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Original article
Characterization of a new regulatory CD4
+
T cell
subset in primary Sjo
¨gren’s syndrome
Alessia Alunno
1,
*, Maria Grazia Petrillo
2,
*, Giuseppe Nocentini
2
, Onelia Bistoni
1
,
Elena Bartoloni
1
, Sara Caterbi
1
, Rodolfo Bianchini
2
, Chiara Baldini
3
,
Ildo Nicoletti
4
, Carlo Riccardi
2
and Roberto Gerli
1
Abstract
Objective. CD4
+
CD25
low
GITR
+
T lymphocytes expressing FoxP3 and showing regulatory function have
been recently described in healthy donors (HD). The objective of the study was to investigate their pres-
ence and role in patients with primary SS (pSS).
Methods. CD4
+
CD25
low
GITR
+
cells circulating in peripheral blood (PB) of patients with pSS were isolated
by MACS technique, their phenotype was studied by flow cytometry and real-time PCR, and their function
was studied by in vitro co-culture. CD4
+
CD25
low
GITR
+
cells infiltrating salivary glands (SGs) were revealed
by immunohistochemistry.
Results. Results indicated that conventional CD4
+
CD25
high
regulatory T cells (Tregs) are decreased,
whereas CD4
+
CD25
low
GITR
+
cells are expanded in the PB of pSS as compared with HD. Phenotypic
analysis demonstrated that CD4
+
CD25
low
GITR
+
cells display Treg markers, including FoxP3, TGF-band
IL-10, and functional experiments demonstrated that they exert a strong inhibitory activity against autolo-
gous effector cells. CD4
+
CD25
low
GITR
+
cells were detectable in great number in the SG inflammatory
infiltrate. Interestingly, PB CD4
+
CD25
low
GITR
+
cell expansion was evident only in patients with inactive
disease, while conventional CD4
+
CD25
high
Treg number was not associated with disease activity.
Conclusion. The present data demonstrate that circulating CD4
+
cells expressing GITR, but with low levels of
CD25 (CD4
+
CD25
low
GITR
+
), are detectable in pSS patients. These cells, displaying Treg phenotype and func-
tion, are present in SG inflamed tissues and are expanded in the PB of subjects with inactive disease. Data
suggest that the expansion of CD4
+
CD25
low
GITR
+
cells in pSS may represent a counter-regulatory attempt
against autoimmune-driven inflammation and may provide a new target for future treatment strategies.
Key words: regulatory T cells, Sjo
¨gren’s syndrome, GITR.
Introduction
Primary SS (pSS) is a systemic autoimmune disorder pri-
marily characterized by chronic inflammation of the exo-
crine glands. Extraglandular manifestations occur in many
patients and may involve almost any organ [13]. Similar
to other CTDs, such as SLE, B cell hyperactivity is a char-
acteristic feature of the disease, as demonstrated by cir-
culating autoantibodies and hypergammaglobulinaemia
[46]. However, analysis of inflamed tissue in the salivary
glands (SGs) shows a predominance of T cells surround-
ing ductal epithelial cells, with a prevailing CD4
+
pheno-
type and signs of activation [7]. Notably, despite B cell
hyperactivity predominates, T cells are thought to play a
key role not only in the development of tissue damage but
also in the systemic derangement of immune response of
pSS [8, 9].
Primary SS has been long thought to be a T helper 1
(Th1)-mediated condition, although observations on this
topic have become contradictory over time. More re-
cently, these discrepancies found a possible explanation
1
Rheumatology Unit and
2
Section of Pharmacology, Toxicology and
Chemotherapy, Department of Clinical and Experimental Medicine,
University of Perugia,
3
Rheumatology Unit, University of Pisa and
4
Laboratory of Confocal Microscopy and Image Analysis, Department
of Clinical and Experimental Medicine, University of Perugia, Italy.
Correspondence to: Roberto Gerli, Rheumatology Unit, Department of
Clinical and Experimental Medicine, University of Perugia, Via Enrico
Dal Pozzo, I-06122 Perugia, Italy. E-mail: gerlir@unipg.it
*Alessia Alunno and Maria Grazia Petrillo contributed equally to this
study.
