Deficiency of innate-like T lymphocytes in chronic obstructive pulmonary disease
Szabó et al. Respiratory Research
Deficiency of innate-like T lymphocytes in chronic obstructive pulmonary disease
Mariann Szabó 1 4
Veronika Sárosi 1 4
Zoltán Balikó 1 4
Kornélia Bodó 0 3
Nelli Farkas 2
Tímea Berki 0 3
Péter Engelmann 0 3
0 Department of Immunology and Biotechnology, Clinical Center, University of Pécs , Szigeti u. 12, Pécs H-7643 , Hungary
1 Division of Pulmonology, 1st Department of Internal Medicine, Clinical Center, University of Pécs , Rákóczi u. 2, Pécs H-7623 , Hungary
2 Department of Bioanalysis, Medical School, University of Pécs , Szigeti u. 12, Pécs H-7643 , Hungary
3 Department of Immunology and Biotechnology, Clinical Center, University of Pécs , Szigeti u. 12, Pécs H-7643 , Hungary
4 Division of Pulmonology, 1st Department of Internal Medicine, Clinical Center, University of Pécs , Rákóczi u. 2, Pécs H-7623 , Hungary
Background: Based on the phenotypic and functional characteristics unconventional T-lymphocytes such as invariant natural killer T (iNKT) cells and mucosal-associated invariant T (MAIT) cells link the innate and adaptive immune responses. Up to now data are scarce about their involvement in pulmonary disorders including chronic obstructive pulmonary disease (COPD). This study explores simultaneously the frequencies of iNKT and MAIT cells in the peripheral blood and sputum of stable and exacerbating COPD patients. Methods: By means of multicolor flow cytometry frequencies of total iNKT and MAIT cells and their subsets were enumerated in peripheral blood and sputum samples of healthy controls, and COPD patients. In addition, gene expression of TCR for iNKT, MAIT cells, and CD1d, MR1 were assessed by qPCR in the study cohorts. Results: Percentages of total iNKT and MAIT cells were dramatically dropped in blood, and reduced numbers of iNKT cells were observed in the sputum of COPD patients. Furthermore decreased DN and increased CD4+ iNKT subsets, while increased DN and decreased CD8+ MAIT subpopulations were measured in the blood of COPD patients. Reduced invariant TCR mRNA levels in COPD patients had confirmed these previous findings. The mRNA expression of CD1d and MR1 were increased in stable and exacerbating COPD patients; however both molecules were decreased upon antibiotic and systemic steroid treatments. Conclusions: Our results support the notion that both invariant T-cell populations are affected in COPD. Further detailed analysis of invariant T cells could shed more light into the complex interactions of these lymphocyte groups in COPD pathogenesis.
COPD; Emphysema; iNKT cells; MAIT cells; CD1d; MR1
Chronic obstructive pulmonary disease (COPD) is the third
leading cause of death worldwide. Pathogenesis of COPD is
characterized by the persistent airflow limitations and
associated with chronic inflammation of the airways [
Several mechanisms underlie the symptoms of COPD;
however the major ones are the emphysema,
bronchiolitis, and mucus hypersecretion [
]. Cigarette smoking is
one of the main causes of the disease, but other
environmental contaminants (e.g. pesticides), industrial dust and
microbial infections (e.g. Haemophilus influenzae,
Pseudomonas aeruginosa, Mycobacterium tuberculosis,
Moraxella catarrhalis) are also highly responsible in this
disease burden. Next to the environmental conditions
endogenous mechanisms (e.g. genetic and epigenetic
factors) also play inevitable role in the development of
COPD. Another favorable concept claims for the
autoimmune origin of COPD that idea is based on the
dysregulated inflammatory process in this disorder [
In the course of COPD chronic airway inflammation is
characterized by the massive involvement of activated
leukocytes. Neutrophils, eosinophils, macrophages and
different subsets of lymphocytes infiltrate the lungs.
