RESEARCH ARTICLE | JULY 01 2011

Reduction of the Peripheral Blood CD56bright NK Lymphocyte Subset in
FTY720-Treated Multiple Sclerosis Patients 
Trina A. Johnson; ... et. al

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J Immunol (2011) 187 (1): 570–579.
https://doi.org/10.4049/jimmunol.1003823

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 The Journal of Immunology

Reduction of the Peripheral Blood CD56bright NK

Lymphocyte Subset in FTY720-Treated Multiple Sclerosis
Patients

Trina A. Johnson,*,† Barbara L. Evans,* Bryce A. Durafourt,* Manon Blain,*

Yves Lapierre,‡ Amit Bar-Or,*,†,‡ and Jack P. Antel*,‡
FTY720 (fingolimod) treatment of multiple sclerosis (MS) results in lymphopenia due to increased recruitment into and decreased
egress from secondary lymphoid organs of CCR7+ lymphocytes. Although absolute numbers of NK lymphocytes were reported as
being unaltered in FTY720-treated MS patients (MS-FTY), such analyses did not detect a change in a minor subset. Because

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expression of CCR7 has been described on CD56bright NK cells, a minority population of NK cells, we investigated the effect of
FTY720 treatment on the phenotype and function of human NK cells in the peripheral circulation of MS patients. MS-FTY
patients displayed a decreased proportion of peripheral CD56brightCD62L+CCR7+ NK cells compared with untreated MS and
healthy donors. In vitro treatment with FTY720-P increased migration of untreated donor NK cells to CXCL12 while reducing the
response to CX3CL1 with similar migration responses seen in NK cells from MS-FTY patients. FTY720-P inhibited sphingosine
1-phosphate–directed migration of CD56bright and CD56dim NK cells subsets from untreated healthy donors. IL-12– and IL-15–
stimulated NK cells from MS-FTY patients displayed similar capacity to produce IFN-g, TNF, IL-10, and MIP-1a cytokines/
chemokines compared with NK cells from untreated healthy donors and displayed comparable levels of degranulation in response
to K562 tumor cells compared with untreated donors. Subset alterations and function of NK cell populations will need to be
considered as part of assessing overall immunosurveillance capacity of patients with MS who will receive sustained FTY720
therapy. The Journal of Immunology, 2011, 187: 570–579.

F
TY720 is a sphingosine 1-phosphate (S1P) receptor ago-
nist with demonstrated efficacy in reducing inflammatory
events in the CNS of multiple sclerosis (MS) patients (1).
MS patients receiving FTY720 oral therapy (MS-FTY) demon-
strate marked peripheral blood lymphopenia that reflects re-
distribution rather than destruction of this cell population (2) (3).
The rapid onset (within days) of the lymphopenia in MS-FTY
patients likely reflects that initial exposure to the agent enhances
initial lymphocyte trafficking into secondary lymphoid organs
(SLOs) by sensitizing cells to chemokine cues (4). Entry into
SLOs primarily involves CCR7-expressing cells responding to the
CCR7 ligands (5). Restricted re-entry to the circulation of CCR7+
lymphocyte populations (naive and central memory T cells,
B cells) is attributed to FTY720-mediated internalization of S1P1
on lymphocytes (6). Entry of Ag-reactive T cells into the circu-
lation following influenza vaccination appears unimpaired in
*Neuroimmunology Unit, Montreal Neurological Institute, McGill University, Mon-
treal, Quebec H3A 2B4, Canada; †Experimental Therapeutics Program, Montreal
Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada; and
‡
Department of Neurology and Neurosurgery, Montreal Neurological Institute,
McGill University, Montreal, Quebec H3A 2B4, Canada
Received for publication December 1, 2010. Accepted for publication April 20, 2011.
Statistical analysis and all experiments within were performed by T.A.J. J.P.A. 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. All authors have seen and agree with
the contents included in this manuscript.
Address correspondence and reprint requests to Dr. Jack P. Antel, Montreal Neuro-
logical Institute, 3801 University, Room 111, Neuroimmunology Unit, Montreal,
Quebec H3A 2B4, Canada. E-mail address: jack.antel@mcgill.ca
Abbreviations used in this article: MFI, mean fluorescence intensity; MS, multiple
sclerosis; MS-FTY, FTY720-treated MS patients; SLO, secondary lymphoid organ;
S1P, sphingosine 1-phosphate; UNTX, untreated healthy donor; UNTX MS, un-
treated multiple sclerosis patient.
Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00
FTY720-treated MS patients, suggesting that re-entry restriction is
not universal (7).
To further define the impact of long-term FTY720 therapy on
immunosurveillance capacity, we addressed how FTY720 treatment
of MS patients alters the distribution of NK cell subsets and what
effect it has on the functional capacity of the NK cells circulating in
these patients. NK cells are constituents of the innate immune
system that act to control microbial infections and cancer and
comprise ∼10% of the circulating lymphocyte pool in peripheral
blood (8). Human NK cells are CD32 lymphocytes that express a
series of distinct molecules, including CD16, NKp46, and CD56;
CD56 is not expressed by murine NK cells (9). NK cell pre-
cursors arise from the bone marrow and undergo maturation pro-
cesses that lead to an increase in effector functions and alterations
in the expression of both integrins and chemotactic receptors. This
allows their redistribution from bone marrow to lymphoid and
nonlymphoid compartments. Whereas murine NK cells have been
classified as CD27dull (mature tissue or blood-resident effector NK
cells) and as CD11bdull (lymphoid-resident, immature NK cells),
human NK cells can be divided into two main corresponding
subsets based on levels of CD56 expression, that is, CD56dim and
CD56bright, which differ in their functional and homing properties
(10). CD56dim NK cells constitute ∼90% of peripheral blood
NK cells, express perforin, and are considered to be cytotoxic
effectors. CD56bright NK cells, which make up the remaining 10%
of peripheral blood NK cells, lack perforin but can, upon stimu-
lation, including with IL-12/IL-15, produce a range of regulatory
and effector cytokines (11). Recent studies indicate that the
CD56dim NK cell subset can also be a source of proinflammatory
cytokines (12, 13). Alterations in peripheral blood NK cell subset
numbers and functions have been linked to disease activity in MS
(14, 15). Treatment of MS patients with daclizumab (IL-2Ra mAb
therapy) leads to expansion of the IL-10–producing CD56bright

