Rheumatology 2013;52:352–362

RHEUMATOLOGY doi:10.1093/rheumatology/kes267
Advance Access publication 11 October 2012

Original article
Decreased Th17 and Th1 cells in the peripheral
blood of patients with early non-radiographic axial
spondyloarthritis: a marker of disease activity in
HLA-B27+ patients

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Marı́a-Belén Bautista-Caro1, Irene Arroyo-Villa1, Concepción Castillo-Gallego1,
Eugenio de Miguel1, Diana Peiteado1, Amaya Puig-Kröger2, Emilio Martı́n-Mola1
and Marı́a-Eugenia Miranda-Carús1

Abstract
Objective. To examine the frequency and phenotype of Th17 cells in the peripheral blood of patients with
early non-radiographic axial SpA (early nrSpA).
Methods. CD4+ T cells were isolated from the peripheral blood of 30 early nrSpA patients, 11 AS patients
and 41 age- and sex-matched healthy controls by Ficoll–Hypaque gradient and magnetic negative selec-
tion. After polyclonal stimulation, the frequency of Th17 and Th1 cells and of cells producing TNF-a or
IL-10 was determined by cytometry and concentrations of IL-17, IL-22, IFN-g, TNF-a, IL-10 and IL-4 were
measured by ELISA.
Results. Early nrSpA but not AS patients demonstrated a significantly lower percentage of circulating
Th17, Th1 and Th17/Th1 cells, together with lower CD4-derived IL-17 and IFN-g secretion, as compared
with controls. In contrast, the percentage of circulating cells producing IL-10 or TNF-a, and the secretion
of CD4-derived IL-10, TNF-a, IL-22 and IL-4 in early nrSpA were not different from controls. All Th17 cells
were CD45RO+CD45RA and CCR6+. The frequency of circulating Th17, Th1 and Th17/Th1 was nega-
tively correlated with BASDAI, BASFI, ASDAS-CRP, ASDAS-ESR, AS quality of life (ASQOL) and patient’s
CLINICAL
SCIENCE

global assessment in HLA-B27+ but not in HLA-B27 early nrSpA patients. A positive correlation between
circulating Th17 cells and BASDAI was observed in AS.
Conclusion. A decreased percentage of Th17, Th1 and Th17/Th1 cells is apparent in peripheral blood
CD4+ T cells from early nrSpA. Th17, Th1 and Th17/Th1 cell numbers are related to disease activity
indices in HLA-B27+, but not in HLA-B27, early nrSpA patients.
Key words: spondyloarthritis, cytokines and inflammatory mediators, T cells, inflammation, lymphocytes.

Introduction SpondyloArthritis International Society (ASAS) criteria,

The term SpA designates a group of diseases character-
ized by inflammatory back pain, sacroiliitis, enthesitis,
asymmetric peripheral arthritis, dactylitis and uveitis.
According to the newly developed Assessment of
1
Department of Rheumatology, Hospital Universitario La Paz-IdiPAZ
and 2Laboratorio de Inmuno-Oncologı́a, Hospital General Universitario
Gregorio Marañón, Madrid, Spain.
Submitted 11 April 2012; revised version accepted 23 August 2012.
Correspondence to: Marı́a-Eugenia Miranda-Carús, Department of
Rheumatology, Hospital La Paz, Paseo de la Castellana 261, 28046
Madrid, Spain. E-mail: mmiranda.hulp@salud.madrid.org
SpA can be classified based on clinical grounds as pre-
dominantly axial SpA or predominantly peripheral SpA
[1, 2], with or without the presence of associated diseases
such as psoriasis, IBD or a triggering infection. AS is the
prototype of axial SpA, and its diagnosis is usually
delayed 6–8 years after the onset of symptoms due to
the late appearance of sacroiliitis on plain radiographs in
a majority of patients [1]. Therefore the ASAS classified
those patients with axial SpA who do not demonstrate
definite radiographic changes in the sacroiliac joints as
non-radiographic axial SpA (nrSpA), in an attempt to im-
prove previous criteria, especially for application in recent

