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B
Degradation1
Departments of
*
Medicine (Rheumatology) and
Microbiology/Immunology, Vanderbilt University Medical School, Nashville, TN 37232; and
Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B
(inhibitor of NF-B) degradation, leading to lower levels of both
nuclear AP-1 and NF-B. Expression of an IB mutant resistant to
signal-induced degradation in transgenic T cells caused markedly
deficient effector cytokine (IL-4, IFN-
) production after primary
TCR stimulation despite a detectable level of nuclear NF-B. A TCR
response element from the IFN- promoter, despite lacking detectable
NF-B/Rel sites, was also unresponsive to TCR ligation. Nuclear
induction of AP-1 proteins in response to T cell activation was
diminished in transgenic T cells. Costimulation induced by
anti-CD28 mAb increased IL-2 production, but failed to reverse the
defects in effector cytokine production. Taken together, these data
indicate that impaired NF-B/Rel signaling in T cells interferes with
the signal transduction pathways required for efficient induction of
effector cytokine production. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and activation-induced apoptosis Refs. (1, 2, 3, 4, 5);
reviewed in Refs. (6, 7, 8, 9, 10). Of note, Ag-specific T cells can proliferate
or produce IL-2 yet exhibit a dramatic block of effector function in
some in vivo models of T cell tolerance (11, 12, 13, 14), whereas in others a
complete block of IL-2 production is observed (reviewed in 15).
Despite the wealth of information regarding cell surface proteins
required for a TCR-MHC interaction to provide growth signals Refs. (16)
and (17); reviewed in Refs. (7), (18), and (19), it is unclear what
alterations in transcription regulation are sufficient to render normal
T lymphocytes unresponsive to TCR engagement.
In this regard, prior in vitro studies have indicated that three
inducible transcription factor families are of particular importance in
T cell activation: NF-ATs, the AP-1 family of basic leucine zipper
proteins, and the NF-B/Rel family reviewed in Refs. (20, 21, 22, 23). In
vitro models of anergy induction using CD4+ T cell clones
have focussed on the AP-1 family (24, 25, 26). This focus is consistent
with the discovery that B lymphocytes rendered tolerant in vivo exhibit
a failure to activate AP-1 (27). However, these B cells manifest a
unique profile in which both IB degradation and AP-1 induction
are inhibited, yet constitutive activation and nuclear translocation of
NF-AT proteins remain intact, and low levels of RelA and c-Rel are
present in the nuclei of tolerant cells (27). Similar alterations may
be present in a model of superantigen-induced CD4+ cell
tolerance (28). Such findings raise the question of whether functional
unresponsiveness could result from inhibition of a single transcription
factor family or instead requires the coordinate dysregulation of
multiple signal transduction pathways.
To address this question we have created transgenic
(Tg)3 mice whose T cells express
IB(
N) (29), an IB mutant that is refractory to
signal-induced degradation (29, 30, 31). Such targeted inhibition impaired
the development of a normal population in the CD8+ lineage
with only a modest effect on the deployment of their CD4+
counterparts (29). This initial study did not investigate the effect of
costimulation or the competence to express effector cytokines such as
IL-4 or IFN-, which may be regulated independently from IL-2.
Although proliferation in response to mitogenic stimuli and IL-2 was
inhibited by the mutant IB transgene, expression of the
activation markers IL-2R and CD69 by T lymphoblasts was normal.