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003823
The Journal of Immunology
subset of NK cells, which may act to regulate the proliferation of
potentially autoreactive T lymphocytes, a suggested mechanism
for the efficacy of this drug (16, 17).
Human lymph node NK cells outnumber blood NK cells by 10:1,
whereas in mice there is an estimated 1:1 ratio (12). This difference
has been attributed to the expression of CCR7 on the human
CD56bright NK cell subset but not on murine NK cells. Entry
of lymphocytes to lymph nodes can be directed via expression of
CCR7, in response to the CCR7 ligands CCL19 and CCL21,
produced in the lymph node. The CD56bright NK cell subset has
been shown to express CCR7, allowing their accumulation in
lymph nodes (18). Additionally, CD62L is expressed at high levels
on CD56bright NK cells found in lymph nodes (19) and has been
shown to be required for NK cell entry into lymph nodes (20).
Lymphoid cell egress from lymph nodes depends on essential
chemotactic cues from S1P. Murine studies show that, whereas
egress from lymph nodes in response to S1P for lymphocytes is
regulated via S1P1 (21), egress of NK cells is regulated through
both S1P1 and S1P5 (9, 22), the dominant SIPR expressed by the
majority of this cell type, CD27dull NK cells (9). NK cells are lost
from the blood, spleen, and lung in S1P5-deficient mice and ac-
cumulate in bone marrow and lymph node (9). S1P1-deficient mice
show a small accumulation of NK cells in lymph nodes (22). NK
cell maturation is associated with upregulated expression of S1P5.
As such, the highest expression of S1P5 is seen in human CD56dim
NK cells, with lower expression evident in CD56bright NK cells (9).
Additional chemokine receptors involved in the trafficking of
NK cells include CXCR4 and CX3CR1, responsive to migration-
directing cues delivered by their ligands CXCL12 and CXC3L1
(fractalkine), respectively (23). CX3CL1 has been shown to attract
NK cells to CNS lesions in the experimental autoimmune en-
cephalomyelitis model (24–26). CXCL12 contributes to NK cell
homing to bone marrow and lymph nodes, as well as to inflam-
matory sites (27). It has been suggested that initial retention of NK
cells in bone marrow is the result of CXCR4/CXCL12 interactions,
and S1P1/S1P5/S1P-mediated signals are needed to overcome this
interaction and allow egress from bone marrow (22).
Although the relative proportion of NK cells within the overall
circulating lymphoid population is increased in FTY720-treated MS
patients, previous studies have reported that absolute numbers of
NK lymphocytes in FTY720-treated mice and humans are unaltered
(9, 28). In the present study we investigated the relationship of NK
cell subset redistribution in FTY720-treated patients based on ex-
pression of CCR7. We provide data supporting the prediction that
there would be a relative depletion of CCR7-expressing CD56bright
NK cells given that these cells have increased sensitivity to che-
mokine ligands that promotes ingress into lymph nodes. Because
CD56bright NK cells are reported to have a relative overexpression
of S1P1 compared with S1P5, we determined their relative response
to S1P itself and whether their response was inhibited by FTY720.
The functional properties of the NK cells remaining in the circu-
lation of FTY720-treated patients was measured in terms of 1)
sensitivity to effects of FTY720 on subsequent chemokine-directed
migration in vitro using CXCL12, CX3CL1, or S1P, 2) IL-12– and
IL-15–induced production of cytokines and chemokines, IFN-g,
TNF-a, IL-10, and MIP-1a, and 3) degranulation in response to
tumor target cells.
Materials and Methods
Donors
Peripheral venous blood was obtained with informed consent from un-
treated healthy donors (UNTX), untreated MS patients (UNTX MS), and
MS-FTY patients. MS-FTY patients were participants in the extension
phases of phase II and III clinical trials for relapsing-remitting multiple
571
sclerosis (1.25 mg fingolimod [FTY720] once daily with subsequent de-
crease to 0.5 mg daily). The number of patients contributing to each ex-
periment is indicated in individual figure legends. Patients participating in
the extension phases of fingolimod clinical trials were treated for between 2
and 6 y. All had expanded disability status scores scores of ,2.0. This study
was approved by the Institutional Review Board of McGill University.
Cell isolation
PBMCs were isolated from venous blood samples using a Ficoll density
gradient (GE Healthcare, Baie d’Urfe, QC, Canada). For most studies, total
NK cells were isolated by depleting CD3+ lymphocytes using CD3+
MACS (Miltenyi Biotec, Auburn, CA) followed by CD56+ selection for
NK cells. For CD107 mobilization assays, NK cells were isolated using
the NK cell negative isolation kit as per the manufacturer’s instructions
(Miltenyi Biotec). All experiments were performed in RPMI 1640 com-
plete medium (containing 10% FCS with antibiotics and glutamine; all
from Invitrogen, Carlsbad, CA) except for migration experiments, which
were performed in RPMI 1640 complete medium supplemented as in-
dicated below. FTY720-P was a gift from Novartis Pharma, and vehicle
control consisted of DMSO containing 50 mM HCl.
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Flow cytometry
PBMCs were analyzed for expression of surface markers using flow
cytometry, according to standard procedures for staining. The following
anti-human Abs were used in various combinations: CD16-FITC, CD56-PE
or Alexa 488, CCR7-PE or Alexa Fluor 647, NKp46-allophycocyanin,
CD3-PerCP, CD62L-PE, CXCR4-PE, CX3CR1-PE, or the appropriate
isotype control (all from BD Biosciences). After staining, cells were
washed twice and fixed in 1% formaldehyde containing PBS. Results were
analyzed with FlowJo software (Tree Star, Ashland, OR). To analyze
CD56 expression, CD56+CD32 isolated NK cells were stimulated for 1 or
18 h in vitro in RPMI 1640 complete media with 1 mM FTY720-P, fol-
lowed by surface CD56 and CD3 staining and flow cytometry.
Migration assay
CD56+CD32 NK cells were pretreated for 30 min in vitro with either
vehicle or FTY720-P (1 mM) in RPMI 1640 complete media (containing
antibiotics and glutamine) supplemented with either 2.5% FCS (CXCL12/
CX3CL1 migrations) or 0.1% BSA (S1P migrations) and seeded at
250,000 per well on top of 5-mm ChemoTx migration chambers (Neuro
Probe, Gaithersburg, MD). For CXCL12/CX3CL1 migrations, wells below
the filter were filled with either RPMI 1640 (2.5% FCS) media alone or
media with chemokine, 10 ng/ml CXCL12 (SDF-1a) (Sigma-Aldrich, St.
Louis, MO), or 10 ng/ml CX3CL1 (fractalkine). For S1P migrations, wells
below the filter were filled with either RPMI 1640 (0.1% BS) media alone
or media with 1 or 10 nM S1P (Sigma-Aldrich). NK cells were allowed to
migrate for 4 h in a humidified incubator (37˚C, 5% CO2). Cells were
removed from individual chambers and stained with combinations of
Abs, including anti-human CD56-Alexa 488, CD3-PerCP, and NKp46-
allophycocyanin (BD Biosciences). Cells were washed three times be-
fore analysis on a flow cytometer (FACSCalibur; BD Biosciences).
Quantification of cell number was assessed by 30-s timed acquisition of
total cell number in a fixed volume of 1% formaldehyde in PBS. Each
condition was performed in triplicate for at least three donors. Migration is
expressed either as 1) migration index, which is a fold migration over
baseline, referring to the total number of CD56+CD32 NK cells migrating
to the specific chemokine over the total number of CD56+CD32 NK cells
migrating toward media alone; or 2) percentage change in migration,
which is the percentage change in the number of cells migrating to the
chemokine as compared with cells migrating to media.
Cytokine production by intracellular cytokine staining
CD56+CD32 NK cells were isolated from either MS-FTY or untreated
donors and treated in vitro for 24 h with a combination of IL-12 (10 ng/ml)
and IL-15 (100 ng/ml). Monensin (2 mM) was added 5 h prior to har-
vesting (Sigma-Aldrich). Cells were stained with anti-CD56 and anti-CD3
Abs. Intracellular cytokine staining for IFN-g (IFN-g-PE), TNF-a (TNF-
a-PE), IL-10 (IL-10-PE), or MIP-1a (CCL3, MIP-1a-PE) (BD Biosci-
ences) was then performed following paraformaldehyde/saponin fixation
and permeabilization. Cells were gated on CD56+CD32 NK cells.
Degranulation (CD107a mobilization) assay
The CD107a mobilization assay was done as previously described (29). NK
cells (effectors) isolated by negative selection were preincubated for 30
min in media, vehicle, or 1 mM FTY720-P. Following treatment, monensin
572 PERIPHERAL NK CELLS IN FTY720-TREATED MS PATIENTS

