Article

Natural killer T-cell characterization through gene expression profiling: An account of versatility bridging T helper type 1 (Th1), Th2 and Th17 immune responses

Department of Immunology, Max Planck Institute for Infection Biology, Berlin, Germany.
Immunology (Impact Factor: 3.8). 02/2008; 123(1):45-56. DOI: 10.1111/j.1365-2567.2007.02701.x
Source: PubMed
ABSTRACT
Natural killer T (NKT) cells constitute a distinct lymphocyte lineage at the interface between innate and adaptive immunity, yet their role in the immune response remains elusive. Whilst NKT cells share features with other conventional T lymphocytes, they are unique in their rapid, concomitant production of T helper type 1 (Th1) and Th2 cytokines upon T-cell receptor (TCR) ligation. In order to characterize the gene expression of NKT cells, we performed comparative microarray analyses of murine resting NKT cells, natural killer (NK) cells and naïve conventional CD4+ T helper (Th) and regulatory T cells (Treg). We then compared the gene expression profiles of resting and alpha-galactosylceramide (alphaGalCer)-activated NKT cells to elucidate the gene expression signature upon activation. We describe here profound differences in gene expression among the various cell types and the identification of a unique NKT cell gene expression profile. In addition to known NKT cell-specific markers, many genes were expressed in NKT cells that had not been attributed to this population before. NKT cells share features not only with Th1 and Th2 cells but also with Th17 cells. Our data provide new insights into the functional competence of NKT cells which will facilitate a better understanding of their versatile role during immune responses.

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Available from: ncbi.nlm.nih.gov
Natural killer T-cell characterization through gene expression
profiling: an account of versatility bridging T helper type 1 (Th1),
Th2 and Th17 immune responses
Introduction
Natural killer T (NKT) cells constitute a unique lympho-
cyte population. Unlike conventional CD4
+
T helper (Th)
cells which are reactive to major histocompatibility com-
plex (MHC) class II-associated peptides expressed by anti-
gen-presenting cells, NKT cells recognize lipids in the
context of CD1d molecules.
1–3
NKT cells are either CD4
+
or CD4
CD8
, and express a skewed range of T-cell
receptor (TCR) variable region genes and the natural
killer (NK) cell marker NK11 (NKR-P1C). In mice, most
NKT cells express an invariant Va14-Ja18 TCR combined
with a limited set of Vb chains.
4
Accordingly, these NKT
cells
5
have been termed invariant (i) NKT cells.
6
These
iNKT cells recognize alpha-galactosylceramide (aGalCer),
the model glycosphingolipid antigen.
2
Upon activation
through TCR ligation, iNKT cells release abundant
T helper type 1 (Th1) cytokines such as interferon (IFN)-
c and tumour necrosis factor (TNF)-a as well as Th2
cytokines such as interleukin (IL)-4, IL-10 and IL-13.
7–10
Prompt and simultaneous expression of Th1 and Th2
cytokines by the same cell type is a hallmark of iNKT
cells.
In fact, iNKT cells shape a wide range of immune
responses: they control tissue destruction, autoimmunity,
antitumour responses, host defence, allergy and inflam-
mation.
11–21
Accordingly, iNKT cell activation has
both beneficial
22–24
and harmful
25,26
consequences. Such
divergent responses may be attributed to their functional
heterogeneity as well as their tissue distribution. However,
Marcus Niemeyer,
1
Alexandre
Darmoise,
1
Hans-Joachim
Mollenkopf,
2
Karin Hahnke,
1
Robert Hurwitz,
3
Gurdyal S. Besra,
4
Ulrich E. Schaible
1
* and Stefan
H. E. Kaufmann
1
1
Department of Immunology,
2
Core Facility
Microarray and
3
Core Facility Biochemistry/
Protein Purification, Max Planck Institute for
Infection Biology, Berlin, Germany, and
4
School of Biosciences, University of Birming-
ham, Edgbaston, Birmingham, UK
doi:10.1111/j.1365-2567.2007.02701.x
Received 30 March 2007; revised 24 May
2007; accepted 24 July 2007.
*Present address: Infectious and Tropical
Diseases, Immunology Unit, London School
of Hygiene & Tropical Medicine, London,
UK.