Because the cytosolic retention molecule IBß is degraded in
response to TCR stimuli when cells are costimulated through CD28 (32),
costimulatory signals could potentially lead to normal T cell
activation despite inhibition of IB degradation. In this regard,
previous results suggested that the inhibition of NF-B/Rel signaling
was not absolute, in that NF-B/Rel proteins were still present at
low levels in freshly isolated nuclei of Tg thymocytes (29). Moreover,
CD28 may signal induction of T cell effector functions through
NF-B-independent mechanisms (33). Accordingly, we have investigated
the nature of T cell responses when selective impairment of IB
degradation was targeted to the T lineage. The result was a defect in
the production of effector cytokines (IL-4, IFN-) that could not be
reversed by costimulation or IL-2 despite the spontaneous development
of CD44high and CD62Llow cells in vivo and
despite increased IL-2 production by T cell costimulation. In addition,
induction of nuclear c-Jun, JunB, and NF-ATc following TCR stimulation
was impaired. Taken together, these findings indicate that inhibiting
the degradation of one IB in vivo is sufficient to produce a state
of lymphocyte unresponsiveness even though low levels of nuclear
NF-B are induced. The resulting cytokine production defect
preferentially affects effector functions and differs from the
phenotypes produced by the absence of individual NF-B/Rel
subunits (34, 35).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IB(N)-Tg mice expressing a mutant IB targeted to the
T lineage using the lck proximal promoter and human CD2
locus control region have been described previously (27). Progeny
derived from two separate founders were backcrossed to C57BL/6 mice for
use in these studies. Dist.IFN- luciferase mice bear a transgene
encoding luciferase under the control of the distal (-98 to -72)
IFN- promoter AP-1-like element and a minimal IFN- promoter TATA
element; they were described in detail previously (36). Briefly, the
2.8-kb transgene contains a head-to-tail tetramer (5' to 3') of the
distal conserved IFN- AP-1-like element (-98 to -72 bp), the
IFN- minimal promoter (-39 to +64), and the luciferase reporter
gene from the plasmid PRL-luc (37). The mice used in this study
resulted from backcrossing onto a C57BL/6 genetic background. All mice
were housed in microisolators under specific pathogen-free conditions.
Cell preparation and culture
Cells from the indicated sources were cultured, and B
cell-depleted suspensions of spleen and lymph node cells were prepared
for mobility shift analyses, as described previously (27). Two
independent experimental methods were used to prepare and stimulate
cytokine production, as indicated in the figure legends. Similar
results were obtained with each method. In method 1, splenocytes
(2.5 x 106/ml) were stimulated without fractionation,
or CD4+ T cells (1 x 106/ml) were
stimulated using APCs (1 x 106/ml) through culture
with plate-bound Abs against CD3 (10 µg/ml of 145-2C11) in the
presence or the absence of an activating hamster mAb against mouse CD28
(10 µg/ml; clone 37.51, PharMingen, San Diego, CA) as previously
described (27, 36). CD4+ T cells (purity, 
90–95%) and
syngeneic APCs were purified (subtraction of CD8+ T cells,
NK cells, and class II-expressing cells from pooled splenocytes and
lymph node cells, or negative selection with mAbs against CD4 and CD8,
respectively) as described previously (36). In method 2,
CD4+ T cells were purified from splenocytes by positive
selection using magnetic beads derivatized with Abs against mouse CD4
and Detachabead (Dynal, Lake Success, NY). Unfractionated splenocytes
and CD4+ T cells were cultured in RP/10F in flat-bottom
96-well microtiter plates (5 x 105 cells/0.2 ml) that
had been coated with anti-CD3 (1 µg/ml in PBS). Where indicated,
an activating Ab against mouse CD28 (PharMingen) was included in the
culture (10 µg/ml).
Gel mobility shift and immunoblot analyses
Nuclear fractions were prepared from single cell suspensions by
high salt extraction in the presence of protease inhibitors (31). Gel
mobility shift assays of NF-B/Rel proteins were performed using a
double-stranded 32P-labeled oligonucleotide modified from
B enhancer sequences in the IL-2R promoter (B-pd; upper
strand, 5'-CAACGGCAGGGGAATTCCCCTCTCCTT) (31). DNA binding reaction
mixtures (20 µl) contained 4 µg of nuclear extract, 2 µg of
double-stranded poly(dI-dC), and 10 µg of BSA buffered in 20 mM HEPES
(pH 7.9), 5% glycerol, 1 mM EDTA, 1% Nonidet P-40, and 5 mM DTT.