(10 mM) was added and NK effector cells (200,000 cells) were plated onto
a confluent layer of K562 targets cells (American Type Culture Collection)
incubated in RPMI 1640 at a 1:1 E:T ratio. Five microliters of PE-
conjugated CD107a Abs or concentration matched PE-conjugated IgG1
isotype control was added to each well. Cells were mixed and spun at 900
rpm for 1 min and incubated for 4 h at 37˚C and 5% CO2. Cells were
washed, surface stained with NKp46-allophycocyanin, and CD16-FITC
and analyzed by flow cytometry. Actively degranulating cells were iden-
tified as NKp46+CD16+CD107a+ NK cells.

RNA isolation, reverse transcription, and real-time PCR

Total RNA isolation from CD56+CD32 MACS-isolated NK cells, cDNA
synthesis, and real-time PCR cells were carried out as previously described
(30). Expression levels for S1PR1 (encoding for S1P1) and S1PR5 (S1P5)
and 18S for all cell types were determined by real-time PCR using Assays-
on-Demand primers and probe sets (Applied Biosystems, Foster City, CA).
For studies comparing S1PR expression in NK cells from MS patients
receiving FTY720 and controls, relative expression levels were expressed
as arbitrary units derived by interpolation of the threshold cycle of each
sample to a standard curve generated from 10-fold serial dilutions of

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mRNA derived from healthy donor PBMCs (S1P1and S1P5). Relative
quantities of 18S mRNA derived from standard curves were also de-
termined from the same cell types and used to normalize the data.
Statistical analyses
Results represent data from at least three independent biological experi-
ments. After ANOVA, a Student t test was used. Statistical analyses were
performed using Prism 5.0 (GraphPad Software). Probability values of
,0.05 were considered to represent statistically significant differences.
Figures show means 6 SE.

