By
From the * Department of Microbiology and Immunology; Rheumatology Division, Department of
Medicine; and the § Howard Hughes Medical Institute, Vanderbilt University Medical Center,
Nashville, Tennessee 37232
Members of the nuclear factor (NF)-B/Rel family transcription factors are induced during
thymic selection and in mature T lymphocytes after ligation of the T cell antigen receptor
(TCR). Despite these findings, disruption of individual NF-
B/Rel genes has revealed no intrinsic defect in the development of mature T cells, perhaps reflecting functional redundancy.
To circumvent this possibility, the T cell lineage was targeted to express a trans-dominant form
of I
B
that constitutively represses the activity of multiple NF-
B/Rel proteins. Transgenic
cells expressing this inhibitor exhibit a significant proliferative defect, which is not reversed by
the addition of exogenous interleukin-2. Moreover, mitogenic stimulation of splenocytes leads
to increased apoptosis of transgenic T cells as compared with controls. In addition to deregulated T cell growth and survival, transgene expression impairs the development of normal T cell
populations as evidenced by diminished numbers of TCRhi CD8 single-positive thymocytes.
This defect was significantly amplified in the periphery and was accompanied by a decrease in CD4+ T cells. Taken together, these in vivo findings indicate that the NF-
B/Rel signaling
pathway contains compensatory components that are essential for the establishment of normal
T cell subsets.
Nuclear translocation of members of the nuclear factor
(NF)- Despite these findings, mice deficient for individual NF To circumvent this critical issue of functional redundancy, we targeted to the T lineage a trans-dominant form
of I Production of Transgenic Mice Expressing I Antibodies and Fluorochrome-conjugated Reagents.
Polyclonal antibodies specific for RelA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The c-Rel-specific antiserum was provided by Dr. N. Rice (National Cancer Institute, Bethesda, MD).
Rabbit antisera generated against amino acids 289-317 of human
I Cell Preparation.
Single cell suspensions from thymus, spleen,
and lymph node were prepared by crushing the organs in complete media (RPMI-1640 supplemented with 10% fetal bovine
serum, 2 mM L-glutamine, and 0.1% penicillin-streptomycin)
followed by hypotonic lysis of erythrocytes. PBLs were purified
from heparinized blood by density gradient centrifugation on Ficoll-HyPaque (22). Splenic T lymphocytes were depleted of B
cells by chromatography through nylon wool columns, followed
by panning on immobilized antibodies against mouse IgM (22,
23). In brief, single cell suspensions from pooled spleens were
added to preequilibrated nylon wool columns in RPMI-1640
supplemented with 5% fetal bovine serum at 37°C. After 45 min
at 37°C, the nonadherent cells were eluted from the column into
plastic Petri dishes (Fisher Scientific) coated with goat anti-mouse
IgM (10 µg/ml) and goat IgG (40 µg/ml) to remove residual B
lymphocytes. The resultant population was <10% B220+ and
75-90% T cells as determined by flow cytometry.
Immunoprecipitations and Western Blot Analyses.
Cytosolic extracts
were prepared from single cell suspensions cultured in the presence or absence of PMA (50 ng/ml) and ionomycin (1 µM) for
30 min using published methods (18), except that the detergent
lysis buffer was supplemented with a mixture of protease inhibitors (24). Where indicated, the translation inhibitor cycloheximide was added at 50 µg/ml for 30 min before stimulation. Lysates were clarified by centrifugation and equilibrated in ELB buffer (50 mM N-2-hydroxyethylpiperazine-N Gel Shift Analysis and DNA-Protein Cross-linking.
Nuclear fractions were prepared from single cell suspensions by high-salt extraction in the presence of protease inhibitors (18). Gel mobility
shift assays were performed by using a 32P-labeled oligonucleotide
duplex derived from Quantitation of IL-2 in Culture Supernatants.
Production of IL-2
was measured by ELISA of supernatants from thymocytes and splenocytes after mitogenic stimulation. The ELISAs were performed
using plates precoated with anti-mouse IL-2 according to the instructions of the manufacturer (Endogen, Boston, MA). IL-2 levels were quantitated by comparison to standards supplied by the
manufacturer.
Proliferation Assays.
Thymocyte suspensions were counted
and plated (2 × 105 cells per 100 µl of media) in microtitre wells
pretreated overnight with either PBS or anti-CD3 mAb (10 µg/ml)
(145-2C11, PharMingen; or 29B, GIBCO BRL). Triplicate samples were cultured for 48 h at 37°C in the presence of PMA and
ionomycin or Con A (2.5 µg/ml). Tritiated thymidine (1 µCi in
100 µl of media) was added to each well for the final 8 h before
determination of radioisotope incorporation into DNA. To quantitate T cell proliferation, pooled spleen and lymph node suspensions were depleted of B cells and macrophages by chromatography through nylon wool columns, then used in proliferation assays, as above, in the presence or absence of an activating hamster mAb against mouse CD28 (10 µg/ml; PharMingen, clone
37.51). To distinguish the proliferative capacity of lymphoblasts
from small lymphocytes, splenocytes were cultured for 40 h in the
absence or presence of Con A (2.5 µg/ml) and then treated for 1 h
with 5-bromo-2 TdT-mediated dUTP-Biotin Nick End Labeling (TUNEL) Assays
and Indirect Immunofluorescence.
Surface staining of single cell suspensions was performed using optimized concentrations of fluorochrome-conjugated purified mAbs (0.25 µg per 106 cells) (27).
Samples of 1-2 × 104 cells were analyzed using a FACScan® Plus
flow cytometer (Beckton-Dickinson, Mountain View, CA) with uniform gates and quadrants. To generate samples for quantitative TUNEL assays, single cell suspensions were cultured in the presence of either Con A (2.5 µg/ml) or immobilized anti-CD3 mAb
(10 µg/ml). After 40 h of culture, cells were harvested and fractions were stained with PE-conjugated mAb against CD4 or CD8
(PharMingen). To perform TUNEL assays, PE-stained cells were
fixed with paraformaldehyde and permeabilized with 70% EtOH
at The induced trans-activation of NF-
To determine whether the I To investigate the subunit composition of the affected
complexes, we performed DNA-protein cross-linking experiments. These studies revealed a striking decrease in nuclear RelA and c-Rel in extracts from Tghi mice as compared with nontransgenic (NTg) controls (Fig. 1 D, lanes 2 and 6). Similar results were obtained using antibodies specific for c-Rel and RelA in supershift analyses (data not
shown). In keeping with these results using Tghi thymocytes, nuclear expression of inducible NF- Prior in vitro studies have suggested a central role
for NF-
Prior studies have shown that c-Rel-deficient T cells develop normally but exhibit impaired mitogenic responses (14).
This proliferative defect was fully reversed by exogenous
IL-2 (14). Evidence of a transgene-dependent block in IL-2
production by Tghi thymocytes led us to investigate their
proliferative response to the T cell mitogens Con A, combinations of PMA and ionomycin, or immobilized antibody to the TCR (anti-CD3). As shown in Fig. 3, Tghi
thymocytes manifested a dramatic decrease in the proliferation induced by each of these mitogens (A-C; control). We
reasoned that this growth defect might be attributed to the
20-fold difference in IL-2 concentrations in these cultures
(see Fig. 2). However, as shown in Fig. 3 A addition of exogenous IL-2 to Tghi thymocytes failed to restore proliferation to normal levels. Similar results were obtained with
unfractionated (data not shown) or B cell-depleted splenocytes (Fig. 3 D). These results are in sharp contrast with
those obtained using c-Rel-deficient mice (14), presumably reflecting an additional contribution by RelA (Fig. 1 D).