Similar reactions were performed using a labeled AP-1 oligonucleotide
(Promega, Madison, WI). Nucleoprotein complexes were then resolved on
native 5% polyacrylamide gels and visualized by autoradiography. Small
scale nuclear extracts for immunoblot experiments were prepared from
5 x 106 cells as previously described (38, 39).
Nuclear proteins were fractionated by SDS-PAGE, transferred to
nitrocellulose membranes, and incubated with specific primary Abs
directed against c-Jun, JunB, and NF-ATc (all from Santa Cruz
Biotechnology, Santa Cruz, CA). Membranes were then washed, incubated
with secondary horseradish peroxidase-conjugated second Abs, and
developed using an enhanced chemiluminescent system according to the
manufacturer’s instructions (Amersham, Arlington Heights, IL).
Analysis of cytokine and luciferase activities
ELISAs for the detection IL-4 and IFN- were performed using
Ab pairs (PharMingen, Sorrentino, CA) according to the manufacturer’s
recommended procedures as described previously (36, 40). The lower
limits of sensitivity of the ELISAs were 10 pg/ml (IL-4) and 1 U/ml
(IFN-), using as a reference standard mouse IFN- with a sp. act.
of 107 U/mg protein (PharMingen). As the upper limits of
sensitivity were 2 ng/ml (IL-4) and 300 U/ml (IFN-), additional
quantitation was performed on serially diluted samples when indicated.
To measure transcriptional activity directed by a minimal
IFN--derived promoter, stimulated cells were harvested from
duplicate cultures, washed twice in PBS, then suspended (30 min at
25°C) in 50 µl of lysis buffer (Luciferase Assay Kit, Promega,
Madison, WI). The supernatant fluid was harvested, and duplicate
20-µl aliquots were assayed for luciferase activity after a 15-s
incubation with 100 µl of luciferase reagent (Promega) in a
luminometer (Turner TD20/20; Promega). The results are expressed as the
mean relative light units per 106 cells produced by the
replicates minus the blank value (background measurement using
luciferase reagent alone).
Enumeration of apoptotic cells
To generate samples for quantitative TUNEL assays, single-cell suspensions were cultured for 40 h in the presence of plate-bound anti-CD3 mAb (10 µg/ml) and anti-CD28 mAb (10 µg/ml) as indicated, harvested, and stained with PE-conjugated mAb against CD4 or CD8. Cells were fixed with paraformaldehyde, permeabilized with 70% ethanol at -20°C, washed in PBS supplemented with 1% BSA, then resuspended in reaction mixtures containing terminal deoxynucleotidyltransferase (Life Technologies, Gaithersburg, MD) and biotin-16-dCTP (Boehringer Mannheim, Indianapolis, IN). After incubation at 37°C for 30 min, cells were washed in PBS/1% BSA, stained with FITC-conjugated avidin, and analyzed by flow cytometry. Control reactions lacking terminal deoxynucleotidyltransferase were performed to quantify nonspecific staining.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B/Rel signaling
Normal activation of CD4+ T cells leads to increased
IL-2 production, which may be regulated independently from effector
proteins such as IL-4 and IFN-. The NF-B/Rel protein family binds
to functionally important IL-2 promoter sequences (32, 36; reviewed in
Ref. 21), and induction of this family of transcriptional activators is
essential for the production of IL-2 in response to TCR cross-linking
(29, 41, 42). However, the induction of proteins such as IL-2R/CD25
and CD69 by TCR stimulation of IB(N)-Tg T cells was inhibited
minimally (29). This selective inhibition raised the question of
whether key effector cytokine genes such as IFN- and IL-4, whose
promoters may be regulated by NF-B binding (43, 44), would be
activated normally despite inhibition of NF-B/Rel signaling. To
investigate the effect of this targeted inhibitor on the ability of
primary T cells to produce effector cytokines, we stimulated cells with
immobilized Abs against the TCR (Fig. 1
).
