Pharmacological Reports 68 (2016) 483489

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Pharmacological Reports
journal homepage: www.elsevier.com/locate/pharep

Original research article

Epicutaneous (EC) immunization with type II collagen (COLL II)

induces CD4+ CD8+ T suppressor cells that protect from
collagen-induced arthritis (CIA)
Katarzyna Marcinska a, Monika Majewska-Szczepanik a, Agata Lazar b,
Paulina Kowalczyk a, Dominika Biaa a, Dorota Wozniak a, Marian Szczepanik a,*
a
b

Department of Medical Biology, Jagiellonian University Medical College, Krakow, Poland

Department of Pathomorphology, Jagiellonian University Medical College, Krakow, Poland

A R T I C L E I N F O

Article history:
Received 20 April 2015
Received in revised form 10 November 2015
Accepted 11 November 2015
Available online 25 November 2015
Keywords:
Epicutaneous immunization
CD4+CD8+RORgt+ suppressor T cells
Collagen-induced arthritis
Cytokines

A B S T R A C T

Background: We have shown previously that epicutaneous (EC) immunization with protein antigen
induces T suppressor cells that alleviate inammatory response in contact hypersensitivity reactions, in
an animal model of multiple sclerosis, and in TNBS-induced colitis.
Methods: DBA/1 mice were EC immunized with type II collagen (COLL II) spread over a gauze patch on
days 0 and 4. On day 7, patches were removed and mice were intradermally (id) immunized with COLL II
in CFA to induce collagen-induced arthritis (CIA).
Results: Our work shows that EC immunization with 100 mg of COLL II prior to CIA induction reduces
disease severity as determined by macroscopic evaluation. Reduced disease severity after EC
immunization with COLL II correlates with milder histological changes found in joint sections.
Experiments with the three non-cross-reacting antigens COLL II, ovalbumin (OVA) and myelin basic
protein (MBP) showed that skin-induced suppression is antigen non-specic. Transfer experiments
show that EC immunization with COLL II induces suppressor cells that belong to the population of CD4+
CD8+ double positive lymphocytes. Flow cytometry experiments showed increased percentage of CD4+
CD8+ RORgt+ cells in axillary and inguinal lymph nodes isolated from mice patched with COLL II.
Conclusion: Maneuver of EC immunization with a protein antigen that induces suppressor cells to inhibit
inammatory responses may become an attractive, noninvasive, needle-free therapeutic method for
different clinical situations.
2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights
reserved.

Introduction

Abbreviations: Ab, antibody; ALNC, axillary and inguinal lymph node cells; antiCPP, Anti-cyclic citrullinated peptide; CFA, complete Freunds adjuvants; CHS,
contact hypersensitivity reactions; CIA, collagen-induced arthritis; COLL II, type II
collagen; EAE, experimental autoimmune encephalomyelitis; EC, epicutaneous;
FBS, fetal bovine serum; id, intradermally; IFA, incomplete Freunds adjuvants; Ig,
immunoglobulin; IL, interleukin; ip, intraperitoneally; iv, intravenous; LPS,
lipopolysaccharide; MBP, myelin basic protein; MPO, myeloperoxidase; NK, natural
killer; NKT, natural killer T cell; OVA, ovalbumin; PBS, phosphate buffered saline;
RA, rheumatoid arthritis; RC, rabbit complement; TGF-b, transforming growth
factor beta; Tc, T cytotoxic cell; Th, T helper cell; TNBS-induced colitis, the 2,4,6trinitrobenzene sulfonic acid-induced colitis; TNP-Ig, TNP conjugated mouse
immunoglobulins; Ts, T suppressor cell.
* Corresponding author.
E-mail addresses: mmszczep@cyf-kr.edu.pl, marian.szczepanik@uj.edu.pl
(M. Szczepanik).

