International Journal of
Molecular Sciences
Article
Regulatory Cell Populations in Relapsing-Remitting
Multiple Sclerosis (RRMS) Patients: Effect of Disease
Activity and Treatment Regimens
Maria Rodi 1,† , Nikolaos Dimisianos 2,† , Anne-Lise de Lastic 1 , Panagiota Sakellaraki 1 ,
George Deraos 3 , John Matsoukas 3 , Panagiotis Papathanasopoulos 2 and Athanasia Mouzaki 1, *
1
2
3
*
†
Division of Hematology, Department of Internal Medicine, Faculty of Medicine, University of Patras,
Patras GR-26500, Greece; marodi_biol@yahoo.gr (M.R.); delastic@gmail.com (A.-L.d.L.);
gsakel@upatras.gr (P.S.)
Department of Neurology, Faculty of Medicine & University Hospital, University of Patras,
Patras GR-26500, Greece; ndimisianos@yahoo.gr (N.D.); papat@upatras.gr (P.P.)
Eldrug S.A., Pharmaceutical Company, Platani, Patras GR-26504, Greece; gderaos@upatras.gr (G.D.);
imats@upatras.gr (J.M.)
Correspondence: mouzaki@upatras.gr; Tel.: +30-2610-969-123
These authors contributed equally to this work.
Academic Editors: Christoph Kleinschnitz and Sven Meuth
Received: 13 July 2016; Accepted: 19 August 2016; Published: 25 August 2016
Abstract: Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS)
of autoimmune etiology that results from an imbalance between CNS-specific T effector cells and
peripheral suppressive mechanisms mediated by regulatory cells (RC). In this research, we collected
blood samples from 83 relapsing remitting MS (RRMS) patients and 45 healthy persons (HC), to assess
the sizes of their RC populations, including CD4+ CD25high Foxp3+ (nTregs), CD3+ CD4+ HLA− G+ ,
CD3+ CD8+ CD28− , CD3+ CD56+ , and CD56bright cells, and how RC are affected by disease activity
(acute phase or remission) and types of treatment (methylprednisolone, interferon, or natalizumab).
In addition, we isolated peripheral blood mononuclear cells (PBMC) and cultured them with
peptides mapping to myelin antigens, to determine RC responsiveness to autoantigens. The results
showed decreased levels of nTregs in patients in the acute phase ± methylprednisolone and
in remission + natalizumab, but HC levels in patients in remission or receiving interferon.
Patients + interferon had the highest levels of CD3+ CD4+ HLA− G+ and CD3+ CD8+ CD28− RC,
and patients in the acute phase + methylprednisolone the lowest. Patients in remission had the
highest levels of CD3+ CD56+ , and patients in remission + natalizumab the highest levels of CD56bright
cells. Only nTregs responded to autoantigens in culture, regardless of disease activity or treatment.
The highest suppressive activity was exhibited by nTregs from patients in remission. In conclusion, in
RRMS disease activity and type of treatment affect different RC populations. nTregs respond to myelin
antigens, indicating that it is possible to restore immunological tolerance through nTreg induction.
Keywords: multiple sclerosis; Tregs; HLA-G; iNKT; NKbright ; methylprednisolone; natalizumab;
interferon; myelin oligodendrocyte glycoprotein; MOG; myelin basic protein; MBP
1. Introduction
Multiple sclerosis (MS) is a chronic autoimmune/inflammatory disease of the central nervous
system (CNS) that results in the demyelination of neurons leading to axonal loss and the accumulation
of disability [1]. MS usually affects young adults aged between 20 and 40 years; women are
affected at least twice as often as men [2]. The course of MS can follow four clinical patterns
that include relapsing remitting MS (RRMS, which accounts for 80%–90% of MS cases at onset),
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secondary progressive MS (SPMS), primary progressive MS (PPMS) and progressive relapsing MS
(PRMS) [3,4]. MS is characterized by effector T-cell (Teff) and macrophage infiltrates that are triggered
by CNS-specific CD4+ T-cells, and autoantibody production [5]. The main autoimmune etiology of
MS consists of activated IFN-γ-producing T helper 1 (Th1) cells, that recognize peptides of the myelin
sheath, including myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte
glycoprotein (MOG) [3], as well as IL-17-producing Th17 cells, a subset of CD4+ T-cells shown to
be involved in the pathogenesis of autoimmune diseases [6]. B-cells also play a significant role
in the pathogenesis of MS not only because they produce autoantibodies, but because they can
stimulate autoreactive CD4+ T-cells directly, through self-antigen presentation and the production of
proinflammatory cytokines [7]. Current theories concerning the pathogenesis of MS involve genetic
and environmental factors, as well as immune dysregulation. Autoreactive T-cells and antibodies are
found in both patients with autoimmune diseases and healthy individuals, so their mere presence is
not enough for the development of an autoimmune response. The breakdown of immune tolerance to
CNS self-antigens in genetically susceptible individuals is considered a key event in the development
of MS [4,5,8].
Experimental reports have demonstrated the important role of regulatory T-cells (Tregs) in CNS
autoimmunity [8–10]. The types of Tregs shown to exert regulatory activities in the CNS include natural
Tregs (nTregs), which are CD4+ T-cells that arise in the thymus and constitutively express the CD25 cell
marker (CD25high ) and the transcription factor forkhead box protein P3 (Foxp3) and inducible Tregs
(iTregs), which can be induced in the periphery during an autoimmune or inflammatory response,
and may or may not express Foxp3 [8–10]. iTregs include T helper 3 (Th3) cells, which originate
from naive T-cells that are either CD4+ or CD8+ and secrete TGF-β, type 1 Tregs (Tr1) cells, which are
derived from CD4+ precursors and secrete IL-10, and CD8+ CD28− cells that render antigen presenting
cells (APC), mainly dendtritic cells (DCs), tolerant through cell-cell contact, can secrete IL-10, TGF-β,
IFN-γ, CCL4, and directly kill CD4+ Teffs and APCs [8–11]. Although Foxp3 is considered a specific
marker of nTregs, activated Teffs and Tr1 cells can transiently express Foxp3 [12,13].