As shown in Fig. 1A, an unfractionated population of
splenocytes from IB(N)-Tg mice was dramatically impaired in
its generation of both IFN- and IL-4. This result was recapitulated
when purified CD4+ T cells were used instead of
unfractionated splenocytes (Fig. 1B). Thus, these responses
of wild-type cells induced in vitro by anti-CD3 cannot be
attributed to cytokine production by NK (IFN-) or NK1.1+
T (IL-4) cells (45). Moreover, the defect of Tg T cells compared with
controls is unlikely to be due only to enhanced apoptosis or decreased
proliferation (see below). These findings indicate that the population
of CD4+ T lymphocytes that developed with a defect in
signal-induced degradation of IB was unable to activate the
expression of key effector molecules.
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The above data suggested that efficient production of effector
cytokines by CD4+ T cells may depend on the NF-B/Rel
signaling pathway. However, maximum rates of IB degradation
require coordinate signaling through the TCR and CD28 (32, 46). Of
note, a significant pool of NF-B/Rel dimers is retained in the
lymphocyte cytoplasm by alternative inhibitory proteins such as
IBß and IB
, rather than by IB (32, 47). In contrast to
IB, IBß is not degraded in response to TCR signaling alone,
requiring the combination of CD28 and a TCR signal (32), while
hypophosphorylated IBß can protect NF-B from IB and
shepherd NF-B into the nucleus (48, 49). Moreover, CD28 can signal
through NF-B-independent pathways (33). Indeed, stimulatory
anti-CD28 Abs proved able to partially reverse the defect in IL-2
production by IB(N) T cells (Fig. 2A). In light of these
findings, we investigated whether ligation of CD28 would either restore
normal production of effector cytokines by IB(N) T cells or
enhance their nuclear induction of NF-B/Rel proteins. Unfractionated
splenocytes and CD4+ T cells from individual Tg or control
mice were stimulated with immobilized anti-TCR Abs, alone or in
combination with activating Abs against CD28. We found that T cells
from the Tg mice were deficient in their production of effector
cytokines even when activated by the combination of costimulatory and
TCR signaling (Fig. 2). Similar results were obtained in experiments
with splenocytes (Fig. 2B) and purified CD4+ T
cells (Fig. 2C).
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B(N)-Tg mice was only modestly higher than that among
cells from littermate controls after stimulation with anti-CD3 or a
combination of anti-CD3 and anti-CD28 (Fig. 3A). Moreover, the number of
Tg T cells recovered after 2 days was greater than half that in non-Tg
(NTg) controls, while the decrease in cytokine production was far
greater (a 10- to 20-fold difference). It was also possible that only a
small minority of cells from IB(N) mice respond to TCR
stimulation. However, 40% of Tg cells acquired blast morphology after
combined stimulation through the TCR and CD28 compared with 57% of
wild-type control cells (Fig. 3B). It is thought that the
bulk of effector cytokine production immediately after polyclonal
stimulation under these conditions reflects activity of a pre-existing
population of resting CD44high (or CD62Llow or
CD45RBlow) CD4+ cells (50). As such, these
results could have arisen if the activation defect of IB(N) Tg
T cells blocked formation of such cells in vivo. However, the observed
steady state frequencies of CD44high and
L-selectinlow CD4+ T cells in spleen (data not
shown) and lymph nodes (Fig. 3C) of young Tg mice were no
less than those in their NTg littermates. Thus, the observed decrease
in cytokine production is not simply due to a diminished population of
responder cells stemming from enhanced apoptosis. We conclude that
despite increased IL-2 production, costimulation is unable to reverse
the inhibitory effect of the IB(N) protein on the effector
cytokine production capacity of T cells.
