Download Enriched CD161high CCR6+ γδ T Cells in the Cerebrospinal Fluid

ORIGINAL CONTRIBUTION
Enriched CD161high CCR6ⴙ ␥␦ T Cells
in the Cerebrospinal Fluid of Patients
With Multiple Sclerosis
Lucas Schirmer, MD; Veit Rothhammer, MD; Bernhard Hemmer, MD; Thomas Korn, MD
Objective: To investigate the expression of CD161
Results: ␥␦ T cells were increased in both blood and CSF
Design: Flow cytometry analysis of CD161 and CCR6
expression and intracellular cytokine staining for interleukin 17 and interferon-␥ on human ␥␦ T cells in blood
and CSF samples.
of patients with CIS/MS in relapse as compared with controls with noninflammatory disease. The fraction of
CD161high CCR6⫹ ␥␦ T cells was significantly higher in
the CSF of patients with CIS/MS in relapse than of those
with systemic autoimmune disorders or controls with noninflammatory disease. The CD161high CCR6⫹ doublepositive ␥␦ T-cell population was further enriched in the
CSF in relation to blood in patients with CIS/MS in relapse but not in patients with infectious disease or the
other control groups. The CD161high CCR6⫹ ␥␦ T-cell
population was characterized by its capacity to produce
interleukin 17.
(KLRB1) and CCR6 on human ␥␦ T cells in blood and cerebrospinal fluid (CSF) of patients with a clinically isolated
syndrome (CIS) and multiple sclerosis (MS) in relapse.
Setting: Department of Neurology, Klinikum rechts der
Isar, Technische Universität München, a tertiary referral center.
Patients: Twenty-six patients with CIS/MS in active re-
lapse, 10 patients with other autoimmune disorders, 12
patients with neuroinfectious diseases, and 15 patients
with noninflammatory neurological diseases.
Conclusion: Interleukin 17–producing CD161high CCR6⫹
␥␦ T cells might contribute to the compartmentalized inflammatory process in the central nervous system of patients with MS.
Main Outcome Measures: Frequencies of CD161high
and CCR6⫹ ␥␦ T cells in blood and CSF samples of patients with CIS/MS in relapse and control patients.
A
Author Affiliations:
Department of Neurology,
Klinikum rechts der Isar,
Technische Universität
München, Munich, Germany.
JAMA Neurol. 2013;70(3):345-351. Published online
December 17, 2012. doi:10.1001/2013.jamaneurol.409
UTOREACTIVE HELPER T SUB-
type 17 (TH17) cells are important inducers of immunopathology in various
animal models of organspecific autoimmunity.1 In addition, TH17
cells appear to be enriched in the peripheral blood and cerebrospinal fluid (CSF) of
patients with multiple sclerosis (MS).2 However, TH17-associated effector cytokines including interleukin 17A (IL-17A), IL-17F,
IL-21, and IL-22 are also produced by ␥␦
T cells.3 Based on a series of recent studies
in murine model systems, it is believed that
␥␦ T cells adopt distinct functional phenotypes according to the signature cytokines
that they produce. Both the interferon-␥
(IFN-␥)–producing (␥␦T1 cells) and IL17–producing subset of ␥␦ T cells (␥␦T17
cells) seem to be committed to their respective phenotype already during thymic
development.4 Elevated fractions of T cells
with a ␥␦ T-cell receptor have been observed in blood samples of patients with MS
and ␥␦ T cells were found to occur in early
JAMA NEUROL/ VOL 70 (NO. 3), MAR 2013
345
MS lesions.5-7 In human peripheral blood,
␥␦ T cells compose only a small population of less than 5% of the entire CD3⫹ Tcell population. As opposed to CD4⫹ TH and
CD8⫹ cytotoxic T cells with conventional
␣␤ T-cell receptors, ␥␦ T cells are not HLA
antigen restricted and are capable of responding rapidly to pattern recognition receptor signals as a first line of defense.