Results
Phenotypic properties of NK cells found in the circulation of
FTY720-treated MS patients
As expected, the total unfractionated lymphocyte count was mark-
edly reduced in the FTY720-treated patients. The average lym-
phocyte count for patients on FTY720 therapy was 0.44 3 109
cells/l (60.2 SD), whereas the lower limit of normal was 0.8 3
109 cells/l. As illustrated in Fig. 1A, MS-FTY patients feature a
decrease in the CD3+ T cell subset in PBMCs and a relative in-
crease in the proportion of NK (CD32, NKp46+) cells compared
with an untreated control. As shown for the overall cohort of
donors in Fig. 1B, PBMCs from MS-FTY patients contained a
significantly higher frequency of cells that expressed the NK cell
markers CD16 (47.55 6 5.46% SE, n = 8) compared with PBMCs
from UNTX patients (18.11 6 2.51% SE, n = 8, p = 0.0007) or
UNTX MS patients (15.36 6 2.58% SE, n = 5, p = 0.0043) and
NKp46 (32.84 6 3.54% SE, n = 8) compared with UNTX
(12.60 6 2.05% SE, n = 11, p = 0.0015) or UNTX MS patients
(14.41 6 2.23% SE, n = 5, p = 0.0031), as well as CD56 (38.74 6
3.33% SE, n = 9) compared with UNTX (16.15 6 2.29% SE,
n = 12, p = 0.0004) or UNTX MS patients (13.25 6 1.75% SE, n =
5, p = 0.001).
As shown for representative donors in Fig. 2A and the overall
cohort of MS-FTY patients and untreated donors in Fig. 2B,
within the CD56+ NK cell population (gated on NKp46+CD32
cells), the proportion of CD56bright NK cells was substantially
lower for MS-FTY patients (1.58 6 0.34%, n = 10) compared with
UNTX (6.13 6 1.26%, n = 12, p = 0.0009) or UNTX MS patients
(6.38 6 1.68%, n = 8, p = 0.0048), resulting in a relative increase
in the proportion of CD56dim NK cells for MS-FTY patients. As
shown in Fig. 2C for representative donors and as an overlay in
Fig. 2D, the loss of CD56bright NK cells results in the concurrent
loss of CD56+NKp46+CD62Lhi NK cells.
We confirmed that CD56bright NK cells from control donors can
be distinguished from CD56dim NK cells by expression of CCR7
(Fig. 3A). For the untreated control, ∼60% of CD56bright NK cells
express CCR7 whereas ,20% of CD56dim NK cells express
FIGURE 1. MS-FTY patients display an increased proportion of cir-
culating NK cells. A, Representative FACS profile showing a decreased
frequency of CD3+ lymphocytes and an increased frequency of NKp46+
NK cells in the total lymphocyte population of a representative MS-FTY
patient compared with an untreated healthy control. B, Mean percentage of
CD16+ cells or (C) NKp46+ cells or (D) CD56+ cells in the total PBMC
population of untreated healthy donors, untreated MS patients, and
FTY720-treated MS patients (n = 8 for UNTX, n = 8 for MS-FTY, and n =
5 for UNTX MS patients). Error bars represent SE. *p , 0.05, **p , 0.01.
CCR7. As FTY720 redistributes CCR7+ cells, we confirmed that
the FTY720-treated MS patients had a significant loss of CCR7
expressing NK cells (Fig. 3B, left). As shown for a representative
donor in Fig. 3B (right), the remaining CD56bright NK cells in MS-
FTY patients display similar mean fluorescence intensity (MFI)
for CCR7 as do the NK cells in control donors.
To assess further phenotypic and functional characteristics of
peripheral blood NK cells in MS-FTY patients compared with
untreated donors, we isolated CD32CD56+ NK cells by immu-
nomagnetic bead positive selection. As shown in Fig. 3C for
representative donors and averaged for the overall cohort in Fig.
3D, the loss in the relative proportion of CD56bright NK cells was
maintained in the population isolated by positive selection. These
cells were used for further assays and were $90% CD56+CD32.
CD56 expression is unaltered by FTY720 treatment in vitro
To address whether the decreased numbers of CD56bright NK cells
in the periphery of MS-FTY patients could result from FTY720
downregulating expression of CD56 in NK cell subsets, we treated
isolated CD56+CD32 NK cells from healthy donors in culture for
1 or 18 h to either vehicle or 1 mM FTY720-P. Levels of CD56
The Journal of Immunology
FIGURE 2. MS-FTY patients show loss of the circulating CD56bright
NK cell population. A, Representative FACS profile of a UNTX control
and MS-FTY patient, depicting a decreased frequency of CD56bright NK
cells from the gated CD32NKp46+ population in MS-FTY patients. B,
Percentage of NKp46+ NK cells from UNTX or UNTX MS donors or MS-
FTY patients that express a CD56bright phenotype (n = 12 UNTX, n = 8 for
UNTX MS, and n = 10 for MS-FTY patients). **p , 0.01, ***p , 0.001.
Error bars represent SE. C, Representative FACS profile of a UNTX
control and MS-FTY patient, depicting a decreased frequency of
CD56brightCD62Lhi NK cells from the gated CD32NKp46+ population in
MS-FTY patients. D, Overlay histogram of CD56brightCD62Lhi cells (gated
with box in C) from a representative UNTX donor and MS-FTY patient
showing loss of CD62LhiCD56bright NK cells in MS-FTY patients. FSc,
forward scatter.
expression at these time points were analyzed by flow cytometry
gating for CD56+CD32 populations. As shown in Fig. 4A for
a representative untreated donor, CD56 expression was not mod-
ulated after 1 or 18 h treatment in either media alone (UNTX),
vehicle (VEH), or 1 mM FTY720-P (FTY). The proportion of
CD56bright and CD56dim NK cell populations was unaltered in any
of the conditions as displayed in an overlay histogram in Fig. 4B
and quantitatively in Fig. 4C (n = 4).
Functional properties of NK cells exposed to FTY720 in vitro
or derived from FTY720 treated patients: chemokine-directed
migration in response to CXCL12 and CX3CL1
The previously reported main functional effect of FTY720 is to
increase the sensitivity of lymphocytes to chemokine cues for
migration (4). As shown in Fig. 5A and 5B for a representative
untreated donor, both CD56dim and CD56bright NK cells express
573
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FIGURE 3. MS-FTY patients have a decreased frequency of circulating
