Angiogenic T cell expansion correlates with severity of peripheral vascular damage in systemic sclerosis
Angiogenic T cell expansion correlates with severity of peripheral vascular damage in systemic sclerosis
Mirko Manetti 0 1
Sara Pratesi 1
Eloisa Romano 1
Silvia Bellando-Randone 1
Irene Rosa 0 1
Serena Guiducci 1
Bianca Saveria Fioretto 1
Lidia Ibba-Manneschi 0 1
Enrico Maggi 1
Marco Matucci-Cerinic 1
0 Department of Experimental and Clinical Medicine, Section of Anatomy and Histology, University of Florence , Florence , Italy , 2 Department of Experimental and Clinical Medicine, Section of Internal Medicine, Azienda Ospedaliero-Universitaria Careggi (AOUC), University of Florence , Florence , Italy
1 Editor: Masataka Kuwana, Keio University , JAPAN
The mechanisms underlying endothelial cell injury and defective vascular repair in systemic sclerosis (SSc) remain unclear. Since the recently discovered angiogenic T cells (Tang) may have an important role in the repair of damaged endothelium, this study aimed to analyze the Tang population in relation to disease-related peripheral vascular features in SSc patients. Tang (CD3+CD31+CXCR4+) were quantified by flow cytometry in peripheral blood samples from 39 SSc patients and 18 healthy controls (HC). Circulating levels of the CXCR4 ligand stromal cell-derived factor (SDF)-1α and proangiogenic factors were assessed in paired serum samples by immunoassay. Serial skin sections from SSc patients and HC were subjected to CD3/CD31 and CD3/CXCR4 double immunofluorescence. Circulating Tang were significantly increased in SSc patients with digital ulcers (DU) compared either with SSc patients without DU or with HC. Tang levels were significantly higher in SSc patients with late nailfold videocapillaroscopy (NVC) pattern than in those with early/active NVC patterns and in HC. No difference in circulating Tang was found when comparing either SSc patients without DU or patients with early/active NVC patterns and HC. In SSc peripheral blood, Tang percentage was inversely correlated to levels of SDF-1α and CD34+CD133+VEGFR-2+ endothelial progenitor cells (EPC), and positively correlated to levels of vascular endothelial growth factor and matrix metalloproteinase-9. Tang were frequently detected in SSc dermal perivascular inflammatory infiltrates. In summary, our findings demonstrate for the first time that Tang cells are selectively expanded in the circulation of SSc patients displaying severe peripheral vascular complications like DU. In SSc, Tang may represent a potentially useful biomarker reflecting peripheral vascular damage severity. Tang expansion may be an ineffective attempt to compensate the need for increased angiogenesis and EPC function. Further studies are required to clarify the function of Tang cells and investigate the mechanisms responsible for their change in SSc.
Data Availability Statement: All relevant data are
within the paper.
Funding: The study was supported by grants from
the University of Florence (Progetti di Ricerca di
Ateneo to MM-C). The funder had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
Systemic sclerosis (SSc) is an orphan connective tissue disease characterized by early
generalized microvascular damage and specific immunologic abnormalities evolving into fibrosis of
the skin and internal organs [1±3]. In SSc, detailed assessment of skin microcirculation by
nailfold videocapillaroscopy (NVC) allows the detection of a variety of morphological changes
reflecting the severity and progression of peripheral microvascular injury, including giant
capillaries, microhemorrhages, loss of capillaries with the appearance of avascular areas, and
abnormal capillary shapes evocative of disturbances in post-ischemic vascular repair and
2, 4, 5
Although the mechanisms underlying endothelial cell damage and defective repair remain
incompletely understood, a large body of evidence suggests that a profound impairment of
either neoangiogenesis or endothelial progenitor cell (EPC)-driven vasculogenesis is a key
feature in SSc [6±10]. In fact, the dysregulation of multiple cellular and molecular pathways, that
are required for post-ischemic endothelial cell and EPC compensatory responses, prevents
peripheral vascular recovery [2, 11±16]. This often results into severe peripheral vascular
manifestations such as digital ulcers (DU) and gangrene [
Recent studies suggest that a specific T cell population, termed angiogenic T cells (Tang),
may promote the formation of new blood vessels and enhance the repair of damaged
]. Tang cells are characterized by the co-expression of CD3, platelet-endothelial cell
adhesion molecule-1 (CD31), and the receptor for the CXC chemokine stromal cell-derived
factor-1 (SDF-1)/CXCL12 (CXCR4 or CD184) [
]. In vitro experiments showed that Tang
cells may foster postnatal vasculogenesis and endothelial repair by stimulating early EPC
differentiation and endothelial cell proliferation and functions possibly through the secretion of
high levels of proangiogenic factors, such as vascular endothelial growth factor (VEGF),
interleukin (IL)-8, IL-17 and matrix metalloproteinase (MMP)-9 . Indeed, it has been
demonstrated that CD3+CD31+CXCR4+ Tang cells constitute the central cluster of EPC colonies
during cultures of human peripheral blood mononuclear cells, and that Tang depletion could
abrogate EPC differentiation and functionality [
]. Moreover, in vivo studies also highlighted
the relevance of the Tang cell subset in the process of new capillary formation in a mouse
model of hind limb ischemia [
]. Of note, it has been recently reported that altered circulating
Tang cell frequencies may be associated with cardiovascular disease in rheumatoid arthritis
(RA), systemic lupus erythematosus (SLE) and antineutrophil cytoplasmic antibody
(ANCA)associated vasculitis [18±20].