Other, less studied, populations of regulatory cells include CD4+ or CD8+ T-cells that lack Foxp3
expression but express the human leukocyte antigen G (HLA-G) [8,14], natural killer T regulatory cells
(iNKT) and CD56bright NK cells, that suppress autoreactive Teffs through direct cytotoxicity and/or
cytokine secretion [15,16].
Several studies have addressed the issues of numbers and function of Tregs in MS patients,
focusing mainly on CD4+ CD25high T-cells. These studies have found no differences in the frequencies
of Tregs between MS patients and controls or between patients at different disease stages (acute
phase or remission) or with different disease subtypes (clinically isolated syndrome (CIS), RRMS,
SPMS, PPMS), although they found loss of Treg suppressive activity [17–19]. Studies investigating
CD4+ CD25high Foxp3+ Tregs in MS patients showed functional impairment [20–22] linked to reduced
Foxp3 expression [20,22] and reduced frequency of Foxp3+ cells [22], whereas other investigators
found decreased levels during remission, which were restored to normal levels during the acute phase
of the disease [23].
Interferon β (IFN-β) has long been established as the first line immunomodulatory treatment
for patients with RRMS. Although the exact mechanism of action for preventing relapses remains
elusive, several studies have suggested that this could be mediated by restoring the levels and function
of Tregs [24–28]. MS relapses are treated with high-dose, short-term intravenous glucocorticoids
(e.g., methylprednisolone). It has been suggested that glucocorticoids mitigate relapses by restoring
the suppressive function of Tregs [29,30].
Natalizumab is a monoclonal antibody against the α4 subunit of α4β1 (very late antigen-4,
VLA-4) and α4β7 integrins, located on the surface of lymphocytes, that acts by blocking their
binding to their endothelial receptors, vascular cell adhesion molecule-1 (VCAM-1) and mucosal
addressin-cell adhesion molecule-1 (MadCAM-1), respectively. This blocking inhibits the infiltration of
autoreactive T-cells into the CNS through the blood-brain barrier (BBB) and, thus, suppresses CNS
Int. J. Mol. Sci. 2016, 17, 1398
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tissue inflammation [31]. Natalizumab has been approved for the prophylactic treatment of RRMS
patients who have failed to respond to first-line therapies or have severe, breakthrough disease [32,33].
Although natalizumab has been shown to affect immune cell responses in more ways than just
preventing infiltration of T-cells into the CNS, very little is known regarding its effects on Tregs.
In this work we assessed the frequencies of peripheral blood CD4+ CD25+ Foxp3+ (nTregs),
+
CD3 CD4+ HLA− G+ , CD3+ CD56+ (iNKT cells), CD8+ CD28− (CD8+ Tregs) and CD56bright cells in
RRMS patients and healthy controls, to determine the effects of disease activity (acute or stable disease)
and treatment regimens (methylprednisolone, interferon, natalizumab) thereof. In addition, we tested
regulatory cell responses to various antigenic peptides mapping to myelin epitopes, to assess their
function in patients at different states of the disease and under different therapies.
2. Results
2.1. CD4+ CD25high Foxp3+ T-Cells (nTregs) in RRMS
The levels of nTregs in peripheral blood of MS patients and HC, are shown in Figure 1. nTreg levels
were lower in acute-phase patients with no treatment (AP-noRx) or under methylprednisolone
(AP-MP), compared to all other patient groups and HC. nTregs were restored to normal levels in
acute-phase patients under interferon β treatment (AP-IFN), whereas patients in remission under no
treatment (Rem-noRx) showed intermediate levels between HC and AP-noRx. Patients in remission
under natalizumab (Rem-NATA) had nTreg levels comparable to those of AP-noRx and AP-MP patients
(Figure 1D).
2.2. Other Regulatory Cell Populations in RRMS
The levels of the other regulatory cell populations studied in the peripheral blood of MS patients
and HC, are shown in Figures 2–4. The percentage of CD3+ CD4+ HLA− G+ T-cells was higher in AP-IFN
patients compared to all other groups, with the difference reaching statistical significance between
AP-IFN and HC, AP-MP, and Rem-NATA patients (Figure 2).
The CD3+ CD8+ CD28− T-cells (CD8+ Tregs) showed the same pattern, with the AP-IFN patients
having the highest levels of CD8+ Tregs in all groups (Figure 3). The opposite occurred with CD3+ CD56+
cells (iNKT cells), with the AP-IFN patients showing the lowest frequency (Figure 4). The frequency
of CD56bright cells was higher in all patient groups compared to HC, but only the difference between
Rem-NATA patients and HC was statistically significant (Figure 4).
2.3. Effect of Culture with Peptides on Regulatory Cell Function
Peripheral blood mononuclear cells (PBMC) were isolated from RRMS patients (AP-noRx, n = 5;
AP-MP, n = 5; AP-IFN, n = 6; Rem-noRx; n = 5, Rem-NATA, n = 14) and controls (HC, n = 19) and
cultured in the presence or absence of peptides mapping to myelin antigens (see M and M), to assess
which RC populations responded to autoantigens. It was observed that only nTregs responded to the
peptides by proliferation and cytokine secretion, whereas the other RC populations studied did not
respond to the peptides (data not shown).
