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PANDR-Binding Hsp60 self epitopes induce an interleukin-10mediated immune response in rheumatoid arthritis.

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ARTHRITIS & RHEUMATISM
Vol. 60, No. 7, July 2009, pp 1966–1976
DOI 10.1002/art.24656
© 2009, American College of Rheumatology
Pan–DR-Binding Hsp60 Self Epitopes
Induce an Interleukin-10–Mediated Immune Response
in Rheumatoid Arthritis
Huib de Jong,1 Floris F. P. Lafeber,1 Wilco de Jager,1 Margje H. Haverkamp,1 Wietse Kuis,1
Johannes W. J. Bijlsma,1 Berent J. Prakken,2 and Salvatore Albani3
Objective. Human Hsp60 is expressed in the
joints of patients with rheumatoid arthritis (RA) and
can elicit a regulatory T cell response in the peripheral
blood and synovial fluid. However, Hsp60 can also
trigger strong proinflammatory pathways. Thus, to understand the nature of these Hsp60-directed responses
in RA, it is necessary to study such responses at the
molecular, epitope-specific level. This study was undertaken to characterize the disease specificity and function of pan–DR-binding Hsp60–derived epitopes as
possible modulators of autoimmune inflammation in
RA.
Methods. Lymphocyte proliferation assays (using
3
H-thymidine incorporation and carboxyfluorescein diacetate succinimidyl ester [CFSE] staining) and measurement of cytokine production (using multiplex immunoassay and intracellular staining) were performed
after in vitro activation of peripheral blood mono-
nuclear cells from patients with RA, compared with
healthy controls.
Results. A disease (RA)–specific immune recognition, characterized by T cell proliferation as well as increased production of tumor necrosis factor ␣ (TNF␣),
interleukin-1␤ (IL-1␤), and IL-10, was found for 3 of
the 8 selected peptides in patients with RA as compared
with healthy controls (P < 0.05). Intracellular cytokine
staining and CFSE labeling showed that CD4ⴙ T cells
were the subset primarily responsible for both the T cell
proliferation and the cytokine production in RA. Interestingly, the human peptides had a remarkably different
phenotype, with a 5–10-fold higher IL-10:TNF␣ ratio,
compared with that of the microbial peptides.
Conclusion. These results suggest a diseasespecific immune-modulatory role of epitope-specific T
cells in the inflammatory processes of RA. Therefore,
these pan–DR-binding epitopes could be used as a tool
to study the autoreactive T cell response in RA and
might be suitable candidates for use in immunotherapy.
Dr. Prakken’s work was supported by a Vidi innovation grant
from The Netherlands Organization for Scientific Research, and by the
Dutch Arthritis Foundation.
1
Huib de Jong, MD, Floris F. P. Lafeber, PhD, Wilco de
Jager, PhD, Margje H. Haverkamp, MD, Wietse Kuis, MD, PhD,
Johannes W. J. Bijlsma, MD, PhD: University Medical Centre Utrecht,
Utrecht, The Netherlands; 2Berent J. Prakken, MD, PhD: University
Medical Centre Utrecht, Utrecht, The Netherlands and EUREKA
Institute for Translational Medicine, Syracuse, Italy; 3Salvatore Albani, MD, PhD: Arizona Arthritis Center, University of Arizona,
Tucson and EUREKA Institute for Translational Medicine, Syracuse,
Italy.
Drs. Prakken and Albani contributed equally to this work.
Dr. Albani holds a patent for the peptide technology via the
University of California, San Diego, for which he receives no compensation.
Address correspondence and reprint requests to Berent J.
Prakken, MD, PhD, Wilhelmina Children’s Hospital and University
Medical Centre Utrecht, Lundlaan 6, 3584 EA Utrecht, The Netherlands. E-mail: b.prakken@umcutrecht.nl.
Submitted for publication December 7, 2007; accepted in
revised form April 7, 2009.
Rheumatoid arthritis (RA) is a systemic autoimmune disease that is characterized by chronic synovial
inflammation of the peripheral joints (1–3). The joint
inflammation in RA results from uncontrolled activation
from both innate and adaptive immunity and is characterized by the infiltration of CD4⫹ T cells, macrophages, and B cells (4). Although the resulting chronic
inflammation is mainly aspecific (5), the presence of
large numbers of clonally expanded CD4⫹ T cells (6,7)
and the association with certain HLA–DR alleles (8,9)
are indicative of an ongoing antigen-driven T cell response in RA (10–13). These autoreactive T cell responses most likely target more than one antigen. Many
autoantigens, including proteoglycans (14,15), type II
collagen (16), gp-39 (17), p205 (18), and citrullinated
1966
IL-10–MEDIATED, PAN–DR-BINDING Hsp60–INDUCED RESPONSE IN RA
proteins (19), have been proposed to play a role in the
pathogenesis of RA (20).
However, until now, a single causative antigen
has not been identified in RA, which excludes the
possibility of immune therapy through specific deletion
of the autoaggressive T cells. As an alternative, epitopespecific immune therapy via the mechanism of bystander
suppression or infection tolerance has been proposed
(21). The major advantage of such peptide-specific
immunotherapy would be that it targets specific T cells,
instead of inducing nonspecific immune suppression.
