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Journal of Immunological Methods 250 (2001) 29–43
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Application of differential display to immunological research
Manir Ali*, Alexander F. Markham, John D. Isaacs
Molecular Medicine Unit, University of Leeds, Clinical Sciences Building, St. James’ s Hospital, Leeds LS9 7 TF, UK
Abstract
The majority of immunological processes are mediated by cell-to-cell contact or receptor–ligand interactions that transmit
intracellular signals and affect the regulation of transcription in the nucleus. As a consequence, precursor cells develop into
their respective lineages and cells differentiate further during an immune response. In order to study changes in normal cells
or even cells that have been isolated from diseased tissue, a number of approaches have been developed. One such method,
differential display (DDRT-PCR), is a versatile technique for the analysis of gene expression that is based on RT-PCR and
denaturing polyacrylamide gel electrophoresis. This technique is applicable to multiple samples of clonal or purified cell
populations as well as to complex tissues and can be used to provide mRNA fingerprints. However, the main purpose of
DDRT-PCR is to isolate differentially regulated genes in biological systems. The method is carried out without prior
hypothesis as to which genes should be examined and so increases the possibility of identifying completely novel and
unexpected changes in transcription. A major drawback has been the isolation of false positive clones and the need to
confirm the results of analysis by another method. This makes DDRT-PCR labour intensive. A number of strategies have
been recommended to reduce these problems, including reverse-northern analysis as a confirmatory step for screening
putative differentials. In order to reduce the number of gel fingerprints that would be required to cover all the mRNAs in a
cell, several focused approaches have been suggested. These include targeted differential display for the isolation of
multigene families that have conserved protein domains or gene signatures and subtractive differential display whereby one
population is subtracted from the other prior to screening. The purpose of this review is to provide some guidance to the
immunologist who might wish to apply DDRT-PCR in their research. A number of examples where DDRT-PCR has been
used successfully in immunological research are included.  2001 Elsevier Science B.V. All rights reserved.
Keywords: Gene expression; Differential display; Transcriptional changes; Gene isolation
1. Differential display RT-PCR (DDRT-PCR)
Differential display is a powerful tool for the
comparison of gene expression between two or more
Abbreviations: RT-PCR, reverse transcription polymerase chain
reaction; mRNA, messenger ribonucleic acid; cDNA, complementary deoxyribonucleic acid
*Corresponding author. Tel.: 144-113-206-6215; fax: 144113-244-4475.
E-mail address: [email protected] (M. Ali).
mRNA populations (Liang and Pardee, 1992; Welsh
et al., 1992). The first part of this technique is an
adaptation of existing technologies, namely PCR and
denaturing polyacrylamide gel electrophoresis, that
have previously been used to provide DNA fingerprints of hybrid cell lines (Ledbetter et al., 1990) and
particular genomic regions (Welsh and McClelland,
1990; Williams et al., 1990). As with all gene
expression technologies, the essential element of the
technique involves the identification (and confirmation) of transcripts that are truly differentially ex-
0022-1759 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved.
PII: S0022-1759( 01 )00304-0
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M. Ali et al. / Journal of Immunological Methods 250 (2001) 29 – 43
pressed. It is this aspect of the method that is
particularly time-consuming and labour intensive.
A schematic for the basic DDRT-PCR protocol is
shown in Fig. 1. Briefly, RNAs extracted from the
sources to be compared are reverse transcribed with
one of a possible set of four degenerate oligonucleotide primers (dT) 12 VC, (dT) 12 VA, (dT) 12 VG or
(dT) 12 VT where V is C, A or G (Liang et al., 1993).
First-strand cDNA is used as a template in the PCR
with the original oligo(dT) primer mixture and a
decamer sequence that has been randomly generated.
It is advisable that the decamer primer has a GC
content .50% and is non-palindromic. The reaction
is carried out in the presence of radiolabelled nucleotide that will incorporate into the accumulating PCR
products. The complex mixture of cDNAs are then
resolved by electrophoresis through a denaturing
polyacrylamide gel and visualised by autoradiography (see Fig. 2). Various parameters in the PCR can
be altered to increase the number of displayed bands
on a gel. For example, the number of PCR cycles or
the concentration of decamer primer can be increased
or the annealing temperature can be lowered
Fig. 2. Autoradiograms of DDRT-PCR gels. RNA isolated from a
T cell clone that had undergone peptide-induced anergy (I) and
control resting cells (II) were taken through DDRT-PCR analysis.
Duplicate PCR reactions were carried out for each of the
conditions to be compared. The image shows that bands 1 and 2
correspond to cDNAs whose transcripts are down-regulated during
T-cell anergy, whereas band 3 depicts cDNAs that are derived
from up-regulated transcripts during this treatment.
Fig. 1. Schematic of DDRT-PCR protocol.
