Distinct and Overlapping Effector Functions of Expanded
Human CD4+, CD8a+and CD4-CD8a-Invariant Natural
Killer T Cells
Vincent O’Reilly1, Shijuan G. Zeng1¤a, Gabriel Bricard2¤b, Ann Atzberger1, Andrew E. Hogan3, John
Jackson1, Conleth Feighery1, Steven A. Porcelli2, Derek G. Doherty1*
1Department of Immunology and Institute of Molecular Medicine, Trinity College Dublin, St. James’s Hospital, Dublin, Ireland, 2Department of Microbiology and
Immunology, Albert Einstein College of Medicine, Bronx, New York, United States of America, 3Obesity Immunology Group, Education and Research Centre, St. Vincent’s
University Hospital and University College Dublin, Dublin, Ireland
CD1d-restricted invariant natural killer T (iNKT) cells have diverse immune stimulatory/regulatory activities through their
ability to release cytokines and to kill or transactivate other cells. Activation of iNKT cells can protect against multiple
diseases in mice but clinical trials in humans have had limited impact. Clinical studies to date have targeted polyclonal
mixtures of iNKT cells and we proposed that their subset compositions will influence therapeutic outcomes. We sorted and
expanded iNKT cells from healthy donors and compared the phenotypes, cytotoxic activities and cytokine profiles of the
CD4+, CD8a+and CD42CD8a2double-negative (DN) subsets. CD4+iNKT cells expanded more readily than CD8a+and DN
iNKT cells upon mitogen stimulation. CD8a+and DN iNKT cells most frequently expressed CD56, CD161 and NKG2D and
most potently killed CD1d+cell lines and primary leukemia cells. All iNKT subsets released Th1 (IFN-c and TNF-a) and Th2 (IL-
4, IL-5 and IL-13) cytokines. Relative amounts followed a CD8a.DN.CD4 pattern for Th1 and CD4.DN.CD8a for Th2. All
iNKT subsets could simultaneously produce IFN-c and IL-4, but single-positivity for IFN-c or IL-4 was strikingly rare in CD4+
and CD8a+fractions, respectively. Only CD4+iNKT cells produced IL-9 and IL-10; DN cells released IL-17; and none produced
IL-22. All iNKT subsets upregulated CD40L upon glycolipid stimulation and induced IL-10 and IL-12 secretion by dendritic
cells. Thus, subset composition of iNKT cells is a major determinant of function. Use of enriched CD8a+, DN or CD4+iNKT
cells may optimally harness the immunoregulatory properties of iNKT cells for treatment of disease.
Citation: O’Reilly V, Zeng SG, Bricard G, Atzberger A, Hogan AE, et al. (2011) Distinct and Overlapping Effector Functions of Expanded Human CD4+, CD8a+and
CD4-CD8a-Invariant Natural Killer T Cells. PLoS ONE 6(12): e28648. doi:10.1371/journal.pone.0028648
Editor: Johan K. Sandberg, Karolinska Institutet, Sweden
Received October 21, 2011; Accepted November 11, 2011; Published December 12, 2011
Copyright: ? 2011 O’Reilly et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the Irish Health Research Board (PHD/2004/2), Science Foundation Ireland (05/RFP/BIC0015) and the National
Institute of Health (RO1 AI45889). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
¤a Current address: Bioprocessing Technology Institute, Singapore, Singapore
¤b Current address: INSERM U851, Faculty of Medicine, University Hospital Lyon-South, Lyon, France
Invariant natural killer T (iNKT) cells are cytotoxic T
lymphocytes that express NK cell markers and a T cell receptor
(TCR) composed of an invariant a-chain (Va24Ja18 in humans
and Va14Ja18 in mice) paired with one of a limited number of b-
chains. iNKT cells recognize glycolipid antigens presented by the
major histocompatibility complex class I-like molecule CD1d
[1,2]. They can recognize a number of self and bacterial
glycolipids [3,4] but the most potent activator of iNKT cells
known to date is the marine sponge-derived glycolipid a-
galactosylceramide (a-GalCer) . Upon activation with a-
GalCer, iNKT cells can kill a wide range of tumor cell lines
[6,7] and secrete a diverse range of growth factors and cytokines
that activate and polarize adaptive immune responses [1,2,8–12].
Activated iNKT cells can also interact directly with other cells of
the immune system and can induce the maturation of dendritic
cells (DC) into antigen-presenting cells (APC) [13–15] and of B
cells into antibody-secreting plasma cells [16,17]. Therapeutic
activation of iNKT cells in murine models can prevent tumor
growth, ameliorate autoimmune disease and protect against
microbial infection [6,18–20]. Numerical and functional iNKT
cell deficiencies have been reported in a number of human
diseases [21–25], but clinical trials that have targeted iNKT cells
in humans have to date been somewhat disappointing [26–30].
