| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Division of Molecular Immunology, National Institute for Medical Research, The Ridgeway, London, United Kingdom
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
There has been much interest in the signals that distinguish interactions between MHC class I peptide complexes with TCR/CD8 molecules and MHC class II peptide complexes with TCR/CD4 molecules and lead to commitment to the CD8 and CD4 lineages, respectively. A quantitative signaling model has been suggested (1, 2) in which different strengths of signal are delivered by these respective encounters, based in part on the observation that the coreceptors CD4 and CD8 interact with different efficiencies with the intracellular tyrosine kinase p56lck (Lck) (3). Thus, coengagement of TCR plus CD4 with an MHC class II/peptide complex has been suggested to deliver a stronger signal than coengagement of TCR plus CD8 with an MHC class I/peptide complex. An alternative view is that the nature of the signal that specifies differentiation to the CD8 lineage is qualitatively different from that which specifies differentiation to the CD4 lineage. In support of this are observations that receptor engagements that act as antagonists for peripheral T cells, such as TCRs interacting with certain MHC/peptide combinations or CD3 ligation by F(ab')2 mAb fragments, are particularly good at inducing differentiation to the CD8 lineage for both MHC class I- and class II-restricted TCRs (4, 5, 6, 7). In contrast, signals that behave as weak agonists for peripheral T cells are generally efficient at inducing differentiation to the CD4 lineage, again regardless of the primary MHC specificity of the TCR (8, 9, 10, 11, 12, 13). Whether these signals specifying lineage commitment differ in degree or substance, at some point there must be a divergence in the signal cascade that ultimately results in the establishment of distinct differentiation outcomes. To date, the point at which the signaling pathways diverge and result in positive selection to the CD4 or CD8 lineage has not been described.
To try and understand the biochemical basis of thymocyte differentiation, we have established an experimental system in which we can mimic CD4 or CD8 lineage commitment signals. We use either bispecific F(ab')2 Abs (BsAbs)5, which coligate CD3 with CD4, to induce differentiation to the CD4 subset (13) or monospecific CD3 F(ab')2 to induce CD8 differentiation (6, 14) in neonatal thymus organ culture (NTOC). Here we examine the contribution of the mitogen-activated protein (MAP) kinase (MAPK) signaling pathway on the induction of CD4 and CD8 thymocyte differentiation. Using the specific MEK inhibitors, PD98059, (15) and UO126 (16), we show that conditions that fully inhibit differentiation of mature CD4 SP cells, whether induced by endogenous ligands or by BsAbs, do not affect differentiation of functional CD8 cells. This is despite the fact that F(ab')2 reagents that preferentially induce commitment to the CD8 lineage do activate ERK kinases. Furthermore, inhibition of ERK activity modifies a CD4 differentiation signal to one appropriate for CD8 lineage commitment. Although the presence of the MEK inhibitor does not block commitment to the CD8 lineage, continued presence of the drug blocks TCR up-regulation, suggesting that MEK activity is important for the final stages of maturation for both lineages. Therefore, by identifying discrete requirements for MEK activity in lineage commitment vs maturation our results may resolve some of the conflicting reports regarding the role of MAPK induction in the differentiation of thymocytes. From our data we would suggest that differential activation of the ERK1/2 kinases is an important branch point in the biochemical signals that are required for lineage specification in the thymus.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The ß2m-deficient (ß2mneg) (17) and I-Aß-deficient (MHC class IIneg) (18) mice have been described and were intercrossed to obtain ß2mneg x class IIneg (MHCneg) mice. TCR transgenic mice backcrossed onto a RAG-1-deficient (RAG-1neg) background (19) were as follows: MHC class I-restricted F5 TCR (20), specific for influenza nucleoprotein restricted by H-2Db and MHC class II-restricted A18 TCR (21), specific for C5 in the context of I-Ek. Male studs, homozygous for the TCR transgenes on the appropriate backgrounds (RAG-1negß2mneg for F5 and RAG-1neg for A18), were bred with RAG-1negß2mneg or RAG-1neg females, respectively, yielding neonates from which thymus lobes were obtained.