! The Author 2012. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

onset SpA [1]. This facilitates the conduct of research in
early disease and early initiation of treatment, which is
pivotal to achieving good response and preventing
chronic disability.
Numerous experimental data indicate that IL-17A may
be implicated not only in human RA but also in SpA [3]. It
was initially established that IL-17A plays an important
role in the pathogenesis of animal models of inflammatory
arthritis: IL-17-deficient mice demonstrate a markedly
attenuated form of CIA [4], neutralization of IL-17 during
the induction of rat adjuvant arthritis suppresses the onset
of disease [5] and anti-IL-17 therapy in established CIA
significantly reduces severity [6]. More recent observa-
tions indicate that joint ankylosis in a murine model is sig-
nificantly associated with the number of popliteal lymph
node T cells producing IL-17 [7], and Th17 cells are ex-
panded in SpA-prone HLA-B27-transgenic rats [8]; in add-
ition, blockade of IL-17 improves the onset of tarsal
ankylosis in a murine model [9] and ameliorates the exa-
cerbated uveitis in mice with proteoglycan-induced arth-
ritis and spondylitis [10].
In human studies, increased levels of IL-17 and Th17
cells have been detected in the peripheral blood and SF of
SpA [11–17]. In addition, AS seems to be associated with
genetic polymorphisms of the IL-23 receptor, and the pro-
tective R381Q gene variant is characterized by impaired
Th17 responses [18, 19]. Furthermore, an anti-IL17A mAb
has shown promising results in the treatment of RA, PsA
and AS [20–23].
To our knowledge there are no published studies on
Th17 cell frequencies in early nrSpA. This is attributable
to the recent definition of this concept and the absence of
previous appropriate and accepted classification criteria
[1]. Existing reports on Th17 biology in undifferentiated
SpA are limited to detection of this cytokine in serum
and SF by ELISA [12, 13], with no data on the frequency
of circulating Th17 cells.
Our objective was to examine the frequency and pheno-
type of Th17 cells in the peripheral blood of early nrSpA
patients. Our early SpA clinic allowed the study of T cells
from patients with early disease of <2 years, and who had
not received DMARDs, steroids or biologics, thereby mini-
mizing the interference of perpetuating inflammatory feed-
back loops and of drugs with ex vivo T cell responses. We
observed that patients with early nrSpA demonstrate a
decreased percentage of circulating Th17, Th1 and Th17/
Th1 cells and, interestingly, Th17, Th1 and Th17/Th1 cell
numbers are clearly correlated with disease activity indices
in HLA-B27+ but not HLA-B27 early nrSpA.
Patients and methods
Patients
Peripheral blood was obtained from 30 early nrSpA, 11 pa-
tients with AS fulfilling modified New York criteria [24] and
41 age- and sex-matched healthy controls. Early nrSpA
patients fulfilled ASAS criteria [1] and the inclusion criteria
were inflammatory back pain with or without asymmetric
peripheral arthritis, disease duration of 3–24 months, age
www.rheumatology.oxfordjournals.org
Decreased circulating Th17 and Th1 cells in early nrSpA
of 18–45 years and being treatment naı̈ve for disease-
modifying drugs, corticosteroids and biologics. La Paz
University Hospital in Madrid, Spain, has an early SpA
unit that receives patients from eight primary care centres,
corresponding to an area of 242 784 inhabitants, as part
of the ESPERANZA programme, a nationwide health man-
agement programme designed to provide excellence in
care for early SpA [25]; this facilitated recruitment of
early nrSpA patients for the present study. At the baseline
visit, medical history, physical examination, ESR, CRP,
HLA-B27, BASDAI, BASFI, ASDAS-CRP, ASDAS-ESR,
the AS Quality of Life (ASQOL) scale, patient’s global as-
sessment and pelvis conventional X-ray were recorded for
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each patient. Among early nrSpA patients 9 were female
and 21 were male; their mean (S.D.) age was 34.9 (7.2)
years, median 35.6 years, maximum 45 years and min-
imum 19 years; 15 (50%) tested positive for HLA-B27.
The mean (S.D) disease duration was 12.7 (5.6) months,
median 13 months, maximum 22 months and minimum
3 months. ASAS criteria fulfilled by HLA-B27+ and HLA-
B27 early nrSpA patients are summarized in Table 1. The
study was approved by the Hospital La Paz-IdiPAZ Ethics
Committee, and all subjects provided written informed
consent according to the Declaration of Helsinki.
Among AS patients, two were taking only NSAIDs and
nine were receiving anti-TNF. There were 1 female and 10
males, their mean (S.D.) age was 49.5 (11.6) years, median
46.0 years, maximum 71 years and minimum 33 years; 10
(90.1%) tested positive for HLA-B27. The mean (S.D.) dis-
ease duration was 21.3 (13.4) years, median 23 years,
maximum 51 years and minimum 3 years.
None of the patients had associated psoriasis, IBD or
history of a preceding infection.
Isolation of CD4+ T cells
Mononuclear cells were isolated from human blood by
density centrifugation over Ficoll–Paque Plus (GE Health-
care, Chalfont St Giles, UK). CD4+ T cells were subse-
quently purified by negative selection in an Automacs
(Miltenyi Biotec, Bergisch Gladbach, Germany) using the
CD4+ T Cell Isolation Kit II from Miltenyi Biotec, containing