A reason for the low efficacy of iNKT cells in human
immunotherapy may lie in their multifunctionality. a-GalCer-
activated iNKT cells can rapidly and simultaneously secrete large
amounts of Th1 and Th2 cytokines, such as interferon-c (IFN-c),
tumor necrosis factor- a (TNF-a), IL-4 and IL-13 [1,8,9] and can
be induced under certain conditions to release the regulatory T
cell (Treg) cytokine IL-10 and the Th17 cytokines IL-17 and IL-22
[10–12,31]. This multiplicity of cytokine production, which
includes cytokines with opposing or mutually-inhibitory roles in
immune responses, may be counter-productive in therapeutic
applications where polarized adaptive immunity is desired. For
example, the antitumor activity of iNKT cells is associated with
their secretion of Th1 cytokines, however, Th2/Treg cytokines
PLoS ONE | www.plosone.org1December 2011 | Volume 6 | Issue 12 | e28648
released by iNKT cells may dampen antitumor immunity and
even promote tumor growth [19,22,32–34]. To overcome this
problem, several groups have synthesized a-GalCer analogues that
can selectively skew iNKT cell responses towards Th1 [35,36] or
Within iNKT cells there are distinct subsets based on CD4 and
CD8 expression and most studies have focused on iNKT cells with
CD4+CD8b2(CD4+) and CD42CD8b2(CD42) phenotypes, the
main subsets seen in mice [1,2]. However, it has become clear that
human CD42iNKT cells in humans can be sub-classified into
CD42CD8a2b2(double-negative or DN) and CD42CD8a+
(comprising CD8a+b2and CD42CD8a+b+cells) subsets [39–
44]. These iNKT cell subsets are reported to have distinct
immunological properties, with CD4+iNKT cells releasing both
Th1 and Th2 cytokines and CD8a+and DN iNKT cells exhibiting
Th1 phenotypes [9,15,39,45–48] and cytotoxic activity [39–44].
Altered iNKT cell subset frequencies and functions have been
described in humans with disease [21,22,24,44,48,49]. Phase I
clinical studies in cancer patients involving in vivo administration of
a-GalCer or infusion of ex vivo expanded and activated iNKT cells
have generally not considered the subset composition of iNKT
cells being activated [26–30], which we hypothesize could play an
important role in clinical outcome. In the present study we
expanded iNKT cells ex vivo from healthy individuals and
systematically compared the phenotypes, cytotoxic activities and
cytokine profiles of the CD4+, DN and CD8a+iNKT subsets. We
report that all expanded iNKT cell subsets can kill CD1d+target
cells and release Th1 and Th2 cytokines, but CD8a+iNKT cells
display the most potent Th1/cytolytic activity while CD4+iNKT
cells release the most Th2 cytokines. All iNKT cell subsets
similarly upregulate CD40L upon activation and induce cytokine
secretion by DC, while only the CD4+iNKT cell subset releases
IL-9 and IL-10. Thus, CD8a+iNKT cells may be ideal candidates
for future iNKT cell-based cancer therapies, whereas the
immunoregulatory properties of CD4+iNKT cells could be
beneficial for the treatment of inflammatory or autoimmune
Materials and Methods
This study was approved by the Research Ethics Committee of
St. James’s Hospital and the Adelaide and Meath Hospitals
incorporating the National Children’s Hospital (SJH/AMNCH),
Dublin. Informed written consent was obtained from all study
participants except when anonymised used buffy coat packs
(obtained from the Irish Blood Transfusion Service) were used.
Antibodies and flow cytometry
Fluorochrome-conjugated monoclonal antibodies (mAb) specific
for human CD1d, CD3, CD4, CD8a, CD8b, CD11c, CD14,
CD25, CD56, CD107a, CD154 (CD40L), CD161, HLA-DR,
NKG2D, the Va24 and Vb11 chains that form the TCR present
on iNKT cells and the complementarity-determining region 3 of
the invariant Va24Ja18 TCR chain (6B11) were obtained from
BD Biosciences (Oxford, UK), Immunotools (Friesoythe, Ger-
many), eBioscience (Hatfield, UK), Biolegend (San Diego, CA) or
Beckman Coulter (Galway, Ireland). Cells were stained with mAbs
in phosphate buffered saline containing 1% bovine serum albumin
and 0.02% sodium azide and analysed using a CyAn ADP flow
cytometer (Beckman Coulter, High Wycombe, UK) and FlowJo
software (Treestar, Ashland, OR). For FoxP3 staining, the FoxP3
staining buffer kit (eBioscience) was used following manufacturer’s
instructions. For CD40L staining, cells were co-cultured for
6 hours with CD1d+APC pulsed with a-GalCer (see below) in the
presence of anti-CD40L mAb and 1 mM monensin (Sigma-
Aldrich, Poole, UK) as described previously . Cells were then
stained with 6B11, CD4 and CD8a and analysed by flow
cytometry. Single stained controls were used to set compensation
parameters and fluorescence-minus-one controls were used to set
Ex vivo expansion of iNKT cells
Peripheral blood mononuclear cells (PBMC) were prepared
from unselected buffy coat packs by density gradient centrifugation
over Lymphoprep (Nycomed Pharma, Oslo, Norway). iNKT cells
were enriched from PBMC by magnetic bead separation using
6B11 coated magnetic beads (Miltenyi Biotec, Bergisch-Gladbach,
Germany). iNKT cells were then purified by cell sorting of
CD3+Va24+Vb11+cells using a MoFloTMXDP Cell Sorter
(Beckman Coulter). Sorted iNKT cells were expanded by culturing
1,000 iNKT cells in iNKT cell medium (RPMI 1640 containing
0.05 mM L-glutamine, 10% HyClone FCS, 1% penicillin-
streptomycin, 1% fungizone 25 mM HEPES, 50 mM 2-mercap-
toethanol, 1 mM sodium pyruvate, 1% non-essential amino acids
mixture and 1% essential amino acids mixture; Gibco-BRL,
Paisley, UK and Thermo-Scientific, Logan, UT) and stimulating
them with 1 mg/ml phytohemaggluttinin-P (PHA-P; Sigma-
Aldrich, Dublin, Ireland) and 250 U/ml IL-2 (R&D Systems,
Abingdon, UK) in the presence of an excess (26105) irradiated
allogeneic PBMC prepared from two donors. After 24 hours and
again after 48 hours, medium was replaced with fresh iNKT cell
medium containing 250 U/ml IL-2. Cells were expanded for a
minimum of 3 weeks before being used in experiments.