Antibodies
Bi- and monospecific F(ab')2 Abs dimerized
through Fos or Jun leucine zippers were prepared as described
previously (22). V regions with specificity for CD3
,
CD4, or CD8
were derived from 145.2C11, GK1.5, and YTS169. mAbs were
purified and conjugated to FITC or biotin in our own laboratory unless
stated otherwise. Rabbit anti-ERK antiserum 122 was a gift from
Prof. Chris Marshall (Institute of Cancer Research, London, U.K.).
Thymus organ culture
Neonatal (day of birth) thymus lobes were cultured for 7 days as
previously described (6, 13) with the indicated Abs or
medium only. MEK inhibitors PD98059 (Calbiochem, La Jolla, CA) or UO126
(gift from Dr. J. M. Trzaskos, DuPont Merck, Wilmington, DE)
were added to lobes in culture medium 1–3 h before Ab addition. Unless
otherwise stated in the figure legends, inhibitor was replenished
either daily or every other day for the first 4 days of culture, after
which lobes were transferred to fresh filters in culture medium without
Ab or inhibitor for the final 2–3 days of culture to allow
re-expression of down-modulated molecules. Single-cell suspensions
prepared from lobes after culture were stained with FITC-CD8
(YTS169.4), PE-CD4 (Boehringer Mannheim, Indianapolis, IN), biotin-HSA
(YBM5.10), or biotin-Vß11 (KT11.5, for F5 TCR),
-Vß8.3 (F23.1, for A18 TCR), or -pan-TCR-ß (H57.597, for
ß2mneg)-specific Abs,
followed by streptavidin-Red 670 (Life Technologies, Grand Island, NY),
and 20,000 live events (gated on forward and side scatter profiles)
were analyzed on a FACScan (Becton Dickinson, Mountain View,
CA).
Proliferation assay
Dendritic cells (DCs) were expanded by culture of C57BL/10 bone marrow in medium supplemented with GM-CSF for 7 days (23). Single-cell suspensions were obtained from equivalent numbers of thymus lobes cultured with 5 µg/ml CD3Fos-F(ab')2, 2 µg/ml CD3/CD4 BsAb, or medium with and without 50 µM MEK inhibitor PD98059 as described above. Thymocytes were stimulated in triplicate wells in 96-well round-bottom tissue culture plates with 2.5 x 104 H-2b DCs/well and a titration of NP68 peptide for 3 days at 37°C in medium. Cultures were pulsed with 37 kBq [3H]thymidine/well for the last 18 h of culture, harvested, and counted in a beta counter with scintillation. Data are presented as stimulation index obtained by dividing the mean counts per minute obtained by culture with Ag by the mean counts per minute from medium alone wells for each group. CD69 expression was analyzed on thymocyte subpopulations harvested from wells after 24-h culture and stained with anti-Vß11-FITC (KT11.5), CD8-PE (PharMingen, San Diego, CA), CD4-APC (PharMingen), and CD69-bio (PharMingen) plus strepavidin Red 613 (Life Technologies, BRL) for analysis on a FACS Calibur (Becton Dickinson).
ERK kinase assay
Thymocytes from MHCneg mice (95% DP, 5%
DN cells) were kept at 4°C on ice. Cells were preincubated with 100
µM MEK inhibitor PD98059 for 30 min at 37°C in medium before being
stimulated for 7 min with Abs (5 µg/ml). Cells (1 x
107 thymocytes/sample) were washed in PBS
containing 400 µM Na3VO4,
5 mM EDTA, and 10 mM NaF; pelleted at 13,000 rpm; and lysed for 1
h at 4°C in 1% Triton X-100, 150 mM NaCl, 50 mM Tris (pH 7.5), 5 mM
EDTA, 10 mM NaF, 10 mM disodiumpyrophosphate, 200 mM PMSF, 5 mM
leupeptin, 1.5 mM pepstatin A, and 1 mM
Na3VO4. Postnuclear
supernatants were precipitated at 4°C with 4 µl of rabbit
anti-ERK antisera 122 and 20 µl of protein A-Sepharose.
