through it, followed by the magnetically labeled cell suspension and more PBS-BSA. The wash volume was collected and contained the cd TCR+ depleted population (CD3+cd TCR). The column was removed from the magnetic separator and the bound cell fraction was eluted with 3 washes of PBS-BSA. The collected volumes were pooled and contained the cd TCR+ enriched population. The cells present in the cd TCR+ depleted and cd TCR+ enriched populations were counted and stained with fluorescent labelled antibodies, to assess by flow cytometry, the cell yield, CD3+cd TCR and cd TCR+ T cell purity. 2.8. Statistical analysis Data is presented using means ± standard deviation (SD) values from triplicate or quadruplicate samples. 343 A non-parametric method, the Mann–Whitney U test was used to assess statistical significance between groups of data. Correlations between sets of data were calculated using the Spearman rank correlation. 3. Results 3.1. Absolute cd TCR+ and Vc9+/Vd2+ cell counts in HCWs, Non-HCWs and TB patients The absolute numbers of cd TCR+, Vc9+/Vd2+, CD4+ and CD8+ T cells in whole blood from TB free HCWs, HCWs who developed TB, Non-HCWs and TB patients was assayed using flow cytometry. Firstly, the results obtained indicated that the absolute numbers of CD4+ and CD8+ T cells present in whole blood of HCWs who remained healthy, HCWs who developed TB, Non-HCWs and TB patients were not significantly different (p > 0.05, Mann–Whitney U test) (data not shown). However, as shown by Fig. 1(a), the cd TCR+ T cells were mainly composed of the Vc9+/ Vd2+ phenotype and were at significantly higher quantities in the blood of HCWs and TB patients compared with Non-HCWs (HCWs, p = 0.001; TB, p = 0.003, Mann–Whitney U test). These results suggest that the presence of large numbers of cd TCR+ cells which are mainly composed of the Vc9+/Vd2+ phenotype, does not afford protection against the eventual development of TB. The results also suggest that the two groups of HCWs, in prolonged contact with patients presenting with active TB, have similar absolute counts of cd TCR+ and Vc9+/Vd2+ T cells as those presented by the TB patient group. Interestingly, significantly higher numbers of cd TCR+ were present in TB patients with advanced disease while Vc9+/Vd2+ (cd TCR+, p = 0.03; Vc9+/Vd2+, p = 0.85, Mann–Whitney U test) cell counts remained similar in both minimal and advanced TB groups (Fig. 1(b)). Therefore the participation of other T cell phenotypes which express the cd TCR+ receptor must be involved in this increase in number. 3.2. T cell activation in HCWs, Non-HCWs and TB patients Ex vivo lymphocytes from subjects were stimulated with PPD and Mtb and the expression of CD25 on T cells was studied. The percentage of PPD or Mtb activated CD4+, CD8+ and cd TCR+ cells expressing CD25 correlated (CD4+, R = 0.729, CD8+, R = 0.792, cd TCR+, R = 0.741, Spearman rank correlation) with the expression of HLA-DR in the subject groups (data not shown). The expression of CD25+ on CD4+, CD8+ and cd TCR+ cells after stimulation with antigen was considered to identify an activated cell population 344 D.J. Ordway et al. / FEMS Immunology and Medical Microbiology 43 (2005) 339–350 Fig. 1. The numbers of cd TCR+ and Vc9+/Vd2+ cells were assessed in peripheral blood samples as described in Section 2. There were no significant differences in absolute counts of cd TCR+ (j) and Vc9+/Vd2+ cells (h) cells between HCWs who subsequently developed TB and HCWs who remained healthy. (a) Ex vivo blood samples from HCWs without TB (HCWs w/o TB) (n = 9), HCWs with TB (HCWs w/TB) (n = 6), Non-HCWs (Non-HCWs) (n = 15) and TB patients (TB) (n = 20) were tested for the presence of cd TCR+ and Vc9+/Vd2+ cells. (b) TB patients with advanced disease (n = 10) (Adv TB) show increased cd TCR+ (d) but not Vc9+/Vd2+ (s) cells compared to TB patients with minimal disease (Min TB) (n = 10). Higher numbers of cd TCR+ were present in TB patients with advanced disease compared to those with minimal disease (statistical differences: *p = 0.03) while Vc9+/Vd2+ cell counts remained similar in both minimal and advanced