From the Committee on Cancer Biology, the
¶ Committee on Immunology, and the Departments of
§ Pathology and ** Medicine, University of
Chicago, Chicago, Illinois 60637 and
Pfizer Global
Research and Development, Groton, Connecticut 06340
Received for publication, December 13, 2002
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ABSTRACT |
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Peripheral CD8+ T
cells circulate in a quiescent naive state until they are primed by
specific antigen and differentiate into effector cells. In the effector
state, CD8+ T cells acquire cytolytic activity and produce
increased levels of cytokines such as interferon- Upon exit from the thymus, CD4 and CD8 single-positive T cells are
thought to enter a naïve state in which they remain until they
encounter specific antigen appropriately presented by
antigen-presenting cells. The engagement of
TCRs1 by peptide/major
histocompatibility complexes in conjunction with costimulatory ligands
initiates signaling events leading to IL-2 production, cell cycle
progression, and differentiation into effector cells. The
differentiation fate of T cells undergoing activation is also
influenced by exogenous cytokines. For CD4+ T cells, IL-12
and IL-4 promote T helper 1 and 2 differentiation, respectively (1-3).
Acquisition of these effector phenotypes appears to require cell cycle
progression and correlates with epigenetic modification of
lineage-specific gene loci (4, 5). For CD8+ T cells,
although T cytotoxic 1 and 2 phenotypes can be similarly induced,
priming with TCR and CD28 ligation in the absence of cytokines is
sufficient to promote differentiation into cytolytic effector cells
that produce IFN- In addition to the development of cytolytic activity and
cytokine-producing capacity, effector CD8+ T cells exhibit
several additional functional alterations compared with naïve
CD8+ T cells. Effector cells exhibit a lower threshold for
TCR-mediated signaling (7, 8) and display a decreased dependence on
CD28 costimulation (9). Phenotypically they up-regulate expression of
CD44, down-regulate expression of CD62L, and also change expression of
chemokine receptors thought to be important for homing to specific tissues (10, 11). Effector but not naïve CD8+ cells
become susceptible to inhibition by CTLA-4 (12, 13) and to induction of
anergy (14) and appear to become inhibitable by other negative
regulatory pathways, such as PD-1 (15, 16), and killer inhibitory
receptors (17, 18). Thus, the primed effector CD8+
phenotype is rather complex and extends beyond effector function per se.
Although the naïve T cell state may have previously been viewed
as "quiescent" and relatively inert, two lines of recent data have
begun to change that perspective. First, several investigators have
demonstrated that signaling through the TCR from self peptide/class I
major histocompatibility complexes is necessary to maintain the
survival of naïve CD8+ T cells in the periphery
(19). Thus, the prevention of apoptotic death in naïve cells is
an active process. Second, the transcription factor LKLF has been shown
to be preferentially expressed in naïve T cells and
down-regulated after activation (20-22). In mice, post-thymic LKLF-deficient T cells fail to enter a naïve resting state and rapidly die (23), suggesting that the naïve state may need to
be actively maintained.
We hypothesized that many of the functional distinctions between
naïve and effector CD8+ T cells may be explained by
differential expression of specific genes. To approach this hypothesis,
a gene array strategy was pursued using naïve and 5-day-primed
2C TCR transgenic x RAG2 Cells--
2C/RAG2 [3H]Thymidine Incorporation--
3 × 104 purified CD8+ T cells were stimulated with
3 × 104 mitomycin C-treated P815.B7-1 cells in a
96-well microtiter plate. At various times after plating, 1 µCi of
[methyl-3H]thymidine (Amersham Biosciences) was added to
the cells. After a 6-hour pulse, the plates were frozen until
harvested. The wells were harvested for determination of
[3H]thymidine incorporation using a Packard Filtermate
Harvester and TopCount-NXT (PerkinElmer Life Sciences).
Cell Counts--
At each day of priming, cells grown in a
24-well plate were collected from three wells and stained with trypan
blue. The average number of trypan blue-negative cells was
calculated. For days 1 and 2, there was the possibility that remaining
live P815.B7-1 cells (H-2d) would prevent accurate
counting of T cells. Therefore, cells were dually stained with
PE-anti-CD8 (BD Biosciences) and fluorescein isothiocyanate-anti-Kd (BD Biosciences). After the
exclusion of propidium iodide-positive cells, the percentage of
CD8-positive and Kd-positive cells was determined. After
day 1 of priming, no viable Kd-positive cells remained in
the culture. To obtain the number of live T cells, the percentage of
CD8 positive cells was then multiplied by the number of
trypan-blue-negative cells. Two days after priming, >98% of live
cells stained positive for CD8 (data not shown).
Fluorescence-activated Cell Sorter Analysis--
To confirm the
naïve and primed states of the cells, they were stained for
cell surface markers CD44 (PE-anti-CD44, BD Biosciences) and CD62L
(PE-anti-CD62L, BD Biosciences). Potential nonspecific binding was
blocked with anti-Fc receptor monoclonal antibody 2.4G2.
Single-color analysis was carried out using FACScanTM and
CellQuestTM software (BD Biosciences, San Jose, CA).
Expression of CD44 and CD62L was verified prior to each experiment.