In this regard, there was a 50% decrease in the frequency of
T cells positive for IL-2R
It is now well recognized that programmed cell
death can occur in response to primary stimulation through
the TCR (34) or after activated T cells are exposed to
certain proapoptotic agents (37). Moreover, evidence from
studies with established cell lines suggests that NF-
In contrast with c-Rel-deficient mice, our results with
I
While T cell subsets are established during the differentiation of immature precursors in the thymus, the ultimate
outcome of T cell development is the establishment of normal populations in the periphery (47). To investigate further the significance of NF-
Since its original discovery in B cells, NF- During the process of T cell activation, nuclear
translocation of NF- In addition to
growth signal transduction, prior studies have suggested
that members of the NF- In light of the observed steady-state decrease in Tghi T cell
populations (Fig. 6), the NF- The thymic phase
of T cell development involves an orderly progression from
double-negative to double-positive thymocytes, which then
undergo selection and emigration. In this biologic context,
our data provide three new lines of evidence for the involvement of NF- There are very few precedents for a signal transduction
pathway which preferentially potentiates development of
mature CD8+ T cells (60). We consider it unlikely that
the observed asymmetry is due to subset-specific differences in transgene expression, because RT-PCR and immunoblotting experiments revealed comparable steady-state
expression of I In summary, we have found that corepression of RelA
and c-Rel in transgenic mice leads to multiple defects in
the T lineage. Specifically, transgenic T cells expressing
IB1/Rel transcription factor family is activated
in thymocytes and mature T lymphocytes after engagement
of the TCR (1). The prototypic form of NF-
B is a heterodimeric complex containing NF-
B1 (p50) or NF-
B2
(p52), either of which may combine with a trans-activating subunit. The major trans-activating subunits of NF-
B that
are induced during T cell activation are c-Rel and RelA
(p65) (5). In quiescent T lymphocytes, these various forms
of NF-
B are sequestered in the cytoplasm by virtue of
their association with a set of inhibitory proteins that includes I
B
(6, 7). During normal T cell activation, I
B
is rapidly degraded via the ubiquitin-proteasome pathway,
thus permitting the nuclear import of NF-
B (6, 7). Binding sites for NF-
B have been found in many genes involved in T cell effector function, including those that encode regulatory cytokines and receptors (5). These findings
raise the possibility that NF-
B regulates homeostatic mechanisms in the T lineage, such as those involved in cell cycle
control or programmed cell death. Moreover, NF-
B is induced in developing thymocytes through interactions with
stromal cells and during thymic selection (4, 8). Taken together, these in vitro studies suggest an important role for
NF-
B/Rel proteins in the development of mature T cells.
B/Rel subunits exhibit no intrinsic defect in the establishment of normal populations in the T lineage (9). For
example, p50-deficient mice express a normal number and
distribution of T cells (9). Although mice lacking the RelA
subunit of NF-
B die in utero, progenitor cells derived
from these animals give rise to a normal T cell repertoire
(10, 11). Targeted disruption of the gene encoding RelB, a
constitutively expressed member of the NF-
B/Rel family,
leads to a profound reduction in APCs rather than an intrinsic defect in the T lineage (12, 13). In contrast, c-Rel-
deficient T cells are impaired in their ability to produce the
growth factor IL-2, resulting in decreased proliferation in
response to mitogens. Despite this proliferative effect, T cell
development in c-Rel
/
mice appears unaffected (14).
These unexpected findings have raised unresolved questions concerning whether NF-
B plays any significant role
in the generation of mature T cells (15). However, all of
these gene disruption experiments are complicated by the potential for functional redundancy. Indeed, prior in vitro
studies have shown that complexes containing either c-Rel
or RelA have the potential to stimulate transcription from
the same promoter (16, 17).
B
under tissue-specific control of the proximal lck
promoter (18, 19). This inhibitor, termed l
B
(
N), lacks
sequences required for signal-dependent degradation and
functions as a constitutive repressor of multiple NF-
B/Rel
proteins in transfected B and T lymphocytes (6, 7, 18, 20).
Consistent with these in vitro studies, signal-dependent induction of the c-Rel and RelA trans-activating subunits of
NF-
B was profoundly impaired in transgenic thymocytes. This dual block in the NF-
B/Rel signaling pathway was
associated with (a) a proliferative defect that is refractory to
IL-2, (b) increased sensitivity of T cells to apoptosis after
mitogenic stimulation, and (c) an abnormal distribution of
T cell subsets. Taken together, these findings demonstrate
that NF-
B plays a significant role in the development and
homeostatic control of mature T cells.
B
(
N).
The cDNA
encoding an NH2-terminally truncated form of human I
B
(amino acids 37-317) fused to the FLAG epitope (18) was cloned
into p1017-lck (20). An SpeI fragment encompassing this tissuespecific transcription unit was isolated by agarose gel electrophoresis and micro-injected (Transgenic Core Facility, Vanderbilt
Cancer Center, Nashville, TN) into the pronuclei of C57BL/6 × DBA/2 zygotes together with a BamHI-XbaI fragment containing the locus control region from the human CD2 gene (21).
Mice were maintained in accordance with federal and state government regulations after institutional approval. Four independent founders with both fragments integrated in the germline
were crossed with C57BL/6 mice for these studies. Northern blot
analysis of organ RNAs and immunoblotting with purified B cells
confirmed the T lineage specificity of transgene expression (data
not shown).
B
have been described (18). Fluorochrome-conjugated antibodies against TCR-
(Cy-Chrome), CD3 (r-PE), CD4 (r-PE), CD8
(FITC or r-PE), CD25 (FITC), CD45R/B220 (FITC),
CD69 (FITC), and H-2Kb (FITC) were obtained from PharMingen (San Diego, CA) or GIBCO BRL (Gaithersberg, MD). Avidin and streptavidin-FITC were obtained from PharMingen.
-2-ethanesulfonic
acid [Hepes] pH 7.0, 250 mM NaCl, 5 mM EDTA, 1 mM
dithiothreitol, and 0.1% NP40). Immunoprecipitations were performed on cytoplasmic extracts (500 µg) using 20 µl of agarose
beads coupled to antibodies specific for either RelA, c-Rel, or the
FLAG epitope. After three washes with ELB buffer, proteins were
either eluted with cognate peptide (RelA, FLAG) or SDS, fractionated by SDS-PAGE, and transferred to polyvinylidene difluoride membranes (Dupont/NEN Life Science, Wilmington, DE).
Membranes were blocked (1 h at room temperature) with Trisbuffered saline containing 0.1% Tween-20 and 5% powdered milk (BLOTTO), then incubated with a rabbit antiserum specific for I
B
. Immunoreactive polypeptides were detected with donkey anti-rabbit IgG conjugated to horseradish peroxidase using
enhanced chemiluminescence (Dupont).