Precipitates were washed twice in kinase buffer (30 mM Tris (pH 8), 20
mM MgCl2, and 2 mM MnCl2) and
resuspended in 30 µl of kinase buffer plus 10 µM cold ATP, 0.4
mg/ml myelin basic protein, and 1.2 MBq
[
-32P]ATP) at 30°C for 30 min. The
reaction was stopped by addition of 2x reducing sample buffer and was
resolved on a 12.5% SDS-PAGE gel. Gels were dried and exposed to
x-ray film.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The role of the MAP kinase pathway in thymocyte differentiation has been investigated by the expression of both dominant negative ras (24) and mek1 (25, 26) constructs in transgenic mice and more recently by using a gain-of-function mutation of erk2, Mek1, and the specific pharmacological inhibitor of MEK, PD98059 (27, 28) with significantly different conclusions. Although these reports agree that activation of the ERK pathway appears unnecessary for negative selection to occur, the dominant negative experiment (25) and one study using the inhibitor PD98059 (28) suggested that ERK activation was required for differentiation from DP to mature SP cells, while another study (27) indicated that inhibition of ERK activity differentially influenced the ratio of CD4:CD8 mature T cells that were produced rather than T cell maturation per se. Interestingly, it was also shown that introduction of constitutively active forms of MEK (28) or hypersensitive mutants of ERK (27) specifically promoted CD4 SP cell differentiation rather than differentiation of both lineages.
We chose to examine the influence of the MAP kinase pathway in an experimental system we established previously, using in vitro culture of neonatal thymus lobes. Using a variety of transgenic and knockout mouse strain combinations we start with a situation where thymocytes have expanded normally as far as the DP subset and are then either completely arrested in their differentiation or during the culture period can differentiate into only a single lineage, unless artificially induced with mono- or bispecific F(ab')2 reagents to differentiate to a particular lineage (6, 13, 14). This protocol has enabled us to directly examine the role of the MAP kinase pathway in the commitment decision for differentiation from DP to CD4 or CD8 SP lineages without unduly influencing the transition from DN to DP thymocytes, a stage of differentiation known to require activation of the MAPK pathway (29) and which may be particularly relevant when monitoring differentiation in fetal thymus lobes.
We examined the influence of PD98059 on the differentiation of neonatal thymus lobes from mouse strains that could efficiently differentiate thymocytes into only CD4+, CD8+, or neither lineage. Thymus lobes that differentiate to the CD8 lineage only were taken from F5/RAG-1neg mice, which have an MHC class I-restricted TCR (20); thymus lobes that differentiate to the CD4 lineage only were obtained from A18A/RAG-1neg mice, which express an MHC class II-restricted TCR (21), or from ß2mneg mice (17), which lack class I molecules on which to select CD8 SP cells; and finally, thymus lobes from F5/RAG-1neg/ß2mneg mice were used, which lack both mature T cell subsets.
Inhibition of MEK has several clear effects on thymus differentiation,
as shown by the data represented both as FACS profiles from individual
lobes in Fig. 1
and as cell numbers and
percentages from pools of lobes in Table I. First, production of mature
(HSAlow) CD8+ thymocytes in
response to endogenous ligands, e.g., in
F5/RAG-1neg lobes is not inhibited and can be
enhanced (from 12 to 27%) by inhibiting MEK during differentiation
(Fig. 1A). Moreover, significant enhancement of CD8 SP
maturation in the presence of PD98059 was observed even in the absence
of class I MHC ligands
(F5/RAG-1neg/ß2mneg
lobes, see Table I). The presence of bovine ß2m
in the culture medium probably promotes stabilization of a few MHC
class I molecules as some mature CD8+ cells can
be recovered from these cultures; the numbers are significantly reduced
when cultured with medium supplemented with
ß2mneg mouse serum M. A.
