Download CD161 defines the subset of FoxP3+ T cells capable of producing

From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
Regular Article
IMMUNOBIOLOGY
CD161 defines the subset of FoxP31 T cells capable of producing
proinflammatory cytokines
Anne M. Pesenacker,1 David Bending,1 Simona Ursu,1 Qiong Wu,1 Kiran Nistala,2 and Lucy R. Wedderburn1
1
Rheumatology Unit, UCL Institute of Child Health, University College London, London, United Kingdom; and 2Rheumatology Unit, Department of Medicine,
University College London, London, United Kingdom
Regulatory FoxP31CD41 T cells (Treg) are vital for maintaining the balance between
tolerance, adequate immune response, and autoimmunity. Despite this immunoregulatory
role, it has been shown that Treg may also produce proinflammatory cytokines. Here we
• CD161 defines
present a distinct population of Treg, defined by CD161 expression, as the major source of
proinflammatory FoxP31
FoxP31 Treg-derived proinflammatory cytokines. CD1611 Treg can be followed throughcells that have classic Treg
signatures, yet share effector out development, from thymus and cord blood to healthy child and adult samples. CD1611
Treg display anergy, are suppressive in cocultures with conventional T cells (Tconv), and
T-cell properties.
possess a predominantly demethylated Treg-specific demethylated region of the FOXP3
• CD1611 Treg proinflammatory
locus. In addition to the production of interleukin (IL) 17A, interferon g, and IL-2, CD1611
phenotype is stable upon Treg FoxP31 cells share markers with Tconv, including expression of the transcription factors
expansion and thus should be retinoic acid-related orphan receptor Cv2 (RORCv2) and T-cell-specific T-box transcription
considered in therapeutic
factor (Tbet). Expression of CD161 and enrichment for cytokine production are stable
strategies using Treg.
characteristics of CD1611 Treg upon both short- and longer-term culture in vitro.
Additionally, CD1611 Treg are highly enriched within the inflammatory environment
of childhood arthritis, suggesting a role in disease. Our data therefore demonstrate that CD1611FoxP31 T cells are a novel Treg
subset, found in health and disease, which display high proinflammatory potential but also exhibit hallmark Treg characteristics.
(Blood. 2013;121(14):2647-2658)
Key Points
Introduction
The maintenance of immune tolerance is a key function of the
immune system, mediated in part by the proper functioning of regulatory cells. One important population of regulatory CD41 T cells
(Treg) is defined by expression of the transcription factor FoxP3,
which is essential for their suppressive capacity toward effector cells.
Treg are thought to arise either early in ontogeny in the thymus
(natural Treg) or by induction in the periphery (induced Treg). Given
the position of FoxP3 as a master regulator of the Treg program,
genetic mutations to the FOXP3 gene can lead to severe autoimmunity, as exemplified by the multiple immunopathologies
exhibited by patients with immunodysregulation, polyendocrinopathy, enteropathy, X-linked syndrome.1,2
In recent years, several different subsets of FoxP31 Treg have been
identified. Memory Treg have been proposed, defined by lack
of CD45RA expression3 and/or CC chemokine receptor (CCR) 6
expression.4 CCR6 is also an important chemokine receptor found
predominantly on effector T helper (Th) 17 cells. Similarly, Treg
expressing the Th1-associated chemokine receptor CXC chemokine
receptor 3 have been identified.5 Thus, it has been proposed that differing chemokine receptor expression patterns on Treg confers the
ability of such cells to colocalize with, and therefore regulate,
different types of immune response.6
Many links between Treg and Th17 cells have been established,
possibly indicating their coevolutionary development.7 We have
previously shown that Treg and Th17 cells have a reciprocal
relationship at the site of inflammation in autoimmune childhood
arthritis,8 and a higher frequency of Treg is associated with a milder
disease course.9 The mere presence of Treg, however, does not
always ensure tolerance, as functionality is vital. A central function
of Treg is suppression of inflammatory responses, and it was
previously thought that a hallmark of Treg was their lack of cytokine
production.10,11 However, recent studies have demonstrated that
a small proportion of Treg is able to produce proinflammatory cytokines.12,13 Following ex vivo isolation, these cytokine-producing
Treg were still able to suppress in vitro and showed demethylation of
the Treg-specific demethylated region (TSDR) of the FOXP3 gene,
a feature that is widely considered to be an epigenetic requisite for
a stable Treg program.12,14
Other studies, which took the approach of expanding or stimulating Treg in vitro, suggested that interferon g (IFN-g) or
Submitted August 4, 2012; accepted January 19, 2013. Prepublished online as
Blood First Edition paper, January 25, 2013; DOI 10.1182/blood-2012-08443473.
The online version of this article contains a data supplement.
Part of the work presented in abstract form at the annual congress of the
British Society of Immunology 2011, Liverpool, United Kingdom, December 8,
2011.
BLOOD, 4 APRIL 2013 x VOLUME 121, NUMBER 14
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked “advertisement” in accordance with 18 USC section 1734.
© 2013 by The American Society of Hematology
2647
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2648
PESENACKER et al
interleukin (IL) 17A could be induced, depending on culture
conditions.15,16 These in vitro manipulated Treg were still able to
suppress conventional T cells (Tconv) proliferation. These interesting
observations imply that the boundaries between Tconv and Treg
programs may be more blurred than previously thought and raise
several important questions, in particular, how such cells arise in vivo
in humans and their functional relevance in health and disease. In
addition, a marker to identify such “hybrid” Treg cells would be of
great interest to those who wish to employ Treg therapies.
In this paper, we have identified the lectin-like receptor, CD161,
as the marker of FoxP31 Treg in humans that harbor proinflammatory potential. CD161 expression has previously been linked to Th17
cells,17 but we believe this to be the first report that definitively links
CD161 expression to the population of FoxP31 cells that display
inflammatory signatures. We report the novel findings that CD1611
Treg are bona fide suppressors and display epigenetic modifications
associated with stable FoxP3 expression. The cytokine-producing
phenotype of CD1611FoxP31 cells persists over longer-term Treg
expansion. CD1611 Treg exist in healthy individuals across a wide
range of ages and tissues. Additionally, we show that such cells are
increased at the sites of inflammation in children with autoimmune
arthritis. We propose that such cells may play important roles in
dictating the balance between immunity and tolerance at sites of
infection and inflammation. However, given their inflammatory
potential, we suggest that CD1611 Treg need to be taken into
account in Treg preparation that may be used for adoptive transfer
cellular therapies in human.
Materials and methods
Human samples and cells
Blood samples were obtained from 39 healthy adult volunteers and 12 healthy
children (aged 1-10 years; male/female, 7:4), all with no known autoimmune
or genetic conditions. Umbilical cord blood (UCB) samples were obtained
from placental cords after normal delivery of healthy infants. Forty-eight
patients with juvenile idiopathic arthritis (JIA; aged 3-16 years; male/female, 8:
36) were included in this study, of which 37 had oligoarticular JIA (O-JIA) and
11 polyarticular JIA (P-JIA) as classified according to internationally agreed
criteria.18 Synovial fluid (SF) mononuclear cells, peripheral blood mononuclear cells (PBMC), and UCB mononuclear cells were prepared by density
gradient centrifugation using LymphoPrep (Axis-Shield) with SF samples
undergoing pretreatment with hyaluronidase (10 U/mL; Sigma-Aldrich).8
Human thymus samples were obtained after surgical removal in children
undergoing corrective cardiac surgery. Thymocytes were isolated by gentle
tissue disruption followed by sieving into RPMI 1640 supplemented with 10%
fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin (Life
Technologies) and used immediately. The study had full ethical approval
from the Local Research Ethics Committee; in accordance with the Declaration
of Helsinki, fully informed consent was obtained from parents of all children
included (with age-appropriate child assent) and from adult donors.