TB groups (p = 0.85, Mann–Whitney U test). and was analyzed in HCWs, Non-HCWs and TB patients (Fig. 2). As expected, PPD preferentially activated cells of the CD4+ T cell phenotype, while CD8+ and cd TCR+ cells were preferentially activated by Mtb. HCWs who developed TB showed no significant differences (p > 0.10, Mann–Whitney U test) when ex vivo cells were stimulated with PPD and Mtb. HCWs who developed TB showed significantly reduced percentages of CD8+CD25+ after cells were stimulated with Mtb (p = 0.02, Mann–Whitney U test). In HCWs the percentage of CD8+CD25+ and cd TCR+CD25+ was significantly increased (CD8+CD25+, p = 0.029; cd TCR+CD25+, p = 0.028, Mann–Whitney U test) in comparison to Non-HCWs. TB patients demonstrated significantly reduced percentages of CD25 positive CD4+ and CD8+ T cells but not of cd TCR+ cells after PPD and Mtb stimulation (CD4+CD25+, p = 0.01; CD8+CD25+, p = 0.046; cd TCR+CD25+, p = 0.79, Mann–Whitney U test) compared to HCWs. TB pa- tients with advanced disease showed reduced mean percentages of CD25 expression on CD4+ and CD8+ T cells, but not on cd TCR+ cells (p = 0.003, p = 0.022, p = 0.474, Mann–Whitney U test) in comparison to minimal disease TB patients. 3.3. Intracellular IFN-c and IL-4 in HCWs and in TB patients The relationship of the in vitro responses to PPD and Mtb by CD4+, CD8+ and cd TCR+ cells from HCWs who remained healthy, HCWs who progressed to active TB and patients presenting with minimal and advanced TB was studied by evaluating the percentage of these populations with intracellular IFN-c. Of ten HCWs tested for the production of IL-4, six showed increased percentages of IL-4 positivity in CD8+ and cd TCR+ cells after stimulation with Mtb [30]. All six of these HCWs with increased percentages of IL-4 progressed D.J. Ordway et al. / FEMS Immunology and Medical Microbiology 43 (2005) 339–350 345 Fig. 2. Expression of CD25 on PBMC stimulated with mycobacterial antigens. PBMC were stimulated for 6 days with PPD, Mtb or no antigen (No Ag). Expression of CD25 was assessed on CD4+, CD8+ and cd TCR+ lymphocytes from HCWs without TB (HCWs w/o TB) (n = 9), HCWs with TB (HCWs w/TB) (n = 6), patients with minimal (Min TB) (n = 10) and advanced TB (Adv TB) (n = 10) were tested as described in Section 2. Individual results are expressed with the mean (horizontal bar). HCWs showed significantly increased CD25 expression on CD8+ and cd TCR+ cells in comparison to Non-HCWs (statistical differences: *p = 0.029; **p = 0.028). HCWs who developed TB showed significantly reduced CD25 expression on CD8+ T cells compared to HCWs without TB (statistical differences: ***p = 0.020). 346 D.J. Ordway et al. / FEMS Immunology and Medical Microbiology 43 (2005) 339–350 to develop active TB within 2–4 years of the immunological testing (Fig. 3(b)) [30]. HCWs who developed TB showed moderate percentages of T cells making IFNc, particularly cd TCR+ cells. HCWs who did not develop TB showed higher percentages of IFN-c in T cells and no IL-4 in CD8+ and cd TCR+ cells. PBMC from TB patients were stimulated for 6 days with PPD and Mtb and assayed for intracellular IFNc and IL-4 by flow cytometry (Fig. 3(a) and (b)). Interferon-gamma in the group of TB patients demonstrated lower mean percentage of cells when stimulated with PPD (CD4+, 10.51 ± 3.75; CD8+, 4.20 ± 1.55; cd TCR+, 0.33 ± 0.28) and with Mtb (CD4+, 6.70 ± 1.76; CD8+, 9.76 ± 5.79; cd TCR+, 2.10 ± 1.50) when compared to HCWS or Non-HCWs. The higher percentages of IFN-c positive T cells in cultures from HCWS or Non-HCWs was significant (p < 0.050) following PPD and Mtb PBMC stimulation for all T cell populations (data not shown). TB patients with advanced disease showed significantly lower percentages of IFN-c staining in all T cell subsets (p < 0.032) to PPD and Mtb in comparison to TB patients with minimal disease (Fig. 3(a)). TB patients with advanced disease showed significantly increased percentages of IL-4 staining following stimulation with Mtb in CD4+ (p = 0.003, Mann–Whitney U test), CD8+ (p = 0.001, Mann–Whitney U test) Fig. 3. Intracellular IFN-c and IL-4 in T cell