Ideally, epitopes for antigen-specific immune therapy
should fulfill at least 3 important stipulations as follows:
1) the epitopes must be present at the site of inflammation; 2) the epitopes should be specifically up-regulated
at these local sites during inflammation; 3) the epitopes
must be recognized by T cells in a majority of patients,
preferably irrespective of their HLA background.
One of the best-studied groups of antigens that
trigger T cell responses in RA is the family of heat-shock
proteins (HSPs). The HSPs are highly conserved proteins that are essential for cell function. They are
expressed during inflammatory conditions and are dominantly recognized by the immune system, thus fulfilling
important criteria for candidate antigens for immune
therapy. Indeed, a peptide derived from the DnaJ HSP
has shown promise in a phase I study in patients with
RA, with a phase II trial currently being performed
(22,23). For the present study, we focused on another
HSP, namely, Hsp60.
For several reasons, Hsp60 can be considered to
be a prime candidate for antigen-specific immune therapy in RA (24). First, experiments in the adjuvantinduced arthritis model suggest that Hsp60 might play a
crucial role in the immune regulation of arthritis (25).
Second, in RA, as well as in juvenile idiopathic arthritis
(JIA), Hsp60 of microbial origins as well as those of
endogenous origins are targets of immune responses
(26,27). In the spontaneous remitting form of JIA
(oligoarticular JIA), T cells reactive to self Hsp60 at the
onset of disease are associated with disease remission
(28) and have a striking regulatory phenotype (29). In
RA, T cells stimulated with human Hsp60 display a
marked suppressive phenotype, compared with T cells
stimulated with homologous mycobacterial Hsp65 (26).
Thus, Hsp60 self-reactive T cells may play a role in
modulating chronic arthritis (adjuvant-induced arthritis,
RA, and JIA), underscoring their potential as candidate
antigens to modulate the immune response in chronic
arthritis (24,30).
Based on the data obtained in experimental
1967
models, it can be expected that Hsp60 will induce both
proinflammatory and down-regulatory mechanisms,
which may be dependent on recognition of different
epitopes derived from the same protein, since, until now,
T cell epitopes from Hsp60 recognized by peripheral
blood mononuclear cells (PBMCs) from a broad population of RA patients have not been identified. Recently,
we identified pan–DR-binding epitopes of both the
human and the mycobacterial Hsp60 protein (31,32).
The goal of this study was to characterize the disease
specificity and function of these Hsp60-derived epitopes
as contributors to the modulation of an autoimmune
inflammatory loop that may be self-reverberating in a
manner independent of its trigger. Our study presents
novel findings of 8 pan–DR-binding Hsp60 T cell
epitopes that induce a disease-specific antiinflammatory
T cell response in PBMCs from RA patients, underlining
the potential of these epitopes as targets for antigenspecific immunotherapy. Furthermore, these newly identified epitopes could be used as a tool to study the
autoreactive T cell response in ongoing RA.
PATIENTS AND METHODS
Patients and cells. Heparinized blood samples were
collected following the standards of the Declaration of Helsinki, from patients with RA (n ⫽ 20), patients with osteoarthritis (OA) (n ⫽ 20), and healthy control subjects (n ⫽ 20)
(with the latter 2 groups of individuals being sex- and agematched to the RA patients). The patients with RA met the
American College of Rheumatology (formerly, the American
Rheumatism Association) 1987 revised criteria for the classification of RA (33). The patients’ characteristics are listed in
Table 1. The mean duration of RA was 6.7 years (range 1–19
years). The patients with OA had been given the diagnosis in
at least 1 joint and did not have infections or any immunityinterfering disease. The local medical ethics review board
approved the study.
PBMCs were isolated by Ficoll density-gradient centrifugation. Viable cells, checked by trypan blue exclusion,
were cultured in RPMI 1640 culture medium supplemented
with 100 units/ml penicillin/streptomycin, 2 mM L-glutamine
(all from Invitrogen, Carlsbad, CA), and 10% heat-inactivated
human AB-positive serum (Sanquin Bloodbank, Amsterdam,
The Netherlands).
Selection of pan–DR-binding peptides. With the use of
a matrix-based computer algorithm predicting the pan–DRbinding epitopes of a given protein sequence, we selected 4
homologous pairs of peptides (p1–p8) (Table 2). Each pair
consisted of a microbial Hsp60 epitope with its human analog.