Flow cytometry and cell sorting
Five- to 8-color flow cytometry was performed with directly conjugated
antibodies (supplemental Table 1; see the Blood Web site) using standard
techniques19; dead cells were excluded using LIVE/DEAD Fixable blue Dead
Cell Stain (Life Technologies). Intracellular staining for cytokines and
transcription factors was performed using a FoxP3 buffer kit (eBioscences).
Cytokine production was assessed after 3-hour stimulation of cells at 37°C in
the presence of phorbol 12-myristate 13-acetate (PMA) (0.05 mg/mL),
ionomycin (0.5 mg/mL), and brefeldin A (5 mg/mL) (Sigma-Aldrich).
Cell sorting was performed with a MoFlo XDP cell sorter (Beckman
Coulter) after preenrichment of CD41 T cells by immunomagnetic negative
BLOOD, 4 APRIL 2013 x VOLUME 121, NUMBER 14
selection (Stemcell Technologies). Gates were set on single lymphocyte
population; dead cells were excluded based on their uptake of 4,6 diamidino2-phenylindole stain (Sigma-Aldrich). Treg were defined as CD41
CD25hiCD127lo cells; Tconv were defined as CD41CD25–CD127hi cells.
Purity and FoxP3 expression was assessed on all sorted cells. Unless specified,
sorted subsets reaching >90% purity based on FoxP31 (Treg) or FoxP3–
(Tconv) staining were used in assays.
Real-time polymerase chain reaction (RT-PCR)
Messenger RNA (mRNA) was extracted from sorted cells according to the
manufacturer’s instructions (Trizol; Life Technologies) and converted to
complementary DNA using priming with random hexamers.19 RT-PCR was
performed using SYBR-green (Biorad) and primers for FOXP3, RORCv2
(variant 2), TBX21 (Tbet), IL23R, and ACTB (b-actin) (supplemental Table 2;
primer design using Primer320). Amplification was performed on the
Rotorgene6000 (Corbett Life Science) with the following cycling conditions:
95°C for 5 minutes, 45 cycles of 95°C for 30 seconds, 60°C for 30 seconds,
and 72°C for 30 seconds, followed by 5 minutes at 72°C. Melt curve and
SYBR-green emission data were collected. Relative concentrations were
calculated using a standard curve; values were normalized to amplification
products of b-actin.
In vitro proliferation and suppression assays
To assess in vitro proliferation or anergy, sorted cells (CD1611 Treg [CD41
CD25hiCD127loCD1611], CD161– Treg [CD41CD25hiCD127loCD161–],
and/or Tconv [CD41CD25–CD127hi]) were carboxyfluorescein diacetate
succinimidyl ester (CFSE) labeled. Briefly, cells were labeled in 1 mM CFSE
solution for 10 minutes at room temperature in the dark, followed by addition
of an equal volume of FBS for 10 minutes at room temperature before
multiple washes with medium. Labeled cells cultured at 5 3 104 per well in Vbottom 96-well plates (CoStar), coated with 1 mg/mL anti-CD3 (clone UCHT1;
R&D systems) 6 5 mg/mL anti-CD28 (clone CD28.2; BD Biosciences)
antibodies, in culture medium (RPMI 1640 supplemented with 10% FBS and
100 U/mL penicillin/streptomycin), at 37°C and 5% CO2. After 5 days, cells
were restimulated as described previously. CFSE dilution, IL-17A, and IFN-g
production were assessed by flow cytometry.
Suppression assays were performed with 5 3 104 CFSE-labeled Tconv
and unlabeled CD1611 or CD161– Treg at a 1:1 ratio under the same
conditions as the proliferation assay. CFSE dilution, IL-17A, and IFN-g production were assessed by flow cytometry. Cytokine protein levels in culture
supernatants were assessed by multiplex immunoassay.21 Suppression of proliferation was calculated using the FlowJo %-divided function. The condition
Tconv alone was set as 0% suppression.
CD161 expression stability
Sorted CD1611 Treg or CD161– Treg subpopulations were CFSE labeled
and spiked at a frequency of 5% to 7% into cultures of 1 3 105 autologous
PBMC in U-bottom 96-well plates (CoStar), precoated with 1 mg/mL antiCD3 and 5 mg/mL anti-CD28, without cytokine or in the presence of IL-2, IL6 (BD), or IL-12 (R&D; all 10 ng/mL), at 37°C in 5% CO2. After 3 days, cells
were restimulated, and CD161 expression and cytokine production were
assessed by flow cytometry.
For Treg expansion, 5 3 104 sorted Treg were cultured in V-bottom plates
coated with 1 mg/mL anti-CD3 and 5 mg/mL anti-CD28, in the presence of
100 U/mL IL-2 (Roche) for 4, 6, or 10 days. IL-2 was renewed every 2 days.
At the end of the culture, CD161 and FoxP3 expression and cytokine production were assessed by flow cytometry.
FOXP3 TSDR methylation analysis
DNA was extracted from sorted cell subsets (purity of >80%) using the DNeasy
blood and tissue kit and bisulfite treated using the Epitect Bisulfite Kit (both
Qiagen) according to the manufacturer’s instructions. A 336–base pair segment
containing the TSDR of FOXP3 was amplified using published primers22 using
platinum high-fidelity Taq (Life Technologies), followed by cloning of PCR
products (TOPO-TA kit). Twenty to 35 clones per subset were sequenced, and
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
BLOOD, 4 APRIL 2013 x VOLUME 121, NUMBER 14
CD161 DEFINES PROINFLAMMATORY FOXP31 T CELLS
2649
Figure 1. CD161 expression by regulatory T cells. Expression of CD161 on CD41FoxP31 T cells was assessed by flow cytometry. Representative fluorescence-activated cell
sorter (FACS) plots of FoxP3 expression gated on CD41 lymphocytes (left plot) and CD161 staining gated on FoxP31CD41 lymphocytes (right plot) in healthy adult (n 5 39; left
panels) and healthy child (n 5 12; right panels) blood samples (summary plot far right) (A), and in cord blood (UCB; n 5 5; left panels) and mature CD4 single positive thymocytes,
defined by CD1a–CD31CD41CD8– (n 5 4; right panels) (B). (C) No difference in FoxP3 protein levels between CD161–FoxP31 and CD1611FoxP31 Treg. Representative
histograms overlay, left (white, CD161–FoxP31; black, CD1611FoxP31), with summary plot, right, of FoxP3 staining intensity (MFI) on CD161–FoxP31 (clear symbols) and CD1611
FoxP31 (filled symbols) CD41 lymphocytes for healthy adult (n 5 37) and healthy child (n 5 12) samples. (D) Nonlinear regression between CD1611 Treg and age in healthy child and
adult samples (n 5 31), 1-site–binding hyperbola. Horizontal bars represent the medians in all summary plots. Statistical analysis by Wilcoxon matched pairs test. **P , .01; *P , .05.
from these, the percentage of clones displaying methylated cytosine guanine
dinucleotide (CpG) for each site and total average were determined.