Cytokine Production by ELISA--
For cell stimulation, 96-well
flat-bottom plates (Costar) were coated with varying amounts of
anti-CD3 (2C11) and 1 µg/ml anti-CD28 (PV-1) (a gift from Carl June,
University of Pennsylvania) overnight at 4 °C. Supernatant was
collected and assessed by ELISA.
Chromium Release Assay--
To assess cytolytic activity, 2 × 103 51Cr-labeled targets were plated with
2 × 105 naïve or effector CD8+ T
cells in a 96-well V-bottom plate (ICN Biomedicals, Costa Mesa, CA).
After 4 h of incubation at 37 °C, 50 µl of supernatant was transferred to a LumaPlate-96 (PerkinElmer Life Sciences) and allowed
to dry overnight. Plates were then counted using TopCount-NXT (PerkinElmer Life Sciences).
Preparation of RNA for Gene Chip Array Analysis--
Total RNA
was isolated from purified T cells. Both naïve and primed
CD8+ T cells were first subjected to Ficoll-Hypaque to
remove dead cells. Cells remaining in the interface were isolated and
washed before being lysed in Trizol® (Invitrogen). Total
RNA was then treated with DNase I (Invitrogen) to minimize
contamination from genomic DNA. Further purification was carried out
using the RNeasy® Clean-Up protocol (Qiagen, Valencia,
CA). RNA was quantitated using the DU-530 spectrophotometer (Beckman).
Gene Chip Array Analysis--
Two replicate experiments were
performed using independent batches of total RNA. Biotin-labeled
in vitro transcripts for hybridization of Affymetrix
oligonucleotide arrays were prepared from 5 µg of total RNA according
to Affymetrix protocols. Briefly, cDNA was made with the
SuperScriptTM Choice System (Invitrogen) using 1 µl of
110 µM oligo(dT)/T7 primer
(5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24).
The double-stranded cDNA was extracted with
phenol:chloroform:isoamyl alcohol, precipitated with 0.5 volumes of 7.5 M ammonium acetate and 2.5 volumes of 100% ethanol.
In vitro transcription with biotin-labeled ribonucleotides
was performed with the Enzo BioArray High Yield RNA Transcript Labeling
Kit (Enzo Diagnostics, New York, NY). Labeled in vitro
transcripts were purified over RNeasy® mini columns
(Qiagen, Valencia, CA) according to the manufacturer's instructions.
12.5 µg of purified, labeled transcript was used to hybridize with
each Affymetrix array according to the manufacturer's instructions.
The average intensities for each probe set were obtained, and the
average intensity of each chip was scaled to the arbitrary value of 300 to allow comparisons among the various chips. After the minimum
intensity value was set to 50, the ratio of the average probe set
intensities between experiments from effector and the corresponding
naïve cells was calculated. If a probe set was called absent in
the chips from all the experiments, it was excluded from further
calculations. The average of the two ratios was calculated, and genes
that expressed greater than 3-fold were manually sorted into functional
categories. Genes with absolute expression levels less than 100 in all
experiments, as well as ESTs and unknown genes, were excluded from
further analysis. Visualization of the intensity and ratio was
performed using Spotfire® Array ExplorerTM
(Spotfire, Somerville, MA) software. A list of differentially expressed
genes represented on the microarray is provided in the Supplemental
Information at http://www.jbc.org.
RT-PCR--
Gene chip array results were confirmed by
semiquantitative RT-PCR. 20 µg of total RNA was used for first strand
cDNA synthesis. Samples were normalized based on equivalent
SDS-PAGE and Western Blotting--
Cells were lysed in either
Triton lysis buffer (50 mM Tris, pH 7.6, 150 mM
NaCl, 0.5% Triton X-100, 5 mM EDTA, 1 mM
trypsin inhibitor, 1 mM benzamidine, 1 mM
sodium orthovanadate (Na3VO4), 10 µg/ml
aprotinin, 25 µM
p-nitrophenyl-p'-guanidinobenzoate, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride), or
RIPA buffer (1× phosphate-buffered saline, 1% Igepal CA-630, 0.5%
sodium deoxycholate, 0.1% SDS, 5 mM EDTA, 1 mM
trypsin inhibitor, 1 mM benzamidine, 1 mM
Na3VO4, 10 µg/ml aprotinin, 25 µM
p-nitrophenyl-p'-guanidinobenzoate, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride).
Cytosolic lysates from 3 to 5 × 106 naïve and
primed 2C/RAG2
For immunoblotting, polyclonal antibodies were diluted 1:250, and
monoclonal antibodies were diluted 1:500-1:1000 with 1% bovine serum
albumin in Tris-buffered saline/Tween 20. Antibodies against
most molecules were obtained from Santa Cruz Biotechnology. The
anti-Gelsolin antibody was a gift from David Kwiatkowski (Harvard University). Antibodies against the following proteins were obtained from the indicated sources: calpactin (annexin I) (BD Transduction Laboratories, San Diego, CA); calcyclin (annexin II) (Swant,
Bellinzona, Switzerland); and Lck, Fyn, and PP1 Characteristics of Naive and Effector CD8+ T
Cells--
We used the 2C TCR transgenic model (24) to study the
functional differences between naïve and effector
CD8+ T cells. These mice were crossed into the
RAG2
Phenotypically, naïve 2C T cells were CD44lo and
CD62Lhi, whereas the majority of effector cells were
CD44hi and CD62Llo (Fig. 1B).