Correspondence: Dr S. H. E. Kaufmann,
Department of Immunology, Max Planck
Institute for Infection Biology, Charite
´
platz
1, Campus Charite
´
Mitte, 10117 Berlin,
Germany. Email: kaufmann@mpiib-
berlin.mpg.de
Senior author: Stefan H. E. Kaufmann
Summary
Natural killer T (NKT) cells constitute a distinct lymphocyte lineage at
the interface between innate and adaptive immunity, yet their role in the
immune response remains elusive. Whilst NKT cells share features with
other conventional T lymphocytes, they are unique in their rapid, con-
comitant production of T helper type 1 (Th1) and Th2 cytokines upon
T-cell receptor (TCR) ligation. In order to characterize the gene expres-
sion of NKT cells, we performed comparative microarray analyses of mur-
ine resting NKT cells, natural killer (NK) cells and naı
¨
ve conventional
CD4
+
T helper (Th) and regulatory T cells (Treg). We then compared
the gene expression profiles of resting and alpha-galactosylceramide
(aGalCer)-activated NKT cells to elucidate the gene expressi on signature
upon activation. We describe here profound differences in gene expres-
sion among the various cell types and the identification of a unique NKT
cell gene expression profile. In addition to known NKT cell-specific mark-
ers, many genes were expressed in NKT cells that had not been attributed
to this population before. NKT cells share features not only with Th1 and
Th2 cells but also with Th17 cells. Our data provide new insights into the
functional competence of NKT cells which will facilitate a better under-
standing of their versatile role during immune responses.
Keywords: transcriptomics; T helper cells; regulatory T cells; natural killer
T cells; natural killer cells
Abbreviations: aGalCer, alpha-galactosylceramide; iNKT cell, invariant natural killer T cell; Th, T helper (CD4
+
CD25
);
Treg, regulatory T cell (CD4
+
CD25
+
).
2007 Max Planck Society Journal compilation 2007 Blackwell Publishing Ltd, Immunology, 123, 45–56 45
IMMUNOLOGY ORIGINAL ARTICLE
Page 1
the mechanisms determining the type of iNKT cell
response and the cytokine profile determining the con-
tribution of iNKT cells to immune responses remain
elusive.
Analysis of gene expression patterns and genomic pro-
filing have become useful tools for investigating the bio-
logical functions of distinct cell types and characterizing
the functional profiles that distinguish a unique pheno-
type, or differentiation or activation stage. Comparative
expression profiling reveals overlapping and unique signa-
tures of distinct cell types and hence underlines differ-
ences and similarities between functionally related cell
types or differentiation steps. We embarked on compara-
tive analysis of the gene expression profiles of iNKT
cells versus NK cells, conventional CD4
+
T cells (Th,
CD4
+
CD25
cells), and regulatory T cells (Treg, CD4
+
CD25
+
cells) to obtain basic information about the func-
tional competence of iNKT cells in innate and adaptive
immune responses. Moreover, analysis of resting versus
activated iNKT cells was undertaken in order to shed
light on their functional plasticity during an immune
response. Our findings reveal a unique gene expression
profile of resting NKT cells in comparison to NK cells,
Th cells and Treg cells, and multiple effector functions
linking activated NKT cells not only to the known Th1
and Th2 phenotypes but also to the Th17 phenotype.
Materials and methods
Mice
We used 7- to 12-week-old C57BL/6 (H-2
b
) wild-type
mice, and Va14-Ja18 transgenic (tg) mice backcrossed
(> 10 generations) on a C57BL/6 background.
27,28
Mice
were bred in our facility at the Bundesinstitut fu
¨
r Risik-
obewertung (BfR) in Berlin and kept under specific path-
ogen-free (SPF) conditions in filter bonnet cages with
food and water ad libitum. All experiments were con-
ducted according to the German Animal Protection Law.
Antibodies
Antibodies against murine CD4 (clone RM4-5), Fas
ligand (FasL) (clone K-10), IL-4 (clone 11B11), IFN-c
(clone R46A2), TNF-a (clone MP6-XT22), chemokine
(C-X-C motif) receptor 4 (CXCR4) (clone 2B11), macro-
phage-1 antigen (CD11b/CD18) (Mac-1) (clone M1/70),
B220 (clone RA3-6B2), CD11c (clone HL3) and CD8
(clone 53-67) were isolated from hybridoma cell lines.
The CD1d/aGalCer tetramers were produced as previ-
ously described.
29
Antibodies against murine NK11
(clone PK136), CD3 (clone 145-2C11), CD25 (clone
PC61), lymphocyte function-associated antigen-1 (LFA-1)
(clone M17/4) and streptavidin-PE-Cy7 were obtained
from BD Bioscience (Heidelberg, Germany). The mono-
clonal antibody (mAb) against murine IL-17 (clone TC11-
18H101) was obtained from BioLegend (San Diego, CA).
Isolation and purification of cells by magnetic antibody
cell sorting (MACS) and fluorescence-activated cell
sorting (FACS)
Isolation of spleen and liver lymphocytes, red blood cell
lysis and tetramer staining were performed as described
elsewhere.
