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FEMS Immunology and Medical Microbiology 43 (2005) 339–350
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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.
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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
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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).
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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.
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