Statistical tests
Statistical analysis was performed using Prism 4 for Macintosh (GraphPad)
software. Data are shown as mean 6 standard error of the mean (SEM). The
Mann-Whitney, Wilcoxon matched pairs test, or paired t test were applied
to compare 2 groups; the 1-way analysis of variance (ANOVA) with
Bonferroni’s post hoc tests was used to compare 3 or more means. Best fit of
correlation was measured by the root mean square. All P values , .05 were
considered statistically significant. In the figures, P values are displayed
according to the following scheme: ***P , .001; **P , .01; *P , .05.
Results
CD1611FoxP31 cells exist at different development stages
Within the CD41 T-cell lineage, expression of CD161 has been
shown to define a population of cells that contains Th17 cells, as
well as “ex-Th17” cells, which have lost their initial IL-17A
production and gained IFN-g production.23,24 However, CD161
expression has not been typically associated with Treg. During our
study of human Treg, we observed a subpopulation that also
expresses CD161 and is present at a median frequency of 14% of
FoxP31CD41 peripheral blood T cells in healthy adult donors
(Figure 1A). Comparable results were seen with 3 different FoxP3
antibody clones, and CD1611FoxP31 cells were CD25hiCD127lo
(supplemental Figure 1). Interestingly, this population was also
observed in the blood of healthy children, although at a significantly lower frequency (median, 8.1%) than in adults (Figure 1A).
To assess when these CD1611FoxP31 cells arise during
development of the immune system, mature thymocytes and
UCB were analyzed as sources of immunologic inexperienced and
neonatal peripheral T cells. CD1611FoxP31 cells were clearly
demonstrated in both cord blood CD41 T cells and also in mature
CD4 single positive thymocytes (defined by CD1a–CD31CD41
CD8–; Figure 1B). Together these data show that CD1611FoxP31
cells are present within the CD4 T-cell population at different stages
of development.
It has been previously shown that expression of FoxP3 protein
in human T cells may be induced by cell activation and that
recently activated “FoxP3 intermediate” CD41 T cells may not have
suppressive function.25 To investigate whether the CD1611FoxP31
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2650
PESENACKER et al
BLOOD, 4 APRIL 2013 x VOLUME 121, NUMBER 14
Figure 2. CD1611FoxP31CD41 T cells are suppressive and anergic in vitro. Treg were sorted by flow cytometry. (A) Sorting strategy for subpopulations of Treg defined
by CD25hiCD127lo, and CD161 expression (a, CD161–; b, CD1611), with representative sort purity of $90% purity for both subsets for FoxP3 and CD25 (dot plots) and
CD161 staining (overlay histogram). (B) Both CD1611 and CD161– Treg are anergic in vitro. Cells were sorted, labeled with CFSE, and stimulated in vitro for 5 days with antiCD3 antibody. Representative histograms showing CFSE dilution of sorted CD161– Treg (left), CD1611 Treg (middle), and Tconv (CD25–CD1271, right) from healthy adult
(1 of 2). Numbers above the bars represent the percentage of divided cells. (C) CD1611 Treg suppress proliferation and cytokine production. Sorted Tconv were labeled with
CFSE, mixed with unlabeled Treg subpopulations as shown, and stimulated for 5 days as described, before analysis by flow cytometry. Representative plots show CFSE
dilution, and the production of IFN-g (upper panels) or IL-17A (lower panels) by Tconv; summary plots at day 5 for the % suppression of division of Tconv in the various
cultures (n 5 4; statistical analysis, 1-way ANOVA). (D) IFN-g and IL-17A protein levels were assessed in the supernatants from cocultures by Luminex assay. Data in dot
plots and histograms are gated on CD41 lymphocytes; all summary plots show mean 6 SEM. ***P , .001; ns, not significant.
population has lower expression of FoxP3 compared with CD161–
FoxP31 cells, we compared the mean fluorescence intensity (MFI) of
FoxP3 protein in the 2 populations. In adult donors, there was no
difference in intensity of FoxP3 staining (MFI) between CD1611 and
CD161–FoxP31 cells, whereas in child donors, CD1611FoxP31
cells actually had a slight but significantly higher median level of
FoxP3 protein than CD161– cells (representative histogram and
summary data; Figure 1C).
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
BLOOD, 4 APRIL 2013 x VOLUME 121, NUMBER 14
CD161 DEFINES PROINFLAMMATORY FOXP31 T CELLS
2651
Figure 3. CD1611FoxP31 regulatory T cells share phenotypic features with memory effector T cells. (A) Production of cytokines ex vivo in Treg subpopulations was
assessed by intracellular flow cytometric staining after stimulation with PMA and ionomycin. Dot plots show intracellular cytokine staining for IL-17A (top left), IFN-g (top right),
and IL-2 (bottom left) gated on FoxP31CD161– and FoxP31CD1611 peripheral blood Treg, respectively, in 1 representative healthy adult, with summary plots (bottom right)
of staining on healthy adult (n 5 13-19) and child (n 5 4-6) peripheral blood samples. (B) Representative staining showing double positive cytokine production (left, IL-17A vs
IFN-g; right, IFN-g vs IL-2) comparing CD161–FoxP31 to CD1611FoxP31 cells. (C) Expression of effector cell markers on CD1611FoxP31 cells. Representative plots
showing expression of CCR6 (top), IL-23R (middle), and CD45RO (bottom) on FoxP31CD161– and FoxP31CD1611 Treg, respectively, with summary plots (right) for
healthy adult (n 5 16-28) and child (n 5 6-8) peripheral blood samples. (D) Transcription factor expression in CD1611FoxP31 cells. Fold change in mRNA expression of
FOXP3, RORCv2, and TBX21 (Tbet) in sorted CD1611 Treg relative to sorted CD161– Treg assayed by RT-PCR (n 5 3-6; top). RORCv2 and Tbet protein expression was
assessed by flow cytometry ex vivo. Representative FACS plots of expression of RORCv2 (middle left) and Tbet (middle right) in CD161–FoxP31 (open histogram) compared
with CD1611FoxP31 cells (filled histogram) and isotype (gray histogram); summary graphs of FACS data are shown below (n 5 7). Data in dot plots and histograms are
derived from gated CD41FoxP31 lymphocytes. In scatter plots, horizontal lines represent median; in bar graph, bars represent mean 6 SEM with Wilcoxon matched pairs
test between cell subsets, and between groups 1-way nonparametric ANOVA (Kruskal-Wallis with Dunn’s test). ***P , .001; **P , .01; *P , .05.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2652
PESENACKER et al
When these data were analyzed by frequency of CD1611
FoxP31 cells within the CD41 population, across the whole age
range, a rise in frequency was seen through childhood, which
plateaued during the adult years; these data were best described by
a 1-site–binding hyperbola (Figure 1D).
CD1611FoxP31 cells behave like classic Treg in vitro
Although FoxP3 expression is one important feature of Treg, we
wished to assess the functional capacity of these subpopulations. To
assess the regulatory status of CD1611FoxP31 cells, we investigated whether these cells were anergic when cultured alone in vitro,
and whether they had the capacity to suppress the proliferation of
conventional CD4 T cells (Tconv). Cells were sorted according to the
schematic in Figure 2A to generate Tconv (CD41CD25–CD127hi)
and 2 populations of putative Treg (CD41CD25hiCD127loCD1611
and CD41CD25hiCD127loCD161–; sort purities of all subsets
>90% [representative sort, Figure 2A]).