Submitted 19 December 2012; revised version accepted
28 March 2013.
!The Author 2013. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com
RHEUMATOLOGY
Rheumatology 2013;52:13871396
doi:10.1093/rheumatology/ket179
Advance Access publication 14 May 2013
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SCIENCE
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by the discovery of IL-17-producing cells (Th17) [10, 11].
In fact, it is now evident that these cells represent a main
pathogenic effector subset involved in the induction of
inflammation and autoimmunity in general and pSS glan-
dular damage in particular [1214]. An exciting aspect of
Th17-cell homeostasis is the reciprocal relationship with
regulatory T cells (Tregs), whose imbalance is believed to
play a major role in the development of autoimmune dis-
eases [1517]. However, it is noteworthy that there is not
yet universal consensus on a definition of Tregs. They
were originally identified by high surface expression of
CD25 molecule (CD25
high
), but further studies indicated
that this prerequisite did not fit the evidence of regulatory
functions exerted by CD4
+
CD25
cells [18, 19]. Tregs
were subsequently identified by the forkhead box protein
P3 (Foxp3) transcriptional factor, which controls their de-
velopment and suppressive function and represents to
date the most specific marker of human Tregs [20, 21].
It is not surprising, therefore, that the few reports on Tregs
circulating in the peripheral blood (PB) of pSS, employing
high CD25 expression and/or FoxP3 expression as iden-
tification markers, are rather contradictory [2228]. In this
setting, it is intriguing the description of CD25
FoxP3
+
T
cells with suppressive activity in the PB of SLE patients,
which appears to confirm that CD25 expression is not
mandatory to recognize Tregs [18, 29, 30].
Among proposed markers of Tregs, glucocorticoid-
induced TNF receptor-related protein (GITR) is a surface
molecule able to identify T cells with regulatory activity in
murine models [31]. Although GITR expression correlates
with that of CD25, CD25
GITR
+
cells exerting suppressive
activity have been described in mice [32]. In this context,
we recently described a T cell subset circulating in healthy
subjects, characterized by high levels of GITR but low
levels of CD25 on cell membrane (CD4
+
CD25
low
GITR
+
)
[33]. CD4
+
CD25
low
GITR
+
cells express Treg markers,
including Foxp3, TGF-band IL-10, exert suppressive ac-
tivity towards effector T cells and are expanded in tumour-
positive lymph nodes from breast cancer patients [33, 34].
Interestingly, the observation that in vitro suppressive ac-
tivity of these cells can be strongly inhibited by an anti-
GITR-blocking antibody suggests that GITR may confer
regulatory properties independently on CD25 expression
[35]. The aim of this study was the evaluation of
CD25
low
GITR
+
cells in PB and target organs of pSS pa-
tients. Possible correlations with clinical and serological
parameters were also verified.
Patients and methods
Study population
Forty patients with pSS classified according to Euro-
American criteria [36] and 20 sex- and age-matched
healthy donors (HD) were studied. At the time of enrol-
ment, clinical and serological records were collected
and disease activity was calculated using the EULAR
Sjo
¨gren’s syndrome disease activity index (ESSDAI) [37].
Thirty-eight patients were females and two were males.
Mean age was 53 ± 14 (mean ± S.D.) years and disease
duration 10 ± 5 years. All patients displayed ANA positiv-
ity. Thirty-eight out of forty patients displayed anti-SSA
positivity and, of these, 32 patients displayed also anti-
SSB antibodies. Twenty-nine patients experienced
extraglandular manifestations, including articular, visceral
and haematological involvement. Twenty patients were
receiving HCQ 200 mg/day and, among them, one patient
was also taking MTX 15 mg/week for severe articular in-
volvement. The other 20 patients were taking only topic
medications for sicca symptoms. None of the patients
was taking CSs. The study was approved by local ethical
committee (Umbria Ethic Committee; CEAS) and written
informed consent was obtained from pSS patients and HD
according to the Declaration of Helsinki.