Mycobacterial Hsp65 was used as a matrix for microbial
Hsp60. This search was based on the capacity to bind to the
HLA–DR1, DR4, and DR7 binding cleft. Threshold values for
the consecutive scores were ⱖ1.570 for DRB1*0101, ⱖ2.617
for DRB1*0401, and ⱖ9.106 for DRB1*0701. Peptide pairs
p1/p2 and p3/p4 were chosen for the microbial epitopes (p1
1968
DE JONG ET AL
Table 1. Clinical characteristics of the patients with rheumatoid arthritis (RA) and the disease and healthy control groups*
Parameter
Patients with established RA
Patients with OA
Healthy controls
No. of patients
Age, mean ⫾ SD (range) years
Sex, no. male: no. female (% male:% female)
No. (%) RF positive
No. (%) with bone erosions
No. (%) with rheumatoid nodules
ESR, mean ⫾ SD (range) mm/hour
CRP, mean ⫾ SD (range) mg/liter
Disease duration, mean ⫾ SD (range) years
Medication, no. (%)
NSAID use
DMARD use
MTX use
Prednisone use
20
55 ⫾ 16 (23–81)
7:13 (35:65)
10 (50)
11 (55)
4 (20)
31 ⫾ 26 (2–91)
42 ⫾ 28 (⬍5–86)
6.7 ⫾ 5 (1–19)
20
60 ⫾ 10 (42–77)
7:13 (35:65)
–
–
–
3 (1–7)†
–
–
20
53 ⫾ 14 (23–75)
6:14 (30:70)
–
–
–
–
–
–
18 (90)
19 (95)
12 (60)
3 (15)
17 (85)
0
0
0
–
–
–
–
* The 3 groups were not significantly different from each other in age and sex distribution. OA ⫽ osteoarthritis; RF ⫽ rheumatoid factor
(determined by Rose-Waaler test and by latex agglutination); ESR ⫽ erythrocyte sedimentation rate; CRP ⫽ C-reactive protein; NSAID ⫽
nonsteroidal antiinflammatory drug; DMARD ⫽ disease-modifying antirheumatic drug; MTX ⫽ methotrexate.
† Tested in only 6 of the patients with OA.
and p3) on the basis of an optimal pan–DR-binding score, and
peptide pairs p5/p6 and p7/p8 were chosen for the pan–DRbinding score of the human epitopes (p6 and p8). The sequential use of a combined DR1/DR4/DR7 algorithm can be used
to identify broadly crossreactive DR-binding peptides (34). In
vitro major histocompatibility complex (MHC) binding studies
have confirmed that Hsp60 peptides identified by the computer algorithm were indeed able to bind to a diverse range of
HLA–DR molecules (31). The pan–DR-binding peptides were
synthesized by automated, simultaneous, multiple-peptide synthesis, with a purity ⬎95%, as described previously (35).
Lymphocyte proliferation assays. Cells were cultured
(2 ⫻ 105 cells in 200 ␮l per well) in triplicate in roundbottomed 96-well plates (Nunc, Roskilde, Denmark) for 120
hours at 37°C in 5% CO2 with 100% relative humidity, in the
absence or presence of 20 ␮g/ml Hsp60 peptides. Concanavalin
A (2.5 ␮g/ml; Calbiochem, San Diego, CA) and diphtheria
toxoid and tetanus toxoid (5 Lf/liter; RIVM, Bilthoven, The
Netherlands) were used as positive controls. A mouse class
II–restricted epitope, ovalbumin (OVA) 323–339, was used as
a negative control. During the last 16 hours of culture, 1 ␮Ci
3
H-thymidine (ICN Biomedicals, Amsterdam, The Netherlands) was added to each well. Cells were harvested, and the
incorporated radioactivity was measured by liquid scintillation
counting, with results expressed in counts per minute. The
magnitude of the proliferative response was expressed as the
stimulation index (SI), which is calculated as the mean counts
per minute of cells cultured with antigen divided by the mean
Table 2. Characteristics of the peptides identified*
Predicted binding capacity, arbitrary units
Peptide/type
Sequence
DR1
DR4
DR7
Overall score
p1/myc 254–268
p2/hum 280–294
p3
myc 216–230
myc 216–230
p4/hum 242–256
p5/myc 210–224
p6/hum 236–250
p7
myc 507–521
myc 507–521
p8/hum 535–549
myc 256–270
hum 282–296
GEALSTLVVNKIRGT
GEALSTLVLNRLKVG
42.4
12.5
17.0
0.9
276.9
8.7
3
1
PYILLVSSKVSTVKD
PYILLVSSKVSTVKD
AYVLLSEKKISSIQS
EAVLEDPYILLVSSK
KCEFQDAYVLLSEKK
3.6
132.0
0.2
28.5
40.8
14.3
4.2
2.8
0.4
3.6
26.7
29.8
7.0
15.5
96.8
3
3
1
2
3
IAGLFLTTEAVVADK
IAGLFLTTEAVVADK
VASLLTTAEVVVTEI
ALSTLVVNKIRGTFK
ALSTLVLNRLKVGLQ
1.8
10.5
12.0
ND
ND
0.3
1.3
3.3
ND
ND
3.7
18.7
68.0
ND
ND
1
2
3
ND
ND
* The origin and amino acid sequences of peptides p1–p8, mycobacterial (myc) Hsp65 256–270, and human (hum) Hsp60 282–296 are listed. The
core sequence of each peptide is underlined. A computer algorithm predicted the core sequences of the peptides on the basis of their
HLA–DR1*0101 (DR1), DR1*0401 (DR4), and DR1*0701 (DR7) binding capacity. A binding score of ⱖ1.570 for DR1*0101, ⱖ2.617 for
DR1*0401, and ⱖ9.106 for DR1*0701 was assumed to be positive. Peptides with a pan–DR-1 binding score of 3 were selected for analysis, and their
human and mycobacterial equivalents were also tested. ND ⫽ not determined.