Both CD1611 and CD161– Treg populations were anergic
(proliferation assessed by CFSE dilution) when cultured alone upon
T-cell stimulation (Figure 2B). Suppression of proliferation of Tconv
by Treg is considered to be a definitive property of classic Treg. The
capacity of CD161– Treg and CD1611 Treg to suppress proliferation
was equivalent as assessed by CFSE dilution of Tconv (Figure 2C),
and upon titration of Treg, no difference was seen (data not shown). In
addition, both Treg subpopulations suppressed production of IFN-g,
but not IL-17A (intracellular cytokine staining, Figure 2C; secreted
cytokine in the supernatant, Figure 2D). Together these data suggest
that the proposed CD1611 Treg population that we have identified,
with the phenotype CD1611FoxP31CD127loCD25hiCD41, can be
classified as regulatory in that it shows both suppressive capacity and
anergy in vitro.
CD1611FoxP31 T cells share phenotypic features with memory
effector T cells
As part of their phenotype, Treg typically are not proinflammatory.
However, some reports suggest that a small proportion of Treg can
make proinflammatory cytokines.12,13,15 Interestingly, ex vivo cytokine staining of healthy PBMC showed a higher frequency of cells
producing proinflammatory cytokines within the CD1611FoxP31
population than the CD161–FoxP31 population (Figure 3A). The
CD1611FoxP31 population had a significantly higher proportion of
cells that produce IL-17A, IFN-g, and surprisingly IL-2, with low or
absent cytokine production within the CD161–FoxP31 population.
A proportion of CD1611FoxP31 cells expressed more than 1
cytokine (Figure 3B). These distinct functional features were clearly
apparent in blood from both adults and children, where the majority
of cytokine-producing Treg is defined by CD161 expression. CD127
has been shown to inversely correlate with FoxP3 expression and
suppressive functions in Treg26: cytokine-producing CD1611
FoxP31 cells were also CD127 low (supplemental Figure 2A).
Because intracellular cytokine staining may not correlate precisely
with secretion, we investigated the capacity of Treg to release the proinflammatory cytokine IL-17. Following cytokine capture, intracellular
FoxP3 staining was performed, and cells were analyzed based on their
expression of CD161 (supplemental Figure 2B). Captured IL-17A
amount was comparable with intracellular cytokine staining, showing
that IL-17A is produced and secreted by FoxP31CD1611 cells.
Furthermore, CD1611 Treg produced cytokines during suppression
assays (supplemental Figure 2C).
Given the evidence for developmental and phenotypic relationships between Treg and Th17 cells, surface markers known to be
BLOOD, 4 APRIL 2013 x VOLUME 121, NUMBER 14
expressed on Th17 cells, such as CCR6, IL-23R,27 and the memory
marker CD45RO,28 were investigated. In both adults and children,
proportions of cells expressing CCR6 and IL-23R were significantly
increased in CD1611FoxP31 compared with CD161–FoxP31 cells,
whereas the majority of CD1611FoxP31 cells were within the
CD45RO1 population, even in the blood of young children (Figure 3C).
We next investigated whether the proinflammatory potential of
CD1611FoxP31 cells is associated with expression of transcription
factors typical of Th1 or Th17 cells. RT-PCR for TBX21 (Tbet) and
RORCv2 revealed a significant increase of the Th17-associated transcription factor RORCv2 (mean fold increase of 6.4, P , .05) and
a trend toward increased Tbet mRNA (mean fold increase of 2.0),
linked to IFN-g production, in CD1611FoxP31 compared with
CD161–FoxP31 cells (Figure 3D, top). There was no difference in
the amount of FOXP3 mRNA between the 2 populations. Significant
increases in protein levels of both RORCv2 and Tbet in CD1611
FoxP31 cells were also confirmed by flow cytometry (Figure 3D,
middle and bottom by MFI). Furthermore, supporting previous
evidence,29 in vitro antigen challenge (Candida albicans, hCMV)
elicited cytokine-polarized responses in CD1611FoxP31 but not
CD161–FoxP31 cells (supplemental Figure 3).
Together these data suggest that CD1611FoxP31 cells are
distinct in their proinflammatory cytokine production and show a
memory phenotype with overlap with both Th17 and Th1 programs.
CD1611 Treg use a diverse TCR-Vb repertoire
CD161 expression has been associated with invariant T-cell receptor
(TCR) expression on natural killer T and some CD81 populations,
which express a limited range of TCR-Vb chain proteins.30,31 To test
the TCR-Vb repertoire on CD1611FoxP31 cells, flow cytometry
staining for TCR-Vb families was performed. The expression of 24
TCR-Vb families was investigated within 5 different T-cell subsets:
total CD41, CD161–FoxP31CD41, CD1611FoxP31CD41,
CD161–FoxP3–CD41, and CD1611FoxP3–CD41 lymphocytes
(Figure 4). TCR-Vb families examined by the panel of available
antibodies covered a mean of 70.18% (6 standard deviation of
8.268) of all analyzed cells, representing the majority of cells within
each subset investigated. The TCR-Vb repertoire was comparable
between all subsets tested, indicating that CD1611FoxP31 cells do
not express an invariant TCR but have a diverse repertoire comparable with CD161–FoxP31 and Tconv.
To investigate the relationship between CD1611 and CD161–
Treg, the clonality of TCR was tested by sequence analysis across the
variable diversity joining junction.32 CD1611 and CD161– Treg
were sorted, and CDR3 regions of TCR-Vb2 cloned and sequenced
(supplemental Figure 4). Both Treg subsets showed clonality, a total
of 345 sequences (170 CD161– Treg, 175 CD1611 Treg), with 122
clonal sequences for CD161– and 118 clonal sequences for CD1611
Treg, which were predominantly nonoverlapping (2 sequences found
in both subsets). These data raise the possibility that CD1611 Treg
may have a distinct origin from CD161– Treg.
CD1611 Treg have a predominantly demethylated TSDR of
FOXP3, and their phenotype is stable in vitro
To investigate the stability of CD161 expression on Treg, CD161–
and CD1611 Treg were sorted, labeled, and followed in an in vitro
culture system where they were present at a frequency of 5% to 7% of
unlabeled total PBMC (for schematic, see Figure 5A). Unstimulated
(Figure 5C) and anti-CD3/CD28 stimulated with or without cytokine
treatment conditions (Figure 5D) were tested in both systems. No
differences in viability between conditions were seen (data not shown).
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
BLOOD, 4 APRIL 2013 x VOLUME 121, NUMBER 14
CD161 DEFINES PROINFLAMMATORY FOXP31 T CELLS
2653
Figure 4. CD1611 regulatory T cells do not express an invariant TCR. Frequency of TCR-Vb expression was assessed using a flow cytometry panel to detect 24 Vb
families, in healthy adult CD41 T cells, CD161–FoxP31CD41, CD1611FoxP31CD41, CD1612FoxP3–CD41, and CD1611FoxP3–CD41 cell populations as shown (n 5 5).
Data were gated on the specific population and then each TCR-Vb. Pie charts show proportion of all cells positive for a specific TCR-Vb (which identify a mean of 70.18% of all
cells within respective subset).
CD1611 Treg (filled histogram and bars) retained expression of
CD161 (Figure 5C-D), whereas CD161– cells (clear histograms and
white bars) did not acquire CD161 expression (supplemental Figure 5).
CD161 expression was stable under all conditions, although CD161
expression per cell (MFI) decreased slightly at 3 days (Figure 5C-D).