Furthermore, effector cells contained an average 4-fold increase in
total RNA content (naïve: 5.1 ± 3.4 µg/107
cells; effector: 19.6 ± 2.7 µg/107 cells).
Primed effector 2C T cells generated in this manner routinely produced
the cytokines IL-2 (Fig. 2A)
and IFN- Gene Array Analysis--
RNA from naïve and effector
CD8+ T cells was used to hybridize to Affymetrix Mu11k
chips containing ~11,000 known mouse genes and ESTs as described
under "Experimental Procedures." Fig.
3 shows a dot plot of the expression
pattern of individual genes in naïve versus effector
CD8+ T cells from the first of two independent
experiments. Most genes were not significantly expressed in these T
cells and thus clustered near the origin of the plot. Most genes that
were expressed in 2C T cells were expressed comparably in both the
naive and effector populations and thus fell along a line with a
slope of one. However, a number of genes was expressed at greater
levels in either the effector population (above the
diagonal) or in the naive population (below the
diagonal).
The expression data from two replicate experiments were averaged, and
known full-length genes whose levels in each T cell population differed
by 3-fold or greater were defined to be differentially expressed as
described under "Experimental Procedures." Approximately 177 known
genes met these criteria (Fig. 4 and the
Supplemental Information at http://www.jbc.org). RNA expression of
68 known genes increased with priming, while that of 109 known genes
decreased. Although the ratios of RNA expression level for some genes
differed between the two experiments, the expression patterns of the
vast majority of genes were concordant.
As expected, expression of genes related to T cell effector function
and surface receptors associated with activation increased upon
priming. Primed effector CD8+ T cells expressed markedly
increased levels of CTLA-2
Of note, mRNA levels of genes involved in generating metabolic
potential increased (Fig. 4C). Expression of genes encoding
Unexpectedly, more genes encoding signal transduction proteins and
transcription factors were down-regulated than were up-regulated upon T
cell priming. Among the few up-regulated mRNAs were those encoding
the heterotrimeric G
Expression of genes related to cell cycle regulation was varied,
presumably because of a fraction of T cells completing cell cycle
progression post-activation. Interestingly, transcripts for
HMG-14, a non-histone chromosomal protein that binds
nucleosomes, and GST1-Hs, a G1 to S phase
transition protein, were in higher abundance in naïve cells
(Fig. 4G). In addition, putative negative regulators of the
cell cycle or antiproliferative molecules, such as TSC22,
GADD45, MyD116, G0S8, BTG1,
and TOB1, were overexpressed in naive cells and decreased
following differentiation. These genes are attractive candidates for
contributing to the quiescent phenotype of naive T cells.
Interestingly, genes involved in protein folding (BIP,
HSP90, HSP105, calnexin) were more
highly expressed in naïve cells (Fig. 4H), as were
genes involved in translation (EIF-1 Confirmation of Gene Array Data by Semiquantitative RT-PCR--
A
subset of 40 genes of interest was selected to re-examine mRNA
expression levels by semiquantitative RT-PCR. Complementary DNA from
independent batches of naïve and effector 2C CD8+ T
cells were used in these analyses. Naïve and effector
cDNA were normalized based on
As stated earlier, our gene array experiments showed higher expression
of BTG1 and TOB1, which are attractive candidates
for contributing to naive cell quiescence (25, 26). These genes are
members of the BTG/TOB (recently renamed
APRO) (27) family, which consists of at least six family
members, any of which could have correlated to the gene chip array
oligonucleotides. RNA expression of BTG-1, -2,
and -3 and TOB-1 and -2 was examined
in our 2C T cells by semiquantitative RT-PCR using gene-specific
primers. Interestingly, expression of only TOB1 and
TOB2 was found to be down-regulated greater than 5-fold in
the primed effector state, making them attractive candidate
anti-proliferative genes in naive CD8+ T cells (Fig.
5A).
Based on confirmed down-regulation of TOB1 and
TOB2 expression in primed effector cells, RT-PCR analysis
was performed to analyze expression of TSC-22 and
G0S8/Rgs2 as well (Fig. 5B). TSC-22 was originally described in a screen to identify
genes induced by TGF- Correlating RNA and Protein Expression of a Subset of
Genes--
Theoretically, post-transcriptional mechanisms could
contribute to the regulation of expression of specific gene products. Therefore, we examined expression of selected molecules by Western blot
analysis in naive and effector 2C T cells. Expression of additional
genes and proteins not represented on the chips but related to genes of
interest were also examined in detail.
The total protein content of primed CD8+ T cells was only
~1.5 times that of an equal number of naïve 2C cells
(naïve: 198 ± 6.8 µg/ml/106 cells;
effector: 295 ± 20 µg/ml/106 cells), suggesting
that changes in protein levels greater than 1.5-fold on a per-cell
basis would be meaningful.