B and R. Z., (M.A.B. and R.Z., unpublished observation). Inclusion of
MEK inhibitor was able to significantly increase the percentage
of mature CD8+ cells that differentiates from
2.5% to 11% (Table I,
F5/RAG-1neg/ß2mneg).
|
|
B and Table I),
and for cells expressing polyclonal TCRs from
ß2mneg mice (Fig. 1C). Indeed, inhibition of MEK is even able to block CD4 SP
thymocyte differentiation induced by artificial stimuli such as CD3/CD4
BsAb (Fig. 1A, F5/RAG-1neg mice, and
Table I,
F5/RAG-1neg/ß2mneg
mice). Moreover, in addition to inhibiting differentiation of
CD4+ cells, the effect of MEK inhibition on lobes
cultured with CD3/CD4 BsAb is to change a stimulus that normally
promotes efficient CD4+ differentiation to one
that induces differentiation of CD8+ cells (Fig. 1, A and B, and Table I). Finally, induction of
CD8+ cell differentiation by culture of lobes
with CD3Fos-F(ab')2, which we have shown to be an
efficient inducer of CD8 maturation (6), is frequently
enhanced in the presence of PD98059 (Table I), confirming that
inhibition of MEK does not inhibit but, rather, promotes
differentiation to the CD8+ lineage.
We confirmed that the effect on thymocyte maturation was a direct
consequence of inhibiting MEK kinase activity by using a second
recently described, noncompetitive inhibitor of MEK, UO126
(16), in cultures of thymus lobes from
F5/RAG-1neg/ß2mneg
mice. As illustrated in Fig. 2, UO126
inhibition of MEK has the same consequence as inhibition by PD98059,
even though the former blocks MEK kinase activity on downstream
targets, while the latter blocks MEK activation by Raf. UO126 shows
more pronounced inhibition of
CD4+ cells than PD98059, consistent with its
ability to block constitutively active MEK, and yet still enhances
differentiation of CD8+ cells.
|
and 2 and Table I).
A direct comparison on TCR up-regulation of
F5/RAG-1neg/ß2mneg
lobes cultured in PD98059 continuously or for the first 3 days only is
shown in Fig. 3. Whereas removal of the
inhibitor has little influence on the degree to which
CD4+ differentiation is inhibited or
CD8+ differentiation is enhanced, there was a
clear inhibition of TCR up-regulation on mature
CD8+ cells if the inhibitor was present
throughout culture. This observation suggests that whereas MEK kinase
activity may not be required for CD8 lineage commitment, it is required
for some of the final maturation stages that result in the
characteristic phenotype of mature CD8 SP cells. These data may also
explain the failure to observe the generation of CD8 SP thymocytes in
the presence of dominant negative ras (24) and
mek1 (25, 26) constructs in transgenic mice or
in the presence of PD98059 in studies where TCR up-regulation is used
to define maturity (28).
|
50
µM; indeed, cell yields were frequently increased in cultures that
had been incubated with PD98059 (Table I). We considered whether the
increase in CD8 SP cells arose as a result of PD98059 protecting the DP
cells from apoptosis during culture, particularly in the presence of
the CD3/CD4 BsAb, which we have previously found to cause apoptosis of
some DP thymocytes (13). However, when lobes are cultured
with another compound that inhibits apoptosis in DP cells,
N-acetyl cysteine (30), we found no change in
the ratio of CD4:CD8 SP cells that arise (data not shown). In contrast,
inhibition of MEK dramatically skews the CD4:CD8 ratio in favor of CD8
SP cells, indicating that inhibition of MEK specifically influences
lineage commitment in addition to any influence it may have on cell
survival. Mature CD8 SP cells that differentiate in the presence of MEK inhibitor are functional
Given that we could show that continuous culture in MEK inhibitor prevented full T cell maturation and resulted in CD8+ T cells that had low levels of TCR, it was important to ascertain that the cells that differentiated in cultures from which inhibitor had been removed for the final few days were functional.
