Alternative Spliced CD1D Transcripts in Human Bronchial
Kambez Hajipouran Benam, Wai Ling Kok, Andrew J. McMichael, Ling-Pei Ho*
MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom
CD1d is a MHC I like molecule which presents glycolipid to natural killer T (NKT) cells, a group of cells with diverse but critical
immune regulatory functions in the immune system. These cells are required for optimal defence against bacterial, viral,
protozoan, and fungal infections, and control of immune-pathology and autoimmune diseases. CD1d is expressed on
antigen presenting cells but also found on some non-haematopoietic cells. However, it has not been observed on bronchial
epithelium, a site of active host defence in the lungs. Here, we identify for the first time, CD1D mRNA variants and CD1d
protein expression on human bronchial epithelial cells, describe six alternatively spliced transcripts of this gene in these
cells; and show that these variants are specific to epithelial cells. These findings provide the basis for investigations into a
role for CD1d in lung mucosal immunity.
Citation: Benam KH, Kok WL, McMichael AJ, Ho L-P (2011) Alternative Spliced CD1D Transcripts in Human Bronchial Epithelial Cells. PLoS ONE 6(8): e22726.
Editor: Johan K. Sandberg, Karolinska Institutet, Sweden
Received April 14, 2011; Accepted July 3, 2011; Published August 11, 2011
Copyright: ? 2011 Benam 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: The study was funded by the Medical Research Council (UK). 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: Lingfirstname.lastname@example.org
CD1d is a member of the CD1 family of transmembrane
glycoproteins. It presents self and foreign glycolipid antigens to a
group ofT lymphocytepopulationcalled naturalkillerT cells(NKT
cells) . In this context, CD1d shows high structural homology to
the MHC class I genes which encodes a type I integral membrane
protein (a heavy chain) with three extracellular domains: a1, a 2,
and a3 . Like HLA A–C, CD1d protein non-covalently
associates with b2-microglobulin (b2m); but unlike the MHC gene,
its expression on professional antigen presenting cells (APC) is
limited in genetic and allelic variation, and is thought to pre-
dominantly engage the semi-invariant T cell receptor (TCR)
expressed by NKT cells. However, the consequence of this
interaction is distinct and wide-ranging. When activated, NKT
cells produce large amounts of cytokines (including TH1, TH2 and
TH17- related cytokines) [2,3,4,5], and much more rapidly than do
conventional T cells. They have the capacity to critically modulate
immunity by interacting with T cells, NK cells, macrophages and B
cells. They regulate the development of a number of inflammatory
diseases – best shown in animal models of Type I diabetes mellitus,
multiple sclerosis, asthma and infectious diseases [6,7,8,9,10,11,12].
In the lungs, divergent effects have been noted after NKT cell
activation. We have shown that NKT cells participate in immune
responses in the lungs, and are protective during influenza virus
infection [11,12]. Their numbers increased in the lungs within
three days of influenza virus infection, and activated NKT cells
can amplify the innate immune response and decrease early viral
load. De Santo et al also showed that NKT cells can reduce the
recruitment of ‘myeloid-derived suppressor cells’ which allowed
increased proliferation of influenza virus-specific CD8 T-cell
responses . CD1d-deficient mice that lack NKT cells also
succumb to other bacterial infection of the lungs like pseudomonas
aeruginosa . However, NKT cells could also be pathogenic in
non-infectious conditions – for example in the development of
airway hyper-responsiveness in animal models of asthma .
Despite these studies, it is not clear how NKT cells arrive in the
andhowtheyareactivated inthe lungs.Intra-nasaladministrationof
a glycolipidligand showed a quick expansion of the cells inthe lungs,
suggesting that a CD1d-expressing cell population could present
antigen to NKT cells in this site . One possibility is dendritic
within the airways. Another possibility is the airway epithelium.
Potentially, CD1d expression on bronchial epithelium could be
involved in activation of NKT cells or promote engagement of NKT
cells with bronchial epithelium and enhance the role of bronchial
epithelium in mucosal immunity. To date, CD1d expression has not
been observed in primary or bronchial epithelial cell lines but it has
been reported on epithelial and parenchymal cells in liver, kidney,
intestine and skin [13,14,15,16,17]. The function of CD1d on these
structural cells is not clear, although freshly isolated intestinal
epithelial cells pulsed overnight with a glycolipid were capable of
activating NKT cells . Here, we provide the first report on
CD1D mRNA and CD1d protein expression in human bronchial
epithelial cells, and describe six alternatively-spliced transcripts of
this gene in these cells. This provides a basis for investigations into a
role for CD1d in lung mucosal immunity.