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B/Rel signaling by IB(N) is refractory
to costimulation
In light of the failure of costimulation to reverse the functional
unresponsiveness of Tg T cells, we investigated whether these
observations could be correlated with the biochemical status of
NF-B/Rel induction. T cells were stimulated with immobilized
anti-TCR Abs, alone or in combination with activating Abs against
CD28. Nuclear extracts from these T cell preparations were analyzed by
gel mobility shift analysis to determine the relative levels of nuclear
NF-B/Rel proteins. These experiments revealed that costimulation
with anti-CD28 was insufficient to reverse the observed inhibition
of NF-B/Rel induction by the IB mutant. Importantly, the
results indicate that a low level of NF-B/Rel proteins was able to
bypass this inhibitory effect (Fig. 4),
leading to detectable levels of NF-B complexes reminiscent of the
low levels of nuclear c-Rel and RelA in nuclei of tolerant B cells
(27). These mobility shift experiments also demonstrated a shift in the
ratio of faster migrating complexes (previously shown to represent
p50/NF-B1 without c-Rel or RelA) relative to the slower mobility
NF-B complexes (previously shown to represent p50/NF-B1 with
c-Rel or RelA). As such, these observations imply that even incomplete
inhibition of the NF-B/Rel pathway can lead to a defect in effector
function.
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Costimulation only partially reversed the defect in IL-2
production by T cells from IB(N) mice, and IL-2 signaling may
influence the development of unresponsiveness or the activation of
effector cytokine production (51). Therefore, we directly investigated
whether exogenous IL-2 would reverse the defect in activation of
effector cytokine genes in IB(N) T cells. The results (Fig. 5) show that exogenous IL-2 was unable to
restore effector cytokine production by Tg T cells to the level
generated by comparably treated cells from NTg littermates. We conclude
that selective inhibition of IB degradation (27) in T cells can
induce a functional defect that is not simply a consequence of
deficient IL-2 production.
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B site
The above findings indicate that inhibition of IB
degradation attenuated the activation of certain target cytokine genes
by a mechanism refractory to exogenous IL-2. Since the IFN- gene
contains a potentially functional NF-B site (44), it was possible
that the observed decrease in IFN- production was due to a
requirement for a crucial NF-B-dependent regulatory sequence. Thus,
the observed decrease in effector cytokine production might represent
either a general inhibition of T cell activation by IB or another
direct transcriptional effect of NF-B/Rel proteins. To help
distinguish between these possibilities, we crossed IB(N) and
dist.IFN- reporter gene Tg mice (36). T cells from these latter mice
transcribe a luciferase gene that is under the control of a minimal
IFN- promoter and an AP-1-like element that lacks an NF-B site.
This imperfect TRE element binds AP-1, but not NF-B/Rel
transcription factors (52). The activity of this minimal reporter gene
mimics much of the specific regulation of the IFN- gene despite the
absence of an NF-B binding site (36). Activation of the luciferase
gene by the dist.IFN- promoter was abrogated in
IB(N)-expressing T cells compared with that in cells from
littermates expressing only wild-type IB (Fig. 6A).