On these premises, the aim of the present study was to investigate the Tang cell population
and to verify its possible correlation with the peripheral vascular features of SSc.
Materials and methods
Patients and controls
Peripheral blood samples were obtained from 39 consecutive patients fulfilling the 2013 ACR/
EULAR classification criteria for SSc [
] and 18 age-matched and sex-matched healthy
controls (HC). Patients with SSc were further classified as limited cutaneous SSc (lcSSc; n = 24) or
diffuse cutaneous SSc (dcSSc; n = 15) according to LeRoy et al. [
]. At the time peripheral
blood was drawn, the presence of DU was recorded. NVC was performed on all 10 fingers by a
single rheumatologist, and images were scored blindly by two experienced examiners who
divided patients into three NVC patterns (i.e., early, active and late) [
]. At the time of
sampling, patients were not on immunosuppressive medications, corticosteroids or other
potentially disease-modifying drugs. Before blood sampling, they were washed out for 10 days from
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oral vasodilating drugs and for 2 months from intravenous prostanoids. Fresh venous blood
samples were drawn and either immediately processed for flow cytometric analysis or left to
clot for 30 minutes before centrifugation at 1,500 g for 15 minutes for serum separation.
Serum samples were stored in aliquots at −80ÊC until used. Paraffin-embedded sections of
lesional forearm skin biopsies were obtained from 7 patients with early dcSSc (disease duration
<2 years from first non-Raynaud symptom) and 6 age-matched and sex-matched HC, as
described elsewhere [
]. The study was conducted in compliance with the Declaration of
Helsinki and was approved by the local institutional review board at the Azienda
OspedalieroUniversitaria Careggi (AOUC), Florence, Italy (AOUC 69/13). All subjects provided written
Flow cytometric analysis of Tang cells and EPC
Circulating Tang cells were characterized by flow cytometry. Briefly, 300 μl of fresh whole
peripheral blood was stained with anti-CD3 allophycocyanin-H7 (APC-H7), anti-CD31
fluorescein isothiocyanate (FITC) and anti-CXCR4 phycoerythrin (PE) antibodies or
isotypematched control IgG antibodies (all from BD Biosciences, San Diego, CA, USA) according to
the manufacturer's instructions. Subsequently, red blood cell lysis was performed and cells
were analyzed. At least 30,000 CD3+ T lymphocytes events were acquired using a FACSCanto
II flow cytometer (BD Biosciences) and analyzed using FACSDiva software (BD Biosciences).
CD3+ T lymphocytes double-positive for CD31 and CXCR4 were considered Tang cells. For
the evaluation of circulating EPC, CD34+ cells were enriched from peripheral blood
mononuclear cells from SSc patients and HC using the CD34 MicroBead Kit (Miltenyi Biotec, Bergisch
Gladbach, Germany) and stained with anti-CD34 FITC, anti-VEGF receptor-2 (VEGFR-2)
Peridinin Chlorophyll Protein-Cy5.5 (PerCP-Cy5.5) (both from Miltenyi Biotec) and
antiCD133 PE (BD Biosciences) antibodies. At least 5,000 events in the CD34+ enriched
population gate were acquired at low rate and EPC were identified as CD34+CD133+VEGFR-2+ cells.