It has been suggested that both IL-12 and IL-2 have an effect on
CD161 expression.33,34 IL-12 is linked to IFN-g production by
T cells35; IL-2 is an important survival factor for Treg36,37; and IL-6,
transforming growth factor b (TGF-b)1IL-1b1IL-23 can drive IL17A production.38,39 IL-6 and IL-12 are also abundant at inflammatory
sites, for example, within affected joints of children with arthritis.23,40
Therefore, we tested if we could further “skew” the phenotype of
CD1611 Treg by culturing in the presence of cytokines. Addition of
IL-12, IL-6, IL-2, or TGF-b1IL-1b1IL-23 to the culture did not alter
stability of CD161 expression, although a trend toward increased
CD161 levels was demonstrated when IL-12 was present (Figure 5D,
top). Additionally, CD1611FoxP31 cells maintained their cytokine
phenotype, producing IL-17A and IFN-g without further stimulation
(Figure 5C, right and far right) and after T-cell activation (Figure 5D,
bottom). Addition of cytokines to the culture system showed that as
expected IL-12 enhanced IFN-g production and limited IL-17A
production, whereas IL-2, IL-6, and TGF-b1IL-1b1IL-23 had little
effect (Figure 5D, bottom).
Recently, the importance of epigenetic modifications for Treg
stability has emerged.14,22 DNA demethylation at the TSDR of the
FOXP3 locus has been deemed crucial for Treg stability. CD161–
Treg and CD1611 Treg both showed a predominantly demethylated TSDR (Figure 5E). Twelve CpG islands within the TSDR of the
FOXP3 locus were analyzed for sorted Tconv, CD161– Treg, and
CD1611 Treg (top to bottom, purity .80%). Across 3 male
individuals, the TSDR of Tconv showed full methylation (96.4% [6
0.8]), CD161– Treg showed a small residual methylation (7.4% [6
2.5]), and CD1611 Treg showed 28.5% (62.1).
Together these data suggest that CD1611 Treg are a stable
subset with predominantly demethylated TSDR at the FOXP3 locus
and that they retain CD161 expression upon TCR stimulation,
whereas CD161– Treg did not gain CD161 expression.
The ex vivo characteristics of CD1611 Treg are maintained
during longer-term in vitro culture
To test CD1611 Treg characteristics in longer-term culture, Treg
were sorted to high purity and cultured under Treg expansion conditions (plate-bound anti-CD3, anti-CD28, and IL-2 every 2 days)
with or without cytokines (IL-12 for Th1-like skewing; TGF-b1IL1b1IL-23 as IL-17A–skewing conditions). There was no difference
in FoxP3 stability between CD161– and CD1611 Treg, and the
frequency of CD1611FoxP31 cells was constant throughout the
10-day culture, irrespective of conditions (Figure 6A). Furthermore,
CD1611FoxP31 cells maintained their capacity to produce IL17A and IFN-g, whereas cytokine production remained negligible
in CD161–FoxP31 cells. Cytokines added had little effect, with
a small increase in IFN-g and slight decrease in IL-17A in the presence
of IL-12 (Figure 6B). This demonstration of persistence of CD1611
FoxP31 Treg proinflammatory potential should be of interest
to investigators attempting to expand Treg ex vivo for potential
therapeutic applications.
CD1611 Treg are enriched in the inflammatory environment
We have previously shown that Treg are greatly enriched within the
highly inflammatory environment of the synovial space in JIA. We
hypothesized that Treg expressing CD161 may be present within the
synovial Treg population. We therefore investigated CD161
expression on Treg in O-JIA (persistent oligoarticular [affected joint
count stable ,5] and extended oligoarticular [affected joint count
increased to >5 after 6 months]) and the more severe clinical type PJIA (at onset, >5 joints involved): these JIA subtypes show localized
inflammation restricted to the joints without systemic or major organ
involvement.18 PBMC of JIA patients showed no difference to the
age-matched healthy control in CD161 frequency of FoxP31
(Figure 7A, right). However, cells from the SF of affected joints
(SF mononuclear cells) had a significantly higher CD1611
frequency within the FoxP31 population (median, 23.7%) compared
with JIA PBMC (median, 10.8%) and healthy samples (median adult,
14.0%; child, 8.1%; Figure 7A), concurring with our previous
findings in JIA, of skewed CD41 populations at the inflamed
site.19,41 Synovial CD1611FoxP31 cells had the same phenotype
and were potent producers of proinflammatory cytokines as the
healthy control CD1611 Treg (Figure 3; JIA data not shown).
In children with an extended O-JIA disease course, there are fewer
FoxP31 cells than in those with the mild self-remitting persistent
O-JIA.8,9 When split into clinical subgroups, data in our study
replicated our previous findings for both CD161–FoxP31 and
CD1611FoxP31 cells (Figure 7B). Additionally, the fold increase
of the CD1611 Treg frequency (persistent O-JIA, 7.6; extended OJIA, 5.4; and P-JIA, 11.0) is much greater than the change in CD161–
Treg (2.6, 2.5, and 3.0, respectively). In our previous study, we also
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2654
PESENACKER et al
BLOOD, 4 APRIL 2013 x VOLUME 121, NUMBER 14
Figure 5. CD1611 regulatory T cells are stable in vitro and possess a predominantly demethylated TSDR. (A-D) Sorted Treg subpopulations were labeled with CFSE,
“spiked” back into autologous PBMC at a frequency of 5% to 7%, and cultured for 3 days on plates coated with or without anti-CD3/CD28 and stimulated with either medium
alone or cytokines as shown. Schematic of experimental setup for in vitro stability analysis is shown in panel A; representative overlay histograms of CD161 expression gated
on CFSE1 cells and summary plots of CD161 MFI at day 0 (n 5 5) (B), after 3-day culture in medium alone (n 5 3) (C), and after 3-day culture on anti-CD3/CD28–coated
plates 6 cytokines (n 5 3) (D, top); cytokine production by the labeled CD161– or CD1611 Treg was also assessed on day 3 (C; D, bottom). Data in histograms are events gated
on CD41CFSE1 live cells. For both histograms and graphs, clear symbols represent CD161– Treg, and filled symbols CD1611Treg. In bar graph, bars represent means 6 SEM
with Wilcoxon matched pairs test between cell subsets, and between groups 1-way nonparametric ANOVA (Kruskal-Wallis with Dunn’s test). **P , .01; *P , .05. (E) Both CD161–
Treg and CD1611 Treg cells contain a predominantly demethylated FOXP3 TSDR. DNA was extracted from sorted Tconv, CD161– Treg, and CD1611 Treg (purity .80%) and
bisulfite treated, and the TSDR was amplified and then cloned. Twenty to 35 individual clones were sequenced, and the percentage of methylated CpG islands determined. Panel E
shows a schematic of the FOXP3 locus representing the location of the 12 CpG islands analyzed (positions 13878 to 14082), with mean % methylation of each CpG island
displayed (left) and the average for all islands (right; blue, 100%, and yellow, 0%; 1 of 3 representative individuals shown).
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
BLOOD, 4 APRIL 2013 x VOLUME 121, NUMBER 14
CD161 DEFINES PROINFLAMMATORY FOXP31 T CELLS
2655
Figure 6. CD1611 regulatory T cells and cytokine-producing phenotype persist upon Treg expansion. Treg expansion over 10 days with or without cytokine treatment
(n 5 4). (A) Representative flow cytometry plots of FoxP3 expression at day 10 with stimulation alone for CD161– and CD1611 cells (left), summary plots for FoxP3
expression overall (middle), and frequency of CD161 in FoxP31 cells (right). Horizontal lines represent the median; diamonds, stimulation alone; crosses, 1IL-12; and
triangles, 1TGF-b, IL-1b, and IL-23. (B) Representative flow cytometry plots showing cytokine production (IL-17A vs IFN-g) of CD161–FoxP31 (left) and CD1611FoxP31
cells (right) with summary plots showing mean 6 SEM for IL-17A single-, IFN-g single-, and IL-17A1IFN-g double-producing cells for CD161– (white bars) and CD1611
FoxP31 (black bars).
showed a reciprocal relationship between the frequency of
FoxP31 and IL-17A–producing CD4 lymphocytes in the inflammatory joint compartment.8 Analysis of our data in this study
for CD161– and CD1611FoxP31 populations demonstrated
that only the CD161–FoxP31 cells show this reciprocal relationship (r 5 20.457; P 5 .015) but not CD1611FoxP31
(r 5 0.179; P 5 not significant) (Figure 7C). Together, these
data demonstrate that CD1611FoxP31 Treg are highly enriched
within an inflammatory environment.