Cell isolation and flow cytometry
Peripheral blood mononuclear cells (PBMCs) were iso-
lated from heparinized venous blood by gradient separ-
ation (Ficoll-Hypaque). For surface and intracellular
staining, a number of antibodies listed in supplementary
data, flow cytometry section, available at Rheumatology
Online, were used. Samples were analysed using
FACScalibur flow cytometer and CellQuestPro software
(Beckton Dickinson).
Magnetic cell sorting
For functional assays, real-time (rt) PCR and flow cytome-
try, CD4
+
T cells were magnetic sorted by negative selec-
tion using human T CD4 isolation kit II and LD columns
(Miltenyi Biotec). Purity of CD4
+
cells was about >98%. A
detailed description of the procedure is available as sup-
plementary data, magnetic cell sorting section, available
at Rheumatology Online.
RNA extraction and rtPCR
Total RNA was isolated with the RNeasy Mini extraction
kit (Qiagen) and reverse transcribed with Quantitect re-
verse transcription kit (Qiagen) as previously described.
rtPCR was performed in a Chromo 4 Four-Color Real-
Time System (Bio-Rad formerly MJ Research) as
described elsewhere [33].
Functional assays
HD and pSS CFSE-labelled CD4
+
CD25
GITR
effector T
cells (0.25 10
5
cells/well) were co-cultured with un-
labelled effector (0.75 10
5
cells/well) or with unlabelled
CD25
+
cell-depleted CD4
+
T cells (0.75 10
5
cells/well) in
RPMI medium as previously described [33]. Suppressive
activity of CD4
+
CD25
low
GITR
+
cells was calculated by
comparing the cell proliferation rate of effector cells co-
cultured with effector cells alone with that of effectors co-
cultured with CD25
+
cell-depleted CD4
+
T cells.
Immunofluorescence and immunohistochemistry of
SG specimens
We first sought to investigate the presence of different
Treg cell subsets within the FoxP3
+
area of mononuclear
cell infiltrate in SS-MSG by double IF. In particular, we
tried to distinguish between GITR-expressing CD25
high
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and CD25
T cells. To our knowledge, neither IF nor
immunohistochemistry (IHC) has been previously em-
ployed to identify GITR expression in MSG. Moreover, in
previous reports investigating conventional Treg cells in
SS-MSG, FoxP3 expression has been assessed only by
immunohistochemistry [12, 22, 24, 27].
In this setting, we failed to achieve good quality staining
for CD25 and Foxp3 using IF and observed low signal
intensity despite high primary antibody concentration.
On the other hand, double staining for GITR and CD25
was of poor quality mainly due to technical incompatibility
of the two antibody staining protocols.
Therefore, we were forced to apply IHC, knowing that
the visual impact would have been lower than IF.
Haematoxylin-eosin staining was performed on minor
labial salivary gland (MSG) samples, each including at
least 5/6 lobules. Diagnosis of pSS was confirmed as pre-
viously described [38, 39]. Serial sections of five MSG
were cut at 3 mm thickness, deparaffinized and rehy-
drated. Envision flex target retrieval solution high pH
(DAKO) was used for antigen retrieval. Then slides were
put on TechMate 500 immunostainer and incubated for
30C at room temperature with mouse anti-human
FoxP3 (Abcam, 1:5), mouse anti-human CD25 (R&D,
1:10) and rabbit anti-human GITR (Acris, 1:50). The anti-
bodies were detected by the DAKO REAL 3-step detec-
tion system for ALP or horseradish peroxidase (HRP).
Briefly, following primary antibody incubation, biotinylated
either anti-rabbit or anti-mouse secondary antibody was
added to the system for 15 min. Subsequently, a 15-min
incubation with streptavidin-conjugated AP or HRP was
performed. Finally, an appropriate chromogen subset
was added and slides were counterstained with haema-
toxilin. Slides were mounted and analysed with Olympus
AX70 microscope.
