Sahmoudi et al. BMC Immunology
(2018) 19:33
https://doi.org/10.1186/s12865-018-0266-8
RESEARCH ARTICLE
Open Access
Immune activation and regulatory T cells in
Mycobacterium tuberculosis infected lymph
nodes
Karima Sahmoudi1,2, Hassan Abbassi3, Nada Bouklata4, Mohamed Nouredine El Alami3, Abderrahmane Sadak2,
Christopher Burant5, W. Henry Boom6, Rajae El Aouad1, David H. Canaday6† and Fouad Seghrouchni1*†
Abstract
Background: Lymph node tuberculosis (LNTB) is the most frequent extrapulmonary form of tuberculosis (TB).
Studies of human tuberculosis at sites of disease are limited. LNTB provides a unique opportunity to compare local
in situ and peripheral blood immune response in active Mycobacterium tuberculosis (Mtb) disease. The present study
analysed T regulatory cells (Treg) frequency and activation along with CD4+ T cell function in lymph nodes from
LNTB patients.
Results: Lymph node mononuclear cells (LNMC) were compared to autologous peripheral blood mononuclear cells
(PBMC). LNMC were enriched for CD4+ T cells with a late differentiated effector memory phenotype. No differences
were noted in the frequency and mutifunctional profile of memory CD4+ T cells specific for Mtb. The proportion of
activated CD4+ and Tregs in LNMC was increased compared to PBMC. The correlation between Tregs and activated
CD4+ T cells was stronger in LNMC than PBMC. Tregs in LNMC showed a strong positive correlation with Th1
cytokine production (IL2, IFNγ and TNFα) as well as MIP-1α after Mtb antigen stimulation. A subset of Tregs in
LNMC co-expressed HLA-DR and CD38, markers of activation.
Conclusion: Further research will determine the functional relationship between Treg and activated CD4+ T cells at
lymph node sites of Mtb infection.
Keywords: Tuberculosis, Lymphadenitis, Lymph node, Treg cells, conventional T cell, activation
Background
Mycobacterium tuberculosis (Mtb) infection is a major
global health problem with approximately 10.4 million
cases and 1.4 million deaths from tuberculosis (TB) in
2015 [1]. Furthermore, one-third of the world’s population is thought to be infected by Mtb. Extra pulmonary
TB represents approximately 20% of clinical TB disease.
Lymph node tuberculosis (LNTB) is the most frequent
extrapulmonary form [1].
Cellular immune responses play a pivotal role in control of Mtb infection with CD4+ T cells having the central role. After infection CD4+ T cells undergo activation
manifested by expression of surface molecules including
* Correspondence: fseghrouchni@yahoo.fr
†
David H. Canaday and Fouad Seghrouchni contributed equally to this work.
1
Laboratory of Cellular Immunology, National Institute of Hygiene, 27,
Avenue Ibn Batouta, PB 769, 11400 Rabat, Morocco
Full list of author information is available at the end of the article
HLA-DR and CD38 [2, 3]. Functionally, CD4+ T cells
control infection by producing Th1 and Th17 cytokines
[4]. Polyfunctional T cells, defined by their ability to
co-express more than one cytokine, have been associated
with protection against Mtb disease [4–6].
At sites of infection, immune responses are modulated
by T regulatory cells (Tregs) [7, 8]. Tregs express CD3,
CD4, high levels of CD25, low levels of the IL-7 receptor
α-chain (CD127) and the intracellular marker forkhead
box p3 (FoxP3) [9].
The relationship between Tregs and immune activation at sites of Mtb disease is not clear [10, 11]. The objective of the present study was to evaluate the
interaction between Tregs and the function and activation of CD4+ T cells in lymph node vs. the peripheral
blood compartments in persons with LNTB.
© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Sahmoudi et al. BMC Immunology
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Methods
Subjects and preparation of immune cells
Eighteen patients (5 men, 13 women, age range 17-60
years) were recruited in the Hassan II University
Hospital of Fes (Morocco) among patients with cervical
lymphadenitis. Active LNTB was diagnosed by history,
physical examination, and lab studies by experienced clinicians. The diagnosis of LNTB was based on a combination of clinical symptoms, pathology and response to
TB drug therapy. Clinical symptoms associated with
lymphadenitis included local lymphadenopathy, weight
loss, fever, sweats, and anorexia. Histopathological evidence consisted of the presence of a granulomatous lesion with caseation in excisional biopsy specimens.
Pulmonary radiography and HIV serology were performed to exclude pulmonary TB and HIV infection respectively. All LNTB cases were newly diagnosed and
none had received anti-TB chemotherapy before sample
collection. Tuberculin skin test results were positive
(induration ≥ 10 mm) for 15 out of 18 patients (83%).
All patients were BCG vaccinated, and none reported
contact with a case of pulmonary TB.
For all patients, the affected lymph node was in the
neck and was surgically removed. In addition, 10 ml of
peripheral blood was collected before starting anti-TB
treatment. One portion of the lymph node was used for
histological examination, and the other for isolation of
lymph node mononuclear cells (LNMC) for immunologic studies.
Biopsy specimens were crushed gently in tissue culture
medium. LNMC were spun and separated using
Ficoll-Hypaque density centrifugation. Peripheral blood
mononuclear cells (PBMC) were isolated from heparinized venous blood under endotoxin-free conditions by
Ficoll-Hypaque (SIGMA) density centrifugation. Cells
were cryopreserved and stored in liquid nitrogen until
shipment by a cryoshipper to Case Western Reserve
University for immunological studies.
Phenotypic and functional study of T cells
PBMC and LNMC (106/ tube) were stimulated with a
pool of 34 overlapping peptides from Mtb-antigen
ESAT6/CFP10 at 6.25 ug/ml per peptide (New England
peptide, Gardner, MA), M. tuberculosis CDC1551 whole
cells lysate (Mtb lysate) (BEI Resources) or staphylococcal enterotoxin B (SEB, 2 μg/ml, Sigma) overnight at 37
°C in 5% CO2. Unstimulated PBMC and LNMC served
as negative controls. Anti-CD28/CD49d (1 μg/ml each,
eBioscience and Biolegend) was added to each tube during stimulation and brefeldin A (5 μg/ml, Sigma) was
added 2 hr later. After stimulation, cells were washed
with PBS and surface stained with anti-CCR7-PE-Cy7
(BD Bioscience) for 15 min at 37 °C, then live dead yellow (Invitrogen), anti-CD14-BV570, anti-CD4-APC/Cy7,
anti-CD8-BV510 (all Biolegend) and anti-CD45RA-PE/
TR (Invitrogen) were added and incubated at RT for 25
min. Afterward cells were washed, permeabilized (Cytofix/Cytoperm Kit, BD Pharmingen) according to the
manufacturer’s instructions and stained for intracellular
expression with anti-CD3-PerCP, anti-IFNγ-Alexa700,
anti-IL2-APC, anti-TNFα- Pacific Blue, anti-IL17-BV711
(all Biolegend) and anti-MIP-1α-FITC (R&D). Cells were
then washed, fixed in 1% paraformaldehyde and 1x106
total events collected from each sample on an LSR-II
flow cytometer (BD). Net responding cells for each cytokine were calculated by subtracting the no antigen condition (medium only) from the antigen simulated result.
The analysis of all functional markers expressed after
stimulation were done on viable total memory CD4+
and CD8+ T cells. Memory phenotype was determined
by CCR7 and CD45RA expression. Naïve T cells were
CD45RA+/CCR7+, central memory (CM) cells were
CD45RA-/CCR7+, effector memory (EM) cells were
CD45RA-/CCR7-and effector cells (E) were CD45RA
+/CCR7-.