subsets from HCWs and TB patients. Intracellular IFN-c and IL-4 was assessed in T cell subsets from HCWs w/o TB (n = 4), HCWs w/TB (n = 6) and TB patients with minimal (n = 10) (Min TB) and advanced disease (n = 10) (Adv TB). PBMC from donors were stimulated for 6 days with PPD, Mtb or incubated without antigen. Subsets of each population were stained for expression of CD4+, CD8+ and cd+ TCR+ cell surface markers in combination with intracellular IFN-c (a) and IL-4 (b). The non-stimulated cells showed low IFN-c and IL-4 staining with <0.06% positive for either intracellular cytokine. Results are expressed as the groups mean percentage of cells staining positive for IFN-c or IL-4 ±SD. HCW with TB showed increased IL-4 in CD8+ T-cells (statistical differences: ***p = 0.002) compared to HCWs without TB. TB patients with advanced disease showed increased IL-4 CD4+ (statistical differences: *p = 0.003), CD8+ and cd TCR+ cells (statistical differences: **p = 0.001) in comparison to TB patients with minimal disease. D.J. Ordway et al. / FEMS Immunology and Medical Microbiology 43 (2005) 339–350 and cd TCR+ cells (p = 0.001, Mann–Whitney U test) in comparison to TB patients with minimal TB (Fig. 3(b)). Increased IL-4 production in CD8+ and cd TCR+ cells after stimulation with Mtb in TB patients correlated with disease severity (CD8+, R = 0.585; cd TCR+, R = 0.866, Spearman rank correlation). 3.4. T-cell cytotoxic responses in HCWs and TB patients Gamma delta TCR+ cells were separated magnetically from Mtb stimulated PBMC of the donor groups and assessed for purity by flow cytometry. The percent purity of cd TCR+ cells was 96.32% (n = 50) and these cells along with the whole PBMC and CD3+ cd TCR cells were utilized in a 6 h cytotoxicity assay. The cd TCR+ cells ability to cause target cell lysis of antigen pulsed macrophages was assessed in HCWs who developed TB and HCWs who remained healthy (Fig. 4). PBMC from HCWs who remained healthy and HCWs who developed TB did not show any differences in percentage of target cell lysis (p = 0.905, Mann–Whitney U test). However, HCWs who subsequently developed TB demonstrated significantly lower percentages of CD3+ cd TCR cell lysis of target cells (p = 0.003, Mann– Whitney U test) while separated cd TCR+ cells showed significantly increased percentages of cytotoxicity (p = 0.001, Mann–Whitney U test). A correlation (R = 0.728, Spearman rank correlation) between the increase of cd TCR+ cell and decrease of CD3+cd TCR cell cytotoxicity was present in HCWs. Fig. 4. Cytotoxic responses of HCWs and TB patients after cell stimulation with Mtb. Cytotoxic responses of cd TCR+ cells, CD3+ cd TCR cells and PBMC from HCWs without TB (HCWs w/o TB) (n = 9) and HCWs with TB (HCWs w/TB) (n = 6) and TB patients with minimal (Min TB) (n = 10) and advanced disease (Adv TB) (n = 10) were tested. Mtb stimulated cd TCR+ cells, CD3+ cd TCR cells and PBMC effector cells from donors were incubated with Mtb stimulated cells, and unstimulated target cells in a 6 h cytotoxicity assay. Results are expressed as percentage (%) of cytotoxicity. HCWs with TB showed lower CD3+cd TCR cell lysis of target cells (statistical differences: *p = 0.003) while separated cd TCR+ cells showed increased percentages of cytotoxicity compared to HCWs without TB (statistical differences: **p = 0.001). 347 PBMC stimulated with Mtb from HCWs, NonHCWs and TB patients showed minor differences in the mean percentage cytotoxicity (±SD, HCWs, 45 ± 9; Non-HCWs, 30 ± 7; TB, 33 ± 7). Cytotoxicity was significantly increased in HCWs (p = 0.01, Mann– Whitney U test) in comparison to the other donor groups. Minimal TB patients demonstrated increased (p = 0.001, Mann–Whitney U test) CD3+cd TCR cytotoxic responses. There were no statistically significant differences (p = 0.130, Mann–Whitney U test) between the cd TCR+ cytotoxic responses in TB patients with minimal or advanced disease states. However, TB patients with advanced disease who had high percentages of cd TCR+ cytotoxic responses, showed low percentages of CD3+cd TCR cell cytotoxic responses. A correlation (R = 0.613, Spearman rank correlation) between the percentage increase of cd TCR+ cell and percentage decrease of CD3+ cd TCR cell cytotoxicity was present in TB patients. 