B enhancer sequences in the IL-2 receptor
promoter (
B-pd) (5
-CAACGGCAGGGGAATTCCCCTCTCCTT-3
) (24). DNA-binding reaction mixtures (20 µl)
contained 4 µg of nuclear extract, 2 µg double-stranded poly (dI-
dC), and 10 µg BSA buffered in 20 mM Hepes (pH 7.9), 5%
glycerol, 1 mM EDTA, 1% NP40, and 5 mM dithiothreitol. Resultant nucleoprotein complexes were resolved on a native 5%
polyacrylamide gel and visualized by autoradiography (25). For
DNA-protein cross-linking analysis, photoreactive derivatives of
the radiolabeled
B probe were synthesized in the presence of
5-bromo-2
-deoxyuridine triphosphate (BrdUTP). DNA binding reaction mixtures were irradiated at 300 nm for 30 min using
a Fotodyne UV transilluminator, diluted to 200 µl with ELB buffer, and immunoprecipitated with the indicated Rel-specific antibodies and protein A-agarose (20 µl). Immune complexes were washed three times in ELB buffer, heat denatured in SDS sample buffer, resolved by SDS-PAGE, and detected by autoradiography.
-deoxyuridine (BrdU; 100 µM). Fractions of
BrdU-positive CD4+ or CD8+ cells were measured as described
(26). In brief, after staining with either anti-CD4-rPE or antiCD8-rPE, cells were permeabilized with 95% EtOH, fixed with
paraformaldehyde, and treated with 50 U of DNase I (BoehringerMannheim Biochemicals, Indianapolis, IN). After a 10 min incubation at room temperature, cells were stained with a FITC-conjugated mAb against BrdU (Beckton-Dickinson, Mountain View,
CA) and analyzed by flow cytometry. Significant BrdU uptake was restricted to cells in the lymphoblast gate as determined by
relative light-scattering characteristics (data not shown).
20°C. After washing in PBS supplemented with 1% BSA
(PBS/BSA), cells were resuspended in reaction mixtures containing terminal deoxynucleotidyl transferase (TdT; GIBCO BRL) and
biotin-16-dCTP (Boehringer-Mannheim Biochemicals). After incubation at 37°C for 30 min, cells were washed in PBS/BSA, stained with FITC-conjugated avidin, and analyzed by flow cytometry (28). Control reactions lacking TdT were performed to
generate a background profile for assessment of nonspecific staining. For restimulation experiments (29), pooled spleen and lymph
node cell suspensions were depleted of B cells and macrophages
by chromatography through nylon wool columns, cultured for 24 h
in the presence of Con A (5 µg/ml), washed with methyl-
-Dmannoside (10 mg/ml), then incubated in complete media containing IL-2 (100 µm/ml) for an additional 24 h. Viable cells
were recovered by fractionation on Ficoll-HyPaque step density
gradients and replated (5 × 105 cells/ml) in the presence of immobilized anti-CD3 antibodies (0.005-0.5 µg/ml) or mouse
TNF-
(1 or 50 ng/ml; R&D Systems, Minneapolis, MN). Cell
death was then quantitated using forward light scatter and 7-amino
actinomycin D staining characteristics, as described (29).
Inhibition of Nuclear c-Rel and RelA Activity in T Lineage
Cells.
B-responsive
genes in the T lineage is mediated primarily by c-Rel and
RelA (reviewed in 5). Prior in vitro studies have shown
that c-Rel and RelA have the capacity to activate transcription from an overlapping set of promoters (16, 17), thus
suggesting compensatory functions. To bypass the potential
for functional redundancy, we created transgenic mice expressing a truncated form of I
B
that is refractory to signal-induced degradation (18). Transgene expression was specifically targeted to the T lineage using the proximal lck promoter (20), thus facilitating studies to assess the role of
the NF-
B/Rel signaling pathway in mouse T lineage cells.
Four founder lines expressing this constitutive repressor,
termed I
B
(
N), were classified as Tglo (n = 2) or Tghi (n = 2) based upon the magnitude of transgene expression. The I
B
(
N) protein was readily detected in both thymus
and spleen from transgenic mice, albeit at different apparent
steady-state levels in these unfractionated cell populations
(Fig. 1 A, lanes 3 and 6). Importantly, this difference was
negligible when immunoblotting experiments were performed with comparable numbers of T lineage cells from
each organ (data not shown).
Fig. 1.
Abrogated expression of NF-B/Rel in I
B
(
N)
transgenic mice. (A) Transgeneencoded I
B
expressed in
thymocytes and splenocytes. Cytoplasmic extracts from unfractionated cell suspensions were subjected to immunopurification
using immobilized anti-FLAG M2 antibodies and resolved by SDSPAGE. Resolved proteins were
transferred to polyvinylidene difluoride membranes and probed
with an antiserum directed
against human I
B
(amino acids 289-317) in conjunction with an enhanced chemiluminescence detection system (Amersham Corp., Arlington Heights,
IL) (18). NTg, nontransgenic;
Tglo, low expressor; Tghi, high
expressor. (B) Assocation of endogenous I
B
(E) and transgene-encoded I
B
(
N) with
the RelA subunit of NF-
B. Isolated thymocytes from the indicated sources were cultured for
0.5 h in the presence of cycloheximide (50 µg/ml) to arrest
translation and then treated with
PMA and ionomycin for 0.5 h as
indicated. Proteins were immunoprecipitated from cytosolic extracts using a RelA-specific
antiserum and subjected to immunoblot analysis as in A. Similar results were obtained after
immunoprecipitation with c-Rel-specific antibodies (data not shown). (C) Gel mobility
shift analysis of nuclear NF-
B/Rel proteins. Thymocyte suspensions were prepared from
the indicated sources and cultured for 0.5 h in the presence or absence of PMA/ionomycin. Equal amounts of nuclear extracts were added to DNA binding mixtures containing a
32P-labeled
B probe (
B-pd) (24). DNA-protein complexes were resolved on nondenaturing polyacrylamide gels and visualized by autoradiography. (D) DNA-protein crosslinking assay of nuclear NF-
B/Rel proteins. DNA binding reaction mixtures (as in C)
were irradiated with UV light, fractionated by SDS-PAGE, and visualized by autoradiography (lanes 1-6) (18, 24, 25). Alternatively, portions of reaction mixtures generated for
lane 2 were subjected to immunoprecipitation using the indicated antiserum before gel electrophoresis (lanes 7-9). Subunit identities are indicated. (E) Gel mobility shift analysis
of nuclear NF-
B/Rel proteins in splenic T cells. B cell-depleted T cell populations
(<10% B220+; >80% CD3+) were prepared from splenocytes pooled from three NTg
and four Tghi mice. Cells were cultured for 1 h in the presence or absence of PMA/ionomycin (lanes 5-8) in parallel with resting and stimulated thymocytes pooled from the same
mice (lanes 1-4). Equal amounts of nuclear extracts were added to DNA binding mixtures
containing a 32P-labeled
B probe (
B-pd) (24). DNA-protein complexes were resolved
on nondenaturing polyacrylamide gels and visualized by autoradiography. The major inducible complexes (p50-RelA and p50-c-Rel) are indicated with an arrow; the constitutive,
higher mobility complexes consist of p50 homodimers (30).