Effector Molecules, Surface Receptors, Metabolic Proteins, and
Cytoskeletal Molecules--
In accordance with the gene array results,
increased expression of granzyme A was seen by RT-PCR in primed 2C
cells (data not shown). As an antibody against granzyme A was not
available, granzyme B was examined by RT-PCR as well as by Western blot
(Fig. 6). As expected, both mRNA and
protein for granzyme B were detected at higher levels in primed T
cells, consistent with the role of granzymes in granule-mediated
cytolysis by effector CD8+ T cells. Expression of
CD62L mRNA was confirmed by semiquantitative RT-PCR
(Fig. 6). Decreased expression of CD62L protein was also observed by
flow cytometry (Fig. 1B). Aldolase C and Signaling Molecules and Transcription Factors--
We were
interested in signal transduction enzymes that might be differentially
expressed in naive and effector T cells as a potential mechanism for
the increased TCR sensitivity and decreased CD28 dependence of the
latter population. Both Lck and Fyn were expressed comparably at both
the mRNA and the protein level in naïve and primed T cells
(Fig. 7). Gene array results revealed diminished expression of mRNA encoding 14-3-3 family members, which are adapter proteins that interact with serine-phosphorylated targets (recently reviewed in Refs. 34, 35). 14-3-3
The increased expression of ERK1 observed by gene array was
confirmed by RT-PCR and protein analysis (Fig. 7). This result prompted
examination of ERK2 as well as of JNK-pathway enzymes. As
shown in Fig. 7, expression of ERK2 and JNK1 protein was comparable in
naïve and effector 2C cells, although mRNA appeared to be modestly decreased in the effector cell population. In contrast, JNK2
was comparably expressed at the mRNA level but was substantially up-regulated at the protein level. Similarly, N-Ras, Rac1, and RhoA were all expressed comparably at the mRNA level but were expressed at significantly higher levels in effector cells at the
protein level (Fig. 7).
The decreased expression of several phosphatase transcripts by gene
array analysis was also of interest and was investigated further. MKP-1
is a dual specificity phosphatase that dephosphorylates ERK1/2 (36).
The decreased expression of MKP-1 mRNA in primed 2C cells was
confirmed by RT-PCR analysis (Fig. 7A). However, very little
MKP-1 protein was detected in naïve T cells, whereas substantially greater levels were detected in primed effector cells.
Similarly, expression of PP1
Based on the observation that several enzymes in mitogen-activated
protein kinase signaling pathways were up-regulated in effector
T cells at the protein level, it was anticipated that basal expression
of transcription factor targets also might show distinct patterns of
expression. Indeed, basal Fos B expression was approximately equal in
primed T cells at the mRNA level, but at the protein level effector
cells showed marked up-regulation compared with naïve cells
(Fig. 7). In contrast, c-Fos mRNA was decreased in primed cells,
with c-Fos protein levels remaining constant. In accordance with the
gene array data, mRNA levels of Jun B and Ets-2 were also decreased
after priming. However, protein levels were either equivalent or
augmented. In summary, the results of these analyses comparing mRNA
and protein levels between naive and effector CD8+ T cells
suggest that expression of many signaling molecules and transcription
factors can be can be regulated at the post-transcriptional level.
The goals of this study were to understand sets of genes that
define a naïve or effector CD8+ T cell population
and to determine whether differences in gene expression could explain
their distinct functional characteristics. Of the ~11,000 known genes
and ESTs we examined, 177 known genes were expressed at relatively
higher levels in either naïve or effector cells. Surprisingly,
a greater number of known genes was found to be down-regulated (109 of
177) than up-regulated (68 of 177) upon differentiation into the
effector state. This observation counters the notion that naïve
cells are inert and supports the concept that the naïve
phenotype is actively maintained. The process of maintaining T cell
quiescence could be intrinsic to the T cells themselves or could occur
by a T cell-extrinsic mechanism.
In contrast to previous gene array studies (21, 37, 38), we used cells
generated from TCR transgenic x RAG2 Despite using equivalent cell numbers, effector CD8+ T
cells yielded approximately four times more total RNA than
naïve cells. This likely represents a combination of greater
ribosomal RNA and to some extent mRNA, although these were not
distinguished in our study. However, total protein content in effector
cells was increased only 1.5 times over that of naïve cells.
This increase in total protein content was proportional to the increase
in cell diameter and mean fluorescence of forward scatter (data not
shown), suggesting that protein concentration remained comparable.
Upon anti-CD3 and anti-CD28 stimulation, naïve cells produced
ample IL-2 but only low amounts of IFN- Based on initial gene array data, we were attracted to the hypothesis
that down-regulation of protein phosphatases could explain increased
TCR sensitivity in differentiated effector cells. PP1 As controls in our experiments, protein levels were examined for a
panel of signaling molecules that were comparably expressed between
naive and effector CD8+ T cells. This included JNK2, N-Ras,
Rac 1, and Rho A. Surprisingly, despite comparable mRNA levels
confirmed by RT-PCR, expression of these molecules was found to be
markedly up-regulated at the protein level. For JNK2, our result is
consistent with those of the Flavell group who reported that JNK
activation was detected late during CD4+ T cell
differentiation (41). Our findings underscore the complex regulation of
expression and activity of specific genes through increased
translation, post-translational modification, or protein stability.