To identify Tregs and activated T cells, PBMC and
LNMC were surface stained with live dead yellow (Invitrogen), anti-CD3-APC/Cy7, anti-CD4-PerCP, anti-CD8
-Alexa700, anti-CD25-APC, anti-HLA-DR-FITC, antiCD38-PE/Cy7(all Biolegend), and anti-CD127-BV6
50(BD Bioscience), and incubated at RT for 25 min.
After, cells were washed, permeabilized and intracellular
stained with, anti-FoxP3-PE (eBioscience) or isotype-m
atched negative control for gating purposes.
Plots were analyzed using FlowJo software (version
6.1.1; Tree Star, Ashland, OR, USA). Boolean analysis on
Flow Jo and SPICE (NIAID, NIH, USA) was used to assess cytokine polyfunctionality.
Statistical analysis
Statistical analysis was performed using the paired
Student’s t test for comparisons of PBMC and LNMC. P
value for comparison of polyfunctional profile was done
using SPICE software. Significance of correlations was
analyzed by the nonparametric Spearman test. A p value
of ≤ 0.05 was considered significant. Data were analyzed
by Statistical Package for the Social Sciences (SPSS) software (IBM) and the GraphPad Prism software, version
5.00 for Windows (GraphPad Software, San Diego).
Results
Phenotypic and cytokine profile of CD4+ T cells in LNMC
and PBMC
We first compared memory phenotypes of T lymphocyte subsets in LNMC and PBMC. LNMC were significantly enriched for CD4+ T cells [80.7±5.6 % vs.
68.0±6.6 % (p = 0.0015)] (Fig. 1a). CD8+ T cells were
concomitantly lower in LNMC compared to PBMC
Sahmoudi et al. BMC Immunology
(2018) 19:33
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Fig. 1 Phenotypic and functional T-cell subset distribution in PBMC and LNMC. Relative frequencies of CD4+T cells (a) and memory subsets of
CD4+ T cells (b) among all CD3+ T cells. Means from 18 subjects are shown and error bars represent standard deviations
[17.6±5.8 % vs. 27.4±11.7 % (p = 0.0055)] (Additional
file 1). Memory subsets of CD4+ T cells were defined
according to expression of CD45RA and CCR7 and
only the effector subset was modestly increased in
LNMC compared to PBMC (p = 0.0002) (Fig. 1b).
T cell functional profiles in response to Mtb antigens
were compared between LNMC and PBMC. IFNγ, IL2,
TNFα, IL17, and MIP-1α production by CD4+ T cells in
PBMC and LNMC in response to Mtb RVL were not
different (Fig. 2a). Similarly CD4+ T cell responses in response to ESAT6/CFP10 peptides were not different between LNMC and PBMC (Additional file 2).
No significant differences were observed in the polyfunctionality of total memory CD4+ T cells in LNMC
Fig. 2 Cytokine expression of memory CD4+T cells after Mtb antigen RVL stimulation (a) and polyfunctional composition of total memory CD4+T
cells after stimulation with Mtb lysate, ESAT6/CFP10, and SEB (b). Results from 11 subjects are shown. Plots are gated on viable memory CD4+T
cells. The pie charts depict the average polyfunctional profile of only the responding cells for each specific stimuli from all subjects. All possible
combinations of responses are represented by the arcs with pie arc color legend on the figure
Sahmoudi et al. BMC Immunology
(2018) 19:33
when compared to PBMC for the same stimuli (Fig. 2b
all three rows with side to side comparison). Polyfunctionality of total memory CD4+ T cells in response to
ESAT6/CFP10 peptides and Mtb lysate were significantly
different from that in response to SEB in both PBMC
and LNMC indicating a difference in Mtb-specific cells
as a group (Fig. 2b, top 4 pies vs. bottom 2 comparison),
but no differences were found in the polyfunctionality in
response to Mtb-specific antigens, ESAT6/CFP10 peptides and Mtb lysate (Fig. 2b top two and middle two
comparison).