[View Larger Versions of these Images (35 + 55K GIF file)]
B
(
N) protein integrated
into the endogenous NF-
B/Rel signaling pathway, coimmunoprecipitation studies were performed with Relspecific antisera. These experiments revealed the presence
of I
B
(
N) in latent NF-
B/Rel complexes containing
the transactivating subunits c-Rel (data not shown) and RelA
(Fig. 1 B). Levels of the endogenous form of I
B
associated with RelA were dramatically decreased; however, prolonged exposure of the immunoblots revealed residual
amounts of this inhibitor (Fig. 1 B, lane 3; data not shown).
In contrast with wild-type I
B
(Fig. 1 B, lanes 1 and 2),
I
B
(
N) was resistant to degradation in thymocytes treated
with PMA and ionomycin (lanes 3 to 6), a combination
that mimics activation through the TCR. To investigate the integrity of the NF-
B/Rel signaling pathway, mobility shift analyses were performed with nuclear extracts from
cells treated with PMA and ionomycin. Under these stimulatory conditions, wild-type but not Tghi thymocytes expressed high levels of nuclear NF-
B/Rel activity (Fig. 1 C,
lanes 2 and 6), whereas thymocytes from Tglo animals expressed intermediate levels of nuclear NF-
B (lane 4). In
addition to being dose-dependent, the observed signaling defect was selective, in that induction of AP-1 activity and
c-jun RNA was unimpaired in transgenic thymocytes (data
not shown).
B complexes
was also decreased in splenic T cells from Tghi mice when
compared with NTg controls (Fig. 1 E, lanes 6 and 8). Taken together, these findings indicate that I
B
(
N) specifically represses signal-dependent activation of nuclear
RelA and c-Rel, which are normally induced during thymic selection and T cell activation (4, 8, 30).
B/Rel proteins in transcriptional regulation of
the IL-2 gene (31, 32). Consistent with these findings, IL-2
production is impaired in T cells derived from c-Rel-deficient mice (14). Considering the inhibitory effects of
I
B
(
N) on c-Rel DNA binding activity, we next investigated whether IL-2 gene expression was compromised in
transgenic thymocytes. For these studies, thymocytes from
Tghi mice and their NTg littermates were cultured in the
absence or presence of PMA and ionomycin, followed by
ELISA to quantitate the concentration of IL-2 secreted into
culture supernatants. As shown in Fig. 2, wild-type thymocytes produced 14,100 pg/ml of IL-2, reflecting at least
a 3,000-fold increase in cytokine expression relative to the
basal level. In contrast, IL-2 production by comparable
numbers of activated Tghi thymocytes was drastically reduced to 645 pg/ml. In companion studies, we found that
IL-2 production by mitogen-stimulated Tghi splenocytes
was also attenuated relative to controls (8% of control; data
not shown). These findings demonstrate that the block imposed on the DNA binding activities of c-Rel and RelA
(see Fig. 1) is associated with a profound deficit in IL-2
production, thus establishing the presence of a significant
functional defect in the NF-
B/Rel signaling pathway that
affects the expression of downstream genes.
Fig. 2.
IL-2 production by thymocytes from IB
(
N) transgenic
mice. Thymocytes from either NTg or Tghi mice were cultured in triplicate for 48 h in media alone (open bars) or treated with combinations of
PMA (50 ng/ml) and ionomycin (1 µg/ml) (stippled bars). Supernatants
from these cultures and IL-2 standards were assayed by ELISA. IL-2 production by unstimulated NTg thymocytes was below the detection limit of
the assay (3-5 pg IL-2/ml). Results represent the mean values (± SEM)
from five separate Tghi mice and five NTg littermates, as analyzed in three
separate experiments.
[View Larger Version of this Image (17K GIF file)]
B
(
N).
chain (CD25), which has in its 5
-flanking DNA a site for RelA/p50 binding (5, 33).
However, Tghi-derived cells that were able to undergo blast
transformation and enter S phase expressed normal levels of
CD25 (data not shown). We conclude that the failure of
exogenous IL-2 to restore normal proliferation of Tghi T
cells is likely due to the reduced expression of a competence factor other than the IL-2R
chain.
Fig. 3.
Failure of IL-2 to rescue the proliferative defect in IB
(
N) mice. Thymocytes from either NTg (open bars) or Tghi (closed bars) mice were
treated for 40 h with (A) combinations of PMA (50 ng/ml) and ionomycin (1 µg/ml). (B) Con A (2.5 µg/ml), or (C) plate-bound anti-CD3 (10 µg/ml),
in the presence or absence of IL-2 (100 U/ml) as indicated. Cells were pulsed for an additional 8 h with tritiated thymidine and harvested for scintillation
counting. The results are shown as the mean of tritiated thymidine incorporation (± SEM) for eight Tghi mice and eight NTg littermates (four independent experiments). (D) Splenocytes depleted of B cells and macrophages were analyzed under similar conditions using plate-bound anti-CD3 (10 µg/ml), agonistic antibodies against CD28 (10 µg/ml), and IL-2 (100 U/ml) as indicated.
[View Larger Versions of these Images (17 + 16 + 18 + 21K GIF file)]
B may
either inhibit (38, 39) or enhance their susceptibility to apoptosis (40). Accordingly, we next investigated the influence of I
B
(
N) transgene expression on entry into the
apoptotic pathway after primary activation of resting T cells
with mitogens. When the prevalence of apoptotic T cells was determined by TUNEL assays after mitogenic stimulation with Con A, we observed that expression of the
I
B
(
N) transgene led to a two-fold increase in the frequency of T cell apoptosis (Fig. 4 A). This enhancement in
programmed cell death applied to both CD4+ and CD8+
T cells. Similar results were obtained using immobilized antiTCR antibodies as the stimulus, albeit with a more pronounced effect on the CD8+ subset (Fig. 4 B). Selective
gating on lymphoblasts revealed a doubling in the frequency of apoptotic CD8+ T cells derived from the transgenic mice as compared with controls. Thus, the increased
sensitivity to apoptosis observed under these experimental
conditions cannot be attributed solely to the failure of these
cells to undergo blast transformation. We conclude that the
expression of I
B
(
N) in transgenic T cells is associated with enhanced apoptosis of T cells in response to primary
TCR stimulation (35, 36).
Fig. 4.
Increased apoptosis among activated T cells from IB
(
N)
transgenic mice. (A) Splenocytes from either NTg (open bars) or Tghi
(closed bars) mice were cultured in the presence of Con A (2.5 µg/ml).
After 40 h in culture, cells were divided equally, stained with PE-labeled
antibodies against CD4 or CD8, and subjected to TUNEL analysis. The
data represent the mean percentage (± SEM) of TUNEL-positive cells in
each T cell subset. The mean was calculated using data from 19 individual
Tghi mice or an equal number of NTg controls (10 independent experiments with 6-8-wk-old mice). The increased frequencies of apoptotic cells among transgenic CD4+ and CD8+ cells were statistically significant
when compared with nontransgenic CD4+ or CD8+ cells (P <0.001).
(B) Splenocytes were activated using plate-bound anti-CD3, then processed for TUNEL assays as in A. Results represent cumulative data from
12 individual Tghi mice or an equal number of NTg controls (six independent experiments with 6-8-wk-old mice). The increased frequencies
of apoptotic cells among transgenic CD4+ and CD8+ cells were statistically significant when compared with nontransgenic CD4+ or CD8+ cells
(P
0.001).
[View Larger Version of this Image (14K GIF file)]
B
(
N) Perturbs Development of Mature T Cell Lineages.