Generation of monocyte derived DC
Monocytes were enriched to .90% purity from PBMC isolated
from buffy coat packs by positive selection using CD14 Microbe-
ads (Miltenyi Biotec). The monocytes were allowed to differentiate
into immature DC (iDC) by culturing them at densities of 106
cells/ml using 3 ml/well of a 6-well plate (Corning, Amsterdam,
Netherlands) in complete RPMI medium (RPMI 1640 containing
0.05 mM L-glutamine, 1% penicillin-streptomycin, 1% fungizone,
25 mM HEPES made with low-endotoxin fetal calf serum)
containing 50 ng/ml granulocyte-macrophage colony-stimulating
factor (GM-CSF) and 70 ng/ml IL-4. Cells were cultured for 6
days, replacing with fresh medium containing cytokines on day 3.
On day 6, iDC were removed from the wells by aspiration using a
wide-gauge Pasteur pipette and washed with warm RPMI
medium. Flow cytometry was used to verify that differentiation
into iDC had taken place and cells expressed HLA-DR and
CD11c but not CD14.
In vitro stimulation of iNKT cells with a-GalCer
iNKT cells were stimulated in vitro with a variety of a-GalCer-
pulsed APC, including iDC, CD1d-transfected HeLa  or C1R
 cells (hereafter referred to as HeLa-CD1d and C1R-CD1d) or
the CD1d+T cell line, Jurkat. As controls, mock-transfected HeLa
and C1R cells (HeLa-mock and C1R-mock) and the CD1d2cell
line K562 were also used. In addition, PBMC from two consenting
patients with B cell chronic lymphocytic leukemia (B-CLL) were
obtained with ethical permission from the Haematology Clinic at
St. James’s Hospital, Dublin.
a-GalCer (KRN7000) was purchased from Funakoshi Co. Ltd,
(Tokyo, Japan) and reconstituted in 100% DMSO at a
concentration of 1 mg/ml followed by heating to 80uC for
2 minutes, sonication for 15 minutes and vortexing for 5 minutes.
This concentrated stock was aliquoted and stored at 270uC. For
CD4+, CD8a+and CD42CD8a2iNKT Cells
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use in iNKT cell assays, stock a-GalCer was thawed followed by
heating to 80uC for 2 minutes, sonication for 10 minutes and
vortexing for 1 minute. Dilution to the required concentration was
then made in 37uC pre-warmed iNKT cell medium followed by
heating to 80uC for 2 minutes, sonication for 5 minutes and
vortexing for 1 minute. Required dilutions thereafter were made
in 37uC pre-warmed medium followed by vigorous vortexing. The
above-mentioned cell lines were pulsed for 18 hours with the
appropriate concentrations of a-GalCer to allow for antigen
processing and presentation. Medium was then removed before
the addition of iNKT cells as described below.
Cytolytic degranulation of iNKT cells cultured with a-GalCer-
pulsed APC was examined by flow cytometric analysis of CD107a
expression by iNKT subsets after electronically gating on CD4+, DN
and CD8a+iNKT cells. iNKTcells and target cells were co-cultured
for 4 hours at 1:1 ratios in the presence of anti-CD107a FITC mAb
and monensin(25 mM)was added after1 hour topreventproteolysis
of the mAb conjugate upon reinternalization of CD107a.
To confirm that CD107a expression directly correlated with
target cell death, the flow cytometry-based Total Cytotoxicity and
Apoptosis Detection Kit (Immunochemistry Technologies, Bloo-
mington, MN) was used to quantify target cell death. Target cells
were incubated with carboxyfluorescein succinimidyl ester (CFSE)
following the manufacturer’ instructions and incubated with sorted
iNKTcell subsetsataratioof5:1for4 hoursat37uCwith 5%CO2.
After incubation, cells were harvested and 7-aminoactinomycin D
(7-AAD), which is excluded by viable cells but can penetrate cell
membranes of dying or dead cells, was added and cells were
analysed immediately by flow cytometry. CFSE+cells which stained
positive for 7-AAD are deemed to be killed target cells.
Analysis of cytokine secretion
To measure cytokine secretion by iNKT subsets, 105sorted
CD4+, DN or CD8a+iNKT cells were stimulated with medium
alone, equal numbers of iDCs or HeLa-CD1d (pulsed with a-
GalCer or vehicle) or 10 ng/ml of phorbol mristate acetate (PMA)
and 1 mg/ml of ionomycin (both from Sigma-Aldrich) for 24 hours.
The Human Th1/Th2/Th9/Th17/Th22 13plex FlowCytomix
Multiplex kit (eBioscience) was used to measure levels of IFN-c,
TNF-a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13,
IL-17a and IL-22 in the cell supernatants, following manufacturer’s
instructions. Results were acquired by flow cytometry and analysed
using FlowCytomix pro 2.2 software (eBioscience). In some
experiments, cell supernatants were assayed for selected cytokines
by enzyme-linked imunosorbent assays (ELISA) using antibody
pairs purchased from R&D Systems (Abingdon, UK).
Intracellular analysis of cytokine production by iNKT
iNKT cells were stimulated for 4 hours with equal numbers of
HeLa-CD1d cells pulsed with aGalCer or vehicle as described
above in the presence of brefeldin A (10 mg/ml, Sigma-Aldrich) to
promote intracellular accumulation of cytokines. Cells were
harvested and stained for cell surface expression of 6B11, CD4
and CD8a and intracellular expression of IFN-c and IL-4 using
fluorochrome-conjugated mAb obtained from BD Biosciences or
eBioscience and analysed by flow cytometry.