30
MACS with MicroBeads (Miltenyi Biotec,
Bergisch Gladbach, Germany) was performed according
to the manufacturer’s protocol to enrich cell populations
by positive selection. Subsequently, enriched cells were
sorted using a FACS-Diva cell sorter (BD Bioscience). All
manipulations were performed on ice. Viable cells were
detected by staining with propidium iodide (PI) or
4
0
,6-diamidino-2-phenylindole dihydrochloride (DAPI).
The purity of sorted fractions was verified by FACS analy-
sis. At least 5 · 10
5
cells were isolated by FACS and fur-
ther used for microarray analysis. FACS was repeated four
times and duplicate samples of each sorted cell type were
used for two independent microarray studies. To avoid
gene expression resulting from handling and purification
of cells, all procedures were performed at 4. Moreover,
to reduce the overall purification time, MACS enrichment
preceded FACS. Furthermore, to avoid non-specific
effects, we performed all incubations in the presence of
blocking antibodies (anti-Fc receptor plus rat serum).
Although we cannot exclude the possibility that these
preparations might have impacted on the results, we con-
sider the chances of this to be minimal. This is consistent
with our observation that cells did not undergo general
apoptosis, as indicated by the lack of apoptosis markers.
In vivo activation of NKT cells
NKT cell activation was performed by injecting a single
dose of 2 lgofaGalCer intravenously (i.v.) into the tail
vein. Mice were killed 1 hr later and spleen cells were
isolated.
FACS analysis
To confirm microarray data at the protein level, flow
cytometric analyses were performed as follows. Single cell
suspensions were incubated with rat serum and anti-CD16/
CD32 (anti-Fc receptor) mAb for 5 min at 4 to block non-
specific antibody binding. Next, fluorescent dye-conjuga-
ted antibodies and phycoerythrin (PE)-conjugated CD1d/
aGalCer tetramers were added and cultures were incubated
for 50 min on ice in the dark on a rocking platform. Viable
cells were detected by staining cells with PI or DAPI. The
Cytofix/Cytoperm kit (PharMingen, San Diego, CA) was
used for intracellular cytokine staining. Tetramers were
prepared and loaded with lipids as described elsewhere.
29
46 2007 Max Planck Society Journal compilation 2007 Blackwell Publishing Ltd, Immunology, 123, 45–56
M. Niemeyer et al.
Page 2

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Supplementary material
The following supplementary material is available for this
article:
Figure S1. Purity of the second experimental set of
selected lymphocyte populations from C75BL/6 mice:
NKT, NK, conventional Th and Treg cells. Upper left:
sorted NKT cells. Upper right: sorted NK cells. Cells were
stained with antibodies against CD3 and NK1.1 and
CD1d/aGalCer tetramers. NKT cells were sorted as
NK1.1
+
and tetramer
+
cells among CD3
+
lymphocytes.
NK cells were sorted as NK1.1
+
and tetramer
)
cells
among CD3
)
lymphocytes. Percentages of positive cells
are indicated in the quadrants. Dead cells were excluded
by PI staining and only viable cells were sorted. Lower
left: sorted CD4
+
CD25
)
T cells. Lower right: sorted
CD4
+
CD25
+
T cells. Cells were stained with antibodies
against CD3, CD4 and CD25. T cells were sorted as CD4
+
and either CD25
)
or CD25
+
cells among CD3
+
lympho-
cytes. Percentages of positive cells are indicated in the
quadrants. Dead cells were excluded by PI staining and
only viable cells were sorted.
Figure S2. Correlation blots of log ratios deduced from 2
independent experimental sets of RNA from NKT cells,
NK cells, CD4
+
CD25
)
T cells (conventional Th) and
CD4
+
CD25
+
regulatory T cells (Treg). Colour-swap dye-
reversal ratio profiles were combined in an error-weighted
fashion (Rosetta Resolver) to create ratio experiments for
the respective experimental sets and the two experimental
sets were compared by using the Rosetta Resolver ‘‘com-
pare function’’ to decide how similar or dissimilar they
were. Correlated signatures are depicted in yellow and
anti-correlated signatures are depicted in pink. Ratios
shown in blue were unchanged between both sets and
red or green ratios were only present in set 1 or set 2,
respectively.
Table S1. Genes upregulated in activated NKT cells.
Depicted are fold change expression differences measured
in 2 independent microarray analyses (Sort I and Sort II),
P values, sequence names, NCBI accession numbers and
names of the genes. With the exception of IL17 in Sort II
only highly significant identified genes with P <005 and
with a minimum of 2-fold change in expression were
considered to be regulated.