B
(
N) transgenic animals demonstrate a proliferative
defect that is refractory to IL-2, as well as enhanced T cell
apoptosis. These novel defects raised the possibility that establishment of T cell populations might be abnormal in
I
B
(
N) transgenic mice. To investigate this possibility,
we first measured the prevalence of CD4 and CD8 singlepositive (SP) cells among thymocytes. Thymic cellularity
was unaffected by the transgene and FACS® profiles indicated normal frequencies of CD4
CD8
and CD4+CD8+
thymocytes (Fig. 5 A). Thus, despite significant inhibition
of NF-
B/Rel activity (see Fig. 1 C), TCR
and
chain
gene rearrangement proceed normally, as does the expansion of immature thymocytes to the double-positive stage
(43). In contrast, Tghi CD8 SP thymocytes bearing high
levels of TCR-
were decreased 40% relative to NTg
controls (P <0.0001), whereas TCRhi CD4 SP cells were
unaffected (Fig. 5 B). There was no difference in the staining intensity for CD8 present on any thymocyte subset, which excludes the possibility that I
B
(
N) downregulated the CD8 gene. Furthermore, expression of the class I
MHC antigen Kb on Tghi thymocytes and splenocytes was
normal, as were mRNA levels for the H-2K antigen. As
such, the observed decrease in CD8 SP cells does not appear to be due to inadequate class I MHC expression (44,
45). Given that the TCRhi subset of thymocytes is associated with repertoire selection (46), we conclude that T cell
development in the thymus of Tghi mice is partially impaired.
Fig. 5.
Decreased thymic CD8+ cells in IB
(
N) transgenic mice. (A) A representative FACS® profile from three-color indirect immunofluorescence experiments. Thymocytes from NTg and Tghi mice were analyzed by flow cytometry for surface expression of CD4 and CD8 on cells bearing high density TCR-
/
(TCR high) or medium to low density receptors (TCR med/low). Gating of cells based on TCR-
/
expression is shown to
the left, together with the percentage of total cells in each gate. Dual parameter fluorescence histograms (CD4 versus CD8) derived from the gated subsets
(TCRmed/lo and TCRhi, respectively) are shown on the right. Numbers represent the percentage of gated thymocytes in each quadrant. Mean cell counts were equivalent in NTg and Tg hi thymuses (99 [± 9.1] × 106 versus 97.7 [± 10.1] × 106 respectively). (B) Subset distribution of the TCRhi thymocyte
populations in NTg (open bars) and Tg hi (closed bars) mice. Mean cell populations (± SEM) or TCRhi cells in the CD4+ CD8
and CD4
CD8+ quadrants are shown (n = 10). There was no significant difference in mean numbers of CD4
CD8
or CD4+ CD8+ cells.
[View Larger Versions of these Images (35 + 15K GIF file)]
B/Rel proteins in this process,
we measured the prevalence of T cells in spleen, lymph
node, and blood. As shown in Fig. 6 A, T cell numbers
were significantly reduced at each of these peripheral sites
in Tghi animals as compared with NTg littermates. With respect to mature T cell subsets, CD8+ T cells were substantially decreased in the periphery of Tghi animals relative to
controls (Fig. 6 B). A significant loss of CD4+ cells was also
observed in spleen and blood, although this defect was consistently less severe (Fig. 6 C). Similar evidence for skewing
of CD4/CD8 ratios was obtained by gating specifically on
/
-TCR-bearing cells in the spleen, lymph nodes, and
blood (data not shown). Thus, the asymmetric effect of
I
B
(
N) on the populations of positively selected SP cells
in the thymus is amplified in the periphery. An altered ratio
of CD4/CD8 subsets was also observed in the Tglo lines of
mice. However, consistent with the biochemical data (see
Fig.1 C), these CD8+ T cells were less profoundly affected
(50% of normal numbers) relative to Tghi mice (Fig. 6, legend), demonstrating a dose-dependent effect of the transgene. Because endogenous I
B
is still present in this
transgenic system (see Fig.1 B), our results may underestimate the quantitative significance of NF-
B/Rel proteins
in determining T cell levels. Notwithstanding this uncertainty, these findings provide strong in vivo evidence that
an intact NF-
B/Rel signaling pathway is essential for acquisition of a normal proportion of CD8+ T cells.
Fig. 6.
Altered T cell subsets in IB
(
N) transgenic mice. Cells
from the indicated peripheral sites were stained with fluorochrome-conjugated mAbs against CD3 (A), CD8 (B), or CD4 (C) and analyzed by
flow cytometry. Mean cell counts were equivalent in NTg and Tg hi
spleens (77.9 [± 7.2] versus 72.7 [± 5.0] × 106 cells, respectively; P >0.5),
and reduced in Tghi lymph nodes (9.25 [± 1.6] × 106 cells) relative to
NTg samples (15.0 [± 2.6] × 106 cells; P = 0.09). Therefore, the data are
presented as the percentage of cells (± SEM) expressing these surface
markers in spleen (n = 22), lymph node (n = 11), or blood (n = 9). Differences between NTg (open bars) and Tghi (closed bars) samples were significant at P <0.0001 (spleen) and P <0.001 (blood, lymph node) with the
exception of CD4+ lymph node cells. Analysis of T cells at each of these
sites failed to reveal the CD8lo population previously linked to superantigen-induced apoptosis and CD8+ T cell loss (26). Evidence of T cell hyperactivation (50) was absent. Splenic cellularity was normal in two independent Tglo lines (n = 9). CD4+ and CD8+ cells represented 17.5 ± 1.6% and 5.0 ± 0.8% of Tglo splenocytes (CD4/CD8 ratio = 4.6 ± 0.9).
In contrast, CD4+ and CD8+ cells represented 20.1 ± 1.0% and 10.0 ± 0.55% of splenocytes from littermate controls. The observed differences in
CD8+ cell numbers and CD4/CD8 ratios were significant at P <0.001 and
P <0.05, respectively.
[View Larger Version of this Image (16K GIF file)]
B has been
widely implicated in the transcriptional control of genes involved in T cell activation and growth (5). This signal
transduction pathway is tightly coupled to the TCR and is
induced during selection of double-positive thymocytes (1-
4, 8). Despite these findings, mice deficient for p50, c-Rel,
or RelB have a normal number and distribution of T cell
subsets (9), thus raising questions concerning the significance of NF-
B/Rel proteins in the establishment and maintenance of a normal T cell compartment (15). However, these gene disruption experiments are complicated by the
potential for functional redundancy and, as shown for RelA,
can lead to early embryonic lethality (10). To circumvent
these technical problems, we targeted to the T lineage a
truncated form of I
B
that constitutively represses the nuclear expression of multiple NF-
B dimers. When expressed
in transgenic thymocytes, this modified inhibitor dramatically attenuated nuclear import of RelA and c-Rel (Fig.
1, C and D). These two Rel-related polypeptides represent the principal trans-activating subunits of NF-
B that are induced in response to TCR ligation (1, 5, 8, 30). Based on these biochemical findings, we investigated whether simultaneous arrest of RelA and c-Rel in the T cell compartment is associated with novel phenotypic alterations relative
to those reported in prior gene targeting studies.