Discussion
It is widely considered that an important function of Treg is the
suppression of disproportionate immune responses. Thus, Treg are an
appealing target to use therapeutically. However, there are still many
unknown elements to Treg therapy. Of topical relevance is the
controversy surrounding the proinflammatory potential of Treg,
which is present in expanded Treg populations, particularly in inflammatory settings.
In this report, we have demonstrated the existence of a distinct
population of FoxP31CD41 Treg, defined by the expression of
the Th17-associated marker CD161, which shows proinflammatory
potential, but also characteristic signatures and behavioral properties
of classic Treg. This population can be followed throughout life;
CD1611FoxP31 cells can be found already within the immunologic
inexperienced lymphocyte population of UCB and emerge as mature
single positive thymocytes along with natural Treg.
CD1611 Treg and CD161– Treg are diverse in their TCR-Vb
family repertoire. Importantly, CD1611 Treg fulfill the hallmark
features of classic Treg as they suppress the proliferation of Tconv to
the same extent as CD161– Treg and show a predominantly
demethylated TSDR of the FOXP3 locus. Overall methylation levels
were in keeping with those for previously published Treg
populations3,22,42; although in 3 individuals tested, CD1611 Treg
displayed a less demethylated TSDR overall compared with CD161–
Treg. The functional consequences of this potentially intermediate
demethylation state warrant future investigation.
We therefore report, to our knowledge for the first time, that CD161
expression defines the major source of cytokine-producing FoxP31
T cells. We report that CD1611 Treg share some characteristics with
Tconv phenotypes, with shared cytokine and chemokine receptors,
memory markers, as well as the presence of Tconv-associated
transcription factors RORCv2 and Tbet. Therefore, CD161 defines
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2656
PESENACKER et al
BLOOD, 4 APRIL 2013 x VOLUME 121, NUMBER 14
Figure 7. CD161 expression by regulatory T cells is enriched in the inflammatory environment. (A) CD161 status of Treg was analyzed on T cells from peripheral blood
(PB) and joint (SF) of children with JIA. Representative FACS plots of FoxP3 expression on CD41 lymphocytes and CD161 staining on FoxP31CD41 lymphocytes from JIA
PB and JIA SF; summary plot (right) shows data for healthy adult PB (n 5 39), healthy child PB (n 5 12), JIA PB (n 5 24), and JIA SF (n 5 41) samples. Data in dot plots are
gated on CD41 lymphocytes (left plots) and then (right plots) on FoxP31 T cells; horizontal lines represent median with Kruskal-Wallis test with Dunn’s multiple comparison
test. ***P , .001; **P , .01; *P , .05; ns, not significant. (B) Frequency of CD1611 Treg in different clinical JIA phenotypes was analyzed by flow cytometry. Frequency of
CD161– Treg (clear) and CD1611 Treg (black) within CD41 T cells; data are divided into clinical subgroups, persistent O-JIA (pers), extended O-JIA (ext), and P-JIA (poly)
for PB (n 5 8, n 5 8, and n 5 5, respectively) and SF (n 5 15, n 5 11, and n 5 11, respectively) samples. Bars represent mean 6 SEM. (C) The inverse relationship between
Th17 and Treg is specific to the CD161– Treg population. Correlation between SF CD161– Treg (left, clear symbols) and CD1611 Treg (right, filled symbols) with IL-17A1
CD41 T cells within the same sample (n 5 28).
a population of “hybrid Treg” that has both inflammatory and
suppressive potentials. Furthermore, we have established that
cytokine-producing CD1611FoxP31 cells are stable on expansion in vitro, whereas CD161–FoxP31 cells do not upregulate
cytokine production, even in Th1- or Th17-skewing conditions.
The precise role in vivo of cytokine-producing CD1611FoxP31
cells is worthy of further characterization.
Our data are supported by recent findings of cytokine-producing
Treg that were generated by in vitro stimulation, expansion, or
cloning, which were still suppressive.12,13,15,16 However, in those
studies, no potential origin or definitive way of identifying these cells
without ex vivo stimulation or permeabilization was given. Here
we suggest that CD161 defines the major source of Treg with
inflammatory potential. CD161-expressing Tconv are the precursors
for Th17, and Th1 cells that have converted from Th17 cells are
exclusively within the CD1611 population.17,23,24 Our data again
support a link between CD161 and IL-17 production, which appears
to be the case in Treg as well.
This growing recognition that Treg may have proinflammatory
potential has significant implications for therapeutic adoptive transfer
of human Treg because standard sorting methods to purify Treg
would include such cells. Our data suggest that the inclusion of
a depletion step, to remove CD1611 Treg, could reduce dangers of
such proinflammatory cells in certain conditions. However, so-called
specialized Treg have been shown to have the ability to regulate
specific Th cell responses in mouse models and thus may have
potential benefit in some conditions.5
Since their discovery, Treg have been subdivided into various
groups. One functionally distinct Treg subset described was based on
HLA-DR protein expression.11 Different mechanisms of suppression
were shown, and the authors demonstrated the importance of both
subsets, with DR1 Treg initiating early contact-dependent and DR–
late FoxP3-associated suppression. Later, it was identified that IL17–secreting Treg lie within the DR– population.15 However, in
healthy individuals, most Treg are DR– (mean of 83%; A.M.P.,
unpublished observations). Here we have demonstrated that CD161
expression accounts almost exclusively for IL-17A–producing Treg.
Another basis for defining Treg subsets is by memory markers.3,4
CD1611 Treg fall within the CD45RO1CD45RA– population
described by Miyara et al3 and the CCR61 population described by
Kleinewietfeld et al4 and therefore can be considered memory Treg.
CD161 expression on Tconv or CD81 T cells has been associated
with autoimmunity and chronic infection in previous reports. CD161
is enriched on T cells within a multitude of autoimmune conditions.17,24,43,44 Furthermore, we have now shown the enrichment of
CD1611 Treg within the inflammatory joint of autoimmune
childhood arthritis, implicating them in disease. In contrast, studies
in HIV and AIDS have shown decreased frequency and levels of
CD161 on T cells.45,46 Treg expressing CD161 have not been
considered in these studies as a potentially important contributor and
may warrant future attention.