Statistical analysis was carried out using Prism GraphPad
Version 5.0. The Kruskal-Wallis test was used to compare
unpaired data in 3 groups and Dunn’s multiple comparison tests
were performed post-hoc to compare individual groups within an
experiment. *, ** and *** represent p,0.05, p,0.01 and p#0.001
Human CD4+iNKT cells expand more readily than CD8a+
and DN iNKT cells in response to mitogen stimulation
The distributions of CD4+, DN and CD8a+iNKT cells in
freshly-isolated PBMC and among in vitro expanded iNKT cells
from healthy donors were examined by flow cytometric analysis of
Va24+Vb11+CD3+cells. Since iNKT cells generally comprise
,0.1% of total peripheral T cells [21,25] (Fig. 1A), phenotypic
analysis of fresh iNKT cells required prior enrichment of iNKT
cells from PBMC by magnetic bead separation followed by
immediate phenotyping for CD4 and CD8a (Fig. 1B). The
enriched iNKT cells were then further purified by cell sorting and
expanded in vitro with PHA-P, irradiated feeders and IL-2 for three
weeks. This method resulted in a several thousand-fold increase in
iNKT cell numbers with $98% expressing Va24+Vb11+
phenotypes (Fig. 1C). The mean percentages of fresh iNKT cells
from 8 donors that expressed CD4+, DN and CD8a+phenotypes
were 15.669.0%, 48.6615.4% and 33.5618.1%, respectively
(Fig. 1D). Virtually all iNKT cells were negative for CD8b. The 20
lines of expanded iNKT cells generated during the course of this
study had highly variable distributions of the CD4+, DN and
CD8a+subsets with means of 46.266.1%, 37.864.6% and
14.563.1%, respectively (Fig. 1 E and F). Thus, while CD4+
iNKT cells generally comprise a minority of human peripheral
iNKT cells, this subtype expanded more readily with mitogen
stimulation in the presence of IL-2. As previously reported 
significant numbers of double positive CD4+CD8a+iNKT cells
were also observed in freshly-isolated iNKT cell preparations
(Fig. 1B). Polyclonal lines of iNKT cells were used in flow
cytometry-based functional assays where electronic gating of
CD4+, DN and CD8a+expression allowed analysis of the
individual subsets. For cytoxicity and cytokine secretion assays,
expanded iNKT subsets were further sorted giving highly pure
(.98%) populations of CD4+, DN and CD8a+iNKT cells
Expanded CD4+, DN and CD8a+iNKT cells are
iNKT lines from up to 5 healthy donors were stained with mAbs
specific for the Va24Ja18 TCR (6B11), CD4, CD8a and either
the NK cell markers CD56, CD161 or NKG2D or the IL-2
receptor a-chain CD25. The proportions of gated CD4+, DN and
CD8a+iNKT cells that express these markers are shown in
Figure 2A. CD56 was found to be expressed by significant
numbers of CD8a+iNKT cells but only by a minority of CD4+
iNKT cells, and on DN iNKT at intermediate frequencies. CD161
and NKG2D were present on most (.80%) CD8a+and DN but
on lower frequencies of CD4+iNKT cells. Thus, expanded CD8a+
and DN iNKT cells more frequently express receptors typically
found on NK cells than CD4+iNKT cells. In contrast, significantly
higher percentages of CD4+
compared to DN and CD8a+iNKT cells (Fig. 2A).
Since activated, but not resting, helper T cells express CD40L
(CD154) , iNKT lines from 3 healthy donors were co-
cultured for 6 hours with HeLa-CD1d cells pulsed with varying
concentrations of a-GalCer (0–1,000 ng/ml) in the presence of
monensin and anti-CD40L mAb. Figure 2B shows that CD40L
expression was induced on similar percentages of CD4+, DN
iNKT cells expressed CD25
CD4+, CD8a+and CD42CD8a2iNKT Cells
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and CD8a+iNKT cells in a dose-dependant manner upon
activation, indicating that these iNKT cell subsets have similar
potential for interacting with other cells, such as B cells and
Since CD25 was found to be expressed by significant numbers
of expanded CD4+iNKT cells (Fig. 2A), we next determined the
frequencies of iNKT cell subsets that express the natural Treg cell
phenotype, FoxP3+CD25+(Fig. 2C). A significant proportion of
Figure 1. Isolation and expansion of human peripheral blood iNKT cells and analysis of CD4 and CD8a expression. A, Representative
flow cytometry dot plot showing Va24 and Vb11 TCR chain expression by freshly-isolated human PBMC. B, Va24 and Vb11 TCR chain expression by
PBMC after enrichment using anti-iNKT cell magnetic beads but without expansion (left panel) and CD4 and CD8a expression by gated Va24+Vb11+
cells (right panel). Plots are representative of PBMCs from 8 healthy donors. C, Va24 and Vb11 TCR chain expression by an expanded sorted iNKT cell
line. Numbers in plots show percentages of cells in each quadrant. D and E, Distribution of CD4+, CD8a+and DN cells among freshly-isolated iNKT
cell lines from 8 randomly obtained healthy donors (D) and among expanded iNKT cells from 20 donors (E). Horizontal lines show means; p values in
shaded boxes show comparisons of CD4+, CD8a+and DN iNKT cell frequencies using the Kruskal-Wallis test; asterisks indicate significant differences
between individual groups (indicated by bars) using post hoc Dunn’s multiple comparison tests; **p,0.01; ***p,0.001 . F, Flow cytometric sorting of
purified CD4+, CD8+and DN iNKT populations from polyclonal iNKT cell lines.