B is associated with transcriptional activation of the genes encoding IL-2 and IL-2R
(CD25),
which together are required for normal growth signal transduction (48). Recent gene disruption experiments have
demonstrated that c-Rel-deficient T cells exhibit a defect
in IL-2 production, which leads to a diminished proliferative response (14). In these prior studies, exogenous IL-2
restored T cell proliferation, indicating that the IL-2 signaling pathway is intact in c-Rel-deficient lymphocytes (14).
Although the mitogenic response of T lineage cells expressing I
B
(
N) is also impaired, we have found that this response cannot be fully restored by exogenous IL-2. Given
the biochemical differences between the two in vivo systems, these data suggest that RelA contributes a unique
function(s) in the T cell activation program that is not compensated for by c-Rel. One potential function involves the
control of IL-2R
gene expression, which is normal in
c-Rel
/
mice (14). Indeed, prior in vitro studies have suggested that the activity of the IL-2R
promoter is under
RelA control (51). However, we have found that T lymphoblasts derived from Tghi mice express normal levels of
surface IL-2R
and that thymocytes from these animals respond to IL-2-mediated growth signals, albeit at lower levels. Taken together, these data suggest that T cells in our
experimental system acquire functional IL-2 receptors, thus
raising the possibility that NF-
B regulates genes important for growth signal transduction in T cells following engagement of the IL-2R (52, 53).
B/Rel family of proteins contribute to the regulation of programmed cell death. For example, high levels of c-Rel expression are associated with
apoptosis in the developing avian embyro and bone marrow cells (40). In contrast, more recent studies indicate that
NF-
B protects embryonic fibroblasts and transformed T cells
from TNF-
-induced apoptosis (38, 39). The data presented here indicate that NF-
B can also protect primary T lymphocytes from death after stimulation through the
TCR. These findings may be related to recent studies demonstrating that CD3 stimulation leads to rapid T cell apoptosis in vivo (35, 36). Of note, apoptosis can also be induced by secondary stimulation of T cells activated by
treatment with a mitogen and IL-2 (29, 37, 54). Consistent
with our primary stimulation data (Fig. 4), we have found
that nylon wool-purified T cells from transgenic spleens
exhibit enhanced apoptosis using this secondary stimulation model (data not shown). However, Tghi T cells also exhibit
a proliferative defect that cannot be rescued by IL-2 (Fig.
3), a finding that complicates the interpretation of any results obtained using these latter experimental conditions.
B-dependent mechanism of
protection described here may exert a significant influence
on lymphocyte homeostasis by inducing the expression of
one or more downstream genes that affect T cell survival.
In this regard, the expression of FasL and anti-apoptotic
gene products such as Bcl-2 and Bcl-xL has been linked to
the regulation of T cell population size (29, 35, 36, 54).
However, levels of Bcl-2, Bcl-x, and FasL RNA in resting
and stimulated cells from Tghi and NTg animals were comparable (data not shown). Prior in vitro studies have also
shown that the death of activated T cells may result from
IL-2 withdrawal or inadequate costimulation through cell
surface CD28 (54, 58, 59). However, our preliminary studies indicate that TCR-mediated apoptosis of transgenic T cells cannot be reversed by either exogenous IL-2 or agonistic
antibodies directed against CD28 (data not shown). As such,
the mechanism by which NF-
B protects normal T cells
from TCR-induced apoptosis may involve a distinct pathway.
B in T Cell Development.
B in T cell development. First, the
number of TCRhi CD4
CD8+ cells in the thymus was decreased, despite the unabated accumulation of immature
thymocytes. Second, mature T cell subsets were further compromised in the periphery. Finally, an unexpected but
striking finding to emerge from these studies was an asymmetric effect of the transgene on the deployment of CD4+
and CD8+ cells.
B
(
N) in the CD4+ and CD8+ lineages
(data not shown). Moreover, transgenic thymocytes exhibited a dramatic reduction (>95%) in the synthesis of IL-2
(Fig. 2), which is produced almost exclusively by the CD4+
CD8
subset (3, 64, 65). Consistent with this finding, we
have also found that transgenic splenocytes produce reduced levels of IL-2 relative to controls (8% of control; data
not shown). Finally, NF-
B induction was arrested in a
predominantly CD4+ population of Tghi T cells (Fig. 1 E).
These data strongly suggest that the I
B
(
N) transgene
blocks NF-
B signaling in the CD4+ T cell compartment.
Despite these findings with CD4+ cells, transgene expression
led to a preferential reduction in CD8+ T cell numbers in
the thymus and periphery (Figs. 5 and 6). We conclude
that the function of the NF-
B/Rel signaling pathway in
lymphocyte homeostasis may be polarized with respect to
the deployment of specific T cell subsets. Although the
mechanism involved remains unclear, one possibility is that
CD8+ cells in the intact animal are more dependent on
NF-
B for protection against apoptosis (Fig. 4 B).
B
(
N) exhibited decreased IL-2 production, a proliferative defect that is refractory to exogenous IL-2, and enhanced apoptosis following mitogenic stimulation. Whereas
the double-negative and double-positive thymocyte subsets
in these mice appeared normal, numbers of TCRhi CD8 SP
thymocytes were significantly decreased. This deficit in CD8+
T cells was exacerbated in the periphery and accompanied
by a decrease in CD4+ T cells and skewed CD4/CD8 ratio. These findings were not apparent in prior gene targeting studies (9), thus suggesting that RelA and c-Rel
serve compensatory functions in homeostasis. Taken together, the data provide clear evidence for the involvement of NF
B/Rel proteins in T lineage development. Furthermore,
the observed asymmetric population dynamics suggest that
the mechanisms controlling CD4+ and CD8+ T cell numbers may involve distinct NF-
B signaling requirements.
Address correspondence to Mark Boothby or Dean Ballard, Department of Microbiology and Immunology, Vanderbilt University Medical School, Nashville, Tennessee 37232-2363.
Received for publication 13 January 1997 and in revised form 24 March 1997.
The authors acknowledge A. Cherrington and W. Armistead for technical assistance; J. Wright and C. Pettipher (Vanderbilt Cancer Center Transgenic Core) for DNA microinjections; A. Lackey (Cytometry Associates, Brentwood, TN) and J. Price (Vanderbilt Cancer Center Core) for expert flow cytometry analyses; L. Glimcher, E. Oltz, T. Aune, G. Miller, D. Perkins, T. Laufer, J. Chen, L. Van Kaer, E. Robey, D. Kioussis, L. Kelley, and P. Fink for helpful discussions; and E. Vance for help with manuscript preparation. D.W. Ballard is an investigator of the Howard Hughes Medical Institute (HHMI). Supported by National Institutes of Health grant AI-33839 and the Howard Hughes Medical Institute (D.C. Scherer, J.A. Brockman, D.W. Ballard), and by National Institutes of Health grant AI-36997, GM-42550, and the Leukemia Society of America (A.L. Mora, M.R. Boothby).1. |
Jamieson, C.,
P.G. McCaffrey,
A. Rao, and
R. Sen.
1991.
Physiologic activation of T cells via the T cell receptor induces NF-kappa B.
J. Immunol.
147:
416-420
|
2. |
Kang, S.-M.,
A.-C. Tran,
M. Grilli, and
M.J. Lenardo.
1992.
NF-![]() |
3. | Chen, D., and E. Rothenberg. 1993. Molecular basis for developmental changes in interleukin-2 gene inducibility. Mol. Cell. Biol. 13: 228-238 [Abstract]. |
4. |
Sen, J.,
L. Venkataraman,
Y. Shinkai,
J.W. Pierce,
F.W. Alt,
S.J. Burakoff, and
R. Sen.
1995.