a cocktail of biotin-conjugated antibodies against CD8,
CD14, CD16, CD19, CD36, CD56, CD123, g/d TCR and
glycophorin A, followed by anti-biotin microbeads. Iso-
lated CD4+ T cells were 98% pure and free of detectable
CD14+ monocytes, CD8+ T cells, CD56+ NK cells, CD19+
B cells and g/d T cells.
T cell stimulation
Immediately after isolation, CD4+ T cells were cultured
and stimulated in 24-well plates (106 cells/well) containing
RPMI 1640 medium (Lonza, Allendale, NJ, USA) with 10%
fetal calf serum (FCS), 2 mM L-glutamine, 50 U/ml penicil-
lin, 50 mg/ml streptomycin and 50 mM 2-mercapto-ethanol
(complete RPMI medium). Two different activation strate-
gies were undertaken. T cells were stimulated for 16 h with
phorbol myristate acetate (PMA) (10 nM) and ionomycin
(2 mM) in the presence or absence of 4 mM monensin (all
three from Sigma-Aldrich, St Louis, MO, USA). In addition,
353
Marı́a-Belén Bautista-Caro et al.
TABLE 1 ASAS classification criteria for axial SpA [1] in 30 patients with early nrSpA
ASA classification criteria
Definite sacroiliitis—modified New York criteria [24]
Sacroiliitis on MRI
Inflammatory back pain
Arthritis
Enthesitis (heel)
Uveitis
Dactylitis
Psoriasis
Crohn’s disease/ulcerative colitis
Good response to NSAIDs
Family history for SpA
HLA-B27
Elevated CRP
cells were cultured for 1–4 days in the presence of a soluble
anti-CD3 IgE subclass mAb (T3/4.E, Sanquin, Amsterdam,
The Netherlands, formerly Central Laboratory of the
Netherlands Red Cross Blood Transfusion Service)
(0.5 mg/ml) plus anti-CD28 (1 mg/ml) (BD Pharmingen, San
Diego, CA, USA) and anti-CD49d mAbs (1 mg/ml) (BD
Pharmingen), and restimulated for the last 6 h with PMA
and ionomycin, with or without monensin.
Intracellular cytokine staining, surface staining and
flow cytometry
Fluorochrome-conjugated mAbs from BD Pharmingen
were used to examine the expression of the phenotyp-
ical markers CD3, CD4, CD8, CCR6, CCR4, CD45RO,
CD45RA, CD25 and CD127. Surface IL-23R was detected
with a biotinylated goat anti-human IL-23R antibody (R&D
Systems, Abingdon, UK), followed by an FITC-labelled
avidin (BD Pharmingen). For intracellular cytokine staining,
T cells were washed with PBS/2% FCS/0.01% NaN3, per-
meabilized for 10 min with FACS permeabilizing solution 2
(BD Pharmingen), washed again and incubated on ice for
1 h with an allophycocyanin-labelled anti-IFN-g mAb
(clone B27; BD Pharmingen), a phycoerythrin (PE)-labelled
anti-IL17A mAb (clone eBio64DEC17; eBioscience, San
Diego, CA, USA), an FITC-labelled anti-TNF-a mAb
(clone Mab11, BD Pharmingen), a PE-labelled anti-IL10
mAb (clone B-T10, Miltenyi Biotech) or fluorochrome-
labelled isotype control mAbs. FoxP3 was detected after
permeabilization with a FoxP3 staining set (Miltenyi
Biotec) and incubation with an anti-FoxP3-AlexaFluor
488 mAb (clone 236A/E7) (eBioscience). After washing
once with PBS/2%FCS/0.01%NaN3 and once with PBS,
cells were resuspended in 1% paraformaldehyde and
analysed in a FACSCalibur flow cytometer using
CellQuest software (BD Biosciences, San José, CA, USA).
ELISAs
Cell-free culture supernatants were collected and stored
at 80 C. ELISAs for IL-17A and IL-22 were performed
using kits from eBioscience. ELISAs for TNF-a, IL-10, IL-4
354
HLA-B27 (n = 15) HLA-B27+ (n = 15)
0 0
15 7
15 15
5 5
1 2
2 1
0 0
0 0
0 0
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10 13
4 5
0 15
0 3
and IFN-g were performed using kits from BD Biosciences
following the manufacturer’s instructions.
Statistical analysis
Comparison between groups was by Mann–Whitney
U-test. When appropriate, Bonferroni correction for mul-
tiple comparisons was applied. Correlations were ana-
lysed using Spearman’s rank correlation coefficients. All
analyses were performed using Prism version 5.0 software
(GraphPad Software, La Jolla, CA, USA).
Results
Expression of IL-17 and IL-22 by early nrSpA and by
AS CD4+ T cells
The frequency of circulating CD4 T cells expressing IL-17
(Th17 cells) was significantly decreased among early
nrSpA patients [median 0.63, interquartile range (IQR)
0.38–1.15%] in comparison with healthy controls
(median 1.03, IQR 0.71–1.52%) (Fig. 1A). There were no
differences between HLA-B27+ and HLA-B27 patients
(Fig. 1A). In contrast, the percentage of Th17 cells in the
periperal blood of AS patients was not different from con-
trols (Fig. 1A). All of the Th17 cells expressed the memory
phenotypical marker CD45RO, were negative for CD45RA
and positive for CCR6 expression (Fig. 1B). The expres-
sion of IL-23R and CCR4 could not be analysed together
with IL-17, since these two molecules are down-regulated
upon stimulation with PMA/ionomycin. Importantly, the
proportion of circulating total CD4+ T cells was not differ-
ent among early nrSpA, AS and control subjects.
In parallel, the concentration of IL-17 detected by ELISA
in culture supernatants of stimulated early nrSpA CD4+
T cells (median 1.34, IQR 0.45–2.23 ng/ml) was signifi-
cantly decreased when compared with healthy controls
(median 2.10, IQR 1.57–4.00 ng/ml) (Fig. 1C), and this
was apparent not only when cells were stimulated over-
night with PMA/ionomycin (see above data) but also after
stimulation for 4 days with anti-CD3/CD28/CD49d
(median 1.61, IQR 0.61–3.62 ng/ml for early nrSpA vs
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 Decreased circulating Th17 and Th1 cells in early nrSpA