Statistical analysis
SPSS 13.0 package was used for statistical analysis and
MannWhitney Utest was used. Spearman correlation
coefficient and binary logistic regression were used to
assess the presence of any association between T cell
population percentages and clinical variables. P-values
<0.05 were considered significant.
Results
Reduction of CD4
+
CD25
high
T cells, but expansion of
CD4
+
CD25
low
GITR
+
T cells, in the PB of pSS patients
The proportion of conventional Tregs, as identified by high
expression of CD25 molecule (CD25
high
), was initially eval-
uated in the PB of pSS patients and HD (Fig. 1A and B).
Despite the physiological variability of circulating Treg cell
number, the mean value of circulating CD4
+
CD25
high
con-
ventional Tregs in SS was significantly lower than that
detected in HD (Fig. 1C), in line with the data from the
majority of previously published studies [24, 25, 28]. We
next analysed the percentage of cells characterized by
GITR expression and low levels of CD25 (CD4
+
CD25
low
GITR
+
), recently described in HD [33]. Interestingly, and
in striking contrast to the findings observed on conven-
tional CD4
+
CD25
high
Tregs, the mean percentage of
CD4
+
CD25
low
GITR
+
cells was significantly increased in
the PB of pSS with respect to HD (Fig. 1D). In this case
also, high variability of CD4
+
CD25
low
GITR
+
cell number
was observed.
Phenotypic characterization of CD4
+
CD25
low
GITR
+
cells in pSS
We recently demonstrated in HD that CD4
+
CD25
low
GITR
+
cells have a regulatory phenotype expressing FoxP3, at
both mRNA and protein level [33]. Since circulating acti-
vated T cells are found in autoimmune disorders and GITR
can also represent a marker of T cell activation [40, 41],
we moved to a phenotypic characterization of CD4
+
CD25
low
GITR
+
cells in pSS in order to verify whether
they showed a Treg phenotype. Intracellular FoxP3 ex-
pression was nearly undetectable in CD4
+
CD25
GITR
effector T cells, as evaluated in freshly isolated cells by
flow cytometry (Fig. 2A and B) and in magnetic sorted
cells by rtPCR (Fig. 2C), but it was consistently expressed
in CD4
+
CD25
low
GITR
+
cell subset obtained from both pa-
tients and HD, resulting significantly different from FoxP3
expression in CD4
+
CD25
GITR
effector T cells.
Moreover, CD4
+
CD25
low
GITR
+
cells also express TGF-b
and IL-10 mRNA (Fig. 3). Interestingly enough, mRNA ex-
pression of FoxP3, TGF-band IL-10 appeared signifi-
cantly higher in pSS than in HD CD4
+
CD25
low
GITR
+
cells. This demonstrates that CD4
+
CD25
low
GITR
+
cells
from pSS patients are not activated T cells and display
a Treg phenotype as demonstrated for the same T cell
subset from HD [33].
Functional suppressive activity by CD4
+
CD25
low
GITR
+
cells towards effector T lymphocytes
Next, we verified the in vitro functional activity of
CD4
+
CD25
low
GITR
+
cells from pSS towards autolo-
gous effector T cells, compared with that observed in
HD. For this purpose, we adopted a previously described
culture system in which CFSE-labelled effector T lympho-
cytes, obtained by depletion of both CD25
+
and GITR
+
cells, are co-cultured with CD25
+
cell-depleted CD4
+
cells [33]. The CD25
+
cell-depleted CD4
+
cell fraction con-
tained physiological levels of CD4
+
CD25
low
GITR
+
cells.
Thus, to test their suppressive activity, the proliferation
of CFSE-labelled effector T lymphocytes co-cultured
with effector T cells was compared with that of CFSE-
labelled effector T lymphocytes co-cultured with CD25
+
cell-depleted CD4
+
cells. Our data demonstrated that
CD4
+
CD25
low
GITR
+
T cells from pSS subjects exert sup-
pressive activity on the proliferation of the respective au-
tologous effector T cells, confirming that they are Treg
cells (Fig. 4B right panel and 4C). Moreover, their inhibi-
tory activity, apparently similar to that exerted by HD
CD4
+
CD25
low
GITR
+
T cells (Fig. 4A right panel and 4C),
suggests that they are fully active.