Expression and induction of nuclear factor-![]() |
5. |
Baeuerle, P.A., and
T. Henkel.
1994.
Function and activation of
NF-![]() |
6. |
Finco, T., and
A.S. Baldwin.
1995.
Mechanistic aspects of
NF-![]() |
7. |
Verma, I.M.,
J.K. Stevenson,
E.M. Schwarz,
D. Van Antwerp, and
S. Miyamoto.
1995.
Rel/NF-![]() ![]() |
8. |
Moore, N.C.,
J. Girdlestone,
G. Anderson,
J.J.T. Owen, and
E.J. Jenkinson.
1995.
Stimulation of thymocytes before and
after positive selection results in the induction of different
NF-![]() |
9. |
Sha, W.C.,
H.C. Liou,
E.I. Tuomanen, and
D. Baltimore.
1995.
Targeted disruption of the p50 subunit of NF-![]() |
10. |
Beg, A.A.,
W.C. Sha,
R.T. Bronson,
S. Ghosh, and
D. Baltimore.
1995.
Embryonic lethality and liver degeneration in
mice lacking the RelA component of NF-![]() |
11. |
Baeuerle, P.A., and
D. Baltimore.
1996.
NF-![]() |
12. |
Weih, F.,
D. Carrasco,
S.K. Durham,
D.S. Barton,
C.A. Rizzo,
R.P. Ryseck,
S.A. Lira, and
R. Bravo.
1995.
Multiorgan inflamamtion and hematopoietic abnormalities in mice
with a targeted disruption of RelB, a member of the NF-![]() |
13. | Burkly, L., C. Hession, L. Ogata, C. Reilly, L.A. Marconi, D. Olson, R. Tizard, R. Cate, and D. Lo. 1995. Expression of relB is required for the development of thymic medulla and dendritic cells. Nature (Lond.). 373: 531-536 [Medline]. |
14. | Köntgen, F., R.J. Grumont, A. Strasser, D. Metcalf, R. Li, D. Tarlinton, and S. Gerondakis. 1995. Mice lacking the c-rel proto-oncogene exhibit defects in lymphocyte proliferation, humoral immunity, and interleukin-2 expression. Genes Dev. 9: 1965-1977 [Abstract]. |
15. | Ghosh, S.. 1995. Transcriptional regulation in lymphocyte differentiation. The Immunologist. 3: 168-169 . |
16. |
Tan, T.H.,
G.P. Huang,
A. Sica,
P. Ghosh,
H.A. Young,
D.L. Longo, and
N.R. Rice.
1992.
![]() |
17. | Garoufalis, E., I. Kwan, R. Lin, A. Mustafa, N. Pepin, A. Roulston, J. Lacoste, and J. Hiscott. 1994. Viral induction of
the human beta interferon promoter: modulation of transcription by NF-![]() |
18. |
Brockman, J.A.,
D.C. Scherer,
T.A. McKinsey,
S.M. Hall,
X. Qi,
W.Y. Lee, and
D.W. Ballard.
1995.
Coupling of a
signal response domain in I![]() ![]() ![]() |
19. | Lewis, D.B., C.C. Yu, K.A. Forbush, J. Carpenter, T.A. Sato, A. Grossman, D.H. Liggitt, and R.M. Perlmutter. 1989. Interleukin 4 expressed in situ selectively alters thymocyte development. J. Exp. Med. 173: 89-100 [Abstract]. |
20. | Scherer, D.C., J.A. Brockman, H.H. Bendall, G.M. Zhang, D.W. Ballard, and E.M. Oltz. 1996. Corepression of RelA and c-Rel inhibits immunoglobulin kappa chain gene transcription and rearrangement in precursor B lymphocytes. Immunity. 5: 563-574 [Medline]. |
21. |
Greaves, D.R.,
F.D. Wilson,
G. Lang, and
D. Kioussis.
1989.
Human CD2 3![]() |
22. | Kruisbeck, A.M. 1992. Isolation and fractionation of mononuclear cell populations. Curr. Prot. Immunol. 3.1.1-3.1.5. |
23. |
Casey, L.S.,
A.H. Lichtman, and
M. Boothby.
1992.
IL-4 induces IL-2 receptor p75 ![]() |
24. |
Ballard, D.W.,
W.H. Walker,
S. Doerre,
P. Sista,
J.A. Molitor,
E.P. Dixon,
N.J. Peffer,
M. Hannink, and
W.C. Greene.
1990.
The v-rel oncogene encodes a ![]() ![]() |
25. | Singh, H.R., R. Sen, D. Baltimore, and P.A. Sharp. 1986. A nuclear factor that binds to a conserved sequence motif in transcriptional control elements of immunoglobulin genes. Nature (Lond.). 319;154-157. |
26. | Dillon, S.R., V.L. MacKay, and P.J. Fink. 1995. A functionally compromised intermediate in extrathymic CD8+ T cell deletion. Immunity. 3: 321-333 [Medline]. |
27. | Holmes, K., and B.J. Fowlkes. 1992. Immunofluorescence and cell sorting: preparation of cells and reagents for flow cytometry. Curr. Prot. Immunol. 5.3.1-5.2.11. |
28. | Kelley, L.L., W.F. Green, G.G. Hicks, M.C. Bondurant, M.J. Koury, and H.E. Ruley. 1994. Apoptosis in erythroid progenitors deprived of erythropoietin occurs during the G1 and S phases of the cell cycle without growth arrest or stabilization of wild-type p53. Mol. Cell. Biol. 14: 4183-4192 [Abstract]. |
29. | Zheng, L., G. Fisher, R.E. Miller, J. Peschon, D.H. Lynch, and M.J. Lenardo. 1995. Induction of apoptosis in mature T cells by tumor necrosis factor. Nature (Lond.). 377: 348-351 [Medline]. |
30. |
Weih, F.,
D. Carrasco, and
R. Bravo.
1994.
Constitutive and
inducible Rel/NF-![]() |
31. |
Verweij, C.L.,
M. Geerts, and
L.A. Aarden.
1991.
Activation
of interleukin 2 gene transcription via the T-cell surface molecule CD28 is mediated through an NF-![]() |
32. | Lai, J.-H., G. Horvath, J. Subleski, J. Bruder, P. Ghosh, and T.-H. Tan. 1995. RelA is a potent transcriptional activator of the CD28 response element within the interleukin-2 promoter. Mol. Cell. Biol. 15: 4260-4271 [Abstract]. |
33. | Cross, S.L., N.F. Halden, M.J. Lenardo, and W.J. Leonard. 1989. Functionally distinct NF-kappa B binding sites in the immunoglobulin kappa and IL-2 receptor alpha chain genes. Science (Wash. DC). 244: 466-469 [Medline]. |
34. | Nossal, G.J.V.. 1994. Negative selection of lymphocytes. Cell. 76: 229-239 [Medline]. |
35. | Sytwu, H.-K., R.S. Liblau, and H.O. McDevitt. 1996. The roles of Fas/APO-1 (CD95) and TNF in antigen-induced programmed cell death in T cell receptor transgenic mice. Immunity. 5: 17-30 [Medline]. |
36. | Tucek-Szabo, C.L., S. Andjelic, E. Lacy, K.B. Elkon, and J. Nikolic-Zugic. 1996. Surface T cell Fas receptor/CD95 regulation, in vivo activation, and apoptosis: activation-induced death can occur without Fas receptor. J. Immunol. 156: 192-200 [Abstract]. |
37. | Zheng, L., S.A. Boehme, J.M. Critchfield, J.C. Zuniga-Pflucker, M. Freedman, and M.J. Lenardo. 1994. Immunological tolerance by antigen-induced apoptosis of mature T lymphocytes. Adv. Exp. Med. Biol. 365: 81-89 [Medline]. |
38. |
Van Antwerp, D.J.,
S.J. Martin,
T. Kafri,
D.R. Green, and
I.M. Verma.