(Guimaraes et al., 1995). Differentially displayed
bands are excised from the gels and reamplified by
PCR in the absence of radionucleotide. The amplified products are then ligated into a plasmid
vector, transformed into bacteria and screened for the
presence of insert DNA by PCR using flanking
vector primer sequences. These cDNAs can be tested
to confirm differential gene expression and in our
experience reverse-northern analysis provides a high
throughput approach. Thus, PCR amplified cDNAs
are immobilised on nylon filter membranes and
screened with radiolabelled complex cDNA mixtures
M. Ali et al. / Journal of Immunological Methods 250 (2001) 29 – 43
derived from the sources to be compared. Recombinant clones are selected and plasmid DNA extracted
prior to sequencing and database searching.
Recent developments have incorporated safer,
non-radioactive methods for the global analysis of
gene expression. These have included silver staining
for DNA detection after polyacrylamide gel electrophoresis (Gottschlich et al., 1997; Kociok et al.,
1998), fluorescent-labelled oligonucleotides for PCR
with analysis by ABI sequencers (Bauer et al., 1993;
Ito et al., 1994; Luehrsen et al., 1997; Smith et al.,
1997a) and the use of biotinylated primers with
streptavidin coated beads for capture (Korn et al.,
1992; Tagle et al., 1993; Rosok et al., 1996).
Alternatively, agarose gel electrophoresis followed
by ethidium bromide staining and UV illumination
for detecting cDNAs is another option (Rompf and
Kahl, 1997; Jefferies et al., 1998; Gromova et al.,
1999).
Other methods available for studying global transcription include serial analysis of gene expression
(SAGE, Velculescu et al., 1995) and hybridisation
with cDNA microarrays (Lennon and Lehrach,
1991). Although DDRT-PCR is not as high throughput as SAGE or cDNA microassays, which have
been prepared by robotics, it has the advantage of
being applicable to small amounts of total RNA as
starting material. SAGE provides a thorough quantitative analysis of total gene expression, but is less
suitable for comparing multiple samples and moreover provides less extensive sequence information
for each cDNA. Typically 11 base pair short sequence tags are generated compared with 100–350
base pairs from DDRT-PCR. The hybridisationbased differential screening of cDNA filters with
radiolabelled total cDNAs has been assisted by
recent developments in image analysis software that
allow for a side-by-side comparison of multiple
samples and have improved the detection of differentials. Such differences, however, are likely to be
biased towards higher abundance transcripts. In
contrast, DDRT-PCR frequently identifies rare
species that are amplified before the cDNA populations are compared. Nowadays, limitations in the use
of small amounts of RNA can themselves be overcome using RNA amplification methods (Van Gelder
et al., 1990; Eberwine et al., 1992). For this, firststrand cDNA is synthesised using an oligo(dT)
31
primer that has a sequence extension at its 59-end for
T7 RNA polymerase binding. Following secondstrand synthesis and ethanol precipitation the sample
is transcribed in vitro using the RNA polymerase.
This procedure has been shown to generate RNA that
reflects the starting population (Poirier et al., 1997).
1.1. Applications
Clonal cell populations provide an ideal template
for differential display because of the homogeneity
of the starting materials. This contrasts with complete tissue specimens from different individuals that
render analysis more complex because of the presence of multiple cell types combined with population
polymorphism. For analysing such non-clonal populations, including peripheral blood mononuclear cells
and primary cell cultures, a number of individuals
ought to be studied for each condition to be evaluated, so that only changes that are consistently
different between the samples are highlighted. Similarly, when analysing a clonal population such as a T
cell clone before or after a specific treatment,
consistent differences should be sought in a variety
of clones. The source of human tissue specimens can
either be large samples taken at postmortem examination or small biopsies taken during routine surgical
procedures. Another source of material is archival
tissue that has been fixed in paraformaldehyde and
embedded in paraffin wax. Furthermore, the recent
introduction of laser-capture microscopy and dissection (Emmert-Buck et al., 1996) has meant that
frozen tissue sections can now be microdissected to
obtain a subset of cells for differential display
analysis (Chuaqui et al., 1997). Other developments
have seen the use of animal models to study specific
biological questions in vivo. For example, in order to
isolate imprinted genes that are specifically transcribed when inherited from one parent but not the
other, mRNA isolated from the tissues of the two
parental strains of mice, reciprocal F 1 hybrids and
pooled backcross progeny can be analysed (Hagiwara et al., 1997).
The differential display technique has been valuable in providing expression profiles for detecting the
optimal fraction of gene expression during a time
course or even to provide a transcriptional overview
between multiple mRNA populations (Liang and
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M. Ali et al. / Journal of Immunological Methods 250 (2001) 29 – 43
Pardee, 1992; Welsh et al., 1992). However, the main
use of DDRT-PCR has been to identify and isolate
differentially expressed transcripts in biological systems. The technique has the ability to highlight lower
abundance transcripts (Guimaraes et al., 1995), since
abundant species are likely to be saturated during
PCR and appear the same on a gel. After sequencing
a differentially regulated fragment and database
searching, the cDNA may prove to be either part of a
known transcript that has or has not have been
previously implicated in the biological question
under study, or it could be part of an uncharacterised
message.