IL-10–MEDIATED, PAN–DR-BINDING Hsp60–INDUCED RESPONSE IN RA
1969
Figure 1. Peptide-induced T cell proliferation of peripheral blood mononuclear cells from patients with rheumatoid arthritis, disease controls
(patients with osteoarthritis), and healthy controls. The results are expressed as the median stimulation index in response to peptides p1–p8,
mycobacterial (m) Hsp65 256–270, human (h) Hsp60 282–296, and negative control peptide ovalbumin (ova) 323–339. Data are presented as box
plots, where the boxes represent the 25th to 75th percentiles, and the lines within the boxes represent the median. Each dot represents the results
for 1 patient. (The mean stimulation indices are listed in the text.) ⴱ ⫽ P ⬍ 0.05.
cpm of cells cultured without antigen. If the variation between
the results obtained in triplicate exceeded 2 standard deviations, the stimuli were excluded.
Blocking assays. Anti–HLA–DR (clone B8.11.2) and
anti–HLA class I monoclonal antibody (mAb) (clone W6/32;
negative control) were obtained from Dr. F. Claas (Department of Immunohematology, Leiden University Medical Cen-
tre, Leiden, The Netherlands). PBMCs (1 ⫻ 106/ml) were
incubated together with these mAb against HLA–DR, for 1 hour.
After this incubation, the lymphocyte proliferation
assay was performed as described above. The blocking capacity
of the mAb was evaluated by calculating the percent reduction
in the proliferative response obtained in the blocked condition
versus that obtained in the unblocked condition.
1970
DE JONG ET AL
Carboxyfluorescein diacetate succinimidyl ester
(CFSE) staining. PBMCs (5 ⫻ 106) were stained with CFSE
(Molecular Probes, Eugene, OR) for 5 minutes in RPMI.
CFSE-labeled PBMCs were then cultured in the presence of a
stimulus for 168 hours. Subsequently, the PBMCs were stained
for CD4 and CD3 (both from BD Biosciences, San Jose, CA),
with results analyzed on a flow cytometer (FACSCalibur; BD
Biosciences).
Cytokine assays. For analysis of antigen-specific cytokine production, PBMCs from the patients with RA were
cultured as described above. Culture supernatants were harvested after 72 hours and stored at ⫺80°C until further
analyzed. Extracellular cytokines and chemokines were measured using a multiplex immunoassay (Luminex, Austin, TX)
as previously described (36–38). In summary, the antibodycoated microspheres were incubated for 60 minutes with
standards or culture medium (25 ␮l) in 96-well, 1.2-␮m filter
plates (Millipore, Amsterdam, The Netherlands). Plates were
washed, and a cocktail of biotinylated detection antibodies was
added for 60 minutes. After repeated washings, streptavidin–
phycoerythrin was added for an additional 10 minutes. Beads
were washed twice, and the fluorescence intensity was measured.
Measurements and analysis of the data from all assays
were performed using the Bio-Plex system in combination with
Bio-Plex Manager software, version 4.1, using 5-parametric
curve fitting (Bio-Rad, Hercules, CA). The concentrations of
the following soluble mediators were measured: interleukin-1␣
(IL-1␣), IL-1␤, IL-2, IL-4, IL-6, IL-8, IL-10, IL-13, IL-15,
IL-17, IL-23, tumor necrosis factor ␣ (TNF␣), and interferon-␥
(IFN␥). The detection range for all cytokines was 1.2–5,000
pg/ml, except IL-8 and IL-23, which had a detection range of
2.4–10,000 pg/ml (38).
None of the above-mentioned cytokines is made exclusively by T cells. Therefore, we performed intracellular
fluorescence-activated cell sorting (FACS) experiments to
investigate which cells were producing these cytokines. Intracellular staining for IL-10, IFNy, TNF␣, and IL-4, as compared
with isotype controls (all from BD Biosciences), and surface
marker staining for CD3 and CD4 were performed after 48
hours of culture with the peptide of interest. The peptidespecific cytokine production was defined as the percentage of
CD3⫹CD4⫹ T cells producing the cytokine of interest after
epitope stimulation minus the percentage found in the nonstimulated condition and after correction for cytokine staining.
Statistical analysis. For statistical analysis, SPSS software, version 10.05 (SPSS, Chicago, IL) was used. Group
differences were statistically evaluated using the MannWhitney U test and the Kruskal-Wallis test. For the analysis of
peptide-induced cytokine production, the geometric mean was
calculated. Correlations between disease parameters and immune responses were calculated with Spearman’s test.
RESULTS
Recognition of pan–DR-binding epitopes in RA
patients. PBMCs from 20 randomly selected RA patients (see Table 1 for demographic and clinical characteristics) were tested for their proliferative response
(using 3H-thymidine incorporation) to the peptide pairs
of peptides p1–p8 (Table 2). Based on observations
published earlier, an SI that is twice that of the background value is defined as a positive proliferative response (31,39). The proliferative response obtained
without adding the antigen (defined as the background
value) ranged from 170 cpm to 1,000 cpm (mean 395 cpm).