Absolute blood cell counts and evaluation of Tang cell phenotype
Trucount Tubes (BD Biosciences) were used for determining the absolute numbers of Tang
cells in SSc patients and HC. Fresh whole peripheral blood (100 μl) was added into the
Trucount Tube and then stained with anti-CD3 Pacific Blue, anti-CD8 PerCP-Cy5.5 (Miltenyi
Biotec), anti-CD4 PE-Cy7, anti-CD31 FITC, anti-CXCR4 PE and anti-CD28 allophycocyanin
(APC) antibodies (all from BD Biosciences). After 15 minutes, red blood cells were lysed with
NH4Cl and then cells were analyzed using a FACSCanto II flow cytometer (BD Biosciences)
equipped with FACSDiva software (BD Biosciences). The absolute cell count was obtained
multiplying the number of positive cell events by the number of Trucount Tube beads and
subsequently dividing by the number of Trucount bead events.
Immunofluorescence staining of skin sections
For antigen retrieval, paraffin-embedded serial skin sections (5 μm thick) were deparaffinized
and boiled for 10 minutes in 10 mM sodium citrate buffer (pH 6.0). After washing in
phosphate buffered saline (PBS), the sections were incubated in 2 mg/ml glycine for 10 minutes to
quench autofluorescence and then blocked for 1 hour at room temperature with 1% bovine
serum albumin in PBS. The slides were subsequently incubated overnight at 4ÊC with a
mixture of prediluted mouse monoclonal anti-CD3 (catalog number ab7507, Abcam, Cambridge,
UK) and rabbit polyclonal anti-CD31 (1:50 dilution; catalog number ab28364, Abcam) or
rabbit monoclonal anti-CXCR4 (1:100 dilution; catalog number ab124824, Abcam) antibodies.
The day after, skin sections were extensively washed in PBS and incubated with a mixture of
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Alexa Fluor-488-conjugated goat anti-mouse IgG and Rhodamine Red-X-conjugated goat
anti-rabbit IgG (both 1:200 dilution; Molecular Probes, Eugene, OR, USA) for 45 minutes at
room temperature in the dark. Irrelevant isotype-matched and concentration-matched mouse
and rabbit IgG (Sigma-Aldrich, St. Louis, MO, USA) were used as negative controls. Nuclei
were counterstained with 40,6-diamidino-2-phenylindole (DAPI) (Chemicon International,
Temecula, CA, USA). The immunostained sections were then observed under a Leica
DM4000 B microscope (Leica Microsystems, Mannheim, Germany). Fluorescence images
were captured using a Leica DFC310 FX 1.4-megapixel digital color camera equipped with the
Leica software application suite LAS V3.8 (Leica Microsystems).
Determination of SDF-1α, VEGF, MMP-9, IL-8 and IL-17 serum levels
Levels of SDF-1α, VEGF, MMP-9, IL-8 and IL-17 in serum samples were measured by
commercial quantitative colorimetric sandwich enzyme-linked immunosorbent assay (Human
CXCL12/SDF-1α Quantikine ELISA Kit, Human VEGF Quantikine ELISA Kit and Human
IL-17 DuoSet ELISA Kit, all from R&D Systems, Minneapolis, MN, USA; Human IL-8/NAP-1
INSTANT ELISA Kit and Human MMP-9 Platinum ELISA Kit, both from eBioscience, San
Diego, CA, USA) according to the manufacturer's instructions. The detection range was
0.156±10.0 ng/ml for SDF-1α, 31.2±2,000 pg/ml for VEGF, 0.23±15.0 ng/ml for MMP-9, 15.6±
1,000 pg/ml for IL-8 and 15.6±1,000 pg/ml for IL-17. Serum samples were diluted 1:250 for the
MMP-9 assay. Concentrations were calculated using a standard curve generated with specific
standards provided by the manufacturer. Each sample was measured in duplicate.
Data are expressed as the median and interquartile range (IQR). Differences between two
independent groups were determined using the nonparametric Mann±Whitney U test.
Correlations were evaluated by nonparametric Spearman's rank correlation analysis. For all tests, a
two-sided p-value less than 0.05 was considered significant. Data analyses were performed
using SPSS 24.0 software (SPSS, Chicago, IL, USA).