Inhaled environmental factors trigger the engagements
of pattern recognition receptors. Their activation leads
to the recruitment of leukocytes through the release of
various chemokines, and pro-inflammatory cytokines
]. These secreted compounds possess important role
in the progressive airflow limitations mediated by
fibrosis and enhanced inflammation [
Adaptive immune cells such as T lymphocytes seem to
have a central role in COPD [
]. Recently, different T cell
subsets are in the center of intensive research in this
]. Emerging data suggests that innate-like T
lymphocytes such as invariant natural killer T (iNKT) cells
and mucosal-associated invariant T (MAIT) cells are also
involved in the pathogenesis of COPD [
]. iNKT and
MAIT cells represent unique, unconventional T cell
populations characterized by the co-expression of unique TCR
and NK cell receptors. Human iNKT cells express the
invariant Vα24-Jα18 TCR α-chain combined with the
limited diversity V β11 chain, while human MAIT cells
express the distinctive Vα7.2-Jα33 TCR α-chain. Based on
the CD4 and CD8 co-receptor expression three iNKT cell
subgroups (CD4+, DN, CD8+) can be distinguished. DN
iNKT cells produce mainly Th1 cytokines, while CD4+
iNKT cells secrete both Th1 and Th2 cytokines [
MAIT cells can be further divided into a minor DN and
major CD8+ subpopulations. These innate T-lymphocytes
are restricted by non-classical antigen presenting
molecules. iNKT cells recognize diverse bacterial
glycolipids presented by CD1d, while MAIT cells engaged by
microbial vitamin B derivates (e.g. riboflavin, folic acid)
presented by the highly conserved non-polymorphic class
1b antigen presenting molecule MHC-1 related protein 1
It has been reported that both subgroups have
biased frequencies and functions in several
immunemediated disorders such as autoimmune diseases, and
]. The available data are scarce about
the potential role of invariant T subpopulations in the
pathogenesis of COPD. So far, more data discuss the
role of NKT cells in COPD ; however the
published results are rather inconsistent in terms of
proportional changes of NKT cells in COPD patients.
Relatively limited information corresponds to the role
of MAIT cells in COPD [
Taken this information into account our aim was to
characterize simultaneously the proportions and subsets
of these two invariant T cell populations in peripheral
blood and induced sputum samples of stable COPD and
exacerbating COPD (AECOPD) patients.
Patient and control groups
We recruited 38 volunteers: 17 healthy controls (mean
age ± SD, 50.6 ± 9.3 years), 11 stable COPD and 10
exacerbating COPD patients into the study. Subjects
with any known autoimmune or malignant disease in
the last 5 years were excluded from the study. Enrolled
COPD patients were characterized according to the
international GOLD guidelines: post-bronchodilator
FEV1/FVC ratio was <70% and mild (GOLD1: FEV1 ≥
80% predicted), moderate (GOLD2: 50% ≤ FEV1 < 80%
predicted), severe (GOLD3: 30% ≤ FEV1 < 50%
predicted), very severe (GOLD4: FEV1 < 30% predicted)
stages were applied as disease categories. All enrolled
AECOPD patients were in severe or very severe stages,
while stable COPD patients were in moderate, severe
and very severe stages. Blood samples were taken as
standard procedures in vacuum blood collecting tubes.
First blood drawn was executed in the morning at the
Division of Pulmonology within 24 h after the admission
in the Department of Emergency, University of Pécs,
where they received treatments (inhalative β antagonist
and corticosteroids). Second blood drawn and sputum
induction of AECOPD patients was executed 72 h
later of the first blood drawn in the morning at the
Division of Pulmonology. Healthy controls and stable
COPD patients were the subject of simultaneous
blood drawn and sputum induction. COPD patients
were treated with standard inhalative medications
(LABA, LAMA, ICS + LABA) based on the GOLD
guidelines. Exacerbating patients received systemic
corticosteroids and antibiotics next to the regular
inhalative medications. To uncover any treatment
effects the AECOPD patient samples were analyzed
separately at the beginning of medications and during
the ongoing medications, marked in datasets as
AECOPD (a) or AECOPD (b), respectively. Basic
clinical characteristics of COPD patients are detailed
in Table 1. The study was approved by Regional
Research Ethics Committee of the Faculty of Medicine
at the University of Pécs (5414/2014) and written
informed consent forms were obtained from all donors.