Five of the 8 peptides induced a positive T cell
proliferative response in RA patients; these 5 peptides
were p1 (mean ⫾ SD SI 2.5 ⫾ 0.5), p2 (mean ⫾ SD SI
4.5 ⫾ 0.8), p3 (mean ⫾ SD SI 3.3 ⫾ 0.4), p6 (mean ⫾ SD
SI 2.1 ⫾ 0.4), and p8 (mean ⫾ SD SI 2.1 ⫾ 0.5) (see
Figure 1 for median values; detailed results for individual patients and for each peptide tested after in vitro
activation in comparison with medium activation are
available upon request from the corresponding author).
Interestingly, all human peptides (with the exception of
p4) caused a more vigorous proliferative response in
PBMCs from RA patients compared with that induced
by their microbial analogs. Overall, the proliferative
response to the HSP epitopes was low, which was as
expected given the fact that these responses reflect a
primary immune response of T cells with a low precursor
frequency directed toward peptides with a high degree
of homology to self. The mean ⫾ SD SI in response to
whole human Hsp60 was 3.2 ⫾ 0.49, and that to the
whole mycobacterial Hsp60 was 3.9 ⫾ 0.58.
Notably, one of the peptides identified by the
computer algorithm, p1 (mycobacterial Hsp65 254–268)
(Table 2), represents only a 2–amino acid frameshift
from mycobacterial Hsp65 256–270, the latter of which
functions as a protective T cell epitope in the adjuvantinduced arthritis model (40). Mycobacterial Hsp65 256–
270 also induced T cell proliferation of the PBMCs from
RA patients (mean ⫾ SD SI 3.9 ⫾ 0.8), while the human
analog of this protective epitope, human Hsp60 282–296,
was also recognized by PBMCs from RA patients
(mean ⫾ SD SI 3.1 ⫾ 0.6) (Figure 1).
No significant correlations were found between
disease duration or the amount of inflammation and the
proliferative responses. The irrelevant control peptide
(OVA 323–339) did not induce significant T cell proliferation of PBMCs from RA patients. If an HSP peptide
induced a positive proliferative response in vitro in an
individual patient, the proliferative response was also
significantly different between cultures with the HSP
peptide and cultures with the control peptide (OVA
323–339) in that individual (results available upon request from the corresponding author). Thus, 5 of the 8
pan–DR-binding epitopes and 2 closely related epitopes
derived from the protective epitope in the adjuvant-
IL-10–MEDIATED, PAN–DR-BINDING Hsp60–INDUCED RESPONSE IN RA
1971
Figure 2. Response of peripheral blood mononuclear cells (PBMCs) from 6 randomly selected patients with rheumatoid arthritis (RA) to
mycobacterial (myc) Hsp60, human (hum) Hsp60, and human peptides p1–p4, in terms of production of pro- and antiinflammatory cytokines.
Production of extracellular cytokines in the supernatants of stimulated PBMCs (compared with cultures without antigen) was measured by multiplex
immunoassay. A, Stimulation with the peptides resulted in a clear induction of interleukin-1␤ (IL-1␤), IL-6, IL-10, and tumor necrosis factor ␣
(TNF␣). The human peptides p2 and p4 induced fewer proinflammatory cytokines, such as IL-1␤ and TNF␣, and almost equal production of IL-10
compared with their microbial homologs. Each column represents the results from an individual patient. B, Ratio of IL-10 to TNF␣ deduced from
epitope-specific cytokine production by PBMCs from 6 patients with established RA. IL-10 and TNF␣ values of 0 were replaced with a value of 0.5
to allow calculation of the ratios. Bars show the median and SEM. The human peptides p2 and p4 induced relatively more IL-10 compared with the
microbial peptides, which resulted in a higher IL-10:TNF␣ ratio.
induced arthritis model induced T cell proliferation of
PBMCs from RA patients.
Disease (RA)–specific response to the pan–DRbinding Hsp60 peptides. We next investigated whether
recognition of the pan–DR-binding Hsp60 epitopes in
PBMCs from RA patients is disease specific. We thus
compared the results obtained from RA patients with
the proliferative response of PBMCs from disease controls (patients with OA) and healthy control subjects. A
positive proliferative response of the PBMCs to the
pan–DR-binding peptides was observed in a higher
percentage of RA patients compared with healthy controls or OA patients. Responses to the control peptide
(OVA 323–331) and tetanus toxoid were not different
between the 3 study groups.
In supernatants cultured with p1, PBMCs from
50% of the RA patients showed a positive reaction to
the peptide (SI ⬎2), compared with only 5% of the
healthy controls and 20% of the OA patients. A similar
pattern was seen in response to p2, with the percentages
of RA patients versus healthy controls and OA patients
showing a positive response being 75% versus 45% and
65%, respectively, while in response to p3, these percentages were 78% versus 45% and 50%, respectively, to
p6, 62% versus 5% and 5%, respectively, to p7, 22%
versus 5% and 0%, respectively, and to p8, 33% versus
0% and 0%, respectively. Similar trends were visible in
response to the mycobacterial Hsp65 peptide 256–270,
in which PBMCs from 65% of the RA patients showed
a positive reaction compared with 25% of healthy controls and 5% of OA patients (P ⬍ 0.001 for RA versus
OA). In cultures with human Hsp60 282–296, these
percentages for PBMCs showing a positive response
were 63% versus 40% and 10%, respectively.