It is important to realise that there are essentially
two types of PCR products that contribute to the
amplification reaction during DDRT-PCR. These are
accounted for by either oligo dT–decamer or decamer–decamer priming (oligo dT–oligo dT priming
although possible has not frequently been reported).
It is the former that gives rise to most of the cDNAs
that are derived from the 39-untranslated portion of
transcripts. The high frequency of novel transcripts
isolated by differential display could in part be
accounted for by the lack of complete 39-untranslated
regions for many known genes included in databases.
To obtain more sequence information, researchers
have often screened cDNA libraries by hybridisation
(Liang and Pardee, 1992; Sun et al., 1994) or used
PCR-based methods to amplify cDNA fragments
(Zeiner and Gehring, 1994; Sompayrac et al., 1995).
The recent introduction of rTth DNA polymerase for
long-distance differential display (Jurecic et al.,
1998; Di Sepio et al., 1998) because it amplifies
0.5–2.0 kilobase fragments instead of 100–350 bp
ought to highlight cDNA that contain part of their
coding regions as well as the 39-untranslated portions.
1.2. Reducing false positives
Most investigators have found that the major
limitation with DDRT-PCR has been the high rate at
which false positive clones have been isolated (Debouck, 1995; Wan et al., 1996). There are several
reasons for this that were not apparent from the
original protocol and now, there are a number of
strategies that can be adopted to reduce this problem.
From the outset, the total RNA used in these
experiments needs to be high quality and free from
chromosomal DNA (Liang et al., 1993). Before use
in the DDRT-PCR, an aliquot of RNA should be
tested by PCR using oligonucleotide primers that
span an intronic sequence, to detect genomic DNA.
If present, this can be removed with DNase. If
enough RNA is available it is advisable to check its
integrity by electrophoresis of an aliquot through a
formaldehyde agarose gel with ethidium bromide
staining. RNA bands corresponding to 28S and 16S
ribosomal RNA should be visible under UV illumination. A further option is purification of the mRNA
from total RNA using oligo(dT) columns. Before
embarking on a comparative study between two or
more RNA populations, the samples need to be standardised or normalised with respect to each other.
The usual way to do this has been to determine
the optical absorbance of the RNA sample at 260 nm,
so that equal amounts are used in the analysis.
However, RNA samples can also be normalised
against a housekeeping transcript such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or bactin by quantitative RT-PCR.
For the selection of bands that are consistent in
differential display gels, it is worth considering the
use of paired RNA samples from duplicate experiments (Sompayrac et al., 1995) and / or performing
the DDRT-PCR reaction in duplicate (Liang et al.,
1993; Zhao et al., 1995). A number of researchers
have reported that the use of some decamer primer
sequences contributes to a lack of reproducibility
because of mispriming. To resolve this, either a
‘hot-start’ can be used in the PCR or the use of
anchor primers as long as 22 nucleotides has been
shown to give reliable results (Linskens et al., 1995;
Zhao et al., 1995; Diachenko et al., 1996).
Another reason for the isolation of false positives
has been that the displayed bands are usually composed of more than a single cDNA entity (Callard et
al., 1994; Li et al., 1994), representing not only the
differentially regulated transcript but also constitutively expressed sequences. The complexity of
cDNA bands has been shown using SSCP gels
(Mathieu-Daude et al., 1996) and restriction enzyme
digestion followed by gel electrophoresis (Smith et
al., 1997b). It has been suggested that a useful first
step is direct sequencing of the cDNA band (Linskens et al., 1995; Wang and Feuerstein, 1995;
M. Ali et al. / Journal of Immunological Methods 250 (2001) 29 – 43
Yoshikawa et al., 1995), so that only bands that give
an unreadable sequence due to cDNA heterogeneity
can be cloned to isolate the differentially expressed
sequence.
1.3. Confirming altered gene expression
There are a number of options for the confirmation
of putative differentially-regulated sequences that
have been derived from an eluted DDRT-PCR band.