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GITR expressing Treg cells in SS
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FIG.1Circulating CD4
+
CD25
high
and CD4
+
CD25
low
GITR
+
T cells in HD and pSS patients.
Flow cytometric analysis of anti-GITR and anti-CD25-stained CD4
+
T lymphocytes, isolated from a representative healthy
donor (A, right panel) and a representative pSS patient (B, right panel), is shown. On the left, the respective isotypic
control is shown. As previously demonstrated [33], CD4
+
CD25
GITR
+
are considered to be CD4
+
CD25
low
GITR
+
because
low levels of CD25 are detected. The percentage of CD4
+
CD25
high
(C) and CD4
+
CD25
low
GITR
+
(D) in CD4
+
T lympho-
cytes purified from PB of 20 HD and 40 pSS patients is shown. Mean percentages (horizontal lines) of CD4
+
CD25
high
and
CD4
+
CD25
low
GITR
+
are significantly different in HD and pSS.
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FIG.2CD4
+
CD25
low
GITR
+
T cells from HD and pSS patients express comparable levels of FoxP3.
Flow cytometric analysis of freshly isolated PBMC from a representative pSS patient is shown (A). FoxP3
+
cell per-
centage was calculated in CD4
+
CD25
GITR
and CD4
+
CD25
low/
GITR
+
cells from four HD and four pSS (B). FoxP3
+
cells within CD4
+
CD25
low
GITR
+
resulted significantly different from that of CD4
+
CD25
GITR
cells in both HD and pSS
patients. FoxP3
+
cell percentage was not different in CD4
+
CD25
low
GITR
+
cells from HD and pSS patients. FoxP3 mRNA
expression (fold increase of mRNA levels in CD4
+
CD25
low
GITR
+
T cells over the respective effector CD4
+
CD25
-
GITR
population arbitrary set equal to 1) (C).
FIG.3pSS-CD4
+
CD25
low
GITR
+
T cells express higher levels of TGF-band IL-10 compared with those of HD.
mRNA expression of TGF-b(A) and IL-10 (B) was evaluated by rtPCR. Gene expression is shown as fold increase of
mRNA levels in the positively sorted CD4
+
CD25
low
GITR
+
T cells (empty column, HD; black columns, pSS) over mRNA
levels in respective effector (CD4
+
CD25
GITR
) T cells, arbitrary set equal to 1. Columns indicate mean ± S.E.M. of four
subjects.
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GITR expressing Treg cells in SS
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Detection of FoxP3
+
, GITR
+
and CD25
+
cells in pSS
MSG inflamed tissue
In order to verify the presence of CD25
low
GITR
+
T cells in
inflamed pSS MSG, serial sections from five pSS patients
were stained with haematoxylineosin, anti-FoxP3, anti-
CD25 and anti-GITR. The first section was stained with
haematoxylineosin to quantify the number of foci present
in the glandular tissue. The second section was stained
with anti-FoxP3 and the third one with anti-CD25 and anti-
GITR. Each examined biopsy specimen exhibited similar
staining patterns. Focal periductal inflammatory infiltrates
of mononuclear cells were evident in salivary tissue
(Fig. 5A). FoxP3 was expressed by several mononuclear
cells and the staining was essentially localized in lympho-
cytic infiltrates (Fig. 5B and C). Fig 5D demonstrates the
presence of GITR
+
and CD25
+
cells in the area enriched of
FoxP3
+
cells, with distinct evidence of GITR
+
CD25
cells.
It is likely that these cells are the above described
CD25
low
GITR
+
T cells that express so low levels of
CD25 to be detectable by rtPCR, but not by flow cytome-
try or immunohistochemistry. Results confirm the involve-
ment of this Treg-cell subset in inflammatory infiltrate of
pSS MSG.