Distribution of activated T cells and Tregs in LNMC and
PBMC
We measured the frequency of activated conventional
CD4+ T cells and Tregs in LNMC and PBMC. Activation was based on the expression of HLA-DR and CD38.
Tregs were identified as CD4+, CD25+, FoxP3+ and
CD127dim. An increased proportion of activated CD4+
HLA-DR+CD38+ T cells (p < 0.0001) and Treg CD4+
CD25+FoxP3+CD127dim (p = 0.0089) in LNMC compared to PBMC was observed (Fig. 3). A positive correlation between the frequency of total activated CD4+ T
cells and Tregs was observed within LNMC (r = 0.676,
p = 0.008) and PBMC (r = 0.549, p = 0.018) (Fig. 4).
We compared, among Tregs in LNMC, the proportion
of cells expressing activation markers. More than 90% of
CD25+ FoxP3+CD127dim cells expressed HLA-DR and/
or CD38. Furthermore, the predominant Treg subpopulation in both LNMC and PBMC was the one expressing
HLA-DR and CD38 simultaneously (Fig. 5).
Treg correlation with Mtb-antigen induced cytokine
production
We determined the correlation between the proportion
of Tregs and cytokine producing CD4+ T cells after
overnight Mtb-antigen activation in LNMC and PBMC.
There was a positive correlation between the proportion
Page 4 of 7
of Tregs and Th1 cytokine production (IL2 and IFNγ) as
well as MIP-1α following stimulation by Mtb lysate antigen within LNMC but not among PBMC (Table 1). A
similar correlation was found after stimulation by Mtb
ESAT6/CFP10 peptides while no correlation was found
after SEB mitogen stimulation (data not shown).
Discussion
The outcome of the immune response to Mtb results
from the simultaneous involvement of activating and
regulatory mechanisms [8, 11]. The nature of the relationship between conventional T cells and Tregs during
active TB is still not clear although this interaction has
been studied in different forms of TB [10, 11]. Increased
expression of activation markers on T cells [12, 13] and
higher levels of Tregs [7, 8] have been described in peripheral blood of patients with TB. Nevertheless, local immune responses may differ from those in peripheral
blood and exploring this interaction in the site of active
infection will give important clues about their involvement in protection or pathogenesis [14, 15]. The present
study sought to evaluate the relationships between Tregs
and conventional CD4+ T cells in lymph nodes and peripheral blood during TB lymphadenitis.
We found a higher proportion of CD4+ T cells in
LNMC compared to PBMC. This is in agreement with
previous studies reporting an increase in the proportion
of CD4+ T cells in the blood of TB patients compared
to uninfected controls and a much higher number of
CD4+ T cells at the site of infection [16, 17]. Except for
modest elevation in effector cells in LNMC, no difference was found in the relative frequency of memory
CD4+ T cell subsets between PBMC and LNMC.
Others have shown that Mtb-specific CD4+ T cells in
bronchoalveolar and pleural fluids are mainly of the
memory phenotype [18, 19]. In LNMC we find that up
to 40% of CD4+ T cells were naïve.
Fig. 3 Frequency distribution of HLA-DR+ CD38+ activated (a) and FoxP3+CD25+CD127- (b) regulatory CD4+T cells in LNMC and PBMC
Sahmoudi et al. BMC Immunology
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Fig. 4 Ex vivo frequencies of Tregs correlated with the ex-vivo frequencies of CD4+ activated cells in PBMC (a) and LNMC (b)
Th1 and Th17 Mtb–specific T cells contribute to the
defense against progressive Mtb infection. Particularly,
TB lymphadenitis was characterized by elevated frequencies of Th1 and Th17 cells in peripheral blood [20].
When we measured intracellular production of Th1 cytokines (IFNγ, IL2 and TNFα), IL17 and MIP-1α, we
were surprised to not find an increase in the frequencies
of Mtb-specific cytokine producing cells in LNMC vs.