CD4+, CD8a+and CD42CD8a2iNKT Cells
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(12.164.1%) and CD8a+(10.463.8%) expressed FoxP3+CD25+
phenotypes (Fig. 2D). However, functional studies were not carried
out to test whether these cells are true regulatory iNKT cells.
iNKT cells(mean34.766.9%) comparedtoDN
Expanded CD4+, DN and CD8a+iNKT cells kill CD1d+
target cell lines and primary CD1d+leukemia cells, with
CD8a+iNKT cells displaying the most potent cytotoxic
Cytolytic degranulation by iNKT cell subsets in response to a-
GalCer presented by K562, Jurkat and mock- and CD1d-
transfected HeLa cells was assayed by analysis of CD107a
externalization. Figure 3A shows that degranulation by CD4+,
DN and CD8a+iNKT cells only occurred when CD1d was
present, since CD107a expression was undetectable when the
CD1d-negative cell lines K562 cells (Fig. 3A, left panel) or HeLa-
mock (not shown) were used as targets. All three iNKT cell subsets
showed an a-GalCer dose-dependent increase in CD107a
expression in response to the CD1d-transfected HeLa cells and
the CD1d+Jurkat cell line (Figure 3A, centre and right panels).
Optimal CD107a expression by all 3 iNKT subsets occurred when
100 ng/ml a-GalCer was used. CD8a+iNKT cells consistently
expressed CD107a at higher frequencies than the other iNKT cell
Figure 2. Surface phenotypes of expanded iNKT cell subsets. A, Percentage expression of CD56, CD161, NKG2D and CD25 by electronically-
gated CD4+, CD8a+and DN cells within expanded iNKT cell lines. Horizontal lines show means; p values in shaded boxes show multiple comparisons
of cell frequencies using the Kruskal-Wallis test; asterisks indicate significant differences between individual groups (indicated) using post hoc Dunn’s
multiple comparison tests; *p,0.05; ***p,0.001. B, Induced cell-surface expression of CD40L by gated iNKT subsets after co-culture of iNKT cell lines
with equal numbers of HeLa-CD1d pulsed with various concentrations of a-GalCer. Results show mean (6SEM) percentages of CD40L+iNKT cell
subsets within lines from 3 donors. C, Representative flow cytometry dot plots showing cell-surface CD25 and intracellular FoxP3 expression by
electronically-gated CD4+, CD8a+and DN iNKT cell subsets from 3 donors which were allowed to rest by culturing for 5 days in the absence of IL-2.
Quadrants delineating positivity and negativity for CD25 and FoxP3 were set using fluorescence-minus-one control mAb staining, omitting CD25 or
FoxP3. Numbers on plots show percentages of cells in each quadrant. D, Frequencies of CD4+, CD8a+and DN iNKT cells within 3 iNKT cell lines that
CD4+, CD8a+and CD42CD8a2iNKT Cells
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subsets, while CD4+iNKT cells expressed this marker at the
lowest frequencies (p,0.01 for HeLa-CD1d and Jurkat; Kruskal-
We also investigated if iNKT cell subsets displayed cytolytic
degranulation when cultured with PBMC from 2 B-CLL patients.
Almost all of the lymphocytes expressed the CD19+CD5+
leukaemia phenotype and 0% and 10.1% were positive for
CD1d (data not shown). iNKT cells from 3 donors exhibited
undetectable degranulation when cultured with the CD1d-
negative sample. When cultured with the CD1d+sample, surface
expression of CD107a by CD8a+iNKT cells in all three iNKT cell
lines was increased almost 3-fold by the addition of a-GalCer (7.8
to 21.4%) (Fig. 3B). In contrast, CD107a expression was only
slightly induced on CD4+and DN iNKT cells.
To confirm that CD107a expression by iNKT cell subsets
correlated with target cell death, we performed cytotoxicity assays
employing CFSE-labelled C1R-CD1d target cells pulsed with
100 ng/ml a-GalCer or vehicle co-cultured for 4 h with highly-
purified CD4+, CD8a+and DN iNKT cells (Figure 1C) at E:T
ratios of 5:1. Target cell death was then assessed by staining with
7-AAD. Figure 3C shows that CD4+, DN and CD8a+iNKT cells
killed means of 13.0%, 23.0% and 33.2% of a-GalCer-primed
target cells, respectively (p,0.05; Kruskal-Wallis). Collectively,
these data indicate that CD4+, CD8a+and DN iNKT cells are all
capable of antitumor cytotoxicity and that CD8a+iNKT cells
consistently display the most potent cytotoxic activity, while CD4+
iNKT cells are the least potent and DN iNKT cells have
intermediate cytolytic activity.
Expanded CD4+, DN and CD8a+iNKT cells have distinct
cytokine secretion profiles
Highly-purified populations of CD4+, DN and CD8a+iNKT
cells (Fig. 1F) were stimulated with medium alone, with equal
numbers of iDCs or HeLa-CD1d cells (with and without 100 ng/
ml a-GalCer), or with PMA and ionomycin for 24 hours. Cell-
supernatants were then harvested and assayed for IFN-c, TNFa,
Figure 3. Antitumor cytotoxicity by CD4+, CD8a+, and DN iNKT cells. A, Expression of cell surface CD107a by iNKT cell subsets after co-
culturing with CD1d2K562 cells, CD1d-transfected HeLa cells or CD1d+Jurkat cells, which were first pulsed for 18 hours with 0–1000 ng/ml a-GalCer.