The differences in peptide recognition by PBMCs
from RA patients and those from healthy controls
reached significance (P ⬍ 0.05) for peptides p1, p2, p6,
and mycobacterial Hsp65 256–270, while the difference
between RA and OA patients reached significance for
peptides p6, p7, mycobacterial Hsp65 256–270, and
human Hsp60 282–296 (Figure 1). Thus, some of the
peptides elicited proliferative T cell responses in PBMCs
from RA patients but not in PBMCs from healthy
controls or those from OA patients, underscoring the
disease-specific recognition of these epitopes in RA.
The human pan–DR-binding epitopes induced a
more favorable IL-10:TNF␣ ratio compared with that
induced by their microbial counterparts. PBMCs from 6
patients with RA, being representative of the whole
group of RA patients, were cultured with or without the
various stimuli. After 72 hours in culture supernatants,
the cells were harvested, and cytokine production was
measured with a multiplex immunoassay (36,38). Based
on the profile of proliferative responses and the corre-
1972
sponding data on the protective epitopes in the
adjuvant-induced arthritis model (p1 having close homology with a protective epitope in adjuvant-induced
arthritis), we decided to focus on peptide pairs p1/p2 and
p3/p4 for these further studies. As shown in Figure 2A,
after culture of PBMCs from RA patients, the levels of
IL-1␤, IL-2, IL-6, IL-10, IL-15, IL-17, IL-23, TNF␣, and
IFN␥ were determined in the supernatants in response
to both whole Hsp60 proteins and to peptides p1–p4.
(Each column in Figure 2 represents the results in
PBMCs from an individual patient.)
We detected peptide-specific production of IL1␤, IL-6, IL-10, IL-15, and TNF␣ by PBMCs from most
of the RA patients studied. The production of IL-1␣,
IL-4, and IL-13 was below the limit of detection (⬍1.2
pg/ml). Production of IL-17, IL-23, and IFN␥ in supernatants with each of the epitope stimuli was similar to
that in the unstimulated condition.
We next investigated which cells produced these
cytokines. Intracellular FACS staining showed that
CD3⫹CD4⫹ T cells from RA patients produced IFN␥,
IL-10, and TNF␣ after 48 hours of stimulation with
peptides p1 and p2 (see Figure 3 for a representative
example). The documented cytokine production by
CD4⫹ T cells obviously does not exclude the possibility
that other cells are also responsible for producing these
cytokines upon peptide stimulation. However, we failed
to detect any significant peptide-specific cytokine production by non–T cells (detailed results available upon
request from the corresponding author). Taken together, these data suggest that CD4⫹ T cells are likely to
be primarily responsible for peptide-specific cytokine
production.
Notably, when the extracellular cytokine data
were analyzed further, the amount of proinflammatory
cytokines (IL-1␤, TNF␣, and IL-6) produced in response
to the mycobacterial peptides was elevated compared
with that produced in response to their human homologs. We chose to calculate the ratio of antiinflammatory cytokines to proinflammatory cytokines. IL-10
was chosen as the antiinflammatory cytokine for this
calculation, because it is known as one of the most
important counterregulatory cytokines. TNF␣ was chosen as the proinflammatory cytokine for this calculation
because it is the most abundant proinflammatory cytokine in RA (41).
The ratios of IL-10 to TNF␣ (median ⫾ SEM) in
PBMCs from an individual RA patient in response to
peptides p1–p4, as shown in Figure 2B, were 0.14 ⫾ 0.02
with p1, 5.17 ⫾ 1.8 with p2, 0.16 ⫾ 0.02 with p3, and
2.02 ⫾ 0.72 with p4. Thus, the IL-10:TNF␣ ratio in
DE JONG ET AL
Figure 3. Peptide-specific production of interferon- ␥ (IFN ␥ ),
interleukin-10 (IL-10), and tumor necrosis factor ␣ (TNF␣), but not
IL-4, by CD3⫹CD4⫹ T lymphocytes. After 48 hours of culture with
medium (unstimulated), with peptides p1 and p2, or with tetanus
toxoid (TT) as a control, peripheral blood mononuclear cells were
harvested and surface stained for CD3 and CD4, and subsequently
stained intracellularly for IFN␥ and IL-10 or for TNF␣ and IL-4.
Results, as assessed by flow cytometry, showed significant production
of IFN␥, IL-10, and TNF␣, but not IL-4, by CD4⫹ T lymphocytes in
response to peptides p1 and p2, at levels above those in the unstimulated and control conditions.
response to the human peptides was more than 4 times
higher than that with the microbial peptides, reflecting a
more antiinflammatory phenotype.
The proliferative response was caused by CD4⫹
proliferating T cells and was HLA–DR restricted. We
have already shown that CD3⫹CD4⫹ T cells produced
epitope-specific production of IL-10, IFN␥, and TNF␣.