PBMC. LNMC were generated from a large block of excised lymph node tissue that included granulomatous
and non-granulomatous areas which may have affected
our ability to detect higher frequencies of Mtb-specific
cells in LNMC than PBMC.
Polyfunctional T cells are correlated with protection in
some studies [5, 6, 21] and with disease activity in others
[22, 23]. In our case, no differences were observed in the
proportion and polyfunctional qualities of the
Mtb-responsive CD4+ T cells between LNMC and
PBMC. Comparing polyfunctionality in CD4+ T cells
responding to Mtb-antigen vs. SEB showed a significant
difference with SEB eliciting a more TNFα dominated
response. This supports that the Mtb-specific responses
were different from those of the whole memory CD4+ T
cell pool.
To analyze the interaction between conventional activated CD4+T cells and Tregs in the lymph node during
active TB, we measured the frequency of CD4+T cells expressing CD38 and HLA-DR. These immune activation
markers were described as substantially elevated in subjects with active TB [24]. CD4+ T cells were more activated in lymph node compared to blood. The increased
proportion of activated T cells in LNMC likely reflects
more exposure to Mtb antigens. The selective accumulation is likely the result of both active recruitment and local
expansion of T cells at this site of Mtb replication [25, 26].
Tregs were identified by selecting CD4+ cells with
high-level expression of CD25, low-level expression of
CD127, and expression of FoxP3. Tregs were also increased in LNMC in response to local immune activation possibly to control immune induced damage [3, 26,
27]. In accordance with the previous study [10], our data
reveal a positive correlation between Treg and CD4+ activated cells within PBMC but this correlation was stronger in LNMC. Tregs co-expressing HLA-DR and CD38
Fig. 5 Distribution of activation markers within CD4+CD25+FoxP3+CD127dim Tregs in PBMC and LNMC
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Table 1 Correlation of the ex vivo frequencies of Tregs with the
frequencies of CD4+T cells expressing IFNγ, IL2, TNFα, IL17 and
MIP1α following Mtb lysate antigen stimulation in PBMC and
LNMC
infection regulates the immune response. Whether this is
advantageous to the host or not remains to be determined.
Additional files
T reg
PBMC
r
LNMC
p
r
p
IFN-γ
-0.19
0.60
0.66
0.04
IL2
-0.04
0.90
0.71
0.02
TNF-α
-0.02
0.96
0.48
0.16
IL17
-0.23
0.52
0.45
0.12
MIP-1α
-0.04
0.91
0.95
<0.001
markers were frequent in the LNMC and PBMC. The
ex-vivo MHC II expressing Tregs are believed to be a functionally mature distinct Treg subset implicated in
contact-dependent in vitro suppression [28]. A positive
correlation between Treg proportion and frequency of
memory Mtb-specific CD4+ T cells expressing each individual measured Th1 function was observed in LNMC but
not in PBMC. The predominance of activated Tregs in
LNMC and the correlation of Tregs with activated CD4+
T cells and CD4+ T cells expressing Th1 cytokines suggests
a regulatory activity specific and enhanced in the lymph
node. An alternate hypothesis could be that the Treg are
just expanding at tissue sites but not necessarily for regulatory purposes. The absence of an expected higher proportion of Th1 producing CD4+T cells in LNMC compared to
PBMC supports the hypothesis that the Treg/CD4 correlation reflected a negative feedback for excess Th1 cytokine
production by increasing suppressive Tregs. Recent data
suggest that IFNγ increases Treg suppressive function for
control of Th1 responses [29, 30]. A limitation of the study
is that we did not have any blood or lymph node material
in healthy BCG-vaccinated or latently infected individuals
available to contrast to the TB lymphadenitis subjects.
Contrasting these cohorts in future studies as well as further research on the function of Tregs and their modulation of immune responses in TB lymphadenitis are needed.