Results show mean (6SEM) percentage expression by iNKT cell subsets within 4 iNKT cell lines (3 for Jurkat). p values in shaded boxes show Kruskal-
Wallis comparisons of CD107a expression by CD4+, CD8a+, and DN iNKT cells; asterisks indicate significant differences between CD4+and CD8a+iNKT
cells using post hoc Dunn’s multiple comparison tests; *p,0.05; **p,0.01. B, Surface CD107a expression by iNKT subsets in response to primary B-
CLL cells pulsed with 100 ng/ml of a-GalCer. C, Cytolytic killing of CD1d-transfected C1R cells pulsed with 100 ng/ml a-GalCer or vehicle by highly-
purified CD4+, DN or CD8a+iNKT cells. Target cells were labelled with CFSE before addition of iNKT cells and death was then analysed by staining with
7-AAD. Data are expressed as means (6SEM) of experiments involving iNKT cell lines from 3 healthy donors. p,0.05 (Kruskal-Wallis); *p,0.05
comparing CD4+and CD8a+iNKT cells (post hoc Dunn’s test).
CD4+, CD8a+and CD42CD8a2iNKT Cells
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IL-1b, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-17a
and IL-22 secretion using the FlowCytomix Multiplex kit (Fig. 4).
When stimulated with a-GalCer-pulsed iDC or HeLa-CD1d cells
or with PMA and ionomycin, all iNKT cell subsets released IFN-c,
with CD8a+iNKT cells secreting the highest levels, CD4+iNKT
cells secreting the lowest levels and DN iNKT cells secreting
intermediate levels. a-GalCer pulsed iDCs or HeLa-CD1d cells
induced low-level secretion of TNF-a, with significantly higher
levels induced by PMA and ionomycin, and again, CD8a+iNKT
cells secreted the highest and CD4+iNKT cells secreted the lowest
levels. Significant IL-6 secretion was detected when all iNKT cell
subsets were co-cultured with a-GalCer-pulsed HeLa-CD1d cells
but not when stimulated with a-GalCer-pulsed iDC or PMA and
ionomycin, suggesting that IL-6 emanated from HeLa cells, but
only when cultured with iNKT cells. Slight but consistent increases
in IL-17a secretion were observed when DN iNKT cells were
stimulated with a-GalCer pulsed HeLa-CD1d cells or PMA and
ionomycin. IL-4, IL-5 and IL-13 were predominantly secreted by
activated CD4+iNKT cells, while DN iNKT cells secreted
intermediate levels and CD8a+iNKT cells secreted the lowest
levels. However, contradicting previous reports [9,46] DN and
CD8a+iNKT cells were nevertheless capable of secreting these
Th2 cytokines. A novel finding in the present study is the release of
IL-9 by iNKT cells and only activated CD4+iNKT cells were
capable of secreting this cytokine under the conditions used. CD4+
iNKT cells were the only iNKT cell subset to secrete IL-10 after
stimulation with PMA and ionomycin but not with a-GalCer
pulsed HeLa-CD1d cells. However, co-cultures of iDC with all
three iNKT cell subsets secreted IL-10, suggesting that this
cytokine mainly emanates from iNKT-cell-matured DC. CD4+,
DN and CD8a+iNKT cells all secreted IL-2 when stimulated with
a-GalCer-pulsed HeLa-CD1d cells or PMA and ionomycin, with
the CD4+subset secreting slightly higher levels compared to DN
and CD8a+iNKT cells. All three iNKT subsets stimulated IL-
12p70 secretion by DC, while CD4+and CD8a+iNKT cells, only,
induced low-level secretion of IL-1b (Fig. 4). No IL-22 was
detected in supernatants of any iNKT cell subsets with any
stimulation (data not shown). These data show that, under these in
vitro conditions, a-GalCer-stimulated CD4+, DN and CD8a+
iNKT cells can release Th1 and Th2 cytokines with CD8a+iNKT
cells biased towards a Th1 phenotype, CD4+iNKT cells
predominantly secreting Th2 cytokines and DN iNKT cells
exhibiting an intermediate Th1/Th2 phenotype. Low-level IL-17
is restricted to DN iNKT cells, while IL-9 and IL-10 are only
released by CD4+iNKT cells.
CD4+, DN and CD8a+iNKT cells exhibit distinct patterns
of single and dual expression of IFN-c and IL-4
iNKT cells were co-cultured with a-GalCer pulsed HeLa-CD1d
cells for 4 hours followed by cell-surface mAb staining of CD4 and
CD8a and intracellular staining of IFN-c and IL-4. The
frequencies of IFN-c and IL-4 single-positive and double-positive
cells were determined by flow cytometry after electronically gating
on the CD4+, DN and CD8a+populations (Fig. 5A). CD8a+
iNKT cells displayed the highest frequencies of IFN-c expression,
with intermediate and low frequencies of DN and CD4+iNKT
cells, respectively, producing IFN-c (Fig. 5B left panel). All three
subsets had similar frequencies of IL-4-producing cells (Fig. 5B
right panel). Interestingly, while significant numbers of all 3 iNKT
cell subsets simultaneously produced IFN-c and IL-4, single-
positivity for IFN-c was rare in the CD4+iNKT cell subset and
single-positivity for IL-4 was rare in CD8a+iNKT cells. In
contrast, stimulated DN iNKT cells had an intermediate
phenotype with significant percentages producing IFN-c or IL-4
only, in addition to double-positive IFNc+IL-4+cells (Fig. 5A and
C). Thus, CD8a+iNKT cells had the highest ratio of IFN-c/IL-4
producing cells followed by DN iNKT cells, with CD4+iNKT
cells having the lowest (Fig. 5D).