Association between disease activity and PB
CD4
+
CD25
low
GITR
+
cell expansion in pSS
Taking into account the wide range of expansion of circu-
lating CD4
+
CD25
low
GITR
+
cells in pSS and their ability to
suppress proliferation of effector T cells, we wondered
whether this may be somehow related to general disease
activity. For this purpose, patients were subdivided in
two groups according to ESSDAI values. Patients with
ESSDAI 42(n= 30), whose only manifestation of disease
was a mild stable polyclonal hypergammaglobulinaemia,
were considered inactive, whereas patients with
ESSDAI >2(n= 10) were classified as active. As Fig. 6
shows, disease activity did not influence the number of
circulating CD4
+
CD25
high
Treg cells, which was
decreased in the group of patients with active as well as
inactive disease with respect to controls. Intriguingly,
however, the proportion of PB CD4
+
CD25
low
GITR
+
cells
in subjects with active disease was similar to that
observed in HD, whereas their number was significantly
increased in the PB of inactive patients. Spearman correl-
ation coefficients were derived and binary logistic regres-
sion was performed to identify possible association
between T cell population percentages and clinical vari-
ables but no significant differences were observed.
Discussion
It is recognized that the fine balance between Treg and T
effector cells regulating immune homeostasis is often dis-
rupted in autoimmune disorders, with evidence of altered
number and/or function of these T cell subsets [1517]. In
this regard, indeed, it is thought that dysfunction or de-
pletion of Tregs, which are physiologically involved in con-
trol and prevention of autoimmune response, may closely
relate to the pathogenesis of several chronic inflamma-
tory/autoimmune rheumatic diseases, including SLE and
RA [4245]. Thus, it is conceivable that closer knowledge
on the pathogenic role of Tregs in these disorders may
help in the development of new therapeutic strategies
[46]. Studies on this topic in humans, however, are often
hampered by confounding factors due to concurrent
chronic treatments that may alter number, phenotype
and function of T cell subsets. In this setting, pSS repre-
sents an interesting model of autoimmune disease that
shares features of CTD with SLE and of localized chronic
inflammation with RA. Moreover, an additional and
FIG.4Inhibitory activity exerted by CD4
+
CD25
low
GITR
+
T
cells pSS patients is similar to that of HD.
CFSE-labelled effector T cells, activated with cross-linked
anti-CD3 antibody, were co-cultured with unlabelled ef-
fector (CD4
+
CD25
GITR
) (left panels in Aand B) or un-
labelled CD25-depleted (including CD4
+
CD25
GITR
and
CD4
+
CD25
/low
GITR
+
) cells (right panels in Aand B) at 1:3
cell ratio. The inhibition of proliferation resulted equal to
27.7% in the representative HD (A) and to 41.5% in the
representative pSS patients (B). Proliferation was evalu-
ated after 4 days. (C) Histogram shows percentage in-
hibition of proliferation by CD4
+
CD25
low
GITR
+
cells
observed in four HD and four pSS patients, indicated by
progressive numbers.
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fundamental element provided by studies on pSS is the
fact that the majority of these patients do not require im-
munosuppressive treatments, due to the frequent benign
course of the disease.
Surprisingly, studies on Tregs in pSS are few and have
yielded controversial results. They mainly investigated cir-
culating conventional CD25
high
Treg cells and, in agree-
ment with our data, a decreased PB cell number
compared with HD was found in three out of six available
studies [24, 25, 28]. In one of the other three studies,
CD4
+
CD25
high
cell percentage similar to HD was
observed in a very small cohort of pSS patients, but de-
scription of clinical features and cellular functional data
were not provided [26]. In contrast, two studies found
increased numbers of circulating CD4
+
CD25
high
cells in
pSS. However, one of these enrolled a significant percent-
age of patients undergoing immunosuppressive treatment
[23] and the other showed, in parallel, a normal number of
FoxP3
+
cells [27], thereby suggesting a PB expansion of
CD25
+
FoxP3
T cells. In this context, it is noteworthy that,
in human SLE, many circulating CD4
+
FoxP3
+
cells, with
poorly defined identity, have been shown to be CD25
low
/
CD25
[45, 47]. The dissociated expression of CD25 and
Foxp3, which is frequently observed in CD4
+
cells from
CTD [26, 48], favours some important considerations.