Sputum induction and processing
Sputum samples were collected from the blood donors.
Sputum was induced by inhalation of hypertonic salt
aerosol (4% NaCl) generated by a MiniPlus Compressor
Nebulizer (Apex Medical Corp., Taipei, Taiwan) according
to the ERS guidelines [
]. After the induction sputum
was processed within 30 min, an equal volume of 0.1%
dithiothreitol solution (DTT, Sigma) was administrated.
Samples were mixed with end-over-end rotator and
incubated at 37 °C for 15 min to make firm homogenization.
The samples were then diluted with PBS and filtered
through a 50-μm pore size cell strainer. Filtered samples
were centrifuged at 1000 rpm for 5 min and then cell
viability was assessed by trypan blue exclusion test. Samples
were further processed for flow cytometry measurements.
Peripheral blood and processed sputum samples of healthy
controls and COPD patients were stained with various
fluorochrome-coupled mouse monoclonal antibodies
specific for human leukocyte surface markers:
anti-Vα24Jα18-FITC, a-Vα7.2-FITC, a-CD8-PE, a-CD4-PerCPCy5.5,
a-CD161-PerCPCy5.5, a-CD3-APC (Biolegend, San Diego,
CA, USA). After incubation erythrocytes were lysed
with FACS Lysing Solution (BD Biosciences). Flow
cytometry data were acquired with BD FACSCalibur
flow cytometer and the analysis were performed with
FCS Express 4 software (De Novo Software, Glendale,
RNA isolation and cDNA synthesis
Peripheral blood mononuclear cells (PBMCs) were
obtained from fresh blood of healthy individuals and COPD
patients by Ficoll (Amersham Pharmacia Biotech Europe,
Uppsala, Sweden) gradient centrifugation following
standard protocol. Isolated PBMCs were resuspended and frozen
in RNALater solution (Ambion, Thermo Fisher Scientific,
Waltham, MA, USA) and stored according to the
manufacturer’s instructions until sample processing.
RNA extraction was performed using NucleoSpin® RNA
isolation kit (Macherey-Nagel GmbH, Düren, Germany)
according to the manufacturer’s guidelines (including
DNase I digestion). Isolated RNA was re-suspended in
nuclease free water, quantified at 260 nm and the quality
of total RNA was confirmed by 1% agarose gel analysis.
The cDNA was constructed from total RNA with High
Capacity cDNA reverse transcription kit (Thermo Fisher
Scientific) in 20 μl reactions using random hexamers
following the manufacturer’s protocol. The resulting
cDNA was stored at −20 °C.
Quantitative real-time PCR
Target gene expression was measured by real-time
PCR using Maxima SYBRGreen MasterMix (Applied
Biosystems) with an ABI Prism 7500 instrument
(Applied Biosystems). The PBMC cDNAs were used as
a template for the amplification reactions. All samples
were tested in duplicates. Primers were designed using
Primer Express software (Thermo Fisher Scientific)
considering the exon-intron boundaries for all target
genes. In the case of invariant TCR α chain primers
were designed around the flanking of Variable, Joining
and Constant rearrangement regions (Table 2).
Thermal profile started at 95 °C for 10 min, 40 cycles of
35 s at 95 °C, 35 s at 60 °C, 1 min at 72 °C.
Statistical analysis was performed with GraphPad Prism
version 5 (GraphPad Software Inc., La Jolla, CA, USA).
Variables were expressed as medians and all whiskers
represent 1.5 interquartile range. In the case of
nonGaussian distribution the effect of treatments were
analyzed by Kruskal-Wallis-test. One way-ANOVA analysis
was performed on the data with normal distributions.