FIG. 1 Expression of IL-17A and IL-22 by early nrSpA and AS CD4+ T cells.

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CD4+ T cells were isolated from the peripheral blood of 41 healthy controls (HCs), 30 early nrSpA (e-nrSpA) patients (15
HLA-B27 and 15 HLA-B27+) and 11 AS patients (1 HLA-B27 and 10 HLA-B27+) and stimulated with PMA/ionomycin for
16 h or with anti-CD3/CD28/CD49d for 4 days. (A) Percentage of CD4+ T cells expressing IL-17A (Th17 cells) after a 16-h
stimulation, as determined by flow cytometry. As the CD4 molecule is down-regulated upon stimulation with PMA, shown
is CD3 expression on isolated CD4+ T cells. (B) Representative flow cytometry dot plots showing expression of CCR6,
CD45RO and CD45RA vs IL-17A in isolated CD4+ T cells. (C) Secretion of IL-17A to the culture medium of CD4+ T cells
stimulated for 16 h with PMA/ionomycin (left panel) or for 4 days with anti-CD3/CD28/CD49d (right panel). (D) Secretion of
IL-22 to the culture medium of CD4+ T cells stimulated for 16 h with PMA/ionomycin (left panel) or for 4 days with
anti-CD3/CD28/CD49d (right panel). Box plots represent the median and interquartile range of all studied subjects,
whiskers represent the maximum and minimum values. *P < 0.05 vs HC; y P < 0.05 vs early nrSpA.

www.rheumatology.oxfordjournals.org 355
Marı́a-Belén Bautista-Caro et al.
2.86, 2,05–6.52 ng/ml for controls) (Fig. 1C). Secretion of
CD4-derived IL-17 was not different in HLA-B27+ vs
HLA-B27 early nrSpA patients. In addition, the amount
of IL-17 secreted by CD4 T cells of AS patients was not
different from controls (Fig. 1C).
As several reports describe the production of IL-17 by
other cell populations [26–29], we additionally isolated g/d
T cells, CD8+ T cells and NK cells from patients and con-
trols. We could not detect any IL-17 by cytometry or
ELISA in these cell subsets from the peripheral blood of
early nrSpA, AS or healthy subjects. Moreover, after thor-
ough depletion of CD4+ T cells from peripheral blood
mononuclear cells, no IL-17 could be detected in culture
supernatants after short or long-term stimulation periods,
in agreement with work published by Shen et al. [15].
We additionally measured the secretion of IL-22, a cyto-
kine secreted by Th17 cells [3], in culture supernatants of
stimulated CD4 T cells. No differences were observed
among early nrSpA, AS and control subjects, after stimu-
lation for 16 h or 4 days (Fig. 1D).
Expression of IFN-g by early nrSpA and by AS CD4+
T cells
The frequency of circulating cells producing IFN-g (Th1)
was significantly decreased among early nrSpA patients
(median 9.07, IQR 6.74–13.52%) in comparison with
healthy controls (median 14.12, IQR 10.79–18.12%)
(Fig. 2A). Among early nrSpA patients there were no dif-
ferences between HLA-B27+ and HLA-B27 subjects
(Fig. 2A). In contrast, the percentage of Th1 cells in the
periperal blood of AS patients was not different from con-
trols (Fig. 2A).
In parallel, the concentration of IFN-g detected by
ELISA in culture supernatants of stimulated early nrSpA
CD4+ T cells (median 8.41, IQR 4.95–13.91 ng/ml) was
significantly decreased as compared with healthy controls
(median 17.67, IQR 7.94–26.11 ng/ml) (Fig. 2B), and this
was apparent not only when cells were stimulated over-
night with PMA/ionomycin (see above data), but also after
stimulation for 4 days with anti-CD3/CD28/CD49d
(median 208.7, IQR 30.0–328.0 ng/ml for early nrSpA vs
296.3, 174.5–934.1 ng/ml for controls) (Fig. 2B).
Secretion of CD4-derived IFN-g was not different in
HLA-B27+ vs HLA-B27 early nrSpA patients. In addition,
the amount of IFN-g secreted by CD4 T cells of AS pa-
tients was not different from controls (Fig. 2B).
Frequency of Th17/Th1 cells in early nrSpA and in AS
A population of cells simultaneously producing IL-17 and
IFN-g (Th17/Th1 cells) [30] was detected in the peripheral
blood of controls, early nrSpA and AS patients, and their
frequency was significantly lower in early nrSpA (median
0.11, IQR 0.05–0.22%) as compared with controls
(median 0.20, IQR 0.13–0.28%) (Fig. 2C). The percentage
of Th17/Th1 cells was not different in HLA-B27+ vs
HLA-B27 early nrSpA patients. In addition, the percent-
age of Th17/Th1 cells in the periperal blood of AS patients
was not different from controls (Fig. 2C).
356
Expression of TNF-a, IL-10 and IL-4 by early nrSpA
CD4+ T cells
We were next interested in determining whether early
nrSpA is characterized by a generalized defect in cytokine
production. To this end, the expression of additional cyto-
kines was investigated in these patients. Interestingly, the
frequency of circulating cells producing TNF-a or IL-10
(Fig. 3A and B), together with the concentration of
TNF-a, IL-10 and IL-4 in supernatants of stimulated
CD4+ T cells (Fig. 3C) were comparable in early nrSpA
and control subjects, and there were no differences be-
tween HLA-B27+ vs HLA-B27 patients. In addition, the
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frequency of circulating CD4+ T cells expressing both
IL-17 and TNF-a was not different in early nrSpA as com-
pared with controls (Fig. 3D).
Frequency of circulating CD25+CD127CD4+ T cells
and of FoxP3+ CD4+ T cells in early nrSpA and in AS
Numerous reports indicate that there is a close relation-
ship between Th17 cells and CD4+CD25+ Tregs [3].
Therefore we decided to examine the numbers of this Th
subset in our patients. The frequencies of circulating
CD25+CD127CD4+ T cells and of FoxP3+ CD4+ T cells
were not different among early nrSpA, AS and control
subjects (Fig. 4A and B).
Relation of circulating Th17, Th1 and Th17/Th1 cells
with clinical and analytical parameters in early nrSpA
and AS
We then decided to analyse the relationship of the circu-
lating Th17, Th1 and Th17/Th1 frequencies with clinical
and analytical data. Interestingly, in HLA-B27+ early
nrSpA patients, the percentage of peripheral blood Th17
cells, Th1 cells and Th17/Th1 cells was negatively corre-
lated with the following parameters: BASDAI, BASFI,
ASDAS-CRP, ASDAS-ESR, ASQOL and patient’s global
assessment (Fig. 5). In contrast, these parameters were
not correlated with circulating Th17, Th1 or Th17/Th1
numbers in HLA-B27 early nrSpA subjects (supplemen-
tary Fig. S1, available as supplementary data at
Rheumatology Online), even though the percentage of cir-
culating Th17 cells was not different between HLA-B27+
and HLA-B27 patients (Fig. 1A). Importantly, no differ-
ences in BASDAI, BASFI, ASDAS-CRP, ASDAS-ESR,
ASQOL or patient’s global assessment were observed be-
tween HLA-B27+ and HLA-B27 patients. Strikingly, in AS
patients, a significant correlation was observed between
the percentage of circulating Th17 cells and the BASDAI
index, but this was a positive correlation as opposed to
the negative correlation seen in early nrSpA (Fig. 6A). In
addition, a good positive correlation between the percent-
age of circulating Th1 cells and the BASDAI index was
apparent in AS, although it did not reach statistical signifi-
cance (P = 0.08) (Fig. 6B). No such correlations were found
in AS with BASFI, ASDAS-CRP, ASDAS-ESR, ASQOL or
patient’s global assessment.
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 Decreased circulating Th17 and Th1 cells in early nrSpA