To delineate the effects of disease state and treatment regimens on nTreg responses, the numbers
of CD4+ CD25− and CD4+ CD25+ cells were measured in cultures ± peptides, to calculate the ratio of
effector to suppressor cells, as well as the percentage of CD4+ CD25+ Foxp3+ nTregs. The net change
of effector/suppressor ratios between cultures ± peptides was also calculated, to assess any shift
towards an effector or suppressor phenotype. Positive changes reflected an effector shift whereas
negative a suppressor shift (Figure 5, left column). These changes were accompanied by corresponding
changes in cytokine ratios, with an increase in anti-inflammatory cytokines when a suppressor shift
was observed (Figure 5, right column).
Effector/suppressor ratios in cultures-peptides showed a reverse trend to that of nTregs in
peripheral blood (Figure 6). The higher ratios were observed in AP-noRx, AP-MP, and Rem-NATA
Int.Int.
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Overall, all groups of RRMS patients displayed a suppressive phenotype during co-cultures
patients,
andorthe
lower
in AP-IFN
patients
(Figureto6A).
observation
wasPEP2
confirmed
by a were
strong
with one
more
antigenic
peptides,
similarly
the This
HC group.
Peptides
and PEP3
+ CD25+ Foxp3+ T-cells (r = −0.64,
inverse
correlation
between
effector/suppressor
ratios
and
CD4
relatively more tolerogenic for HC subjects and PEP1 and PEP3 for RRMS patients (Figure 7).
p < 0.0001)
(Figure
6B).
When
the percentages
of co-cultures with a suppressive change in effector/suppressor ratio
Overall,
all
groups
of
RRMS
patients displayed
suppressive
phenotype
co-cultures
with
were calculated, no statistical differences
betweenapatient
groups
and HC during
were found,
with the
one
or
more
antigenic
peptides,
similarly
to
the
HC
group.
Peptides
PEP2
and
PEP3
were
relatively
exception of the Rem-noRx patient group that showed a more than two-fold higher percentage of
more
tolerogenic
for HC
subjects to
and
PEP3
for RRMS
patients
(Figure
suppressive
cultures
compared
thePEP1
otherand
patient
groups
and HC
(Figure
8). 7).
+
Figure
Flow
cytometricanalysis
analysistotodetermine
determineCD4
CD4++CD25
CD25++Foxp3
Figure
1. 1.
Flow
cytometric
Foxp3+RC
RClevels
levelsininhuman
humanperipheral
peripheral
blood.
A
representative
analysis
is
shown
for
one
healthy
control
(A1–C1)
and
one
relapsing
blood. A representative analysis is shown for one healthy control (A1–C1) and one relapsingremitting
remitting
(RRMS)patient
patient(A2–C2).
(A2–C2). The
The white
white blood
blood cells
MSMS
(RRMS)
cells (WBC)
(WBC) were
were gated
gatedon
onlymphocytes,
lymphocytes,based
basedonon
forward
and
side
light
scatter
(A1,A2)
analyzed
for CD4
and CD25
expression
(B1,B2);
The
forward
and
side
light
scatter
(A1,A2)
andand
analyzed
for CD4
and CD25
expression
(B1,B2);
The double
doublecells
positive
werefurther
analyzed
for Foxp3 (C1,C2);
expression
(C1,C2);
The
in the
dot
positive
were cells
analyzed
for further
Foxp3 expression
The
numbers
in numbers
the dot plots
indicate
plots
indicate
the
percentage
of
gated
cells
expressing
the
relevant
marker.
The
tables
underneath,
the percentage of gated cells expressing the relevant marker. The tables underneath, show the absolute
show the
absolute
number
of cells
in each population
analyzed;
Bottom
The results
of the
number
of cells
in each
population
analyzed;
Bottom graph:
The results
of graph:
the analysis
of all patients
analysis of all patients (n = 83) and controls (HC, n = 45). AP-noRx (n = 13), patients in the acute phase of
(n = 83) and controls (HC, n = 45). AP-noRx (n = 13), patients in the acute phase of the disease without
the disease without treatment; AP-MP (n = 17), patients in the acute phase under treatment with
treatment; AP-MP (n = 17), patients in the acute phase under treatment with methylprednisolone;
methylprednisolone; AP-IFN (n = 12), patients in the acute phase under treatment with interferon β;
AP-IFN (n = 12), patients in the acute phase under treatment with interferon β; Rem-noRx (n = 15),
Rem-noRx (n = 15), patients in remission receiving no treatment; Rem-NATA (n = 26), patients in
patients in remission receiving no treatment; Rem-NATA (n = 26), patients in remission under treatment
remission under treatment with natalizumab. * p = 0.04, ** p = 0.01, *** p = 0.002.
with natalizumab. * p = 0.04, ** p = 0.01, *** p = 0.002.
Int. J. Mol. Sci. 2016, 17, 1398
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+CD4
−G++RC levels in human peripheral
+ CD4
++HLA
Figure
Flow cytometric
cytometric analysis
analysisto
todetermine
determineCD3
CD3
HLA−
Figure 2.
2. Flow
G RC levels in human peripheral
blood.
A
representative
analysis
is
shown
for
one
healthy
control
blood. A representative analysis is shown for one healthy control (A1–D1)
(A1–D1) and
and one
one RRMS
RRMS patient
patient
(A2–D2);
The
WBC
were
gated
on
lymphocytes,
based
on
forward
and
side
light
scatter
(A2–D2); The WBC were gated on lymphocytes, based on forward and side light scatter (A1,A2)
(A1,A2) and
and
analyzed
CD3(B1,B2),
(B1,B2),CD4
CD4(C1,C2)
(C1,C2)and
and
HLA-G
expression
(D1,D2);
numbers
indot
theplots
dot
analyzed for CD3
HLA-G
expression
(D1,D2);
TheThe
numbers
in the
plots
indicate
the percentage
of gated
cells expressing
the relevant
marker.