Both analyses were followed by Dunn’s or Bonferroni
post hoc tests, respectively. p < 0.05 was denoted as
Decreased frequency of total iNKT cells and biased proportions of iNKT subgroups in stable COPD and AECOPD patients
iNKT cells are rare lymphoid cells in human peripheral
blood. We compared the overall proportions of iNKT cells
in the blood samples of healthy controls, stable COPD
and AECOPD patients by applying multicolor flow
cytometry (Fig. 1a). After gating on lymphocytes iNKT cells were
defined according to the staining with
anti-Vα24-Jα18FITC and a-CD3-APC antibodies. iNKT subsets were
characterized by a-CD8-PE and a-CD4-PerCPCy5.5
antibodies (Additional file 1: Figure S1A). We assessed that
whether the effects of the systemic steroid and antibiotic
treatments compromise the proportions of iNKT cell
subsets. We found significant decrease in the total iNKT
population of stable COPD and AECOPD patients
compared to healthy controls (Fig. 1a and b). Furthermore, we
observed biased frequencies of CD4+ and DN iNKT
subpopulations, since CD4+ iNKT cells were elevated (Fig. 1c),
while DN iNKT subpopulation had a significant decrease
in the AECOPD patients compared to healthy controls
(Fig. 1d). Minor CD8+ iNKT population did not indicate
any characteristic changes (data not shown). There was no
significant difference between the iNKT cells of stable
COPD and AECOPD patient groups. Regarding to smoker
and non-smoker (non-recent smoker individuals who
stopped smoking minimum two years before sample
collection) status we observed significant differences in
the iNKT population of stable COPD patients. Smoker
Amplicon size (bp)
aUpper and lower sequences represent forward and reverse primers, respectively
patients have significantly reduced iNKT cell
frequencies compared to non-recent smokers (Additional file 2:
Figure S2A). In addition we compared the frequencies
of total iNKT cells from smoker HC, stable COPD
and AECOPD individuals. We found significant
decrease in iNKT proportions of AECOPD patients
compared to HC cohort, while iNKT cells from
smoker stable COPD patients evidenced a
nonsignificant decrease compared to the smoker HC
group (Additional file 2: Figure S2C). Smoking-related
information of healthy subjects and COPD patients
are detailed in Table 3.
Decreased proportions of total MAIT cells and biased DN,
CD8+ MAIT subpopulations in stable COPD and AECOPD patients
After gating on CD3+ lymphocytes MAIT cells were
defined by staining with anti-Vα7.2-FITC and
aCD161-PerCPCy5.5 antibodies. DN and CD8+ MAIT
cell subsets were characterized by a-CD8-PE staining
(Additional file 1: Figure S1B). Indeed, MAIT cells are
more frequent in human peripheral blood compared
to iNKT cells. Measured iNKT cell percentages were
exclusively below 0.5%, while MAIT cells evidenced
2.5% high frequencies in the blood samples of healthy
controls (Figs. 1 and 2). Similarly to iNKT cells we
observed significantly dropped MAIT cell numbers in
the peripheral blood of stable COPD and AECOPD
patients compared to healthy controls (Fig. 2a and b).
We found non-significant increase of DN MAIT
subpopulation compared to the controls (Fig. 2c).
Furthermore, the proportions of CD8+ MAIT cells
were significantly decreased in COPD patient groups
compared to healthy controls (Fig. 2d). There was
no difference of total and DN or CD8+ MAIT cells
between stable COPD and AECOPD patients. Besides, we
measured significantly decreased total MAIT cell
proportions in smoker, stable COPD patients compared to the
non-recent smoker populations (Additional file 2:
Figure S2B). In addition we compared the frequencies
of total MAIT cells from smoker HC, stable COPD and
AECOPD individuals. We found significant decrease in
MAIT cell proportions of both smoker stable and
AECOPD patients compared to smoker HC cohort
(Additional file 2: Figure S2D).