We next wondered whether the proliferative response to
the peptides was the result of proliferating CD4⫹ T
cells. Proliferating PBMC populations were monitored
by fluorescent dye (CFSE) incorporation. Representative examples are shown in Figure 4. After 168 hours in
culture, it was possible to identify a circumscript population of lymphocytes that had proliferated as a result of
exposure to the stimuli. The lymphocytes responding to
either the HSP peptides or to whole mycobacterial
IL-10–MEDIATED, PAN–DR-BINDING Hsp60–INDUCED RESPONSE IN RA
Figure 4. Peptide-specific proliferation of CD4⫹ T cells. Peripheral
blood mononuclear cells from patients with rheumatoid arthritis were
stained with carboxyfluorescein diacetate succinimidyl ester (CFSE)
after 168 hours of culture in the presence of either medium, mycobacterial (myc) Hsp60, mycobacterial peptide p3, or tetanus toxoid as
control. The whole lymphocyte population is indicated by the solid
line, while the CD3⫹CD4⫹ lymphocyte population is indicated by the
grey area. The peptide-specific lymphocyte populations proliferating in
response to mycobacterial Hsp60 and mycobacterial peptide p3, in
contrast to that with the control tetanus, are almost all CD4 positive.
Hsp65 were almost exclusively CD4⫹ T cells. In contrast, following stimulation with tetanus toxoid, approximately one-half of the proliferating PBMCs were
CD4⫹. Control experiments showed that the
CD4⫹CFSElow population was indeed also CD3⫹,
which was further proof of the T cell–specific proliferation. Moreover, when CD4⫹ T cells were depleted prior
to stimulation, the immune response was abolished
(further details are available upon request from the
corresponding author).
As described above, the peptides were identified
by their theoretical capacity to bind to multiple
HLA–DR molecules. Their pan–DR-binding capacity
was also confirmed by in vitro MHC binding assays (31).
To further confirm that the documented proliferative
response of PBMCs from RA patients to the novel
epitopes was indeed HLA–DR dependent, we performed lymphocyte stimulation assays in the presence of
blocking anti–HLA–DR antibodies. For these experiments, peptides p2 and p3 were chosen, because they
induced the highest proliferative responses in PBMCs
from RA patients. The response to the whole human
and microbial proteins could be blocked by 70–75%,
while in cultures with peptides p2 and p3, an inhibition
of 47% and 64%, respectively, was induced by the
anti–HLA–DR mAb, resulting in normalization of the
SI. Thus, the proliferation of PBMCs from RA patients,
when exposed to the pan–DR-binding peptides, is a
result of a class II MHC–restricted CD4⫹ T cell response.
DISCUSSION
The discovery of T cells with a regulatory capacity has yielded new hopes for antigen-specific immune
1973
therapy in autoimmune diseases such as RA (42). However, unlike in the experimental models, knowledge of
specific antigenic targets for immune therapy in humans
is still limited. HSPs are candidate antigens for such
immune therapy, because they are immunodominant
proteins that are up-regulated at sites of inflammation.
Recently, phase I and phase II immunotherapy trials
with epitopes derived from DnaJ HSP and Hsp60
showed great promise in RA (refs. 23 and 43 and
Koffeman E, et al: unpublished observations) and type
II diabetes mellitus (23,44), respectively. Over the years,
a large amount of data has been gathered, in experimental arthritis models as well as in patients with JIA and
patients with RA, suggesting that Hsp60 is a potential
target for immunotherapy in arthritis (24). Whole
Hsp60, however, can activate both the adaptive and the
innate arms of the immune system, which underscores
the importance of identifying suitable T cell epitopes for
immune therapy, thus bypassing the risks of nonspecific
activation. Such Hsp60 T cell epitopes would have the
additional advantage that they could also be used as a
tool to study the arthritis-specific T cell repertoire,
which would thus lead to better understanding of the
pathogenesis of RA.
Although immune recognition of whole mycobacterial and whole human Hsp60 in PBMCs and synovial
fluid mononuclear cells from patients with RA is well
documented (26,36,45–48), no Hsp60 T cell epitope that
is recognized in a majority of RA patients has yet been
identified. By using a computer algorithm that can
predict the potential pan–DR-binding motifs (34), we
identified 8 potential T cell epitopes derived from both
microbial and human Hsp60. Four of these epitopes
from both microbial origin (p1 and p3) and human
origin (p2 and p6) were recognized by HLA–DR–
restricted CD4⫹ T cells in ⬎50% of the RA patients.
The response was significantly different in comparison
with that in healthy controls, for p1, p2, and p6. It is of
interest that some T cell proliferation could also be
detected (but to a lower extent) in PBMCs from OA
patients, possibly reflecting an inflammatory process in
that disease. Based on analysis of the extracellular
cytokines produced by PBMCs from RA patients after
exposure to the human peptides, an elevated IL-10:
TNF␣ ratio was observed in response to the human
peptides as compared with that in response to the
microbial homologs. This proliferative response, as well
as the cytokine response, was driven solely by CD4⫹ T
cells.
The antigen-specific responses observed (proliferation of T cells as well as elevated cytokine levels)
1974
were robust but modest, fitting a primary immune
response of T cells with a low precursor frequency.
Because these T cells are specific for self or almost-self
antigens, it is expected that these cells are under the
control of peripheral tolerance and thus do not proliferate vigorously (31,49).