The finding that CD1611 Treg are enriched at sites of inflammation is interesting. The persistence and overrepresentation of
CD1611 Treg compared with CD161– Treg might be detrimental for
the regulation of autoimmune responses. We propose that upon an
acute insult, CD1611FoxP31 cells may have a role in dictating the
balance between tolerance and immunity, with the ability to
produce proinflammatory cytokines and also to suppress an overly
active immune response. CD1611 Treg may be recruited via
CD161 itself, as well as through CCR6, to inflammatory areas.6,47
Additionally, the CD161 ligand lectin-like transcript 1 can be
induced upon activation of many cell types, possibly in response to
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
BLOOD, 4 APRIL 2013 x VOLUME 121, NUMBER 14
CD161 DEFINES PROINFLAMMATORY FOXP31 T CELLS
inflammation, which may equip such cells for activation of CD161expressing T cells.48
It remains unclear which signals direct the nature of the
cytokines produced by CD1611FoxP31 cells, but it is likely that
the cytokine milieu is central in determining their cytokineproduction profile, as demonstrated for some Treg.12,15 Similarly, it
remains to be seen what the direct effects of CD161 ligation are on
Treg and whether this is involved in the manifestation of the
proinflammatory profile. To address this in the in vitro setting is
technically challenging because the stimulatory antibody against
CD161 is sterically hindered by the antibodies used for sorting (A.M.
P., unpublished observations), which means functional analysis on
purified populations with present reagents is very limited. Because
CD1611 Treg are a minor population, stimulation on bulk Treg
cultures does not constitute a sensitive enough assay to determine the
functionality of CD161 (A.M.P., unpublished observations), and more
robust in vitro modeling systems will need to be established to address
this area.
An in vivo system to test CD1611 Treg functionality would be
valuable. Unfortunately, this is not readily available because human
CD161 does not have an exact equivalent in mice. Although CD161
is the only natural killer receptor protein 1 family member encoded in
humans, in mice there are several different and functionally diverse
natural killer receptor protein 1 receptors, with different expression
profiles and a maximum of 45% homology compared with human
CD161 (reviewed in Yokoyama et al49). It therefore remains to be
determined whether similar or related markers of Treg with proinflammatory potential exist, if at all, in other species.
In conclusion, we report the novel discovery that CD161
defines the major source of inflammatory cytokines by Treg.
CD1611FoxP31 T cells can be found throughout development
and show an effector-like phenotype. Despite a low frequency in
healthy environments, such cells are highly enriched at the site of
inflammation in autoimmune JIA. CD1611 Treg may therefore be
pivotal in dictating the balance between tolerance, appropriate
immune response, and autoimmunity. Because the field currently
lacks a clear understanding of the functions of CD161, especially
on T cells, further work into the biology of CD161-expressing Treg
2657
is necessary; additionally, given the knowledge that CD1611
FoxP31 cells display persistent proinflammatory characteristics,
we suggest that the CD1611 population should be taken into
account in Treg isolation protocols to be used for adoptive transfer
therapies.
Acknowledgments
The authors thank volunteers, patients, and their parents for
contribution of samples, as well as hospital staff who made this
study possible. The authors also thank A. Furmanski, T. Crompton,
and G. Davies for contributing thymus samples; and A. Eddaoudi and
flow cytometry facility staff for cell sorting. Group members are
thanked for their help in sample preparation and advice.
This work was supported by Nuffield Oliver Bird Programme
(A.M.P.), Arthritis Research UK (Foundation Fellow 19761
[D.B.], Career Progression fellowship 19392 [K.N.], and Vacation
Scholarship [Q.W.]), and SPARKS UK (08ICH09) (S.U.).
Authorship
Contribution: All authors approved the final version to be published
and had input in revising it for intellectual content and style; A.M.P.
had full access to all the data in the study and takes responsibility for
the integrity of the data and the accuracy of the data analysis; A.M.P.,
D.B., K.N., and L.R.W. were responsible for the experimental
design; A.M.P., D.B., Q.W., and S.U. carried out the acquisition of
data; and A.M.P., D.B., and L.R.W. performed analysis and interpretation of the data.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Anne M. Pesenacker, UCL Institute of Child
Health, 30 Guilford Street, London, WC1N 1EH; e-mail:
[email protected].
References
1. Wildin RS, Smyk-Pearson S, Filipovich AH.
Clinical and molecular features of the
immunodysregulation, polyendocrinopathy,
enteropathy, X linked (IPEX) syndrome. J Med
Genet. 2002;39(8):537-545.
2. Fontenot JD, Gavin MA, Rudensky AY. Foxp3
programs the development and function of CD41
CD251 regulatory T cells. Nat Immunol. 2003;
4(4):330-336.
3. Miyara M, Yoshioka Y, Kitoh A, et al. Functional
delineation and differentiation dynamics of human
CD41 T cells expressing the FoxP3 transcription
factor. Immunity. 2009;30(6):899-911.
4. Kleinewietfeld M, Puentes F, Borsellino G, et al.
CCR6 expression defines regulatory effector/
memory-like cells within the CD25(1)CD41 T-cell
subset. Blood. 2005;105(7):2877-2886.
perspective. Nat Rev Immunol. 2009;9(12):
883-889.
inflammation and cancer. J Immunol. 2011;
186(7):4388-4395.
8. Nistala K, Moncrieffe H, Newton KR, et al.
Interleukin-17-producing T cells are enriched in
the joints of children with arthritis, but have
a reciprocal relationship to regulatory T cell
numbers. Arthritis Rheum. 2008;58(3):875-887.
14. Floess S, Freyer J, Siewert C, et al. Epigenetic
control of the foxp3 locus in regulatory T cells.
PLoS Biol. 2007;5(2):e38.
9. de Kleer IM, Wedderburn LR, Taams LS, et al.
CD41CD25bright regulatory T cells actively
regulate inflammation in the joints of patients with
the remitting form of juvenile idiopathic arthritis.
J Immunol. 2004;172(10):6435-6443.
10. Yagi H, Nomura T, Nakamura K, et al. Crucial role
of FOXP3 in the development and function of
human CD251CD41 regulatory T cells. Int
Immunol. 2004;16(11):1643-1656.
5. Campbell DJ, Koch MA. Phenotypical and
functional specialization of FOXP31 regulatory
T cells. Nat Rev Immunol. 2011;11(2):119-130.
11. Baecher-Allan C, Wolf E, Hafler DA. MHC class II
expression identifies functionally distinct human
regulatory T cells. J Immunol. 2006;176(8):
4622-4631.
6. Yamazaki T, Yang XO, Chung Y, et al. CCR6
regulates the migration of inflammatory and
regulatory T cells. J Immunol. 2008;181(12):
8391-8401.
12. Dominguez-Villar M, Baecher-Allan CM, Hafler
DA. Identification of T helper type 1-like, Foxp31
regulatory T cells in human autoimmune disease.
Nat Med. 2011;17(6):673-675.
7. Weaver CT, Hatton RD. Interplay between the
TH17 and TReg cell lineages: a (co-)evolutionary
13. Kryczek I, Wu K, Zhao E, et al. IL-171 regulatory
T cells in the microenvironments of chronic
15. Beriou G, Costantino CM, Ashley CW, et al. IL-17producing human peripheral regulatory T cells
retain suppressive function. Blood. 2009;113(18):
4240-4249.
16. McClymont SA, Putnam AL, Lee MR, et al.
Plasticity of human regulatory T cells in healthy
subjects and patients with type 1 diabetes.
J Immunol. 2011;186(7):3918-3926.
17. Cosmi L, De Palma R, Santarlasci V, et al. Human
interleukin 17-producing cells originate from
a CD1611CD41 T cell precursor. J Exp Med.
2008;205(8):1903-1916.
18. Petty RE. Growing pains: the ILAR classification
of juvenile idiopathic arthritis. J Rheumatol. 2001;
28(5):927-928.