CCR7+ NK cells. A, Representative FACS profile of CD56+CD32 gated
NK cells from a UNTX control and a MS-FTY patient, depicting CCR7
expression on CD56bright NK cells (box gate). MS-FTY patients display
a decrease in the frequency of CCR7+CD56bright NK cells. B, Histogram
overlay of CCR7 expression on total CD56+CD32 NK cells (left) or
CD56brightCD32 NK cells (right) from a representative UNTX control or
MS-FTY patient, depicting loss of CCR7 expression on NK cells from an
MS-FTY patient. Similar MFI of CCR7 expression on the circulating
CD56bright NK cells is seen in both groups. C, Representative FACS profile
showing the phenotypic properties of cells recovered after immuno-
magnetic isolation using CD3 depletion and anti-CD56 Ab-coated im-
munomagnetic beads from a representative UNTX control and MS-FTY
patient. Almost all cells are CD32 and CD56+. The representative MS-
FTY patient depicts relative absence of the CD56bright NK population
(outlined in boxes). D, Percentage of CD56+CD32 NK cells from UNTX
donors or MS-FTY patients that expressed a CD56bright phenotype after
immunomagnetic bead isolation (n = 4 UNTX, n = 5 MS-FTY patients).
Error bars represent SE. *p , 0.05. ISO, isotype control.
the chemokine receptors CXCR4 and CX3CR1 for the chemokine
ligands CXCL12 and CX3CL1, respectively.
Fig. 5C shows the results of in vitro treatment of CD32CD56+
NK cells from untreated donors or MS-FTY patients in vitro with
vehicle or 1mM FTY720-P and assessing chemokine-directed mi-
gration through microchemotaxis chambers to CXCL12. NK cells
from untreated donors that were treated in vitro with 1 mM
FTY720-P displayed a significant increase in migration to
574
FIGURE 4. FTY720 treatment does not modulate CD56 expression
in vitro. CD56+CD32 NK cells from a UNTX donor were incubated either
with no treatment, vehicle, or 1 mM FTY720-P for 1 or 18 h in vitro. A,
Representative FACS profile showing the percentage CD56bright NK cells
in the total untreated or vehicle-treated or FTY720-treated CD56+CD32
NK cells of a healthy donor. B, Representative overlay of CD56 expression
in untreated donor NK cells incubated with either no treatment, vehicle, or
1 mM FTY720-P for 1 or 18 h in vitro, as compared with isotype control.
Complete overlay of MFI is seen for all three conditions. C, Mean per-
centage of CD56+CD32 NK cells that were CD56bright after 1 or 18 h
incubation with no treatment, vehicle, or 1 mM FTY720-P (n = 4 untreated
healthy donors). Error bars represent SE. FTY-P, 1mM FTY720-P; iso,
isotype control; no tx, no treatment; VEH, vehicle.
CXCL12 of 68.1 6 6.6% (n = 4) over baseline migration to media
alone compared with vehicle-treated NK cells from the same
donors (50.4 6 6.6%, n = 4) over baseline (p = 0.027). The
percentage increase in migration over baseline of NK cells derived
from MS-FTY patients either after vehicle treatment (60.2 6
14.6%, n = 3) or treatment with 1 mM FTY720-P (69.0 6 11.5%,
n = 3) was not significantly different from migration results ob-
served for the untreated donors.
Fig. 5D shows the results of in vitro treatment of CD56+CD32
NK cells from untreated donors or MS-FTY patients in vitro with
vehicle or 1 mM FTY720-P and assessing chemokine-directed
migration to CX3CL1. NK cells from untreated donors that were
treated in vitro with 1 mM FTY720-P displayed reduced response
to CX3CL1 (14.0 6 5.1% increase in migration over baseline, n =
3) compared with vehicle-treated NK cells from the same donors
(36.6 6 6.0%, n = 3, p = 0.016). Percentage migration over
baseline of NK cells derived from MS-FTY patients displayed
a similar pattern with a significantly reduced response after
PERIPHERAL NK CELLS IN FTY720-TREATED MS PATIENTS
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FIGURE 5. Migration responses to CXCL12 and CX3CL1 chemokine
for NK cells from MS-FTY patients and untreated donors. A, Represen-
tative overlay of CXCR4 and (B) CX3CR1 expression in CD56bright and
CD56dim NK cell subsets from an untreated donor compared with isotype-
stained NK cells. Both CD56dim and CD56bright NK cells express the
chemokine receptors CXCR4 and CX3CR1 for their respective ligands
CXCL12 and CX3CL1. C, Percentage change in migration over baseline
(migration to media alone) of CD56+CD32 NK cells isolated from un-
treated donors or MS-FTY patients preincubated with either vehicle or
1 mM FTY720-P showing increased migration for the untreated controls
toward CXCL12 chemokine after FTY720-P pretreatment. MS-FTY
patients do not show significantly different migration results compared
with those observed for the untreated donors D, Percentage change in
migration of CD56+CD32 NK cells isolated from untreated healthy donors
or MS-FTY patients preincubated with either vehicle or 1 mM FTY720-P
showing migration toward CX3CL1 chemokine (n = 3 for UNTX and MS-
FTY groups). Error bars represent SE. *p , 0.05. bright, CD56bright NK
cells; dim, CD56dim NK cells; FTY-P, 1 mM FTY720-P; VEH, vehicle.
treatment with 1 mM FTY720-P (13.4 6 4.8%, n = 3) compared
with the vehicle treatment (40.2 6 10.8%, n = 3, p = 0.004).
Human NK cells express S1P1 and S1P5 and display increased
migration in the presence of the S1P1 agonist SEW2871
In Fig. 6A, we confirm that human CD56+CD32 NK cells taken
from both healthy donors and MS-FTY patients express mRNA
transcripts for the S1P receptors S1P1 and S1P5. To assess the
affect of S1P1-specific agonism on migration to CXCL12, we
exposed CD56+CD32 NK cells from healthy controls to vehicle or
the S1P1 agonist SEW2871, then assessed migration toward me-
dia alone or CXCL12 in vitro. As shown in Fig. 6B, total CD56+
CD32 NK cells from healthy controls migrated significantly more
to CXCL12 when treated with 100 nM SEW2871 (2.43 6 0.20,
n = 6) than with low-dose 10 nM SEW2871 (1.99 6 0.21, n = 4,
p = 0.015) or vehicle (1.98 6 0.18, n = 8, p = 0.004). NK cells
from each condition that had migrated were immunostained with