Smoker, stable COPD patients have significantly
increased DN and decreased CD8+ MAIT
subpopulations (Additional file 3: Figure S3A and B). In smoker
AECOPD population DN MAIT cells were
nonsignificantly increased, while CD8+ MAIT cells were
significantly reduced compared to the non-smoker
AECOPD population (Additional file 3: Figure S3C and
D). We did not find any proportional difference in
MAIT cells between the smoker or non-smoker healthy
populations (Additional file 4: Figure S4B).
Decreased iNKT and MAIT cell proportions in the induced sputum of COPD patients
We aimed to assess whether the invariant T cells are
differently represented in the airways compared to the
peripheral blood. Applying the same staining set-up we
measured the frequencies of iNKT and MAIT cells in
the induced sputum of healthy subjects and COPD
patients. Albeit the frequencies of iNKT cells were
higher among the lymphocytes in the sputum than in
peripheral blood, but we found a significant decrease of
this invariant T cell population in the AECOPD
patients compared to the healthy controls (Fig. 3a).
iNKT cells evidenced non-significant decrease in the
stable COPD patients.
In the case of sputum MAIT cells we have not found
differences between healthy controls and COPD
patients; however we observed a non-significant trend of
decrease in MAIT cell proportions in stable COPD and
AECOPD patients compared to the control population
Decreased mRNA expression profile of the iNKT and MAIT
TCRs in COPD patients
Besides the flow cytometry analysis we assessed the
mRNA expression levels of the canonical invariant TCR
in iNKT and MAIT cells. Being in agreement with the
flow cytometry-based observations we measured
significant decrease of the Vα24-Jα18 invariant alpha chain in
iNKT cells of AECOPD patients compared to the healthy
controls. Interestingly, we observed significant difference
between stable COPD and AECOPD (b) patients in the
mRNA levels of iNKT TCR (Fig. 4a).
Furthermore, we evaluated the expression levels of
Vα7.2 MAIT TCR in the aforementioned study groups.
Significantly decreased expression pattern of Vα7.2-Jα33
TCR mRNA message was determined in all COPD
patients compared to the healthy controls (Fig. 4b).
Biased mRNA expression patterns of CD1d and MR1 antigen presenting molecules in COPD patients
Next we assessed the CD1d and MR1 mRNA expression
levels. We found that CD1d expression is significantly
increased in stable COPD and AECOPD (a) patients
compared to the healthy controls. In contrast, upon
treatment the CD1d expression of AECOPD (b) patients
was not significantly different from the healthy control
group; however it was significantly decreased compared
to stable COPD patients (Fig. 5a).
Moreover we checked the expression levels of the
MAIT cell-engaged MR1 molecules in the study groups.
Interestingly, we found that MR1 expression levels were
significantly increased in stable COPD and AECOPD (a)
patients, while there was no significant change of MR1
expression in the treated hospitalized AECOPD (b)
patients compared to the healthy controls (Fig. 5b).
Exposure to cigarette smoke initiates a chronic
inflammatory response hallmarked by cellular infiltration in
]. It is suggested that the proportions of
neutrophils are inversely correlated with the extension of
emphysema, while the amount of infiltrating
macrophages and T cells are positively correlated with the level
of lung destruction. In the course of COPD pathogenesis
involved T cell subgroups and their pathogenic role are
just partly uncovered [
]. Recent information claims that
a large number of T cells reside in the inflamed lung and
mostly CD8+ T cells are responsible for the development
of emphysema [
In this attempt we undertake the task to characterize
and enumerate the distinct innate-like T lymphocyte
subsets (namely invariant NKT and MAIT cells) in the
peripheral blood and sputum samples of COPD patients.