These results do suggest that increased expression of one or more of
these signaling molecules at the protein level could conceivably
contribute to the increased TCR sensitivity as seen in the effector state.
We observed that several putative antiproliferative genes were more
highly expressed in naïve cells, including TSC-22,
TOB1, TOB2, and G0S8. It is possible
that the functions of these genes, alone or in combination, may
contribute to the quiescence of naïve cells. TSC-22,
a gene whose mRNA expression was found to be decreased in our
effector cells as well as two other studies comparing naïve and
activated lymphocytes (21, 22), binds DNA and is thought to be a
transcriptional regulator. Target genes of TSC-22 are unknown to date;
however, it has been shown that it is a transcriptional repressor alone
or in conjunction with a closely related homolog, THG-1 (30).
Consistent with our observed down-regulation of TOB1 and
TOB2 following differentiation out of the naive state, Tob1
was recently shown to inhibit T cell proliferation and cytokine production (42). G0S8/RGS2 has been shown to be an
immediately early response gene induced upon T cell activation (33,
43). It is involved in inhibiting Gq Transcripts of genes involved in metabolism were present at higher
levels in effector cells relative to naïve cells. In
particular, we observed up-regulation of eight of 10 glycolytic
enzymes, suggesting that glycolysis may play a significant role in the
function of effector T cells. Preliminary results have revealed that
glucose uptake and glycolytic rate are markedly increased in resting
effector compared with resting naive CD8+ T cells (data not
shown). It has been shown that proliferating cultured rat thymocytes
rely heavily on glycolysis rather than aerobic respiration even in the
presence of normal O2 levels (46-48). It is also
conceivable that effector cells utilize glycolysis to avoid byproducts
of oxidative phosphorylation, such as reactive oxygen species, which
can inhibit proliferation (49) or lead to cellular apoptosis (50).
A final category of genes of interest up-regulated in CD8+
effector cells is that of cytoskeletal molecules. Calcyclin,
calpactin, vimentin, and gelsolin were
notably expressed at higher levels following differentiation.
Preliminary results have indicated that several additional molecules
that regulate actin cytoskeletal dynamics, including Vav,
Wiscott-Aldrich Syndrome protein, and LIM kinase 1 are also expressed
at increased levels in effector cells (data not shown). There is some
debate as to whether the role of a dynamic actin cytoskeleton in
receptor segregation and immune synapse formation is primarily to
support signal transduction amplification or rather to promote directed
execution of T cell effector function (51, 52). The observed increase
in cytoskeletal proteins in effector cells supports the latter
hypothesis and should be testable by examining the role of individual
cytoskeletal regulators in TCR signaling versus cytolysis.
In summary, our study illustrates the complexity of naïve T
cell regulation, emphasizing the need to examine both mRNA and protein levels to make conclusions regarding the expression of particular molecules in a specific cell population. Further studies are
necessary to determine the role of naive T cell genes in limiting T
cell activation, as well as the function of augmented glycolysis, cytoskeletal dynamics, and signaling proteins in effector T cell activities.
. They also
exhibit increased T cell receptor sensitivity, decreased CD28
dependence, and become inhibitable by CTLA-4 and other negative
regulatory pathways. We hypothesized that one mechanism by which these
two states are regulated is via differential expression of specific
genes. To this end, basal gene expression profiles of naïve and
effector 2C TCR transgenic x RAG2
/
CD8+ T
cells were analyzed using Affymetrix arrays representing 11,000 genes.
Of the 177 differentially expressed known genes, 68 were expressed at
higher levels in effector cells, but 109 were more abundant in
naïve cells, supporting the notion that the naive state is not
passive. Expression of genes related to metabolism, actin cytoskeletal
dynamics, and effector function increased with priming, whereas
expression of putative anti-proliferative genes decreased.
Semiquantitative reverse transcription-PCR was utilized as a
secondary validation for selected transcripts, and Western blot
analysis was used to examine protein expression for molecules of
interest. Surprisingly, for 24 genes examined, 12 showed discordant protein versus mRNA expression. In summary, our study
indicates that: 1) not only does the expression of some genes in naive
CD8+ T cells become up-regulated upon priming, but the
expression of other genes is down-regulated as well and 2) the
complexities of T cell differentiation include regulation at the
post-transcriptional level.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(6). Acquisition of lytic activity by
CD8+ cells also requires cell cycle progression (6),
implying a similar requirement for epigenetic modification for effector
cytotoxic lymphocyte-specific gene expression.
/
CD8+ T cells. Of
11,000 genes represented, ~177 known genes were differentially expressed. Counter to expectations, approximately half of the differentially expressed genes were present at decreased levels in
effector cells, supporting the notion that the naïve state is
not inert. RNA expression of genes related to metabolism, actin cytoskeletal dynamics, and T cell effector function increased with
priming. Semiquantitative RT-PCR was utilized as a secondary validation
for selected transcripts, and Western blot analysis was used as a
tertiary validation for molecules of interest. However, although
differential protein expression correlated with differential RNA
expression in some instances, this was not observed for many signaling
molecules and transcription factors. Comparable RNA but increased
protein levels, or decreased RNA with comparable or increased protein
levels, were frequently observed. These results suggest that
post-transcriptional events, such as regulated translation, protein
stability, and post-translational modification, may be common and are
likely to contribute to the control of peripheral T cell differentiation.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice have been described
(9). Naïve 2C/RAG2
/
CD8+ T cells
were isolated using a negative selection protocol. Spleens from
2C/RAG2
/
mice were macerated and washed once with
Dulbecco's modified Eagle's medium containing 10% fetal calf serum.