Rheumatoid arthritis (RA) is a chronic inammatory disease

that affects about 1% of the adult population and occurs twice as
frequently among women than men [1]. The onset may appear at
any age, but the peak of incidence comes in the 25 55 age range
[2]. Because the disease affects persons in economically productive
age ranges, it presents an ever-increasing economic and social
problem affecting the quality of life of the patients and their ability
to work.
Non-steroid anti-inammatory drugs commonly used in RA
therapy through their anti-inammatory and analgesic activity,
principally relieve the symptoms of RA, with minimal impact on
the disease process, and concurrent side effects [3]. Additionally,
the treatment of RA includes drugs modifying the disease process,
e.g. sulfasalazin, methotrexate and cyclosporin A. These drugs

http://dx.doi.org/10.1016/j.pharep.2015.11.004
1734-1140/ 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.

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K. Marcinska et al. / Pharmacological Reports 68 (2016) 483489

apart from their therapeutic effects may cause a number of severe

side effects [4]. Finally, RA therapy uses biological drugs, which can
interfere with the immune mechanisms underlying the RA
pathology, such as, e.g. cytokine inhibitors. These drugs face
barriers, e.g. limited availability, high price and inducement of nonspecic immunosuppression [5].
Therefore, there is huge need to develop new therapies, noninvasive and free of side effects, specically eliminating the
undesired inammatory reaction accompanying RA. Throughout
the world, efforts have been made to develop a vaccine which
would prevent the onset of autoimmune diseases, including RA.
Our previous work showed that epicutaneous (EC) immunization of mice with different protein antigens applied on the skin
in the form of a gauze patch or cream emulsion induces a state of
subsequent tolerance that inhibits both Th1 and Tc1-mediated
contact hypersensitivity (CHS) [69]. Further study showed that
maneuver of EC immunization with protein antigen also
suppressed NK cell dependent CHS [10]. This was also found
in an experimental autoimmune encephalomyelitis (EAE) where
EC immunization with myelin basic protein (MBP) reduced
disease severity and decreased disease incidence [11,12]. Furthermore, using allogeneic skin graft experimental model, we
showed that EC immunization with a protein antigen delays graft
rejection [13]. Then we showed that EC immunization with
protein antigen TNP-Ig can also alleviate TNBS-induced colitis in
mice [14].
Our recent study shows that EC immunization with type II
collagen (COLL II) reduces disease severity as determined
macroscopically [15]. Decreased disease severity observed after
EC immunization with COLL II was conrmed by reduced MPO
(myeloperoxidase) activity in joint tissue and with decreased
production of anti-citrullinated protein (anti-CPP) and anti-type II
collagen IgG2a antibodies. Observed protection from disease was
transferable with TCRab+ lymphocytes. Both in vitro and in vivo
experiments show that IL-17A plays an important role in ECinduced suppression of CIA. Moreover, EC application of COLL II at
the rst signs of CIA also results in suppression of disease [15].
The current study shows that similarly to CHS and EAE, skininduced suppression is antigen-non-specic and is mediated by
CD4+ CD8+ double positive suppressor cells and that these cells
express transcription factor RORgt.

Materials and methods

Mice
Male DBA/1 mice 8 12 weeks old were from the breeding unit
of the Department of Medical Biology, Jagiellonian University,
School of Medicine. Mice were fed autoclaved food, and water. All
experiments were conducted according to guidelines of the Animal
Use and Care Committee of the Jagiellonian University School of
Medicine.
Reagents
Bovine type II collagen, complete (CFA) and incomplete (IFA)
Freunds adjuvants were obtained from Chondrex Inc. (Redmond,
WA, USA). LPS (from Escherichia coli 026:B6), OVA (Grade V) and
guinea pig MBP were purchased from Sigma (St. Louis, MO, USA).
RPMI 1640 and fetal bovine serum (FBS) were from Life
Technologies (Grand Island, NY, USA). Low-tox rabbit complement
(RC) was from Pel-Freeze Biologicals (Brown Deer, WI, USA).
Protein A was from Pharmacia Fine Chemicals (Piscataway, NJ,
USA), whereas Sepharose 4 Fast Flow was from Pharmacia LKB
Biotechnology AB (Uppsala, Sweden).