Demographic and clinical characteristics of SSc patients are summarized in Table 1. Peripheral
blood samples from 39 SSc patients and 18 HC were analyzed by flow cytometry, quantifying
Tang cell population by means of their CD3, CD31 and CXCR4 expression (Fig 1A and 1B).
The percentage of circulating CD3+CD31+CXCR4+ Tang cells in total CD3+ T cells was not
different between the whole SSc patient cohort (median 29.9, IQR 22.3−36.2) and HC (median
25.2, IQR 23.3−33.5) (Fig 1C). No difference in circulating Tang cells was detected between
patients with lcSSc (median 30.4, IQR 23.4−36.4) and those with dcSSc (median 28.3, IQR 21.0
However, an interesting association between Tang cell levels and the extent of peripheral
microvascular damage was found. First, subgroup analysis revealed that Tang cells were
significantly increased in SSc patients with DU (median 35.5, IQR 32.2−42.5) compared either with
SSc patients without DU (median 23.3, IQR 18.5−26.6) or with HC (p<0.0001 for both) (Fig
2A and 2B). Furthermore, Tang cell percentage was significantly higher in SSc patients with
late NVC pattern (median 34.9, IQR 25.0−42.0) than in those with early/active NVC patterns
(median 26.5, IQR 20.4−32.9) and in HC (p = 0.01 and p = 0.04, respectively) (Fig 2C). On the
contrary, no difference in circulating Tang cell percentages was found when comparing either
SSc patients without DU or patients with early/active NVC patterns and HC (Fig 2B and 2C).
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No significant association was found with the other clinicodemographic and laboratory
parameters of SSc patients.
As displayed in Table 2, similar results were obtained when the Tang cell population was
expressed as absolute numbers of circulating CD3+CD31+CXCR4+ cells.
Moreover, we evaluated the proportions of circulating Tang cell subsets in SSc patients and
HC on the basis of the expression of the CD4, CD8 and CD28 antigens (Fig 3A±3C). The
percentages of CD4+, CD8+, CD28+ and CD28null cells in total CD3+CD31+CXCR4+ Tang cells
were similar in SSc patients and HC (Table 3). In addition, no difference in the proportions of
the analyzed cell subsets in total CD3+CD31+CXCR4+ Tang cells was found between SSc
patient subgroups according to the presence of DU and NVC patterns (Table 3).
Next, we quantified circulating SDF-1α, a chemokine which may play a major role in the
mobilization and trafficking of CXCR4-bearing Tang cells. In SSc peripheral blood, the
percentage of Tang cells exhibited a strong inverse correlation with the levels of SDF-1α
(Spearman's rho = -0.78, p<0.0001) (Fig 4A). This result is made even more significant by the fact
that such a correlation was not found in HC (Fig 4B). Interestingly, when the relationship
between Tang cells and SDF-1α levels was examined in the SSc patient subgroups, a significant
inverse correlation was found only in patients with DU (Spearman's rho = -0.52, p = 0.03) and
in those with late NVC pattern (Spearman's rho = -0.74, p<0.0001).
Since previous studies have shown that Tang cells may be linked to the EPC population [
], the possible relationship between circulating Tang cells and CD34+CD133+VEGFR-2+
EPC was explored. Consistent with previous reports [
], the percentage of circulating
CD34+CD133+VEGFR-2+ EPC in total CD34+ cells was significantly decreased in SSc patients
(median 0.39, IQR 0.05−0.80) respect to HC (median 1.60, IQR 0.55−2.80) (p = 0.001). As
displayed in Fig 5A and 5B, Tang cells exhibited a significant negative correlation with EPC levels
in SSc (Spearman's rho = -0.33, p = 0.04), but not in HC.
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Fig 1. Angiogenic T cells (Tang) in peripheral blood of healthy controls (HC) and systemic sclerosis
(SSc) patients. (A) Gating strategy used for the flow cytometric enumeration of Tang. Gated CD3+ T
lymphocytes were analyzed for CD31 and CXCR4 expression by flow cytometry. Tang population was
identified as CD3+CD31+CXCR4+ cells in the lymphocyte gate. Quadrants were set according to the
fluorescence signal provided by the isotype controls. (B) Representative CD31 versus CXCR4 dot plots of a
HC and a SSc patient. (C) Percentages of circulating CD3+CD31+CXCR4+ Tang cells in total CD3+ T cells
from HC (n = 18) and SSc patients (n = 39). Data are shown as box plots. Each box represents the 25th to
75th percentiles. Lines inside the boxes represent the median. Lines outside the boxes represent the 10th and
the 90th percentiles. Differences were evaluated by Mann±Whitney U test.