CD8+ T cells were enriched using StemSepTM
Enrichment Mixture for murine CD8+ T cells (Stem Cell
Technologies, Vancouver, British Columbia, Canada) according to the
manufacturer's instructions. To generate primed cells, 105
freshly purified 2C/RAG2
/
CD8+ T cells were
plated with 5 × 105 P815.B7-1 cells pretreated with
mitomycin C (50 µg/ml/107 cells for 90 min at 37 °C;
Sigma) in 1.5 ml of Dulbecco's modified Eagle's medium in each
well of a 24-well plate. On the fifth day, cells were harvested and
subjected to Ficoll-Hypaque centrifugation to remove cell debris. In
some experiments, cells primed for a second 4-5-day period were used
as indicated.
-actin expression by RT-PCR. For each PCR amplification, 1 µl of
cDNA was used in a 50-µl reaction. To ensure that amplification
remained within the linear range, 1:5 serial dilutions of cDNA were
made. RT-PCR for
-actin (25 cycles) was used as a control for
mRNA abundance. For other genes, the number of cycles ranged from
25 to 45. Annealing temperatures varied, since the
Tm of each primer differed, and amplification
was carried out at 72 °C. RT-PCR for each gene was performed several
times using different batches of cDNA. For sequences of
gene-specific primers, refer to the Supplemental Information at
http://www.jbc.org.
/
CD8+ T cells were subjected
to SDS-PAGE on 10-15% acrylamide gels. Lysates from equal numbers of
cells were loaded on the same blot. Total protein content in
naïve and primed cells was measured using DC Protein Assay
Reagent (Bio-Rad).
(Upstate
Biotechnology, Lake Placid, NY). Appropriate secondary antibodies were
diluted 1:2500-3000 with Tris-buffered saline/Tween 20:
horseradish peroxidase (HRP)-
-goat IgG (Zymed
Laboratories Inc.); HRP-
-rabbit IgG (Amersham Biosciences); HRP-
-mouse IgG (Amersham Biosciences).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
background to ensure a monoclonal population of
CD8+ cells (9). Naïve cells were freshly isolated
from the spleens of 4- to 6-week-old mice by negative selection. A
subset of purified 2C/RAG2
/
T cells was taken to
generate effector CD8+ T cells in vitro through
a 5-day stimulation with mitomycin C-treated P815.B7-1 cells. As
illustrated in Fig. 1A, this
stimulation resulted in vigorous thymidine incorporation that peaked on
day 3, with an expansion of viable T cell numbers that lagged behind by
24 h. By day 5, the cells re-entered a resting state, at which
time they did not proliferate or spontaneously produce cytokines. At later times, without restimulation, the T cells began to die, presumably due to the lack of viable antigen-presenting cells (data not
shown). The primed cells used in this study were taken on the fifth day
after stimulation.
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Fig. 1.
Phenotypic characterization of naïve
and effector 2C CD8+ T cells. A,
[3H]Thymidine incorporation and parallel cell counts
during priming of 2C T cells with P815.B7-1. Naïve (day 0)
cells were differentiated with mitomycin C-treated P815-B7.1 cells in a
96-well plate as described under "Experimental Procedures." On each
day of differentiation, cells were pulsed for 6 h with 1 µCi of
[methyl-3H]thymidine and then frozen and harvested. In
parallel, naïve 2C CD8+ T cells were plated with
mitomycin C-treated P815-B7.1 cells at a 1:5 ratio in a 24-well plate.
Beginning on day 2, cells were collected and counted on each day of
differentiation by the trypan blue exclusion method. B,
surface expression of CD44 and CD62L on naïve and effector 2C
CD8+ T cells. Naïve and effector cells were stained
with PE-conjugated anti-CD44 and anti-CD62L. Expression was analyzed by
flow cytometry. The y-axis represents cell number.
(Fig. 2B) and acquired antigen-specific
cytolytic activity (Fig. 2C). Interestingly, the increased
IL-2-producing capability of primed cells was reflected by a shift in
the dose response to anti-CD3 mAb, whereas the increased IFN-
production was reflected predominantly by an increased magnitude of
response. These results suggest that at least two molecular mechanisms
may account for the greater cytokine-producing capability of primed
effector CD8+ T cells.
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Fig. 2.
Functional characterization of naïve
and effector 2C CD8+ T cells. A
and B, IL-2 and IFN- production of naïve (
)
and effector (
) 2C CD8+ T cells following stimulation
with increasing amounts of anti-CD3 (2C11) and anti-CD28 (PV-1; 1 µg/ml). Supernatant from cells were collected after 20 h and
analyzed for cytokine production by ELISA. C, cytolysis.