Table S2. Primer sequences for real-time qPCR. The
NCBI accession numbers, the primer sequences and the
primer names are depicted, whereby the extensions Fwd
and Rev denote the forward and reverse primers for given
genes.
This material is available as part of the online article
from: http://www.blackwell-synergy.com/.
Please note: Blackwell Publishing are not responsible
for the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
56 2007 Max Planck Society Journal compilation 2007 Blackwell Publishing Ltd, Immunology, 123, 45–56
M. Niemeyer et al.
Page 12
  • Source
    • "Recent advances in NKT cell biology suggest that it is probably the most heterogeneous member of T lymphocyte family and hence exerts contrasting effects . NK1.1 À NKT cells, producing Th17 cytokine (IL-17), have been identified in mice [6]; however, IL-17 gene expression by both NK1.1 À and NK1.1 + NKT cells was observed upon activation in vitro [31]. IL-17-producing NK1.1 À NKT subset frequently occurs in lung and recruits neutrophil to the site of inflammation [6]. "
    [Show abstract] [Hide abstract] ABSTRACT: Natural killer T (NKT) cells are a unique subset of glycolipid-reactive T lymphocytes that share properties with natural killer (NK) cells. These lymphocytes can produce array of cytokines and chemokines that modulate the immune response, and play a pivotal role in cancer, autoimmunity, infection and inflammation. Owing to these properties, NKT cells have gained attentions for its potential use in antitumor immunotherapies. To date several NKT cell-based clinical trials have been performed in patients with cancer using its potent ligand α-galactosylceramide (α-GalCer). However, inconsistent therapeutic benefit, and inevitable health risks associated with drug dose and NKT cell activation have been observed. α-GalCer-activated NKT cells become anergic and produce both Th1 and Th2 cytokines that may function antagonistically, limiting the desired effector functions. Besides, various co-stimulatory and signaling molecules such as programmed death-1 (PD-1; CD279), casitas B-cell lymphoma-b (Cbl-b) and CARMA1 have been shown to be implicated in the induction of NKT cell anergy. In this Review, we discuss the role of such key regulators and their functional mechanisms that may facilitate the development of improved approaches to overcome NKT cell anergy. In addition, we describe the evidences indicating that tailored-ligands can optimally activate NKT cells to obtain desired immune responses.
    Full-text · Article · Dec 2013 · Human immunology
  • Source
    • "NKT cells are implicated in liver injury; their primary function is to mediate a balance between local production of Th1 pro-inflammatory and Th2 anti-inflammatory cytokines [179]. IFNγ, IL12, TNFα, and TNFβ are among the Th1 pro-inflammatory/anti-fibrogenic cytokines released by NKT cells, and IL4, IL10, and IL13 are among those of the Th2 anti-inflammatory/profibrogenic response [180]. NASH is associated with a relative imbalance favoring a Th1 pro-inflammatory response [181, 182]. "
    [Show abstract] [Hide abstract] ABSTRACT: Mounting epidemiological evidence points to an association between metabolic syndrome and non-alcoholic steatohepatitis (NASH), an increasingly recognized new epidemic. NASH pathologies include hepatocellular ballooning, lobular inflammation, hepatocellular injury, apoptosis, and hepatic fibrosis. We will review the relationship between insulin resistance and inflammation in visceral obesity and NASH in an attempt to shed more light on the pathogenesis of these major metabolic diseases. Moreover, we will identify loss of the carcinoembryonic antigen-related cell adhesion molecule 1 as a unifying mechanism linking the immunological and metabolic abnormalities in NASH.
    Full-text · Article · Nov 2013 · Seminars in Immunopathology
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    • "(33, 34). However, by using the microarray assay on mouse NKT cells, production of IL-17 by both NK1.1+ and NK1.1− cells upon activation in vitro has also been reported (35). "
    [Show abstract] [Hide abstract] ABSTRACT: We demonstrate that CD161 is a highly up-regulated gene in human interleukin (IL) 17 T helper cell (Th17) clones and that all IL-17-producing cells are contained in the CD161(+) fraction of CD4(+) T cells present in the circulation or in inflamed tissues, although they are not CD1-restricted natural killer T cells. More importantly, we show that all IL-17-producing cells originate from CD161(+) naive CD4(+) T cells of umbilical cord blood, as well as of the postnatal thymus, in response to the combined activity of IL-1 beta and IL-23. These findings implicate CD161 as a novel surface marker for human Th17 cells and demonstrate the exclusive origin of these cells from a CD161(+)CD4(+) T cell progenitor.
    Full-text · Article · Sep 2008 · Journal of Experimental Medicine
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