It is necessary to note that NKT cells are phenotypically
and functionally diverse lymphocytes. These innate-like
T cells contain the extensively characterized
CD1drestricted invariant NKT cells (type I or iNKT cells),
CD1d-restricted non-invariant (type II) and CD1d
independent (type III) NKT cells [
]. In fact, the role of
NKT cells in pulmonary disorders is quite controversial
that could be due to their diversity and the inconsistent
application of diagnostic reagents to identify them [
All these obstacles detain the objective comparisons of
independent experimental results. Furthermore, several
line of evidence proved that the aforementioned type I
and type II NKT cells frequently unfold opposing effects
on each other in certain pathological conditions [
There are several staining procedures to analyze NKT
cells, but among those only the Vα24-Jα18 / CD3 or
αGalCer (exogenous model antigen)-loaded CD1d
tetramer / CD3 co-staining seems reliable to identify
equivocally the iNKT cells [
Based on the Vα24-Jα18 / CD3 co-staining our results
are partly concordant with the previous findings [
concerning decreased iNKT cells in stable COPD and
exacerbating COPD patients. We measured a significant
drop of total iNKT cells in the peripheral blood of
COPD patients compared to healthy subjects.
Furthermore our qPCR analysis confirmed these findings and
supported the notion that total iNKT population was
decreased in COPD. Indeed, in the aforementioned
] several different staining patterns have been
performed to analyze iNKT cells in the peripheral blood
of COPD patients. According to our flow cytometry and
qPCR results we can conclude that blood iNKT cell
numbers are decreased in COPD patients.
On the other hand it has been reported that CD4+
iNKT subset was decreased in stable COPD and
AECOPD patients [
]. In contrast, we found that the
frequencies of CD4+ iNKT cells have been significantly
increased in the peripheral blood of AECOPD patients
and non-significantly elevated in the peripheral blood of
stable COPD patients. Other iNKT cell subsets (DN,
CD8+) have not been enumerated [
]. In our AECOPD
study cohort the DN iNKT population evidenced
decreased proportions, while CD8+ iNKT cells did not
demonstrate any difference compared to the healthy
controls. Conversely to the aforementioned publication
] we did not observe significant differences of
circulating iNKT cells in stable COPD and AECOPD patients.
To the best of our knowledge, there is no available
publication about the distribution of the DN and CD8+
iNKT cell subsets in COPD patients.
By the means of CD3 / CD56 co-staining classifies the
mixed population of NKT-like lymphocytes consist of
iNKT, type II NKT cells and other T cells. Therefore the
measured frequencies of NKT-like cells can be falsely
interpreted due to their complexity [
]. By means of CD3 /
CD56 double-labeling several publications reported biased
proportions of NKT-like cells in COPD patients [
Indeed, these datasets should be handled cautiously
according to the previously described considerations.
All aforementioned publications reported about
elevated proportions of NKT-like cells in the sputum of
COPD patients. In contrast we observed decreased
iNKT cell frequencies in the sputum of COPD patients
compared to controls similarly to those measured in
peripheral blood. Indeed, an explanation for the discrepancy
among our data and others could be the different
staining methods and detection of mixed populations of
NKT-like cells [
One additional study enumerated low amounts of
iNKT cells in bronchoalveolar lavage and sputum
samples of control, allergic and COPD patients [
There was no significant difference among the iNKT
proportions isolated from controls and COPD patients.
Indeed, in this study iNKT cells and not NKT-like cells
were analyzed, however the staining combination was
different from our approach applying specific antibodies
against both α and β chain of the iTCR (Vα24-Jα18 and
Vβ11, respectively). This data was verified by qPCR
analysis targeting the invariant TCR. Another study
concordantly found elevated number of iNKT cells and
NKT-like cells in the sputum of COPD patients and in
the cigarette smoke induced mouse COPD model [
In this report human iNKT cells were identified based
on the αGalCer-derivative loaded CD1d tetramer
staining. According to Chi et al. [
CD1d tetramer enhanced the specificity but also
increased the variations and could not be observed
differences between blood iNKT from COPD patients and
healthy controls applying this staining approach;
however these authors did not measure the proportions
of iNKT cells in the sputum.