The importance of iNKT cells in the prevention of disease and
as potential therapeutic targets was first recognized with
observations that mice lacking CD1d or iNKT cells are
predisposed to developing cancer, autoimmune and infectious
disease [6,51] and with the discovery of a-GalCer and its
beneficial effects in murine models of disease [5,18,19,20]. The
promising results in murine models have led to clinical trials in
humans with various cancers, involving i.v. injection of a-GalCer
 or a-GalCer-pulsed APC [26,30]. These studies showed that
iNKT-based immunotherapy is well tolerated, but the biological
responses were only marginal. Subsequent therapeutic strategies
involved the transfer of ex vivo expanded autologous iNKT cells
alone or in combination with a-GalCer-pulsed APC [27,28]. The
expanded iNKT cells used in all studies were polyclonal mixtures
of CD4+, DN and CD8a+iNKT cells. Since these iNKT cell
subsets differ in their immunoregulatory properties and may even
be mutually-inhibitory, it may be beneficial to adoptively transfer
enriched iNKT cell subsets selected for desired properties.
Although previous studies have compared the effector functions
of different iNKT cell subsets [9,15,39–45,47,48], many have been
restricted to the analysis of fresh rather than expanded peripheral
blood iNKT cells; many have compared CD4+and CD42iNKT
cells only, omitting the distinction between CD8a2b2(DN) and
CD8a+b2(CD8a+) subsets; most did not use DC as APC for a-
GalCer as was used in many clinical studies; and most were limited
to the analysis of Th1 and Th2 cytokines only.
In the present study, we have systematically compared the
phenotypes, cytolytic activities and cytokine profiles of ex vivo
expanded human peripheral CD4+, DN and CD8a+iNKT cells.
Expanded rather than fresh iNKT cells were studied to provide
information that is relevant to immunotherapy, which employs
expanded iNKT cells. All 3 populations were present in PBMC,
with DN iNKT cells generally being the predominant subtype
followed by CD8a+iNKT cells and CD4+iNKT cells being the
least abundant. Expansion of sorted iNKT cells from 20 randomly
selected donors in vitro resulted in lines containing .98%
Va24+Vb11+cells with variable expression of CD4 and CD8a+.
While all 3 iNKT cell subsets expanded vigorously, CD4+iNKT
cells were the predominant subset in our expanded lines,
indicating that this subset expands most readily using our methods.
Phenotypic analysis of expanded CD4+, DN and CD8a+iNKT
cells revealed that, similar to iNKT cells in resting PBMC [9,44],
CD8a+and DN iNKT cells more frequently express cell-surface
receptors such as CD56, CD161 and NKG2D that are typically
found on NK cells. In accordance with this NK-like phenotype
and as previously reported [39–44], CD8a+and DN iNKT cells
displayed more potent cytotoxicity than CD4+iNKT cells against
a number of CD1d+tumor cell lines, with consistently higher
proportions expressing cell-surface CD107a and killing target cells.
However, these cells were not capable of natural cytotoxicity, since
killing was dependent on the presence of CD1d and a-GalCer.
Compared to CD4+and CD8a+iNKT cells, DN iNKT cells
displayed intermediate cytotoxic phenotypes. When PBMC from
two B-CLL patients were used as targets, only CD8a+iNKT cells
displayed cytotoxic phenotypes. Thus, while all iNKT cell subsets
possess cytolytic activity, CD8a+iNKT cells are the most potent
killers and are likely to be superior inducers of cell-mediated
CD4+, CD8a+and CD42CD8a2iNKT Cells
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CD4+, CD8a+and CD42CD8a2iNKT Cells
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Figure 4. Cytokine secretion by iNKT cell subsets. Highly-purified populations of CD4+, DN and CD8a+iNKT cells were cultured in medium
alone,with iDCor HeLa-CD1d cells in the absence or presence of 100 ng/mla-GalCer, or with PMA andionomycin for 24 hours. Levelsof IFN-c, TNFa, IL-
1b, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-17a and IL-22 in cell supernatants were measured using the FlowCytomix Multiplex kit. Results show
mean (6SEM) cytokine levels in experiments involving iNKT cell lines from 3 healthy donors. p values in shaded boxes show Kruskal-Wallis comparisons
of cytokine levels released by CD4+, CD8a+, and DN iNKT cells; *p,0.05 comparing CD4+and CD8a+iNKT cells using post hoc Dunn’s test.
Figure 5. Single and dual production of IFN-c and IL-4 production by iNKT subsets. A, Flow cytometry plots showing IFN-c and IL-4
expression by iNKT cells after 4 hours of co-culture with a-GalCer pulsed HeLa-CD1d cells. 6B11+cells were electronically gated followed by gating on
CD4+, DN and CD8a+cells. Plots are representative of experiments involving iNKT cell lines from 3 healthy donors. B, IFN-c and IL-4 production by
iNKT cell subsets stimulated with HeLa-CD1d cells pulsed with various concentrations of a-GalCer. Graphs show mean (6SEM) percentages of cells
expressing IFN-c (left) and IL-4 (right) out of iNKT cell lines from 4 healthy donors. C, Mean (6SEM) percentages of CD4+, DN and CD8a+iNKT cells
that expressed IFN-c only, IFN-c and IL-4 and IL-4 only after stimulation with HeLa-CD1d cells pulsed with 100 ng/ml a-GalCer (n=2). D, Ratios of IFN-
c/IL-4-expressing CD4+, DN and CD8a+iNKT cells after stimulation with a-GalCer-pulsed HeLa-CD1d cells (n=2).
CD4+, CD8a+and CD42CD8a2iNKT Cells
PLoS ONE | www.plosone.org9 December 2011 | Volume 6 | Issue 12 | e28648
immunity. In support of this notion, CD8a+iNKT cells are the
most efficient at transactivating conventional CD8+T cells in co-
cultures with PBMC .