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promoter binds AP-1, but not
NF-B transcription factors, these data raise the question of whether
the defect in induction of this promoter element may result from
diminished induction of AP-1 transcription factors. To investigate this
possibility, gel mobility shift assays of AP-1 binding activity were
performed using nuclear extracts derived from unstimulated and
activated cells (Fig. 6
B). These experiments indicated that
AP-1 activity in IB(N) T cells was diminished compared with
that in wild-type controls. To extend this observation, additional
nuclear extracts prepared from resting and activated primary wild-type
and Tg T cells were analyzed for levels of c-Jun, JunB, and NF-ATc by
Western blotting. We found that induction of nuclear c-Jun, JunB, and
NF-ATc protein was inhibited in T cells with impaired NF-B signaling
(Fig. 6C). Although CD28 costimulation increased levels of
nuclear JunB and NF-ATc in wild-type T cells, it did not restore levels
of these transcription factors in Tg T cell nuclei to normal. Thus, the
defect in effector cytokine production induced initially by inhibition
of NF-B signaling can be correlated with diminished induction of
critical transcription factors that regulate IFN- gene
transcription. These findings suggest that the observed defect of IL-4
and IFN- production by these lymphocytes is not simply a consequence
of deficient IL-2 production and includes a propagation of inhibitory
effects on T cell activation. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B
degradation, through expression of a trans-dominant
inhibitor, IB(N), is sufficient to generate an
unresponsiveness that is manifested as impaired effector cytokine
production after primary stimulation of normal CD4+ T
cells. Importantly, this unresponsive state was achieved despite the
induction of low levels of nuclear NF-B and was not reversed by
either costimulation through CD28 or provision of exogenous IL-2. An
impaired IFN- response was also manifested at the transcriptional
level. Inhibition of NF-B/Rel signaling blocked induction of a TCR
response element from the IFN- promoter despite the absence of an
NF-B site and also decreased the nuclear induction of AP-1 binding
activity as well as that of c-Jun, JunB, and NF-ATc. In contrast, the
low level of NF-B/Rel proteins that escaped the inhibitory effect of
IB(N) appeared sufficient to permit significant IL-2
production by T cells upon costimulation. This latter observation
suggests that activation of signaling pathways other than NF-B/Rel
(33) makes quantitatively important contributions to CD28 costimulation
of IL-2 production by primary T cells.
These experiments focussed on primary ex vivo stimulation of cells to
reflect analyses of lymphocytes that have been exposed in vivo to
conditions that induce unresponsiveness (12, 14, 27, 28). It is thought
that the bulk of the IFN- and IL-4 responses under these conditions
derives from pre-existing CD44high CD4+ T cells
(50; reviewed in 54). The observation that similar numbers of
CD44high, CD62Llow, and CD45RBlow
(data not shown) CD4+ cells were present in our wild-type
and IB(N) samples suggests that the acquisition of these
phenotypic characteristics does not depend on NF-B/Rel signaling.
Accordingly, our data cannot distinguish between an impairment of
signaling during stimulation in vivo and the interesting possibility
that the signaling defect prevented development or survival of a
population of T cells in vivo that has the capacity to produce IFN-
within 24–48 h after in vitro stimulation. Of note, c-Jun is induced
equally in naive and effector T cells following antigenic stimulation
(55), indicating that CD4+ T cells of each functional type
induce this transcription factor. In contrast, c-Jun induction in cells
from IB(N) mice was impaired, suggesting that some inhibition
of signaling during the in vitro stimulation was exerted. It is
unlikely that preferential apoptosis of CD44high cells
could account for the observed defect in cytokine production (Fig. 3).
It also was conceivable that the defect in effector cytokine production
reflects some block in proximal activation, an event that could be
bypassed using PMA/ionomycin. However, we have found that although the
inhibition of NF-B/Rel signaling had little effect on the prevalence
of early apoptotic cells compared with controls, IL-4 and IFN-
production by Tg cells remained profoundly inhibited (unpublished
observations). In summary, our data indicate that an initial
impairment of NF-B signaling leads to a defect in effector cytokine
production through inhibition of signaling at a differentiation step in
vivo, during reactivation in vitro, or both.
The ability of a targeted perturbation in one signaling pathway to create such a spectrum of defects is a striking outcome. There has been little previous evidence that partial inhibition of one transcriptional pathway can cause lymphocyte activation defects refractory to costimulation and IL-2. Other correlates of T lymphocyte unresponsiveness induced in anergy models in tissue culture systems have included diminished or aberrant activation of signal-transducing kinases such as ZAP-70, the Ras/Raf/mitogen-activated protein kinase kinase (MAPKK) cascade, c-Jun N-terminal kinase (JNK), and extracellular signal-related kinase (ERK) (25, 26, 56, 57). While informative mutations in humans indicate that ZAP-70 kinase activity is required for CD4+ T cell activation, the precise roles of other elements in these pathways remain uncertain, because defects in the Ras/Raf/MAPKK pathway block T cell development (58, 59, 60, 61). These impairments in signal transduction lead to defective activation of the AP-1 family of basic leucine zipper transcription factors in anergized CD4+ T cell clones (24, 25, 26). However, it is unclear whether impaired activation of AP-1-like proteins is sufficient to render primary CD4+ T cells unresponsive and unable to perform effector functions. Gene-targeting experiments have created mice whose T cells lack the c-Fos and c-Jun transcription factors, but such mice exhibit apparently normal T cell activation, as do mice whose T cells express a dominant negative MAP/ERK kinase kinase (MEKK) (52, 62, 63). Thus, it remains to be determined whether a primary defect in AP-1 activation will inhibit the effector functions of mature T cells.