When screening large numbers of differentials, reverse-northern analysis is preferred (Nedivi et al.,
1993; Mou et al., 1994). A number of variations of
this method exist. For the preparation of gridded
cDNA filters, researchers have either grown and
lysed bacterial colonies on nylon membranes (Zhang
et al., 1996) or applied the DNA directly after PCR
amplification or plasmid extraction. The PCR products to be immobilised may be derived using flanking vector primers (Von Stein et al., 1997) or after
partial sequencing of the clones a combination of
specific and decamer oligonucleotides (Martin et al.,
1998). We prefer the latter which avoids amplifying
the polyadenylate tail in the insert DNA. The gridded
membranes are incubated with total cDNAs that we
derived from the mRNAs of the samples to be
compared. These cDNAs can be radiolabelled with
a-[ 32 P]-dCTP or a-[ 33 P]-dCTP and after hybridisation screening, the images can be developed on
autoradiograms or phosphorscreens. For standardisation of reverse-northerns, equal amounts of RNA can
be used from the outset to make complex probes and
the grids can be normalised against a constitutively
expressed control such as a housekeeping transcript.
It is essential that appropriate densitometry scanning
software be used to assign a numerical value to each
dot on the grids. Without the use of quantitation, this
hybridisation-based procedure is insufficiently sensitive to detect minor changes in expression of rare
transcripts.
An interesting alternative to reverse-northerns that
requires large amounts of RNA and testing of each
differential band one at a time, is affinity capturing
of the cDNA on northern blots (Li et al., 1994;
Denovan-Wright et al., 1999). This procedure isolates the truly differentially-regulated transcript as a
cDNA fragment that can be cloned directly from the
33
northern membrane. Methods such as ribonuclease
protection assays, specific reverse-transcription PCR,
northern blot analysis, quantitative real-time PCR
and in situ hybridisation are useful for analysing
single differentials and the latter three techniques
provide useful extra information towards characterisation of the corresponding transcript.
Northern blotting has been problematic in confirming differential gene expression for isolated clones
(Liang and Pardee, 1992; Sun et al., 1994). This may
be due to genuine false positives such as constitutively expressed transcripts that have come through
the differential analysis. However, subtle differences
that are highlighted in DDRT-PCR are also difficult
to detect. Furthermore, a northern hybridisation
analysis sometimes does not give a signal for the
clone to be analysed. This may reflect the isolation
of rare transcripts or co-migrating cDNA contaminants. However, DNA bands less than 150 base pairs
are difficult to label to probe Northerns. Northern
blot analysis is useful for determining the size of the
full-length mRNA transcript from which the cDNA
is derived and also provides information about
alternatively spliced variants.
A more recent development has been the use of
real-time PCR to monitor gene expression by directly
observing the accumulation of double-stranded DNA
product after each cycle (Higuchi et al., 1992;
Wittwer et al., 1997a,b). This is achieved by detecting the fluorescence caused by laser light excitation
of the newly synthesised double-stranded DNA,
which has undergone SYBR-green dye incorporation. This method requires specific primers for the
gene of interest and a housekeeping control gene but
is particularly useful for providing quantitative numerical values for gene expression.
The least favoured method for comparative gene
expression analysis is in situ hybridisation, since it is
time-consuming and requires considerable optimisation. Only absolute changes in gene expression can
be confirmed by visual inspection of tissue slides or
whole mounts (Toki et al., 1998; Bryant et al.,
1999). Even with advances in image analysis software there is no easy way to quantify differences and
one must often resort to a subjective interpretation.
Nevertheless, in situ hybridisation provides useful
information about which cell type transcribes the
gene of interest in complex tissues.
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M. Ali et al. / Journal of Immunological Methods 250 (2001) 29 – 43
1.4. Examples in immunology
The vertebrate immune system consists of differentiated cells derived from either myeloid or
lymphoid precursors that originate from haematopoietic stem cells in the bone marrow. The myeloid
progenitors give rise to granulocytes, macrophages,
platelets and erythrocytes whereas B and T cells are
developed from the lymphoid progenitors. The cells
of the immune system combat infection once it has
entered the surface epithelial layer that protects the
host from invading pathogens. During the initial
stages of infection the innate immune system, which
includes the complement system in plasma and
phagocytic macrophages in tissues, becomes activated and triggers the inflammatory process. However, there is also a second line of defence provided
by the lymphocytes which make up the adaptive
response, consisting of T cell and antibody-mediated
immunity. Although the adaptive immune response
is also dependent on cells of the innate system, this
reaction is specific, efficient and has immunological
memory so that there is a rapid response upon
subsequent reinfection. Whilst a vast amount of
research has resulted in new discoveries regarding
the mechanisms involved in the development and
differentiation of immune cells, as well as the
interactions between cells during normal and abnormal immune responses, there are still many fundamental aspects that require investigation particularly at the molecular level. Thus one strategy has
been to compare gene expression in order to identify
new molecules in a particular immunological context.
Differential display has already been applied with
success in immunological settings and there are
numerous reports in the literature. The technique has
been used on purified populations derived from
human peripheral blood mononuclear cells (Azzoni
et al., 1996; Ruegg et al., 1996; Ishaq et al., 1998) as
well as spleen tissue (Blaser et al., 1998), thymus
(Poirier et al., 1999) and bone marrow derived cells
(Chen et al., 1998; Weiler et al., 1999) including
those that have been isolated from transgenic mice.