The current study will help develop an optimal approach
to such supplementary exploration.
Conclusion
This study has important original findings regarding local
immune responses in active LNTB. We found no Th1,
Th17 or MIP-1α production differences by Mtb-specific
CD4+ T cells in total LNMC vs. PBMC. Tregs were positively correlated with Th1 expressing Mtb-specific CD4+
T cells in LNMC but not in PBMC. Activated HLA-DR
+CD38+Tregs were more abundant, suggesting modulation by Tregs of immune responses in LNMC. We suggest
that increased Tregs at the lymph node site of active Mtb
Additional file 1: CD8+ T-cell subset distribution in PBMC and LNMC.
Relatives frequencies of CD8+ T cells among all CD3+ T cells. Means from
18 subjects are shown and error bars representing standard deviations.
(PPTX 51 kb)
Additional file 2: Cytokine expression of memory CD4+T cells after
ESAT6/CFP10 stimulation. Results from 11 subjects are shown. Plots are
gated on viable memory CD4+T cells. (PPTX 91 kb)
Abbreviations
LNTB: Lymph node TB; TB: Tuberculosis; Mtb: Mycobacterium tuberculosis;
Treg: T regulatory cells; LNMC: Lymph node mononuclear cells;
PBMC: Peripheral blood mononuclear cells; CD127: IL-7 receptor α-chain;
FoxP3: The intracellular marker forkhead box p3; Mtb lysate: M. tuberculosis
CDC1551 whole cells lysate; SEB: Staphylococcal enterotoxin B; N: Naïve T
cells; CM: Central memory cells; EM: Effector memory cells; E: Effector cells
Acknowledgements
The authors would like to thank all patients for their participation. They also
would like to thank all the health and laboratory professional that facilitate
the realization of this work.
Funding
This study was supported by the Hassan II Academy for Sciences and techniques
of Morocco (IMMGEN project), NIH AI108972, NIH AI080313, NIH P30 AI036219,
NIAID Contract No. HHSN266200700022C / NO1-AI-70022, and VA GRECC.
Availability of data and materials
All the raw data were kept in the experiment book, or as electronic files such
as acquired by flow cytometer will be available on request.
Authors’ contributions
This study was designed by KS, WHB, RE, DHC and FS. HA, NB and MN were
the key investigators that assisted in design of the clinical aspects of the
study then recruited, treated, and collected clinical data on the subjects. .
Data analysis, interpretation and statistical analysis were performed by KS, AS,
CB, DHC and FS. KS, DHC and FS wrote the first draft and AS, WHB, RE, FS
and DHC contributed to the final manuscript. All authors read and approved
the final manuscript.
Ethics approval and consent to participate
The study protocol was approved by the two Ethical Committees for
Biomedical Research; Faculty of Medicine and Pharmacy, Mohammed V
University of Rabat (Morocco) and University Hospitals of Cleveland
Institutional Review Board (US). Informed consent was obtained from each
patient according to the National Ethics Committee.
Consent for publication
Not applicable
Competing interests
The authors declare that they have no competing interests.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Author details
1
Laboratory of Cellular Immunology, National Institute of Hygiene, 27,
Avenue Ibn Batouta, PB 769, 11400 Rabat, Morocco. 2Faculty of Sciences,
University Mohammed V Agdal, Rabat, Morocco. 3Department of ENT,
Maxillo- facial, Reconstructive and Plastic Surgery, University Hospital Hassan
II, Fes, Morocco. 4National Reference Laboratory of Mycobacteriology, the
National Institute of Hygiene, Rabat, Morocco. 5Case Western Reserve
Sahmoudi et al. BMC Immunology
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University School of Nursing, Cleveland, USA. 6TB Research Unit and Division
of Infectious Diseases, Case Western Reserve University, University Hospitals
of Cleveland and Cleveland VA, Cleveland, OH, USA.
20.
Received: 19 January 2018 Accepted: 11 October 2018
21.
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