Hence, ␥␦ T cells are assumed to link innate with adaptive immune responses.8 It
is possible that ␥␦ T cells also play a role in
lymphoid stress surveillance responses, implying their potential significance in tumor immunology and autoimmunity.9
However, it is unclear whether specific subsets of ␥␦ T cells, ie, ␥␦T1 vs ␥␦T17 cells,
are preferentially involved either in distinct autoimmune disorders or anatomical
niches that are affected by these autoimmune disorders. Indeed, increased fractions of IL-17–producing ␥␦ T cells have
been recovered from the peripheral blood
of patients with ankylosing spondylitis10 and
from psoriasis lesions.11
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Author Aff
Departmen
Klinikum r
Technische
München, M
Table. Clinical Characteristics of Patients With CIS/MS in Relapse, Other Neurological Autoimmune Disorders,
Neuroinfectious Diseases, and Noninflammatory Neurological Diseases
Patient No./
Sex/Age, y
EDSS Score
(at Relapse)
Diagnosis
WBC
Count, /µL
CSF Cell
Count, /µL
CSF to Serum Albumin
Ratio, ⴛ10ⴚ3/L
CSF
3
11
9
16
7
4
5
7
3
4
12
7
6
6
8
16
6
4
11
8
3
7
4
3
7
6
5.6
3.9
5.3
5.6
4.9
4.6
4.8
6.3
5.7
6.3
5.7
4.7
4.7
3.8
6.9
4.8
7.0
8.2
2.9
6.0
5.0
3.6
5.6
5.4
7.9
7.5
OCB
OCB
OCB
OCB
OCB
OCB
OCB
OCB
Polyclonal
OCB
OCB
OCB
OCB
Polyclonal
OCB
OCB
OCB
NA
OCB
OCB
OCB
OCB
OCB
OCB
OCB
OCB
Other Autoimmune Disorders
7500
8200
7200
6900
6000
5800
5900
4800
9400
6300
4
3
61
3
64
6
161
4
8
10
4.9
4.2
13.4
5.4
39.7
15.8
7.5
6.1
32.5
12.7
OCB
Polyclonal
Polyclonal
Polyclonal
Polyclonal
Polyclonal
Polyclonal
Polyclonal
Identical
OCB
Neuroinfectious Diseases
9100
7000
7200
8300
6600
6700
5500
6700
4300
10 800
13 100
6900
37
350
97
11
27
12
11
215
2
48
14
37
8.8
4.6
7.0
4.3
4.8
4.2
73.6
13.0
8.1
13.7
5.4
11.6
NA
Polyclonal
Polyclonal
Polyclonal
Polyclonal
OCB
OCB
NA
Polyclonal
Polyclonal
Polyclonal
Polyclonal
CIS/MS in Relapse
2.0
14 300
3.0
6700
1.0
5700
3.5
7900
1.0
6700
3.0
9400
3.0
12 600
3.0
13 100
1.0
6500
3.0
13 700
2.5
5200
3.0
8100
2.5
5500
2.0
11 300
3.0
8500
3.0
10 300
1.0
9600
1.0
9500
1.0
7400
3.0
6500
7.0
7600
2.5
10 100
4.0
4300
3.0
4600
2.5
8900
3.0
5200
1/F/47
2/F/25
3/M/32
4/M/26
5/F/22
6/F/25
7/F/21
8/F/38
9/M/23
10/F/25
11/F/35
12/F/31
13/F/32
14/F/29
15/F/42
16/F/53
17/M/34
18/M/29
19/F/34
20/M/49
21/M/33
22/M/31
23/F/47
24/M/35
25/F/19
26/M/34
CIS
CIS
CIS
CIS
CIS
CIS
CIS
CIS
CIS
CIS
CIS
CIS
CIS
CIS
CIS
CIS
CIS
CIS
MS in relapse
MS in relapse
MS in relapse
MS in relapse
MS in relapse
MS in relapse
MS in relapse
MS in relapse
27/F/51
28/F/69
29/F/75
30/M/68
31/M/66
32/F/21
33/M/27
34/M/48
35/F/63
36/M/70
SAPHO syndrome
Tolosa-Hunt syndrome
Temporal arteritis
Cerebral vasculitis
Neurosarcoidosis
Neurolupus
APMPPE
Guillain-Barre syndrome
Guillain-Barre syndrome
Guillain-Barre syndrome
37/F/78
38/F/35
39/F/82
40/F/40
41/M/38
42/F/24
43/F/43
44/F/24
45/M/36
46/M/56
47/M/72
48/M/51
Meningitis (viral)
Meningitis (viral)
Meningitis (viral)
Meningitis (viral)
Meningitis (viral)
Meningitis (unknown etiology)
Meningitis (bacterial)
Meningitis (bacterial)
Plexus neuritis (unknown etiology)
Radiculitis (viral)
Facial nerve palsy (viral)
Neurosyphilis
(continued)
By using the surface markers CD161 and CCR6 that
have been associated with production of IL-17 in other
immune cell subsets,12,13 we herein characterized ␥␦ Tcell subpopulations in fresh blood and CSF samples of
patients with a clinically isolated syndrome (CIS) or with
MS in relapse in comparison with inflammatory and noninflammatory control groups. Overall, we found increased fractions of ␥␦ T cells in both peripheral blood
and CSF samples of patients with CIS/MS in relapse. Only
in patients with CIS/MS in relapse, but not in controls,
the CD161high CCR6⫹ ␥␦ T-cell subset was enriched in
the CSF as compared with peripheral blood. By intracellular cytokine staining, we determined that the CD161high
CCR6⫹ ␥␦ T-cell subpopulation was characterized by the
capacity to produce IL-17 whereas CD161low CCR6⫺ ␥␦
T cells produced large amounts of IFN-␥ but no IL-17.