Naïve (
) and effector (
) 2C CD8+ T cells were
assessed for their cytolytic ability in a 4-hour chromium release
assay. P815-B7.1 and EL4 (syngeneic control) cells were labeled with
51Cr-labeled sodium chromate for 1.5 h, washed, and
plated with naïve or effector 2C CD8+ T cells.
Effector:target ratio is 100:1. The amount of 51Cr present
in 50 µl of supernatant was quantified.
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Fig. 3.
Dot plot of gene expression profiles of
naïve and effector 2C CD8+ T cells.
Represented on the gene chip microarrays were 11,000 genes and ESTs.
Expression level of a gene was quantified in arbitrary units as
determined by the screen. Each gene is represented by a single dot.
Genes that were expressed at equivalent levels between naïve
and effector 2C CD8+ T cells lie close to the line, whose
slope is 1. This dot plot is representative of one of two independent
gene array screens.
View larger version (33K):
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Fig. 4.
Bar graph representation of gene
expression profiles of naïve and effector 2C
CD8+ T cells. Expression levels from two
independent screens were averaged, and ratios of the mean value are
shown. Genes whose expression increased after priming have a positive
change in gene expression (red). Genes whose expression
decreased after priming have a negative change in gene expression
(green).
, Granzyme A,
SPI-3, and CCPI genes, all of which encode
proteins involved in cytolysis (Fig. 4A). Increased basal
expression of cytokine genes was not detected, supporting the notion
that the effector T cell population was resting at the time of
collection. Genes encoding cell surface receptors known to
correlate with naive and effector states changed as expected. The RNA
level of CD62L was decreased in effector CD8+ T
cells compared with naive cells, whereas the levels of Ly6.2 and CTLA-4 were increased (Fig. 4B).
-enolase, pyruvate kinase, triosephosphate isomerase,
hexokinase II, 6-phosphofructokinase type C (all participants in the
glycolytic pathway) was induced in effector CD8+ T cells.
Some housekeeping genes such as Apex, which is involved in
base excision repair and also regulates DNA binding activity of
transcription factors, and DDOST, which is involved in the transfer of an oligosaccharide onto nascent polypeptides from the
endoplasmic reticulum, were down-regulated. Expression of several
molecules associated with cytoskeletal regulation was also increased in
primed cells. Although the levels of actin transcripts did not change
appreciably upon priming, mRNAs for gelsolin,
calcyclin, and calpactin were markedly
up-regulated (Fig. 4D).
subunit, ERK-1, and
molecules involved in inositol phospholipid signaling, such as
myoinositol-1 (or -4) monophosphatase (IMP) and an
inositol 1,4,5-trisphosphate receptor (p400) (Fig. 4E).
However, many genes were found to be expressed at lower levels in
primed cells, including those encoding phosphatases (MKP-1,
PRL-1, and PP2A) and putative adapters
(SOCS-3 and 14-3-3). Primed effector cells also
displayed lower levels of the transcription factor genes Jun
B, c-Fos, FOG, Ikaros,
c-Jun, Ets-1, and Ets-2. (Fig.
4F). Interestingly, basal expression of the Fos family
member Fos B was much increased in effector cells over
naïve cells, as was that of GATA-3, a transcription
factor required for T helper 2 development.
, EIF-4
). Additional
miscellaneous genes are listed in Fig. 4I.
-actin expression as determined by
RT-PCR because
-actin mRNA and protein expression were similar
between naïve and effector cells. Of these 40 genes, 35 gave
similar results to those seen by gene array, yielding a concordance
rate of 88% (Table I). Thus, results
obtained using oligonucleotide arrays are not necessarily identical to
those obtained by gene-specific RT-PCR, emphasizing the importance of
confirmatory screens.
Gene array confirmation by RT-PCR
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Fig. 5.
Semiquantitative RT-PCR of putative
anti-proliferative genes. RNA expression was confirmed by
semiquantitative RT-PCR as described under "Experimental
Procedures." Each reaction contained decreasing amounts of cDNA
(1:5 serial dilution) as indicated by the descending
triangle. A, BTG/TOB
family. B, TSC-22 and G0S8. Both
TSC-22 and G0S8 were represented on the
microarrays, as indicated (arbitrary units of expression). Values
represent averages from two independent screens.
(28). Expression of TSC-22 has been
shown to be down-regulated in transformed versus normal
cells (29, 30), and introduction of TSC-22 into tumor cells
can induce apoptosis (31, 32). G0S8 was identified by its
overexpression at the G0-G1 transition of the
cell cycle (33). As shown in Fig. 5B, expression of both of
the genes was diminished in effector compared with naive T cells,
supporting a potential role for TSC-22 and G0S8 in regulating
the naive state as well.
-enolase expression was also assessed by semiquantitative RT-PCR and Western blotting. Effector cells displayed greater RNA and protein levels of
both genes relative to naïve cells. Ca2+-binding
proteins that interact with the cytoskeleton (calcyclin, calpactin, and
annexin V) were also significantly increased in effector cells at both
the mRNA and the protein level (Fig. 6). Collectively, these
results suggest that both glycolysis and actin cytoskeletal activity
may be up-regulated in effector CD8+ T cells, perhaps to
meet the needs of specific effector functions.
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Fig. 6.