CD56 fluorescent Abs and quantified for the number of CD56bright
and CD56dim NK cells that had migrated in response to CXCL12.
Both CD56bright and CD56dim NK cells are seen to migrate in re-
sponse to CXCL12. CD56dimCD32 NK cells from healthy con-
trols migrated significantly more to CXCL12 when treated with
100 nM SEW2871 (3.33 6 0.24, n = 5) than with vehicle (2.7 6
0.24, n = 8, p = 0.011) (Fig. 6C). CD56brightCD32 NK cells from
healthy controls migrated significantly more to CXCL12 when
The Journal of Immunology
FIGURE 6. NK cells are sensitized to CXCL12-directed migration in an
S1P1-dependent manner. A, CD56+CD32 NK cells were isolated from
either UNTX donors (n = 4) or MS-FTY (n = 3) patients and subjected to
RNA extraction, reverse transcription, and real-time PCR for S1P1 and
S1P5. NKs in the UNTX group showed greater expression of S1P5 com-
pared with S1P1. B–D, CD56+CD32 isolated NK cells from healthy
donors were pretreated with vehicle, 10 nM SEW2871, or 100 nM
SEW2871 before migration toward 10 ng/ml CXCL12 in vitro. The mi-
gration index was calculated for (B) total CD56+CD32 NK cells, (C)
CD56dim NK cells, or (D) CD56bright NK cells migrating to chemokine (n =
4). Error bars represent SE. *p , 0.05, **p , 0.01. SEW, SEW2871;
VEH, vehicle.
treated with 100 nM SEW2871 (1.77 6 0.09, n = 4) versus vehi-
cle (1.41 6 0.06, n = 4, p = 0.001) (Fig. 6D).
S1P-dependent migration of NK cell subsets
As shown in Fig. 7A, total NK cells (CD56+CD32) migrate to 1 nM
S1P. Subset analysis of the overall migrated cell population in-
dicated that there was an increase in the migration index for both
CD56dim cells (Fig. 7B) and CD56bright NK cells (Fig. 7C) in re-
sponse to 1 nM S1P. This effect was abolished by pretreatment of
these cells with 1 mM FTY720-P.
Cytokine production by IL-12/IL-15–stimulated NK cells
We treated CD56+CD32 cells from control donors in vitro with
IL-12 and IL-15 cytokine for 24 h and performed intracellular
575
staining for TNF-a, IFN-g, IL-10, and MIP-1a. Cells were also
immunostained for CD56 expression. The addition of IL-12 or
IL-15 alone results in very little production of any cytokine/
chemokine, whereas the addition of IL-12 and IL-15 signifi-
cantly increased cytokine/chemokine production.
As shown in Fig. 8, there was no significant difference in the
overall production of any of the cytokines/chemokine tested in
total CD56+CD32 NK cells isolated from MS-FTY patients
compared with untreated healthy donors, suggesting the relative
overabundance of CD56dim NK cells in both groups masks any
small changes that may be seen due to the loss of the minority
population of CD56bright NK cells in MS-FTY patients.
Cytotoxicity of NK cells from MS-FTY patients
In the peripheral circulation, CD56dim NK cells are the cytolytic
subset of NK cells. To determine whether long-term FTY720
treatment alters the ability of this population to degranulate in
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response to target cells, we examined CD107a mobilization as a
degranulation indicator in response to the K562 tumor target cell
line. NK cells isolated by negative selection from untreated or
MS-FTY patients were incubated in the absence or presence of
target K562 cells, and CD107a and CD16 expression were
assessed on NKp46+ cells. Degranulating cells were defined as
CD16+/CD107a+ cells. In Fig. 9A, representative plots from one
untreated individual (UNTX) and one MS-FTY patient show that
FIGURE 7. NK cell migration in response to S1P is inhibited by
FTY720-P. CD56+CD32 isolated NK cells from UNTX donors were
pretreated with vehicle or 1 mM FTY720-P before migration toward 1 or
10 nM S1P in vitro. The migration index was calculated for (A) total
CD56+CD32 NK cells, (B) CD56dim NK cells, or (C) CD56bright NK cells
migrating to S1P (n = 3). Increased migration is no longer apparent in the
presence of 1 mM FTY720-P. Error bars represent SE. *p , 0.05. FTY-P,
1 mM FTY720-P; VEH, vehicle.
576
FIGURE 8. NK cells from MS-FTY display similar
IFN-g, TNF, IL-10, or MIP-1a production after
stimulation with IL-12 and IL-15 in vitro versus un-
treated controls. A, Mean percentage of CD56+CD32
NK cells from UNTX donors or MS-FTY patients
that are IFN-g+ after no treatment or IL-12 with or
without IL-15 stimulation for 24 h in vitro (n = 7
UNTX, n = 10 MS-FTY). B, Mean percentage of
CD56+CD32 NK cells from UNTX donors or MS-
FTY patients that are TNF+ after no treatment or IL-
12 with or without IL-15 stimulation for 24 h in vitro
(n = 8 UNTX, n = 11 MS-FTY). C, Mean percentage
of CD56+CD32 NK cells from UNTX donors or MS-
FTY patients that are IL-10+ after no treatment or IL-
12 with or without IL-15 stimulation for 24 h in vitro
(n = 3 UNTX, n = 5 MS-FTY). D, Mean percentage
of CD56+CD32 NK cells from UNTX donors or MS-
FTY patients that are MIP-1a+ after no treatment or
IL-12 with or without IL-15 stimulation for 24 h
in vitro (n = 3 UNTX, n = 5 MS-FTY). Error bars
represent SE. no tx, no treatment.
NK cells from an untreated individual in the absence of K562
target demonstrate minimal spontaneous degranulation (2.0%) and
no effect by vehicle (1.8%) or FTY720-P (1.7%) acute treatment.
In the presence of K562 target cells, NK cells from a representa-
tive donor displayed similar degranulation profiles for untreated
(12.6%), vehicle (12.5%), and FTY720-P–treated (12.1%) con-
ditions. NK cells from MS-FTY patients displayed comparable
levels of degranulation whether they were not pretreated (16.6%),
pretreated with vehicle (16.4%), or pretreated with FTY720-P
(14.8%). As expected, the MFI for CD16 decreases as degra-
nulation occurs.
In Fig. 9B, for the overall cohort of untreated control, the effect
of no treatment, vehicle, or 1mM FTY720-P on degranulation
(CD107a+ cells) is shown. No significant difference in CD107a+
cells was noted for untreated control NK cells in contact with
K562 target cells either in the presence of no treatment (no tx,