Primary human bronchial epithelial cells and airway
epithelial cell lines express CD1D
Three sets of primers (‘‘A/A’’, ‘‘B/B’’ and ‘‘C/C’’) were
designed to specifically amplify CD1D transcript within its coding
PLoS ONE | www.plosone.org1August 2011 | Volume 6 | Issue 8 | e22726
exons (Table 1, and Figure 1A). ‘‘A/A’’ spanned a2 exon covering
most of a1 and a3 exons, and ‘‘B/B’’ amplified transmembrane
(TM) and most of a3 and cytoplasmic tail (T) exons. We first
determined if human bronchial epithelial cells expressed CD1D,
using RNA derived from ex vivo primary human bronchial
epithelial cells obtained by bronchoscopic brushing. These brushed
cells comprised 90% epithelial cells, as shown by pancytokeratin
and ß tubulin IV immunofluorescense staining (Figure 1B), and
werefound to express CD1D (Figure 1C,right panel). SinceCD1D-
expressing cells may have contaminated this population of primary
epithelial cells, we proceeded to examine CD1D expression in pure
airway epithelial cells using the Beas2B cell line. RT-PCR detected
CD1D transcripts in Bea2B (Figure 1D). The Jurkat cell line, which
naturally expresses CD1d protein, was used as a positive control.
Absence of contaminating DNA was shown by including a reaction
where no reverse-transcriptase was used (‘No RT’).
Expression of CD1d protein on epithelial cells was
observed with some but not all CD1d mAbs
Using three mouse anti- human CD1d mAbs (clones CD1d 43,
CD1d 51.1.3 and NOR 3.2), we showed surface CD1d expression
on Beas2B, primary brushed bronchial and NHBE cells with all
three mAbs; but, A549 – a Type II human pneumocyte, stained
positive with one Ab only (Figure 2A–C). The clone 51.1.3 had
been previously reported to partially cross-react with CD1b .
To verify CD1d detection, Beas2B cells were stained with an anti-
CD1b mAb. We found no CD1b expression on Beas2B, whereas
the positive control, monocyte-derived dendritic cells (MD-DCs)
distinctly expressed CD1b (Figure 2B).
The epitopes for these CD1d mAbs are currently unknown.
However, the differential expression raised possibility of the
existence of variants and prompted design of further primer sets to
examine this prospect.
RT-PCR of primary bronchial epithelial and cell line
confirms expression of CD1D in bronchial epithelium and
reveals six variant transcripts
To explore the possibility of splice variants, primers were
designed to probe the exon boundaries (Figure 3A) (primer pairs
‘‘C/C’’, ‘‘D/D’’ and ‘‘D/E’’). Using primer pair ‘‘C/C’’, which
amplifies a 572–bp product containing parts of the signal peptide
(S) and a2 exons, and the whole a1 exon of CD1D, we unex-
pectedly, observed a 305-bp product in brushed primary bronchial
epithelial cells (Figure 3B, left panel), which raised the possibility of
an alternatively spliced variant. This was also the case in the
Beas2B cell line (Figure 3B, right panel), where both the full-length
and 305-bp products were amplified. Direct nucleotide sequencing
confirmed the shorter amplicon to be CD1D with a spliced a1
exon, which we called ‘‘V1’’ (Figure 3B, bottom panel). To ensure
that V1 is not a non-specific amplification, RT-PCR using the C/
C primer was repeated on cDNA reverse transcribed using a
CD1D-specific primer (GGACGCCCTGATAGGAAGTT) rath-
er than oligo dT. The latter also showed the presence of V1
(Figure 3B, right panel).