Studies have also used mutated (Jin et al., 1997;
Semizarov et al., 1998; Verkoczy et al., 1998) or
transfected cell lines (Amson et al., 1996) and even
clones that have undergone various cytokine (Sun et
al., 1998) or drug treatments (Nocentini et al., 1997).
Previously unidentified molecules in immune cells
that have been isolated by differential display include
secreted proteins (Jin et al., 1997; Blaser et al.,
1998), cell surface markers (Ruegg et al., 1996;
Nocentini et al., 1997; Chen et al., 1998; Xu et al.,
1998), molecular chaperones and signalling molecules (Semizarov et al., 1998), nuclear receptors
(Ishaq et al., 1998) and transcription factors (Sun et
al., 1998; Garcia-Domingo et al., 1999). Here we
describe a number of examples of the use of
differential display in lymphocyte and myeloid cell
research.
1.5. Myeloid cell development and differentiation
The differentiation of myeloid precursors that
leads to the development of granulocytes is mediated
in part by the interaction of cytokines or ligands with
receptors on the surface of myeloid cells. Thus, mast
cells are derived from myeloid precursors that differentiate in the tissues to form two different types,
MMC (immature mucosal mast cells) and CTMC
(connective tissue mast cells). Murine bone marrowderived cells can be cultured in vitro either with
stem-cell factor (SCF) or interleukin-3 (IL-3) to
produce differentiated cells that are phenotypically
similar to MMC or CTMC respectively. In order to
discover transcripts that may be specific for each cell
type, Chen et al. (1998) carried out a differential
display screen and identified a novel cell surface
molecule in the SCF-derived mast cells that they
called Pactolus.
The human myeloid leukemia cell line HL60 can
be differentiated into macrophages upon activation of
the protein kinase C (PKC) receptor by PMA
(phorbol 12-myristate 13-acetate). To isolate transcripts that are differentially-regulated after signalling through the PKC pathway, the HL60 cell line
was compared with the variant cell line HL525 that
is PKCb deficient (Semizarov et al., 1998). The
transcript for protein kinase X (PRKX) that is
downstream of PKCb in the signalling cascade was
isolated and subsequently shown to be specific to the
myeloid lineage.
Weiler et al. (1999) compared gene expression
between the promyelocyte cell lines EML and
MPRO that are derived from murine bone marrow
M. Ali et al. / Journal of Immunological Methods 250 (2001) 29 – 43
and are temporally related by 48 hours of differentiation respectively. Out of fifteen cDNA isolates, three
were confirmed to be differentially-expressed by
northern blotting. One of these, the molecule D3,
possesses a putative nuclear localisation signal.
However, its precise role in myeloid differentiation
has yet to be elucidated.
1.6. Innate immunity
Circulating monocytes of the innate immune system respond to the endotoxin, bacterial lipopolysaccharide (LPS) that binds to the LPS receptor (CD14)
on the cell surface. In order to identify transcripts
that may be involved in the regulation of LPS
responsiveness, macrophage-derived cell lines from
the bone marrow of LPS responsive mice, C3H / HeN
and the LPS hyporesponsive strain, C3H / HeJ that
has a mutation in the LPS receptor, were grown in
LPS-free media and then used to analyse gene
expression differences by differential display (Jin et
al., 1997, 1999). Of the four transcripts confirmed to
be differentially-expressed, the protease, matrix
metalloproteinase-9 (MMP9) and anti-protease, secretory leukocyte protease inhibitor (SLPI) were
identified in the mutant cell line. Surprisingly, the
expression of MMP9 was also found to be greater in
primary macrophages derived from the mutant mice
in response to LPS when compared to normal mice
(Jin et al., 1999). This paradoxical observation
suggests that different pathways are regulated by the
LPS response. SLPI originally identified in epithelial
cells as an inhibitor of leukocyte serine proteases
was also found to be inducible by LPS in wild type
macrophages. This response was however suppressed
by IFNg. Jin et al. (1997) went on to suggest that
SLPI may function as an antagonist of bacterial LPS.
A study by Kang et al. (1998) to find new
components in the insect immune system investigated gene expression in the larvae of the moth,
Trichoplusia ni, after bacterial challenge with Enterobacter cloacae. They identified a peptidoglycan
recognition protein, PGRP that was subsequently
shown to have homologues in human and mouse and
whose expression was restricted to organs in the
immune system. This example illustrates the common origin of the vertebrate and invertebrate innate
immune systems.
35
1.7. Lymphocyte development
Throughout development of mature cells in the
lymphoid lineage, recombination-activating gene
(RAG) expression varies in a stage-specific manner.