4. Discussion It has been demonstrated that HCWs with repeated exposure to Mtb demonstrate an early increased percentage of IL-4 production in CD8+ and cd TCR+ cells prior to the onset of disease [30] compared to those HCWs who remained healthy. In the present report, we demonstrate that these same HCWs who developed TB, have a reduced activation of CD8+ T cells and lysis of Mtb infected macrophages by CD3+ cd TCR cells. The reduced percentage of CD3+ cd TCR cell cytotoxicity in HCWs who developed TB was associated with an increase in percentage of cd TCR+ cell cytotoxicity. At the other end of the clinical TB spectrum, TB patients with advanced disease displayed decreased percentage of CD8+ and CD4+ T cell activation and increased percentages of IL-4 in CD8+ and cd TCR+ cells which correlated with disease severity. These patients also showed augmented cd TCR+ cytotoxic responses when CD3+ cd TCR cell cytotoxic responses were reduced. This study suggests that repetitive exposure to Mtb may lead to early signs of decreased ab T cell immune responses which may lead to progression of disease. Studies on TB infection in HCWs in Portugal [13] show there is a high rate of hospital HCWs with TB due to the obvious lack of infection control facilities. There may also be failure of HCWs to seek early treatment, and lack of disclosure of the numbers of such staff with occupationally-acquired disease. The six out of ten HCWs who went onto develop TB in our study were not assayed by RFLP for bacterial identification. Therefore we can not identify the source of Mtb transmission. Occupational acquisition of TB in our study supports the study by Alonso-Echanove et al. [19] in that among 348 D.J. Ordway et al. / FEMS Immunology and Medical Microbiology 43 (2005) 339–350 our HCWs who developed TB, the common job categories were nurses (4) and clinicians (2) who had worked for more than 5 years in the infectious disease wards. Our results show that the absolute number of circulating peripheral blood cd TCR+ and Vc9+/Vd2+ subset is increased in HCWs with repeated exposure to Mtb compared to Non-HCWs. These findings support other studies which demonstrate that cd TCR+ and Vc9+/Vd2+ T cell subsets are expanded by Mtb [29,38,39] and are present in higher quantities in healthy PPD+ donors [25]. Increased cd TCR+ cells and Vc9+/ Vd2+ T cell subsets in the peripheral blood have been shown to be increased in other intracellular parasitic infections, such as malaria [40], visceral leishmaniasis [41], listeriosis [42], and tularaemia [43]. The increase in the cd TCR+ cell subset has been associated with a protective immune response, although increased quantities of these cells does not stop the disease process. Tularaemia is an acute febrile disease and studies have demonstrated that the proportion of circulating human cd TCR+ cells increases after the first week of onset of disease and remains elevated for more than one year [44]. The authors suggest that the prolonged course of elevation may suggest a role for cd TCR+ cell immunoregulation or modulation of an inflammatory response. Thus it is plausible that our HCWs maintained elevated cd TCR+ cells/functions until the time of onset of TB. The slow growth and chronic nature of Mtb and other mentioned intracellular parasites, may result in inhibition of numbers and/or function of CD4+ and CD8+ T cells, with an increase in cd TCR+ cells. However, in our study, HCWs who were repeatedly exposed to aerosolised Mtb showed increased numbers of peripheral blood cd TCR+ and Vc9+/Vd2+ T cell subsets and this response did not prevent the development of TB disease. Absolute numbers of cd TCR+ and Vc9+/ Vd2+ subsets could not be utilised for diagnostic purposes as HCWs who developed TB and those who did not demonstrated similar numbers of these cells. Although the absolute numbers of these sub-populations are similar to those reported by others for HCWs and Non-HCWs [32,45], they are