Monoclonal antibodies and hybridoma

Protein A and Sepharose 4 Fast Flow were used to obtain
puried IgG from supernatants of hybridoma cell cultures
according to the directions of the manufacturer with our
modications [16]. The following protein A afnity puried rat
anti-mouse monoclonal antibodies (mAb) were used: anti-CD4
(clone TIB 207) and anti-CD8 (clone TIB 105.3) from late Dr. C.A.
Janeway, Jr., Yale University, New Haven, CT, USA.
Induction and gross assessment of CIA
Immunization and arthritis evaluation were performed as
described previously [11]. DBA/1 mice were injected id at the base
of the tail with 100 mg COLL II emulsied in CFA on day 0 and
boosted with 100 mg COLL II in IFA on day 21 after the rst
immunization. To accelerate the development of arthritis, 40 mg of
LPS in sterile PBS was intraperitoneally (ip) injected on day
28. Animals were observed daily for the presence of arthritis and
the clinical severity of disease was scored for each paw on a scale of
0 4 [15]. The criteria for the grading were as follows: 0no evidence
of erythema and swelling; 1mild erythema and swelling of the
wrist or the ankle; 2moderate erythema and swelling from the
wrist to the metacarpal joints or from the ankle to the metatarsal
joints; 3severe erythema and swelling of the entire paw including
digits; 4maximal erythema and swelling of the paw.
Histology of the ankle joint
On day 67 after immunization, the mice were sacriced and the
hind paws were xed in 10% buffered formalin, decalcied and
embedded in parafn. Joint sections (5 7 mm) were prepared and
stained with hematoxylin and eosin (H&E). The histological
evaluation was performed under the light microscope.
EC immunization with protein antigen
EC immunization was performed by applying to the shaved skin
of the mouse dorsum a 1 cm2 gauze patch soaked with a solution
containing COLL II (100 mg/mouse) in a volume of 100 ml PBS on
day 0. The patch was secured by adhesive tape wrapped around the
midsection. Positive control mice were patched with PBS alone.
The patch was left in place from day 0 until day 4, when it was
replaced with a fresh patch [15]. On day 7, mice were id immunized
with COLL II in CFA as described above.
To test antigen specicity of skin-induced suppression, mice
were patched with COLL II or non-cross-reacting antigens OVA or
MBP prior to CIA induction.
In some experiments, lymph organs isolated from donors EC
immunized with COLL II were used as a source of suppressor cells.
Phenotype of suppressor cells
To determine the phenotype of EC-induced suppressor cells in
vivo, axillary and inguinal lymph node cells (ALNC) isolated from
mice EC treated with COLL II were incubated in PBS on ice with
puried anti-CD4 or anti-CD8 mAbs (1 mg Ab/106 cells), or with
PBS alone for 45 min. The cells were washed and incubated with a
predetermined dilution of RC for 60 min at 37 8C, and then washed
and resuspended in PBS. Next, cells were counted and cell viability
was assessed by trypan blue exclusion. 2.5  107 of EC-induced
suppressor cells treated with RC alone (suppression control), or
2.5  107 of cell aliquots of EC-induced suppressor cells treated
with appropriate mAb and RC were transferred intravenously (iv)
into naive recipients that underwent CIA induction. Mice were
observed daily and scored for arthritis as described above.