The tables
underneath
indicate
the percentage
of gated
cells expressing
the relevant
marker.
The tables
underneath
show
show
the absolute
number
each population
analyzed;
graph:
Theof
results
of the
the absolute
number
of cellsofincells
eachinpopulation
analyzed;
BottomBottom
graph: The
results
the analysis
analysis
of
all
patients
(n
=
83)
and
controls
(HC,
n
=
45).
For
abbreviations
see
legend
of
Figure
1.
of all patients (n = 83) and controls (HC, n = 45). For abbreviations see legend of Figure 1. * p = 0.05,
***pp==0.05,
p =p 0.01,
*** p = 0.006.
0.01,*****
= 0.006.
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−
Figure
cytometric analysis
analysis to
to determine
determine CD3
CD3++CD8
CD8++CD28
Figure 3.
3. Flow
Flow cytometric
CD28−RC
RClevels
levelsinin human
human peripheral
peripheral
blood.
blood. AArepresentative
representativeanalysis
analysisisisshown
shownfor
forone
onehealthy
healthycontrol
control(A1–D1)
(A1–D1) and
and one
one RRMS
RRMS patient
patient
(A2–D2);
The
WBC
were
gated
on
lymphocytes,
based
on
forward
and
side
light
scatter
(A1,A2)
(A2–D2); The WBC were gated on lymphocytes, based on forward and side light scatter (A1,A2)and
and
analyzed
analyzedfor
forCD3
CD3(B1,B2),
(B1,B2),CD4
CD4(C1,C2),
(C1,C2), and
andCD28
CD28 (D1,D2)
(D1,D2) expression.
expression. The
Thenumbers
numbersin
inthe
thedot
dotplots
plots
indicate
of gated
gatedcells
cellsexpressing
expressingthe
therelevant
relevant
marker.
The
tables
underneath,
show
indicate the
the percentage
percentage of
marker.
The
tables
underneath,
show
the
the
absolute
number
of cells
in each
population
analyzed;
Bottom
graph:
The results
the analysis
absolute
number
of cells
in each
population
analyzed;
Bottom
graph:
The results
of theofanalysis
of all
of
all patients
(n =and
83)controls
and controls
n =For
45).abbreviations
For abbreviations
see legend
of Figure
p = 0.05.
patients
(n = 83)
(HC, (HC,
n = 45).
see legend
of Figure
1. * p1.= *0.05.
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+CD56+ and CD56bright RC levels in human
Figure 4.4. Flow
Flowcytometric
cytometricanalysis
analysistotodetermine
determineCD3
CD3
+ CD56
+ and CD56bright RC levels in human
Figure
peripheral
blood.
A
representative
analysis
is
shown
for
one
healthy
control(A1–C1)
(A1–C1)and
andone
oneRRMS
RRMS
peripheral blood. A representative analysis is shown for one healthy control
patient
in
the
acute
phase
under
interferon
(AP
-IFN)
treatment
(A2–C2);
The
WBC
were
gated
on
patient in the acute phase under interferon β (AP-IFN) treatment (A2–C2); The WBC were gated
lymphocytes,
based
on
forward
and
side
light
scatter
(A1,A2),
and
analyzed
for
CD3
and
CD56
on lymphocytes, based on forward and side light scatter (A1,A2), and analyzed for CD3 and CD56
expression(B1,B2);
(B1,B2);The
The
CD56
positive
cells further
were further
analyzed
for the of
intensity
of CD56
expression
CD56
positive
cells were
analyzed
for the intensity
CD56 expression
bright
bright ) (CD56
) (C1,C2);
Theinnumbers
in the
dot plots
the of
percentage
ofexpressing
gated cells
expression
(CD56
(C1,C2); The
numbers
the dot plots
indicate
theindicate
percentage
gated cells
expressing
the
relevant
marker.
The
tables
underneath,
show
the
absolute
number
of
cells
in each
the relevant marker. The tables underneath, show the absolute number of cells in each population
population
analyzed;
Bottom
graphs:
The
results
of
the
analysis
of
all
patients
(n
=
83)
and
controls
analyzed; Bottom graphs: The results of the analysis of all patients (n = 83) and controls (HC, n = 45).
+; (Right)
bright. For abbreviations see legend of Figure 1. * p = 0.05.
+ CD56
+ ; (Right)
bright . For
CD56
(HC, n
= 45).
(Left)
CD3+CD56
(Left)
CD3
CD56
abbreviations
see legend of Figure 1. * p = 0.05.
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+ CD25
+ /CD4
+ CD25
−)
+CD25
+/CD4
+CD25
−) after
Figure 5. Left
ratios
(CD4
Leftcolumn:
column:Net
Net%%changes
changesofofregulatory/effector
regulatory/effector
ratios
(CD4
72
h culture
of PBMC
MS patients
HC with
antigenic
peptides
PEP1-4
or cP7;PEP1-4
Right column:
after
72 h culture
of of
PBMC
of MSand
patients
andthe
HC
with the
antigenic
peptides
or cP7;
Net
% column:
changes Net
in the
corresponding
anti-inflammatory/inflammatory
cytokine ratios ([IL-4
+ IL-10]/
Right
% changes
in the corresponding
anti-inflammatory/inflammatory
cytokine
ratios
[IFN-γ
TNF-α + IL-17A])
in the+culture
supernatants.