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Fig 2. Circulating levels of angiogenic T cells (Tang) correlate with severity of peripheral vascular
damage in systemic sclerosis (SSc) patients. (A) Representative CD31 versus CXCR4 dot plots of a SSc
patient without digital ulcers (DU) and a SSc patient with DU. (B) Percentages of circulating
CD3+CD31+CXCR4+ Tang cells in total CD3+ T cells from HC (n = 18), SSc patients without DU (n = 21) and
SSc patients with DU (n = 18). (C) Percentages of circulating CD3+CD31+CXCR4+ Tang cells in total CD3+ T
cells from HC (n = 18), SSc patients with early/active nailfold videocapillaroscopy (NVC) patterns (n = 20) and
SSc patients with late NVC pattern (n = 19). Each box represents the 25th to 75th percentiles. Lines inside the
boxes represent the median. Lines outside the boxes represent the 10th and the 90th percentiles. Circles
indicate outliers. Differences were evaluated by Mann±Whitney U test.
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We also investigated whether in SSc patients and HC the Tang population could correlate
with circulating levels of proangiogenic factors which have been reported to be secreted by
Tang cells, namely VEGF, MMP-9, IL-8 and IL-17 [
] (Fig 6A±6H). This analysis revealed
that in SSc the percentage of circulating Tang cells was positively correlated with the levels of
VEGF (Spearman's rho = 0.51, p = 0.001) and MMP-9 (Spearman's rho = 0.37, p = 0.02) (Fig
6A and 6C), but not with the levels of IL-8 and IL-17 (Fig 6E and 6G). None of the analyzed
factors was found to be significantly correlated with Tang cells in HC (Fig 6B, 6D, 6F and 6H).
Finally, the possible presence of Tang cells in the biopsies of the forearm skin affected by
the disease was investigated by immunofluorescence (Fig 7A±7D). As displayed in Fig 7B,
CD3+CD31+ T lymphocytes were frequently observed in SSc dermal perivascular
inflammatory infiltrates, while they could not be detected in HC skin. Moreover, CD3/CD31 and CD3/
CXCR4 double immunofluorescence performed on serial skin sections clearly demonstrated
the presence of perivascular CD3+CD31+CXCR4+ Tang cells in SSc dermis (Fig 7C and 7D).
This is the first study investigating the possible involvement of the recently identified Tang cell
population in SSc. Our findings demonstrate that Tang cells are selectively expanded in the
circulation of SSc patients displaying severe peripheral vascular complications like DU. In fact,
patients with advanced derangement of the dermal capillary network and DU had circulating
levels of Tang cells significantly higher than HC, both in absolute numbers and as a percentage
of T cells. On the contrary, Tang levels did not differ between HC and SSc patients with
moderate capillary damage and lack of DU. Thus, our data suggest that circulating Tang cells
might represent a novel biomarker closely reflecting the severity of SSc-related peripheral
The maintenance of endothelial homeostasis and the formation of new blood vessels are of
paramount importance in the control of human health and disease. They are finely tuned and
controlled by a complexity of soluble and cellular components. In this context, Tang are a
unique T cell subset endowed with proangiogenic functions capable of sustaining endothelial
repair by interacting with EPC and endothelial cells [17±20]. Tang, first described by Hur et al.
in 2007 [
], are CD3+CD31+CXCR4+ cells required for in vitro colony formation and
differentiation of early hematopoietic EPC. Furthermore, it appears that Tang cells may enhance
endothelial proliferation and functions by either cell contact-dependent or paracrine
]. In particular, Tang secrete a wide array of proangiogenic factors, including VEGF,
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Fig 3. Analysis of circulating angiogenic T cell (Tang) subsets in healthy controls (HC) and systemic
sclerosis (SSc) patients. (A-C) The proportions of CD4+, CD8+, CD28+ and CD28null cells in total
CD3+CD31+CXCR4+ Tang cells were assessed by flow cytometry. Representative dot plots of a HC and a SSc
patient for each Tang cell subset are shown.