For ‘‘D/D’’ and ‘‘D/E’’ primers, the forward primer in both sets
originates in the first half of the a2 exon, while the reverse primers
Rev. D and Rev. E originate in T (tyrosine tail) and 39-UTR of exon
7, respectively (Figure 3A). RT-PCR with ‘‘D/D’’ amplified six
products with varying lengths (Figure 3C). Only one (Non-Spliced;
NS) corresponded to expected non-spliced transcript (636-bp). To
examine whether the other amplified DNAs were spliced forms of
CD1D, the bands were cut out from the gel and nucleotide
sequence determined. This confirmed all as PCR products for
CD1D (‘‘V2–6’’). In addition, sequencing revealed the splice
junctions for three of the variants: ‘‘V4’’, ‘‘V5’’, and ‘‘V6’’
(Figure 3D). V4 and V5 were deficient in a3 (279 bases) and a3-
TM (379 bases), respectively; whereas ‘‘V6’’ represented a variant
with double splicing events, trimming out the second half of a2 (last
133outof 279 bases)and most (274 out of279bases)of the a3 exon.
To compare the relative amount of variants, qPCR using SYBR
Green methods or Taqman probe was carried out for ‘‘V1’’, ‘‘V3–
6’’, using the primer and probe sets described in Table 1. Multiple
primers for ‘‘V2’’ produced late Ct on amplification plot and were
inconsistent. We believe this reflected very low level of gene
expression and therefore did not test V2’s relative levels. We tested
these primers in NHBE primary cells (Figure 4A, left panel). This
allowed us to confirm the variants in primary human bronchial
epithelial cells and also show that relative to the alpha-1 deficient
splice variant (‘‘V1’’) (Figure 4A), ‘‘V4’’ and ‘‘V6’’ were most
highly expressed. Relative to the full length transcript (FL), ‘‘V1’’
was 9.2 fold lower (Figure 4A, right panel). Characteristics and
potential implications of ‘‘V1–6’’ are summarized in Table 2.
To exclude possibility of DNA defects as the source for these
variants, genomic DNA from Beas2B were amplified using two of
the primers and showed to have only one band (Figure 4B).
Table 1. Primers and probes used in this study.
Target genePrimer or Probe sequence (59R39)
CD1D For. A: CTCGTCCTTCGCCAATAGC
Rev. A: ACCACATCCAGGGTTGCTC
For. B: AGCCTGTATGGGTGAAGTGG
Rev. B: GGACGCCCTGATAGGAAGTT
For. C: CTGCTGTTTCTGCTGCTCTG
Rev. C: GACTCAAGGAGGCCACTGAC
For. D: CTGGGAACGCCTCAAATAAC
Rev. D: ACAGGACGCCCTGATAGGA
Rev. E: GAAAGCTGCCTCATGACTGTT
CD1DFor. V1 (SYBR): GCGCTGAAGATCCCTTGGA
Rev. V1 (SYBR): TACATGGAAGAAGTTATTTGAGGCG
For. V3 (SYBR): CAAGAGGCCCCACTTTGGT
Rev. V3 (SYBR): GAGACATGGCACACCAGCAG
For. V4 (Taqman): AACAGTGCAGTGGCTCCTTAATG
Rev. V4 (Taqman): TCCCACCTTGCTTCTTCAGTTC
Probe V4 (Taqman): CCCAATTTGTCAGTGGCCTCCTTGAGT
For. V5 (SYBR): GAGGCCCCACTTTGGGTAAA
Rev. V5 (SYBR): CAGGACGCCCTGATAGGAACT
For. V6 (Taqman): TTTGGAGCAGGTGGGAGCTA
Rev. V6 (Taqman): CGGGAGGTAAAGCCCACAAT
Probe V6 (Taqman): AGTCCTGGCGTGCTTGCTGTTCCTC
HPRTFor. (Taqman): GACTTTGCTTTCCTTGGTCAGG
Rev. (Taqman): AGTCTGGCTTATATCCAACACTTCG
Probe (Taman): TTTCACCAGCAAGCTTGCGACCTTGAT
For.: Forward primer; Rev.: Reverse primer; SYBR: real time PCR amplification by
SYBR Green method; Taqman: real time PCR amplification by Taqman assay
using FAM-, TAMRA-conjugated primers; V1–6: CD1D spliced variants.