Although previous reports have focussed on the AP-1 family as a
critical step in the transcriptional regulation of clonal anergy
induction in vitro, IB degradation and NF-B/Rel signaling are
abnormal in lymphocytes rendered tolerant in vivo (27, 28). Moreover,
the Ras/Raf/MEKK pathways may regulate IB degradation through
MEKK1, and NF-B induction may, in turn, influence transcriptional
activation through both NF-B and AP-1 sites (64, 65, 66). Alternatively,
IL-2 responsiveness may be crucial for the acquisition of T cell
competence to produce effector cytokines (51). In this regard it is
interesting to note that although T cells from IB(N) mice can
be induced to express normal levels of cell surface expression
IL-2R, -ß, and -c subunits, the IL-2-dependent induction of
STAT proteins is significantly
decreased.4 Taken together,
these findings suggest that degradation of the different IBs may be
a key regulatory target in determining the characteristics of a T cell
response to TCR engagement. In principle, independent regulation of the
cytosolic retention of different trans-activating subunits
of NF-B may lead to different patterns of unresponsiveness in T
cells. Specifically, when only c-Rel was eliminated from the
NF-B/Rel signaling pathway, the primary defect was the absence of
IL-2 production, and almost all other defects were normalized if
exogenous IL-2 was present (34, 42). When only the RelA component of
signaling was impaired, IL-2 production was normal, and cells exhibited
no increase in TCR-induced apoptosis, but T cells exhibited a
proliferative defect refractory to the endogenously produced IL-2 (35).
Finally, T cells with impaired IB degradation affecting both the
RelA and c-Rel trans-activators exhibited defective
production of effector cytokines (IL-4, IFN-) and enhanced
TCR-induced apoptosis despite costimulation or the provision of
exogenous IL-2. Taken together, these observations can be reconciled if
differential regulation of c-Rel and RelA lead to different
patterns of CD4+ T cell unresponsiveness. However, it
is not clear what was the prevalence of CD44high
CD4+ cells in c-Rel or RelA-deficient samples, and the
patterns of cytokine production could not be compared with those of
IB(N) cells in the same experiments. Although inhibition of
IB degradation leading to coordinate, but incomplete, cytosolic
retention of RelA and c-Rel has been demonstrated in vivo (27), it also
is not clear whether selective defects in the nuclear induction of
specific members of the NF-B/Rel family occur. Notwithstanding these
issues, the present findings demonstrate that the inhibition of
IB degradation, resulting in an incomplete blockade of NF-B
translocation to the nucleus, could suffice to inhibit an effector
function of CD4+ T cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mark Boothby, Department of Microbiology and Immunology, AA-4214 Medical Center North, Vanderbilt University School of Medicine, Nashville, TN 37232-2363. E-mail address:
3 Abbreviations used in this paper: Tg, transgenic; NTg, nontransgenic; IB, inhibitor of NF-B; MEKK, MAP/ERK kinase kinase.
4 A. L. Mora, J. Youn, A. D. Keegan, and M. Boothby. NF-B participation in the lymphokine-dependent proliferation of T lymphoid cells. Submitted for publication.
Received for publication September 28, 1998. Accepted for publication February 26, 1999.