IL-8, IL-17, granulocyte-colony stimulating factor, and MMP-9 [
]. Altogether, these
properties give Tang the ability to stimulate angiogenesis in vitro and promote neovascularization of
ischemic tissues in vivo, as demonstrated in a hind limb ischemia experimental model [
Growing evidence indicates that impaired angiogenesis and vasculogenesis critically
contribute to the pathogenesis and clinical manifestations of SSc [
2, 9, 11, 12
]. An abnormal
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DU, digital ulcers; NVC, nailfold videocapillaroscopy.
Values are the percentage of cells in total CD3+CD31+CXCR4+ Tang cells expressed as median (IQR).
expression of several proangiogenic and angiostatic factors has been reported in SSc tissues
and peripheral blood [
7, 12, 25, 26
]. Moreover, a number of in vitro and in vivo studies showed
that either dermal microvascular endothelial cells or bone marrow-derived circulating EPC are
defective in SSc [8±16]. The present findings provide the first evidence that Tang cells may be
part of this complex scenario. Indeed, differential percentages and absolute numbers of
circulating Tang cells were detected in SSc patients according to the severity of peripheral
vasculopathy. Furthermore, our immunohistologic analyses revealed that Tang cells are present in
perivascular inflammatory infiltrates of early SSc skin lesions. It appears that Tang are
endowed with a high capacity of adhesion to endothelial cells and transendothelial migration
through the endothelial junctions using CD31, as well as a great capacity to invade the
ischemic tissue using MMP-9 . It has also been postulated that Tang cells expressing high levels
of CXCR4 home to areas of ischemia where SDF-1 level is high [
]. Of note, previous studies
from our group have shown that dermal expression of SDF-1 is strongly increased in the early
phase of SSc [
]. In addition, in our patients the percentage of Tang cells was inversely
correlated to the circulating levels of SDF-1α. Thus, in SSc the SDF-1/CXCR4 axis might play a
major role in the homing of Tang cells to the affected skin.
The expansion of Tang cells observed in SSc patients with significant microvascular
involvement and reduction of the blood flow might be a reaction to an inefficient angiogenesis
and EPC function. This hypothesis is supported by the evidence that the levels of Tang cells
were inversely correlated with those of EPC in the circulation of SSc patients. Furthermore, in
SSc patients circulating Tang levels exhibited a positive correlation with the levels of VEGF
and MMP-9, two soluble mediators that have been implicated in SSc-related angiogenic
disturbances and that can be secreted even by Tang themselves [
11, 12, 17, 28
In line with our findings, ANCA-associated vasculitis patients with relapsing disease course
showed an expansion of circulating Tang [
]. Moreover, another study reported that the
percentage of circulating CD8+ Tang cells was significantly increased in SLE patients when
compared to HC [
]. Conversely, lower Tang cell numbers have been associated with vascular
disease in RA and hypertensive patients [
]. Of note, a very recent study revealed the
existence of two different subsets of Tang cells based on CD28 expression, which may be endowed
with opposite functions . In particular, it appears that the CD28null Tang subset displays a
senescent phenotype and might exert cytotoxic and inflammatory rather than protective effects
on endothelial cells [
]. Interestingly, the percentage of CD28null Tang cells was found to
be especially increased in patients with traditional cardiovascular risk factors and patients with
SLE, but not in RA patients . However, when we investigated Tang cell subsets on the basis
of the expression of CD4, CD8 and CD28 antigens, we could not find any significant difference
in the proportions of CD4+, CD8+, CD28+ and CD28null Tang cells either between SSc patients
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Fig 4. Correlation analysis of the percentage of circulating angiogenic T cells (Tang) with serum
levels of stromal cell-derived factor-1α (SDF-1α) in systemic sclerosis (SSc) patients and healthy
controls (HC). (A) In SSc patients, the percentage of CD3+CD31+CXCR4+ Tang cells is inversely correlated
with circulating levels of SDF-1α. (B) No significant correlation is observed in HC. Data are shown as
scatterplot, each dot representing a subject. Spearman's rho correlation coefficient and p value are indicated.
NS, not significant.
and HC or between SSc patient subgroups stratified according to the severity of peripheral
vasculopathy. Thus, the expansion of Tang cells observed in SSc patients with more severe
peripheral vascular complications seems not attributable to a specific Tang subpopulation.