Cells were harvested from NTOCs from either
F5/RAG-1neg mice, which differentiated in medium
with and without PD98059, or from
F5/RAG-1neg/ß2mneg
mice, which were also induced to differentiate with CD3/CD4 and
CD3Fos-F(ab')2 reagents in the presence or the
absence of PD98059, and were stimulated with APCs plus antigenic
peptide, NP68. Proliferation was measured on day 3 of culture, and the
specific stimulation index obtained in the presence of peptide compared
with that in medium controls is shown in Fig. 4A. In all cases CD8 SP cells
that differentiated in the presence of the MEK inhibitor proliferated
specifically to peptide Ag. Examination of CD69 up-regulation after
culture for 24 h confirmed that 
20–30% of the
CD8+ cells were activated in response to peptide
in all cases (Fig. 4B), whereas there was no activation of
CD4+ cells, as expected (data not shown).
Therefore, the magnitude of the responses reflected the efficiency with
which CD8 cells differentiated in the presence of the inducing agents
and the inhibitor. In agreement with the increased percentage of CD8 SP
observed by FACS analysis (Fig. 4B), the greatest
proliferation was observed from thymus lobes that had been stimulated
to differentiate to CD8 SP by incubation with
CD3Fos-F(ab')2 plus PD98059. However, CD8 SP
cells that arose after culture with either medium plus inhibitor or
CD3/CD4 plus inhibitor were also responsive to peptide. These data
confirm that CD8+ cells that mature in the
presence of MEK inhibitors are capable of responding to Ag.
|
CD3 ligation in the absence of costimulation has been shown to act as an anergic signal for T cell clones (31). Furthermore, the biochemical signals resulting from ligation of CD3 by F(ab')2 Abs on T cell clones (32) or mature thymocytes (6) have been shown to resemble those induced by antagonist peptides. It has been reported that T cell clones exposed to antagonistic peptides fail to activate ERK kinases, in contrast to clones exposed to antigenic peptides (33, 34, 35). Given that inhibition of MEK activity did not interfere with induction of CD8 differentiation by CD3Fos-F(ab')2, we investigated whether ligation of receptors on DP thymocytes with CD3Fos-F(ab')2 or CD3/CD4 BsAb would induce activation of the ERK kinases.
Thymocytes from MHCneg mice were used in the
assay for ERK activity to exclude any contribution from mature SP
thymocytes. Thymocyte suspensions were pretreated for 30 min in medium
alone or in medium containing MEK inhibitor and then stimulated with
Abs at 37°C for 7 min. As shown in Fig. 5, activation
of ERK kinase activity, as measured by phosphorylation of MBP, is
stimulated by incubation of thymocytes with intact 2C11 Ab,
CD3Fos-F(ab')2 or CD3/CD4 BsAb, and in all cases
this activity is reduced to background levels by preincubation of cells
with the inhibitor PD98059. There was some variability in the extent to
which the different stimuli induced ERK kinase activity, and in the
experiment shown in Fig. 5, CD3Fos-F(ab')2 gave
stronger activation of ERK than CD3/CD4 BsAb. However, over five
independent experiments, in which the extent of ERK activation after
stimulation varied between 2- to 5-fold above the background level,
there was no clear distinction in the ability of one stimulus to induce
higher levels of ERK kinase activity than the other. These data agree
with a recent report that showed that, despite its ability to
antagonize T cell responses, CD3Fos-F(ab')2
induced similar levels of ERK activation to that induced by CD3/CD4
BsAb in T cell clones at early time points (36).