CD1d Expression on Bronchial Epithelium
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CD1D spliced variants are expressed on epithelial cell
lines but not in antigen presenting cells, and fibroblast
Although we have shown existence of CD1D variants in human
bronchial epithelial cells, it is unclear if the same set of variants is
observed in other cells. Unlike other CD1 family members (CD1A,
CD1C, and CD1E), CD1D splicing has only been marginally
studied in two reports [20,21]. One study found eight variants in
peripheral blood mononuclear cells (PBMCs), but using nested
PCR which reduced the reliability of the results . Neither
study compared the expression of the spliced variants across
different cells. To address this, we examined CD1D variants in
several cell lines representing epithelium from various sites
(Beas2B, 16HBE14O2, NKer, HaCat, HCT116 and TZM-bl;
sources described in Methods) compared to antigen presenting
cells (THP-1), T cells (MOLT4) and fibroblasts (MRC5 and
IMR90). Using a primer set that identified variants ‘‘V2–6’’ (‘‘D/
D’’), we found similar splicing patterns among bronchial epithelial
cells 16HBE14O2and Beas2B, normal keratinocytes (NKer) and
colon epithelial cells (HCT116) (all expressed full length and ‘‘V2’’
to ‘‘V6’’ variants). This suggests potential similarity in splicing
machinery at resting state across different mucosa-lining epithelial
cells, though cancerous transformation or immortalised nature of
cell lines may account this phenomenon The fibroblasts only
expressed ‘‘V6’’ (MRC5) and ‘‘V3’’, ‘‘V4’’ and ‘‘V6’’ (IMR90).
The primary cells PBMC and dendritic cells (DCs) also expressed
all variants but possibly to a different relative amounts (Figure 4D).
The a3–deficient CD1D variant (‘‘V4’’), when present, was the
most intensely PCR-amplified transcript in all cell types.
In this paper, we show that CD1d is expressed on bronchial
epithelial cells, both in primary and airway epithelial cell lines, and
that there are at least six spliced variants likely specific to epithelial
cells. We felt that examining the function of these epithelial CD1d
transcripts was beyond the scope of this paper so it is unclear
currently how these proteins function in the context of the lungs.
However, it is possible to speculate on the potential implications of
CD1d expression on these structural cells. Due to the large surface
area of the airway epithelium, one prospect of CD1d expression
here is the rapid presentation of both self and exogenous
glycolipids to infiltrating NKT cells. This could maintain NKT
cells proliferation in situ and prolong their protective effect in the
lungs. The opposite is also possible – that of enhancing NKT cell’s
harmful effect e.g. airway hyper-responsiveness observed in
murine models of asthma. In the ovalbumin asthma model,
Figure 1. Identification of CD1D transcript in human respiratory epithelial cells. Panel A: Graphical representation of CD1D mRNA; coding
exons in orange and non-coding exons in open boxes. Primer annealing sites are illustrated by horizontal arrows, with the expected amplicon size for
each pair. Panel B: Brushed bronchial cells immunostained with pancytokeratin and b–tubulin IV, identifying epithelial cells and cilia respectively;
‘‘isotype control’’ - no primary Ab treatment. The 2006magnification shows ß tubulin IV staining in green and nuclei in blue (DAPI). Panel C: Agarose
gel on the right demonstrates RT-PCR on these cells. Panel D: CD1D expression on Beas2B and Jurkat cell lines.
CD1d Expression on Bronchial Epithelium
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CD1d2/2mice do not develop airway hyper-responsiveness
A potentially interesting part of our results is the observation of
the same profile of transcripts in epithelial but not haematopoietic
cells. The epithelial cell lines from skin, lungs, cervix and colon
showed ‘‘V2’’ to ‘‘V6’’ expression (‘‘V2’’ and ‘‘V3’’ very faint in
the cervical epithelial cell line, TZM-bl). The exception to this is
A549, a type II alveolar epithelial cell line which only expressed
‘‘V4’’ and ‘‘V5’’. Type II alveolar epithelial cells have the unique
property of producing surfactants, which regulate the surface
tension of the alveoli and also to contribute to host defence. In the
latter, two subsets of surfactant (SP-A and SP-D, part of collectins
family) act as opsonins to facilitate elimination of pathogens by
alveolar macrophages . The CD1D transcripts on these cells
(‘‘V4’’ and ‘‘V5’’, discussed later as potential ß2m-independent
and soluble forms respectively) could serve as innate host defence
molecules rather than antigen-presenting molecules.