So far the identification of MAIT cells is more
straightforward compared to those of NKT cells. Since
the available antibody reagents such as a-Vα7.2, a-CD3
and a-CD161 only determines one specific staining
algorithm to identify specifically MAIT cells. According
to the available information MAIT cells are decreased in
the peripheral blood of COPD patients compared to
]. In this report decreased proportions of
DN and CD8+ MAIT cell subpopulations were evaluated
in the COPD study cohorts. Indeed, we found a
significant drop of total MAIT cells frequencies in COPD
patients. Contrary to the aforementioned observation
] the proportions of CD8+ MAIT cells were
significantly decreased, however the DN MAIT cell population
showed a trend for non-significant increase in COPD.
Furthermore we evaluated Vα7.2-Jα33 TCR mRNA by
qPCR in the control and patient cohorts. We
independently found similar decrease of MAIT TCR message in
the COPD patients.
MAIT cells showed a slight non-significant decrease in
the sputum of COPD patients that is concordant with
others findings [
]. This particular study emphasizes
that ICS treatments in COPD impaired MAIT cell
numbers. In fact, due to limited number of studies we cannot
rule out the ICS effect on MAIT cells in COPD however,
in our study nearly half of stable COPD patients did not
receive ICS treatments and evidenced declined
frequencies of MAIT cells and decreased Vα7.2-Jα33 TCR
mRNA. MAIT cell frequencies were not significantly
different between ICS treated or non-treated stable COPD
patients and those from AECOPD patients under strict
ICS therapy (Additional file 4: Figure S4A). In addition a
novel observation claims that exposure to cigarette
smoke can reduce CD8+ MAIT cells in healthy
individuals and MS patients [
]. In contrast, we have
compared the total MAIT cells (and subsets) of our
nonsmoker vs. smoker healthy cohorts, but we did not find
any significant differences (Additional file 4: Figure S4B).
Unique highly conserved antigen-presenting molecules
such as CD1d and MR1 for iNKT and MAIT cells have a
broad tissue and cellular expression patterns in human.
CD1d is most typically expressed on various hematopoetic
antigen presenting cells (APCs, eg. dendritic cells,
macrophages, B cells, DP thymocytes) and intestinal epithelial
cells, while MR1 is expressed in a wide variety of tissues
including lung, liver, spleen, thymus and small intestine,
colon and peripheral blood leukocytes [
knowledge about the changes of these antigen presenting
molecules is lacking in COPD pathogenesis. Human alveolar
macrophages have evidenced a decreased expression of
HLA-DR molecules in COPD patients, however there was
no such difference in peripheral mononuclear cells [
Recently the MR1 expression of in vitro cultured
pulmonary macrophages has been enumerated upon incubation
with nontypeable Haemophilus influenzae (NTHi). MR1
was up-regulated in the NTHi exposed macrophages;
however fluticazone and budesonide treatments decreased
the MR1 level in the NTHi exposed cells [
]. In our
data-set elevated expression of CD1d and MR1 mRNA
were measured in stable and AECOPD patients, while the
systemic steroid treated AECOPD patients evidenced a
dropped pattern for both non-polymorphic
antigenpresenting molecules similarly to the previous in vitro
]. These results could be explained by the
activation status of the APCs. On the other hand it is
possible that the APCs try to compensate the loss of
stimulatory signals due to the decreased amount of
innate-like T cells by the over-expression of these unique
antigen presenting molecules.
To best of our knowledge first we examined
simultaneously the proportions of iNKT and MAIT cells in the
same COPD patient groups. In concert, with other
studies we found decreased invariant T cell numbers in
the peripheral blood of COPD patients, whether this is
due to ICS treatments or directly related to disease
pathogenesis needs to further investigated. In contrast to
most studies, we observed all subpopulations of iNKT
and MAIT cells. These subpopulations have unique
functional differences that might correspond to the
disease progression. Furthermore we evaluated the
expression of iNKT and MAIT iTCR and CD1d/MR1
molecules that evidenced characteristic bias in COPD.