([IL-4 ++IL-10]/[IFN-γ
+ TNF-α
IL-17A])
in the culture supernatants.
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Figure 6. (A) Effector/suppressor ratios of HC and patient groups after cultures of PBMC in the
Figure 6. (A) Effector/suppressor ratios of HC and patient groups +after high
cultures+ of PBMC in the
absence of antigenic peptides. Note the complementarity with the CD4 CD25
Foxp3 frequencies
+ CD25high Foxp3+ frequencies
absence
of
antigenic
peptides.
Note
the
complementarity
with
the
CD4
Figure
6. 1.(A)
ratios of
HC and patient groups
cultures
of the
PBMC
in the
in
Figure
* pEffector/suppressor
= 0.05; and (B) correlation
of effector/suppressor
ratiosafter
in culture,
with
percentage
+CD25
highFoxp3
+ frequencies
in Figure
1. * pof=antigenic
0.05; CD4
and
(B) correlation
of
effector/suppressor
in culture,
with
the percentage
+CD25
highNote
+ Tregs
absence
peptides.
complementarity
thepratios
CD4
of
corresponding
Foxp3the
(Spearman r =with
−0.64,
< 0.0001).
+ CD25
in Figure 1. * CD4
p = 0.05;
and high
(B) correlation
of effector/suppressor
ratios
with the percentage
of corresponding
Foxp3+ Tregs
(Spearman r = −
0.64,inpculture,
< 0.0001).
of corresponding CD4+CD25highFoxp3+ Tregs (Spearman r = −0.64, p < 0.0001).
Figure 6. (A) Effector/suppressor ratios of HC and patient groups after cultures of PBMC in the
absence of antigenic peptides. Note the complementarity with the CD4+CD25highFoxp3+ frequencies
in Figure 1. * p = 0.05; and (B) correlation of effector/suppressor ratios in culture, with the percentage
of corresponding CD4+CD25highFoxp3+ Tregs (Spearman r = −0.64, p < 0.0001).
Figure 7. Mean changes in effector/suppressor ratios in HC (A) and RRMS patients (B), after culture
with the antigenic peptides (PEP1-4, cP7) (whiskers are min to max).
7. Mean
changes
in effector/suppressor ratios
(A)(A)
andand
RRMS
patients
(B), after
FigureFigure
7. Mean
changes
in effector/suppressor
ratiosininHC
HC
RRMS
patients
(B),culture
after culture
with the antigenic peptides (PEP1-4, cP7) (whiskers are min to max).
with the antigenic peptides (PEP1-4, cP7) (whiskers are min to max).
When the percentages of co-cultures with a suppressive change in effector/suppressor ratio were
calculated, no statistical differences between patient groups and HC were found, with the exception
Figure 7. Mean changes in effector/suppressor ratios in HC (A) and RRMS patients (B), after culture
of the Rem-noRx patient group that showed a more than two-fold higher percentage of suppressive
with the antigenic peptides (PEP1-4, cP7) (whiskers are min to max).
cultures compared to the other patient groups and HC (Figure 8).
Figure 8. Percentage of cultures showing a suppressive shift in HC and patient groups, after culture
with the antigenic peptides (PEP1-4, cP7). Note the significant increase in Rem-noRx patients.
Figure 8. Percentage of cultures showing a suppressive shift in HC and patient groups, after culture
with the antigenic peptides (PEP1-4, cP7). Note the significant increase in Rem-noRx patients.
8. Percentage
of cultures
showingaasuppressive
suppressive shift
in HC
and and
patient
groups,groups,
after culture
Figure 8.Figure
Percentage
of cultures
showing
shift
in HC
patient
after culture
with the antigenic peptides (PEP1-4, cP7). Note the significant increase in Rem-noRx patients.
with the antigenic peptides (PEP1-4, cP7). Note the significant increase in Rem-noRx patients.
Int. J. Mol. Sci. 2016, 17, 1398
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3. Discussion
The role of Tregs in the pathogenesis of MS and other autoimmune diseases, is investigated by
many groups, mainly because of their putative therapeutic potential [8–10]. In earlier years, the only
marker used to differentiate Tregs from effector CD4+ T-cells was the constitutive expression of CD25
(IL-2Rα chain) [34], although it was already recognized that the pool of CD4+ CD25+ Tregs could
include recently activated Teffs. However, CD4+ T-cells that express high levels of CD25 (CD25high )
exhibit the highest suppressive activity and represent a small percentage (2%–4%) of CD4+ T-cells [34].
Earlier studies of CD4+ CD25high Tregs in MS, showed similar frequencies of these cells in the peripheral
blood between MS patients and healthy controls [17–19,28], but reported reduced suppressive activity
using various functional assays [18,19,28]. In the CSF, compared to blood, CD4+ CD25high Tregs were
reported to be similar [19], or elevated [28] in MS patients. Subsequent studies used additional markers
to characterize nTregs from the CD4+ CD25high pool, the most important of which was the expression
of Foxp3. Foxp3 is considered a specific marker for nTregs, although Teffs and Tr1 cells can transiently
express Foxp3 upon activation [13], through STAT-5 signaling cytokines [12].