Against expectation, we observed only very low/undetectable
levels on full length mRNA in PBMC and DCs (Figure 4C). There
have been very few studies on the variety of CD1D mRNAs in
these cells and their relative expression to the full-length transcript.
Kojo et al mentioned existence of multiple CD1D transcripts in
PBMCs, however, the level of expression was so low that they
required nested PCR to identify this; only three healthy volunteers
were studied, among these, one had extremely low/undetectable
levels of non-spliced full-length CD1D. Our low expression is in
keeping with their finding, and the low/undetectable expression
by one-round of PCR in MOLT-4 and THP cells support the
finding in primary hematopoietic cells Since the CD1d protein has
been identified in these cell populations, one possibility is that the
mAbs used detected proteins translated by the variants rather than
the full length CD1D.
Some potential functions of the different variants could be
gleaned from knowledge of the various regions that are spliced
(Table 2). Lipid-binding groove in CD1d is formed by a1 and a2
domains [26,27]. The protein for ‘‘V1’’, deficient in a1, is unlikely
to participate in antigen loading and presentation. Hydrophobic
amino acid residues encoded by transmembrane (TM) exon are
Figure 2. Human respiratory epithelial cells express CD1d protein. Panel A: Flow cytometry analysis of CD1d expression on Beas2B and A549
epithelial cell lines, using three anti-human CD1d mAbs – clones 42, 51.1.3, and NOR3.2. Panel B: CD1b staining on Beas2B cells and CD1b-expressing
monocyte-derived dendritic cells (DC). Panel C: Flow cytometry analysis of CD1d expression on primary human lung epithelial cells (top panel) and
normal human bronchial epithelial (NHBE; bottom panel) cells.
CD1d Expression on Bronchial Epithelium
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required for insertion of the CD1d protein into plasma membrane.
The TM exon is spliced out in ‘‘V2’’ and ‘‘V5’’. In addition, ‘‘V3’’
and ‘‘V6’’ as a consequence of shift in reading frames contain early
stop codons in a3 and TM, respectively. So, it is likely that
proteins coded by these variants will be either secreted (soluble
isoform) or retained intracellularly. There is precedence for this -
Woolfson et al showed expression of CD1A and CD1C TM-
deficient transcripts, and detected corresponding soluble protein
isoforms . Similarly, soluble HLA-G, an MHC class I
molecule which has limited polymorphism and shares high
structural similarity with CD1D , is encoded by a TM-
deficient variant [30,31], and was recently shown to activate NK
cells via NFkB signalling .
‘‘V4’’ is the most highly expressed transcript in NHBE cells; and
is potentially an interesting variant which could have epithelial-
specific function. Its protein product is predicted to lack ß2m
binding ability but it has intact TM and cytoplasmic tail (T) exons,
so it can produce a cell surface-expressed b2m-free CD1d isoform.
On human intestinal epithelial cells, the major form of CD1d is a
non-glycosylated, ß2m-independent molecule, although these cells
also express a ß2m-associated, fully glycosylated form of CD1d
. The importance of ß2m association with CD1d with respect
to the CD1d biosynthetic pathway is not established. Association
of ß2m with CD1d could have a role in regulating the extent of
CD1d glycosylation and the maturity of the attached carbohydrate
side chains . Although, in most cases, forms of MHCI protein
that do not associate with ß2m do not fold their antigen presenting
domains properly, cells from ß2m -deficient mice are able to
stimulate proliferation of CD1d-restricted T cell clones ,
suggesting presence of a CD1d variant that do not rely on ß2m for
antigen presentation and function.