Previous studies [9,15,39,45–48] have shown that all subsets of
activated iNKT cells are capable of releasing Th1 cytokines. The
present study also places CD8a+iNKT cells as the most potent
inducers of Th1 immunity, being capable of releasing the greatest
amounts of IFN-c and TNF-a upon stimulation with PMA and
ionomycin or a-GalCer presented by iDC or HeLa-CD1d cells
and being the cells that most readily produce IFN-c in the absence
of IL-4. Collectively, our data suggest that CD8a+iNKT cells are
superior inducers of antitumor and antiviral immunity.
In contrast to CD8a+iNKT cells, we found that CD4+iNKT
cells release higher amounts of the Th2 cytokines IL-4, IL-5 and
IL-13 upon stimulation with a-GalCer presented by iDC or HeLa-
CD1d cells or with PMA and ionomycin. This predominance of
Th2 cytokine secretion by CD4+iNKT cells is well documented
[9,15,39,45,46,47,48] but we also clearly show by the FlowCyto-
mix Multiplex kit, ELISA and intracellular flow cytometry that
CD8a+and DN iNKT cells can also release these cytokines under
the same stimulatory conditions, albeit at lower levels and lower
frequencies. Analysis of dual IFN-c and IL-4 production by
activated iNKT cell subsets revealed that significant proportions of
all three iNKT cell subsets could simultaneously produce both
cytokines, but at markedly different ratios. Thus, iNKT cells are
likely to regulate the Th1/Th2 balance of immune responses by
modulating the relative levels of IFN-c and IL-4, or IFN-c/IL-4
ratios, rather than simply turning on or off the production of either
cytokine. While potential beneficial clinical effects of each cytokine
are likely to be modified by simultaneous production of the other
[19,22,32,33,48], it is noteworthy that single positivity for IFN-c
was almost exclusively found in the CD8a+and DN iNKT cell
subsets and single positivity for IL-4 was restricted to CD4+and
DN iNKT cells. Thus, immunotherapy that exploits the
immunoregulatory properties of iNKT cells may benefit from
the use of sorted iNKT cell subsets with more polarized cytokine
We report here for the first time that human CD4+iNKT cells
but not CD8a+nor DN iNKT cells release IL-9 in response to
pharmacological or glycolipid stimulation. IL-9, a key cytokine in
the recruitment and activation of mast cells, is produced by subsets
of Th1, Th17 and Treg cells as well as the recently-defined Th9
subset of CD4+T cells . IL-9 production by a subset of non-
invariant (type 2) murine CD1d-restricted CD4+NKT cells is
associated with IgE production  and antigen-induced
pulmonary inflammation and mast cell infiltration . In view
of the reported (but disputed) infiltration of CD4+iNKT cells into
the lungs of humans with bronchial asthma , it is possible that
CD4+iNKT cell-derived IL-9 may play a role in the pathogenesis
in some cases.
Murine and human iNKT cells have recently been reported to
produce the Th17 cytokines IL-17 and IL-22 in response to a-
GalCer stimulation [10,11]. Under our stimulatory conditions
human DN iNKT cells, only, were capable of releasing IL-17 but
IL-22 was not detected in the supernatants of any activated iNKT
cell subset. We also confirm reports [34,39,47,56] that purified
CD4+, but not DN or CD8a+iNKT cells, release IL-10 and many
can express CD25+FoxP3+phenotypes, suggestive of a Treg cell
phenotype. The suppressive role of IL-10 in Th1 and Th2 cell-
mediated immunity will provide a further way by which iNKT
cells can modulate adaptive immunity and iNKT cell-derived IL-
10 is thought to suppress tumor immunity [22,34] and protect
against diabetes .
Rapid release of multiple cytokines is likely to have early
influences on innate immune responses, however, iNKT cells can
also contribute to adaptive immunity via stimulatory interactions
with DC [13,14,58], B cells [16,17], conventional T cells  and
NK cells [43,59]. Here we found that CD4+, DN and CD8a+
iNKT cells similarly upregulate CD40L upon activation and
similarly induce IL-10 and IL-12 by iDC, whereas CD4+and
CD8a+iNKT cells, only, induce IL-1b production. While all
iNKT cell subsets can induce cytokine production by DC,
previous studies [15,40] have shown that CD8a+iNKT cells can
also kill DC, resulting in reduced IL-12 secretion and skewed Th2
iNKT cells can be beneficial or detrimental in different disease
settings [5,6,18–22,48] resulting from cytotoxicity- and cytokine-
biased immune activities. Consequently, several groups are
attempting to modulate the effector profiles of iNKT cells for
therapeutic application, by modifying the cytokine environment
[10,53,60], mode of activation [11,61] and structures of glycolipid
antigens [35,36,37,38,58]. Our data show that therapeutic
manipulation of iNKT cells may necessarily require the sorting
of iNKT cells into functionally-distinct subsets. As well as selecting
for desired effector functions, sorting of iNKT cell subsets could
also allow selection of iNKT cells with distinct adhesion  and
homing receptors [9,42,46,47] that will promote optimal localiza-
tion to the relevant sites.
We thank the Irish Blood Transfusion Service for providing buffy coat
packs and Tony McElligott for providing the blood samples from B-CLL
patients. Thanks to Padraic Dunne, Margaret Dunne, Bozgana Mangan,
Laura Madrigal-Estebas, Jacinta Kelly, Cliona O’Farrelly and Graham
Pidgeon for helpful discussions.
Conceived and designed the experiments: VO DGD JJ CF. Performed the
experiments: VO SGZ GB AA AEH. Analyzed the data: VO DGD.
Contributed reagents/materials/analysis tools: SAP. Wrote the paper: VO
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PLoS ONE | www.plosone.org11December 2011 | Volume 6 | Issue 12 | e28648