Indeed, a better understanding of cellular interactions
between invariant T cells and APCs might lead for future
cellular-based therapies in COPD [
Additional file 1: Figure S1. Representative flow cytometry dot plots
reveal the gating strategy to identify iNKT cells and their DN, CD4+, CD8+
subsets (A). Representative flow cytometry dot plots and a histogram
demonstrate the gating strategy to identify MAIT cells and their DN, CD8+
subsets (B). (PDF 199 kb)
Additional file 2: Figure S2. Percentages of total iNKT (A) and MAIT (B)
cells were enumerated in the peripheral blood of non-smoker and
smoker populations in stable COPD patient cohort. Data present here
were derived from six non-smoker and five stable COPD blood donors.
Percentages of total iNKT (C) and MAIT (D) cells were enumerated in the
peripheral blood of smoker populations in HC, stable COPD and AECOPD
patient cohorts. Data present here were derived from five-five smoker
HC, smoker stable COPD and smoker AECOPD blood donors. Boxes show
interquartile ranges (IQR) whiskers represent lowest and highest values,
horizontal lines indicate median. (PDF 158 kb)
Additional file 3: Figure S3. Percentages of DN (A) and CD8+ (B)
MAIT cells were evaluated in the peripheral blood of non-smoker and
smoker populations in stable COPD patient cohort. Data present here
were derived from six non-smoker and five smoker stable COPD
blood donors. Percentages of DN (C) and CD8+ (D) MAIT cells were
measured in the peripheral blood of non-smoker and smoker
populations in the AECOPD patient cohort. Data present here were
derived from five non-smoker and five smoker AECOPD blood
donors. Boxes show interquartile ranges (IQR) whiskers represent
lowest and highest values, horizontal lines indicate median. Asterisks
represent significant p (* < 0.05) values. (PDF 129 kb)
Additional file 4: Figure S4. Percentages of total MAIT cells were
evaluated in the peripheral blood of inhaled corticosteroid treated (ICS) or
non-treated (No ICS) stable COPD and AECOPD patients (A). Data present here
were derived from five/six stable COPD patients under no ICS/ICS therapy,
and ten AECOPD blood donors all receiving ICS therapy. Percentages of total
MAIT cells were measured in the peripheral blood of non-smoker and smoker
populations of healthy controls (B). Data present here were derived from
eleven non-smoker and five smoker healthy control blood donors. Boxes show
interquartile ranges (IQR) whiskers represent lowest and highest values,
horizontal lines indicate median. (PDF 65 kb)
AECOPD: Acute exacerbating chronic obstructive pulmonary disease;
APC: Antigen presenting cell; COPD: Chronic obstructive pulmonary disease;
DTT: Dithiothreitol; HLA: Human leukocyte antigen; ICS: Inhalative
corticosteroids; iNKT: Invariant natural killer T cells; iTCR: Invariant T-cell
receptor; LABA: Long acting β agonist; LAMA: Long acting muscarinic
agonist; MAIT: Mucosal-associated invariant T cells; MS: Multiple sclerosis;
NTHi: Nontypeable Haemophilus influenzae; PBS: Phosphate buffered saline
We are grateful to the contributions provided by Krisztina Tóth and Viktória
Vasa (University of Pécs, Hungary). The present scientific contribution is
dedicated to the 650th anniversary of the foundation of the University of
We acknowledge the financial support of the Hungarian Respiratory Society
and the Medical School Research Foundation, University of Pécs
Availability of data and materials
The dataset supporting the conclusions of this article is included within the
article and its additional files.
MSZ and PE designed and performed the experiments, data analysis and
manuscript writing. ZB, SV contributed in patient collection and study
coordination. KB performed the experiments. NF performed statistical
analysis. TB contributed in reagents and study coordination. All authors read
and approved the final manuscript.
Ethics approval and consent to participate
The study was approved by Regional Research Ethics Committee of the
Faculty of Medicine at the University of Pécs (5414/2014) and written
informed consent forms were obtained from all donors.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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