Verification of gene array results by
semiquantitative RT-PCR and Western blotting of effector function-,
metabolism-, and cytoskeleton-related genes. A, the
average numbers (in arbitrary units of expression) from two independent
gene array screens of naïve and effector 2C CD8+ T
cells. NOC, not represented on chip. B, RNA
expression was verified by semiquantitative RT-PCR as described under
"Experimental Procedures." Naïve and effector cDNA were
normalized based on -actin expression. Each reaction contained
decreasing amounts of cDNA (1:5 serial dilution) as indicated by
the descending triangle. C, analysis of protein
expression by Western blotting. Equal numbers of naïve and
effector cells were lysed using Triton or RIPA lysis buffer. Lysates
from 3 × 106 cells were loaded for each sample.
mRNA was
confirmed to be down-regulated by RT-PCR in primed cells. In contrast,
protein levels were comparable, suggesting the possibility of
post-transcriptional regulation of this molecule.
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Fig. 7.
Verification of gene array results by
semiquantitative RT-PCR and Western blotting of adapter proteins,
signaling molecules, and transcription factors. A, the
average numbers (in arbitrary units of expression) from two independent
gene array screens of naïve and effector 2C CD8+ T
cells. NOC, not represented on chip. B,
verification of gene expression by semiquantitative RT-PCR as described
in Fig. 6. C, analysis of protein expression by Western
blotting as described in Fig. 6.
mRNA was markedly diminished in
primed cells, whereas protein levels were similar.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice, enabling us
to examine CD8+ T cells with a single specificity. The use
of a clonal T cell population minimizes potential differences in gene
expression that can be attributed to variation in activation state.
Although effector CD8+ T cells were generated in
vitro in response to mitomycin C-treated P815-B7.1 cells, there
were no viable P815-B7.1 cells by day 5 of differentiation and every
effort was made to eliminate contamination from other cell types.
However, even after further purification by Ficoll-Hypaque
centrifugation, some erythrocytes may have been inadvertently included
in the preparation of the naïve cells, which could
theoretically contribute to certain mRNA species despite lacking
nuclei and active transcription. However, potential erythrocyte contamination in the naïve population is unlikely to explain the majority of differentially expressed genes observed.
. In contrast, effector cells
produced high levels of both IL-2 and IFN-
. Thus, IFN-
was
produced at an increased magnitude following differentiation. In
contrast, effector cells required 10-fold less anti-CD3 mAb to produce
the same level of IL-2 as naïve cells, indicating a shift in
the TCR dose response. Thus, the greater IL-2 production seen with
effector cells is reflected by increased TCR sensitivity. These results
suggest that two molecular mechanisms likely explain the increased
functional responsiveness of CD8+ effector cells. For
IFN-
(and likely for cytolytic activity as well), epigenetic
modification of specific gene loci probably contributes to the
increased magnitude of gene expression following differentiation.
Chromatin remodeling at the IFN-
locus has been described in
antigen-experienced CD4+ T cells (4, 5). However, the
mechanism to explain increased TCR sensitivity in effector cells is not
understood and could be explained by changes in expression of signaling
molecules that are responsive to TCR engagement.
and
MKP-1 mRNA were all expressed at lower levels following
differentiation. However, analysis of protein expression revealed
comparable or increased expression of MKP-1 and PP1
by Western
blotting, demonstrating discordance between mRNA and protein data.
These results are consistent with the known property of
phosphatase-kinase interactions in regulating stability of the
phosphatase (39, 40) and imply that mRNA levels of phosphatases do
not necessarily predict the quantity of protein present due to
post-translational modification. Several transcription factors
(c-Fos, JunB, and Ets-2) were also found to be down-regulated in effector compared with naive
CD8+ T cells at the mRNA level but were expressed
comparably at the protein level. It is conceivable that protein
stability is similarly prolonged for these factors due to distinct
intermolecular interactions or other post-translational effects.
function, which
could interfere with cell surface signaling to PLC
and inositol
lipid signaling (44). Although peripheral T cells lacking
Rgs2 have been shown to exhibit a defect in proliferation
(45), whether overexpression of Rgs2 also limits cell cycle
progression in lymphocytes has not been examined.
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ACKNOWLEDGEMENTS |
---|
We thank Mark Neveu for facilitating the gene array experiments and Janel Washington for assistance with mouse breeding.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant R01 AI47919 and a Burroughs Wellcome Fund Clinical Scientist Award in Translational Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains supplemental information on each
differentially expressed gene as determined by the two gene array
screens and sequences of gene-specific primers used to verify
differential gene expression by RT-PCR.
To whom correspondence should be addressed: University of
Chicago, 5841 S. Maryland Ave., MC2115, Chicago, IL 60637. Tel.: 773-702-4601; Fax: 773-702-3701; E-mail:
tgajewsk@medicine.bsd.uchicago.edu.
Published, JBC Papers in Press, February 11, 2003, DOI 10.1074/jbc.M212741200
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ABBREVIATIONS |
---|
The abbreviations used are: TCR, T cell receptor; IFN, interferon; RT, reverse transcription; PE, phycoerythrin; ELISA, enzyme-linked immunosorbent assay; IL, interleukin; EST, expressed sequence tag; mAb, monoclonal antibody; HRP, horseradish peroxidase.
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