Other markers used for the characterization of Tregs include CD39 (an ectoenzyme that
degrades ATP to AMP), CTLA-4 (Cytotoxic T-lymphocyte antigen 4, a CD28-family receptor)
and GITR (Glucocorticoid-induced tumor necrosis factor receptor, a member of the TNF receptor
superfamily) [23,35]. Huan et al. [20] reported identical levels of CD4+ CD25+ Tregs in RRMS patients
and HC, but reduced expression of Foxp3 in RRMS patients. Reduced levels of CD4+ CD25high Foxp3+
Tregs in the blood of RRMS (but not SPMS) patients, with an associated increase of Tregs in the CSF,
were also observed by Venken et al. [22]. Michel et al. [36] reported that the CD4+ CD25high pool of
T-cells was depleted of cells expressing IL-7 receptor α-chain (CD127), a marker present on activated
T-cells but not on Tregs. CD4+ CD25high CD127low Tregs exhibited similar suppressive functions in
RRMS patients and HC. Dalla Libera et al. [23] reported decreased numbers of Tregs (defined by CD25,
Foxp3, CD39, CTLA-4, and GITR expression) in RRMS patients during remission, which were restored
to normal levels in the acute phase, concluding that Tregs are not involved in causing clinical attacks,
but retain functionality and are increased during acute phase to restore homeostasis. Bjerg et al. [37]
studied RRMS patients in remission and found that CD4+ CD25high CD127low Foxp3+ Tregs were higher
than HC in the group of patients with less severe disease (lower EDSS score and shorter disease
duration) and lower in patients with more severe disease.
Other studies addressed the issue of the effect of immunomodulating and immunosuppressive
treatments on Tregs. One study with IFNβ-1α-treated MS patients, showed increased numbers
of CD4+ CD25high Tregs (CTLA-4+ and GITR+ ) and functional enhancement after six months of
treatment [38]. Other studies with RRMS patients treated with IFNβ (β-1α or β-1β), found improved
frequency and function of CD4+ CD25+ Foxp3+ Tregs [27,28]. Natalizumab treatment was reported to
have no effect on CD4+ CD25high Foxp3+ Tregs frequency or activity [21,39], with the exception of one
case study that reported increased numbers and restored function of nTregs in a patient treated with
natalizumab after relapsing following stem cell transplantation [40]. Treatment with glucocorticoids
after an acute attack was reported to induce an increase in CD4+ CD25high [29] and Foxp3+ Treg
frequency and function [30].
The marked diversity in the observations between various studies regarding population sizes,
disease characteristics, comparators, Treg identification markers, and methodology used for Treg
isolation and functional assays, make a direct comparison of the results very difficult. Our results
showed decreased levels of CD4+ CD25high Foxp3+ Tregs in patients in the acute phase (except when
under IFNβ treatment) and an up-regulation during remission (except when under natalizumab).
This is in contrast to the observations by Dalla Libera et al. [23] who reported lower levels during stable
disease and restoration to control levels during acute clinical attacks. However, the authors did not
clarify how many of their patients were under prophylactic treatment, if any, and they also used mRNA
expression of Foxp3 and other Treg markers within the CD4+ T-cells, which are also expressed by
activated Teffs and Tr1 cells [13]. Haas et al. [19] found decreased suppressive activity of CD4+ CD25high
Int. J. Mol. Sci. 2016, 17, 1398
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Tregs in their RRMS patients in the acute phase. In a minor subset of patients entering remission,
suppressive function of Tregs displayed an increase, though not statistically significant. The majority of
the studies included patients in remission, so there was no acute phase arm to compare [17,18,20,22,37].
Reduced frequency of nTregs during the acute phase can be explained by their migration to sites of
inflammation in the CNS, as previously suggested [22,41,42].
Our finding of increased frequency of nTregs under IFNβ treatment is consistent, since most
studies verify it [22,24,26–28,38,43]. The fact that our patients were in the acute phase of the disease
indicates that IFNβ treatment per se increases nTreg numbers regardless of disease activity. In addition,
the effector/suppressor cell ratio of the patients under IFNβ treatment was similar to the HC group
(cf. Figure 6) and their nTregs retained their function (cf. Figure 8). Nevertheless, these events were
not by themselves sufficient to protect these patients from relapsing, perhaps because higher numbers
of nTregs and/or nTregs with higher suppressive potential are needed, compared to HC, to ameliorate
the disease by counteracting the activity of the autoreactive clones.
We also found that treatment with natalizumab does not restore the frequency of nTregs, a finding
corroborated by others [21,39]. Patients in remission under natalizumab had also a significantly higher
effector/suppressor cell ratio compared to HC (cf. Figure 6), probably reflecting the inhibition of Teff
infiltration into the CNS through the BBB, although their nTregs retained their function (cf. Figure 8).
It appears that in this group of patients peripheral blood nTreg levels, per se, are not reflective of
disease activity and remission is due to natalizumab activity [31] and, perhaps, the effect of other RC
populations that were elevated (see below).
Regarding the effect of glucocorticoids (methylprednisolone), we did not find any increase in
nTregs compared to untreated acute phase patients, as indicated by others [29,30]. The reduced
frequency of nTregs in patients in the acute phase under methylprednisolone treatment is, perhaps,
due to nTreg migration to sites of inflammation in the CNS [22,41,42] that is reversed when the patients
enter remission. Again, in these patients nTregs retained their function (cf. Figure 8).
In our study we also examined the frequencies of other RC phenotypes, which may play a role
in regulating autoimmune diseases. HLA− G+ and CD8+ Tregs were elevated mainly in the AP-IFN
group, whereas iNKT cells were markedly down-regulated in the same group. HLA− G+ Tregs have
been recently identified as a novel subset of naturally, thymus-derived regulatory T-cells, found in
the blood and sites of inflammation to modulate inflammatory responses [14,44]. CD8+ Tregs are also
important in immune regulation and treatment with glatiramer acetate, another first-line therapy for
RRMS, has been shown to enhance their function [45]. Natalizumab-treated patients in our study,
while having lower levels of Foxp3+ Tregs, showed higher levels of HLA− G+ and CD8+ Tregs, as well
as CD56bright NK-cells, a subset of NK-cells with suppressive activity [46], the expansion of which is
proposed as a mechanism of action of a new therapy for RRMS with a monoclonal antibody to CD25
(daclizumab) [47].