at variance with the studies of others who claim that the number of these cells are similar in both TB free and TB patients [25]. TB patients in our study exhibited similar high numbers of circulating peripheral blood cd TCR+ and Vc9+/ Vd2+ subsets when compared to HCWs, supporting other reports [33]. Tuberculosis patients with advanced disease had higher numbers of cd TCR+ while Vc9+/ Vd2+ subsets remained the same when compared to TB patients with minimal disease. This data supports other studies [33,46] implicating other phenotypes which express cd TCR+ cells bearing CD56+ and/or CD16+ molecules, resulting in an increase in these subsets cytotoxicity as pulmonary involvement becomes more severe. It is feasible that during prolonged antigenic stimulation of the immune system or during the slow tuberculosis disease process, a gradual decrease in the lytic activity of the main phenotypes, CD4+ and CD8+ T cells occurs resulting in an increase of cd TCR+ cytotoxicity. Gamma delta TCR+ cells have also been shown to be involved in other important roles, such as regulation of inflammation, and rapid immobilisation resulting in an influx of cells into the local site of infection taking place in acute infection. Studies by Saunders et al. [47] have demonstrated that isolates of M. avium that are less virulent and induce protective T cell specific immunity are unaffected by the absence of cd TCR+ cells, while highly virulent strains that induce poor protective immunity in the absence of cd TCR+ cells lead to reductions in neutrophil influx and tissue damage in the lungs of mice. This latter study highlights the possibility that in mice infected with highly virulent strains inducing poor protective immunity, cd TCR+ cells may also compensate for reduced CD4+ and CD8+ T cell protective immunity in the lungs by increasing the influx of inflammatory cells to the lungs. In our study, HCWs who developed TB, were not assessed for bacterial identification therefore we can not rule out that they could have been infected with an M. tuberculosis strain of higher virulence, resulting in poor induction of protective immunity and increased cd TCR+ responses. In conclusion, our results suggest that individuals with multiple exposure to Mtb show an early compensatory mechanism involving an increase in numbers and lytic responses of cd TCR+ cells. However these cd TCR+ responses do not protect such TB contacts from the subsequent development of TB infection. As the disease progresses, and pulmonary involvement becomes more severe, cd TCR+ T cell compensation becomes more predominant, and these responses thus provide a measure of the extent of clinical TB. Acknowledgements This work was supported by a grant from the European Union (IC18*CT970236), Comissão Nacional de Luta contra a SIDA (001074) and Fundação Calouste Gulbenkian project ‘‘Pesquisa de novos marcadores de imunidade celular para a detecção precoce da infecção por M. tuberculosis’’. We would like to thank Dr. Ian Orme and Dr. Mercedes Gonzalez Juarrero, Colorado State University, Colorado, for reviewing the manuscript and Dr. D. Smith, from the London School of Hygiene and Tropical Medicine, UK, for donating the Mtb H37Rv strain, the Centro de Malária e Outras Doenças Tropicais (CMDT/IHMT), the Department of Microbiology at IHMT and the Hospital Egas Moniz, Lisbon, Portugal for their collaboration. We would like to thank the Serviço de Doenças Infecciosas e Parasitárias, Serviço de Pneumologia and Serviço de Medi- D.J. Ordway et al. / FEMS Immunology and Medical Microbiology 43 (2005) 339–350 cina II of the Hospital Egas Moniz, Lisbon, Portugal, Pneumologia IV of the Pulido Valente Hospital, Lisbon, Portugal and the Hospital Dr. José Maria Antunes Junior, Torres Vedras, Portugal for their collobration. We would like to thank Cristina Graça Lobo for her dedication on this project, although she is no longer with us. Lastly we would like to thank all the participants in the study. References [1] Direcção Geral de Saúde. (2002) Tuberculose em Portugal 2000, Lisboa. 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