Regardless of the transcripts, identification by antibodies
confirms the presence of protein on the surface of bronchial
epithelial cells. The CD1d antibody, clone 42, has been widely
used as a blocking antibody to prevent activation of NKT by
CD1d-expressing cells [35,36,37], therefore flow cytometry results
Figure 3. Expression of six CD1D alternatively spliced CD1D variants in human respiratory epithelial cells. Panel A: Schematic
illustration of primer pairs ‘‘C/C’’, ‘‘D/D’’, and ‘‘D/E’’ annealing sites on CD1D mRNA, and corresponding full-length product sizes. Panel B: RT-PCR on
primary bronchial epithelial cells detected a 305-bp band. Same primers amplified the expected full-length (572-bp) product plus a shorter amplicon
(305-bp) both in oligo dT– and CD1D specific primer–reverse transcribed cDNA from Beas2B. Direct nucleotide sequencing of the smaller band (lower
panel) identified a CD1D variant lacking a1 exon (‘‘V1’’). Panel C: Primer pairs ‘‘D/D’’ and ‘‘D/E’’ detected five other CD1D variants (‘‘V2–6’’) in Beas2B.
Panel D: Direct nucleotide sequencing of ‘‘V4–6’’ revealed the splicing junction in these variants.
CD1d Expression on Bronchial Epithelium
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verify presence of (at least) one functional CD1d (likely full length
protein) with the ability to associate with ß2m on the surface
human bronchial epithelial cells.
In summary, we have identified for the first time, CD1D
expression on human bronchial epithelial cells and detection of at
least some of the variant protein by CD1d monoclonal antibodies.
This provides a basis for further investigations for the role of this
molecule in lung immune responses.
Materials and Methods
Cells and culture conditions
Fresh primary human bronchial epithelial cells were obtained
from the primary bronchus of a patient undergoing bronchoscopy
for a localised lung tumour. Samples were obtained on the contra-
lateral (normal) side to the tumour, by brushing the primary
bronchus using a bronchoscope brush (KeyMed fiberoptic
Figure 4. Relative expression of CD1D transcripts in human respiratory epithelial cells and cell-type pattern of variant expression.
Panel A: Relative abundance of different CD1D variants by qPCR in NHBE cells compared to ‘‘V1’’(left), and between ‘‘V1’’ and full length (FL). Panel B:
PCR using ‘‘C/C’’ and ‘‘D/D’’ primers in genomic DNA from Beas2B cell line. Panel C: Pattern of expression for CD1D variant in different human cell
lines and primary cells; GAPDH was used as a control.
Table 2. Predicted properties of CD1d proteins coded by different mRNA transcripts in human respiratory epithelial cells.
Variant Spliced sequenceFrame shift Stop codon position Membrane insertionAg presentation Predicted MW (kDa)
V2 TMOut-of-frame 78thbase in exon 7Unlikely Likely36.1
V3Part of a2Out-of-frame1stbase in a3 exonUnlikelyUnlikely21.4
a3 In-frame UnchangedLikelyLikely27.4
a3-TMOut-of-frame78thbase in exon 7Unlikely Likely25.8
V6 Part of a2+a3Out-of-frame 25thbase in TM exonUnlikelyUnlikely18.9
V1–6: CD1D spliced variants; NS: Non-spliced.
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bronchoscope; Olympus; Japan). The cells were dislodged into
complete RPMI-1640 (Invitrogen, UK) by rigorous agitation of
the brush within the container and used within 6 hours. The
patient provided written informed consent and the study was
approved by the Oxfordshire Research Ethics Committee.
Primary normal human bronchial epithelial cells (NHBE cells)
were purchased from Lonza (Walkersville, Inc), and grown in
serum-free and hormone and growth factor supplemented
bronchial epithelial growth media (BEGM) (Lonza, Walkersville,
Inc.), under submerged conditions.
All cell lines were purchased from the American Type Culture
Collection(Manassas, VA), apartfrom NKer, a human keratinocyte
line immortalized using human papilloma virus-16 E6 protein
which was a kind gift from Dr E O’Toole, (Northwestern University
Medical School, Chicago, USA). Beas-2B was cultured in F12
Nutrient Mixture Kaighn’s Modification (F12K); A549 (human
type II alveolar epithelial cell line), 16HBE14O2(differentiated
SV40-transformed human bronchial epithelial), TZM-bl (CD4-
expressing adenocarcinoma-derived human cervical epithelial cell
line), HaCat (spontaneously immortalised human keratinocyte),
HCT116 (human colon carcinoma epithelial) and NKer were
maintained in Dulbecco’s modified Eagle medium (DMEM)
(Invitrogen, Carlsbad, CA). Jurkat (an immortalized T cell line),
THP1 (a human monocytic/macrophage cell line) and MOLT4 (a
human leukemic T lymphoblast cell line) were cultured in RPMI-
1640. All cell culture media were supplemented with 10% FBS
(Invitrogen), 2 mML-glutamine (Invitrogen), 50 U/ml penicillinG,
and 50 mg/ml streptomycin (Invitrogen) (complete RPMI 1640).