K. Marcinska et al. / Pharmacological Reports 68 (2016) 483489

485

Fig. 1. EC immunization with COLL II prior to CIA induction alleviates disease severity. (a) Scheme of EC immunization and CIA induction. Mice were EC treated with PBS or
COLL II (100 mg/mouse) on days 0 and 4. On day 7, mice were id immunized with COLL II in CFA and boosted with 100 mg COLL II in IFA on day 21 after the rst immunization.
To accelerate the development of arthritis, 40 mg of LPS in sterile PBS was intraperitoneally (ip) injected on day 28. (b) Mice were patched with PBS (Group A) or COLL II (Group
B) prior to CIA induction. Disease severity was evaluated macroscopically. Each group contained seven to nine mice. Data are presented as the mean arthritis score in each
group  SE. Statistical signicance between groups emerged after day 28: p < 0.01 for Group B vs. Group A.

Staining of lymphoid cells and ow cytometry analysis

Statistical analysis

ALNC isolated from mice EC treated with PBS or COLL II were

washed and re-suspended in 100 ml of PBS plus FBS. The cells were
pre-incubated with 0.5 mg of anti-mouse CD16/CD32 mAb for
15 min at 4 8C followed by incubation with appropriate uorochrome conjugated mAb (30 min, 4 8C). After wash, the cells were
resuspended in 100 ml of 1% paraformaldehyde prior to FACS
analysis. For FoxP3 staining, after wash, ALNC were xed with
xation and permeabilization buffer (eBioscience) followed by
incubation with anti-mouse/rat FoxP3-PE antibody at 4 8C for
30 min. The cells were re-suspended in 100 ml of 1% paraformaldehyde prior to FACS analysis. The monoclonal antibodies used in
this study were from BioLegend (anti-TCRb-APC-Cy7, anti-PD-1PerCP-Cy5.5 and anti-CD4-PerCP-Cy5.5), eBioscience (anti- CD16/
CD32, anti-FoxP3-PE and anti-DX5-FITC), and from BD Biosciences
(anti-CD4-PE-Cy7, anti-CD8-FITC, anti-CD25-APC and anti-TCRbFITC).

Data in graphs are shown as mean  SE. Statistical signicance

was estimated by Mann-Whitney test, Kruskal-Wallis test and
unpaired ttest. Statistical signicance was set at p < 0.05.
Results
Epicutaneous application of COLL II prior to CIA induction
inhibits joint inammation
DBA/1 mice were patched with 100 mg of COLL II or treated with
PBS alone as described in Materials and methods (Fig. 1a). On day 7,
patches were removed, the mice were id immunized with COLL II
and CFA on day 0, then boosted with COLL II in IFA on day 21 and
nally treated ip with LPS. Data presented in Fig. 1b show that EC
immunization with COLL II prior to induction of CIA reduces
disease severity (Group B vs. Group A). Macroscopic disease
evaluation was conrmed by histology (Fig. 2).

Fig. 2. Microscopic evaluation of inammatory response in the joints. (a) The histological activity index (HAI). (b,c) Joint sections stained with H&E. Histology showing
granulation tissue and inammatory inltration (A), synovial hyperplasia (C), pannus formation (D) and cartilage and bone erosion (F).

K. Marcinska et al. / Pharmacological Reports 68 (2016) 483489

486

Fig. 3. Antigen specicity of skin-induced suppression. Mice were EC treated with PBS (Group A) or COLL II (Group B) or OVA (Group C) or MBP (Group D) prior to CIA
induction. Disease severity was evaluated macroscopically. Each group contained seven to nine mice. Data are presented as the mean arthritis score in each group  SE.
Statistical signicance between groups emerged after day 28: p < 0.05 for Groups B, C and D vs. Group A.

Skin-induced suppression is antigen non-specic

EC immunization with COLL II induces CD4+ CD8+ RORgt+ cells

Our previous work on skin-induced suppression showed that

this phenomenon was antigen-non-specic both in Th1 and Tc1
CHS responses and an animal model of multiple sclerosis
[6,9,11]. In our current work, we tested the antigen specicity
of EC-induced suppression of CIA. Data presented in Fig. 3 show
that EC immunization with three non-cross-reacting antigens
COLL II, OVA and MBP prior to CIA induction inhibits disease
severity (Groups B, C and D vs. Group A respectively).