CD25 can be induced on T cell surface by activation
and does not appear essential for human peripheral
Treg-cell maintenance in vivo [18]. Therefore, speculations
FIG.6Circulating CD4
+
CD25
high
and CD4
+
CD25
low
GITR
+
T cells in HD and pSS patients according to disease activity.
Flow cytometric analysis of anti-GITR and anti-CD25-stained CD4
+
T lymphocytes was performed. Percentage of
CD4
+
CD25
high
(A) and CD4
+
CD25
low
GITR
+
(B) in CD4
+
T cells purified from freshly isolated PB of 20 HD (n= 30), patients
with inactive disease identified by an ESSDAI 42(n= 20) and patients with active disease identified by an ESSDAI >2
(n= 10) was evaluated. Bars indicate mean ± S.E.M.
FIG.5CD4
+
CD25
low
GITR
+
T cells are present in pSS salivary gland inflammatory infiltrate.
Haematoxylin and eosin staining of the mononuclear cell infiltrate (A;5magnification). FoxP3 expression revealed by
immunohistochemistry within the mononuclear cell infiltrate in a consecutive section of MSG (B;5magnification). In a
consecutive section, the area identified by the rectangle in panel B is shown at 20magnification (C). The same area
stained with anti-CD25 (brown) and anti-GITR (blue) antibodies is depicted at 50magnification (D). Asterisks identify
GITR
+
CD25
cells. Results were similar in the other four MSG analysed.
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GITR expressing Treg cells in SS
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on altered proportions of Tregs simply based on pheno-
typic analysis of this molecule must be made with caution
in conditions characterized by T cell activation, such as
systemic autoimmune disorders. Although also Foxp3
could be induced at least transiently upon T cell activa-
tion, it represents a key regulator of development and
suppressive activity of Tregs and, therefore, it is con-
sidered at present one of the most reliable markers of
these cells [20]. Unfortunately, its intracellular localization
makes difficult its utilization to separate Tregs for thera-
peutic purposes [49]. On the basis of the described uncer-
tainties on phenotypic identification of Tregs, we analysed
a recently described CD4
+
CD25
low
GITR
+
cell subset, able
to exert strong suppressive activity in humans [33], in pSS
patients and found a consistent PB expansion of these
cells. In agreement with the data observed in HD,
CD4
+
CD25
low
GITR
+
cells circulating in pSS display a
Treg phenotype, characterized by simultaneous expres-
sion of FoxP3, TGF-band IL-10. Levels of expression of
these Treg markers are significantly higher than those
observed in healthy donors, possibly suggesting that
they represent activated Treg cells [50, 51]. The hypoth-
esis is in line with the possibility that effector T cells are
partly activated in autoimmune diseases [52], thus eliciting
Treg activation, and may explain why Treg cells of pSS do
not seem to suppress autologous effector T cells at levels
higher than Treg cells from HD. The demonstration of
the suppressive activity of CD4
+
CD25
low
GITR
+
cells is
of particular relevance in view of the fact that the
majority of the published studies investigating PB Treg
cells in pSS do not examine their functional activity
[22, 2527].
The increased frequency of T lymphocytes with pheno-
type and activity of Treg cells, we observed in the PB of
pSS, appears to be in contrast with the findings usually
described in autoimmune disorders of both animal models
and humans, where deficiency in Tregs number and/or
function has been postulated to be critical in triggering
autoimmunity development [26]. In addition, it is of interest
to note that sialadenitis is one of the most common auto-
immune manifestations observed in experimental animals
with manipulated Tregs. Thus, Treg-cell deficiency should
be expected in human pSS.