FIG. 2 Expression of IFN-g by early nrSpA and AS CD4+ T cells.

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(A) Percentage of CD4+ T cells expressing IFN-g (Th1 cells) after 16 h of stimulation. (B) Secretion of IFN-g to the culture
medium of CD4+ T cells stimulated for 16 h (left panel) or for 4 days (right panel). (C) Percentage of CD4+ T cells
expressing both IL-17A and IFN-g (Th17/Th1 cells) after 16 h of stimulation. *P < 0.05 vs HC. See Fig. 1 for details and
definitions.

www.rheumatology.oxfordjournals.org 357
Marı́a-Belén Bautista-Caro et al.
FIG. 3 Expression of IL-10, TNF-a and IL-4 by early nrSpA
CD4+ T cells.
(A, B) Percentage of CD4+ T cells expressing IL-10 or
TNF-a after 16 h of stimulation. (C) Secretion of IL-10,
TNF-a and IL-4 by CD4+ T cells. (D) Percentage of CD4+
T cells simultaneously expressing IL-17A and TNF-a.
See Fig. 1 for details and definitions.
Discussion
Several previous studies indicated that Th17 cells may
play an important role in the pathogenesis of SpA
[7–23], but limited data exist on Th17 biology in patients
with early nrSpA. We detected a significantly decreased
frequency of circulating Th17 cells in early nrSpA, and
there were no differences between HLA-B27+ and
HLA-B27 patients. Furthermore, the frequency of circu-
lating Th1 cells, of circulating Th17/Th1 cells and the se-
cretion of CD4-derived IL-17 and IFN-g were also
decreased in early nrSpA. This was not attributable to a
generalized defect of cytokine production, since the fre-
quency of circulating cells expressing TNF-a or IL-10, to-
gether with the secretion of CD4-derived TNF-a, IL-10,
IL-4 and IL-22, were not different in early nrSpA patients
as compared with healthy controls. That is, CD4+ T cells of
358
FIG. 4 Percentage of circulating CD25+CD127CD4+
T cells and FoxP3+CD4+ T cells in early nrSpA and AS.
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(A) Percentage of CD25+CD127 cells among circulating
CD4+ T cells in healthy controls (HCs) (n = 41), in all of our
early nrSpA patients as a group (n = 30), in the subgroup of
HLA-B27early nrSpA (n = 15), in HLA-B27+ early nrSpA
(n = 15) and in AS patients (n = 11). (B) Percentage of CD4+
T cells expressing FoxP3. See Fig. 1 for details and
definitions.
early nrSpA patients demonstrated a selectively
decreased secretion of both IL-17 and IFN-g. In contrast,
our patients with AS of long duration demonstrated ex-
pression of IL-17 and IFN-g that was comparable to that
observed in healthy controls, suggesting that the findings
in early nrSpA may be related to the early disease stage
and/or different medication. Previous studies on the fre-
quency of Th17 cells in the peripheral blood of AS patients
have yielded conflicting results: while some have
described increased Th17 frequencies [11, 15, 16],
others have reported frequencies comparable to those
observed in control subjects [29], and the latter are con-
sistent with our findings.
To date, limited information exists regarding Th17 biol-
ogy in patients with early nrSpA, due in part to the previ-
ous absence of appropriate classification criteria [1]. Our
finding that circulating Th17 cells are decreased in early
nrSpA naı̈ve for treatment with DMARDs, steroids or
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 Decreased circulating Th17 and Th1 cells in early nrSpA

FIG. 5 Relationship of Th17, Th1 and Th17/Th1 periperal blood cell frequencies with clinical parameters in HLA-B27+
early nrSpA patients.