JAMA NEUROL/ VOL 70 (NO. 3), MAR 2013
346
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Table. Clinical Characteristics of Patients With CIS/MS in Relapse, Other Neurological Autoimmune Disorders,
Neuroinfectious Diseases, and Noninflammatory Neurological Diseases (continued)
Patient No./
Sex/Age, y
49/F/70
50/F/76
51/F/64
52/M/47
53/M/56
54/M/71
55/M/47
56/M/54
57/F/55
58/F/54
59/F/37
60/F/21
61/F/35
62/M/62
63/M/75
Diagnosis
Meningeoma
Meningeosis carcinomatosis
Parkinson disease
Myopathia (unknown etiology)
Epileptic seizure
Transient double vision (unknown etiology)
Transient paresthesia (unknown etiology)
Transient hypesthesia (unknown etiology)
AION
Subarachnoid hemorrhage
Ganglioglioma
Medulloblastoma
Amaurosis fugax
Polyneuropathia
Oculomotor palsy
EDSS Score
(at Relapse)
WBC
Count, /µL
Noninflammatory Diseases
17 100
6700
8500
5900
5500
5400
7100
6200
6900
6300
7400
7000
5300
8300
6300
CSF Cell
Count, /µL
CSF to Serum Albumin
Ratio, ⴛ10ⴚ3/L
CSF
4
4
4
14
4
13
3
4
3
57
22
30
13
5
3
5.6
19.9
6.1
7.4
10.8
15.4
6.3
9.3
5.7
6.0
4.6
5.4
4.3
8.4
15.7
NA
NA
OCB
NA
Polyclonal
NA
Polyclonal
Polyclonal
Polyclonal
Polyclonal
OCB
NA
NA
Polyclonal
Polyclonal
Abbreviations: AION, anterior ischemic optic neuropathy; APMPPE, acute posterior multifocal placoid pigment epitheliopathy; CIS, clinically isolated syndrome;
CSF, cerebrospinal fluid; EDSS, Expanded Disability Status Scale; MS, multiple sclerosis; NA, not applicable; OCB, oligoclonal band; SAPHO, synovitis, acne,
pustulosis, hyperostosis, and osteitis; WBC, white blood cell.
SI conversion factor: To convert WBC count to ⫻109/L, multiply by 0.001.
METHODS
SUBJECTS
We examined fresh peripheral blood and CSF samples from 26
patients with CIS/MS in relapse. The patients were in active relapse and not yet receiving steroid treatment. The control groups
included 10 patients with other autoimmune disorders, 12 patients with neuroinfectious diseases, and 15 patients with noninflammatory neurological diseases (Table). The patients were
between 19 and 82 years of age (mean [SD] 44 [25] years for
all patients and 33 [4] years in the CIS/MS in relapse group)
(Table). In patients with CIS/MS in relapse, the mean (SD) Expanded Disability Status Scale score at the time of blood and
CSF sampling was 2.6 (1.3). This study was approved by the
local ethics committee and informed consent was obtained from
all patients.
was carried out overnight at 4⬚C with antibodies to IFN-␥–
PacificBlue (clone B27; Invitrogen) and IL-17A–APC (clone
eBio64CAP17; eBiosciences). IgG isotype controls were run in
parallel. A live/dead staining was applied (Invitrogen) to exclude dead cells in all cases.
DATA ANALYSIS
All cells were analyzed using an FACS cytometer (CYAN; Beckman Coulter) and FlowJo Software (Treestar). For statistical evaluation, the nonparametric Kruskal-Wallis test with a Dunn multiple-comparison post hoc test was applied to compare different
␥␦ T-cell populations between patients with CIS/MS in relapse
and control patients. A Wilcoxon signed rank test was applied
for comparison of ␥␦ T-cell fractions between peripheral blood
and CSF samples. All tests were classified as significant if P was
⬍.05. GraphPad PRISM (Graph Pad Software Inc) software was
used for statistical calculations and graph generation.