Of the identified epitopes, the microbial peptide
p1 (mycobacterial Hsp65 254–268) and its human homolog, peptide p2, are of special interest, because the
sequence of p1 is a 2–amino acid frameshift from
mycobacterial Hsp65 256–270, a protective epitope in
multiple models of experimental arthritis (40). Importantly, we could also document T cell recognition of this
mycobacterial Hsp65 256–270 epitope in RA, as well as
T cell recognition of its human homolog, human Hsp60
282–296. In experimental arthritis, this protective
epitope exerts its protective effect by inducing crossreactive (self-reactive) T cells. In the RA population, we
observed a correlation between the mycobacterial peptides and their human homologs for the peptide pairs
p1/p2 (Spearman’s ␳ ⫽ 0.47, P ⬍ 0.05) and Hsp65
256–270/Hsp60 282–296 (Spearman’s ␳ ⫽ 0.80, P ⬍
0.05). This may indicate that crossreactive T cells between human and microbial Hsp60 peptides are present
in RA patients. Taking into account the specific recognition of both the microbial and the human epitopes,
this suggests that also in RA, either p1 (mycobacterial
Hsp65 254–268) or the original protective epitope identified in experimental arthritis (mycobacterial Hsp65
256–270) may be a candidate antigen for immune therapy.
In order to determine the peptide recognition in
a broad population of patients, we chose to perform a
cross-sectional study with randomly selected patients
with RA. As has been described in earlier studies, the
development of responsiveness to Hsp60 is associated
with disease duration (45,48). Obviously, in this study,
we could not address this issue. Further research on both
patients with early RA and patients with RA of longer
duration should be done in order to draw any conclusions regarding the question of whether the peptide
responses quantitatively or qualitatively change during
the course of the disease and/or whether they may be
correlated with disease outcome.
One might argue that the documented recognition of several pan–DR-binding T cell epitopes in RA is
a mere reflection of the known association of RA with
certain HLA types, such as HLA–DR1 and DR4. However, as was shown by Southwood et al (34), the use of a
combined DR1/DR4/DR7 algorithm can identify
broadly crossreactive DR-binding peptides. Conse-
DE JONG ET AL
quently, the peptides selected by the model are able to
bind to multiple HLA–DR types, as has been shown in
in vitro MHC binding studies (31). To further confirm
this, we performed HLA typing of the patients and
related their HLA type to the proliferative response of
the pan–DR-binding epitopes. As expected, no correlation was found between the HLA type and the quality or
quantity of the immune response induced (results not
shown). In addition, when the shared epitope (HLA–
DR4) was examined in more detail, no significant differences were found when comparing the proliferative
response of PBMCs obtained from DR4⫹ patients with
RA with that of PBMCs obtained from DR4⫺ patients
with RA. Therefore, the association between certain
HLA types cannot be the explanation for the diseasespecific recognition of the pan–DR-binding epitopes in
RA.
Earlier studies in patients with JIA and patients
with RA, using whole Hsp60 proteins, suggested that T
cell recognition of human Hsp60 is associated with a
regulatory phenotype (26,29,31). Interestingly, in our
study, the human Hsp60 epitopes also induced a more
antiinflammatory T cell response, as depicted by marked
changes in the IL-10:TNF␣ ratio. This again underlines
the potential role of these peptides in the ongoing
inflammatory process. Whether these cells play an antiinflammatory role in vivo obviously could not be determined in this study.
We previously showed that DnaJ P1, a peptide
derived from another HSP, induces proinflammatory
responses in PBMCs from patients with early RA and
can be used to modulate autoimmune inflammation in
these patients (23). The results from the present study
show that the phenomenon of disease- and
inflammation-specific recognition of HSP-derived
epitopic peptides, which we first described in patients
with RA using DnaJ P1, is, in reality, more complex and
involves peptides that are functionally similar to DnaJ
P1 (proinflammatory) as well as others that are quite
different (50). Thus, our study contributes to a better
understanding of the mechanism that can be manipulated for therapeutic purposes. The major advance is
that it provides evidence of more DnaJ P1–like peptides
that could be used in the same way and could, perhaps,
capture a broader patient population. In addition, our
findings appear to identify other peptides that probably
have a different mechanism of action.
Further studies are needed to evaluate the capacity of these epitopes to induce regulatory T cells in vitro
and ex vivo in relation to specific disease characteristics
of patients with RA. This should also yield important
IL-10–MEDIATED, PAN–DR-BINDING Hsp60–INDUCED RESPONSE IN RA
information on which specific epitope is most suitable
for immune therapy in RA. Based on the present data,
as well as the findings in experimental arthritis and the
preliminary phase I study with HSP DnaJ P1 in humans,
it seems that a peptide in the region of 254–270 of
mycobacterial Hsp65 could be an ideal candidate for an
immune therapy trial in patients with RA.
16.
17.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Prakken had full access to all of
the data in the study and takes responsibility for the integrity of the
data and the accuracy of the data analysis.
Study conception and design. De Jong, Kuis, Bijlsma, Prakken, Albani.
Acquisition of data. De Jong, Lafeber, de Jager, Haverkamp, Bijlsma.
Analysis and interpretation of data. De Jong, de Jager, Prakken.
18.
19.
20.
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