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 C. M. Tato, A. Villarino, J. H. Caamano, M. Boothby, and C. A. Hunter Inhibition of NF-{kappa}B Activity in T and NK Cells Results in Defective Effector Cell Expansion and Production of IFN-{gamma} Required for Resistance to Toxoplasma gondii J. Immunol., March 15, 2003; 170(6): 3139 - 3146. [Abstract] [Full Text] [PDF]
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M. Soutto, F. Zhang, B. Enerson, Y. Tong, M. Boothby, and T. M. Aune A Minimal IFN-{gamma} Promoter Confers Th1 Selective Expression J. Immunol., October 15, 2002; 169(8): 4205 - 4212. [Abstract] [Full Text] [PDF]
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 J. Caamano and C. A. Hunter NF-{kappa}B Family of Transcription Factors: Central Regulators of Innate and Adaptive Immune Functions Clin. Microbiol. Rev., July 1, 2002; 15(3): 414 - 429. [Abstract] [Full Text] [PDF]
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 C. M. Tato and C. A. Hunter Host-Pathogen Interactions: Subversion and Utilization of the NF-{kappa}B Pathway during Infection Infect. Immun., July 1, 2002; 70(7): 3311 - 3317. [Full Text] [PDF]
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J. R. Maxwell, C. Ruby, N. I. Kerkvliet, and A. T. Vella Contrasting the Roles of Costimulation and the Natural Adjuvant Lipopolysaccharide During the Induction of T Cell Immunity J. Immunol., May 1, 2002; 168(9): 4372 - 4381. [Abstract] [Full Text] [PDF]
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H. Ren, A. Schmalstieg, N. S. C. van Oers, and R. B. Gaynor I-{kappa}B Kinases {alpha} and {beta} Have Distinct Roles in Regulating Murine T Cell Function J. Immunol., April 15, 2002; 168(8): 3721 - 3731. [Abstract] [Full Text] [PDF]
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X.-Y. Zhou, Y. Yashiro-Ohtani, M. Nakahira, W. R. Park, R. Abe, T. Hamaoka, M. Naramura, H. Gu, and H. Fujiwara Molecular Mechanisms Underlying Differential Contribution of CD28 Versus Non-CD28 Costimulatory Molecules to IL-2 Promoter Activation J. Immunol., April 15, 2002; 168(8): 3847 - 3854. [Abstract] [Full Text] [PDF]
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K.-J. Malmberg, V. Arulampalam, F. Ichihara, M. Petersson, K. Seki, T. Andersson, R. Lenkei, G. Masucci, S. Pettersson, and R. Kiessling Inhibition of Activated/Memory (CD45RO+) T Cells by Oxidative Stress Associated with Block of NF-{kappa}B Activation J. Immunol., September 1, 2001; 167(5): 2595 - 2601. [Abstract] [Full Text] [PDF]
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A. L. Mora, J. Youn, A. D. Keegan, and M. Boothby NF-{{kappa}}B/Rel Participation in the Lymphokine-Dependent Proliferation of T Lymphoid Cells J. Immunol., February 15, 2001; 166(4): 2218 - 2227. [Abstract] [Full Text] [PDF]
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J. Caamano, C. Tato, G. Cai, E. N. Villegas, K. Speirs, L. Craig, J. Alexander, and C. A. Hunter Identification of a Role for NF-{kappa}B2 in the Regulation of Apoptosis and in Maintenance of T Cell-Mediated Immunity to Toxoplasma gondii J. Immunol., November 15, 2000; 165(10): 5720 - 5728. [Abstract] [Full Text] [PDF]
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F. Zhang, M. Rincon, R. A. Flavell, and T. M. Aune Defective Th Function Induced by a Dominant-Negative cAMP Response Element Binding Protein Mutation Is Reversed by Bcl-2 J. Immunol., August 15, 2000; 165(4): 1762 - 1770. [Abstract] [Full Text] [PDF]
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