Our previous work showed that EC immunization with COLL II

induces TCRab+ suppressor cells that inhibit CIA development and
also reduce its severity [15]. To characterize the skin-induced
suppressor cells we employed two approaches, ow cytometry and
adoptive cell transfer. Data presented in Fig. 4 show that EC
immunization with COLL II does not affect the percentage of
TCRab+ CD4+ CD25+FoxP3+, NKT, TCRab+ CD4+ PD-1+ and TCRab+
CD8+ PD-1+ cells (Fig. 4ad, Group B vs. Group A). To determine the

B
DX5+

CD25+FoxP3+
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15

Fig. 4. Expression of surface and intracellular markers by EC-induced suppressor cells. DBA/1 mice were EC treated with PBS or COLL II. On day +7, ALNC were isolated and
stained with proper monoclonal antibodies prior to FACS analysis. (a) Treg cell identication. ALNC were stained for surface markers with anti-CD4-PerCP-Cy5.5, anti-TCRbFITC and anti-CD25-APC mAbs. Samples were then intracellulary stained with anti-FoxP3-PE mAb using a mouse Treg staining kit prior to analysis. (b d) Surface markers
expression. ALNC were stained with anti-DX5-FITC and anti-TCRb-APC-Cy7 mAbs or with anti-TCRb-APC-Cy7, anti-PD-1-PerCP-Cy5.5, anti-CD4-PE-Cy7 and anti-CD8- FITC
mAbs prior to analysis. Statistical signicance: p = NS for Group B vs. Group A.

K. Marcinska et al. / Pharmacological Reports 68 (2016) 483489

involvement of CD4 and CD8 cells in skin-induced suppression,

ALNC were treated with anti-CD4 or anti-CD8 mAbs and RC or
complement alone prior to adoptive transfer into recipient mice
that underwent CIA induction. Data presented in Fig. 5a show that
depletion of either CD4 or CD8 cells abrogates skin-induced
suppression (Groups C and D vs. Group B). Our previous work on
skin-induced suppression in animal model of CHS and EAE
showed that patching with protein antigen induces CD4+ CD8+
double positive suppressor T cells [6,11]. To test if EC immunization with COLL II induces such cell population, we performed
another experiment. Data presented in Fig. 5b show that mixing
CD4-depleted cells with CD8-depleted cell population prior to
adoptive transfer does not restore suppressor activity of skininduced suppressor cells (Group E vs. Group B). Additionally, FACS
analysis of ALNC showed that EC immunization with COLL II
increases percentage of CD4+ CD8+ cells when compared with PBS
treated mice (Fig. 6a; Group B vs. Group A). Our previous work
showed that IL-17A is involved in skin-induced suppression in CIA
model [15]. Moreover, we found increased percentage of TCRab+
RORgt+ lymphocytes in ALNs. Thus, we decided to evaluate
percentage of CD4+ CD8+ RORgt+ cells in ALNs isolated from mice
patched with COLL II. Data presented in Fig. 6b and c show that EC
immunization with COLL II increases percentage of CD4+ CD8+
RORgt+ cells when compared with PBS patched mice (Group B
vs. Group A).
Discussion
We have previously shown that EC immunization with protein
antigen induces an antigen non-specic suppression of either Th1
or Tc1-mediated CHS responses [6,7,9], alleviates inammatory
response in EAE [18] and TNBS-induced colitis [14]. Additionally,
our work employing allogeneic skin grafts have shown that EC
immunization with an uninvolved protein antigen delays graft
rejection in an antigen non-specic manner [13]. Finally, our
clinical studies have demonstrated for the rst time the effectiveness of skin-induced suppression in a group of multiple sclerosis
patients [17,18].
The current work shows that patching with COLL II prior to CIA
induction alleviates disease severity. The macroscopic disease
evaluation correlates with histological changes. To determine
antigen specicity of EC-induced suppression, mice were EC
immunized with three non-cross-reacting antigens COLL II, OVA
and MBP prior to CIA induction with COLL. Data presented in Fig. 3