16.3 6 1.3%, n = 4), pretreatment with vehicle (VEH, 15.7 6
1.3%, n = 4), or FTY720-P (FTY, 15.0 6 1.3%, n = 4). Addi-
tionally, rates of spontaneous degranulation (absence of K562
target cells) were not significantly different in all conditions. Fig.
9C demonstrates the overall cohort for both untreated controls
and MS-FTY patients, which were averaged showing comparable
levels of degranulation between groups. NK cells, not pretreated,
showed similar degranulation in the presence of K562 target cells
for the untreated controls (16.3 6 1.3%, n = 4) and the MS-FTY
patients (16.4 6 1.2%, n = 3). As with the untreated controls, MS-
FTY patients showed similar spontaneous degranulation in the
absence of K562 cells.
Discussion
This study focused on the phenotypic and functional properties of
NK cell lymphocyte populations isolated from the circulation of
MS-FTY patients and the short-term in vitro effects of FTY720 on
NK cells isolated from MS-FTY and untreated donors. Daily oral
FTY720 therapy is demonstrated to produce a significant reduction
in T and B cells in the systemic circulation resulting from a re-
distribution of these cells (3, 31). FTY720 retains CCR7+ lym-
phocytes in secondary lymphoid compartments, resulting in an
increased relative proportion of CCR72 populations in the blood
of MS-FTY patients (3). Our recent analysis of the residual cir-
PERIPHERAL NK CELLS IN FTY720-TREATED MS PATIENTS
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culating CD8+ lymphocyte populations in FTY720- treated
patients indicated persistence of cells (late effector memory
CCR72CD27+CD28+) with a reduced capacity to respond to
chemokine signals that would contribute to recruitment of these
cells into tissues, including the CNS (32). Such changes in the
adaptive immune system may imply an increased reliance on the
innate immune system for immunosurveillance in FTY720-treated
patients.
For our phenotypic analysis of NK cells, we focused on ex-
pression of NKp46, CD56, and CD16. Whereas NKp46 is ex-
pressed solely on NK cells, CD56 and CD16 are expressed addi-
tionally on other subsets (CD56+ NKT cells CD16+ monocytes)
(33). In our experiments, NK cells were identified as NKp46+
or CD56+ CD32 cells. We confirmed the previously reported
relative increase in the proportion of NK cells in the peripheral
circulation of MS-FTY patients (28). We took advantage of
CD56bright being a marker for the regulatory subset of NK cells in
humans to demonstrate redistribution of NK cell subsets that
would not have been evident in animal studies (9) or previous hu-
man studies (28) that did not take into account the minor circu-
lating subset of CD56bright NK cells. We now document a loss of
CD56bright NK cells (CD62L hi) and a relative increase in the
proportion of CD56dim NK cells in the periphery of MS-FTY
patients. Such a finding could reflect either a downregulation
of CD56 from the surface of the CD56bright NK cells, or a re-
distribution of the CD56bright NK cell population by action of the
drug, or both. We directly demonstrate that short-term FTY720
treatment does alter expression of CD56 in vitro, thus supporting
our hypothesis that the decrease in the CD56bright NK cell pop-
ulation in the circulation of MS-FTY patients reflects a re-
distribution of the CD56bright NK cells.
As previously reported, CCR7 is detected on 60% of human
CD56bright and only a minority of CD56dim NK cells (34, 35). It is
the CCR7-expressing population that is depleted from the sys-
temic circulation of the MS-FTY group. We have previously
shown that CCR7 status underlies the redistribution of CD8+
T lymphocyte subsets in MS-FTY patients (3, 32). T lymphocyte
egress from lymphoid organs is mainly dependent on S1P1 with
S1P1R (36–38). Recent animal based studies have identified S1P5
as being required for NK cell exit from bone marrow and spleen,
The Journal of Immunology
FIGURE 9. NK cells isolated from MS-FTY patients or FTY720-treated
healthy donor NK cells display functional degranulation in vitro. A,
Representative FACS profile showing the proportion of untreated, vehicle,
or 1 mM FTY720-P preincubated NKp46+ NK cells that express CD107a
when cultured alone or with K562 cells. The degranulation response
(CD107a+, gated box) in the presence of K562 cells was comparable under
all conditions tested. B, Percentage of CD107a+NKp46+ NK cells from the
cohort of untreated donors obtained after preincubation with no treatment,
vehicle, or 1 mM FTY720-P and with or without coculture with K562 cells
(n = 4 UNTX donors). C, Percentage of CD107a+NKp46+ NK cells from
the cohort of untreated donors or MS-FTY patients after 4 h incubation
with or without K562 cells. Degranulation responses were similar between
UNTX and MS-FTY groups in all treatments (n = 4 UNTX, n = 3 MS-
FTY). Error bars represent SE. *p , 0.05.
as well as for allowing their circulation in the blood, spleen, and
lung (9). Animal-based studies report that S1P5 is resistant to
FTY720-induced downmodulation; FTY720 treatment of animals
continues to allow NK cell egress (9). As mentioned, CD56bright
NK cells, unlike CD56dim NK cells, do not show preferential
expression of S1P5. Our data indicate that both CD56bright and
CD56dim NK cells respond to S1P; pretreatment with FTY720-P
inhibits this migration.
We and others have previously investigated the mechanism of
action for FTY720 to alter lymphocyte sensitivity to chemokine
signals. Our previous studies of in vitro treatment with FTY720-P
577
highlighted a role for FTY720 in increasing the sensitivity of ef-
fector memory CD8+ lymphocytes to the chemokines CXCL12
and CCL2 when modeled using microchemotaxis assays (32).
NK cells express the chemokine receptors CXCR4 and CX3CR1,
which respond to their cognate ligand chemokines CXCL12
(SDF-1a) and CX3CL1 (fractalkine) with increased expression on
the CD56bright NK cells compared with the CD56dim NK cells.