SAMPLE PREPARATION
AND FLOW CYTOMETRY
RESULTS
Erythrocytes in fresh EDTA blood were lysed and leukocytes
were washed with 2% fetal calf serum/phosphate-buffered saline. Fresh CSF samples were directly washed and processed.
Ex vivo staining was carried out with the following antibodies: pan-␥␦-TCR-FITC (clone Immu510; Beckman Coulter),
CD161-PE (clone 191B8; Miltenyi Biotec Inc), CCR6-PE-Cy7
(clone 53103; R&D Systems), and CD3-APC-Cy7 (clone SK7;
BD Biosciences) for 30 minutes at 4⬚C. For functional analysis, fresh healthy donor peripheral blood mononuclear cells were
isolated using a density Ficoll-Paque (Biocoll; Biochrom) gradient centrifugation; 5 ⫻ 105 cells/well were then stimulated
in T-cell medium14 for 4 hours in the presence of phorbol 12–
myristate 13–acetate (50 ng/mL; Sigma), ionomycin (1 ␮g/
mL; Sigma), and monensin (GolgiStop, 1 ␮g/mL; BD Biosciences). On stimulation, surface staining was carried out with
antibodies to pan-␥␦-TCR-FITC, CD161-PE, CCR6-PeCy7, and
CD3-ECD (clone UCHT1; Beckman Coulter) followed by fixation and permeabilization using cytofix/cytoperm and perm/
wash buffer (BD Biosciences). Intracellular cytokine staining
␥␦ T cells have been implicated in lymphoid stress surveillance responses and, thus, might play a role in various human autoimmune disorders. Therefore, we determined the fractions of ␥␦ T cells in the blood and CSF of
patients with active MS as compared with control subjects. By ex vivo surface staining, we found significantly
higher fractions of ␥␦ T cells in both blood and CSF
samples of patients with CIS/MS in relapse as compared
with controls with noninflammatory diseases (mean [SD],
blood: 7.4% [5.2%] of CD3⫹ T cells vs 3.6% [2.8%]; CSF:
2.4% [1.6%] vs 1.2% [0.8%]) (Figure 1A). The fraction
of ␥␦ T cells in the peripheral blood is independent of
sex but decreases with age.15,16 Thus, we wished to focus
on functionally defined ␥␦ T-cell subsets and compare
them between distinct anatomical compartments within
each group. To determine the potential functional phenotype of ␥␦ T cells, we used surface markers that were
JAMA NEUROL/ VOL 70 (NO. 3), MAR 2013
347
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A
CSF
Blood
104
104
103
103
γδ TCR
12.9
4.7
102
102
101
101
100
100
100
101
102
103
104
100
101
102
103
104
CD3
γδ T Cells in % of CD3 + T Cells (Blood)
P < .05
P < .05
7.5
γδ T Cells in % of CD3 + T Cells (CSF)
24
16
8
0
Noninflammatory Infectious
B
5.0
2.5
0.0
Other
CIS/MS Relapse
Autoimmune
Noninflammatory Infectious
Blood
Other
CIS/MS Relapse
Autoimmune
CSF
104
104
103
103
4.5
34.6
102
101
101
CD161
102
100
100
100
101
102
103
104
100
101
102
103
104
CCR6
P < .05
P < .05
45
CD161high CCR6 + Cells
in % of γδ T Cells (CSF)
CD161high CCR6 + Cells
in % of γδ T Cells (Blood)
45
30
15
0
30
15
0
Noninflammatory Infectious
Other
CIS/MS Relapse
Autoimmune
Noninflammatory Infectious
Other
CIS/MS Relapse
Autoimmune
Figure 1. ␥␦ T cells and ␥␦ T-cell subsets in the blood and cerebrospinal fluid (CSF) of various populations of patients. A, Contour plots with parent gate on CD3⫹
T cells. ␥␦ T-cell frequencies (in percentage) are shown in the peripheral blood and CSF of patients with clinically isolated syndrome/multiple sclerosis in relapse
(CIS/MS in relapse) as compared with control groups (Kruskal-Wallis followed by a Dunn post hoc test). TCR indicates T-cell receptor. B, Contour plots with
parent gate on ␥␦ T cells. CD161high CCR6⫹ ␥␦ T-cell frequencies (in percentage) in blood and CSF samples of patients with CIS/MS in relapse and control patient
groups are plotted (Kruskal-Wallis followed by a Dunn post hoc test). Significant differences between groups are marked by a capped line.