487

show that EC immunization with any of tested non-cross-reacting

protein antigens prior to CIA induction inhibits disease severity.
This nding is in line with our previous studies both in CHS and
EAE [6,7,11] showing lack of antigen specicity of skin-induced
suppression.
In classic T cell-mediated suppression both suppressor and
effector cells are specic for the same antigen, i.e. the suppression
is antigen specic and limited to that antigen. The non-specic
tolerance observed in our model possibly is similar to the
phenomenon known as bystander suppression or determinant
spreading [19] in which suppressor cells elicited by a particular
antigen secrete antigen non-specic cytokines that inhibit the
immune responses of other antigen-specic T cells. In the model of
mucosal tolerance, orally introduced antigen appears to interact
with gut associated lymphoid tissue to generate T suppressor cells
that produce anti-inammatory cytokines TGF-b, IL-4 and IL-10
[20], and mediate bystander suppression [19]. It was previously
shown that oral tolerance is dependent on antigen-specic
activation of the T suppressor cells. Once antigen-specic
triggering occurs, suppression is mediated by non-specic
suppressive cytokines produced by the T suppressor cells
[21]. In the case of skin-induced suppression of inammatory
response in CIA, immune response to COLL II was inhibited by all
tested antigens applied epicutaneously. Thus, the presence of the
antigen responsible for inducing T suppressor cells is not required
during elicitation of the T suppressor effect [22].
This might suggest that skin-induced suppressor cells work
either via different mechanisms that are involved in bystander
suppression in oral tolerance, or an antigen deposited on the skin
persists for a long period of time in peripheral lymphoid organs.
Our previous work shows that skin-induced suppression in CIA
model does not induce IL-4, IL-10, IL-17E and TGF-b that
potentially could be involved in suppression of T-cell-mediated
immune response [15]. Unexpectedly, patching with COLL IIinduced release of IL-17A. In vivo treatment of skin-tolerized mice
with anti-IL-17A antibodies abolished protection form CIA
[15]. These results suggest that investigated suppression is
different from mucosal tolerance as it does not involve IL-4,
IL-10 and TGF-b but requires the presence of IL-17A.
Our nding is in contrast with previous reports showing that
IL-17, secreted by a variety of immune and nonimmune cells
including CD4+ Th17 T cells is an important contributor to the
pathogenesis of several autoimmune diseases [23]. Several studies
in patients and animal models showed the implication of IL-17 in

Fig. 5. Phenotype of skin-induced suppressor cells. (a) Recipient mice received all ALNC (Group B) or CD4-depleted (Group C) or CD8-depleted (Group D) ALNC isolated from
donors patched with COLL II. In positive control mice did not receive any cell transfer (Group A). Then, all experimental groups underwent CIA induction followed by
macroscopic evaluation of the disease. Each group contained seven to nine mice. Data are presented as the mean arthritis score in each group  SE. Statistical signicance
between groups emerged after day 28: p < 0.05 for Group B vs. Group A. (b) Recipients were injected with all ALNC (Group B) or CD4-depleted (Group C) or CD8-depleted (Group D)
ALNC or CD4-depleted ALNC reconstituted with CD8-depleted ALNC (Group E) isolated from donors patched with COLL II. The positive control group did not receive any cell transfer
(Group A). Then, all experimental groups underwent CIA induction followed by macroscopic evaluation of the disease. Each group contained seven to nine mice. Data are presented
as the mean arthritis score in each group  SE. Statistical signicance between groups emerged after day 28: p < 0.001 for Group B vs. Group A.