19. Moncrieffe H, Nistala K, Kamhieh Y, et al. High
expression of the ectonucleotidase CD39 on
T cells from the inflamed site identifies two distinct
populations, one regulatory and one memory
T cell population. J Immunol. 2010;185(1):
134-143.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2658
BLOOD, 4 APRIL 2013 x VOLUME 121, NUMBER 14
PESENACKER et al
20. Rozen S, Skaletsky H. Primer3 on the WWW for
general users and for biologist programmers.
Methods Mol Biol. 2000;132:365-386.
21. de Jager W, Prakken BJ, Bijlsma JW, et al.
Improved multiplex immunoassay performance in
human plasma and synovial fluid following
removal of interfering heterophilic antibodies.
J Immunol Methods. 2005;300(1-2):124-135.
22. Baron U, Floess S, Wieczorek G, et al. DNA
demethylation in the human FOXP3 locus
discriminates regulatory T cells from activated
FOXP3(1) conventional T cells. Eur J Immunol.
2007;37(9):2378-2389.
23. Nistala K, Adams S, Cambrook H, et al. Th17
plasticity in human autoimmune arthritis is driven
by the inflammatory environment. Proc Natl Acad
Sci USA. 2010;107(33):14751-14756.
24. Cosmi L, Cimaz R, Maggi L, et al. Evidence of the
transient nature of the Th17 phenotype of CD41
CD1611 T cells in the synovial fluid of patients
with juvenile idiopathic arthritis. Arthritis Rheum.
2011;63(8):2504-2515.
25. d’Hennezel E, Yurchenko E, Sgouroudis E, et al.
Single-cell analysis of the human T regulatory
population uncovers functional heterogeneity and
instability within FOXP31 cells. J Immunol. 2011;
186(12):6788-6797.
26. Liu W, Putnam AL, Xu-Yu Z, et al. CD127
expression inversely correlates with FoxP3 and
suppressive function of human CD41 T reg cells.
J Exp Med. 2006;203(7):1701-1711.
27. Annunziato F, Cosmi L, Santarlasci V, et al.
Phenotypic and functional features of human
Th17 cells. J Exp Med. 2007;204(8):1849-1861.
28. Plebanski M, Saunders M, Burtles SS, et al.
Primary and secondary human in vitro T-cell
responses to soluble antigens are mediated by
subsets bearing different CD45 isoforms.
Immunology. 1992;75(1):86-91.
29. Duhen T, Duhen R, Lanzavecchia A, et al.
Functionally distinct subsets of human FOXP31
Treg cells that phenotypically mirror effector Th
cells. Blood. 2012;119(19):4430-4440.
30. Exley M, Porcelli S, Furman M, et al. CD161
(NKR-P1A) costimulation of CD1d-dependent
activation of human T cells expressing invariant V
alpha 24 J alpha Q T cell receptor alpha chains.
J Exp Med. 1998;188(5):867-876.
31. Chen YT, Kung JT. CD1d-independent
developmental acquisition of prompt IL-4
gene inducibility in thymus CD161(NK1)CD44lowCD41CD8- T cells is associated with
complementarity determining region 3-diverse
and biased Vbeta2/Vbeta7/Vbeta8/Valpha3.2
T cell receptor usage. J Immunol. 2005;175(10):
6537-6550.
32. Wedderburn LR, King DJ. Analysis of the T-cell
receptor repertoire of synovial T-cells. Methods
Mol Med. 2007;136:97-116.
33. Azzoni L, Zatsepina O, Abebe B, et al. Differential
transcriptional regulation of CD161 and a novel
gene, 197/15a, by IL-2, IL-15, and IL-12 in NK and
T cells. J Immunol. 1998;161(7):3493-3500.
34. Poggi A, Costa P, Tomasello E, et al. IL-12induced up-regulation of NKRP1A expression
in human NK cells and consequent NKRP1Amediated down-regulation of NK cell activation.
Eur J Immunol. 1998;28(5):1611-1616.
35. Chan SH, Kobayashi M, Santoli D, et al.
Mechanisms of IFN-gamma induction by natural
killer cell stimulatory factor (NKSF/IL-12). Role of
transcription and mRNA stability in the synergistic
interaction between NKSF and IL-2. J Immunol.
1992;148(1):92-98.
36. Furtado GC, Curotto de Lafaille MA,
Kutchukhidze N, et al. Interleukin 2 signaling is
required for CD4(1) regulatory T cell function.
J Exp Med. 2002;196(6):851-857.
37. de la Rosa M, Rutz S, Dorninger H, et al.
Interleukin-2 is essential for CD41CD251
regulatory T cell function. Eur J Immunol. 2004;
34(9):2480-2488.
38. Bettelli E, Carrier Y, Gao W, et al. Reciprocal
developmental pathways for the generation of
pathogenic effector TH17 and regulatory T cells.
Nature. 2006;441(7090):235-238.
39. Veldhoen M, Hocking RJ, Atkins CJ, et al.
TGFbeta in the context of an inflammatory
cytokine milieu supports de novo differentiation of
IL-17-producing T cells. Immunity. 2006;24(2):
179-189.
40. de Jager W, Hoppenreijs EP, Wulffraat NM, et al.
Blood and synovial fluid cytokine signatures in
patients with juvenile idiopathic arthritis: a crosssectional study. Ann Rheum Dis. 2007;66(5):
589-598.
41. Hunter PJ, Nistala K, Jina N, et al. Biologic
predictors of extension of oligoarticular juvenile
idiopathic arthritis as determined from synovial
fluid cellular composition and gene expression.
Arthritis Rheum. 2010;62(3):896-907.
42. Wehrens EJ, Mijnheer G, Duurland CL, et al.
Functional human regulatory T cells fail to control
autoimmune inflammation due to PKB/c-akt
hyperactivation in effector cells. Blood. 2011;
118(13):3538-3548.
43. 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.
44. Annibali V, Ristori G, Angelini DF, et al. CD161
(high)CD81T cells bear pathogenetic potential in
multiple sclerosis. Brain. 2011;134(pt 2):542-554.
45. Kulkarni A, Paranjape R, Thakar M. Expansion of
defective NK cells in early HIV type 1C infection:
a consequence of reduced CD161 expression.
AIDS Res Hum Retroviruses. 2012;28(1):
100-105.
46. Singleterry WL, Henderson H, Cruse JM.
Depletion of pro-inflammatory CD161(1) double
negative (CD3(1)CD4(-)CD8(-)) T cells in AIDS
patients is ameliorated by expansion of the gd
T cell population. Exp Mol Pathol. 2012;92(1):
155-159.
47. Poggi A, Costa P, Zocchi MR, et al. NKRP1A
molecule is involved in transendothelial migration
of CD41 human T lymphocytes. Immunol Lett.
1997;57(1-3):121-123.
48. Germain C, Meier A, Jensen T, et al. Induction of
lectin-like transcript 1 (LLT1) protein cell surface
expression by pathogens and interferon-g
contributes to modulate immune responses. J Biol
Chem. 2011;286(44):37964-37975.
49. Yokoyama WM, Plougastel BF. Immune functions
encoded by the natural killer gene complex. Nat
Rev Immunol. 2003;3(4):304-316.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2013 121: 2647-2658
doi:10.1182/blood-2012-08-443473 originally published
online January 25, 2013
CD161 defines the subset of FoxP3+ T cells capable of producing
proinflammatory cytokines
Anne M. Pesenacker, David Bending, Simona Ursu, Qiong Wu, Kiran Nistala and Lucy R.
Wedderburn
Updated information and services can be found at:
http://www.bloodjournal.org/content/121/14/2647.full.html
Articles on similar topics can be found in the following Blood collections
Immunobiology (5489 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society
of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.