recently associated with production of IL-17 in other immune cell populations. We focused on CCR6 and CD161,
which seem to be robust markers for IL-17 production
in human TH cells. Herein, we detected significantly higher
frequencies of CD161high CCR6⫹ ␥␦ T cells in the CSF of
patients with CIS/MS in relapse (mean [SD], 16.5% [11%])
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as compared with CSF samples from patients with other
autoimmune disorders (mean [SD], 5.9% [5.4%]) and patients with noninflammatory disease (mean [SD], 6.5%
[6%]) (Figure 1B). The fraction of CD161high CCR6⫹ ␥␦
T cells was also elevated in the CSF of patients with neuroinfectious disease (mean [SD], 9.5% [7.3%]). However, when we compared paired samples of peripheral
blood and CSF within the same group, we found CD161high
CCR6⫹ ␥␦ T cells to accumulate in CSF only in patients
with CIS/MS in relapse (P = .02) (Figure 2A and B), but
not in other control groups including patients with viral
or bacterial meningitis (Figure 2B). Notably, the CD161high
␥␦ T-cell population (ie, not the double-positive population) even decreased from blood to CSF in the group
with noninflammatory disease (P = .001). These data suggest that compartmentalized inflammatory conditions in
the central nervous system (CNS) might be associated
with an increase in the CSF fraction of CD161high CCR6⫹
␥␦ T cells and that this specific ␥␦ T-cell subset is particularly enriched in the CSF in relation to peripheral blood
in patients with active CNS-specific autoimmune diseases such as CIS/MS in relapse. In our cohort, we did
not find a correlation between the Expanded Disability
Status Scale score of the patients with CIS/MS in relapse
with the frequency of either the entire ␥␦ T-cell population or the fraction of the CD161high CCR6⫹ ␥␦ T cells
in the peripheral blood or CSF (data not shown).
Intracellular cytokine staining revealed that IL-17–
producing ␥␦ T cells were predominantly found in the
CD161high CCR6⫹ double-positive subpopulation (mean
[SD], 4.1% [2.5%] IL-17–only producers; 5.9% [8.1%]
IL-17 and IFN-␥ double producers; and 58.4% [7.7%] IFN␥–only producers) (Figure 2C). The CD161low CCR6⫺ compartment contained only 0.1% of IL-17 producers but was
characterized by the production of large amounts of IFN-␥
(mean [SD], 85.4% [7.7%]). CD161⫺ CCR6⫺ ␥␦ T cells
only produced low amounts of IFN-␥ (mean [SD], 47.9%
[17.9%]) and no IL-17 (mean [SD], 0.11% [0.14%]). Taken
together, our results suggest that CD161high CCR6⫹ ␥␦ T
cells that have the capacity to produce IL-17 might preferentially accumulate in the CSF in autoimmune inflammatory processes that target the CNS.
COMMENT
In the present study, we found that CD161high CCR6⫹ ␥␦
T cells are increased in the CSF of patients with infectious
CNS diseases and with CIS/MS in relapse. However, only
patients with CIS/MS in relapse showed a preferential enrichment of the CD161high CCR6⫹ ␥␦ T-cell subset in the
CSF as compared with the systemic compartment. Similar
to TH cells, the combined expression of CD161 and CCR6
defined a subset of IL-17–producing ␥␦ T cells in adult human individuals. It is unclear whether CSF-derived ␥␦ T
cells upregulate CD161 and CCR6 on local inflammatory
stimuli in the CNS or whether peripherally activated
CD161high CCR6⫹ ␥␦ T cells are recruited to the CSF. However, the fact that in viral meningitis, which is usually subsequent to or accompanied by a systemic infection, no clear
enrichment of CD161high CCR6⫹ ␥␦ T cells was detected
in CSF vs blood supports the hypothesis that local inflam-
matory stimuli in the CSF compartment might maintain
the CD161high CCR6⫹ ␥␦ T-cell population more efficiently in the CSF than in the blood of patients with MS.
In the current understanding, ␥␦ T cells that populate
the secondary lymphoid tissue (noncanonical ␥␦ T cells)
are committed to functionally distinct subsets already in
the thymus. In a series of studies in murine systems, transcriptional networks and surface markers of IFN-␥–
producing ␥␦T1 cells vs IL-17–producing ␥␦T17 cells have
been identified.17,18 It is likely that similar developmental
pathways are operational for ␥␦ T cells in humans as well.