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The left panels show correlations of Th17, the central panels show correlations of Th1 and the right panels show cor-
relations of Th17/Th1 cells with the BASDAI index (A), BASFI index (B), ASDAS-CRP (C), ASDAS-ESR (D), ASQOL (E) and
patient’s global assessment (F) in HLA-B27+ early nrSpA patients (n = 15). Each filled square represents one patient.

www.rheumatology.oxfordjournals.org 359
Marı́a-Belén Bautista-Caro et al.
FIG. 6 Relationship of Th17 (A) and Th1 (B) periperal
blood cell frequency with the BASDAI index in AS
patients.
biologics is striking and not easy to explain from a
pathomechanistic perspective. Th17 cells are found in
the facet joints of AS patients [29], and it is possible that
in early disease, recirculation of Th17 cells through the
peripheral blood is limited due to sequestration at the in-
flammatory site. As an abundance of IL-17-producing
neutrophils has been observed in AS facet joints [29],
and mast cells seem to be the major source of IL-17 in
SpA synovial membranes [28], it has been suggested that
these cell types could play a leading role in the pathogen-
esis of SpA [31]. It has been described that additional
sources of IL-17, such as g/d T cells and NK cells, may
also be pathogenic [26, 27]. In this context we could not
detect any IL-17 expression by g/d T cells, CD8+ T cells or
NK cells from the peripheral blood of healthy controls, AS
or early nrSpA patients, consistent with work published by
Shen et al. [15].
Interestingly, we also observed decreased frequency
of circulating Th1 cells in early nrSpA, with no difference
between HLA-B27+ and HLA-B27 patients, whereas the
percentage of Th1 cells in the peripheral blood of our pa-
tients with AS was comparable to that observed in con-
trols. Previous work has described either decreased
[32–34] or normal [15] circulating Th1 frequencies in AS.
Again, our data indicate that patients with early disease
are characterized by a distinct immunological status.
We additionally observed that the frequency of circulat-
ing CD4+CD25+CD127 T cells and CD4+FoxP3+ T cells in
early nrSpA patients was not different from control sub-
jects. To our knowledge, this is the first report on the fre-
quency of this T cell subset in patients with early nrSpA.
Tregs may be implicated in the pathogenesis of autoim-
mune and inflammatory conditions and their numbers
have been found altered in the peripheral blood of patients
with SLE and RA [35]. Previous work by Appel et al. [36]
indicated that the frequency of CD4+FoxP3+ in the periph-
eral blood of established SpA, including 11 subjects with
undifferentiated SpA, is not different from controls, which
is consistent with our results.
Our most striking finding was the good negative correl-
ation between clinical parameters of disease activity or
function and the numbers of Th17, Th1 and Th17/Th1
cells in the peripheral blood of HLA-B27+ early nrSpA pa-
tients. This is especially remarkable given the difficulty in
assessing clinical activity in SpA due to the lack of a uni-
versally accepted gold standard [37], and given that some
of the currently used indices measure only one aspect of
360
the disease or are fully patient or physician oriented.
Among HLA-B27+ early nrSpA patients, the circulating
Th17, Th1 and Th17/Th1 frequencies were negatively cor-
related with the BASDAI, BASFI, ASDAS-CRP, ASDAS-
ESR and ASQOL indices, and with patient’s global as-
sessment. Of note, these correlations were not detected
in HLA-B27 early nrSpA subjects, and reasons to explain
this observation are not apparent to us at present. In con-
trast, and more remarkably, the BASDAI index was posi-
tively correlated with circulating Th17 cells in AS patients,
even though Th17 cells were not overrepresented in their
peripheral blood. That is, those AS patients who demon-
strated the highest disease activities tended to show
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higher levels of circulating Th17 cells. This was in contrast
to the observation in early nrSpA, where the highest dis-
ease activities were associated with the lowest rates of
circulating Th17 cells. This observation reinforces the find-
ing that Th17 cell biology is different in early nrSpA vs AS,
and indicates that further investigation is needed on this
matter. A negative correlation between patient’s global
assessment of disease activity and circulating Th1 cells
was previously reported by Baeten et al. [33] in patients
with long-term established SpA, which confirms that
values of certain Th cell subsets in peripheral blood can
be used as alternative markers of disease activity.
Conclusions
In summary, we have observed a significantly decreased
frequency of circulating Th17, Th1 and Th17/Th1 cells in
early nrSpA patients together with a preserved frequency
of circulating CD4+ T cells producing IL-10 or TNF-a; in
addition, the frequency of circulating CD4+CD25+CD127
T cells and CD4+FoxP3+ T cells in early nrSpA was not
different from that observed in control subjects. The cir-
culating Th17, Th1 and Th17/Th1 frequencies were nega-
tively correlated with BASDAI, BASFI, ASDAS and ASQOL
indexes, and with patient’s global assessment, in
HLA-B27+ but not in HLA-B27 early nrSpA patients.
This indicates that further studies are needed to fully
understand the complex biology of Th17 cells in early
nrSpA. In addition, the circulating Th17 frequency can
be considered as a surrogate measure of disease activity
in HLA-B27+ patients with early nrSpA.
Rheumatology key messages
. The frequency of circulating Th17, Th1 and Th17/
Th1 cells is decreased in patients with early nrSpA.
. Circulating Th17, Th1 and Th17/Th1 frequencies are
negatively correlated with disease activity in HLA-
B27+ early nrSpA.
Acknowledgements
M.-E.M.-C. conceived and designed the experiments; M.-
B.B-C. and I.A.-V. performed the experiments; M.-E.M.-
C., M.-B.B.-C., I.A.-V., E.dM., C.C.-G., D.P.L. and
E.M.-M. analysed the data; E.dM., C.C.G., D.P. and
www.rheumatology.oxfordjournals.org