1996.
Suppression of TNF-![]() ![]() |
39. |
Beg, A.A., and
D. Baltimore.
1996.
An essential role for NF![]() ![]() |
40. | Abbadie, C., N. Kabrun, F. Bouali, J. Smardova, D. Stehelin, B. Banderbunder, and P.J Enrietto. 1993. High levels of c-Rel expression are associated with programmed cell death in the developing avian embryo and in bone marrow cells in vitro. Cell 75: 899-912 [Medline]. |
41. |
Jung, M.,
Y. Zhang,
S. Lee, and
A. Dritschlo.
1995.
Correction of the radiation senstivity in ataxia-telangiectasia by a
truncated I![]() ![]() |
42. |
Grilli, M.,
M. Pizzi,
M. Memo, and
P.F. Spano.
1996.
Neuroprotection by aspirin and sodium salicylate through blockade of NF-![]() |
43. | Huesman, M., B. Scott, P. Kisielow, and H. Von Boehmer. 1991. Kinetics and efficacy of positive selection in the thymus of normal and T cell receptor transgenic mice. Cell. 66: 533-540 [Medline]. |
44. | Matsuyama, T., T. Kimura, M. Kitagawa, K. Pfeffer, T. Kawakami, N. Watanabe, T.M. Kundig, R. Amakawa, K. Kishihara, A. Wakeham, et al . 1993. Targeted disruption of IRF-1 or IRF-2 results in abnormal type I IFN gene induction and aberrant lymphocyte development. Cell. 75: 83-97 [Medline]. |
45. |
White, L.C.,
K.L. Wright,
N.J. Felix,
H. Ruffner,
L.F.L. Reis,
R. Pine, and
J.P.-Y. Ting.
1996.
Regulation of LMP2
and TAP1 genes by IRF-1 explains the paucity of CD8+ T
cells in IRF-1 ![]() ![]() |
46. | Jameson, S.C., K.A. Hogquist, and M.J. Bevan. 1995. Positive selection of thymocytes. Annu. Rev. Immunol. 13: 93-126 [Medline]. |
47. | Sprent, J. 1989. T lymphocytes and the thymus. In Fundamental Immunology. W.E. Paul, editor. Raven Press, NY. 69-93. |
48. | Crabtree, G.R.. 1989. Contingent genetic regulatory events in T lympyhocyte activation. Science (Wash. DC). 243: 355-361 [Medline]. |
49. | Schorle, H., T. Holtschke, T. Hunig, A. Schimpl, and I. Horak. 1991. Development and function of T cells in mice rendered interleukin-2 deficient by gene targeting. Nature (Lond.). 352: 621-624 [Medline]. |
50. |
Willerford, D.M.,
J. Chen,
J.A. Ferry,
L. Davidson,
A. Ma, and
F.W. Alt.
1995.
Interleukin-2 receptor ![]() |
51. |
Doerre, S.,
P. Sista,
S.C. Sun,
D.W. Ballard, and
W.C. Greene.
1993.
The c-rel proto-oncogene product represses
NF-![]() |
52. | Brach, M.A., H.J. Gruss, D. Riedel, R. Mertelsmann, and F. Herrmann. 1992. Activation of NF-kappa B by interleukin 2 in human blood monocytes. Cell Growth Diff. 3: 421-427 [Abstract]. |
53. |
Arima, N.,
W.A. Kuziel,
T.A. Grdina, and
W.C. Greene.
1992.
IL-2-induced signal transduction involves the activation of nuclear NF-kappa B expression.
J. Immunol.
149:
83-91
|
54. | Van Parijs, L., A. Ibraghimov, and A.K. Abbas. 1996. The role of costimulation and Fas in T cell apoptosis and tolerance. Immunity. 4: 321-328 [Medline]. |
55. | Veis, D.J., C.M. Sorenson, J.R. Shutter, and S.J. Korsmeyer. 1993. Bcl-2-deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair. Cell. 75: 229-240 [Medline]. |
56. | Ma, A., J.C. Pena, B. Chang, E. Margosian, L. Davidson, F.W. Alt, and C.B. Thompson. 1995. Bcl-x regulates the survival of double-positive thymocytes. Proc. Natl. Acad. Sci. USA. 92: 4763-4767 [Abstract]. |
57. | Motoyama, N., F. Wang, K.A. Roth, H. Sawa, K.-I. Nakayama, K. Nakayama, I. Negishi, S. Senju, Q. Zhang, S. Fujii, and D.Y. Loh. 1995. Massive cell death of immature hematopoietic cells and neurons in Bcl-x-deficient mice. Science (Wash. DC). 267: 1506-1510 [Medline]. |
58. | Nagata, S., and P. Golstein. 1995. The Fas death factor. Science (Wash. DC). 267: 1449-1456 [Medline]. |
59. | Boise, L.H., and C.B. Thompson. 1996. Hierarchical control of lymphocyte survival. Science (Wash. DC). 274: 67-68 [Medline]. |
60. | Arpaia, E., M. Shahar, H. Dadi, A. Cohen, and C.M. Roifman. 1994. Defective T cell receptor signaling and CD8+ thymic selection in humans lacking ZAP-70 kinase. Cell. 76: 947-958 [Medline]. |
61. | Elder, M.E., D. Lin, J. Clever, A.C. Chan, T.J. Hope, A. Weiss, and T.G. Parslow. 1994. Human severe combined immunodeficiency due to a defect in ZAP-70, a T cell tyrosine kinase. Science (Wash. DC). 264: 1596-1599 [Medline]. |
62. | Chan, A.C., T.A. Kadlecek, M.E. Elder, A.H. Filipovich, W.L. Kuo, M. Iwashima, T.G. Parslow, and A. Weiss. 1994. ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency. Science (Wash. DC). 264: 1599-1601 [Medline]. |
63. | Park, S.Y., K. Saijo, T. Takahashi, M. Osawa, H. Arase, H. Hirayama, K. Miyake, H. Nakauchi, T. Shirasawa, and T. Saito. 1995. Developmental defects of lymphoid cells in Jak3 kinase-deficient mice. Immunity. 3: 771-782 [Medline]. |
64. |
Fischer, M.,
I. McNeil,
T. Suda,
J.E. Cupp,
K. Shortman, and
A. Zlotnik.
1991.
Cytokine production by mature and
immature thymocytes.
J. Immunol.
146:
3452-3456
|
65. | Rincon, M., and R.A. Flavell. 1996. Regulation of AP-1 and NFAT transcription factors during thymic selection of T cells. Mol. Cell. Biol. 16: 1074-1084 [Abstract]. |