In mice, there is strong evidence that ␥␦T17 cells are selfrenewing in the peripheral immune compartment and are
only produced by the embryonic but not the adult thymus.19 This is unclear in humans but the most convincing data that ␥␦T17 cells might be involved in human disease conditions were collected in a study on bacterial
meningitis in young children.20 Our data in patients with
active MS suggest that CD161high CCR6⫹ ␥␦ T cells that
have the potential to produce IL-17 are still detectable in
young adult individuals. Similarly, ␥␦T17 cells were reported to be present either systemically or within lesions
in patients with Mycobacterium tuberculosis infection,21 ankylosing spondylitis, and psoriasis.10,11 Some of these reports used the expression of the IL-23 receptor as a marker
for ␥␦T17 cells. It is true that the expression of the IL-23
receptor faithfully reports the capacity of ␥␦ T cells to produce IL-17, which has been elegantly shown in murine
IL-23 receptor reporter models.14,22 However, in our hands,
none of the commercially available antibodies to the human IL-23 receptor was either sensitive or specific enough
to faithfully stain those ␥␦ T cells that were to express the
molecule based on either real-time messenger RNA analysis or intracellular cytokine staining for IL-17. Therefore,
we relied on alternative markers, ie, CD161 and CCR6,
that have been proven to be associated with the production of IL-17.12,23 Herein, we show that CCR6 expression
together with CD161 on ␥␦ T cells discriminates between IL-17–producing (CD161high CCR6⫹) and IL-17–
negative (CD161low CCR6⫺) ␥␦ T cells.
Since we found an increase in CD161high–expressing ␥␦
T cells in the CSF under inflammatory conditions (be it
infectious or autoimmune), we hypothesize that the expression of the C-type lectin-like receptor CD161 might
indicate target tissue tropism of ␥␦ T cells into the CSF
compartment under inflammatory conditions. Although
binding to its ligand LLT1 might promote the entry of
CD161-positive immune cells into specific tissue niches,
it is unclear whether CD161 has a direct role in the trafficking of immune cell populations to sites of inflammation.24,25 Nevertheless, CD161high CD8⫹ T cells were recently found to markedly contribute to intralesional
immune cell infiltrates in MS lesions24 and CD161⫹ TH cells
appear to accumulate in inflammatory gut lesions.26 Moreover, the CD161 gene encoding the CD161 (KLRB1) receptor was identified as a possible susceptibility candidate gene in MS by the International Multiple Sclerosis
Genetics Consortium.27
The expression of the chemokine receptor CCR6 has
been linked to the capacity to produce IL-17 in human
TH17 cells.12,13 Binding of CCR6 to its ligand CCL20, which
is produced by epithelial cells including the plexus
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A
Blood
B
CSF
14.5
82.7
17.3
CD161high CCR6 + Cells
in % of γδ T Cells
85.5
36
Noninflammatory
diseases
Infectious diseases
30
24
20
12
10
0
0
Blood
CSF
Blood
CSF
CCR6
12.7
46.2
53.8
24
Cell Count
CD161high CCR6 + Cells
in % of γδ T Cells
87.3
Other autoimmune
disorders
45
16
30
8
15
0
CD161
CIS/MS Relapse
P < .05
0
Blood
CSF
Blood
CSF
C
104
24.6
9.49
CD161high CCR6 +
103
CD161low CCR6 –
102
CD161– CCR6 –
CD161
101
100
38.7
100
101
102
103
104
CCR6
CD161– CCR6 –
104
CD161high CCR6 +
CD161low CCR6 –
47.9 (17.9)
0.1 (0.1)
104
104
85.4 (7.7)
58.4 (7.7)
0.1 (0.1)
103
103
102
102
102
101
101
101
IFN-γ
103
5.9 (8.1)
0.1 (0.1)
100
100
101
102
103
104
0.1 (0.1)
100
100
101
102
103
4.1 (2.5)
100
104
100
101
102
103
104
IL-17
Figure 2. Cerebrospinal fluid (CSF) accumulation and functional phenotype of CD161high CCR6⫹ ␥␦ T cells. A, Histograms with parent gate on ␥␦ T cells. Differential
expression of CCR6 (upper panel) and CD161 (lower panel) on ␥␦ T cells in peripheral blood and CSF. B, Scatterplots showing differences in CD161high CCR6⫹ ␥␦
T-cell frequencies between blood and CSF in patients with clinically isolated syndrome/multiple sclerosis in relapse (CIS/MS in relapse) and control patient groups
(Wilcoxon signed rank test). (C) Contour and dot plots with parent gate on ␥␦ T cells (contour plot) and subpopulations of ␥␦ T cells according to their CD161 and
CCR6 status (lower panel). Note that interleukin 17 (IL-17)–producing ␥␦ T cells are exclusively found in the CD161high CCR6⫹ subpopulation. All numbers are given
as a percentage of parent population (parts A and C), and in dot plots (part C), numbers are given as mean (SD). Three independent experiments.