Cells were cultured at 37uC and 5% CO2.
Monocyte-derived dendritic cells (DCs) were generated as
previously described . All DCs were checked for maturation
markers (CD83, CD86, and HLA-DR; eBioscience, USA) and
determined to be CD1b+and CD1d+and CD142at time of use.
DNA preparation, RNA isolation and cDNA preparation
Genomic DNA was purified using DNeasy Blood & Tissue Kit
(Qiagen, UK). Total RNA was extracted by lysing the cells in 1 ml
TRIzol reagent (Ambion, UK) or using RNeasy Mini Kit (Qiagen,
UK), following the manufacturers’ protocols. After extraction with
either protocol, RNA was incubated with DNase I (Qiagen, UK)
for 15 minutes at room temperature to remove residual contam-
inating DNA, and the enzyme inactivated by heating at 65uC for
5 minutes. Isolated total RNA was reverse transcribed using
SuperScriptH Reverse Transcriptase III kit (Invitrogen, UK), ac-
cording to manufacturer’s protocols. cDNA synthesis was primed
by mixing 1 ml of either oligo dT (50 mM), or gene-specific primer
(1 mM), and 1 ml of dNTP mix (10 mM) with up to 5 mg total
Reverse transcription PCR and real time quantitative PCR
Primers and probes were designed using either Primer ExpressH
software version 3.0 (Applied Biosystems, UK) or the online
Primer3 application (http://frodo.wi.mit.edu/primer3/). For real
time PCR analysis, the melting temperatures of probes were 10uC
higher than the corresponding primers, and the amplicons had a
length of 50–150-bp.
For RT-PCR, two microlitres cDNA was added to 48 ml reaction
mix containing 2 U FastStart Taq DNA Polymerase (Roche, UK),
5 ml 106 PCR buffer, 2 mM MgCl2, 1 mM of each dNTP and
1 mM of forward and reverse primers and amplified by GeneAmpH
PCR System 2700 thermal cycler (Applied Biosystems, UK).
Real time PCR was performed using 7500 Fast real time PCR
system (Applied Biosystems, UK). Reactions contained 2 ml cDNA
added to 10 ml 26Universal or SYBR Green Master Mix (Applied
Biosystems, UK) and appropriate CD1d forward and reverse
primers, and probes where used (Eurogentec, UK). PCR conditions
for SYBR Green assay were as followed: 50uC for 2 minutes, 95uC
for 10 minutes and 45 cycles of 95uC for 15 seconds and 60uC for
1 minute. Taqman assay conditions were optimised at 95uC for
20 seconds and 45 cycles of 95uC for 3 seconds and 60uC for
The results were quantified using 22DDCtmethod , where
DCt (CtCD1D variant2Cthousekeeping gene) was calculated initially, and
then DDCt was obtained by subtracting DCt of reference CD1D
variant (e.g. V1) from that of a given CD1D variant (e.g. V3, V4,
etc). Comparable amplification efficiencies for both housekeeping
genes and CD1D variants was verified by serial dilution of two
highly abundant CD1D variants against HPRT housekeeping
Primer and probe sequences are depicted in Table 1.
antibody-binding sites were blocked by incubation with blocking
buffer (5% FCS, 1% BSA in PBS); and stained with pancytokeratin
(PCK) mAb (Abcam, UK), or b-tubulin IV (BioGenex, UK), and
then Alexa FluorH 633-conjugated goat anti-mouse IgG (Invitrogen,
Purified PCR products were sequenced using the BigDyeR
Terminator v3.1 cycle sequencing kit (Applied Biosystems, UK)
and analyzed using a 3730 ABI capillary electrophoresis system
(Applied Biosystems, UK).
The authors thank Dr. Simon Brackenridge for technical discussions.
Conceived and designed the experiments: LPH. Performed the experi-
ments: KHB WLK. Analyzed the data: LPH KHB. Wrote the paper: LPH.
Discussed data: AJM.
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