The apparently conflicting data of the present study de-
serve some consideration. As mentioned above, pSS pre-
sents features of a chronic inflammatory disease,
essentially localized at exocrine gland tissues. MSG infil-
trate is mainly represented by T cells with selective local-
ization of different Th-cell subsets [53]. In agreement with
previously published reports [12, 22], we showed that
many MSG infiltrating lymphocytes express the Treg
master regulator FoxP3. Our results also demonstrated
that many infiltrating T cells display GITR without evidence
of CD25 on their surface, thereby confirming the presence
of the CD25
low
GITR
+
Treg-cells in the inflamed glandular
tissue. The presence of these T cells at the site of inflam-
mation raises the question of their functional activity at this
location. In this context, it is worth noting that the degree
of mononuclear cell infiltrate appears to positively
correlate with both FoxP3
+
and proinflammatory Th17-
cell number in mild and moderate inflammation, while
Th17-cell expansion strongly predominates on Treg-cell
number in the advanced MSG lesions [12, 22, 27]. In con-
sequence, it is conceivable to imagine that the loss of
down-modulatory/proinflammatory T cell balance may
lead to diffuse inflammatory infiltrates and consequent
glandular tissue damage. This may be the result of the
well-known developmental plasticity of Treg and Th17
cells that can lead to the conversion of FoxP3
+
Treg
cells into IL-17-producing cells in the context of an inflam-
matory cytokine milieu enriched of TGF-band IL-6 [54].
Unfortunately, although we showed a strong suppressor
capacity of circulating CD25
low
GITR
+
cells, their low
number in MSG infiltrates hampers the possibility to con-
firm their functional properties at tissue level. Notably,
however, pSS also displays features of a classical CTD
with aberrant immune activation, circulating autoantibo-
dies and possible diffuse inflammatory extraglandular
organ involvement. Interestingly, these features appear
to be more frequent in patients with more diffuse inflam-
matory MSG infiltration [55]. Thus, PB expansion of
CD4
+
CD25
low
GITR
+
cells in pSS may be the reflection of
a general, rather than local, counter-regulatory attempt
exerted by the immune system against the predominant
proinflammatory cytokine effects driven by autoimmune
phenomena. The present findings, showing that the ex-
pansion of this Treg subset is essentially observed in pa-
tients with inactive disease, seem to support this
hypothesis.
In conclusion, the present results demonstrated PB ex-
pansion in pSS subjects of a recently identified T cell
subset with regulatory properties, characterized by high
GITR, but low CD25, surface expression. These cells dis-
play FoxP3, express TGF-band IL-10, key cytokines
involved in Treg suppressive mechanisms, and are func-
tionally active, being able to inhibit effector T cell prolifer-
ation. Although the presence of these T cells in MSG
infiltrate may suggest they exert a role in the local patho-
genic mechanism of the disease, we were unable to verify
their function that, otherwise, may be influenced by the
inflammatory microenvironment. However, unlike conven-
tional CD4
+
CD25
high
Tregs, which were reduced and did
not correlate with disease activity, PB expansion of
CD4
+
CD25
low
GITR
+
cells was observed only in patients
with inactive disease, thus further supporting their in vivo
regulatory role. On the basis of these findings, we believe
that the identification of a surface marker such as GITR,
able to recognize T cells with regulatory properties, may
represent an interesting target for future therapeutic
proposals.
Rheumatology key messages
.In pSS patients, CD4
+
CD25
low
GITR
+
cells display
the phenotype and function of Treg cells.
.In pSS, a great number of CD4
+
CD25
low
GITR
+
cells
infiltrates salivary glands.
.CD4
+
CD25
low
GITR
+
cell number doubles in inactive
pSS compared with active pSS and healthy donors.
1394 www.rheumatology.oxfordjournals.org
Alessia Alunno et al.
at GOT (Consortium) on July 11, 2013http://rheumatology.oxfordjournals.org/Downloaded from
Funding: This work was supported by the ‘Fondazione
Cassa di Risparmio di Perugia’.
Disclosure statement: The authors have declared no con-
flicts of interest.
Supplementary data
Supplementary data are available at Rheumatology
Online.
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