epithelium in the CNS, was proposed to guide CCR6⫹ T
cells into the inflamed CNS.28 We and others have found
that CCR6 strongly segregates with the capacity to pro-
duce IL-17 in noncanonical murine ␥␦ T cells,14,18 and
in young human patients with meningitis, IL-17–
producing ␥␦ T cells were strongly enriched in the
JAMA NEUROL/ VOL 70 (NO. 3), MAR 2013
350
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CD161⫹ CCR6⫹ T-cell receptor ␥9␦2 compartment.20
Herein, we found that CD161high CCR6⫹ ␥␦ T cells are
enriched in the CSF of patients with CIS/MS in relapse,
suggesting that this subset of ␥␦ T cells is not only associated with infectious diseases but might also play a
role in the immunopathology of CNS-specific autoimmune disorders as well.
In conclusion, in adult individuals, CD161high CCR6⫹
␥␦ T cells were specifically enriched in the CSF of patients with CIS/MS in relapse in comparison with systemic autoimmune disorders and noninflammatory neurological diseases. The CD161high CCR6⫹ subpopulation
of ␥␦ T cells is characterized by their IL-17-producing
capacity. Hence, we assume that CD161high CCR6⫹ ␥␦ T
cells might be important in the immunopathology of MS
and represent a therapeutic target in compartmentalized autoimmune reactions in the CNS.
5.
6.
7.
8.
9.
10.
11.
Accepted for Publication: August 17, 2012.
Published Online: December 17, 2012. doi:10.1001/2013
.jamaneurol.409
Correspondence: Thomas Korn, MD, Department of Neurology, Klinikum rechts der Isar, Technische Universität München, Ismaninger Str 22, 81675 Munich, Germany ([email protected]).
Author Contributions: Study concept and design: Schirmer,
Rothhammer, Hemmer, and Korn. Acquisition of data:
Schirmer, Rothhammer, and Korn. Analysis and interpretation of data: Schirmer, Rothhammer, and Korn. Drafting of the manuscript: Schirmer and Korn. Critical revision
of the manuscript for important intellectual content: Schirmer,
Rothhammer, Hemmer, and Korn. Statistical analysis:
Schirmer, Rothhammer, and Korn. Obtained funding:
Schirmer, Hemmer, and Korn. Administrative, technical, and
material support: Rothhammer, Hemmer, and Korn. Study
supervision: Korn.
Conflict of Interest Disclosures: None reported.
Funding/Support: Dr Schirmer was supported by a Du
Pré Grant of the Multiple Sclerosis International Federation and intramural funding of the medical faculty of the
Technische Universität München. Dr Korn is the recipient of a Heisenberg grant from the DFG and is supported by other grants of the DFG. Dr Hemmer was supported by a grant from the German Ministry for Education
and Research (“German Competence Network Multiple
Sclerosis,” Control-MS).
Additional Contributions: We thank Verena Grummel
and Tobias Keiner for skillful technical assistance.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
REFERENCES
26.
1. Korn T, Bettelli E, Oukka M, Kuchroo VK. IL-17 and Th17 cells. Annu Rev Immunol.
2009;27:485-517.
2. Brucklacher-Waldert V, Stuerner K, Kolster M, Wolthausen J, Tolosa E. Phenotypical and functional characterization of T helper 17 cells in multiple sclerosis.
Brain. 2009;132(pt 12):3329-3341.
3. Korn T, Petermann F. Development and function of interleukin 17-producing ␥␦
T cells. Ann N Y Acad Sci. 2012;1247(Jan):34-45.
4. Ribot JC, deBarros A, Pang DJ, et al. CD27 is a thymic determinant of the bal-
27.
28.
JAMA NEUROL/ VOL 70 (NO. 3), MAR 2013
351
ance between interferon-gamma- and interleukin 17-producing gammadelta T
cell subsets. Nat Immunol. 2009;10(4):427-436.
Wucherpfennig KW, Newcombe J, Li H, Keddy C, Cuzner ML, Hafler DA. Gamma
delta T-cell receptor repertoire in acute multiple sclerosis lesions. Proc Natl Acad
Sci U S A. 1992;89(10):4588-4592.
Selmaj K, Brosnan CF, Raine CS. Colocalization of lymphocytes bearing gamma
delta T-cell receptor and heat shock protein hsp65⫹ oligodendrocytes in multiple sclerosis. Proc Natl Acad Sci U S A. 1991;88(15):6452-6456.
Poggi A, Zocchi MR, Costa P, et al. IL-12-mediated NKRP1A up-regulation and
consequent enhancement of endothelial transmigration of V delta 2⫹ TCR gamma
delta⫹ T lymphocytes from healthy donors and multiple sclerosis patients.