Our study has some limitations. First, the study design is cross-sectional and therefore
prospective analyses will be required to ascertain whether the proportion of circulating Tang cells
may vary in SSc patients with progression of peripheral vascular damage. Furthermore, our
findings need to be replicated in larger and independent cohorts of SSc patients. Notwithstanding
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Fig 5. Correlation analysis of the percentages of circulating angiogenic T cells (Tang) and endothelial
progenitor cells (EPC) in systemic sclerosis (SSc) patients and healthy controls (HC). (A) In SSc
patients, the percentage of CD3+CD31+CXCR4+ Tang cells is inversely correlated with that of
CD34+CD133+VEGFR-2+ EPC. (B) No significant correlation is observed in HC. Data are shown as
scatterplot, each dot representing a subject. Spearman's rho correlation coefficient and p value are indicated.
NS, not significant.
these limitations, the strength of our study remains the novelty of our findings in a
well-characterized and homogeneous group of SSc patients.
In summary, we found that Tang cells relate to the severity of peripheral vascular disease in
SSc patients. Considering the relevance of Tang cells in the control of angiogenesis and
endothelial and EPC homeostasis, it may be of interest to further investigate the feasibility of these
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Fig 6. Correlation analysis of the percentage of circulating angiogenic T cells (Tang) and serum levels
of proangiogenic factors in systemic sclerosis (SSc) patients and healthy controls (HC). Scatterplots
of the correlation analysis between CD3+CD31+CXCR4+ Tang cells and serum levels of (A, B) vascular
endothelial growth factor (VEGF), (C, D) matrix metalloproteinase (MMP)-9, (E, F) interleukin (IL)-8 and (G,
H) IL-17 are shown. Each dot represents a subject. Spearman's rho correlation coefficient and p value are
indicated. NS, not significant.
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Fig 7. Angiogenic T cells (Tang) are present in systemic sclerosis (SSc) dermal perivascular inflammatory infiltrates. (A)
Representative microphotograph of skin sections from SSc patients stained with hematoxylin and eosin. Perivascular inflammatory
cells from the boxed area are shown at higher magnification in the inset. (B) Representative fluorescence microphotograph of skin
sections from SSc patients double immunostained for the pan-T lymphocyte marker CD3 (green) and CD31 (red), and
counterstained with 40,6-diamidino-2-phenylindole (DAPI; blue) for nuclei. Arrows indicate CD3+CD31+ T lymphocytes. (C, D)
Representative fluorescence microphotographs of serial skin sections from SSc patients double immunostained for CD3 (green)
and CD31 (red; C) or CXCR4 (red; D). Arrows indicate CD3+CD31+CXCR4+ T lymphocytes (Tang). Insets: higher magnification
views of immunopositive lymphocytes from the respective panels. Scale bars are indicated in each panel.
cells as a potential surrogate biomarker of cardiovascular risk factors and therapeutic target for
SSc patients. Finally, further studies are required to clarify the function of Tang cells and
investigate the mechanisms responsible for their change in SSc.
Conceptualization: Mirko Manetti, Sara Pratesi, Enrico Maggi, Marco Matucci-Cerinic.
Data curation: Mirko Manetti, Sara Pratesi, Eloisa Romano, Irene Rosa, Lidia
Ibba-Manneschi, Marco Matucci-Cerinic.
Formal analysis: Mirko Manetti, Sara Pratesi, Eloisa Romano, Irene Rosa, Lidia
Funding acquisition: Marco Matucci-Cerinic.
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Investigation: Mirko Manetti, Sara Pratesi, Eloisa Romano, Silvia Bellando-Randone, Irene
Rosa, Serena Guiducci, Bianca Saveria Fioretto, Lidia Ibba-Manneschi, Enrico Maggi,
Methodology: Mirko Manetti, Sara Pratesi, Eloisa Romano, Silvia Bellando-Randone, Irene
Resources: Mirko Manetti, Lidia Ibba-Manneschi, Enrico Maggi, Marco Matucci-Cerinic.
Supervision: Mirko Manetti, Marco Matucci-Cerinic.
Validation: Enrico Maggi.
Visualization: Mirko Manetti, Sara Pratesi, Lidia Ibba-Manneschi, Marco Matucci-Cerinic.
Writing ± original draft: Mirko Manetti, Sara Pratesi, Lidia Ibba-Manneschi, Enrico Maggi,
Writing ± review & editing: Mirko Manetti, Sara Pratesi, Eloisa Romano, Silvia
Bellando-Randone, Irene Rosa, Serena Guiducci, Bianca Saveria Fioretto, Lidia Ibba-Manneschi, Enrico
Maggi, Marco Matucci-Cerinic.
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