In order to identify transcripts that are co-expressed
with RAG1, Verkoczy et al. (1998) compared gene
expression in B cell line derivatives of OCI LY8,
that was originally isolated from a patient with B
lineage large cell lymphoma. The molecule hBRAG
(human B-cell RAG-associated gene) that encodes a
transmembrane spanning glycoprotein was identified
in the cell line expressing high levels of RAG1.
hBRAG was subsequently shown to be B cell
specific suggesting that it may play a role in B cell
development.
During the maturation of B and T cells in the bone
marrow and thymus respectively, there are a number
of mechanisms that eliminate most of the non-functional, self-reactive and transformed cells. However,
a few of these cells escape and are found in the
periphery. In order to isolate molecules that may be
restricted to the immunoblast fraction of blood
mononuclear cells that contains early lineage precursors, Ruegg et al. (1996) fractionated human
peripheral blood mononuclear cells (PBMCs) by
density centrifugation before embarking on a differential display screen. The previously unidentified
molecule B4B was found in a subset of immature B
cells that do not express the cytoplasmic m chain and
so potentially lack productive rearrangements of the
immunoglobulin loci. The progenitor B cell that
expresses B4B was subsequently found to be more
abundant in the bone marrow than the peripheral
blood. B4B contains four putative transmembranespanning domains and has been shown, after transient over-expression in COS-7 cells, to induce
growth arrest. This suggests that B4B may contribute
to the elimination of non-functional B cells from the
peripheral circulation by blocking cell cycle events.
The thymus determines the proportion of T cells
that enter the peripheral circulation with CD4 or
CD8 cell surface markers. During this procedure
double negative thymocytes develop into double
positives and then mature into CD4 or CD8 single
positive cells. In order to gain an insight into the
molecular process of positive selection in the
thymus, enriched populations of CD8 1 thymocytes
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M. Ali et al. / Journal of Immunological Methods 250 (2001) 29 – 43
and their immediate progenitors CD4 1 CD8 1 were
isolated from transgenic mice and purified by negative selection and cell sorting (Poirier et al., 1999).
These selected populations were compared by differential display. A number of molecules were
identified including mIAN-1 (murine immune-associated nucleotide-1) that was expressed in CD8 1 cells.
Further work showed that mIAN-1 was also transcribed in CD4 1 thymocytes. The expression of
mIAN-1 correlated with CD3-mediated signalling
and the molecule was shown to have homology to
the plant protein, aig1, that is induced after bacterial
infection.
In order to discover unique molecules that would
distinguish between Th1 cells that activate macrophages as well as trigger inflammatory diseases and
Th2 cells that contribute to the allergic response, a
differential display screen was carried out on eight
different cloned lines of mouse origin (Xu et al.,
1998). Four IFNg-producing Th1 clones were compared with four Th2 clones that produced IL-4 and
IL-5. Of the ten transcripts that were confirmed to be
differentially-regulated by reverse-northern analysis,
the cell surface marker ST2L was abundant in Th2
cells. ST2L is restricted to this subset and anti-mouse
ST2L has allowed pure populations to be sorted.
Furthermore the in vivo administration of the antibody caused collagen-induced arthritis to worsen and
induced resistance to Leishmania major infection in
mice. Xu et al. (1998) suggest that ST2L may be a
useful therapeutic target for allergic disease.
1.8. Lymphocyte differentiation
There are several mechanisms by which potentially auto-reactive T cells that have escaped deletion
in the thymus can be silenced in the peripheral
circulation. One such mechanism, anergy, is a state
of non-responsiveness that is characterised by an
inability of the T cells to produce IL-2. Korthauer et
al. (2000) identified a single transcript out of 64
isolates that was consistently more abundant in 4
different murine models of T cell anergy when
comparing anergic versus responsive Th1 clones.
This molecule GRP1 (general receptor of phosphoinositides 1) was induced in anergic cells and is
located in the plasma membrane where it mediates
adhesion to integrins.
A study that compared gene expression in un-
treated human peripheral blood mononuclear cells
with OKT-3 activated cells identified a novel nuclear
receptor RXRa in unstimulated cells (Ishaq et al.,
1998). The increased expression of RXRa correlated
with a block in the G 1 phase of the cell cycle so that
in activated cells the transition from G 1 to S phase
caused down-regulation of RXRa.
To look for transcripts that are specific to activated
T cells, Blaser et al. (1998) purified CD8 1 cells from
the spleens of mice infected with LCMV
(lymphocytic choriomeningitis virus) and compared
these with uninfected spleens by differential display.
The transcript LGALS1 (lectin, galactose-binding,
soluble) was identified in activated CD8 1 cells.
LGALS1 monomers are secreted and act by inhibiting proliferation of CD8 1 T cells. In vitro activated
CD4 1 T cells but not B cells were also found to
produce this autocrine negative factor.