J Immunol. 1999;162(7):4349-4354.
Martin B, Hirota K, Cua DJ, Stockinger B, Veldhoen M. Interleukin-17-producing
gammadelta T cells selectively expand in response to pathogen products and
environmental signals. Immunity. 2009;31(2):321-330.
Hayday AC. Gammadelta T cells and the lymphoid stress-surveillance response.
Immunity. 2009;31(2):184-196.
Kenna TJ, Davidson SI, Duan R, et al. Enrichment of circulating IL-17-secreting
IL-23 receptor-positive gammadelta T cells in patients with active ankylosing
spondylitis. Arthritis Rheum. 2012;64(5):1420-1429.
Cai Y, Shen X, Ding C, et al. Pivotal role of dermal IL-17-producing ␥␦ T cells in
skin inflammation. Immunity. 2011;35(4):596-610.
Cosmi L, De Palma R, Santarlasci V, et al. Human interleukin 17-producing cells originate from a CD161⫹CD4⫹ T cell precursor. J Exp Med. 2008;205(8):1903-1916.
Acosta-Rodriguez EV, Rivino L, Geginat J, et al. Surface phenotype and antigenic specificity of human interleukin 17-producing T helper memory cells. Nat
Immunol. 2007;8(6):639-646.
Petermann F, Rothhammer V, Claussen MC, et al. ␥␦ T cells enhance autoimmunity by restraining regulatory T cell responses via an interleukin-23dependent mechanism. Immunity. 2010;33(3):351-363.
Cairo C, Armstrong CL, Cummings JS, et al. Impact of age, gender, and race on
circulating ␥␦ T cells. Hum Immunol. 2010;71(10):968-975.
Michishita Y, Hirokawa M, Guo YM, et al. Age-associated alteration of ␥␦ T-cell
repertoire and different profiles of activation-induced death of V␦1 and V␦2 T
cells. Int J Hematol. 2011;94(3):230-240.
Turchinovich G, Hayday AC. Skint-1 identifies a common molecular mechanism
for the development of interferon-␥-secreting versus interleukin-17-secreting ␥␦
T cells. Immunity. 2011;35(1):59-68.
Haas JD, González FH, Schmitz S, et al. CCR6 and NK1.1 distinguish between
IL-17A and IFN-gamma-producing gammadelta effector T cells. Eur J Immunol.
2009;39(12):3488-3497.
Haas JD, Ravens S, Düber S, et al. Development of interleukin-17-producing ␥␦ T
cells is restricted to a functional embryonic wave. Immunity. 2012;37(1):48-59.
Caccamo N, La Mendola C, Orlando V, et al. Differentiation, phenotype, and function of interleukin-17-producing human V␥9V␦2 T cells. Blood. 2011;118(1):
129-138.
Peng MY, Wang ZH, Yao CY, et al. Interleukin 17-producing gamma delta T cells
increased in patients with active pulmonary tuberculosis. Cell Mol Immunol. 2008;
5(3):203-208.
McGeachy MJ, Chen Y, Tato CM, et al. The interleukin 23 receptor is essential
for the terminal differentiation of interleukin 17-producing effector T helper cells
in vivo. Nat Immunol. 2009;10(3):314-324.
Maggi L, Santarlasci V, Capone M, et al. CD161 is a marker of all human IL-17producing T-cell subsets and is induced by RORC. Eur J Immunol. 2010;40
(8):2174-2181.
Annibali V, Ristori G, Angelini DF, et al. CD161(high)CD8⫹T cells bear pathogenetic potential in multiple sclerosis. Brain. 2011;134(pt 2):542-554.
Rosen DB, Cao W, Avery DT, et al. Functional consequences of interactions between human NKR-P1A and its ligand LLT1 expressed on activated dendritic cells
and B cells. J Immunol. 2008;180(10):6508-6517.
Kleinschek MA, Boniface K, Sadekova S, et al. Circulating and gut-resident human Th17 cells express CD161 and promote intestinal inflammation. J Exp Med.
2009;206(3):525-534.
Hafler DA, Compston A, Sawcer S, et al; International Multiple Sclerosis Genetics Consortium. Risk alleles for multiple sclerosis identified by a genomewide
study. N Engl J Med. 2007;357(9):851-862.
Reboldi A, Coisne C, Baumjohann D, et al. C-C chemokine receptor 6-regulated
entry of TH-17 cells into the CNS through the choroid plexus is required for the
initiation of EAE. Nat Immunol. 2009;10(5):514-523.
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