Our results from the functional assays showed that only nTregs, isolated from RRMS patients
and HC, exhibited suppressive activity when cultured with various MOG or MBP peptides. This does
not mean that the other RC populations studied in this work are not functional, merely that they do
not respond to this type of antigenic stimulation. Under these culture conditions, nTregs from RRMS
patients exhibited suppressive activity equal to nTregs isolated from HC or, as in the case of nTregs
from patients in remission without therapy, higher than in HC. These results indicate that the reduction
in nTreg suppressive activity reported in earlier studies [18,19] can be attributed to the type of stimulus
used for the functional assays, and that it is possible to restore immunological tolerance through nTreg
induction when suitable antigenic stimuli are employed.
Int. J. Mol. Sci. 2016, 17, 1398
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4. Materials and Methods
4.1. Study Subjects
Eighty three patients with clinically definite MS of the relapsing-remitting type (RRMS), according
to the 2005 revised McDonald’s criteria [48], were included in the study. Historical data and neurologic
examination were used to assess their disease course, current status, disease duration, and level of
disability, according to the expanded disability status scale-EDSS [49]. The demographic and clinical
characteristics of MS patients are presented in Table 1. The patients were divided into five groups
based on the disease status (acute phase, AP, or remission, Rem) and the type of treatment they
were receiving at the time their blood samples were drawn (no treatment, noRx; methylprednisolone,
MP; interferon β, IFN or natalizumab, NATA). The groups were: (a) patients in the acute phase
of the disease who were treatment-naive (AP-noRx, n = 13); (b) patients in the acute phase under
treatment with methylprednisolone (AP-MP, n = 17); (c) patients in the acute phase under treatment
with interferon β (AP-IFN, n = 12); (d) patients in remission receiving no treatment (Rem-noRx,
n = 15); and (e) patients in remission under treatment with natalizumab (Rem-NATA, n = 26). To note,
in patients with clinically definite RRMS the acute phase is defined as a clinical relapse, i.e., a new onset
of a neurological dysfunction of the kind seen in MS that can be either a subjective report (symptom)
or an objective observation (sign). Usually it is both, since a new symptom indicates a dysfunction that
can be identified by the neurologist. The duration of the new symptom or sign has to be longer than
24 h, and usually lasts for days or weeks. It is not necessary to perform an MRI to verify the relapse;
it is, per se, a clinical event.
Forty five age- and sex-matched, healthy individuals (Table 1), with no history of neurological or
autoimmune disease and no concomitant signs or symptoms of infection or inflammation, served as
a healthy control group (HC). All subjects signed an informed consent form before blood collection.
The study protocol was approved by the Scientific Review Board and Ethics Committee of Patras
University Hospital (Reg# 451/17.10.08, 17 October 2008). The Hospital abides by the Helsinki
declaration on ethical principles for medical research involving human subjects.
Table 1. Demographic and clinical characteristics of MS patients and controls.
Group
N (M/F)
Age (Years)
Disease Duration (Years)
EDSS
HC
RRMS patients
AP-noRx
AP-MP
AP-IFN
Rem-noRx
Rem-NATA
45 (19/26)
83 (33/50)
13 (4/9)
17 (8/9)
12 (4/8)
15 (6/9)
26 (11/15)
36.31 ± 9.26
37.53 ± 10.65
35.54 ± 9.46
32.82 ± 10.32
33.75 ± 9.03
42.47 ± 13.44
39.35 ± 8.71
NA
4.31 ± 3.80
1.76 ± 2.66
1.13 ± 1.55
3.62 ± 1.79
5.75 ± 3.86
6.93 ± 3.24
NA
2.41 ± 1.89
1.61 ± 1.74
1.55 ± 1.23
2.83 ± 1.55
2.36 ± 2.09
3.34 ± 1.82
Data are given as mean ± SD. HC, healthy controls; AP, patients in the acute phase of the disease; Rx, treatment;
Rem, patients in remission; MP, methylprednisolone; IFN, interferon β; NATA, natalizumab; NA, not applicable;
N, number of subjects; M, male; F, female; EDSS, expanded disability status scale.
4.2. Cells and Cultures
Whole blood samples (10 mL) were collected from MS patients and HC in heparinized BD
Vacutainers (BD, Plymouth, UK). Peripheral blood mononuclear cells (PBMC) were isolated by ficoll
(BIOCHROM AG, Berlin, Germany) density gradient centrifugation. The cells were cultured for 72 h
in RPMI 1640 medium (GIBCO BRL, Gaithersburg, MD, USA), containing 10% fetal bovine serum and
1% penicillin/streptomycin at a concentration of 106 cells/mL, in the presence or absence of peptides
PEP1-4 or cP7 (see Section 4.3), at a concentration of 10 pg/mL/106 cells. At the end of the culture
period, PBMC were harvested and the culture supernatants were collected for the determination
of cytokine concentrations (see Section 4.5). PBMC were washed with PBS and their numbers and
phenotypes were determined by flow cytometry.
Int. J. Mol. Sci. 2016, 17, 1398
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4.3. Peptides Mapping to MOG and MBP Myelin Antigens
The peptides used for the functional assays are shown in Table 2. Peptides PEP1-4 are MOG35–55
antigenic epitopes prepared as described [50]. Peptide cP7 is an MBP87–99 antigenic epitope prepared
as described [51].