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K. Marcinska et al. / Pharmacological Reports 68 (2016) 483489

Fig. 6. EC immunization with COLL II increases percentage of CD4+ CD8+ RORgt+ cells.
DBA/1 mice were EC treated with PBS or COLL II. On day +7 ALNC were isolated and stained with proper monoclonal antibodies prior to FACS analysis. (a) Expression of CD4
and CD8 coreceptors. ALNC were stained with anti-CD4-PE-Cy7 and anti-CD8-FITC mAbs prior to FACS analysis. (b, c) RORgt expression. ALNC were stained with anti-TCRbAPC-Cy7, anti-CD4-PE-Cy7 and anti-CD8- FITC mAbs. After xation and permeabilization, ALNC were incubated with anti-ROR-gt-PE mAb prior to analysis. Statistical
signicance: p < 0.05 for Group B vs. Group A.

RA. Additionally, the induction of CIA was clearly suppressed in

IL-17-decient mice [24] and spontaneous arthritis no longer
developed in mice decient in both IL-1 receptor antagonist and IL17 [25]. However, our nding is in line with data of Geha et al. [26]
who have shown that EC immunization with OVA induces a Th17
response. It has been also reported that IL-17A can act in some
cases as an anti-inammatory agent that ameliorates DSS-induced
colitis [27] and suppresses inammation in a murine model of
asthma [28]. More recently, a population of IL-17A-producing
CD161+ FoxP3+ Treg cell population has been characterized
[29]. These cells are highly enriched within the inammatory
environment of childhood arthritis [29]. This observation has been
conrmed by other groups showing the existence of IL-17Aproducing CD4+ CD25hi CD127l8CD161+ CD45RA Treg in humans
[30]. This population of Treg cells is functionally suppressive, has
phenotypic/molecular characteristics to other subpopulations of
Treg cells, and retains suppressive function following IL-17
induction. These CD161+ FoxP3+ Treg cells accumulate in inamed
joints of patients with inammatory arthritis and are the
predominant IL-17-producing Treg cell population at these sites
[30]. These data strongly support our nding that IL-17A may be
involved in the negative regulation of inammatory responses
during CIA.

Additionally, ow cytometry experiments show that EC

immunization with COLL II does not affect the percentage of
TCRab+ CD4+ CD25+ FoxP3+, NKT, TCRab+ CD4+ PD-1+ and TCRab+
CD8+ PD-1+ cell populations.
Finally, adoptive cell transfer of CD4 or CD8-depleted lymphoid
cells suggests involvement of these cells in skin-induced suppression. However, transfer of mixed CD4-depleted cells with CD8depleted cell population did not restore suppressor activity of skininduced suppressor cells suggesting that skin-induced T suppressor cells co-express both CD4 and CD8 coreceptors. These data are
supported by ow cytometry experiment showing that EC
immunization with COLL II increases percentage of CD4+ CD8+
cells in ALNs. This is in line with our previous work in CHS and EAE
models [6,12] showing that skin-induced suppressor cells belong
to the population of TCRab+ CD4+ CD8+ double positive cell
population. As we found that IL-17A is involved in skin-induced
suppression of inammatory response in CIA, we evaluated the
percentage of CD4+ CD8+ RORgt+ cells in ALN isolated from mice
patched with COLL II. This experiment showed that EC immunization with COLL II increases percentage of CD4+ CD8+ RORgt+ cells
when compared with PBS patched mice.
In summary, induction of suppression via EC immunization
with protein antigen can inhibit cell-mediated immune responses

K. Marcinska et al. / Pharmacological Reports 68 (2016) 483489

in an animal model of RA, as we previously have demonstrated in

CHS [610], allogeneic skin graft rejection [13], TNBS-induced
colitis [14] and EAE [11,12]. This method therefore may be useful
for the treatment of a variety of inammatory diseases. Maneuver
of EC immunization with a protein antigen that induces suppressor
cells to inhibit inammatory responses may become an attractive,
noninvasive, needle-free therapeutic method for different clinical
situations.
Conict of interest statement
The authors declare no conict of interest.
Acknowledgments
This work was supported by grant from the National Science
Center 2011/03/B/NZ6/00801 to MS.
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