In vitro treatment with FTY720-P promoted migration of un-
treated donor NK cells to CXCL12, an effect also observed with
human CD8+ T cells (32). We postulate that the lack of increased
response in the MS-FTY patient-derived NK cells reflects either
the effects of chronic FTY720 therapy or a relative lack of re-
sponse by CD56dim NK cells. Our observation that this chemo-
kine-directed migration was enhanced by the S1P1 agonist
SEW2871 suggests that the FTY720-P effect was mediated via
S1P1. We found that in vitro treatment with FTY720-P reduced
the migration response of NK cells to CX3CL1, with such an
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effect being maintained even on cells derived from patients on
chronic therapy. Our data on the differential effects of FTY720 on
individual chemokine signals parallel studies in mice that showed
FTY720 promotes lymph node homing and in vitro migration to
some chemokines, including CXCL12, but has no effect on others,
including CX3CL1 (39).
Although not present in large numbers in the peripheral circu-
lation, activated CD56bright NK cells are a major contributor to
cytokine production. Using an intracellular staining method, we
stimulated NK cells with the monokines IL-12 and IL-15, a stim-
ulus known to induce cytokine production from NK cells (11). As
previously reported we note that both CD56bright and CD56dim NK
cells are capable of producing cytokines, and thus any loss of
cytokine production that may have been expected by the loss of
CD56bright NK cells from the overall NK population is masked by
the continued production of cytokine from CD56dim NK cells, and
no overall differences in cytokine production are evident by
comparing the percentages of total NK cells producing cytokine.
With regard to cytotoxic functions, CD56dim resting NK cells are
cytotoxic, whereas CD56bright NK cells require activation by IL-2
to become cytolytic (40). Because the numbers of CD56bright NK
cells in the periphery are altered, we sought to confirm whether
this has any outcome on cytotoxic function of the remaining
CD56dim population. As reviewed by Segal (15), clinical or ra-
diological disease activity in patients with MS has been linked to
the frequency and functional competence of circulating NK cells.
These early experiments did not specifically isolate NK cells but,
rather, they examined the cytotoxic potential of PBLs; as such,
they take into account the differential cytotoxic capabilities of
CD56dim and CD56bright subsets.
We opted to perform CD107a mobility assays to determine the
degranulation capabilities of total NKp46+ NK cells isolated from
either untreated donors or MS-FTY patients in the presence or
absence of FTY720. In a recent study, it was shown that S1P can
protect K562 cells from NK cell-mediated cell lysis by acting
through S1P1 on the tumor target cells. FTY720, acting on the
tumor cells, can reverse this protection, suggesting that FTY720
may promote tumor targeting (41). The authors implicated an
effect on the target cell rather than on the NK cell. Our results
indicate that CD56dim NK cells isolated from MS-FTY patients
have retained the ability to directly target foreign cells and thus
retain their immunosurveillance capabilities.
The overall consequence of a redistribution of CCR7+CD56bright
NK cells to the lymph node may be minimal since most CD56bright
NK cells already act within regional lymph nodes in their regu-
latory capacity (42, 43). As mentioned, treatment of MS patients
with daclizumab (IL-2Ra mAb therapy) leads to expansion of the
578
IL-10–producing CD56bright subset of NK cells, which may act to
regulate the proliferation of potentially autoreactive T lympho-
cytes. In the context of inflammation within target tissues, how-
ever, loss of circulating CD56bright NK cells may have conse-
quences. In the periphery, most NK cells (∼90%) are CD56dim
CD16+, whereas at sites of inflammation CD56brightCD162 NK
cells predominate (10) and can synergize with monocytes to
promote a proinflammatory environment (44). The presence of
NK cells at inflamed sites, including the CNS, has been shown
to be important for controlling experimental autoimmune en-
cephalomyelitis (45, 46). With the recent U.S. Food and Drug
Administration approval for the use of FTY720 as a sustained
therapy for MS patients, the impact of the observed redistribution
and function of NK cell populations on immunosurveillance
and disease susceptibility, as revealed in larger treatment cohorts,
may soon be determined.
Acknowledgments
We thank members of the Experimental Therapeutics Program and staff of
the Montreal Neurological Institute’s Clinical Research Unit for expert
technical support. We thank Novartis Pharma, Inc., for the donation of
FTY720-P for in vitro studies.
Disclosures
A.B.-O. has received personal compensation for consulting, serving on
scientific advisory boards, and/or speaking activities from Bayer, Bayhill
Therapeutics, Berlex, Biogen Idec, BioMS, Diogenix, Eli-Lilly, Genentech,
GlaxoSmithKline, Guthy-Jackson/Greater Good Foundation, Merck-Serono,
Novartis, Ono, Roche, Sanofi Aventis, Teva Neuroscience, and Wyeth and
has received research funding from Bayhill Therapeutics, Biogen Idec,
Genentech, Merck-Serono, and Teva Neuroscience. J.P.A. has received com-
pensation for serving on data safety monitoring and/or advisory boards for
Bayhill Therapeutics, Biogen Idec, Eli-Lilly, Genentech, GlaxoSmithKline,
Merck Serono, Novartis, Sanofi Aventis, and Teva Neuroscience, has re-
ceived research funding from Novartis for studies unrelated to the current
report, and receives an honorarium for serving as coeditor for the Multiple
Sclerosis Journal. Y.L. has received compensation for serving on advisory
boards for Bayer Schering Pharma, Biogen Idec, Merck Serono, Novartis
Pharma, and Teva Neuroscience. The remaining authors have no financial
conflicts of interest.
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