E.M.-M. contributed reagents, materials and analysis
tools; M.-E.M.-C., M.-B.B.-C., I.-A.-V. and E.dM. wrote
the manuscript.
Funding: This work was supported by Ministerio de
Ciencia e Innovación grant SAF 2009-07100; by the
Fundación Española de Reumatologı́a—ESPERANZA
programme; and by the RETICS programme, RD08/0075
(RIER) from Instituto de Salud Carlos III (ISCIII). The fun-
ders had no role in study design, data collection and ana-
lysis, decision to publish or preparation of the manuscript.
Disclosure statement: The authors have declared no
conflicts of interest.
Supplementary data
Supplementary data are available at Rheumatology
Online.
References
1 Rudwaleit M, van der Heijde D, Landewé R et al. The
Assessment of SpondyloArthritis International Society
classification criteria for peripheral spondyloarthritis and
for spondyloarthritis in general. Ann Rheum Dis 2011;70:
25–31.
2 Rudwaleit M, van der Heijde D, Landewé R et al. The
development of Assessment of SpondyloArthritis
International Society classification criteria for axial spon-
dyloarthritis (part II): validation and final selection. Ann
Rheum Dis 2009;68:777–83.
3 Lubberts E. Th17 cytokines and arthritis. Semin
Immunopathol 2010;32:43–53.
4 Nakae S, Nambu A, Sudo K, Iwakura Y. Suppression of
immune induction of collagen-induced arthritis in
IL-17-deficient mice. J Immunol 2003;171:6173–7.
5 Bush KA, Farmer KM, Walker JS, Kirkham BW. Reduction
of joint inflammation and bone erosion in rat adjuvant
arthritis by treatment with interleukin-17 receptor IgG1 Fc
fusion protein. Arthritis Rheum 2002;46:802–5.
6 Lubberts E, Koenders MI, Oppers-Walgreen B et al.
Treatment with a neutralizing anti-murine interleukin-17
antibody after the onset of collagen-induced arthritis
reduces joint inflammation, cartilage destruction, and
bone erosion. Arthritis Rheum 2004;50:650–9.
7 Tsurui H, Nakae S, Shirai T, Sudo K, Hirose S. Ankylosing
enthesitis associated with up-regulated IFN-gamma and
IL-17 production in (BXSB x NZB) F(1) male mice: a new
mouse model. Mod Rheumatol 2009;19:316–22.
8 Glatigny S, Fert I, Blaton MA et al. Proinflammatory Th17
cells are expanded and induced by dendritic cells in
spondylarthritis-prone HLA-B27-transgenic rats. Arthritis
Rheum 2012;64:110–20.
9 Ebihara S, Ono M. Inhibition of IL-17 by antibody admin-
istration improves onset of tarsal ankylosis in a murine
model. Arthritis Rheum 2011;63:S528.
10 Kezic JM, Glant TT, Rosenbaum JT, Rosenzweig HL.
Neutralization of IL-17 ameliorates uveitis but damages
photoreceptors in a murine model of spondyloarthritis.
Arthritis Res Ther 2012;14:R18.
www.rheumatology.oxfordjournals.org
Decreased circulating Th17 and Th1 cells in early nrSpA
11 Bowness P, Ridley A, Shaw J et al. Th17 cells expressing
KIR3DL2+ and responsive to HLA-B27 homodimers are
increased in ankylosing spondylitis. J Immunol 2011;186:
2672–80.
12 Romero-Sanchez C, Jaimes DA, Londoño J et al.
Association between Th-17 cytokine profile and clinical
features in patients with spondyloarthritis. Clin Exp
Rheumatol 2011;29:828–34.
13 Singh R, Aggarwal A, Misra R. Th1/Th17 cytokine profiles
in patients with reactive arthritis/undifferentiated spondy-
loarthropathy. J Rheumatol 2007;34:2285–90.
14 Leipe J, Grunke M, Dechant C et al. Role of Th17 cells in
human autoimmune arthritis. Arthritis Rheum 2010;62:
Downloaded from https://academic.oup.com/rheumatology/article/52/2/352/1832610 by guest on 14 October 2023
2876–85.
15 Shen H, Goodall JC, Hill Gaston JS. Frequency and
phenotype of peripheral blood Th17 cells in ankylosing
spondylitis and rheumatoid arthritis. Arthritis Rheum 2009;
60:1647–56.
16 Jandus C, Bioley G, Rivals JP et al. Increased numbers of
circulating polyfunctional Th17 memory cells in patients
with seronegative spondylarthritides. Arthritis Rheum
2008;58:2307–17.
17 Wang X, Lin Z, Wei Q, Jiang Y, Gu J. Expression of IL-23
and IL-17 and effect of IL-23 on IL-17 production in
ankylosing spondylitis. Rheumatol Int 2009;29:1343–7.
18 Di Meglio P, Di Cesare A, Laggner U et al. The IL-23R
R381Q gene variant protects against immune-mediated