Table 2. Peptides used for the functional assays.
Peptide
Description
PEP1
Rat MOG35–55 epitope conjugated with
oxidized mannan
PEP2
Rat MOG35–55 epitope conjugated with
reduced mannan
PEP3
Human MOG35–55 epitope conjugated with
oxidized mannan
PEP4
Human MOG35–55 epitope conjugated with
reduced mannan
cP7
Citrullinated human MBP87–99 epitope:
Cyclo(87–99) [Cit91 , Ala96 , Cit97 ] MBP87–99
Sequence
H–Met35 –Glu–Val–Gly–Trp–Tyr–Arg–Ser–Pro–Phe–
Ser–Arg–Val–Val–His–Leu–Tyr–Arg–Asn–Gly–Lys55 –OH
H–Met35 –Glu–Val–Gly–Trp–Tyr–Arg–Pro–Pro–Phe–
Ser–Arg–Val–Val–His–Leu–Tyr–Arg–Asn–Gly–Lys55 –OH
Cyclo(87–99) Val–His–Phe–Phe–Cit91 –Asn–Ile–Val–
Thr–Ala96 –Cit97 –Thr–Pro
4.4. Flow Cytometry
Whole blood cells or PBMC were labeled using the following monoclonal antibodies: CD3-PC5
(UCHT1, Beckman Coulter-BC, Paris, France), CD4-FITC (13B8.2, BC), CD8-FITC (B9.11, BC), CD28-PE
(B-T3, Abcam, Cambridge, UK), CD25-PE (M-A251, Becton Dickinson-BD Biosciences-Pharmigen,
San Diego, CA, USA), CD56-PE (N901, BC), HLA-G-PE (MEM-G/9, Abcam), and PE-Cy5 Conjugated
anti-human Foxp3 (PCH101, eBiosciences, San Diego, CA, USA). All procedures were performed
according to the manufacturers’ instructions. For intracellular staining of Foxp3, the cells were labeled
with CD4-FITC and CD25-PE antibodies and were stained intracellularly for Foxp3, after fixation and
permeabilization. Whole blood samples were treated with BD Pharm Lyse buffer 1× (BD) for 15 min,
after staining, to lyse red blood cells.
Flow cytometry was performed on an EPICS Coulter XL-MCL Flow Cytometer (BC). At least
20,000 events were acquired for extracellular and 100,000 events for intracellular staining. Data analysis
was performed using the FlowJo V7.5 software (Tree Star Inc., Ashland, OR, USA).
4.5. Cytokines Measured in Culture Supernatants
Both inflammatory (IFN-γ, TNF-α, and IL-17A) and anti-inflammatory (IL-4 and IL-10)
cytokines were measured in the supernatants of PBMC cultured with or without peptides.
Measurement of cytokine levels was performed on a BD FACSArray Bioanalyzer using the cytometric
bead array (CBA) assay (human Th1/Th2/Th17 Cytokine Kit, BD Biosciences). The ratio of
anti-inflammatory/inflammatory cytokines was calculated in all cultures and the net change was
determined to assess the shift towards a suppressor or effector phenotype after culture with
each peptide.
4.6. Statistical Analysis
Comparisons between different groups of patients and controls were performed using the
one-way analysis of variance (ANOVA) or the Kruskal-Wallis test, depending on the normality of the
distribution of values. When the null hypothesis of the Kruskal-Wallis or ANOVA test was rejected,
the Mann-Whitney test with Bonferroni correction was employed for the pairwise comparisons of the
groups. The p values were calculated two-tailed and in all cases considered statistically significant if
p was ≤0.05. Data were analyzed using the GraphPad Prism v.5.03 (San Diego, CA, USA).
Int. J. Mol. Sci. 2016, 17, 1398
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5. Conclusions
In conclusion, in RRMS patients, different regulatory cell populations show a varied distribution
according to the phase of the disease and treatment regimens. nTreg levels were lower in the
patients in the acute phase of the disease before therapy or under methylprednisolone treatment,
but were restored to HC-levels in patients in the acute phase receiving interferon. Patients in
remission receiving no treatment had HC-levels of nTregs, whereas under natalizumab-treatment
their nTreg levels were decreased to acute phase levels. Patients receiving interferon had the
highest levels of CD3+ CD4+ HLA− G+ and CD8+ Tregs, whereas patients in the acute phase receiving
methylprednisolone the lowest. Patients in remission had the highest levels of iNKT cells and patients
in remission under natalizumab treatment the highest levels of CD56bright cells. nTregs, but not the
other regulatory cells studied, exhibited suppressive activity when cultured with at least one myelin
peptide antigen, i.e., they retained their function when they recognized their cognate antigen.
Acknowledgments: This work was supported by the Greek General Secretariat of Research and Technology
“Cooperation” grant 09SYN-21-609 (O.P. Competitiveness & Entrepreneurship, EPAN II) to Athanasia Mouzaki
and John Matsoukas, by a Du Pre’ grant from Multiple Sclerosis International Federation (MSIF) to George Deraos
and an unconditional grant by Genesis Pharma to Nikolaos Dimisianos.
Author Contributions: Nikolaos Dimisianos and Panagiotis Papathanasopoulos were responsible for patient
selection and sample collection; Maria Rodi and Panagiota Sakellaraki performed the assays; George Deraos
and John Matsoukas provided the peptides; Maria Rodi, Nikolaos Dimisianos , Anne-Lise de Lastic and
Athanasia Mouzaki analyzed the data; Athanasia Mouzaki, Nikolaos Dimisianos and Maria Rodi wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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