Importantly, the data clearly indicate that at these concentrations of
MEK inhibitor the kinase activity induced by these stimuli is reduced
to background levels. Therefore, it appears that despite the
observation that ERK kinase activity is induced by stimuli that promote
both CD4 and CD8 maturation, this activity is critical for
differentiation of CD4, but not CD8, SP thymocytes.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
These data are in agreement with the studies of Sharp et al. (27), who showed with a gain-of-function ERK transgene that activation of ERK promoted differentiation of CD4 SP cells. In addition, they showed that culture of day 17 thymus lobes with the same MEK inhibitor used in this study, PD98059, produced a reversal in the CD4:CD8 SP ratio resulting from a reduction in the number of CD4 cells. The latter suggested that MEK activity was important for positive selection of CD4, but not CD8, cells. In the current study we use neonatal rather than fetal thymus lobes and can confirm that when starting with a thymus containing differentiated DP cells, two independently acting MEK inhibitors, PD98059 and UO126, specifically block selection of DP thymocytes to the CD4 lineage. CD4 differentiation was significantly arrested by the inhibitor whether the selecting ligand was endogenous MHC:peptide complex or whether we used CD3/CD4 BsAbs to induce maturation.
In contrast to CD4 differentiation, CD8 cell maturation was generally enhanced in the presence of the MEK inhibitor both to endogenous ligand and to stimulation with either CD3Fos-F(ab')2 or with CD3/CD4 BsAb. Furthermore, the CD8 SP cells that arose not only had a mature phenotype (HSAlow TCRhigh), but were fully responsive to Ag. The differential sensitivity of the CD4 vs the CD8 lineage to the effects of the inhibitors suggest that the primary influence is on the thymocytes themselves, particularly when differentiation is induced by F(ab')2 reagents that only bind thymocytes. However, we cannot rule out the possibility that the MEK inhibitors influence the expression of genes in thymic stromal cells, such as Notch or its ligands (37), which may have additional influence on differentiation of CD8 cells.
It is unlikely that the levels of MEK inhibitor we added to the thymus
lobes were sufficient to completely block MEK activity during the first
few days of culture, and therefore, it is possible that low levels of
ERK activation are also required for development of mature CD8 SP
cells. Involvement of the Ras 
MEK ERK signal transduction
pathway in thymocyte-positive selection has been suggested from the
experiments of Alberola-Ila et al. (25, 26), who showed
that dominant negative forms of Ras or MEK, both individually and
particularly in combination, could completely inhibit positive
selection of both SP lineages. However, we also showed that activation
of the ERK pathway is involved in processes other than the lineage
commitment signal. For example, we found that although daily addition
of the MEK inhibitor did not impede production of
HSAlow CD8 SP cells, these cells had not fully
up-regulated TCR levels to those normally found in mature T cells. In
contrast, replenishing the MEK inhibitor daily while the inducing Abs
were present during the first few days of culture, but removing both Ab
and inhibitor for the last 3 days of culture, allowed differentiation
of TCRhigh CD8 cells and yet still specifically
blocked CD4 SP differentiation. These data suggest that high levels of
ERK activity may not be required for the CD8 lineage commitment
decision, but may be essential for other aspects of maturation such as
up-regulation of the TCR, and the continuous presence of a DN form of
the enzyme or the pharmacological inhibitor (28) would not
permit these later maturation stages to occur.
From our earlier studies we observed that the signals that favored CD4 SP cell differentiation, namely limited coligation of CD3 with the coreceptors, behave as weak agonists for peripheral T cells, whereas the signals that favor CD8 commitment behave as T cell antagonists (6). If this is generally true, it is perhaps not surprising that activation of the ERK kinase pathway is required for CD4 differentiation but not for CD8 differentiation, as a number of groups have shown that stimulation of mature T cells with antagonist peptides does not lead to activation of Ras/ERK kinases, unlike activation with agonist ligands (33, 34, 35). In view of this, we were surprised to observe that treatment of thymocytes with CD3Fos-F(ab')2 was able to efficiently stimulate ERK kinase activity, as CD3Fos-F(ab')2 behaves as a powerful antagonist for peripheral T cells and T cell clones (6, 32). However, a recent report (36) has shown that although stimulation of a T cell clone with either CD3Fos-F(ab')2 or CD3/CD4 BsAb resulted in similar levels of ERK-1 and ERK-2 activity at early time points; the ERK activity after CD3Fos-F(ab')2 treatment decayed more rapidly, similar to what we previously showed for ZAP-70 activation in thymocytes in response to these stimuli (6).