diseases by impairing IL-23-induced Th17 effector re-
sponse in humans. PLoS One 2011;6:e17160.
19 Sarin R, Wu X, Abraham C. Inflammatory disease pro-
tective R381Q IL23 receptor polymorphism results in
decreased primary CD4+ and CD8+ human T-cell func-
tional responses. Proc Natl Acad Sci USA 2011;108:
9560–5.
20 Genovese MC, Van den Bosch F, Roberson SA et al.
LY2439821, a humanized anti-interleukin-17 monoclonal
antibody, in the treatment of patients with rheumatoid
arthritis: a phase I randomized, double-blind,
placebo-controlled, proof-of-concept study. Arthritis
Rheum 2010;62:929–39.
21 Hueber W, Patel DD, Dryja T et al. Effects of AIN457,
a fully human antibody to interleukin-17A, on psoriasis,
rheumatoid arthritis, and uveitis. Sci Transl Med 2010;2:
52ra72.
22 McInnes I, Sieper J, Braun J et al. Anti-interleukin 17A
monoclonal antibody secukinumab reduces signs and
symptoms of psoriatic arthritis in a 24-week multicenter,
double-blind, randomized, placebo-controlled trial.
Arthritis Rheum 2011;63:S306.
23 Baeten D, Sieper J, Emery P et al. The anti-IL17A
monoclonal antibody secukinumab (AIN457) showed
good safety and efficacy in the treatment of active
ankylosing spondylitis. Ann Rheum Dis 2011;70(Suppl. 3):
127.
24 van der Linden S, Valkenburg HA, Cats A. Evaluation of
diagnostic criteria for ankylosing spondylitis. A proposal
for modification of the New York criteria. Arthritis Rheum
1984;27:361–8.
25 Muñoz-Fernández S, Carmona L, Collantes E et al.
A model for the development and implementation of a
361
Marı́a-Belén Bautista-Caro et al.
national plan for the optimal management of early spon-
dyloarthritis: the Esperanza Program. Ann Rheum Dis
2011;70:827–30.
26 Cua DJ, Tato CM. Innate IL-17-producing cells: the sen-
tinels of the immune system. Nat Rev Immunol 2010;10:
479–89.
27 Ito Y, Usui T, Kobayashi S et al. Gamma/delta T cells are
the predominant source of interleukin-17 in affected joints
in collagen-induced arthritis, but not in rheumatoid arth-
ritis. Arthritis Rheum 2009;60:2294–303.
28 Noordenbos T, Yeremenko N, Gofita I et al. Interleukin-
17-positive mast cells contribute to synovial inflammation
in spondylarthritis. Arthritis Rheum 2012;64:99–109.
29 Appel H, Maier R, Wu P et al. Analysis of IL-17(+) cells in
facet joints of patients with spondyloarthritis suggests that
the innate immune pathway might be of greater relevance
than the Th17-mediated adaptive immune response.
Arthritis Res Ther 2011;13:R95.
30 Acosta-Rodriguez EV, Napolitani G, Lanzavecchia A,
Sallusto F. Interleukins 1beta and 6 but not transforming
growth factor-beta are essential for the differentiation of
interleukin 17-producing human T helper cells. Nat
Immunol 2007;8:942–9.
31 Yeremenko N, Baeten D. IL-17 in spondyloarthritis: is the
T-party over? Arthritis Res Ther 2011;13:115.
362
32 Rudwaleit M, Siegert S, Yin Z et al. Low T cell produc-
tion of TNFalpha and IFNgamma in ankylosing
spondylitis: its relation to HLA-B27 and influence of the
TNF-308 gene polymorphism. Ann Rheum Dis 2001;60:
36–42.
33 Baeten D, Van Damme N, Van den Bosch F et al. Impaired
Th1 cytokine production in spondyloarthropathy is
restored by anti-TNFalpha. Ann Rheum Dis 2001;60:
750–5.
34 Ergin A, Syrbe U, Scheer R et al. Impaired peripheral Th1
CD4+ T cell response to Escherichia coli proteins in pa-
tients with crohn’s disease and ankylosing spondylitis.
J Clin Immunol 2011;31:998–1009.
Downloaded from https://academic.oup.com/rheumatology/article/52/2/352/1832610 by guest on 14 October 2023
35 Chavele KM, Ehrenstein MR. Regulatory T-cells in sys-
temic lupus erythematosus and rheumatoid arthritis.
FEBS Lett 2011;585:3603–10.
36 Appel H, Wu P, Scheer R et al. Synovial and peripheral
blood CD4+FoxP3+ T cells in spondyloarthritis.
J Rheumatol 2011;38:2445–51.
37 Machado P, Landewé R, Lie E et al. Assessment of
SpondyloArthritis International Society. Ankylosing
Spondylitis Disease Activity Score (ASDAS):
defining cut-off values for disease activity states
and improvement scores. Ann Rheum Dis 2011;
70:47–53.
www.rheumatology.oxfordjournals.org