Cytokine-receptor interactions on the cell surface
induce a cascade of intracellular signalling events
that eventually lead to the activation of some genes
and the inhibition of others in the nucleus. In an
effort to distinguish differences between the similar
pathways that are induced by IL-2 and IL-12, Azzoni
et al. (1996) isolated a purified population of T and
NK cells by negative selection of human peripheral
blood mononuclear cells and treated these cells with
the recombinant cytokines. Differential display was
used to identify a number of transcripts that were
more abundant in the IL-2 treated cells. These
molecules included c-fos and junB that are members
of the AP-1 transcription factor family and egr-1
which is a member of the family of immediate early
transcription factors.
Likewise, to reveal transcripts that may be affected by signalling through the IL-9 receptor, Sun et al.
(1998) treated the murine helper T cell line, D10,
with IL-9. After differential display the cytokineinducible transcript, mrg1 (melanocyte-specific gene
(msg1) related gene) was isolated. This molecule is
also transcribed in activated cells and is an immediate early transcriptional activator which, when overexpressed in vitro, has been shown to induce cellular
transformation.
1.9. Apoptosis
In order to study the molecular pathways that are
affected by p53-induced apoptosis, Amson et al.
M. Ali et al. / Journal of Immunological Methods 250 (2001) 29 – 43
(1996) created LTR6 cells by transfecting a temperature sensitive mutant of p53 into the myeloid
leukemia cell line M1. At the permissive temperature
the p53 variant functions like wild-type and induces
programmed cell death of the murine M1 cells.
Using differential display analysis, ten differentiallyregulated transcripts were identified within the first
hour of apoptotic induction. These transcripts included phospholipase C b4, ZFM1 and the vertebrate
homologue of the Drosophila seven in absentia gene
(Siah).
Another cell death mechanism is the Fas / FasL
system that renders susceptibility to apoptosis by a
receptor-mediated process. For example, when B
cells are stimulated with CD40L this induces Fas
expression and renders the cells susceptible to cell
death. However, when signalling also occurs through
the B cell receptor the cells become resistant to
Fas-mediated apoptosis. In order to study the mechanism of resistance to Fas-killing, splenic B cells
¨ mouse were incubated with
purified from a naıve
CD40L alone as well as with anti-IgM and the cells
analysed by differential display (Schneider et al.,
1999). Amongst the eight transcripts that were
confirmed to be differentially-expressed, the previously unreported molecule FAIM (Fas apoptosis
inhibitory molecule) was discovered in the Fas
resistant B cells.
To identify transcripts that are induced by
glucocorticoids, Nocentini et al. (1997) treated the T
cell hybridoma cell line, 3DO, with dexamethasone,
for analysis by differential display. A previously
unidentified member of the TNF / NGF receptor
family was discovered. This molecule GITR
(glucocorticoid-induced TNF receptor family-related
gene) is a transmembrane protein that is abundant in
dexamethasone-treated cells as well as activated T
cells. Transient over-expression of GITR in vitro
protects the cells against apoptosis that is mediated
through the T cell receptor by anti-CD3 monoclonals. Other apoptotic signals such as those induced by Fas ligation, incubation with dexamethasone or UV illumination do not protect these cells.
The findings suggest that GITR is involved in the
regulation of T cell receptor-induced apoptosis.
Finally, Garcia-Domingo et al. (1999) studied the
apoptotic process in pre-B cells by analysing transcripts induced over a time course of cell death.
When cultured in IL-7 deficient growth media the
37
IL-7 dependent murine pre-B cell line, WOL-1
begins to apoptose. The novel transcript, DIO-1
(death inducer-obliterator-1) that has a putative
nuclear localisation signal and transcription activation domain was isolated. When over-expressed in
vitro DIO-1 was shown to translocate into the
nucleus to trigger apoptosis.
1.10. Refinements to the original DDRT-PCR
method
An estimate of the number of gel fingerprints that
are required to display all 15 000 transcripts (Alberts
et al., 1994) that are supposedly expressed within a
given cell type can be made. If we assume that each
differential display reaction generates 50 mRNA
species, there are four such reactions for each gel run
and under ideal conditions each transcript is only
represented once, then we would require a minimum
of 75 gels. Bearing in mind that under real conditions this is probably an underestimate, a more
focused DDRT-PCR approach may be required. A
number of techniques that are modifications of the
original differential display protocol (Liang and
Pardee, 1992) are available and may be more useful.