We have proposed a model of lineage commitment that suggests that the
discriminatory signals that direct differentiation to either the CD4 or
CD8 subsets are a function of the extent to which Src family kinases,
particularly Lck, are activated at the same time as the TCR is engaging
ligand in the thymus (6, 14). Thus, engagements of the TCR
that stimulate significant activation of Lck are most appropriate for
directing cells to the CD4 SP lineage, whereas limited activation of
Lck results in commitment to CD8. The data presented here would suggest
that one consequence of stimulating Lck and a requirement for
commitment to the CD4 lineage would be a progression of signals through
the Ras MEK ERK pathway. In contrast, although artificial
stimuli that result in CD8 differentiation may also activate the ERK
kinases, this activation and presumably the immediate downstream
consequences on gene transcription are not required for the CD8
commitment decision.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 U.B. and M.A.B. contributed equally to this work.
3 Current address: Institut für Virologie and Immunobiologie, Versbacher Strasse 7, D-97078 Wurzburg, Germany.
4 Address correspondence and reprint requests to Dr. Rose Zamoyska, Division of Molecular Immunology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, U.K. E-mail address:
5 Abbreviations used in this paper: BsAbs, bispecific F(ab')2 Ab; NTOC, neonatal thymus organ culture; MAP, mitogen-activated protein; ERK, BsAbs, bispecific F (ab')2 Ab extracellular signal-related kinase; HSA, heat stable Ag; SP, single positive; MAPK, MAP kinase; MEK, MAPK/ERK kinase; RAG, recombinase-activating gene; DC, dendritic cells; DN, double negative; DP, double positive.
Received for publication January 19, 1999. Accepted for publication May 5, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
ñiga-Pflücker, J. B. Bolen, A. M. Kruisbeek. 1989. Engagement of CD4 and CD8 expressed on immature thymocytes induces activation of intracellular tyrosine phosphorylation pathways. J. Exp. Med. 170:1671.This article has been cited by other articles:
|
|
 |
|
  N. J. Rowbotham, A. L. Hager-Theodorides, M. Cebecauer, D. K. Shah, E. Drakopoulou, J. Dyson, S. V. Outram, and T. Crompton Activation of the Hedgehog signaling pathway in T-lineage cells inhibits TCR repertoire selection in the thymus and peripheral T-cell activation Blood, May 1, 2007; 109(9): 3757 - 3766. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. L. Rudd, A. Tua-Smith, and D. B. Straus Lck SH3 Domain Function Is Required for T-Cell Receptor Signals Regulating Thymocyte Development Mol. Cell. Biol., November 1, 2006; 26(21): 7892 - 7900. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. V. Nguyen, Y. Ke, E. E. Zhang, and G.-S. Feng Conditional Deletion of Shp2 Tyrosine Phosphatase in Thymocytes Suppresses Both Pre-TCR and TCR Signals J. Immunol., November 1, 2006; 177(9): 5990 - 5996. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. J. Priatel, X. Chen, S. Dhanji, N. Abraham, and H.-S. Teh RasGRP1 Transmits Prodifferentiation TCR Signaling That Is Crucial for CD4 T Cell Development J. Immunol., August 1, 2006; 177(3): 1470 - 1480. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Liu, B. J. Taylor, G. Sun, and R. Bosselut Analyzing Expression of Perforin, Runx3, and Thpok Genes during Positive Selection Reveals Activation of CD8-Differentiation Programs by MHC II-Signaled Thymocytes J. Immunol., October 1, 2005; 175(7): 4465 - 4474. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Lombardi, D. Burzyn, J. Mundinano, P. Berguer, P. Bekinschtein, H. Costa, L. F. Castillo, A. Goldman, R. Meiss, I. Piazzon, et al. Cathepsin-L Influences the Expression of Extracellular Matrix in Lymphoid Organs and Plays a Role in the Regulation of Thymic Output and of Peripheral T Cell Number J. Immunol., June 1, 2005; 174(11): 7022 - 7032. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Nishida, Y. Matsuki, T. Ono, T. Oguma, K. Tsujimoto, M. Sato, and T. Tadakuma The Novel Murine CD4+CD8+ Thymocyte Cell Line Exhibits Lineage Commitment into Both CD4+ and CD8+ T Cells by Altering the Intensity and the Duration of Anti-CD3 Stimulation In Vitro J. Immunol., June 1, 2004; 172(11): 6634 - 6641. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Aliahmad, E. O'Flaherty, P. Han, O. D. Goularte, B. Wilkinson, M. Satake, J. D. Molkentin, and J. Kaye TOX Provides a Link Between Calcineurin Activation and CD8 Lineage Commitment J. Exp. Med., April 19, 2004; 199(8): 1089 - 1099. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Norment, L. Y. Bogatzki, M. Klinger, E. W. Ojala, M. J. Bevan, and R. J. Kay Transgenic Expression of RasGRP1 Induces the Maturation of Double-Negative Thymocytes and Enhances the Production of CD8 Single-Positive Thymocytes J. Immunol., February 1, 2003; 170(3): 1141 - 1149. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Lucas, L. O. Atherly, and L. J. Berg The Absence of Itk Inhibits Positive Selection Without Changing Lineage Commitment J. Immunol., June 15, 2002; 168(12): 6142 - 6151. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Hayashi, N. Abe, T. Watanabe, M. Obinata, M. Ito, T. Sato, S. Habu, and M. Satake Overexpression of AML1 Transcription Factor Drives Thymocytes into the CD8 Single-Positive Lineage J. Immunol., November 1, 2001; 167(9): 4957 - 4965. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. M. Schaeffer, C. Broussard, J. Debnath, S. Anderson, D. W. McVicar, and P. L. Schwartzberg Tec Family Kinases Modulate Thresholds for Thymocyte Development and Selection J. Exp. Med., October 2, 2000; 192(7): 987 - 1000. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Basson, T. J. Wilson, G. A. Legname, N. Sarner, P. D. Tomlinson, V. L. J. Tybulewicz, and R. Zamoyska Early Growth Response (Egr)-1 Gene Induction in the Thymus in Response to TCR Ligation During Early Steps in Positive Selection Is Not Required for CD8 Lineage Commitment J. Immunol., September 1, 2000; 165(5): 2444 - 2450. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T.-K. Yu, E. G. Caudell, C. Smid, and E. A. Grimm IL-2 Activation of NK Cells: Involvement of MKK1/2/ERK But Not p38 Kinase Pathway J. Immunol., June 15, 2000; 164(12): 6244 - 6251. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Bommhardt, Y. Scheuring, C. Bickel, R. Zamoyska, and T. Hunig MEK Activity Regulates Negative Selection of Immature CD4+CD8+ Thymocytes J. Immunol., March 1, 2000; 164(5): 2326 - 2337. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Lin, N. S. Longo, X. Wang, J. A. Hewitt, and K. M. Abraham Lck Domains Differentially Contribute to Pre-T Cell Receptor (TCR)- and TCR-{alpha}/{beta}-regulated Developmental Transitions J. Exp. Med., February 21, 2000; 191(4): 703 - 716. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. L. Sharp and S. M. Hedrick Commitment to the CD4 Lineage Mediated by Extracellular Signal-Related Kinase Mitogen-Activated Protein Kinase and Lck Signaling J. Immunol., December 15, 1999; 163(12): 6598 - 6605. |