One such method, RNA arbitrarily primed PCR
(RAP-PCR, Welsh et al., 1992) tries to target the
coding regions of transcripts by using one or two
arbitrary primers instead of oligo(dT). Primer design
packages have been developed that can be used to
select a number of efficient primers for the amplification of the coding regions of genes (Pesole et al.,
1998; Consalez et al., 1999). Another approach
called targeted differential display uses an oligonucleotide primer that directs the amplification of
multigene family members with conserved protein
domains. The Prosite database provides a list of
proteins that have common domains and sequence
motifs. The oligonucleotide used in the PCR can
either be a specific primer that is used at a low
temperature or, as is more often the case, a degenerate primer mixture for use at higher stringencies
(Stone and Wharton, 1994). There are numerous
examples of this refined differential display approach
in the literature. For example, primers have been
targeted to amplify AUUUA sequences that are
predominantly found in the 39-untranslated regions
of cytokine, proto-oncogene and transcription factor
mRNAs (Dominguez et al., 1998; Utans-Schneitz et
38
M. Ali et al. / Journal of Immunological Methods 250 (2001) 29 – 43
al., 1998). Furthermore, degenerate oligonucleotides
have also been designed for the amplification of a
superfamily of small G proteins (Liu et al., 1999),
the homeotic domains of the Dlx gene family (Ryoo
et al., 1997) and signal peptide sequences that lie
near the 59-ends of selected mRNAs (Tohonen et al.,
1998). There are also reports of primers against
protein kinase and zinc finger domains (Stone and
Wharton, 1994; Donohue et al., 1995; Chuaqui et al.,
1997) as well as the conserved regions in nuclear
hormone receptors (Yoshikawa et al., 1995). Examples of the use of targeted differential display also
includes the analysis of parasite (Hagen et al., 1997;
Michalski and Weil, 1999), fungal (Birch, 1998) and
plant genes (Martens and Forkmann, 1999). The
scope for the use of this technique is enormous
providing that a specific question is asked from the
outset.
An alternative directed approach is subtractive
differential display (Lee and Welch, 1997; Wang et
al., 1997; Burger et al., 1998; Pardinas et al., 1998;
Wang and Uhl, 1998) (see Fig. 3). Although this
Fig. 3. Subtractive differential display.
technique has limitations in that it only allows a
comparative analysis between two mRNA populations, it provides a more thorough investigation than
the original differential display method. This is
achieved by carrying out an enrichment step for the
isolation of differentially regulated transcripts, using
a subtractive procedure prior to screening. In order to
avoid the technically demanding subtraction of
cloned cDNA libraries (Zimmerman et al., 1980;
Wieland et al., 1990; Lee et al., 1991), the PCRbased method of suppression subtractive hybridisation (SSH) can be used (Diatchenko et al., 1996; Von
Stein et al., 1997). Essentially this procedure relies
on synthesising double-stranded cDNA from the
mRNA populations to be compared, followed by
restriction enzyme digestion to create discrete fragments. The subtractive procedure is a two-way
comparison so that each cDNA population is subtracted one from the other in turn. For suppression
subtraction, a different set of adaptors is ligated to
two aliquots of tester cDNA that contain the differentially expressed sequences of interest and these
are both hybridised independently with an excess of
driver cDNA, which does not contain these sequences. Consequently, unhybridised differentially
expressed cDNAs are obtained that are present only
in the tester cDNAs and absent from the driver. The
two cDNA aliquots are then mixed prior to PCR with
adaptor-specific primers. This gives rise to an enriched population that can be analysed by differential
display with radiolabelled nucleotides and polyacrylamide gel electrophoresis. There are a number
of variations to this technique that incorporate the
restriction enzyme digestions of double-stranded
cDNA followed by adaptor ligation but avoid the use
of the subtractive step. These include ordered differential display (ODD, Matz et al., 1997), AFLPbased mRNA fingerprinting (Money et al., 1996),
and RFLP-coupled domain-directed differential display (Fischer et al., 1995; Tahtiharju et al., 1997).
Alternatively, cDNA representational difference
analysis (RDA, Hubank and Schatz, 1994) uses the
subtraction procedure but ligates the PCR-amplified
enriched cDNA directly into a plasmid vector for
immediate analysis of the selected clones, thus
avoiding the radioactive labelling and polyacrylamide gel electrophoresis steps.
Recently, the molecular analysis of RNA mole-
M. Ali et al. / Journal of Immunological Methods 250 (2001) 29 – 43
cules that bind to a specific RNA-binding protein has
been suggested as another method of enrichment
(Trifillis et al., 1999). This method, called specific
nucleic acids associated with proteins (SNAAP)
utilises a fusion molecule of the RNA-binding
protein of interest tagged to a glutathione-S-transferase domain. The hybrid is incubated with cellular
mRNA and protein bound mRNAs are isolated on
glutathione–Sepharose for analysis by differential
display. This elegant method has potential and ought
to be particularly applicable to diseases where such
regulatory proteins are involved.
39
abnormal or deficient immune response to combat
disease.
Acknowledgements
Thanks to Tracy Smart for her patience, encouragement and support. The authors are funded by the
West Riding Medical Research Trust, MRC, the
Wellcome Trust, YCR and the Candlelighter’s Trust.
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